Minima Medica 2022 by Malta Medical Students' Association

2022 ISSUE

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Editorial: The Role of the Doctor in Time of Crisis

M D, F

Foreword Message

Pro f

an Calleja A e .J

C li n

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I am very delighted to be once again invited by the Malta Medical Students’ Association (MMSA) to write the editorial for this issue of Minima Medicamenta. It brings back nostalgic memories of when I myself was the Editor of the ‘Murmur’, which at the time was the MMSA’s student magazine, the content of which was more of a lighter nature. MMSA has made giant leaps, and as evidenced by this particular issue, medical students are getting involved in authoring high quality scientific work. While the coronavirus pandemic is still raging on, the war in Ukraine is sadly causing human tragedies on a daily basis. In addition, thousands of international medical students have been caught up in the middle of this crisis, forced to interrupt their studies and flee the country, with no transcript or any evidence that they were studying medicine. There is a big shortage in university places leading to a medical doctorate in other countries, while at the same time, transferring to a different university is quite complicated due to differences in curriculums, the documentation required and additional costs. In situations like these, one has to pause and appreciate the medical education that is available on our island and that is so often taken forgranted. The University of Malta is a highly regarded and historic university and has a long history of medical education.The MD Programme offered by the Faculty of Medicine and Surgery and awarded by the University of Malta has been accredited by the Association of Medical Schools in Europe and ASIIN, a German accreditation institution. As a result of these accreditations of the MD degree, University of Malta medical graduates have more opportunities for mobility and lifelong learning at an international level. The engagement of medical students in medical education is of vital importance. Afterall, according to Rudolf Virchow (1821-1902): “Medical education does not exist to provide students with a way of making a living, but to ensure the health of the community.” A doctor’s role is not only to diagnose and cure, but also to inform and educate society. Medicine is not only a science; it is also an art. It deals with the very processes of life, which must be understood before they may be guided.

Prof. Jean Calleja Agius

MD, FRCOG, FRCPI, MSc Clinical Embryology (Leeds), PhD (London) page 1

olas Gale h c i a N

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Foreword Message M e dic al

c Edu

I am extremely pleased to launch MMSA’s Minima Medica Journal, which is back for a new edition in 2022! Ever since its inaugural edition, Minima Medica has been as a way for medical students to publish their research in a peer-reviewed journal. It also serves as a stepping stone for those who wish to enter the vast world of research but don’t know where to start. The 2022 edition of this journal includes 10 articles covering a large variety of medical topics, with each article being supervised and reviewed by two separate academics from the University of Malta, helping to strengthen the reliability and validity of Minima Medica as a reputable research journal. This journal would not have been possible without the outstanding work of the team around it. I am extremely thankful for Daniela Chatlani, the SCOME Publications Coordinator, who has been coordinating the entire process leading up to the words you will be reading shortly, from releasing and vetting article applications, to the coordination of the SCOME Research Conference at which this journal is being released. I would also like to thank Rebekah St John, the SCOME Assistant, whose unwavering commitment to the standing committee has shone through during every part of the year, and especially throughout this process. The MMSA PRO, Martina Formosa, as well as PR coordinator Martina Baldacchino, should also be celebrated, as without their unique designing capabilities, this journal would not have existed. Finally, I would like to thank the many authors for putting the time and effort into writing and submitting the articles you are about to read, as well as the academics who found the time to review each and every article. It is because of you that this journal is what it is. I hope that every single reader, be they a student, academic, or otherwise, finds this journal to be a fruitful and interesting read, and that they enjoy delving deeper into some of the many marvels that the field of medicine has to offer.

Nicholas Galea

Medical Education Officer page 2

in SC

Foreword Message

la Chatla e i n an

E P u b li c a ti o n s

Dear readers, It is with great pleasure that I was given the opportunity to launch this year's edition of Minima Medica. Ever since its revival back in 2020, this journal has proved to be a success with multiple medical students publishing each year. A wide variety of literature reviews have been published this year, ranging from anatomy to physiology to psychiatry. This reflects the great variety of specialties in medicine and how each one of them is equally as important as the other. I would like to thank Prof. Calleja Aguis for taking her time to write the foreword for this year's edition as well as this year's SCOME officer Nicholas Galea and SCOME assistant Rebekah St John, for all their assistance and guidance. Lastly, I would like to thank all the contributing authors, without which this journal would not be possible.

Daniela Chatlani

SCOME Publications Coordinator

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Sprengel’s Deformity: A Case report and Review of the Literature

Sprengel’s deformity or congenital elevation of the scapula is a rare congenital disorder where the scapula is abnormally elevated and dysplastic (1). The condition was first described in 1863, and named after Otto Gerhard Karl Sprengel, who reported on four cases in 1891 (2). Although it is considered to be the most common congenital deformity of the shoulder, its incidence is unknown since a vast number of cases remain undiagnosed (3). The condition is more common in females, with a female-to-male ratio of 3:1 (4). Although the deformity typically affects one shoulder, it may also be bilateral, and occurs in approximately 10 to 30% of cases (5).

The scapula is normally found between the 2nd and 7th thoracic vertebrae on the posterior thoracic wall. Sprengel’s deformity occurs due to failure of the affected scapula to migrate caudally during early foetal development (6). The underlying cause for this to occur is currently unknown, and is often sporadic; however, an autosomal dominant inheritance pattern of the deformity may be present in rare cases, referred to as Corno’s disease (7). It has been proposed that oligohydramnios or neural crest defects are the root cause of the deformity (2), causing a transient interruption in embryonic blood supply via the subclavian artery (4). During embryogenesis, the scapula normally forms at the level of the cervical spine, then moves caudally to its normal position during the third month of gestation (8). In Sprengel’s deformity, this migration fails to occur (1). Moreover, an omovertebral bone may be present in 19 – 47% of cases (9), extending between the affected scapula and the spinous processes; transverse processes; or laminae of vertebrae C4 to C7. This further impedes the normal caudal migration of the scapula, producing the abnormal elevation. The connection may be fibrous, osseous or cartilaginous (6,9).

Sprengel’s deformity is most commonly graded according to the clinical Cavendish classification, which groups the deformity based on the degree of elevation and associated deformity (1,3). Table 1: Cavendish Classification

Grade Grade 1

Description Very mild deformity observed. When patient is dressed, the deformity is almost invisible.

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Grade 2

The deformity is still mild but noticeable as a bump. The superomedial portion of the scapula is convex, forming the bump.

Grade 3

Moderate deformity, with 2 – 5 cm of visible elevation of the affected shoulder.

Grade 4

Severe deformity with > 5 cm elevation of the affected shoulder, accompanied by neck webbing.

In addition, the radiographic Rigault classification is utilised to grade the deformity on xrays, based on the vertebral level of the scapula to indicate the grade (10).

Table 2: Rigault Classification

Grade

Description

Grade I

Superomedial scapular angle lower than T2 but above T4 transverse process.

Grade II

Superomedial angle positioned between C5 and T2 transverse process.

Grade III

Superomedial angle above C5 transverse process.

In approximately 75% of cases, other abnormalities are present which may form part of a syndrome (11) and which must be considered when dealing with such patients. The following have all been reported in the context of this deformity (1,4,11): - Klippel-Feil Syndrome (20 to 42% of patients (2); - Congenital torticollis; - Defects of the cervical vertebrae; - Kyphoscoliosis or isolated scoliosis; - Spina bifida; - Clavicle hypoplasia. Furthermore, atrophy or hypoplasia of the shoulder girdle musculature may be a feature of this deformity (1). With severe deformities, the inferior angle of the scapula is rotated medially, thereby directing the glenoid fossa downwards (6). This, together with the abnormal regional muscles, limits the range of motion of the affected shoulder, especially abduction (12).

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Sprengel’s deformity is frequently noted at infancy due to the cosmesis, especially if grade III or IV, as well as due to the restricted movement at the glenohumeral joint (11). A portion of cases are diagnosed later in life incidentally, during an unrelated physical examination or radiological study, as was the case with our patient. A standard postero-anterior (PA) projection of the chest or an antero-posterior (AP) shoulder radiograph should be taken as a first-line modality to diagnose and assess the severity of the deformity (1,12). It may be necessary to obtain an x-ray of the cervical spine, in order to assess the omovertebral bone attachment and for any related bony anomalies (12). Further imaging may be done using computed tomography (CT) or magnetic resonance imaging (MRI) for pre-operative planning (6,13). Pre-natal diagnosis is possible using ultrasound and is recommended for screening in Corno’s disease (14). Mild cases are typically managed non-operatively, using physiotherapy (2). Surgical correction of Sprengel’s deformity is the management of choice for severe deformities with cosmetic and functional limitations. The ideal age range for surgical intervention is between the ages of three to eight (2); however older individuals may still be operated without issue. Two surgical approaches are most commonly used, namely the modified Green and the Woodward procedures, which both transfer muscle origins and include an osteotomy (15). According to Gonen, 2010 and Borges, 1996 an improvement in range of motion by up to 50 degrees may be seen post-operatively with either procedure (16,17).

A 60-year-old man presented to the emergency department complaining of new onset lower limb oedema, affecting the right leg and extending up to the right shin. Further examination revealed occasional shortness of breath. A chest x-ray was requested, and no cardiovascular or pulmonary abnormalities were shown (Figure 1). Sprengel’s deformity of the left shoulder was noted incidentally, with presence of an omovertebral bone, extending from the medial angle of the scapula to C6 (marked by a red arrow on figures 1 and 2). Interestingly, the patient reported a history of spina bifida, which is commonly associated with this deformity, as discussed prior (1,11). The left shoulder was significantly raised with some restriction of movement present. Based on the radiographs available, this case would classify as a grade II deformity using the Rigault classification (10). An x-ray of the cervical spine (Figure 2) and left shoulder had been acquired two years prior, which clearly demonstrated the omovertebral bone as well as a mild cervical scoliotic curvature (4), which is also often found in individuals with Sprengel’s deformity. No relevant further testing was performed and the patient was sent home later that day, considering that the deformity was asymptomatic and not pertinent to the presenting complaint.

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Figure 1: Cropped PA chest x-ray showing the deformity (arrow marking omovertebral bone)

Figure 2: AP cervical spine x-ray showing the omovertebral bone (arrow) page 7

This case report and brief review of the relevant literature outlines the nature of Sprengel’s deformity: a relatively rare musculoskeletal disorder with varying degrees of severity. This particular case demonstrated that most cases of Sprengel’s deformity are in fact detected incidentally and do not necessitate any intervention or follow-ups. With severe cases, detection often occurs at infancy and may require correction.

1. Bickle I, Trajcevska E. Sprengel deformity. https://radiopaedia.org/articles/sprengel-deformity. Accessed 08/23, 20

Available

at:

2. Pediatric Orthopaedic Society of North America. Sprengel's Deformity. Available at: https://posna.org/Physician-Education/Study-Guide/Sprengel-s-Deformity. Accessed 08/22, 2021. 3. Guillaume R, Nectoux E, Bigot J, Vandenbussche L, Fron D, Mézel A, et al. Congenital high scapula (Sprengel's deformity): Four Cases. Diagn Interv Imaging 2012;93(11):878-883. 4. Jones T. Sprengel's Deformity. 2021; https://www.orthobullets.com/pediatrics/4038/sprengels-deformity. 2021.

Available Accessed

at: 08/24,

5. Thacker M, M., Feldman D, S. Sprengel Deformity. 2020; Available https://emedicine.medscape.com/article/1242896-overview. Accessed 08/25, 2021.

at:

6. Dilli A, Ayaz U, Damar C, Ersan O, Hekimoglu B. Sprengel Deformity: Magnetic Resonance Imaging Findings in Two Pediatric Cases. J Clin Imaging Sci 2011;1(13). 7. Gomez A, Cadogan M. Renzo Corno. 2020; Available at: https://litfl.com/renzo-corno/. Accessed 08/22, 2021. 8. Tanaka S, Sakamoto R, Kanahashi T, Yamada S, Imai H, Yoneyama A, et al. Shoulder girdle formation and positioning during embryonic and early fetal human development. PLOS ONE 2020;15(9):e0238225. 9. Habana JA, Knipe H. Omovertebral bone . 2021; Available https://radiopaedia.org/articles/omovertebral-bone. Accessed 08/22, 2021.

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10. Kamal Y. Sprengel-deformity: An update on the surgical management. Pulsus J Surg Res 2018;2(2):64-68. 11. National Organization for Rare Disorders. Sprengel Deformity. Available at: https://rarediseases.org/rare-diseases/sprengel-deformity/. Accessed 08/21, 2021.

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12. Thacker M, M., Feldman D, S. Sprengel Deformity Workup. 2020; Available at: https://emedicine.medscape.com/article/1242896-workup. Accessed 08/24, 2021. 13. Yamada K, Suenaga N, Iwasaki N, Oizumi N, Minami A, Funakoshi T. Correction in malrotation of the scapula and muscle transfer for the management of severe Sprengel deformity: static and dynamic evaluation using 3-dimensional computed tomography. J PediatrOrthop 2013;33(2):205-211. 14. Chinn D. Prenatal ultrasonographic diagnosis of Sprengel's deformity. J Ultrasound Med2001;20(6):693-697. 15. da Silva Reginaldo S, de Macedo RR, de Andrade Amaral R, Cardoso ALP, Araújo HRS, Daher S. Sprengel's Deformity: Surgical Correction by a Modified Green Procedure. Revista Brasileira de Ortopedia (English Edition) 2009;44(3):208-213. 16. Emel G, Umit S, Sukru S, Bulent B, Yalim A, Erbil A. Long-term results of modified Green Method in Sprengtel's Deformity. J Child Ortho 2010;4(4):309-314. 17. J. L, Borges, A. S, B. C, Torres, J. R, Bowen. Modified Woodward Procedure for Sprengel Deformity of the shoulder: long-term results. J Pediatr Orthop 1996;16(4):508-513.

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Craniofacial Microsomia List of abbreviations: CFM Craniofacial microsomia HFM Hemifacial microsomia GS Goldenhar syndrome TMJ Temporomandibular joint OSA Obstructive sleep apnoea CNS Central nervous system MDO Mandibular distraction osteogenesis OAVS Oculoauriculovertebral syndrome

CFM is one of the main terms used to describe the congenital abnormality characterized by underdeveloped facial features arising from the first and second branchial arches. Other terms used to describe such a condition include hemifacial microsomia (HFM), first and second branchial arch syndrome, Goldenhar syndrome (GS), otomandibular dysostosis and oculoauriculovertebral syndrome. (1) (2) In 1989, Cohen et al (3) came up with the term oculoauriculovertebral spectrum to include all of the different phenotypical variations that can be seen in this condition. Even though there is no definite diagnostic criteria for CFM, affected patients will all suffer from some degree of hypoplasia affecting the facial tissues namely skeletal and soft tissue, ear, orbit and facial nerve. (4)

CFM is the second most common facial anomaly after cleft lip and palate. It is most often quoted as affecting between 1 in 3500 to 1 in 5600 newborns in the United States but this may be an underestimate because there is no clear-cut criteria for diagnosis of this condition. For reasons that are idiopathic, the disorder is 50% more prevalent in males than in females. Most cases are unilateral (85%) with the right side being affected the most in a 3:2 ratio. (5)(6)(7) According to EUROCAT data, congenital defects of the ear, face and neck including CFM have a prevalence of 1.53 per 10,000 births per year in Malta. (8)

During the 4th week of gestation, neural crest cells from the neural tube migrate to branchial arches, which are a series of five paired swellings of mesenchyme. These branchial arches are made up of ectoderm, mesoderm and endoderm and will give rise to the various structures making up the face. The first and second branchial arches are those associated with CFM.

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They give rise to skeletal, muscular, vascular and neural structures innervated by trigeminal (cranial nerve V) for the first pharyngeal arch and the facial nerve (cranial nerve VII) for the second pharyngeal arch. The first arch consists of the mandibular and maxillary processes, which give rise to structures associated mostly with the oral jaw including the maxilla, mandible, zygoma, trigeminal nerve, muscles of mastication, malleolus of incus and the anterior portion of ear. The second arch consists of the hyoid arch which gives rise to structures associated mainly with jaw support, including the hyoid bone, stylus process, facial nerves and muscles, most of the ear and stapes. (7) For these structures to form, cellto-cell communication must be maintained. Disruption of such a communication may result in hypoplasia or aplasia of the affected structure. (9)

CFM has a multifactorial type of inheritance with both intrinsic and extrinsic causes. There are three leading hypotheses explaining the aetiology of CFM.

The stapedial artery maintains blood flow between the first and second branchial arches. (7) In 1973, Poswillo (10) conducted a series of experiments on mice where he administered triazine, a teratogen in the 6th week of gestation. This caused a haematoma in the stapedial artery leading to a hypoxic environment and as a result caused a disruption in communication between the two pharyngeal arches. This vascular insult created phenotypes which are similar to those observed in CFM. Furthermore, the different degrees of occlusion in the artery could be the reason behind the wide range of phenotypes that exist.

In 1992, Cousley and Wilson (11) developed a hypothesis which stated that hemifacial microsomia (HFM) could be a result of abnormal development of Meckel’s cartilage. Meckel’s cartilage develops from the first branchial arch and forms the cartilaginous part of the mandibular arch and therefore, provides a sort of scaffold for normal lower jaw development. It is also associated with middle ear formation. Since abnormalities of the middle ear and lower jaw are associated with HFM, it was hypothesized that interference by vascular events of Meckel’s cartilage could be the cause of HFM.

Neural crest cells play an extremely important role in craniofacial development. Their migration from the neural tube to the pharyngeal arches is necessary for the normal formation of facial structures. Thus, abnormal development, death or abnormal migration of these cells can lead to CFM. (12)

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These three pathogenic models are interrelated with each other. Meckel’s cartilage is derived from neural crest cells and any vascular insult can disrupt neural crest cell or Meckel’s cartilage development. Likewise, anomalies in neural crest cells may lead to abnormal blood flow in the craniofacial area. Therefore, these three hypotheses may interact with each other to produce the HFM phenotype. (13)

There are numerous environmental factors which may be responsible for the pathogenic models. Risk factors include teratogen exposure such as thalidomide, vasoactive drug use such as ibuprofen, gestational smoking, second trimester vaginal bleeding, multiple gestation, the use of assisted reproductive technology as well as maternal diabetes mellitus. Such risk factors may lead to the disruption in embryonic blood flow for example, a local haemorrhage in stapedial artery which can lead to a hypoxic environment and damage to surrounding tissues. (14) (15)

Whilst most cases seem to be sporadic with no previous family history, growing evidence suggests that there is a genetic predisposition. Previously it was believed that only 2% of cases have a familial history however, this is probably an underestimate since some mild cases may have gone undiagnosed or misdiagnosed. (5) A later study by Kaye and colleagues (16) has suggested that up to 44% of cases have a familial basis with a recurrence rate of 2-3% in first degree relatives. Data has suggested an autosomal dominant inheritance with incomplete penetrance for this congenital anomaly. Research has indicated many possible chromosomal abnormalities that are responsible for HFM. The main pathogenic genes often quoted in literature are summarized in Table 1. Causes such as 5p deletions, duplication of 14q23.1 and abnormalities of chromosomes 18 and 22 have been frequently implicated as causes of CFM. (17)

Table 1 showing the main pathogenic genes associated with CFM

Gene/s

Position on chromosome

Type of mutation

Crkl

22q11.2

Deletion (18) (19) (20) (21)

OTX2, SIX6, SIX1

14q22.3

Duplication (22)

NXD2, IRX4, IRX2

5p15.33-pter

Deletion (23)

GSC

14q32

Deletion (24)

OTX2

14q23.1

Duplication (25) (26)

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CFM is generally diagnosed clinically however, radiographic tests such as MRI, CT scans, panoramic radiography and TC3D as well as genetic tests for possible pathogenic genes and mutations can aid in confirming the diagnosis. (27) Throughout the scientific community there still seems to be no agreement on the establishment of clear and specific diagnostic criteria for CFM. However, many clinicians suggest that hypoplasia of one or more structures arising from the first and second branchial arches is required for diagnosis of CFM. In 1993, Cousley (28) proposed the following minimum diagnostic criteria: 1. Homolateral lower jaw and ear defects 2. Asymmetrical mandibular and auricle defects together with a. Two or more secondarily related abnormalities b. Positive familial history of CFM

The heterogenic nature of the disease presentation led to the creation of many classification systems. These classification systems proved to be useful in determining the course of action after diagnosis since the treatment and surgical plan depends on the severity of the deformity. The first accepted classification system for CFM was proposed by Pruzansky (29) in 1969 where he concentrated on the underdevelopment of the mandible. Based on radiographic findings he grouped mandibular hypoplasia into three possible presentations. In 1988, Kaban and colleagues (30) added on this system by also describing the position of the temporomandibular joint (TMJ). Later on, classification systems which described and included all the parts of the face were formulated. In 1987, the SAT system was developed by David and colleagues (31) to include skeletal, auricular and soft tissue abnormalities. This classification system was further modified by Vento and colleagues (32) in 1991 to create the OMENS (Orbit, mandible, ear, nerve, soft tissue) classification system and later on was termed OMENS+ by Horgan et al (33) in 1995 to include other abnormalities apart from those affecting the craniofacial region. The latter is probably the most widely accepted classification system used by physicians for CFM. It includes all the possible craniofacial defects such as orbital deformation, underdevelopment of the mandible, auricular defects, nerve and soft tissue defects. The OMENS system uses a scoring system where each section is scored between 0 and 3 with 3 being the most severe. In a total deformity score, the greater the score, the greater the severity of the CFM phenotype. A visual representation of the OMENS+ was created in 2007 (34) and later adjusted in 2011(35) so as to try to standardize diagnosis of CFM.

