Neuroprotection
Method and Protocols
Edited by Swapan K. Ray
Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC, USA
Editor
Swapan K. Ray
Department of Pathology, Microbiology, and Immunology
University of South Carolina School of Medicine
Columbia, SC, USA
ISSN 1064-3745ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-0716-3661-9ISBN 978-1-0716-3662-6 (eBook) https://doi.org/10.1007/978-1-0716-3662-6
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Preface
Neuroprotection to the diseases and injuries of the central nervous system (CNS), which includes brain and spinal cord, remains a formidable challenge. All the major CNS diseases and injuries after the onset are ongoing, difficult to control with only symptomatic treatments, regrettably incurable, and thus harbinger of early death. It is important that we know and use the contemporary and appropriate methods for understanding the histological, cellular, and molecular mechanisms of both the neuropathogenesis and neuroprotection in preclinical models of the major CNS diseases and injuries to translate this knowledge to the clinical settings. The good news is that we at the present are gaining better understanding than the past of the complex histological, cellular, and molecular mechanisms of neuropathogenesis as well as of neuroprotection in preclinical models of the most visible CNS diseases and injuries due to the advancements in our ability to design creative molecular biology methodology with the advent of the state-of-the-art technology. This volume of the Methods in Molecular Biology (MMB) on “Neuroprotection” is a collection of the cuttingedge chapters mostly on modern protocols (methods) and a few on comprehensive conceptions (reviews) aimed primarily at graduate students, medical students, postdoctoral fellows, and new principal investigators for providing them with opportunities for exploring neurodegenerative and neuroprotective mechanisms in a large number of preclinical models or some clinical samples of the prominent CNS diseases and injuries for discovering and recommending the best drugs and devices to this point for exploring neuroprotection in humans.
This volume of MMB on “Neuroprotection” is broadly organized with its chapters in three categories in this thematic order: chapters that describe methods (Chapters 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) and some reviews (Chapters 13, 14, and 15) for understanding neuropathology and neuroprotection in the CNS diseases and injuries as a whole; chapters that describe mostly methods and some reviews on neuropathology and neuroprotection in specific CNS diseases in an alphabetic order such as Alzheimer’s disease or AD (Chapters 16, 17, 18, 19, 20, 21, 22, 23, and 24 on methods and Chapters 25 and 26 on reviews), amyotrophic lateral sclerosis (no muscle nourishment in spinal cord areas due to scarring) or ALS (Chapters 27 and 28 on methods), Huntington’s disease or HD (Chapter 29 on method), multiple sclerosis or MS (Chapters 30 and 31 on reviews), and Parkinson’s disease or PD (Chapters 32, 33, 34, and 35 on methods and Chapter 36 on review); and chapters that describe examining and understanding neuropathology and neuroprotection in specific CNS injuries such as spinal cord injury or SCI (Chapter 37 on method), traumatic brain injury or TBI (Chapters 38 and 39 on methods and Chapter 40 on review), and ischemic brain injury or just ischemia (Chapter 41 on method). All chapters are carefully edited and thoroughly revised (in some cases repeatedly) for significant improvements and bringing them to the acceptable final forms. Altogether 41 chapters in this volume will give our readers an ample opportunity to gain expertise in conducting various protocols and expand knowledge on neuropathology and neuroprotection in the major CNS diseases and injuries. Each method chapter in this volume of MMB on “Neuroprotection” contains an Abstract followed by four distinct sections: (1) Introduction, (2) Materials, (3) Methods, and (4) Notes. The “Abstract” is a summary of interesting laboratory technique(s) for revealing mechanisms of neurodegeneration and neuroprotection in a CNS disease or injury.
(1)“Introduction”describes the background information, principle of the procedure with referencestotheworkof the author(s) and other investigators in the field, outlines the plan formajoraspectsofthemethod(s), and briefly states results to show feasibility and validity of theprocedures.(2)“Materials” is an essential part of the protocol chapter describing the maincomponents,chemicals, biochemicals, buffers, solutions, supplies, and major equipmentneededtoconduct the procedures successfully. This section also discreetly declares the specificrequirementsfor storage, stability, purity, temperature and light sensitivity, harmful effectsofanyreagentsand solutions, treatment, protection, and disposal of biological and chemicalwastes.(3)“Methods” is the next section that systematically and categorically describeshowtoexecute the individual steps in the protocol and achieve results, points out detailsofthepractical pointers, and clarifies logical aspects of these steps in a proper arrangement.(4)“Notes” can be a significant savior to any novice user of the protocol as thissectionopenlypoints out the possible sources of the problems and disappointments, findsreasonablesolutions to the problems, and clearly provides pointers to detect and defeat thedifficultiestoattain success with the protocol.
As I mentioned above, a few review chapters are also included in this volume of MMB on “Neuroprotection” to provide our readers with current conceptions on the specific topics in some of the major CNS diseases and injuries. Each review chapter in general contains Abstract, Introduction, Subtitles (focused on specific parts of the subject matter), and Conclusions (with future directions).
