Clinics March 2013 by Clinics

Volume 68 Number 3 - March/2013

CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Anna Sara Shafferman Levin Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Antonio Egidio Nardi Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Berenice Bilharinho Mendonça Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Carlos Serrano Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Edmund Chada Baracat Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Emilia Inoue Sato Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Fulvio Alexandre Scorza Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Geraldo Busatto Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Gustavo Franco Carvalhal Faculdade de Medicina da Pontifıćia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Ivete Bedin Prado Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Ludhmila Abrahao Hajjar Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Mauricio Etchebehere Universidade Estadual de Campinas Campinas, SP, Brazil Naomi Kondo Nakagawa Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Olavo Pires de Camargo Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Oswaldo Keith Okamoto Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Patricia Rieken Macedo Rocco Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sandro Esteves ANDROFERT - Andrology & Human Reproduction Clinic Campinas, SP, Brazil Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Silvia Vanessa Lourenço Faculdade de Odontologia da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Sophie Françoise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil Suely Kazue Nagahashi Marie Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Thelma Suely Okay Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Vale´ria Aoki Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Walter Gomes Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India

Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ernest Eugene Moore University of Colorado Denver Denver, CO, USA

Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil

Artur Brum-Fernandes Universite´ de Sherbrooke Que´bec, Canada´

Euclides Ayres Castilho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ke-Seng Zhao Southern Medical University Guangzhou, China

Adauto Castelo Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ademar Lopes Fundaçaõ Antoˆnio Prudente, Hospital do Caˆncer Saõ Paulo, SP, Brazil

Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil

Alberto Azoubel Antunes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Daniel Romero Munõz Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Alexandre Roberto Precioso Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany

Andrea Schmitt University of Goettingen Goettingen, Germany Arnaldo Valdir Zumiotti

Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Fa´bio Biscegli Jatene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK

Francisco Laurindo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Marcelo Zugaib Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan

Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello

Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Mauro Perretti William Harvey Research Institute London, UK

Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

Pedro Puech-Leaõ Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Milton de Arruda Martins Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA

Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA

Philip Cohen University of Houston Health Center Houston, Texas, USA

Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA

Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´

Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil

Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Roberto Chiesa San Raffaele Hospital

Milan, Italy Ronald A. Asherson Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica Samir Rasslan Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Board of Governors Alberto Jose´ da Silva Duarte Aluisio Augusto Cotrim Segurado Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonc¸a Carlos Roberto Ribeiro de Carvalho Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Dalton de Alencar Fischer Chamone Daniel Romero Mun˜oz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euripedes Constantino Miguel Fa´bio Biscegli Jatene Flair Jose´ Carrilho Gerson Chadi Gilberto Luis Camanho Irene de Lourdes Noronha Irineu Tadeu Velasco Ivan Cecconello

Jorge Elias Kalil Jose´ Antonio Franchini Ramires Jose´ Antonio Sanches Jose´ Eduardo Krieger Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Lenine Garcia Branda˜o Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Miguel Srougi Milton de Arruda Martins Nelson de Luccia Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hila´rio Nascimento Saldiva

Editorial Director Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Paulo Marcelo Gehm Hoff Pedro Puech-Lea˜o Remo Susanna Ricardo Ferreira Bento Ricardo Nitrini Roberto Kalil Roberto Zatz Roger Chammas Samir Rasslan Sandra Josefina Ferraz Ellero Grisi Selma Lancman Tarcı´sio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Venaˆncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob

Editorial Assistants Nair Gomes Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Ariane Maris Gomes Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Editorial Office: Rua Dr. Ovı´dio Pires de Campos, 225 - 6 ˚ Andar CEP 05403-010 Saõ Paulo/SP Tel.: +55-11-2661-6235 Email: clinics.office@gmail.com Website: www.scielo.br/clinics Submission: http://mc04.manuscriptcentral.com/clinics-scielo Indexations: LILACS; MEDLINE; PubMed; PubMed Central; SciELO; Science Citation Index Expanded (ISI Web of Knowledge); Scopus; Ulrich’s Periodical Directory; Qualis/Capes - Classified as an International Circulation Journal in Medicine. Clinics. Saõ Paulo: Scientific Journal of Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clıńicas da FMUSP, 1946–2004. 1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo. CDD 610

PUBLICATION INFORMATION AND EDITORIAL POLICIES CLINICS publishes peer-reviewed articles of interest to clinicians and researchers in the medical sciences. CLINICS is registered with PubMed Central and SciELO and complies with the policies of funding agencies, such as the Wellcome Trust, the Research Councils UK - (RCUK), the National Institutes of Health (NIH), and the German Research Foundation (DFG), which request or require deposition of the published articles that they fund into publicly available databases. CLINICS supports the position of the International Committee of Medical Journal Editors (http:// www.icmje.org/) on trial registration. All trials initiated after January 1, 2012 must be prospectively registered (before patient recruitment begins) in a publicly accessible registry. Trials initiated before January 1, 2012 must be registered before submission to our journals. See the ICMJE FAQ regarding trial registration for further details. Visit http://www.who.int/ictrp/ network/list_registers/en/index.html for the WHO’s list of approved registries. CLINICS suggests: http://www.clinicaltrials. gov as a user friendly site.

clinical, and surgical research. Original studies must conform to the following format: Title page:

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Publication Fees CLINICS uses a business model in which expenses are recovered in part by charging a publication fee to the authors or research sponsors for each published article. Our 2013 prices are as follows: fast track: US$ 1,500.00; original articles, review articles and rapid communications: US$ 1,200.00. Invited reviews, editorials and letters to the editors: no charge. * The exchange rate for payments in Brazil-Real is the commercial exchange rate of the day the articles is accepted. Clinics uses the Banco do Brasil currency conversion tool. Manuscripts involving human subjects or the use of laboratory animals must clearly state adherence to appropriate guidelines and approval of protocols by their institutional review boards. Photographs that may identify patients or other human participants of studies shall be acceptable only when a legally valid consent form is signed by the participating patient, other human participant, or his/her legally constituted representative. Manuscripts should be digitalized using a Word *.doc-compatible software program and submitted online in English. Authors are strongly advised to submit the manuscript in its final form to a spell check for English (US). Submissions with excessive spelling or syntax mistakes as well as articles in which the meaning is not sufficiently clear shall be returned to authors for correction. Authors are also strongly advised to use abbreviations sparingly whenever possible to avoid jargon and improve the readability of the manuscript. All abbreviations must be defined the first time that they are used. Only terms or expressions that are used at least 5 times throughout the text should be abbreviated. Never use abbreviations that spell common English words, such as FUN, PIN, SCORE and SUN. Please make sure to submit your manuscript in the exact format that is described below. Failure to do so will cause the submission to be returned to you during the preliminary examination by the Editorial Office.

Manuscripts are invited in the following categories: ORIGINAL STUDY: Complete original studies should be submitted in this category. Three sections are offered: basic,

Title (up to 250 characters); Running title (up to 40 characters, letters and spaces); Full address of corresponding author only; Authors’ names (without titles or degrees). Authors should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Such participation must be declared in this section of the manuscript.

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Abstract: Abstracts are limited to 250 words and structured into objectives, method, results, and conclusions. Citations or abbreviations (except internationally recognized abbreviations, such as weights, measures, and physical or chemical abbreviations) are not permitted. Authors are strongly encouraged not to display numerical statistical information but to merely state what is significantly different (or not) between the described parameters. Keywords: For keywords, 3–6 items from the Medical Subject Headings (MeSh) should be used. Introduction: The introduction should set the purpose of the study, provide a brief summary (not a review) of previous relevant studies, and state the new advances in the current investigation. The introduction should not include data or conclusions from the work being reported. A final sentence summarizing the novel finding to be presented is permissible. Materials and Methods: This section should briefly give clear and sufficient information to permit the study to be repeated by others. Standard techniques only need to be referenced. Previously published methods may be briefly described following the reference. Ethics: When reporting experiments on human subjects, indicate whether the procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, which was revised in 1983. When reporting experiments on animals, indicate whether the institution’s guide, a national research council’s guide, or any national law on the care and use of laboratory animals was followed. Results: The results section should be a concise account of the new information that was discovered, with the least personal judgment. Do not repeat in text all the data in the tables and illustrations but briefly describe what these data comprise. Discussion: The discussion should include the significance of the new information and relevance of the new findings in light of existing knowledge. Only unavoidable citations should be included. Citation to review articles are not encouraged in this section. Acknowledgments: This section should be short, concise, and restricted to acknowledgments that are necessary.

References in text: CLINICS adopts the Vancouver format. Cite references in the text using Arabic numerals in the order of appearance, within parentheses, (1) after the previous word, with spacing as in this example: ‘‘Diabetes (2), hypertension (3,4) and alcoholism (5–9) are complex medical problems (10).’’ Under exceptional circumstances, authors’ names may appear in text: Single author: ‘‘Einstein (11) proposed a new theory …’’, Two authors: ‘‘Watson & Crick (12) reported on the structure of …’’, or Three or more authors: ‘‘Smith et al. (13) described …’’ Reference List: Only citations that appear in the text should be referenced. References must be restricted to directly relevant published works, papers, or abstracts. Unpublished papers, unless accepted for publication, should not be cited. Work that is accepted for publication should be referred to as ‘‘in press’’ and a letter of acceptance of the journal must be provided. Authors are responsible for the accuracy and completeness of their references and for correct text citation. Usually the total number of references should not exceed 35. For up to six authors, list all authors. For more than 6 authors, list first six authors followed by ‘‘et al.’’. Tables and Figures: The maximum number of tables and/or figures is six tables and/or figures. Tables: Should be constructed using the table feature in your word processor or using a spreadsheet program such as Excel. The tables should be numbered in order of appearance in the text, using Arabic numerals. Each table should have a title and an explanatory legend, if necessary. All tables must be referenced and succinctly described in the text. Under no circumstances should a table repeat data that are data presented in an illustration. Statistical measures of variation (i.e., standard deviation or standard error) should be identified, and decimal places in tabular data should be restricted to those with mathematical and statistical significance. Figures: Photographs, illustrations, charts, drawings, line graphs, etc are all defined as figures. Number figures consecutively using Arabic numerals in order of appearance. Figure legend(s) should be descriptive and should allow examination of the figure without reference to text. Images must be of professional quality and uploaded as *.tiff files. Generally, figures will be reduced to fit one column of text. The actual magnification of all photomicrographs should be provided, preferably by placing a scale bar on the print. Line graphs and charts should never be sent as *.jpeg illustrations. We recommend preparing line graphs and charts as ExcelH files and copying these files into a Word *.doc sheet.

FAST TRACK ARTICLES: Fast-track articles should follow the same format described above for original studies. The Editorial Office will produce a first-action response in the shortest possible time and will publish accepted fast track articles in the next available issue. Only one article may be submitted as fast track in any calendar year by any author or co-author. In the Comments section, the authors must explain the justification for fast-track publication. Rejection by journals with a higher impact factor than ours is an acceptable reason for requesting fast-track status. However, the reviewers’ reports from the previous submission

must be included in the current submission. Information contained in the comments is limited to the editor and shall remain confidential. No publication fee discount is allowed for accepted fast track articles. REVIEW ARTICLES: Review articles should cover themes that are relevant to medical practice. Spontaneously submitted reviews are welcome; however, potential authors should bear in mind that they are expected to have expertise in the reviewed field. The sections should be arranged as follows:

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Title page: As described in the Original Study section. Manuscript: Abstract, keywords and text should be arranged to cover the subject that is being reviewed. If appropriate, the method of reference collection should be described. The use of headings, subheadings, and paragraph titles is encouraged to improve clarity. Abbreviations, acknowledgments, tables and figures should be formatted as described in the Original Study section. The number of references is at the discretion of the authors. No publication fee discount is allowed for spontaneously submitted review articles that are accepted for publication.

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Title page: As described in the Original Study section. Manuscript: Rapid communications are limited to 1,500 words, not including the reference list, abstract and keywords. Authors should format rapid communications based on the subject at hand. Abstracts are limited to 250 words and structured into objectives, method, results, and conclusions. Citations or abbreviations (except internationally recognized abbreviations, such as weights, measures, and physical or chemical abbreviations) are not permitted. For keywords, 3-6 items from the Medical Subject Headings (MeSh) should be used.

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Currently CLINICS does not accept: case reports, technical notes, retrospective studies, translations and validations of questionnaires, and articles referring to first demonstration in Brazil. Peer Review: Manuscripts are reviewed by at least two expert consultants. Accepted manuscripts are edited to comply with the journalâ&#x20AC;&#x2122;s format, remove redundancies, and improve clarity and understanding without altering meaning. The edited text will be presented to authors for approval. Submission: A copyright transfer form, signed by all authors, must be submitted by fax (55-11-2661-7524) or by mail as soon as the manuscript is submitted. Any financial or other relationships that may lead to a conflict of interest must be

disclosed in the copyright transfer form. If the editor considers this conflict of interest relevant to the paper, a footnote will be added to show the equity interest in or affiliation with the identified commercial firm(s). When the authors are satisfied that the manuscript complies with the journal format, our site should be accessed using the website www.clinics.org.br. The system will guide authors through the manuscript submission process and will prompt authors to input information into specific fields as they are submitting their manuscript. The editorial office and authors will be automatically notified of the submission. Progress of the manuscript through the Editorial Officeâ&#x20AC;&#x2122;s procedures will be available to authors at all times.

ISSN-1807-5932

CLINICS CONTENTS Clinics 2013 68(3):281–435

EDITORIAL

Connective tissue diseases following silicone breast implantation: where do we stand? Juliane G Hortolam, Joze´lio Freire de Carvalho, Simone Appenzeller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

CLINICAL SCIENCES

99mTc-thymine scintigraphy may be a promising method in the diagnosis of breast cancer Monica Pires Ribeiro, Sergio Augusto Lopes de Souza, Flavia Paiva Proenc¸a Lobo Lopes, Paulo Henrique Rosado-de-Castro, Lea Mirian Barbosa da Fonseca, Bianca Gutfilen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

Etiology of liver cirrhosis in Brazil: chronic alcoholism and hepatitis viruses in liver cirrhosis diagnosed in the state of Espı´rito Santo Patricia Lofego Gonçalves, Maria da Penha Zago-Gomes, Carla Couzi Marques, Ana Tereza Mendonça, Carlos Sandoval Gonçalves, Fausto Edmundo Lima Pereira. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

Prima-1 induces apoptosis in bladder cancer cell lines by activating p53 Camila B. Piantino, Sabrina T. Reis, Nayara I. Viana, Iran A. Silva, Denis R. Morais, Alberto A. Antunes, Nelson Dip, Miguel Srougi, Katia R. Leite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

Is there a relationship between endothelial nitric oxide synthase gene polymorphisms and ankylosing spondylitis? Ismail Sari, Yusuf Ziya Igci, Gercek Can, Ali Taylan, Dilek Solmaz, Bulent Gogebakan, Servet Akar, Zeynep Eslik, Giray Bozkaya, Nurullah Akkoc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

Objective evaluation of plantar hyperhidrosis after sympathectomy Nelson Wolosker, Augusto Ishy, Guilherme Yazbek, Jose´ Ribas Milanez de Campos, Paulo Kauffman, Pedro Puech-Lea˜o, Fa´bio Biscegli Jatene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Giant cell arteritis: a multicenter observational study in Brazil Alexandre Wagner Silva de Souza, Karine Yoshiye Kajiyama Okamoto, Fabiano Abrantes, Bruno Schau, Ana Beatriz Santos Bacchiega, Samuel Katsuyuki Shinjo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

Comparison of 7.2% hypertonic saline - 6% hydroxyethyl starch solution and 6% hydroxyethyl starch solution after the induction of anesthesia in patients undergoing elective neurosurgical procedures Liujiazi Shao, Baoguo Wang, Shuangyan Wang, Feng Mu, Ke Gu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Adverse reactions to antituberculosis drugs in Manguinhos, Rio de Janeiro, Brazil Glauciene Santana Damasceno, Lusiele Guaraldo, Elyne Montenegro Engstrom, Mariza Miranda Theme Filha, Reinaldo Souza-Santos, Ana Gloria Godoi Vasconcelos, Suely Rozenfeld . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

Primary myelofibrosis: risk stratification by IPSS identifies patients with poor clinical outcome Bruno Deltreggia Benites, Carolina Silva Costa Lima, Irene Lorand-Metze, Marcia Torresan Delamain, Gislaine Borba Oliveira, Daiane de Almeida, Carmino Antonio de Souza, Jose Vassallo, Katia Borgia Barbosa Pagnano . . . . 339

Electrical impedance tomography to evaluate air distribution prior to extubation in very-low-birthweight infants: a feasibility study Felipe de Souza Rossi, Ana Cristina Zanon Yagui, Luciana Branco Haddad, Alice D’Agostini Deutsch, Celso Moura Rebello . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

Zinc, vitamin A, and glutamine supplementation in Brazilian shantytown children at risk for diarrhea results in sex-specific improvements in verbal learning Aldo A. M. Lima, Michelle P. Kvalsund, Paula P. E. de Souza, I´talo L Figueiredo, Alberto M. Soares, Rosa M. S. Mota, Noe´lia L. Lima, Relana C. Pinkerton, Peter P. Patrick, Richard L. Guerrant, Reinaldo B. Oria´ . . . . . . . . . . . . . . . . 351

Association between muscle strength and the cardiopulmonary status of individuals living with HIV/AIDS Vagner Raso, Roy J. Shephard, Jorge Casseb, Alberto Jose´ da Silva Duarte, Paulo Roberto Santos Silva, Ju´lia Maria D’Andre´a Greve. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

The ‘silence’ of silent brain infarctions may be related to chronic ischemic preconditioning and nonstrategic locations rather than to a small infarction size Chao Feng, Xue Bai, Yu Xu, Ting Hua, Xue-Yuan Liu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

Conjoined twins – twenty years’ experience at a reference center in Brazil Ana Cristina Aoun Tannuri, Julio Americo Pereira Batatinha, Manoel Carlos Prieto Velhote, Uenis Tannuri . . . . . . 371

Fetuin-A levels in hyperthyroidism Baris¸ Onder Pamuk, Hamiyet Yılmaz, Tugba Topcuoglu, Oktay Bilgir, Ozlem C ¸ alan, Gulseren Pamuk, Derun Taner Ertugrul . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

BASIC RESEARCHES

The influence of sleep deprivation and obesity on DNA damage in female Zucker rats Neuli M. Tenorio, Daniel A. Ribeiro, Tathiana A. Alvarenga, Ana Carolina C. Fracalossi, Viviane Carlin, Camila Hirotsu, Sergio Tufik, Monica L. Andersen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385

Blockade of CXCR1/2 chemokine receptors protects against brain damage in ischemic stroke in mice Larissa Fonseca da Cunha Sousa, Fernanda Matos Coelho, David Henrique Rodrigues, Alline Cristina Campos, Lucı´ola da Silva Barcelos, Mauro Martins Teixeira, Milene Alvarenga Rachid, Antonio Lu´cio Teixeira . . . . . . . . . . . . . . . . . . . 391

Hemodynamic and ventilatory response to different levels of hypoxia and hypercapnia in carotid body-denervated rats Joa˜o Paulo J. Sabino, Mauro de Oliveira, Humberto Giusti, Mogens Lesner Glass, Helio C. Salgado, Rubens Fazan Jr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

REVIEWS

Current strategies for preventing renal dysfunction in patients with heart failure: a heart failure stage approach Victor Sarli Issa, Lu´cia Andrade, Edimar Alcides Bocchi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

What has changed in brachial plexus surgery? Marcelo Rosa de Rezende, Gustavo Bersani Silva, Emygdio Jose´ Leomil de Paula, Rames Mattar Junior, Olavo Pires de Camargo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

READERS OPINIONS

Eat as much as you burn - a good diet and eating less should be more important than an intense exercise program for decreasing morbidity and mortality Mustafa Cakar, Sevket Balta, Sait Demirkol, Omer Kurt, Hakan Sarlak, Zekeriya Arslan . . . . . . . . . . . . . . . . . . . . 419

Hyperventilation increases the rate of the rise in arterial blood desflurane concentration during induction - a Gas ManH simulation Junyong In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421

Which device should be chosen for the percutaneous closure of post-traumatic ventricular septal defects? Sait Demirkol, Sevket Balta, Mustafa Cakar, Ugur Kucuk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423

Medical information technologies can increase quality and reduce costs Sigfrido Burgos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

RAPID COMMUNICATIONS

Can fasting plasma glucose and glycated hemoglobin levels predict oral complications following invasive dental procedures in patients with type 2 diabetes mellitus? A preliminary case-control study Ana Carolina Fragoso Motta, Cristiane Aparecida Nogueira Bataglion, Maria Cristina Foss-Freitas, Milton Cesar Foss, Marilena Chinali Komesu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

Design and baseline characteristics of a coronary heart disease prospective cohort: two-year experience from the strategy of registry of acute coronary syndrome study (ERICO study) Alessandra C. Goulart, Itamar S. Santos, Debora Sitnik, Henrique L. Staniak, Ligia M. Fedeli, Carlos Alberto Pastore, Nelson Samesima, Marcio S. Bittencourt, Alexandre C. Pereira, Paulo A. Lotufo, Isabela M. Bensenor . . . . . . . . . . 431

ERRATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

EDITORIAL

Connective tissue diseases following silicone breast implantation: where do we stand? Juliane G Hortolam, Joze´lio Freire de Carvalho, Simone Appenzeller State University of Campinas, Faculty of Medical Science, Department of Medicine, Rheumatology Unit, Campinas/SP, Brazil.

(anti-SSB/La) and anticollagen II (6-10). Antibodies and symptoms were more evident in patients who had possessed implants for a longer time period (10). In conclusion, silicone breast implants may act as a foreign body and elicit autoantibody production. However, thus far, no clear link between the presence of autoantibodies and symptoms has been observed.

Silicone, which is considered biologically inert, has been used in many different types of medical devices, including tubing; breast, joint and penile implants; artificial heart valves; and intraocular lenses, among others. After a silicone implant is placed, a mild reaction to the foreign body is generally observed, followed by encapsulation (1). Case reports of women with silicone breast implants and connective tissue disease (CTD), especially scleroderma, began to appear in English-language medical literature in the 1980s. Currently, it is widely believed that there is no association between silicone breast implants and autoimmune disease (1). However, when a patient presents with autoimmune symptoms after silicone breast implantation, the question often arises again. Although several authors have reported the appearance of CTDs (e.g., scleroderma, Still’s disease, systemic lupus erythematosus (SLE), Sjogren’s Syndrome and dermatomyositis) after silicone breast implantation, several casecontrol studies do not show such an association. However, there are a few points that deserve attention. Silicone breast implants act as a foreign body, and inflammatory responses to silicone, such as granulomatous skin reactions to injected silicone, synovitis around silicone prosthetic joints and lymphadenopathy proximal to silicone prostheses, have been observed (2,3). Microscopic evidence of silicone has been observed far from the original site (e.g., in the liver), suggesting that a small quantity of silicone particles detaches and migrates through the lymphatic or circulatory system to other organs (1,4–6). They may act as adjuvants and start an inflammatory process in joints or activate the immune system and stimulate the production of autoantibodies (1,2). The activation of the immune system has been demonstrated by the presence of antibodies to silicone and the presence of autoantibodies in the sera of patients with silicone breast implants. Although several studies have detected increased titers of anti-silicone antibodies, no association with clinical findings has been observed (6-10). Significantly more patients with silicone breast implants present with positive anti-DNA, Sjogren syndrome antigen B

& AUTHOR CONTRIBUTIONS All authors participated in the preparation and correction of the text.

& REFERENCES 1. Levy Y, Rotman-Pikielny P, Ehrenfeld M, Shoenfeld Y. Silicone breast implantation-induced scleroderma: description of four patients and a critical review of the literature. Lupus. 2009;18(13):1226-32, http://dx. doi.org/10.1177/0961203309347795. 2. Groff CD, Schned AR, Taylor TH. Silicone-induced adenopathy eight years after metacarpophalangeal arthroplasty. Arthritis Rheum. 1981;24(12):1578-81, http://dx.doi.org/10.1002/art.1780241220. 3. Cristie AJ, Weinherger KA, Dietrich M. Silicone lymphadenopathy and synovitis. Complications of silicone elastomer finger joint prostheses, JAMA 1977;237(14):1463-4. 4. Anderson DR, Schwartz J, Cottrill CM, McClain SA, Ross JS, Magidson JG, Klainer A, Bisaccia E. Silicone granuloma in acral skin in a patient with silicone-gel breast implants and systemic sclerosis. Int J Dermatol. 1996;35(1):36-8. 5. Sergott TJ, Limoli JP, Baldwin CM Jr, Laub DR. Human adjuvant disease, possible autoimmune disease after silicone implantation: a review of the literature, case studies. Plast Reconstr Surg. 1986;78(1):104-14. 6. Iannello S, Belfiore F. Silicone breast prosthesis and rheumatoid arthritis: a new systemic disease: siliconosis. A case report and a critical review of the literature. Minerva Med. 1998;89(4):117-9. 7. Goldblum RM, Pelley RP, O’Donell AA, Pyron D, Heggers JP. Antibodies to silicone elastomers and reactions to ventriculoperitoneal shunts. Lancet 1992;340(8818):510-3, http://dx.doi.org/10.1016/01406736(92)91710-P. 8. Teuber SS, Rowley MJ, Yoshida SH, Ansari AA, Gershwin ME. Anticollagen autoantibodies are found in women with silicone breast implants. J Autoimmun. 1993;6(3):367-77, http://dx.doi.org/10.1006/ jaut.1993.1031. 9. Karlson EW, Hankinson SE, Liang MH, Sanchez-Guerrero J, Colditz GA, Rosenau BJ, et al. Association of silicone breast implants with immunologic abnormalities: a prospective study. Am J Med. 1999;106(1):11-19. 10. Zandman-Goddard G, Blank M, Ehrenfeld M, Gilburd B, Peter J, Shoenfeld Y. A comparison of autoantibody production in asymptomatic and symptomatic women with silicone breast implants. J Rheumatol 1999;26(1):73-7.

Email: appenzellersimone@yahoo.com Tel.: 55 19 3289-1818 Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(03)E01

281

CLINICAL SCIENCE

99mTc-thymine scintigraphy may be a promising method in the diagnosis of breast cancer Monica Pires Ribeiro, Sergio Augusto Lopes de Souza, Flavia Paiva Proenc¸a Lobo Lopes, Paulo Henrique Rosado-de-Castro, Lea Mirian Barbosa da Fonseca, Bianca Gutfilen Universidade Federal do Rio de Janeiro, Faculdade de Medicina, Hospital Universita´rio Clementino Fraga Filho, Departamento de Radiologia, Rio de Janeiro/RJ, Brazil.

OBJECTIVE: Mammography has been established as the gold standard for the detection of breast cancer, and imaging techniques such as ultrasonography, magnetic resonance imaging, scintigraphy and positron emission tomography may be useful to improve its sensitivity and specificity. The objective of this study with breast scintigraphy was to evaluate the uptake of 99mTc-thymine in mammary lesions. METHODS: A total of 45 patients were included in this study. Thirty-three patients (73%) were subjected to surgery or percutaneous biopsy, providing histopathological data. The other 12 patients who remained under surveillance received clinical examinations and biannual mammography with a normal follow-up of at least three years, the data from which were used for comparison with the scintimammography results. RESULTS: The majority of patients (64.4%) had clinically impalpable lesions with a mammogram diagnosis of microcalcifications, impalpable nodules, or focal asymmetry. Of the studied lesions, 87% were smaller or equal to 20 mm in diameter, and 22% had malignant histopathological findings. Scintigraphy with 99mTc-thymine had a sensitivity of 70%, a specificity of 85.7%, positive and negative predictive values of 58.3% and 90.9%, respectively, and an accuracy of 82.2%. CONCLUSIONS: The results of this study are consistent with those previously reported by other authors. The good specificity and high negative predictive value of this technique and the absence of uptake in the heart indicate that it may be a promising complementary method in clinical practice and that it may contribute to reducing unnecessary benign biopsies. KEYWORDS: Breast Scintigraphy; 99mTc-thymine; Breast Cancer. Ribeiro MP, Souza SA, Lopes FP, Castro PH, Fonseca LM, Gutfilen B. 99mTc-thymine scintigraphy may be a promising method in the diagnosis of breast cancer. Clinics. 2013;68(3):283-289. Received for publication on September 25, 2012; First review completed on October 3, 2012; Accepted for publication on October 19, 2012 E-mail: bgutfilen@hucff.ufrj.br Tel.: 55 21 2562-6274 / 55 21 2562-6273

trials and observational studies (3-5). Mammographic screening reduces the mortality from breast cancer in women between 40 and 69 years of age by 20 to 35% (4,6). However, not all malignancies are detected using this screening method. Major factors that lead to false-negative findings using mammography include breast density, implants, severe dysplastic disease, and significant architectural distortion following breast surgery or radiation therapy (7-10). Approximately 90 to 95% of women with abnormal mammogram findings do not have breast cancer. The relative inefficiency reflected by this statistic highlights the necessity of new diagnostic methods that can improve the sensitivity and specificity of mammography (10,11). The development of improved non-invasive diagnostic methods for the screening of breast lesions is needed, which would reduce the requirement for invasive procedures such as breast biopsy for a precise diagnosis (10,11). Conventional biopsies, in addition to the potential for the involvement of hospitalization, anesthesia, and clinical complications, are unnecessary in 80% of cases and may induce changes in the

& INTRODUCTION Breast cancer is a major public health problem, the incidence and mortality of which are continuously increasing. Breast cancer represents the most common malignancy in women, affecting more than one million individuals every year worldwide (1,2). An early diagnosis of breast cancer may result in a better prognosis. Thus, regular screenings are recommended by health systems around the world. Mammography has been established as the gold standard method for the detection of breast cancer (3-5). The efficacy of cancer screening using mammography has been demonstrated in both randomized

Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(03)OA01

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as: Category 0 - need additional imaging evaluation; Category 1 - negative; Category 2 - benign finding; Category 3 - probably benign finding - follow-up in a short time frame is suggested; Category 4 - suspicious abnormality, biopsy considered; Category 5 - highly suggestive of malignancy, appropriate action needed; Category 6 - known biopsy - proven malignancy. The average age of the patients studied was 49.9 years and ranged from 26 to 80 years. All patients were informed about the procedure and, after acceptance for inclusion in the study, signed a written informed consent. The research protocol was approved by the research ethics committee of the institution. To evaluate breast lesions, 370 MBq (10 mCi) of 99mTcthymine was injected intravenously into the forearm, contralateral to the breast with the lesion. Patients were placed in a prone position on a foam mattress, and the studied breast was positioned to make it accessible. The other breast remained compressed to the mattress. The homolateral arm of the affected breast was placed above the patientâ&#x20AC;&#x2122;s head in a position as comfortable as possible. The surface of the collimator (a device of the gamma camera that is capable of collimating radiation to produce the patientâ&#x20AC;&#x2122;s images) touched the side of the body with the studied breast. Counts were acquired in a 15% window centered at 140 keV. The acquisition of images was initiated 15 minutes after the administration of 99mTc-thymine, and each acquisition lasted for 10 minutes. Both breasts were evaluated. The first lateral image obtained was of the breast without lesions, followed by the breast with the suspected tumor. Following image acquisition, the patient was placed in the supine position with the hands behind the head. Planar images were analyzed separately by two nuclear medicine physicians and, when the opinions were discordant, the results were determined by consensus. Using a qualitative analysis of images, scintigraphy was considered positive when there was a high uptake of the radiopharmaceutical in a segment of the breast. Scintigraphy was considered negative in cases where there was a homogeneous distribution of the radiopharmaceutical in both breasts. The data were evaluated by the analysis of the sensitivity, specificity, positive and negative predictive values, and measures of accuracy (confidence interval of 95%; 95% CI) for scintigraphy in relation to the standard for histology (positive and negative). The histopathological samples were obtained from study patients with breast mammography results with BI-RADSH classifications of category 4 or 5, which included 33 patients. Twelve patients with BI-RADSH classification of category 3 had been recommended for monitoring with biannual mammography. We used the follow-up period of three years and the radiological gold standard for comparison with the outcome of breast scintigraphy in these patients. The degree of agreement was evaluated using the Kappa coefficient. The criterion for determination of significance was 5%.

breast parenchyma that hinder subsequent mammographic readings (4,10). In addition, procedures such as percutaneous or surgical biopsy can cause anxiety, inconvenience, and discomfort to the patient and can add medical costs to both the patient and the health care system (10-12). In the last decade, progress in pharmacology combined with evolving technology has enabled the establishment of scintimammography as a non-invasive technique for the localization and staging of malignant tumors of the breast using molecules such as Sestamibi and Tetrofosmin labeled with technetium-99m (99mTc) (13-15). Other molecules such as 99mTc(V) dimercaptosuccinic acid (DMSA) and 99mTclabeled alkyl triphenyl phosphonium (99mTc-Mito10MAG3) are also being studied to improve detection (16,17). The quality of scintimammography is not compromised by breast density, which limits the effectiveness of mammography particularly in young women. Breast scintigraphy has a major indication for select groups of patients with dense breasts; patients using hormone therapy; patients suspected of having recurrent disease resulting in architectural distortions secondary to previous surgery; patients undergoing chemotherapy or radiotherapy or who have breast implants; and patients at high risk for breast cancer (15,18-21). Scintimammography is also useful for the study of breast cancer multicentricity (13,15). A recent meta-analysis of 42 studies that used ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), scintimammography, and positron emission tomography (PET) determined that MRI seemed to be a more useful complement to existing surveillance methods to assess patients with suspected recurrent and/or metastatic breast cancer (17). Although MRI can be applied in these patient populations, its usage is limited by higher costs and a higher rate of falsepositive results compared with mammography (4). In previous reports, we presented the use of 99mTcthymine in the evaluation of nodules and the thickening of breast tissue. The pattern of 99mTc-thymine uptake could differentiate malignant lesions of the breast from benign lesions and breast densities in which there is uptake of the radiopharmaceutical (22). We also compared the uptake of 99mTc-thymine and 99mTc-sestamibi in malignant breast lesions and found that 99mTc-thymine exhibited higher sensitivity, specificity, accuracy, and positive predictive values compared to 99mTc-sestamibi (19). Here, we evaluated the use of 99mTc-thymine for the study of malignant microcalcifications and initial palpable and impalpable malignant lesions of the breast.

& METHODS AND MATERIALS Study subjects consisted of 45 patients of the gynecology and mastology services of two public hospitals. Inclusion criteria were: (I) patients who detected changes in their breast self-examination, (II) patients with microcalcifications detected using mammography consisting of asymmetry or focal nodules smaller or equal to 25 mm in diameter, and (III) patients with BI-RADSH categories 3, 4, or 5. We excluded patients based on the following criteria: (I) breast lesions greater than 25 mm in diameter, (II) prior chemo or hormone therapy, (III) breast surgery or prior homolateral radiotherapy, and (IV) pregnancy or lactation. The BI-RADS system includes categories that are used to standardize interpretation of mammograms among radiologists (23). BI-RADS assessment categories can be summarized

& RESULTS Thirty-three of the 45 patients studied (73%) were subjected to surgery or percutaneous biopsy, providing histopathological data. The patients (n = 12) that remained under surveillance received clinical examinations and

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were first described as myocardial perfusion tracers due to their property of accumulating in cells with higher mitochondrial activity and demonstrating perfused myocardium in contrast with ischemic and fibrotic regions. Afterwards, they were applied to detect tumors based on the principle that tumors such as breast cancer generally have higher mitochondrial activity than the surrounding cells. Despite promising initial results, breast scintigraphy with 99mTc-sestamibi is not yet included in the routine diagnosis of breast cancer. The sensitivity of the method was reported to range from 84 to 96% in initial studies, and the specificity ranged from 86 to 100% (13,26,27). However, in more recent publications, particularly in multicenter studies in which a large number of palpable and impalpable lesions were studied, sensitivity and specificity were both reduced (28-30). Tiling et al. (31) reported that tumor size appears to be crucial in limiting the capacity of scintigraphy to detect breast cancer, and a number of studies have also identified a correlation between the sensitivity of breast scintigraphy and the diameter of the tumor (29,31). In a modified meta-analysis of the use of 99mTc-sestamibi for the diagnosis of initial breast cancer in 2,727 lesions, Khalkhali and Itti (32) stated that the overall sensitivity and specificity of breast scintigraphy were 83.3% and 81.3%, respectively. The overall sensitivity reported included palpable and impalpable lesions, which likely explains the difference in reported results in this and other reviews (32). In a multicenter study to assess the effectiveness of breast scintigraphy with 99mTc-sestamibi in 1243 women with 33% palpable lesions, Sampalis et al. (33) concluded that breast scintigraphy was highly effective and potentially useful as a complement to mammography for the early detection of breast cancer. These authors reported a sensitivity and specificity of 93% and 87%, respectively, with a negative predictive value of 98% and a positive predictive value of 58% (33). We previously described a technique for the labeling of thymine, a thymidine precursor, with 99mTc. (19,22). Here, we obtained a sensitivity of 70%, a specificity of 85.7%, an accuracy of 82.2%, and positive and negative predictive values of 58.3% and 90.9%, respectively. It is noteworthy that the total number of patients was 45, which were divided into two groups following BI-RADSH 3 classification: one (n = 33) with histopathological results and the other (n = 12) without biopsy. The false-positive results in most of the other studies using 99mTc-sestamibi resulted from the presence of fibroadenoma, atypical hyperplasia, fibrocystic disease, and inflammatory disease (26,31,34,35). In our study, the four false-positive results also consisted of fibrocystic breast changes (two cases), fibroadenoma (one case), and one case of atypical hyperplasia. Patients with atypical hyperplasia have been shown to have an increased relative risk for breast cancer and a higher incidence of positive breast scintigraphy (36). The results of 99mTcsestamibi scintigraphy are least useful in cases of breast microcalcifications and in situ carcinoma, as reported by Gommans et al. (37). Moreover, unlike 99mTc-thymine, the myocardial uptake of the 99mTc-sestamibi is relatively high, which leads to greater background activity due to the proximity to breast tissues. Despite this, intravenously administered 99mTc-thymine has the potential advantage of being used in radioguided surgeries to detect impalpable breast lesions without conventional Radioguided Occult

biannual mammography with a normal follow-up of at least three years, the data from which were used for comparison with the scintimammography results. A majority of patients (29/45; 64.4%) had clinically impalpable lesions with a mammogram diagnosis of microcalcifications, impalpable nodules, or focal asymmetry. Sixteen patients (35.6%) had palpable lesions. Clustered microcalcifications, which were or were not associated with a nodule, were present in 24/45 patients (53%). Among the studied lesions, 87% were smaller or equal to 20 mm in diameter (41/47). Two patients had two lesions. Ten (22%) of the 45 patients studied had malignant histopathological findings (11 lesions - eight cases of infiltrating ductal carcinoma, one case of in situ ductal carcinoma and invasive lobular carcinoma, and one case of cribriform type intraductal carcinoma. All patients with confirmed breast cancer were between 42 and 75 years old. In comparison with breast mammography, 7/10 of the cancer cases were correctly identified by breast scintigraphy with 99mTc-thymine. In three patients (3/10), breast scintigraphy showed false-negative results (a palpable nodule with normal mammogram, an irregular nodule suspected for malignancy, and clustered microcalcifications on mammography). Thirty patients with negative breast scintigraphy results had histopathology and/or radiological follow-up for three years. Four patients who had positive breast scintigraphy had benign histopathological biopsies. The false-positive results were: two cystic nodules, one fibroadenoma, and an atypical hyperplasia. In one patient (BI-RADSH 3) with a positive breast scintigraphy, histopathological examination was not performed by decision of the mastology service, which followed the patient for three years. Table 1 shows the patientsâ&#x20AC;&#x2122; characteristics, Table 2 lists the measures of accuracy of the patients studied, and Table 3 shows the patients sorted by BI-RADS. We observed a significant concordance (Kappa = 0.520, standard error = 0.147, p = 0.0002) between the diagnosis by scintigraphy and the pattern of histology, but it was of a moderate degree. Figure 1 illustrates a normal distribution of 99mTc-thymine scintigraphy in the anterior view with a high liver uptake. We can observe that there is no heart uptake. Figure 2 is an example of a negative scintigraphy image obtained with 99mTc-thymine. A homogenous distribution of the radiopharmaceutical can be observed in both breasts. Figure 3 shows an example of a positive scintigraphy image obtained with 99mTc-thymine in which an increased uptake of the radiopharmaceutical is observed in the region of the right breast nodule.

& DISCUSSION Although the efficacy of mammography has been demonstrated, perfect sensitivity or specificity is not achieved in women undergoing screening. As such, the issue of adverse consequences for women who do or do not have breast cancer has been a source of growing attention (15,24,25). If breast cancer screening is to be successful, the majority of cancers among screened women must be detected when tumors are small and before the occurrence of distant or nodal metastases. To achieve this, a combination of imaging modalities may be superior to any single screening technique (9). 99mTc-sestamibi and 99mTc-tetrofosmin

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Table 1 - Patient characteristics.

Patient

Age

Location

Side

1 2 3 4

80 58 52 42

UOQ Retroareolar UOQ LIQ

R R L L

5 6 7

52 50 55

UOQ UOQ UIQ

L L L

8 9 10 11

46 43 67 50

L L L R

12 13 14 15 16 17 18 19

54 61 68 56 30 61 56 55

20 21* 22 23 24 25 26 27 28 29 30 31 32

65 36 67 58 39 75 70 45 68 54 61 55 61

LIQ UOQ Retroareolar Asymmetry on UOQ, UIQ UIQ and UOQ UOQ UOQ UIQ Retroareolar UOQ UOQ Asymmetry on LIQ and UIQ UOQ LIQ LOQ UOQ UIQ UOQ LIQ LIQ UOQ UOQ UOQ UOQ Subareolar

R R+L L R R L L R L L L R L

33 34

26 67

UOQ Retroareolar

35 36

63 62

R R L L L L L L

Microcalcifications

BI-RADSH

Yes

Lesion size on mammography (mm)

Scintigraphy

Biopsy

Histopathology

9.2

NEG POS NEG POS

Yes Yes

4 4 4 5

3 4 4

Yes Yes

NEG POS NEG

Follow-up NEG NEG

3 4 3 3

18 and 7 18

NEG POS NEG NEG

Follow-up POS Follow-up Follow-up

Fibrocystic disease IDC (15 mm) Fibrocystic disease In situ ductal carcinoma and invasive lobular carcinoma (20 mm) Fibrocystic disease Usual ductal hyperplasia IDC (18613 mm) -

4 3 4 4 3 4 3 3

17 12 15 25 25

NEG NEG NEG NEG NEG NEG NEG NEG

NEG Follow-up NEG NEG Follow-up NEG Follow-up Follow-up

Fibrocystic disease Fibrocystic disease Fibrocystic disease -

NEG POS (R) NEG NEG POS POS NEG NEG NEG NEG NEG NEG POS

Follow-up NEG NEG NEG NEG POS NEG NEG POS Follow-up NEG POS POS

NEG NEG

Fibroadenoma Fibrocystic disease Fibrocystic disease AEH IDC (10 mm) IDC (25 mm) IDC (10 mm) Cribriform IDC (20 mm) Fibroadenoma Fibrocystic disease, adenosis Fibrocystic disease, adenosis, usual ductal hyperplasia Fibrocystic disease, adenosis Adenosis, usual ductal hyperplasia IDC (20 mm) IDC (25 mm) IDC (23 mm) Fibrocystic disease

Yes Yes

Yes Yes (L) Yes

Yes Yes Yes Yes

4 4 4 4 4 5 4 4 5 4 4 4 4

R R

Yes

4 4

Asymmetry, LIQ UOQ and LOQ

L L

Yes

4 4

NEG NEG

UOQ

Yes

POS

NEG

UOQ

Yes

NEG

39 40 41 42 43 44 45

58 44 48 49 48 36 50

Asymmetry Asymmetry UOQ UOQ UIQ and LIQ UIQ and LIQ LIQ

R L L R R L L

4 4 4 4 4 4 4

POS NEG POS NEG NEG NEG POS

POS POS POS NEG NEG Follow-up Follow-up

Yes Yes

20 (R) 13 15

Abbreviations: UOQ, upper outer quadrant; UIQ, upper inner quadrant; LOQ, lower outer quadrant; LIQ, lower inner quadrant; AEH, atypical epithelial hyperplasia; IDC, invasive ductal carcinoma; NEG, negative; POS, positive; R, right; L, left. * Lesions in both breasts.

Lesion Localization (ROLL) and Sentinel Node and Occult Lesion Localization (SNOLL) techniques. In nearly all reports of breast scintigraphy, 99mTcsestamibi or 99mTc-tetrofosmin was used. The 99mTcthymine results in this study are consistent with those

reported by other authors (30,38), even though the majority of patients in this study had impalpable lesions. To be an acceptable complementary method to mammography, breast scintigraphy must provide high specificity. We obtained a high level of specificity and a high negative

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Table 2 - Measures of accuracy of scintigraphy using 99mTc-thymine for the diagnosis of breast cancer. Measures Sensitivity Specificity Positive Predictive Value Negative Predictive Value Accuracy

LL 95%

UL 95%

70.0 85.7 58.3 90.9 82.2

41.6 74.1 30.4 81.1 71.1

98.4 97.3 86.2 100 93.4

LL 95%: Lower limit of 95%; UL: Upper limit of 95%.

predictive value in this breast scintigraphy study. These make this test attractive because of the potential to reduce unnecessary biopsies. Other studies have reported a better sensitivity but a lower specificity than those reported in this study (39). Although the early detection of breast cancer needs to be improved with the development of equipment that can discover smaller lesions, the employment of 99mTcthymine seems to be more advantageous than other radiopharmaceuticals because it is a precursor to a DNA nitrogenous base that plays an important role in tumoral growth. Some of the main characteristics of the metabolism of tumor cells are the increase of glycolysis and mitochondrial activity, the synthesis of nucleic acids, and the production of proteins previous to mitosis. Unlike methods that simply demonstrate an increase in mitochondrial activity, which may occur in tissues that are not under cell division, 99mTcthymine has the potential to demonstrate the synthesis of nucleic acids related to tumor growth. This effect increases the sensitivity and specificity of tumor detection. Recently, Sun et al. (40) imaged DNA synthesis with PET using 18F- 1-(2’-deoxy-2’-fluoro-beta-D-arabinofuranosyl) thymine (FMAU), a pyrimidine analogue that is phosphorylated by thymidine kinase and incorporated into DNA. Their results demonstrated that 18F-FMAU was selectively retained in the DNA of proliferating cells and was resistant to degradation (40). We have similarly shown that 99mTcthymine is incorporated into cellular DNA (22). Unlike PET and PET-CT, which require expensive equipment with limited availability worldwide, conventional gamma cameras are able to perform SPECT imaging and are much more readily available. Therefore, the use of 99mTc-thymine as a radiopharmaceutical for SPECT imaging has the potential for broader use. It is important to note that our study has limitations; for instance, our study included a great number (87%) of lesions smaller or equal to 20 mm and few patients with malignant lesions. These factors could explain the lower sensitivity that we report for scintigraphy. Further studies are needed to provide a greater understanding of the application of this technique in these two groups. Nonetheless, the high level of specificity and the high negative predictive value of this technique indicate that the use of breast scintigraphy with 99mTc-thymine may be a promising complementary method,

Figure 1 - Scintigraphy shows a normal distribution of 99mTcthymine in anterior view with high liver uptake and no heart uptake.

particularly in selected patients. It may be a useful adjunct method in high-risk patients such as those with prior breast cancer, a very strong family history of breast cancer, lobular cancer in situ, or Paget disease. Scintimammography may also be helpful for the study of borderline lesions of BI-RADS categories 3 and 4 or patients under evaluation for recurrence and adjuvant chemotherapy. Therefore, scintimammography

Table 3 - Patients sorted by BI-RADS category.

BI-RADS 3 BI-RADS 4 BI-RADS 5

Number of patients

Age (mean¡SD)

8 34 3

52.1¡11.1 54.3¡11.1 61.7¡17.4

MicroLesion size on calcifications mammography in (%) mm (mean¡SD) 38% 44% 33%

Figure 2 - A 52-year-old woman with a focal asymmetric density in the lower internal quadrant of the right breast on mammography (BI-RADSH 3). This image shows a negative 99mTcthymine scintigraphy (no uptake).

18.5¡8.0 17.1¡4.7 17.1¡11.2

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10.

11.

12.

13.

14.

Figure 3 - A 58-year-old patient with a nodule (15 mm) in the right breast and a mammographic BI-RADSH 4 (a nodule in the right upper quadrant + microcalcifications). Breast scintigraphy with 99mTc-thymine shows uptake in right breast. Histopathological findings showed an infiltrating ductal carcinoma.

15. 16.

17.

may be a valuable tool in clinical practice that can reduce the number of unnecessary benign biopsies.

18.

& ACKNOWLEDGMENTS This study was supported by grants from Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).

19.

20.

& AUTHOR CONTRIBUTIONS Ribeiro MP contributed to the screening of the patients, design and writing of the paper. Souza SA, Fonseca LM, and Gutfilen B contributed to the execution of scintigraphy, discussion of the results, design and writing of the paper. Lopes FP contributed to the discussion of the results, design and writing of the paper. Castro PH contributed to the design and writing of the paper.

21.

22.

23.

& REFERENCES

24.

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99mTc-thymine scan in breast cancer Ribeiro MP et al. 37. Gommans GM, van der Zant FM, van Dongen A, Boer RO, Teule GJ, de Waard JW. (99M)Technetium-sestamibi scintimammography in nonpalpable breast lesions found on screening X-ray mammography. Eur J Surg Oncol. 2007;33(1):23-7. 38. Itti E, Ahdoot H, Khalkhali I. Scintimammography for the Diagnosis of Breast Cancer. . Journal of Womenâ&#x20AC;&#x2122;s Imaging. 2002;4(2):66-72, http://dx. doi.org/10.1097/00130747-200205000-00005. 39. Prats E, Banzo J, Merono E, Herranz R, Carril JM. 99mTc-MIBI scintimammography as a complement of the mammography in patients with suspected breast cancer. A multicentre experience. Breast. 2001;10(2):109-16. 40. Sun H, Sloan A, Mangner TJ, Vaishampayan U, Muzik O, Collins JM, et al. Imaging DNA synthesis with [18F]FMAU and positron emission tomography in patients with cancer. Eur J Nucl Med Mol Imaging. 2005;32(1):15-22, http://dx.doi.org/10.1007/s00259-004-17138.

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289

CLINICAL SCIENCE

Etiology of liver cirrhosis in Brazil: chronic alcoholism and hepatitis viruses in liver cirrhosis diagnosed in the state of Espı´rito Santo Patricia Lofego Gonçalves,I Maria da Penha Zago-Gomes,I Carla Couzi Marques,I Ana Tereza Mendonça,I Carlos Sandoval Gonçalves,I Fausto Edmundo Lima PereiraII I

Universidade Federal do Espı´rito Santo (UFES), Hospital Universita´rio Cassiano A Moraes Serviço de Gastroenterologia, Vito´ria/ES, Brazil. II Universidade Federal do Espı´rito Santo (UFES), Nućleo de Doenças Infecciosas do Centro de Cieˆncias da Sau´de, Vito´ria/ES, Brazil.

OBJECTIVES: To report the etiology of liver cirrhosis cases diagnosed at the University Hospital in Vitoria, Espirito Santo, Brazil. METHODS: The medical charts of patients with liver cirrhosis who presented to the University Hospital in Vitoria were reviewed. Chronic alcoholism and the presence of hepatitis B or C infections (HBV and HCV, respectively) were pursued in all cases. RESULTS: The sample consisted of 1,516 cases (male:female ratio 3.5:1, aged 53.2¡12.6 years). The following main etiological factors were observed: chronic alcoholism alone (39.7%), chronic alcoholism in association with HBV or HCV (16.1%), HCV alone (14.5%) and in association with alcoholism (8.6%) (total, 23.1%), and HBV alone (13.1%) and in association with alcoholism (7.5%, total 20.6%). The remaining etiologies included cryptogenic cases (9.8%) and other causes (6.0%). The mean patient age was lower and the male-to-female ratio was higher in the cirrhosis cases that were associated with alcoholism or HBV compared with other causes. Intravenous drug abuse and a history of surgery or blood transfusion were significantly associated with HCV infection. Hepatocellular carcinoma was present at the time of diagnosis in 15.4% of cases. Chronic alcoholism associated with HCV infection was significantly associated (p,0.001) with reduced age (at the time of cirrhosis diagnosis) and increased prevalence of hepatocellular carcinoma (p = 0.052). CONCLUSION: Alcoholism, HCV and HBV are the main factors associated with liver cirrhosis in the state of Espirito Santo. Chronic alcoholism associated with HCV infection reduced the age of patients at the time of liver cirrhosis diagnosis. KEYWORDS: Liver Cirrhosis; Hepatitis B Virus; Hepatitis C Virus; Alcoholism. Gonçalves PL, Zago-Gomes MP, Marques CC, Mendonça AT, Gonçalves CS, Pereira FE. Etiology of liver cirrhosis in Brazil: chronic alcoholism and hepatitis viruses in liver cirrhosis diagnosed in the state of Espı´rito Santo. Clinics. 2013;68(3):291-295. Received for publication on July 3, 2012; First review completed on August 31, 2012; Accepted for publication on November 1, 2012 E-mail: felp@ndi.ufes.br Tel.: 55 27 3335-7210

morphological features, but these reports did not include information about the etiology of the associated chronic liver disease (1). Additionally, three studies reported mortality from liver cirrhosis; these studies were based on the analysis of death certificates, and none investigated the etiology (2-4). In Vitoria, the capital of the state of Espirito Santo, the University Hospital Cassiano A Moraes (HUCAM) is a referral hospital for patients with liver disease. Since 1993, all patients presenting with chronic liver disease to the HUCAM Gastroenterology outpatient clinic have been evaluated for chronic alcoholism, hepatitis B (HBV, evaluated with HBsAg) and hepatitis C (HCV, evaluated with anti-HCV). The aim of this study was to review the cases of liver cirrhosis diagnosed at HUCAM between 1993 and 2011 to identify the etiology of cirrhosis, particularly as it related to alcoholism and HBV and HCV. This investigation was justified for many reasons: (a) in PubMed, there is a lack of

& INTRODUCTION There are few reports on the etiology of liver cirrhosis in Brazil. A systematic search in PUBMED (January 2012, search limited to human studies) using the keywords ‘‘liver cirrhosis Brazil’’ yielded 409 publications; however, none focused on the etiology of cirrhosis in relation to chronic alcoholism and hepatitis B and C infection in all patients. There are reports of anatomic studies of liver cirrhosis in large series of autopsies with descriptions of patient

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Table 1 - Etiology in 1,516 liver cirrhosis cases.

Etiology*

N (%) [M:F ratio]

Chronic alcoholism HCV HCV plus alcoholism HBV HBV plus alcoholism Other causes Cryptogenic

602 (39.7) 221 (14.5) 131 (8.6) 199 (13.1) 114 (7.5) 92 (6.0) 149 (9.8)

[13.3:1] [0.97:1] [7.2:1] [4.4:1] [66:1] [0.4:1] [1.4:1]

Age (mean¡SD)

p-value** (MxF)

All cases

Male

Female

51.6¡10.8# 56.9¡11.7 50.3¡11.8## 51.7¡13.8 50.2¡11.4### 55.0¡17.7 60.1¡14.1

51.8¡10.8 55.6¡12.1 49.8¡9.8 51.0¡13.7 50.3¡11.4 57.0¡17.4 59.9¡13.8

0.6¡10.7 58.1¡11.3 52.9¡10.1 54.7¡14.3 45.5¡6.3 54.2¡17.9 60.4¡14.5

0.508 0.108 0.249 0.143 0.554 0.495 0.840

Five cases were HBV and HCV positive, and three had chronic alcoholism and HBV and HCV infection. M = male; F = female; HCV = hepatitis C virus; HBV = hepatitis B virus. **Age comparison between males and females. # Mean patient age for chronic alcoholism and HBV versus HCV, Other causes and cryptogenic: p,0.05; chronic alcoholism versus HBV, p = 0.461. ## Mean patient age for HCV versus HCV plus alcoholism, p,0.001. ### Mean patient age for HBV versus HBV plus alcoholism, p = 0.338.

information published on the etiology of liver cirrhosis in Brazil; (b) the study population is representative of liver cirrhosis occurring in the state because HUCAM is a referral hospital that treats patients from all municipalities; and (c) although there are geographical variations in the environmental factors associated with liver cirrhosis, the state of Espirito Santo has a population with demographic characteristics that are similar to the Brazilian population at large.

This research was approved by the Ethics Committee of the Health Sciences Center of the Federal University of Espirito Santo.

& RESULTS The male-to-female ratio of the 1,516 patients included in this study was 3.5:1, and the average age was 53.2¡12.6 years (52.4¡12.0 years for males and 56.1¡13.9 years for females, p,0.001). The ethnic distribution based on skin color was 69.5% Caucasian and 29.8% Afro-descent, with no gender distribution difference. The main etiologies of liver cirrhosis, the mean patient ages and the gender distribution according to the etiology are summarized in Table 1. Chronic alcoholism was the most frequent etiology (39.7%), followed by HCV alone (14.5%) and HBV alone (13.1%). Other causes of cirrhosis included non-alcoholic steatohepatitis (67 cases, 4.4%), primary biliary cirrhosis (12 cases, 0.8%) and autoimmune hepatitis (10 cases, 0.7%); sclerosing cholangitis, secondary biliary cirrhosis and metabolic diseases were less frequent, totaling 0.1% of cases. In 9.8% of cases, the cirrhosis was considered cryptogenic. In 16.1% of the cases, chronic alcoholism was associated with HCV or HBV infection. Both the average patient age and male-to-female ratio differed significantly between the cases of alcoholic cirrhosis and cryptogenic cirrhosis or cirrhosis associated with viruses (p,0.001). Moreover, when viral infection was associated with chronic alcoholism, the demographic profile of HBV- or HCV-associated cirrhosis changed; patient age at the time of diagnosis decreased in the HCV cases

& PATIENTS AND METHODS The study population consisted of 1,516 patients with liver cirrhosis who presented to the Gastroenterology Department of HUCAM between December 1993 and December 2011. We only included those cases that simultaneously performed the evaluations of chronic alcoholism and hepatitis B and C infections. All of the laboratory testing was performed with commercial kits at the routine hospital laboratory. Persistent infection with HBV and HCV was assessed by the evaluating HBsAg and anti-HCV antibodies in serum, respectively. HCV infection was confirmed by detecting the virus in the plasma with a polymerase chain reaction (PCR). According to criteria used by the HUCAM Gastroenterology Department, patients were diagnosed with chronic alcoholism when the ethanol ingestion was .80 g per day in men and .40 g per day in women for ten years or longer. Liver cirrhosis was diagnosed according to clinical data in addition to either a positive radiologic or pathologic result (i.e., computerized tomography [CT] scan, nuclear magnetic resonance imaging, ultrasonography, endoscopy or liver biopsy). In addition, the patient age, gender, intravenous drug abuse, history of surgery and blood transfusion, sexual risk behavior (defined as more than one sexual partner per year without protection) and the presence of associated hepatocellular carcinoma (HCC) at the time of diagnosis of cirrhosis were recorded in all cases. HCC was diagnosed using the following criteria: (a) typical pattern in two imaging methods; (b) one suggestive image plus increased plasma levels of alpha-fetoprotein (.200 ng/ml); or (c) histopathology (5). The statistical analysis was performed using the SPSS for Windows, version 1.9 (IBM, USA). A p-value of ,0.05 was considered significant.

Table 2 - Risk factors associated with HCV, HBV or chronic alcoholism associated with liver cirrhosis. Risk factors Blood transfusion Yes No Surgery Yes No I.V. drug abuse Yes No *

292

HCV

HBV

Alcoholism

p-value*

172 161

31 271

47 509

,0.001

191 141

75** 137

107 449

,0.001

75 137

5 297

18 537

,0.001

HCV versus HBV or Alcoholism; **HBV versus alcoholism, p,0.001 for surgery and p.0.05 for blood transfusion or I.V. drug abuse.

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Liver Cirrhosis in Brazil Gonc存alves PL et al.

(p,0.001), although the observed age reduction was not significant in the HBV cases (p = 0.338). However, the male-tofemale ratio increased in HBV and HCV cases (p,0.001). The patient history of surgery or blood transfusion and intravenous drug abuse in relation to disease etiology is summarized in Table 2. These factors were significantly associated with cirrhosis related to HCV infection compared with frequencies in cirrhosis associated with alcoholism or HBV (p,0.001). Sexual risk behavior did not show any relationship with etiology (data not shown). At the time of the cirrhosis diagnosis, 15.4% of patients were simultaneously diagnosed with HCC (Table 3). This association was significantly higher in the cirrhotic cases associated with HBV and HCV, as well as the cryptogenic cases, compared with the alcoholic cirrhosis cases (p,0.001). However, this association increased when viral infection was associated with chronic alcoholism (HBV or HCV alone 81/420, HBV and alcoholism or HCV and alcoholism 63/ 245, p = 0.052).

than for those patients considered at risk of developing chronic liver disease (11). It is difficult to compare the data on the etiology of cirrhosis presented herein with the data observed worldwide because there are few reports on the etiology of cirrhosis in which the three main risk factors (alcoholism, HBV and HCV infections) were investigated simultaneously. A systematic literature review on alcohol abuse and chronic liver disease showed that 32% of cirrhosis was associated with chronic alcoholism (12). In Europe, particularly in Ireland, UK (6), Italy (13) and Eastern Europe (14), chronic alcoholism is recognized as an important cause of liver cirrhosis. Reduced alcohol consumption, paralleling a decreased incidence of and mortality from liver cirrhosis, has been reported in several European countries (14), confirming chronic alcoholism as an important etiological factor of liver cirrhosis. In Latin America, studies in Mexico (8) and Chile (15) showed that alcohol was the most frequent cause of cirrhosis in 39.5% and 46.3% of cases, respectively. However, it is possible that there is an overdiagnosis of alcoholic cirrhosis in some countries that have underestimated HBV and HCV infection, as it was recently emphasized with respect to the etiology of liver cirrhosis in Mexico (16). A review of published papers on the impact of HBV and HCV on the etiology of liver cirrhosis and hepatocellular carcinoma worldwide demonstrated a large variation in the occurrence of HBV- and HCV-associated liver cirrhosis. The contribution of HBV in the etiology of cirrhosis ranged from 5% in Japan to 57% in China, Mongolia, South Korea and Taiwan. The frequencies of HCV-related cirrhosis ranged from 16% in Africa to 62% in Japan (17). In this study, HBV and HCV alone or in association with chronic alcoholism was present in 43.7% of cases (13.1% and 7.5%, respectively, for HBV and 14.5% and 8.6%, respectively, for HCV). In the MEDLINE literature search, no reports were found on the prevalence of HBV or HCV in cases of liver cirrhosis in Brazil. However, two national surveys showed a common association between HCV and HBV infection and HCC but with variable proportions in different regions of the country (18,19). Our data showing a high prevalence of HBVassociated cirrhosis further support the existing data that demonstrate the prevalence of HBV in the state of Espirito Santo (20). Moreover, HBV-associated HCC is frequently diagnosed at the University Hospital in Vitoria. The contribution of HBV and HCV in the etiology of liver cirrhosis in other Brazilian regions may be similar to that observed in the two national surveys of hepatocellular carcinoma; a country-wide high prevalence of HCV and the increased frequency of HBV in areas where high or intermediate chronic HBV infection has been reported, particularly in the north and central west regions and in the state of Espirito Santo. In cirrhotic cases of viral etiology, the mean patient age was decreased when those cases were associated with chronic alcoholism. The average patient age was significantly lower in the cases with HCV plus chronic alcoholism compared with cases of HCV alone, which supported the premise that chronic alcoholism was an accelerating factor in the evolution of fibrosis in chronic hepatitis C (21,22). In relation to HBV, the effects of associated chronic alcoholism in progression of liver fibrosis are less clear. Chronic alcoholism facilitates the proliferation of the virus in experimental models of HBV infection (23); however, few studies have investigated the progression of lesions in

& DISCUSSION We believe that this is the first Brazilian study to investigate the etiology of liver cirrhosis associated with alcoholism and hepatitis B and C viruses in a significant sampling of patients. When compared with the data reported in the literature (6-8), our study population had a lower mean patient age and higher male-to-female ratio. These differences may be related to the uniqueness of our study population; there were a large number of cirrhotic cases associated with chronic alcoholism and HBV. Importantly, when associated with these two conditions, cirrhosis usually occurs more often at an earlier age and in men (8,9). In fact, as summarized in Table 1, the mean patient age was lower, and the male-to-female ratio was higher in those cases associated with alcoholism or HBV compared with the other etiologies, including HCV, as well as in the cryptogenic cases (p,0.05). The results demonstrated that alcohol abuse was the main etiology of cirrhosis in this case series; this observation supported prior reports of the high prevalence of alcohol abuse in the state of Espirito Santo (27.8% in men and 10.8% in women in metropolitan Vitoria in 2008 according to the Health Ministry) (10). Furthermore, in this study, the criteria defining patients as chronic alcoholics were even stricter Table 3 - Presence of hepatocellular carcinoma at the time of diagnosis among 1,516 liver cirrhosis cases. Etiology Chronic alcoholism HBV (all cases) HBV alone HBV plus ethanol HCV (all cases) HCV alone HCV plus ethanol Cryptogenic Other causes *

Associated hepatocellular carcinoma N of HCC/N of cirrhosis (%) 49/602 (8.1)* 88/313 (28.1) 51/199 (25.6) 37/114 (32.4) 56/352 (15.9) 30/221 (13.5) 26/131 (19.8) 30/149 (20.1) 9/92 (9.7)

Chronic alcoholism versus HBV (all cases) or HCV (all cases) or cryptogenic etiology. p,0.001; chronic alcoholism versus other causes, p = 0.595.

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differences in environmental and behavioral factors associated with the etiologies of liver cirrhosis, particularly hepatitis viruses and chronic alcoholism. Further studies on the etiology of liver cirrhosis from other Brazilian regions are needed to improve the understanding of the etiology of end-stage liver disease in our country.

patients with chronic hepatitis B associated with chronic alcoholism (24). Alcohol abuse in patients with chronic hepatitis B has been associated with increased mortality (25) and increased risk for HCC (26). Moreover, studies in Japan (27) and Brazil (28) have demonstrated a significantly lower mean age of patients when HBsAg-positive HCC was associated with chronic alcoholism compared with the HBsAg-positive cases without chronic alcoholism. In cases reported here, the mean patient age at the time of diagnosis was decreased in the cases of HBV infection associated with chronic alcoholism compared with cases of HBV infection alone. However, the difference was not statistically significant, which suggested that chronic alcoholism impacted the progression to liver cirrhosis in patients with chronic hepatitis B less than in patients with chronic hepatitis C. In evaluating the risk factors for viral infection, the history of surgery and blood transfusion and the use of intravenous drugs were significantly associated with hepatitis C virus infection; this correlation supported other findings in the literature (29). Among other etiological factors associated with liver cirrhosis, the most frequent included non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis and autoimmune hepatitis. More rare etiologies included sclerosing cholangitis, secondary biliary cirrhosis and metabolic diseases. The frequency of cirrhosis associated with NASH reflected the increasing prevalence of steatohepatitis associated with obesity and dyslipidemia, which has been observed worldwide (30). Further investigations are needed to verify whether the observed low frequencies of cirrhosis associated with autoimmune injury are caused by difficulties in diagnostic resources or by the low prevalence of these conditions in this community. The prevalence of primary biliary cirrhosis and autoimmune hepatitis varies worldwide (31,32). The prevalence of cryptogenic cirrhosis, which was similar to that observed in Mexico (8), was higher than that observed in developed countries, such as the United States and Japan (7,33). The prevalence of cryptogenic cases may be at least partially related to the underdiagnosis of some etiologies in the first ten years of this study because of limited diagnostic capabilities and particularly, steatohepatitis, given its recent rapidly increasing prevalence. The presence of hepatocellular carcinoma at the time of diagnosis in 15.4% of cirrhosis supports the high risk of tumor development in patients with liver cirrhosis (26). As shown worldwide (26), our observations confirmed that patients with cirrhosis associated with HBV or HCV were at high risk for developing hepatocellular carcinoma, with the risk increasing in association with chronic alcoholism. In conclusion, this analysis evaluated a large number of liver cirrhosis cases that were diagnosed at the University Hospital in the state of Espirito Santo and simultaneously investigated chronic alcoholism and HBV and HCV infection. We showed that chronic alcohol consumption alone was the most frequent etiologic factor and that when associated with HBV or HCV, decreased the age at diagnosis and increased the frequency of associated hepatocellular carcinoma. These results confirmed that alcohol abuse was an important factor in worsening the evolution of chronic hepatitis B and C infection. Brazil is a large country; therefore, the data presented here may not be applicable to other states in the country given the regional

& ACKNOWLEDGMENTS This research was supported by intramural funding from the Infectious Diseases Unit at the Health Sciences Center, Federal University of Espirito Santo.

& AUTHOR CONTRIBUTIONS Gonçalves PL reviewed the files, analyzed the data and wrote the draft of the manuscript. Zago-Gomes MP, Mendonça AT and Marques CC participated in the file review. Gonçalves CS analyzed the data. Pereira FE planned the study, participated in the result analysis and reviewed the final manuscript.

& REFERENCES 1. Montenegro MR, Da Silva LC, Pontes JF. An evaluation of the problem of hepatic cirrhosis as seen in Sao Paulo, Brazil. I. Criteria for classification and incidence. Gastroenterology. 1957;33(2):178-91. 2. Puffer RR, Griffith GW. Caracteristicas de la mortalidad urbana. Washington, DC: Organazicion Panamericana de La Salud; 1968 (Scientific Publication 151). 3. Guimara˜es C, Pacheco-de-Souza JM, Jorge MH, Laurenti R, Gotlieb SL, Santo AH, et al. [Mortality of adults 15 to 74 years of age in Saõ Paulo, Botucatu and Saõ Manoel (Brazil), 1974/1975]. Rev Saude Publica. 1979;13(Suppl 2):1-73. 4. Lessa I. Cirrhosis of the liver in Brazil: mortality and productive years of life lost prematurely. Rev Panam Salud Publica/Pan Am J Public Health. 1997;1:125-32. 5. Bruix J, Sherman M. Practice Guidelines Committee, American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma. Hepatology. 2005;42(5):1208-36. 6. Fleming KM, Aithal GP, Solaymani-Dodaran M, Card TR, West J. Incidence and prevalence of cirrhosis in the United Kingdom, 1992-2001: a general population-based study. J Hepatol. 2008;49(5):732-8. 7. Michitaka K, Nishiguchi S, Aoyagi Y, Hiasa Y, Tokumoto Y, Onji M. The Japan Etiology of Liver Cirrhosis Study Group. Etiology of liver cirrhosis in Japan: a nationwide survey. J Gastroenterol. 2010;45(1):86-94. 8. Meńdez-Sańchez N, Aguilar-Ramı´rez JR, Reyes A, Dehesa M, Juo´rez A, Castn˜eda B et al. Etiology of liver cirrhosis in Mexico. Ann Hepatol. 2004;3(1):30-3. 9. Fattovich G, Bortolotti F, Donato F. Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors. J Hepatol. 2008;48(2):335-52. 10. Brasil, Ministe´rio da Sau´de. Indicadores de fatores de risco e de proteçaõ. Disponı´vel online:,http://tabnet.datasus.gov.br/cgi/dh.exe?idb2009/g05. def. 11. O’Shea RS, Dasarathy S, McCullough AJ. Practice Guideline Committee of the American Association for the Study of Liver Diseases; Practice Parameters Committee of the American College of Gastroenterology. Alcoholic liver disease. Hepatology. 2010;51(1):307-28. 12. Rehm J, Taylor B, Mohapatra S, Irving H, Baliunas D, Patra J, et al. Alcohol as a risk factor for liver cirrhosis: a systematic review and metaanalysis. Drug Alcohol Rev. 2010;29(4):437-45. 13. Corrao G, Zambon A, Torchio P, Arico` S, La Vecchia C, di Orio F. Attributable risk for symptomatic liver cirrhosis in Italy. Collaborative Groups for the Study of Liver Diseases in Italy. J Hepatol. 1998;28(4):608-14. 14. Zaton´ski WA, Sulkowska U, Manćzuk M, Rehm J, Boffetta P, Lowenfels AB, et al. Liver cirrhosis mortality in Europe, with special attention to Central and Eastern Europe. Eur Addict Res. 2010;16(4):193-201. 15. Alonso FT, Garmendia ML, Aguirre M, Searle J. Analisis de la tendencia de la mortalidade por cirrosis hepa´tica en Chile: Anõs 1990 a 2007. Rev Med Chile. 2010;138(10):1253-8. 16. Torres-Poveda K, Burguete-Garcıá AI, Madrid-Marina V. Liver cirrhosis and hepatocellular carcinoma in Mexico: impact of chronic infection by hepatitis viruses B and C. Ann Hepatol. 201;10(4):556-8. 17. Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol. 2006;45(4):529-38. 18. Gonçalves CS, Pereira FE, Gayotto LC. Hepatocellular carcinoma in Brazil: report of a national survey (Floriano´polis, SC, 1995). Rev Inst Med Trop Sao Paulo.1997;39(3):165-70.

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19. Carrilho FJ, Kikuchi L, Branco F, Gonc存alves CS, Mattos AA, Brazilian HCC Study Group. Clinical and epidemiological aspects of hepatocellular carcinoma in Brazil. Clinics. 2010;65(12):1285-90. 20. Yoshida CF, Camargo IF, Mercadante LA, Gaspar AM, Gomes DF, Schatzmayr HG. Hepatitis B serological patterns of asymptomatic carriers in an endemic region and evaluation of blood plasma as a source of hepatitis B vaccine. Vaccine. 1986;4(4):253-6. 21. Gitto S, Micco L, Conti F, Andreone P, Bernardi M. Alcohol and viral hepatitis: a mini-review. Dig Liver Dis. 2009;41(1):67-70. 22. Mueller S, Millonig G, Seitz HK. Alcoholic liver disease and hepatitis C: a frequently underestimated combination. World J Gastroenterol. 2009; 15(28):3462-71. 23. Larkin J, Clayton MM, Liu J, Feitelson MA. Chronic ethanol consumption stimulates hepatitis B virus gene expression and replication in transgenic mice. Hepatology. 2001;34(4 Pt 1):792-7. 24. Corrao G, Torchio P, Zambon A, Ferrari P, Arico` S, di Orio F. Exploring the combined action of lifetime alcohol intake and chronic hepatotropic virus infections on the risk of symptomatic liver cirrhosis. Collaborative Groups for the Study of Liver Diseases in Italy. Eur J Epidemiol. 1998;14(5):44756. 25. Marcellin P, Pequignot F, Delarocque-Astagneau E, Zarski JP, Ganne N, Hillon P, et al. Mortality related to chronic hepatitis B and chronic

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CLINICAL SCIENCE

Prima-1 induces apoptosis in bladder cancer cell lines by activating p53 Camila B. Piantino, Sabrina T. Reis, Nayara I. Viana, Iran A. Silva, Denis R. Morais, Alberto A. Antunes, Nelson Dip, Miguel Srougi, Katia R. Leite Faculdade de Medicina da Universidade de Sa˜o Paulo, Laboratory of Medical Investigation, Urology Department – LIM55, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: Bladder cancer represents 3% of all carcinomas in the Brazilian population and ranks second in incidence among urological tumors, after prostate cancer. The loss of p53 function is the main genetic alteration related to the development of high-grade muscle-invasive disease. Prima-1 is a small molecule that restores tumor suppressor function to mutant p53 and induces cancer cell death in various cancer types. Our aim was to investigate the ability of Prima-1 to induce apoptosis after DNA damage in bladder cancer cell lines. METHOD: The therapeutic effect of Prima-1 was studied in two bladder cancer cell lines: T24, which is characterized by a p53 mutation, and RT4, which is the wild-type for the p53 gene. Morphological features of apoptosis induced by p53, including mitochondrial membrane potential changes and the expression of thirteen genes involved in apoptosis, were assessed by microscopic observation and quantitative real-time PCR (qRTPCR). RESULTS: Prima-1 was able to reactivate p53 function in the T24 (p53 mt) bladder cancer cell line and promote apoptosis via the induction of Bax and Puma expression, activation of the caspase cascade and disruption of the mitochondrial membrane in a BAK-independent manner. CONCLUSION: Prima-1 is able to restore the transcriptional activity of p53. Experimental studies in vivo may be conducted to test this molecule as a new therapeutic agent for urothelial carcinomas of the bladder, which characteristically harbor p53 mutations. KEYWORDS: Bladder cancer; p53; Apoptosis; Prima-1. Piantino CB, Reis ST, Viana NI, Silva IA, Morais DR, Antunes AA, et al. Prima-1 induces apoptosis in bladder cancer cell lines by activating p53. Clinics. 2013;68(3):297-303. Received for publication on August 20, 2012; First review completed on October 3, 2012; Accepted for publication on November 4, 2012 E-mail: camilapiantino@hotmail.com Tel.: 55 11 3061-7183

particular, p53 loss of function has been related to the development of high-grade muscle-invasive disease (5). As a key gatekeeper of the G1/S checkpoint for cell-cycle progression, p53 is indispensable for maintaining genomic stability and keeping urothelial growth in check. Structural and functional p53 defects are found in over one-half of human urothelial carcinomas, contributing to genomic instability and consequently, numerous chromosomal aberrations. p53 mutations first affect one allele, followed by a second that may be a mutation, deletion or silencing by methylation of the additional wild-type allele, which disables the function of this tumor suppressor gene (6). Consistent with this action, the expression of p21 (WAF1), an important p53 downstream target, is downregulated in the majority of urothelial carcinomas that harbor p53 mutations (7). Several codons in the p53 gene appear to be preferentially mutated, including codons 280 and 285 (8). The specific mutation of these codons is rare in other epithelial tumors; thus, it has been suggested that they result from a urothelium-specific carcinogen or carcinogenic event. Prima-1 (p53 reactivation and induction of massive apoptosis 1) is a low–molecular weight compound identified

& INTRODUCTION Bladder cancer (BC) represents 3% of all carcinomas in the Brazilian male population and ranks second in incidence among urological tumors, after prostate cancer (1), with 73,310 new cases and 14,880 deaths estimated for 2012 in the USA (2). Tobacco smoking, occupational exposure to aromatic amines, arsenic, radiation exposure from the radiation therapy of neighboring organs and the therapeutic use of alkylating agents are the main risk factors for the development of urothelial tumors (3). A large number of genetic changes have been associated with the genesis and progression of BC, and many of them appear to abrogate the G1/S cell cycle checkpoint (4). In

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serum (Sigma, St. Louis, MO, USA) in a 5% CO2 atmosphere at 37˚C. RNA concentrations were determined by measuring the absorbance at 260/280 nm using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA). cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Reactions were incubated at 25˚C for 10 min, followed by 37˚C for 120 min and 85˚C for 5 min. The cDNA was stored at 220˚C until use.

in a cell-based chemical library screen at the National Cancer Institute (National Institutes of Health, Bethesda, MD). This compound has been demonstrated to suppress the growth of cells that harbor mutant p53 by restoring the sequencespecific DNA-binding, wild-type conformation and transcriptional transactivation of mutant p53. Prima-1 has been shown to induce apoptosis in human tumor cells in a p53dependent manner and suppress the growth of human tumor xenografts carrying mutant p53 (9). In this study, we examined the effects of Prima-1 on the T24 and RT4 BC cell lines, which express a mutant and wildtype p53 gene, respectively, and analyzed whether this compound was able to induce p53 activity after UVC damage. To test this hypothesis, we evaluated whether exposing p53 wild-type (RT4) and p53 mutant (T24) cells to Prima-1 and ultraviolet radiation (UVC) alters the expression of p53 downstream genes that are related to apoptosis induction.