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CFM has a wide phenotypic presentation with a broad spectrum of manifestations usually associated with structures originating from the first and second branchial arches. While some cases may present with just some mild facial asymmetry and so are quite easy to miss, other cases may be very severe with gross facial asymmetry, microtia and extracranial presentations such as cardiac and renal problems. The main clinical presentations observed in CFM are summarized in Table 2. Table 2 Clinical manifestations of CFM according to the system affected(36)

System affected Ocular

Auricular and auditory

Maxillofacial and oral region

Feature/s and clinical manifestations Upper eyelid colobomas Epibulbar dermoids Anophthalmia/microphthalmia Orbital dystopia Microtia Anotia External auditory canal atresia Pharyngotympanic tube dysfunction Preauricular skin tags Conductive hearing loss Mandibular asymmetry - hypoplastic jaw, agenesis of condyle and ramus, aplasia of TMJ Underdeveloped muscles of mastication Delayed dental development and hypodontia Malocclusion Macrostomia Feeding difficulties

Neurologic

Facial nerve palsy Sensorineural hearing loss Impaired extraocular movements Asymmetrical palatal elevation

Respiratory

Upper airway obstruction Obstructive sleep apnoea (OSA)

Cardiac

Tetralogy of Fallot Septal defects Situs inversus

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Renal

Vertebral

CNS

Developmental

Renal agenesis Double ureter Hydronephrosis Hydroureter Crossed renal ectopia Hemivertebrae Scoliosis Fusion of vertebrae Occipitalization of atlas Spina bifida Compression of brain and spinal cord Cervical spine instability Neural tube defects Corpus callosum agenesis or hypoplasia Intracranial lipomas Hydrocephaly Microcephaly Arnold-Chiari malformation Ventriculomegaly Cerebral hypoplasia Speech and language delay Neuropsychomotor delay Intellectual disability Social problems

Since CFM is an abnormality which presents with a wide array of phenotypic presentations, the treatment plan of such a condition should be tailored personally to each patient. No single surgical protocol exists and the management plan should be adapted according to the patients’ age and severity of abnormalities. (7) Developing a sustainable and long-term treatment plan is not an easy task and should be done within a multi-disciplinary team since the condition affects almost all functional aspects of life including breathing, communication, feeding, growth, speech, development and quality of life. (37) An overview of a treatment plan according to the different life-stages is summarized in Table 3.

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Table 3 Overview of evaluations and treatments in CFM patients according to life-stage Life Stage Antenatal

Neonate

Infancy and early childhood

Mixed dentition stage (6-12 years)

Evaluations

Common surgeries/treatments

Prenatal diagnosis using ultrasound Polyhydramnios

Preparation of the neonate team for life-threatening emergencies immediately after birth

Respiratory status Renal and cardiac consultations Assess facial asymmetry Oral health

Airway procedures eg. tracheostomy Cardiac and renal corrective surgeries if life-threatening Eyelid colobomas repair

Hearing status Feeding and growth Assess facial asymmetry Oral health

Removal of preauricular skin tags Cleft lip/palate and gross macrostomia repair Hearing augmentation (hearing aids) Eye drops and eye bandaging if facial nerve palsy present Mandibular distraction osteogenesis (MDO) or costochondral grafting in cases where there is respiratory compromise due to craniofacial malformation Removal of lipodermoids and epibulbar choristomas

Obstructive sleep apnoea (OSA) and airway problems Psychosocial aspects Orthopaedic consultation Assess facial asymmetry Occlusion Oral health

Ear reconstruction – autologous or alloplastic Facial reanimation surgery/tarsorrhaphy Mandibular and maxillary surgery to fix hypoplastic mandible through: Orthodontic appliances Costochondral grafts to reconstruct the temporomandibular joint (TMJ) and condyle-ramus unit MDO Aural atresia repair Soft tissue augmentation using fat grafts or vas

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Adolescence and adulthood

OSA symptoms Psychosocial health Oral health Occlusion

Corrections and retouching of previous surgeries and secondary defects

Further research is required to comprehend better the molecular mechanisms of CFM so that the exact pathogenesis of CFM is elicited. Small-molecule drugs as well as CRISPR/CAS9-based genome editing are two potential preventative and treatment measures that however, require plenty of more research and studies. (13)

CFM is a term used to describe the congenital anomaly associated with hypoplasia of facial structures originating from the 1st and 2nd branchial arches. It is documented as being one of the most common craniofacial anomalies with a broad spectrum of phenotypical presentations and different degrees of severity. The aetiology of CFM is multifactorial with environmental and genetic causes. Due to differences in severity between patients suffering from CFM, clinicians created classification systems so as to better classify CFM patients making it easier to develop a standardized plan of action depending on patient class. Since clinical manifestations are not limited only to the craniofacial area, the best treatment plan should take a holistic approach within a multidisciplinary team that will cater to the patient’s needs throughout his or her life.

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1. Grabb WC. The first and second branchial arch syndrome. Plast Reconstr Surg. 1965 Nov;36(5):485–508. 2. Converse JM, Coccaro PJ, Becker M, Wood-Smith D. On hemifacial microsomia. The first and second branchial arch syndrome. Plast Reconstr Surg. 1973 Mar;51(3):268–79. 3. Cohen MM, Rollnick BR, Kaye CI. Oculoauriculovertebral spectrum: an updated critique. Cleft Palate J. 1989 Oct;26(4):276–86. 4. Poswillo D. The aetiology and pathogenesis of craniofacial deformity. Dev Camb Engl. 1988;103 Suppl:207–12. 5. Brandstetter KA, Patel KG. Craniofacial Microsomia. Facial Plast Surg Clin N Am. 2016 Nov;24(4):495–515. 6. Cousley RRJ, Calvert ML. Current concepts in the understanding and management of hemifacial microsomia. Br J Plast Surg. 1997 Oct;50(7):536–51. 7. Birgfeld C, Heike C. Craniofacial Microsomia. Clin Plast Surg. 2019 Apr;46(2):207–21. 8. European Platform on Rare Disease Registration [Internet]. [cited 2019 Oct 30]. Available from: https://eu-rd-platform.jrc.ec.europa.eu 9. Johnston MC, Bronsky PT. Prenatal Craniofacial Development: New Insights On Normal and Abnormal Mechanisms. Crit Rev Oral Biol Med. 1995 Jan;6(1):25–79. 10. Poswillo D. The pathogenesis of the first and second branchial arch syndrome. Oral Surg Oral Med Oral Pathol. 1973 Mar;35(3):302–28. 11. Cousley RR, Wilson DJ. Hemifacial microsomia: developmental consequence of perturbation of the auriculofacial cartilage model? Am J Med Genet. 1992 Feb 15;42(4):461–6. 12. Beleza-Meireles A, Clayton-Smith J, Saraiva JM, Tassabehji M. Oculo-auriculo-vertebral spectrum: a review of the literature and genetic update. J Med Genet. 2014 Oct;51(10):635– 45. 13. Chen Q, Zhao Y, Shen G, Dai J. Etiology and Pathogenesis of Hemifacial Microsomia. J Dent Res. 2018 Nov;97(12):1297–305. 14. Werler MM, Sheehan JE, Hayes C, Padwa BL, Mitchell AA, Mulliken JB. Demographic and Reproductive Factors Associated with Hemifacial Microsomia. Cleft Palate Craniofac J. 2004 Sep;41(5):494–500.

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15. Werler MM, Sheehan JE, Hayes C, Mitchell AA, Mulliken JB. Vasoactive exposures, vascular events, and hemifacial microsomia. Birt Defects Res A Clin Mol Teratol. 2004 Jun;70(6):389–95. 16. Kaye CI, Martin AO, Rollnick BR, Rollnick R, Nagatoshi K, Israel J, et al. Oculoauriculovertebral anomaly: Segregation analysis. Am J Med Genet. 1992 Aug 1;43(6):913–7. 17. Heike CL, Luquetti DV, Hing AV. Craniofacial Microsomia Overview. :32. 18. Miller KA, Tan TY, Welfare MF, White SM, Stark Z, Savarirayan R, et al. A Mouse Splice-Site Mutant and Individuals with Atypical Chromosome 22q11.2 Deletions Demonstrate the Crucial Role for Crkl in Craniofacial and Pharyngeal Development. Mol Syndromol. 2014;5(6):276–86. 19. Xu J, Fan YS, Siu VM. A child with features of Goldenhar syndrome and a novel 1.12 Mb deletion in 22q11.2 by cytogenetics and oligonucleotide array CGH: Is this a candidate region for the syndrome? Am J Med Genet A. 2008 Jul 15;146A(14):1886–9. 20. Digilio MC, McDonald-McGinn DM, Heike C, Catania C, Dallapiccola B, Marino B, et al. Three patients with oculo-auriculo-vertebral spectrum and microdeletion 22q11.2. Am J Med Genet A. 2009 Dec;149A(12):2860–4. 21. Tan TY, Collins A, James PA, McGillivray G, Stark Z, Gordon CT, et al. Phenotypic variability of distal 22q11.2 copy number abnormalities. Am J Med Genet A. 2011 Jul;155(7):1623–33. 22. Zielinski D, Markus B, Sheikh M, Gymrek M, Chu C, Zaks M, et al. OTX2 Duplication Is Implicated in Hemifacial Microsomia. Herault Y, editor. PLoS ONE. 2014 May 9;9(5):e96788. 23. Ala-Mello S, Siggberg L, Knuutila S, von Koskull H, Taskinen M, Peippo M. Further evidence for a relationship between the 5p15 chromosome region and the oculoauriculovertebral anomaly. Am J Med Genet A. 2008 Oct 1;146A(19):2490–4. 24. Kelberman D, Tyson J, Chandler D, McInerney A, Slee J, Albert D, et al. Hemifacial microsomia: progress in understanding the genetic basis of a complex malformation syndrome. Hum Genet. 2001 Dec;109(6):638–45. 25. Ballesta-Martínez MJ, López-González V, Dulcet LA, Rodríguez-Santiago B, Garcia-Miñaúr S, Guillen-Navarro E. Autosomal dominant oculoauriculovertebral spectrum and 14q23.1 microduplication. Am J Med Genet A. 2013 Aug;161(8):2030–5. 26. Ou Z, Martin DM, Bedoyan JK, Cooper ML, Chinault AC, Stankiewicz P, et al. Branchiootorenal syndrome and oculoauriculovertebral spectrum features associated with duplication of SIX1 , SIX6 , and OTX2 resulting from a complex chromosomal rearrangement. Am J Med Genet A. 2008 Oct 1;146A(19):2480–9. page 19

27. Universidad de Chile, Véliz M S, Agurto V P, Hospital Luis Calvo Mackenna, Leiva V N, Universidad de Chile. Microsomía hemifacial. Revisión de la literatura. Rev Fac Odontol [Internet]. 2016 Feb [cited 2019 Oct 8];27(2). Available from: http://aprendeenlinea.udea.edu.co/revistas/index.php/odont/article/view/17643 28. Cousley RRJ. A comparison of two classification systems for hemifacial microsomia. Br J Oral Maxillofac Surg. 1993 Apr;31(2):78–82. 29. PRUZANSKY S. Not all dwarfed mandibles are alike. Birth defects. 1969;5:120-9. 30. Kaban LB, Moses MH, Mulliken JB. Surgical correction of hemifacial microsomia in the growing child. Plast Reconstr Surg. 1988 Jul;82(1):9–19. 31. David DJ, Mahatumarat C, Cooter RD. Hemifacial Microsomia: A Multisystem Classification. Plast Reconstr Surg. 1987 Oct;80(4):525–33. 32. Vento AR, Labrie RA, Mulliken JB. The O.M.E.N.S. Classification of Hemifacial Microsomia. Cleft Palate Craniofac J. 1991 Jan;28(1):68–77. 33. Horgan JE, Padwa BL, Labrie RA, Mulliken JB. OMENS-Plus: Analysis of Craniofacial and Extracraniofacial Anomalies in Hemifacial Microsomia. Cleft Palate Craniofac J. 1995 Sep;32(5):405–12. 34. Gougoutas AJ, Singh DJ, Low DW, Bartlett SP. Hemifacial microsomia: clinical features and pictographic representations of the OMENS classification system. Plast Reconstr Surg. 2007 Dec;120(7):112e–20e. 35. Birgfeld CB, Luquetti DV, Gougoutas AJ, Bartlett SP, Low DW, Sie KCY, et al. A phenotypic assessment tool for craniofacial microsomia. Plast Reconstr Surg. 2011 Jan;127(1):313–20. 36. Alfi D, Lam D, Gateno J. Branchial Arch Syndromes. Atlas Oral Maxillofac Surg Clin. 2014 Sep;22(2):167–73. 37. Heike CL, Hing AV, Aspinall CA, Bartlett SP, Birgfeld CB, Drake AF, et al. Clinical care in craniofacial microsomia: A review of current management recommendations and opportunities to advance research: AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS). Am J Med Genet C Semin Med Genet. 2013 Nov;163(4):271–82.

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Skin Cancer in Organ Transplant Patients - Epidemiology and Risk Factors

Skin cancers are the commonest malignancies seen in organ transplant recipients (OTRs) due to the permanent need for immunosuppression (Euvrard et al., 2003; Ulrich et al., 2004), 95% of which are nonmelanoma skin cancers (NMSC), namely squamous cell carcinomas (SCC) and basal cell carcinomas (BCC). Other frequently seen skin cancers include Kaposi sarcoma (KS), Merkel cell carcinoma (MCC), and malignant melanoma (MM) (Mittal & Colegio, 2017). Whilst in the general population the incidence of BCC is greater than that of SCC, OTRs show a reversal of this ratio (Ulrich et al., 2008). Immunosuppressants have such a significant role that OTRs are 65 to 250 times more likely to develop SCC for example, when compared to the general population (Lindelöf et al., 2000). Approximately half of all malignancies seen in patients following a solid organ transplant are in fact skin cancers, and these are more severe when compared to skin cancers in non-transplant patients, as they are more aggressive and tend to metastasize early (Greenberg & Zwald, 2011). In OTRs, the sensation of pain associated with a cutaneous SCC is thought to be a warning signal for invasive tumour and has been associated with an increased risk of overall mortality in these patients. Melanoma-related mortality was also reported to be 2-5 times higher in OTRs when compared with nonrecipients (Robbins et al., 2015). SCC and BCC tend to appear 8-10 years after OTRs undergo transplantation, specifically in sun-exposed regions of the skin (Ulrich et al., 2008).

Risk factors for skin cancer in OTR are similar to those in individuals with a healthy immune system, the most important of which are fair skin (Fitzpatrick skin types I-III), eyes and hair, as well as sun exposure. Sun exposure has a snowball effect in regards to skin cancer, as increased amounts of ultraviolet (UV) radiation exposure across one’s life is one of the most significant carcinogens that causes skin cancer (Euvrard et al., 2003). UV B and A are present in ground level sunlight, however UVB is more readily absorbed by DNA leading to gene mutations, thus making it more carcinogenic than UVA (de Gruijl, 2000).

Proving that sun exposure is one of the most important risk factors for skin cancer, NMSC lesions are ordinarily seen only on UV exposed skin areas. Furthermore, NMSC are more abundant in individuals living in countries with a sunnier climate. Therefore one of the most crucial factors that plays a role in decreasing the risk of skin cancer is sun protection (Euvrard et al., 2006). page 21

With regards to UV radiation in OTRs, the severe carcinogenic effect of UV radiation plays an even greater role when compared to the general population. UV radiation itself causes both local and systemic immunosuppression (Yu et al., 2014), so when combining this with the lifelong immunosuppression treatment that OTRs need, these patients have a much higher risk for developing malignancies as their immune system is rendered almost ineffective to carry out tumour immune surveillance (Mittal & Colegio, 2017). It is therefore crucial for OTRs to receive sufficient education about sun protection and UV avoidance as much as possible.

Another important risk factor is age, as studies have shown that there is a higher prevalence of NMSC in older patients receiving transplants (Otley et al., 2005), as older patients have been exposed to UV for longer and have a waning immunity.

History of previous skin cancer, male sex and genetic polymorphisms are also risk factors for skin cancer in OTR. Mutations in the p53 tumour suppressor gene are the most common mutations found in skin cancers, thus making it a good marker to estimate cancer risk (Page et al., 2006). Tumour protein 53 (TP53) mutations allow tumour cells to withstand apoptosis and proliferate, while destroying healthy nearby keratinocytes. Other genes that are commonly mutated include cyclin kinase inhibitor 2A mutations (CDKN2A), Ras and NOTCH1 (Que et al., 2018). CDKN2A mutations affect the cell cycle by damaging the control proteins responsible for the cell cycle progression, differentiation, senescence and apoptosis (Brown et al., 2004). Ras mutations affect cellular signal transduction, and NOTCH1 mutations act as a rate-limiting step for carcinogenesis, most notably SCC (South et al., 2014).

Specific underlying diseases were also shown to have an effect on skin cancer risk in OTRs. It was noted that there was an associated decreased skin cancer risk in kidney transplant recipients with underlying diabetes, however an increased skin cancer risk in kidney transplant recipients with underlying polycystic kidney disease (Kaufmann et al., 2005). When observing liver transplant recipients there was an associated increased skin cancer risk in patients with underlying cholestatic liver disease and cirrhosis (Otley et al., 2005).

Immunosuppression also causes OTRs to be more vulnerable to viruses when compared to the general population, and this is of particular importance when considering Human papillomavirus (HPV) infections which are another risk factor for skin cancer (Mittal & Colegio, 2017). HPV is most notably associated with SCC, however does not seem to be linked with BCC. Cutaneous SCC was in fact thought to have originated both from DNA damage due to UV radiation as well as HPV exposure (Ally et al., 2013). In fact, studies showed that HPV DNA is found in 11-32% of normal skin, however is found in up to 90% of cutaneous SCCs in OTRs (Nindl et al., 2007). page 22

In non-white transplant recipients, tumours are more often located in non-sun-exposed areas and are, in most cases, associated with HPV infection. Epstein-Barr virus (EBV), Hepatitis B virus (HBV), hepatitis C virus (HCV), Kaposi sarcoma herpes virus (KSHV), human T cell lymphotropic virus type 1 (HTLV-1), and Merkel cell polyomavirus (MCPyV) are other relevant viruses in this setting (Schiller & Lowy, 2021).

OTRs also have an increased risk for malignancy due to the direct influence of the immunosuppressive treatment that the patient is on (Mittal & Colegio, 2017). Apart from the choice of immunosuppressive agent, the duration and intensity of treatment affects the development of consequent skin cancer (Bouwes et al., 1996). Exposure to immunosuppression has been shown to play a significant role, as studies show that a longer exposure has been associated with increased number of lesions (Euvrard et al., 2006). This is seen in heart transplant recipients who tend to have a twice as high risk for developing skin cancer when compared to renal transplant recipients (Wu & Orengo, 2002), due to the older age of such patients upon receiving the transplant, as well as due the more intensive immunosuppressant treatment that is necessary (Zwald & Brown, 2011). Furthermore, certain specific immunosuppressive drugs also have direct carcinogenic actions along with their action on impairing the immune system. An example of such a drug is Azathioprine, as studies have suggested that Azathioprine exposure combined with UV exposure can lead to DNA damage which may in turn lead to malignancy (Jiyad et al., 2016; O'Donovan et al., 2005). Cyclosporine is another commonly used immunosuppressant drug, which has been reported to have potential adverse side effects leading to skin cancer (Muellenhoff & Koo, 2012). However, newer immunosuppressive drugs such as Mycophenolate, Tacrolimus, Sirolimus and Everolimus have proven to be much less carcinogenic, implying a more hopeful outlook for OTRs, despite these drugs being much more expensive.