The major CNS diseases and injuries are colloquially called neurological disorders or neurodegenerative disorders, all of which are associated with degeneration of the neural cells (neurons and glia) at varying degrees causing deficiencies in neurological functions and behaviors. To inspire beginners in the neuropathology and neuroprotection field with the simplest pointers, each of the major CNS diseases and injuries (with chapters in this volume) is briefly defined below in just one sentence.
AD named after the German neuropathologist Alois Alzheimer is a neurodegenerative brain disorder manifested with formation of abnormal amyloid plaques and neurofibrillary tangles, killing nerve cells first in the hippocampus and later in the cerebral cortex, causing loss of connections between the nerve cells, gradually wiping out memory and thinking skills, and eventually causing dementia or obliterating the cognitive functioning of the AD patients to deter to carry out the easiest tasks. ALS described first by the French neurologist Jean-Martin Charcot is also called Lou Gehring’s disease or motoneuron diseases (MND) as it is a neurodegenerative spinal cord disorder that primarily and increasingly kills motoneurons with generally no known cause (sporadic) or rarely a genetic cause (hereditary) such as a mutation in the gene encoding the antioxidant enzyme superoxide dismutase 1 (SOD1), leading to loss of voluntary (intentional) muscle movement and paralysis. HD named after the American physician George Huntington is an inherited neurodegenerative brain disorder that causes neurons in some parts of the brain to die due to presence of the mutant huntingtin protein with a repeat of many glutamine residues (36 or more) resulting from its defective gene with cytosine, adenine, and guanine (CAG) repeat many more times than normal huntingtin gene (the function of which is essential for development and vesicle trafficking in a cell), impairing voluntary movement of the HD patients manifested with unrestrained dance-like movements (chorea), abnormal body postures, tremors, rigidity, slow or abnormal eye movements, impaired gait and balance, difficulty with speech and swallowing, and problems with emotion, thinking, and personality. MS, which is first welldefined as “scle ´ rose en plaques” (the French name of MS) on the white matter of brain and spinal cord by again the French neurologist Jean-Martin Charcot, is the most common
neurologicaldisorderthat predominantly affects women, and it is now widely recognized as anautoimmunedemyelinating and neurodegenerative disease in which the myelin and myelin-producingoligodendrocytes become the targets of T cell-mediated autoimmune response,resultingindepletion of the white matter, progressive axonal damage, neurodegeneration,andlossofneuronal function. PD, which is first described as a “shaking palsy” bytheEnglishphysician James Parkinson, is an ongoing neurodegenerative movement disorderoftheCNS,especially of the brain with loss of its substantia nigra neurons that producedopamine(aneurotransmitter required by the dopaminergic neurons for regulatingmotorfunction,motivation, and sexual arousal), leading to onset of tremor, muscle rigidity,slownessinmovement (bradykinesia), stiffness in the limbs or the trunk of the body, impairedbalance,lossof smell, sleep dysfunction, mood disorders, excess salivation, and constipation.
SCI is a devastating and progressive neurological disorder that mostly affects young people when they experience a primary injury in any part of the spinal cord due to car accident, fall, sports mishap, or violent event. TBI, which is also an advancing neurological disorder, is defined by neuropathogenesis in the brain and deficiency in the brain functions following an external traumatic impact injury. Ischemic brain injury or ischemia is a neurological disorder that occurs if the blood flow to any part of the brain is suppressed or interrupted by a buildup of plaques (atherosclerosis), preventing brain tissue in that area from receiving oxygen and nutrients, triggering degeneration of the brain tissue, and causing neurological problems and paralysis.
None of the major CNS diseases and injuries are yet curable. Therefore, we must continue to use available modern methods and innovative ideas to understand their neuropathology at greater depths to figure out the most promising targets for neuroprotection. All the chapters presented in this volume of the MMB are expected to be highly useful to the beginners as well as to the new and seasoned investigators in the field of CNS diseases and injuries for designing and performing their experiments, gaining novel insights, and making new contributions to the field for the benefits of the future generations.
Finally, I would like to thank all the authors from around the globe for contributing their thoughtful method chapters and thought-stimulating review chapters on major CNS diseases and injuries to this volume of MMB on “Neuroprotection” for helping other researchers, especially new investigators, stay interested and involved in this complex and ever-intriguing field and continue to carry out groundbreaking research. I would also like to thank the MMB series Editor, Dr. John M. Walker, who is a Professor Emeritus at University of Hertfordshire in the United Kingdom, for emboldening me for pursuing this lengthy mega project and taking this volume to the finish line for its final production by the Springer Nature. Additionally, my special thanks go to the Associate Editor, Anna Rakovsky, and all other Editorial Staffs at the Springer Nature for their dedication and diligence in publishing this elegant volume that we all are proud of.