Quantitative Real-Time PCR (qRT-PCR) and Gene Expression The expression level of 13 genes (Table 1) was analyzed by qRT-PCR using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). Target sequences were amplified in a 10 ml reaction containing 5 ml of TaqMan Universal PCR Master Mix, 0.5 ml of a TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA, USA) (primers and probes, see Table I), 1 ml of cDNA and 3.5 ml of DNase-free water. The PCR cycling conditions were as follows: 2 min at 50 ˚C, 10 min at 95 ˚C and 40 cycles of 15 seconds at 95 ˚C and 1 min at 60 ˚C. A TaqMan B2M (Applied Biosystems, Foster City, CA, USA) (b2 microglobulin) Assay was used as an endogenous control. We used the DDCT method to calculate the relative expression of the 13 target genes using the following formula: DDCT = (CT of the target gene in T24 and RT4 cells after 1, 18 and 192 h after exposure to 50 mM Prima-1 – the CT of the endogenous control under the same conditions) – (CT of the target gene in T24 and RT4 cells without Prima-1 treatment - CT of the endogenous control under the same conditions). The gene expression fold change was calculated as 2-DDCT.

& MATERIALS AND METHODS Cell Lines and Treatment The human urothelial carcinoma T24 (p53 mt) cell line was kindly provided by Dr. Salvadori (Botucatu, Brazil). RT4, a primary urothelial carcinoma bladder cell line from a Caucasian (p53 wt) was purchased from EACC (Salisbury, UK). The cell lines were cultured in McCoy’s medium supplemented with 20% (v/v) heat-inactivated fetal bovine serum (Sigma, St.Louis, MO, USA) under an atmosphere of 5% CO2 at 37 ˚C. For the experiments, T24 and RT4 cells were seeded in 25 cm2 tissue culture flasks and maintained at 37 ˚C/5% CO2 overnight to allow the cells to adhere. On the next day, the cells were exposed to 253.7 nm of UVC for 2 h to induce DNA damage. UVC radiation causes specific DNA lesions, and in addition to being a powerful tool for scientific studies, it is a methodology that is easily implemented in the laboratory because the majority of germicidal lamps emit UVC light (10). After exposure, the cells were treated with 50 mM Prima-1 (Sigma, St.Louis, MO, USA) for 1 and 192 h.

Evaluation of the mitochondrial membrane potential by DePsipher The DePsipher Kit (Trevigen, Gaithersburg, MD, USA) was used to measure the mitochondrial membrane potential under a fluorescence microscope (11,12). T24 and RT4 cells (26105/ml) were seeded in a 24 well plate 24 h prior to the experiments. Then, the plate was exposed to 253.7 nm of UVC for 2 h for the induction of DNA damage. Prima-1 (50 mM) was added, and 18 h later, the cells were washed with PBS and detached from the cell culture wells with trypsin. As a control, we used T24 and RT4 cell lines exposed to UVC for 2 h in the absence of treatment. The

RNA Isolation and cDNA Synthesis Following the above experiment, total RNA was isolated with the mirVanaTM Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. As a control, we used T24 and RT4 cell lines cultured in McCoy’s medium supplemented with 20% (v/v) heat-inactivated fetal bovine Table 1 - Assay identifiers (IDs) used for qRT-PCR. Gene symbol CASP2 CASP3 CASP7 CASP9 BBC3 (PUMA) BAK1 BAX MDM2 PMAIP1 (NOXA) B2M CDKN1A (p21) TP53 APAF1 CYC1 (cytochrome-c)

Gene name

Assays IDs

2, apoptosis-related cysteine peptidase 3, apoptosis-related cysteine peptidase 7, apoptosis-related cysteine peptidase 9, apoptosis-related cysteine peptidase BCL2 binding component 3 BCL2-antagonist/killer 1 BCL2-associated X protein Mdm2 p53 binding protein homolog phorbol-12-myristate-13-acetate-induced protein 1 b2-microglobulin cyclin-dependent kinase inhibitor 1A (p21, Cip1) tumor protein p53 apoptotic peptidase activating factor 1 cytochrome c-1

Hs00234982_m1 Hs00234385_m1 Hs00169152_m1 Hs00964603_m1 Hs00248075_m1 Hs00940249_m1 Hs00180269_m1 Hs01066930_m1 Hs00382168_m1 Hs99999907_m1 Hs00355782_m1 Hs01034249_m1 Hs00559441_m1 Hs00357717_m1

caspase caspase caspase caspase

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cells were incubated in the dark with 5 mg/ml DePsipher (5.59.6.69-tetrachloro-1.19.3.39-tetraethyl-benzimidazolyl carbocyanin iodide) solution for 30 min at 37 ˚C, washed with reaction buffer with stabilizer, placed on a glass slide and covered with a glass coverslip. The stained cells were observed under an inverted fluorescence microscope using a fluorescein long-pass filter (fluorescein and rhodamine). DePsipher visualizes the potential-dependent accumulation in mitochondria, which is indicated by a fluorescence emission shift from red (590 nm) to green (530 nm).

cell line, the same expression level was detected after 18 h in the p53 mt T24 cell line.

Evaluation of the Mitochondrial Membrane Potential with DePsipher In T24 cells treated with 50 mM Prima-1, there was a disruption in the DYm that was similar to what was observed in RT4 cells exposed to UVC. This result was different from that found for T24 cells that did not exhibit a disruption in DYm in the absence of Prima-1 (Figure 2).

Apoptosis analysis

Morphological analysis

Following the conditions previously reported, the morphological aspects of the cells were analyzed and photographed under a microscope.

After a 2 h of exposure to UVC radiation followed by 192 h of 50 mM Prima-1 treatment, we observed similar morphological changes in the T24 and RT4 cell lines (Figure 3).

Ethics

& DISCUSSION

This project was submitted to the research ethics committee of the Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo and approved at the August 5th, 2009 meeting (Protocol No. 0718/09).

In this study, we showed that Prima-1 induces apoptosis in a p53 mutant bladder cancer cell line by reestablishing p53 transcriptional activity. Noxa was induced at 0 and 1 h in T24 cells, while in RT4 cells, Noxa expression was detected at 1 and 18 h. Puma was not detected in RT4 cells but was overexpressed in T24 cells at 18 h. Bak and Bax oligomerize, altering the permeability of the mitochondrial membrane by facilitating the release of cytochrome c and activating caspases. In our model, only Bax was expressed by T24 cells after 18 h, and Bak was not expressed at any time after treatment. In contrast, Bak was expressed by the p53 wt RT4 cells, while Bax was not expressed. A recent study showed that p53 participates in the intrinsic cell death pathway via the induction of Bax, Puma and Noxa (13). Thus, we can argue that in our model, in which T24 cells were exposed to UVC and Prima-1, intrinsic apoptosis pathway induction was promoted by the restoration of the p53 transcriptional function and the induction of Noxa, Puma and Bax expression. Noxa and Puma are proteins transcribed by Pmaip 1 and Bbc3, respectively, which are important p53-induced genes that are considered essential mediators of the apoptotic arm of the p53 pathway (14,15). In studies with knockout mice, some authors have demonstrated that Puma plays an important role in p53mediated apoptosis in a wide range of cell types (15-17), while in this model of apoptosis, the role of Noxa is restricted to fibroblasts (18). The dramatic effect that the loss of Puma has on the sensitivity of different cell types in relation to p53-mediated cell death is particularly revealing (16,19), indicating that Puma is a crucial mediator of apoptosis in response to p53. Lomonosova et al. (20) demonstrated that other BH3 proteins along with p53, such as Puma, could overcome the dependence on Bax and Bak in mediating cell death. Thus, an explanation for our data may be that we observed the expression of Puma but not Bax and that the isolated expression of Bak, which was independent of Bax, is sufficient to induce apoptosis. In contrast, in the p53 wt RT4 cell line, we observed the occurrence of apoptosis independent of Puma expression. A difference in the described mechanism of apoptosis induction in this cell line has been described for cisplatin, which, along with the phosphorylation and stabilization of p53, activates the expression of Fas, Puma and caspase-10 (21). A critical event for apoptosome assembly is the release of cytochrome c from the mitochondria, a step tightly regulated by members of the B cell lymphoma 2 (Bcl-2)

& RESULTS Gene Expression Profile The gene expression profiles after Prima-1 exposure to both cell lines are presented in Figure 1.

Prima-1 and Apoptosis Pmaip 1 and Bbc3 are important genes induced by p53 that transcribe Noxa and Puma, respectively, and are essential for apoptosis induction. Noxa was found to be induced at 0 and 1 h in the T24 cells, while Noxa expression was detected at 1 and 18 h in the RT4 cells. Puma was not detected in RT4 cells but was overexpressed in T24 cells at 18 h. Bak and Bax oligomerize and alter the permeability of mitochondrial membranes by facilitating the release of cytochrome c to activate caspases. In our model, only Bax was expressed by the T24 cells after 18 h, while Bak was not expressed. However, Bak was expressed in the p53 wt RT4 cells, while Bax was not expressed. Our analysis revealed that caspase 9, Apaf-1 and cytochrome-c, which are involved in the activation of the caspase cascade, were progressively overexpressed in the RT4 cells; however, in the T24 cells, these genes were more expressed at 0 h and upregulated at 1 h; an even greater increase was observed at 18 h. Caspase 2 is considered an orphan caspase and thought to be an additional initiating caspase. We observed progressive caspase 2 overexpression in the T24 and RT4 cells. An analysis of the effector caspases 7 and 3 also demonstrated a progressive upregulation in both cell lines, with a peak at 18 h.

Prima-1 and p53 Activation To demonstrate that the apoptosis induction was related to the reestablishment of p53 transcriptional activation, we evaluated the expression of MDM2 and p21, which are important p53-regulated genes. We found that p21 was expressed in RT4 cells at low levels compared with T24 cells after Prima-1 treatment, and the same pattern of expression was noted for MDM2. An evaluation of p53 expression in cell lines exposed to UVC and Prima-1 revealed that while p53 was overexpressed immediately after exposure in the p53 wt RT4

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Figure 1 - Profile of MDM2, Puma, Bax, Noxa, Casp9, Casp7, Casp3, Casp2, p53, Apaf-1, p21 and cytochrome-c gene expression in T24 (p53 mt) and RT4 (p53 wt) cells after UVC radiation and treatment with Prima -1 for 1 and 18 h, compared with the control.

domain (BH3-only). Once the BH3-only subfamily is activated, the inhibitory effects of the antiapoptotic Bcl-2 protein family are overcome, enabling the oligomerization of Bak-Bax within the mitochondrial outer membrane (MOM). Our results demonstrated differences in the involvement of Bak and Bax, and the former was shown to participate in apoptosis in RT4 cells and the latter in T24

protein family (22). This family is comprised of three subfamilies, depending on the number of Bcl-2 homolog (BH) domains that they contain. The antiapoptotic subfamily consists of members containing BH4 domains, and Bcl-2 is the most well-known protein in this class. The two other subfamilies are proapoptotic in nature, either lacking the BH4 domain (Bax, Bak) or solely containing a BH3

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Figure 2 - DYm evaluation by DePsipher (A) RT4 cells exposed to UVC in the absence of Prima-1. (B) RT4 cells exposed to UVC and treated with Prima-1 for 18 h. (C) T24 cells exposed to UVC in the absence of Prima-1. (D) T24 cells exposed to UVC and treated with Prima-1 for 18 h.

cells. Alternative routes reported by others help us understand these discrepant results. Lei et al. (23) showed that gossypol, a natural polyphenolic compound found in cotton seeds and used in infertility testing, is subsequently able to induce mitochondrial membrane rupture to release cytochrome c, and this effect is independent of Bax and Bak activation. Green et al. (24) postulated the existence of two alternative routes for cytochrome c release. The first is that the activation and oligomerization of Bax interacts with other mitochondrial membrane proteins, leading to release of cytochrome-c, and the second involves the opening of mitochondrial pores and a nonspecific release of cytochrome-c. In an experimental study with bak-/-/bax-/- mice, Claveria et al. (25) found that the occurrence of programmed cell death was preserved. In a study published by Katrhyn et al. concerning deconstructing p53 transcriptional networks in tumor suppression, the lack of Bax and Puma was not sufficient to diminish apoptosis in some cell types, which may explain the occurrence of apoptosis in the RT4 cell line even in their absence (13). Caspase-9 is an initiator caspase and is thus involved in the initiation of the proteolytic cascade, which is associated with the intrinsic apoptosis pathway (26,27). Once activated, caspase-9 cleaves and activates the effector caspases 3 and 7, which are required for proteolysis and subsequent cell death (28). Our analysis revealed the effective initiators of apoptosis, i.e., caspase 9, apaf-1 and cytochrome-c, responsible for the activation of the caspase cascade. We demonstrated that there was an upregulation of these genes in the p53 wt RT4 cell line and the p53 mt Prima-1-treated T24

cells. There was a peak in the expression of caspase-9 in the T24 cell line after UVC radiation exposure, followed by a gradual decrease in its expression levels, while in the RT4 cells, the peak occurred later. Our analysis showed that the effectors caspases, caspase 7 and 3, progressively increased in cell lines, with a peak at 18 h for both. These data suggest that Prima 1 was able to effectively induce apoptosis in a p53 mutant bladder cancer cell line. Caspase 2, which is considered an orphan caspase, is considered another initiating caspase. This protein is the most evolutionarily conserved of all caspases (29), and although there are numerous reports concerning the role of caspase-2 in apoptosis, there is much controversy regarding this protein, making it difficult to correctly place caspase-2 in the apoptotic cascade; hence, its role in apoptosis remains unclear, making it the orphan of the caspase family. Although caspase-2 shares some substrate specificity with executioner caspases (30,31), in many ways, it is considered an initiator caspase. Caspase-2 has been reported to cleave the pro-apoptotic Bcl2 family member Bid (32). This action is generally considered to be the mode through which caspase-2 initiates apoptosis. We also observed progressive caspase 2 overexpression in the T24 and RT4 cell lines, confirming the data published by Shen et al. (2008), who demonstrated that caspase 2 plays an important role in the intrinsic apoptosis pathway mediated by p53 promoting the translocation of Bax to the mitochondria, a key step for the release of cytochrome c (33). The loss of mitochondrial membrane potential in the T24 strain occurred after 18 h of treatment with Prima-1,

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Figure 3 - Microscopic images of T24 cells prior to UVC exposure (A) and 192 h after exposure and treatment with Prima-1 (B) Microscopic images of RT4 cells prior to UVC exposure (C) and 192 h after exposure and treatment with Prima-1 (D). A and C (100 x). B and D (400 x).

demonstrating that this mechanism is a direct target of this compound and confirming the findings of Shen et al. (33). The morphological features observed in the T24 and RT4 cell lines after 24 h of treatment with Prima-1 demonstrates morphological changes, which became more pronounced with increasing treatment time. To demonstrate that apoptosis induction was related to the reestablishment of p53 transcriptional activation, we evaluated the expression of other important genes regulated by p53: MDM2 and p21. We found that p21 was expressed at lower levels in RT4 compared with T24 cells upon Prima1 treatment, and the same was observed for MDM2. These results suggest that Prima-1 was able to restore p53 function because after treating the T24 cells (p53 mt), we observed an increase in the expression of p21 and MDM2, which are important genes activated by p53. The gradual increase in the expression of p21 after UVC radiation exposure and subsequent Prima-1 treatment is further evidence for the restoration of p53 because the activation of p53 induces the expression of many proteins, including p21 (34). Messina et al. studied the effect of Prima-1 in thyroid cancer cells and also demonstrated an increase in p21 expression following treatment with this compound (35). In evaluating the p53 expression in cell lines exposed to UVC and Prima-1, we found that p53 was overexpressed immediately after treatment in the p53 wt RT4 cell line, and the same level of expression was detected after 18 h in p53 mt T24 cell line. However, the p53 expression level in T24 cells after cell damage was significantly lower than that found in RT4 cells. Our findings also revealed that Prima-1 acts to increase the p53 mRNA levels in p53 wt cell lines. The mechanism involved in this phenomenon

has not been previously reported and requires additional study. There are no data in the English literature demonstrating Prima-1 activity in bladder cancer cell lines. p53 mutations are highly associated with high-grade invasive urothelial carcinomas, and it is well known that there is a lack of suitable therapeutic options for these cases (36). The possibility of restoring p53 activity may be of great interest, and the possibility of using it intravesically is tempting. We have previously reported a preliminary study evaluating Prima-1 as a potential treatment in an in vivo model of orthotopic urothelial carcinoma. Although this treatment was not effective, there were no side effects, and more studies are necessary to consider this compound as an option in clinical practice (37). In conclusion, we demonstrated that Prima-1 is able to reactivate p53 function in p53-mutated bladder cancer cell line by promoting apoptosis, inducing the expression of Bax and Puma, activating the caspase cascade and disrupting the mitochondrial membrane independent of Bak in the T24 cell line (p53 mt).

& ACKNOWLEDGMENTS This work was supported by the State of SaË&#x153;o Paulo Research Foundation (FAPESP).

& AUTHOR CONTRIBUTIONS Piantino CB was responsible for the development and execution of the study. Reis ST was responsible for the real-time experiments. Viana NI and Morais DR cooperated in the maintenance of cell cultures. Silva IA

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collaborated in the preparation of solutions and dilutions. Antunes AA, Dip Junior N and Srougi M collaborated in project development. Leite KRM generally supervised the project.

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Is there a relationship between endothelial nitric oxide synthase gene polymorphisms and ankylosing spondylitis? Ismail Sari,I Yusuf Ziya Igci,II Gercek Can,III Ali Taylan,IV Dilek Solmaz,I Bulent Gogebakan,II Servet Akar,I Zeynep Eslik,II Giray Bozkaya,V Nurullah AkkocI I

Dokuz Eylul University, School of Medicine, Department of Internal Medicine, Division of Rheumatology, Izmir, Turkey. II Gaziantep University, School of Medicine, Department of Medical Biology, Gaziantep, Turkey. III Izmir Ataturk Training and Research Hospital, Department of Rheumatology, Izmir, Turkey. IV Izmir Tepecik Training and Research Hospital, Department of Rheumatology, Izmir, Turkey. V Izmir Bozyaka Training and Research Hospital, Department of Biochemistry, Izmir, Turkey.

OBJECTIVE: Nitric oxide is produced by endothelial nitric oxide synthase, and its production can be influenced by polymorphisms of the endothelial nitric oxide synthase gene. Because candidate genes responsible for susceptibility to ankylosing spondylitis are mostly unknown and available data suggest that there may be problems related to the nitric oxide pathway, such as endothelial dysfunction and increased asymmetric dimethylarginine, this study aimed to assess the association of common endothelial nitric oxide synthase gene polymorphisms with ankylosing spondylitis. METHODS: One hundred ninety-four unrelated Turkish ankylosing spondylitis patients and 113 healthy without apparent cardiovascular disease, hypertension or diabetes mellitus were included. All individuals were genotyped by PCR-RFLP for two single-nucleotide polymorphisms, namely 786T.C (rs2070744, promoter region) and 786 Glu298Asp (rs1799983, exon 7). Variable numbers of tandem repeat polymorphisms in intron 4 were also studied and investigated by direct electrophoresis on agarose gel following polymerase chain reaction analysis. The Bath ankylosing spondylitis metrology index of the patients was calculated, and human leukocyte antigen B27 was studied. RESULTS: All studied polymorphisms satisfied Hardy-Weinberg equilibrium. Sex distributions were similar between the patient and control groups. No significant differences were found in the distributions of allele and genotype frequencies of the studied endothelial nitric oxide synthase polymorphisms between patients and controls. There were no correlations between endothelial nitric oxide synthase polymorphisms, disease duration, Bath ankylosing spondylitis metrology index or human leukocyte antigen B27. CONCLUSION: The results presented in this study do not support a major role of common endothelial nitric oxide synthase polymorphisms in Turkish ankylosing spondylitis patients. KEYWORDS: Ankylosing Spondylitis; Endothelial Nitric Oxide Synthase; Nitric Oxide; Inflammation; Atherosclerosis. Sari I, Igci YZ, Can G, Taylan A, Solmaz D, Gogebakan B, et al. Is there a relationship between endothelial nitric oxide synthase gene polymorphisms and ankylosing spondylitis? Clinics. 2013;68(3):305-309. Received for publication on September 25, 2012; First review completed on October 22, 2012; Accepted for publication on November 4, 2012 E-mail: ismailsari35@gmail.com Tel.: 90 2324123725

important role in determining susceptibility and disease severity. HLA–B27 is the major gene associated with AS. Several non-MHC genes have also been linked to the disease (1). In recent years, there has been considerable interest regarding early atherosclerosis and AS (2-4). In this respect, several studies have suggested an increased prevalence of endothelial dysfunction, an early step in the pathogenesis of atherosclerosis (2,3,5,6), in AS. It is well-known that nitric oxide (NO) is essential in the maintenance of vascular tonus (7) and that the presence of endothelial impairment (reduced vascular relaxation) may suggest a problem regarding the NO pathway. In addition, a considerable number of studies have reported that asymmetric dimethylarginine (ADMA),

& INTRODUCTION Ankylosing spondylitis (AS) is a chronic inflammatory disease of the spine and sacroiliac joints. Although the pathogenesis of AS is not known, genetic factors play an

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temperatures and product sizes were as follows: Glu298Asp: (forward) 59-GTCACGGAGACCCAGCCAATG-39 and (reverse) 59-GCCCTTCTTGAGAGGCTCAGGGAT-39, 61.4 ˚C, 325 bp; 786T.C: (forward) 59-AGCTAGTGGCCTTTCTCCAGCCC-39 and (reverse) 59-CCCAGCCCCAATTTCCTGGAACC-39, 61.4 ˚C, 335 bp; and VNTR repeat region: (forward) 59-GCCTTGGCTGGAGGAGGGGA-39 and (reverse) 59-TGGGGGAGAAGCAGCAGCCA-39, 57.1 ˚C, 242 bp. For the Glu298Asp and 2786T.C regions, PCR products were digested using MboI (Fermentas, Vilnius, Lithuania) and HpaII (New England Biolab, Hitchin, UK) restriction endonuclease enzymes, respectively. Restriction fragments were separated by electrophoresis on a gel composed of 3% agarose. For the Glu298Asp region, the T allele resulted in 195 and 130 bp bands, while the G allele resulted in single band of 225 bp. For the 2786T.C region, the C allele resulted in 167, 46 and 122 bp bands, while the T allele resulted in 122 and 213 bp bands. The 27 bp VNTR repeat region in intron 4 was analyzed by direct electrophoresis on 3% agarose gel after PCR amplification. The 4b allele amplicon size was 242 bp, whereas the 4a allele amplicon size was 215 bp. HLA B27 analysis for the patient group was performed using a commercially available SSP-typing kit (Olerup; QIAGEN Vetriebs GmbH, Wien, Austria) according to the manufacturer’s recommendations. SSP-typing results were visualized using 2% agarose gel electrophoresis.

an endogenous NO inhibitor, is significantly increased in the blood of AS patients (8-10). Both endothelial impairment and increased ADMA concentrations suggest the possibility of a problem related to the NO pathway in AS. NO is produced by endothelial NO synthase (eNOS), and its production can be influenced by polymorphisms of the eNOS gene (7). Because candidate genes responsible for susceptibility to AS are mostly unknown and available data suggest the possibility of a problem related with the NO pathway, this study aimed to assess the association of common eNOS gene polymorphisms with AS.

& METHODS Sample size, patients and controls Sample size was calculated using the results of previous studies that investigated eNOS gene polymorphisms in Turkish patients with inflammatory rheumatic diseases based on a = 0.05 and a power of 80% (11,12). The minimum minor allele frequencies required for this estimation for each group were as follows: Exon 7 (G-894T), 96 subjects; promoter (T-786C), 94 subjects; and intron 4 (variable number of tandem repeat polymorphisms, VNTR), 58 subjects. We included 194 unrelated AS patients diagnosed according to the modified New York criteria (13). Patients without a history of hypertension (current anti-hypertensive treatment and/or observation of blood pressure levels .140/90 mmHg), diabetes mellitus (participants who reported having ever been told by a physician that they have diabetes mellitus or who reported taking insulin or pills to lower blood glucose levels) or coronary artery disease (history of myocardial infarction, angina pectoris or coronary artery angioplasty) were recruited consecutively from the rheumatology outpatient clinics of the Dokuz Eylul University and three training hospitals (Ataturk, Bozyaka and Tepecik) located in Izmir city. A total of 113 healthy controls subject undergoing the same exclusion criteria as the patients and who did not have any first-degree relatives with diagnoses of AS or related spondyloarthropathies were recruited from the relatives of health professionals and blood donors. Spinal mobility was assessed by the Bath Ankylosing Spondylitis Metrology Index (BASMI) (14). Patients were also evaluated with the Bath Ankylosing Spondylitis Functional Index (BASFI) (15) and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) (16). The Ministry of Health of Turkey Ethics Committee approved the study, and all individuals provided informed consent prior to blood collection in compliance with the principles of the Declaration of Helsinki.

Statistical analysis MedCalc software, version 12.3.0.0 (MedCAlc, Mariakerke, Belgium), was used to estimate sample size. The rest of the statistical analysis was performed on SPSS v. 16.0 software (SPSS Inc, Chicago, IL). Data are expressed as the means ¡ SDs for continuous variables or as percentages of the total for categorical variables. Pearson x2 or Fisher’s exact tests were used to assess intergroup significance, and a Student’s t test was used to determine differences in means. The distribution of the control genotypes was checked for the Hardy–Weinberg equilibrium. The overall distributions of alleles and genotypes for each polymorphism were compared between cases and controls using x2 analyses. Associations between alleles and genotypes and other variables were examined by x2 tests. An analysis of covariance (ANCOVA) was used to control for confounding variables. A double-tailed p-value of , 0.05 was considered statistically significant.

& RESULTS There were 194 AS patients (139 male [M], 55 female [F]; 41.5¡10.8 years) and 113 healthy controls (69 M, 44 F; 38.6¡11.1 years). Sex distributions were similar between the patient and control groups (p = 0.07); however, age was significantly lower in the control group compared with the patients (p = 0.03). Disease duration for the patients was 14.9¡9.5 years. HLA-B27 was positive in 72% of patients. The mean BASMI (0-10), BASFI (0-10) and BASDAI (0-10) values were 2.8¡2.4, 3.2¡2.3 and 3.2¡2.5, respectively. The clinical and demographical features of the patient and control groups are given in Table 1. All studied polymorphisms satisfied the Hardy-Weinberg equilibrium in the controls (eNOS T-786C: X2(1) = 1.96, p = 0.16; eNOS 4b4a VNTR: X2(1) = 2.74, p = 0.09; eNOS Glu298Asp: X2(1) = 1.21, p = 0.27).

Genotyping For all subjects, peripheral blood samples were collected into sterile tubes with EDTA. Genomic DNA was extracted from whole blood using standard proteinase K digestion and the salt-chloroform method. The PCR-RFLP analysis method was used to evaluate the association between three polymorphisms in the eNOS gene and AS. Three PCR primer sets were used for amplification of each polymorphic region, including the Glu298Asp (rs1799983, exon 7), 2786T.C (rs2070744, promoter region) and 4b4a (intron 4, 27 bp repeat) regions. The primers were designed using the Primer3 algorithm via the primer-BLAST interface on the NCBI BLAST web site. PCR primer sequences, annealing

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eNOS polymorphisms and ankylosing spondylitis Sari I et al.

Table 1 - Clinical and demographic characteristics of the patients and controls. AS patients (n = 194)

Healthy controls (n = 113)

p-value

41.5¡10.8 139/55 72 14.9¡9.5 2.8¡2.4 3.2¡2.3 3.2¡2.5

38.6¡11.1 69/44

0.03 0.07

Age (years) Sex, M/F HLAB27 positivity, % Disease duration (years) BASMI (0-10) BASFI (0-10) BASDAI (0-10)

Continuous data are presented as the means ¡ standard deviations. BASMI = Bath Ankylosing Spondylitis Metrology Index, BASFI = Bath Ankylosing Spondylitis Functional Index, BASDAI = Bath Ankylosing Spondylitis Disease Activity Index.

Genotype distributions

Correlation analysis

The frequencies of the T/C polymorphisms at position 786 in the promoter region (patients vs. controls) were as follows: TT (46.3% vs. 34.8%), TC (46.3% vs. 53.6%) and CC (7.4% vs. 11.6%) (p = 0.12). The frequencies of polymorphisms in exon 7 (Glu298Asp; patients vs. controls) were as follows: GG (57.5% vs. 50.9%), GT (35.8% vs. 43.8%) and TT (6.7% vs. 5.4%) (p = 0.36). The frequencies of 4b4a VNTR (intron 4) polymorphisms (patients vs. controls) were as follows: bb (71.7% vs. 65.2%), ba (26.7% vs. 33.9%) and aa (1.6% vs. 0.9%) (p = 0.3). After controlling for age and sex, the distributions of the genotypes (T-786C, Glu298Asp and 4b4a VNTR) were still not significantly different between the patients and controls (p = 0.06, 0.43 and 0.38, respectively).

The correlation analysis showed that eNOS gene polymorphism T-786C was not correlated with the variables of disease duration, BASMI, BASFI, BASDAI or HLAB27 (p = 0.2, 0.1, 0.4, 0.4 and 0.8, respectively). These variables were also not correlated with the Glu298Asp (p = 0.3, 0.3, 0.2, 0.4 and 0.6, respectively) or 4b4a VNTR (p = 0.9, 0.6, 0.4, 0.4 and 0.2, respectively) polymorphisms.

& DISCUSSION In this study, we showed that the frequencies of common eNOS gene polymorphisms were not different between AS patients and controls. We also observed that these polymorphisms were not associated with disease duration, activity, function, severity or HLAB27. NO is a molecule that plays an important role in a variety of physiologic functions, including the regulation of blood vessel tone, inflammation, mitochondrial functions and apoptosis (17). In mammals, NO can be generated by three different isoforms of the enzyme NO synthase, including the neuronal, inducible and endothelial forms (18). All three NOS isozymes have regulatory functions in the cardiovascular (CV) system (18). The most important isoform is eNOS, which keeps blood vessels dilated, controls blood pressure and has numerous other vasoprotective and antiatherosclerotic effects (18). Endothelium-derived NO synthase is encoded by the eNOS gene on chromosome 7 (7). In recent years, several polymorphisms of the eNOS gene and their associations with various diseases have been

Allelic distributions eNOS T-786C (patients vs. controls) was distributed as T (69.4% vs. 61.6%) and C (30.6% vs. 38.4%) (p = 0.07). Glu298Asp (patients vs. controls) was distributed as G (75.4% vs. 72.8%) and T (24.6% vs. 27.2%) (p = 0.5). 4b4a VNTR (patients vs. controls) was distributed as b (85.1% vs. 82.1%) and a (14.9% vs. 17.9%) (p = 0.36). There were still no significant differences between the groups regarding T786C, Glu298Asp and 4b4a VNTR alleles after controlling for age and sex (p = 0.08, 0.44 and 0.48, respectively). A summary of the allele and genotype frequencies of the three investigated eNOS gene polymorphisms in AS patients and healthy controls is given in Table 2.

Table 2 - Allele and genotype frequencies of the eNOS gene polymorphisms in patients with AS and healthy controls. Alleles % (n) eNOS -786

Genotypes % (n)

Patients (n = 188) Controls (n = 112) p-value

69.4 (261) 61.6 (138)

30.6 (115) 38.4 (86)

46.3 (87) 34.8 (39)

46.3 (87) 53.6 (60) 0.12

7.4 (14) 11.6 (13)

eNOS 27 bp VNTR

Patients (n = 191) Controls (n = 112) p-value

85.1 (325) 82.1 (184)

14.9 (57) 17.9 (40)

71.7 (137) 65.2 (73)

26.7 (51) 33.9 (38) 0.3

1.6 (3) 0.9 (1)

eNOS+894 (Glu298Asp) Patients (n = 193) Controls (n = 112) p-value

0.07

0.36 G

75.4 (291) 72.8 (163)

24.6 (95) 27.2 (61)

57.5 (111) 50.9 (57)

35.8 (69) 43.8 (49) 0.36

6.7 (13) 5.4 (6)

0.5

Note that no significant differences were found in the distributions of allele and genotype frequencies of the studied eNOS gene polymorphisms between patients and controls.

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We acknowledge that there were some limitations in our study. (1) We did not study the surrogate markers of atherosclerosis or biomarkers such as carotid intima-media thickness, flow-mediated dilatation and ADMA in this study. Upon consideration, the use of these parameters may be more appropriate for drawing conclusions about eNOS polymorphisms in AS. (2) The inclusion of AS patients with CV risk factors may be more useful for understanding the significance of these polymorphisms in patients with or without CV conditions. In conclusion, despite these limitations, the results presented in this study do not provide support for a major role of common eNOS polymorphisms in Turkish AS patients. Further replication studies in different populations with larger numbers of patients are needed to confirm our results.

studied. In particular, a single nucleotide polymorphism in the promoter region (T-786C), a 894G .T polymorphism leading to amino acid substitution at position 298 (Glu298Asp, rs1799983) in exon 7, and the 4b4a polymorphism (a VNTR) located in intron 4 of the eNOS have received the most attention due to their functional relevance to eNOS activity (19). It has been shown that the T-786C polymorphism reduces eNOS gene promoter activity and affects eNOS protein expression, while the Glu298Asp polymorphism causes a structural change in the eNOS protein associated with impaired eNOS activity (20,21). In addition, it has also been shown that the 27bp-VNTR polymorphism reduces the plasma concentration of NO (22). The dysregulation of eNOS caused by these gene polymorphisms leads to decreased NO production and is thought to contribute to the pathogenesis of several diseases, including inflammatory disorders. In this study, we investigated three common eNOS gene polymorphisms (T-786C, Glu298Asp = G894T and 4b4a VNTR) that have been shown to be associated with eNOS activity. It has been reported that these polymorphisms are associated with CV risk factors and CV disease (7). In the current study, to avoid the confounding effects of these variables on our parameters, we excluded subjects with hypertension, diabetes mellitus and CVD. We also performed statistical adjustments for age and sex to minimize the effects of demographics on our results. Because of its relationship with inflammation, several studies have investigated the association of eNOS gene polymorphisms with inflammatory rheumatic diseases, including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis and Behcet’s disease (11,12,17,23,24). Some of these studies have yielded evidence of an association of the eNOS polymorphisms with either the pathogenesis of rheumatic diseases or extraarticular manifestations (12,17,23,24). It is well known that there is a strong genetic basis for AS, and some genes, particularly HLAB27, have already been implicated in disease pathogenesis. In recent years, several other non-MHC genes, including genes involved in intracellular antigen processing and cytokine production (especially genes in the IL–17–IL–23 pathway), have also been shown to be related to AS (1). However, it is not known whether AS is associated with eNOS gene polymorphisms. In the current study, we investigated eNOS gene polymorphisms in AS patients for the following reasons. (1) Previous studies have reported an increased frequency of impaired vascular relaxation (which is known to be mediated by NO) (2-4,6) and higher levels of ADMA (8-10). Although ADMA impairs post-NO production (unrelated to eNOS polymorphisms), it is responsible for the decreased NO activity (8-10). Based on these findings, we suggest that the NO pathway may be impaired and that this might be associated with genetic polymorphisms in the eNOS gene (2). Some reports have revealed an association between eNOS gene polymorphisms and inflammatory diseases (11,12,17,23,24). Because the pathogenesis of the genes is not fully understood in such inflammatory diseases, we wondered whether there was a link between eNOS gene polymorphisms and AS. We did not find any differences between patients and controls regarding common eNOS gene polymorphisms. We also did not find any relationship between eNOS gene polymorphisms, HLAB27, disease severity, function or activity.

& ACKNOWLEDGMENTS The funding of this study was supported by ‘‘The Society for Research and Education in Rheumatology Turkey’’.

& AUTHOR CONTRIBUTIONS Sari I wrote the manuscript, designed the study and performed the statistical analysis of results. Igci YZ, Gogebakan B and Eslik Z carried out the laboratory analyses. Akar S and Akkoc N helped with the general design of the manuscript and contributed to the discussion section. Taylan A, Can G and Bozkaya G collected the patient data. Solmaz D contributed to the statistical analysis and collected the patient data.

& REFERENCE 1. Reveille JD. Genetics of spondyloarthritis--beyond the MHC. Nat Rev Rheumatol. 2012;8(5):296-304, http://dx.doi.org/10.1038/nrrheum.2012. 41. 2. Bodnar N, Kerekes G, Seres I, Paragh G, Kappelmayer J, Nemethne ZG, et al. Assessment of subclinical vascular disease associated with ankylosing spondylitis. J Rheumatol. 2011;38(4):723-9, http://dx.doi. org/10.3899/jrheum.100668. 3. Gonzalez-Juanatey C, Vazquez-Rodriguez TR, Miranda-Filloy JA, Dierssen T, Vaqueiro I, Blanco R, et al. The high prevalence of subclinical atherosclerosis in patients with ankylosing spondylitis without clinically evident cardiovascular disease. Medicine (Baltimore). 2009;88(6):358-65. 4. Sari I, Okan T, Akar S, Cece H, Altay C, Secil M, et al. Impaired endothelial function in patients with ankylosing spondylitis. Rheumatology (Oxford). 2006;45(3):283-6. 5. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362(6423):801-9, http://dx.doi.org/10.1038/362801a0. 6. Azevedo VF, Pecoits-Filho R. Atherosclerosis and endothelial dysfunction in patients with ankylosing spondylitis. Rheumatol Int. 2010;30(11):1411-6, http://dx.doi.org/10.1007/s00296-010-1416-3. 7. Cooke GE, Doshi A, Binkley PF. Endothelial nitric oxide synthase gene: prospects for treatment of heart disease. Pharmacogenomics. 2007;8(12):1723-34, http://dx.doi.org/10.2217/14622416.8.12.1723. 8. Kemeny-Beke A, Gesztelyi R, Bodnar N, Zsuga J, Kerekes G, Zsuga M, et al. Increased production of asymmetric dimethylarginine (ADMA) in ankylosing spondylitis: association with other clinical and laboratory parameters. Joint Bone Spine. 2011;78(2):184-7, http://dx.doi.org/10. 1016/j.jbspin.2010.05.009. 9. Sari I, Kebapcilar L, Alacacioglu A, Bilgir O, Yildiz Y, Taylan A, et al. Increased levels of asymmetric dimethylarginine (ADMA) in patients with ankylosing spondylitis. Intern Med. 2009;48(16):1363-8, http://dx. doi.org/10.2169/internalmedicine.48.2193. 10. Erre GL, Sanna P, Zinellu A, Ponchietti A, Fenu P, Sotgia S, et al. Plasma asymmetric dimethylarginine (ADMA) levels and atherosclerotic disease in ankylosing spondylitis: a cross-sectional study. Clin Rheumatol. 2011;30(1):21-7, http://dx.doi.org/10.1007/s10067-010-1589-x. 11. Oksel F, Keser G, Ozmen M, Aksu K, Kitapcioglu G, Berdeli A, et al. Endothelial nitric oxide synthase gene Glu298Asp polymorphism is associated with Behcet’s disease. Clin Exp Rheumatol. 2006;24(5 Suppl 42):S79-82. 12. Sinici I, Kalyoncu U, Karahan S, Kiraz S, Atalar E. Endothelial nitric oxide gene polymorphism and risk of systemic sclerosis: predisposition effect of T-786C promoter and protective effect of 27 bp repeats in Intron 4. Clin Exp Rheumatol. 2010;28(2):169-75.

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13. van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum. 1984;27(4):361-8, http://dx.doi.org/ 10.1002/art.1780270401. 14. Jenkinson TR, Mallorie PA, Whitelock HC, Kennedy LG, Garrett SL, Calin A. Defining spinal mobility in ankylosing spondylitis (AS).The Bath AS Metrology Index. J Rheumatol. 1994;21(9):1694-8. 15. Calin A, Garrett S, Whitelock H, Kennedy LG, Oâ&#x20AC;&#x2122;Hea J, Mallorie P, et al. A new approach to defining functional ability in ankylosing spondylitis: the development of the Bath Ankylosing Spondylitis Functional Index. J Rheumatol. 1994;21(12):2281-5. 16. Garrett S, Jenkinson T, Kennedy LG, Whitelock H, Gaisford P, Calin A. A new approach to defining disease status in ankylosing spondylitis: the Bath Ankylosing Spondylitis Disease Activity Index. J Rheumatol. 1994;21(12):2286-91. 17. Nagy G, Koncz A, Telarico T, Fernandez D, Ersek B, Buzas E, et al. Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus. Arthritis Res Ther. 2010;12(3):210, http://dx.doi.org/10.1186/ar3045. 18. Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33(7):829-37, 837a-837d, http://dx.doi.org/ 10.1093/eurheartj/ehr304. 19. Thameem F, Puppala S, Arar NH, Stern MP, Blangero J, Duggirala R, et al. Endothelial nitric oxide synthase (eNOS) gene polymorphisms and their association with type 2 diabetes-related traits in Mexican

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Americans. Diab Vasc Dis Res. 2008;5(2):109-13, http://dx.doi.org/10. 3132/dvdr.2008.018. Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, et al. T-786--.C mutation in the 5â&#x20AC;&#x2122;-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999;99(22):2864-70, http://dx.doi.org/10.1161/01.CIR.99. 22.2864. Yoshimura M, Yasue H, Nakayama M, Shimasaki Y, Sumida H, Sugiyama S, et al. A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese. Hum Genet. 1998;103(1):65-9, http://dx.doi.org/10.1007/s004390050785. Tsukada T, Yokoyama K, Arai T, Takemoto F, Hara S, Yamada A, et al. Evidence of association of the ecNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun. 1998;245(1):190-3, http://dx.doi.org/10.1006/bbrc.1998.8267. Lee YH, Lee HS, Choi SJ, Ji JD, Song GG. Associations between eNOS polymorphisms and susceptibility to systemic lupus erythematosus: a meta-analysis. Inflamm Res. 2012;61(2):135-41, http://dx.doi.org/10.1007/ s00011-011-0397-3. Melchers I, Blaschke S, Hecker M, Cattaruzza M. The -786C/T singlenucleotide polymorphism in the promoter of the gene for endothelial nitric oxide synthase: insensitivity to physiologic stimuli as a risk factor for rheumatoid arthritis. Arthritis and rheumatism. 2006;54(10):3144-51, http://dx.doi.org/10.1002/art.22147.

CLINICAL SCIENCE

Objective evaluation of plantar hyperhidrosis after sympathectomy Nelson Wolosker,I Augusto Ishy,II Guilherme Yazbek,I Jose´ Ribas Milanez de Campos,II Paulo Kauffman,I Pedro Puech-Lea˜o,I Fa´bio Biscegli JateneII I

Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Division of Vascular and Endovascular Surgery, Saõ Paulo/SP, Brazil. Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Division of Thoracic Surgery, Saõ Paulo/SP, Brazil.

OBJECTIVE: The aim of the present study was to prospectively, randomly, blindly, and objectively investigate how surgery affects plantar sudoresis in patients with palmar and plantar hyperhidrosis over a one-year period using a sudorometer (VapoMeter). METHODS: From February 2007 to May 2009, 40 consecutive patients with combined palmar hyperhidrosis and plantar hyperhidrosis underwent video-assisted thoracic sympathectomy at the T3 or T4 ganglion level (15 women and 25 men, with a mean age of 25 years). RESULTS: Immediately after the operation and during the one-year follow-up, all of the patients were free from palmar hyperhidrosis episodes. Compensatory hyperhidrosis of varying degrees was observed in 35 (87.5%) patients after one year. Only two (2.5%) patients suffered from severe compensatory hyperhidrosis. There was a large initial improvement in plantar hyperhidrosis in 46.25% of the cases, followed by a progressive regression of that improvement, such that only 30% continued to show this improvement after one year. The proportion of patients whose condition worsened increased progressively (from 21.25% to 47.50%), and the proportion of stable patients decreased (32.5% to 22.50%). This was not related to resection level; however, a lower intensity of plantar hyperhidrosis prior to sympathectomy correlated with worse evolution. CONCLUSION: Patients with palmar hyperhidrosis and plantar hyperhidrosis who underwent video-assisted thoracic sympathectomy to treat their palmar hyperhidrosis exhibited good initial improvement in plantar hyperhidrosis, which then decreased to lesser degrees of improvement over a one-year period following the surgery. For this reason, video-assisted thoracic sympathectomy should not be performed when only plantar hyperhidrosis is present. KEYWORDS: Sweating; Hyperhidrosis; Video-Assisted Thoracic Sympathectomy; Autonomic Ganglia. Wolosker N, Ishy A, Yazbek G, de Campos JR, Kauffman P, Puech-Lea˜o P, et al. Objective evaluation of plantar hyperhidrosis after sympathectomy. Clinics. 2013;68(3):311-315. Received for publication on August 15, 2012; First review completed on September 10, 2012; Accepted for publication on November 7, 2012 E-mail: nwolosker@yahoo.com.br Tel.: 55 11 2151-5423

evaluations. The patients reported good initial improvement in PLH immediately after surgery; however, this improvement subsided over the one-year period following surgery (5,6). Tests for the objective quantification of sweating have been developed (7,8,9,10). Recently, a device known as a VapoMeter has been used to quantify the diffusion of water vapor through the skin under controlled conditions of temperature and humidity. We used this device to assess the outcomes of treatment for primary palmar HH by videoassisted thoracoscopic sympathectomy (VATS) in 40 patients (11). There are no studies of long-term sweating of the feet using objective evaluation of sudoresis in patients with palmoplantar HH submitted to thoracic sympathectomy. The aim of the present study was to prospectively and objectively investigate how surgery affects plantar sudoresis in the feet of patients with palmo-plantar HH over a oneyear period using a sudorometer (VapoMeter).

& INTRODUCTION Plantar hyperhidrosis (PLH) is a very common complaint (1) and is usually associated (more than 80% of cases) with palmar hyperhidrosis (PH) (2). Patients undergoing thoracic sympathectomy to treat palmo-plantar hyperhidrosis (HH) recover with anhidrosis of the hands in more than 95% of cases (3), thereby improving their quality of life (QOL) (4). The few studies on the evolution of feet sweating after thoracic sympathectomy have been based on subjective

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was revealed to the patients only after completing one year of follow-up. Six patients exhibited slight/weak pleural adherences, which were detached using an endoscopic scalpel. Only one patient had a right-side residual pneumothorax (2.5%) during the immediate postoperative period, which amounted to less than 10% and did not require pleural drainage. One patient had paresthesia (2.5%) in the upper right limb, which regressed within the first month of followup. Compensatory HH was observed in varying degrees in 35 patients (87.5%) after one year. Only two (2.5%) patients suffered from severe compensatory HH. The patients’ PLH complaints were investigated using a VapoMeter, a noninvasive, portable instrument (Delfin Technologies Ltd., Kuopio, Finland). This device measures transepidermal water loss (TEWL) using a closed measurement chamber that eliminates external interference from air currents. The instrument is placed on the skin and maintains contact with the skin surface for approximately 10 seconds. The evaporation rate is calculated from the rise in relative air humidity inside the closed chamber of the device, and this value is quantified in g/m2/h (increase in the water mass per unit of evaporation area per unit of time). The measurements were performed at the HH outpatient clinic in a climate-controlled room at a temperature between 21 and 24 ˚C, independent of the season of the year. All of the patients remained at rest for 20 to 30 minutes prior to the measurements to reduce external influences. The measurements were made on the plantar face (Figure 1). Objective quantification of plantar sweating was performed using the VapoMeter prior to the operation and at one week, one month, six months, and one year after the operation. In the control group, we performed only one quantification of sweating in the same location. The principal investigator performed all of the evaluations. We performed two types of objective assessments of PLH. First, we evaluated the average plantar sweating in each group of patients before surgery and at seven days, 30 days, six months, and one year after surgery. Second, each foot was evaluated, and the changes in the measurements for sweating were compared over time. In this second analysis, we compared the values obtained at the time points after the surgery with the values obtained prior to surgery. We considered sudoresis to be improved for each period when the values were 25% lower than the initial value for the same foot; worse when the values were 25% greater than the initial value; and stable when the values changed by less than 25% from the original measurement. Finally, we studied the relationship between the level of resection and the evolution of the amount of sudoresis for each foot after surgery. We empirically considered 41.5 g/ m2/h as the maximum reference value for normal sudoresis (25% greater than baseline).

& METHODS From February 2007 to May 2009, 40 patients with combined palmar and plantar hyperhidrosis underwent VATS (15 women and 25 men, with a mean age of 25 years) in a prospective, randomized, blind, and controlled study according to the guidelines of the Ethics Committee for Analysis of Research Projects on Human Experimentation at our institution. None of these patients exhibited HH in other parts of the body. In addition to these patients with HH, we also evaluated 20 healthy participants (10 men and 10 women) without any previous history of hyperhidrosis to form a control group (CON), which served as a reference and comparison standard when following the subjects who underwent the operation. The demographic data are described in Table 1. The patients underwent VATS at two levels: 20 patients at the T3 ganglion level and 20 patients at the T4 level. All of the procedures were performed by the same surgical team using stable surgical techniques throughout the study. The patients were informed that they would be subjected to one of two levels of VATS routinely used in our group: T3 or T4. Randomization was performed by a computergenerated list prior to the beginning of the protocol. This list remained confidential and under possession of a single person who did not participate in the study. The inclusion of patients in groups T3 and T4 was announced to the team immediately prior to the operation. All patients underwent surgery under general anesthesia in a semi-seated position while inclined at 45 ˚. Two 5 mm mini-incisions were made: the first at the fourth intercostal space on the anterior axillary line, into which the thoracoscope was introduced, and the second at the second intercostal space on the medial axillary line, into which an electric or harmonic cautery was introduced. For sympathectomy at the T3 ganglion level, after identification of the sympathetic chain, the patients underwent resection of the chain (sympathicotomy) on the body of the third (R3) and fourth ribs (R4), followed by thermoablation of the segment isolated between them (the ribs). For the T4 ganglion level, the patients underwent sympathicotomy on the body of the fourth (R4) and fifth ribs (R5), followed by thermoablation of the segment isolated between them (the ribs). The same procedure was performed on the contralateral chain at the same level in all cases. Immediately after the operation and during the one-year follow-up, none of the patients reported any episodes of palmar hyperhidrosis. There was no mortality, and it was not necessary to convert the videothorascopic procedure into an open surgery in either of the groups. The outpatient evaluation, quantification of sweat, and application of the QOL questionnaire were performed by an independent observer who did not participate in the operation and had no knowledge of the level of surgical intervention. The team responsible for the operative procedures did not participate in any phase of the outpatient evaluations. The level of the thermoablated ganglion Table 1 - Demographic data from patients in both groups.

Age (mean ¡ SD) Body mass index Gender (male:female)

Control

Plantar hyperhidrosis undergoing T3

Plantar hyperhidrosis undergoing T4

25.8¡7.0 22.3¡2.9 10:10

25.1¡5.5 21.8¡1.4 7:13

25.0¡7.1 23.0¡1.4 8:12

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Objective evaluation of plantar hyperhidrosis. Wolosker N et al.

Table 3 - Evolution of the amount of sudoresis for each foot over time.

IMPROVED STABLE WORSE

7 Days

1 Month

37 (46.25) 26 (32.5%) 17 (21.25)

31 (38.75) 19 (23.75%) 30 (27.5)

6 Months

12 Months

29 (36.25) 24 (30.00) 20 (25.00%) 18 (22.50%) 31 (38.75) 38 (47.50)

Friedman p = 0.1738.

Analyses of the amount of sweating for each foot at one week, one month, six months, and one year after the operation are presented in Table 3. A total of 37 feet (46.25%) showed an initial improvement in plantar hyperhidrosis, followed by a progressive regression of the improvement, such that the improvement was sustained in only 30% of feet after one year. The proportion of feet whose condition worsened increased progressively (from 21.25% to 47.50%), and the proportion of stable patients decreased (32.5% to 22.50%). Table 4 displays the changes in the amount of sweat for each foot according to the resection level (T3 and T4) at one week, one month, six months, and one year after the operation. Both groups showed similar changes in the amount foot sweating over time, the first month being the only exception, which was not maintained. Table 5 presents the changes in sudoresis for each foot according to the amount of sweat prior to surgery (either less than 41.5 g/m2/h or greater than 41.5 g/m2/h) at the different time points after the operation. After one week, we observed that the feet that previously exhibited more sweating (greater than 41.5 g/m2/h) had a higher rate of improvement and stability and a lower rate of deterioration compared with the feet that exhibited less sweating. After one year, only five feet with less sweating had improved, and 54.2% were worse. For the group with the most sweating, 32.2% improved, 44.6% worsened, and 23.2% were stable.

Figure 1 - Location for the measurement of the feet.

Statistical Analysis For the variable age, a one-way analysis of variance (ANOVA) test was used. For gender, the x2 test was used. For the pre-operative and post-operative (at one week, one month, six months, and one year) evaporation rates of the feet in the T3 and T4 ganglia groups, a two-way ANOVA was used. The Bonferroni multiple comparisons test was applied when a statistically significant difference was detected. The Friedman test was used to assess the association between HH outcomes at seven days, one month, six months, and one year. The chi-square frequency test was used for comparisons between T3 and T4 at each time point. The level of statistical significance was set to 5%.

& DISCUSSION The surgical method of choice for treating HH is VATS, which most frequently involves resections of the third or fourth ganglion for the treatment of PH (12).

& RESULTS Objective quantifications of average plantar sweating prior to the operation and at one month, six months, and one year after the operation are presented in Table 2 according to the resection level. There were significant differences between the control group (27.69) and patients with HH (64.04 and 56.84) (p,0.01) (Table 2). However, there was no difference between the levels of resection in the different periods.

Table 4 - Changes in the amount of sudoresis for each foot according to the resection level (T3 and T4) over time (in this table, patients are analyzed, not individual feet). Time point 7 Days

1 Month

Table 2 - Mean plantar sweating before and after sympathectomy.

BEFORE 1 MONTH 6 MONTHS 12 MONTHS

Control

27.69

64.04 53.60 60.82 86.78

56.84 66.44 73.71 64.98

6 Months

12 Months

Status

T3 Freq. (%)

T4 Freq. (%)

p-value

Improved Stable Worse Improved Stable Worse Improved Stable Worse Improved Stable Worse

20 (50.0) 10 (25.0) 10 (25.0) 15 (37.5) 14 (35.0) 11 (27.5) 17 (42.5) 9 (22.5) 14 (35.0) 10 (25.0) 8 (20.0) 22 (55.0)

17 (42.5) 16 (40.0) 7 (17.5) 16 (40.0) 5 (12.5) 19 (47.5) 12 (30.0) 11 (27.5) 17 (42.5) 14 (35.0) 10 (25.0) 16 (40.0)

0.340

p-value obtained from the chi-square frequency test.

313

0.040

0.509

0,398

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Table 5 - Changes in the amount of sudoresis for each foot over time according to the amount of sudoresis prior to surgery. Values less than or greater than 41.5 were compared. Time point 7 Days

1 Month

6 Months

12 Months

Status

Less than 41.5 Freq. (%)

Greater than 41.5 Freq. (%)

p-value

Improved Stable Worse Improved Stable Worse Improved Stable Worse Improved Stable Worse

8 (33.3) 5 (20.8) 11 (45.9) 7 (29.2) 7 (29.2) 10 (41.6) 7 (29.2) 5 (20.8) 12 (50.0) 5 (20.8) 6 (25.0) 13 (54.2)

29 (51.8) 21 (37.5) 6 (10.7) 24 (42.9) 12 (21.4) 20 (35.7) 22 (39.3) 15 (26.8) 19 (33.9) 18 (32.2) 13 (23.2) 25 (44.6)

0.002

0.498

0.400

0.579

p-value obtained by the chi-square test.

When the resection level is higher (T3), hands will exhibit less sweating; when the resection level is lower, there is a reduced likelihood that the patient will present with compensatory HH (13,14,15). This procedure is safe and has a low complication rate (16,17,18,19), as confirmed by our study, where the patients did not experience any repercussions. In this case series, 40 consecutive bilateral sympathectomies were performed, and we obtained full success in all cases in terms of improving hand sudoresis. Compensatory HH (13,14) is the most frequent and important problem related to VATS. At level T3 and especially at level T4, HH is less severe than at level T2 (15). Usually, patients with HH have excessive sweating in more than one location. In this study, we analyzed 40 patients with HH in the hands and feet, which is the most common combination. In many situations, PLH levels can be as great as or greater than PH levels (20). In patients with PLH as the primary complaint, lumbar sympathectomy is the treatment of choice (21), which was not the case in this study because PH was the principal complaint of every patient, whereas PLH was secondary. In this study, we obtained data through an objective method previously used and validated for the measurement of sweating in humans (22). In previously published studies that evaluated the results of sympathectomy in the treatment of PLH, most of the data were based on the patientsâ&#x20AC;&#x2122; subjective reports (4,5). Although the subjective data were most often associated with the perception of sudoresis and with satisfaction after surgery, objective data for comparison with previously published studies (11) were rarely obtained. Because we observed that there was great variability in the extent of sweating between the two feet of a single individual (there was disagreement in 15 patients), we decided to evaluate the variations of sweating by foot and not by individual. We initially analyzed the average of the objective values for each group and observed that on average, the TEWL rates for the T3 and T4 ganglion groups were higher than those in the control group, which provides objective evidence. After surgery, on average, we did not observe significant changes over time regarding the persistence of PLH. These analyses were not significant because the standard deviation in each case was high. Additionally, with this type of analysis, we could not determine the individual repercussions of feet sudoresis over time, which

is why we decided to analyze the changes in sudoresis for each foot. We used the original measurement for each foot and compared it with the subsequent measurement for each patient using the initial measurement as the point of reference. We defined three possibilities for each case: improvement when the levels were 25% higher than the initial levels, worsening when they were 25% lower than the initial levels, and stability when the values were intermediate. In our series, 46.25% of the patients had an improvement in their foot HH one week after VATS, and 30% reported improvement one year after VATS. This objective result was similar to the results of previous subjective studies. In a previous study by our group (5), we found a significant improvement in 23.4% of patients and only one case of cure (1.4%) based on subjective data. Hsu et al. (23) reported better results, with improvement in 64% of patients after sympathectomy at the T2 level using a subjective method of analysis. This large initial improvement, which has also been described by other authors (5,6), and the subsequent worsening have no convincing anatomical or physiological explanations. The improvements were likely a result of stress reduction caused by the patientsâ&#x20AC;&#x2122; postoperative palmar anhidrosis, which may have caused an improvement in the patientsâ&#x20AC;&#x2122; emotional states. This new situation may break the negative feedback loop that might be leading to plantar sweating. The worsening of PLH over time (an increase in the proportion of patients with no improvement from 21.25% to 47.50%) may be related to the return of the emotional stress that was initially lessened because of the high degree of satisfaction obtained from postoperative palmar anhidrosis and to the effect of compensatory HH. After analyzing the effect of thoracic sympathectomy on PLH and taking the resection level into consideration, we observed a decrease in improvement in the T3 group from 50% to 25% and a decrease in the T4 group from 42.5% to 35.0%. A comparison of PLH prior to surgery and at one month after treatment revealed that a lower intensity of HH before sympathectomy resulted in a poorer outcome. Similar data were observed for patient satisfaction after sympathectomy in a previous study, in which a worse preoperative QOL for patients undergoing sympathectomy to treat primary

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Objective evaluation of plantar hyperhidrosis. Wolosker N et al. 8. Kuwabara S, Tamura N, Yamanaka Y, Misawa S, Isose S, Bae JS, et al. Sympathetic sweat responses and skin vasomotor reflexes in carpal tunnel syndrome.Clin Neurol Neurosurg. 2008;7:691-5, http://dx.doi. org/10.1016/j.clineuro.2008.04.004. 9. Tetteh HA, Groth SS, Kast T, Whitson BA, Radosevich DM, Klopp AC, et al. Primary Palmoplantar Hyperhidrosis and Thoracoscopic Sympathectomy: A New Objective Assessment Method. Ann Thorac Surg. 2009;87(1):267-75, http://dx.doi.org/10.1016/j.athoracsur.2008.10.028. 10. Krogstad AL, Skymne A, Pegenius G, Elam M, Wallin BG. No compensatory sweating after botulinum toxin treatment of palmar hyperhidrosis. Br J Dermatol. 2005;152(2):329-33. 11. Ishy A, de Campos JR, Wolosker N, Kauffman P, Tedde ML, Chiavoni CR, et al. Objective evaluation of patients with palmar hyperhidrosis submitted to two levels of sympathectomy: T3 and T4. Interact Cardiovasc Thorac Surg. 2011;12(4):545-8, http://dx.doi.org/10.1510/ icvts.2010.252015. 12. Liu Y, Yang J, Liu J, Yang F, Jiang G, Li J, et al. Surgical treatment of primary palmar hyperhidrosis: a prospective randomized study comparing T3 and T4 sympathicotomy. Eur J Cardiothorac Surg. 2009;35(3):398402, http://dx.doi.org/10.1016/j.ejcts.2008.10.048. 13. Yazbek G, Wolosker N, de Campos JR, Kauffman P, Ishy A, Puech-Leao P. Palmar hyperhidrosis–which is the best level of denervation using video-assisted thoracoscopic sympathectomy: T2 or T3 ganglion? J Vasc Surg. 2005;42(2):281-5, http://dx.doi.org/10.1016/j.jvs.2005.03.041. 14. Yazbek G, Wolosker N, Kauffman P, Campos JR, Puech-Leaõ P, Jatene FB. Twenty months of evolution following sympathectomy on patients with palmar hyperhidrosis: sympathectomy at the T3 level is better than at the T2 level. Clinics. 2009;64(8):743-9, http://dx.doi.org/10.1590/ S1807-59322009000800006. 15. Wolosker N, Yazbek G, Ishy A, de Campos JR, Kauffman P, Puech-Leao P. Is sympathectomy at T4 level better than at T3 level for treating palmar hyperhidrosis? J Laparoendosc Adv Surg Tech A. 2008;18(1):102-6, http://dx.doi.org/10.1089/lap.2007.0030. 16. Cameron AE. Specific complications and mortality of endoscopic thoracic sympathectomy. Clin Auton Res. 2003;13 Suppl 1:I31-5. 17. de Campos JR, Kauffman P, Wolosker N, Munia MA, de Campos Werebe E, Andrade Filho LO, et al. Axillary hyperhidrosis: T3/T4 versus T4 thoracic sympathectomy in a series of 276 cases. J Laparoendosc Adv Surg Tech A. 2006;16(6):598-603, http://dx.doi.org/10.1089/lap.2006.16. 598. 18. Munia MA, Wolosker N, Kauffman P, de Campos JR, Puech-Leaõ P. A randomized trial of T3-T4 versus T4 sympathectomy for isolated axillary hyperhidrosis. J Vasc Surg. 2007;45(1):130-3, http://dx.doi.org/10.1016/ j.jvs.2006.09.011. 19. Munia MA, Wolosker N, Kaufmann P, de Campos JR, Puech-Leaõ P. Sustained benefit lasting one year from T4 instead of T3-T4 sympathectomy for isolated axillary hyperhidrosis. Clinics. 2008;63(6):771-4. 20. Lin TS, Fang HY. Transthoracic endoscopic sympathectomy in the treatment of palmar hyperhidrosis–with emphasis on perioperative management (1,360 case analyses). Surg Neurol. 1999;52(5):453-7, http:// dx.doi.org/10.1016/S0090-3019(99)00111-1. 21. Loureiro M de P, de Campos JR, Kauffman P, Jatene FB, Weigmann S, Fontana A. Endoscopic lumbar sympathectomy for women: effect on compensatory sweat. Clinics. 2008;63(2):189-96. 22. De Paepe K, Houben E, Adam R, Wiesemann F, Rogiers V. Validation of theVapoMeter, a closed unventilated chamber system to assess transepidermal water loss vs. the open chamber Tewameter. Skin Res Technol. 2005;11(1):61-9, http://dx.doi.org/10.1111/j.1600-0846.2005.00101.x. 23. Hsu CP, Chen CY, Lin CT, Wang JH, Chen CL, Wang PY. Video-assisted thoracoscopic T2 sympathectomy for hyperhidrosis palmaris. J Am Coll Surg. 1995;179(1):59-64. 24. Wolosker N, Yazbek G, de Campos JR, Munia MA, Kauffman P, Jatene FB, et al. Quality of life before surgery is a predictive factor for satisfaction among patients undergoing sympathectomy to treat hyperhidrosis. J Vasc Surg. 2010;51(5):1190-4, http://dx.doi.org/10.1016/j.jvs. 2009.11.078. 25. Maccarty CS. Surgical procedures on the sympathetic nervous system for peripheral vascular disease. In: Allen EV, Barker NW, Hines EA, editors. Peripheral vascular diseases, 3rd ed. Saunders, Philadelphia; 1962. p. 805-18.

hyperhidrosis reflected a better postoperative improvement in QOL (24). The innervations of the legs and feet are well known. The preganglionic fibers arise from the spinal medulla at its lower thoracic segment and from the first and second lumbar segments (T10 to L2), and they subsequently cross over to the corresponding ganglions of the sympathetic chain. These fibers progress down the chain and form synapses with the postganglionic neurons located between the fourth lumbar ganglion and third sacral ganglion, from which the postganglionic fibers arise and become incorporated into the nerve branches of the sacral plexus to innervate the lower extremities (25). Although the results presented in this case series cannot be explained by our current scientific knowledge, it is important to have such data so that comprehensive information can be provided to patients prior to VATS. Patients with palmar and plantar hyperhidrosis who underwent VATS to treat their palmar hyperhidrosis exhibited good initial improvement in plantar hyperhidrosis, which was not sustained over a one-year period following surgery. For this reason, VATS should not be considered when only plantar hyperhidrosis is present.

& AUTHOR CONTRIBUTIONS Wolosker N designed and conceived the study, performed the data analyses, and contributed to the discussion. Ishy A designed the study, collected the data and performed the surgeries. Yazbek G, de Campos JR and Kauffman P conducted the analyses and contributed to the discussion. Puech-Lea˜o P and Jatene FB conceived the study and contributed to the discussion.

& REFERENCES 1. Reisfeld R, Nguyen R, Pnini A. Endoscopic thoracic sympathectomy for hyperhidrosis: experience with both cauterization and clamping methods. Surg Laparosc Endosc Percutan Tech. 2002;12(4):255-67, http://dx. doi.org/10.1097/00129689-200208000-00011. 2. Yamashita N, Tamada Y, Kawada M, Mizutani K, Watanabe D, Matsumoto Y. Analysis of family history of palmoplantar hyperhidrosis in Japan. J Dermatol. 2009;36(12):628-31, http://dx.doi.org/10.1111/j. 1346-8138.2009.00732.x. 3. Kao MC, Lin JY, Chen YL, Hsieh CS, Cheng LC, Huang SJ. Minimally invasive surgery: video endoscopic thoracic sympathectomy for palmar hyperhidrosis. Ann Acad Med Singapore. 1996;25(5):673-8. 4. de Campos JR, Kauffman P, Werebe EC, Andrade Filho LO, Kusniek S, Wolosker N, et al. Quality of life, before and after thoracic sympathectomy: report on 378 operated patients. Ann Thorac Surg. 2003;76(3):88691, http://dx.doi.org/10.1016/S0003-4975(03)00895-6. 5. Neumayer C, Panhofer P, Zacherl J, Bischof G. Effect of endoscopic thoracic sympathetic block on plantar hyperhidrosis. Arch Surg. 2005;140(7):676-80, http://dx.doi.org/10.1001/archsurg.140.7.676. 6. Wolosker N, Yazbek G, Milanez de Campos JR, Kauffman P, Ishy A, Puech-Leao P. Evaluation of plantar hyperhidrosis in patients undergoing video-assisted thoracoscopic sympathectomy. Clin Auton Res. 2007;17(3):172-6, http://dx.doi.org/10.1007/s10286-007-0420-5. 7. Asahina M, Yamanaka Y, Akaogi Y, Kuwabara S, Koyama Y, Hattori T Measurements of sweat response and skin vasomotor reflex for assessment of autonomic dysfunction in patients with diabetes. J Diabetes Complications. 2008;22:278-83, http://dx.doi.org/10.1016/j. jdiacomp.2007.03.009.

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CLINICAL SCIENCE

Giant cell arteritis: a multicenter observational study in Brazil Alexandre Wagner Silva de Souza,I Karine Yoshiye Kajiyama Okamoto,I Fabiano Abrantes,I Bruno Schau,II Ana Beatriz Santos Bacchiega,II Samuel Katsuyuki ShinjoIII I Universidade Federal de Saõ Paulo (Unifesp/EPM), Rheumatology Division, Saõ Paulo/SP, Brazil. II Universidade do Estado do Rio de Janeiro (UERJ), Rheumatology Division, Rio de Janeiro/RJ, Brazil. III Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo (HCFMUSP), Rheumatology Division, Saõ Paulo/SP, Brazil.

OBJECTIVE: To describe demographic features, disease manifestations and therapy in patients with giant cell arteritis from referral centers in Brazil. METHODS: A retrospective cohort study was performed on 45 giant cell arteritis patients from three university hospitals in Brazil. Diagnoses were based on the American College of Rheumatology classification criteria for giant cell arteritis or temporal artery biopsy findings. RESULTS: Most patients were Caucasian, and females were slightly more predominant. The frequencies of disease manifestations were as follows: temporal headache in 82.2%, neuro-ophthalmologic manifestations in 68.9%, jaw claudication in 48.9%, systemic symptoms in 44.4%, polymyalgia rheumatica in 35.6% and extracranial vessel involvement in 17.8% of cases. Aortic aneurysms were observed in 6.6% of patients. A comparison between patients with biopsy-proven giant cell arteritis and those without temporal artery biopsies did not yield significant differences in disease manifestations. All patients were treated with oral prednisone, and intravenous methylprednisolone was administered to nearly half of the patients. Methotrexate was the most commonly used immunosuppressive agent, and low-dose aspirin was prescribed to the majority of patients. Relapses occurred in 28.9% of patients, and aspirin had a protective effect against relapses. Females had higher prevalences of polymyalgia rheumatica, systemic manifestations and jaw claudication, while permanent visual loss was more prevalent in men. CONCLUSIONS: Most of the clinical features of Brazilian giant cell arteritis patients were similar to those found in other studies, except for the high prevalence of neuro-ophthalmic manifestations and permanent blindness in the Brazilian patients. Aspirin had a protective effect on relapses. KEYWORDS: Giant Cell Arteritis; Glucocorticoids; Methotrexate; Multicenter Study; Vasculitis. Souza AW, Okamoto KY, Abrantes F, Schau B, Bacchiega AB, Shinjo SK. Giant cell arteritis: a multicenter observational study in Brazil. Clinics. 2013;68(3):317-322. Received for publication on July 21, 2012; First review completed on August 26, 2012; Accepted for publication on November 9, 2012 E-mail: alexandre_wagner@uol.com.br Tel.: 55 11 5576-4239

Giant cell arteritis (GCA) is a systemic chronic vasculitis of large- and medium-sized vessels that mainly affects the cranial branches of arteries originating from the aortic arch in individuals who are more than 50 years of age (1,2). The highest annual incidence rates of GCA are found in Scandinavian countries and the northern United States, where there are many people of Scandinavian descent; the GCA incidence rates are lower in southern European countries, such

as in Italy and Spain. The lowest incidence rates have been reported in Japan, Turkey and Israel and among native Alaskans (2-5). GCA case series have been published from different countries (e.g., Saudi Arabia, Mexico, Tunisia and India) and population groups (e.g., African American, Asian and Hispanic patients from the United States) (3-6). To our knowledge, the only data on the epidemiology of GCA in the Brazilian population are two case reports and a small study evaluating anterior ischemic optic neuropathy (AION) in GCA patients (7-9). Therefore, this study aimed to describe the demographic features, disease manifestations, therapy and outcomes of Brazilian patients with GCA.

& MATERIALS AND METHODS

& INTRODUCTION

Study design and protocol

No potential conflict of interest was reported.

We performed a retrospective cohort study to evaluate Brazilian patients who fulfilled the American College of

DOI: 10.6061/clinics/2013(03)OA06

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as the means, medians, standard deviations, ranges or as 95% confidence intervals (CIs). Comparisons between groups were performed using a chi-square test or Fisher’s exact test for categorical data and Student’s t-test or MannWhitney U-test for continuous variables. Univariate and multivariate logistic regression models analyzed associations with relapses, and results are expressed as odds ratios (ORs) and 95% CIs.