Skin cancer may be a very serious condition, and is significantly more likely to occur in OTRs who are on lifelong immunosuppressants. Not only are OTRs more likely to develop skin cancers, but such malignancies are more aggressive when compared to skin cancers in non transplant patients. Important risk factors for the development of skin cancer in OTRs include: skin type; UV radiation exposure (which is one of the most significant carcinogens in relation to skin cancer); older age; male gender; history of previous skin cancer; mutations such as TP53, Ras, CDKN2A and NOTCH1; underlying disease; HPV and other viruses such as EBV, HBV, HCV, KSHV, HTLV-1, and MCPyV; and the effects of the specific immunosuppressive drugs used. The importance of skin cancer prevention should be emphasised in OTRs especially due to their increased risk, and sufficient education should be delivered to such patients regarding sun protection and UV avoidance.

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1. Ally MS, Tang JY, Arron ST. Cutaneous human papillomavirus infection and Basal cell carcinoma of the skin. J Invest Dermatol 2013; 133: 1456– 1458. 2. Bouwes Bavinck JN, Hardie DR, Green A, et al. The risk of skin cancer in renal transplant recipients in Queensland, Australia. A follow-up study. Transplantation 1996; 61: 715– 721. 3. Brown V.L., Harwood C.A., Proby C.M., et al. p16INK4a and p14ARF tumor suppressor genes are commonly inactivated in cutaneous squamous cell carcinoma. J Invest Dermatol. 2004; 122: 1284-1292 4. de Gruijl FR. Photocarcinogenesis: UVA vs UVB. Methods Enzymol. 2000;319:359-66. doi: 10.1016/s0076-6879(00)19035-4. PMID: 10907526. 5. Euvrard S, Kanitakis J, Claudy A. Skin cancers after organ transplantation. N Engl J Med. 2003 Apr 24;348(17):1681-91. doi: 10.1056/NEJMra022137. PMID: 12711744. 6. Euvrard S, Kanitakis J, Decullier E, Butnaru AC, Lefrançois N, Boissonnat P, Sebbag L, Garnier JL, Pouteil-Noble C, Cahen R, Morelon E, Touraine JL, Claudy A, Chapuis F. Subsequent skin cancers in kidney and heart transplant recipients after the first squamous cell carcinoma. Transplantation. 2006 Apr 27;81(8): 1093-100. doi: 10.1097/01.tp.0000209921.60305.d9. PMID: 16641592. 7. Greenberg JN, Zwald FO. Management of skin cancer in solid-organ transplant recipients: A multidisciplinary approach. Dermatol Clin 2011; 29: 231– 241. Ix. 8. Jiyad, Z., C. M Olsen, M. T Burke, N. M Isbel, and A. C Green. "Azathioprine and Risk of Skin Cancer in Organ Transplant Recipients: Systematic Review and Meta‐Analysis." American Journal of Transplantation 16.12 (2016): 3490-503. Web. 9. Kauffman HM, Cherikh WS, Cheng U et al. Maintenance immunosuppression with target-ofrapamycin inhibitors is associated with a reduced incidence of de novo malignancies. Transplantation 2005; 80: 883– 889. 10. Lindelöf B. , Sigurgeirsson B. , Stern R.S. , et al. Incidence of skin cancer in 5356 patients following organ transplantation. Br J Dermatol. 2000; 143: 513-519 11. Mittal, A., and O. R Colegio. "Skin Cancers in Organ Transplant Recipients." American Journal of Transplantation 17.10 (2017): 2509-530. Web. 12. Muellenhoff MW, Koo JY. Cyclosporine and skin cancer: an international dermatologic perspective over 25 years of experience. A comprehensive review and pursuit to define safe use of cyclosporine in dermatology. J Dermatolog Treat. 2012 Aug;23(4):290-304. doi: 10.3109/09546634.2011.590792. Epub 2011 Sep 21. PMID: 21936704.

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13. Nindl I, Gottschling M, Stockfleth E. Human papillomaviruses and non-melanoma skin cancer: Basic virology and clinical manifestations. Dis Markers 2007; 23: 247– 259 14. O'Donovan P, Perrett CM, Zhang X, et al. Azathioprine and UVA light generate mutagenic oxidative DNA damage. Science. 2005; 309(5742): 1871- 1874. 15. Otley CC, Cherikh WS, Salasche SJ, McBride MA, Christenson LJ, Kauffman HM. Skin cancer in organ transplant recipients: effect of pretransplant end-organ disease. J Am Acad Dermatol. 2005 Nov;53(5):783-90. doi: 10.1016/j.jaad.2005.07.061. Epub 2005 Sep 22. PMID: 16243126. 16. Page A, Navarro M, Suarez-Cabrera C, et al. Protective role of p53 in skin cancer: Carcinogenesis studies in mice lacking epidermal p53 [published correction appears in Oncotarget. 2017 Mar 28;8(13):22304]. Oncotarget. 2016;7(15):20902-20918. doi:10.18632/oncotarget.7897 17. Que SKT, Zwald FO, Schmults CD. Cutaneous squamous cell carcinoma: Incidence, risk factors, diagnosis, and staging. J Am Acad Dermatol. 2018 Feb;78(2):237-247. doi: 10.1016/j.jaad.2017.08.059. PMID: 29332704. 18. Robbins HA, Clarke CA, Arron ST, et al. Melanoma Risk and Survival among Organ Transplant Recipients. J Invest Dermatol. 2015;135(11):2657-2665. doi:10.1038/jid.2015.312 19. Schiller JT, Lowy DR. An Introduction to Virus Infections and Human Cancer. Recent Results Cancer Res. 2021;217:1-11. doi:10.1007/978-3-030-57362-1_1 20. South A.P. , Purdie K.J. , Leigh I.M. et al. NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis. J Invest Dermatol. 2014; 134: 2630-2638 21. Ulrich C, Schmook T, Sachse MM, Sterry W, Stockfleth E. Comparative epidemiology and pathogenic factors for nonmelanoma skin cancer in organ transplant patients. Dermatol Surg. 2004 Apr;30(4 Pt 2):622-7. doi: 10.1111/j.1524-4725.2004.30147.x. PMID: 15061846. 22. Ulrich, C., J. Kanitakis, E. Stockfleth, and S. Euvrard. "Skin Cancer in Organ Transplant Recipients—Where Do We Stand Today?" American Journal of Transplantation 8.11 (2008): 2192-198. Web. 23. Wu, J. J, & Orengo, I. F. (2002). Squamous Cell Carcinoma in Solid-Organ Transplantation. Dermatology Online Journal, 8(2). 24. Yu SH, Bordeaux JS, Baron ED. The immune system and skin cancer. Adv Exp Med Biol 2014; 810: 182– 191. 25. Zwald F.O. , Brown M. Skin cancer in solid organ transplant recipients: advances in therapy and management: part I. Epidemiology of skin cancer in solid organ transplant recipients. J Am Acad Dermatol. 2011; 65: 253-261 page 25

CHARCOT MARIE TOOTH DISEASE Charcot-Marie-Tooth disease is a clinically heterogenous disorder with a prevalence of about 1 in every 2500. This disease is characterised by a progressive neuropathy which may present with a range of phenotypes. The classical phenotype of the disease generally includes distal muscular atrophy and skeletal deformities but varies with severity and age of onset. Electrophysiology is especially important for the current classification system of CMT, differentiating between CMT1, CMT2 and intermediate CMT subtypes. Over 60 causative genes have been identified for this disorder, owing to the advances that have been made in molecular techniques. Such techniques, especially Next-generation sequencing, have since revolutionised genetic testing and greatly expanded our knowledge of individual phenotypes and genotypes. Having said this, CMT is a continuously evolving field. In fact, although CMT is not associated with any particular treatment, clinical trials related to potential therapeutics are currently underway after presenting with positive outcomes in animal models. Although new techniques are being developed, diagnosis remains particularly focused on both the clinical and neurophysiological features, which in turn guides the application of Nextgeneration sequencing. Moreover, further research concerning epidemiology, as well as outcome measures is required to gain yet a better understanding of CMT and its subtypes, in hopes of developing specific treatment options and potentially even finding a cure so to subsequently improve the patients’ quality of life.

CMT NGS SAP HNPP CHN DSS PMP22 MFN-2 MPZ GJB1 WES WGS P0 UPR NSAIDs cAMP NT3 CMTNS

Charcot Marie Tooth Next- Generation Sequencing Sensory Action Potential Hereditary Neuropathy with Liability to Pressure Palsy Congenital Hypomyelinating Neuropathy Déjèrine-Sottas Syndrome Peripheral Myelin Protein 22 Mitofusin-2 Myelin Protein Zero Gap Junction Protein Beta 1 Whole Exome Sequencing Whole Genome Sequencing Protein 0 Unfolded Protein Response Nonsteroidal Anti-Inflammatory Drugs Cyclic AMP Neurotrophin 3 CMT Neuropathy Scale page 26

Charcot Marie Tooth (CMT) disease represents a spectrum of hereditary neuropathies which are clinically and genetically heterogeneous. With an estimated prevalence of 1 in every 2,500 people (1), CMT is in fact among the most common inherited neuropathies (2). Since its discovery over 120 years ago, substantial progress has been made in the discovery of causative genes. Most mutations in this disease primarily influence Schwann cells and peripheral axons (3). These may include radial or axonal transport proteins, transcription factors for Schwann cell differentiation, molecular chaperones or cytoskeletal, endosomal and mitochondrial proteins. Depending on the protein affected, the mutation can either cause axonal loss or demyelination (4), and this explains why clinical features, onset and severity tend to vary between subtypes of CMT. The length-dependent deterioration of sensory and/or motor fibres typically characterizes the disease and leads to what is known as the classical CMT phenotype. This includes loss of sensation, depressed tendon reflexes, distal muscular atrophy and skeletal deformities (5). Symptoms tend to initially affect the lower limbs, particularly the intrinsic foot musculature, but can also progress to the peroneal and anterior tibialis muscles and eventually to the distal upper limbs (6). Similarly, gait difficulties are common due to the foot deformities that develop, including hammer toes and pes cavus (7). The pathophysiology of these skeletal abnormalities, particularly pes cavus, has been thought to be because of the loss of innervation of intrinsic foot muscles and can sometimes even progress to pronounced osseous changes (8,9). Having said this, the slow progression of the disease, typically presenting within the first 2 decades of life, leads to no significant alteration in life expectancy (10). Furthermore, the classical CMT phenotype has also been described to include features seen on electrophysiology and nerve biopsy, particularly absent/reduced sensory action potential (SAP) amplitudes, reduced nerve conduction velocities (below 38m/s) and onion bulb formation along with demyelination and remyelination features (11).

Figure 1 – Patients presenting with the classical CMT phenotype. A/B: muscular atrophy in the lower limb. C/D/E: foot deformities (pes cavus, callosities and high arches). F: wasting of the intrinsic muscles of the hand Taken from (Pareyson and Marchesi 2009).

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Being that the clinical features of CMT subtypes are considered similar, ancillary diagnostic tests have been identified to assist in guiding genetic testing (12). Initially CMT was classified into demyelinating, axonal and intermediate forms on the basis of electrophysiology by using nerve conduction speeds in upper-limb motor nerves, specifically the ulnar and median nerves (5,13). Velocities less than 38m/s indicate demyelinating forms, specifically CMT1 and CMT4, while velocities greater than 38m/s indicate axonal forms, specifically CMT2. CMT1 is considered an AD demyelinating disorder, while CMT4 is typically considered an AR demyelinating disorder. Further differentiation can then be achieved by sural nerve biopsy since CMT subtypes are found to show distinct myelin malformations. Demyelinating forms mainly exhibit onion bulb formations in Schwann cells, whereas axonal forms exhibit axonal degeneration and regeneration which characterises the subtype (10). Intermediate velocities usually indicate X-linked CMT (CMTX1) or dominant-intermediateCMT. This subtype can present with features of both demyelinating and axonal types and is considered the second most prevalent subtype of CMT (10). CMTX1 typically presents differently for males and females, with males exhibiting specific symptoms, including strokelike symptoms, namely dysarthria and ataxia, as well as transient white matter hyperintensities on peripheral nerve MRI (9,14). The third most frequent type of CMT is Hereditary Neuropathy with liability to Pressure Palsy (HNPP), typically presenting with recurring, temporary motor and sensory mononeuropathies which may lead to palsies in nerves or plexi (15). On biopsy, HNPP patients exhibit characteristic tomaculae, described as an excess of myelin ensheathing axons (12). CMT3 is the subtype used to describe a group of early onset disorders; Congenital Hypomyelinating Neuropathy (CHN) and Déjèrine-Sottas syndrome (DSS). CHN, typically causing hypotonia in infants, is characterised by a defect in Schwann cells wherein peripheral nerves exhibit a lack of myelin and a very limited amount of basal lamina onion bulbs. On the other hand, DSS which is said to be the worst subtype of the disease, is typically characterised by an early onset, generally in infancy and exhibits delayed motor milestones as well as nerve hypertrophy (14). Being an AR disorder, CMT4 is typically considered more severe than majority of CMTs. It contains all AR demyelinating subtypes, typically having an early onset with an involvement of proximal musculature. In fact, patients typically lose their ability to walk at a relatively early stage. Phenotype of CMT4 can sometimes also be complicated by vocal cord paralysis and sensorineural hearing loss (10). Lastly, CMT5 is an AD disorder involving pyramidal features such as hyperreflexia (typically causing patients to be Babinski sign positive) or even spastic paraplegia. CMT6, on the other hand, has an early onset as well as a phenotype complicated by optic atrophy, commonly leading to vision loss (5).

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Figure 2 – Flow chart classifying the main CMT subtypes based on electrophysiology, inheritance patterns and/or clinical features. CMT: Charcot Marie Tooth Disease; NCV: Nerve Conduction Velocity; AD: Autosomal Dominant; AR: Autosomal Recessive.

CMT subtype

Prominent characteristic of the subtype

CMT1

AD Demyelinating form. CMT1A is the most common subtype of CMT. Intermediate NCV.

CMTX1

Exhibit features of both demyelinating and axonal types.

HNPP

Exhibit recurring, temporary motor and sensory mononeuropathies.

CMT2

NCV >38m/s, therefore axonal form. Patients exhibit both sensory and motor symptoms.

CMT3

Early onset. Includes Congenital Hypomyelinating Neuropathy (CHN) and Déjèrine-Sottas syndrome (DSS).

CMT4

AR Demyelinating form. Early onset and more severe. Patients exhibit involvement of proximal musculature.

CMT5

AD disorder characterised by pyramidal features.

CMT6

Early onset disorder characterised by optic atrophy.

Table 1: Summarised overview of CMT subtypes. CMT: Charcot Marie Tooth Disease; NCV: Nerve Conduction Velocity; AD: Autosomal Dominant; AR: Autosomal Recessive; HNPP: Hereditary Neuropathy with liability to Pressure Palsy

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The discovery of the first causative gene for CMT in 1991 has since led to advances in genetic testing wherein a genetic diagnosis is now achievable in approximately 70% of patients (10). With the use of NGS, studies have reported that in the majority of CMT patients, the etiology is a mutation in one of the following genes: the peripheral myelin protein 22 (PMP22), Mitofusin-2 (MFN-2), myelin protein zero (MPZ) or gap junction protein beta 1 (GJB1) gene (16) (figure 3). Whole exome sequencing (WES) and whole genome sequencing (WGS) are also considered very useful for identifying causative genes, thereby improving the diagnostic yield, especially when NGS panels do not detect any of the known pathogenic genes (17,18).

Figure 3: A pie chart demonstrating the breakdown of CMT cases presenting at a UK clinic (Murphy et al. 2012; Davidson et al. 2012). Adapted from (Rossor et al. 2013).

Figure 4: Location and function of some of the genes mentioned. Adapted from (Rossor et al. 2013)

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A multidisciplinary approach, along with ancillary services are currently the best method for managing CMT patients. Rehabilitation and instrument use, such as assistive orthopedic devices, plantars, ankle-foot orthoses and occupational therapy have been reported necessary for quality of life improvement and prevention of further complications. In fact, physical therapy has been found beneficial for both gait retraining as well as for maintaining a good posture (22,23).

Now that studies have found pain to be a significant symptom of CMT, whether neuropathic or non-neuropathic, treatment prescribed to ease this symptom is essential for patient management. Examples may include nonsteroidal anti-inflammatory drugs (NSAIDs), anticonvulsants and tricyclic anti-depressants. While NSAIDs generally aid back or lower limb pain, the latter two drugs typically help treat the neuropathic pain that is associated with the disease (24). Non-pharmacological pain management for CMT is also possible and includes maintaining an appropriate BMI and participating in regular physical activity of low intensity (25). In fact, exercise decreases the sensation of pain, ameliorates ambulation and reduces lower-limb weakness (26,27).

Orthopedic procedures used to manage CMT, specifically foot surgeries, include tendon transfers, osteotomies and plantar fasciotomies (28). Although they do not ameliorate lower limb strength or sensation, they have been found to improve gait difficulties by correcting complications such as hammertoes and pes cavus (29).Apart from foot deformities, surgical treatment may also be used to increase upper limb mobility or treat scoliosis which is exhibited in approximately 15-25% of cases (30).

Presently, although CMT is not associated with any particular disease modifying treatments, many preclinical trials on animal models are underway, encouraging advances in potential therapeutics which can possibly be made use of by CMT patients in the near future (12). Progesterone antagonists have been found advantageous in rat models bearing a PMP22 overexpression. These agents aim to downregulate PMP22 which is essential to combat the consequences caused by the novel duplication(1). In fact, research done on transgenic CMT1A rats reported improvements in both clinical features and neuropathology (31).

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Through its effects on cAMP, ascorbic acid has been found to similarly reduce PMP22 expression in transgenic mice and has now advanced to the controlled trial phase. In fact, in a particular animal study, longer lifespans were even reported (32). Having said this, some studies have noted no therapeutic effect in humans, with minimal changes noted on the CMT neuropathy scale (CMTNS) (33–36). For this reason, further research is required about it’s pharmacokinetic properties, especially due to side effects observed when large dosages are administered (37). Neurotrophin 3 (NT3) has also demonstrated positive results for CMT1A models. In one study, when NT3 was applied to axons of mice ensheathed by CMT1A human Schwann cells, significant axonal regeneration was observed, leading to improvements in sensation and myelination of sural nerve (10). It has been reported that the mode of action for NT3 is through its effect on autocrine survival which promotes nerve growth (38). Mutations in myelin structural genes (as are MPZ and PMP22) are thought to potentially cause a build-up of misfolded proteins or a stimulation of the UPR. Therapeutics such as curcumin have therefore been developed to target these pathomechanisms (29). In fact curcumin, has been seen to reduce ER stress by releasing the misfolded P0 or mutant PMP22 proteins from the ER of Schwann cells of CMT1B animal models (39). This decreases Schwann cell apoptosis, indirectly increasing axon myelination in demyelinating CMTs including those caused by PMP22 and MPZ gene abnormalities (40). New techniques such as Induced Pluripotent Stem Cells (iPSCs) and RNAi are also currently under investigation. They can potentially be used for disease modeling so to effectively test new therapeutics. iPSCs are typically derived from skin biopsies of CMT patients and are artificially reprogrammed. Being human cell models bearing pathophysiological abnormalities, results using this technology could possibly be more beneficial than animal models when developing therapeutics (41).