Columbia, SC, USA
Swapan K. Ray
1 TUNEL-n-DIFL Method for Detection and Estimation of Apoptosis
Specifically in Neurons and Glial Cells in Mixed Culture and Animal Models of Central Nervous System Diseases and Injuries 1 Swapan K. Ray
2 Isolation of Capillaries from Small Amounts of Mouse Brain Tissue 27
Junqiao Mi, Aili Sun, Laura Hartel, Christina Dilling, Patrick Meybohm, and Malgorzata Burek
3 Isolation of Extracellular Vesicles Using Formulas to Adapt Centrifugation to Different Centrifuges
Ramon Handerson Gomes Teles, Daniela Engelmayr, Patrick Meybohm, and Malgorzata Burek
4 High-Resolution Respirometry for Mitochondrial Function in Rodent Brain
Aishika Datta, Deepaneeta Sarmah, Bijoyani Ghosh, Nikita Rana, Anupom Borah, and Pallab Bhattacharya
5 Quantification of Neuronal Dendritic Spine Density and Lengths of Apical and Basal Dendrites in Temporal Lobe Structures Using Golgi-Cox Staining
Vivek Dubey, Aparna Banerjee Dixit, Manjari Tripathi, P. Sarat Chandra, and Jyotirmoy Banerjee
6 Quantification of Neuroinflammatory Markers in Blood, Cerebrospinal Fluid, and Resected Brain Samples Obtained from Patients 67
Arpna Srivastava, Aparna Banerjee Dixit, Manjari Tripathi, P. Sarat Chandra, and Jyotirmoy Banerjee
7 Targeted Modification of Epigenetic Marks Using CRISPR/dCas9-SunTag-Based Modular Epigenetic Toolkit
Min Kyung Song and Yoon-Seong Kim
8 Elevated Plus Maze for Assessment of Anxiety and Memory in Rodents .
Ravi Chandra Sekhara Reddy Danduga and Phani Kumar Kola
81
93
9 Drosophila melanogaster Neuromuscular Junction as a Model to Study Synaptopathies and Neuronal Autophagy 97
Anushka Chakravorty, Vasu Sheeba, and Ravi Manjithaya
10 Cell-Based Assay to Detect the Autoantibody Serostatus in Patients with Neuromyelitis Optica Spectrum Disorder (NMOSD) 121 Pallavi Chatterjee, Suparna Saha, and Debashis Mukhopadhyay ix
11 Using Small Molecules for Targeting Heavy Metals in Neurotoxicity and Neuroinflammation .
Pronama Biswas and Sunil S. More
12 Chromatographic Separation and Quantitation of Sphingolipids from the Central Nervous System or Any Other Biological Tissue 149
Swapan K. Ray and Somsankar Dasgupta
13 Role of Network Pharmacology in Prediction of Mechanism of Neuroprotective Compounds 159
Saima, S. Latha, Ruchika Sharma, and Anoop Kumar
14 Role of Serotonergic System in Regulating Brain Tumor-Associated Neuroinflammatory Responses .
Surojit Karmakar and Girdhari Lal
15 Impacts of Omega-3 Fatty Acids, Natural Elixirs for Neuronal Health, on Brain Development and Functions 209 Archana S. Rao, Ajay Nair, K. Nivetha, Bibi Ayesha, Kapadia Hardi, Vora Divya, S. M. Veena, K. S. Anantharaju, and Sunil S. More
16 Microglial Uptake of Extracellular Tau by Actin-Mediated Phagocytosis . . . . . . . 231
Hariharakrishnan Chidambaram, Smita Eknath Desale, Tazeen Qureshi, and Subashchandrabose Chinnathambi
17 Internalization and Endosomal Trafficking of Extracellular Tau in Microglia Improved by α-Linolenic Acid
Smita Eknath Desale, Hariharakrishnan Chidambaram, Tazeen Qureshi, and Subashchandrabose Chinnathambi
18 Understanding Actin Remodeling in Neuronal Cells Through Podosomes 257 Tazeen Qureshi, Smita Eknath Desale, Hariharakrishnan Chidambaram, and Subashchandrabose Chinnathambi
19 Quantitative Investigation of Neuroprotective Role of ROR1 in a Cell Culture Model of Alzheimer’s Disease
Kaushik Chanda and Debashis Mukhopadhyay
20 microRNA Isolation, Expression Profiling, and Target Identification for Neuroprotection in Alzheimer’s Disease
Saleem Iqbal and Debnath Pal
21 Quantitative Measurement of Tau Aggregation in Genetically Modified Rats with Neurodegeneration.
YouJin Lee and Eric M. Morrow
22 Detection and Characterization of Apoptosis-Related Proteins in Hippocampal Neurodegeneration: From mRNA Expression to Protein Quantification