Rheumatology (ACR) Criteria for GCA (10) and who were under regular follow-up in three tertiary university hospitals (Universidade Federal de Sa˜o Paulo, Universidade de Sa˜o Paulo and Universidade do Estado do Rio de Janeiro). The study was approved by the institutional ethics committees, and data were collected between 2009 and 2010. Patients’ medical records were reviewed to collect data according to a standardized protocol. Demographic features included age at diagnosis and at study date, gender, ethnicity and information on ancestry. We recorded information about systemic symptoms, temporal and occipital headache, jaw claudication, neuro-ophthalmic manifestations of GCA, partial or complete vision loss secondary to GCA neuroophthalmic manifestations, neurological manifestations, such as transient ischemic attacks, ischemic stroke and peripheral neuropathy, symptoms of polymyalgia rheumatica (PMR), involvement of large- and/or middle-sized vessels and erythrocyte sedimentation rate (ESR) at disease onset. Neuro-ophthalmic manifestations of GCA were defined as amaurosis fugax, diplopia, AION or posterior ischemic optic neuritis (PION) and central retinal artery occlusion. A positive temporal artery biopsy was considered when vasculitic findings, such as inflammatory infiltrate on an arterial wall with or without granuloma and multinucleated giant cells, were described upon pathological examination of temporal artery specimens. The investigation of extracranial artery involvement was performed by computed tomography angiography or magnetic resonance angiography when patients presented vascular signs and symptoms, such as limb claudication, vascular bruits or a decrease in arterial pulses, or when imaging studies performed for other purposes displayed arterial abnormalities. When the aorta and/or its main branches were affected by aneurysms, stenosis or arterial occlusions, this was regarded as extracranial artery involvement. Comorbidities and/or complications of secondary glucocorticoid therapy presented by GCA patients were recorded. Disease relapse was considered when the patient presented typical manifestations of GCA associated with an increased ESR (. 50 mm/1st hour) after excluding secondary causes, such as infections. Deaths that occurred during the follow-up period were recorded. The GCA disease course was classified (according to the presence or absence of relapses) as monophasic or relapseremitting. We evaluated medical therapies, including prednisone use, pulse therapy with methylprednisolone, methotrexate or another immunosuppressive agent, antiplatelet therapy and statins. Patients with active GCA and visual symptoms were initially treated with high-dose prednisone (1 mg/kg/ day) with intravenous pulse methylprednisolone (1 g/day for three days), while patients presenting uncomplicated forms of GCA were initially treated with prednisone (40 mg/day). Prednisone tapering began after the resolution of active GCA symptoms and was performed at the physician’s discretion. Methotrexate was added after the first relapse as a steroid-sparing agent and to prevent more disease relapses. If patients could not tolerate methotrexate, it was replaced by another immunosuppressive agent, such as azathioprine or oral cyclophosphamide.

& RESULTS Demographic features and GCA manifestations The median ages of the 45 GCA patients at the time of study and at disease onset were 78.0 (95% CI: 73.2–78.4) and 73.0 (95% CI: 68.7–73.7) years, respectively. In 23 (51.1%) patients, diagnoses of GCA were made between 70 and 79 years of age, and no patients had onset of disease manifestations before age 50 or after age 90. The mean follow-up period was 54.9¡41.9 months. The female-tomale ratio was 1.8:1.0, and most GCA patients were Caucasians (86.7%), while 13.3% were mestizos. Among Caucasian GCA patients, 69.2% were of Portuguese, 25.6% of Italian and 5.2% of Spanish descent. Table 1 describes the prevalence of GCA manifestations in this sample of Brazilian patients. Although all patients fulfilled the ACR criteria for GCA, a temporal artery biopsy was performed on only 18 (40.0%) patients, and it displayed features of vasculitis in 16 of those (88.8%) cases. No significant differences in demographic features, disease manifestations or ESR were found between patients with biopsy-proven GCA and those classified as GCA according to the ACR criteria (Table 2). All patients with neuroophthalmic manifestations developed AION, and it was preceded by amaurosis fugax in 11 cases. No patients developed diplopia, PION or central retinal artery occlusions. In all but one patient with permanent visual loss, amaurosis developed at disease onset in patients presenting neuro-ophthalmic manifestations who did not show improvement in visual acuity after commencing glucocorticoids. Only one patient developed permanent visual loss (unilateral amaurosis) during a relapse. Among patients with extra-cranial artery involvement, aortic aneurysms were found in three patients (6.6%), the thoracic aorta was involved in two patients (4.4%), and the abdominal aorta Table 1 - Manifestations of giant cell arteritis among Brazilian patients. Manifestation Systemic manifestations, n (%) Fever, n (%) Anorexia, n (%) Weight loss, n (%) Temporal headache, n (%) Occipital headache, n (%) Jaw claudication, n (%) Anterior ischemic optic neuritis, n (%) Amaurosis fugax, n (%) Permanent visual loss, n (%) Unilateral visual loss, n (%) Bilateral visual loss, n (%) Polymyalgia rheumatica, n (%) Extracranial vessel involvement, n (%) Neurologic manifestations, n (%)

Statistical analysis SPSS software (version 15.0) was used to carry out the statistical analyses. Categorical data are presented as total numbers and percentages, and numerical data are presented

n - number of patients.

318

Frequency 20 14 13 17 37 6 22 31 11 16 10 6 16 8

(44.4) (31.1) (28.9) (37.8) (82.2) (13.3) (48.9) (68.9) (24.4) (35.5) (22.2) (13.3) (35.6) (17.8) 3 (6.7)

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Giant cell arteritis in Brazil Souza AW et al.

Table 2 - Comparison between patients with biopsy-proven GCA and those classified according the ACR criteria for GCA. Variables Median age at study, years Females, n (%) Caucasians, n (%) Temporal headache, n (%) Occipital headache, n (%) Jaw claudication, n (%) Polymyalgia rheumatic, n (%) Systemic manifestations, n (%) Neuro-ophthalmic manifestations, n (%) Extracranial vessel involvement, n (%) Neurological manifestations, n (%) Mean ESR at diagnosis, mm/hour

Biopsy-proven GCA (N = 16)

GCA based on ACR criteria (N = 29)

p-value

80.5 (56-87) 12 (75.0) 14 (87.5) 13 (81.3) 0 (0.0) 7 (43.8) 3 (18.8) 7 (43.8) 13 (81.3) 3 (18.8) 2 (12.5) 90.1¡41.7

78.0 (58-86) 17 (58.6) 25 (86.2) 24 (82.8) 6 (20.7) 15 (51.7) 13 (44.8) 13 (44.8) 18 (62.1) 5 (17.2) 1 (3.4) 71.3¡25.7

0.141 0.221 0.642 0.600 0.058 0.608 0.075 0.944 0.160 0.600 0.285 0.075

Numerical data are presented as the medians and ranges or as the means ¡ standard deviations; ACR – American College of Rheumatology; ESR – erythrocyte sedimentation rate; GCA – giant cell arteritis; n – number of patients.

6.04; 95% CI: 1.18–30.87; p = 0.031). After adjusting for age, gender and ethnicity in a multivariate model, the beneficial effects of aspirin use to prevent relapses (OR 0.02; 95% CI: 0.001–0.60; p = 0.023), as well as the higher risk for relapses associated with jaw claudication (OR 21.86; 95% CI: 1.32– 360.28; p = 0.031) and the involvement of extra-cranial arteries (OR 45.91; 95% CI: 1.40–1503.54; p = 0.032), remained significant.

was involved in one patient (2.2%). The dissection of the thoracic aorta was observed in only one patient (2.2%) with an aortic aneurysm. Stenosis of extra-cranial arteries was found in five (11.1%) patients with GCA. Subclavian arteries were involved in four cases (8.8%), common carotid arteries were involved in two (4.4%), and the common iliac arteries and superior mesenteric artery were involved in one patient each (2.2%). No patients developed cervical artery stenosis. Peripheral neuropathy was the only neurologic manifestation observed, and no patients developed stroke during the follow-up period. The mean ESR value at disease onset was 78.4¡33.4 mm/1st hour.

Outcomes

All patients were treated with oral corticosteroids at disease onset and during any relapses; intravenous methylprednisolone was administered to 23 (51.1%) patients. Oral weekly methotrexate was prescribed to 21 (46.7%) patients at a median dose of 15 mg (95% CI: 14.8–18.5). An alternative immunosuppressive agent was prescribed to four GCA patients (8.8%); two patients took oral cyclophosphamide, and two took azathioprine. Aspirin was prescribed to 32 (71.1%) and statins to 16 (35.6%) patients.

During follow-up, 41 (91.1%) GCA patients developed at least one complication related to long-term corticosteroid use, including systemic hypertension (66.7%), dyslipidemia (37.8%), osteoporosis (37.8%), diabetes (33.3%), cataracts (20.0%) or glaucoma (8.9%). Coronary heart disease was observed in only two patients (4.4%) during the follow-up period. Four GCA patients (8.9%) developed cancer during the study period; lymphoma was diagnosed in two patients, and skin basal cell carcinoma and breast cancer were diagnosed in one patient each. Four GCA patients (8.9%) died during the study, including three from neoplasia (two with lymphoma and one with breast cancer) and one with sepsis due to severe pneumonia.

Relapses

Comparisons between genders

Most GCA patients had a monophasic disease course, and relapses were observed in 13 (28.9%) patients. The median number of relapses was 1.0 (95% CI: 0.99–1.93). PMR was the most common reactivation form (occurring in six relapses). AION occurred in five relapses, new onset of temporal headache occurred in three, extra-cranial vasculitis occurred in two, and polyarthritis occurred in one. Jaw claudication, PMR and the involvement of large vessels were the GCA manifestations most commonly found during disease course among relapsing patients compared with patients with monophasic disease. Patients with monophasic disease used aspirin more frequently than relapsing patients, and no significant relapse differences were found in GCA patients treated with methylprednisolone intravenous pulse, methotrexate or statins (Table 3). Univariate analysis showed that aspirin had a protective effect against relapses (OR 0.19; 95% CI: 0.04–0.80; p = 0.024). A higher risk for relapse was found in GCA patients who presented PMR (OR 8.03; 95% CI: 1.89–34.10; p = 0.005), jaw claudication (OR 10.5; 95% CI: 1.96–55.99; p = 0.006) and the involvement of extra-cranial arteries (OR

Female GCA patients had a higher prevalence of PMR, systemic manifestations and jaw claudication than male patients. Moreover, they developed more osteoporosis and dyslipidemia during the course of the disease as complications due to chronic steroid therapy. Permanent visual loss was significantly more prevalent in men with GCA than women (Table 4).

Therapy

& DISCUSSION To the best of our knowledge, this is the first study addressing the features of Brazilian GCA patients. Jaw claudication, PMR and extra-cranial vessel involvement were more commonly found in patients with relapsing disease. Furthermore, female patients had a higher prevalence of PMR, systemic manifestations and jaw claudication than men, while the latter developed permanent visual loss more frequently. Finally, antiplatelet therapy was associated with a lower relapse rate. The purpose of this study was to describe GCA features in a country where GCA is uncommon.

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Table 3 - Comparison between monophasic and relapsing patients with giant cell arteritis. Variables Median age at onset, years Females, n (%) Non-Caucasians, n (%) Polymyalgia rheumatica, n (%) Systemic manifestations, n (%) Temporal headache, n (%) Occipital headache, n (%) Jaw claudication, n (%) Neuro-ophthalmic manifestations, n (%) Permanent visual loss, n (%) Extra-cranial vessel involvement, n (%) Mean ESR at diagnosis, mm/hour Methotrexate use, n (%) Intravenous pulse with MP, n (%) Statins use, n (%) Aspirin use, n (%) Deaths, n (%)

Relapsing patients (n = 13)

Non-relapsing patients (n = 32)

p-value

70.0 (54-80) 11 (84.6) 3 (21.1) 9 (69.2) 7 (53.8) 11 (84.6) 2 (15.4) 11 (84.6) 7 (53.8) 3 (23.1) 5 (38.5) 69.3¡44.6 8 (61.5) 5 (38.5) 6 (46.2) 6 (46.2) 2 (15.4)

75.5 (55-82) 18 (56.3) 3 (9.4) 7 (21.9) 13 (40.6) 26 (81.3) 4 (12.5) 11 (34.4) 24 (75.0) 13 (40.6) 3 (9.4) 82.7¡27.2 13 (40.6) 18 (56.3) 10 (31.3) 26 (81.3) 2 (6.3)

0.065 0.072 0.220 0.003* 0.419 0.789 0.796 0.002* 0.165 0.265 0.021 0.346 0.202 0.279 0.344 0.019 0.329

Numerical data are presented as the medians and ranges or as the means ¡ standard deviations; ESR - erythrocyte sedimentation rate; n - number of patients; MP – methylprednisolone.

The median age of disease onset among Brazilian GCA patients was 73.0 years, which falls within the previously described peak of incidence between 70 and 80 years of age (4,11). The age of disease onset of approximately half of our patients was clustered between 70 and 79 years. However, we found no increase in GCA incidence rate in patients between 80 and 89 years of age, and no cases of GCA were diagnosed after the age of 90 years. This differs from the literature, in which the risk of developing GCA increases with advancing age and the incidence of GCA is 20-fold higher among individuals older than 90 years than in those between 50 and 60 years (12). Similarly to other studies, Brazilian GCA patients were predominantly female (1,2). The reason for this is not completely understood; sex-based hormonal differences could be a possible explanation because androgens seem

to play a protective role against autoimmunity and men are exposed to higher androgen levels than women throughout their lives. Due to the post-menopausal onset of GCA, estrogens are less likely to be involved in the pathogenesis (13,14). No significant differences in GCA manifestations between genders have been found in large series evaluating patients with GCA; only an increased prevalence of headache in women was observed in the study performed by Machado et al. (15). However, the higher prevalence of PMR and systemic manifestations among female patients and the higher prevalence of permanent visual loss among the men in our study are in accordance with the results observed in studies that specifically addressed sex differences in GCA (13,16,17). The prevalences of most GCA manifestations in Brazilian patients are similar to those described by other studies; the

Table 4 - Comparison between males and females with giant cell arteritis. Variables Mean age at onset, years Non-Caucasians, n (%) Polymyalgia rheumatica, n (%) Systemic manifestations, n (%) Temporal headache, n (%) Occipital headache, n (%) Jaw claudication, n (%) Neuro-ophthalmic manifestations, n (%) Permanent visual loss, n (%) Extra-cranial vessel involvement, n (%) Mean ESR at diagnosis, mm/hour Complications due to steroid therapy, n (%) Systemic hypertension, n (%) Diabetes, n (%) Dyslipidemia, n (%) Osteoporosis, n (%) Cataracts, n (%) Glaucoma, n (%) Coronary heart disease, n (%) Cancer, n (%) Deaths, n (%)

Females (n = 29)

Males (n = 16)

p-value

70.3¡6.5 6 (20.7) 14 (48.3) 17 (58.6) 26 (89.7) 4 (13.8) 20 (69.0) 19 (65.5) 6 (20.7) 5 (17.2) 80.3¡34.4 28 (96.6) 22 (75.9) 12 (41.4) 15 (51.7) 15 (51.7) 4 (13.8) 2 (6.9) 2 (6.9) 3 (10.3) 3 (10.3)

72.9¡9.1 0 (0.0) 2 (12.5) 3 (18.8) 11 (68.8) 2 (12.5) 2 (12.5) 12 (75.0) 10 (62.5) 3 (18.8) 74.2¡31.8 13 (81.3) 8 (50.0) 3 (18.8) 2 (12.5) 2 (12.5) 5 (31.3) 2 (12.5) 0 (0.0) 1 (6.3) 1 (6.3)

0.323 0.051 0.016* 0.010* 0.079 0.903 ,0.001 0.511 0.005* 0.899 0.579 0.084 0.078 0.123 0.009 0.009 0.161 0.527 0.283 0.644 0.644

Numerical data are presented as the medians and ranges or as the means ¡ standard deviations; ESR - erythrocyte sedimentation rate; n - number of patients.

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Limitations of this study include its retrospective design, the relatively small number of patients and the fact that it was based on referral centers. Most limitations may be explained by the rarity of GCA in Brazil. In this setting, essentially all recognized patients with GCA are referred to tertiary centers for follow-up and management. Referral delays and previous treatment with high-dose corticosteroids are some reasons for the relatively low frequency of temporal artery biopsy (40%) in GCA patients observed in this study. Another limitation of this study is the underestimation of extra-cranial arterial involvement in GCA, as its screening in our patients relied only on signs and symptoms of vascular manifestations and finding vascular abnormalities by chance on imaging studies performed for other purposes. It is now well known that extra-vascular involvement can occur asymptomatically, even at the time of GCA diagnosis (33). In conclusion, GCA in Brazil is mostly found among female Caucasians, and the majority of clinical features are similar to those found in other studies, except for the low frequency of systemic symptoms and high prevalence of neuro-ophthalmic manifestations, including permanent blindness. A significant protective effect of aspirin against disease relapses was observed.

only exception is the lower prevalence of systemic manifestations, such as fever, anorexia and weight loss, in Brazilian patients (1-3,6,15). Due to the retrospective nature of this study, systemic manifestations may have been undervalued and not indicated in the medical records. The high prevalence of neuro-ophthalmic manifestations in this study may be due to referral bias because most GCA patients were referred by ophthalmologists (i.e., all 31 patients with neuro-ophthalmic manifestations). Thus, the high rate of permanent visual loss may reflect the higher prevalence of visual symptoms, in addition to reflecting the delay in recognition of disease features and the low threshold for the diagnosis of GCA due to its rarity in Brazil. Salvarani et al. (18) showed that most cases of permanent visual loss develop prior to the start of corticosteroid therapy. AION was the leading neuro-ophthalmic manifestation in our patients, and it has been reported in approximately 90% of patients with visual symptoms due to GCA (18,19). Amaurosis fugax is recognized as a risk factor for the cranial ischemic complications of GCA (20), and it preceded AION in one-third of our cases. The prevalence of aortic aneurysms in our patients was lower than the 9.5–18.0% previously described (21,22). Despite the low prevalence of aortic aneurysms in this study, one patient developed an aneurysm dissection in the thoracic aorta, and this life-threatening complication reaffirms the importance of evaluating GCA patients for large vessel involvement. The prevalence of arterial stenosis in our study is similar to the 13–14% previously described, and it involved mainly subclavian and common carotid arteries (22,23). In this study, corticosteroids were the most prescribed therapy for active GCA. Although a small trial that included biopsy-proven GCA patients found that intravenous methylprednisolone led to a higher rate of sustained remission, a lower daily dose of prednisone and lower cumulative dose of glucocorticoid, in this study, it was reserved mostly for patients with neuro-ophthalmic manifestations (24). Methotrexate was prescribed to our patients after the first relapse based on a meta-analysis that showed the benefit of methotrexate in preventing relapses and reducing exposure to corticosteroids (25). The frequency of disease relapses in our study was slightly lower than the 30–50% described by other studies (26-29). Although PMR and temporal headache are common features observed in relapsing GCA (26), we found an unexpectedly high frequency of AION among relapsing patients. We also found higher prevalences of PMR, jaw claudication and extra-cranial vessel involvement as GCA manifestations during the course of the disease in relapsing patients. However, in a study that specifically addressed relapses in patients with GCA, no significant differences were found in the disease manifestations of relapsing and non-relapsing patients (26). Two retrospective cohort studies (30,31) found that antiplatelet therapy with low-dose aspirin reduces cranial ischemic complications of GCA, including visual loss and cerebrovascular accidents. Most of our patients were treated with low-dose aspirin, and we observed a protective effect of aspirin on disease relapses. We speculate that the prevention of cranial complications may have accounted for the lower rate of relapses in patients treated with aspirin. Narva´ez et al. (32) did not find any effect of aspirin on either the prevention of cranial ischemic complications or GCA relapse rates.

& ACKNOWLEDGMENTS The authors thank Dr. Paulo Massabki and Professor Luis Eduardo Coelho Andrade for their contributions to the development of this study.

& AUTHOR CONTRIBUTIONS Souza AW contributed to the study design, performed the statistical analysis and wrote the manuscript. Okamoto KY contributed to the study design and participated in the collection of data and manuscript revision. Abrantes F, Schau B and Bacchiega AB participated in the collection of data and revised the manuscript. Shinjo SK contributed to the study design, participated in the collection of data and revised the manuscript.

& REFERENCES 1. Salvarani C, Cantini F, Hunder GG. Polymyalgia rheumatica and giantcell arteritis. Lancet. 2008;372(9634):234-45. 2. Salvarani C, Cantini F, Boiardi L, Hunder GG. Polymyalgia rheumatica and giant-cell arteritis. N Eng J Med. 2002;347(4):261-71. 3. Borchers AT, Gershwin ME. Giant cell arteritis: a review of classification, pathophysiology, geoepidemiology and treatment. Autoimmun Rev. 2012;11(6-7):A544-54. 4. Gonzales-Gay MA, Vazquez-Rodriguez TR, Lopez-Diaz MJ, MirandaFilloy JA, Gonzalez-Juanatey C, Martin J, et al. Epidemiology of giant cell arteritis and polymyalgia rheumatica. Arthritis Rheum. 2009;61(10):145461. 5. Richards BL, March L, Gabriel SE. Epidemiology of large-vessel vasculitis. Best Pract Res Clin Rheumatol. 2010;24(6):871-83. 6. Alba MA, Mena-Madrazo JA, Reyes E, Flores-Suarez LF. Giant cell arteritis in Mexican patients. J Clin Rheum. 2012;18(1):21-7. 7. dos Anjos DA, dos Anjos RF, de Paula WD, Sobrinho AB. F-18 FDG PET/ CT in giant cell arteritis with polymyalgia rheumatica. Clin Nucl Med. 2008;33(6):402-4. 8. Godoy P, Araujo S de A, Paulino E Jr, Lana-Peixoto MA. Coronary giant cell arteritis and acute myocardial infarction. Arq Bras Cardiol. 2007;88(4):e84-7. 9. Monteiro ML. Anterior ischemic optic neuropathy: a comparison of the optic disc area of patients with the arteritic and non-arteritic forms of the disease and that of normal controls. Arq Bras Ophthalmol. 2006; 69(6):805-10. 10. Hunder GG, Bloch DA, Michel BA, Stevens MB, Arend WP, Calabrese LH, et al. The American College of Rheumatology 1990 criteria for the classification of giant cell arteritis. Arthritis Rheum. 1990;33(8):1122-8. 11. Lopez-Diaz MJ, Llorca J, Gonzalez-Juanatey C, Pena-Segredo JL, Martin J, Gonzalez-Gay MA. Implication of the age in the clinical spectrum of giant cell arteritis. Clin Exp Rheumatol. 2008;26(3 Suppl 49):S16-22. 12. Bengtsson BA, Malmvall BE. The epidemiology of giant cell arteritis including temporal arteritis and polymyalgia rheumatica. Incidences of

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24. Mazlumzadeh M, Hunder GG, Easley KA, Calamia KT, Matteson EL, Griffing WL, et al. Treatment of giant cell arteritis using induction therapy with high-dose glucocorticoids: a double-blind, placebo-controlled, randomized prospective clinical trial. Arthritis Rheum. 2006;54(10):3310-8. 25. Mahr AD, Jover JA, Spiera RF, Hernańdez-Garcıá C, FernandezGutie´rrez B, Lavalley MP, et al. Adjunctive methotrexate for treatment of giant cell arteritis: an individual patient data meta-analysis. Arthritis Rheum. 2007;56(8):2789-97. 26. Martinez-Lado L, Calvinõ-Dıáz C, Pin˜eiro A, Dierssen T, VazquezRodriguez TR, Miranda-Filloy JA, et al. Relapses and recurrences in giant cell arteritis: a population-based study of patients with biopsy-proven disease from northwestern Spain. Medicine (Baltimore). 2011;90(3):18693. 27. Proven A, Gabriel SE, Orces C, O’Fallon WM, Hunder GG. Glucocorticoid therapy in giant cell arteritis: duration and adverse outcomes. Arthritis Rheum. 2003;49(5):703-8. 28. Salvarani C, Macchioni PL, Tartoni PL, Rossi F, Baricchi R, Castri C, et al. Polymyalgia rheumatica and giant cell arteritis: a 5-year epidemiologic and clinical study in Reggio Emilia, Italy. Clin Exp Rheumatol. 1987;5(3):205-15. 29. Andersson R, Malmvall BE, Bengtsson BA. Long-term corticosteroid treatment in giant cell arteritis. Acta Med Scan. 1986;220(5):465-9. 30. Lee MS, Smith SD, Galor A, Hoffman GS. Antiplatelet and anticoagulant therapy in patients with giant cell arteritis. Arthritis Rheum. 2006;54(10):3306-9. 31. Nesher G, Berkun Y, Mates M, Baras M, Rubinow A, Sonnenblick M. Low-dose aspirin and prevention of cranial ischemic complication in giant cell arteritis. Arthritis Rheum. 2004;50(4):1332-7. 32. Narvaéz J, Bernad B, Go´mez-Vaquero C, Garcıá-Go´mez C, Roig-Vilaseca D, Juanola X. Impact of antiplatelet therapy in the development of severe ischemic complications and in the outcome of patients with giant cell arteritis. Clin Exp Rheumatol. 2008;26(3 Suppl 49):S57-62. 33. Prieto-Gonza´lez S, Arguis P, Garcıá-Martıńez A, Espı´gol-Frigole´ G, Tavera-Bahillo I, Butjosa M, et al. Large vessel involvement in biopsyproven giant cell arteritis: prospective study in 40 newly diagnosed patients using CT angiography. Ann Rheum Dis. 2012;71:1170-6.

different clinical presentations and eye complications. Arthritis Rheum. 1981;24(7):899-904. Narvaez J, Nolla-Sole´ JM, Valverde-Garcıá J, Roig-Escofet D. Sex differences in temporal arteritis and polymyalgia rheumatica. J Rheumatol. 2002;29(2):321-5. Schuurs AH, Verheul HA. Effects of gender and sex steroids on the immune response. J Steroid Biochem. 1990;35(2):157-72. Machado EB, Michet CJ, Ballard DJ, Hunder GG, Beard CM, Chu CP, et al. Trends in incidence and clinical presentation of temporal arteritis in Olmsted County, Minnesota, 1950-1985. Arthritis Rheum. 1988;31(6):7459. Gonzalez-Gay MA, Garcia-Porrua C, Amor-Dorado JC, Llorca X. Influence of age, sex, and place of residence on clinical expression of giant cell arteritis in Northwest Spain. J Rheumatol. 2003;30(7):1548-51. Nir-Paz R, Gross A, Chajek-Shaul T. Sex differences in giant cell arteritis. J Rheumatol. 2002;29(6):1219-23. Salvarani C, Cimino L, Macchioni P, Consonni D, Cantini F, Bajocchi G. Risk factors for visual loss in an Italian population-based cohort of patients with giant cell arteritis. Arthritis Rheum. 2005;53(2):293-7. Gonza´lez-Gay MA, Garcıá-Porruá C, Llorca J, Hajeer AH, Branãs F, Dababneh A, et al. Visual manifestations of giant cell arteritis. Trends and clinical spectrum in 161 patients. Medicine (Baltimore). 2000;79(5):283-92. Nesher G, Berkun Y, Mates M, Baras M, Nesher R, Rubinow A, et al. Risk factors for cranial complications of GCA. Medicine (Baltimore). 2004;83(2):114-22. Gonzalez-Gay MA, Garcia-Porrua C, Pin˜eiro A, Pego-Reigosa R, Llorca J, Hunder GG. Aortic aneurysm and dissection in patients with biopsyproven giant cell arteritis from Northwestern Spain: a population-based study. Medicine (Baltimore). 2004;83(6):335-41. Nuenninghof DM, Hunder GG, Christianson TJ, McClelland RL, Matteson EL. Incidence and predictors of large-artery complication (aortic aneurysm, aortic dissection, and/or large-artery stenosis) in patients with giant cell arteritis: a population-based study over 50 years. Arthritis Rheum. 2003;48(12):3522-31. Klein RG, Hunder GG, Stanson AW, Sheps SG. Large artery involvement in giant cell (temporal) arteritis. Ann Intern Med. 1975;83(6):806-12.

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CLINICAL SCIENCE

Comparison of 7.2% hypertonic saline - 6% hydroxyethyl starch solution and 6% hydroxyethyl starch solution after the induction of anesthesia in patients undergoing elective neurosurgical procedures Liujiazi Shao, Baoguo Wang*, Shuangyan Wang, Feng Mu, Ke Gu Department of Anesthesiology, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing, China. *corresponding author.

OBJECTIVE: The ideal solution for fluid management during neurosurgical procedures remains controversial. The aim of this study was to compare the effects of a 7.2% hypertonic saline - 6% hydroxyethyl starch (HS-HES) solution and a 6% hydroxyethyl starch (HES) solution on clinical, hemodynamic and laboratory variables during elective neurosurgical procedures. METHODS: Forty patients scheduled for elective neurosurgical procedures were randomly assigned to the HSHES group or the HES group. After the induction of anesthesia, patients in the HS-HES group received 250 mL of HS-HES (500 mL/h), whereas the patients in the HES group received 1,000 mL of HES (1000 mL/h). The monitored variables included clinical, hemodynamic and laboratory parameters. Chictr.org: ChiCTR-TRC12002357 RESULTS: The patients who received the HS-HES solution had a significant decrease in the intraoperative total fluid input (p,0.01), the volume of Ringer’s solution required (p,0.05), the fluid balance (p,0.01) and their dural tension scores (p,0.05). The total urine output, blood loss, bleeding severity scores, operation duration and hemodynamic variables were similar in both groups (p.0.05). Moreover, compared with the HES group, the HS-HES group had significantly higher plasma concentrations of sodium and chloride, increasing the osmolality (p,0.01). CONCLUSION: Our results suggest that HS-HES reduced the volume of intraoperative fluid required to maintain the patients undergoing surgery and led to a decrease in the intraoperative fluid balance. Moreover, HS-HES improved the dural tension scores and provided satisfactory brain relaxation. Our results indicate that HS-HES may represent a new avenue for volume therapy during elective neurosurgical procedures. KEYWORDS: Hypertonic Saline; Hydroxyethyl Starch; Neurosurgery; Fluid Management. Shao L, Wang B, Wang S, Mu F, Gu K. Comparison of 7.2% hypertonic saline - 6% hydroxyethyl starch solution and 6% hydroxyethyl starch solution after the induction of anesthesia in patients undergoing elective neurosurgical procedures. Clinics. 2013;68(3):323-328. Received for publication on September 27, 2012; First review completed on October 22, 2012; Accepted for publication on November 9, 2012 E-mail: wbgttyy@sina.com Tel.: 86 10 62856766

formation of cerebral edema, which is detrimental during neurosurgical procedures. The main goal of intraoperative fluid management is to achieve hemodynamic homeostasis via the administration of small fluid volumes while avoiding fluid overload (2). Currently, increasing investigation into the ideal solution for volume therapy during neurosurgical procedures is being conducted around the world. Since small volume resuscitation with hypertonic saline (HS) in patients suffering from hemorrhage shock was first described in the 1980s (3), HS with or without a colloid such as dextran or hydroxyethyl starch (HES) has emerged as an attractive alternative in fluid management in a variety of surgical practices (4). For example, a wealth of evidence from cardiac surgeries has shown that HS with or without a colloid can exert significant beneficial effects in maintaining hemodynamic stability and a positive fluid balance (5-9).

& INTRODUCTION Intraoperative fluid management is a critical component of perioperative care in neurosurgical practice (1). Patients undergoing elective neurosurgical procedures sometimes require large volumes of intravenous fluid to maintain hemodynamic homeostasis during surgery. However, these volumes may leave patients with an excessive fluid load that increases risks for various complications such as the

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pressure (CVP) at 8-12 mmHg (10.7-16.0 cmH2O) and the mean arterial pressure (MAP) at $65 mmHg. Packed red blood cells (PRBC) were transfused if the hemoglobin concentration fell below 10 g/L. No additional colloid was used during the operation.

Moreover, similar benefits from HS with or without HES have also been demonstrated in aortic aneurysm surgery (10-12) and abdominal hysterectomy (13,14). However, to our knowledge in the clinical setting of neurosurgical practice, HS is mainly used to control intracranial hypertension (ICP) due to its hyperosmolar nature (15), and little is known about the possible role of HS-HES in intraoperative fluid management during elective neurosurgical procedures. In the prospective randomized clinical study presented here, we compared the effects of a 7.2% HS - 6% HES solution (HS-HES) and a 6% HES solution (HES) in terms of the clinical, hemodynamic and laboratory measurements in patients undergoing elective neurosurgical procedures. Our hypothesis was that HS-HES could reduce the volume of intraoperative fluid required to maintain the patients undergoing surgery and lower the dural tension during the operation.

Measurements For all of the patients, each set of measurements included clinical, hemodynamic and laboratory measurements. The clinical variables included the volumes of Ringer’s solution and PRBC infused, intraoperative total urine output, blood loss, operative duration, intraoperative bleeding severity score and dural tension score. The fluid balance was calculated as the difference between the intraoperative total input (randomized solution, 20% mannitol, Ringer’s solution and PRBC) and the output (intraoperative total urine output and blood loss). The blood loss was estimated according to the difference between the volume of fluid collected in the graduated suction bottles and surgical drapes and the volume of any washout fluids used during the operation. The intraoperative bleeding severity was assessed in a blinded manner by the neurosurgeons according to the following grading scale: I. Mild bleeding and no suctioning of blood was required; II. Mild bleeding with occasional suctioning required, but the surgical field was not obscured; III. Moderate bleeding with frequent suctioning required, and the bleeding obscured the surgical field after the suction was removed; IV. Severe bleeding with constant suctioning required, the bleeding appeared again before it was removed by the suction and the surgical field was severely obscured, making surgery impossible. The dural tension was used to estimate the degree of brain relaxation and was determined immediately after the opening of the dura by the neurosurgeons who were blinded to the group assignments. The dural tension scores were assigned using the following scale as described by Cold et al. (16) with a minor modification: I. Normal dural tension: the neurosurgeon easily opened the dura mater; II. Increased dural tension: the dura mater could be opened without additional procedures to lower the ICP; III. Markedly increased dural tension: additional procedures were necessary to lower the ICP to open the dura mater. The hemodynamic measurements included the heart rate (HR), MAP (via the radial artery) and CVP (via the right internal jugular vein), which were monitored continuously using an anesthesia monitor (GE-Ohmeda S/5, USA). The laboratory measurements consisted of the hemoglobin concentration (Hb), platelet count (Plt), hematocrit (Hct), coagulation parameters (prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen concentration (Fbg)), plasma electrolyte concentrations (sodium, potassium, chloride and calcium) and osmolality. All of the hemodynamic and laboratory measurements were documented before the HS-HES or HES infusion (T0) and then again at 30 min (T1), 60 min (T2), 70 min (T3, following the administration of mannitol), 120 min (T4) and 180 min (T5) after the start of infusion.

& MATERIALS AND METHODS Study population Approval for the study was obtained from the Ethics Committee of Beijing Sanbo Brain Hospital, Capital Medical University, and written informed consent was obtained from all of the study participants. This study enrolled 40 consecutive American Society of Anesthesiologists (ASA) ASA I-II patients scheduled for elective neurosurgical procedures. The exclusion criteria were age ,18 years or .80 years; clinical signs of significantly increased ICP such as severe headache, blurred vision and/or papilledema; history of cardiac, pulmonary and renal dysfunction; fluid or electrolyte disturbances; preoperative coagulation disorders; and preoperative treatment with diuretics and/or osmotic agents. The protocol was registered at Chictr.org (ChiCTR-TRC-12002357).

Anesthetic management Patients were premedicated with intravenous midazolam (0.05 mg/kg) 10 min before the induction of anesthesia. General anesthesia was induced with intravenous fentanyl (2 mg/kg), vecuronium bromide (0.1 mg/kg) and propofol (2 mg/kg). After the induction of anesthesia, radial arterial catheters and right internal jugular venous catheters were inserted. After endotracheal intubation, the lungs were mechanically ventilated with intermittent positive pressure ventilation to maintain the end-tidal CO2 pressure at 4.04.67 kPa (30-35 mmHg) during the operation. Anesthesia was maintained with isoflurane (end-tidal minimum alveolar concentration of 1-1.2%) combined with an intravenous bolus of fentanyl or vecuronium if necessary.

Intraoperative fluid management After stabilization at a steady hemodynamic state after the induction of anesthesia, the patients were randomly assigned to receive 250 mL of a 7.2% HS - 6% HES solution (HS-HES group, 500 mL/h) or 1,000 mL of a 6% HES solution (HES group, 1,000 mL/h) through a central venous line. Both solutions were supplied by Fresenius Company (Bad Homburg, Germany). All of the patients in both groups were routinely given 250 mL of 20% mannitol over 10 min at 1 hr after the start of volume expansion. Ringer’s solution was used as a maintenance fluid during the study period at a rate targeted for maintaining the central venous

Statistical analysis Statistical analysis was performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). All of the data are presented as the mean ¡ SD. For the clinical parameters, the differences between the HS-HES and HES groups were analyzed using a x2 test or Fisher’s exact test (categorical

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Effect of HS-HES in neurosurgical patients Shao L et al.

treatment also led to a significant decrease in the dural tension scores compared with HES infusion (p,0.05, Table 2). The changes in HR, MAP and CVP were similar in both groups during the study period (Table 3). Specifically, the HR and CVP in both groups significantly increased after the volume infusion and then decreased over time (Table 3), whereas the MAP was lower at T4 and T5 than at T0 in each group. There were no significant differences between the groups in terms of the Hb, Plt, Hct, PT, APTT and Fbg values and the plasma potassium or calcium concentrations, but the plasma levels of sodium and chloride and the osmolality were markedly increased in the HS-HES group compared with the HES group (Table 4). After the volume expansion, the Hb, Plt, Hct and Fbg values and the plasma calcium concentration in both groups decreased significantly by varying degrees, while the PT and APTT values increased significantly during the study period. The plasma potassium concentration displayed biphasic changes with an increase at the early time points and a decrease at the later time points after HS-HES or HES infusion. Furthermore, in the HS-HES group, the concentrations of plasma sodium and chloride peaked immediately at the end of the HS-HES infusion (T1) and remained higher than that of the HES group throughout the study period. The plasma osmolality was significantly higher after HS-HES infusion (T1-5) than prior to HS-HES infusion (T0).

Table 1 - Demographic data for the patients treated with HS-HES (n = 20) or HES (n = 20).

Age (yrs) Sex (M/F) Weight (kg) Brain lesion Glioma Meningioma

HS-HES group

HES group

39¡12 11/9 60¡10

40¡13 10/10 58¡11

19 1

17 3

The data are presented as the mean ¡ SD. HS-HES: 7.2% hypertonic saline - 6% hydroxyethyl starch solution, HES: 6% hydroxyethyl starch solution.

variables), a Mann-Whitney U test (intraoperative bleeding severity scores and dural tension scores) and an unpaired Student’s t test (age, weight, operation duration, intraoperative total fluid input, volume of Ringer’s solution, PRBC, total urine output, blood loss and fluid balance). For variables with multiple measurements (hemodynamic and laboratory variables), a repeated measures analysis of variance was used to evaluate the effects of time and group assignment, while two-way analysis of variance was used to compare the difference within the groups. Significance was established at p,0.05.

& RESULTS As shown in Table 1, there were no significant differences between the two groups with respect to demographic data (age, sex, weight and surgical procedures). With regard to the clinical measurements, the two groups did not significantly differ by operation duration, volume of PRBC, intraoperative total urine output, blood loss or intraoperative bleeding severity scores (Table 2). The percentages of patients who required a PRBC transfusion in the HS-HES group and the HES group were 25% (5/20) and 20% (4/20), respectively (Table 2). Compared with HES infusion, HS-HES infusion reduced the total fluid input (p,0.01, Table 2) and the volume of Ringer’s solution (p,0.05, Table 2) required during the operation. Moreover, the fluid balance of the HS-HES group was significantly lower than the HES group (p,0.01, Table 2), and HS-HES

& DISCUSSION In the present study, we compared the effects of HS-HES and HES in terms of the clinical, hemodynamic and laboratory measurements in patients undergoing elective neurosurgical procedures. The main findings were as follows: 1) Administration of 250 mL of HS-HES was as efficacious as 1,000 mL of HES in maintaining hemodynamic homeostasis during the operation. 2) HS-HES treatment significantly reduced the intraoperative total fluid input and the volume of Ringer’s solution required to maintain the patients undergoing surgery and led to a significant decrease in the intraoperative fluid balance. 3) The majority of the reduction in total fluid input (61%) was due to the difference in the volume of HES (1,000 mL) and HS-HES (250 mL) initially administered. A reduction in the administration of Ringer’s solution accounted for 39% of the difference. 4) HS-HES infusion markedly reduced the dural tension scores and provided more satisfactory brain relaxation for the operations. 5) HS-HES infusion significantly increased the plasma concentrations of sodium and chloride and increased the plasma osmolality. These findings provide substantial evidence that the HS-HES solution is an attractive choice for fluid management during neurosurgical procedures. The ideal solution for intraoperative volume therapy is controversial. In neurosurgical procedures, achieving hemodynamic homeostasis by the administration of small fluid volumes is of major interest for avoiding fluid overload. HS has been used in the clinic for several decades; its osmotic and volume-expanding properties make it effective as a reliable solution for intraoperative volume therapy. Thus, there is great interest in the use of HS during operations. Moreover, HS is usually used in association with a colloid in most research studies because a wealth of evidence has

Table 2 - Clinical parameters of the patients treated with HS-HES (n = 20) or HES (n = 20).

Operation duration (min) Total fluid input (mL) HS-HES HES 20% mannitol Ringer’s solution PRBC Percentage of patients requiring a PRBC transfusion Total urine output (mL) Blood loss (mL) Fluid balance (mL) Dural tension scores (Grade I/II/III) Bleeding severity scores (Grade I/II/III/IV) *

HS-HES group

HES group

350¡129 4261¡674** 250 0 250 3687¡659* 74¡141 25% (5/20)

360¡96 5498¡676 0 1000 250 4178¡639 70¡152 20% (4/20)

1430¡572 591¡296 2240¡706** 14/5/1* 8/9/3/0

1585¡653 470¡245 3443¡553 6/11/3 9/8/3/0

p,0.05 versus the HES group, p,0.01 versus the HES group. The data are presented as the mean ¡ SD. PRBC: packed red blood cell.

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Table 3 - Hemodynamic parameters of the patients treated with HS-HES (n = 20) or HES (n = 20).

HR (beats/min) MAP (mmHg) CVP (cmH2O) #

Group

Before infusion

HS-HES HES HS-HES HES HS-HES HES

67¡10 67¡13 76¡13 83¡16 8¡3 8¡2

Time after HS-HES or HES infusion (min) 30

120

180

71¡13 68¡13 79¡14 78¡11 11¡3## 12¡3##

75¡12## 72¡15# 74¡11 82¡10 12¡3## 12¡2##

78¡13## 77¡15## 71¡11 78¡10 14¡3## 13¡3##

71¡14 75¡11## 67¡9## 74¡11## 10¡4## 9¡2##

73¡16# 77¡13## 68¡9## 69¡11## 8¡3 9¡3##

p,0.05 versus the measurement before infusion, p,0.01 versus the measurement before infusion. The data are presented as the mean ¡ SD. HR: heart rate, MAP: mean arterial pressure, CVP: central venous pressure.

shown that a colloid such as HES can prolong the hemodynamic efficiency of HS (17). Previous studies on models of hemorrhagic shock have indicated that HS infusion in a volume equivalent to 25% of the total blood lost can restore hemodynamic stability (18,19), whereas an HES infusion can replace the blood lost for a volume expansion at close to a 1:1 ratio (20,21). In the present study, our data showed that there were no significant differences in the hemodynamic parameters between the groups after the infusion of 250 mL of HSHES or 1,000 mL of HES, which corroborate the above results. Moreover, our results indicated that HS-HES could reduce the intraoperative total fluid input and the volume of Ringer’s solution required to maintain the patients undergoing surgery, which was in agreement with the evidence that HS can be used as the fluid for small-volume resuscitation in patients with hypovolemic shock (22). However, in the present study, the majority of the total fluid input reduction was due to the initial fluid load. Thus, because the magnitude of the hemodilution and CVP were

similar in the present study, we assumed that the volume of the plasma expansion was similar in both groups in spite of the different volumes of fluid administered. Multiple mechanisms are involved in the volume-expanding properties of HS, including the compartment redistribution with fluid shifts to the vascular bed (8), the positive effects on cardiac output (23) and the hormonal and immunologic effects (24). Fluid balance during an operation and the postoperative period are of considerable importance to organic function, especially to cerebral function in neurosurgical procedures. In the present study, we investigated the effect of HS-HES on the fluid balance during surgery and found that HS-HES infusion could significantly reduce the intraoperative fluid balance. Although we did not collect any data on the effect of HS-HES on the postoperative fluid balance, the results from other authors have demonstrated that a near-zero fluid balance was observed in patients who were given an HS-dextran infusion 48 hrs after cardiac surgery (8,25), which suggests that the maximal effect of HSHES on the reduction of the fluid balance might appear

Table 4 - Laboratory parameters of the patients treated with HS-HES (n = 20) or HES (n = 20). Group

Time after HS-HES or HES infusion (min)

Before infusion 30

Hb (g/L) Plt (6107/L) Hct (%) PT (s) APTT (s) Fbg (g/L) Na+ (mmol/L) Cl- (mmol/L) K+ (mmol/L) Ca2+ (mmol/L) Osm (mOsm/kg H2O)

HS-HES HES HS-HES HES HS-HES HES HS-HES HES HS-HES HES HS-HES HES HS-HES HES HS-HES HES HS-HES HES HS-HES HES HS-HES HES

129.1¡11.6 138.8¡19.9 208.2¡53.7 220.4¡42.9 38.4¡3.2 41.3¡5.0 10.4¡0.9 10.5 ¡0.7 26.3¡5.8 24.3¡3.7 2.0¡0.4 2.0¡0.6 138.7¡2.7 139.2¡2.5 107.6¡1.4 106.3¡3.0 3.8¡0.3 3.9¡0.3 2.1¡0.1 2.2¡0.1 300.7¡5.8 301.0¡5.7

60 ##

110.4¡10.4 116.2¡18.7## 197.4¡43.7## 205.2¡37.6## 33.0¡3.2## 34.7¡4.9## 12.0¡0.7## 11.8¡0.7## 33.4¡7.1## 30.8¡4.9## 2.0¡0.7 1.8¡0.6 152.5¡4.4**## 139.2¡2.1 122.4¡4.3**## 109.8¡2.2## 3.5¡0.4## 3.7¡0.3## 1.9¡0.1## 1.9¡0.2## 323.5¡8.2**## 300.1¡5.8

109.9¡11.4 113.7¡29.0## 197.0¡45.1## 188.0¡36.0## 32.8¡3.4## 32.5¡4.7## 11.9¡0.8## 12.2¡0.7## 33.4¡8.0## 35.3¡7.0## 1.8¡0.3 1.6¡0.5## 147.4¡2.7**## 138.0¡4.8 118.5¡3.2**## 110.0¡3.6## 4.1¡0.5## 3.8¡0.4 1.9¡0.1## 1.8¡0.3## 316.5¡5.4**## 302.9¡6.0

** #

70 ##

120 ##

101.7¡11.0 100.2¡16.7## 184.4¡43.3## 173.3¡37.7## 30.6¡3.2## 30.3¡4.6## 12.2¡0.9## 12.7¡0.8## 37.6¡9.4## 40.6¡8.7## 1.7¡0.3## 1.5¡0.5## 137.7¡8.3** 131.4¡2.8## 113.3¡3.3**## 105.2¡2.4 4.3¡0.6## 3.9¡0.5 1.8¡0.1## 1.7¡0.2## 328.7¡8.7**## 313.5¡7.4##

180 ##

110.4¡11.8 105.7¡17.9## 204.4¡48.0 191.3¡38.9## 33.3¡3.5## 31.7¡4.8## 11.8¡0.7## 12.2¡1.1## 33.8¡7.7## 36.1¡6.9## 1.8¡0.3 1.7¡0.6## 141.9¡2.5**# 134.8¡2.6## 114.9¡3.0**## 107.3¡2.9 4.8¡0.7## 4.3¡0.5## 1.9¡0.1## 1.8¡0.1## 320.9¡5.3**## 306.7¡7.5##

107.9¡14.1## 106.9¡19.5## 200.2¡46.4 198.6¡49.3## 32.5¡3.9## 32.3¡5.5## 12.0¡0.8## 12.2¡1.0## 33.1¡8.5## 36.7¡7.1## 1.8¡0.3 1.6¡0.5## 141.5¡2.5**# 136.3¡3.1## 116.0¡2.9**## 109.2¡3.3## 4.4¡0.5## 4.3¡0.4## 1.9¡0.1## 1.9¡0.2## 315.8¡5.8**## 307.1¡8.2##

p,0.01 versus the HES group, p,0.05 versus the measurement before infusion, p,0.01 versus the measurement before infusion. The data are presented as the mean ¡ SD. Hb: hemoglobin concentration, Plt: platelet count, Hct: hematocrit, PT: prothrombin time, APTT: activated partial thromboplastin time, Fbg: fibrinogen concentration, Osm: osmolality.

326

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Effect of HS-HES in neurosurgical patients Shao L et al.

they were higher in the HS-HES group than in the HS group. By contrast, the plasma chloride levels remained higher than normal throughout the study period. In the later study periods (T3-5), the plasma sodium and chloride concentrations showed transient decreases in comparison with those concentrations at T2 before they gradually increased. In the present study, the blood sample taken after the administration of mannitol (T3) was analyzed because previous studies had indicated that mannitol can provide a transient volume expansion and subsequent diuretic action (27), which may explain the changes in the plasma concentrations of sodium and chloride at T3-5. The plasma potassium concentrations in both groups displayed biphasic changes with increases at the earlier time points and decreases at the later time points. The concentrations never exceeded the normal clinical range, which might be due to the multiple additive effects such as hemodilution from volume expansion at the earlier time points and the diuretic effect from the mannitol at the later time points. Moreover, we found that the plasma calcium concentration decreased in both groups. Two reasons may account for the hypocalcaemia: intraoperative bleeding can lead to the loss of calcium; and hemodilution occurred from the volume expansion of the HS-HES or HES treatments, which did not contain calcium. However, even though no neurologic deficits were found in the present study, it is still necessary to monitor the electrolyte concentrations when an HS-HES solution is infused. Not surprisingly, a clear increase in plasma osmolality was observed after the HS-HES infusion but not after the HES infusion. This increase is one of the known mechanisms involved in the beneficial effects of HSHES treatment. In conclusion, a 7.2% HS - 6% HES solution infused after the induction of anesthesia is an attractive choice for fluid management during neurosurgical procedures. HS-HES infusion can reduce the volume of intraoperative fluid required to maintain the patients undergoing surgery and can lead to a decrease in the intraoperative fluid balance. Furthermore, the majority of the total fluid input reduction (61%) was due to the difference in the volume of HES (1,000 mL) and HS-HES (250 mL) initially administered. A reduction in the administration of Ringerâ&#x20AC;&#x2122;s solution accounted for 39% of the difference. Moreover, HS-HES decreased the dural tension scores and provided satisfactory brain relaxation. Our results indicate that HS-HES may represent a new avenue for volume therapy during neurosurgical procedures.

during the postoperative period. Further studies investigating the effect of HS-HES on the postoperative fluid balance in patients following neurosurgical procedures are needed. In our study, we used dural tension scores as an alternative tool to indirectly monitor ICP. We found that HS-HES reduced the dural tension scores and provided more satisfactory brain relaxation for the operation. In clinical practice, the ICP is not measured routinely during elective neurosurgical procedures (26), which is also a limitation of our present study. Thus, dural tension scores are always used to partially reflect the ICP in neurosurgical research and to compare the various interventions because evidence has shown a strong positive correlation between the degree of cerebral swelling and ICP (16,27-29). In the present study, the improvement in the dural tension scores in the HS-HES group may have arisen from the effect of HS on controlling the ICP. HS reduces ICP in various types of intracranial diseases, particularly following head trauma with increased ICP (30). This effect exerted by HS-HES infusion can mainly be attributed to its hyperosmotic properties. HS-HES infusion produces an osmotic gradient between the intravascular and intracellular/interstitial compartments when the blood-brain barrier is intact, leading to a reduction of brain volume and therefore reducing the ICP (31). Additional effects such as decreasing the formation of CSF may also contribute to the reduction of ICP arising from HS-HES infusion (32). Moreover, we found that HS-HES may exert additional effects to control ICP even among patients who had already received mannitol, which corroborated the evidence suggesting that HS remained effective for mannitol-resistant ICP. Opening the dura mater is a critical moment during a craniotomy because the swelling brain may protrude through the craniotomy site, which can seriously jeopardize surgical access and increase the risk of cerebral ischemia with a possible worsening of the outcome (16,28). Therefore, satisfactory brain relaxation, as reflected by a reduction in the dural tension scores in the HS-HES group, facilitates the surgical process and reduces the potential risk for poor outcomes. In the present study, we did not find a marked diuretic effect after HS-HES infusion compared with HES infusion, although some studies have shown that HS can act as a diuretic (33). We speculated that HS-HES infusion significantly increased the plasma osmolality, which stimulated the release of antidiuretic hormones and led to the absorption of free water by the kidneys (34). Further study is needed to explain the effect of HS-HES on the hormones regulating water metabolism. Interference with blood coagulation is another important issue that deserves attention during volume therapy (35). In the present study, although the coagulation parameters in both groups showed significant changes from the baseline values, they were still within the clinically tolerated ranges, which indicated that there was no or only slight coagulation impairment after HS-HES infusion. The finding that there was no significant difference in the intraoperative bleeding severity scores between the two groups supported the above conclusions. Electrolyte abnormalities are common complications after the infusion of hyperosmotic drugs (15). In the present study, hypernatremia and hyperchloremia occurred immediately after HS-HES infusion. Normal plasma sodium levels were present at the end of study period, although

& AUTHOR CONTRIBUTIONS Shao L wrote the manuscript and was responsible for the data collection. Wang B is the corresponding author, conceived and designed the study and was responsible for the data collection, analysis and interpretation, as well as the preparation of the manuscript. Wang S, Mu F and Gu K were responsible for the patient recruitment and the data collection and analysis.

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20. Barros JM, Do NPJ, Marinello JL, Braz LG, Carvalho LR, Vane LA, et al. The effects of 6% hydroxyethyl starch-hypertonic saline in resuscitation of dogs with hemorrhagic shock. Anesth Analg. 2011;112(2):395-404, http://dx.doi.org/10.1213/ANE.0b013e3181f2e9b2. 21. James MF, Latoo MY, Mythen MG, Mutch M, Michaelis C, Roche AM, et al. Plasma volume changes associated with two hydroxyethyl starch colloids following acute hypovolaemia in volunteers. Anaesthesia. 2004;59(8):738-42, http://dx.doi.org/10.1111/j.1365-2044.2004.03811.x. 22. Sapsford W. Hypertonic saline dextran--the fluid of choice in the resuscitation of haemorrhagic shock? J R Army Med Corps. 2003;149(2):110-20. 23. Kien ND, Kramer GC. Cardiac performance following hypertonic saline. Braz J Med Biol Res. 1989;22(2):245-8. 24. Rizoli SB, Rhind SG, Shek PN, Inaba K, Filips D, Tien H, et al. The immunomodulatory effects of hypertonic saline resuscitation in patients sustaining traumatic hemorrhagic shock: a randomized, controlled, double-blinded trial. Ann Surg. 2006;243(1):47-57, http://dx.doi.org/ 10.1097/01.sla.0000193608.93127.b1. 25. Oliveira SA, Bueno RM, Souza JM, Senra DF, Rocha-e-Silva M. Effects of hypertonic saline dextran on the postoperative evolution of Jehovahâ&#x20AC;&#x2122;s Witness patients submitted to cardiac surgery with cardiopulmonary bypass. Shock. 1995;3(6):391-4. 26. Dinsmore J. Anaesthesia for elective neurosurgery. Br J Anaesth. 2007;99(1):68-74. 27. Wu CT, Chen LC, Kuo CP, Ju DT, Borel CO, Cherng CH, et al. A comparison of 3% hypertonic saline and mannitol for brain relaxation during elective supratentorial brain tumor surgery. Anesth Analg. 2010;110(3):903-7, http://dx.doi.org/10.1213/ANE.0b013e3181cb3f8b. 28. Petersen KD, Landsfeldt U, Cold GE, Petersen CB, Mau S, Hauerberg J, et al. Intracranial pressure and cerebral hemodynamic in patients with cerebral tumors: a randomized prospective study of patients subjected to craniotomy in propofol-fentanyl, isoflurane-fentanyl, or sevofluranefentanyl anesthesia. Anesthesiology. 2003;98(2):329-36, http://dx.doi. org/10.1097/00000542-200302000-00010. 29. Iversen BN, Rasmussen M, Cold GE. The relationship between intracranial pressure and the degree of brain swelling in patients subjected to infratentorial surgery. Acta Neurochir (Wien). 2008;150(4):337-44, http://dx.doi.org/10.1007/s00701-008-1461-1. 30. White H, Cook D, Venkatesh B. The role of hypertonic saline in neurotrauma. Eur J Anaesthesiol Suppl. 2008;42:104-9, http://dx.doi. org/10.1017/S0265021507003420. 31. Elkins MR, Bye PT. Mechanisms and applications of hypertonic saline. J R Soc Med. 2011;104 (Suppl 1):S2-5. 32. Gemma M, Cozzi S, Tommasino C, Mungo M, Calvi MR, Cipriani A, et al. 7.5% hypertonic saline versus 20% mannitol during elective neurosurgical supratentorial procedures. J Neurosurg Anesthesiol. 1997;9(4):329-34, http://dx.doi.org/10.1097/00008506-199710000-00007. 33. Ziai WC, Toung TJ, Bhardwaj A. Hypertonic saline: first-line therapy for cerebral edema? J Neurol Sci. 2007;261(1-2):157-66, http://dx.doi.org/10. 1016/j.jns.2007.04.048. 34. Peters HP, Robben JH, Deen PM, Wetzels JF. Water in health and disease: new aspects of disturbances in water metabolism. Neth J Med. 2007;65(9):325-32. 35. Schramko AA, Suojaranta-Ylinen RT, Kuitunen AH, Kukkonen SI, Niemi TT. Rapidly degradable hydroxyethyl starch solutions impair blood coagulation after cardiac surgery: a prospective randomized trial. Anesth Analg. 2009;108(1):30-6, http://dx.doi.org/10.1213/ane.0b013e31818c12 82.

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CLINICAL SCIENCE

Adverse reactions to antituberculosis drugs in Manguinhos, Rio de Janeiro, Brazil Glauciene Santana Damasceno,I Lusiele Guaraldo,II Elyne Montenegro Engstrom,I Mariza Miranda Theme Filha,I Reinaldo Souza-Santos,I Ana Gloria Godoi Vasconcelos,I Suely RozenfeldI I

Escola Nacional de Sau´de Pu´blica Sergio Arouca/Fiocruz, Rio de Janeiro/RJ, Brazil. II Instituto de Pesquisa Clı´nica Evandro Chagas/Fiocruz, Rio de Janeiro/ RJ, Brazil.

OBJECTIVES: This study aimed to characterize and estimate the frequency of adverse reactions to antituberculosis drugs in the population treated at the Centro de Sau´de Escola Germano Sinval Faria, a primary health care clinic in Manguinhos, Rio de Janeiro City, and to explore the relationship between adverse drug reactions and some of the patients’ demographic and health characteristics. METHODS: This descriptive study was conducted via patient record review of incident cases between 2004 and 2008. RESULTS: Of the 176 patients studied, 41.5% developed one or more adverse reactions to antituberculosis drugs, totaling 126 occurrences. The rate of adverse reactions to antituberculosis drugs was higher among women, patients aged 50 years or older, those with four or more comorbidities, and those who used five or more drugs. Of the total reactions, 71.4% were mild. The organ systems most affected were as follows: the gastrointestinal tract (29.4%), the skin and appendages (21.4%), and the central and peripheral nervous systems (14.3%). Of the patients who experienced adverse reactions to antituberculosis drugs, 65.8% received no drug treatment for their adverse reactions, and 4.1% had one of the antituberculosis drugs suspended because of adverse reactions. ‘‘Probable reactions’’ (75%) predominated over ‘‘possible reactions’’ (24%). In the study sample, 64.3% of the reactions occurred during the first two months of treatment, and most (92.6%) of the reactions were ascribed to the combination of rifampicin + isoniazid + pyrazinamide (Regimen I). A high dropout rate from tuberculosis treatment (24.4%) was also observed. CONCLUSION: This study suggests a high rate of adverse reactions to antituberculosis drugs. KEYWORDS: Tuberculosis; Drug Toxicity; Patient Treatment Refusal; Primary Health Care. Damasceno GS, Guaraldo L, Engstrom EM, Theme Filha MM, Souza-Santos R, Vasconcelos AG, et al. Adverse reactions to antituberculosis drugs in Manguinhos, Rio de Janeiro, Brazil. Clinics. 2013;68(3):329-337. Received for publication on June 26, 2012; First review completed on July 18, 2012; Accepted for publication on November 9, 2012 E-mail: glaucienesantana@uol.com.br Tel.: 55 21 2598-2631

previous factors and one of the key obstacles to controlling the disease (2). In Brazil, ‘‘...tuberculosis is neither an emerging nor a reemerging public health problem. It is a problem that has been present and resident for a long time’’ (3). Brazil ranks 19th among countries with the most tuberculosis cases in the world today (5). Nationwide, some 5,000 to 6,000 deaths from tuberculosis are recorded annually (6). Recently, the number of new cases in Brazil has declined significantly, from 51.44/100,000 in 1999 to 37.12/100,000 in 2008 (7). Nonetheless, in 2008, incidence in the Rio de Janeiro municipal area was 68.64/100,000 (8), the highest in the country, while in some of the municipality’s most underserved areas, such as the Manguinhos district, annual incidence reached 145/100,000 (9). Manguinhos has Rio de Janeiro’s third-lowest Human Development Index, is considered the city’s worst district in terms of basic sanitation, and houses a large number of former prison inmates. Its population profile features a high proportion of chemical- and alcohol-dependent individuals

& INTRODUCTION Tuberculosis is the chronic infectious disease that kills the most individuals worldwide. Since 1993, the World Health Organization (WHO) (1,2) has considered tuberculosis a global emergency. In 2005, there were an estimated eight million new cases and approximately two million deaths per year worldwide, with only half the cases reported to the WHO (1). The main factors aggravating the situation are social inequality, the spread of AIDS, an aging population, major migratory movements (3), improper health care (4), and a lack of information, which is associated with these

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tuberculosis, and considered new cases between January 2004 and December 2008. During the study, 291 cases of tuberculosis were identified in the SINAN, and 370 cases were identified in the case log book. After matching the datasets and excluding duplicate records and cases that were reported more than once because of relapse or return after dropout, 279 cases remained for patient record analysis. After the inclusion criteria were applied, 176 cases remained eligible for the study. The records of 103 patients were excluded for the following reasons: not being considered new cases of pulmonary tuberculosis (relapse or return after dropout, n = 25); residency in other districts of Rio de Janeiro (n = 14); other types of tuberculosis (n = 12); not having completed at least 30 days of treatment (n = 13); transfer for follow-up to another clinic (n = 13); changes of diagnosis (n = 20); and incomplete records that made it impossible to identify the closure status (n = 6; Figure 1). Patients were followed up until case closure by cure, dropout, failure/multidrug-resistant tuberculosis (MDRTB), or death.

and a large street population that is characterized by extreme poverty and little schooling. These problems accentuate the difficulties of controlling tuberculosis (10). The Ministry of Health (MoH) considers that compliance with the appropriate recommended therapeutic regimen makes tuberculosis curable in essentially 100% of cases. Using antituberculosis drugs correctly for an adequate period ensures a cure (6). Although they are effective at combating Mycobacterium tuberculosis, tuberculosis drugs can cause adverse reactions that may jeopardize treatment and contribute to nonadherence (11). An adverse drug reaction (ADR) consists of ‘‘a response to a drug that is noxious and unintended and occurs at doses normally used in man for the prophylaxis, diagnosis, or therapy of disease’’ (12). Treatment dropout impairs control of the disease, hinders its cure, and increases treatment time and costs (4). In Manguinhos and other communities in major urban centers, the tuberculosis incidence is much higher than the national and local means; accordingly, this higher incidence deserves detailed study. This study aimed to characterize and estimate the frequency of adverse reactions to antituberculosis drugs in the population treated at the Centro de Sau´de Escola Germano Sinval Faria (CSEGSF), a primary health care clinic in Manguinhos, Rio de Janeiro City. In addition, the study explored the relationship between adverse drug reactions and some of the patients’ demographic and health characteristics.

Information sources, data collection, and study variables For data collection, a patient record data collection form and a form-completion instructions manual were developed in an electronic format using Access (v. 2.0, 2003). The two instruments were standardized and tested. The information drawn from the patient records was complemented with project sources (13). Data were collected by the lead researcher in June and July 2010. To ensure quality at this stage, two other authors (LG and SR) collected data again from 10% of the patient records. At this stage, no major divergences were found. Some aspects of data recording that had no impact on the estimates were standardized. The following variables were considered: Adverse drug reaction (ADR): Information drawn from the progress sheets in patients’ records, with the respective dates. Case closure status: Information drawn from data collected for the project (13). The variable categories and definitions (6) were as follows: Dropout: ‘‘Any patient who fails to attend the clinic for more than 30 consecutive days after the scheduled return date. In cases of supervised treatment, the 30-day limit is counted from the last drug administration.’’ Failure/MDR-TB: ‘‘Persistently positive sputum smear tests at the end of treatment. Also classified as ‘Failure’ are patients who are strongly positive (++ or +++) at start of treatment and maintain that status until the fourth month, as well as all those who start treatment positive, then are negative, then once again positive for two consecutive months, as of the fourth month of treatment.’’ Death: ‘‘Classified upon learning of the patient’s death during treatment, regardless of cause.’’ Cure: ‘‘Cases initially positive for pulmonary TB: discharge by cure will be given when, on completing treatment, the patient returns two negative sputum smear tests, one during the follow-up stage and one upon completion of treatment (cure). Discharge by cure will also be given when treatment is deemed complete based on clinical and radiological criteria and complementary examinations.’’

& METHODS This study forms part of the project ‘‘Fatores associados aos desfechos do tratamento da tuberculose em um centro de atenc¸a˜o ba´sica de sau´de: uma ana´lise sob a o´tica antropolo´gica e epidemiolo´gica’’ (13) [Factors associated with tuberculosis treatment outcomes at a primary health care clinic: an anthropological and epidemiological analysis], which is financed by the Global Fund to Fight Tuberculosis, Aids and Malaria and conducted at CSEGSF/ENSP/Fiocruz/RJ. Information was drawn from the Rio de Janeiro compulsory notifications database (SINAN/SMS RJ), patient records, the CSEGSF case log book, and the computerized patient register. These sources served as the basis for project researchers (13) to define the tuberculosis ‘‘cases’’ used for the study of adverse reactions.

Study design and population This study was descriptive via retrospective review of patient medical records. The population consisted of new pulmonary tuberculosis cases treated at the CSEGSF outpatient department. According to the MoH Manual Te´cnico para o Controle da Tuberculosis, a case of tuberculosis is any individual with a diagnosis confirmed by sputum smear test or culture and for whom the physician establishes a diagnosis of tuberculosis based on clinical and epidemiological data and the results of complementary examinations. A new case is someone with tuberculosis who has never undergone antituberculosis chemotherapy, has done so for less than 30 days, or was treated for tuberculosis more than five years ago (14). Included in this study were children, adults, and older adults of both sexes who were residing in the Manguinhos district of Rio de Janeiro, diagnosed with pulmonary

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Adverse reactions to antituberculosis drugs Damasceno GS et al.

Figure 1 - Flowchart of cases of tuberculosis (TB) included in the study, CSEGSF/ENSP/FIOCRUZ-RJ, 2004-2008.

Associated comorbidities: Information drawn from patient records. All comorbidities were recorded for subsequent analysis using the Naranjo algorithm (15), a tool for evaluating the causal relationship between drug use and adverse reaction. The date of occurrence was that of the first recording of each comorbidity during the patient’s treatment period. The International Classification of Diseases (ICD-10) was attached to the electronic form so that the ICD codes described in the record form could be identified and the morbidities described in the record form could be assigned an ICD code. All prior diseases, such as hypertension, diabetes, AIDS, and alcoholism, recorded by the medical team were considered. Any symptoms or signs that might be ADRs were also recorded. Sex and age: information was drawn from patient records. Age was recorded as a continuous variable and subsequently categorized into the following age groups: 0 to 19 years, 20 to 49 years, and 50 years or older. Drugs used: Information was drawn from patient record progress sheets, with prescription dates. Antituberculosis drugs (rifampicin, isoniazid, pyrazinamide) and other drugs, both continuous- and occasional-use, were considered. The following categories were created for the antituberculosis drugs: prescribed (P), suspended by patient (SP), suspended by medical practitioner (SM), dropout (D), completion of therapy (C), and non-protocol doses (P*). For the analysis of the total number of antituberculosis and other drugs used, two categories were created: up to four and five or more. Drugs used during treatment were counted, and repeat prescriptions were disregarded.

Data analysis Two components were considered in the analysis: patients and ADRs. The data on patients and ADRs are given in absolute values and as proportions. The mean and median behavior of the continuous variables was also noted. Proportions were compared using the Pearson chi-squared (x2) test. Patients were classified by occurrence of ADR with the aid of the Naranjo algorithm, which contains ten questions, each of which receives a score. The questions specifically address the compatibility between when the reaction appeared and when the drug was taken; the nature of the reaction; the drug’s pharmacological characteristics; and alternative causes (15). The algorithm enables ADRs to be classified as ‘‘definite’’, ‘‘probable’’, ‘‘possible’’, or ‘‘doubtful’’. In the case of doubtful reactions, patients were classified as not experiencing an ADR. Recording the dates of interest (prescription of each drug; appearance of the ADR; comorbidities identified) made it possible to rigorously identify the time relation between prior exposure to the drugs and the subsequent appearance of adverse reactions. ADRs were classified by the organ system affected according to WHO terminology (WHO-ART, 2005) (16) and described by type, period of occurrence, and severity. Severity classifications were based on the MoH definitions (6), as follows: mild effects, in which there is no immediate modification of the standard regimen, and major effects, which may entail interruption or alteration of the treatment. The identification of ADRs not described by MoH was based on the scientific literature (17,18,19,20).

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proportion of ADRs than other age groups (52.4% vs. 22.2% among the 0- to 19-year-old subjects). The stratum with four or more comorbidities showed a higher proportion of ADRs than others (80.0% vs. 20.6% for no comorbidities). The stratum using five or more drugs showed a higher proportion of ADRs (61% vs. 31.6% among users of up to four drugs; Table 1). Of the 176 patients studied, 73 (41.5%) developed one or more ADRs, for a total of 126 occurrences (Tables 1 and 2). Of the 73 patients with ADRs, 39 developed only one ADR, while 34 patients developed two or more ADRs during the study period. Approximately 78% (98/126) of the ADRs are described in the MoH epidemiological surveillance guide (Guia de Vigilaˆncia Epidemiolo´gica) (6). Of the ADRs found in this study, 71.4% were considered mild reactions, 6.4% were major, and 22.2% were considered ‘‘other’’. Of the latter, the most frequent (32.1%) was dizziness (Table 2). No death from antituberculosis ADR was observed. Of the total patients with ADRs, 48 (65.8%) received no drug treatment for the ADR, and three (4.1%) had one of the antituberculosis drugs suspended because of an adverse reaction. The organ systems most affected by ADRs were the gastrointestinal tract (29.4%), the skin and appendages (21.4%), the central and peripheral nervous systems (14.3%), and the musculoskeletal system (10.3%). Disturbances of the urinary system, the liver and gall bladder, the female reproductive system, the white and red blood cells and reticuloendothelial system, metabolism and nutrition, and the overall state of health and psychiatric disturbances were also found. Each of these disturbance types comprised between 0.8 and 5.5% of the cases analyzed. Table 3 describes the ADRs according to the treatment period during which they occurred and according to the severity of the ADR and the related drugs. ADRs may be associated with more than one drug, which share an array of undesirable side-effects, attributable in part to common pharmacokinetic processes. In the study sample, 81 (64.3%) of the reactions occurred during the first two months of treatment, and most reactions (92.6%) were imputed to the combination of rifampicin + isoniazid + pyrazinamide (Regimen I). The severest ADRs tended to occur from the third month of treatment onwards (p,0.05). The likelihood of an adverse reaction being caused by the drugs was analyzed using the Naranjo algorithm. The results showed a predominance of probable reactions (75%), followed by possible reactions (24%) and definite reactions (1%). A high rate of dropout from TB treatment (24.4%) was observed (43/176). The dropout rate was higher among the men than women (29.4% vs. 18.3%) and higher among younger people (37% were from 0 to 19 years old vs. 23.6% in the 20 to 49 years group and 21.1% in the 50 years or older group), although the differences were not statistically significant. Dropout was also observed to be greater (p,0.05) among those with no recorded comorbidities (38.7% vs. 18.5% with one to three comorbidities and 13.3% with four or more comorbidities), those who used up to four drugs (32.7% vs. 10.7% of users of five or more drugs), and those who developed no adverse reactions to the antituberculosis drugs (34.0% vs. 13.0% among those with an ADR).

To explore the relationships between the ADRs and the characteristics of the study population, a multiple correspondence analysis (MCA) was used. This is a descriptive or exploratory multivariate technique. The analysis was essentially graphic and was conducted by observing proximity among points in a multidimensional space representing the spatial configuration of the categories of variables analyzed (21). The analyses were performed using the statistical package SPSS for Windows, Version 17.0. Correspondence analysis was performed using the software XLSTAT 4.0.

Ethical considerations The project was approved by the Oswaldo Cruz Foundation (CEP/ENSP/Fiocruz) ethics committee for research with human subjects under Number 0038.0.031.000-09.

& RESULTS The 176 patients selected were included in the analyses. Their ages ranged from 1 to 85 years (mean = 32.4 years; median = 29 years). Of the participants, 113 (64.2%) were male, 128 (72.7%) were 20 to 49 years old, 161 (91.5%) had up to three recorded comorbidities, and 117 (66.5%) used up to four drugs (Table 1). Of the total patient population, 126 (71.6%) were cured, while 43 (24.4%) dropped out of treatment, five (2.8%) died from tuberculosis, one (0.6%) died from other causes, and one (0.6%) had multidrugresistant tuberculosis. The mean number of drugs used was 4.46 (median = 3). The most frequent comorbidities among the patients studied were lifestyle-related problems (tobacco and/or alcohol; 23.4%), hypertension (12.6%), diabetes (11.7%), and HIV (10.8%). In the study sample, the proportion of ADRs among women was greater than among men (54.0% vs. 34.5%). Patients aged 50 years or older displayed a higher Table 1 - Population characteristics of new tuberculosis cases by occurrence of adverse reactions (ADRs) to antituberculosis drugs, CSEGSF/ENSP/FIOCRUZ-RJ, 20042008. Patient Characteristics

Without ADRs N (%)

Gender Male Female* Age group 0 to 19 years 20 to 49 years * 50 years or older * Comorbidities # None 1 to 3** 4 or more** Number of drugs 0 to 4 5 or more** Total

With ADRs N (%)

Total N (%)

74 (65.5) 29 (46.0)

39 (34.5) 34 (54.0)

113 (64.2) 63 (35.8)

21 (77.8) 72 (56.2) 10 (47.6)

6 (22.2) 56 (43.8) 11 (52.4)

27 (15.3) 128 (72.7) 21 (12.0)

50 (79.4) 50 (51.0) 3 (20.0)

13 (20.6) 48 (49.0) 12 (80.0)

63 (35.8) 98 (55.7) 15 (8.5)

80 (68.4) 23 (39.0) 103 (58.5)

37 (31.6) 36 (61.0) 73 (41.5)

117 (66.5) 59 (33.5) 176 (100.0)

p,0.05; p,0.01. # By International Statistical Classification of Diseases code number. **

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Table 2 - Frequency of adverse drug reactions (ADRs) to antituberculosis drugs by type and severity, CSEGSF/ENSP/ FIOCRUZ-RJ, 2004-2008. Description of adverse reactions

(%)

Minor adverse reactions Gastric irritation (nausea, vomiting), abdominal pain Itchy skin Arthralgia or arthritis Headache Sweat and urine orange Behavior change (euphoria, insomnia, anxiety, and drowsiness) Peripheral neuropathy (burning of the extremities) Fever Major adverse reactions Exanthema Hepatotoxicity (vomiting, hepatitis, changes in liver function tests) Thrombocytopenia, leukopenia, eosinophilia, hemolytic anemia, agranulocytosis, vasculitis Other1 Total

90 37 20 13 6 6 4 3 1 8 3 3 2 28 126

71.4 29.3 15.9 10.3 4.8 4.8 3.1 2.4 0.8 6.4 2.4 2.4 1.6 22.2 100.0

ADRs not described in the MoH Guia de Vigilaˆncia Epidemiolo´gica: adenopathy, hallucinations, amenorrhea, anemia, increased uric acid, increased menstrual flow, painful urination, rash, weakness, jaundice, desquamative (scaly) lesions on the knees, urticarial lesions, malaise (sickness), other psychotic disorders, allergic reactions, and dizziness.

adults, had up to three comorbidities, used up to four drugs, and evolved to cure. One of the main findings of this study was that 42% of the patients presented antituberculosis ADRs, three-quarters of which were classified as ‘‘probable’’. Of the total ADRs, 78% were described in the MoH epidemiological surveillance guide (Guia de Vigilaˆncia Epidemiolo´gica), and 71% were considered mild. One-third of the reactions affected the gastrointestinal system, although essentially all systems were affected. The study showed a high rate of ADRs, similar to that found by a study in Sa˜o Paulo city (22) (49.1%) among patients treated at a teaching hospital outpatient department. In Canada, a retrospective study of 1,061 patients

The graph analysis appears to show two groups, each represented by one ellipse. The small ellipse shows the group of individuals who dropped out of treatment, are male, used up to four drugs, had no comorbidities, and did not develop ADRs (A, M, F1, C1, and N). The bigger ellipse shows the group of individuals who were cured, were aged 20 to 49 years, used more than five drugs, presented one to three comorbidities, and developed ADRs (C, I2, F2, C2, and S; Figure 2).