Studies associated with Charcot Marie Tooth disease have increased dramatically recently, partly due to the advances made in genetic testing and sequencing. Increasing the genetic spectrum underlying the disease and gaining a better understanding of the disease’s pathophysiology has led to the emergence of potential therapeutics, some of which are now progressing to the clinical trial phase. Overall, CMT is an evolving disease and new technologies are constantly being developed to help guide research towards definitive cures. Having said this, under the circumstances, clinicians are still supporting patients and treating them symptomatically in hopes of alleviating as much

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1.Pareyson D, Saveri P, Pisciotta C. New developments in Charcot-Marie-Tooth neuropathy and related diseases. Curr Opin Neurol. 2017;30(5):471–80. 2. Braathen GJ. Genetic epidemiology of Charcot-Marie-Tooth disease. Acta Neurol Scand, Suppl. 2012;(193):iv–22. 3. Gutmann L, Shy M. Update on Charcot-Marie-Tooth disease. Curr Opin Neurol. 2015 Oct;28(5):462–7. 4. Azevedo H, Pupe C, Pereira R, Nascimento OJM. Pain in Charcot-Marie-Tooth disease: an update. Arq Neuropsiquiatr. 2018 Apr;76(4):273–6. 5. Vallat J-M, Mathis S, Funalot B. The various Charcot-Marie-Tooth diseases. Curr Opin Neurol. 2013 Oct;26(5):473–80. 6. Burns J, Ryan MM, Ouvrier RA. Evolution of foot and ankle manifestations in children with CMT1A. Muscle Nerve. 2009 Feb;39(2):158–66. 7. Baets J, De Jonghe P, Timmerman V. Recent advances in Charcot-Marie-Tooth disease. Curr Opin Neurol. 2014 Oct;27(5):532–40. 8. Berciano J, Gallardo E, García A, Pelayo-Negro AL, Infante J, Combarros O. New insights into the pathophysiology of pes cavus in Charcot-Marie-Tooth disease type 1A duplication. J Neurol. 2011 Sep;258(9):1594–602. 9. Ramchandren S. Charcot-Marie-Tooth Disease and Other Genetic Polyneuropathies. Continuum (Minneap Minn). 2017;23(5, Peripheral Nerve and Motor Neuron Disorders):1360–77. 10. Pareyson D, Marchesi C. Diagnosis, natural history, and management of Charcot-Marie-Tooth disease. Lancet Neurol. 2009 Jul;8(7):654–67. 11. Reilly MM, Murphy SM, Laurá M. Charcot-Marie-Tooth disease. J Peripher Nerv Syst. 2011 Mar;16(1):1–14. 12. Szigeti K, Lupski JR. Charcot–Marie–Tooth disease. Eur J Hum Genet 17, 703–710 (2009) [Internet]. 2009 Mar 11 [cited 2021 Feb 22]; Available https://doi.org/10.1038/ejhg.2009.31

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16. Saporta ASD, Sottile SL, Miller LJ, Feely SME, Siskind CE, Shy ME. Charcot-Marie-Tooth disease subtypes and genetic testing strategies. Ann Neurol. 2011 Jan;69(1):22–33. 17. Choi B-O, Koo SK, Park M-H, Rhee H, Yang S-J, Choi K-G, et al. Exome sequencing is an efficient tool for genetic screening of Charcot-Marie-Tooth disease. Hum Mutat. 2012 Nov;33(11):1610–5. 18. Gonzaga-Jauregui C, Harel T, Gambin T, Kousi M, Griffin LB, Francescatto L, et al. Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy. Cell Rep. 2015 Aug 18;12(7):1169–83. 19. Murphy SM, Laura M, Fawcett K, Pandraud A, Liu Y-T, Davidson GL, et al. Charcot-Marie-Tooth disease: frequency of genetic subtypes and guidelines for genetic testing. J Neurol Neurosurg Psychiatr. 2012 Jul;83(7):706–10. 20. Davidson GL, Murphy SM, Polke JM, Laura M, Salih MAM, Muntoni F, et al. Frequency of mutations in the genes associated with hereditary sensory and autonomic neuropathy in a UK cohort. J Neurol. 2012 Aug;259(8):1673–85. 21. Rossor AM, Polke JM, Houlden H, Reilly MM. Clinical implications of genetic advances in Charcot-Marie-Tooth disease. Nat Rev Neurol. 2013 Oct;9(10):562–71. 22. Shy ME. Therapeutic strategies for the inherited neuropathies. Neuromolecular Med. 2006;8(1–2):255–78. 23. Carter GT, Weiss MD, Han JJ, Chance PF, England JD. Charcot-Marie-Tooth disease. Curr Treat Options Neurol. 2008 Mar;10(2):94–102. 24. Backonja M-M. Use of anticonvulsants for treatment of neuropathic pain. Neurology. 2002 Sep 10;59(5 Suppl 2):S14-7. 25. Schenone A, Nobbio L, Monti Bragadin M, Ursino G, Grandis M. Inherited neuropathies. Curr Treat Options Neurol. 2011 Apr;13(2):160–79. 26. Kilmer DD. Response to aerobic exercise training in humans with neuromuscular disease. Am J Phys Med Rehabil. 2002 Nov;81(11 Suppl):S148-50. 27. Young P, De Jonghe P, Stögbauer F, Butterfass-Bahloul T. Treatment for Charcot-Marie-Tooth disease. Cochrane Database Syst Rev. 2008 Jan 23;(1):CD006052. 28. Ward CM, Dolan LA, Bennett DL, Morcuende JA, Cooper RR. Long-term results of reconstruction for treatment of a flexible cavovarus foot in Charcot-Marie-Tooth disease. J Bone Joint Surg Am. 2008 Dec;90(12):2631–42. 29. Saporta MA. Charcot-Marie-Tooth disease and other inherited neuropathies. Continuum (Minneap Minn). 2014 Oct;20(5 Peripheral Nervous System Disorders):1208–25. 30. Karol LA, Elerson E. Scoliosis in patients with Charcot-Marie-Tooth disease. J Bone Joint Surg Am. 2007 Jul;89(7):1504–10.

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31. Sereda MW, Meyer zu Hörste G, Suter U, Uzma N, Nave K-A. Therapeutic administration of progesterone antagonist in a model of Charcot-Marie-Tooth disease (CMT-1A). Nat Med. 2003 Dec;9(12):1533–7. 32. Kaya F, Belin S, Bourgeois P, Micaleff J, Blin O, Fontés M. Ascorbic acid inhibits PMP22 expression by reducing cAMP levels. Neuromuscul Disord. 2007 Mar;17(3):248–53. 33. Shy ME, Blake J, Krajewski K, Fuerst DR, Laura M, Hahn AF, et al. Reliability and validity of the CMT neuropathy score as a measure of disability. Neurology. 2005 Apr 12;64(7):1209–14. 34. Burns J, Ouvrier RA, Yiu EM, Joseph PD, Kornberg AJ, Fahey MC, et al. Ascorbic acid for Charcot-Marie-Tooth disease type 1A in children: a randomised, double-blind, placebocontrolled, safety and efficacy trial. Lancet Neurol. 2009 Jun;8(6):537–44. 35. Pareyson D, Reilly MM, Schenone A, Fabrizi GM, Cavallaro T, Santoro L, et al. Ascorbic acid in Charcot-Marie-Tooth disease type 1A (CMT-TRIAAL and CMT-TRAUK): a double-blind randomised trial. Lancet Neurol. 2011 Apr;10(4):320–8. 36. Lewis RA, McDermott MP, Herrmann DN, Hoke A, Clawson LL, Siskind C, et al. High-dosage ascorbic acid treatment in Charcot-Marie-Tooth disease type 1A: results of a randomized, double-masked, controlled trial. JAMA Neurol. 2013 Aug;70(8):981–7. 37. Toth C. Poor tolerability of high dose ascorbic acid in a population of genetically confirmed adult Charcot-Marie-Tooth 1A patients. Acta Neurol Scand. 2009 Aug;120(2):134–8. 38. Sahenk Z, Nagaraja HN, McCracken BS, King WM, Freimer ML, Cedarbaum JM, et al. NT-3 promotes nerve regeneration and sensory improvement in CMT1A mouse models and in patients. Neurology. 2005 Sep 13;65(5):681–9. 39. Khajavi M, Shiga K, Wiszniewski W, He F, Shaw CA, Yan J, et al. Oral curcumin mitigates the clinical and neuropathologic phenotype of the Trembler-J mouse: a potential therapy for inherited neuropathy. Am J Hum Genet. 2007 Sep;81(3):438–53. 40. Patzkó A, Bai Y, Saporta MA, Katona I, Wu X, Vizzuso D, et al. Curcumin derivatives promote Schwann cell differentiation and improve neuropathy in R98C CMT1B mice. Brain. 2012 Dec 1;135(Pt 12):3551–66. 41. Saporta MA, Dang V, Volfson D, Zou B, Xie XS, Adebola A, et al. Axonal Charcot-MarieTooth disease patient-derived motor neurons demonstrate disease-specific phenotypes including abnormal electrophysiological properties. Exp Neurol. 2015 Jan;263:190–9.

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Connectomics: A Journey Through Time. Connectomics is a relatively new field in neuroscience which tackles the intricate neural circuitry of the brain (1). Several technologies have been constructed along the years to aid in visualising the central nervous system and in taking on the challenge of obtaining a structural map of the brain’s neural connections (2). This review goes over the major milestones reached in this field from the first full connectome ever obtained, the Brainbow technique, and eventually, the collaborative approach launched by the National Institutes of Health, i.e. the Human Connectome Project. This ambitious project aims to establish how brain connectivity underlies brain function by compiling brain imaging data and freely sharing this with researchers from all over the world (3). Enhancing our knowledge of brain structure and function, possibly through such collaborations, will hopefully allow insight into the processes underlying behaviour, and possibly, a better understanding of brain diseases and disorders in the future (4).

Alzheimer’s disease (AD) Caenorhabditis elegans (C. elegans) Connectome Coordination Facility (CCF) Human Connectome Project (HCP) Lifespan Connectome Studies (LCS) Magnetic Resonance Imaging (MRI) Massachusetts General Hospital (MGH) National Institutes of Health (NIH) Resting state-functional magnetic resonance imaging (rfMRI) Spectral and photophysical protein variants (XFPs) Task-based functional magnetic resonance imaging (tfMRI) University of California Los Angeles (UCLA) Washington University, the University of Minnesota (UMinn) and Oxford University consortium (Wu-Minn-Ox)

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This century has been renowned for its major leaps in science, particularly the accomplishment of the Human Genome Project in sequencing the entire human genome in 2001 (5,6). Analogously, a major challenge which neuroscientists are confronting involves mapping the connections of the brain with the hope of unravelling the secrets of the human mind (1). The desinence of the word ‘connectomics’ is the same as that of the word ‘genomics’, and as genomics involved the sequencing of the entire set of genes (the genome) of several species, connectomics aims to map all of neuronal connections of the brain (the connectome) to allow a better understanding of human brain function (7). ‘Connectomics’ was better defined by Lichtman and Sanes as “a branch of biotechnology concerned with applying the techniques of computer-assisted image acquisition and analysis to the structural mapping of sets of neural circuits or to the complete nervous system of selected organisms using high-speed methods, with organizing the results in databases, and with applications of the data” (8). Therefore, the analysis of connectomes requires new developments in technology (1). Novel biotechnologies were, in fact, developed for this endeavour aimed at generating circuit diagrams at macroscopic, mesoscopic or microscopic resolutions (2). Continuous innovation in new technologies is expected to help pave the way for a more indepth understanding of the brain’s complex neural connections. Understandably, along with the study of the normal human connectome comes the study of its disturbances, so-called connectopathies, which are most definitely associated with numerous neurological (9) and psychiatric disorders (8). Hence, obtaining connectome data will facilitate the study of brain disorders, including schizophrenia, autism and epilepsy (10–12), from a network perspective (13) and possibly result in improved treatments from which society will benefit greatly (14). Furthermore, through collaborations, researchers can accelerate the progress towards understanding the processes that underlie brain function and brain circuit formation, and lead to the heightening of our knowledge of growth, plasticity (15), consciousness and intelligence (14).

The ground-breaking paper, “The structure of the nervous system of the nematode Caenorhabditis elegans”, also known as “the mind of a worm” (16), was the first ever documentation of the entire nervous system of an animal. This paper signified the end of the first phase of a project initiated around twenty years prior by Sydney Brenner. This project ambitiously began to confront the enigma that is the brain, and through this, Brenner and his colleagues founded the field of connectomics, a field which was yet to be named at their time (17). Brenner believed that by knowing the structure of the nervous system and the pattern of its connections, two further problems would remain: the genetic control of the nervous system and the control of the nervous system over behaviour (18). Although a structural description of the neural circuitry would not be fully sufficient as an answer to these problems, it was essential (19). page 37

In 1963, Brenner acquired a culture of the nematode Caenorhabditis elegans (C. elegans) and left it in the hands of Nichol Thomson for him to section, fix and examine under the light microscope. To their satisfaction, a clear visual of the nervous system appeared, and C. elegans was chosen for this project. The identical cellular makeup of every individual organism, along with only 300 neurons making up its nervous system, its transparent body, good genetics, and short life cycle of around three and a half days made C. elegans ideal for this mission (1,17). The next step involved obtaining a detailed synaptic map and the only way this could be done was through electron microscopy (14). Despite Brenner’s attempts at obtaining machines and devising contraptions to facilitate the process, eventually, the reconstruction had to be done by hand (16). This involved marking glossy prints of electron micrographs from the series of sections obtained by Thomson, thus generating trails of coloured numbers, each indicating a different neuron. Finally, synapses were recorded and positioned on neuron maps which were presented (17). This approach did not produce a full-scale three-dimensional representation, but rather skeleton maps which were sufficient to produce a proper circuit diagram of the nematode. The project took 15 years to complete (17).

Cajal’s revolutionary method of labelling neurons to detect cellular elements making up the neural circuit had one major limitation: it was only applicable to a small quantity of cells. The thought behind it, however, remained intriguing (20). In 2007, a team led by Jeff Lichtman and Joshua Sanes at Harvard University, designed two genetic strategies, eloquently named ‘Brainbow’, for the stochastic expression of multiple spectral and photophysical protein variants (XFPs) on a single transgene. The expression of three different XFPs in combination would theoretically result in the coloration of neurons in one of ten hues. Yet if these three XFPs are expressed to different degrees in different neurons, a much greater number of hues will result. The stochastic expression of colour was accomplished through a mechanism based on the Cre/loxP recombination scheme. Cre recombinase selectively catalyses recombination between a pair of so-called loxP sites, each consisting of 34-nucleotide sequences. Subsequently, a DNA segment that lies in between two loxP sites of the same orientation is excised, whilst a DNA segment lying in between two loxP sites of opposite orientation is inverted (21). Hence, this technique allows for the discrimination of individual neurons on the basis of colour in over 100 colours rather than just two or three (20). The ability to delineate individual neurons and trace their processes within their native context in the central and peripheral nervous systems of transgenic mice, expedites the study of how these neurons interact. Therefore, it has allowed researchers to make a huge leap towards their ultimate goal which aims to comprehend how neural circuits underlie behaviour (22). page 38

Just like any other technique, the Brainbow method has its limitations. Primarily, a spectrum of 100 colours is still insufficient to view full regions of the nervous system (1,21). Moreover, due to the diffraction-limited resolution of light microscopy, fluorescence images appear at a relatively low resolution when observing more complex neuronal wiring systems. Hence, thinner sections are required. Fortunately, however, various solutions have already emerged, such as the use of Array Tomography which allows quantitative, large-field volumetric imaging of large amounts of XFPs and tissue molecular structure at high spatial resolution (23). Despite its limitations, Brainbow contributed extensively to the field of connectomics since obtaining partial connectomes can also be of great insight into the complexity of the brain. In addition, partial connectomes can also be of great use in the study of mouse models of psychiatric disorders as it is being suspected that defects in neuronal connections, whether they concern pattern, number, or proportion, might underlie behavioural disorders, such as schizophrenia and autism. These disorders might arise due to genetic and environmental factors that can affect several developmental events, resulting in a quantitative or qualitative defect in circuitry, resulting in so-called ‘connectopathies’. The review of quantitative and qualitative properties of brain circuitry through Brainbow labelling might be adequate to put this hypothesis up to the test (21).

Recent advances in neuroimaging have made the thorough study of in-vivo human brain connectivity possible across large numbers of individuals (24). By systematically compiling brain imaging data from numerous subjects and freely sharing this valuable information with others around the world, researchers can obtain insight into how brain connectivity underlies brain function in a shorter period (3). This will hopefully lead to improved diagnoses and treatments of brain disorders in the future (4). With these principal aims in mind, in 2009, the NIH Blueprint for Neuroscience Research inaugurated a $30 million project: the Human Connectome Project (HCP), with the intention of mapping the brain circuitry using revolutionary non-invasive brain imaging technologies (3). In 2010, Blueprint awarded two research consortia with a sum of $40 million to collaboratively compile a wiring diagram of the human brain in high resolution (25). The WuMinn-Ox consortium aimed to comprehensively map the connectivity diagrams in each of 1200 healthy adults – twin pairs and their siblings, with a genetically-informative design, using ground-breaking techniques of non-invasive neuroimaging. This includes four magnetic resonance-based modalities, including a new 3 Tesla MRI scanner, as well as a new 7 Tesla scanner, along with magnetoencephalography and electroencephalogram (24).

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The data acquired concerning the anatomical and functional connections between different regions of the brain of each individual would then be related to data obtained from behavioural testing. Through the comparison of connectomes and genetic information between identical twins and fraternal twins, the relative involvement of genes and environmental factors in the formation of brain circuitry could be determined. Moreover, further clarification of the organisation of brain networks could be obtained. After elaborate analysis using sophisticated tools, the data could then become accessible on the web through a customised Connectome Database Neuroinformatics Platform (4). In collaboration, the MGH/Harvard-UCLA consortium focused on optimising MRI technology. This meant enhancing the resolution of diffusion MRI to unprecedented heights for even more accurate imaging of the structural connections of the brain (26).

Figure 1: Connectivity in the occipital lobe generated from an average connectome from 24 individuals using Human Connectome Project data. Adapted from (21) CC-BY http://creativecommons.org/licenses/by/4.0/

Other initiatives around the world have been making even more connectome data available over the years, such as the Human Brain Project in Europe which aims to achieve the reconstruction of the multi-scale organisation of the brain, using simulation-based approaches along with productive loops of experiments and big-data analysis (27).

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Studies contributing to the HCP are housed within the Connectome Coordination Facility (CCF) (28). All CCF studies are classified under three main categories: Healthy Adult Connectome, Lifespan Connectome Studies and Connectomes in relation to Disease (29). The study mentioned previously, titled HCP Young Adult, took on the challenge of charting the neuronal pathways from 1200 healthy young adults, using novel neuroimaging technology (30). Lifespan Connectome Studies (LCS) aim to extend the data gathered from HCP Young Adult to healthy humans of other age groups, to create a high-quality dataset to be used for comparison. Currently, there are four research projects concerned with LCS (31). Two of these large-scale brain imaging studies include the Human Connectome Projects in Development and Aging. These projects are collecting structural, rfMRI, tfMRI, diffusion, and perfusion MRI in participants from five to 100+ years of age. The result of this project will provide a rich, novel, and multi-modal dataset consistently collected across a wide age range (32). Earlier developmental stages are being researched by the Developing Human Connectome Project, which is researching prenatal and neonatal brain development, and the Lifespan Baby Connectome project which is studying brain development in children from birth to the age of five (28). The third category of studies in the HCP: connectomes in relation to human disease, was funded by the NIH for HCP-style data collection to be applied to cohorts at risk for or already suffering from brain diseases or disorders. Over 10 disease connectome studies have been funded, and more are anticipated (28). These studies address many brain diseases and disorders, including schizophrenia and Alzheimer’s disease (AD). Both schizophrenia and AD have been shown to have associated changes in brain connectivity throughout the course of their progression. Therefore, obtaining a large, accessible connectivity dataset from both healthy individuals and patients would allow further understanding of the correlation between these changes in connectivity and disease symptoms. In turn, this will benefit the prediction of prognosis and the treatment of the disease (33).