Kajal Rawat, Vipasha Gautam, Arushi Sandhu, and Lekha Saha
23 Isolation and Detection of Pathological Tau Species in a Tauopathy Mouse Model 317
Abhay Kumar Singh, Karthikeyan Selvarasu, and Siva Sundara Kumar Durairajan
24 Enzyme Inhibition Assays for Monoamine Oxidase
329 Bijo Mathew, Jong Min Oh, Della Grace Thomas Parambi, Sachithra Thazhathuveedu Sudevan, Sunil Kumar, and Hoon Kim
25 Role of Amyloid Beta in Neurodegeneration and Therapeutic Strategies for Neuroprotection. .
Priyam Ghosh, Kavita Narang, and Parameswar Krishnan Iyer
26 Amyloid Beta–Mediated Neurovascular Toxicity in Alzheimer’s Disease . . . . . . . 355 Sayani Banerjee and Sugato Banerjee
27 Fecal Microbiota Transplantation in Amyotrophic Lateral Sclerosis: Clinical Protocol and Evaluation of Microbiota Immunity Axis
Elena Niccolai, Ilaria Martinelli, Gianluca Quaranta, Giulia Nannini, Elisabetta Zucchi, Flavio De Maio, Giulia Gianferrari, Stefano Bibbo ` , Giovanni Cammarota, Jessica Mandrioli, Luca Masucci, and Amedeo Amedei
28 Integrated Multi-Omics Analysis and Validation in Yeast Model of Amyotrophic Lateral Sclerosis 397 Saiswaroop Rajaratnam, Sai Sanwid Pradhan, Ashwin Ashok Naik, and Venketesh Sivaramakrishnan
29 Imaging and Assay of the Dynamics of Cytotoxic Huntingtin (HTT) Protein Aggregates Regulated by lncRNAs 421 Kaushik Chanda and Debashis Mukhopadhyay
30 Astrocyte Activation and Drug Target in Pathophysiology of Multiple Sclerosis
Preeti Bisht, Charul Rathore, Ankit Rathee, and Atul Kabra
31 Promises of Lipid-Based Nanocarriers for Delivery of Dimethyl Fumarate to Multiple Sclerosis Brain
Sreya Subhash, Nishtha Chaurawal, and Kaisar Raza
32 Chrysin for Neurotrophic and Neurotransmitter Balance in Parkinson’s Disease 477
Alagudurai Krishnamoorthy, Riddhi Upadhyay, and Murugan Sevanan
33 Establishment of a 6-OHDA Induced Unilaterally Lesioned Male Wistar Rat Model of Parkinson’s Disease
Namrata Kumari and Pratibha Mehta Luthra
34 Evaluating Motor Dysfunction and Oxidative Stress Induced by Trichloroethylene in Wistar Rats 499 Rajnish Srivastava, Kanupriya Chauhan, and Ramaish Sharma
35 Neurobehavioral Analysis to Assess Olfactory and Motor Dysfunction in Parkinson’s Disease
Samir Ranjan Panda, Pallabi Panja, Ujjawal Soni, and V. G. M. Naidu
36 Ion Channels and Metal Ions in Parkinson’s Disease: Historical Perspective to the Current Scenario
Bhupesh Vaidya, Dibya S. Padhy, Hem C. Joshi, Shyam S. Sharma, and Jitendra Narain Singh
37 Creating a Reproducible Model of Spinal Cord Injury in Rats: A Contusion Approach
Syed Shadab Raza
38 Weight-Drop Method for Inducing Closed Head Diffuse Traumatic Brain Injury 569
Bhagawati Saxena, Bhavna Bohra, and Krishna A. Lad
39 iDISCO Tissue Clearing Whole-Brain and Light Sheet Microscopy for High-Throughput Imaging in a Mouse Model of Traumatic Brain Injury
Hannah Flinn, Leonardo Cruz-Pineda, Laura Montier, Philip J. Horner, and Sonia Villapol
40 Insights from Rodent Models for Improving Bench-to-Bedside Translation in Traumatic Brain Injury
Tulasi Pasam and Manoj P. Dandekar
41 Rat Model of Middle Cerebral Artery Occlusion
Syed Shadab Raza Index
589
623
Contributors
AMEDEO AMEDEI • Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy; Internal Interdisciplinary Medicine Unit, Careggi University Hospital, Florence, Italy
K. S. ANANTHARAJU • Department of Chemistry, Dayananda Sagar College of Engineering, Bangalore, India
BIBI AYESHA • School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India
JYOTIRMOY BANERJEE • Department of Biophysics, AIIMS, New Delhi, India
SAYANI BANERJEE • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Kolkata, India
SUGATO BANERJEE • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Kolkata, India
PALLAB BHATTACHARYA • Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, India
STEFANO BIBBO ` • Digestive Disease Center, A. Gemelli University Hospital IRCCS, Catholic University of Sacred Heart, Rome, Italy
PREETI BISHT • University Institute of Pharma Sciences, Chandigarh University, Ajitgarh, Punjab, India
PRONAMA BISWAS • School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, Karnataka, India
BHAVNA BOHRA • Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad, India
ANUPOM BORAH • Cellular and Molecular Neurobiology Laboratory, Department of Life Science and Bioinformatics, Assam University, Silchar, Assam, India
MALGORZATA BUREK • Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, University Hospital Wu¨rzburg, Wurzburg, Germany
GIOVANNI CAMMAROTA • Digestive Disease Center, A. Gemelli University Hospital IRCCS, Catholic University of Sacred Heart, Rome, Italy
ANUSHKA CHAKRAVORTY • Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India
KAUSHIK CHANDA • Department of Neuroscience, UF Scripps Biomedical Research, Jupiter, FL, USA
P. SARAT CHANDRA • Department of Neurosurgery, AIIMS, New Delhi, India
PALLAVI CHATTERJEE • Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, A CI of Homi Bhabha National Institute, Kolkata, India
KANUPRIYA CHAUHAN • Moradabad Educational Trust Group of Institutions Faculty of Pharmacy, Moradabad, Uttar Pradesh, India
NISHTHA CHAURAWAL • Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Ajmer, Rajasthan, India
HARIHARAKRISHNAN CHIDAMBARAM • Neurobiology Group, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India; Department of Neurochemistry, National Institute
xiii
xivContributors
ofMentalHealthand Neuro Sciences (NIMHANS), Institute of National Importance, Bangalore,Karnataka, India
SUBASHCHANDRABOSE CHINNATHAMBI • Neurobiology Group, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India; Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences (NIMHANS), Institute of National Importance, Bangalore, Karnataka, India