& DISCUSSION The cases of tuberculosis treated at the CSEGSF/Ensp/ Fiocruz were predominantly patients who were men,

Table 3 - Adverse drug reactions (ADRs) to antituberculosis drugs by severity, drugs involved, and treatment period, CSEGSF/ENSP/FIOCRUZ-RJ, 2004-2008. Up to 2nd month N (%)

3rd month onwards N (%)

Total ADRs N (%)

64 (71.1) 3 (37.5)

26 (28.9) 5 (62.5) *

90 (91.8) 8 (8.2)

50 (92.6) 16 (42.1) 7 (50.0) 3 (33.3) 2 (100.0) 1 (100.0) 2 (40.0) 81 (64.3)

4 (0.8) 22 (57.9) 7 (50.0) 6 (66.7) 1 (100.0) 1 (100.0) 1 (100.0) 3 (60.0) 45 (35.7)

54 (42.9) 38 (30.2) 14 (11.1) 9 (7.1) 2 (1.59) 1 (0.8) 1 (0.8) 1 (0.8) 1 (0.8) 5 (4.0) 126 (100.0)

# *

Type of ADR Minor Major Drugs R+H+Z R+H R H R+Z Z R+E R+H+E R+H+Z+E Other combinations1 Total

* p,0.05. #Twenty-eight ADRs were excluded because they are not described in the MoH Guia de Vigilaˆncia Epidemiolo´gica. Half of the ADRs occurred during the first two months of treatment. R = rifampicin; H = isoniazid; Z = pyrazinamide; E = ethambutol. 1 Other combinations include: rifampicin + ethinylestradiol; rifampicin + isoniazid + paracetamol + sulfadiazine + sulfamethoxazole + omeprazole; rifampicin + efavirenz + metronidazole + sulfamethoxazole; rifampicin + isoniazid + efavirenz + sulfadiazine + sulfamethoxazole + pyrimethamine; isoniazid with alcohol intake. D – dropout; C – cure; N – no ADR; S – ADR present; F – female; M – male; I1 – aged 0 to 19 years.; I2 – aged 20 to 49 years; I3 – aged 50 or older years; F1 – used up to four drugs; F2 – used five or more drugs; C1 – no comorbidity; C2 – one to three comorbidities; C3 – more than three comorbidities.

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Figure 2 - Graphic display of patient characteristics according to the occurrence of adverse drug reactions (ADRs) to antituberculosis drugs, the Centro de SauÂ´de Escola Germano Sinval Faria/ENSP/FIOCRUZ-RJ, from 2004 to 2008 through the two dimensions of correspondence analysis.

found a 30% rate of adverse reactions to antituberculosis drugs (23). However, prospective studies in Brazil have shown that the rate adverse reactions to antituberculosis drugs may be still higher, reaching 83.5% of patients (24). The positive associations between ADR and female sex and advanced age may have differing interpretations, as found in other studies (11,25,26). Regarding female sex, the mechanism is unknown; however, previous interpretations include hormonal oscillations at different stages of life that modify drug responses; interactions between antituberculosis and contraceptive drugs that may favor the appearance of ADRs (17,19); or greater attendance at health services, which would facilitate the detection of ADRs. Regarding age, the older population is more prone to develop ADRs, particularly the most severe forms (11,23,25,26). Patients older than 60 years are likely to experience intoxications from these drugs (14) because both hepatic and renal metabolisms can be impaired with age, favoring the accumulation of metabolites and the occurrence of ADRs (27). Tuberculosis may be concomitant with other diseases, thus increasing the number of drugs patients use and

predisposing them toward adverse reactions. In the study sample, patients who used more than five drugs in the course of their treatment developed more ADRs. Kishore et al. (11) observed in India that nearly half the patients who developed ADRs were prescribed more than five drugs. In terms of severity, a higher frequency of mild reactions was observed. This is consistent with the findings of a study conducted in SaË&#x153;o Paulo city, in which 81% of the ADRs were considered mild, and the most common ADRs were abdominal pain (20.4%) and arthralgia (16.4%) (22). Regarding the nature of the ADRs, gastrointestinal disturbances, skin conditions, and joint pains were the most commonly identified problems. Gastrointestinal disturbances, which are characterized by gastric irritation, nausea, vomiting, and abdominal pain, were present in the epidemiological studies (11,22,23,25) at frequencies ranging from 14.28% (11) to 29.7% (22). It is particularly important to note reactions that impair the liver because they may require interrupting treatment and may even lead to death. In a national study of 297 patients in SaË&#x153;o Paulo, 3.7% of the patients (n = 11) had their drug regimen altered because of adverse reactions, and hepatotoxicity was responsible for

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prospective follow-up of the participants, as will be discussed below. From the correspondence analysis, it was possible to identify two groups. The first comprised predominantly men, younger patients, those with fewer comorbidities and adverse reactions, those using fewer drugs, and those who dropped out of treatment. Interestingly, higher dropout rates among men and among young patients were also observed in another retrospective study of 481 smearpositive patients conducted in Cuiaba´ (Mato Grosso) from 1998 to 2000 (25). The second group included predominantly women, older patients, those with more comorbidities and adverse reactions, those using more drugs, and those who evolved to cure. It is plausible that the second group frequented the clinic more assiduously, which would enable the adverse reactions to be more readily identified and recorded. The group comprising mainly men, young patients, and those with fewer comorbidities may have dropped out of treatment; accordingly, fewer adverse reactions were recorded. This latter group may have been more irregular in the treatment (failing to take the drugs for a few days), may have presented milder adverse reactions more often in the first two months, and may not have returned to the clinic for subsequent appointments. Thus, those who dropped out of treatment would have demographic characteristics that would warrant supposing that the ADRs may have been the reason for dropout. There may, however, be other more important reasons, such as the socioeconomic situation itself, in the case of young patients. Regarding the study’s limitations, it should be noted that the retrospective design, handwritten patient records, and patients’ failure to attend appointments may all have undermined the credibility of the information on the drugs prescribed, dosages and frequency of drug use by the patients, and the patients’ clinical progression. Nonetheless, given the overall quality of the records and the completeness of the information observed during data collection, it can be said that generally, the validity of the records is assured and did not impair the general information about exposure to the drugs. Regarding the information on social variables, such as schooling, little information was entered in the records: no information on level of schooling was available for 71.6% (126/176) of the patients studied. The information in the clinic’s patient records was sufficient for the study of ADRs, and the estimates are compatible with expected values. The fact that the clinic in question engages in technology development, research, and teaching in the health field certainly contributed to our ability to obtain information of acceptable quality and quantity. Electronic patient records and prescriptions and improvements in patient data recording may be introduced in the future and would contribute to improving the quality of research at the clinic. Furthermore, regarding methodological considerations, it should be noted that the differences in the study designs hindered comparisons of their results. It should also be remembered that in many countries, the main tuberculosis treatment regimen includes ethambutol (which was only recently adopted in Brazil) and streptomycin, which are considered first-line drugs. For analysis purposes, the information on comorbidities was considered only once, at the date of the first entry in the patient record. Additionally, the comorbidities and ADRs

63.7% of the changes (22). Compared with other studies (11,22,23,25), our sample showed a low percentage of hepatotoxicity (2.4%), which may be explained in part by the fact that complementary examinations to detect evidence of hepatic alterations could not be performed on the 43 patients (24.4%) who dropped out of treatment. Only 30.1% of the antituberculosis ADRs were treated at CSEGSF. ADRs must be identified and treated to prevent similar reactions, reduce health service costs, and minimize patient suffering. ADRs are known to occur predominantly in the early months of tuberculosis treatment (22,25,26,28), although severe effects, such as hepatotoxicity, may occur later, after the fifth month of treatment (29). Similarly, in our study, most (64.3%) of the ADRs occurred in the first two months of treatment, which may be explained by the fact that the three drugs associated with most of the ADRs were used simultaneously during that period. Establishing the probability that the relationship between drug and ADR is causal (causality) depends largely on the information obtained, particularly regarding alternative causes, such as other diseases and the use of other drugs. Our findings permit most (75%) of the adverse reactions to be characterized as probably occasioned by the drugs. A retrospective study of 326 patients in India found an estimate similar to ours (72.5%) (11). Meanwhile, a retrospective study of 1,061 patients in Canada classified 51.7% as likely and 20.9% as definitely drug-caused (23). Instruments for analyzing causality (30,31) are grounded in basic considerations, such as temporality, pharmacological plausibility, and alternative causes. For that reason, the Naranjo probabilities scale (15), a method that has been previously validated, in widespread use for several decades, and applied in both prospective and retrospective studies, was used in this study. Even given the use of tools to ensure that causality is judged objectively, the quality of the information obtained in each study and the source of scientific evidence can explain the variations between estimates. The drugs for tuberculosis are effective and should be used in the correct dosages for a period of six months. One factor of concern is the rate of treatment dropout before the end of that period, which impairs the drugs’ effectiveness and can lead to multiple-drug resistance. For that reason, we were surprised at the percentage of patients who dropped out of treatment (24.4%). In 2001, the national mean dropout rate was approximately 12% (32), and it reached 27.3% in some state capitals (33). The WHO target is to reduce that rate to 5% (34). The high dropout rate reflects the tuberculosis scenario in many localities in Brazil, where social problems such as hunger, extreme poverty, violence, and drug abuse are themselves risk factors for treatment dropout [36]. It is thus possible that for these patients, adherence to tuberculosis treatment involves other issues that require more complex, systemic solutions. Losses from patients who dropped out of treatment between January 2004 and December 2008 prevented an analysis of the association between dropout and ADRs. To explore the hypothesis that the adverse effects of the treatment may lead to dropout would entail actively finding the cases that failed to attend the clinic. That would be extremely difficult in the context of this study, in which the time lag would make it difficult to trace losses. Alternatively, approaches could be developed to include

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and participated in the planning, collection, analysis, interpretation of data, and critical revision of intellectual content. All authors read and approved the final manuscript.

were assumed to be concomitant, regardless of the date when the comorbidities were recorded. That is because the retrospective design and degree of irregularity in attending appointments made it impossible to guarantee whether acute conditions recorded at a time inconsistent with (previous or subsequent to) the occurrence of the ADR actually occurred at the time of the ADR. For chronic conditions, such as hypertension or diabetes, that assumption does not entail errors. For acute, symptomatic conditions, however, comorbidities may have been overestimated, and ADRs may have been underestimated. A literature search found no studies addressing the association between dropout and the incidence of adverse reactions, in part because observational studies of adverse reactions to tuberculosis treatment are still scarce in Brazil. Future studies should examine that association, although the difficulties of such an examination are not insignificant. In retrospective studies, such as the present one, testing associations is problematical because the occurrence of ADRs among patients who dropped out of treatment is recorded only for the period during which the patient remains in treatment, and that time varies from individual to individual. In prospective studies, testing the association between adverse reactions and dropout would raise ethical issues and entail modifying the definition of the outcome variable. Here, dropout from treatment was considered to be failure to attend an appointment for 30 days or more, which was based on the MoH criteria. A prospective approach could consider the intention to drop out of treatment or the failure to attend an appointment for a shorter period. In addition, detecting patients who dropped out of treatment could also yield useful information about the reasons for dropout, which may include the occurrence of ADRs. To conclude, this study suggests a high rate of ADRs, which were detected particularly in one subgroup of patients. Various measures can be taken to reduce those figures, particularly measures to increase awareness among patients and health personnel. Regarding the latter, it would be necessary to introduce changes to the work process and how production is organized, with a view toward spreading technical knowledge about the occurrence of ADRs and establishing mechanisms so that ADRs are appropriately recorded, treated and controlled. From a research standpoint, prospective studies in the population of the Manguinhos district and others with similar social and economic profiles could produce new information to help control adverse reactions and increase adherence to tuberculosis treatment.

& REFERENCES 1. Hijjar MA, Proco´pio MJ, Freitas LMR, Guedes R, Bethem EP. Epidemiologia da tuberculose: importaˆncia no mundo, no Brasil e no Rio de Janeiro. Pulmaõ RJ. 2005;14(4):310-14. 2. Fundo Global da Tuberculose. Falta de informaçaõ ainda e´ o maior desafio. http://www.fundoglobaltb.org.br/download/Pesquisa_FGDATAUFF_tuberculose_jan-2010.PDF. 3. Ruffino-Netto A. Tuberculose: a calamidade negligenciada. Revista da Sociedade Brasileira de Medicina Tropical. 2002;35(1):51-8, http://dx. doi.org/10.1590/S0037-86822002000100010. 4. Mendes AM, Fensterseifer LM. Tuberculose: porque os pacientes abandonam o tratamento? Bol Pneumol Sanit. 2004;12:25-36. 5. Ministuˆ`rio da Sauˆ¤de. Brasil reduz casos novos de tuberculose. http:// portal.saude.gov.br/portal/aplicacoes/noticias/default.cfm?pg = dspDetalhe Noticia&id_area = 124&CO_NOTICIA = 12351. 6. Ministuˆ`rio da Sauˆ¤de. Guia de Vigilaˆncia Epidemiolo´gica 7 ed. Brası´lia: Ministe´rio da Sau´de; 2009, http://portal.saude.gov.br/portal/arquivos/ pdf/gve_7ed_web_atual.pdf. 7. Ministe´rio da Sau´de. Incideˆncia de tuberculose cai 27,58% em 10 anos. Brası´lia: Ministe´rio da Sau´de; 2009. http://www.infectologia.org.br/ default.asp?site_Acao = &paginaId = 134&mNoti_Acao = mostraNoticia& noticiaId = 10014. 8. Ministe´rio da Sau´de. Rio e´ o estado com maior incideˆncia de tuberculose, diz ministe´rio. Brası´lia: Ministe´rio da Sau´de; 2010. http://g1.globo.com/Noticias/Brasil/0,,MUL1542625-5598,00-RIO+E+ O+ESTADO+COM+MAIOR+INCIDENCIA+DE+TUBERCULOSE+DIZ+ MINISTERIO.html. 9. Mendes JM, Fonseca LS, Lourenço MC, Ferreira RMC, Saad MHF. Um estudo retrospectivo dos aspectos epidemiolo´gicos da tuberculose na comunidade do Complexo de Manguinhos localizado em a´rea urbana do Rio de Janeiro, Brasil, 2000-2002. J Bras Pneumol. 2007;33(4):443-7. 10. Santos AL. Diagno´stico do abandono do tratamento da tuberculose na a´rea de abrangeˆncia do Centro de Sau´de Escola Germano Sinval Faria. [Professional Masters Thesis in Family Health]. [Rio de Janeiro]: Universidade Estaćio de Sa´; 2009. 94p, http://portal.estacio.br/media/ 2267956/antenor%20completa.pdf. 11. Kishore PV, Subish P, Pradip O, Shankar PR. Pattern of Adverse Drug Reactions Experienced by Tuberculosis Patients in a Tertiary Care Teaching Hospital in Western Nepal. Pak J Pharm Sci. 2008;21(1):51-6. 12. World Health Organization. International drug monitoring: the role of national centres. Geneva: World Health Organization; 1972, http:// whqlibdoc.who.int/trs/WHO_TRS_498.pdf. 13. Engstrom EM, Ferreira J, Alves LC. Fatores associados aos desfechos do tratamento da tuberculose em um centro de atençaõ ba´sica de sau´de: uma ana´lise sob a o´tica antropolo´gica e epidemiolo´gica. 2009. 14. Ministe´rio da Sau´de. Manual tećnico para o controle da tuberculose: caderno de atençaõ ba´sica – No. 6. Brası´lia: Ministe´rio da Sau´de; 2002, http://bvsms. saude.gov.br/bvs/publicacoes/caderno_atencao_basica.pdf. 15. Naranjo CA, Busto U, Sellers EM, Sandor P, Ruiz I, Roberts EA, et al. A method for estimating the probability of adverse drug reactions. Clin Pharmacol Ther. 1981;30(2):239-45, http://dx.doi.org/10.1038/clpt.1981.154. 16. World Health Organization, WHO-ART Critical Term List, Current Critical Term List, World Health Organization, Uppsala Monitoring Centre (UMC), 2005. http://www.umc-products.com/graphics/3149.pdf. 17. Drugdex Evaluations. Micromedex 2.0 Health Care Series. Thomson Reuters. http://www.thomsonhc.com/micromedex2/librarian. 18. Kasper DL. Harrison’s Principles of Internal Medicine. 16th ed. New York: McGraw-Hill; 2005. 2800p. 19. Hardman JG, Limberd LE, Molinoff PB, Ruddon RW, Gilman AG, Goodman and Gilman’s. The Phamacological Basis of Therapeutics. 9th ed. New York: Mc Graw-Hill, 1996. Section IX, Chemotherapy of microbial diseases; pp. 1027-224. 20. Dukes MNG, Aronson JK. Meyler’s side effects of drugs. 14th ed. New York: Elsevier; 2000. 1780p. 21. Oliveira RVC, Silva AM, Assis SG, Santos NC. Ana´lise de correspondeˆncia mu´ltipla e ana´lise de agrupamento na reduçaõ de dimensionalidade de indicadores de eventos de vida. Rev. Bras Estat. 2006;67(226):95-116. 22. Viera DEO, Gomes M. Efeitos adversos no tratamento da tuberculose: experieˆncia em serviço ambulatorial de um hospital-escola na cidade de Saõ Paulo. J Bras Pneumol. 2008;34(12):1049-55. 23. Marra F, Marra CA, Bruchet N, Richardson K, Moadebi S, Elwood RK, et al. Adverse drug reactions associated with first-line anti-tuberculosis drug regimens. Int J Tuberc Lung Dis. 2007;11(8):868-75. 24. Maciel ELN, Guidoni LM, Favero JL, Hadad DJ, Molino LP, Dietze R. Efeitos adversos causados pelo novo esquema de tratamento da tuberculose preconizado pelo Ministe´rio da Sau´de do Brasil. J Bras Pneumol. 2010;36(2):232-38.

& ACKNOWLEDGMENTS We would like to thank the postgraduate program in Epidemiology at the Escola Nacional de Sau´de Pu´blica Se´rgio Arouca (ENSP/Fiocruz), which made this study based on the main author’s MSc thesis possible; the project of the Global Fund to Fight Tuberculosis, Aids and Malaria; and Professor Dora Chor for her critical reading of the manuscript.

& AUTHOR CONTRIBUTIONS Damasceno GS was responsible for the literature review, data collection, data analysis, and drafting the manuscript. Guaraldo L participated in the design, planning, collection, analysis, interpretation of data, and critical revision of intellectual content. Engstrom E participated in planning the study. Theme Filha M conducted the field activities. Souza Santos R participated in the planning and preparation of the data entry program. Vasconcelos AG participated in the statistical analysis and interpretation of data. Rozenfeld S was responsible for conceiving and designing the study

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Adverse reactions to antituberculosis drugs Damasceno GS et al.

25. Yee D, Valiquette C, Pelletier M, Parisien I, Rocher I, Menzies D. Incidence of serious side effects from firstline anti-tuberculosis drugs among patients treated for active tuberculosis. Am J Respir Crit Care Med. 2003;167(11):1472-7, http://dx.doi.org/10.1164/rccm.200206-626OC. 26. Ormerod LP, Horsfield N. Frequency and type of reactions to antituberculosis drugs: observations in routine treatment. Tuber Lung Dis. 1996;77(1):37-42, http://dx.doi.org/10.1016/S0962-8479(96)90073-8. 27. Rozenfeld S. Prevaleˆncia, fatores associados e mau uso de medicamentos entre idosos: uma revisa˜o. Cad. Sau´de Pu´blica. 2003;19(3):717-24, http:// dx.doi.org/10.1590/S0102-311X2003000300004. 28. Schaberg T, Rebhan K, Lode H. Risk factors for side-effects of isoniazid, rifampin and pirazinamide in patients hospitalized for pulmonary tuberculosis. Eur Respir J. 1996;9(10):2026-30, http://dx.doi.org/10. 1183/09031936.96.09102026. 29. Pande JN, Singh SP, Khilnani GC, Khilnani S, Tandon RK. Risk factors for hepatotoxicity from antituberculosis drugs: a case-control study. Thorax. 1996;51(2):132-6, http://dx.doi.org/10.1136/thx.51.2.132.

30. Karch FE & Lasagna L. Adverse drug Reactions. A critical review. JAMA. 1975;234(12):1236-41. 31. WHO, The Uppsala Monitoring Centre (UMC). Causality Assessment of Suspected Adverse Reactions. http://www.who-umc.org. 32. Ruffino-Netto A. Programa de Controle da Tuberculose no Brasil: situaçaõ atual e novas perspectivas. Informe Epidemiolo´gico do SUS, 2001;10(3):129-38. 33. Ferreira SMB, Silva AM, Botelho C. Abandono do tratamento da tuberculose pulmonar em Cuiaba´ – MT- Brasil. J Bras Pneumol. 2005; 31(5):427-35. 34. WHO, Global Tuberculosis Control: surveillance, planning, financing: WHO report 2008. http://www.who.int/tb/publications/global_ report/2008/pdf/fullreport.pdf. 35. Natal S, Valente J, Gerhardt G, Penna ML. Modelo de prediçaõ para o abandono do tratamento da tuberculose pulmonar. Boletim de Pneumologia Sanita´ria. 1999;7(1):65-77.

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Primary myelofibrosis: risk stratification by IPSS identifies patients with poor clinical outcome Bruno Deltreggia Benites,I Carolina Silva Costa Lima,I Irene Lorand-Metze,I Marcia Torresan Delamain,I Gislaine Borba Oliveira,I Daiane de Almeida,I Carmino Antonio de Souza,I Jose Vassallo,II Katia Borgia Barbosa PagnanoI I

Universidade Estadual de Campinas, Centro de Hematologia e Hemoterapia, Hemocentro, Campinas/Sa˜o Paulo, Brazil. Campinas, Laboratory of Investigative and Molecular Pathology, CIPED, Campinas/SP, Brazil.

Universidade Estadual de

OBJECTIVES: To evaluate whether risk scores used to classify patients with primary myelofibrosis and JAK-2 V617F mutation status can predict clinical outcome. METHODS: A review of clinical and laboratory data from 74 patients with primary myelofibrosis diagnosed between 1992 and 2011. The IPSS and Lille scores were calculated for risk stratification and correlated with overall survival. RESULTS: A V617F JAK2 mutation was detected in 32 cases (47%), with no significant correlation with overall survival. The patients were classified according to the scores: Lille - low, 53 (73.%); intermediate, 13 (18%); and high, 5 (7%); and IPSS – low, 15 (26%); intermediate-1, 23 (32%); intermediate-2, 19 (26%); and high, 15 (31%). Those patients presenting a higher risk according to the IPSS (high and intermediate-2) had a significantly shorter overall survival relative to the low risk groups (intermediate-1 and low) (p = 0.02). CONCLUSIONS: These results emphasize the importance of the IPSS prognostic score for risk assessment in predicting the clinical outcome of primary myelofibrosis patients. KEYWORDS: Primary Myelofibrosis; IPSS; Prognosis. Benites BD, Lima CS, Lorand-Metze I, Delamain MT, Oliveira GB, Almeida D, et al. Primary myelofibrosis: risk stratification by IPSS identifies patients with poor clinical outcome. Clinics. 2013;68(3):339-343. Received for publication on October 11, 2012; First review completed on October 13, 2012; Accepted for publication on November 15, 2012 E-mail: kborgia@unicamp.br Tel.: 55 19 3521-8740

The diagnostic criteria for this disease have been revised since the discovery in 2005 of the acquired mutation V617F in the JAK2 gene, which is found in 50-60% of patients with PMF (4). The JAKs, which include JAK1, JAK2, JAK3 and TYK, are a family of cytoplasmic tyrosine kinases that are essential for cytokine signaling and gene transcription. The JAK2V617F mutation results in the constitutive activation of JAK2 tyrosine kinase and its downstream targets, which leads to increased signaling of associated cytokine receptors and the subsequent proliferation of hematopoietic cells harboring these receptors (5). The clinical significance of the mutation is evident in that homozygosis for JAK2 V617F results in a more symptomatic illness relative to heterozygous patients. However, the prognostic significance of the mutation is controversial, including its implication in poor clinical outcomes and the risk of progression to leukemia (6). Until recently, the most widely used prognostic classification of PMF was the Lille score, which categorizes patients into three risk groups based on the hemoglobin level and leukocyte count at diagnosis (7). However, the Lille score fails to clearly discriminate between intermediate- and highrisk prognostic categories. Recognition of these limitations led to a multicenter study aimed at building a new International Prognostic Scoring

& INTRODUCTION Primary myelofibrosis (PMF) is a clonal hematopoietic stem cell disorder characterized by bone marrow fibrosis, extra-medullary hematopoiesis with splenomegaly and leukoerythroblastosis in the peripheral blood (1,2). The clinical manifestations of PMF include severe anemia, marked hepatosplenomegaly and constitutional symptoms. Ineffective erythropoiesis and extra-medullary hematopoiesis are the main causes of anemia and organomegaly, respectively. Approximately 20% of patients diagnosed with PMF may present progression to acute leukemia, but most patients die of other conditions, such as cardiovascular events, or as a consequence of cytopenias, such as infections or bleeding (3).

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System (IPSS) for PMF, in which seven European and American centers contributed data from more than 1,000 PMF patients. In the multivariate analysis, the initial features independently associated with a poor prognosis were an age older than 65 years, the presence of constitutional symptoms, hemoglobin ,10 g/dL, WBC count .256109/L and the presence of blasts in the peripheral blood. These prognostic factors formed the basis for four risk groups with clear-cut, non-overlapping survival curves: no factors (low risk), one factor (intermediate risk-1), two factors (intermediate risk-2) or three or more factors (high risk) (8). The IPSS demonstrated a higher discriminatory power than previous scoring systems and showed a high degree of replicability and predictive accuracy. The IPSS is based on prognostic factors recorded at the diagnosis of PMF that are not necessarily stable over the course of the disease. To address this shortcoming, a dynamic prognostic model (DIPSS) was subsequently developed, and it utilizes the same prognostic variables used in IPSS but can be applied at any time during the course of the disease (9). The DIPSS assigns two, rather than one, adverse points for hemoglobin ,10 g/dL, and the risk categorization is accordingly modified: low (0 adverse points), intermediate-1 (1 or 2 points), intermediate-2 (3 or 4 points) and high (5 or 6 points). As many patients are elderly at diagnosis or present several comorbidities, most PMF patients are managed with supportive care only. Thus, a high degree of prognostic certainty is desired to permit the recommendation of more aggressive or high-risk therapeutic procedures, and the patientâ&#x20AC;&#x2122;s classification by prognostic score enables better therapeutic planning, especially for patients who are younger and eligible for bone marrow transplantation. Furthermore, the development of methods to assess and validate measures for clinical outcome becomes crucial in the era of targeted therapies, in which the therapeutic potential of JAK inhibitor molecules has emerged (10-12). Thus, the aim of our study was to analyze the clinical and laboratory data of consecutive PMF cases diagnosed in a single center, focusing on the Lille and IPSS prognostic scores and comparing their applicability to predict a poor outcome.

Table 1 - Risk models for myelofibrosis applied in the current study. Risk model

Risk factors

Point per factor

Lille

Hb ,10 g/dL WBC ,4 or .306109/l

1 1

IPSS

Age .65 years Hb ,10 g/dL WBC .256109/l Circulating blasts $1% Constitutional symptoms

1 1 1 1

Risk stratification Low: 0 points Intermediate: 1 points High: 2 points Low: 0 points Intermediate-1: 1 points Intermediate-2: 2 points High: 3 or more points

and/or unexplained persistent fever or excessive sweating) and myelofibrosis grade on a bone marrow biopsy based on the Baumeister scale and karyotype obtained from the marrow aspirate. JAK-2 mutation status was analyzed at diagnosis or during the follow-up. The Lille and IPSS prognostic scores were also calculated (Table 1).

Statistical analysis Overall survival (OS) was calculated from the diagnosis until the last follow-up or death, and transformation-free survival (TFS) was calculated from diagnosis until the progression to acute myeloid leukemia, last evaluation or death. Survival curves were calculated using the log-rank test (software Winstat 3.11 Statistics for Windows, version 3.1, Kalmia Co. Inc). Correlations between thrombosis and the JAK2 mutation and between the myelofibrosis grade in the bone marrow and IPSS were evaluated using the chisquare test.

& RESULTS Patient features at presentation The main clinical and laboratory data of the 74 patients in this study are summarized in Table 2. The median age at diagnosis was 71.5 years. The hemoglobin level was ,10 g/ dL in 13 patients, the WBC was .256109/l in eight patients and splenomegaly was found in 31 patients (42%). Cytogenetic analysis was performed in 34 patients (46%), and one case showed a partial deletion of chromosomes 13 and 15. The myelofibrosis grade based on the Baumeister scale was available in 54 patients: 13 (24%) were classified as grade 2, 27 (50%) as grade 3, and 14 (26%) as grade 4.

& PATIENTS AND METHODS Seventy-four consecutive patients with primary myelofibrosis diagnosed between January 1992 and August 2011 were included in this study for retrospective analysis. The local ethics committee approved this study. All cases were reclassified according to the WHO classification. In all cases, the presence of increased reticulin and/or collagen bone marrow content without any apparent cause (such as chronic myeloid leukemia, PV, myelodysplasia, lymphoproliferative disorders, scleroderma and primary pulmonary hypertension) was required in addition to the presence of features typical of this disease, including palpable splenomegaly, leukoerythroblastosis or histological demonstration of myeloid metaplasia. The variables selected for their prognostic significance were assessed at diagnosis and included the following: peripheral blood counts (hemoglobin, total leukocytes, platelets and circulating blasts), splenomegaly, constitutional symptoms (weight loss greater than 10% of the baseline value in the year preceding the PMF diagnosis

Treatment Sixty-six patients (89%) received hydroxyurea (HU), which was discontinued in six cases: three patients switched Table 2 - Clinical data of the primary myelofibrosis patients (n = 74). Demographics/Characteristics Age (median and range) Presence of V617V JAK2 mutation Splenomegaly at diagnosis Hb (g/dL) (median and range) Leucocytes/mm3 (median and range) .30,000/mm3 ,4000/mm3 Platelets (median and range)

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N 71.5 32 31 12.2 11.4

(31-92) (47%) (42%) (5.4-16.9) (0.9-47.3) 2 6 456 (76-1.545)

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Primary myelofibrosis - risk stratification Benites BD et al.

Figure 1 - Overall survival of patients according to V617F JAK2 mutation status.

to anagrelide because of toxicity (lower limb ulcers), and three patients switched to thalidomide and prednisone because of therapeutic failure. Three patients were treated only with supportive care (transfusion). Three patients received no specific treatment during the course of this study because they presented as clinically stable and had blood counts similar to the normal range. A splenectomy was performed in two patients, and three others received splenic radiotherapy to control the symptoms. Only one patient was eligible for allogeneic bone marrow transplantation, and he achieved complete remission, which was maintained by the end of this study (15 months of disease-free survival). Thrombotic events were observed in 13 (18%) patients (arterial occlusion in seven and venous thrombosis in six); only one event occurred during thalidomide treatment. There was no other relationship between these events and the therapy applied or any other clinical or laboratory variable.

However, in the IPSS data, patients classified in the highrisk groups (high and int-2 scores) showed a significantly lower overall survival than patients in the low-risk groups (int-1 and low) (p = 0.02) (Figure 2). There was no significant difference in overall survival when patients were stratified according to myelofibrosis grade (p = 0.81). We did not find a correlation between myelofibrosis grade and IPSS score (p = 0.59).

& DISCUSSION Myelofibrosis is a myeloproliferative stem cell disorder that is curable exclusively by allogeneic hematopoietic stem cell transplantation. It also has substantial patient morbidity and mortality, which results in an important need for improved therapies. Recent advances in the understanding of the pathogenetic mechanisms underlying this disease have led to many clinical trials evaluating novel therapies, such as ruxolitinib, which is the first JAK2 inhibitor approved by the FDA for the treatment of patients with intermediate- or high-risk myelofibrosis (13-15). Clinical trials of these compounds have demonstrated improvement

V617F JAK2 mutation analysis A V617F JAK2 mutation was detected in 32 cases (47%). Patients with the mutation presented a shorter overall survival (39% vs. 77%), although this difference was not significant (p = 0.448) (Figure 1).

Table 3 - Risk stratification according to the Lille and IPSS scores.

Risk stratification and clinical endpoints According to the Lille score, most patients presented a low risk (73.5%) (Table 2). The IPSS classification is described in Table 3. During the analysis period, 15 deaths were recorded: nine due to infectious complications, three after a blast crisis (occurring a median of 45 months after the diagnosis), and three others by causes not related to the disease. According to the Lille classification, no significant difference was observed in overall survival (Figure 1).

IPSS Low Intermediate-1 Intermediate-2 High Lille Low Intermediate High

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N 15 23 19 15

(26%) (32%) (26%) (31%)

53 (73.5%) 13 (18%) 5 (7%)

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Figure 2 - Overall survival of patients according to the IPSS.

in constitutional symptoms and splenomegaly in patients with both mutated and wild-type JAK2 PMF (16,17). However, the development and validation of useful tools to determine the prognosis and estimate survival is necessary for better management of these patients. Although the association was not significant, our findings suggest that positivity for the JAK2 (V617F) mutation in our group of patients may be associated with poorer survival. Published data regarding the role of the JAK2 (V617F) mutation in the prediction of the survival of PMF patients reveal contradictory results. Campbell et al. showed that patients positive for the mutation had a shorter overall survival, even after correction for confounding factors (p = 0.01) (18). However, a recent study showed that a positive qualitative test for the V617F mutation does not predict survival but that patients with a lower V617F allele burden presented a worse survival, which most likely indicates the presence of an overriding V617F-negative clone that confers a more aggressive disease phenotype (19,20). Although important for the differential diagnosis of PMF with ET, the myelofibrosis grade based on the Baumeister scale was not correlated with survival or the IPSS score. Most of our patients presented with grade 3 or 4 (76%). Our study also demonstrated that the IPSS is an appropriate method for identifying patients with a worse overall survival, which agrees with data from other studies (8,9,21). Patients classified as high-risk (high and intermediate-2 scores) showed a significantly lower overall survival than low-risk patients (intermediate-1 and low scores). In addition to demonstrating the efficacy of the IPSS for prognosis evaluation in myelofibrosis, our results also demonstrate the limitations of the Lille score in determining risk for patients with a more severe disease. The Lille score model was initially designed using 195 patients with PMF.

As described in other studies, the Lille score is capable of identifying a well-defined group of patients with a good prognosis but fails to clearly identify patients with a very poor prognosis and those with an intermediate prognosis. This issue may be partially explained by the reliance of the score on the hemoglobin level because leukopenia and leukocytosis greater than 306109/L are infrequent at the time of the PMF diagnosis (8,22). In fact, in our population, only two patients presented leukocytosis greater than 306109/L, and six presented with leukocytosis ,46109/L at diagnosis. These findings corroborate the evidence indicating that the IPSS must be evaluated for all patients recently diagnosed as having PMF. In addition to predicting survival, it is a helpful tool to evaluate therapeutic options; it is easy to calculate and requires the evaluation of only simple clinical and laboratory data. Other clinical trials should be encouraged to better evaluate the influence of the IPSS score in predicting the response to the new drugs available for the treatment of PMF. In summary, this study confirmed the importance of the IPSS for risk factor stratification. Adjunctive treatment with hydroxyurea is able to control low-risk patients in the cellular phase of the disease. However, patients with intermediate- and high-risk disease are candidates for other therapeutic approaches, such as bone marrow transplantation or experimental drug therapies.

& AUTHOR CONTRIBUTIONS Benites BD contributed to the study conception and design, data collection, analysis and interpretation. Pagnano KB and Lorand-Metze I contributed to the study conception and design, data analysis and interpretation. Lima CS contributed to data collection, analysis and interpretation. Delamain MC, Oliveira GB, Souza CA and Vassallo J contributed to data analysis and interpretation. Almeida D contributed to the JAK2 mutation analysis. All authors helped in the collection or assembly of data, analysis and

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Primary myelofibrosis - risk stratification Benites BD et al. 11. Verstovsek S, Kantarjian H, Mesa RA, Pardanani AD, Cortes-Franco J, Thomas DA, et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med. 2010;363(12): 1117-27. 12. Pardanani A, Gotlib JR, Jamieson C, Cortes JE, Talpaz M, Stone RM, et al. Safety and efficacy of TG101348, a selective JAK2 inhibitor, in myelofibrosis. J Clin Oncol. 2011;29(7):789-96. 13. Koschmieder S, Koppelle A, Seifert H. Ruxolitinib for myelofibrosis. N Engl J Med. 2012 May 24;366(21):2031-2; author reply 2032-4. 14. Deisseroth AB, Kaminskas E, Grillo J, Chen W, Saber H, Lu H, et al. U.S. Food and Drug Administration approval: ruxolitinib for the treatment of patients with intermediate and high-risk myelofibrosis. Clin Cancer Res. 2012;18(12):3212-7. 15. Mascarenhas J, Hoffman R. Ruxolitinib: the first FDA approved therapy for the treatment of myelofibrosis. Clin Cancer Res. 2012;18(11):3008-14. 16. Harrison C, Kiladjian JJ, Al-Ali HK, Gisslinger H, Waltzman R, Stalbovskaya V, et al. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med. 2012;366(9):787-98. 17. Tefferi A, Litzow MR, Pardanani A. Long-term outcome of treatment with ruxolitinib in myelofibrosis. N Engl J Med. 2011;365(15):1455-7. 18. Campbell PJ, Griesshammer M, Dohner K, Dohner H, Kusec R, Hasselbalch HC, et al. V617F mutation in JAK2 is associated with poorer survival in idiopathic myelofibrosis. Blood. 2006;107(5):2098-100. 19. Tefferi A, Lasho TL, Huang J, Finke C, Mesa RA, Li CY, et al. Low JAK2V617F allele burden in primary myelofibrosis, compared to either a higher allele burden or unmutated status, is associated with inferior overall and leukemia-free survival. Leukemia. 2008;22(4):756-61. 20. Guglielmelli P, Barosi G, Specchia G, Rambaldi A, Lo Coco F, Antonioli E, et al. Identification of patients with poorer survival in primary myelofibrosis based on the burden of JAK2V617F mutated allele. Blood. 2009;114(8):1477-83. 21. Tefferi A, Jimma T, Gangat N, Vaidya R, Begna KH, Hanson CA, et al. Predictors of greater than 80% 2-year mortality in primary myelofibrosis: a Mayo Clinic study of 884 karyotypically annotated patients. Blood. 2011;118(17):4595-8. 22. Cervantes F, Pereira A. Prognostication in primary myelofibrosis. Curr Hematol Malig Rep. 2012r;7(1):43-9.

interpretation of data and critical revision of the article for important intellectual content.

& REFERENCES 1. Cervantes F, Pereira A. Advances in the understanding and management of primary myelofibrosis. Curr Opin Oncol. 2011;23(6):665-71. 2. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114(5):937-51. 3. Tefferi A. Myelofibrosis with myeloid metaplasia. N Engl J Med. 2000;342(17):1255-65. 4. Abdel-Wahab O. Genetics of the myeloproliferative neoplasms. Curr Opin Hematol. 2011;18(2):117-23. 5. Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia. 2010;24(6):1128-38. 6. Barosi G, Bergamaschi G, Marchetti M, Vannucchi AM, Guglielmelli P, Antonioli E, et al. JAK2 V617F mutational status predicts progression to large splenomegaly and leukemic transformation in primary myelofibrosis. Blood. 2007;110(12):4030-6. 7. Dupriez B, Morel P, Demory JL, Lai JL, Simon M, Plantier I, et al. Prognostic factors in agnogenic myeloid metaplasia: a report on 195 cases with a new scoring system. Blood. 1996;88(3):1013-8. 8. Cervantes F, Dupriez B, Pereira A, Passamonti F, Reilly JT, Morra E, et al. New prognostic scoring system for primary myelofibrosis based on a study of the International Working Group for Myelofibrosis Research and Treatment. Blood. 2009;113(13):2895-901. 9. Passamonti F, Cervantes F, Vannucchi AM, Morra E, Rumi E, Pereira A, et al. A dynamic prognostic model to predict survival in primary myelofibrosis: a study by the IWG-MRT (International Working Group for Myeloproliferative Neoplasms Research and Treatment). Blood. 2010;115(9):1703-8. 10. Stein BL, Crispino JD, Moliterno AR. Janus kinase inhibitors: an update on the progress and promise of targeted therapy in the myeloproliferative neoplasms. Curr Opin Oncol. 2011;23(6):609-16.

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Electrical impedance tomography to evaluate air distribution prior to extubation in very-low-birthweight infants: a feasibility study Felipe de Souza Rossi,I Ana Cristina Zanon Yagui,I Luciana Branco Haddad,I Alice D’Agostini Deutsch,I,II Celso Moura RebelloI I

Hospital Israelita Albert Einstein, Departamento Materno-infantil, Saõ Paulo/SP, Brazil. II Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Instituto da Criança, Departamento de Pediatria, Disciplina de Pediatria Neonatal, Saõ Paulo/SP, Brazil.

OBJECTIVES: Nasal continuous positive airway pressure is used as a standard of care after extubation in verylow-birth-weight infants. A pressure of 5 cmH2O is usually applied regardless of individual differences in lung compliance. Current methods for evaluation of lung compliance and air distribution in the lungs are thus imprecise for preterm infants. This study used electrical impedance tomography to determine the feasibility of evaluating the positive end-expiratory pressure level associated with a more homogeneous air distribution within the lungs before extubation. METHODS: Ventilation homogeneity was defined by electrical impedance tomography as the ratio of ventilation between dependent and non-dependent lung areas. The best ventilation homogeneity was achieved when this ratio was equal to 1. Just before extubation, decremental expiratory pressure levels were applied (8, 7, 6 and 5 cmH20; 3 minutes each step), and the pressure that determined the best ventilation homogeneity was defined as the best positive end-expiratory pressure. RESULTS: The best positive end-expiratory pressure value was 6.3¡1.1 cmH20, and the mean continuous positive airway pressure applied after extubation was 5.2¡0.4 cmH20 (p = 0.002). The extubation failure rate was 21.4%. X-Ray and blood gases after extubation were also checked. CONCLUSION: This study demonstrates that electrical impedance tomography can be safely and successfully used in patients ready for extubation to suggest the best ventilation homogeneity, which is influenced by the level of expiratory pressure applied. In this feasibility study, the best lung compliance was found with pressure levels higher than the continuous positive airway pressure levels that are usually applied for routine extubation. KEYWORDS: Infant; Premature; Electric Impedance; Continuous Positive Airway Pressure. Rossi FS, Yagui AC, Haddad LC, Deutsch AA, Rebello CM. Electrical impedance tomography to evaluate air distribution prior to extubation in very-low-birth-weight infants: a feasibility study. Clinics. 2013;68(3):345-350. Received for publication on October 30, 2012; First review completed on November 18, 2012; Accepted for publication on November 18, 2012 E-mail: celso.rebello@uol.com.br Tel.: 55-11-8392-2041

applied using nasal prongs is the most common type of NIV performed on extreme preterm infants (3-5). For very-low-birth-weight (VLBW) infants who undergo invasive ventilation, extubation failure is unpredictable and occurs in approximately 20 to 30% of patients (6). The use of CPAP in these patients is the standard of practice that aims to reduce this complication, but different approaches and interfaces are used among hospitals (7-10). Electrical impedance tomography (EIT) provides realtime bedside information that can be used to estimate regional lung ventilation (11-14). This technique has been used to study ventilation patterns in adults and neonates, but it has not yet been used to evaluate preterm infants before extubation (15-20). Recently, differences in tidal ventilation were assessed for volume-targeted ventilation in preterm infants (14). Variations in positive end-expiratory pressure (PEEP) levels and the resulting effect on air

& INTRODUCTION Mechanical ventilation is crucial for the stabilization and care of extreme preterm infants. It can be applied invasively, through an endotracheal tube, or non invasive ventilation (NIV), with the use of non-invasive interfaces, which is associated with a lower morbidity rate in preterm infants (1,2). Nasal continuous positive airway pressure (CPAP)

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A thorax x-ray and ABG analysis were performed six to twelve hours after extubation. Evidence for atelectasis was specifically checked in the x-ray images. Oxygen index (OI), alveolar-arterial oxygen difference (A-a), arterial/alveolar ratio (a/A) and pH and paCO2 levels were calculated and compared. The mean and standard deviation were used to describe data. Continuous data were analyzed using Student-t test or the Mann-Whitney test as appropriate. R statistical software (version 2.10.1) was used for all analyses. p#0.05 was considered statistically significant.

distribution in preterm infants during spontaneous assisted conventional ventilation were not previously studied. In this study, we used EIT in VLBW during assisted mechanical ventilation immediately before extubation. This study was designed to determine the feasibility of evaluating the PEEP level associated with a more homogeneous air distribution within the lungs before extubation by using electrical impedance tomography. We used EIT to determine the PEEP level associated with the best air distribution in the lungs of these patients, which corresponds to the best ventilation homogeneity (VH) and follows the global inhomogeneity index concept previously described by Zhao et al., i.e., a ratio of 1 ventilation between the dependent and non-dependent lung (21,22).

& RESULTS After the non-invasive, radiation-free and harmless EIT method was explained, one family declined to consent. Five other eligible infants were also not enrolled because they were extubated without the presence of a member of the study team at the NICU. The demographic characteristics of the fourteen patients included in this study are shown in Table 1. The mean value and standard deviation of the body weight were 940¡205 g, gestational age was 28.1¡2.6 weeks and age at EIT evaluation was 7.5¡10.8 days. Extubation failure was observed in three infants (21.4%). One patient was reintubated for central apnea without evident deterioration of lung compliance. Reintubation because of respiratory distress after tube removal occurred in two patients. Both patients were ventilated for more than four weeks prior to EIT evaluation and were diagnosed as having bronchopulmonary dysplasia. The mean PEEP level determined by the EIT that resulted in the best VH was 6.3¡1.1 cmH2O, which was significantly different from 5.2¡0.4 cmH2O, which was the CPAP empirically applied after extubation (p = 0.002). The two patients in which extubation failed had a best PEEP = 6 cmH2O and were extubated to a CPAP of 5 and 6 cmH20, respectively. Of all EIT signals collected, 70.2% of the data were considered appropriate to be analyzed. ABG analysis, oxygenation indices, the x-ray pattern and the other preliminary outcomes are presented in Table 2. No side effects, either related to the use of the EIT device in these infants, including skin lesions secondary to electrodes, or related to the infant manipulation, were found. Figure 2 corresponds to EIT images representing the loss of ventilation in the dependent zone after a PEEP reduction in one of the studied patients.

& MATERIAL AND METHODS Study population This study was performed in the Hospital Israelita Albert Einstein NICU in Sa˜o Paulo after approval by the institutional review board and the institution’s ethics committee. Every VLBW infant (birth weight ,1,500 g), regardless of the prior respiratory disease that determined invasive ventilation, was submitted to the following ventilatory parameters before extubation: synchronous intermittent mandatory ventilation plus a pressure support mode (SIMV+PS; Servo-i - Maquet Getinge Group, Sweden), FiO2#0.35, PEEP#6 cmH20, and respiratory rate #15 breaths/min. Exclusion criteria included congenital malformations, intracranial hemorrhage of grades III and IV (23) and previous pneumothorax. The patients were assigned to the routine CPAP of 5 to 6 cmH20 applied after tube removal at the discretion of the attending staff, as is the routine procedure at our institution. As this was a feasibility study, a convenience sample of fourteen patients was chosen.

EIT measurements and data analysis A 16-electrode EIT belt was specially designed to fit VLBW infants who had thorax diameters as small as 17 cm. The EIT used an EnlightH technology model DX-1800 apparatus that sampled at 50 Hz (Dixtal, Sao Paulo, Brazil). EIT images were continuously recorded and reconstructed based on a 3-D finite element mesh under decremental PEEP levels (8, 7, 6 and 5 cmH20; 3 minutes for each step). The ventilation ratio between the dependent and the non-dependent lung areas (upper/lower ratio, U/L) was continuously calculated. Because of the variability of the impedance change caused by the typical respiratory pattern of VLBW infants during spontaneous assisted breathing, as well as variability arising from the patients’ movements, only stable respiratory cycles defined by at least two of the researchers were used to calculate the PEEP that promoted the best ventilation homogeneity (VH), which was considered an upper/lower ratio of 1, as previously described (Figure 1).

& DISCUSSION This feasibility study showed that it is possible to evaluate air distribution during spontaneous assisted ventilation in VLBW infants with the use of EIT and also determined the PEEP that results in the best VH. To our knowledge, the use of dynamic images to evaluate different PEEP levels and their effects on preterm lungs in infants receiving assisted conventional mechanical ventilation has never been reported before. Similarly to other studies using controlled ventilation, both in adult anesthetized patients with healthy lungs and in animal models of lung injury (21,22,24), we applied an index to define a more homogeneous air distribution between lung areas. However, there is a concern that heterogeneity is a physiological phenomenon in patients who are to be extubated and who also present minimal or no lung collapse (25).

Preliminary outcome evaluation Extubation failure was defined as the need for reintubation within 72 hours after tube removal. Patients were reintubated if they needed FiO2$0.6 to maintain arterial oxygen saturation between 88-94%, PaCO2$70 mmHg or if they presented apnea that required bag mask ventilation for stabilization.

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EIT and CPAP pressure after extubation Rossi FS et al.

Figure 1 - The ‘‘functional map’’ (FM) screen used to evaluate upper/lower ventilation ratio (U/L) during image acquisition and off-line analysis and to determine ventilation homogeneity (VH). U/L (regional ventilation ratio) displays the calculated ratio at a certain window of data collection. Of note is the variability of the impedance presented in the lower black box, which was detected during the first half of data acquisition.

The use of EIT to describe air distribution during mechanical ventilation in preterm infants has only recently been reported, in which the age of the infant during volumetargeted ventilation (14) and the application of surfactant (26) impact the distribution of tidal ventilation. In the small and heterogeneous population studied, the mean value of the best PEEP determined by EIT during assisted ventilation was higher than the usual pressures applied with CPAP after tube removal at our NICU. Assuming that extubation failure may be reduced if the

Table 1 - Demographic characteristics of the 14 newborns included in the study. Prenatal Infant Sex Apgar 5 steroids 1 2 3 4 5 6 7 8 9 10 11 12 13 14

F F M M F M M M F M M F F F

10 8 10 10 9 9 6 6 5 8 9 6 8 8

Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes No

BW (g) GA (wk) 860 1055 1060 1290 1175 1185 675 990 930 920 560 880 855 730

26.0 33.0 29.9 30.4 28.6 31.6 24.4 28.0 26.4 25.6 25.0 26.7 30.7 27.1

EITd (days)

Surfactant

1 1 5 5 1 6 29 8 2 1 35 8 1 2

No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Table 2 - Secondary outcomes among the 14 newborns included in the study. Mean¡SD Atelectasis after extubation (%) 0.0(0.0%) Oxygen index 4.4¡2.2 Alveolar-arterial oxygen difference 129¡64 (mmHg) Arterial/alveolar ratio 0.315¡0.121 7.354¡0.070 pH (-logH+) PaCO2 (mmHg) 39.2¡9.6

BW birth weight, GA gestational age at birth, EITd day of life when EIT was performed.

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Maximum Minimum 8.0 237.9

1.6 43.5

0.580 7.470 62.9

0.130 7.240 28.0

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Figure 2 - Panels A and B show an example of ventilatory modification that was detected by electrical impedance tomography after PEEP reduction. The best ventilation homogeneity is in panel A, with an upper/lower ratio (U/L) of 1.05. After PEEP reduction, panel B presents a U/L ratio of 2.22 with collapse of the dependent area of the left lung. In both situations, the right lung was more aerated than the left lung.

interpretation of impedance variation in these extreme patients (34,35). The electrodes also need to be gentle on the skin of VLBW infants who are particularly prone to lesions and must also have a size capable of fitting around the infants’ very small thoraxes. The spatial resolution of EIT images is affected by and directly related to the number of electrodes that are used (11,36). Mechanical ventilation, which is used worldwide to increase the survival of many neonates in need, still relies on empirical protocols. The possibility to study ventilation in real time at the bedside without radiation and without high costs using EIT in these tiny and unstable patients deserves future trials.

pressures that determine the best VH are efficiently applied using non-invasive ventilation, the maintenance of a desired CPAP level is highly desired. If this is true, however, in contrast to invasive ventilation, part of the pressure may leak through the mouth, esophagus or nasal prong. The EIT can be used to evaluate breathing patterns of non-ventilated neonates (16,27), and it could also possibly be used to titrate CPAP levels in extubated patients. However, this was not explored in our protocol. Despite these limitations, our results may be used as a reference for future studies that will evaluate EIT application in preterm infants who are considered ready for extubation. Although different CPAP apparatuses were used, previous data suggest that this factor does not influence the extubation success rate (9,28,29). The extubation failure found in this small sample of patients was in agreement with previous studies (6,7,10,25,29). Variability of the VH index during spontaneous breathing was a hallmark observed in all patients studied (Figure 1). EIT evaluation performed with the use of muscle relaxation and sedation during controlled ventilation in animals and patients does not address this variability because all of the respiratory cycles behave similarly (13,15,21,22,24,30). Previous studies evaluating neonates have also described marked impedance variations during both assisted and unassisted respirations. These studies used very short and stable EIT data to evaluate and characterize air distribution among different lung areas (14,17,31). In contrast to this concept, we collected and analyzed the largest number of cycles that were unaffected by movements or artifacts. In doing so, almost 30% of the raw data was not used for interpretation. Our strategy is more time-consuming and can be biased by individual interpretation, but may better reflect the usual respiratory pattern of the neonates. A tool capable of automatically discarding impedance variations unrelated to air movement could facilitate EIT interpretation in spontaneous breathing neonates and is highly desired. One interesting finding was the absence of a trend favoring non-dependent ventilation at lower levels of PEEP. This was in contrast to results in previous EIT studies that utilized controlled ventilation. There are at least two explanations for this observation. First, as the neonates were considered ready for extubation, their respiratory compliance was likely close to normal, which may have minimized the effects of lung recruitment observed in sicker lungs (30,32). Second, we studied only a narrow PEEP window because we were aware of the possible deleterious effects of using very high or low PEEP for the VLBW infants. A recent study showed that spontaneous non-ventilated preterm infants tended to ventilate non-dependent areas more than dependent areas (33) whereas ventilated infants had higher ventilation in the middle third of the chest (14). The other analyzed outcomes of atelectasis after extubation and blood gas analysis did not show differences among the patients but only represent preliminary data because the sample studied was too small for clinical inferences. EIT is still being adapted to generate high-quality images and information in small patients. Specific algorithms for image reconstruction must be developed to allow the correct

& ACKNOWLEDGMENTS The authors thank Prof. Dr. Marcelo Britto Passos Amato and the Experimental Pneumology Laboratory – LIM 09, University of Sa˜o Paulo, for support during this study.

& AUTHOR CONTRIBUTIONS Rossi FS and Rebello CM participated in original concept and writing of the manuscript and contributed to data acquisition. Yagui AC and Haddad LC contributed to data acquisition. Deutsch AA participated in the original concept and writing of the manuscript.

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CLINICAL SCIENCE

Zinc, vitamin A, and glutamine supplementation in Brazilian shantytown children at risk for diarrhea results in sex-specific improvements in verbal learning Aldo A. M. Lima,I Michelle P. Kvalsund,II Paula P. E. de Souza,I I´talo L Figueiredo,I Alberto M. Soares,I Rosa M. S. Mota,I Noe´lia L. Lima,I Relana C. Pinkerton,I Peter P. Patrick,III Richard L. Guerrant,I,II Reinaldo B. Oria´I I

Federal University of Ceara´, School of Medicine, Clinical Research Unit and Institute of Biomedicine/Center for Global Health and Department of Physiology and Pharmacology, Fortaleza/CE, Brazil. II University of Virginia, School of Medicine, Center for Global Health, Division of Infectious Diseases, Charlottesville, VA, USA. III University of Virginia, School of Medicine, Kluge Children’s Hospital, Department of Pediatrics, Charlottesville, VA, USA.

OBJECTIVE: To identify the impact of supplemental zinc, vitamin A, and glutamine, alone or in combination, on long-term cognitive outcomes among Brazilian shantytown children with low median height-for-age z-scores. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in children aged three months to nine years old from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for cognitive testing (total of 167 children) were: (1) placebo, n = 25; (2) glutamine, n = 23; (3) zinc, n = 18; (4) vitamin A, n = 19; (5) glutamine+zinc, n = 20; (6) glutamine+vitamin A, n = 21; (7) zinc+vitamin A, n = 23; and (8) glutamine+zinc+vitamin A, n = 18. Neuropsychological tests were administered for the cognitive domains of non-verbal intelligence and abstraction, psychomotor speed, verbal memory and recall ability, and semantic and phonetic verbal fluency. Statistical analyses were performed using SPSS, version 16.0. ClinicalTrials.gov: NCT00133406. RESULTS: Girls receiving a combination of glutamine, zinc, and vitamin A had higher mean age-adjusted verbal learning scores than girls receiving only placebo (9.5 versus 6.4, p = 0.007) and girls receiving zinc+vitamin A (9.5 versus 6.5, p = 0.006). Similar group differences were not found between male study children. CONCLUSIONS: The findings suggest that combination therapy offers a sex-specific advantage on tests of verbal learning, similar to that seen among female patients following traumatic brain injury. KEYWORDS: Malnutrition; Cognition; Zinc; Vitamin A; Glutamine; Child. Lima AA, Kvalsund MP, Souza PP, Figueiredo IL, Soares AM, Mota RM, et al. Zinc, vitamin A, and glutamine supplementation in Brazilian shantytown children at risk for diarrhea results in sex-specific improvements in verbal learning. Clinics. 2013;68(3):351-358. Received for publication on September 12, 2012; First review completed on September 28, 2012; Accepted for publication on November 19, 2012 E-mail: rbo5u@virginia.edu Tel.: 55 85 3366-8239

(2), and subsequent poor school performance during the developmental childhood years (3). While these long-term negative outcomes have been well documented, our understanding of measures that could prevent or reverse these cognitive short falls is very limited. Micronutrients, such as zinc, vitamin A, and glutamine, hold promise to avert these deficits due to these compounds’ potential role in protecting the brain and in intestinal development (4,5). Numerous clinical zinc supplementation trials have revealed decreased diarrheal mortality and morbidity in children following supplementation (6). In addition, a pooled analysis of 37 supplementation trials regarding the impact of zinc supplementation on growth shortfalls revealed a highly significant impact on linear growth in prepubertal children (7). The mechanisms by which zinc acts to protect against diarrhea and growth deficits are not

& INTRODUCTION The vicious cycle of diarrhea/malnutrition in early childhood acts synergistically to impair the child’s full developmental potential. A series of cohort studies from our group, enrolling children under diarrhea and enteric disease surveillance in the first two years of life, has documented significant growth shortfalls (1), blighted cognitive potential

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fully understood, but they are likely multi-factorial, including anti-bactericidal and antioxidant effects (8,9), with improved mucosal immune response and recovery (10). Zinc is also involved in neurogenesis, neuronal migration, and synaptogenesis (11,12), and oral zinc supplementation improved behavioral and hippocampal development in a murine model of under-nutrition early in life (13). Vitamin A deficiency is a well-recognized cause of preventable blindness, but it now is also being recognized for its maintenance of intestinal epithelial integrity and the immune response (14). These effects could explain the reductions of the mortality and severity of diarrheal episodes seen following vitamin A supplementation in children. Further, while the importance of vitamin A in embryogenesis and congenital abnormalities has been well documented, very little is known about the importance of post-natal vitamin A deficiency in the healthy functioning of the central nervous system. Studies of retinoid knockout mice and mice subjected to post-natal vitamin A deprivation have suggested that retinoid signaling pathways play critical roles in higher cognitive functioning in the central nervous system (15). No studies have been conducted regarding the impact of vitamin A supplementation alone on cognitive outcomes in humans. Glutamine is a major energy source for the intestinal mucosa, and it plays a key role in nucleic acid biosynthesis (16). Glutamine supplementation has been associated with improved gastrointestinal function in a variety of clinical scenarios, including ameliorating mucosal atrophy after parenteral nutrition (17), improving gut mucosal healing following cancer therapy (18), and enhancing enteric immune responses (16). Glutamine is also found abundantly in the central nervous system, where it acts as a precursor of neurotransmitter amino acids, an oxidative energy source, and an ammonia detoxifier (19). To the best of our knowledge the impact of glutamine supplementation on cognition has never been investigated in children. This prospective, randomized, double-blind, placebocontrolled trial investigated the impact of supplemental zinc, vitamin A, and glutamine, alone or in combination, on long-term cognitive outcomes in a group of high-risk children identified as below the median height-for-age zscore (HAZ) in a slum community in Fortaleza-Ceara´, Brazil.

Study Design and Baseline Evaluation This study was a prospective, double-blind, randomized, placebo-controlled trial and was registered as a clinical trial (NCT00133406) with the US National Library of Medicine (http://www.clinicaltrials.gov). Enrollment/recruitment was conducted from June 2000 to August 2004. After parental consent was obtained, the parents and children were interviewed by an experienced field study team (one nurse and two community health workers) to collect demographic information, including age, sex, birth weight, level of maternal education, family income, and baseline anthropometric information. Weight was measured using a calibrated digital weighing scale with 100 g precision (Tanita Solar Scale, Tanita Corporation of American Inc., Arlington, IL, USA). Height was measured in the supine position for children younger than 24 months of age and standing for children aged 24 months old or older using an anthropometric rod with an accuracy of 0.1 cm. HAZ, weight-for-age z-scores (WAZ), and weight-for-height zscores (WHZ) were calculated using the Epi-Info anthropometric software (Center for Disease Control, Atlanta, GA, USA) as markers of physical development and nutritional status. These anthropometric z-scores are the numbers of standard deviations greater or less than the median values for the National Center for Health Statistics (NCHS) and International Reference Population (21).

Sample Size Calculation A sample size was calculated to allow for appropriate power to detect group differences in cognitive scores between children receiving zinc plus vitamin A compared with children receiving no vitamin A or zinc, averaging the difference in children receiving glutamine and children not receiving glutamine. Using a two-tailed test with type I error = 5%, 35 children per cell were calculated to be sufficient to have 80% power to detect a difference of 0.36 (small to medium effect size) and 90% power to detect a difference of 0.42 (medium effect size).

Nutritional Intervention and Surveillance Three hundred and fourteen children were randomized with regard to receiving vitamin A (100,000 IU retinyl palmitate if ,12 months old or 200,000 IU retinyl palmitate if $12 months old every four months), one dose at 0, four, and eight months of the study protocol; zinc (40 mg twice weekly); or both for one year. Half of each group received glutamine (16 g daily given over ten days started during the first month of the study protocol) (22), of whom one hundred and sixty-seven were available and eligible for cognitive testing. Only trained health care agents of the study surveillance team administered all of the micronutrients. The health workers administered the micronutrients during home visits with direct observation of intake. Lglutamine was obtained from Rexim (Courbevoie, France); L-glycine was obtained from Spectrum Chemicals (Gardena, CA, USA); zinc acetate was obtained from Spectrum Chemicals; and vitamin A (retinyl palmitate in vegetable oil with 40 IU of alpha-tochopherol as an antioxidant) was obtained from Hoffman-La Roche (Basel, Switzerland). Isomolar glycine (8.3 g/daily) was used as a placebo for glutamine. Tanjal juice was used as the placebo for zinc, and the same amounts of alpha-tocopherol and vegetable oil were used as the placebo for vitamin A. Tocopherol was chosen as the placebo for vitamin A because it is also a

& METHODS Population This study was undertaken in the urban shanty compound community of Parque Universita´rio, Fortaleza, the capital of the state of Ceara´ in northeast Brazil. All children (identified through a community census) between three months and nine years of age and below the median heightfor-age z-score (HAZ, population median = -0.06) were eligible to participate. The latter eligibility criterion was selected because of the high predictive value of HAZ scores in identifying children with chronic, persistent diarrheal episodes in the first two years of life (20). The exclusion criteria included children being exclusively breast-fed, participants in any study over the previous two years, and children having illnesses with fevers greater than 38 ˚C at the time of enrollment.

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(UFC), Fortaleza, Ceara´, Brazil; Conselho Nacional de E´tica em Pesquisa (CONEP) at the Ministry of Health, Brası´lia, Brazil; and the University of Virginia IRB for Health Services Research. In addition, the study was followed and approved by the Data Safety and Management Board (DSMB) at the National Institutes of Health.

fat-soluble vitamin, and the tocopherol-dosing preparation is similar to vitamin A capsule dosing. The vitamin A and placebo capsules were the same color, size, and taste. A computer-generated random number list assigned the children to one of eight treatment arms, including: 1) placebo; 2) glutamine; 3) zinc; 4) vitamin A; 5) glutamine+zinc; 6) glutamine+vitamin A; 7) zinc+vitamin A; or 8) glutamine+zinc+vitamin A. The treatment groups and oral treatment regimens, including dose, frequency, and duration of supplementation, are outlined in Table 1. A member of the field study team blinded to treatment group administered the supplementations and visited each child twice weekly to assess tolerance and any adverse effects of supplementation. No significant differences in the rates of adverse events were found among the treatment groups.

Statistical Analysis The data were double-entered by independent persons and validated using Excel software, version 4.0. All of the data were analyzed using the SPSS statistical software package (SPSS, Inc. Chicago, IL, USA). A one-way analysis of variance with post-hoc least significant difference (LSD) tests for twoby-two group comparisons was used to identify simple main effect differences in age at study entry, age at cognitive testing, time between cognitive testing and study enrollment, sex, birth weight, maternal education, family income, and nutritional parameters at study entry and conclusion, including weight-for-age WAZ, HAZ, and WHZ z-scores, as well as performance on the neuropsychological assessments. The treatment groups were further categorized into four micronutrient exposure groups, including zinc, vitamin A, zinc+vitamin A, or glutamine, and compared with regard to these variables to identify the impact of simple single or combination micronutrient exposures on study variables. An analysis of covariance was used to adjust for the influence on cognitive performance in terms of sex and the time between study enrollment and cognitive assessment. Continuous variables in tables are presented as the means¡standard deviations. Categorical variables are presented as percentages of the total population. A p-value ,0.05 was considered statistically significant.

Neuropsychological Assessment Children who completed the supplementation protocol and were at least five years of age (appropriate age for testing) underwent five cognitive tests at an average of four years later (range 1.4-6.6). The following tests with Brazilian adaptations were included: the Test of Non-Verbal Intelligence, 3rd Edition (TONI-3); the Wide Range Assessment of Memory and Learning, 2nd Edition (WRAML-2); Verbal Learning and Delayed Recall; the Weschler Intelligence Scale for Children (WISC) Coding A (for children ages 5-7) and Coding B (for children ages 8-13); and the Developmental Neuropsychological Assessment (NEPSY) Verbal Fluency test. The cognitive assessment chosen covered the cognitive domains of non-verbal intelligence and abstraction, psychomotor speed, verbal memory and recall ability, and semantic and phonetic verbal fluency. These tests have been used in previous studies in this population (2) and in other non-Englishspeaking populations (23). All of the tests were administered in a standardized manner and scored by a Brazilian developmental psychologist who was blinded to treatment group. The tests were administered in a silent room located in a school facility within the community.

& RESULTS A flow chart of the study children is provided in Figure 1. A total of 324 children were identified and screened for study eligibility, of whom 201 (64%) completed nutrient supplementation during the 12-month study protocol. Among children completing the supplementation protocol, 167 (83%) children underwent the cognitive examinations. The most common reason for not undergoing the cognitive testing was that the child was younger than five years of age and thus too young to undergo the tests (n = 30). Children

Ethical Approval The study protocol and informed consent form were approved by the Comiteˆ de E´tica em Pesquisa e Complexo Hospitalar (COMEPE) at the Federal University of Ceara´

Table 1 - Treatment groups and oral treatment regimens of the study participants. A randomized, double-blind, and placebo-controlled clinical trial of zinc, vitamin A, and glutamine supplementation in Brazilian shanty-town children from Fortaleza, CE, northeastern Brazil, during June 2000 and August 2004, with follow-up between June 2000 and December 2007. Treatment Group

Oral Treatment Regimen

Placebo Glutamine Zinc Vitamin A Glutamine+Zinc Glutamine+Vitamin A Zinc+Vitamin A Glutamine+Zinc+Vitamin A

Placebo* (Placebo glutamine+Placebo zinc+Placebo vitamin A) Glutamine**+Placebo zinc+Placebo vitamin A Zinc{+Placebo glutamine+Placebo vitamin A Vitamin A{+Placebo zinc+Placebo glutamine Glutamine**+Zinc{+Placebo vitamin A Glutamine**+Vitamin A{+Placebo zinc Zinc{+Vitamin A{+Placebo glutamine Glutamine**+Zinc{+Vitamin A{

Isomolar glycine doses (8.3 g/day) over ten days started at one month of the study protocol; zinc vehicle (Tanjal juice) twice weekly for twelve months; placebo retinol = vitamin E (tocopherol 40 IU) each for four months at 0, four, and eight months. ** Oral glutamine (16.2 g/day) for ten days starting at one month of the study protocol. { Zinc (zinc acetate) 40 mg twice weekly for 12 months. { Vitamin A (retinyl palmitate) 100,000 IU for children ,12 months or 200,000 IU for children at least 12 months old, given as one dose at 0, four, and eight months of the study protocol.

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Figure 1 - Flow of Study Children to Micronutrient Supplementation Groups and Cognitive Testing.

versus combination glutamine, zinc, and vitamin A group difference in WAZ scores almost reached significance at the end of the study (p = 0.06).

who completed the cognitive testing did not differ significantly from children who did not undergo cognitive testing with regard to sex, birth weight or length, or baseline WAZ or WHZ. However, the children who completed cognitive testing were significantly older (p,0.001), weighed more and were taller at baseline (both p-values ,0.001), and had better baseline HAZ scores (p = 0.026) than those who did not complete the cognitive testing. Table 2 outlines the demographic and other characteristics of the children who completed cognitive testing, overall and by treatment group. The study children ranged from five to 13 years of age at the time of cognitive testing. Slightly more than half of the study children were female (53%). Only 30 children (18%) had mothers with more than a primary school education. Data on family income were available for 123 children, of whom 59 (48%) were living in a household earning the Brazilian minimum wage of 415 Brazilian Reais per month (equivalent to 265 USD per month) or less. No significant group differences were identified regarding age at study entry, age at cognitive testing, time between cognitive testing and study entry, sex, birth weight, maternal education, or family income. Regarding nutritional parameters, no significant group differences were identified at study entry or conclusion regarding HAZ, WAZ, or WHZ scores, with the exception that children in the combination glutamine, zinc, and vitamin A group had higher WAZ scores at study entry compared with placebo children (p = 0.04). This placebo

Cognitive Performance The mean age-adjusted cognitive test scores for all eight treatment arms are presented in Table 3. No significant group differences were found regarding the age-adjusted TONI-3 Intelligence Quotient (IQ), WISC-III Coding, WRAML-2 Delayed Verbal Learning, or NEPSY Verbal Fluency mean scores. z-scores for semantic and phonetic fluency among children aged 6-12 years old were also compared across treatment groups to identify any differential impact of supplementation group on verbal fluency categories. No significant group differences were identified based on verbal fluency subtest z-scores. However, children receiving combination glutamine, zinc, and vitamin A performed significantly better on WRAML-2 verbal learning than the placebo group, with mean age-adjusted scores of 9.28 (SD 3.41) and 7.28 (SD 3.34), respectively (p = 0.02). After controlling for time between study entry and cognitive assessment, sex stratification revealed that this improvement in verbal learning was highly significant among the girls (p = 0.007) but not among the boys (p = 0.43) who received combination glutamine, zinc, and vitamin A versus placebo (Table 4). In addition, female children receiving combination glutamine, zinc, and vitamin A performed significantly better on verbal learning than female children

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Table 2 - Demographics and other characteristics of the study participants. A randomized, double-blind, and placebocontrolled clinical trial of zinc, vitamin A, and glutamine supplementation in Brazilian shanty-town children from Fortaleza, CE, northeastern Brazil, during June 2000-August 2004, with follow-up between June 2000 and December 2007. Placebo (n = 25)

Glutamine (n = 23)

Age at Study Entry (months) 48.8¡29.8 Age at Cognitive Testing 8.43¡2.02 (years) Time between Cognitive 4.42¡1.46 Testing and Study Entry (years) Female n (%) 12 (48) Birth weight 2.93¡0.63 5 (20) Maternal Education (, primary school) n (%) 9 (53) Family Income.than Minimum Wage n (%) Nutritional Parameters at Study Baseline Weight-for-Age z score -1.00¡1.10 Height-for-Age z score -1.26¡0.97 Weight-for-Height z score -0.25¡1.13 *

Zinc (n = 18)

Glutamine+ Zinc+Vitamin Glutamine+Zinc+ Total Vitamin Glutamine+ A (n = 19) Zinc (n = 20) Vitamin A (n = 21) A (n = 23) Vitamin A (n = 18) (n = 167)

53.9¡28.2 62.2¡26.0 43.3¡25.5 55.2¡28.3 8.73¡2.18 9.20¡2.20 7.55¡2.14 8.76¡1.87

54.8¡29.0 8.61¡1.99

59.5¡26.1 8.98¡2.30

56.0¡25.5 8.34¡1.86

54.1¡27.4 8.58¡2.08

4.29¡1.31 4.09¡1.15 3.99¡1.48 4.22¡1.34

4.10¡1.32

4.09¡1.15

3.73¡1.33

4.13¡1.33

9 (39) 11(61) 9 (47.4) 9 (45) 3.18¡0.45 3.13¡0.68 3.10¡0.52 3.19¡0.41 2 (9) 6 (33) 3 (16) 2 (11)

9 (43) 3.13¡0.54 6 (29)

16 (70) 3.22¡0.37 3 (14)

13 (72) 3.2¡0.42 3 (18)

88 (53) 3.13¡0.51 30 (18)

11(61)

8 (50)

6 (35)

67 (55)

-0.85¡0.81 -1.01¡0.91 -0.33¡1.00

-0.99¡0.98 -1.07¡0.95 -0.42¡0.93

-0.41¡0.80* -0.82¡0.69 0.25¡1.12

-0.67¡0.94 -0.95¡0.90 -0.07+0.99

11 (65)

6 (60)

4 (31)

9 (60)

-0.42¡0.84 -0.55¡0.82 -0.47¡1.04 -0.54¡0.92 -0.69¡0.89 -0.80¡1.01 -0.86¡0.79 -0.95¡0.90 0.10¡0.87 -0.07¡0.82 0.15¡1.01 0.12¡0.93

Combination Zinc+Vitamin A+Glutamine versus Placebo, p = 0.04.

receiving zinc and vitamin A alone (p = 0.006). These group differences persisted after controlling for differences in baseline WAZ scores. However, within the placebo group, the girls had worse verbal learning scores than the boys (p,0.05). No significant between group differences were identified before or after sex stratification on the WRAML-2 delayed verbal learning score. This combination group effect also almost reached statistical significance compared with female children receiving glutamine and zinc alone (p = 0.08). The treatment arms were also categorized into four groups based on micronutrient exposure (i.e., zinc, vitamin A, combination zinc+vitamin A, or glutamine) to assess the differential impact of exposure to a specific single micronutrient or combination of micronutrients on cognitive test performance. The results of cognitive testing revealed no significant between group differences based on micronutrient exposure categories.

& DISCUSSION This study found a superior impact of combination glutamine, zinc, and vitamin A supplementation on the WRAML-2 verbal learning in a group of children with low to median HAZ scores (and thus at high risk for the cognitive deficits seen in children with childhood diarrhea and malnutrition). These findings were statistically significant overall, but sex stratification identified highly significant differences only between female children receiving combination therapy and girls receiving zinc and vitamin A alone or placebo. Supplementation was found to be beneficial in girls compared with the placebo group. Thus, the combined intervention with glutamine, zinc, and vitamin A was more important to the female group because the girls were more affected than the boys. No other differences were identified between supplementation treatment groups or following the categorization of treatment groups into one of four exposure groups.