Despite the major advancements in the field of connectomics, some still question whether the efforts being put into the reconstruction of a connectome on the scale of a mammalian brain will be worth the large financial investment required, since one cannot be sure that the information obtained will be of any value. Many also argue that with the great amount of variability in the circuitry of the brain, it might not even be possible to relate structure to function. And if possible, it could be that our minds are no match for such complexity. It is important to point out that connectomics does not dismiss the value of the information gained through physiological and pharmacological studies. It merely highlights the fact that the study of brain function without knowledge of its structure will be just as successful as the study of genetics without knowledge of the genome. Whilst connectomics data may not be the answer to every problem related to the understanding of the human mind, it may provide insight which might not be discovered otherwise. Through the comparison of healthy and diseased brains, young and old, and human and other primate brains, further knowledge could be gained of the foundations of psychiatric diseases, the developmental changes in circuitry, and a better understanding of intelligence. page 41

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Mercury-Induced Heavy Metal Toxicosis: A Review Most heavy metals in the environment can cause conditions collectively referred to as heavy metal toxicosis or poisoning. Such conditions are influenced by the quantity involved, pathophysiology and even radioactivity of the heavy metal (1–3). Cases of heavy metal poisoning in humans have been documented since antiquity (4), becoming more relevant over the last century due to global industrialisation, greatening the risk of occupational and environmental exposure. This concerns pesticide use, industrial runoff or effluent, spills, and leakages, amongst others. Bioaccumulation can subsequently occur once toxic heavy metals contaminate the environment, which negatively impacts different organisms, including humans. In recent times, protocols to phase out all heavy metal use and improve occupational and environmental health and safety have started implementation (5,6). Consequently, notable decreases in heavy metal emissions are now being observed in some geographic regions (Figure 1). From the heavy metals that exist, arsenic, cadmium, lead, mercury, and thallium are consistently mentioned in literature for their toxic effects (2,7–10). Physiologically important heavy metals, otherwise known as trace minerals, are also known to cause human poisoning (8,9). The term ‘heavy metal toxicosis’ encompasses the signs and symptomatology observed in most cases of poisoning by heavy metals, however, unique pathophysiological mechanisms between different aetiological heavy metals make a heavy metal toxicosis caused by one heavy metal different from another (Table 1). For the purpose of this review, the discussion will only tackle heavy metal toxicosis induced by mercury (Figure 2). Heavy metal toxicosis has a worldwide incidence and this is especially true for mercury, with all humans considered to have some degree of exposure to this heavy metal throughout their lifetime. Due to its ubiquity, high potency, and eco-biological effects, mercury has been ranked as one of the top ten chemicals that are of major concern to public health by the World Health Organisation (11). This is reflected in the findings of a 2020 report which gathered exposure and poisoning case data from fifty-five regional poison centres that served the entire population of the United States and its territorial extent for the year of 2019. A total of 2,337 single exposures to mercury, in any chemical form, had been documented, with most cases being unintentional (89.4%), in adults aged 20 or over (69.1%), and with mercury thermometers being the commonest exposing source (44.1%) (12). A smaller scale study done in Beijing, China also reported similar demographic findings (13). Countries which have a large or significant proportion of workforce in the mining or chemical plant industry have higher rates of mercury-induced toxicosis, these include mainly Asian countries such as Bangladesh, China, and India, and African countries such as Burkina Faso, South Africa, Tanzania, and Zimbabwe (10,11,14–18). Other countries like Iraq and Japan have increased prevalence of mercuryrelated disease due to previous industrial incidents (10,11,18). Higher incidence of mercury page 45

poisoning is also observed in countries where there is less strict regulation of the occupational sector and consumer market, such as Egypt and Somalia (19,20). Conditions related to chronic mercury exposure are more common in countries with large fishing populations and high fish consumption, such as Brazil, Canada, China, Colombia, and Greenland (11). The toxic effects of heavy metals like mercury are attributed to their physicochemical similarity to trace minerals required by the human body, enabling them to undergo redox reactions and form coordination complexes, displacing physiologically important metals from their ligands. This also allows binding to other cellular components where metals are not normally found, such as nucleic acids, structural proteins, and enzymes, interrupting normal function. Coordinate bonding is also of pharmacodynamic importance, particularly in chelation therapy, which is currently the main treatment modality for mercury-induced heavy metal toxicosis (mercury poisoning).

Figure 1: Trends in heavy metal emissions across the 33 EEA member countries. (Taken from European Environment Agency, 2019)

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Table 1: The symptomatic and pathological variation exhibited in different heavy metal toxicoses as induced by some of the most toxic heavy metals currently known. (Adapted from Henningsson et al., 1993; Wössmann et al., 1999; Patrick, 2002; Ibrahim et al., 2006; Siu et al., 2009; Park and Zheng, 2012; Maret and Moulis, 2013; Afal and Wiener, 2014; Jaishankar et al., 2014; Senthilkumaran et al., 2017; Bjeloševič et al., 2018; CDC, 2018; Ganguly et al., 2018; Azeh Engwa et al., 2019; WHO, 2019; Kemnic and Coleman, 2021; Rajkumar and Gupta, 2021)

The quantity of mercury within the natural environment has been elevated by anthropogenic pollution (35), bioaccumulating in prone aquatic ecosystems that include predatory fish such as shark, tilefish, swordfish, king mackerel and tuna, freshwater fish such as pike, bass, muskellunge and walleye, and shellfish (Figure 3) (36–38), which are the main natural source for humans to acquire mercury poisoning (11,18,39). This has led to recommendations being made for children and pregnant or breastfeeding women to avoid the consumption of such fish to reduce the risk of impaired neurodevelopment of the child or foetal brain (11,18). Occupational roles within the mining, hydroelectric, chemical, and agricultural industries (36,40), and consumer products and appliances, such as glass thermometers, barometers, certain fluorescent lamps, dental amalgams, button cells, old television sets, laboratory preparations, certain vaccines, topical products, and traditional medication or practices also pose risk for mercury poisoning (7,41,42). A gradual phase out process (Figure 1) has reduced the incidence of mercury poisoning, however lack of strict regulation in some areas has allowed for some mercury-containing products to remain commercially available, and considerable risk is still present (43).

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Figure 2: A glass ampoule containing pure elemental mercury. Being liquid at room temperature enables mercury to seep through any potential cracks that may form in such apparatus if mishandled, increasing the likelihood of an exposure incident.

Figure 3: The mercury cycle, depicting the physical and chemical transformations of mercury in different environmental reservoirs, and its bioaccumulative properties. (Taken from NHDES, 2019)

Like other metals, part of the chemical nature of mercury is the ability to form organic and inorganic compounds. Physicochemical differences between the pure metal, its organic compounds, and its inorganic compounds, can significantly influence the toxicology of mercury, from the exposing source to the symptoms manifested (Table 2). This may complicate diagnosis and thus highlights the need to collect a detailed history and correlate this with the clinical presentation and subsequent investigatory findings.

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In the natural environment, organic and inorganic forms (compounds) of mercury are mostly found, acting as different, interlinked mercury reservoirs influenced by both natural and artificial processes, comprising the mercury cycle (Figure 3) (36–38). Mercury in pure metallic form is very rarely found in the natural environment, and large quantities of it are instead artificially formed (37). The physicochemical properties of the encountered form dictate the place of absorption and bloodstream entry, hence, many potential aetiologies attributable to mercury poisoning (26). Being a volatile liquid metal at room temperature (Figure 2), pure elemental mercury emits colourless, odourless vapours, making inhalation the most likely route of exposure. This also makes it possible for mercury to be aspirated via syringe, being implicated in cases of self-harm, where a mercury bolus would have been intravenously administered. In such cases, the bolus itself does not cause systemic toxicity, instead it acts as an embolus, causing physical blockage of blood vessels at the injection site and in more vulnerable areas such as the lungs (44). Elemental mercury is only able to cause systemic poisoning by inhalation due to the thinness and dense vascularisation of the pulmonary mucosa, thus any inhaled vapours are easily absorbed into the circulation (44). The toxicodynamics of organomercury compounds gained relevance in environmental toxicology much more recently, particularly with methylmercury due to its significant bioaccumulative properties within food chains, enabling predatory species to accumulate large amounts of the compound, which are then consumed by humans (45). Therefore, organomercury exposure typically happens by ingestion (46). Poisoning by inorganic mercury is comparatively rarer since potential sources of exposure are quite limited, but usually occurs either by ingestion or contact because of its continued use in topical products in certain regions (26). Organomercury compounds are the most capable of traversing the bodily mucosae due to their lipophilic nature that allows them to dissolve in oily sebaceous skin secretions and traverse cell membranes, enabling significant transdermal, pulmonary, and gastrointestinal absorption (44). Certain compounds, such as dimethylmercury, have also demonstrated the ability to permeate through latex gloves (2,47). Larger organomercury compounds are less toxic to humans as their larger carbohydrate chains reduce the ability of the molecule to cross cellular membranes (44). If ingested, the mercury atoms of organomercury compounds may be liberated from their molecules as divalent cations (Hg2+) by the acidic environment in the stomach or by intestinal bacteria (2). Such cations are identical to those liberated from toxic mercuric salts. Inorganic mercuric salts exhibit efficient gastrointestinal absorption, owing to the ability of Hg2+ ions to utilise non-specific metal transporters within the gastrointestinal mucosa for their uptake (48). In the bloodstream, atoms of mercury and organomercury compounds can cross continuous capillaries, including those of the blood-brain barrier and placenta, due to a greater lipid solubility. This may either occur by simple diffusion or via a transporter (46). The mercuric cation is water soluble and can only pass through more porous fenestrated or sinusoidal capillaries (49). Intracellularly, it is the inorganic and organic forms of mercury that mainly cause pathology in relation to sulfhydryl binding (Figure 4). Elemental mercury instead may bind other moieties, including phosphoryl, carboxyl, and amide side groups (50).

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Figure 4: Schematic representation of the biomolecular basis of mercury poisoning as caused by organic and inorganic mercury. Note: NADP+: nicotinamide adenine dinucleotide phosphate; FAD: flavin adenine dinucleotide; Hg2+: mercury (II); Se: selenium; MeHg: methylmercury; HgSe: mercury selenide. (Adapted from Holmgren and Lu, 2010)

Table 2: Pathophysiological variability of different forms, sources, and exposures of mercury results in different symptomatic manifestations that may complicate diagnosis, also applicable to other heavy metal toxicoses. Sites written in italics indicate a less likely mode of absorption. (Adapted from Haddad and Stenberg, 1963; Bradberry et al., 1996; McKinney, 1999; Boyd et al., 2000; Ibrahim et al., 2006; Chan, 2011; Park and Zheng, 2012; Jaishankar et al., 2014; Kim et al., 2015)

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Exposure routes depend on the source and form of mercury involved, generally including inhalation, ingestion, contact, and rarely intravenous injection in self-harm or murder cases (2,24,26,48,58). Organic mercury is the most frequently involved form, being almost ubiquitous in the natural environment and highly bioaccumulative (Figure 3) (45,46). Thus, toxic amounts could easily be ingested, as demonstrated by previous incidents concerning the consumption of food and drink with excessive levels of methylmercury (59,60). Inorganic salts of mercury are less accessible to the public, however, sales of certain topical products such as skin lightening creams and ointments, cosmetic soaps, eye make-up, mascara, and cleaning products containing these salts mean that contact exposure is still a relevant means of acquiring mercury poisoning (26).

The chemical form influences how much and where mercury can enter and leave the circulation (Table 2). Once taken up by tissues, intracellular mercury may be converted into the inorganic ion, Hg2+, through oxidation or demethylation (24). The pathophysiology of mercury poisoning usually involves this mercuric cation, or else an organomercury compound should this be implicated in the exposure (Figure 4). Both forms bind sulfhydryl groups of intracellular thiol molecules such as cysteine, thioredoxin, glutathione, albumin and S-adenosylmethionine, impairing function (24). With regards to thioredoxin (Figure 4) and glutathione, their respective antioxidant-generating redox reactions, present in all living cells of the human body, become blocked (61,62). Inorganic mercury additionally sequesters cellular selenium, forming mercury selenide and inhibiting thioredoxin reductase selenoenzyme biosynthesis (Figure 4). This decreases antioxidant recycling, rendering the cell unable to reduce reactive oxygen species and control oxidative damage (51,63,64). Consequently, cellular components such as mitochondria, lipids, microtubules, ribosomes, endoplasmic reticula, and genes become oxidatively altered or damaged, and homeostatic processes involving membrane potential, proteins and calcium become disrupted (24). Tissue load and rate of oxygen consumption determine the extent of the damaging effects, therefore tissues such as brain tissue are more vulnerable (65). In the brain these processes lead to neurotoxic build-up of serotonin, glutamate, and aspartate due to a degraded microtubular structure (24). Inhibition of Sadenosylmethionine, a catecholamine-O-methyltransferase cofactor, further deteriorates neural physiology, causing catecholamine accumulation, hence leading to adrenergic symptoms that are likened to a phaeochromocytoma (22,23,30). Other manifestations of mercury poisoning include renal impairment, autoimmunity, and skin irritation (24,26), however these mostly occur when certain forms of mercury are involved.

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As mentioned earlier, mercury may chemically convert from one form to another in both extracellular and intracellular compartments, linking different pathophysiological processes together, and in turn, signs and symptoms may be mixed (Table 2) (2,24). Poisoning by organomercury is typically associated with neurological symptoms collectively known as Minamata disease (66,67). Inorganic mercury poisoning may also cause neurological symptoms, however it is more likely to cause renal damage than other forms of mercury poisoning (24,26,68). Additionally, there is now also evidence to support that inorganic mercury poisoning results in decreased hepatic and bone marrow function since these tissues possess capillary sinusoids (69,70). Organic and inorganic mercury also cause autoimmunity as part of the poisoning, whereby antibody production has been observed for myelin basic protein and glial fibrillary acidic protein in the brain, and the glomerular basement membrane of renal nephrons (71,72). These can be good indicators in the clinical presentation and investigatory findings (66,67). Elemental and inorganic mercury are more likely to cause an acute form of mercury poisoning that is more site-specific rather than systemic, as it is more difficult to reach pathology-inducing levels in the blood from their most common exposure routes. For instance, the application of topical products containing inorganic mercury can cause mild cutaneous symptoms such as irritation, suggested to be due to mercuric salt accumulation in sweat glands, sebaceous glands, and hair follicles (56). These acute exposures are also thought to have long-term implications, including a predisposition to Alzheimer’s disease and Young’s syndrome (26,73).

Symptom-based diagnosis of mercury poisoning is not recommended due to its variability and non-specificity (Table 2) (7). Diagnostic efforts should be focused on physical findings, appropriate history of when and how the exposure came to be, determination of the exposing agent, and identification of raised mercury body levels. Blood mercury biomarker indices may only be considered in combination with other clinical findings and if the exposure is either recent, chronic, or involves organic forms of mercury, as only these conditions have clinically significant blood mercury levels (74). The stability of mercury is greater in urine and hair; hence biomarker indices of such samples would be more indicative of possible poisoning. Furthermore, an association between urine mercury levels, memory, and language aptitude is also deemed to be diagnostically significant (75). Autoantibody levels of anti-glomerular basement membrane antibodies are suggested to be specific to mercury poisoning as well (75). The management procedure for mercury poisoning is similar to other heavy metal toxicoses. The patient should first be isolated from the intoxicating source to prevent further inhalation or ingestion of, or physical contact with mercury, and subsequently decontaminated by removing contaminated clothing and washing, with possible lavage (24,39,75–79). This may be followed by further investigation to determine the extent of bodily dispersal and assess the state of exposed mucocutaneous tissue.

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Chelation therapy is the primary treatment modality for mercury poisoning (Table 3) (9). Chelating agents are ions or molecules that possess atoms or side groups which can coordinate bond with a metal atom or ion by donating a pair of electrons, therefore acting as a ligand (9). Essentially, such agents exploit the ability of a metal ion, such as the mercuric ion, Hg2+, to bind to electron donor molecules within the body, such as sulfhydryl side groups, by possessing these side groups themselves (Figure 5). These actions stabilise the charged metal ion in a preferably inert and non-toxic complex with the chelating agent, favouring bodily excretion mainly via the renal and biliary systems (9,80). Various chelating agents are now available for administration, possessing different electron donor groups, with combination therapy also being common (Table 3) (9,79). In severe mercury poisoning, patient stabilisation under vital organ monitoring is required. This may involve artificial ventilation, plasmapheresis, haemodialysis, haemoperfusion, intravenous therapy and possible mineral and antioxidant supplementation to mobilise the metal (9,34,75– 79,81,82). Intervention may however not always be required, for example, since pure elemental mercury exhibits poor gastrointestinal absorption, it can be left to pass naturally from the body along with faeces under careful monitoring in cases where this has been ingested (54).

Figure 5: Simplified ball-and-stick model representing the mechanism of action of chelating agents. A molecule of dimercaprol is depicted where one of its sulfhydryl groups (labelled, SH) is donating an electron pair (• •) to a metal atom (M) bound to a protein via a side group containing sulfur (S). This restores the original protein side group and isolates the metal atom from the protein. (Adapted from Wikimedia Commons)

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Table 3: The viability of chelation therapy for treating mercury poisoning in comparison to various other metal toxicoses. Note: Cr: Chromium; Mn: Manganese; Fe: Iron; Co: Cobalt; Cu: Copper; Zn: Zinc; Ag: Gold; Cd: Cadmium; Sn: Tin; Au: Gold; Hg: Mercury; Tl: Thallium; Pb: Lead; Bi: Bismuth. (Adapted from Aaseth et al., 2016; Rafati Rahimzadeh et al., 2017; Kim et al., 2019)

In this review, the physicochemical influences, pathophysiology, clinical presentation, diagnosis, and management of mercury-induced heavy metal toxicosis have been outlined and discussed. Emphasis is to be made on how mercury poisoning can easily be prevented and its incidence reduced with proper regulation, enforcement and education when it comes to consumerism, occupational safety, and environmental health. Failure to do so will lead to significantly worse outcomes for both humans and the environment due to the reactive nature of mercury, and therefore merits a global cooperative effort.

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74. Carrier G, Bouchard M, Brunet RC, Caza M. A toxicokinetic model for predicting the tissue distribution and elimination of organic and inorganic mercury following exposure to methyl mercury in animals and humans. II. Application and validation of the model in humans. Toxicol Appl Pharmacol. 2001 Feb 15;171(1):50–60. 75. Ye B-J, Kim B-G, Jeon M-J, Kim S-Y, Kim H-C, Jang T-W, et al. Evaluation of mercury exposure level, clinical diagnosis and treatment for mercury intoxication. Ann Occup Environ Med. 2016 Jan 22;28:5. 76. Malbrain ML, Lambrecht GL, Zandijk E, Demedts PA, Neels HM, Lambert W, et al. Treatment of severe thallium intoxication. J Toxicol Clin Toxicol. 1997;35(1):97–100. 77. Patrick L. Lead toxicity, a review of the literature. Part 1: Exposure, evaluation, and treatment. Altern Med Rev. 2006 Mar;11(1):2–22. 78. Rafati-Rahimzadeh M, Rafati-Rahimzadeh M, Kazemi S, Moghadamnia AA. Current approaches of the management of mercury poisoning: need of the hour. Daru. 2014 Jun 2;22:46. 79. Rafati Rahimzadeh M, Rafati Rahimzadeh M, Kazemi S, Moghadamnia A-A. Cadmium toxicity and treatment: An update. Caspian J Intern Med. 2017;8(3):135–45. 80. Aaseth J, Crisponi G, Anderson O. Chelation Therapy in the Treatment of Metal Intoxication. 1st ed. Elsevier; 2016. 81. Vengamma B, Naveen T, Naveen V, Rao JV. Lead encephalopathy in adults. J Neurosci Rural Pract. 2014;5(2):161. 82. Wani AL, Ara A, Usmani JA. Lead toxicity: a review. Interdiscip Toxicol. 2015 Jun;8(2):55–64. 83. Wikimedia Commons. File:Dimercaprol-3D-balls.png - Wikimedia Commons [Internet]. [cited 2021 Jan 22]. Available from: https://commons.wikimedia.org/wiki/File:Dimercaprol-3Dballs.png

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Vitamin D and its fundamental role for the immune system Vitamin D (25-(OH)2-D3) is not just a vitamin but it has both endocrine and paracrine functions (Nair & Maseeh, 2012). 25-(OH)2-D3 plays a fundamental role for the immune response and protection. Vitamin D target tissues which contain a specific vitamin D nuclear receptor (VDR). VDRs are present in more than 30 cell types particularly those responsible for calcium homeostasis, immune function, endocrine, hematopoiesis and tumors (Kongsbak et al., 2013). BsmI (rs1544410), ApaI, and TaqI are fragment length polymorphisms (RFLP) located next to each other in the region of intron 8 and exon 9 on the vitamin D receptor gene (VDR) (Colombini et al; 2016). These polymorphisms, as will be discussed below have been commonly associated with several diseases.