LEONARDO CRUZ-PINEDA • Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Academic Institute, Houston, TX, USA
MANOJ P. DANDEKAR • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India
RAVI CHANDRA SEKHARA REDDY DANDUGA • Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai, India
SOMSANKAR DASGUPTA • Department of Neuroscience and Regenerative Medicine, Institute of Molecular Medicine and Genetics, Augusta University, Augusta, GA, USA
AISHIKA DATTA • Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, India
FLAVIO DE MAIO • Department of Laboratory and Infectious Sciences, A. Gemelli University Hospital IRCCS, Rome, Italy
SMITA EKNATH DESALE • Neurobiology Group, Division of Biochemical Sciences, CSIRNational Chemical Laboratory, Pune, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India; Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences (NIMHANS), Institute of National Importance, Bangalore, Karnataka, India
CHRISTINA DILLING • Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, University Hospital Wu¨rzburg, Wurzburg, Germany
VORA DIVYA • School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India
APARNA BANERJEE DIXIT • Dr B R Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India
VIVEK DUBEY • Department of Biophysics, AIIMS, New Delhi, India
SIVA SUNDARA KUMAR DURAIRAJAN • Molecular Mycology and Neurodegenerative Disease Research Laboratory, Department of Microbiology, Central University of Tamil Nadu, Thiruvarur, India
DANIELA ENGELMAYR • Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, University Hospital Wu¨rzburg, Wurzburg, Germany; Graduate School of Life Sciences, Julius-Maximilians-University Wu¨rzburg, Wurzburg, Germany
HANNAH FLINN • Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Academic Institute, Houston, TX, USA
VIPASHA GAUTAM • Department of Pharmacology, Research Block B, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
BIJOYANI GHOSH • Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, India
PRIYAM GHOSH • Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, Assam, India
GIULIA GIANFERRARI • Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
KAPADIA HARDI • School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India
LAURA HA RTEL • Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, University Hospital Wu¨rzburg, Wurzburg, Germany
PHILIP J. HORNER • Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Academic Institute, Houston, TX, USA
SALEEM IQBAL • Axe Molecular Endocrinology and Nephrology, CHUL Research Center and Laval University, Quebec City, QC, Canada
PARAMESWAR KRISHNAN IYER • Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, Assam, India; Center for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India; Jyoti and Bhupat Mehta School of Health Sciences and Technology, Indian Institute of Technology Guwahati, Guwahati, Assam, India
HEM C. JOSHI • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Mohali, Punjab, India
ATUL KABRA • University Institute of Pharma Sciences, Chandigarh University, Ajitgarh, Punjab, India
SUROJIT KARMAKAR • National Centre for Cell Science (NCCS), Ganeshkhind, Pune, Maharashtra, India
HOON KIM • Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon, Republic of Korea
YOON-SEONG KIM • RWJMS Institute for Neurological Therapeutics, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ, USA
PHANI KUMAR KOLA • Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA
ALAGUDURAI KRISHNAMOORTHY • International Institute of Biotechnology and Toxicology, Padappai, Chennai, India
ANOOP KUMAR • Department of Pharmacology, Delhi Pharmaceutical Science and Research University (DPSRU), New Delhi, India
NAMRATA KUMARI • Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India
SUNIL KUMAR • Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Sciences Campus, Kochi, India
KRISHNA A. LAD • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Ahmedabad, Gandhinagar, Gujarat, India
GIRDHARI LAL • National Centre for Cell Science (NCCS), Ganeshkhind, Pune, Maharashtra, India
S. LATHA • Department of Pharmacology, Delhi Pharmaceutical Science and Research University (DPSRU), New Delhi, India
YOUJIN LEE • Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA; Center for Translational Neuroscience, Carney Institute for Brain Science, and Brown Institute for Translational Science (BITS), Brown University, Providence, RI, USA
PRATIBHA MEHTA LUTHRA • Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India
JESSICA MANDRIOLI • Neurology Unit, Department of Neuroscience, Azienda Ospedaliero Universitaria di Modena, Modena, Italy; Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
RAVI MANJITHAYA • Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India; Neuroscience Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India; Chronobiology and Behavioural Neurogenetics Laboratory, Neuroscience Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India
ILARIA MARTINELLI • Neurology Unit, Department of Neuroscience, Azienda Ospedaliero Universitaria di Modena, Modena, Italy; Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena, Italy
LUCA MASUCCI • Department of Laboratory and Infectious Sciences, A. Gemelli University Hospital IRCCS, Rome, Italy; Department of Basic Biotechnological Sciences, Intensivological and Perioperative Clinics, Catholic University of Sacred Heart, Rome, Italy
BIJO MATHEW • Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Sciences Campus, Kochi, India
PATRICK MEYBOHM • Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, University Hospital Wu¨rzburg, Wurzburg, Germany