Table 3 - Mean age-adjusted (scaled) performance on cognitive tests by treatment group. A randomized, double-blind, placebo-controlled clinical trial of zinc, vitamin A, and glutamine supplementation in Brazilian shanty-town children from Fortaleza, CE, northeastern Brazil, during June 2000-August 2004, with follow-up between June 2000 and December 2007. Cognitive Tests

Placebo (n = 25)

Glutamine (n = 23)

IQ 79.1¡9.12 81.8¡7.76 Coding 9.45¡3.51 10.4¡3.13 Verbal Learning 7.28¡3.34 8.43¡2.41 Delayed Verbal 8.20¡2.69 7.78¡1.57 Learning Verbal Fluency 6.22¡2.70 6.67¡2.67 NEPSY Verbal Fluency Subtest Z-scores* Semantic Fluency -1.17¡0.84 -0.63¡0.78 Phonetic Fluency -1.38¡0.67 -1.48¡0.71

Zinc (n = 18)

Vitamin A (n = 19)

Glutamine+ Zinc (n = 20)

Glutamine+ Zinc+Vitamin Vitamin A (n = 21) A (n = 23)

79.9¡6.92 9.67¡3.66 7.61¡2.40 8.00¡1.88

82.2¡5.87 9.84¡3.15 8.74¡2.47 7.79¡2.22

80.4¡8.05 9.75¡3.17 8.15¡2.83 8.35¡2.08

81.91¡7.12 9.71¡3.78 7.9¡2.77 8.71¡2.43

82.5¡9.94 9.78¡4.17 7.57¡2.81 8.52¡2.64

82.1¡9.78 10.8¡3.38 9.28¡3.41** 8.11¡3.08

7.11¡3.59

5.74¡2.35

5.95¡2.81

6.75¡2.65

6.36¡2.95

7.33¡2.63

-0.43¡0.82 -1.04¡1.19

-1.15¡0.49 -1.56¡0.80

-0.63¡0.86 -1.49¡0.70

-0.36¡0.94 -1.18¡0.88

-0.82¡0.73 -1.54¡0.87

-1.24¡1.38 -1.14¡0.83

* Children aged 6-12 years old only; **Comparison of Combination Glutamine+Zinc+Vitamin A versus placebo, p = 0.02. IQ = intelligence quotient. NEPSY = A developmental neuropsychological assessment.

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Table 4 - Mean and standard deviations of age-adjusted performance on cognitive tests by sex after adjusting for time between study enrollment and cognitive evaluation. A randomized, double-blind, placebo-controlled clinical trial of zinc, vitamin A, and glutamine supplementation in Brazilian shanty-town children from Fortaleza, CE, northeastern Brazil, during June 2000-August 2004, with follow-up between June 2000 and December 2007. Cognitive Tests

Placebo

Glutamine

Vitamin A

Zinc

Glutamine+ Zinc

Glutamine+ Vitamin A

Zinc + Vitamin A

Glutamine+ Zinc+Vitamin A

83.2 (2.8) 11.0 (1.2) 7.6 (0.9) 7.5 (11.1) 6.0 (1.0)

81.1 (2.8) 11.2 (1.2) 8.3 (0.9) 6.7 (8.8) 6.0 (1.0)

80.0 (2.5) 10.7 (1.1) 8.3 (0.8) 6.6 (10.8) 7.4 (0.9)

83.7 (2.8) 10.1 (1.2) 7.3 (0.9) 7.1 (10.9) 6.3 (1.0)

83.3 (2.8) 10.4 (1.2) 7.5 (0.9) 7.8 (11.6) 7.1 (1.0)

81.7 (2.1) 9.7 (0.9) 6.5 (0.7)** 6.7 (10.7) 6.2 (0.7)

81.1 (2.1) 11.4 (1.0) 9.5 (0.8) 6.2 (8.7) 7.5 (0.8)

80.9 (2.2) 10.0 (0.9) 8.9 (0.7) 9.3 (0.9) 7.1 (0.8)

82.1 (2.6) 8.6 (1.1) 9.2 (0.9) 7.8 (0.5) 5.5 (0.9)

79.7 (3.1) 8.0 (1.3) 6.6 (1.1) 8.7 (1.1) 6.5 (1.2)

77.7 (2.5) 9.4 (1.1) 8.8 (0.8) 9.0 (0.9) 5.7 (0.9)

80.7 (2.4) 9.2 (1.0) 8.2 (0.8) 9.7 (1.0) 6.5 (0.9)

84.4 (3.2) 10.0 (1.4) 9.9 (1.1) 8.7 (1.0) 6.7 (1.1)

84.8 (3.7) 9.6 (1.6) 9.0 (1.2) 7.4 (0.6) 7.0 (1.3)

Female IQ Coding Verbal Learning Delayed Verbal Learning Verbal Fluency

82.6 (2.4) 10.1 (1.1) 6.4 (0.8)* 5.0 (8.5) 6.2 (0.9) Male

IQ Coding Verbal Learning Delayed Verbal Learning Verbal Fluency

75.9 (2.3) 8.8 (1.0) 8.0 (0.8) 6.7 (0.9) 6.1 (0.8)

Glutamine+Zinc+Vitamin A versus Placebo, p = 0.007. Glutamine+Zinc+Vitamin A versus Zinc+Vitamin A, p = 0.006. IQ = intelligence quotient.

Previous studies from our group have shown improvements in intestinal barrier function after a ten day treatment regimen of glutamine-enriched formula (22). A recent metaanalysis showed that zinc supplementation reduces diarrhea morbidity in children, especially after six months of age (24); both of these effects could be helpful during the critical time window for brain development in the study children, who live in endemic areas of enteric infections and malnutrition. This sex-specific benefit of combination glutamine, zinc, and vitamin A supplementation was an unexpected finding. Studies of recovery following traumatic brain injury (TBI) in children have shown a female recovery advantage in verbal list learning (25,26). In a study of 60 children (aged 6-16 years) with TBI identified through a private rehabilitation center in the midwestern U.S., female children performed significantly better than male children on the California Verbal Learning Test-Childrenâ&#x20AC;&#x2122;s Version, even after controlling for the impact of the severity of head injury (25). In contrast with these findings, there have been studies reporting a sex-specific decrease in intelligence test results in children treated for acute lymphoblastic leukemia, with girls showing lower IQ scores than boys (27,28), in contrast with the specific deficits in verbal learning seen in our study and the verbal intelligence deficits seen in pediatric intracranial hemorrhage and TBI studies. Our study, which included children at high risk for the longterm cognitive deficits seen with childhood diarrhea and malnutrition (based on low baseline HAZ scores), supports the findings of verbal list learning advantages following combination glutamine, zinc, and vitamin A supplementation. There is accumulating evidence that b-estradiol (the primary biological form of estrogen) not only has a neuroprotective role but might also activate restorative processes, improving learning and memory, and promote the formation of synapses in the nervous system during aging and early development, as seen in animal models (29,30). However, estradiol-based hormonal treatment in post-menopausal women has been found to be detrimental, increasing the risk of dementia and stroke (30).

Furthermore, sex differences during cognitive development remain controversial (31). One potential explanation for these contradictory findings is the requirement that bestradiol interact with IGF-1 for its neuroprotective effects (32). This study did not evaluate IGF-1 serum levels in these children (a study that is under way by our group); however, early weight gain and subsequent linear growth were associated with increments in serum IGF-1 levels. The neuroprotective effects of the combination nutritional therapy seen in the girls in this study might be related to improved IGF-1 interactions. More studies are warranted to test this hypothesis. Zinc supplementation trials in human children have not consistently shown improvement in cognitive outcomes. A randomized, controlled zinc supplementation trial in 740 Chinese children aged six to nine years attending three periurban schools found that children receiving micronutrient supplementation plus 20 mg of zinc six days/week for ten weeks had significantly improved fine and gross motor skills, attention, abstract concept formation, and abstract reasoning skills compared with children who received micronutrient supplementation without zinc or who received zinc supplementation alone (23). However, a study of 162 Guatemalan school children aged seven to eight years receiving 20 mg zinc/day for 25 weeks failed to find any significant differences after six months of follow-up in mental concentration or short-term memory compared with children receiving a placebo (24). Similarly, no significant differences in attention were found among 60 Canadian boys with low height percentiles attending first grade (aged 5-7 years) in 47 schools in southern Ontario who received 10 mg of zinc in a 1 mL solution of ZnSO4 added to apple/ orange juice compared with children receiving a similar placebo solution (25). These differences might be related to the prevalence and severity of zinc deficiencies in the study population, the impact of other concurrent micronutrient deficiencies, the simultaneous use of other supplements, the dose and duration of the supplementation, and the sensitivity and appropriateness of the neuropsychological testing employed.

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Combination Micronutrients and Cognition Lima AA et al. RB were responsible for the study design and coordination. All of the authors have read and contributed significantly to the manuscript.

The finding of a verbal learning benefit among female children receiving combination glutamine, zinc, and vitamin A supplementation addresses previous concerns that multiple micronutrient deficiencies present in undernourished children could reduce or obscure the clinical benefits of supplementation with single micronutrients (33). However, it should be noted that the present study did not conduct serial micronutrient serum assessments, and thus, the reasons for the positive effect of the full combination of supplements compared with single supplements or other combinations of supplementation remain speculative. Simultaneous supplementation with multiple micronutrients could act synergistically to improve long-term growth and cognitive outcomes in high-risk children by acting on multiple organ systems, including the gastrointestinal tract, systemic immune system, central nervous system, and neuroendocrine axes. Previous clinical studies of multiple micronutrient supplementation have produced divergent results, with some reporting positive benefits on attention, learning, and IQ, and others reporting no effects (34). Comparisons of these trials are difficult due to variations in population sampling, supplementation regimens (including some macronutrient and energy supplementation), routes of administration, durations of supplementation, and neuropsychological assessment instruments. No previous studies have applied supplementation with a combination of glutamine, zinc, and vitamin A. It is noteworthy that the WAZ at the start of the study was higher in the zinc+vitamin A+glutamine groups compared with the placebo groups, which might have explained the improved verbal learning scores in that group; however, WAZ at start of the study was not a significant predictor of verbal learning scores and therefore cannot be used to explain that improvement. Regarding limitations, we acknowledge that due to the small size of each interventional group at the end of followup, this study lost power and could have incorporated unexpected bias in the statistical analyses; therefore, our findings should be interpreted with caution and should be confirmed with larger study designs. In conclusion, we documented sex-specific improvements in selected cognitive tests after combined nutrient supplementation in at-risk children living in impoverished settings in northeastern Brazil. More studies (now being planned by our group) are warranted to evaluate the potential mechanisms of nutrient-to-nutrient interactions underlying these findings and the potential associations with IGF-1 levels in children.

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& ACKNOWLEDGMENTS The authors would like to thank Sayonara Bezerra de Alencar, Fa´tima Alves, Rosiana Maria de Paula Silva, and Maria Luzia F. Melo for all of their hard work and dedication to the Parque Universita´rio community and the Clinical Research Unit of the Federal University of Ceara´. The study was supported by a Brazilian funding agency, CNPq, and an ICIDR program grant, #5 U01 AI026512, from the NIH, USA.

& AUTHOR CONTRIBUTIONS Kvalsund MP, Souza PP, and Patrick PP conducted and supervised the cognitive assessments. Lima NL coordinated the surveillance team. Soares AM, Mota RM, and Pinkerton RC were responsible for data safety, monitoring, and the statistical analyses. Figueredo IL conducted the data analyses and prepared the manuscript. Lima AA, Guerrant RL, and Oria

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28. Waber DP, Urion DK, Tarbell NJ, Niemeyer C, Gelber R, Sallan SE. Late effects of central nervous system treatment of acute lymphoblastic leukemia in childhood are sex-dependent. Dev Med Child Neurol. 1990;32(3):238-48. 29. Wilson ME, Westberry JM, Trout AL. Estrogen receptor-alpha gene expression in the cortex: sex differences during development and in adulthood. Horm Behav. 2011;59(3):353-7, http://dx.doi.org/10.1016/j. yhbeh.2010.08.004. 30. Azcoitia I, Arevalo MA, De Nicola AF, Garcia-Segura LM. Neuroprotective actions of estradiol revisited. Trends Endocrinol Metab. 2011;22(12):467-73. 31. Ardila A, Rosselli M, Matute E, Inozemtseva O. Gender differences in cognitive development. Dev Psychol. 2011;47(4):984-90, http://dx.doi. org/10.1037/a0023819. 32. Selvamani A, Sohrabji F. The neurotoxic effects of estrogen on ischemic stroke in older female rats is associated with age-dependent loss of insulin-like growth factor-1. J Neurosci. 2010;30(20):6852-61, http://dx. doi.org/10.1523/JNEUROSCI.0761-10.2010. 33. Black MM. Micronutrient deficiencies and cognitive functioning. J Nutr. 2003;133(11 Suppl 2):3927S-3931S. 34. Vazir S, Nagalla B, Thangiah V, Kamasamudram V, Bhattiprolu S. Effect of micronutrient supplement on health and nutritional status of schoolchildren: mental function. Nutrition. 2006;22(1 Suppl):S26-S32, http://dx.doi.org/10.1016/j.nut.2004.07.021.

21. de Onis M, Onyango AW, Borghi E, Garza C, Yang H. Comparison of the World Health Organization (WHO) Child Growth Standards and the National Center for Health Statistics/WHO international growth reference: implications for child health programmes. Public Health Nutr. 2006;9(7):942-947. 22. Lima AA, Brito LF, Ribeiro HB, Martins MC, Lustosa AP, Rocha EM, et al. Intestinal barrier function and weight gain in malnourished children taking glutamine supplemented enteral formula. J Pediatr Gastroenterol Nutr. 2005;40(1):28-35, http://dx.doi.org/10.1097/00005176-20050100000006. 23. Brown L, Sherbenou RJ, Johnsen SK. Test of nonverbal intelligence. 3rd ed. Austin (TX): PRO-ED; 1997. 24. Lazzerini M, Ronfani L. Oral zinc for treating diarrhoea in children. Cochrane Database Syst Rev. 2012;6:CD005436. 25. Donders J, Hoffman NM. Gender differences in learning and memory after pediatric traumatic brain injury. Neuropsychology. 2002;16(4):4919, http://dx.doi.org/10.1037/0894-4105.16.4.491. 26. Donders J, Woodward HR. Gender as a moderator of memory after traumatic brain injury in children. J Head Trauma Rehabil. 2003;18(2):106-15, http://dx.doi.org/10.1097/00001199-200303000-00002. 27. Robison LL, Nesbit ME Jr., Sather HN, Meadows AT, Ortega JA, Hammond GD. Factors associated with IQ scores in long-term survivors of childhood acute lymphoblastic leukemia. Am J Pediatr Hematol Oncol. 1984;6(2):115-21, http://dx.doi.org/10.1097/00043426-198406020-00001.

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Association between muscle strength and the cardiopulmonary status of individuals living with HIV/AIDS Vagner Raso,I,II,III Roy J. Shephard,IV Jorge Casseb,V,VI Alberto Jose´ da Silva Duarte,V,VI Paulo Roberto Santos Silva,VII Ju´lia Maria D9Andreá GreveI,VII I Faculdade de Medicina da Universidade de Saõ Paulo - (FMUSP), Department of Experimental Pathophysiology, Saõ Paulo/SP, Brazil. II Bandeirante University of Saõ Paulo (UNIBAN), Master Program on Body Balance Rehabilitation and Social Inclusion, Saõ Paulo/SP, Brazil. III University of Western Saõ Paulo, (UNOESTE), Medicine and Physical Education School, Saõ Paulo/SP, Brazil. IV University of Toronto, Faculty of Kinesiology & Physical Education, Toronto, ON/Canada. V Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo - (ADEE 3002-HC-FMUSP), Secondary Immunodeficiency Ambulatory, Saõ Paulo/SP, Brazil. VI Faculdade de Medicina da Universidade de Saõ Paulo - (LIM-56-HC-FMUSP), Laboratory of Investigation in Dermatology and Immunodeficiencies, Saõ Paulo/SP, Brazil. VII Faculdade de Medicina da Universidade de Saõ Paulo - (LEM-IOT-HCFMUSP) Movement Studies Laboratory, Orthopedics and Traumatology Institute, Saõ Paulo/SP, Brazil.

OBJECTIVE: The purpose of this study was to compare aerobic function [anaerobic threshold (%V_ O2-AT), respiratory compensation point (%V_ O2-RCP) and peak oxygen uptake (V_ O2peak)] between physically active patients with HIV/AIDS and matched controls and to examine associations between disease status, poor muscle strength, depression (as estimated by the profile of mood states questionnaire) and the aerobic performance of patients. METHODS: Progressive treadmill test data for %V_ O2-AT (V-slope method), RCP and (V_ O2peak) were compared between 39 male patients with HIV/AIDS (age 40.6¡1.4 years) and 28 male controls (age 44.4¡2.1 years) drawn from the same community and matched for habitual physical activity. Within-patient data were also examined in relation to CD4+ counts (nadir and current data) and peak isokinetic knee torque. RESULTS: AT, RCP and (V_ O2peak) values were generally similar for patients and controls. Within the patient sample, binary classification suggested that AT, RCP and (V_ O2peak) values were not associated with either the nadir or current CD4+ count, but treadmill test variables were positively associated with peak isokinetic knee torque. CONCLUSION: The aerobic performance of physically active patients with HIV/AIDS is generally well conserved. Nevertheless, poor muscle strength is observed in some HIV/AIDS patients, which is associated with lower anaerobic power and (V_ O2peak), suggesting the possibility of enhancing the aerobic performance of patients with weak muscles through appropriate muscle-strengthening activities. KEYWORDS: Anaerobic Threshold; Cardiopulmonary Exercise Testing; HIV; Peak Aerobic Power; Muscle Strength. Raso V, Shephard RJ, Casseb J, Duarte AJ, Silva PR, D9Andre´a Greve JM. Association between muscle strength and the cardiopulmonary status of individuals living with HIV/AIDS. Clinics. 2013;68(3):359-364. Received for publication on October 13, 2012; First review completed on November 21, 2012; Accepted for publication on November 22, 2012 E-mail: vraso@usp.br Tel.: 55 11 2661-6041

functional loss following treatment. Most studies have pointed to a loss of muscular strength, with little deterioration of cardio-respiratory fitness (1-2). Nevertheless, some reports have also indicated adverse effects of the disease and treatment with non-nucleoside reverse transcriptase inhibitors on various aspects of cardio-respiratory health. Reported changes include delayed heart rate recovery following exercise (3), increased cardiovascular risk factors (particularly the HDL/LDL cholesterol ratio, secondary to decreased plasma adiponectin levels) (4-5), deteriorating endothelial function (6), atherosclerosis progression (7) and increased risk of myocardial infarction (8). Aerobic exercise has been recommended for patients with HIV/AIDS, both as a possible means of slowing disease progression [with benefits observed by Mustafa et al. (9),

& INTRODUCTION The acute phase of HIV/AIDS is frequently marked by a substantial loss of physical fitness. In planning an appropriate course of rehabilitation, it is important to know which aspects of fitness deteriorate and the persistence of this

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intravenous drug use or blood transfusion. The mean duration of diagnosed infection was 6.1¡0.8 years, with 20 of the 39 patients developing AIDS within 6.0¡0.9 years. HAART had been administered to 18 of the 39 patients for an average of 56.1¡7.5 months, with an adherence of 9.2¡0.3 on a 0 to 10 scale. The control group met the same health criteria as the patients, with the exception that the former were HIVnegative. The two groups were also matched in terms of their age, physical activity level and body mass index.

but not by Stringer et al. (10) or Terry et al. (11)] and also for exercise’s potential to reverse the depressed mood that commonly accompanies both HIV/AIDS infection and highly active antiretroviral therapy (HAART) treatment [with elevations of mood state reported by Ciccolo et al. (12), Neidig et al. (13) and Stringer et al. (10), but not by Terry et al. (11)]. In most reports to date, any changes in an individual’s muscular strength have been considered independently of peak aerobic power. However, this interpretation is not entirely appropriate. The peak oxygen intake that a patient can attain partially depends upon the muscle mass that is activated by the test exercise (14). Furthermore, if the muscles are weak, perfusion of the active tissues is restricted, and lactate begins to accumulate at a low power output, causing local fatigue and limiting the peak effort that can be developed (15,16). To shed further light upon these issues, we took a substantial sample of physically active male patients with HIV/AIDS and compared their aerobic performance with matched control subjects from the same milieu. We also compared values for subsets of the patient sample classified in terms of immune status, exposure to retroviral treatment and current peak isokinetic muscle torque. Our primary hypotheses were that aerobic function would generally be well maintained in physically active patients with HIV/AIDS but that even within a sample engaged in regular endurance activity, aerobic performance might nevertheless be impaired in individual patients with substantial muscle weakness.

Body composition and habitual physical activity

& METHODS

Height and body mass were determined by standard anthropometric techniques. Adiposity was determined using Harpenden skinfold calipers (Baty International, Burgess Hill, West Sussex, UK); central adiposity was represented by the sum of subscapular, mid-axillary, abdominal and suprailiac skinfolds, and peripheral adiposity was represented by the sum of the biceps, triceps, anterior thigh and mid-calf skinfolds. A Portuguese version of the international physical activity questionnaire (IPAQ) was used to estimate habitual physical activity (18,19). Subjects reported the frequency and duration of walking (considered light activity) and bouts of moderate and vigorous physical activity. The information was combined to yield an approximate physical activity score, measured in kJ of energy expended per week. For each of the light [3.3 METs], moderate [4.0 METs] and vigorous [8.0 METs] activities, the duration (minutes per day) was multiplied by the reported frequency (days per week) to yield accumulated totals of MET-min week-1.

Volunteers

Cardiopulmonary parameters

The patient sample consisted of 39 physically active males living with HIV/AIDS. All were volunteers who were recruited from an ambulatory outpatient clinic. The 28 matched controls were drawn from the same city. All subjects were informed about the procedures and risks before giving written consent to participate in the study, which had been approved by the research ethics committee of the Sa˜o Paulo University Hospital (File reference 768/06). The protocol met all of the ethical standards established for this journal (17). An initial telephone call was used to invite 56 volunteers with HIV/AIDS for a screening that focused on their current health status, current drug and cigarette consumption and physical activity. This was followed by a hospital visit for a detailed history and physical examination covering previous and current health status and other tests that included a 12-lead electrocardiogram, questionnaires regarding mood state and ability to perform the basic and instrumental activities of daily living, measures of body composition and routine blood and urine tests. Criteria for exclusion included the following: (1) acute or chronic psychological disturbances; (2) central or peripheral nervous system disorders; (3) musculo-skeletal problems; (4) cardiopulmonary or metabolic disorders; (5) cigarette smoking; (6) surgery or bed rest in the previous three months; and (7) any orthopedic conditions that could limit exercise testing or be exacerbated by exercise testing. Thirty-nine of the 56 volunteers met the criteria for the definitive study. Infection had been acquired through homosexual or heterosexual intercourse in 32 of the 39 patients; in the remainder of the patients, the source of infection was

Peak oxygen uptake ( O2peak) was measured directly using a progressive treadmill protocol (20,21). An automated gas analyzer (CPX/D, Medgraphics, Saint Paul, MN), calibrated against room air and a medically certified gas mixture of 11.9% O2 and 5.12% CO2, provided breath-bybreath data on respiratory gas exchange. A 12-lead electrocardiogram was monitored continuously during testing. Exercise began at a walking velocity of 3.6 km h-1 and with a 1% gradient. The velocity was increased by 1.2 km h-1 in each succeeding minute until volitional exhaustion. O2peak was recorded when one or more of the following criteria were satisfied: 1) respiratory exchange ratio (RER) $1.10; 2) attainment of the age-predicted maximal heart rate; 3) volitional fatigue; and/or 4) signs of exhaustion (unsteady gait, hyperpnea, sweating, facial flushing and grimacing) (2). The test-retest error of peak oxygen intake determinations was ,5%. A computerized regression analysis determined the anaerobic threshold (AT) from a plot of CO2 intake ( CO2) versus O2 intake ( O2) and the onset of excess CO2 output (V-slope method). The respiratory compensation point (RCP) was detected by plotting respiratory minute ventilation against CO2.

Peak muscle torque Peak muscle torque was evaluated using an isokinetic dynamometer (Biodex Multi-Joint System, Shirley, NY). This apparatus was calibrated weekly, according to the manufacturer’s instructions. After one familiarization test, volunteers performed five definitive maximal extensor movements at an angular velocity of 60 ˚?s-1, with verbal encouragement throughout; a two-minute rest interval was

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Cardiopulmonary status in HIV/AIDS Raso V et al.

Table 1 - Characteristics of controls and patients (classified by CD4+ nadir). Individuals living with HIV/AIDS

Control (n = 28) +

CD4 nadir (cells?mm-3) , 200 (n = 17) Age (years) Body height (m) Body composition Body mass (kg) BMI (kg?m-2) CBA (mm) PBA (mm) TBA (mm) HA (MET.min.week-1) Immunological parameters CD4+ (cells?mm-3) CD8+ (cells?mm-3) CD4+ nadir (cells?mm-3) Viral load (log)

44.4¡2.1 1.74¡0.1 80.1¡1.21 26.4¡0.4 ___ ___ ___ 1797¡56

$ 200 (n = 22)

Total (n = 39)

41.9¡1.9 39.6¡2.0 1.69¡0.2 1.73¡0.1 ————————————————————— 71.6¡2.9 74.0¡2.5 24.8¡0.8 24.8¡0.9 52.2¡5.0 71.4¡8.0 24.9¡2.4 30.8¡3.9 77.1¡6.9 102.3¡11.3 1589¡229 1726¡188 ———————————— 433¡67 588¡902 1083¡111 1086¡167 154¡36 344¡482 4.1¡0.3 4.5¡0.2

40.6¡1.4 1.71¡0.1 72.9¡1.9 24.8¡0.6 63.0¡5.2 28.2¡2.4 91.2¡7.2 1666¡144 521¡59 1084¡105 261¡34 4.4¡0.2

Values are means ¡ standard errors of mean; BMI: body mass index; CBA: central body adiposity; PBA: peripheral body adiposity; TBA: total body adiposity; HA: habitual activity; 1 Statistically significant difference between control and HIV/AIDs group with CD4+,200 cells?mm-3 (p,0.05); 2Statistically significant difference between HIV+ CD4+,200 cells?mm-3 and HIV+ CD4+$200 cells?mm-3 (p,0.05).

allowed between sets. The largest of the five readings was noted as the individual’s peak torque [Nm].

For the flow cytometry, 200 mL of whole blood was incubated with 5 mL of appropriate monoclonal antibodies (CD4+, CD8+ [Becton-Dickinson, Miami, FL]) for 20 minutes in the dark and at room temperature. Samples with isotypic control antibodies (IgG1[FITC]/IgG1[PE]/IgG1[PCy-5]) were run in parallel with each sample. A minimum of 5000 cells was analyzed on the Coulter XL-MCL counter (Coulter Corp., Miami, FL) using XL System II software (Coulter Corporation, Miami, FL). The lowest recorded CD4+ count was considered the CD4+ nadir.

Blood collection, leukocyte counts and flow cytometry Subjects refrained from ingesting solid or liquid food containing caffeine or chocolate or cola-based products and avoided even moderate physical activity for 48 hours before blood sampling. The subjects arrived at the laboratory at 7:00 a.m., having fasted overnight, and blood was collected from the median antecubital vein after 30 min of seated rest. Differential leukocyte counts were performed using an automated Cell-Dyn 3500 analysis system (Coulter Corp., Miami, FL).

Statistical analysis All analyses were performed using the Predictive Analytics Software 17.0 for Windows Package (PASW,

Table 2 - Findings during progressive treadmill (TM) test for controls and patients (classified by CD4+ nadir). Shaded areas indicate statistically significant differences. Individuals living with HIV/AIDS +

CD4 nadir (cells?mm-3) Control(a) (n = 28) HR (bpm) AT RCP PHR RPE AT RCP O2 (mL?kg-1?min-1) AT RCP Peak O2 (% peak) AT RCP TM test duration (min) Peak isokinetic torque (Nm?kg-1)

, 200(b) (n = 17)

__ 119¡3 140¡3 179¡3 __ 8.5¡0.2 11.9¡0.3 __

120¡3 141¡4 178¡4 8.8¡¡0.2 11.8¡0.4

$200(c) (n = 22) _____________ 122¡3 147¡3 179¡2 _____________ 9.2¡0.3 12.2¡0.3 _____________

Total (n = 39)

121¡2 144¡2 178¡2 9.0¡0.2 12.0¡0.3

16.4¡0.5 22.6¡0.7 32.2¡0.9 __

17.4¡0.5 24.3¡1.0 34.6¡1.4

17.5¡0.6 23.2¡0.9 33.9¡1.2 _____________

17.5¡0.4 23.7¡0.7 34.2¡0.9

51.3¡1.6 70.5¡1.8 6.7¡0.2 __

50.3¡1.5 70.8¡2.0 9.5¡0.5 242¡11

52.3¡2.0 68.6¡2.3 9.6¡0.6 253¡11

51.4¡1.3 69.6¡1.6 9.6¡0.4 248¡8

p,0.05; Values are means ¡ standard errors of mean; AT: anaerobic threshold; RCP: respiratory compensation point; VO2: oxygen intake; HR: heart rate; PHR: peak heart rate; RPE: rating of perceived exertion; Statistically significant difference between control and HIV+ subgroups.

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Table 3 - Findings during progressive treadmill (TM) test and strength measurements for patients (classified by current CD4+ count, cells?mm-3). Shaded area indicates statistically significant differences. Individuals living with HIV/AIDS +

Current CD4+$200 cells mm-3 (n = 33)

Current CD4 ,200 cells mm-3 (n = 6) HR (bpm) AT RCP PHR RPE AT RCP

________________

8.8¡0.3 (8.0-10.0) 11.5¡0.9 (9.0-15.0)

O2 (mL?kg-1?min-1) AT RCP Peak

16.5¡1.3 (11.4-20.1) 21.6¡1.8 (15.4-28.7) 32.5¡3.3 (20.1-41.7)

O2 (% peak) AT RCP TM test duration (min) Peak isokinetic torque (Nm?kg-1)

51.7¡3.0 67.8¡4.0 8.3¡0.9 205¡13

115¡2 (107-120) 134¡5 (122-157) 172¡8 (136-191)

122¡3 (94-156) 146¡3 (116-178) 179¡2 (161-203) ________________ 9.0¡0.2 (7.0-12.0) 12.1¡0.3 (9.0-15.0) ________________ 17.6¡0.4 (13.4-22.5) 24.0¡0.7 (16.4-31.7) 34.4¡0.9 (25.2-43.4) ________________

(41.0-59.0) (53.0-77.0) (4.0-10.0) (151-241)

51.5¡1.4 (40.9-79.0) 70.1¡1.7 (52.0-92.0) 9.8¡0.4 (6.0-20.0) 257¡8 (150-349)*

*p,0.05; Values are means ¡ standard errors of mean; AT: anaerobic threshold; RCP: respiratory compensation point; VO2: oxygen intake; HR: heart rate; PHR: peak heart rate; RPE: rate of perceived exertion.

adequate sample power to detect such an effect at the 95% confidence level. This remained true with binary subdivision of the 39-patient sample.

Inc., Chicago, IL). Kolmogorov-Smirnov statistics demonstrated that all data were normally distributed. Data are presented as the means ¡ standard errors of the mean. Unpaired Student t-tests were used to compare patients and control groups and also to compare patient subgroups. Univariate regression analyses also investigated associations of peak isokinetic torque with anaerobic threshold, respiratory compensation point and O2 peak. Statistical significance was set at p,0.05 throughout. We determined that a 10% loss of aerobic performance would have practical and clinical significance. Thus, the sample size of 39 patients and 28 controls provided

& RESULTS General characteristics and immunological data for the patients and control subjects are summarized in Table 1. The patients tended to have a slightly lower body mass than the controls, particularly in the subgroup with a lower CD4+ nadir, but the patients did not differ significantly from the controls in terms of age or body mass index. In terms of

Table 4 - Findings during progressive treadmill (TM) test and strength measurements for patients (classified by peak muscle torque at 60o?sec-1). Shaded areas indicate statistically significant differences. Peak isokinetic torque (Nm?kg-1) ,230 (n = 15)

$230 (n = 24)

5.4¡0.2 (3.6-7.2) 7.3¡0.2 (6.0-8.4) 11.3¡0.6 (7.2-15.6)

6.0¡0.2 (4.8-7.2)* 8.3¡0.2 (6.0-10.8)* 13.4¡0.4 (9.6-18.0)*

-1

TM Velocity (km?h ) AT RCP PEAK HR (bpm) AT RCP PHR RPE AT RCP

________________ 122¡4 (94-145) 142¡3 (125-159) 174¡3 (136-195)

120¡3 (94-156) 145¡3 (116-178) 180¡3 (161-203) ________________

9.1¡0.3 (7.0-12.0) 11.9¡0.5 (9.0-15.0)

9.0¡0.2 (7.0-12.0) 12.0¡0.3 (9.0-15.0) ________________

O2 (mL?kg-1?min-1) AT RCP Peak

15.9¡0.7 (11.4-20.0) 20.6¡0.7 (15.4-23.8) 29.8¡1.2 (20.1-40.0)

O2 (% peak) AT RCP TM test duration (min) HA (MET.min.week-1) Peak isokinetic torque (Nm?kg-1)

54.2¡2.7 (41.0-79.0) 70.0¡2.9 (53.0-92.0) 9.0¡0.5 (4.0-13.0) 1667¡222 (714-3999) 203¡7 (150-229)

18.3¡0.4 (15.1-22.5)* 25.4¡0.7 (18.4-31.7)* 36.6¡0.9 (27.7-43.4)* ________________ 49.9¡1.2 (40.9-67.0) 69.6¡1.8 (52.0-87.0) 9.9¡0.6 (6.2-20.0) 1665¡193 (747-3839) 277¡7 (231-349)*

*p,0.05; values are means ¡ standard errors of mean; AT: anaerobic threshold; RCP: respiratory compensation point; VO2: oxygen intake; HR: heart rate; PHR: peak heart rate; RPE: rating of perceived exertion; HA: habitual activity.

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extensor torque of the patients (248ÂĄ8 Nm) was similar to previously reported values for healthy subjects in this age group (21-27). Findings during the progressive treadmill test are summarized in Table 2. Patients and control subjects reached similar peak heart rates, which were relatively high for their age, but the peak oxygen intake values were not outstanding in either group. There was no evidence that the AT, RCP or ( O2peak) was any lower in the patients than in the control subjects, irrespective of the patientsâ&#x20AC;&#x2122; CD4+ nadir. A similar analysis in terms of whether the patients had received HAART revealed no differences in this subgroup (data not shown). Classification of patients based on their current CD4+ counts (Table 3) again revealed no statistically significant differences, with the exception of peak isokinetic torque, which was approximately 20% less in those individuals with a count ,200 cells.mm-3. However, when patients were classified in terms of their peak isokinetic torque (Table 4), the AT, RCP and peak aerobic power were all significantly greater in those individuals with well-preserved muscular strength; stronger subjects were able to progress to a significantly greater treadmill velocity. Linear regression analysis revealed parallel trends among individual subjects (Figure 1); AT and RCP exhibited statistically positive associations with peak isokinetic torque.

& DISCUSSION Our data confirm our initial hypothesis: on average, the ( O2peak) of physically active individuals with HIV/AIDS does not differ from that of control subjects of similar age and with similar physical activity patterns. This is in agreement with previous reports, which have also suggested that after treatment, the cardio-respiratory fitness of physically active individuals with HIV/AIDS generally shows little deterioration (1-2). Nevertheless, our findings also underline that within this apparent normality of aerobic function, individual patients who have substantial muscle strength deficiency also exhibit substantial deficiency of aerobic performance, averaging around 20%, relative to equally active but stronger peers. Because our analysis was cross-sectional, it might be argued that the individuals who developed a low peak isokinetic torque had engaged in less habitual activity during their leisure time or were simply less motivated during exercise testing. The first of these explanations can be largely discounted because the reported activity levels of the two subgroups were equal. The small and statistically non-significant trend to a lower peak heart rate (Table 4) and some association between a poor aerobic performance and high depression scores, as measured by the profile of mood states questionnaire (data not shown), may signal somewhat poorer motivation in those who were depressed. Nevertheless, it seems of importance that lower muscle strength was also linked to a lower current CD4+ count (Table 2). Moreover, although differences in motivation might have influenced the peak oxygen intake, it is not as easy to explain the lower ATs of the weaker individuals (Table 4) simply in terms of differences in individual motivation. A direct effect of local muscle strength upon perfusion of the limbs and, thus, peak oxygen transport offers a more plausible explanation (14). Therefore, we

Figure 1 - Relationship between peak muscle torque (Nm?kg-1) and : VO2 at (A) the anaerobic threshold, (B) the respiratory compensation point and (C) the peak oxygen intake (mL?kg?-1min-1).

reported activity, both groups were quite active, undertaking the equivalent of 60 minutes or more of moderate activity (4 METS) per day. The average peak isokinetic

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conclude that an HIV/AIDS-induced reduction in muscle mass and the resultant loss of muscle strength appears to impair both anaerobic effort (by causing lactate to accumulate at a lower fraction of maximal oxygen intake) (15) and ( O2peak) (because of both earlier fatigue and the lower volume of muscle that is available for activation during treadmill testing) (14). There are a number of limitations to our data. Unfortunately, we were unable to obtain strength data for the control subjects, but the average values observed in the patients were not much lower than those reported in previous normal studies (22-27). It could also be objected that with greater opportunity to practice procedures, higher test scores might have been obtained on both the treadmill and the dynamometer tests; however, this in no way invalidates the differences we have observed between patients with high and low muscular strength values. It should also be emphasized that our patient sample was male, and the sample was deliberately selected in terms of a number of criteria for good health; the sample represented approximately one-sixth of patients attending the HIV/ AIDS clinic. It would be interesting to extend our observations by assessing the effects of muscle weakening upon aerobic function in an unselected group of HIV/AIDS patients, including women and men. The practical clinical implications of our findings point to the following: to ensure good aerobic power in patients with HIV/AIDS, rehabilitation should include a program of progressive muscle strengthening exercises, and such rehabilitation should be continued until the strength of the major muscle groups has been normalized. This proposal now needs to be tested with a group of HIV/AIDS patients with aerobic impairment secondary to poor muscle strength by evaluating how far the patients’ aerobic power can indeed be enhanced by a program that normalizes muscular function.

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& AUTHOR CONTRIBUTIONS

19.

Casseb J, Duarte AJ, DGreve JM and Raso V had full access to all of the data in the study and take responsibility for the integrity of the data and accuracy of the data analysis. DGreve JM, Raso V and Shephard RJ were responsible for the study concept and design and provided critical revision of the manuscript for important intellectual content. DGreve JM, Raso V and Silva PRS were responsible for data acquisition. Raso V was responsible for data analysis and interpretation and performed the statistical analysis. DGreve JM and Raso V drafted the manuscript. Duarte AJS was responsible for administrative, technical or material support and study supervision.

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CLINICAL SCIENCE

The ‘silence’ of silent brain infarctions may be related to chronic ischemic preconditioning and nonstrategic locations rather than to a small infarction size Chao Feng,I Xue Bai,I Yu Xu,II Ting Hua,II Xue-Yuan LiuI I Tongji University, School of Medicine, Shanghai Tenth People’s Hospital, Department of Neurology, Shanghai, China. Medicine, Shanghai Tenth People’s Hospital, Department of Radiology, Shanghai, China.

Tongji University, School of

OBJECTIVE: Silent brain infarctions are the silent cerebrovascular events that are distinguished from symptomatic lacunar infarctions by their ‘silence’; the origin of these infarctions is still unclear. This study analyzed the characteristics of silent and symptomatic lacunar infarctions and sought to explore the mechanism of this ‘silence’. METHODS: In total, 156 patients with only silent brain infarctions, 90 with only symptomatic lacunar infarctions, 160 with both silent and symptomatic lacunar infarctions, and 115 without any infarctions were recruited. Vascular risk factors, leukoaraiosis, and vascular assessment results were compared. The National Institutes of Health Stroke Scale scores were compared between patients with only symptomatic lacunar infarctions and patients with two types of infarctions. The locations of all of the infarctions were evaluated. The evolution of the two types of infarctions was retrospectively studied by comparing the infarcts on the magnetic resonance images of 63 patients obtained at different times. RESULTS: The main risk factors for silent brain infarctions were hypertension, age, and advanced leukoaraiosis; the main factors for symptomatic lacunar infarctions were hypertension, atrial fibrillation, and atherosclerosis of relevant arteries. The neurological deficits of patients with only symptomatic lacunar infarctions were more severe than those of patients with both types of infarctions. More silent brain infarctions were located in the corona radiata and basal ganglia; these locations were different from those of the symptomatic lacunar infarctions. The initial sizes of the symptomatic lacunar infarctions were larger than the silent brain infarctions, whereas the final sizes were almost equal between the two groups. CONCLUSIONS: Chronic ischemic preconditioning and nonstrategic locations may be the main reasons for the ‘silence’ of silent brain infarctions. KEYWORDS: Silent Brain Infarctions; Symptomatic Lacunar Infarctions; Small Vessel Disease; Leukoaraiosis. Feng C, Bai X, Xu Y, Hua T, Liu XY. The ‘silence’ of silent brain infarctions may be related to chronic ischemic preconditioning and nonstrategic locations rather than to a small infarction size. Clinics. 2013;68(3):365-369. Received for publication on September 18, 2012; First review completed on October 30, 2012; Accepted for publication on November 26, 2012 E-mail: superfengc@126.com Tel.: 86 2166302582

caused by the occlusion of small vessels and are usually regarded as SVDs (2). Except for the different signals on diffusion-weighted images (DWIs) during the acute phase of SLIs, the lack of stroke-like symptoms or ‘silence’ is the only prominent feature used to distinguish SBIs from SLIs (1,4). However, the ‘silence’ of SBIs is still confusing to researchers and clinicians. The relatively small sizes of SBIs are the only widely accepted explanation for this peculiar feature (1,5). However, SLIs that result in obvious symptoms can also be small. Several recent studies on the evolution of SLIs indicate that their final sizes are much smaller than those measured for DWIs after the onset of stroke (6-8). Thus, the difference between the sizes of SBIs and SLIs may require further evaluation. The size-related explanation for the ‘silence’ of SBIs is also worth investigating. Previous studies on lacunar infarcts mostly investigated the difference between SLI patients with or without SBIs

& INTRODUCTION Silent brain infarctions (SBIs) are very common in the elderly (1). SBIs are believed to be caused by the occlusion of small vessels and are therefore categorized as a small vessel disease (SVD); SVDs are related to different types of arteriole degeneration (2,3). SBIs are mainly identified by imaging tools (4) and are difficult to distinguish from old symptomatic lacunar infarctions (SLIs), which are also

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(5,9,10); few studies focused on the ‘silence’ of SBIs. Furthermore, no study has compared the evolution of SBIs and SLIs. In this study, four groups were designed according to the presence of SBIs and SLIs. Vertical and horizontal comparisons of the following characteristics were performed among groups: risk factors, locations, evolution of infarctions, leukoaraiosis, atherosclerosis (AS) of relevant vessels, and the NIH Stroke Scale (NIHSS) scores of patients with SLIs. Based on the comparisons, we determined the possible mechanisms responsible for the ‘silence’ of SBIs.

Review and criteria of the MR images All MR images were analyzed by two radiologists blinded to the clinical data. The discrepancies between the interpretations of the two readers regarding the presence of SBIs and SLIs and the leukoaraiosis scores were resolved by a visual consensus. According to the conclusion on the evolution of acute lacunar infarctions, we defined SLI as a ,25-mm-diameter focal lesion that is hyper-intense on a DWI among small penetrating arteries distributed in the thalamus, gangliocapsular regions, corona radiata, and brainstem (7). An SBI was defined as a .3-mm-diameter focal cavitated lesion that is hypo-intense on T1-weighted images, hyper-intense on T2-weighted images, hypo-intense with a hyper-intense boundary on FLAIR images, and without corresponding stroke-like symptoms or neurologic deficits (4). An old SLI was defined as a focal lesion with signals similar to an SBI on T1- and T2-weighted images, with signals similar to cavitations or leukoaraiosis on FLAIR, and with a corresponding history of stroke-like symptoms or neurologic deficits (7,8,11). Old SLIs were analyzed only when the corresponding MR images after the onset of stroke were accessible at our workstation. The diameters of acute SLIs were measured on DWIs, T1-weighted images, and FLAIR images. The diameters of SBIs and old SLIs were measured on T1-weighted images and FLAIR images. All diameters listed for statistical analysis were the average of values measured by the two readers. Leukoaraiosis was scored from 0 to 3 according to Fazekas’ scale (12). Grades 2 to 3 leukoaraiosis were regarded as advanced leukoaraiosis (ad-LA).

& MATERIALS AND METHODS General population of patients This study was based on patients diagnosed with acute lacunar infarction or patients confirmed to have no signs of stroke who consecutively visited the Department of Neurology of the 10th People’s Hospital in Shanghai from December 2011 to August 2012. The patients had neurologic deficits, nonspecific symptoms (such as headaches), and dizziness; alternatively, they had a fear of stroke because of the presence of risk factors. The patients were administered a medical evaluation protocol, including brain magnetic resonance imaging (MRI), electrocardiogram, and blood tests for blood glucose, lipids, and homocysteine. All patients also underwent a vascular assessment involving either computed tomography angiography (CTA) or magnetic resonance angiography (MRA). Patients diagnosed with acute lacunar infarction were evaluated using the NIHSS. All procedures were approved by the Institutional Review Board of our hospital, and written informed consent was obtained from every participant. Based on the presence of SBIs and SLIs on MRIs, 521 patients were enrolled and divided into four groups, namely, SBI, SLI, SBI&SLI (SS), and control groups. These groups consisted of the following patients: 156 patients with only SBIs, 90 patients with only new SLIs, 160 patients with both SBIs and new SLIs, and 115 patients without any identified infarcts. The following patients were excluded from these four groups: patients diagnosed with other types of stroke but not acute lacunar infarction, patients with a history of stroke, and patients with metallic implants. In addition to the MRI results of the four groups listed above, two sets of images were analyzed retrospectively to study the evolution of SBIs and SLIs: 1) previous MR images of the 32 patients in the SBI and SS groups who underwent MRI more than once and 2) previous MR images obtained after the onset of stroke for the other 31 patients with lacunes identified as old SLIs.

Vascular risk factors and vascular assessment Data related to the following factors were collected: hypertension, diabetes mellitus (DM), dyslipidemia, ischemic heart disease (IHD), atrial fibrillation (AF), hyperhomocysteinemia (HHCY), smoking, and CTA or MRA results. Atherosclerotic plaques and stenosis of infarction-related arteries (e.g., middle cerebral and internal carotid arteries for infarctions in the gangliocapsular regions and basilar and vertebral arteries for infarctions in the brainstem) were considered to be possible atherosclerotic sources. AF diagnosed before or during hospitalization was regarded as a possible cardiogenic mechanism.

Statistical analyses All data were analyzed using SPSS (version 18.0, SPSS, Inc., Chicago, IL, USA). ANOVA and chi-squared tests were performed to compare the baseline characteristics and locations. The Student’s t-test was used to compare infarction sizes and NIHSS scores. Multiple logistic regression models were constructed to determine the risk factors for SBIs and SLIs. p,0.05 was considered to indicate a significant difference.

MRI protocol The MR images in this study were primarily obtained using a 1.5 T scanner (Philips, Eindhoven, Noord-Brabant, Netherlands) and a 3.0 T scanner (Siemens, Erlangen, Bavaria, Germany). The MRI protocol consisted of a T1weighted image [repetition time/echo time (TR/TE) = 101/ 1.92 for the 1.5 T scanner and 2000/9 for the 3.0 T scanner], fluid attenuated inversion recovery images (FLAIR) (TR/ TE = 6000/110 for the 1.5 T scanner and 8500/94 for the 3.0 T scanner), DWI (TR/TE = 3393/86 for the 1.5 T scanner and 6000/94 for the 3.0 T scanner) in the axial plane, and a T2weighted image (TR/TE = 1940/120 for the 1.5 T scanner and 4540/96 for the 3.0 T scanner) in the sagittal plane with 16 layers.

& RESULTS Baseline characteristics of the four groups Table 1 lists the baseline characteristics of age, prevalence of different vascular risk factors, AS of relevant arteries, adLA, and the proportions of patients who underwent MRI on the 1.5 T scanner and CTA as the method of vascular assessment. Compared with the control group, the patients in the other three groups were relatively older and had

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Chronic ischemia and the silence of SBIs Feng C et al.

Table 1 - Baseline characteristics of the different groups.

Female (%) Age (years) Hypertension (%) Diabetes (%) Dyslipidemia (%) Hyperhomocysteinemia (%) Ischemic heart disease (%) Atrial fibrillation (%) Current smoking (%) Advanced leukoaraiosis (%) Atherosclerosis (%) CTA (%) 1.5 T (%) a

p,0.05,

SBI (n = 156)

SLI (n = 90)

SS (n = 160)

Control (n = 115)

50.6 68.9¡7.0b 88.5b 19.2 64.1 69.2 b 14.1 a 7.7 25.0 53.8 b 14.7 73.7 72.4

51.1 63.7¡7.3b 80.0b 30.0 b 78.9 b 45.6 a 16.7 a 12.2 a 30.0 a 25.5 b 47.8 b 77.8 74.4

48.1 70.7¡7.0b 86.9b 21.3a 61.3 65.6 b 13.8 a 8.1 35.0 b 60.1 b 30.6 b 69.4 76.3

55.7 58.2¡8.2 31.3 11.3 54.8 30.4 5.2 3.5 15.7 7.8 5.2 79.1 73.9

p,0.01 vs. the control group.

higher prevalence rates of hypertension, HHCY, IHD, and ad-LA. The prevalence rates of DM, dyslipidemia, AF, and AS were also higher in the SLI group than in the control group. Multiple comparisons among the SBI, SLI, and SS groups were also performed, and positive significant results (p,0.05) are shown in Table 2. Compared with the SLI group, patients in the SBI and SS groups were relatively older with higher prevalence rates of HHCY and ad-LA and lower prevalence rates of dyslipidemia and AS. No difference was observed for the prevalence of hypertension among the three groups. However, the durations of hypertension for hypertensive patients in the SBI and SS groups were significantly longer than for those in the SLI group (both p,0.01). The NIHSS scores were higher in the SLI group than in the SS group (p = 0.017).

Size and evolution of infarcts The diameters of the SBIs and SLIs were compared among groups and different time points. The average diameters of the SBIs and SLIs were determined based on all infarcts in the SBI, SLI, and SS groups. The comparisons among different time points were performed based on the MR images of 63 patients, i.e., 32 patients with 67 SBIs and 31 patients with 31 old SLIs obtained at different times. The results indicated that the sizes of the SBIs changed slightly after an average period of 13.4 months, whereas the SLIs shrank considerably after 13.0 months to final sizes nearly equal to those of the SBIs (p = 0.24). The details of this analysis are shown in Table 5.

& DISCUSSION This study revealed that the risk factors for the SBI and SLI groups differed to some extent and that the risk factors of the SS group were similar to those of the SBI group. The main risk factors for SBIs were hypertension, age, and HHCY; the main risk factors for SLIs were hypertension, DM, dyslipidemia, IHD, and AF. Patients with SBIs had long-term hypertension with a high prevalence of ad-LA, whereas the prevalence of AS in relevant arteries was much higher in the SLI and SS groups. Based on these different risk factors and features, the specific mechanisms of arterial occlusion can be speculated to differ between SBIs and SLIs. The classic mechanisms of SVDs, such as arteriolosclerosis caused by long-term hypertension, may be the main pathogenesis of SBIs. However, AS and embolisms are more important for SLIs (5,10,13,14). The specific hypertension and leukoaraiosis-related pathogenesis of SBIs differs from the pathogenesis of SLIs and may be the main reason for the ‘silence’ of SBIs. Leukoaraiosis represents incomplete ischemia caused by the stenosis of small vessels. This condition progresses with age, and long-term hypertension that causes chronic arteriolosclerosis and arteriolar stenosis further aggravates the ischemia (15,16). Furthermore, SBIs are commonly found to coexist with leukoaraiosis (17). Thus, incomplete and chronic ischemia may also be the basis for SBIs. Prior to the appearance of SBIs, the particular region supplied by the arteriole with chronic arteriolosclerosis may be irreversibly and covertly impaired because of ischemia and may gradually lose its function. SBIs form after the threshold of ischemic stress is broken by the progress of ischemia. Thus, the ‘silence’ of SBIs can be explained by chronic ischemic

Risk factors of SBIs and SLIs vs. the control group Multiple regression models were constructed to identify the risk factors for different infarcts. The control group served as a reference, and the SS group was excluded. Sex, age, hypertension prevalence, DM, dyslipidemia, HHCY, IHD, AF, smoking, AS of relevant arteries, and ad-LA were added to the two models. The results are listed in Table 3.

Locations of SBIs and SLIs The locations of the SBIs and SLIs in the SBI, SLI, and SS groups were subsequently investigated, and the results are listed in Table 4. Compared with SLIs, more SBIs were located in the corona radiata (p,0.01) and fewer were located in the internal capsule and brainstem (p,0.01 for both locations).

Table 2 - Positive results of the multiple comparisons. SBI group vs. SLI group Ageb Diabetes Dyslipidemia Hyperhomocysteinemia b Advanced leukoaraiosisb Atherosclerosisb b

SBI group vs. SS group

SLI group vs. SS group

Age Smoking Atherosclerosisb

Ageb Dyslipidemiab Hyperhomocysteinemia b Advanced leukoaraiosisb Atherosclerosisb NIH Stroke Scale scores

p,0.01.

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Table 3 - Risk factors for SBI and SLI vs. the control group. SBI vs. control

Sex Age (years) Hypertension Diabetes Dyslipidemia Hyperhomocysteinemia Ischemic heart disease Atrial fibrillation Smoking Advanced leukoaraiosis Atherosclerosis

SLI vs. control

OR (95% CI)

p-value

OR (95% CI)

p-value

0.723 (0.280-1.871) 1.129 (1.024-1.245) 21.708 (7.668-61.455) 0.959 (0.238-3.862) 1.869 (0.736-4.743) 8.331 (3.138-22.118) 2.139 (0.448-10.222) 2.509 (0.334-18.858) 4.605 (1.154-18.380) 4.357 (1.978-9.598) 0.473 (0.101-2.220)

0.504 0.015 0.000 0.953 0.188 0.000 0.341 0.372 0.031 0.000 0.343

1.019 (0.369-2.813) 1.072 (0.977-1.177) 6.320 (2.679-14.906) 3.654 (1.227-10.883) 3.535 (1.422-8.792) 1.699 (0.686-4.205) 4.163 (1.105-15.691) 7.262 (1.094-48.218) 4.095 (1.258-13.328) 1.179 (0.522-2.664) 13.951 (4.628-42.057)

0.970 0.142 0.000 0.020 0.007 0.252 0.035 0.040 0.019 0.692 0.000

SBIs and instead may be similar to the pattern of large infarctions caused by acute and complete arterial occlusion due to AS or embolism. The benefit of thrombolytic therapy (29) for SLI patients could also support this conclusion. In addition, most patients in the SLI group did not have leukoaraiosis or long-term hypertension. With a different pattern of arterial occlusion due to a different pathogenesis and without the pathological basis of chronic ischemia and neuroprotection of ischemic tolerance, these SLIs resulted in acute symptoms. However, it is difficult to explain the overt symptoms of those SLIs without identified AS or with coexisting SBIs and leukoaraiosis. It is still possible that some of the SLIs might be related to acute and complete occlusion caused by undetectable AS of perforating arteries (27). However, this hypothesis cannot be proven without pathological evidence. Meanwhile, it is not reasonable to attribute all of those SLIs to AS. The different locations can also be a possible reason for the ‘silence’ and ‘overtness’ of the infarcts. This study showed that more SBIs were identified in the corona radiata and basal ganglia; these locations were different from the distribution of SLIs. Compared with the internal capsule and brain stem, where more important conducting bundles and nuclei are concentrated, the corona radiata and basal ganglia are relatively nonstrategic areas, where small infarctions may cause only mild or even no symptoms. This study also confirmed that the average final size of the SLIs was nearly equal to that of the SBIs. Therefore, the amount of brain tissue destroyed by the SLIs may be equal to the amount for the SBIs; consequently, the size-related explanation for the ‘silence’ of SBIs might not be reasonable. The overestimation of the infarction size using DWIs may be caused by acute inflammation and edema, which exacerbate neurologic symptoms (30,31). These pathological changes were also reported to be related to acute and complete

preconditioning and long-term ischemic tolerance. The comparison of the results between the SLI and SS groups in this study supports this explanation. The SS group consisted of significantly older patients than the SLI group, with longer hypertension durations but lower NIHSS scores. The lower NIHSS scores can be attributed to the neuroprotective effect of SBIs similar to the effects of previous transient ischemic attacks (TIA) (18-20). For both TIA and SBIs, ischemic preconditioning and ischemic tolerance may be the most reasonable explanations for this effect (21-23). Another possible mechanism for the ‘silence’ based on chronic ischemia is that the cavity of an SBI may gradually form within several days or even months with the progression of ischemia similar to the chronic progression of leukoaraiosis (15) (i.e., no stroke-onset and neurologic symptoms). In fact, some SBIs are difficult to distinguish from focal leukoaraiosis. In our study on the evolution of infarctions, we observed that several SBIs tended to enlarge over time. However, this trend was not obvious and cannot be supported without frequent follow-up MRIs. However, this study revealed that AS of relevant arteries was a strong risk factor for SLIs. In fact, Fisher and Caplan’s pathological studies in the last century demonstrated that AS in perforating and parent arteries was an important cause for SLIs (14,24). Some recent imaging studies also verified the association between SLIs and AS in parent and other relevant arteries (25-27). In addition, AS in parent arteries without obvious lumen stenosis and in perforating arteries could not be detected by most imaging tools; therefore, it might be underestimated as a common cause for SLIs (27). Our results verified the association between SLIs and AS. However, IHD and AF were also identified as risk factors for single SLIs in this study. It has been suggested that single SLIs could also be caused by a cardiac embolism (10,28). Therefore, the pathogenesis of some SLIs, particularly those without coexisting SBIs, might be different from the chronic and incomplete ischemic pattern of

Table 5 - Size and evolution of the infarcts.

Table 4 - Locations of the SBIs and SLIs.

Basal ganglia Thalamus Internal capsule Corona radiata Brainstem a

p,0.05,

SBI (n = 702)

SLI (n = 250)

176 (25.07%) 93 (13.25%) 51 (7.26%) 308 (43.87%) 74 (10.54%)

48 (19.2%) 25 (10.00%) 60 (24.00%)b 53 (21.20%)b 64 (25.60%)b

Average diameter on T1/FLAIR (mm) Average diameter on a DWI (mm) Previous diameter on T1/FLAIR (mm)c Previous diameter on a DWI (mm)c Duration of two time points of MRI (months)c Follow-up diameter on T1/FLAIR (mm)c a

p,0.01.

368

p,0.05, points.

p,0.01,

SBI

SLI

7.9¡2.8 – 8.0¡2.3 – 13.4¡3.5

13.9¡4.3b 14.2¡5.0 14.0¡3.9b 14.3¡4.5 13.0¡3.3

8.1¡2.3

8.7¡2.7

based on 63 patients with MR images at two time

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ischemia (31) and were not obvious in the pathogenesis of SBIs observed in the current study. Therefore, this difference between the evolution of SBIs and SLIs can also be attributed to the different patterns of ischemia and occlusion emphasized above. In conclusion, this study showed that the ‘silence’ of SBIs can be attributed to chronic ischemic preconditioning and long-term ischemic tolerance but not to the infarction size. The nonstrategic locations of these infarcts can also partly account for the ‘silence’. The strengths of this study included the consecutive study design with a relatively large sample size, multiple groups, and comparisons of different patterns. However, this study also had limitations. First, the study was not based on the general population, and the results may be influenced by the limited representativeness of the sample selected from hospitalized patients. Second, the study was based on clinical data and neuroimaging, and the results of the pathogenesis of SBIs and SLIs were speculated without confirmation by pathological examination. We cannot expound further on the pathogenesis, and more comprehensive studies are required.

& ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (No. 81000492, No. 30971029, and No. 81171163).

& AUTHOR CONTRIBUTIONS Feng C led the study design, data acquisition, data analysis, and manuscript writing. Bai X, Xu Y, and Hua T assisted with the data acquisition, data analysis, and manuscript writing. Liu XY assisted with the study design, data analysis, and manuscript review.

& REFERENCES 1. Vermeer SE, Longstreth WT, Jr., Koudstaal PJ. Silent brain infarcts: a systematic review. Lancet Neurol. 2007;6(7):611-9, http://dx.doi.org/10. 1016/S1474-4422(07)70170-9. 2. Moran C, Phan TG, Srikanth VK. Cerebral small vessel disease: a review of clinical, radiological, and histopathological phenotypes. Int J Stroke. 2012;7(1):36-46. 3. Pantoni L. Cerebral small vessel disease: from pathogenesis and clinical characteristics to therapeutic challenges. Lancet Neurol. 2010;9(7):689701, http://dx.doi.org/10.1016/S1474-4422(10)70104-6. 4. Zhu YC, Dufouil C, Tzourio C, Chabriat H. Silent Brain Infarcts: A Review of MRI Diagnostic Criteria. Stroke. 2011;42(4):1140-5, http://dx. doi.org/10.1161/STROKEAHA.110.600114. 5. Boiten J, Lodder J, Kessels F. Two clinically distinct lacunar infarct entities? A hypothesis. Stroke. 1993;24(5):652-6, http://dx.doi.org/10. 1161/01.STR.24.5.652. 6. Benavente O, Tonarelli C, Pergola S. DWI overestimates the ultimate size of lacunar infarcts: analysis from SPS3. Stroke. 2007;38:487. 7. Koch S, McClendon MS, Bhatia R. Imaging evolution of acute lacunar infarction: leukoariosis or lacune? Neurology. 2011;77(11):1091-5, http:// dx.doi.org/10.1212/WNL.0b013e31822e1470. 8. Moreau F, Patel S, Lauzon ML, McCreary CR, Goyal M, Frayne R, et al. Cavitation After Acute Symptomatic Lacunar Stroke Depends on Time, Location, and MRI Sequence. Stroke. 2012;43(7):1837-42, http://dx.doi. org/10.1161/STROKEAHA.111.647859. 9. Arauz A, Murillo L, Cantu C, Barinagarrementeria F, Higuera J. Prospective study of single and multiple lacunar infarcts using magnetic resonance imaging: risk factors, recurrence, and outcome in 175 consecutive cases. Stroke. 2003;34(10):2453-8, http://dx.doi.org/10. 1161/01.STR.0000090351.41662.91.

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Conjoined twins – twenty years’ experience at a reference center in Brazil Ana Cristina Aoun Tannuri, Julio Americo Pereira Batatinha, Manoel Carlos Prieto Velhote, Uenis Tannuri Faculdade de Medicina da Universidade de Sa˜o Paulo, Pediatric Surgery Division, Pediatric Liver Transplantation Unit and Laboratory of Research in Pediatric Surgery (LIM 30), Sa˜o Paulo/SP, Brazil.

OBJECTIVE: This study reports on the experience of one hospital regarding the surgical aspects, anatomic investigation and outcomes of the management of 21 conjoined twin pairs over the past 20 years. METHODS: All cases of conjoined twins who were treated during this period were reviewed. A careful imaging evaluation was performed to detail the abdominal anatomy (particularly the liver), inferior vena cava, spleen and pancreas, either to identify the number of organs or to evaluate the degree of organ sharing. RESULTS: There were eight sets of ischiopagus twins, seven sets of thoracopagus twins, three sets of omphalopagus twins, two sets of thoraco-omphalo-ischiopagus twins and one set of craniopagus twins. Nine pairs of conjoined twins could not be separated due to the complexity of the organs (mainly the liver and heart) that were shared by both twins; these pairs included one set of ischiopagus twins, six sets of thoracopagus twins and one set of thoraco-omphalo-ischiopagus twins. Twelve sets were separated, including seven sets of ischiopagus twins, three sets of omphalopagus twins, one set of thoracopagus twins and one set of craniopagus conjoined twins. The abdominal wall was closed in the majority of patients with the use of mesh instead of the earlier method of using tissue expanders. The surgical survival rate was 66.7%, and one pair of twins who did not undergo separation is currently alive. CONCLUSION: A detailed anatomic study of the twins and surgical planning must precede separation. A wellprepared pediatric surgery team is sufficient to surgically manage conjoined twins. KEYWORDS: Conjoined Twins; Surgical Separation; Ischiopagus Tripus Conjoined Twins; Omphalopagus Conjoined Twins; Thoracopagus Conjoined Twins. Tannuri AC, Batatinha JA, Velhote MC, Tannuri U. Conjoined twins – twenty years’ experience at a reference center in Brazil. Clinics. 2013;68(3):371-377. Received for publication on October 6, 2012; First review completed on October 26, 2012; Accepted for publication on November 28, 2012 E-mail: uenist@usp.br Tel.: 55 11 3681-2943

individuals are joined by part of their anatomy and share one or more organs. The incidence of conjoined twins ranges from 1:50,000 to 1:100,000 live births. This number could be higher, but most of these pregnancies result in miscarriages and still births; only 18% of all conjoined infants survive (2), and approximately 35% of live births die within the first 24 hours, and only 18% of all conjoined twins survive longer than 24 hours. In Brazil, where abortion is not legal, we believe that the incidence of conjoined twins is likely higher than in most developed countries. Conjoined twins are classified based on the terminology proposed by Spencer and colleagues (3). Based on this terminology, we use the most prominent site of union plus the suffix ‘‘pagus,’’ which is a Greek word meaning ‘‘that which is fixed.’’ Spencer et al. also divided the twins into three major groups: twins with a ventral union, twins with a dorsal union and twins with a lateral union. The first major group includes four types: cephalopagus (head), thoracopagus (chest), omphalopagus (umbilicus) and ischiopagus (hip). The dorsal union includes three types: pygopagus (sacrum), rachipagus (spine) and craniopagus (cranium). The last major group includes just one type of twins that is referred to as parapagus (side) twins.

& INTRODUCTION Conjoined twins have fascinated mankind throughout the centuries because of the rarity of this type of birth; however, conjoined twins have always been a challenge for physicians. Eng and Chang Bunker are likely the most famous pair of conjoined twins. These twins were born in Siam in 1811, taken to the United States by a circus company and exhibited as the curious ‘‘Siamese Twins’’, thus providing the origin of the colloquial term (1). The brothers lived for 63 years without considering separation, and they married sisters and fathered 21 children. As a rare outcome of a monoamniotic and monochorionic gestation, conjoined twins occur when two identical

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The surgical separation of conjoined twins is now the principal aim of all medical teams who treat this uncommon condition. However, separation presents both surgical and anesthetic challenges. In addition, this surgery is sometimes not possible because the anomalies are rare and difficult to manage, even for experienced surgeons. If we share our experiences and learn from others, we can enhance our knowledge and skills for treating conjoined twins. In Brazil, when a diagnosis of conjoined twinning is made, the pregnant mother is usually referred to a tertiary hospital that specializes in obstetric and perinatal care. Although there are several reports in the medical literature about conjoined twinning, only one Brazilian study has been published on this issue; that study was performed in a tertiary perinatology referral university center over a period of 25 years (4). The authors reported the occurrence of 14 pairs of conjoined twins and the successful separation of only one pair of omphalopagus twins. The present study aims to report the experience of one Brazilian hospital over a period of 20 years, focusing on surgical aspects, anatomic investigations and outcomes.

Table 1 - Conjoined twin type and anatomical evaluation. Type Ischiopagus

Thoracopagus

Omphalopagus

& PATIENTS AND METHODS

Craniopagus

This is a retrospective review of all cases of conjoined twins treated between January 1992 and July 2012 at the Pediatric Surgery Division and Liver Transplantation Unit of the Child Institute of the Faculdade de Medicina da Universidade de Sa˜o Paulo. The study was approved by the ethics committee of the institution, and we obtained parental approval to publish the children’s pictures. Patient information was obtained by reviewing medical records and the intranet database of the hospital. Perinatal data included prenatal ultrasound diagnosis, gender, birth weight and the anatomy of the twins. The patients were classified based on the most prominent site of fusion, based on the embryological classification proposed by Spencer (3). Data regarding stillbirths, miscarriages and conjoined twin gestations were excluded. Cases of fetus-in-fetus, considered by some authors to be‘‘incomplete conjoined twins,’’ were also excluded from this study. A careful imaging evaluation was performed for all conjoined twins, as described in Table 1. Angiographic studies of the liver circulation were performed when the computed angiotomography or magnetic resonance imaging studies did not provide consistent information about these important anatomic details. After a careful imaging evaluation, the possibility of separation surgery was determined, and the twins were divided into two groups as follows:

Evaluation Ultrasonography of the abdomen, skull and pelvis Echocardiography Radiography Doppler ultrasound Contrast meal and enema Computed tomography Computed angiotomography Magnetic resonance imaging Micturating uretrocystography Endoscopy Cavography Hepatic venography Ultrasonography of the abdomen and skull Echocardiography Radiography Fetal echocardiography Computed angiotomography Doppler ultrasound of the abdomen Magnetic resonance imaging Ultrasonography of the abdomen and skull Echocardiography Radiography Doppler ultrasound of the abdomen Computed tomography of the brain and skull Computed angiotomography of the brain Complete ultrasonography examination of the abdomen Echocardiography

& RESULTS Twenty-one sets of conjoined twins were analyzed. Most of the pairs were female, with 13 female sets and 8 male sets. The mean birth weight of the twin pairs was 2,921.17¡1,078.05 g. A prenatal diagnosis was made in 19 pregnancies (90.5%). The mean age at separation surgery, excluding the two conjoined twins who underwent emergency separations during the newborn period, was 9 mo 24 d¡4 mo 25 d. The mean weight of the twin sets at the time of the separation surgery, excluding the emergency separations, was 9,656.08¡4,594.02 g. The 21 sets included eight sets of ischiopagus twins, seven sets of thoracopagus twins, three sets of omphalopagus twins, two sets of thoraco-omphalo-ischiopagus twins and one set of craniopagus twins. The data collected for the groups are described above.

Non-operative management Nine pairs of twins were not candidates for separation based on imaging evaluations. The separation procedure was not possible due to the complexity of organs that were shared by both twins, mainly the liver and heart. The decision was made after consulting with the parents and the Ethical Committee of the Institution.

1. Conjoined twins who were not candidates for surgical separation for the reasons described above. 2. Conjoined twins who underwent surgical separation. In this group, the following data were collected and analyzed: the age and weight at the time of the separation surgery, the length of surgery, the duration of anesthesia during the separation surgery, a detailed description of the separation surgery, the type of abdominal wall closure, postoperative complications and death.

Ischiopagus Only one set of ischiopagus twins was not separated due to severe perinatal asphyxia that evolved to death within one day of life. This set had a diaphragmatic hernia with the stomach and spleen occupying one twin’s thorax and the stomach and hepatic lobe occupying the other twin’s thorax; this placement may have caused a pneumothorax in both twins. The twins shared one liver and pelvis; in addition,

Numerical data are presented as the mean¡standard deviation. Statistical analyses were performed using the Student’s t-test.

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Figure 1A - Ischiopagus tripus twins. Note that these twins have two normal legs and a third abnormal leg (patient 14). Figure 1B Newborn thoracopagus conjoined twins who shared a heart, liver and small intestine (patient 1).

with the anesthesiologists and rehearsed. The twins were comfortably positioned on the surgical table, and the procedure was started. Four anesthesiologists were required (two for each twin). Following endotracheal intubation, each twin underwent central vein and radial arterial cannulation for complete monitoring during the operation. A Foley bladder catheter was also inserted. The separation procedure was always performed by a single surgical team. After the separation, the second twin was moved with the corresponding anesthetic equipment and a second team of surgeons to another surgical room for the final reconstruction procedures. The reconstruction of the first twin was completed by the surgical team that performed the separation. After the separation procedure, all of the infants were transferred to the intensive care unit for strict monitoring. Among the 21 sets of conjoined twins, 12 underwent separation surgery (seven ischiopagus, three omphalopagus, one thoracopagus and one craniopagus). The mean anesthesia time for the separation surgery was 8 h 45 min¡4 h 49 min, and the mean surgery time was 6 h 52 min¡4 h 10 min.

they had three legs (classified as ‘‘ischiopagus tripus’’) and severe cardiovascular defects. The other seven sets of ischiopagus twins were separated (Figure 1A).

Thoracopagus Most sets of thoracopagus twins were not separated (6 of 7) because of complex cardiac anomalies, including two hearts sharing a ventricular wall and one shared heart containing four fused atria and two ventricles. The liver was also shared in all sets. Three sets also presented with duodenal sharing. Five of six sets died during the neonatal period (Figure 1B), and the remaining set died within four months.

Thoraco-omphalo-ischiopagus Two sets of complex thoraco-omphalo-ischiopagus twins did not undergo separation. One of these sets of twins died three days after birth due to serious cardiac defects. The second set of thoraco-omphalo-ischiopagus twins presented with four arms, two legs, one bladder, one pelvis, fused small intestines from the terminal portion to the anus and one set of female genitalia. Therefore, the two infants had individual stomachs, duodenums, jejunums and most of the ilea. The twins had horseshoe kidneys and a bicornuate uterus. The livers were fused and drained to the inferior vena cava of just one infant. Furthermore, the portal veins were crossed. For this case, the pediatric surgery group considered separation. However, in addition to liver sharing, one of the infants had complex cardiac anomalies (aorta and pulmonary artery emerging from the right ventricle and aortic coarctation) that led the parents to refuse surgical separation. This decision was supported by the ethical committee of our institution, as previously described, and the ethical committee of the Regional Medical Council of Sa˜o Paulo. At the time of publication, these twins were still alive and growing.

Ischiopagus Six ischiopagus tripus and one ischiopagus tetrapus (with four normal legs) twin pairs underwent separation. The mean anesthesia time was 11 h 35 min¡3 h 57 min, and the mean surgery time was 8 h 29 min¡3 h 09 min. The separation procedure began by making a large longitudinal incision across the anterior abdominal wall. The fused livers were separated along the anterior midline, after verifying that each liver had its own hilum and hepatic veins. Each infant had a normal stomach, duodenum, gallbladder, pancreas and spleen. All sets had shared intestines. In twins with a single anus, a colostomy or ileostomy was performed in one infant, depending on whether the small or large intestine was shared, and an intestinal anastomosis was performed on the second infant who kept the anus. In twins with one anus per twin, the intestines were separated according to the origin of

Operative management When separation was pursued after exhaustive imaging examinations, the technical aspects were always discussed

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intestinal sharing irrigation. A colostomy or ileostomy was performed when the anus was considered nonviable. For the urinary tract, crossed ureters were a common finding (5 of 6 sets); in these cases the ureter of one twin implanted in the bladder of the other twin. In these cases, the ureters were divided close to the bladder of the other twin and then reimplanted in the bladder of the corresponding twin. The separation was completed by adequate and anatomical division of internal and external genital organs and the bones of the pelvis using appropriate orthopedic instruments. The reconstruction phase for each separated twin was performed by a reconstruction of the digestive system with intestinal anastomoses, followed by reimplantation of the divided ureters. The last phase of reconstruction was the closure of the abdominal wall. For all cases of ischiopagus tripus twins, we preferred to use the third abnormal leg for soft-tissue coverage.The bones were excised after careful dissection, and a large flap containing skin and skeletal muscles was obtained for complete closure of the abdominal cavity of one of the twins. The abdominal wall of the other infant was closed with mesh. In the first two pairs of conjoined twins, tissue expanders were used prior to the separation procedure; however, no advantage was noted because mesh was still required to close the abdomen and to cover the viscera. In one of these twin pairs, the abdominal skin became necrotic one week after the separation procedure, most likely due to ischemia caused by the previous placement of the tissue expander. The necrotic skin was excised, and the child completely recovered due to the formation of granulation tissue and wound healing by secondary intention (Figure 2). Based on the experience of these two cases, we abandoned the use of tissue expanders in subsequent cases. However, even in the infant for whom the third leg was used, mesh was needed to complete the abdominal wall closure. Of the seven sets of twins who underwent separation, one set developed sepsis and did not survive. These infants had serious complications during the preparation phase, including pulmonary infection and respiratory insufficiency. In addition, both infants had serious renal dysplasia and renal insufficiency. Despite these complications, the separation procedure was performed after obtaining consent from the family. Among the 12 infants who survived, 10 developed a wound infection (83.3%) or a urinary tract infection. Two patients developed late enteric fistulas due to the exposure of the intestine, despite the presence of the mesh. These infants underwent another operation during which the fistulas were successfully closed and a new mesh was inserted. One infant developed evisceration and needed a second operation to close the abdomen and place a new mesh. One twin developed late sepsis and died.