Most studies have linked low levels of 25-OH-D3 with increased risk for asthma (Nair & Maseeh, 2012). On the other hand Ramadan et al; (2019) highlight that VDR expression in asthmatic children was found to be low. Calcitriol is the active form of Vitamin D and when calctriol binds to VDR receptors it has been seen to regulate lymphocyte and monocyte sensitivity to glucocorticoids by inducing expression of FOXP3. FOXP3, the immune response protein, is the master transcription factor which determines the Treg phenotype (Nair & Maseeh, 2012). According to Marques et al; (2015), FOXP3 gene expression has been seen to decrease in asthmatic patients. In fact, in vitro studies have deduced that vitamin D treatment in asthmatic patients reduces airway proliferation of smooth muscle cells (Nair & Maseeh, 2012). The advantage of vitamin D treatment in asthma has been observed in VDR-knockout mice which in the presence of induced asthma do not show inflammatory responses suggesting how in fact calcitriol may be immunosuppressive as it inhibits signs/symptoms of Th1 autoimmune illness (Wittke et al; 2004). The TaqI VDR genotype is associated with increased risk of asthma primarily when conditions of vitamin D levels are normal (>20 ng/ml) (Papadopoulou et al; 2015).

Rheumatoid Arthritis an autoimmune disease of unknown cause characterized by inflammation of the joints. Vitamin D deficiency is known to result in diffuse musculoskeletal pain (Meena et al; 2018). Vitamin D is known to prompt immunologic tolerance. Kostoglou-Athanassiou et al. (2012) explain how vitamin D deficiency impacts immune tolerance inducing autoimmune diseases including rheumatoid arthritis. As shown in figure 1., Meena et al; (2008) demonstrate how low serum vitamin D in fact leads to high rheumatoid arthritis disease activity by discovering that F allele present in F/F FokI VDR polymorphism are associated with RA. The f variant in the VDR has three more amino acids when compared with the F allele and the FokI alleles differ functionally because VDR affinity and VDR elements transactivation are altered (Meena et al; 2008). page 61

Figure 1: Inverse relationship of serum vitamin D (ng/ml) with Rheumatoid Arthritis Disease Activity (Meena et al; 2008)

MS is a chronic disease which affects the central nervous system and occurs when the immune system attacks the nerve fibers and myelin sheathing in the brain and spinal cord. When studying on the prevalence of MS, Sintzel, Rametta & Reder (2018) have observed how MS is more predominant in northern countries than in the tropics due to the number of hours of sunlight in winter and/or annually. In fact MS patients, like in asthma and rheumatoid arthritis have lower plasma calcitriol levels (Sintzel, Rametta & Reder, 2018). Mansouri et al; (2014) performed several studies which have shown how increased sunlight exposure leads to decreased vitamin D deficiency, decreasing MS risk. In fact they also explain how this decrease in risk is higher when sun exposure in particular occurred during childhood and puberty stage. Other studies have also shown how the birth month is related to MS risk. Individuals who were born in the fall means that their mother when pregnant has been exposed to summer sunlight. These individuals had a lower MS risk. On the other hand, individuals who were born during spring means that their mother when pregnant were less exposed to summer sunlight. These individuals were found to have a higher risk of MS. This indicates an observational relationship between maternal sunlight exposure during pregnancy and risk of MS (Sintzel, Rametta & Reder, 2018). The most recent meta-analysis to date, have studied VDR polymorphisms risk of acquiring MS. It was found that based on geographical location, Asians who had the BsmI variant had an increased risk of MS while ApaI showed lesser risk. Yet no association was found in variants and MS risk in the EU. The Fok1 variant was not related with increased MS risk or by geographical location (Imani et al; 2019).

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Vitamin D deficiency is present in patients’ with Crohn’s disease or ulcerative colitis. Vitamin D maintains a healthy microbiome in the gut because of VDR gut epithelial signaling. This has been seen to inhibit inflammation-induced epithelial cell apoptosis and prevents pathogenic CD8+ T cells proliferation therefore protecting the mucus barrier. Therefore, by affecting the gut microbiome vitamin D can ensure immune function of the host (Sheng et al; 2019).

Sequestration of calcitriol depends on the amount of adipose tissue. The more adipose tissue present results in a reduction in circulating calcitriol plasma levels. Therefore decreased 25-OHD3 plasma levels could stimulate fat accumulation leading to metabolic syndrome. Vitamin D deficiency can lead to obesity. In fact, 1,25-(OH)2-D3 can result in adipocyte apoptosis by competition between VDR and RXR to bind with peroxisome-proliferator activation receptor (PPARγ) which suppresses fat deposition, and stimulating steroid-metabolism enzyme expression of 11β-hydroxysteroid hydroxylase- a steroid-metabolizing enzyme. Together with caloric restriction, Vitamin D can result in weight loss. Berridge (2017) suggests that Vitamin D increases insulin sensitivity as calcitriol leads to the upregulation of the translocation of glucose transporter 4 (GLUT4) and adipocyte insulin signaling. On the other hand vitamin D may also play a role in decreasing the risk of Type 1 Diabetes (T1DM). It does this by destructing insulin-producing pancreatic- β cells which are secreted by cytokines and the free radicals released from inflammatory infiltrates; a process referred to as T-cell dependent. 1,25-(OH)2-D3 downregulates IL-12 production. Therefore IL-12dependent Th1 cells will have suppressed activity, triggering cytotoxic CD8+ lymphocytes and macrophages activity. Risk of T1D is positively related to latitude and inversely proportional to hours of sunlight (Berridge, 2017). Vitamin D deficiency also appears to increase the risk of noninsulin-dependent diabetes (type 2 diabetes, T2DM). As discussed above Vitamin D reduces inflammation. In fact according to Martine & Cambell, 2011, when Vitamin D is not present, chronic inflammation associated with insulin resistance will increase. In fact it has been found that serum calcitriol is inversely proportional to insulin resistance, body fat mass and body mass index (BMI). In fact studies have proven that the highest risk of T1/2DM is higher in winter because circulating levels of calcitriol are at their lowest. BsmI and ApaI are two VDR polymorphisms which have been linked to high fasting glucose levels, hyperinsulinemia, and higher T2D risk. The greatest relationship between levels of vitamin D and T2DM are highest among overweight and obese people (Martin & Campbell, 2011 & Berridge, 2017).

In asthma, rheumatoid arthritis, MS, IBS and diabetes vitamin D supplementation can be given to either prevent or reduce the severity of such conditions. Yet further studies focusing on vitamin D administration and sufficient levels of Vitamin D administration are necessary to conclude this.

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Colombini A, Bruno M, Lombardi G, Croiset SJ, Ceriani C, Buligan C, Barbina M, Banfi G and Cauci S. (2016). BsmI, ApaI and TaqI Polymorphisms in the Vitamin D Receptor Gene (VDR) and Association with Lumbar Spine Pathologies: An Italian Case-Control Study. PLoS One; 11(5): e0155004. Imani D, Razi B, Motallebnezhad M & Rezaei R (2019). Association between vitamin D receptor (VDR) polymorphisms and the risk of multiple sclerosis (MS): an updated meta-analysis. BMC Neurology 339(1044). Kongsbak M, Levring TB, Geisler C & Essen MR (2013). The vitamin D receptor and T cell function. Frontiers in Immunology 4:148, Retrieved from: https://doi.org/10.3389/fimmu.2013.00148 Kostoglou-Athanassiou I, Athanassiou P, Lyraki A, Raftakis I & Antoniadis C (2012). Vitamin D and Rheumatoid Arthritis. Therapeutic Advances in Endocrinology and Metabolism, 3(6), 181– 187. Maalej A, Petit-Teixeira E, Michou L, Rebai A, Cornelis F & Ayadi H (2005). Association study of VDR gene with rheumatoid arthritis in the French population. Genes and Immunity 6:707–711. Mansouri B, Asadollahi S, Heidari K, Fakhri M, Assarzadegan F, Nazari M. Risk factors for increased multiple sclerosis susceptibility in the Iranian population. Journal of Clinical Neuroscience 21:2207–2211. Martin T & Campbell K (2011). Vitamin D and Diabetes. Diabetes Spectrum, 24(2), 113-118. Meena N, Chawla SPC, Garg R, Batta A & Kaur S (2018). Assessment of Vitamin D in Rheumatoid Arthritis and Its Correlation with Disease Activity. Journal of Natural Science, Biology and Medicine 9(1), 54–58. Nair R & Maseeh A (2012). Vitamin D: The “sunshine” vitamin. Journal of Pharmacology and Pharmacokinetics 3(2), 118–126. Papadopoulou A, Kouis P, Middleton N, Kolokotroni O, Karpathios T, Nicolaidou P & Yiallouros PK (2015). Association of vitamin D receptor gene polymorphisms and vitamin D levels with asthma and atopy in Cypriot adolescents: a case–control study. Multidisciplinary Respiratory Medicine volume 26. Ramadana A, Sallamb SF,.Elsheikhc MS, Ishak SR, Abdelsayed MGR, Salahd M, Nazihe R, Khairat R, Ibrahim OM (2019). VDR gene expression in asthmatic children patients in relation to vitamin D status and supplementation. Gene Reports 15: 100387.

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Sintzel MB, Rametta, M & Reder AT (2018). Vitamin D and Multiple Sclerosis: A Comprehensive Review. Neurology and Therapy 7(1): 59–85. Sheng-Mei S, Wen, Yan-Li, Hou, Hai-Bin; Liu, Hai-Xia (2019). Effectiveness of vitamin D for irritable bowel syndrome: A protocol for a systematic review of randomized controlled trial. Medicine , 98(9): pe14723 Wittke A, Weaver V, Mahon BD, August A & Cantorna MT (2004). Vitamin D Receptor-Deficient Mice Fail to Develop Experimental Allergic Asthma. The Journal of Immunology, 173 (5): 34323436.

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The role of Nitric Oxide in Stroke pathophysiology Bilateral common carotid artery occlusion (BCCAO) Calcium/calmodulin-dependent protein kinase type II subunit alpha (CaMKIIα) Cerebral blood flow (CBF) Endothelial nitric oxide synthase (eNOS) Glucagon-like peptide-1 receptor (GLP-1R) Haemorrhagic strokes (HS) Hypoxia-inducible factor 1-alpha (HIF-α) Inducible nitric oxide synthase (iNOS) Inhaled nitric oxide (iNO) Intracerebral haemorrhage (ICH) Ischaemic stroke (IS) L-arginine (L-Arg) Matrix metalloproteinase (MMP-9) Mixed-lineage kinase 3 (MLK3) Neuronal nitric oxide synthase (nNOS) Nitric Oxide (NO) Nitric Oxide Synthase (NOS) N-methyl-D-aspartate (NMDA) Peroxynitrite (ONOO-) Postsynaptic density protein 95 (PSD-95) Subarachnoid haemorrhage (SAH) Transient ischaemic attacks (TIA) Zona occludens (ZO-1)

Stroke is one of the leading causes of death and disability worldwide. Strokes can be classified as being either ischaemic strokes (IS) or haemorrhagic stokes (HS), mini strokes or transient ischaemic attacks (TIA). Ischaemic strokes tend to be the most frequent, with an incidence of about 60-70% (1). Nitric oxide (NO) has been indicated to contribute to the pathogenesis of both ischaemic and haemorrhagic forms of stroke.

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Brain damage in the case of IS and TIA occurs in the form of ischaemic/reperfusion which has a multitude of possible causes such as excitotoxicity, calcium dysregulation, or due to either nitrosative or oxidative stress (2). Moreover, the aetiology is somewhat different in the sense that ISs are thrombotic in nature whereas TIAs are embolic and therefore of short duration or transient. Serum NO levels have been indicated to rise exponentially during the onset of ischaemia in Northern Indian patients (3). NO may either be neuroprotective or neurotoxic in the ischaemic environment, depending on the isoform through which it is produced. There are three different forms of nitric oxide synthase (NOS) the enzyme responsible for the synthesis of NO. These are: endothelial nitric oxide synthase (eNOS); neuronal nitric oxide synthase (nNOS); and inducible nitric oxide synthase (iNOS). Whilst eNOS has been indicated to reduce ischaemic injury, on the other hand, NO produced by either nNOS or iNOS tend to aggravate neuronal damage. nNOS has been shown to increase during the onset of ischaemia in bilateral common carotid artery occlusion (BCCAO) stroke models in mice. However, research still has to be carried out to determine how nNOS may worsen outcome as the administration of L-citrulline was shown to be overall beneficial to improve outcome, despite the concurrent elevation in the levels of nNOS (4). Therefore, the exact mechanism through which nNOS is harmful still requires further research. A death-promoting pathway through which nNOS could potentially lead to neuronal death is via the interaction of NMDA receptors with the postsynaptic density protein 95 (PSD-95), and that is tethered to nNOS. In a series of experiments involving the use of stroke rodent models, administration of nNOS-N1-133 together with the drug ZL006 prevents the dimerization of the nNOS-PDZ-95 complex, were found to both reduce the size of the infarct and improve neurological function (5). A further mechanism that contributes to neuronal death during ischaemia is thought to be through the nitrosylation of the GluR6 subunit of the NMDA receptor as a result of the increased upregulation of nNOS. This interaction is thought to promote the assembly of the complex between the PDZ-95 domain and, GluR6 with mixed-lineage kinase 3 (MLK3), which increases the activation of the downstream signalling molecule c-Jun N-terminal kinase (JNK) and consequently the phosphorylation of the protein c-Jun (6). The therapeutic importance of this mechanism is likely conferred through the suppression of the activity of the downstream pathways potentiated by nitrosylation of GluR6 (7–10). The inhibition of the PDZ-95 domain also has been indicated to have the potential to block this pathway (6). iNOS upregulation has also been indicated to indicate bad outcome following ischaemia. Administration on rats of the iNOS inhibitor s-methylisothiourea on rats has been previously shown to cause a reduction in apoptosis following ischaemia, as well as a combined decrease in the levels of NO and peroxynitrite (ONOO-) (11). This is supported by another study in rats which showed that the administration of the angiotensin 2 type 1 receptor blocker, Losartan, caused a reduction is ischaemic damage in part through attenuation of iNOS (12)

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Despite research convincingly implicates nNOS and iNOS activation with neuronal damage following IS, numerous studies provide contrasting results. A study conducted on rats showed that transient ischaemia exhibited improved outcome when the effected brain area contained a greater number of nNOS-producing neurons that were colocalized with iNOS. The improved outcome might, in this case, be related to the potential in promoting neurogenesis (13). Another study showed that post-conditioning rats with the general anaesthetic isoflurane exerted a protective effect through the increase of hypoxia-inducible factor 1-alpha (HIF-α) and the consequent elevation in iNOS. Inhibition of iNOS prevented this protective effect, indicating that higher levels of iNOS may be neuroprotective. However, there is need for further research (14). On the other hand, upregulation of eNOS has been associated with decreased ischaemic damage. A study conducted on diabetic rats showed that the glucagon-like peptide-1 receptor (GLP-1R) proteins was associated with beneficial outcome as a result of a combined reduction of iNOS and an increase in eNOS (15). L-Citrulline, which may serve as a precursor for NO though promoting generation of L-Arginine, has also been associated with increased beneficial outcome in BCCAO rats. It prevents reduced production of eNOS during ischaemia either in low-dose (40mg/kg) co-administration with glutathione (16), or when administered at a higher dose (100mg/kg) (4). This occurrence may be explained through the mechanism where the combined administration of both L-Citrulline and Glutathione prevent the S-glutathionylation of eNOS (16). S-glutathionylation may cause the uncoupling of eNOS dimers by inhibiting eNOS dimerization (17–19). In contrast, however, a study showed that, initially, ischaemia causes an upregulation of eNOS. However, eNOS levels return to the normal level within a day. The administration of losartan was shown to maintain this elevated level of eNOS, reducing ischaemia induced damage. The suggested mechanism of function of losartan is that it increased the phosphorylation of both eNOS, as well as its downstream products causing the activation of the PI3K/Akt pathway. This may lead to neuroprotective effects with respect to ischaemic injury (12). These studies indicate the need for further research on the association between eNOS mechanisms and beneficial ischaemic damage outcome, and which can lead to the development of a treatment to improve outcomes in patients.

Haemorrhagic stroke is less common than ischaemic stroke. Haemorrhagic strokes are divided into two subgroups: those which are the more common intracerebral haemorrhage (ICH) which makes up around 10-15% of global strokes (20); and the less common subarachnoid haemorrhage (SAH) which accounts for approximately 7% of global strokes. SAH is associated with a relatively high of negative outcome even in among the younger population. (21). SAH has been associated with a number of secondary problems such as vasospasms. These were thought to be a delayed consequence of SAH, and were found to occur approximately 5 days after injury (22). However, more recent research has found that these vasoconstrictions may even begin to occur right after SAH (23). This finding is of great significance due to the high mortality rate of SAH within the first two days following the insult (24). In addition, SAH may cause other secondary pathological effects such as causing thrombus formation and dysfunction in the microcirculation, causing a delayed ischaemia (25– 27). page 68

NO has been indicated to affect both forms of haemorrhage. However, more research has been conducted with respect to SAH. In SAH, NO levels have been shown to decrease between the onset of SAH and lasting several hours (28). However, other research has indicated that there may be a surplus of nitric oxide following this period of NO deficiency (29). This NO deficiency has been linked to reduced blood supply, and consequently leading to vasospasms (30). Therefore, treatment for these vasospasm in animals models has been attempted via NO administration of NO donors (23) as well as NO inhalation (31). Inhaled nitric oxide (iNO) was able to reverse the majority of micro-vasospasms. iNO exerted a dilatory effect in the remainder of microvessels, and was also able to reduce vasospasm in larger vessels, with this effect typically wearing out after one day (31). Both studies indicated an elevated cerebral blood flow (CBF), which is important to restore blood supply (23,31). However, one benefit of using iNO as a treatment is that it has no effect on the systemic blood pressure, making it a safer option (31). The different isoforms of NOS, like in the case of brain ischaemia, have been indicated to be either beneficial or pathological. eNOS deficiency has been indicated in SAH to cause vasospasms (32). This is supported by research indicating that the upregulation of eNOS is beneficial (33). Vasospasm should induce the production of eNOS due to the stress experienced by the endothelium. Research conducted on mice indicated that eNOS was upregulated following the onset of SAH due to an increased phosphorylation. However, eNOS dimers were uncoupled, and this resulted in the production of ONOO- instead of NO, resulting in the decrease in eNOS physiological function (34). Given the beneficial role of eNOS in haemorrhagic pathology, further research may help develop a new form of treatment for the augmentation of eNOS levels. nNOS in both SAH and ICH appears to be pathological. In rat models of SAH, nNOS phosphorylation appeared to be increased at the Ser847 site. This is associated with a decrease in nNOS activity. The mechanism through which this may occur is via the SAHinduced increased intra-cerebral pressure which may stimulate Calcium/calmodulindependent protein kinase type II subunit alpha (CaMKIIα activation), resulting in nNOS phosphorylation (35). The importance of this occurrence is that a decreased activity may be an adaptive mechanism to prevent neurotoxicity, possibly by stimulating the expression of heme oxygenase-1 (36). In rat ICH models, there appeared to be upregulation of nNOS activity. The application of the nNOS inhibitor S-methyl-L-thiocutrulline gave positive results due to the reduction of both neuronal death and behavioural deficits (37). iNOS is apparently upregulated in both SAH (34,38) and ICH (39) mice models. The application of Baicilin, which is a traditional ingredient in Chinese medicine, was shown to reduce iNOS and NOX-2. iNOS and NOX-2 are associated with oxidative damage under the pathological state, aiding in reducing the proinflammatory state, and also contributing to restore the blood-brain barrier (38). Furthermore, Baicilin has also been indicated to reduce infarct volume in SAH (40). This is, in part, due to the inhibition of iNOS, indicating that it typically has a pathological role in SAH. In ICH, iNOS has been shown to increase, and the inhibition of iNOS has been shown to be beneficial to ICH outcome (35,41–43). The presence

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of recombinant osteopontin, which is an inhibitor of iNOS, had a beneficial effect on the outcome of ICH in mice as it reduced brain oedema, as well as increased the number of surviving neurons. The mechanism through which recombinant osteopontin appears to carry out its effect involves reducing phosphorylated Stat-1 levels, possibly by increasing its rate of ubiquitination (44). In addition, recombinant osteopontin reduced matrix metalloproteinase (MMP)-9 levels), which is responsible for causing the disruption of tight junctions by reducing the levels of zona occludens (ZO)-1 (39). This research indicates that iNOS and nNOS inhibitors functioning are promising fields for further research for developing new treatments to improve outcome of HS patients.