JUNQIAO MI • Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, University Hospital Wu¨rzburg, Wurzburg, Germany; Graduate School of Life Sciences, Julius-Maximilians-University Wu¨rzburg, Wurzburg, Germany
LAURA MONTIER • Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Academic Institute, Houston, TX, USA
SUNIL S. MORE • School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, Karnataka, India
ERIC M. MORROW • Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA; Center for Translational Neuroscience, Carney Institute for Brain Science, and Brown Institute for Translational Science (BITS), Brown University, Providence, RI, USA
DEBASHIS MUKHOPADHYAY • Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, A CI of Homi Bhabha National Institute, Kolkata, India
V. G. M. NAIDU • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, Assam, India
ASHWIN ASHOK NAIK • Disease Biology Lab, Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Anantapur, Andhra Pradesh, India
AJAY NAIR • School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India
GIULIA NANNINI • Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
KAVITA NARANG • Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, Assam, India
ELENA NICCOLAI • Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
K. NIVETHA • School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India
JONG MIN OH • Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon, Republic of Korea
DIBYA S. PADHY • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Mohali, Punjab, India
DEBNATH PAL • Department of Computational and Data Sciences, Indian Institute of Science, Bangalore, Karnataka, India
SAMIR RANJAN PANDA • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, Assam, India
PALLABI PANJA • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, Assam, India
DELLA GRACE THOMAS PARAMBI • College of Pharmacy, Department of Pharmaceutical Chemistry, Jouf University, Sakaka, Al Jowf, Saudi Arabia
TULASI PASAM • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India
SAI SANWID PRADHAN • Disease Biology Lab, Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Anantapur, Andhra Pradesh, India
GIANLUCA QUARANTA • Department of Laboratory and Infectious Sciences, A. Gemelli University Hospital IRCCS, Rome, Italy
TAZEEN QURESHI • Neurobiology Group, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India; Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences (NIMHANS), Institute of National Importance, Bangalore, Karnataka, India
SAISWAROOP RAJARATNAM • Disease Biology Lab, Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Anantapur, Andhra Pradesh, India
NIKITA RANA • Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, India
ARCHANA S. RAO • School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India
ANKIT RATHEE • University Institute of Pharma Sciences, Chandigarh University, Ajitgarh, Punjab, India
CHARUL RATHORE • University Institute of Pharma Sciences, Chandigarh University, Ajitgarh, Punjab, India
KAJAL RAWAT • Department of Pharmacology, Research Block B, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
SWAPAN K. RAY • Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC, USA
KAISAR RAZA • Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Ajmer, Rajasthan, India
SYED SHADAB RAZA • Laboratory for Stem Cell & Restorative Neurology, Department of Biotechnology, Era’s Lucknow Medical College and Hospital, Era University, Sarfarazganj, Lucknow, India; Department of Stem Cell Biology and Regenerative Medicine, Era’s Lucknow Medical College Hospital, Era University, Sarfarazganj, Lucknow, India
LEKHA SAHA • Department of Pharmacology, Research Block B, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
SUPARNA SAHA • NINDS, NIH, Bethesda, MD, USA
SAIMA • Department of Pharmacology, Delhi Pharmaceutical Science and Research University (DPSRU), New Delhi, India
ARUSHI SANDHU • Department of Pharmacology, Research Block B, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
DEEPANEETA SARMAH • Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, India
BHAGAWATI SAXENA • Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad, India
KARTHIKEYAN SELVARASU • Molecular Mycology and Neurodegenerative Disease Research Laboratory, Department of Microbiology, Central University of Tamil Nadu, Thiruvarur, India
MURUGAN SEVANAN • Department of Biotechnology, Karunya Institute of Technology and Sciences (Deemed to be University), Coimbatore, India
RAMAISH SHARMA • ISF College of Pharmacy, Moga, Punjab, India
RUCHIKA SHARMA • Centre for Precision Medicine and Pharmacy, Delhi Pharmaceutical Sciences and Research University (DPSRU), New Delhi, India
SHYAM S. SHARMA • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Mohali, Punjab, India
VASU SHEEBA • Neuroscience Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India; Chronobiology and Behavioural Neurogenetics Laboratory, Neuroscience Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India
ABHAY KUMAR SINGH • Molecular Mycology and Neurodegenerative Disease Research Laboratory, Department of Microbiology, Central University of Tamil Nadu, Thiruvarur, India
JITENDRA NARAIN SINGH • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Mohali, Punjab, India
VENKETESH SIVARAMAKRISHNAN • Disease Biology Lab, Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Anantapur, Andhra Pradesh, India
MIN KYUNG SONG • RWJMS Institute for Neurological Therapeutics, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ, USA; College of Nursing Science, Kyung Hee University, Seoul, Republic of Korea