Figure 2A - Ischiopagustripus twins. Note the two normal legs and a third abnormal leg (patient 10). Two tissue expanders were used. Figure B - Twins after separation. Note the complete cicatrization of the abdominal wall.

intestines were separate. During the investigation period, one infant developed a pulmonary hemorrhage and died, leading to an emergency separation. The live infant survived the separation procedure and died of sepsis after 11 months.

Omphalopagus The omphalopagus twins required a shorter duration of anesthesia (mean time of 5 h 12 minยก2 h 3 min) and a shorter duration of surgery (mean time of 3 h 13 minยก1 h 27 min compared with the ischiopagus twins (p = 0.02 and p = 0.005 for anesthesia and surgery time, respectively). Although all sets of twins had a shared liver, none had a complex or shared biliary tract. There were two independent hepatic circulations, and each twin had an inferior vena cava. In addition, we did not encounter any congenital heart defects, and separation was possible in all sets. In all patients, the abdominal wall was closed with mesh, without the use of previously placed tissue expanders. One pair of twins was separated at 11 months of life and had an omphalocele that spontaneously epithelialized; complete skin coverage was achieved without difficulty (Figure 3). In one pair of twins, one of the twins died within one hour of birth. An emergency separation procedure was performed, and the live infant survived the separation

Thoracopagus Only one set of thoracopagus twins underwent separation surgery. This set had a single pericardial sac, and a small part of the anterior ventricular wall was shared between both hearts. However, the two hearts also had serious anomalies, interventricular communication in one heart and a hypoplastic right ventricle in the other heart. The thoracic cavities were shared from the nipple level to the inferior abdomen. These twins shared one liver with two hila, two gallbladders, two stomachs and two duodenums. The

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Additionally, no significant genetic, environmental or demographic associated factors were detected (5). Although the literature advises the use of a multidisciplinary approach for surgical separation to improve survival rates (2,6,7), the separations in our institution were performed by a team composed only of trained pediatric surgeons. Despite this difference, our results did not differ significantly from those of other studies in terms of the final outcomes of the patients and mortality rates. The problems encountered in our series and the lessons learned from our experience during the treatment of these 21 conjoined twins enrich our knowledge regarding surgery in complex pediatric patients. The first difficulty encountered was the anesthesia required for imaging investigations during the pre-separation phase. These imaging investigations included interventional radiological investigations and angiotomographic examinations. These procedures provided invaluable insight into potential or actual problems that may arise during the separation, such as the difficulties encountered in the ischiopagus tripus twins who died one week after surgery (patient 15). As with every surgery, surgical separation of conjoined twins carries its own risks, which may be avoided by reducing the surgery and anesthesia times. Therefore, a meticulous investigation of the twins’ anatomy is as crucial as the improvement of appropriate surgical techniques by training and experience. However, we also learned that, despite carefully studying the twins’ anatomy, unexpected anatomical variations are frequently identified during the surgery; the surgical team must be prepared for these variations. Therefore, the separation must be performed by a team of trained general pediatric surgeons. In our experience, the inclusion of several specialties (orthopedic, plastic, urologic and cardiovascular surgeons) during the separation procedure was often confusing and did not lead to better results. In only one case was a team of neurosurgeons involved because the surgery required neurosurgical expertise that we did not have. There are various controversies regarding the ideal age at which the separation procedure should be performed. Spitz et al. (8) preferred to operate at approximately three months of age, which allows time for detailed investigations to be conducted and enables separation to take place when the body wall can still rapidly expandto close substantial defects. However, a high incidence of postoperative wound infection can occur, and separation is very harmful to the body’s functional reserves. Therefore, we preferred to operate at approximately 10 months of age, despite some psychosocial issues that may occur during the waiting period. Consequently, the mean age of our patients at the time of surgery was 10 months and 9 days. Skin closure always presents a challenge and should be carefully considered before the separation procedure has begun. Many surgeons tend to use tissue expanders (1,9-11) and sometimes mesh (8,11). We opted for mesh instead of tissue expanders because of our personal experience, and data from the literature show that tissue expanders lead to complications in as many as 57.2% of twins in whom they are used (1). These complications include insufficient skin expansion, tissue expander infection, skin necrosis over the expander, exposure of the device and seroma formation. These complications require additional operations and general anesthesia. Moreover, the surgery to place the expanders represents a risk itself and involves unnecessary

Figure 3 - Omphalopagus twins (patient 18). Note the spontaneously epithelialized omphalocele.

procedure. After five days, she presented with a gastric rupture that was repaired with gastric suture. This child recovered very well.

Craniopagus There was one pair of craniopagus twins with serious associated defects. One of the twins had a skull and brain with normal volumes, anorectal agenesis and a recto-vesical fistula. The other twin presented with microcephaly and sirenomelia (‘‘mermaid syndrome’’). Both twins underwent an emergency colostomy during the neonatal period and were referred to us when they were 10 months old. Tomographic and angiotomographic studies of the brain revealed that complete separation would be impossible, and the twin with microcephaly and sirenomelia would have to be sacrificed during separation. After obtaining consent from the family, the separation procedure was performed by a team of neurosurgeons. The twin who survived underwent surgery for his anorectal agenesis six months after the separation. The colostomy was finally closed after two months and the infant recovered very well. Regarding the mortality rate of our series, among the 42 infants (21 pairs of twins), 24 died and 18 were alive at the time this manuscript was written. Because one pair of twins is alive and did not undergo separation and 16 children survived the separation procedure, we conclude that the final survival rate for the procedure was 66.7% (16 infants alive among 24 infants [12 sets of twins] who underwent surgery). The most recent follow-up of these infants indicated that they are living and experiencing normal quality of life. Table 2 summarizes the anatomical details and outcomes of the twins.

& DISCUSSION The present series of patients is impressive because the largest sample of such malformations ever studied included 383 reviewed sets of conjoined twins. That study was published in 2011, and the most important findings included a marked variation in pregnancy outcomes, similarity in the proportion of types of twins among hospitals, significant female predominance and apparently increasing prevalence in South American countries.

375

Gender

F F

F F F

M F

F M F

M F F M M

M F F F

M M

Case

1 2

3 4 5

6 7

8 9 10

11 12 13 14 15

376

16 17 18 19

20 21

Thoracoomphaloischiopagus Craniopagus

Omphalopagus Omphalopagus Omphalopagus Thoracoomphaloischiopagus

Ischiopagus Ischiopagus Ischiopagus Ischiopagus Ischiopagus

Ischiopagus Ischiopagus Ischiopagus

Thoracopagus Thoracopagus

Thoracopagus Thoracopagus Thoracopagus

Thoracopagus Thoracopagus

Type

Large intestine and bladder Liver and duodenum Liver 2 legs, bladder and pelvis, colon, genitalia and anus, just one inferior vena cava. Portal vein of twin 1 drains to portal vein of twin 2. 3 legs, complex cardiopathy Brain vascular connections. Twin 1 with microcephaly and sirenomelia. Twin 2 with anorectal agenesis.

Four legs, liver, large intestine and crossed ureters Third malformed leg, large intestine, anus, scrotum, bladder and liver Third malformed leg, vulva, urethra, bladder, liver, large intestine and crossed ureters Third malformed leg, liver, large intestine and urethra. Crossed ureters Third malformed leg, anus, large intestine, liver and crossed ureters Four normal legs, sigmoid and rectum, anus, liver, Third malformed leg, ileum and large intestine, liver and crossed ureters Third malformed leg, liver, small intestine and anus. Crossed ureters

Heart and liver Pericardium, liver and small intestine

Heart, liver, spleen, and small intestine Heart and liver Heart

Heart, liver and small intestine Heart and liver

Main organs shared

Outcome

Twins died three days after birth Separation with 10 months of life. Twin 1 was sacrificed and twin 2 is alive and well.

Set submitted to separation; one survived Successful separation with 16 months of life Successful separation with 16 months of life Successful separation with 10 months of life Both infants evolved, after separation, with sepsis and bradycardia leading to death Twin 1 died leading to separation surgery. Twin 2 survived Separated with 3 days of life. Successful separation with 11 months of life Parents denied the separation due to high risks. The twins are alive.

Cardiac insufficiency and set death with 11 days of life Perinatal asphyxia and progressive bradycardia due to congenital cardiac defects; set died with 1 day of life Perinatal asphyxia, submitted to ventilation; set died on the 27th day of life Cardiogenic shock and death on the 11th day of life Cardiac insufficiency, pulmonary edema and sepsis by Enterobacter cloacae; died with 28 days of life Bronchopneumonia and hypoxia leading to death at 4 months of life Twin 1 died of sepsis and pulmonary hemorrhage leading to emergency separation, twin 2 died with 11 months of life Successful separation at 9 months of life Set died of perinatal asphyxia within 1 day of life Successful separation at 10 months of life

Table 2 - Conjoined twins treated at the Child Institute of the Faculdade de Medicina da Universidade de SaË&#x153;o Paulo.

Conjoined twins experience in Brazil Tannuri AC et al. CLINICS 2013;68(3):371-377

CLINICS 2013;68(3):371-377

Conjoined twins experience in Brazil Tannuri AC et al.

delays and costs. In a recent review of 12 separated sets of conjoined twins, the author concluded that tissue expanders are not required in most patients (12). In our series, we did not use other reported techniques that are available for providing adequate coverage of the abdominal contents and viscera. The utilization of skin grafts, although skin grafts were recently reported for use in separating conjoined twins, they should not be utilized for covering the abdominal viscera (1). Skin grafts can be appropriately used to cover a granulating surface that develops in a surviving twin. However, we have noted that during the late postoperative period of these separated twins, skin grafts are unnecessary; spontaneous epithelialization occurs if adequate nutritional support is provided to the infant. The other recently reported technique involves creating a pneumoperitoneum during the preoperative period by injecting 500 to 1,500 mL of air every 3 days to increase the abdominal circumference and promote soft tissue and skin expansion (1). Because the literature does not report positive results for this technique (13,14), we think that it may be abandoned. Our results show a high incidence of ischiopagus (38.1%) and thoracopagus (42.8%) twins. Interestingly, the incidence of thoracopagus twins at our facility is similar to that reported in the collaborative study cited above (4). We believe that the low incidence of omphalopagus twins (14.3%) is due to the easier separation of this type at other centers, which leads to fewer transfers of these twins to a reference center. In contrast, there is a high incidence of stillbirth and miscarriage among thoraco-omphalo-ischiopagus twins because of the associated complex cardiac anomalies. However, our incidence of ischiopagus twins is quite different from the 1.8% reported in the collaborative study (5). We have no explanation for this difference. Finally, the least common and perhaps the most difficult type of twin to separate is the craniopagus type because the cranial union often involves a variety of neural and vascular connections. In Brazil, legally allowed abortion should be considered for sets of conjoined twins with poor prognoses, particularly for thoracopagus twins, who are unlikely to be successfully separated and have a low survival rate. In thoracopagus sets, fetal echocardiography plays an important role in determining the anatomy of the hearts and helps the pediatric and surgical teams prepare. Fortunately, a prenatal diagnosis was made in 90.5% of our cases. This diagnosis is very useful for preparing the obstetric and pediatric surgical teams for a successful delivery and for researching the possibility of a surgical separation. The surgical mortality rate of our series was 33.3%, which is in accordance with other published series. In a recent publication from the Philippines, the mortality rate of nine sets of twins was 17.7% (10). Spitz in the United Kingdom reported a mortality rate of 50% in 12 separation procedures, seven of which were performed emergently (6). Based on several publications and the present experience, emergency separations always have dismal outcomes. Therefore, careful deliberation and complete evaluation of the twins before any surgical intervention are important to ensuring that the surgery proceeds smoothly and with good results. The Pediatric Surgery Division of the Child Institute of the University of Sa˜o Paulo Medical School has treated 21

sets of conjoined twins over 20 years, with surgical experience in 12 pairs of patients. Considering the surgical outcomes, it is concluded that a well-prepared pediatric surgery team is sufficient for the surgical management of conjoined twins. Occasionally, emergency separation is needed; low survival rates are expected in this circumstance because of the poor condition of the patients and the limited availability of imaging exams. A good anatomical survey and proper surgical planning must precede the separation, and the twins must be as healthy as possible. Recent advances in imaging techniques for preoperative investigations provide adequate anatomic diagnosis and predict the possibility of separation. Moreover, advances in anesthetic care and postoperative critical care have improved outcomes and survival rates.

& ACKNOWLEDGMENTS This study was initiated by the second author under the supervision of the first author, and it was financed by Fundaçaõ de Amparo a Pesquisa do Estado de Saõ Paulo (project number 2011/08273-0).

& AUTHOR CONTRIBUTIONS Tannuri AC and Batatinha JA reviewed the medical records and prepared the manuscript. Velhote MC and Tannuri U are the surgeons who reviewed the final version of manuscript..

& REFERENCES 1. Jackson OA, Low DW, Larossa D. Conjoined twin separation: lessons learned. PlastReconstr Surg. 2012;129(4):956-63, http://dx.doi.org/10. 1097/PRS.0b013e3182442323. 2. Rode H, Fieggen AG, Brown RA, Cywes S, Davies MR, Hewitson JP, et al. Four decades of conjoined twins at Red Cross Children’s Hospital – lessons learned. S Afr Med J. 2006;96(9 Pt 2):931-40. 3. Spencer R. Anatomic description of conjoined twins: a plea for standardized terminology. J Pediatr Surg. 1996;31(7):941-4, http://dx. doi.org/10.1016/S0022-3468(96)90417-0. 4. Berezowski AT, Duarte G, Rodrigues R, de Carvalho Cavalli R, dos Santos R de O, de Andrade Vicente YA, et al. Conjoined twins: an experience of a tertiary hospital in Southeast Brazil. Rev Bras Ginecol Obstet. 2010;32(2):61-5. 5. Mutchinick OM, Luna-Mun˜oz L, Amar E, Bakker MK, Clementi M, Cocchi G, et al. Conjoined twins: a worldwide collaborative epidemiological study of the International Clearinghouse for Birth Defects Surveillance and Research. Am J Med Genet C Semin Med Genet. 2011;157C(4):274-87, http://dx.doi.org/10.1002/ajmg.c.30321. 6. Spitz L, Kiely EM. Conjoined twins. JAMA. 2003;289(10):1307-10, http:// dx.doi.org/10.1001/jama.289.10.1307. 7. Filler RM. Conjoined twins and their separation. Semin Perinatol. 1986;10(1):82-91. 8. Spitz L, Kiely EM. Experience in the management of conjoined twins. Br J Surg. 2002;89(9):1188-92. 9. Shi CR, Cai W, Jin HM, Chen F, Zhou Y, Zhou DX. Surgical management to conjoined twin in Shanghai area. Pediatr Surg Int. 2006:22(10):791-5, http://dx.doi.org/10.1007/s00383-006-1745-1. 10. Saguil E, Almonte J, Baltazar W, Acosta A, Caballes A, Catangui A, et al. Conjoined twins in the Philippines: experience of a single institution. Pediatr Surg Int. 2009;25(9):775-80, http://dx.doi.org/10.1007/s00383009-2426-7. 11. Cywes S, Millar AJW, Rode H, Brown RA. Conjoined twins: the Cape Town experience. Pediatr Surg Int. 1997;12(4):234-48. 12. Rabeeah A. Conjoined twins – past, present, and future. J Pediatr Surg. 2006;41(5):1000-4, http://dx.doi.org/10.1016/j.jpedsurg.2005.12.045. 13. Yokomori K, Ohkura M, Kitano Y, Nakajo T, Harii K, Tanikaze S. Comprehensive planning of operative strategy for separation of ischiopagustripus twins with particular reference to quality of life. J Pediatr Surg. 1993;28(6):833-7, http://dx.doi.org/10.1016/00223468(93)90338-L. 14. Hung WT, Chen WJ, Chen HT, Hsu TC, Chao CC, Wu TT. Successful separation of ischiopagustripus conjoined twins. J Pediatr Surg. 1986;21(11):920-3, http://dx.doi.org/10.1016/S0022-3468(86)80088-4.

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CLINICAL SCIENCE

Fetuin-A levels in hyperthyroidism Barıs¸ Onder Pamuk,I Hamiyet Yılmaz,I Tugba Topcuoglu,II Oktay Bilgir,II Ozlem C¸alan,III Gulseren Pamuk,IV Derun Taner ErtugrulV I

Izmir Bozyaka Teaching and Research Hospital, Department of Endocrinology and Metabolism, Izmir, Turkey. II Izmir Bozyaka Teaching and Research Hospital, Department of Internal Medicine, Bozyaka, Izmir. III Izmir Bozyaka Teaching and Research Hospital, Department of Biochemistry, Bozyaka, Izmir, Turkey. IV Izmir Bozyaka Teaching and Research Hospital, Department of Family Medicine,Bozyaka, Izmir, Turkey. V Kecioren Teaching and Research Hospital, Department of Endocrinology and Metabolism, Ankara, Turkey.

OBJECTIVE: Fetuin-A is a protein secreted from the liver that inhibits arterial calcification deposition and can contribute to insulin resistance. Hyperthyroidism is also associated with insulin resistance. It is not known whether hyperthyroidism has an effect on fetuin-A levels. METHODS: We measured fetuin-A levels and homeostasis model of assessment-insulin resistance before hyperthyroidism treatment was initiated and after euthyroidism was achieved. A total of 42 patients diagnosed with hyperthyroidism were enrolled in this study. Fetuin-A, insulin, high-sensitivity C-reactive protein, fasting blood glucose, free T3 (fT3), free T4 (fT4), and thyrotropin were measured before and after euthyroidism was established. RESULTS: Basal fasting blood glucose, high-sensitivity C-reactive protein, insulin, c-peptide, homeostasis model of assessment-insulin resistance, fT3, fT4 and fetuin-A levels were significantly decreased after euthyroidism was achieved (Table 1). Basal fasting blood glucose (r:0.407, p:0.008), high-sensitivity C-reactive protein (r:0.523, p,0.0001), insulin (r:0.479, p:0.001), homeostasis model of assessment-insulin resistance (r:0.541, p,0.0001), fT3 (r:0.492, p:0.001) and fT4 (r:0.473, p:0.002) were positively correlated with basal fetuin-A levels. Basal thyrotropin levels were significantly negatively correlated (r:-0.553, p,0.0001) with basal fetuin-A levels. CONCLUSION: Our findings suggest that hyperthyroidism influences fetuin-A levels. KEYWORDS: Fetuin-A; Hyperthyroidism; Insulin Resistance. Pamuk BO, Yılmaz H, Topcuoglu T, Bilgir O, C¸alan O, Pamuk G, et al. Fetuin-A levels in hyperthyroidism. Clinics. 2013;68(3):379-383. Received for publication on December 16, 2012; First review completed on January 8, 2013; Accepted for publication January 26, 2013 E-mail: bopamuk@gmail.com Tel.: +90 232 250-5050

associated with hepatosteatosis and are elevated in patients with insulin resistance (8). Fetuin-A can also be valuable for the prediction of new-onset type 2 diabetes. In the European Prospective Investigation into Cancer and Nutrition Study, circulating fetuin-A levels predicted the incidence of type 2 diabetes during a 7-yr follow-up (9). In the Health, Aging, and Body Composition study, serum fetuin-A levels were also significantly associated with incident diabetes during a 6-yr follow-up (10). Hyperthyroidism is also associated with insulin resistance and elevated blood glucose levels (11-12). However, the link between hyperthyroidism and insulin resistance has not been fully elucidated. To our knowledge, fetuin-A levels have not been measured in hyperthyroidism. In this study, we aimed to investigate the level of fetuin-A in hyperthyroidism.

& INTRODUCTION Fetuin-A is a protein secreted from the liver that inhibits arterial calcium deposition (1). It interacts with calcium and phosphorus in the serum, increasing their solubility and inhibiting calcium crystal precipitation. Fetuin-A-knockout mice have been shown to develop greater soft tissue calcification compared with wild-type control mice (2-3). Lower fetuin-A levels are associated with cardiovascular disease (CVD) events and all-cause mortality in end-stage renal disease (ESRD) (4). Fetuin-A inhibits insulin receptor tyrosine kinase activity in the muscle and liver, thereby inhibiting insulin signaling and introducing insulin resistance in vitro (5). In humans, fetuin-A may be an important link between obesity and insulin resistance (6-7). Fetuin-A concentrations are also

& METHODS Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

A total of 42 patients diagnosed with hyperthyroidism were enrolled in this study. Blood samples were collected before hyperthyroidism treatment was initiated and after euthyroidism was achieved. Serum glucose levels were measured with an autoanalyzer with the manufacturer’s commercially available kits (Cobas 8000, Roche Diagnostics,

No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(03)OA15

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Table 1 - Biochemical results before and after euthyroidism was achieved (mean¡SD). Parameter (reference values)

Hyperthyroid

Euthyroid

p-value

Fetuin-A (ng/mL) FT3 (pg/mL) (2.5-3.9) FT4 (ng/dL) (0.61-1.06) TSH (mIU/mL) (0.41-4.25) Fasting blood glucose (mg/dL) (70110) Insulin (mU/mL) (1.9-23) HOMA-IR hsCRP (mg/L) (0.068-8.2) Calcium (mg/dL) (8.6-10.2)

468.7¡143.5 8.6¡2.3 4.7¡1.6 0.006¡0.014 113.1¡15.6

234.5¡103.8 2.7¡0.3 1.2¡0.4 0.986¡0.467 94.8¡22.2

,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001

13.1¡2.5 3.69¡1.07 6.27¡2.65 10.1¡0.4

8.5¡2.1 2.00¡0.67 1.92¡0.79 9.4¡0.3

,0.0001 ,0.0001 ,0.0001 ,0.0001

normal distribution. A Wilcoxon signed rank test was used to analyze data with a skewed distribution. Pearson’s and Spearman’s analyses were used to identify correlations between study parameters. For all statistics, a two-sided pvalue ,0.05 was considered statistically significant. All analyses were performed with SPSS 15.0 for Windows.

Mannheim, Germany). Free T3, free T4, TSH and insulin levels were measured with a chemiluminescence immunometric assay using a UniCell DXI 800 analyzer (Beckman Coulter Inc., Fullerton, CA, USA). Serum hsCRP levels were determined using an ELISA according to the manufacturer’s instructions (DRG International, Inc., USA). The intra- and inter-assay coefficients of variation were below 10%. Serum fetuin-A levels were measured with a human fetuin-A ELISA kit (BioVendor Laboratory Medicine, Brno, Czech Republic). The analytical sensitivity of the human fetuin-A ELISA kit was 0.35 ng/mL. The intra- and inter-assay coefficients of variation were below 6.5%. The exclusion criteria were previously known diabetes mellitus, atherosclerotic vascular disease, infections, malignancy, amyloidosis, autoimmune diseases, alcohol consumption or smoking history, congestive heart failure, and liver or renal dysfunction. HOMA-IR was calculated using the following formula: HOMA-IR = fasting glucose (mmol/L)6fasting insulin (mU/mL)/22.5. The distribution of continuous variables was determined by the Kolmogorov-Smirnov test. A paired-sample t test was performed to analyze initial and final values with a

& RESULTS Of the 42 hyperthyroid patients (48.5¡15.0 years old), 27 patients were female, and 15 were male. Among the causes of hyperthyroidism, 30 patients had Graves’ disease, 11 patients had toxic adenoma, and 1 patient had two toxic nodules. Euthyroidism was achieved in 3.0¡0.7 (mean¡SD) months. Basal fasting blood glucose, hsCRP, insulin, HOMA-IR, fT3, fT4, calcium and fetuin-A levels were significantly decreased after euthyroidism was achieved (Table 1) (Figure 1). Basal fasting blood glucose (r:0.407, p:0.008), hsCRP (r:0.523, p,0.0001), insulin (r:0.479, p:0.001), HOMA-IR (r:0.541, p,0.0001), fT3 (r:0.492, p:0.001) and fT4 (r:0.473, p:0.002) were positively correlated with basal fetuin-A levels (Figures 2, 3). Basal TSH levels were significantly negatively correlated (r:-0.553, p,0.0001) with basal fetuin-A levels. The basal body mass index (before:

Figure 1 - The comparison of fetuin-A (ng/mL) levels during hyperthyroidism and euthyroidism.

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Figure 2 - The correlation between free FT4 (reference range: 0.61-1.06 ng/dL) and fetuin-A (ng/mL) levels.

Figure 3 - The correlation between TSH (uIU/mL) and fetuin-A (ng/mL) levels.

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27.1¡3.2, after: 28.2¡3.7, p,0.0001) increased significantly after euthyroidism was achieved.

(13). In addition to its functions as an inhibitor of tissue calcification, fetuin-A is an endogenous inhibitor of the insulin receptor tyrosine kinase (14), and fetuin-A-knockout mice exhibit increased insulin sensitivity (15). Recent studies have associated high levels of fetuin-A with an increased risk of type 2 diabetes incidence (16), insulin resistance, hepatosteatosis (8) and metabolic syndrome (7). Li et al. previously showed that fetuin-A plays a protective role in systemic inflammation by activating high-mobility group box 1 (HMGB1) synthesis (20). Hyperthyroidism may also initiate an inflammatory cascade and eventually stimulate fetuin-A synthesis. Insulin resistance in hyperthyroidism may also be related to the increased levels of fetuin-A. Yavuz et al. previously showed that levothyroxine treatment of goiter patients significantly increased hsCRP at 16 weeks (21). In accordance with that study, we found a significant decrease in hsCRP levels with hyperthyroidism treatment. Fetuin-A also has strong antiinflammatory properties (13). In our study, fetuin-A levels decreased with hsCRP levels, which may also be related to the inflammation-fetuin-A interaction. In our study, insulin levels were significantly positively correlated with fetuin-A levels; furthermore, insulin levels and fetuin-A levels were decreased after euthyroidism was achieved. The relationship between fetuin-A and insulin has been studied in both clinical and experimental studies. Fetuin-A binds to insulin receptors in adipose and muscular tissue and inhibits insulin receptor tyrosine kinase activity as well as insulin receptor autophosphorylation in vivo and in vitro (13). Fetuin-A has also been suggested to potentially cause insulin resistance and metabolic syndrome (13). In the present study, fasting glucose levels decreased significantly with hyperthyroidism treatment. Fasting glucose levels were significantly positively correlated with fetuin-A levels. It was previously shown that higher fetuinA concentrations were associated with type 2 diabetes and insulin resistance (24). In another study, fetuin-A levels were significantly correlated with fasting plasma glucose and CRP (25). The present study is in accordance with these findings, as fetuin-A levels decreased significantly as the fasting glucose levels and insulin resistance decreased after hyperthyroidism treatment. Atherosclerosis has been shown to be enhanced in hypothyroidism (26). The anti-atherogenic effects of thyroid hormones may also be related to fetuin-A. Further clinical and experimental studies investigating the effect of hypothyroidism on fetuin-A levels may reveal its influence on atherosclerosis in hypothyroidism. In conclusion, this study showed that fetuin-A levels declined significantly after hyperthyroidism treatment. Fetuin-A levels were also correlated with thyroid hormones and insulin resistance. Further experimental studies are needed to elucidate the molecular link between the hormones regulating tissue calcification and thyroid hormones.

& DISCUSSION In this study, we found that fetuin-A levels decreased significantly with hyperthyroidism treatment. To our knowledge, this is the first study investigating the effect of hyperthyroidism on fetuin-A levels. In this study, hsCRP levels also decreased significantly with the normalization of thyroid hormones. Furthermore, fetuin-A levels were positively correlated with thyroid hormone, HOMA-IR and hsCRP levels. Fetuin-A is a liver-derived blood protein that acts as a potent inhibitor of ectopic mineralization. Monomeric fetuin-A protein binds to small clusters of calcium and phosphate. Therefore, fetuin-A is a mineral carrier protein and a systemic inhibitor of pathological mineralization that complements local inhibitors that act in a cell-restricted or tissue-restricted fashion (13). Fetuin-A deficiency is associated with soft tissue calcification in mice and humans. Fetuin-A is a prominent serum glycoprotein as well as a major noncollagenous component of mineralized bone in mammals. In vitro, fetuin-A can inhibit or stimulate osteogenesis, depending on its concentrations (17). Rasul et al. previously found a positive correlation between fetuinA and bone alkaline phosphatase in type 2 diabetic males. In females, fetuin-A was significantly negatively associated with C-telopeptide levels (18). However, the researchers did not measure thyroid hormones in that study. Thyroid hormones increase bone turnover markers, and hyperthyroidism increases bone turnover. Fetuin-A may be increased in hyperthyroidism through a mechanism related to bone metabolism. Fetuin-A has been studied in several metabolic derangements; however, its role has not been studied in hyperthyroidism. Therefore, we could not compare our results with previous studies. Sato et al. examined the effects of T3 on the expression of calcification-associated genes and found that a physiological concentration of T3 increased the mRNA level of matrix G1a protein (MGP), which is a potent inhibitor of vascular calcification in vivo (19). They also showed that hyperthyroidism upregulated MGP mRNA 4.5 fold and reduced the calcium content of rat aortic smooth tissue by 11%. Fetuin-A also inhibits tissue calcification in a similar fashion; therefore, we hypothesize that there may be an interaction between fetuin-A synthesis and thyroid hormones because we found a positive correlation between these two parameters. To our knowledge, no experimental study in the literature has investigated the effect of thyroid hormones on the mRNA levels of fetuin-A. Hypercalcemia is a wellknown complication of hyperthyroidism, and stimulating fetuin-A synthesis may be an adaptation to prevent ectopic tissue calcification in hyperthyroidism. In a previous study of 32 thyrotoxic teenage girls, 21 (66%) showed some degree of calcification compared with 73 of 600 (12%) in the control group (22). Yamashita et al. previously observed that fibroblast growth factor (FGF)-23 levels (which are also related to soft tissue calcification) decreased significantly with hyperthyroidism treatment (23). However, they did not measure fetuin-A levels, and we therefore cannot compare our results with that study. Fetuin-A levels have been found to be higher in diabetes and inflammatory diseases, such as ankylosing spondylitis

& AUTHOR CONTRIBUTIONS Pamuk BO, Ertugrul DT conceived and designed the study, analyzed and interpreted the data, drafted the article, and critically revised the article for important intellectual content. Yılmaz H, Topcuoglu T, Bilgir O, Calan O, Pamuk G organized data in the tables, assisted the patients during the study, helped in the literature revision, drafted the manuscript, and critically revised the manuscript regarding important intellectual content.

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Hyperthyroidism and fetuin-A Pamuk BO et al. 14. Rauth G, Poschke O, Fink E, Eulitz M, Tippmer S, Kellerer M, et al. The nucleotide and partial amino acid sequences of rat fetuin. Identity with the natural tyrosine kinase inhibitor of the rat insulin receptor. Eur J Biochem. 1992;204(2):523-9. 15. Mathews ST, Singh GP, Ranalletta M, Cintron VJ, Qiang X, Goustin AS, et al. Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene. Diabetes. 2002;51(8):2450-8, http://dx.doi.org/ 10.2337/diabetes.51.8.2450. 16. Ix JH, Biggs ML, Mukamal KJ, Kizer JR, Zieman SJ, Siscovick DS, et al. Association of fetuin-a with incident diabetes mellitus in communityliving older adults: the cardiovascular health study. Circulation. 2012;125(19):2316-22, http://dx.doi.org/10.1161/CIRCULATIONAHA. 111.072751. 17. Binkert C, Demetriou M, Sukhu B, Szweras M, Tenenbaum HC, Dennis JW. Regulation of osteogenesis by fetuin. J Biol Chem. 1999;274(40): 28514-20, http://dx.doi.org/10.1074/jbc.274.40.28514. 18. Rasul S, Ilhan A, Reiter MH, Todoric J, Farhan S, Esterbauer H, et al. Levels of fetuin-A relate to the levels of bone turnover biomarkers in male and female patients with type 2 diabetes. Clin Endocrinol (Oxf). 2012;76(4):499-505, http://dx.doi.org/10.1111/j.1365-2265.2011.04246.x. 19. Sato Y, Nakamura R, Satoh M, Fujishita K, Mori S, Ishida S, et al. Thyroid hormone targets matrix Gla protein gene associated with vascular smooth muscle calcification. Circ Res. 2005;97(6):550-7, http://dx.doi. org/10.1161/01.RES.0000181431.04290.bd. 20. Li W, Zhu S, Li J, Huang Y, Zhou R, Fan X, et al. A hepatic protein, fetuin-A, occupies a protective role in lethal systemic inflammation. PLoS One. 2011;6(2):e16945, http://dx.doi.org/10.1371/journal.pone. 0016945. 21. Yavuz DG, Yazici D, Toprak A, Deyneli O, Aydin H, Yuksel M, et al. Exogenous subclinical hyperthyroidism impairs endothelial function in nodular goiter patients. Thyroid. 2008;18(4):395-400, http://dx.doi.org/ 10.1089/thy.2007.0299. 22. Senac MO, Jr., Lee FA, Gilsanz V. Early costochondral calcification in adolescent hyperthyroidism. Radiology. 1985;156(2):375-7. 23. Yamashita H, Yamazaki Y, Hasegawa H, Yamashita T, Fukumoto S, Shigematsu T, et al. Fibroblast growth factor-23 in patients with Gravesâ&#x20AC;&#x2122; disease before and after antithyroid therapy: its important role in serum phosphate regulation. J Clin Endocrinol Metab. 2005;90(7):4211-5, http://dx.doi.org/10.1210/jc.2004-2498. 24. Song A, Xu M, Bi Y, Xu Y, Huang Y, Li M, et al. Serum fetuin-A associates with type 2 diabetes and insulin resistance in Chinese adults. PLoS One. 2011;6(4):e19228, http://dx.doi.org/10.1371/journal.pone. 0019228. 25. Ou HY, Yang YC, Wu HT, Wu JS, Lu FH, Chang CJ. Serum fetuin-A concentrations are elevated in subjects with impaired glucose tolerance and newly diagnosed type 2 diabetes. Clin Endocrinol (Oxf). 2011;75(4):450-5, http://dx.doi.org/10.1111/j.1365-2265.2011.04070.x. 26. Ichiki T. Thyroid hormone and atherosclerosis. Vascul Pharmacol. 2010;52(3-4):151-6, http://dx.doi.org/10.1016/j.vph.2009.09.004.

& REFERENCES 1. Price PA, Lim JE. The inhibition of calcium phosphate precipitation by fetuin is accompanied by the formation of a fetuin-mineral complex. J Biol Chem. 2003;278(24):22144-52, http://dx.doi.org/10.1074/jbc. M300744200. 2. Schafer C, Heiss A, Schwarz A, Westenfeld R, Ketteler M, Floege J, et al. The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest. 2003;112(3):357-66. 3. Westenfeld R, Jahnen-Dechent W, Ketteler M. Vascular calcification and fetuin-A deficiency in chronic kidney disease. Trends Cardiovasc Med. 2007;17(4):124-8, http://dx.doi.org/10.1016/j.tcm.2007.02.005. 4. Ketteler M, Bongartz P, Westenfeld R, Wildberger JE, Mahnken AH, Bohm R, et al. Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a crosssectional study. Lancet. 2003;361(9360):827-33, http://dx.doi.org/10. 1016/S0140-6736(03)12710-9. 5. Auberger P, Falquerho L, Contreres JO, Pages G, Le Cam G, Rossi B, et al. Characterization of a natural inhibitor of the insulin receptor tyrosine kinase: cDNA cloning, purification, and anti-mitogenic activity. Cell. 1989;58(4):631-40, http://dx.doi.org/10.1016/0092-8674(89)90098-6. 6. Mori K, Emoto M, Yokoyama H, Araki T, Teramura M, Koyama H, et al. Association of serum fetuin-A with insulin resistance in type 2 diabetic and nondiabetic subjects. Diabetes Care. 2006;29(2):468, http://dx.doi. org/10.2337/diacare.29.02.06.dc05-1484. 7. Ix JH, Shlipak MG, Brandenburg VM, Ali S, Ketteler M, Whooley MA. Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study. Circulation. 2006;113(14):1760-7, http:// dx.doi.org/10.1161/CIRCULATIONAHA.105.588723. 8. Stefan N, Hennige AM, Staiger H, Machann J, Schick F, Krober SM, et al. Alpha2-Heremans-Schmid glycoprotein/fetuin-A is associated with insulin resistance and fat accumulation in the liver in humans. Diabetes Care. 2006;29(4):853-7, http://dx.doi.org/10.2337/diacare.29. 04.06.dc05-1938. 9. Stefan N, Fritsche A, Weikert C, Boeing H, Joost HG, Haring HU, et al. Plasma fetuin-A levels and the risk of type 2 diabetes. Diabetes. 2008;57(10):2762-7, http://dx.doi.org/10.2337/db08-0538. 10. Ix JH, Wassel CL, Kanaya AM, Vittinghoff E, Johnson KC, Koster A, et al. Fetuin-A and incident diabetes mellitus in older persons. JAMA. 2008;300(2):182-8, http://dx.doi.org/10.1001/jama.300.2.182. 11. Dimitriadis G, Baker B, Marsh H, Mandarino L, Rizza R, Bergman R, et al. Effect of thyroid hormone excess on action, secretion, and metabolism of insulin in humans. Am J Physiol. 1985;248(5 Pt 1):E593-601. 12. Dimitriadis GD, Raptis SA. Thyroid hormone excess and glucose intolerance. Exp Clin Endocrinol Diabetes. 2001;109 Suppl 2:S225-39, http://dx.doi.org/10.1055/s-2001-18584. 13. Jahnen-Dechent W, Heiss A, Schafer C, Ketteler M. Fetuin-A regulation of calcified matrix metabolism. Circ Res. 2011;108(12):1494-509, http:// dx.doi.org/10.1161/CIRCRESAHA.110.234260.

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BASIC RESEARCH

The influence of sleep deprivation and obesity on DNA damage in female Zucker rats Neuli M. Tenorio,I Daniel A. Ribeiro,II Tathiana A. Alvarenga,I Ana Carolina C. Fracalossi,II Viviane Carlin,II Camila Hirotsu,I Sergio Tufik,I Monica L. AndersenI I Universidade Federal de Saõ Paulo (UNIFESP), Departamento de Psicobiologia, Saõ Paulo/SP, Brazil. Departamento Biocieˆncia, Saõ Paulo/SP, Brazil.

Universidade Federal de Sa˜o Paulo (UNIFESP),

OBJECTIVE: The aim of this study was to evaluate overall genetic damage induced by total sleep deprivation in obese, female Zucker rats of differing ages. METHOD: Lean and obese Zucker rats at 3, 6, and 15 months old were randomly distributed into two groups for each age group: home-cage control and sleep-deprived (N = 5/group). The sleep-deprived groups were deprived sleep by gentle handling for 6 hours, whereas the home-cage control group was allowed to remain undisturbed in their home-cage. At the end of the sleep deprivation period, or after an equivalent amount of time for the home-cage control groups, the rats were brought to an adjacent room and decapitated. The blood, brain, and liver tissue were collected and stored individually to evaluate DNA damage. RESULTS: Significant genetic damage was observed only in 15-month-old rats. Genetic damage was present in the liver cells from sleep-deprived obese rats compared with lean rats in the same condition. Sleep deprivation was associated with genetic damage in brain cells regardless of obesity status. DNA damage was observed in the peripheral blood cells regardless of sleep condition or obesity status. CONCLUSION: Taken together, these results suggest that obesity was associated with genetic damage in liver cells, whereas sleep deprivation was associated with DNA damage in brain cells. These results also indicate that there is no synergistic effect of these noxious conditions on the overall level of genetic damage. In addition, the level of DNA damage was significantly higher in 15-month-old rats compared to younger rats. KEYWORDS: Sleep Deprivation; Obesity; Zucker Rats; DNA Damage; Comet Assay; Gentle Handling; Female. Tenorio NM, Ribeiro DA, Alvarenga TA, Fracalossi ACC, Carlin V, Hirotsu C, et al. The influence of sleep deprivation and obesity on DNA damage in female Zucker rats. Clinics. 2013;68(3):385-389. Received for publication on October 30, 2012; First review completed on November 4, 2012; Accepted for publication on November 22, 2012 E-mail: mandersen@unifesp.br / ml.andersen12@gmail.com Tel.: 55 11 2149-0155

Because of the simplicity and sensitivity of this method, the comet assay has rapidly gained acceptance as a genotoxicity assay (7). The basic principle of the comet assay is the migration of DNA in an agarose matrix under electrophoretic conditions. When viewed under a microscope, a damaged cell has the appearance of a comet, with a head (nuclear region) and a tail that contains DNA fragments or strands migrating towards the anode (8). Previous studies have demonstrated that the comet assay is a suitable tool to investigate genotoxicity in vivo (9,10). The extent of the comet is directly related to the level of DNA damage (11,12). Hence, the comet assay is a useful technique to detect DNA strand breaks in multiple organs and under different conditions, such as sleep deprivation (SD), disease progression, drug abuse, etc. (13-17). For example, we have used this technique to detect DNA breakage in vivo in mediumterm carcinogenesis assays and parasitic infections and following exposure to xenobiotics that are present in the environment (9,15,18). In recent years, increasing attention has been given to the genetic changes that are caused by sleep loss in different contexts of SD (16,19). Recently, we demonstrated that SD

& INTRODUCTION Factors such as sleep curtailment, obesity, and age can exert a wide variety of negative effects on the immune system and induce pathological processes such as genomic damage (1,2). Moreover, these factors are able to reduce the body’s capacity to repair DNA damage, increase the cellular sensitivity to oxidative stress, and promote cell death (3). All of this evidence demonstrates that the repair of oxidative damage in DNA is a dynamic and highly regulated process. When DNA repair is inhibited, cells age prematurely and die more easily (4). One useful method for quantifying DNA damage is the alkaline comet assay (single-cell gel electrophoresis) (5,6).

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induces genetic damage in the blood and brains cells of male rats (16), indicating that lack of sleep leads to severe molecular damage (19). In humans, the shortening of sleep can occur due to various factors, such as social life, artificial light, shift work, or sleep disturbances. Indeed, sleep loss is heightened in societies that have a frenzied lifestyle. Additionally, most sleep disorders, such as sleep apnea and insomnia, lead to sleep deprivation, which disrupts vital biological processes that are necessary for physical health (20). SD and obesity have been linked with comorbidities associated with morbid obesity rather than being causal factors (20,21). However, to the best of our knowledge, no study has investigated the influence of these risk factors on the genome at the single-cell level in this rat strain. Thus, the aim of this investigation was to evaluate overall genetic damage induced by sleep loss in obese female Zucker rats at different time points during their life span.

Experimental Design Lean and obese Zucker rats (3, 6, and 15 months old) were randomly assigned to either the control (CTRL) or the sleepdeprived (SD) group for a total of 12 groups (five animals/ group). The experiments were conducted while the female rats were in the diestrus phase of their estrous cycle. We elected to use rats only during diestrus because this phase was previously shown to be particularly affected by SD (30). The SD groups were handled gently for 6 hours, whereas the CTRL group was allowed to sleep in an adjacent room. The animals were sacrificed immediately after the SD period.

Sample Collection At the end of the SD period, or after an equivalent amount of time for the CTRL groups, the animals were brought to an adjacent room and were euthanized by decapitation between 1 and 3 pm. Blood was collected in sterile tubes containing liquid EDTA, aliquots were removed, and cellular suspensions (,10 ml) were used for the comet assay. In addition, central fragments from the kidney, heart, liver, and brain (prefrontal cortex) were also collected and minced in 0.9% NaCl in preparation for the comet assay. The sub-region of the prefrontal cortex was chosen based on results from our previous studies (16,17,19). The samples were maintained at -80 ˚C until the assays were performed. All of the samples were handled in an identical manner.

& MATERIAL AND METHODS Animals The experiments in this study were performed on female lean and obese adult Zucker rats (3, 6, and 15 months old). The animals were provided by the Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia (CEDEME) of the Universidade Federal de Sa˜o Paulo. The animals were housed in groups of three in standard polypropylene cages in a room that was maintained at 22˚C with a 12:12 h light-dark cycle (lights on at 7 am) and were given free access to food and water. The rats used in this study were maintained and treated in accordance with the guidelines established by the Ethical and Practical Principles for the Use of Laboratory Animals (22). All animal procedures were approved by the University Ethics Committee (Protocol #1268/08). Female rats were chosen because women have been shown to be frequently exposed to stressful events (12) and because their mortality is often increased prior to menopause (11). Obese Zucker rats are frequently used as a model for earlyonset, hyperplastic-hypertrophic obesity (23). Although Zucker rats cannot serve as an exact model of the human condition, these animals have provided important insights into the causes of human obesity and the possible consequences associated with this condition (24). Obesity in this strain is transmitted as a Mendelian autosomal recessive trait. Homozygous animals have been shown to accumulate fat progressively beginning at the 5th week of life (25) and demonstrate physical hypoactivity and hyperphagia. In addition, homozygous animals with access to food ad libitum will exhibit hyperinsulinemia (25,26), insulin resistance, hyperlipidemia (24,27), and hypothermia (27,28).

Comet Assay The protocol used to process the peripheral blood, kidney, liver, heart, and brain cells was a modified version of the protocol outlined by Tice et al. (31). Briefly, a volume of 5 ml of peripheral blood or of the cellular suspensions from the liver, heart, kidney, or prefrontal cortex was added to 120 ml of 0.5% low-melting-point agarose at 37 ˚C. The low-meltingpoint agarose containing cells was then layered onto a slide pre-coated with 1.5% regular agarose and covered with a coverslip. Prior to electrophoresis, the slides were left in an alkaline buffer (pH.13) for 20 min and were then electrophoresed for 20 min at 0.7 V/cm and 300 mA on ice. After electrophoresis, the slides were neutralized in 0.4 M TrisHCl (pH 7.5), fixed in absolute ethanol, and stored until they were analyzed on a fluorescent microscope at 4006 magnification. Independent positive controls, which were cells from the peripheral blood, liver, and brain, were treated in vitro with 10 mM H2O2 (hydrogen peroxide) for 10 min at 4 ˚C to ensure the reproducibility and sensitivity of the assay. All of the positive controls were made using cells from control lean rats. All of the samples from CTRL lean rats were treated randomly, and all analyses were performed blinded.

Total Sleep Deprivation

Analysis of Genotoxicity Data

Animals in the SD group were subjected to a single episode of continuous SD during the first 6 hours of the light phase (7 am to 1 pm) using a gentle handling method. The SD was performed by introducing an object (a pick) into the cage and tapping the cages whenever the animals appeared drowsy. The animals were not disturbed during feeding or drinking (29). We chose this method rather than the platform technique because obese animals may have had difficulty standing on the narrow platforms inside the water tank, which are used in the conventional platform method.

A total of 50 randomly captured comets per animal (25 cells from each slide) (18) were examined blindly by one expert observer. These comets were observed at 4006 magnification using a fluorescent microscope (Olympus). The microscope was connected through a black and white camera to an image analysis system (Comet Assay II, Perceptive Instruments, Suffolk, Haverhill, UK). The instrument had been previously calibrated according to the manufacturer’s instructions. The computerized image analysis system acquires images, computes the integrated

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Sleep loss and obesity on DNA damage in zucker rats Tenorio NM et al.

intensity profiles for each cell, estimates the comet cell components, and evaluates the range of the derived parameters. Undamaged cells have an intact nucleus without a tail, whereas damaged cells have the appearance of a comet (Figure 1). To measure the level of DNA damage, the tail intensity (% migrated DNA) was considered (32).

Table 1 - DNA damage (expressed as tail moment data) in the peripheral blood from lean and obese Zucker rats that underwent sleep deprivation. DNA damage (tail moment)

Age Control

Statistical Methods The results obtained in the comet assay were evaluated statistically as recommended by Wiklund and Agurrel (33). Thus, the tail intensity data were log10-transformed and compared by one-way ANOVA followed by a post-hoc Dunn’s test using Sigma Stat for Windows. According to the single-cell gel (comet) assay expert group, comet assay data should be log-transformed prior to statistical tests because they are not normally distributed (33). Body weight was analyzed using Student’s t-test for independent samples comparing lean and obese rats within each age group. The level of significance was set at a = 0.05 for the experiments in this study.

Sleep Deprivation

Positive control1

Lean

Obese

Lean

Obese

0.7 0.9 0.5 0.5 0.8 0.7¡0.2

0.2 1.0 0.8 0.3 0.5 0.5¡0.3

0.1 0.6 0.5 0.9 0.2 0.5¡0.2

1.0 0.7 0.8 0.5 0.2 0.6¡0.3

3.5 2.3 2.4 5.2 3.7 3.4¡1#

0.9 1.3 0.7 0.5 1.4 0.9¡0.4

0.8 0.2 0.7 0.4 1.2 0.6¡0.4

0.5 0.8 0.3 0.2 0.6 0.4¡0.2

1.4 0.6 0.9 1.4 1.9 1.2¡0.5

3.5 2.3 2.4 5.2 3.7 3.4¡1#

2.7 2.3 1.5 1.7 1.0 1.8¡0.5*

1.8 1.2 2.2 0.8 1.4 1.5¡0.5*

1.5 1.0 0.5 0.7 1.4 1.0¡0.4*

2.8 1.5 0.9 1.3 2.0 1.7¡0.6*

3.5 2.3 2.4 5.2 3.7 3.4¡1#

3 months

Mean¡S.D. 6 months

Mean¡S.D. 15 months

& RESULTS As expected, the adult obese rats had significant increases in body weight compared to the lean group at 3 months old (404.0¡35.8 vs. 205.3¡16.2, p#0.001), 6 months old (544.3¡56.6 vs. 241.4¡23.4, p#0.001), and 15 months old (747.0¡42.3 vs. 259.2¡45.6, p#0.001). However, the mean body weight of the obese group was not significantly different for the SD condition at any age compared to the CTRL condition. No statistically significant differences were identified in the blood cells of the obese rats during the younger phases of life. In addition, SD was not able to induce DNA breakage at these ages. Nevertheless, DNA migration was observed in rats that were 15 months old, irrespective of their obesity status (Table 1). No significant differences were observed among the groups in the kidney or the heart. For the liver, DNA migration was observed in the liver cells of the SD obese rats at every age evaluated. SD alone was not able to induce genetic damage in the liver, as shown in Table 2. A high level of DNA breakage was observed in the brains of 15-month-old rats following SD only. This result suggests that obesity status does not affect genetic damage in brain cells (Table 3). The blood, liver, and brain cells were further assayed with hydrogen peroxide to ensure the sensitivity of the assay. Sensitivity was confirmed by statistically significant differences (p,0.05), which were detected when compared to negative controls on all organs evaluated.

Mean¡S.D. *

p,0.05 when compared to 3 and 6 months; #p,0.05 when compared to all groups. 110 mM H2O2 (hydrogen peroxide) for 10 min at 4 ˚C.

Table 2 - DNA damage (expressed as tail moment data) in the livers of lean and obese Zucker rats that underwent sleep deprivation. DNA damage (tail moment)

Age Control

Sleep Deprivation

Positive control1

Lean

Obese

Lean

Obese

1.3 0.5 0.7 0.4 0.4 0.7¡0.4

1.7 0.6 1.5 1.8 1.6 1.5¡0.5

0.3 0.9 0.4 0.7 0.2 0.5¡0.3

1.7 1.4 1.9 0.8 2.3 1.6¡0.5¥

3.3 6.7 4.5 5.8 7.0 5.4¡1.5#

0.6 1.2 0.4 0.5 1.7 0.9¡0.6

2.4 1.0 1.4 2.0 2.6 1.8¡0.7

0.8 0.8 0.6 0.5 1.2 0.7¡0.2

3.0 3.2 2.1 1.8 1.2 2.2¡0.8¥

3.3 6.7 4.5 5.8 7.0 5.4¡1.5#

0.8 1.0 1.5 0.3 0.6 0.8¡0.5

1.9 2.0 1.4 1.9 0.7 1.5¡0.5

0.5 0.9 1.3 0.4 0.5 0.6¡0.4

1.4 2.4 2.0 1.8 0.7 1.7¡0.7*¥

3.3 6.7 4.5 5.8 7.0 5.4¡1.5#

3 months

Mean¡S.D. 6 months

Mean¡S.D. 15 months

Mean¡S.D.

Figure 1 - Representative comet images of a blood cell from a negative control (A), a cell exposed to sleep deprivation (B), and an H2O2-treated cell (positive control) (C). DNA was stained with ethidium bromide; 406 magnification.

387

p,0.05 when compared to 3 and 6 months; ¥p,0.05 when compared to respective lean rats, #when compared to all groups. 110 mM H2O2 (hydrogen peroxide) for 10 min at 4 ˚C.

Sleep loss and obesity on DNA damage in zucker rats Tenorio NM et al.

CLINICS 2013;68(3):385-389

not necessarily predict the mutagenic potential of all pathological conditions. Moreover, the genotoxicity of SD and obesity status can be modulated in combination with other DNA-damaging agents present in the brain. Altogether, the results from previous studies and this investigation provide evidence that sleep loss is able to induce genetic damage in brain cells, regardless of obesity status or age. In support of these results, Andersen and Ribeiro et al. (16) reported that SD promoted genetic damage in blood and brain cells, particularly following acute exposure. Chang et al. (38) argue that SD predisposes the liver to oxidative stress and phospholipid damage, leading to injury of the genetic apparatus. Nevertheless, our results revealed that the comet assay failed to detect DNA breakage in liver cells of rats subjected to SD but detected DNA damage in liver cells from obese rats. These findings corroborate data that indicate that genotoxicity in liver cells is associated with obesity in Zucker rats (38). These findings are biologically relevant because they demonstrate that obesity may negatively impact the incidence, severity, and clinical course of many types of chronic liver diseases and that obesity may be a causative agent for injuries such as nonalcoholic steatohepatitis (39,40). Moreover, the results of an epidemiological study have shown that there is a strong, positive correlation between body mass index and the incidence of several types of cancer (2). Nevertheless, age and SD did not modulate the noxious effects observed in the liver cells from these rats. The development of genetic damage in target cells depends not only on the initial levels of the induced DNA damage and its repair but also on other contributing factors, including the production of reactive metabolites, their distribution, and their effect on cell proliferation. Moreover, genotoxicity tests detect compounds that induce DNA damage directly or indirectly by various mechanisms. However, no single test is able to detect all genotoxic agents. Thus, for more detailed information on the genotoxic potential of SD on liver cells, a series of tests are necessary. In conclusion, our results revealed that obesity was associated with genetic damage in liver cells, whereas SD was associated with DNA damage in the peripheral blood and brain cells. The results of this study suggest that there is no synergistic effect of these noxious conditions. Because DNA damage is an important event that is related to genomic instability and to several degenerative diseases, the results of this study are a relevant contribution to the evaluation of the potential health risks associated with sleep loss related to chronic diseases such as obesity.

Table 3 - DNA damage (expressed as tail moment data) in the brain tissues of lean and obese Zucker rats that underwent sleep deprivation. DNA damage (tail moment)

Age Control

Sleep Deprivation

Positive control1

Lean

Obese

Lean

Obese

0.6 1.2 0.6 0.5 1.4 0.8¡0.4

0.8 1.2 0.6 0.1 0.2 0.5¡0.5

1.9 1.2 1.0 1.5 1.7 1.5¡0.3

4.4 3.7 1.6 2.4 0.8 2.6¡1.5

4.5 6.2 3.6 7.2 5.5 5.4¡1.4#

1.5 1.3 0.8 0.4 0.6 0.9¡0.5

2.0 1.4 0.5 0.3 0.2 0.8¡0.7

2.5 2.0 1.1 2.4 0.9 1.7¡0.8

3.3 2.7 1.7 1.3 1.9 2.2¡0.7

4.5 6.2 3.6 7.2 5.5 5.4¡1.4#

1.7 0.9 0.4 0.8 0.3 0.8¡0.6

0.1 0.2 0.4 0.3 0.2 0.2¡0.1

3.0 2.7 2.8 2.7 1.8 2.6¡0.4

4.5 2.8 3.2 3.8 4.0 3.7¡0.7*¥

4.5 6.2 3.6 7.2 5.5 5.4¡1.4#

3 months

Mean¡S.D. 6 months

Mean¡S.D. 15 months

Mean¡S.D. *

p,0.05 when compared to 3 and 6 months; ¥p,0.05 when compared to respective lean rats, #when compared to all groups. 110 mM H2O2 (hydrogen peroxide) for 10 min at 4 ˚C.

& DISCUSSION This study evaluated DNA migration induced by experimental sleep loss in obese versus lean rats. DNA migration was detected by the comet assay in peripheral blood cells from 15-month-old rats. Based on the tail moment data, the results of this study demonstrated that the comet assay, under our specific experimental conditions, was able to detect the presence of DNA migration in peripheral blood cells from rats at 15 months of age. DNA migration was not detected at younger ages (3 or 6 months), which was possibly because the time allowed was insufficient for a positive genotoxic response to occur on peripheral blood cells. It seems that age plays an important role in the detection of DNA breakage by the comet assay. The brain was evaluated in this study because it has been shown to be the primary target of DNA damage following SD. In fact, our results demonstrated that extensive genotoxic damage occurred in 15-month-old obese rats subjected to SD. This increase could be partially explained by increased levels of the bigenomic transcripts of oxidative stress (34), which likely contribute to an increase in adenosine triphosphate production during wakefulness (35). Others have also reported that aging impairs the unfolded protein response following SD because of the activation of several pro-apoptotic signaling proteins (36). The results of a study by Kumar et al. suggest that a nitric oxide (NO) mechanism is closely involved in SD-induced behavioral alterations and oxidative damage in mice (37). In this study, obesity did not lead to DNA migration in brain cells. It is important to note that the comet assay does

& ACKNOWLEDGMENTS The authors gratefully acknowledge the invaluable assistance of Marilde Costa and Waldemarks Leite. FUNDING: This work was supported by grants from Associaçaõ Fundo de Incentivo a` Pesquisa (AFIP) and Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (CEPID #98/ 14303-3 to S.T., #11/12325-6 to T.A.A., #10/50129-1 to C.H., #07/ 01228-4 to D.A.R., #07/01228-4 to A.C.C.F., and #09/03360-2 to V.C.). M.L.A., D.A.R., C.T.B., and S.T. are recipients of CNPq fellowships.

& AUTHOR CONTRIBUTIONS Tenorio NM, Tufik S, and Andersen ML were involved in the conception and design of the study. Tenorio NM, Alvarenga TA, and Hirotsu C acquired the data. Tenorio NM, Ribeiro DA, Alvarenga TA, Fracalossi

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AC, and Carlin V analyzed and interpreted the data. Tenorio NM, Ribeiro DA, Alvarenga TA, Fracalossi AC, Carlin V, Hirotsu C, Tufik S, and Andersen ML were responsible for the final approval of the completed article.

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Blockade of CXCR1/2 chemokine receptors protects against brain damage in ischemic stroke in mice Larissa Fonseca da Cunha Sousa,I Fernanda Matos Coelho,I David Henrique Rodrigues,I Alline Cristina Campos,I Lucı´ola da Silva Barcelos,II Mauro Martins Teixeira,I Milene Alvarenga Rachid,III Antonio Lu´cio TeixeiraI I

Universidade Federal de Minas Gerais (UFMG), Laborato´rio de Imunofarmacologia, Departamento de Bioquı´mica e Imunologia, Belo Horizonte/MG, Brazil. II Universidade Federal de Minas Gerais (UFMG), Departamento de Fisiologia, Belo Horizonte/MG, Brazil. III Universidade Federal de Minas Gerais (UFMG), Instituto de Cieˆncias Biolo´gicas (ICB), Departamento de Patologia Geral, Belo Horizonte/MG, Brazil.

OBJECTIVE: Ischemic stroke may result from transient or permanent reductions of regional cerebral blood flow. Polymorphonuclear neutrophils have been described as the earliest inflammatory cells to arrive in ischemic tissue. CXCR1/2 receptors are involved in the recruitment of these cells. However, the contribution of these chemokine receptors during transient brain ischemia in mice remains poorly understood. In this work, we investigated the effects of reparixin, an allosteric antagonist of CXCR1/2 receptors, in a model of middle cerebral artery occlusion and reperfusion in mice. METHODS: C57BL/6J male mice treated with reparixin or vehicle were subjected to a middle cerebral artery occlusion procedure 1 h after the treatment. Ninety minutes after ischemia induction, the monofilament that prevented blood flow was removed. Twenty-four hours after the reperfusion procedure, behavioral changes, including motor signs, were analyzed with the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. The animals were sacrificed, and brain tissue was removed for histological and biochemical analyses. Histological sections were stained with hematoxylin and eosin, neutrophil infiltration was estimated by myeloperoxidase activity and the inflammatory cytokine IL-1b was measured by ELISA. RESULTS: Pre-treatment with reparixin reduced the motor deficits observed in this model of ischemia and reperfusion. Myeloperoxidase activity and IL-1b were reduced in the reparixin-treated group. Histological analysis revealed that ischemic injury was also attenuated by reparixin pre-treatment. CONCLUSIONS: Our results suggest that the blockade of the CXCR1/2 receptors by reparixin promotes neuroprotective effects by reducing the levels of polymorphonuclear infiltration in the brain and the tissue damage associated with middle cerebral artery occlusion and reperfusion. KEYWORDS: Cerebral Ischemia; Neutrophils; Reparixin; CXCR1/CXCR2 Receptors. Sousa LF, Coelho FM, Rodrigues DH, Campos AC, Barcelos LS, Teixeira MM, et al. Blockade of CXCR1/2 chemokine receptors protects against brain damage in ischemic stroke in mice. Clinics. 2013;68(3):391-394. Received for publication on September 12, 2012; First review completed on October 26, 2012; Accepted for publication November 22, 2012 E-mail: laris.fonseca@gmail.com Tel.: 55 31 3409-2651

In the early stages of ischemia, polymorphonuclear neutrophils (PMNs) are the main type of infiltrating cells. Several studies report the later presence of monocytes/ macrophages, activation of resident brain cells and the expression of proinflammatory cytokines, chemokines and adhesion molecules in the injured brain (4). Leukocyte infiltration, especially of PMNs, seems to play a deleterious role during an ischemic event in the central nervous system (5). PMNs are a potential source of radical oxygen species, proteinases and cytokines that may contribute to the brain injury (6). Furthermore, strategies affecting the recruitment of PMNs are of great interest for controlling various inflammatory diseases. Several authors have postulated that the expression of certain chemokines, mainly CXC ligand 8 (CXCL8) in humans and CXCL1 and CXCL2 in rodents, by central nervous system cells is required for PMN accumulation and activation (7). Furthermore, human patients exhibit increased circulating levels of CXCL8 following stroke (8).

& INTRODUCTION Ischemic stroke may result from transient or permanent reductions of regional cerebral blood flow (1). In humans, stroke is the second most common cause of death worldwide (2) and often occurs in the territory supplied by the middle cerebral artery (3). Inflammatory processes have been implicated in the pathophysiology of cerebral ischemia, involving the recruitment and influx of leukocytes from the circulation to the ischemic brain tissue.

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Experimental procedures: The subcutaneous administration of reparixin (30 mg/kg) was performed 60 minutes before cerebral ischemia induction (15). The animals were divided into the following three experimental groups: Sham (i.e., the group in which the arteries were visualized, but there was no occlusion of the middle cerebral artery), Vehicle (i.e., the group pre-treated with the vehicle, phosphate buffer solution, 60 minutes before MCAo) and Reparixin (i.e., the group pre-treated with the drug 60 minutes before MCAo). To evaluate neurological signs secondary to MCAo, the animals were assessed with the SHIRPA battery 24 h after reperfusion (16). Immediately after behavioral testing, all animals were sacrificed, and the whole brain was removed for biochemical and histological analyses. Neutrophil infiltration in the brain was estimated by myeloperoxidase activity test (MPO), and the results were expressed in relative units. The concentration of IL-1b was measured in brain tissue using a commercially available enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions (Duo-Set Kits; R&D Systems, Minneapolis, MN). For histological analysis, a brain fragment was preserved in 10% buffered formalin. Sections (4 mm thick) were subject to routine hematoxylin and eosin staining. Ischemia/reperfusion-induced lesions and the infiltration of inflammatory cells were analyzed qualitatively. Statistical analysis: Data are represented as the means ¡ SEM. One-way ANOVA followed by the Newman-Keuls post-hoc test was used for multiple comparisons. The statistical significance was set at p,0.05.

In the last decade, several studies have suggested that anti-inflammatory strategies could be of paramount importance to prevent brain damage following cerebral ischemia (9,10). Indeed, anti-chemokine approaches successfully conferred neuroprotection in rodent models of brain ischemia (11-13). Reparixin is a chemical derivative of phenyl propionic acids that acts as noncompetitive allosteric antagonist of the chemokine receptors CXCR1 and CXCR2, which bind to the chemokines CXCL1 (previously known as KC) and CXCL2 (previously known as MIP-2). Therefore, the aim of this study was to investigate the effect of reparixin on inflammatory and behavioral outcomes in a model of middle cerebral artery occlusion and reperfusion (MCAo) in mice.

& MATERIAL AND METHODS Animals: Male C57BL/6J mice (8-10 weeks old/20-25 g) obtained from the Animal Care Facilities of the Institute of Biological Science (ICB-UFMG) were maintained in a temperature controlled room (24¡1 ˚C) under standard laboratory conditions with free access to food and water and a 12-h light/12-h dark cycle (lights on at 7 a.m.). All experiment procedures were conducted in accordance with the Animal Ethics Committee (CETEA-UFMG). Drugs: Reparixin (30 mg/kg – Dompe´ Pharma, L’Aquila, Italy), a CXCR1/2 allosteric antagonist, was dissolved in saline and administered subcutaneously. Transient focal cerebral ischemia: Mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (150 mg/kg) and xylazine (10 mg/kg). The surgical procedure was performed on a warming mantle with all animals spontaneously breathing. Transient focal cerebral ischemia was induced by MCAo, using the method previously described for rats by Longa et al., 1989 (10). Briefly, a blunted silicon tip (Silon2 APS Fluido, Petro´polis, Brazil) and a 5-0 nylon monofilament catalyst (Silon2 APSC, Petro´polis, Brazil) were advanced to the level of the carotid bifurcation via the internal carotid artery until light resistance was felt. The monofilament was removed after 90 minutes of occlusion. In the sham group, the arteries were visualized but not disturbed. SHIRPA screening test: The primary SmithKline/ Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) screen consists of a wide range of tests, organized into the following five functional categories: neuropsychiatric state (i.e., spontaneous activity, transfer arousal, touch escape, positional passivity, biting, fear, irritability, aggression, vocalizations), motor behavior (i.e., body position, tremor, locomotor activity, pelvic elevation, gait, tail elevation, trunk curl, limb grasping, wire maneuver, negative geotaxis), reflex and sensory function (i.e., startle response, visual placing, pinna reflex, corneal reflex, toe pinch, righting reflex), autonomic function (i.e., respiratory rate, defecation, urination, palpebral closure, piloerection, skin color, heart rate, lacrimation, salivation, body temperature) and muscle tone and strength (i.e., grip strength, body tone, limb tone, abdominal tone). The SHIRPA provides a basic behavioral and functional profile through the observational assessment of individual performance (14). All measures were performed in a Plexiglas square arena (40 cm 6 40 cm 6 30 cm).

& RESULTS When subjected to MCAo, vehicle-treated mice exhibited higher MPO activity, an index of PMN infiltration in the brain, in comparison with the sham animals. Reparixin significantly prevented the increase of MPO induced by MCAo (Figure 1). Vehicle-treated animals subjected to the MCAo procedures also displayed higher brain levels of IL1-b compared to sham mice. Similar to the MPO results, pretreatment with

Figure 1 - Reparixin reduces neutrophil activity in the brain after middle cerebral artery occlusion/reperfusion (MCAo) in mice. The figure shows myeloperoxidase (MPO) activity in mice not subjected to MCAo (Sham) or subjected to MCAo with preadministration of vehicle (Vehicle) or reparixin (30 mg/kg, s.c.) 60 minutes before MCAo procedures. The results are expressed as the means ¡ SEM (n = 6). * p,0.05 compared to Sham group. # p,0.05 compared to vehicle. (ANOVA followed by NewmanKeuls posthoc test).

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reparixin significantly reduced the levels of this inflammatory cytokine (Figure 2). The ischemia/reperfusion procedure induced an extensive hemorrhagic necrosis in the brain (Figures 3 A-D). This lesion was circumscribed by inflammatory cells, mainly PMNs, surrounded by degenerative tissue (Figures 3 C and D). Corroborating the MPO and IL1-b results, reparixin treatment attenuated the ischemia/reperfusion-induced lesions and the infiltration of inflammatory cells (Figures 3 E and F). Consistent with these pathological results, the MCAo procedure induced motor impairment, which was attenuated by pretreatment with reparixin (Figure 4). Other SHIRPA categories did not change after MCAo.

& DISCUSSION In this work, we demonstrated that the inhibition of CXCR1/2 receptors by reparixin protected the brain after MCAo. The blockade of CXCR1/2 receptors attenuated not only the structural changes in the ischemic brain but also the corresponding behavioral consequences of the brain injury. Tissue damage after ischemia is mediated through several mechanisms, including hypoxia and inflammatory responses (17). The latter is particularly relevant in the context of reperfusion, in which PMNs infiltrate ischemic/reperfused organs, such as the brain, lung and intestine (10, 18, 19). Once in the tissue, neutrophils release enzymes from their granules that may cause serious damage to the inflamed tissue. The peak of PMN infiltration occurs 24 h after the ischemia (20); this was the time point chosen to perform the analyses for this work. Strategies against the recruitment of PMNs have been associated with protective effects in ischemia/reperfusion models (21,19). Considering the role of CXCR1 and CXCR2 in PMN recruitment, their blockade could theoretically protect the brains of mice subjected to MCAo (10). Reparixin is a compound that inhibits the effects of CXCL1 and CXCL2 by allosteric modulation of the CXCR1 and CXCR2 receptors. In rats, reparixin reduced brain PMN infiltration and the associated tissue damage in a cerebral ischemia/ reperfusion model (10). The administration of SB225002, a CXCR2 antagonist, was also associated with reduced

Figure 3 - Histopathological analysis of cerebral sections of sham (A-B), ischemia/reperfusion (C-D) and reparixin-treated mice subjected to middle cerebral artery occlusion/reperfusion (E-F). Hematoxylin and eosin-stained sections. Cerebral cortex of sham mice showing normal tissue (A) and healthy neural cells (B). Cerebral section of an ischemia/reperfusion mouse exhibits large hemorrhagic necrosis (C). Note the inflammatory cells and vacuolated tissue around the infarct area (D). Reparixin-treated animals present focal infarct surrounded by ischemic neural cells (asterisks) (E). The dotted line delineates dark and shrunken neural cells (left) from normal neural cells (right) (F). Original magnifications: A, C and E: x200. B, D and F: x400.

neutrophil infiltration in the brains of rats 24 h after cerebral ischemiaâ&#x20AC;&#x201C;reperfusion (22). Our results are in agreement with these preliminary findings in rats (10,13). In parallel with the decrease of PMN infiltration, as assessed by the MPO assay, reparixin treatment decreased tissue damage and motor compromise. Therefore, this study reinforces the role of PMNs in stroke physiopathology and suggests that reparixin is a promising agent for future experimental and clinical studies. A better understanding of the mechanisms involved in stroke pathogenesis can foster the search for candidate drugs to prevent or attenuate the deficits caused by cerebral ischemia. This is of paramount importance because quality of life for stroke sufferers is largely dependent on the severity of their motor and/or cognitive deficits (23). Members of the IL-1 family of cytokines, such as IL-1b, are considered major effectors of injury in stroke. Inhibiting the signaling of IL-1a and IL-1b with an IL-1 receptor antagonist was protective in experimental models of cerebral ischemia (24). We found that reparixin-pretreated mice had lower brain levels of IL-1b in conjunction with less structural and functional compromise. CXCR1/2 receptor blockade by reparixin pretreatment resulted in a decrease of cerebral damage, as indicated by a reduction of PMN recruitment, IL-1b levels and motor

Figure 2 - Reparixin reduces the levels of IL-1b in the brain after middle cerebral artery occlusion/reperfusion (MCAo) in mice. Bars represent levels of IL-1b (pg/100 mg) measured by ELISA in the brain tissues of mice subjected or not (SHAM) to MCAo and pretreated with vehicle or reparixin (30 mg/kg, s.c.). The results are expressed as the means ÂĄ SEM (n = 6). *p,0.05 compared to Sham group. # p,0.01 compared to Vehicle. (ANOVA followed by Newman-Keuls posthoc test).

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4. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 1999;22(9):391-7, http://dx.doi. org/10.1016/S0166-2236(99)01401-0. 5. Emerich DF, Dean RL 3rd, Bartus RT. The role of leukocytes following cerebral ischemia: pathogenic variable or bystander reaction to emerging infarct?. Exp Neurol. 2002;173(1):168-81, http://dx.doi.org/10.1006/ exnr.2001.7835. 6. Witki-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L. Neutrophils: molecules, functions and pathophysiological aspects. Lab Invest. 2000;80(5):617-53, http://dx.doi.org/10.1038/labinvest.3780067. 7. Campbell IL. Chemokines as plurifunctional mediators in the CNS: implications for the pathogenesis of stroke. Ernst Schering Res Found Workshop. 2004;(45):31-51. 8. Kostulas N, KivisaÂ¨kk P, Huang Y, Matusevicius D, Kostulas V, Link H. Ischemic stroke is associated with a systemic increase of blood mononuclear cells expressing interleukin-8 mRNA. Stroke. 1998;29(2):462-6, http://dx.doi.org/10.1161/01.STR.29.2.462. 9. Barone FC, Feuerstein GZ. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J Cereb Blood Flow Metab. 1999;19(8):819-34, http://dx.doi.org/10.1097/00004647-199908000-00001. 10. Villa P, Triulzi S, Cavalieri B, Di Bitondo R, Bertini R, Barbera S, et al. The interleukin-8 (IL-8/CXCL8) receptor inhibitor repertaxin improves neurological deficits and reduces long-term inflammation in permanent and transient cerebral ischemia in rats. Mol Med. 2007;13(3-4):125-33. 11. Matsumoto T, Ikeda K, Mukaida N, Harada A, Matsumoto Y, Yamashita J, et al. Prevention of cerebral edema and infarct in cerebral reperfusion injury by an antibody to interleukin-8. Lab Invest. 1997;77(2):119-25. 12. Yamasaki Y, Matsuo Y, Zagorski J, Matsuura N, Onodera H, Itoyama Y, et al. New therapeutic possibility of blocking cytokine-induced neutrophil chemoattractant on transient ischemic brain damage in rats. Brain Res. 1997;759(1):103-11, http://dx.doi.org/10.1016/S0006-8993(97)00251-5. 13. Garau A, Bertini R, Colotta F, Casilli F, Bigini P, Cagnotto A, et al. Neuroprotection with the CXCL8 inhibitor reparixin in transient brain ischemia. Cytokine. 2005;30(3):125-31, http://dx.doi.org/10.1016/j.cyto. 2004.12.014. 14. Lalonde R, Dumont M, Staufenbiel M, Strazielle C. Neurobehavioral characterization of APP23 transgenic mice with the SHIRPA primary screen. Behav Brain Res. 2005;157(1):91-8, http://dx.doi.org/10.1016/j. bbr.2004.06.020. 15. Coelho FM, Vinho V, Amaral FA, Sachs D, Costa VV, Rodrigues DH, et al. The chemokine receptors CXCR1/CXCR2 modulate antigeninduced arthritis by regulating adhesion of neutrophils to the synovial microvasculature. Arthritis and Rheumatism. 2008;58(8):2329-37, http:// dx.doi.org/10.1002/art.23622. 16. Rogers DC, Fisher EM, Brown SD, Peters J, Hunter AJ, Martin JE. Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment. Mamm Genome. 1997;(10):711-3, http://dx.doi.org/10.1007/s003359900551. 17. Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L. Neutrophils: molecules, functions and pathophysiological aspects. Lab Invest. 2000;80(5):617-53, http://dx.doi.org/10.1038/labinvest.3780067. 18. Sekido N, Mukaida N, Harada A, Nakanishi I, Watanabe Y, Matsushima K. Prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8. Nature. 1993;365(6447):654-7, http://dx. doi.org/10.1038/365654a0. 19. Souza DG, Bertini R, Vieira AT, Cunha FQ, Poole S, Allegretti M, et al. Repertaxin, a novel inhibitor of rat CXCR2 function, inhibits inflammatory responses that follow intestinal ischaemia and reperfusion injury. Br J Pharmacol. 2004;143(1):132-42. 20. Jiang N, Chopp M, Chawala S. Neutrophil inhibitory factor treatment of focal cerebral ischemia in the rat. Brain Res. 1998;788(1-2):25-34, http:// dx.doi.org/10.1016/S0006-8993(97)01503-5. 21. Souza D G, Cassali G D, Pool S, Teixeira MM. Effects of inhibition of PDE4 and TNF-alpha on local and remote injuries following ischaemia and reperfusion injury. Br J Pharmacol. 2001;134(5):985-94. 22. Brait VH, Rivera J, Broughton BR, Lee S, Drummond GR, Sobey CG. Chemokine-related gene expression in the brain following ischemic stroke: no role for CXCR2 in outcome. Brain Res. 2011;1372:169-79, http://dx.doi.org/10.1016/j.brainres.2010.11.087. 23. Kwa VI, Limburg M, de Haan RJ. The role of cognitive impairment in the quality of life after ischaemic stroke. J Neurol. 1996;243(8):599-604. 24. Brough D, Tyrrell PJ, Allan SM. Regulation of interleukin-1 in acute brain injury. Trends Pharmacol Sci. 2011;32(10):617-622, http://dx.doi.org/10. 1016/j.tips.2011.06.002.