Both nNOS and iNOS have also been associated with worsened outcomes in cases of ischaemic and haemorrhagic stroke. In contrast, eNOS has been implicated to contribute to smaller infarct sizes and better long-term outcomes. Therefore, NOS-targeted treatment which increases levels of eNOS and/or decreasing levels of both nNOS and iNOS could be a possible therapeutic target for treatment.

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8. Tian H, Zhang Q-G, Zhu G-X, Pei D-S, Guan Q-H, Zhang G-Y. Activation of c-Jun NH2-terminal kinase 3 is mediated by the GluR6.PSD-95.MLK3 signaling module following cerebral ischemia in rat hippocampus. Brain Res. 2005 Nov 2;1061(1):57–66. 9. Pei D-S, Wang X-T, Liu Y, Sun Y-F, Guan Q-H, Wang W, et al. Neuroprotection against ischaemic brain injury by a GluR6-9c peptide containing the TAT protein transduction sequence. Brain. 2006 Feb;129(Pt 2):465–479. 10. Li T, Yu H-M, Sun Y-F, Song Y-J, Zhang G-Y, Pei D-S. Inhibition of cerebral ischemia/reperfusion-induced injury by adenovirus expressed C-terminal amino acids of GluR6. Brain Res. 2009 Dec 1;1300:169–176. 11. ArunaDevi R, Ramteke VD, Kumar S, Shukla MK, Jaganathan S, Kumar D, et al. Neuroprotective effect of s-methylisothiourea in transient focal cerebral ischemia in rat. Nitric Oxide. 2010 Jan 1;22(1):1–10. 12. Liu H, Liu X, Wei X, Chen L, Xiang Y, Yi F, et al. Losartan, an angiotensin II type 1 receptor blocker, ameliorates cerebral ischemia-reperfusion injury via PI3K/Akt-mediated eNOS phosphorylation. Brain Res Bull. 2012 Oct 1;89(1-2):65–70. 13. Corsani L, Bizzoco E, Pedata F, Gianfriddo M, Faussone-Pellegrini MS, Vannucchi MG. Inducible nitric oxide synthase appears and is co-expressed with the neuronal isoform in interneurons of the rat hippocampus after transient ischemia induced by middle cerebral artery occlusion. Exp Neurol. 2008 Jun;211(2):433–440. 14. Fang Li Q, Xu H, Sun Y, Hu R, Jiang H. Induction of inducible nitric oxide synthase by isoflurane post-conditioning via hypoxia inducible factor-1α during tolerance against ischemic neuronal injury. Brain Res. 2012 Apr 27;1451:1–9. 15. Zhao L, Xu J, Wang Q, Qian Z, Feng W, Yin X, et al. Protective effect of rhGLP-1 (7-36) on brain ischemia/reperfusion damage in diabetic rats. Brain Res. 2015 Mar 30;1602:153–159. 16. Matsuo K, Yabuki Y, Fukunaga K. Combined l-citrulline and glutathione administration prevents neuronal cell death following transient brain ischemia. Brain Res. 2017 May 15;1663:123–131. 17. Chen C-A, Wang T-Y, Varadharaj S, Reyes LA, Hemann C, Talukder MAH, et al. Sglutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature. 2010 Dec 23;468(7327):1115–1118. 18. Chen C-A, Lin C-H, Druhan LJ, Wang T-Y, Chen Y-R, Zweier JL. Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling. J Biol Chem. 2011 Aug 19;286(33):29098–29107. 19. Popov D. Protein S-glutathionylation: from current basics to targeted modifications. Arch Physiol Biochem. 2014 Oct;120(4):123–130. page 71

20. Aronowski J, Hall CE. New horizons for primary intracerebral hemorrhage treatment: experience from preclinical studies. Neurol Res. 2005 Apr;27(3):268–279. 21. Taylor TN, Davis PH, Torner JC, Holmes J, Meyer JW, Jacobson MF. Lifetime cost of stroke in the United States. Stroke. 1996 Sep;27(9):1459–1466. 22. Mayberg MR, Batjer HH, Dacey R, Diringer M, Haley EC, Heros RC, et al. Guidelines for the management of aneurysmal subarachnoid hemorrhage. A statement for healthcare professionals from a special writing group of the Stroke Council, American Heart Association. Circulation. 1994 Nov;90(5):2592–2605. 23. Lilla N, Hartmann J, Koehler S, Ernestus R-I, Westermaier T. Early NO-donor treatment improves acute perfusion deficit and brain damage after experimental subarachnoid hemorrhage in rats. J Neurol Sci. 2016 Nov 15;370:312–319. 24. Hop JW, Rinkel GJ, Algra A, van Gijn J. Case-fatality rates and functional outcome after subarachnoid hemorrhage: a systematic review. Stroke. 1997 Mar;28(3):660–664. 25. Hansen-Schwartz J, Vajkoczy P, Macdonald RL, Pluta RM, Zhang JH. Cerebral vasospasm: looking beyond vasoconstriction. Trends Pharmacol Sci. 2007 Jun;28(6):252–256. 26. Macdonald RL, Pluta RM, Zhang JH. Cerebral vasospasm after subarachnoid hemorrhage: the emerging revolution. Nat Clin Pract Neurol. 2007 May;3(5):256–263. 27. Pluta RM, Hansen-Schwartz J, Dreier J, Vajkoczy P, Macdonald RL, Nishizawa S, et al. Cerebral vasospasm following subarachnoid hemorrhage: time for a new world of thought. Neurol Res. 2009 Mar;31(2):151–158. 28. Jung CS, Oldfield EH, Harvey-White J, Espey MG, Zimmermann M, Seifert V, et al. Association of an endogenous inhibitor of nitric oxide synthase with cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage. J Neurosurg. 2007 Nov;107(5):945–950. 29. Pluta RM. Delayed cerebral vasospasm and nitric oxide: review, new hypothesis, and proposed treatment. Pharmacol Ther. 2005 Jan;105(1):23–56. 30. Schubert GA, Thome C. Cerebral blood flow changes in acute subarachnoid hemorrhage. Front Biosci. 2008 Jan 1;13:1594–1603. 31. Terpolilli NA, Feiler S, Dienel A, Müller F, Heumos N, Friedrich B, et al. Nitric oxide inhalation reduces brain damage, prevents mortality, and improves neurological outcome after subarachnoid hemorrhage by resolving early pial microvasospasms. J Cereb Blood Flow Metab. 2016;36(12):2096–2107. 32. Pluta RM. Dysfunction of nitric oxide synthases as a cause and therapeutic target in delayed cerebral vasospasm after SAH. Neurol Res. 2006 Oct;28(7):730–737. page 72

33. Sugawara T, Ayer R, Jadhav V, Chen W, Tsubokawa T, Zhang JH. Simvastatin attenuation of cerebral vasospasm after subarachnoid hemorrhage in rats via increased phosphorylation of Akt and endothelial nitric oxide synthase. J Neurosci Res. 2008 Dec;86(16):3635–3643. 34. Sabri M, Ai J, Knight B, Tariq A, Jeon H, Shang X, et al. Uncoupling of endothelial nitric oxide synthase after experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab. 2011 Jan;31(1):190–199. 35. Makino K, Osuka K, Watanabe Y, Usuda N, Hara M, Aoyama M, et al. Increased ICP promotes CaMKII-mediated phosphorylation of neuronal NOS at Ser847 in the hippocampus immediately after subarachnoid hemorrhage. Brain Res. 2015 Aug 7;1616:19–25. 36. Kasamatsu S, Watanabe Y, Sawa T, Akaike T, Ihara H. Redox signal regulation via nNOS phosphorylation at Ser847 in PC12 cells and rat cerebellar granule neurons. Biochem J. 2014 Apr 15;459(2):251–263. 37. Lu A, Wagner KR, Broderick JP, Clark JF. Administration of S-methyl-L-thiocitrulline protects against brain injuries after intracerebral hemorrhage. Neuroscience. 2014 Jun 13;270:40–47. 38. Shi X, Fu Y, Zhang S, Ding H, Chen J. Baicalin Attenuates Subarachnoid Hemorrhagic Brain Injury by Modulating Blood-Brain Barrier Disruption, Inflammation, and Oxidative Damage in Mice. Oxid Med Cell Longev. 2017 Aug 24;2017:1401790. 39. Wu B, Ma Q, Suzuki H, Chen C, Liu W, Tang J, et al. Recombinant osteopontin attenuates brain injury after intracerebral hemorrhage in mice. Neurocrit Care. 2011 Feb;14(1):109–117. 40. Liu Q, Liu J, Wang P, Zhang Y, Li B, Yu Y, et al. Poly-dimensional network comparative analysis reveals the pure pharmacological mechanism of baicalin in the targeted network of mouse cerebral ischemia. Brain Res. 2017 Jul 1;1666:70–79. 41. Kim DW, Im S-H, Kim J-Y, Kim D-E, Oh GT, Jeong S-W. Decreased brain edema after collagenase-induced intracerebral hemorrhage in mice lacking the inducible nitric oxide synthase gene. Laboratory investigation. J Neurosurg. 2009 Nov;111(5):995–1000. 42. Jung K-H, Chu K, Jeong S-W, Han S-Y, Lee S-T, Kim J-Y, et al. HMG-CoA reductase inhibitor, atorvastatin, promotes sensorimotor recovery, suppressing acute inflammatory reaction after experimental intracerebral hemorrhage. Stroke. 2004 Jul;35(7):1744–1749. 43. Sinn D-I, Lee S-T, Chu K, Jung K-H, Kim E-H, Kim J-M, et al. Proteasomal inhibition in intracerebral hemorrhage: neuroprotective and anti-inflammatory effects of bortezomib. Neurosci Res. 2007 May;58(1):12–18. 44. Guo H, Wai PY, Mi Z, Gao C, Zhang J, Kuo PC. Osteopontin mediates Stat1 degradation to inhibit iNOS transcription in a cecal ligation and puncture model of sepsis. Surgery. 2008 Aug;144(2):182–188. page 73

The Role of Biomarkers in the Diagnosis of Acute Aortic Dissection

AD - Aortic Dissection AAD - Acute Aortic Dissections IRAD - International Registry of Aortic Dissections CT Scan - Contrast-enhanced computed tomography MRI - Magnetic resonance imaging DSA - digital subtraction angiography. MiRNA - MicroRNA

Aortic dissection (AD), is characterised by the spontaneous development of false lumen in the innermost layer of the aorta and can present at various sites, with the ascending aorta being the most frequent location for its presentation. (1,2) Aortic dissections have a pathophysiologic sequence that includes aortic wall inflammation, apoptosis of vascular smooth muscle cells, aortic media degeneration due to inflammatory cell infiltration in the aortic media, elastin disruption, and vessel dissection. (1),(3) Consequently, immediate and accurate management and intervention are required, as mortality rates after the rupture exceed 80%.(2),(3) Acute aortic dissection presents a great diagnostic challenge, as its typical signs and symptoms are nonspecific. The most common symptom includes acute onset of tearing chest, as well as pain in the back or abdomen, often described as ‘sharp’. (1,2) Hypertension is a significant factor. Imaging tests are used to make a diagnosis, including transesophageal echocardiography, CT angiography, MRI, and contrast aortography, with recent studies showing that on a posterior-anterior chest x-ray, a widened mediastinum may also be a clinical presentation of aortic dissection. (1)

The majority of Aortic Syndromes are caused by AD, with intramural haematoma and penetrating atherosclerotic ulcers accounting for the remainder. (4) Whilst acute AD and intramural haematoma have similar diagnostic and treatment challenges, the presence of an 'entry tear,' which is defined as a rupture in the intimal layer of the media, can distinguish the two pathologies. (1,3) If an aortic dissection is identified within 14 days after the commencement of the rupture, it is categorised as acute, and if it is diagnosed beyond two weeks, it is classified as chronic. page 74

Dissections could interact with the true aortic lumen via intimal rupture at a distal site, permitting systemic blood flow to be maintained. Serious consequences include aortic valvular dilation and regurgitation, as well as heart failure. Another risk factor is a fatal aortic rupture through the adventitia into the pericardium, right atrium, or left pleural space. Dissection variants are thought to be precursors to classic aortic dissection, which include an intramural hematoma that separates the intima and media without a clear intimal tear or flap, an intimal tear and bulge without the presence of a haematoma or false lumen, as well as dissection, as well as a hematoma caused by atherosclerotic plaque ulceration. The classification of AD is classified by the DeBakey system or the Stanford System. The DeBakey classification is of three types; Type I refers to lesions involving both the ascending aorta and descending aorta, Type II depicts lesions solely in the ascending aorta and Type III for the descending aorta.(3) With reference to the Stanford classification, Type A concerns dissections within the ascending aorta, regardless of anatomical entry location. Stanford Type A, Type I and II need immediate surgical intervention, reducing mortality rates by 20% within 30 days of onset. (1) Stanford Type B depicts lesions occurring in the descending aorta, and such distal AD may be managed medically, lowering mortality rates by 10% within the same time frame if no complications are present.(1,4)

Figure 1: Simplistic diagram highlighting the changes in aortic lumen in different Acute Aortic Syndromes

At present, physicians consider biomarkers an attractive alternative diagnostic tool. Acute AD is depicted by the remodelling of the damaged aortic media via secondary thrombosis and inflammatory reactions. (4) Accordingly, various studies have focussed on biomarkers relating to such thrombotic and inflammatory reactions at the lesion site, as well as injury to the smooth muscle tissue and elastic laminae of the aortic media and interna. (1)

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Figure 2: Diagnostic Algorithm of Potential D-dimer Patients, adapted from Wilcox G. Nursing patients with acute aortic dissection in emergency departments. Emerg Nurse. 2019 May 7;27(3):32–41. Initial studies on biomarkers for AD show that circulating smooth muscle proteins are potential candidates for biomarkers, seeing as they are predominantly found in the aortic medial layer. Smooth muscle myosin heavy chains were investigated, and they showed significant rises in acute AD patients, with levels being 20-fold higher. While biomarkers had good diagnostic accuracy, they were shown to be high in the first 6 hours after onset, resulting in a short diagnostic window (5).

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D-dimer is currently an available biomarker for AD. This molecule is comprised of two cross-linked fibrinogen D fragments. Although D-dimer is formed during fibrinolysis, it is a marker of thrombin activity and fibrin turnover, which reflects both haemostasis and fibrinolysis. D-dimer has an approximated 8-hour half-life and becomes detectable in the blood approximately 2 hours after index thrombus formation. D-dimer however, is not a specific marker for coagulation activity, limiting its utility. This is due to the close relationship between coagulation and inflammation, as well as the involvement of several factors in the coagulation cascade in each of these systems. Increased levels may be observed in conditions where fibrin is formed and later degraded, such as recent surgery, pregnancy, trauma events, heart disease as well as infection. (6,7) The IRAD study on AD found that a cut-off level of 500ng/ml could be applied to both pulmonary embolism and AD within a day of symptom onset. The condition is linked to a rapid rise in D-dimer, with the first 6 hours following onset being the most significant. As a result, dissection during the first 6 hours can be safely ruled out. As a result, routine use of D-dimer helps risk-stratify people with query acute AD (5).

When blood clots are either being produced or broken down due to a damaged blood vessel, as what occurs in Aortic Dissection, a fibrin mesh and platelets form at the site of injury. This protective process against haemorrhage starts half an hour after trauma has occurred, and the vessel constricts due to spasms. (8) Collagen is exposed to the blood and cytokines and inflammatory markers are released via the extracellular matrix. The interactivity between tyrosine kinase receptors, glycoprotein receptors and G-coupled receptor proteins and von Willebrand Factor mediates the adhesion of platelets. (9) During the propagation phase of coagulation, the prothrombinase complex FXa+FVa generates a large amount of thrombin on the activated platelet surface. Thrombin cleaves fibrinogen to fibrin, thus forming a stabilising fibrin network with FXIII. There are three major steps required for D-dimer formation, starting off when a polymerization site on fibrin is exposed, simultaneously when thrombin is cleaved. (8) This encourages the binding of fibrinogen or its monomer fibrin, in order to form thick fibrils. The role of plasmin is the proteolytic degradation of fibrin, and it remains fluid until thrombin cleaves 25% to 30% of plasma fibrinogen, allowing fibrin to polymerize while facilitating thrombin activation of plasma factor XIII. (7) Tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) is required for plasminogen to plasmin conversion. Plasminogen activator inhibitor (PAI)-1, which is a serine protease inhibitor, is an essential fibrinolytic system inhibitor that forms complexes with tPA and uPA quickly. PAI-1, an acute-phase protein, has high variability amongst individuals. Thrombin activatable fibrinolysis inhibitor (TAFI) is activated by thrombin and plasmin and is responsible for fibrinolysis. Thrombin remains bound to fibrin, and as more fibrin page 77

molecules clump, plasma factor XIII bound to fibrinogen is activated. Until a fibrin gel is observed, a complex between soluble fibrin polymers, thrombin, and plasma factor XIII facilitates the formation of factor XIIIa. The second step of forming this biomarker is for Factor XIIIa forms intermolecular isopeptide links between lysine and glutamine residues in the soluble protofibrils and the insoluble fibrin gel to covalently cross-linked fibrin monomers. (9)

Figure 3: The Dynamic Formation of D-dimer, shown in three steps. Adapted from Adam SS, Key NS, Greenberg CS. D-dimer antigen: current concepts and future prospects. Blood. 2009 Mar 26;113(13):2878–87. Until the action of plasmin releases the D-dimer antigen from crosslinked fibrin, it is undetectable. Plasmin formed on the fibrin surface by plasminogen activation cleaves fibrin at specific sites in the final step of D-dimer formation. Fibrin degradation products come in a wide range of molecular weights, including the D-dimer and fragment E complex terminal degradation products of cross-linked fibrin. (10)

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The function of biomarkers is to assess the risk and severity of the disease. This is measured by the sensitivity and specificity of a biomarker in relation to cardiac disease. (11) Sensitivity refers to the ability to correctly detect the disease in patients while specificity is related to the ability to identify patients that do not have the disease. Such screening tests are not diagnostic, however, they are used as identification means for the individual to know if they have a certain condition. (12) D-dimer correlates positively with the extent of the dissection, as a key finding found is that the degree of aortic dissection and the condition of the false lumen were both reflected in the patients' admission D-dimer concentration. (13) The concentration of Ddimer was higher in patients with dissection that extended below the diaphragm level than in patients with dissection that did not extend to this extent. Low D-dimer concentrations are seen to be correlated with a tear that is either limited in the ascending aorta, or a thrombosed false lumen in the descending aorta. (14) Thus far, studies state that there is no established acceptable failure rate for ruling out Acute Aortic Syndromes. This leads to high rates of AAS being misdiagnosed, reaching over 40%. D-dimer as a biomarker has several advantageous roles for Acute Aortic Dissection, due to such a test being less invasive, cost-effective and readily available in comparison to transesophageal echocardiography and MRI. Currently, D-Dimer is functionally used to test for short term outcomes of acute AD patients, with long-term outcomes yet to be explored, due to the fact that D-Dimer as a stand-alone test is inaccurate, due to the low specificity yet high sensitivity. In order for biomarkers to be widely used as a diagnostic test for dissection of the aorta, the D-Dimer biomarker must be paired with a biomarker with high specificity. Studies and meta-analysis reviewed analysed patients with were diagnosed with acute AD, using the ADD-RS classification, of which patients with an Acute Aortic DissectionRisk Score above or equal to 1 had a sensitivity of 95% (95% Confidence Index, 91.5-97.4) and specificity of 26.4% (95% CI, 24.3–28.7) when diagnosing Acute Aortic Dissection. It was found that the difference in concentration of D-d was significantly high, varying greatly from patient groups and is dependent on the extent of dissection, due to degradation fibrin fibres of an extensive false lumen would increase the D-dimer levels (15). Various meta-analyses conclude that 0.5ug/mL D-dimer cut off levels have a high sensitivity yet a relatively low specificity (95%-98% and 40%-60% respectively). Thus, this biomarker is termed sensitive yet non-specific (15,16). A key finding found in this study is that the degree of aortic dissection and the condition of the false lumen were both reflected in the patients' admission D-dimer concentration. [25] The concentration of D-dimer was higher in patients with dissection that extended below the diaphragm level than in patients with dissection that did not extend to this extent. Similarly, the concentration of D-dimer was higher in patients with partially thrombosed or patented false lumen than in patients with fully thrombosed FL (16). page 79

Moreover, low D-dimer concentrations are seen to be correlated with a tear that is either limited in the ascending aorta, or a thrombosed false lumen in the descending aorta. In addition, it was further reported that negative D-dimer results in patients correlated with a shorter dissection rate, yet it was inconclusive whether this related to a good prognosis rate (14). Studies have confirmed that this biomarker has a high sensitivity for Acute Aortic Dissection diagnosis, due to the false lumen thrombosis results in tissue factor release activates the fibrin dissolution system, as well as the coagulation reaction endogenously. as mentioned previously. This study, like the previous studies mentioned in this systematic review, also confirmed that D-dimer levels are significantly more abundant in acute AD positive groups when compared to non-ADD clusters and control patients, further confirming its high diagnostic sensitivity but low specificity (17).