UJJAWAL SONI • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, Assam, India
ARPNA SRIVASTAVA • Department of Neurology, AIIMS, New Delhi, India
RAJNISH SRIVASTAVA • Moradabad Educational Trust Group of Institutions Faculty of Pharmacy, Moradabad, Uttar Pradesh, India
SREYA SUBHASH • Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Ajmer, Rajasthan, India
SACHITHRA THAZHATHUVEEDU SUDEVAN • Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Sciences Campus, Kochi, India
AILI SUN • Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, University Hospital Wu¨rzburg, Wurzburg, Germany
RAMON HANDERSON GOMES TELES • Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, University Hospital Wu¨rzburg, Wurzburg, Germany; Laboratory of Tumor Microenvironment, Department of Cell and Developmental Biology, Institute of Biomedical Sciences (ICB), University of Sao Paulo, Sao Paulo, Brazil; Graduate School of Life Sciences, Julius-Maximilians-University Wu¨rzburg, Wurzburg, Germany
MANJARI TRIPATHI • Department of Neurology, AIIMS, New Delhi, India
RIDDHI UPADHYAY • Department of Biotechnology, Karunya Institute of Technology and Sciences (Deemed to be University), Coimbatore, India
BHUPESH VAIDYA • Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Mohali, Punjab, India
S. M. VEENA • Department of Biotechnology, Sapthagiri College of Engineering, Bangalore, India
SONIA VILLAPOL • Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Academic Institute, Houston, TX, USA
ELISABETTA ZUCCHI • Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
TUNEL-n-DIFL Method for Detection and Estimation of Apoptosis Specifically in Neurons and Glial Cells in Mixed Culture and Animal Models of Central Nervous System Diseases and Injuries
Swapan K. Ray
Abstract
Detection of merely apoptosis does not reveal the type of central nervous system (CNS) cells that are dying in the CNS diseases and injuries. In situ detection and estimation of amount of apoptosis specifically in neurons or glial cells (astrocytes, oligodendrocytes, and microglia) can unveil valuable information for designing therapeutics for protection of the CNS cells and functional recovery. A method was first developed and reported from our laboratory for in situ detection and estimation of amount of apoptosis precisely in neurons and glial cells using in vitro and in vivo models of CNS diseases and injuries. This is a combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL) or simply TUNEL-n-DIFL method for in situ detection and estimation of amount of apoptosis in a specific CNS cell type. An anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) for blue fluorescence, fluorescein isothiocyanate (FITC) for green fluorescence, or Texas Red (TR) for red fluorescence can be used for in situ detection of apoptotic cell DNA, which is earlier labeled with TUNEL using alkali-stable DIG-11dUTP. A primary anti-NeuN (neurons), anti-GFAP (astrocytes), anti-MBP (oligodendrocytes), or antiOX-42 (microglia) IgG antibody and a secondary IgG antibody conjugated with one of the above fluorophores (other than that of ani-DIG antibody) are used for in situ detection of apoptosis in a specific CNS cell type in the mixed culture and animal models of the CNS diseases and injuries.
Key words Apoptosis, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Double immunofluorescence labeling (DIFL), Central nervous system (CNS) cells, CNS diseases and injuries, Mixed culture, Brain and spinal cord sections, TUNEL-n-DIFL method
1 Introduction
Diseases and injuries in the central nervous system (CNS) are associated with the programmed cell death or apoptosis in neurons and glial cells [1–3]. Various in vitro and in vivo models are used to mimic CNS diseases and injuries to device the appropriate therapeutic strategies for prevention of apoptosis in the CNS cells.
Swapan K. Ray (ed.), Neuroprotection: Method and Protocols, Methods in Molecular Biology, vol. 2761, https://doi.org/10.1007/978-1-0716-3662-6_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024
Monocultures of neurons or glial cells such as astrocytes, oligodendrocytes, and microglia are straightforward to discover the factor that causes induction of apoptosis in a specific CNS cell type and therapeutic inhibition of its apoptosis [4–6]. However, monoculture model of a CNS disease or injury misses the interactions and communications between neurons and glial cells that actually determine the amount of apoptosis occurring in a specific CNS cell type. Therefore, using co-culture or mixed culture of neurons and glial cells is a better option than monoculture to mimic a CNS disease or injury [7–11]. Yet, the best option for understanding CNS degeneration and exploring therapeutic efficacy is the employment of the in vivo models using rodents that fairly recapitulate many human CNS diseases and injuries [12–14].
The problem is how to simultaneously detect in situ cell death in a specific CNS cell type in the mixed culture as well as in the brain and spinal cord sections from the animal models of the CNS diseases and injuries to explore efficacy of specific therapeutic opportunities to prevent cell death. To address this problem, an innovative and novel method has been developed in our laboratory by combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL), or TUNEL-n-DIFL, which can successfully be applied (with a change of primary antibody for specific CNS cell type) to the mixed culture and animal models of the CNS diseases and injuries for in situ detection and assessment of amount of apoptosis in a specific CNS cell type before and after therapeutic treatments [15].