Figure 4 - Reparixin prevents motor impairment after middle cerebral artery occlusion/reperfusion (MCAo) in mice. Bars indicate levels of motor deficits as assessed by SHIRPA in mice subjected or not to MCAo (Sham) or subjected to MCAo pretreated with vehicle or reparixin (30 mg/kg, s.c.). The results are expressed as the means ÂĄ SEM (n = 6). * p,0.05 compared to Sham group; ANOVA followed by Newman-Keuls post-test. (ANOVA followed by Newman-Keuls posthoc test).

impairment following MCAo. Further studies are needed to determine if reparixin treatment after ischemia could reverse the brain injury and the associated long-lasting neurological consequences of stroke in mice. The lack of discrimination of cortical versus subcortical damage and of identification of the penumbra area in histopathological analysis must be regarded as limitations of this study and deserve further investigation. In conclusion, these results support the concept that the blockade of inflammatory processes mediated by CXCL8 could be a future intervention for the improvement of the structural and behavioral consequences of human cerebrovascular diseases.

& ACKNOWLEDGMENTS This study was funded by CNPq, Fapemig and Rede IBN-Net.

& AUTHOR CONTRIBUTIONS Sousa LFC performed the surgical procedures to induce transient cerebral ischemia, behavioral tests and immunological assays and drafted the first version of the manuscript. Coelho FM and Rodrigues DH participated in the immunological assays and behavioral tests. Campos AC and Barcelos LS contributed to the analysis and interpretation of the results. Rachid MA performed the histopathological analysis. Teixeira MM and Teixeira AL designed the study and were responsible for editing the manuscript. All authors have read and approved the final version of the manuscript.

& REFERENCES 1. Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J. Leukoc. Biol. 2010;87(5):779-89, http://dx.doi.org/ 10.1189/jlb.1109766. 2. Donnan GA, Fisher M, Macleod M, Davis SM. Stroke. Lancet. 2008; 371(9624):1612-23, http://dx.doi.org/10.1016/S0140-6736(08)60694-7. 3. Durukan A, Tutlisumak T. Ischemic stroke in mice and rats. Methods Mol Biol. 2009;573:95-114, http://dx.doi.org/10.1007/978-1-60761-247-6_ 6.

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Hemodynamic and ventilatory response to different levels of hypoxia and hypercapnia in carotid bodydenervated rats Joaõ Paulo J. Sabino, Mauro de Oliveira, Humberto Giusti, Mogens Lesner Glass, Helio C. Salgado, Rubens Fazan Jr. Universidade de Saõ Paulo, School of Medicine of Ribeiraõ Preto, Department of Physiology, Saõ Paulo/SP, Brazil.

OBJECTIVE: Chemoreceptors play an important role in the autonomic modulation of circulatory and ventilatory responses to changes in arterial O2 and/or CO2. However, studies evaluating hemodynamic responses to hypoxia and hypercapnia in rats have shown inconsistent results. Our aim was to evaluate hemodynamic and respiratory responses to different levels of hypoxia and hypercapnia in conscious intact or carotid body-denervated rats. METHODS: Male Wistar rats were submitted to bilateral ligature of carotid body arteries (or sham-operation) and received catheters into the left femoral artery and vein. After two days, each animal was placed into a plethysmographic chamber and, after baseline measurements of respiratory parameters and arterial pressure, each animal was subjected to three levels of hypoxia (15, 10 and 6% O2) and hypercapnia (10% CO2). RESULTS: The results indicated that 15% O2 decreased the mean arterial pressure and increased the heart rate (HR) in both intact (n = 8) and carotid body-denervated (n = 7) rats. In contrast, 10% O2 did not change the mean arterial pressure but still increased the HR in intact rats, and it decreased the mean arterial pressure and increased the heart rate in carotid body-denervated rats. Furthermore, 6% O2 increased the mean arterial pressure and decreased the HR in intact rats, but it decreased the mean arterial pressure and did not change the HR in carotid body-denervated rats. The 3 levels of hypoxia increased pulmonary ventilation in both groups, with attenuated responses in carotid body-denervated rats. Hypercapnia with 10% CO2 increased the mean arterial pressure and decreased HR similarly in both groups. Hypercapnia also increased pulmonary ventilation in both groups to the same extent. CONCLUSION: This study demonstrates that the hemodynamic and ventilatory responses varied according to the level of hypoxia. Nevertheless, the hemodynamic and ventilatory responses to hypercapnia did not depend on the activation of the peripheral carotid chemoreceptors. KEYWORDS: Arterial Pressure; Heart Rate; Pulmonary Ventilation; Chemoreceptor Cells. Sabino JPJ, Oliveira M, Giusti H, Glass ML, Salgado HC, Fazan Jr R. Hemodynamic and ventilatory response to different levels of hypoxia and hypercapnia in carotid body-denervated rats. Clinics. 2013;68(3):395-399. Received for publication on September 28, 2012; First review completed on November 21, 2012; Accepted for publication on November 25, 2012 E-mail: rfazan@usp.br Tel.: 55 16 3602-3331

increase in pulmonary ventilation (PV) (5-8). Nevertheless, studies in rats have shown conflicting hemodynamic responses to hypoxia. While some studies have demonstrated that hypoxia elicits a decrease in the mean arterial pressure (MAP) combined with an increase in heart rate (HR) (9-15), others have shown an increase in MAP combined with a decrease in HR (16,17). In contrast, while there is general agreement that hypercapnia promotes a hypertensive response (18-21), there is no agreement concerning the HR response to hypercapnia. Studies have reported that bradycardia (22,23) or tachycardia (18) was elicited by hypercapnia. Accordingly, the evident disagreement in the literature concerning the hemodynamic and ventilatory responses to hypoxia and hypercapnia requires further investigation and characterization of the role of the peripheral carotid chemoreceptors in these responses. Therefore, this study aimed to evaluate the hemodynamic and ventilatory

& INTRODUCTION Peripheral arterial chemoreceptors consist of aortic bodies situated in the aortic arch and carotid bifurcations (1), while central chemoreceptors are located on the ventral surface of the medulla (2). The chemoreceptors play an important role in the regulation of ventilation and circulation in response to changes in arterial O2 and/or CO2 (2-4). It has been well established that activation of the chemoreceptors under hypoxia or hypercapnia elicits an

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responses to different levels of hypoxia and hypercapnia in conscious intact or carotid body-denervated (CBD) rats.

Experimental Protocol Each animal was individually placed into the plethysmographic chamber at 25 ˚C and allowed to move freely while the chamber was flushed with humidified air (21% O2 and 79% N2, 1.2 L.min-1) for 40-50 min of acclimatization. After the baseline measurements of respiratory parameters and AP, each animal was subjected to 3 levels of hypoxia (15, 10 and 6% of inhaled O2) and combined hypercapnia and hyperoxia (10% CO2+31% O2). The order of each episode of hypoxia or hypercapnia was chosen randomly, and the episodes were spaced 30 min apart. Hypoxia was achieved by switching the gas flushed through the chamber to 100% N2. Nitrogen flow was adjusted (2, 3.5 or 5 L.min-1) to achieve the desired level of hypoxia (15, 10 or 6%) within approximately 45 s. Hypercapnia plus hyperoxia (10% CO2+30% O2) was achieved by flushing the chamber with 50% CO2 and 50% O2 (2 L.min-1) for 45 s. Each period of chemoreceptor stimulation was held for 1 minute, and the respiratory parameters, MAP and HR were measured. Subsequently, the chamber was opened to atmospheric air, and the animals were given a 30-min recovery period. At the end of the experiment, intravenous KCN was administered to verify the efficacy of carotid body denervation, as described elsewhere (17).

& MATERIALS AND METHODS Experiments were performed on male Wistar rats (270300 g) housed individually with free access to food and water and maintained on a 12:12-h light-dark cycle. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals [Dept. of Health, Education and Welfare, Publication No. (NIH) 85-23, Revised 1985. Office of Science and Health Reports, DRR/NIH, Bethesda, MD]. The experimental protocols used in this research were approved by the Committee of Ethics in Animal Research of the School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo (protocol 053/2010). Two days before the experiments, the rats were anesthetized with tribromoethanol (250 mg.kg-1, i.p., Sigma, St. Louis, MO, USA), and bilateral ligature of the carotid body arteries was performed as described elsewhere (24). Briefly, the carotid artery bifurcations were identified and isolated bilaterally. Then, the carotid body arteries were identified and tightly ligated with a suture line to block the arterial supply to the carotid bodies. The procedure was similar for rats that received the sham operation (intact), except that they did not undergo the ligature. After artery ligation, polyethylene catheters were inserted into the left femoral artery and vein for the direct measurement of arterial pressure (AP) and administration of potassium cyanide (KCN, 160 mg.kg-1, Merck, Darmstadt, Germany), respectively. Both catheters were tunneled subcutaneously and exteriorized through the back of the neck, and the surgical incision sites were closed by sutures. For the experiments, the rats were placed into a 3.9 L PlexiglasH chamber (with sealed exit ports for catheters), which allowed them to move freely. The arterial catheter was connected to a pressure transducer (MLT0380/D, ADInstruments, Sydney, Australia), and the amplified AP signal was fed to an IBM/PC connected to a Power Lab system (ML866, ADInstruments, Sydney, Australia) and continuously sampled (2 kHz). Different gas mixtures were allowed to easily pass through the chamber to change the gas concentration. The gas concentration inside the chamber was continuously measured with an O2 analyzer (GasAlertMax XT, Canada) and a CO2 analyzer (microCapStar CO2 Monitor, PA, USA). A flowmeter gasmixing pump (Cameron, Canada) allowed for the injection of different gas concentrations into the chamber. To measure respiratory parameters, the airflow through the chamber was interrupted for a short time (,1 min), and the inlet and outlet ports were sealed. Pressure oscillations caused by respiratory movements were detected by a differential pressure transducer (ML141, ADInstruments, Sydney, Australia) and were digitally recorded simultaneously with AP. Volume calibration was performed during each measurement by injecting a known air volume (1 mL) into the chamber. The tidal volume (TV) was calculated offline using the formula described by Malan (25). The respiratory frequency (RF) was calculated from the extrapolation of the plethysmographic signal. Pulmonary ventilation (PV) was calculated as the product of TV and RF. MAP and HR were calculated from the arterial pulse pressure.

Statistical Analysis The results are expressed as means¡standard errors of the mean (SEM). Changes in the MAP and PV in response to hypoxia, hypercapnia and KCN were evaluated by the paired t-test. The comparison between CBD and the intact rats was evaluated by Student’s t-test. p,0.05 was considered to be statistically significant.

& RESULTS Hemodynamic responses to KCN in intact and CBD rats The baseline values for MAP and HR were similar between the intact (n = 8) and CBD rats (n = 7) and are shown in Table 1. KCN produced a hypertensive response combined with bradycardia in intact rats but did not change the MAP or HR in CBD rats (Figure 1).

Hemodynamic responses to different levels of hypoxia and hypercapnia Hypoxia with 15% O2 elicited a small but significant hypotensive and tachycardic response that was similar between intact and CBD rats (Figure 2). Hypoxia with 10% O2 did not change the MAP but elicited a tachycardic response in intact rats. In CBD rats, 15% O2 elicited a marked decrease in AP and increased HR (Figure 2). The strongest hypoxia (6% O2) elicited a hypertensive and bradycardic response in intact rats and a decrease in MAP with no change in HR in CBD rats (Figure 2). Hypercapnia (10% CO2+31% O2) promoted an increase in the MAP and a decrease in HR, which was similar in intact and CBD rats (Figure 3).

Response of pulmonary ventilation to hypoxia and hypercapnia The baseline values for PV were similar between the intact and CBD rats (Table 1). The 3 levels of hypoxia

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Acute hypoxia and hypercapnia in rats. Sabino JPJ et al.

Table 1 - Baseline levels of mean arterial pressure (MAP), heart rate (HR) and pulmonary ventilation (PV) in intact and carotid body-denervated (CBD) rats before chemoreceptor stimulation with KCN, hypoxia and hypercapnia. The data are expressed as means¡SEM. INTACT

KCN 15% O2 10% O2 6% O2 10% CO2+31% O2

CBD

INTACT

CBD

MAP

mmHg

bpm

mmHg

bpm

(mL.kg-1.min-1)

100¡2 101¡4 103¡3 109¡6 103¡4

413¡19 411¡17 398¡10 394¡8 401¡12

94¡4 93¡5 99¡4 99¡4 98¡4

426¡25 415¡20 415¡20 427¡23 439¡24

1075¡98 1024¡61 961¡81 918¡105

924¡56 1012¡136 935¡141 983¡111

increased PV in both groups (Figure 2). However, the PV response elicited by 15% inhaled O2 was smaller than the responses elicited by 10% or 6% O2 in both groups. Moreover, peripheral chemoreceptor denervation attenuated the ventilatory responses in CBD rats (Figures 2). Hypercapnia (10% CO2+31% O2) promoted a similar increase in PV in both groups (Figure 3).

finding suggests that the hemodynamic response to this level of hypoxia in rats does not depend on carotid chemoreceptor activation because a similar response was

& DISCUSSION The major finding of this study is that the hemodynamic response varies markedly according to the level of hypoxia (i.e., AP and HR responses can change markedly depending on the level of hypoxia applied). Moreover, this study demonstrated that the hemodynamic and ventilatory responses to the combination of hypercapnia and hyperoxia did not depend on the peripheral carotid chemoreceptors. Hypoxia with 15% O2 produced slight but significant hypotension and tachycardia in intact and CBD rats. This

Figure 2 - Bar graph showing the changes in the mean arterial pressure (MAP, top), heart rate (HR, middle) and pulmonary ventilation (PV, bottom) in response to 3 levels of hypoxia (15%, 10% and 6% of inhaled O2) in intact and carotid bodydenervated (CBD) rats. * p,0.05 compared to 15% O2.+p,0.05 compared to the same level of hypoxia in intact rats.

Figure 1 - Bar graph showing the changes in the mean arterial pressure (MAP) and heart rate (HR) in response to KCN in intact and carotid body-denervated (CBD) rats. *p,0.05 compared to intact rats.

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to counteract a decrease in total peripheral resistance triggered by the effect of hypoxia on skeletal muscle fibers (26) or vasodilation of the blood vessel walls (27,28). The tachycardia in the intact rats could be due to the hypoxiainduced activation of the peripheral chemoreflex, which increases sympathetic activity and enhances the release of plasma catecholamines (33). Studies on conscious intact rats have shown that hypoxia with 8 or 10% O2 for 30 min decreased the MAP and increased HR (9,13,14). In addition, Sugimura et al. (34) also observed that 10% inhaled O2 for 10 min elicited hypotension combined with tachycardia in conscious rats. However, the difference between the results from previous studies (9,13,14,34) and this study might be explained by the difference in the timeframe of exposure to hypoxia among the studies (1 min vs. 10 or 30 min). The hypotensive response caused by 10% O2 in the CBD rats might be explained by attenuation of the sympathetic vasoconstrictor outflow due to the removal of the carotid chemoreceptor drive. It has been shown that hypoxia increases adrenal sympathetic nerve activity and catecholamine secretion (33), while sympathetic activation is not triggered in the absence of peripheral chemoreceptors in CBD rats. The tachycardia observed in the CBD rats exposed to 10% O2 may be due to the baroreflex, which is triggered by a fall in AP (30-32). Although the CBD rats do not have an active peripheral chemoreflex, their baroreflex is well preserved, which explains this reflex tachycardia. Hypoxia with 6% O2 in the conscious intact rats produced marked hypertension and bradycardia, while in the CBD rats, 6% O2 produced hypotension without any change in HR. The findings of this study in conscious intact rats are in line with previous studies (16,17), indicating that maximal peripheral chemoreceptor activation is attained with 6% to 7% of inhaled O2. Previous studies in anesthetized rats have demonstrated that hypoxia promotes hypotension and tachycardia (10-12). Nevertheless, the undesirable effect of anesthesia and the longer exposure of 3 min of hypoxia might explain the discrepancy in these results (10-12) compared to this study. Hypoxia with 6% O2 in conscious CBD rats produced significant hypotension without any change in HR. These findings were similar to the hemodynamic responses observed with 10% O2, except that the increase in baseline HR did not reach statistical significance. Thus, due to the lack of activation of the peripheral chemoreceptors in CBD rats, the hypotensive response might be explained by the direct effect of hypoxia in the blood vessel wall (27,28) and endothelial cells to promote vasodilation (28,35). Hypercapnia (10% CO2+31% O2) increased MAP and decreased HR in intact and CBD rats. These findings indicate that the hemodynamic response to hypercapnia does not involve the peripheral carotid chemoreceptors but involves the central chemoreceptors. These results are in line with the previous findings of Walker and Brizzee (23) in conscious rats.

Figure 3 - Bar graph showing the changes in the mean arterial pressure (MAP, top), heart rate (HR, middle) and pulmonary ventilation (PV, bottom) in response to hypercapnia (10% CO2+31% O2) in intact and carotid body-denervated (CBD) rats. *p,0.05 compared to intact rats.

observed in CBD rats. In line with this study, Marshall and Metcalfe (10-12) also reported hypotension and tachycardia in response to 15% inhaled O2. The hypotensive response with 15% O2 might be explained by the release of adenosine from the skeletal muscle fibers (26) or blood vessel walls (27,28). Adenosine acts on endothelial A1-receptors to stimulate the synthesis and release of NO, which ultimately induces vasodilation (29). The tachycardia observed in intact and CBD rats exposed to 15% O2 might be due to the baroreflex, which is triggered by a drop in AP (30-32). Hypoxia with 10% O2 produced a similar hemodynamic response compared to 15% O2 (i.e., hypotension and tachycardia, except that the hypotensive response did not achieve statistical significance in the intact rats). Nevertheless, the decrease in AP in response to 10% O2 in the CBD rats was markedly greater compared to 10% O2 in the intact rats. The lack of a significant hypotensive response in the intact rats might be explained by sympathetic activation elicited by the peripheral chemoreceptors

Ventilatory responses Hypoxia with 15, 10 and 6% O2 increased PV in the intact rats but attenuated the ventilatory response in CBD rats. These results indicate that in intact rats, PV is partially dependent on the peripheral carotid chemoreceptors because in the absence of this mechanism, the central chemoreceptors may have triggered the blunted response. It is worth mentioning that the findings concerning the PV responses observed in this study are in line with previous

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Acute hypoxia and hypercapnia in rats. Sabino JPJ et al. 12. Marshall JM, Metcalfe JD. Influences on the cardiovascular response to graded levels of systemic hypoxia of the accompanying hypocapnia in the rat. J Physiol. 1989;410:381-94. 13. Hirakawa H, Nakamura T, Hayashida Y. Effect of carbon dioxide on autonomic cardiovascular responses to systemic hypoxia in conscious rats. Am J Physiol. 1997;273(2 Pt 2):R747-54. 14. Murasato Y, Hirakawa H, Harada Y, Nakamura T, Hayashida Y. Effects of systemic hypoxia on R-R interval and blood pressure variabilities in conscious rats. Am J Physiol. 1998;275(3 Pt 2):H797-804. 15. Marcus NJ, Olson EB Jr., Bird CE, Philippi NR, Morgan BJ. Timedependent adaptation in the hemodynamic response to hypoxia. Respir Respir Physiol Neurobiol. 2009;165(1):90-6, http://dx.doi.org/10.1016/j. resp.2008.10.013. 16. Bao G, Randhawa PM, Fletcher EC. Acute blood pressure elevation during repetitive hypocapnic and eucapnic hypoxia in rats. J Appl Physiol. 1997;82(4):1071-8. 17. Barros RC, Bonagamba LG, Okamoto-Canesin R, de Oliveira M, Branco LG, Machado BH. Cardiovascular responses to chemoreflex activation with potassium cyanide or hypoxic hypoxia in awake rats. Auton Neurosci. 2002;97(2):110-5, http://dx.doi.org/10.1016/S1566-0702(02)00050-4. 18. Richardson DW, Wasserman AJ, Patterson JL, Jr. General and regional circulatory responses to change in blood pH and carbon dioxide tension. J Clin Invest. 1961;40:31-43, http://dx.doi.org/10.1172/JCI104234. 19. Kollai M, Koizumi K. Reciprocal and non-reciprocal action of the vagal and sympathetic nerves innervating the heart. J Auton Nerv Syst. 1979;1(1):33-52, http://dx.doi.org/10.1016/0165-1838(79)90004-3. 20. Rose Jr CE, Althaus JA, Kaiser DL, Miller ED, Carey RM. Acute hypoxemia and hypercapnia: increase in plasma catecholamines in conscious dogs. Am J Physiol. 1983;245(6):H924-9. 21. Somers VK, Mark AL, Zavala DC, Abboud FM. Contrasting effects of hypoxia and hypercapnia on ventilation and sympathetic activity in humans. J Appl Physiol. 1989;67(5):2101-6. 22. Fukuda Y, Sato A, Suzuki A, Trzebski A. Autonomic nerve and cardiovascular responses to changing blood oxygen and carbon dioxide levels in the rat. J Auton Nerv Syst. 1989;28(1):61-74, http://dx.doi.org/ 10.1016/0165-1838(89)90008-8. 23. Walker BR, Brizzee BL. Cardiovascular responses to hypoxia and hypercapnia in barodenervated rats. J Appl Physiol. 1990;68(2):678-86. 24. Franchini KG, Krieger EM. Cardiovascular responses of conscious rats to carotid body chemoreceptor stimulation by intravenous KCN. J Auton Nerv Syst. 1993;42(1):63-9, http://dx.doi.org/10.1016/0165-1838(93)90342-R. 25. Malan A. Ventilation measured by body plethysmography in hibernating mammals and in poikilotherms. Respir Physiol. 1973;17(1):32-44, http://dx.doi.org/10.1016/0034-5687(73)90108-4. 26. Marshall JM, Thomas T, Turner L. A link between adenosine, ATPsensitive k+channels, potassium and muscle vasodilatation in the rat in systemic hypoxia. J Physiol. 1993;472:1-9. 27. Mian R, Marshall JM. The role of adenosine in dilator responses induced in arterioles and venules of rat skeletal muscle by systemic hypoxia. J Physiol. 1991;443:499-511. 28. Mian R, Marshall JM. The role of adenosine in mediating vasodilatation in mesenteric circulation of the rat in acute and chronic hypoxia. J Physiol. 1995;489( Pt 1):225-34. 29. Ray CJ, Marshall JM. Measurement of nitric oxide release evoked by systemic hypoxia and adenosine from rat skeletal muscle in vivo. J Physiol. 2005;568(Pt 3):967-78, http://dx.doi.org/10.1113/jphysiol.2005.094854. 30. Kirchheim H R. Systemic arterial baroreceptor reflexes. Physiol Rev. 1976;56(1):100-77 31. Chapleau MW, Hajduczok G, Abboud FM. Mechanisms of resetting of arterial baroreceptors: an overview. Am J Med Sci. 1988;295(4):327-34. 32. Krieger EM, Salgado HC, Michelini LC. Resetting of the baroreceptors. In: International Review of Physiology, University Park Press, Baltimore, ed. Guyton AC, Hall JE. 1982;26:119-46. 33. Biesold D, Kurosawa M, Sato A, Trzebski A. Hypoxia and hypercapnia increase the sympathoadrenal medullary functions in anesthetized, artificially ventilated rats. Jpn J Physiol. 1989;39(4):511-22. 34. Sugimura M, Hirose Y, Hanamoto H, Okada K, Boku A, Morimoto Y, et al. Influence of acute progressive hypoxia on cardiovascular variability in conscious spontaneously hypertensive rats. Auton Neurosci. 2008;141(1-2):94-103, http://dx.doi.org/10.1016/j.autneu.2008.05.008. 35. Marshall JM. Adenosine and muscle vasodilatation in acute systemic hypoxia. Acta Physiol Scand. 2000;168(4):561-73, http://dx.doi.org/10. 1046/j.1365-201x.2000.00709.x. 36. Forster HV, Martino P, Hodges M, Krause K, Bonis J, Davis S, et al. The carotid chemoreceptors are a major determinant of ventilatory CO2 sensitivity, and PaCO2 during eupneic breathing. Adv Exp Med Biol. 2008;905:322-26, http://dx.doi.org/10.1007/978-0-387-73693-8_56. 37. Forster HV, Smith CA. Central CO2 chemoreception in cardiorespiratory control contributions of central and peripheral chemoreceptors to the ventilator response to CO2/H+. J Appl Physiol. 2010;108(4):989-94, http://dx.doi.org/10.1152/japplphysiol.01059.2009.

studies in intact and CBD rats (6,7,23). In addition, recent studies have suggested a significant role for the peripheral chemoreceptors as an important mechanism for the ventilatory response during exposure to hypercapnia (36,37). Moreover, the response of PV to hypercapnia (10% CO2+31% O2) was similar in intact and CBD rats. Thus, the PV response to hypercapnia depends mainly on the central chemoreceptors. It is possible that the severity of hypercapnia makes the peripheral chemoreceptor less important in modulating the ventilatory response. A similar response was observed in previous studies on conscious intact rats, which indicated that hypercapnia with 6 to 7% CO2 increased PV but did not produce a significant difference in the ventilatory response compared to CBD rats (7-8). In summary, this study shows that the hemodynamic and ventilatory responses varied according to the level of hypoxia. Nevertheless, the hemodynamic and ventilatory responses to hypercapnia (10% CO2+31% O2) did not depend on peripheral carotid chemoreceptors.

& ACKNOWLEDGMENTS Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientifico e Tecnolo´gico (CNPq) and Coordenadoria de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES).

& AUTHOR CONTRIBUTIONS Sabino JP collected the data for this manuscript. Oliveira M and Giusti H provided technical support concerning the surgical procedures for vessel cannulation (Oliveira M) and carotid body denervation (Giusti H) and provided support during data acquisition. Glass ML provided valuable insight into data interpretation and manuscript revision. Fazan Jr R provided a substantial contribution to protocol design and data analysis. Salgado HC was the mentor of Sabino JP and was responsible for the conception of the rationale for the development of the study.

& REFERENCES 1. Chugh SS, Chua TP, Coats AJS. Peripheral chemoreflex in chronic heart failure: Friend and foe. Am Heart J. 1996;132(4):900-4, http://dx.doi.org/ 10.1016/S0002-8703(96)90333-6. 2. Schultz HD, Sun SY. Chemoreflex function in heart failure. Heart Fail Rev. 2000;5(1):45-56, http://dx.doi.org/10.1023/A:1009846123893. 3. Somers VK, Mark AL, Abboud FM. Interaction of baroreceptor and chemoreceptor reflex control of sympathetic nerve activity in normal humans. J Clin Invest. 1991;87(6):1953-7, http://dx.doi.org/10.1172/JCI115221. 4. Pitsikoulis C, Bartels MN, Gates G, Rebmann RA, Layton AM, De Meersman RE. Sympathetic drive is modulated by central chemoreceptor activation. Respir Physiol Neurobiol. 2008;164(3):373-9, http://dx.doi. org/10.1016/j.resp.2008.08.010. 5. Grisk O, Exner J, Schmidt M, Honig A. Effects of acute hypoxia and hyperoxia on ventilation in spontaneously hypertensive and normotensive rat. J Auton Nerv Syst. 1996;57(3):177-80, http://dx.doi.org/10. 1016/0165-1838(95)00079-8. 6. Roux JC, Peyronnet J, Pascual O, Dalmaz Y, Pequignot JM. Ventilatory and central neurochemical reorganisation of O2 chemoreflex after carotid sinus nerve transection in rat. J Physiol. 2000;522(Pt 3):493-501, http:// dx.doi.org/10.1111/j.1469-7793.2000.t01-4-00493.x. 7. Serra A, Brozoski D, Hedin N, Franciosi R, Forster HV. Mortality after carotid body denervation in rats. J Appl Physiol. 2001;91(3):1298-306. 8. Mouradian GC, Forster HV, Hodges MR. Acute and chronic effects of carotid body denervation on ventilation and chemoreflexes in three rat strains. J Physiol. 2012;590(Pt 14):3335-47, http://dx.doi.org/10.1113/ jphysiol.2012.234658. 9. Walker BR. Role of vasopressin in the cardiovascular response to hypoxia in the conscious rat. Am J Physiol. 1986;251(6 Pt 2):H1316-23. 10. Marshall JM, Metcalfe JD. Cardiovascular changes associated with augmented breaths in normoxia and hypoxia in the rat. J Physiol. 1988;400:15-27. 11. Marshall JM, Metcalfe JD. Analysis of the cardiovascular changes induced in the rat by graded levels of systemic hypoxia. J Physiol. 1988;407:385-403.

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REVIEW

Current strategies for preventing renal dysfunction in patients with heart failure: a heart failure stage approach Victor Sarli Issa,I Lućia Andrade,II Edimar Alcides BocchiI I Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Institute do Coraçaõ (InCor), Saõ Paulo/SP, Brazil. II Faculdade de Medicina da USP (FMUSP), Laborato´rio de Pesquisa Ba´sica da Disciplina de Nefrologia, Saõ Paulo/SP, Brazil.

Renal dysfunction is common during episodes of acute decompensated heart failure, and historical data indicate that the mean creatinine level at admission has risen in recent decades. Different mechanisms underlying this change over time have been proposed, such as demographic changes, hemodynamic and neurohumoral derangements and medical interventions. In this setting, various strategies have been proposed for the prevention of renal dysfunction with heterogeneous results. In the present article, we review and discuss the main aspects of renal dysfunction prevention according to the different stages of heart failure. KEYWORDS: Heart Failure; Renal Failure; Renal Dysfunction. Issa VS, Andrade L, Bocchi EA. Current strategies for preventing renal dysfunction in patients with heart failure: a heart failure stage approach. Clinics. 2013;68(3):401-409. Received for publication on September 20, 2012; First review completed November 19, 2012; Accepted for publication on November 19, 2012 E-mail: victor.issa@incor.usp.br Tel.: 55 11 2661-5419

mortality or whether it merely serves as a marker of a more severe disease remains unclear. Conditions such as diabetes and hypertension may offer an epidemiological link that associates heart failure and kidney disease. Additionally, kidney disease and heart failure have been suggested not to represent single clinical entities but rather to represent manifestations of a broader vascular injury associated with aging that affects multiple organs (6). In this setting, various strategies have been proposed for the prevention of renal dysfunction with heterogeneous results. In the present article, we review and discuss the main aspects of renal dysfunction prevention according to the different stages of heart failure.

& SCOPE OF THE PROBLEM Renal dysfunction is common during episodes of acute decompensated heart failure and is found in up to 64% (1) of patients at hospital admission. Historical analysis indicates that the mean creatinine level at admission has risen in recent decades (2). Different mechanisms underlying this change over time have been proposed. First, as a consequence of better clinical care and surgical interventions, there has been a shift towards a larger number of patients with advanced heart failure (3), and in these circumstances, patients tend to be older and to accumulate risk factors, such as hypertension and diabetes mellitus (4,5). Additionally, hemodynamic and neurohumoral derangements are exacerbated during episodes of decompensation and further contribute to de novo kidney dysfunction or the worsening of a chronic kidney disease. Medical interventions may also produce a reduction of the glomerular filtration rate (GFR) by reducing blood pressure, inducing hypovolemia or reducing the glomerular perfusion pressure. However, in biological phenomena, epidemiological associations may not reflect causality, and whether worsening renal function itself contributes to the increased

Definitions In 2005, the American Heart Association and the American College of Cardiology proposed a staging classification that incorporated a conceptual change relative to the classical definition of heart failure syndrome, which dealt almost exclusively with symptomatic patients. The new categorization incorporated stages A and B, with stage A identifying patients at risk for heart failure but without structural heart disease or symptoms of the syndrome and stage B encompassing patients with structural heart disease but without symptoms of heart failure. During the course of the syndrome, patients may experience a decrease in kidney function, and strategies for treatment and prevention should take into consideration the burden of epidemiological characteristics and the presence of risk factors in the context of the hemodynamic and neurohumoral events associated with heart failure syndrome.

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Patients with heart failure who are experiencing a decrease in renal function usually have peculiar characteristics, including manifestations of hypervolemia, oliguria and diuretic resistance, in a clinical picture frequently referred to as cardiorenal syndrome. Cardiorenal syndrome has been suggested to encompass disorders of the heart and kidneys, either acute or chronic, in such an association that dysfunction in one organ may induce acute or chronic dysfunction of the other. A categorization into five different types has been proposed (7). Although such a categorization may be helpful in identifying patients with different pathophysiological mechanisms, it includes a heterogeneous group of clinical presentations or diagnoses under the same definition and adds little value to the care of patients with a condition in which the mechanisms remain largely unknown and for which a precise diagnosis may still be elusive in clinical practice. The AKIN and RIFLE criteria have been proposed to stratify patients at risk for the occurrence of renal dysfunction (Figure 1). However, it should be acknowledged that these criteria are mostly derived from cohorts of patients in heterogeneous clinical conditions and represent a general approach to critically ill patients that including cardiac surgery patients, burn patients and liver and bone marrow transplantation patients. Furthermore, a subset of patients who experience an increase of 0.3-0.5 mg/dL in serum creatinine or a decrease in glomerular filtration rate (GFR) of 9-15 ml/min during admission has been recognized as being at increased risk for renal dysfunction (8,9). Different biomarkers have been suggested for the diagnosis of renal dysfunction in the setting of heart failure (Table 1).

Table 1 - Biomarkers in heart failure. GLOMERULAR

FUNCTION

TUBULOINTERSTITIAL

FUNCTION

CREATININE GFR ESTIMATION BUN CYSTATIN C

NAG NGAL KIDNEY INJURY MOLECULE

ALBUMINURIA

FATTY ACID BINDING PROTEIN

INTERLEUKIN 18,

URINARY

EXOSOMES

BUN: blood urea nitrogen; GFR: glomerular filtration rate; NAG: N-ACETYLB-D-GLUCOSAMINIDASE; NGAL: NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN.

are precipitated by renal hypoperfusion; in addition, clinical experience has indicated that interventions directed towards increasing cardiac output often restore organ perfusion, as indicated by clinical parameters such as urine output and mental status. Clinically, worsening renal function typically occurs within days of hospitalization, which suggests a direct causative effect of the hemodynamic derangement observed when initiating treatment for acute decompensated heart failure (ADHF), and patients with progressive pump failure or cardiogenic shock often develop progressive renal impairment that is reversible with advanced support. However, data from clinical trials have challenged this rationale. The ESCAPE trial tested the influence of a pulmonary artery catheterâ&#x20AC;&#x201C;guided therapy in individuals with acute decompensated heart failure and found no correlation between the baseline renal function and cardiac index (10). Furthermore, an improvement in the cardiac index did not result in improved renal function. This notion is additionally supported by other investigations that showed that an improved cardiac index or decreased pulmonary capillary wedge pressure failed to predict improvement in renal function (11). Data from the ADHERE registry indicated that most patients with acute decompensated heart failure present with elevated, rather than low, blood pressure (12) and that the incidence of worsening renal function is similar among patients with

Pathophysiological aspects Classically, the occurrence of renal dysfunction in patients with heart failure has been attributed to a low-flow state in which the decline in glomerular filtration rate is directly determined by reduced cardiac output. Some indirect evidence supports this theory, especially the occurrence of neurohumoral phenomena in patients with heart failure that

Figure 1 - The Acute Kidney Injury Network and Risk, Injury, Failure, Loss, and End-stage kidney disease definitions.

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progressive condition and that interventions should be guided by disease stage, patient clinical presentation and the presence of co-morbidities (Figure 2).

reduced or preserved systolic function (13). Given these findings, other mechanisms have been proposed. An alternative explanation could involve a redistribution of blood from arterial to venous circulation that leads to an effective reduction in renal blood flow. The resulting reduction in arterial effective volume can result in increased sodium and water absorption through stimulation of the sympathetic nervous system, the renin-angiotensin-aldosterone system and vasopressin secretion to preserve renal perfusion and the renal filtration fraction. However, such mechanisms are often difficult to demonstrate in individual patients at the bedside. Modifications of transrenal perfusion pressure have increasingly received attention. Transrenal perfusion pressure is calculated as the mean arterial pressure minus the central venous pressure; therefore, for a patient with volume overload and heart failure, the combination of increased central venous pressure with low systemic pressure may lead to a severe compromise of the net renal perfusion pressure. Systemic pressure may be reduced by a decrease in cardiac output and hypotension; additionally, drugs currently used to treat heart failure patients may potentiate these effects. For example, diuretics may cause hypovolemia, and drugs that inhibit the angiotensin system may induce vasodilation of the efferent circulation and reduction in the glomerular perfusion pressure. Because the kidney is an encapsulated organ, when renal congestion occurs, the increased venous pressure may distend the venules surrounding the distal ends of the tubules, which can obliterate the lumen of the tubule (14). Other studies have simply suggested that the increased central venous pressure could be transmitted back to the renal veins, thereby causing an increase in the renal interstitial pressure (15,16). This possibility is reinforced by the observation that intrarenal and systemic angiotensin II concentrations increase with increasing renal venous pressure (17,18). Elevation of renal venous pressure from the extrinsic compression of the veins has also been suggested as a mechanism contributing to reduced renal function in patients with ascites (19). Non-hemodynamic mechanisms are also involved. Hyperactivation of the neurohumoral and inflammatory axes has been associated with progression or renal and cardiac dysfunction. The activation of RAAS and SNS induces increased afterload, myocardial oxygen consumption, sodium retention and ventricular remodeling and is associated with worse outcomes among patients with heart failure. Accordingly, the activation of these systems may also contribute to the acceleration of renal disease, as indicated by the positive effects of RAAS blockade on renal function in patients with hypertension and diabetes. However, these effects are likely mediated by endothelial dysfunction, oxidative stress, fibrosis and inflammation (20).

General approach As discussed above, extreme variations in volume can reduce the GFR. In hypovolemia, GFR can be reduced by the reduction of effective plasmatic volume and renal hypoperfusion. In contrast, hypervolemic patients may have impaired renal function because of renal venous congestion, reduced renal perfusion pressure or renal compression. Therefore, intense and sudden variations in the plasmatic volume of patients with decompensated heart failure should be strongly avoided. Thus, excessive diuretic therapy has traditionally been seen as deleterious, and in fact, reduced volume has been described in patients with chronic heart failure under diuretic therapy. However, transient elevations in creatinine (possibly associated with modifications of plasmatic volume and blood pressure) have been considered as common in patients with decompensated heart failure and thus considered to not carry the same prognostic significance as persistent elevations, especially in patients with predominant right ventricular failure who show improvements in congestive phenomena (21). Additionally, other interventions currently considered to be standard for the treatment of acute decompensated heart failure may have deleterious effects on renal function. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers can decrease filtration pressure within the glomerulus by inducing systemic hypotension and predominant vasodilation of the glomerular efferent arteriole. Although ACE inhibitors and ARBs remain a mainstay for the treatment of patients with chronic heart failure, their utility during episodes of decompensation has not been clearly demonstrated, and current guidelines do not support the interruption of therapy with ACE inhibitors and ARBs during episodes of acute decompensated heart failure. The influence of beta-blockers on renal function is much less understood. A small study suggested that the initiation of beta-blocker therapy was associated with preserved renal function in heart failure patients with a lower baseline GFR but not in those with a higher baseline GFR (22), although these results have yet to be confirmed. Finally, small retrospective studies have suggested that spironolactone may increase creatinine levels (23,24) and should be used cautiously in patients with reduced GFR. During episodes of acute decompensation, invasive interventions are often required, and clinicians should be aware of the possibility of contrast-induced nephropathy (CIN). Recommendations for the prevention of CIN have been published (25), and additional care should be taken to avoid congestive phenomena during pre-procedure hydration. Frequently, a dose reduction or cessation of the diuretic therapy is performed in place of active intravenous hydration. As generally recommended for patients with chronic heart failure, nonsteroidal anti-inflammatory drugs (NSAIDs) should not be used during an episode of acute decompensation, as NSAIDs induce decreases in prostaglandin E2, which leads to increased sodium and water reabsorption that can result in weight gain and edema. As noted, in individuals with decreased circulating volume, vasodilatory prostaglandins are produced by the kidneys to offset other vasoconstricting autacoids. In a clinical setting in which the renal blood flow depends on prostaglandin

Preventive measures Despite the recognition of the epidemiological and prognostic relevance of renal dysfunction in patients with decompensated heart failure and all of the efforts directed to the discovery of novel and more accurate methods for its diagnosis, few specific interventions are currently available for the prevention of renal dysfunction in this setting. Most of bedside protocols are based on experimental data or on data derived from other clinical conditions. We will briefly review the general approaches currently available and some specific interventions currently proposed or under investigation, with a focus on the concept that heart failure is a

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Figure 2 - Prevention strategies for renal dysfunction according to the heart failure stage.

synthesis, NSAIDs can significantly decrease renal blood flow, with resultant acute renal failure (26).

with a possible trend towards proteinuria reduction (2,31). However, some studies with rosuvastatin indicate a possible negative effect on renal function and proteinuria. The GISSIHF trial (32) and the CORONA trial (33) tested the effects of statin therapy in patients with chronic heart failure and did not demonstrate any influence on the hard end-points. Therefore, even if statins have a positive influence on the renal function of patients with heart failure, an effect on the overall prognosis is unlikely. Adenosine A1 receptor antagonists. Stimulation of the adenosine A1 receptor on the glomerular afferent arteriole reduces renal blood flow and the glomerular filtration rate (GFR), and also increases sodium and water reabsorption through the stimulation of receptors on the proximal tubules. Adenosine receptor antagonists, such as aminophylline and theophylline, have been used in heart failure patients, but arrhythmic effects have hampered their use. Recently, the PROTECT trial tested the effects of rolofylline as a renal protective strategy in 2,033 patients with acute heart failure, and the results indicated no effect of rolofylline in this setting (34). Xanthine oxidase inhibitors. Data derived from experimental and clinical models have suggested that xanthine oxidase inhibitors have positive metabolic effects in the failing myocardium that provide increases in energy delivered by adenosine triphosphate (35). In a small study of patients with ischemic cardiomyopathy, a single intravenous dose of 400 mg of oxypurinol led to a

Specific interventions Statins. Data from studies in patients with chronic kidney disease suggest that statins may preserve renal function (27). An analysis of the CARE (28) study demonstrated a significant difference in the rate of decline with pravastatin in individuals with severe CKD at baseline relative to the placebo. In the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) study (29), the effect of atorvastatin administered at a dose 10 to 80 mg/day on renal function was compared with the ‘‘usual care’’ in previously untreated dyslipidemic patients with coronary heart disease. At the end of the study, creatinine clearance had increased by 12% in the atorvastatin group and 4.9% in the ‘‘usual care’’ patients. In a subanalysis of the Treating to New Targets (TNT) study (30), the effect of intensive lipid reduction on renal function using 10 mg versus 80 mg of atorvastatin in patients with coronary heart disease was evaluated. The expected 5-year decline in renal function was not observed in this study, and the estimated GFR improved in both TNT treatment groups but was significantly greater with atorvastatin at 80 mg than at 10 mg, which suggests a dose-related benefit. Additionally, a meta-analysis of 13 small prospective, controlled trials examining the effect of statins on renal function showed that treatment with statins reduced the rate of decline in the GFR

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reduction in end-systolic volume and an increased left ventricular ejection fraction relative to the placebo. In patients with chronic kidney disease (36), the use of allopurinol has been associated with a reduction in cardiovascular events and increases in the GFR (37,38). However, the effects of allopurinol for renal protection in patients with heart failure are still under discussion. Xanthine oxidase-derived reactive oxygen species accumulation leads to nitric oxide scavenging and endothelial dysfunction but also to various other detrimental metabolic, functional and immunologic effects. Novel data are emerging to suggest direct effects of uric acid on immune stimulation and metabolic interference (39). Renin inhibitors. Despite the current availability of medical treatments for heart failure, the residual rates of hospitalization and death remain high. One putative explanation for these high rates is insufficient RAAS inhibition produced by the triggering of counterregulatory mechanisms of renin release by single-site RAS blockade. Therefore, the use of a specific inhibitor of human renin, such as aliskiren, has been proposed. Aliskiren was tested in addition to ACE inhibitors or ARBs in patients with diabetes mellitus and renal dysfunction; however, an increase in adverse events (increased incidence of renal complications, hyperkalemia, hypotension and non-fatal strokes) and no apparent benefits among the patients randomized to aliskiren prompted the data safety and monitoring board for the study to recommend its termination. Subsequently, the ASPIRE study reported a significant increase in the risk of renal failure, hypotension and hyperkalemia with the combination of aliskiren and an ACE inhibitor or ARB in high-risk post-myocardial infarction patients (40). Ongoing trials will evaluate the effects of aliskiren on the prognosis, ventricular function and renal function in the setting of chronic (41) and acute heart failure (42). Renal denervation. Hyperactivation of the SNS is involved in the pathophysiology of hypertension, renal insufficiency and heart failure. Devices have been developed for ablation of the renal sympathetic nerves by a radiofrequency-emitting catheter inserted percutaneously into the femoral artery in the groin and consecutively advanced to lie in the lumen of each renal artery. Sympathetic nerves enter the human kidneys through the walls of the renal arteries and are thus within reach of the ablative energy. This type of intervention has been reported to be successful in patients with resistant hypertension (43). Renal function, as assessed by serum creatinine, eGFR and cystatin C concentrations, was unchanged from the baseline in both groups at 6 months. Several ongoing clinical trials are investigating the safety and efficacy of renal denervation in patients with CHF, and its application as a preventive measure for renal dysfunction remains speculative (44). Low-dose dopamine. The infusion of low-dose dopamine (4-5 mg kg-1min-1) has been shown to induce renal vasodilation and natriuresis mediated by the stimulation of the dopamine alpha-1 and alpha-2 receptors in the proximal tubule, the thick ascending loop of Henle and the cortical collecting ducts (45). In addition, putative renoprotective effects of dopamine have also been suggested in various clinical settings. In a meta-analysis of 61 trials that randomly assigned 3,359 patients at risk for acute renal failure to receive low-dose dopamine or placebo, dopamine was associated with a 24% increase in urine output, 4% relative decrease in serum creatinine level and 6% relative

increase in measured creatinine clearance (46). In patients with systolic heart failure, low-dose dopamine has also been demonstrated to increase renal blood flow and the GFR. In a clinical trial with 60 patients with acute decompensated heart failure, the combination of infused low-dose furosemide and low-dose dopamine was suggested to be as effective as a high-dose furosemide infusion in terms of the clinical and diuretic response and to be associated with a significantly lower rate of worsening renal function, which suggests a renoprotective effect in this patient population (47). The long-term effects of this intervention in a larger population of patients are yet to be determined. Diuretics. Despite the scarce evidence based on prospective data and clinical trials, diuretics have long been a mainstay in the treatment of episodes of acute decompensated heart failure in the presence of congestive phenomena. Concerns regarding diuretic use involve ototoxicity, hypocalcemia and diuretic-induced hypovolemia; these conditions become particularly important in the setting of ADHF because of the presence of hypotension, right ventricular failure and elevated intra-abdominal pressure as well as the use of high diuretic doses. Thus, a renoprotective strategy could potentially be derived from the continuous infusion of smaller doses of a diuretic. Different diuretic strategies were tested in a clinical trial (48) that assigned patients with acute decompensated heart failure to receive furosemide administered intravenously in a bolus every 12 hours or by continuous infusion and at a low or high dose; the authors concluded that there were no significant differences in the change in renal function when diuretic therapy was administered by bolus compared with continuous infusion or at a high dose compared with a low dose. Human recombinant B-type natriuretic peptide. Nesiritide is a systemic pulmonary vasodilator that is capable of promoting natriuresis and diuresis and of inhibiting the RAAS. Initial experiments in patients without heart failure and outpatients with stable heart failure produced conflicting results regarding the effect of nesiritide on the GFR, with some studies suggesting an improvement in the GFR and others showing no impact (49,50,51). Later, a metaanalysis found that treatment with nesiritide was associated with worsened renal function (52). A clinical trial with 75 patients with acute decompensated heart failure showed that nesiritide had no impact on renal function (53). Recently, a larger clinical trial with 7,141 patients confirmed that nesiritide had a neutral effect on renal function but was associated with an increase in the rate of hypotension (54). These findings do not warrant the use of nesiritide as a renoprotective measure in patients with ADHF. Other similar molecules, such as alpha-human atrial natriuretic peptide (carperitide), are currently under investigation. Calcium sensitizers. This class of drugs includes inotropic agents, such as levosimendan, that also produce arterial and venous vasodilation through the activation of ATP-sensitive potassium channels. Because central venous pressure is an independent predictor of the GFR in patients with heart failure, levosimendan has been proposed as an agent that may be associated with improved renal function (55). Thus, levosimendan infusion has been described to improve the GFR more than infusion of dobutamine, even though both treatment regimens were associated with an increase in urine output (56). Another study suggested a sustained improvement in renal function up to 3 months

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after infusion in patients with advanced heart failure eligible for heart transplantation (57). In the LIDO trial, levosimendan showed greater improvement of serum creatinine levels than dobutamine (58). However, the use of levosimendan in patients with ADHF has been hampered by the results of more recent clinical trials: in SURVIVE (59), the primary endpoint of total mortality was not different between levosimendan and dobutamine; in REVIVE-II (60), the combined clinical endpoint including mortality and symptoms did indicate a benefit of levosimendan over a placebo. Even if the putative beneficial effects of levosimendan on renal function are confirmed, the positive effects on renal blood flow may be offset by the drop in blood pressure and risk of arrhythmia. Ultrafiltration. Ultrafiltration (UF) has been proposed as an alternative to diuretics to obtain faster relief of pulmonary/systemic congestion. This method consists of an extracorporeal circuit through which blood is pumped from a venous access into the filter, to be then returned into the patient (61). During IU, water crosses the semipermeable membrane in the filter by ultrafiltration, which is a process driven by the hydrostatic pressure difference across the filter membrane. Small solutes that pass through the ultrafiltration membrane, such as electrolyte, are removed concurrently. Thus, UF offers isotonic removal and maintains the plasma concentration of low-molecularweight solutes such as sodium and other small solutes. Most of the expected clinical benefit is provided by fluid removal and improvement of pulmonary/systemic congestion; however, neither significant correction of hyponatremia, azotemia, hypo/hyperkalemia or metabolic acidosis/alkalosis, nor a significant removal of highmolecular-weight substances (such as myocardialdepressant factors and cytokines) can be expected. The UNLOAD trial (62) compared UF with diuretic therapy in patients with ADHF; UF produced greater weight loss and a more negative fluid balance and was associated with a reduced readmission rate. However, patients with hypotension, hemodynamic instability or under vasoactive inotropes were excluded; additionally, no improvement in neurohormonal activation accompanied the fluid removal and weight change. Data on the effects of UF on renal function in heart failure are lacking; small observational studies showed no changes in serum creatinine when UF was compared to diuretics in patients with chronic heart failure (63), and in a small case series, 45% of patients underwent hemodialysis during the same or a subsequent hospitalization after UF (64). Thus far, there is no indication that the use of UF in patients with ADHF may prevent the occurrence of renal dysfunction. Arginine vasopressin receptor antagonists. Tolvaptan was compared with a placebo in a randomized, doubleblind trial involving 254 patients with chronic heart failure. When added to standard heart failure therapy, tolvaptan was associated with significant weight reduction secondary to fluid loss relative to the placebo. Among 70 patients with hyponatremia, tolvaptan was associated with improved and often normalized serum sodium concentrations. The Acute and Chronic Therapeutic Impact of a Vasopressin Antagonist in Congestive Heart Failure trial (ACTIV in CHF) was a multicenter, randomized, double-blind, placebo-controlled, parallel-group, dose-ranging phase II feasibility trial that compared three once-daily doses of tolvaptan with a placebo in patients with worsening heart

failure symptoms. Sixty-eight patients (21.3%) had hyponatremia at randomization. All doses of tolvaptan significantly reduced body weight 24 hours after the first dose, with significant increases in urine volume relative to the placebo. Significant hemodynamic changes and significant differences in the 60-day rehospitalization rate, unscheduled visits for heart failure and deaths were not noted between patients treated with tolvaptan and those treated with the placebo. These preliminary findings in patients with heart failure led to the large, multicenter, international Efficacy of Vasopressin Antagonism in Heart Failure Outcome Study with Tolvaptan (EVEREST). This trial was a combination of three studies: two were identically designed to investigate short-term effects on global clinical status and symptoms in patients with heart failure, and the third study was designed to analyze all randomized patients to evaluate outcomes, including time to all-cause mortality and time to first cardiovascular mortality or hospitalization for heart failure. EVEREST was a randomized, double-blind, placebo-controlled study that enrolled 4,133 hospitalized patients with symptomatic left ventricular systolic dysfunction (left ventricular ejection fraction #40%). The patients were randomized to 30 mg of tolvaptan or a placebo once a day for 60 days in addition to their standard heart failure regimen. The results indicated a significant improvement in patient-assessed dyspnea on day 1 in the tolvaptan group relative to the group receiving the placebo. This improvement in dyspnea could be related to the significant reduction in body weight secondary to aquaresis recorded on day 1 in patients receiving tolvaptan relative to the patients receiving the placebo. Overall, tolvaptan had no effect on all-cause mortality. Similarly, no significant difference in the composite measure of death from cardiovascular causes or hospitalization for heart failure was detected between the tolvaptan and placebo groups (65,66,67). Hypertonic saline solution. Hypertonic saline solutions have been studied in different forms of cardiovascular collapse since 1917 (68), and data from experimental shock models demonstrate that the infusion of 7.5% NaCl produces vasodilatation and increased regional blood flow to the coronary (69), renal (70), intestinal and skeletal muscle (71) circulation. Additionally, hypertonic saline improves renal function and myocardial contractility, probably because of a direct cardiac inotropic effect induced by hypertonicity (72,73). In patients, the infusion of 7.5% NaCl has been successfully used in cardiogenic shock arising from right ventricular infarction (74). Small volumes of saline solutions have also been tested in patients with heart failure (75,76), and most studies have focused on the aspects of safety and effectiveness. A randomized trial reported, as a secondary finding in a selective population of patients highly resistant to diuretics, that the infusion of saline solutions with different tonicities was associated with lower creatinine levels (77). However, few previous studies specifically examined the effects of a saline solution on renal function in patients with decompensated heart failure. Recently, hypertonic saline solution administration was reported to attenuate heart failure-induced kidney dysfunction as indicated by improvement in the renal glomerular filtration rate (as measured by serum cystatin C and creatine levels) and tubular function (as measured by the expression of renal tubular proteins aquaporin 2, NHE3 and urea transporter 1). These data indicate that hypertonic

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saline is a promising low-cost therapeutic strategy in patients with decompensated heart failure that warrants further investigation.

14. 15.

& CONCLUSIONS Despite recent improvements in the understanding of pathophysiological phenomena and in diagnostic tools, therapies targeted towards the prevention of renal dysfunction in patients with heart failure are still lacking. Interventions aimed at pivotal pathophysiological points and administered early during the course of the disease may provide better results. However, prospective data in larger populations for most of the new, currently available strategies are still needed.

16. 17.

18. 19. 20.

& AUTHOR CONTRIBUTIONS Issa VS wrote the manuscript. Andrade L and Bocchi EA revised and corrected the text.

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Effects of the adenosine A1 receptor antagonist rolofylline on renal function in patients with acute heart failure and renal dysfunction: results from PROTECT (Placebo-Controlled Randomized Study of the Selective Adenosine A1 Receptor Antagonist Rolofylline for Patients Hospitalized with Acute Decompensated Heart Failure and Volume Overload to Assess Treatment Effect on Congestion and Renal Function). J Am Coll Cardiol. 2011;57(19):1899-907, http://dx.doi.org/10.1016/j. jacc.2010.11.057.

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55. Damman K, Voors AA. Levosimendan improves renal function in acute decompensated heart failure: cause and clinical application. Cardiovasc Drugs Ther. 2007;21(6):403-4, http://dx.doi.org/10.1007/s10557-0076070-y. 56. Yilmaz MB, Yalta K, Yontar C, Karadas F, Erdem A, Turgut OO, et al. Levosimendan improves renal function in patients with acute decompensated heart failure: comparison with dobutamine. Cardiovasc Drugs Ther. 2007;21(6):431-5, http://dx.doi.org/10.1007/s10557-007-6066-7. 57. Zemljic G, Bunc M, Yazdanbakhsh AP, Vrtovec B. Levosimendan improves renal function in patients with advanced chronic heart failure awaiting cardiac transplantation. J Card Fail. 2007;13(6):417-21, http:// dx.doi.org/10.1016/j.cardfail.2007.03.005. 58. Follath F, Cleland JG, Just H, Papp JG, Scholz H, Peuhkurinen K, et al. Efficacy and safety of intravenous levosimendan compared with dobutamine in severe low-output heart failure (the LIDO study): a randomised doubleblind trial. Lancet. 2002;360(9328):196-202, http://dx. doi.org/10.1016/S0140-6736(02)09455-2. 59. Mebazaa A, Nieminen MS, Packer M, Cohen-Solal A, Kleber FX, Pocock SJ, et al. Levosimendan vs dobutamine for patients with acute decompensated heart failure: the SURVIVE Randomized Trial. JAMA. 2007;297(17):1883-91, http://dx.doi.org/10.1001/jama.297.17.1883. 60. Cleland JG, Freemantle N, Coletta AP, Clark AL. Clinical trials update from the American Heart Association: REPAIR-AMI, ASTAMI, JELIS, MEGA, REVIVE-II, SURVIVE, and PROACTIVE. Eur J Heart Fail 2006;8(1):105-10, http://dx.doi.org/10.1016/j.ejheart.2005.12.003. 61. Fiaccadori E, Regolisti G, Maggiore U, Parenti E, Cremaschi E, Detrenis S, et al. Ultrafiltration in heart failure. Am Heart J. 2011;161(3):439-49, http://dx.doi.org/10.1016/j.ahj.2010.09.014. 62. Constanzo MR, Guglin ME, Saltzberg MT, Jessup ML, Bart BA, Teerlink JR, et al. UNLOAD trial investigators. Ultrafiltration versus intravenous diuretics for patients hospitalized for acute decompensated heart failure. J Am Coll Cardiol. 2007;49:675-83, http://dx.doi.org/10.1016/j.jacc.2006. 07.073. 63. Wertman BM, Gura V, Schwarz ER. Ultrafiltration for the management of acute decompensated heart failure. J Card Fail. 2008;14(9):754-9, http://dx.doi.org/10.1016/j.cardfail.2008.07.230. 64. Gheorghiade M, Follath F, Ponikiwski P, Barsuk JH, Blair JE, Cleland JG, et al. Assessing and grading congestion in acute heart failure: a scientific statement from the Acute Heart Failure Committee of the Heart Failure Association of the European Society of Cardiology and endorsed by the European Society of Intensive Care Medicine. Eur J Heart Fail. 2010;12(5):423-33. 65. Gheorghiade M, Gattis WA, O’Connor CM, Adams KF Jr, Elkayam U, Barbagelata A, et al. Effects of tolvaptan, a vasopressin antagonist, in patients hospitalized with worsening heart failure: a randomized controlled trial. JAMA. 2004;291(16):1963-71, http://dx.doi.org/10. 1001/jama.291.16.1963. 66. Konstam MA, Gheorghiade M, Burnett JC Jr, Grinfeld L, Maggioni AP, Swedberg K, et al. Effects of oral tolvaptan in patients hospitalized for worsening heart failure: the EVEREST outcome trial. JAMA 2007;297(12):1319-31, http://dx.doi.org/10.1001/jama.297.12.1319. 67. Gheorghiade M, Konstam MA, Burnett JC Jr, Grinfeld L, Maggioni AP, Swedberg K, et al. Short-term clinical effects of tolvaptan, an oral vasopressin antagonist in patients hospitalized for heart failure. JAMA. 2007;297(12):1332-43, http://dx.doi.org/10.1001/jama.297.12.1332. 68. Penfield WG. The treatment of severe and progressive hemorrhage by intravenous injections. Am J Physiol. 1919;48(1):121-8. 69. Crystal GJ, Gurevicius J, Kim SJ, Eckel PK, Ismail EF, Salem MR. Effects of hypertonic saline solutions in the coronary circulation. Circ Shock. 1994;42(1):27-38. 70. Maningas P. Resuscitation with 7.5% NaCl in 6% dextran-70 during hemorrhagic shock in swine: effects on organ blood flow. Crit Care Med. 1987;15(12):1121-6, http://dx.doi.org/10.1097/00003246-198712000-000 09. 71. Rocha-e-Silva M, Negraes GA, Soares AM, Pontieri V, Loppnow L. Hypertonic resuscitation from severe hemorrhagic shock: patterns of regional circulation. Circ Shock. 1986;19(2):165-75. 72. Ing RD, Nazeeri MN, Zeldes S, Dulchavsky SA, Diebel LN. Hypertonic saline/dextran improves septic myocardial performance. Am Surg 1994;60(7):505-7; discussion 508. 73. Kien ND, Kramer GC. Cardiac performance following hypertonic saline. Braz J Med Biol Res. 1989;22(2):2245-8. 74. Ramires JA, Serrano Ju´nior CV, Ce´sar LA, Velasco IT, Rocha e Silva Ju´nior M, Pileggi F. Acute hemodynamic effects of hypertonic (7.5%) saline infusion in patients with cardiogenic shock due to right ventricular infarction. Circ Shock. 1992;37(3):220-5. 75. Issa VS, Bacal F, Mangini S, Carneiro RM, Azevedo CH, Chizzola PR, et al. Hypertonic saline solution for renal failure prevention in patients with decompensated heart failure. Arq Bras Cardiol. 2007;89(4):251-5. 76. Paterna S, Di Pasquale P, Parrinello G, Amato P, Cardinale A, Follone G, et al. Effects of high-dose furosemide and small-volume hypertonic saline solution infusion in comparison with a high dose of furosemide as a bolus, in refractory congestive heart failure. Eur J Heart Fail 2000;2(3):305-13, http://dx.doi.org/10.1016/S1388-9842(00)00094-5.

35. Hirsch GA, Bottomley PA, Gerstenblith G, Weiss RG. Allopurinol acutely increases adenosine triphospate energy delivery in failing human hearts. J Am Coll Cardiol. 2012;59(9):802- 8, http://dx.doi.org/10.1016/j. jacc.2011.10.895. 36. Baldus S, Mu¨llerleile K, Chumley P, Steven D, Rudolph V, Lund GK, et al. Inhibition of xanthine oxidase improves myocardial contractility in patients with ischemic ardiomyopathy. Free Radic Biol Med. 2006;41(8):1282-8, http://dx.doi.org/10.1016/j.freeradbiomed.2006.07. 010. 37. Goicoechea M, Garcia de Vinuesa S, Verdalles U, Ruiz-Caro C, Ampuero J, Rinco´n A, et al. Effect of allopurinol on chronic kidney disease progression and cardiovascular risk. Clin J Am Soc Nephrol. 2010; 5(8):1388-93, http://dx.doi.org/10.2215/CJN.01580210. 38. Kanbay M, Ozkara A, Selcoki Y, Isik B, Turgut F, Bavbek N, et al. Effect of treatment of hyperuricemia with allopurinol on blood pressure, creatinine clearance, and proteinuria in patients with normal renal function.Int Urol Nephrol. 2007;39(4):1227-33, http://dx.doi.org/10. 1007/s11255-007-9253-3. 39. Doehner W, Landmesser U. Xanthine oxidase and uric acid in cardiovascular disease: clinical impact and therapeutic options Semin Nephrol. 2011;31:433-40. 40. Solomon SD, Shin SH, Shah A, Skali H, Desai A, Kober L, et al. Effect of the direct renin inhibitor aliskiren on left ventricular remodelling following myocardial infarction with systolic dysfunction. Eur Heart J. 2011;32(10):1227-34, http://dx.doi.org/10.1093/eurheartj/ehq522. 41. Krum H, Massie B, Abraham WT, Dickstein K, Kober L, McMurray JJV, et al. Direct renin inhibition in addition to or as an alternative to angiotensin converting enzyme inhibition in patients with chronic systolic heart failure: rationale and design of the Aliskiren Trial to Minimize OutcomeS in Patients with HEart failuRE (ATMOSPHERE) study. Eur J Heart Fail. 2011;13(1):107-14. 42. Gheorghiade M, Albaghdadi M, Zannad F, Fonarow GC, Bohm M, Gimpelewicz C, et al. Rationale and design of the multicentre, randomized, double-blind, placebo-controlled Aliskiren Trial on Acute Heart Failure Outcomes (ASTRONAUT). Eur J Heart Fail. 2011;13(1):1006. 43. Esler MD, Krum H, Sobotka PA, Schlaich MP, Schmieder RE, Boehm M, et al. Renal sympathetic denervation in patients with treatment-resistant hypertension (The Symplicity HTN-2 Trial): a randomised controlled trial. Lancet. 2010;376(9756):1903-9. 44. Sobotka PA, Krum H, Bo¨hm M, Francis DP, Schlaich MP. The Role of Renal Denervation in the Treatment of Heart Failure. Curr Cardiol Rep. 201;14(3):285-92. 45. Seri I, Kone BC, Gullans SR, Aperia A, Brenner BM, Ballermann BJ. Influence of Na intake on dopamine-induced inhibition of renal cortical Na(þ)-K(þ)-ATPase. Am J Physiol. 1990;258(1 Pt 2):F52-60. 46. Friedrich JO, Adhikari N, Herridge MS, Beyene J. Meta-analysis: low dose dopamine increases urine output but does not prevent renal dysfunction or death. Ann Intern Med. 2005;142(7):510-24. 47. Giamouzis G, Butler J, Starling RC, Karayannis G, Nastas J, Parisis C, Rovithis D, Economou D, Savvatis K, Kirlidis T, Tsaknakis T, Skoularigis J, Westermann D, Tscho¨pe C, Triposkiadis F. Impact of dopamine infusion on renal function in hospitalized heart failure patients: results of the Dopamine in Acute Decompensated Heart Failure (DAD-HF) Trial. J Card Fail. 2010;16(12):922-30, http://dx.doi.org/10.1016/j.cardfail. 2010.07.246. 48. Felker GM, Lee KL, Bull DA, Redfield MM, Stevenson LW, Goldsmith SR, et al. Diuretic strategies in patients with acute decompensated heart failure. N Engl J Med. 2011;364(9):797-805. 49. Marcus LS, Hart D, Packer M, Yushak M, Medina N, Danziger RS, et al. Hemodynamic and renal excretory effects of human brain natriuretic peptide infusion in patients with congestive heart failure - A doubleblind, placebo-controlled, randomized crossover trial. Circulation. 1996;94(12):3184-9, http://dx.doi.org/10.1161/01.CIR.94.12.3184. 50. La Villa G, Fronzaroli C, Lazzeri C, Porciani C, Bandinelli R, Vena S, et al. Cardiovascular and renal effects of low dose brain natriuretic peptide infusion in man. J Clin Endocrinol Metab. 1994;78(5):1166 -71, http://dx. doi.org/10.1210/jc.78.5.1166. 51. Jensen K, Eiskjaer H, Carstens J, Pedersen EB. Renal effects of brain natriuretic peptide in patients with congestive heart failure. Clin Sci (Lond). 1999;96:5-15, http://dx.doi.org/10.1042/CS19980105. 52. Sackner-Bernstein JD, Skopicki HA, Aaronson KD. Risk of worsening renal function with nesiritide in patients with acutely decompensated heart failure. Circulation. 2005;111(12):1487-91, http://dx.doi.org/10. 1161/01.CIR.0000159340.93220.e4",-1,"xxx/93220.e4. 53. Witteles RM, Kao D, Christopherson D, Matsuda K, Vagelos RH, Schreiber D, et al. Impact of nesiritide on renal function in patients with acute decompensated heart failure and pre-existing renal dysfunction a randomized, double-blind, placebo-controlled clinical trial. J Am Coll Cardiol. 2007;50(19):1835-40, http://dx.doi.org/10.1016/j.jacc.2007.03. 071. 54. O’Connor CM, Starling RC, Hernandez AF, Armstrong PW, Dickstein K, Hasselblad V, et al. Effect of nesiritide in patients with acute decompensated heart failure. N Engl J Med. 2011;365(1):32-43.

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REVIEW

What has changed in brachial plexus surgery? Marcelo Rosa de Rezende, Gustavo Bersani Silva, Emygdio Jose´ Leomil de Paula, Rames Mattar Junior, Olavo Pires de Camargo Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Institute of Orthopedics and Traumatology, Saõ Paulo/SP, Brazil.

Brachial plexus injuries, in all their severity and complexity, have been extensively studied. Although brachial plexus injuries are associated with serious and often definitive sequelae, many concepts have changed since the 1950s, when this pathological condition began to be treated more aggressively. Looking back over the last 20 years, it can be seen that the entire approach, from diagnosis to treatment, has changed significantly. Some concepts have become better established, while others have been introduced; thus, it can be said that currently, something can always be offered in terms of functional recovery, regardless of the degree of injury. Advances in microsurgical techniques have enabled improved results after neurolysis and have made it possible to perform neurotization, which has undoubtedly become the greatest differential in treating brachial plexus injuries. Improvements in imaging devices and electrical studies have allowed quick decisions that are reflected in better surgical outcomes. In this review, we intend to show the many developments in brachial plexus surgery that have significantly changed the results and have provided hope to the victims of this serious injury. KEYWORDS: Brachial Plexus; Nerve Transfer; Microvascular Decompression Surgery; Free Tissue Flaps; Review. Rezende MR, Silva GB, Paula EJ, Mattar Junior R, Camargo OP. What has changed in brachial plexus surgery? Clinics. 2013;68(3):411-418. Received for publication on October 4, 2012; First review completed on October 22, 2012; Accepted for publication on November 1, 2012 E-mail: marcelo@marcelorosaderezende.com.br Tel.: 55 11 3256-9325

Sa˜o Paulo, 95% of the cases of brachial plexus injury result from motorcycle accidents. It is very important to classify the injury for treatment purposes. It seems most intuitive to us to divide the injuries into upper trunk (Erb-Duchene; C5/C6), extended upper trunk (Erb-Duchene; C5/C6/C7), lower trunk (Dejerine-Klumpke; C8/T1) and swinging limb (all roots). In terms of prognostic factors relating to the level of the injury, Rorabeck and Harris (4) indicated that isolated injuries of the upper trunk have a better prognosis than do isolated lesions of the divisions, upper roots or lower trunk. Complete injuries and pain persisting for more than six months indicate a poor prognosis in terms of neurological recovery, independent of the level of the injury. The first serious approaches towards brachial plexus treatment were begun in the 1940s and 1950s by pioneers such as Seddon (5) and Bateman (6), among others. At that time, the parameter used for surgical indications was an attempt to clinically identify whether the injury was an avulsion or a neuroma with continuity. The first was characterized as an irreversible injury, while the second would have a better prognosis. Among the signs of a poor prognosis was the Claude-Bernard-Horner sign, which was claimed by several authors (4,7) to be very reliable for diagnosing avulsion injuries of the lower roots. Presence of the Tinel sign in the supraclavicular region indicated that graftable roots were possibly present. The concept that reigned until the 1970s in relation to treatments for avulsion injuries was that these were cases

& INTRODUCTION Brachial plexus injuries have been a challenge throughout the history of medical knowledge in the sense of both the need to understand the neural anatomical structures affected and the aim of proposing treatment that may restore function to the injured upper limb. The brachial plexus is formed by the roots from C5 to T1, and it may or may not receive contributions from C4 and T2 (pre- or post-fixed). Its anatomy is complex and is characterized by nerves coming from the plexus that interrelate to form fascicles and finally the nerves that head to all parts of the upper limb. Despite the few studies that have been conducted in Brazil, it is apparent that there has been a large increase in the number of brachial plexus injuries occurring, which is a consequence of significant increases in the use of motorcycles as a means of transport (1,2). American and European studies have demonstrated that 10 to 20% of peripheral nerve injuries are brachial plexus injuries and that 80 to 90% of these injuries are caused by motorcycle and car accidents (3). In our outpatient clinic at the Hospital das Clı´nicas de

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to reach the effector organ (muscle or sensory unit). According to some authors, this growth should not take more than 18 to 24 months. After this time, the fibrotic muscle tissue should be replaced (16). For diagnosing the injury, there needs to be a clinical assessment made in conjunction with an imaging examination and an electrical study on nerve conduction. Among the imaging methods, the standard is myelotomography, which has been shown to be very good for detecting root avulsion and has a sensitivity of 85% and a specificity of 95% (17). Nonetheless, our experience shows that magnetic resonance imaging is now an effective method for demonstrating not only the avulsion but also the level of the injury along the plexus. Therefore, this procedure is now our preferred imaging examination. Electroneuromyography is used with the aim of differentiating plexopathy from root injury and for establishing a prognosis for nerve injuries, including post-reconstruction surgeries. The motor amplitude in electroneuromyography is not a good means for evaluating the injury level, but sensory studies in comparison with the contralateral site provide important information about the injury location and whether avulsion is present.

for amputation procedures or arthrodesis of the shoulder, elbow and wrist, depending on the level of the injury. In cases of amputation, attempts were made to create limb prostheses. In non-avulsion cases, an expectant approach was taken for two years that awaited any possible return to functioning, a state from which attempts to perform orthopedic surgery could be made. In addition, in 1965, Seddon (8) described a surgical procedure for cases of avulsion simply as a means of accelerating the diagnosis and being able to bring forward amputation surgery. Although in the 1990s it was considered prudent to wait five or six months before indicating surgery, the improvements in imaging examinations and electrical studies that have now become established mean that today, there is a tendency to indicate surgery earlier rather than more than three months after the injury because it is now known that nerve regeneration has a better prognosis the earlier it occurs. Bertelli and Ghizoni (7) backed indications of exploratory surgery three to six months after the injury and, similarly to Narakas and Hentz (9), emphasized that cases operated upon more than nine months after the injury have worse prognoses. In surgical procedures for cases of neuroma with continuity, coaptation of the injured stumps was often performed while keeping the patient in a forced position or even through shortening the clavicular bone. This situation only changed with the advent of the use of the nerve graft that was proposed by Seddon (10) in 1963, followed by several studies at the beginning of the 1970s (3). These confirmed that this procedure was effective for reconstructing the loss of a nerve segment. Recently, several studies on the possibility of using neurotrophic factors for increasing the efficacy of the nerve regeneration process have been published, although there remains no standardization regarding use of these factors (11). In addition to repairs on injured nerve structures in the plexus, the technique of neurotization (which consists of suturing a remaining intact nerve to another that is injured) has constituted a major advance for brachial plexus reconstructions. It has evolved from the first descriptions of use of the intercostal nerve (9,12) to the use of many other nerves (13-15), thereby contributing greatly towards improving the results from brachial plexus surgery.

During the surgery It is now possible to conduct studies on the condition of the brachial plexus nerves in greater detail during the surgery (18). With the aid of electrical studies conducted during the operation, it should be possible to achieve greater sureness with regard to making decisions such as whether to preserve an injured neural segment. Electrical stimulation of the brachial plexus can be performed, although there may be ‘‘contamination’’ of the stimulus to other nerve branches, thus making it difficult to make a selective assessment on each nerve. Electroneuromyography examinations can also be performed, despite the logistic difficulty of doing so during the surgery. If a neuroma is identified, there is some doubt regarding whether it should be resected or whether simple neurolysis should be performed. Studying the nerve action potential (NAP) (19) using one electrode proximally to the neuroma and one distally to it enables quantitative assessment of the number of viable nerve fibers and thus assists in decisionmaking. Studying the sensory evoked potential (SEP) using one electrode distally to the nerve root studied and picking up the stimulus in the contralateral cortex makes it possible to diagnose intraforaminal avulsion.