The present studies discussed posed various limitations. There is no established acceptable failure rate for ruling out Acute Aortic Syndromes. This leads to high rates of misdiagnosis, reaching over 40%. In addition, it is often difficult with one cross-sectional CT image to assess the highest aortic width of the surgical site. These parameters alone will not appear to be sufficient in such situations to evaluate the long-term prognosis (18) There is a limitation of the specific epitope on the D‐dimer fragment that can be found for the monoclonal antibody used in a D‐dimer assay, but greater than 20 different monoclonal antibodies are used in 30 different D-dimer tests (15). D-dimer as a biomarker is helpful, yet specificity and sensitivity of the test are sub-par, hence combined detection by using multiple biomarkers would improve diagnosis of Acute Aortic Dissection, especially when there is a lack of imaging equipment available (17). Furthermore, a possible area for future research and literature would be Acute Aortic Dissection in patients of the age of 18 or younger. The majority of the present literature focus solely on patients above the age of 18, thus it is inconclusive whether D-dimer is a suitable and sensitive biomarker for sudden traumatic aortic injury in younger individuals.

MiRNA is small pieces of non-coding strands of RNA which are about 22 nucleotides long. The synthesis of miRNA is done through 2 different pathways which are the canonical pathway and the non-canonical pathway. The former pathway would involve pri-MiRNA synthesis from the genes, then this pri-MiRNA would be converted into preMiRNA through ribonucleotide III (Drosha) and DGCR8 (RNA binding protein DiGeorge Syndrome Critical Region 8). The pre-MiRNA is then transported to the cytoplasm through the exportin 5/RanGTP complex, where the terminal loop would be removed by Dicer forming the mature MiRNA duplex, this would then be loaded onto the argonaute proteins (AGO). On the other hand, the non-canonical pathways are grouped into the Drosha/DGCR8 independent and Dicer independent processes (19). page 80

MiRISC (minimal RNA induced silencing complex) would interact with MRE (MiRNA response elements) which are found on the mRNA. When the mRNA and MRE fully match there would be AGO2 mediated splicing of the mRNA. When the miRNA binds to the 5’UTR on the mRNA it would suppress gene expression and when binding to the promoter region on the mRNA it would promote transcription (19). The miRNAs have been linked to several cardiovascular diseases such as hypertension, heart failure, ischemic heart disease and left ventricular hypertrophy. MiRNA plays an important part in these diseases mentioned because it contributes to cardiac remodelling, therefore fibrosis and hypertrophy of myocytes can lead to the activation of this remodelling process (20).

Recently miRNAs are also being considered to be used as a biomarker for the detection of aortic dissection or even detect a predisposing risk of people who are at risk of developing an aortic dissection such as people with hypertension. Apart from having the aforementioned benefit it also has a higher specificity than biomarkers that are currently being used such as D-dimer. The use of this biomarker would limit the error by which people are misdiagnosed which currently stands at around 25-50% due to offering different miRNAs which helps diagnosis of diseases such as myocardial infarction as opposed to aortic dissection (21). Different miRNAs would have different biological pathways in which they play a role. The miRNA-15a is upregulated in diseases such as heart failure, myocardial ischemia and myocardial reperfusion injury (22). This miRNA has a specificity of 100% and therefore this can be used to track the progress of aortic dissection and also screen people which are prone to have the disease (23). MiRNA-23a is significantly important in cardiac development, myogenesis and hypertrophy, this would increase in cases of aortic dissection (23,24). This same miRNA has increased sensitivity and therefore can differentiate chest pain due to an aortic dissection or without an aortic dissection. Furthermore, it can also be used to monitor any suspected acute aortic dissections.

Studies also investigated the possibility of using the 4miRNAs which are miR-25, miR-29a, miR-155 and miR-26b and using them as a set to diagnose an acute aortic dissection. When combining these different types of miRNAs are tested together this would lead to a specificity of 100%, whilst the currently used biomarker D-dimer has a specificity between 96-60%. MiR-15a has the highest specificity (100%) and therefore this could be used as a prognostic marker, therefore this could be used to track the progress of an acute aortic dissection. Furthermore, this biomarker could be used to diagnose patients which have a subacute aortic dissection. MiR-23a has the highest sensitivity (91.9%), therefore this could be used to differentiate between chest pain which might not be the cause of aortic dissection and monitor an acute aortic dissection (20,23). page 81

The use of these accurate MiRNA serum tests would be less invasive than transesophageal echocardiography which is currently used for the diagnosis of an aortic dissection. Furthermore, the use of serum tests would be less costly to conduct than CT scans which are used for the confirmation of an aortic dissection. Even Though, MiRNAs are very promising in what they offer to the field of medicine there still needs to be further tests done to assess the viability of these miRNAs. Further studies need to be deployed to find the best combination of miRNAs that can be used in tandem (19,20).

MiRNAs have a longer time window than D-dimer, therefore an aortic dissection could be diagnosed before treatment and surgery are too late for an option. Furthermore, serumbased tests would be less invasive than transesophageal echocardiography or CT scans which are more costly to conduct. However, despite all these advantages further experimentation needs to be carried out to test the viability and prognosis of aortic dissections as only limited research was done till now (19,20).

The importance of developing specific diagnostic biomarkers or rapid testing systems is to expedite the process of diagnosing and treating acute AD patients as soon as possible. Similarly, the development of new treatments or medical interventions with the goal of halting the growth of aneurysms and lowering the risks of acute AD results in a better prognosis for patients diagnosed with AA. The creation of such potential therapeutic agents or interventions does, in fact, provide longevity. Indeed, acute and chronic aortic conditions rely primarily on imaging modalities, and the likelihood that imaging will continue to be the primary tool in assessing such patients is high. Many studies, however, agree that studies of serum biomarkers to establish and monitor aortic diseases could be combined with current imaging tools to improve and advance diagnostic precision and patient management overall.

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(1) Morello F, Piler P, Novak M, Kruzliak P. Biomarkers for diagnosis and prognosticstratification of aortic dissection: challenges and perspectives. Biomarkers in medicine 2014;8(7):931-941. (2) Toghill BJ, Saratzis A, Bown MJ. Abdominal aortic aneurysm—an independent disease to atherosclerosis? Cardiovascular Pathology 2017;27:71-75. (3) Mussa FF, Horton JD, Moridzadeh R, Nicholson J, Trimarchi S, Eagle KA. Acute aortic dissection and intramural hematoma: a systematic review. JAMA 2016;316(7):754-763. (4) Cui J, Jing Z, Zhuang S, Qi S, Li L, Zhou J, et al. D-dimer as a biomarker for acute aortic dissection: a systematic review and meta-analysis. Medicine 2015;94(4). (5) Suzuki T, Distante A, Eagle K. Biomarker-assisted diagnosis of acute aortic dissection: how far we have come and what to expect. Curr Opin Cardiol 2010;25(6):541-545. (6) Giannitsis E, Mair J, Christersson C, Siegbahn A, Huber K, Jaffe AS, et al. How to use Ddimer in acute cardiovascular care. European Heart Journal: Acute Cardiovascular Care 2017;6(1):69-80. (7) D-dimer understand the test. (8) Adam SS, Key NS, Greenberg CS. D-dimer antigen: current concepts and future prospects. Blood, The Journal of the American Society of Hematology 2009;113(13):2878-2887. (9) LaPelusa A, Dave HD. Physiology, Hemostasis. 2019. (10) Tsai TT, Trimarchi S, Nienaber CA. Acute aortic dissection: perspectives from the International Registry of Acute Aortic Dissection (IRAD). European journal of vascular and endovascular surgery 2009;37(2):149-159. (11) Ray P, Manach YL, Riou B, Houle TT, Warner DS. Statistical evaluation of a biomarker. The Journal of the American Society of Anesthesiologists 2010;112(4):1023-1040. (12) Evaluating Screening Tests. (13) Nazerian P, Mueller C, Soeiro AdM, Leidel BA, Salvadeo SAT, Giachino F, et al. Diagnostic accuracy of the aortic dissection detection risk score plus D-dimer for acute aortic syndromes: the ADvISED Prospective Multicenter Study. Circulation 2018;137(3):250-258. (14) Nitta K, Imamura H, Kashima Y, Kamijo H, Ichikawa M, Okada M, et al. Impact of a negative D-dimer result on the initial assessment of acute aortic dissection. Int J Cardiol 2018;258:232-236.

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(15) Itagaki R, Kimura N, Mieno M, Hori D, Itoh S, Akiyoshi K, et al. Characteristics and Treatment Outcomes of Acute Type AAortic Dissection With Elevated D‐Dimer Concentration. Journal of the American Heart Association 2018;7(14):e009144. (16) Nazerian P, Mueller C, Soeiro AdM, Leidel BA, Salvadeo SAT, Giachino F, et al. Diagnostic accuracy of the aortic dissection detection risk score plus D-dimer for acute aortic syndromes: the ADvISED Prospective Multicenter Study. Circulation 2018;137(3):250-258. (17) Xiao Z, Xue Y, Yao C, Gu G, Zhang Y, Zhang J, et al. Acute aortic dissection biomarkers identified using isobaric tags for relative and absolute quantitation. BioMed research international 2016;2016. (18) Mori K, Tamune H, Tanaka H, Nakamura M. Admission values of D-dimer and C-reactive protein (CRP) predict the long-term outcomes in acute aortic dissection. Internal Medicine 2016;55(14):1837-1843. (19) O'Brien J, Hayder H, Zayed Y, Peng C. Overview of microRNA biogenesis, mechanisms of actions, and circulation. Frontiers in endocrinology 2018;9:402. (20) Xu Z, Wang Q, Pan J, Sheng X, Hou D, Chong H, et al. Characterization of serum miRNAs as molecular biomarkers for acute Stanford type A aortic dissection diagnosis. Scientific reports 2017;7(1):1-11. (21) Romaine SP, Tomaszewski M, Condorelli G, Samani NJ. MicroRNAs in cardiovascular disease: an introduction for clinicians. Heart 2015;101(12):921-928. (22) Liu L, Liang Z, Lv Z, Liu X, Bai J, Chen J, et al. MicroRNA-15a/b are up-regulated in response to myocardial ischemia/reperfusion injury. Journal of geriatric cardiology: JGC 2012;9(1):28. (23) Dong J, Bao J, Feng R, Zhao Z, Lu Q, Wang G, et al. Circulating microRNAs: a novel potential biomarker for diagnosing acute aortic dissection. Scientific Reports 2017;7(1):1-11. (24) Wang S, He W, Wang C. MiR‐23a Regulates the vasculogenesis of coronary artery disease by targeting epidermal growth factor receptor. Cardiovascular therapeutics 2016;34(4):199208.

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How does your body become your enemy and an eating disorder, your best friend?

The term body image encompasses a wide range of beliefs held by an individual regarding what an ‘ideal body’ should look like in terms of physical appeal. This encompasses what one believes to be the ideal height, weight, shape, muscularity and sexual attractiveness, amongst other features. They summate to create an internalized perception of one’s actual body in comparison to their vision of the ideal body. This perception in turn influences one’s behaviour and may encourage body-modulatory behaviours.

Having a positive body image refers to one being content with their own body and appreciating both its appearance and physical capabilities. Negative body image, or body dissatisfaction on the contrary is when one directs a strong sense of frustration and disliking towards their appearance. It is important to note body image is not absolute. A person throughout their lives can experience both opposite ends of the spectrum and this is not associated with harmful effects in a normal context. However, prolonged periods of body dissatisfaction can severely dampen one’s quality of life and cause both physical and psychological harm (1). Many believe body dissatisfaction is present mostly in females and while it is true that statistically more females tend to adopt a negative body image when compared to males, men and boys are increasingly becoming affected by body dissatisfaction mostly due to the beliefs tied to not being lean and muscular enough. A population study in 2016 in fact found that while less men are affected by body dissatisfaction, the negative impacts relating to psychological distress and quality of life were more striking in males when compared to females (2). With regard to age group, body dissatisfaction is mostly prevalent in adolescents which is due to a multitude of internal and external factors. Among external factors, we find that social media platforms like Instagram have an important role in setting the stage for unrealistic body types which are many times lean bodies with very small waists and pronounced curves (in the case of females) or lean and muscular (mostly in the case of males). They are made to look ‘picture perfect’ within the beliefs of Western society. Many tend to compare themselves with photos of celebrities or even with their own peers, who frequently manipulate their photos to resemble society’s ‘ideal’ body type. Direct links between body image and social media were demonstrated in a study where adolescent page 85

girls were shown photos of themselves which were retouched and reshaped using Instagram filters. It was found that the majority of girls experienced a lower body image after being shown their own modified pictures. This shows what a crucial affect society’s bombardment of ‘ideal body types’ has on the general public (3).

As previously mentioned, the subjective view on one’s body influences behaviours linked to body modification, including dietary intake and exercise. Within the normal range this may sometimes be beneficial in motivating an overweight or obese individual to lead a healthier lifestyle. However, having a negative body image may contribute to bad lifestyle habits which can develop into psychological disorders known as eating disorders. Eating disorders (EDs) are classified by the DSM V as being “persistent disturbance of eating or eating-related behaviour that results in the altered consumption or absorption of food and that significantly impairs physical health or psychosocial functioning”. The more common examples of EDs include anorexia nervosa, which signifies abnormally low body weight, excessive food restriction and a pathological fear of weight gain, and bulimia nervosa which entails recurrent episodes of binge eating (consumption of excessive quantities of food) followed by compensatory behaviours such as purging, consuming laxatives and excessive exercise. A diagnostic criterion for both aforementioned disorders includes body dissatisfaction triggering such symptoms (4). Globally EDs affect roughly 9% of the population with 10,200 people dying annually due to their condition (5). In the UK as many as 1.6 million individuals suffer from EDs and locally in Malta, a study in 2020 identified up to 1675 youths between the ages of 10-16 years affected by such disorders (6). EDs are of medical relevance due to causing mortality and wide-range morbidities (7). The incidence rate of EDs is likely far higher than what statistics show as many individuals with EDs go undiagnosed. As for morbidities, EDs cause a wide range which include both increased risk for other psychological disorders, the most common being personality disorders, depressive disorders and alcoholism (7), as well as physiological abnormalities relating to hormone imbalances caused by malnutrition and stress, including amenorrhea or irregular menstrual cycles in females and ionic imbalances like hypokalemia and alkalosis. Since adolescence is a time of growth and development, EDs negatively impact puberty and may cause delayed or stunted growth, problems in bone mass accretion and bone loss, hair loss, dry skin and in more severe cases, organ damage (8). Essentially, since EDs may be sparked from a desire to improve one’s body image, EDs can be the product of an extreme diet gone wrong and it may be difficult for the individual to admit they may have a problem. This is especially true for less ‘outwardly visible’ EDs such as bulimia nervosa where the person’s BMI is commonly normal or overweight. A person suffering from an ED may experience shame and may want to hide the disorder to the best of their abilities, making this very hard for parents/relatives to spot or for the medical professional to diagnose especially early on. Amongst the first noticeable signs of EDs, parents report noticing their child eating less, taking more frequent trips to the bathroom page 86

especially after a meal, and excessively exercising to the point where they are too lethargic to do their usual daily tasks (9) Frequently people suffering an ED will tend to experience denial and will in fact be reluctant to seek treatment in fear that treating this problem will ultimately result in the person ‘letting go’ and eating in excess leading to weight gain. This pathological fear of weight gain results in reinforcement of ED behaviours which leaves the individual stuck in a vicious cycle and can be devastating to the individual’s relatives and loved ones (9). Moreover, eating disorders are maintained by giving the person a sense of control and effectiveness achieved when a person restricts the amount of food they eat, and this may be done as compensation for a sense of lack of control experienced in the individual’s personal life. Treatment is something such individuals may fear and avoid due to an overwhelming fear of loss of control. Studies in fact found that many start dieting during a stressful and chaotic period in their lifetime (10).

Working on ameliorating one’s body dissatisfaction is an integral part in treating and even preventing EDs, and is also important in preventing relapse, since body image concerns fuel these disorders. If improving one’s body image is not included as one of the therapies, other therapies may prove to be very distressing for the individual and actually increase risk of depressive moods (1). Different therapeutic strategies to tackle body dissatisfaction include the following: 1. Developing coping strategies to deal with weight conversations and possible teasing by peers, which may take the form of role plays and self-reflection. 2. Improving Social media literacy, meaning opening one’s eyes to the unrealistic body standards social media content displays. 3. Cognitive dissonance, a tactic in which the individual challenges their own views and irrational beliefs on what constitutes an attractive body. 4. Behavioural interventions focused on reducing body avoidance through gradual exposure to mirrors, and self-photos and videos. 5. Modifying and preventing negative responses associated with body checking behaviours such as weighing oneself during therapy. 6. Preventing self-depreciation targeted towards one’s body and instead focusing on promoting positive body experiences through strategies like self-compassion therapy. 7. Encouraging self-care directed behaviours.

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In conclusion, one’s internalized perception of themselves plays a central role in their psychological and ultimately in their physical health. Excessive and prolonged concern over one’s body image may spark eating disorders which carry their own extensive set of morbidities. It is important that friends, parents and other relatives of people suffering eating disorders are sensitive towards their problem. All too frequently, parents can engage in blaming behaviours towards themselves or their children and shame their children for their behaviours. This only worsens the problem as the individual feels a sense of shame and guilt that plays a huge role in the amplification of behaviours linked to such disorders. This therefore makes them more reluctant to seek help. It is also important that practicing physicians recognise certain warning signs a patient may show during consultations and tackle such warning signs in a sensitive and compassionate manner. Lastly, it is important to encourage body positivity and following a healthy lifestyle rather than engaging in restrictive diets which may be harmful. Ultimately a healthy lifestyle and satisfaction with one’s appearance is the key to improving overall physical, social and psychological well- being.

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1. McLean SA, Paxton SJ. Body image in the context of eating disorders. Psychiatr Clin North Am. 2019;42(1):145–56. 2. Griffiths S, Hay P, Mitchison D, Mond JM, McLean SA, Rodgers B, et al. Sex differences in the relationships between body dissatisfaction, quality of life and psychological distress. Aust N Z J Public Health. 2016 Dec;40(6):518–22. 3. Kleemans M, Daalmans S, Carbaat I, Anschütz D. Picture perfect: the direct effect of manipulated instagram photos on body image in adolescent girls. Media Psychol. 2016 Dec 15;21(1):1–18. 4. American Psychiatric Association. DSM-5 Diagnostic Classification. Diagnostic and statistical manual of mental disorders. American Psychiatric Association; 2013. 5. DA Economics. The social and economic cost of eating disorders in the United States of America: a report for the strategic training initiative for the prevention of eating disorders and the academy for eating disorders. 2020; 6. As many as 1,675 youngsters in Malta may be affected by an eating disorder Newspoint - University of Malta [Internet]. [cited 2021 Apr 15]. Available from: https://www.um.edu.mt/newspoint/news/2021/01/eating-disorder-study-fsw 7. Demmler JC, Brophy ST, Marchant A, John A, Tan JOA. Shining the light on eating disorders, incidence, prognosis and profiling of patients in primary and secondary care: national data linkage study. Br J Psychiatry. 2020;216(2):105–12. 8. Rome ES, Ammerman S. Medical complications of eating disorders: an update. J Adolesc Health. 2003 Dec;33(6):418–26. 9. Lock J, Grange DL. Help your teenager beat an eating disorder. Guilford Publications; 2015. 10. Froreich FV, Vartanian LR, Grisham JR, Touyz SW. Dimensions of control and their relation to disordered eating behaviours and obsessive-compulsive symptoms. J Eat Disord. 2016 May 3;4:14.

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