& ARTICLE SELECTION CRITERIA The selection criteria for the articles we reviewed were a) original articles describing the different techniques cited throughout the text, and b) articles based on developments of original techniques, mainly those proposing comparisons between operative outcomes. Although the authors privileged more recent studies (edited in the last 10 years), there was no strict time interval for a paper to be considered in this work.

Surgical indication Brachial plexus injuries are generally associated with severe sensory and motor deficits of the upper limb. Therefore, all efforts towards treatment should take into consideration the correct staging of the lesion so that the best type of treatment can be chosen. In our experience, considering that the trauma that led to the brachial plexus injury generally involved high energy, spontaneous regeneration of the injury is unusual. Thus, we very often indicate a surgical procedure as a means of therapy and staging. Even in cases of avulsion, there are options such as neurotization that can be used with the aim of restoring some degree of functionality. Therefore, brachial plexus surgery is important both for performing reconstruction and for resolving doubts.

& DIAGNOSTIC METHODS Before the surgery Staging the brachial plexus injury is of enormous importance for making an indication of exploratory surgery and for helping to plan it. It is known that nerve reconstruction should be performed as early as possible because after this procedure has been accomplished, functional recovery remains dependent on axonal growth

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performing neurotization, given that the chances of reinnervation of the antagonist musculature are thus increased, whereas this outcome is less likely with neurotization alone.

Surgical procedures Decisions regarding brachial plexus surgery should be made as a function of the predefined priorities. Because of the small number of nerve units available, decisions regarding whether to prioritize the shoulder or the elbow often have to be made. In our view, shoulder stability is fundamental for good functioning of the elbow; therefore, the concept of seeking to reconstruct from proximal to distal applies. It should be kept in mind that the results obtained in relation to C8 and T1 are very unfavorable, which provides justification for some authorsâ&#x20AC;&#x2122; opinion that these roots should not be reconstructed (7).

Intra-plexular neurotization There are several options for neurotization of the remaining nerve units, and many of them have been described recently. Radial nerve to axillary nerve â&#x20AC;&#x201C; This was described in 2003. The authors (30-32) demonstrated through an anatomical and clinical study that it was possible to obtain a branch of the radial nerve for suturing to the posterior branch of the injured axillary nerve (Figure 1). Subsequent clinical studies demonstrated good results with this technique (approximately 124 degrees of abduction), especially if in association with neurotization of the spinal accessory nerve with the suprascapular nerve (33). Ulnar nerve to musculocutaneous nerve â&#x20AC;&#x201C; The first description of this technique was provided by Oberlin et al. (34) in 1994. It consists of neurotization of a fascicle of the ulnar nerve to a motor branch of the musculocutaneous nerve that heads towards the biceps. Excellent clinical results have been confirmed by different authors (13,26,27,33,35-39). Our experience with this type of neurotization has also been very good: in most cases, we have achieved a biceps of at least grade 3, which could be given more potential through possible transfer of the flexionpronation musculature (Steindler). The meta-analysis study conducted by Merrel et al. (33) indicated that the best results were achieved when the procedure was performed up to six months after the trauma, while there was a sharp worsening of the results if performed more than 12 months after the trauma.

Neurolysis Nerve reconstructions of the brachial plexus have historically gone through the stages of neurolysis, direct nerve repair, nerve grafts (20) and, lastly, neurotization. Neurolysis consists of promoting dissection of the nerve that presents a neuroma with continuity. This technique is performed with the aim of decompressing any viable fascicles in relation to the fibrotic tissue. Today, it is possible to have a quantitative assessment of the number of viable nerve fibers. This ability is due to the advent of intraoperative electrical studies (18), especially the nerve action potential (NAP), which uses one electrode proximally to the neuroma and one distally to it. This information may help in making the decision regarding whether to perform neurolysis or to resect the neuroma and perform a nerve graft.

Nerve graft Nerve grafting started in 1963 by Seddon (10) who introduced the concept of using nerve grafts to reconstruct nerve losses. Good results were subsequently confirmed by other authors (21-24). Until then, attempts to perform direct suturing of injured nerves were made at any cost despite bone shortening or maintenance of the limb in forced positions, which compromised nerve regeneration. The use of grafts initially required suturing of the ends of the sural nerve individually, which made the procedure very tedious and less precise. However, with the advent of fibrin glue (25), it became possible to form a group of graft ends, thus constituting a single structure. Fewer stitches were needed in the proximal and distal sutures, which made the grafting procedure much simpler and much more secure.

Neurotization The procedure of neurotization consists of transferring an undamaged motor nerve to another nerve that is injured. The nerves used are remainders from the trauma to the brachial plexus and may come from the brachial plexus (intra-plexular) or from elsewhere (extra-plexular). Through neurotization, the conditions for a quality suture close to the effector area (muscle unit) are provided, which diminishes the distance for nerve regeneration. We agree with other authors (26-28) who have affirmed that direct suturing should always be attempted in cases of neurotization because using a graft leads to a worsening of the results from nerve regeneration. Another point in relation to neurotization is that although recent neurotization techniques have generally shown good results, some authors (7,29) have emphasized the importance of primary reconstruction of the brachial plexus whenever possible when

Figure 1 - Radial nerve to axillary nerve.

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Figure 2 - Ulnar nerve to musculocutaneous nerve (Oberlin procedure).

injuries to the suprascapularis nerve and the ease of execution of this procedure. These nerves are close together, and the same route is taken as the one used for exploration of the brachial plexus. The result from this procedure has been shown to be below expectations considering the proximity of the supra- and infraspinatus muscles. Venkatramani et al. (27) reported that there was a gain in shoulder abduction of 66 degrees in 60% of the cases that they studied and that the external rotation was generally unsatisfactory. Even with these results showing partial recovery, Narakas and Hentz (9) reaffirmed that the shoulder range of motion was twice what was obtained through arthrodesis on the shoulder. We believe that the prime function of this type of neurotization is shoulder stabilization, which may ensure a better result with regard to gains in elbow flexion and may provide greater synchronism of gait to avoid swinging limbs. Contralateral C7 to median nerve – This transfer was first described by Gu et al. (46) in 1992, who proposed harvesting the root of the contralateral C7 and transferring this root to

Despite the good results from neurotization of the biceps muscles, double neurotization was proposed (40) and was named Oberlin2 surgery. In this technique, in addition to the usual procedure, a fascicle from the median nerve would be used for neurotization in the motor branch of the brachial muscle. In a study comparing these two techniques, Sungpet et al. (38) did not find any difference regarding the final functional result. Medial pectoral nerve to musculocutaneous nerve – This procedure was described by Brandt and Mackinnon (41) in 2003 and consists of suturing the medial pectoral nerve directly to the musculocutaneous nerve with the aim of restoring the elbow flexion. It should be remembered that the pectoral nerve receives contributions from the lateral and medial fascicle and may thus be compromised in many cases (Figure 2).

Extra-plexular neurotization Phrenic nerve to musculocutaneous nerve – This transfer was first described by Gu et al. (42) in 1990. It can be performed because the phrenic nerve is generally preserved in cases of brachial plexus injury, given that its biggest contribution comes from C3 and C4. To avoid using grafts, the dissection on the phrenic nerve needs to be performed as distally as possible. According to Monreal (43), the phrenic nerve is eminently motor and presents 800 neurons. Some divergences persist regarding the morbidity ensuing from its removal: this removal could lead to breathlessness when making effort. This procedure is not recommended for patients with previous pulmonary diseases or for children under the age of two years. Phrenic nerve to suprascapular nerve – This type of transfer is an alternative to the spinal accessory nerve and can be used for neurotization of another injured nerve. In some clinics, this procedure is used routinely with good results (42), and it can be highlighted that function returns after eight months. Morbidity relating to the removal of the phrenic nerve needs to be taken into consideration. We do not have much experience with this procedure, but because the phrenic nerve is an eminently motor nerve and the distance to the effector area is short, it is reasonable to expect that good results are possible. Spinal accessory nerve to suprascapular - The spinal accessory nerve is a pure motor nerve without approximately 1500 axons (44) (Figure 3). This neurotization was first described in the 1980s (9,12,45) and is perhaps the neurotization that is used most often, given the frequency of

Figure 3 - Medial pectoral nerve to musculocutaneous nerve.

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News in brachial plexus surgery Rezende MR et al.

the median nerve of the injured side by means of a nerve graft (Figure 4). The first problem posed related to the morbidity caused by sectioning the C7 root, but subsequent studies revealed that this morbidity was minimal. Another issue posed was that the graft was from a long nerve and that there was a long distance to the effector area. This concern was confirmed through the results presented that showed that protective sensitivity was recovered in 83% of the cases, but with an unfavorable motor result (15). In view of these points, we have not used this procedure at our clinic. Intercostal nerve to musculocutaneous nerve â&#x20AC;&#x201C; This procedure was described in the 1980s (9) and uses the intercostal nerves, which each present approximately 1200 axons transferred to the musculocutaneous nerve with the aim of restoring shoulder flexion (Figure 5). This surgery implies accessing not only the upper limb but also the thoracic region just below the nipples. In general, at least two intercostal nerves are dissected as far as proximally to the sternal region to gain length, thus favoring not using a graft (28). The study by Merrel et al. (33) confirmed positive functional results with the return of elbow flexion against gravity in more than 65% of cases. This proportion is only lower than the result from neurotization of the ulnar nerve to the musculocutaneous nerve. The intercostal nerve is of mixed type; therefore, identifying the motor branch is fundamental to the success of the procedure. We have considerable experience with this procedure, which allows us to affirm that if it is impossible to use the ulnar nerve as a source of neurotization, the second choice is to take the intercostal nerve to the musculocutaneous nerve.

Figure 5 - Spinal accessory nerve to musculocutaneous nerve.

osteotomy and wrist arthrodesis, were routine procedures (48), along with limb amputation, for which the level was defined according to the degree of functional recovery of the limb. This scenario has changed drastically with improvements in the results from nerve reconstructions, especially through neurotization, which has enabled joint stabilization and a return to functioning for some muscles. This advance, together with conventional free muscle transfers, has made arthrodesis and amputation exceptional approaches in treating brachial plexus injuries. Arthrodesis: Although some clinics advocate using shoulder arthrodesis as a means of stabilizing this joint, we are now giving preference to the transfer of the trapezium to the humerus, as first described by Saha (49). We reserve wrist arthrodesis for situations in which, because of the small number of tendons available for transfer, we can use the carpal flexors and extensors for the finger tendons in performing the arthrodesis.

Complementary orthopedic treatment Until the 1960s, bone surgery had an important role in treating patients with brachial plexus injuries, especially among those with complete injuries. Certain procedures, such as shoulder arthrodesis (47), external derotation

Tendon transfers Trapezium muscle to humerus (shoulder abduction) â&#x20AC;&#x201C; In this type of transfer, the acromial insertion of the trapezium muscle is transposed to the proximal humerus together with a segment of the acromion with the aim of achieving a gain in shoulder abduction. Despite unsatisfactory results in terms of shoulder abduction, we have observed that this transfer has an important role as a shoulder stabilizer. It has positive results, particularly in cases of severe shoulder instability with inferior subluxation of the glenohumeral joint. Trapezium muscle to humerus (external rotation of the arm) â&#x20AC;&#x201C; In this transfer, the lower segment of the trapezium is dissected while maintaining its vascularization and innervation. With complementation using a graft from the fascia lata or by expanding the muscle segment as far as the external border of the acromion using the aponeurotic

Figure 4 - Phrenic nerve to musculocutaneous nerve.

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wheelchair), this technique is contraindicated. We believe that it should be used when other transfers are not possible. Flexor-pronator musculature to humerus – Steindler (elbow flexion) – This technique was described by Steindler in 1918 and consists of transfer of the flexorpronator musculature to the metaphyseal region of the humerus (52). To achieve success in this procedure, it is essential for the C8 and T1 roots to be undamaged. In performing this technique, this musculature is made to act as an elbow flexor. Our experience with this procedure confirms other authors’ experience (53) that indicated that this transfer is efficient for boosting elbow flexion, i.e., when the existing elbow flexor musculature is at least grade 2 (54). This transfer has been shown to be insufficient for achieving functional elbow flexion on its own. Free muscle transfer – The first report of free muscle transfer was made by Tamai et al. (55) in 1970, who reported that this type of procedure was successful in dogs. Specifically for brachial plexus injuries, this type of transfer has been performed using the gracilis muscle to restore elbow flexion (Figure 6). There is no doubt that this procedure represents a major advance in cases of chronic lesions that did not have any prognosis. Such cases have come to be viewed as having very favorable functional results (13,56-58). For this procedure to be successful, not only are vascular sutures needed; additionally, a good donor motor nerve has to be chosen to perform neurotization. The intercostal nerve, the spinal accessory nerve and even fascicles from the ulnar nerve have been used for this procedure, each with advantages and disadvantages. One point that we judge to be important is to always seek direct suturing and avoid using nerve grafts at all costs. One of the points that gives rise to discussion is in relation to the limits between expecting the flexor musculature to become reinnervated or deciding to perform free muscle transfer. In our clinic, based on previous studies that

tissue, the trapezium is inserted into the intertuberous sulcus with the aim of gaining external rotation. Few reports are yet available in the worldwide literature (50) regarding functional results, but based on our experience with this technique, the initial results are very promising. Pectoralis major to biceps (Clark) – This procedure was described by Clark (51) and is an option for gaining elbow flexion when the pectoralis major muscle is preserved. This situation is unusual because its innervation comes from the medial and lateral fascicles, which are very commonly affected in brachial plexus injuries. Another negative factor is that the distal suture of the muscle in the biceps is of the muscle-to-muscle type, which compromises its quality, particularly with regard to adjusting the necessary tension. Latissimus dorsi to elbow flexor – Success in this transfer also depends on the integrity of the muscle and its innervation through the thoracodorsal nerve, which is often injured. When undamaged, this muscle is a very efficient transfer that can result in restitution of flexion strength and even cosmetic improvement regarding the muscle outline in the anterior region of the arm. Our preference has been for a bipolar transfer in which the muscle is fully lifted and then reinserted both proximally and distally, which facilitates adjustment of tension on the transferred muscle. Triceps to biceps – In this type of transfer, the triceps is used to act as an elbow flexor, for which the basic condition is that the C7 root and therefore the triceps should be undamaged. Through this complete distal deinsertion of the tricipital tendon and its transfer to the bicipital tendon, a good elbow flexion result can be obtained after a period of rehabilitation for functional readaptation of the triceps. The major inconvenience of this technique is in relation to the loss of active extension of the elbow, thereby leading to dependence on passive extension through the action of gravity. For patients who depend on elbow extension (with use of crutches or a

Figure 6 - Free muscle transfer (gracilis to biceps): pre- and postoperative results. Note deficit of elbow flexion and satisfactory elbow function afterwards.

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News in brachial plexus surgery Rezende MR et al. 23. Millesi H. Surgical management of brachial plexus injuries. J Hand Surg Am. 1977;2(5):367-78. 24. Millesi H. [Late results of the interfascicular nerve transplant. Restorative surgery of lesions of the brachial plexus]. Rev Med Suisse Romande. 1973;93(7):511-9. 25. Narakas A. The use of fibrin glue in repair of peripheral nerves. Orthop Clin North Am. 1988;19(1):187-99. 26. Rohde RS, Wolfe SW. Nerve transfers for adult traumatic brachial plexus palsy (brachial plexus nerve transfer). HSS J. 2006;3(1):77-82. 27. Venkatramani H, Bhardwaj P, Faruquee SR, Sabapathy SR. Functional outcome of nerve transfer for restoration of shoulder and elbow function in upper brachial plexus injury. J Brachial Plex Peripher Nerve Inj. 2008;3:15, http://dx.doi.org/10.1186/1749-7221-3-15. 28. El-Gammal TA, Fathi NA. Outcomes of surgical treatment of brachial plexus injuries using nerve grafting and nerve transfers. J Reconstr Microsurg. 2002;18(1):7-15, http://dx.doi.org/10.1055/s-2002-19703. 29. Flores LP. Brachial plexus surgery: the role of the surgical technique for improvement of the functional outcome. Arq Neuropsiquiatr. 2011;69(4):660-5, http://dx.doi.org/10.1590/S0004-282X2011000500016. 30. Leechavengvongs S, Witoonchart K, Uerpairojkit C, Thuvasethakul P, Malungpaishrope K. Combined nerve transfers for C5 and C6 brachial plexus avulsion injury. J Hand Surg Am. 2006;31(2):183-9, http://dx.doi. org/10.1016/j.jhsa.2005.09.019. 31. Witoonchart K, Leechavengvongs S, Uerpairojkit C, Thuvasethakul P, Wongnopsuwan V. Nerve transfer to deltoid muscle using the nerve to the long head of the triceps, part I: an anatomic feasibility study. J Hand Surg Am. 2003;28(4):628-32, http://dx.doi.org/10.1016/S0363-5023(03) 00200-4. 32. Leechavengvongs S, Witoonchart K, Uerpairojkit C, Thuvasethakul P. Nerve transfer to deltoid muscle using the nerve to the long head of the triceps, part II: a report of 7 cases. J Hand Surg Am. 2003;28(4):633-8, http://dx.doi.org/10.1016/S0363-5023(03)00199-0. 33. Merrell GA, Barrie KA, Katz DL, Wolfe SW. Results of nerve transfer techniques for restoration of shoulder and elbow function in the context of a meta-analysis of the English literature. J Hand Surg Am. 2001;26(2):303-14, http://dx.doi.org/10.1053/jhsu.2001.21518. 34. Oberlin C, Beal D, Leechavengvongs S, Salon A, Dauge MC, Sarcy JJ. Nerve transfer to biceps muscle using a part of ulnar nerve for C5-C6 avulsion of the brachial plexus: anatomical study and report of four cases. J Hand Surg Am. 1994;19(2):232-7, http://dx.doi.org/10.1016/ 0363-5023(94)90011-6. 35. Rohde RS, Wolfe SW. Nerve transfers for adult traumatic brachial plexus palsy (brachial plexus nerve transfer). HSS J. 2007;3(1):77-82, http://dx. doi.org/10.1007/s11420-006-9027-y. 36. Vekris MD, Beris AE, Johnson EO, Korobilias AV, Pafilas D, Vekris AD, et al. Musculocutaneous neurotization to restore elbow flexion in brachial plexus paralysis. Microsurgery. 2006;26(4):325-9, http://dx. doi.org/10.1002/micr.20246. 37. Teboul F, Kakkar R, Ameur N, Beaulieu JY, Oberlin C. Transfer of fascicles from the ulnar nerve to the nerve to the biceps in the treatment of upper brachial plexus palsy. J Bone Joint Surg Am. 2004;86-A(7):148590. 38. Sungpet A, Suphachatwong C, Kawinwonggowit V, Patradul A. Transfer of a single fascicle from the ulnar nerve to the biceps muscle after avulsions of upper roots of the brachial plexus. J Hand Surg Br. 2000;25(4):325-8, http://dx.doi.org/10.1054/jhsb.2000.0367. 39. Ferraresi S, Garozzo D, Buffatti P. Reinnervation of the biceps in C5-7 brachial plexus avulsion injuries: results after distal bypass surgery. Neurosurg Focus. 2004;16(5):E6. 40. Mackinnon SE, Novak CB, Myckatyn TM, Tung TH. Results of reinnervation of the biceps and brachialis muscles with a double fascicular transfer for elbow flexion. J Hand Surg Am. 2005;30(5):97885, http://dx.doi.org/10.1016/j.jhsa.2005.05.014. 41. Brandt KE, Mackinnon SE. A technique for maximizing biceps recovery in brachial plexus reconstruction. J Hand Surg Am. 1993;18(4):726-33, http://dx.doi.org/10.1016/0363-5023(93)90328-Z. 42. Gu YD, Wu MM, Zhen YL, Zhao JA, Zhang GM, Chen DS, et al. Phrenic nerve transfer for treatment of root avulsion of the brachial plexus. Chin Med J (Engl). 1990;103(4):267-70. 43. Monreal R. Restoration of Elbow Flexion by Transfer of the Phrenic Nerve to Musculocutaneous Nerve after Brachial Plexus Injuries. Hand 2007;2(4):206-11, http://dx.doi.org/10.1007/s11552-007-9050-6. 44. Ricardo M. Surgical treatment of brachial plexus injuries in adults. International Orthopaedics. 2005;29(6):351-4, http://dx.doi.org/10.1007/ s00264-005-0017-3. 45. Allieu Y, Privat JM, Bonnel F. Paralysis in root avulsion of the brachial plexus. Neurotization by the spinal accessory nerve. Clin Plast Surg. 1984;11(1):133-6. 46. Gu YD, Zhang GM, Chen DS, Yan JG, Cheng XM, Chen L. Seventh cervical nerve root transfer from the contralateral healthy side for treatment of brachial plexus root avulsion. J Hand Surg Br. 1992;17(5):518-21, http://dx.doi.org/10.1016/S0266-7681(05)80235-9. 47. Pfeil J, Martini AK. [Indications and results of shoulder arthrodesis and concomitant myoplastic interventions. Follow-up of 60 patients].

indicated that the nerve and muscle receptors were significantly impaired in lesions that were more than 12 months old (59-60), we have chosen to use free muscle transfer. We use the gracilis muscle with very satisfactory results. In conclusion, the treatment of brachial plexus injuries has evolved over recent decades. Many new procedures have been incorporated with known ones, allowing a better perspective for functional recovery after surgical approaches to treat this severe injury.

& AUTHOR CONTRIBUTIONS Rezende MR was the originator and primary contributor to text and data collection. Silva GB was the secondary contributor to text and data collection. Paula EJ contributed to the text formatting and revision. Mattar Junior R and Camargo OP revised the text.

& REFERENCES 1. Andrew T, Wallace WA. Do brachial plexus injuries occur at initial impact in motor-cyclists? Br Med J. 1978;1(6):1668, http://dx.doi.org/10. 1136/bmj.1.6128.1668. 2. Midha R. Epidemiology of brachial plexus injuries in a multitrauma population. Neurosurgery. 1997;40(6):1182-8, http://dx.doi.org/10. 1097/00006123-199706000-00014. 3. Flores LP. [Epidemiological study of the traumatic brachial plexus injuries in adults]. Arq Neuropsiquiatr. 2006;64(1):88-94, http://dx.doi. org/10.1590/S0004-282X2006000100018. 4. Rorabeck CH, Harris WR. Factors affecting the prognosis of brachial plexus injuries. J Bone Joint Surg Br. 1981;63-B(3):404-7. 5. Seddon HJ. A Classification of Nerve Injuries. Br Med J. 1942;2(4260):2379, http://dx.doi.org/10.1136/bmj.2.4260.237. 6. Bateman JE. An operative approach to supraclavicular plexus injuries. J Bone Joint Surg Br. 1949 ;31B(1):34-6. 7. Bertelli JA, Ghizoni MF. Results and current approach for Brachial Plexus reconstruction. J Brachial Plex Peripher Nerve Inj. 2011;6(1):2, http://dx.doi.org/10.1186/1749-7221-6-2. 8. Seddon H. Nerve Injuries. Med Bull (Ann Arbor). 1965;31:4-10. 9. Narakas AO, Hentz VR. Neurotization in brachial plexus injuries. Indication and results. Clin Orthop Relat Res. 1988:43-56. 10. Seddon HJ. Nerve Grafting. J Bone Joint Surg Br. 1963;45:447-61. 11. Fornazari AA, Rezende MR, Mattar Junior R, Taira RI, Santos GB, Paulos RG. Effect of neurotrophic factor, MDP, on ratsâ&#x20AC;&#x2122; nerve regeneration. Braz J Med Biol Res. 2011;44(4):327-31, http://dx.doi.org/10.1590/ S0100-879X2011007500021. 12. Narakas AO. Thoughts on neurotization or nerve transfers in irreparable nerve lesions. Clin Plast Surg. 1984;11(1):153-9. 13. Chuang DC-C. Neurotization and Free Muscle Transfer for Brachial Plexus Avulsion Injury. Hand Clin. 2007;23(1):91-104, http://dx.doi.org/ 10.1016/j.hcl.2007.02.001. 14. Swanson AN, Wolfe SW, Khazzam M, Feinberg J, Ehteshami J, Doty S. Comparison of neurotization versus nerve repair in an animal model of chronically denervated muscle. J Hand Surg Am. 2008;33(7):1093-9, http://dx.doi.org/10.1016/j.jhsa.2008.02.025. 15. Songcharoen P, Wongtrakul S, Spinner RJ. Brachial plexus injuries in the adult. nerve transfers: the Siriraj Hospital experience. Hand Clin. 2005;21(1):83-9, http://dx.doi.org/10.1016/j.hcl.2004.10.002. 16. Feinberg JH, Radecki J, Wolfe SW, Strauss HL, Mintz DN. Brachial plexopathy/nerve root avulsion in a football player: the role of electrodiagnostics. HSS J. 2008;4(1):87-95, http://dx.doi.org/10.1007/ s11420-007-9064-1. 17. Carvalho GA, Nikkhah G, Matthies C, Penkert G, Samii M. Diagnosis of root avulsions in traumatic brachial plexus injuries: value of computerized tomography myelography and magnetic resonance imaging. J Neurosurg. 1997;86(1):69-76, http://dx.doi.org/10.3171/jns.1997.86.1.0069. 18. Flores LP. The importance of the preoperative clinical parameters and the intraoperative electrophysiological monitoring in brachial plexus surgery. Arq Neuropsiquiatr. 2011;69(4):654-9, http://dx.doi.org/10. 1590/S0004-282X2011000500015. 19. Kline DG, Happel LT. Penfield Lecture. A quarter centuryâ&#x20AC;&#x2122;s experience with intraoperative nerve action potential recording. Can J Neurol Sci. 1993;20(1):3-10. 20. Hudson AR, Dommisse I. Brachial plexus injury. Can Med Assoc J. 1977;117:1162-4. 21. Millesi H. Brachial plexus injuries. Management and results. Clin Plast Surg. 1984;11(1):115-20. 22. Millesi H. Brachial plexus injuries. Nerve grafting. Clin Orthop Relat Res. 1988:36-42.

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48. 49. 50. 51. 52. 53. 54.

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55. Tamai S, Komatsu S, Sakamoto H, Sano S, Sasauchi N. Free muscle transplants in dogs, with microsurgical neurovascular anastomoses. Plast Reconstr Surg. 1970;46(3):219-25. 56. Manktelow RT, Zuker RM, McKee NH. Functioning free muscle transplantation. J Hand Surg Am. 1984;9A(1):32-9. 57. Hattori Y, Doi K, Baliarsing AS. A part of the ulnar nerve as an alternative donor nerve for functioning free muscle transfer: a case report. J Hand Surg Am. 2002;27(1):150-3, http://dx.doi.org/10.1053/ jhsu.2002.29484. 58. Hattori Y, Doi K, Ikeda K, Pagsaligan JM, Watanabe M. Restoration of prehension using double free muscle technique after complete avulsion of brachial plexus in children: a report of three cases. J Hand Surg Am. 2005;30(4):812-9, http://dx.doi.org/10.1016/j.jhsa.2004.12.011. 59. Yamasita AC, Mazzer N, Barbieri CH. Desenvolvimento de um software flexı´vel no estudo de regeneraçaõ nervosa perife´rica. Acta Ortop Bras. 2008;16(3):177-9. 60. Costa MP. A utilizaçaõ do tubo de aćido poliglico´lico e FK506 na regeneraçaõ de nervos perife´ricos. Acta Ortop Bras. 2006;14(1):25-9.

Z Orthop Ihre Grenzgeb. 1985;123(5):872-5, http://dx.doi.org/10.1055/s2008-1044770. Parry CB. The management of injuries to the brachial plexus. Proc R Soc Med. 1974;67(6 Pt 1):488-90. Saha AK. Surgery of the paralysed and flail shoulder. Acta Orthop Scand. 1967;(Suppl 97):5-0. Elhassan B, Bishop A, Shin A. Trapezius transfer to restore external rotation in a patient with a brachial plexus injury. A case report. J Bone Joint Surg Am. 2009;91(4):939-44. Clark JM. Reconstruction of biceps brachii by pectoral muscle transplantation. Br J Surg. 1946;34(134):180. Mayer L, Green W. Experiences with the Steindler flexorplasty at the elbow. J Bone Joint Surg Am. 1954;36A(4):775-89. Chen WS. Restoration of elbow flexion by modified Steindler flexorplasty. Int Orthop. 2000;24(1):43-6, http://dx.doi.org/10.1007/s002640050011. Rezende MR. Avaliaçaõ do ganho funcional do cotovelo com a cirurgia de Steindler na lesaõ do plexo braquial. Acta Ortop Bras. 2011;19:154-8, http://dx.doi.org/10.1590/S1413-78522011000300008.

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Eat as much as you burn - a good diet and eating less should be more important than an intense exercise program for decreasing morbidity and mortality Mustafa Cakar,I Sevket Balta,II Sait Demirkol,II Omer Kurt,I Hakan Sarlak,I Zekeriya ArslanII I

Gulhane Medical Academy, Department of Internal Medicine, Ankara/Turkey. II Gulhane Medical Academy, Department of Cardiology, Ankara/Turkey.

Email: drmustafacakar@gmail.com Tel.: 90 312 3044015

exercise, the weight of the animals fed a high-fat diet did not normalize to that of the normal diet control groups. Resistance training led to lower amounts of adipose tissue compared to swimming. Animals with sedentary life conditions fed the low-fat diet clearly had the lowest visceral adiposity. We believe that the relative failure of the success of intense exercise in the swimming and resistance training groups should be discussed in future studies. Additionally, this result suggests that a good diet and lower caloric intake may be more important than an intense exercise program for decreasing morbidity and mortality. However, the obesity induced in rats fed a high-fat diet and submitted to exercise programs may differ from some acquired and hereditary types of obesity observed in humans. The physiologic effects and pathways involved in acquired and hereditary types of obesity may differ, and these differences may affect the success of treatment. We believe that the study results should be evaluated in these respects.

Dear Editor, We read the article ’’The effects of exercise modalities on adiposity in obese rats’’ written by Guilherme Fleury Fina Speretta et al. with great interest (1). The authors concluded that both of the examined exercise modalities improved lipid profile, adiposity and obesity-associated inflammation in rats, suggesting their use as an alternative to control the deleterious effects of a high-fat diet in humans (1). We thank the authors for their contribution of such a well-designed and well-presented study. We believe that these findings will encourage further studies on obesity and the therapeutic role of diet. In the aging and increasingly obese population worldwide, this study deserves to be emphasized in terms of these current needs. Hyperlipidemia and obesity are strongly associated with the development of atherosclerosis, and coronary artery disease, cerebrovascular accidents and peripheral vascular disease are of particular clinical importance (2,3). Success in decreasing serum lipid levels and obesity has led to successful decreases in patient morbidity and mortality (4). Lifestyle changes and exercise are important and, additionally, antihyperlipidemic drugs can successfully reduce the incidence of atherosclerosis and related diseases. It is well known that nearly one hundred patients must be treated to protect only one from any atherosclerotic event (5). Therefore, investigators should work to identify new drugs, treatment approaches, diet therapies and other modalities to decrease serum lipid levels and obesity and subsequently lower the expected mortality and morbidity of these patients. The present study was primarily conducted in three exercise groups of mice, which were divided into animals receiving a normal diet or a high-fat diet. It was clear that the high-fat diet led to higher visceral adiposity. A statistically significant decrease occurred after swimming and high-resistance exercise in animals fed a high-fat diet. However, this decrease was not as large as the increase in adiposity caused by the high-fat diet and, after intense

& REFERENCES 1. Speretta GFF, Rosante MC, Duarte FO, Leite RD, Lino AD de S, Andre RA, et al. The effects of exercise modalities on adiposity in obese rats. Clinics. 2012;67(12):1469-77, http://dx.doi.org/10.6061/clinics/2012(12) 19. 2. Ciolac EG, Greve JMD. Exercise-induced improvements in cardiorespiratory fitness and heart rate response to exercise are impaired in overweight/obese postmenopausal women. Clinics. 2011;66(4):583-9. 3. Martinelli LMB, Mizutani BM, Mutti A, D’elia MPB, Coltro RS, Matsubara BB. Quality of life and its association with cardiovascular risk factors in a community health care program population. Clinics. 2008;63(6):783-8. 4. Carneiro JAO, Santos-Pontelli TEG, Vilac¸a KHC, Pfrimer K, Colafeˆmina JF, Carneiro AAO, et al. Obese elderly women exhibit low postural stability: a novel three-dimensional evaluation system. Clinics. 2012;67(5):475-81, http://dx.doi.org/10.6061/clinics/2012(05)12. 5. Ridker PM, MacFadyen JG, Fonseca FAH, Genest J, Gotto AM, Kastelein JJP, et al. Number needed to treat with rosuvastatin to prevent first cardiovascular events and death among men and women with low lowdensity lipoprotein cholesterol and elevated high-sensitivity C-reactive protein: justification for the use of statins in prevention. Circulation Cardiovascular Quality and Outcomes. 2009;2(6):616-23, http://dx.doi. org/10.1161/CIRCOUTCOMES.109.848473.

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READERS OPINION

Hyperventilation increases the rate of the rise in arterial blood desflurane concentration during induction - a Gas ManH simulation Junyong In Dongguk University, Ilsan Hospital, Department of Anesthesiology and Pain Medicine, Goyang, Gyeonggido/Republic of Korea. Email: dragona1@dumc.or.kr Tel.: 82 31 9617875

Lu et al. described that hyperventilation accelerated the desflurane uptake from the alveolar to the arterial blood (1). It is a very intriguing study of a direct determination of the desflurane concentration in the arterial blood (Ades). In comparison to the end-tidal volatile anesthetic concentration, the arterial concentration of a volatile anesthetic is more likely to reflect its concentration in the brain (1-3). A pharmacokinetic model for volatile anesthetics has been developed and used for over 25 years (4). Therefore, the effect of ventilation on Ades can be simulated with this model. Gas ManH (Version 4.1, Med Man Simulations, Inc., Chestnut Hill, MA, USA) is a computer simulation program which contains a five-compartment system built statistically which simulates the pharmacokinetics for volatile anesthetic uptake and distribution (4) with embedded parameters (5). This program helps users and readers understand anesthetic uptake and distribution with graphs. If the embedded parameters are adjusted prior to a simulation, simulated results can be seen in graphs individually. For the simulations, the necessary parameters and patient data were taken from the report of Lu et al. (1): an anesthesia breathing circuit volume of 6 L; a fresh gas flow of 6 L/min; a functional residual capacity of 2.4 L; 61.8, 62.0, and 58.0 kg patient (hyperventilation, normal ventilation, and hypoventilation, mean values from each group); an alveolar minute ventilation of 3.33, 2.38, and 1.80 L/min, mean values from each group; cardiac output which was derived from the cardiac index of the report of Lu et al. with Du Bois method (5.38, 5.56, and 5.36 L/min, mean values from each group). Other parameters were not manipulated. Therefore, the values for compartment volumes (vessel rich group, muscle, fat, and venous group) and the values for regional blood flow percentages (vessel rich group, muscle, and fat) showed some differences depending on the entered weight. At 0, 1, 3, 5, 10, 20, 30, and 40 minutes, the simulated Ades were recorded. In these simulations, despite having some limitations on the data used (e.g. using mean values of weight and alveolar ventilation) in the report of Lu et al, Gas ManH shows

Figure 1 - Simulated arterial concentration of desflurane (%) time curves under three different ventilatory settings, including hyperventilation, normal ventilation, and hypoventilation in Gas ManH (Version 4.1, Med Man Simulations, Inc., Chestnut Hill, MA, USA).

similar trends with the simulated Ades just as those of Lu et al (1). Hyperventilation seems to produce a higher Adesover-time curve than normal ventilation and hypoventilation. Simulated Ades in the hyperventilation group also seems to increase more rapidly than those in the normal ventilation and the hypoventilation groups during the first 5 minutes (Figure 1).

& REFERENCES 1. Lu CC, Lin TC, Hsu CH, Yu MH, Chen TL, Chen RM, et al. Hyperventilation accelerates the rise of arterial blood concentrations of desflurane in gynecologic patients. Clinics. 2012;67(9):1029-34, http://dx. doi.org/10.6061/clinics/2012(09)08. 2. Lu CC, Tsai CS, Ho ST, Chueng CM, Wang JJ, Wong CS, et al. Pharmacokinetics of desflurane uptake into the brain and body. Anaesthesia. 2004;59(3):216-21, http://dx.doi.org/10.1111/j.1365-2044. 2003.03654.x. 3. Lu CC, Lin TC, Yu MH, Chen TL, Lin CY, Chiang JS, et al. Effects of changes in alveolar ventilation on isoflurane arterial blood concentration and its uptake into the human body. Pharmacology. 2009;83(3):150-6, http://dx.doi.org/10.1159/000187719. 4. Philip JH. Gas Man--an example of goal oriented computer-assisted teaching which results in learning. Int J Clin Monit Comput. 1986;3(3):165-73, http://dx.doi.org/10.1007/BF01716358. 5. Eger EI, 2nd. Anesthetic uptake and action, Baltimore: Williams & Wilkins. 1974.

Copyright Ă&#x; 2013 CLINICS â&#x20AC;&#x201C; This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(03)LE02

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READERS OPINION

Which device should be chosen for the percutaneous closure of post-traumatic ventricular septal defects? Sait Demirkol,I Sevket Balta,I Mustafa Cakar,II Ugur KucukI I

Gulhane Military Medical Academy, School of Medicine, Department of Cardiology, Ankara/Turkey. Medicine, Department of Internal Medicine, Ankara/Turkey.

Gulhane Military Medical Academy, School of

Email: saitdemirkol@yahoo.com Tel.: +90-312-3044281

The percutaneous therapy of structural heart defects has become an alternative approach to surgery in selected patients. The most important considerations before performing the percutaneous closure are whether the defect can be closed via the percutaneous approach and which device should be selected. There has generally been no consensus on the selection of the device. Further studies should be conducted in the development of defect-specific devices, which may result in an improvement in patient outcome.

Dear Editor, We read with great interest the article ‘‘Percutaneous closure of a post-traumatic ventricular septal defect with a patent ductus arteriosus occluder’’ written by Xi EP et al. (1). The authors aimed to report their experiences with three patients who underwent the percutaneous closure of a posttraumatic ventricular septal defect (VSD) with a patent ductus arteriosus (PDA) occluder. They concluded that the closure of a post-traumatic ventricular septal defect using a PDA occluder is feasible, safe, and effective. We believe that these findings will act as a guide for further studies regarding the closure of post-traumatic ventricular septal defects with occluder devices. We wish to make a minor criticism about this study. In the first case, the authors placed a muscular VSD occluder. However, its right plate had an inappropriate configuration; thus, they closed the defect with a PDA occluder. Therefore, they selected the PDA occluder in the two subsequent patients. Although the percutaneous closure of traumatic and postinfarction VSDs can be accomplished with septal occluder devices safely and effectively (2,3), why did they choose the PDA occluder for the other two patients? Additionally, why did they not try to implant a VSD occluder device? In the Discussion section, they indicated that the PDA occluder cannot cause a ventricular outflow tract obstruction. However, all of the patients had muscular VSD, which itself cannot occlude the outflow tract.

& AUTHOR CONTRIBUTIONS Demirkol S contributed to the ideas for the manuscript and the manuscript writing. Balta S contributed to the manuscript writing. Cakar M contributed to literature search. Kucuk U contributed to the critical review of the paper.

& REFERENCES 1. Xi EP, Zhu J, Zhu SB, Yin GL, Liu Y, Dong YQ, et al. Percutaneous closure of a post-traumatic ventricular septal defect with a patent ductus arteriosus occluder. Clinics. 2012;67(11):1281-3, http://dx.doi.org/10. 6061/clinics/2012(11)10. 2. Demkow M, Ruzyllo W, Kepka C, Chmielak Z, Konka M, Dzielinska Z, et al. Primary transcatheter closure of postinfarction ventricular septal defects with the Amplatzer septal occluder- immediate results and up-to 5 years follow-up. EuroIntervention. 2005;1(1):43-7. 3. Holzer R, Balzer D, Amin Z, Ruiz CE, Feinstein J, Bass J, et al. Transcatheter closure of postinfarction ventricular septal defects using a new Amplatzer muscular VSD occluder: results from the US registry. Catheter Cardiovasc Interv. 2004;61(2):196-201, http://dx.doi.org/10. 1002/ccd.10784.

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Medical information technologies can increase quality and reduce costs Sigfrido Burgos University of South Alabama, College of Medicine, Mobile/AL, USA. Email: sigfridoburgos@southalabama.edu Tel.: 001 251 414-8143

provide a renewed push for patient-safety initiatives in a technology-enabled healthcare system (4). EHRs hold considerable promise to potentially reduce injuries by preventing harmful medical errors and mitigate malpractice claims. Modern telecommunications between a physician and a patient are often sufficient to establish the relationship necessary for post-consultation feedback. Additionally, messaging systems may help to prevent medical errors and adverse events by allowing patients to easily vocalize clinically significant concerns that they do not believe warrant a hospital visit (5). As the use of EHRs grows, it stands to reason that failure to adopt a functional EHR system may constitute a deviation from the standard of patient care. This standard is normally defined by reference to what is customary among physicians in the same specialty in similar settings. Once a critical mass of healthcare providers adopts EHRs, others may need to follow. This rationalization assumes that the cost-benefit calculus of adopting medical information technologies is reasonable, so that harm and injuries are prevented at an efficient cost.

Patients and healthcare providers are continually exposed to a healthcare system powered by medical information technologies. One of the first technologies showing promise is electronic health records (EHRs). Basic and advanced EHR systems facilitate access to essential clinical information, such as patient demographic characteristics, patient-physician encounters, laboratory and imaging results, and physiciansâ&#x20AC;&#x2122; notes, and allow the computerized provider-order entry of medications, which checks orders against patient information to flag potential allergic reactions, dosage errors, and drug interactions. Additionally, evolving functionalities allow for the entry of laboratory test orders and nursing orders as well as clinical-decision support, including information about relevant clinical practice guidelines, clinical reminders, and guidance and safety alerts. However, EHRs go beyond documentation because they encompass coding functions, the ability to create and export invoices, the automated creation of consultation and patient letters, electronic prescribing, and task tracking, all of which translate into greater money and time savings for patients and providers (1-3). Today, many EHR systems allow for electronic communications among providers within the same organization, while others allow for secure messaging between healthcare providers and patients. These patient-provider communications vary from requests to refill prescriptions to reports of symptoms requiring prompt medical assessment. In some instances, systems maintained by different entities share information about patients through health information exchange networks. Healthcare providers adopting EHRs have reported improvements in the quality of care they provide. In fact, as momentum gathers, the creation of unique EHR-related national patient-safety goals may

& REFERENCES 1. Jha AK, DesRoches CM, Campbell EG, Donelan K, Rao SR, Ferris TG, et al. Use of electronic health records in U.S. hospitals. N Engl J Med. 2009;360(16):1628-38. 2. Blumenthal D, Glaser JP. Information technology comes to medicine. N Engl J Med. 2007;356(24):2527-34. 3. Shea S, Hripcsak G. Accelerating the use of electronic health records in physician practices. N Engl J Med. 2010;362(3):192-5. 4. Sittig DF, Singh H. Electronic health records and national patient-safety goals. N Engl J Med. 2012;367(19):1854-60. 5. Mangalmurti SS, Murtagh L, Mello MM. Medical malpractice liability in the age of electronic health records. N Engl J Med. 2010;363(21):2060-7.

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Can fasting plasma glucose and glycated hemoglobin levels predict oral complications following invasive dental procedures in patients with type 2 diabetes mellitus? A preliminary case-control study Ana Carolina Fragoso Motta,I Cristiane Aparecida Nogueira Bataglion,I Maria Cristina Foss-Freitas,II Milton Cesar Foss,II Marilena Chinali KomesuI I University of Saõ Paulo, Dental School of Ribeiraõ Preto, Department of Morphology, Stomatology and Physiology, Ribeiraõ Preto/SP, Brazil. II University Saõ Paulo, School of Medicine of Ribeiraõ Preto, Department of Internal Medicine, Division of Endocrinology and Metabolism, Ribeiraõ Preto/SP, Brazil.

OBJECTIVE: To evaluate the effects of the levels of glycemic control on the frequency of clinical complications following invasive dental treatments in type 2 diabetic patients and suggest appropriate levels of fasting blood glucose and glycated hemoglobin considered to be safe to avoid these complications. METHOD: Type 2 diabetic patients and non-diabetic patients were selected and divided into three groups. Group I consisted of 13 type 2 diabetic patients with adequate glycemic control (fasting blood glucose levels ,140 mg/dl and glycated hemoglobin (HbA1c) levels ,7%). Group II consisted of 15 type 2 diabetic patients with inadequate glycemic control (fasting blood glucose levels .140 mg/dl and HbA1c levels .7%). Group III consisted of 18 nondiabetic patients (no symptoms and fasting blood glucose levels ,100 mg/dl). The levels of fasting blood glucose, glycated HbA1c, and fingerstick capillary glycemia were evaluated in diabetic patients prior to performing dental procedures. Seven days after the dental procedure, the frequency of clinical complications (surgery site infections and systemic infections) was examined and compared between the three study groups. In addition, correlations between the occurrence of these outcomes and the glycemic control of diabetes mellitus were evaluated. RESULTS: The frequency of clinical outcomes was low (4/43; 8.6%), and no significant differences between the outcome frequencies of the various study groups were observed (p.0.05). However, a significant association was observed between clinical complications and dental extractions (p = 0.02). CONCLUSIONS: Because of the low frequency of clinical outcomes, it was not possible to determine whether fasting blood glucose or glycated HbA1c levels are important for these clinical outcomes. KEYWORDS: Type 2 Diabetes Mellitus; Oral Infections; Glycemic Control. Motta AC, Bataglion CA, Foss-Freitas MC, Foss MC, Komesu MC. Can fasting plasma glucose and glycated hemoglobin levels predict oral complications following invasive dental procedures in patients with type 2 diabetes mellitus? A preliminary case-control study. Clinics. 2013;68(3):427-430. E-mail: anacfm@usp.br Tel.: 55 16 3602-4109

glucose (4). It has been reported that chronic hyperglycemia accelerates the accumulation of advanced glycated endproducts (AGEs) (5), which results in local tissue alterations that can increase susceptibility to infections (6). It is important to be able to identify patients who have an elevated risk of developing oral complications related to invasive dental procedures. Thus, this study aimed to evaluate the frequency of clinical complications following invasive dental procedures in type 2 diabetic patients based on their levels of glycemic control. Furthermore, this study sought to determine the levels of fasting blood glucose (FBG) and HbA1c that could be considered safe to avoid these complications.

& INTRODUCTION Diabetes mellitus (DM) is a progressive chronic disease that has a high level of morbidity because of the comorbidities that occur during disease evolution, which can compromise patient quality of life (1). One of the most important complications of DM is the observed increase in susceptibility to infections (2), likely due to impaired immunological defenses (3), which have been associated with increased concentrations of plasma

& SUBJECTS AND METHODS Ethics statement

No potential conflict of interest was reported.

This study was approved by the Ethics Committee of the Dentistry School of Ribeira˜o Preto, Sa˜o Paulo University,

DOI: 10.6061/clinics/2013(03)RC01

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Dental School of Ribeira˜o Preto. Prior to performing any dental work, the levels of FBG, HbA1c, and fingerstick capillary glycemia (FCG) were determined in all type 2 diabetic patients. Seven days after the dental procedure, the frequency of clinical complications (surgery site infections and systemic infections) in the three study groups was analyzed. In addition, the presence of tissue necrosis, purulent secretion, pain, edema, and dehiscence/wound breakdown was examined. Symptoms were registered using a 10-cm visual analog scale (VAS; 0 = no pain to 10 = extreme pain). The correlations between clinical outcomes and dental procedures, clinical outcomes and symptoms, and symptoms and type of dental procedure were also evaluated.

Brazil (CAAE #0012.0.138.000-10). All subjects provided written informed consent prior to their participation in this study.

Subjects Type 2 diabetic patients and non-diabetic patients over 35 years of age were selected and divided into three groups. The study cohort consisted of individuals of both genders and a range of ethnicities. Group I consisted of type 2 diabetic patients with adequate glycemic control (FBG levels below 140 mg/dl and HbA1c levels less than 7%). Group II consisted of type 2 diabetic patients with inadequate glycemic control (FBG levels .140 mg/dl and HbA1c levels .7%). Group III was the control group and consisted of non-diabetic patients (no symptoms of diabetes and FBG levels ,100 mg/dl). Diabetes diagnoses were made based on the World Health Organization (WHO) recommendations (7). All diabetic patients included in this study had met the diagnostic criteria for diabetes for at least five years. The following inclusion criteria were used: at least six teeth, a requirement for dental surgery (simple extraction) or complex periodontal treatment (non-surgical scaling and root planning). The need for extraction was evaluated using clinical and radiographic examinations. The requirement for periodontal treatment was evaluated via periodontal screening and recording (PSR), and patients classified as PSR 3 or 4 codes were selected. Subjects were excluded if they presented with co-existing local or systemic infections, severe complications related to diabetes, or if they had received antimicrobial treatment for oral infections in the previous three months.

Statistical analysis Fisher’s exact tests were used to evaluate the differences in clinical outcomes after invasive dental procedures in the three groups, the association between clinical outcome and procedure type, and the effects of gender in each of the three groups. These analyses were performed using SAS software (Statistical Analysis System - SASH 9.0 software; Cary, NC, USA). Differences in age, glycemic control, and symptoms reported by the patients between the three groups were analyzed using Kruskal-Wallis tests with Dunn post hoc tests, and these analyses were performed using R software (R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org). The data were reported as the means and standard deviation (SD), and the level of significance was set at 5% for all analyses.

& RESULTS

Study design

The demographic characteristics and glycemic control of the subjects are shown in Table 1. After reviewing 1,175 charts for inclusion and exclusion criteria, 147 type 2 diabetics were initially selected. However, only 28 subjects were found to be eligible for the current study. Of these, 13 subjects had type 2 diabetes with adequate glycemic control (Group I), and 15 exhibited inadequate glycemic control (Group II). The other 119 patients were excluded for at least

A case-control study was developed and performed between May 2010 and November 2011. The charts from diabetic patients treated at the Diabetes Outpatient Clinics of the University Hospital of the Medical School of Ribeiraõ Preto, Saõ Paulo University, were reviewed, and patients that met the eligibility criteria were referred to the Dental School of Ribeiraõ Preto for dental treatments. Patients for the control group were selected from the clinics of the

Table 1 - Clinical characteristics of type 2 diabetic patients with adequate (Group I) and inadequate glycemic control (Group II) and non-diabetic subjects (Group III) and the frequency of symptom levels (0-5 and 6-10) reported after dental invasive procedures. Variables Gender* Male Female Age (years) Mean ¡ SD Capillary glycemia (mg/dl)** Mean ¡ SD Fasting blood glucose (mg/dL)** Mean ¡ SD HbA1c (%)** Mean ¡ SD Clinical outcomes* No Yes Symptoms (VAS) 0-5 6-10 *

Group I (n = 13)

Group II (n = 15)

Group III (n = 18)

p-value

7 (53.8%) 6 (46.2%)

4 (26.7%) 11 (73.3%)

3 (16.6%) 15 (83.4%)

0.10{

58.53¡6.11

52.86¡7.24

46.53¡7.61

,0.01{

151.33¡39.80

240.93¡72.89

105.26¡22.91

,0.01{

134.76¡25.18

197.33¡71.79

88.73¡9.78

,0.01{

6.5¡0.50

10.22¡1.33

,0.01{

12 (92.3%) 1 (7.7%)

13 (86.6%) 2 (13.3%)

17 (94.4%) 1 (5.6%)

0.81{

12 (92.3%) 1 (7.7%)

14 (93.4%) 1 (6.6%)

17 (94.4%) 1 (5.6%)

0.80{

Values shown as n (%); ** values shown as the means and standard deviation (SD); { Fisher’s exact test; { Kruskal-Wallis test; VAS: visual analog scale.

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Oral complications in type 2 diabetes Motta AC et al.

one of the following reasons: FBG levels .300 mg/dl and/ or HbA1c levels .10%, which are associated with complications of diabetes; not having at least six teeth; or refusing to participate in the study. Eighteen non-diabetic patients were also studied as a control group (Group III). FBG, HbA1c, and FCG levels were higher in type 2 diabetic patients with inadequate glycemic control compared with those with adequate glycemic control. The frequency of clinical complications was low and similar (p = 0.80) in the three groups, and this frequency was independent of glycemic control (Table 1). Of the 46 patients examined here, only four (8.6%) showed oral complications: one in Group I, two in Group II, and one in Group III. These four patients were treated by dental extraction, and their outcomes were all characterized by intra-oral swelling, redness, pus, and wound dehiscence. All 27 patients underwent scaling and root planning, which did not induce any complications. Thus, dental extractions were associated with increased numbers of complications compared with scaling and root planning (p = 0.02) (Table 2). Most patients (43/46; 93.4%) reported pain symptoms ranging from 0 and 5 (VAS), but three patients reported symptoms ranging from 6 to 10 (Table 1). All of the patients who received scaling and root planning had VAS scores of 5 or lower, and all three of the patients who reported symptoms greater than 6 had undergone extractions (of 19 total, 15.7%) (Table 2). However, no significant associations were observed between the clinical outcomes and symptoms (p = 0.81) or between the type of dental procedure and symptoms (p = 0.20).

FCG = 98 mg/dl (Group III patient). Regarding the HbA1c values, the three diabetic patients who had postoperative complications had HbA1c values as follows: 7% (Group I patient) and 9% and 11% (Group II patients). The use of antibiotics has been recommended for type 2 diabetics prior to dentoalveolar surgery (8,9) to prevent surgical site infections and facilitate the healing process. In this study, antibiotics were not used prior to dental procedures in any of the groups, but the frequency of clinical complications was low nonetheless. The similar prevalence of surgical site infections in type 2 diabetic patients with adequate and inadequate glycemic control and in non-diabetics suggests that the rate of infections may not depend on glycemic control, despite the reports of an increased risk of infections in diabetic patients (10). In addition, these results suggest that antibiotic prophylaxis may only be appropriate in specific cases, not during routine practice. All of the patients in the current study were treated with local antibiotic irrigation, and the surgical wounds of all patients were clinically repaired within 10 days. The present study failed to identify a direct relationship between FBG levels or HbA1c values and postoperative complications; thus, it was impossible to suggest FBG or HbA1c values that would be predictive of complications. This study did reveal that dental extractions are more frequently characterized by complications than scaling and root planning procedures, but this effect was not linked to glycemic control. The limitations of this study included the small number of patients and the fact that postoperative FBG values were not evaluated. It is possible that studies with larger sample sizes might be sufficiently powered to assess the impact of glycemic control on the occurrence of postoperative complications.

& DISCUSSION This study evaluated the frequency of postoperative dental complications in patients with type 2 diabetes based on their levels of glycemic control. The results demonstrated a low frequency of complications, and no evidence of an effect of glycemic control on clinical outcomes was observed (Table 1). Of the 46 patients who underwent invasive procedures, only four (8.6%) had surgical site infections, and this outcome occurred in patients from each of the three groups: one patient from Group I, two patients from Group II and one patient from Group III. The following FBG and FCG values were observed in the patients who had postoperative complications: FBG = 137 mg/dl and FCG = 225 mg/dl (Group I patient); FBG = 219 mg/dl; FCG = 300 mg/dl and FBG = 301 mg/dl; FCG = 195 mg/dl (Group II patients); and FBG = 88 mg/dl;

& ACKNOWLEDGMENTS The authors are grateful to Ms Milena Saavedra Lopes Amaral and Maria Aparecida Yoshiko Hirasawa Matuyama for assistance with sample analysis. Dr. Cristiane Aparecida Nogueira Bataglion was supported by a scholarship from the Coordination for the Improvement of Graduated Personnel. The study was funded by the Foundation of Support to Teaching, Research and Assistance of HCFMRP-USP (FAEPA).

& AUTHOR CONTRIBUTIONS Motta AC conceived and designed the study and drafted the manuscript. Bataglion CA performed the study. Foss-Freitas MC, Foss MC, and Komesu MC participated in the design and coordination of this study.

& REFERENCES

Table 2 - Frequency of type 2 diabetic patients presenting outcomes and symptoms based on the type of dental procedure. Procedure

Outcomes* No Yes Symptoms (VAS)* 0-5 6-10 *

1. American Diabetes Association. Diagnosis and Classification of diabetes mellitus. Diabetes Care. 2011;34:S62-S69, http://dx.doi.org/10.2337/ dc11-S062. 2. Foss NT, Polon DP, Takada MH, Foss-Freitas MC, Foss MC. Skin lesions in diabetic patients. Rev Sau´de Pu´blica. 2005;39(4):677-82, http://dx.doi. org/10.1590/S0034-89102005000400024. 3. Shah BR, Hux JE. Quantifying the risk of infectious diseases for people with diabetes. Diabetes care. 2003;26(2):510-3, http://dx.doi.org/10. 2337/diacare.26.2.510. 4. Foss-Freitas MC, Foss NT, Donadi EA, Foss MC. In vitro TNF-a and IL-6 production by adherent peripheral blood mononuclear cells patients evaluated according to the metabolic control. Ann NY Acad Sci. 2006;1079:177-80, http://dx.doi.org/10.1196/annals.1375.027. 5. Vlassara H, Brownlee M, Manogue KR, Dinarello CA, Pasagian A. Cachectin/TNF and IL-1 induced by glucose-modified proteins: role in normal tissue remodeling. Science. 1988;240(4858):1546-8, http://dx.doi. org/10.1126/science.3259727.

p-value**

SRP

Extraction

27 (100%) 0 (0%)

15 (78.9%) 4 (21.1%)

0.02

27 (100%) 0 (0%)

16 (84.2%) 3 (15.8%)

0.20

Values shown as n (%); VAS: visual analog scale; SRP: scaling and root planning; ** Fisher’s exact test.

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non-antimicrobial mechanisms. Adv Dent Res. 1998;12(2):12-26, http:// dx.doi.org/10.1177/08959374980120010501. 9. Tong DC, Rothwell BR. Antibiotic prophylaxis in dentistry: a review and practice recommendations. J Am Dent Assoc. 2000;131(3):366-74. 10. Rao DD, Desai A, Kulkarni RD, Gopalkrishnan K, Rao CB. Comparison of maxillofacial space infection in diabetic and nondiabetic patients. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010;110(4):e7-12.

6. Komesu MC, Tanga MB, Buttros KR, Nakao C. Effects of acute diabetes on rat cutaneous wound healing. Pathophysiology. 2004;11(2):63-7, http://dx.doi.org/10.1016/j.pathophys.2004.02.002. 7. World Health Organization [Internet]. WHO/Diabetes; Disponible: http://www.who.int/topics/diabetes_mellitus/en/. 8. Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, Sorsa T. Tetracyclines inhibit connective tissue breakdown by multiple

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Design and baseline characteristics of a coronary heart disease prospective cohort: two-year experience from the strategy of registry of acute coronary syndrome study (ERICO study) Alessandra C. Goulart,I,II Itamar S. Santos,I,II Debora Sitnik,I Henrique L. Staniak,I Ligia M. Fedeli,I Carlos Alberto Pastore,III Nelson Samesima,III Marcio S. Bittencourt,I Alexandre C. Pereira,I,III Paulo A. Lotufo,I,II Isabela M. BensenorI,II I

Universidade de Saõ Paulo, Hospital Universita´rio, Center for Clinical and Epidemiological Research, Saõ Paulo/SP, Brazil. Universidade de Saõ Paulo, Brazil. III Instituto do Coraçaõ (InCor), Hospital das Clıńicas da Universidade de Saõ Paulo, Brazil.

Faculdade de Medicina,

OBJECTIVES: To describe the ERICO study (Strategy of Registry of Acute Coronary Syndrome), a prospective cohort to investigate the epidemiology of acute coronary syndrome. METHODS: The ERICO study, which is being performed at a secondary general hospital in Sao Paulo, Brazil, is enrolling consecutive acute coronary syndrome patients who are 35 years old or older. The sociodemographic information, medical assessments, treatment data and blood samples are collected at admission. After 30 days, the medical history is updated, and additional blood and urinary samples are collected. In addition, a retinography, carotid intima-media thickness, heart rate variability and pulse-wave velocity are performed. Questionnaires about food frequency, physical activity, sleep apnea and depression are also applied. At six months and annually after an acute event, information is collected by telephone. RESULTS: From February 2009 to September 2011, 738 patients with a diagnosis of an acute coronary syndrome were enrolled. Of these, 208 (28.2%) had ST-elevation myocardial infarction (STEMI), 288 (39.0%) had non-STelevation myocardial infarction (NSTEMI) and 242 (32.8%) had unstable angina (UA). The mean age was 62.7 years, 58.5% were men and 77.4% had 8 years or less of education. The most common cardiovascular risk factors were hypertension (76%) and sedentarism (73.4%). Only 29.2% had a prior history of coronary heart disease. Compared with the ST-elevation myocardial infarction subgroup, the unstable angina and non-ST-elevation myocardial infarction patients had higher frequencies of hypertension, diabetes, prior coronary heart disease (p,0.001) and dyslipidemia (p = 0.03). Smoking was more frequent in the ST-elevation myocardial infarction patients (p = 0.006). CONCLUSIONS: Compared with other hospital registries, our findings revealed a higher burden of CV risk factors and less frequent prior CHD history. KEYWORDS: Coronary Acute Syndrome; Epidemiology; Registries. Goulart AC, Santos IS, Sitnik D, Staniak HL, Fedeli LM, Pastore CA, et al. Design and baseline characteristics of a coronary heart disease prospective cohort: two-year experience from the strategy of registry of acute coronary syndrome study (ERICO study). Clinics. 2013;68(3):431-434. Received for publication on October 9, 2012; First review completed on November 5, 2012; Accepted for publication on November 13, 2012 E-mail: agoulart@hu.usp.br Tel.: 55 11 3091-9241

elevation myocardial infarction (NSTEMI) and ST elevation myocardial infarction (STEMI) (1). According to recent official data, there were 12,340 hospitalizations in the city of Sa˜o Paulo during the 2011 calendar year due to angina pectoris or myocardial infarction (International Classification of Diseases – ICD I20/I21) that were reimbursed by the Brazilian National Health System, with a total 94,857 days of hospitalization (2). However, most medical facilities that assist these patients are classified as secondary care hospitals and do not provide interventional cardiac procedures. ACS registries are more frequently conducted in tertiary-care hospitals (3-4). Hereafter, we will describe the design and baseline characteristics of the ERICO (Estrate´gia de Registro de

& INTRODUCTION Acute coronary syndrome (ACS) is a major cause of mortality and morbidity worldwide. This syndrome is a broad term that includes unstable angina (UA), non-ST

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Insuficieˆncia Coronariana – Strategy of Registry of Acute Coronary Syndrome) study, a hospital-based cohort study of patients with ACS, which is still ongoing in a communityhospital in the city of Sa˜o Paulo.

such as urgent or scheduled percutaneous coronary transluminal angioplasty (PTCA) and/or coronary artery bypass graft surgery (CABG), echocardiogram findings and information about medications are obtained. Questionnaires regarding depressive symptoms (PHQ-9) (8), food-frequency (9), physical activity (10,11), sexual activity and sleep apnea (Berlin questionnaire) (12) are also administered by trained interviewers. Tests for serum creatinine, fasting blood glucose, oral glucose tolerance test, hepatic enzymes, cholesterol levels and microalbuminuria are conducted. Additionally, a more detailed evaluation of the cardiovascular system, including pulse-wave velocity, heart rate variability, carotid artery intima-media thickness and retinography is performed. A new blood sample for biobank storage is obtained. For those participants who are unable to attend the 30-day consultation, information about their cardiovascular evaluation and vital status is obtained by phone.

& METHODS Setting The ERICO study was launched in February 2009 to establish a surveillance strategy for ACS and the quality of care at Hospital Universita´rio, Universidade de Sa˜o Paulo (HU-USP). This institution is a 260-bed teaching community hospital in the borough of Butanta˜ that had a population of 428,000 inhabitants in 2010 and a human development index of 0.716 (5-6). The frequency of patients with ACS in HUUSP is approximately one patient per day. Individuals with ACS are treated in the emergency department, the internal medicine ward or a general intensive care unit. Patients who need an interventional procedure are mostly referred to the Heart Institute at the University of Sa˜o Paulo.

Long-term follow-up Six months and annually after the index event, all participants are contacted by phone to update information about their vital status, cardiovascular history, medications, depressive symptoms, physical activity and sleep apnea.

Case definition Myocardial infarction (MI) was defined by the presence of symptoms consistent with cardiac ischemia within 24 hours of hospital presentation and troponin I levels above the 99th percentile with a test-specific coefficient of variation ,10% (1,7). STEMI was defined by the presence of criteria for MI plus one of the following: (a) persistent ST segment elevation of $1 mm in two contiguous electrocardiographic leads or (b) the presence of a new or presumably new left bundle branch block. NSTEMI was defined by the presence of criteria for MI but not for STEMI. The UA diagnosis required the presence of symptoms consistent with cardiac ischemia 24 hours prior to hospital admission, absence of MI criteria and at least one of the following: (a) history of coronary heart disease (CHD); (b) positive coronary disease stratification test (invasive or noninvasive); (c) transient ST segment changes $0.5 mm in two contiguous leads, new T-wave inversion of $1 mm and/or pseudonormalization of previously inverted T waves; (d) troponin I .0.4 ng/ml (which guarantees a troponin I level above the 99th percentile regardless of the utilized kit); or (e) diagnostic concordance of two independent doctors.

Outcomes Relevant outcomes include the occurrence of: (a) new chest pain, (b) confirmed new ACS event, (c) cardiac revascularization via either PTCA or CABG (not due to the index event), (d) stroke and (e) all-cause and cardiovascular deaths. Each identified event is adjudicated using predefined international criteria (1,7). Vital status is complemented periodically by a hot-pursuit strategy. We routinely check the state death index to look for missing participants. Death certificates are obtained whenever possible.

Statistical Analysis The subjects are classified into three subgroups according to the ACS subtype. Categorical variables are presented as proportions and are compared using Chi-square/Fisher’s exact tests. Continuous variables are presented as means (standard deviations) and are compared using one-way ANOVA, with the Bonferroni post-hoc test for multiple comparisons. The statistical analyses are performed with SPSS version 16.0.

Study protocol All individuals with suspected ACS are invited to participate in our study. Data regarding sociodemographics, main cardiovascular risk factors (hypertension, diabetes, obesity, dyslipidemia, smoking, familial and personal history of CHD, physical inactivity, cocaine use and menopause), medications and a questionnaire for depressive symptoms (Patient Health Questionnaire-PHQ9) are obtained by trained interviewers (8). Three physicians are responsible for the review of all medical charts, for asking participants about all necessary information at hospital admission and for ordering electrocardiograms, laboratory tests (troponin I, MB-creatine kinase, serum glucose, total cholesterol, HDL/LDL-cholesterol, triglycerides and total blood cell count) and in-hospital medical treatment. All participants are asked permission for the storage of blood samples in the HU-USP biobank for further evaluation.

& RESULTS From February 2009 to September 2011, 738 patients diagnosed with ACS were enrolled. Of those, 208 (28.2%) had STEMI, 288 (39.0%) had NSTEMI and 242 (32.8%) had UA (Table 1). The mean age was 62.7 years, 58.5% were men and 77.4% had #8 years of education. The most common primary cardiovascular risk factors were hypertension (76.0%) and physical inactivity (73.4%). Only 29.2% had a prior history of CHD. Compared with the STEMI subgroup, the UA and NSTEMI patients had higher frequencies of hypertension, diabetes, prior CHD and dyslipidemia. Current smoking was more frequent in the STEMI patients. Furthermore, based on the telephone follow-up, our oneyear case-fatality rate for all-cause deaths was 12.1% (89 fatal events/738 ACS cases).

30-day follow-up Participants are reevaluated by a physician 30 days after the event. Additional data on cardiovascular risk stratification,

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CLINICS 2013;68(3):431-434

ERICO study: Design and baseline Goulart AC et al.

Table 1 - Baseline characteristics of ERICO patients according to the diagnosis at discharge. Diagnosis at discharge

Characteristics

Mean age (years) (ยกSD) Age strata (%) #45 46-55 56-65 66-75 .75 Gender (%) Male Female Race (%) White Mixed Black Asiatic Years of Education (%) Illiterate 1-7 .8 Marital status (%) Single Married Divorced Widowed Prior CHD (%) Cardiovascular risk factors* (%) Family history of premature CHD ** Hypertension * Diabetes mellitus * Smoking status * Never Past Current Dyslipidemia * Sedentarism (%)

Non-ST-segment elevation myocardial infarction (n = 288)

ST-segment elevation myocardial infarction (n = 208)

Unstable angina (n = 242)

Total (n = 738)

65.3 (ยก13.3)

59.2(ยก13.2)

62.7(ยก12.5)

62.7(ยก13.2)

15 (5.2) 48 (16.7) 79 (27.4) 70(24.3) 76 (26.4)

26 51 70 34 27

(12.5) (24.5) (33.7) (16.3) (13.0)

16 (6.6) 53(21.9) 66 (27.3) 60 (24.8) 47 (19.4)

57 152 215 164 150

167(58.0) 121 (42.0)

135 (64.9) 73 (35.1)

130 (53.7) 112 (46.3)

432 (58.5) 306 (41.5)

207 (71.9) 55 (19.1) 22 (7.6) 4 (1.4)

151 (72.6) 51 (24.5) 5 (2.4) 1 (0.5)

149 (61.6) 77 (31.8) 15 (6.2) 1 (0.4)

507 (68.7) 183 (24.8) 42 (5.7) 6 (0.8)

41(14.3) 186 (64.8) 60 (20.9)

24 (11.7) 129 (62.6) 53 (25.7)

41(16.9) 148 (61.20) 53 (21.9)

106 (14.4) 463(63.0) 166 (22.6)

31 (10.8) 173 (60.5) 26 (9.1) 56 (19.6) 64 (24.9)

37 (17.9) 138 (66.7) 13 (6.3) 19 (9.2) 30 (15.8)

36 (14.9) 139 (57.7) 21 (8.7) 45 (18.7) 98 (46.7)

31 (14.2) 450 (61.3) 60 (8.2) 120 (16.3) 192 (29.2)

68 (26.1) 219 (77.4) 131 (46.1)

51 (27.6) 123 (61.5) 57 (28.6)

69 (30.8) 206 (86.6) 96 (41.4)

188 (28.1) 548 (76.0) 284 (39.7)

91 (34.3) 93 (35.1) 81(30.6) 141(55.3) 206 (75.7)

53 (26.8) 69 (34.8) 76 (38.4) 81(48.2) 134 (70.5)

80 (36.5) 91 (41.6) 48 (21.9) 124 (62.0) 157 (73.0)

224 253 205 346 497

p-value ,0.001 ,0.001

(7.7) (20.6) (29.1) (22.2) (20.3) 0.054

0.004

0.44

0.01

(32.8) (37.1) (30.1) (55.5) (73.4)

,0.001 0.50 ,0.001 ,0.001 0.006

0.03 0.45

Some proportions might not add up to 100% due to rounding. * All cardiovascular risk factors were based on previous medical history. ** Family history of premature coronary heart disease was defined as coronary heart disease in a first-degree male relative before age 55 or in a firstdegree female relative before age 65.

ERICO participants have high frequencies of cardiovascular risk factors (14,15). A possible reason that explains these differences is the low number of individuals with prior CHD in this cohort, which could be associated with a high frequency of cardiovascular risk factors, as lifestyle changes are more frequent in people with a previous ACS events. The comparison with the Gulf RACE participants revealed a high proportion of current smokers, which is most likely associated with the cultural lifestyle in Middle Eastern countries (16). There are two large studies of ACS patients in Brazil, both with methodological differences compared with ERICO. The BRACE study is a multi-center study designed to evaluate regional differences regarding the use of effective in-hospital treatments of patients with ACS (17). The second study is the Brazilian arm of INTERHEART, an international case-control study designed to ascertain the impact of conventional and emerging cardiovascular risk factors on acute MI (18). The ERICO study has several characteristics that distinguish it from other registries in Brazil. First, it evaluates prognostic factors at the emergency room and 30 days after

& DISCUSSION Here, we describe the design and baseline characteristics of a prospective cohort of patients with an ACS event assisted at a community hospital, with the main objective of identifying prognostic factors associated with early and late case-fatality and survival. At the baseline, a low proportion of ERICO participants had previous ACS events (29.2%). This result contrasted with the findings of: the GRACE (the Global Strategy of Acute Coronary Events) study, which included individuals with ACS events from 184 centers, most located in high-income countries; the multi-center Gulf registry of acute coronary events (Gulf RACE) study conducted in Middle Eastern countries; and the data of the tertiary center-based study in Brazil in which 47.6% of individuals diagnosed with ACS had previous coronary disease (13). We may speculate that these differences occurred because patients with previous ACS events are more prone to seek specialized centers in the case of a new event. The frequencies of the cardiovascular risk factors vary across registries. Compared with the GRACE registry, the

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ERICO study: Design and baseline Goulart AC et al.

CLINICS 2013;68(3):431-434

the event to verify the best time for the association of appropriate factors with late outcomes. Second, each participant in the study has multiple biological samples stored in liquid nitrogen, which can be used for future nested case-control studies addressing new prognostic factors. We intend to evaluate biomarkers associated with the nitration of proteins, such as nitrotyrosine and myeloperoxidase, and biomarkers involved with monocyte/ macrophage activation, such as the monocyte chemoattractant protein 1 (MCP1), ILb1 and netrin-1, in samples collected at hospital admission and after 30 days and determine the relationships of these biomarkers with longterm prognosis. In addition, DNA and RNA markers will be investigated with respect to their predictive accuracy for incident events and will be incorporated into clinical prediction models calibrated for the Brazilian population. Third, the number of exposures included in the study is high. In addition to common cardiovascular risk factors, we are exploring inflammatory biomarkers, DNA extraction, psychosocial factors, diet evaluation via a food-frequency questionnaire, carotid intima-media thickness, pulse-wave velocity, heart rate variability and retinography. Although this is a single-center study, which may not be representative of all of the diversity of the Brazilian population, ERICO can add new information about prognostic factors in ACS related to early and late outcomes in a community hospital, which is an underrepresented scenario for ACS studies.

4. 5.

7. 8.

10.

& AUTHOR CONTRIBUTIONS 11.

Goulart AC conceived and designed the study, was responsible for the data acquisition, analysis and interpretation, drafting of the manuscript, critical revision of the manuscript for important intellectual content, statistical analysis, administrative and technical support, and study supervision. Santos IS conceived and designed the study, was responsible for the data acquisition, analysis and interpretation, drafting of the manuscript, critical revision of the manuscript for important intellectual content, statistical analysis and study supervision. Sitnik D was responsible for the data acquisition and critical revision of the manuscript for important intellectual content. Staniak HL conceived and designed the study, was responsible for the data acquisition, analysis and interpretation and critical revision of the manuscript for important intellectual content. Fedeli LM was responsible for the data acquisition, analysis and interpretation and critical revision of the manuscript for important intellectual content. Pastore CA and Samesima N performed the analysis and interpretation of data (ECG) and critical revision of the manuscript for important intellectual content. Bittencourt MS conceived and designed the study and was responsible for the critical revision of the manuscript for important intellectual content. Pereira AC conceived and designed the study and was responsible for the data analysis and interpretation and critical revision of the manuscript for important intellectual content. Lotufo PA and Bensen˜or IM obtained the funding, conceived and designed the study and were responsible for the critical revision of the manuscript for important intellectual content, statistical analysis, administrative and technical support, study supervision.

12. 13. 14.

15.

16.

17.

& REFERENCES

18.

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ERRATA

& CLINICS 2012;67(11):1281-3 On page 1283, in the fourth paragraph, the VSD, which the authors presented the figure (Figure 1B), described the postinfarction VSD, whereas it should have been post-traumatic VSD, as follows: ‘‘The post-traumatic VSD is almost angulated (Figure 1B), but the congenital VSD or ASD is straight.’’

& CLINICS 2013;68(2):253-62 Replace III Universiti Malaya, Faculty of Medicine, Department of Anatomy, Malaysia Medical Center, Kuala Lumpur, Malaysia For

III

Universiti Kebangsaan Malaysia, Faculty of Medicine, Department of Anatomy, Kuala Lumpur, Malaysia

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