While TUNEL reagents remain the same, cell marker antibody needs to be changed to NeuN (neuronal nuclei), GFAP (glial fibrillary acidic protein), MBP (myelin basic protein), or CD11b (cluster of differentiation 11b)/OX-42 for detection and estimation of apoptosis in neurons, astrocytes, oligodendrocytes, or microglia, respectively, in the mixed culture and CNS tissue sections. TUNEL-n-DIFL method provides consistent qualitative and quantitative results to the investigators who can use a neat experimental design, appropriate TUNEL reagents, an anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin3-acetic acid (AMCA), fluorescein isothiocyanate (FITC), or Texas Red (TR) for detecting DIG incorporated genomic DNA (via a preceding TUNEL); an anti-NeuN, anti-GFAP, anti-MBP, or anti-OX-42 primary IgG antibody for marking neurons, astrocytes, oligodendrocytes, or microglia, respectively; a secondary IgG antibody conjugated with any one of the above fluorophores (other than that of ani-DIG antibody) against the cell marker primary IgG antibody; and evidently some practice. Two different antibodies with two fluorophores of distinct emission maxima (with a difference of at least 40 nm wavelength) can be detected simultaneously, providing discernible colors [16, 17]. Fluorescence colors of AMCA (blue), FITC (green), and TR (red) are easily
distinguishable. Estimation of two fluorescence colors (e.g., AMCA and TR, FITC and TR) gives quantitative values for amount of apoptosis in a specific CNS cell type. After its initial development and reporting [15], this method has successfully been used by many investigators in our laboratories over the years [18–25] as well as in other laboratories around the world [26–33] for generating and reporting their results. As the name says, TUNEL can detect genomic DNA nick ends in cells undergoing apoptosis, necrosis, and any other cell death mechanism involving DNA fragmentation. To validate that CNS cells are undergoing undoubtedly apoptosis, investigators are encouraged to use the cell culture or tissue samples to perform additional experiments such as internucleosomal DNA fragmentation assay for observing DNA laddering as the hallmark of apoptosis [34–36] or a microscopic study for identification and confirmation of characteristic apoptotic features such as cell shrinkage, chromatin condensation, and membrane blebbing [37–39]. DetectionofApoptosisExplicitlyinNeuronsandGlialCells3
This TUNEL-n-DIFL method is acquiescent to versatility. In addition to the CNS cells and tissue sections, TUNEL-n-DIFL method can be applied to other cell type in mixed culture and any other organ tissue section with the use of specific antibody for in situ detecting and estimating apoptosis in a specific cell type in the mixed culture and organ tissue section employed in the experiment, as already reported by some creative investigators [29, 31, 32]. Contemporary trends indicate the increasing use of brain organoids [40–45] and spinal cord organoids [46–49] for understanding the mechanisms of pathogenesis in three dimensions (3D) and development of treatments for neuroprotection in CNS disorders. TUNEL-n-DIFL method is thus well positioned for its application to 3D brain organoids as well as to 3D spinal cord organoids for assessment of neuronal and glial cell apoptosis before and after therapeutic treatments.
The apoptotic mode of cell death needs to be demonstrated and confirmed by the classic internucleosomal DNA fragmentation (DNA laddering) assay, which has also been developed in our laboratory by isolation and agarose gel electrophoresis of the genomic DNA from the CNS tissue of animals with CNS disorder [15]. Appearance of the internucleosomal DNA fragmentation as a DNA ladder of 180 base pairs (bp) on the agarose gel following electrophoresis is the classic biochemical hallmark of apoptosis, while appearance of the DNA smear of random DNA fragmentation on agarose gel is an indication of cell death by necrosis.
2 Materials
Follow all safe practices including use of laboratory coats, gloves, and protective eyeglasses for handling and using all materials in the laboratory. Use ultrapure deionized water (with a sensitivity of 18 MΩ-cm at a room temperature of around 25 °C) and
2.1 Mixed Culture Model of CNS Disease or Injury
analytical-grade chemical reagents for preparation of all solutions for the experiments. Carefully manage and dispose of all chemical and biological waste materials following the regulatory guidelines.
1. Employ neurons and glial cells (astrocytes, oligodendrocytes, or microglia) of animal origin (e.g., mouse, rat) or human origin at proper proportions for studies in mixed cultures or co-cultures in 6-well plates (see Note 1).
2. For growth and experimentation with the mixed culture of CNS cells, use appropriate media and instructions for the CNS cells of mouse [50, 51], rat [52, 53], or human [54–56] origin (see Note 2).
3. Use neurotoxins (see Note 3) such as rotenone and 1-methyl4-phenylpyridinium ion or MPP+ to mimic Parkinson’s disease or PD [21, 55, 57], amyloid beta or Aβ to mimic Alzheimer’s disease or AD [58], mutant form of the superoxide dismutase enzyme 1 or SOD1 to mimic amyotrophic lateral sclerosis or ALS [59], and glutamate to mimic spinal cord injury or SCI [60] in the cell culture models following standard procedures.
2.2 Animal Model of CNS Disease or Injury
2.3
Brain and Spinal Cord Tissue Sectioning
1. Use an animal model of PD [12, 21], AD [61, 62], MS [24, 63], SCI [64, 65], or any other CNS disorder in which you are interested. Develop and well establish the animal model of the CNS disease or injury with its standard behavioral studies or clinical scores before attempting any histological and molecular studies with the brain and spinal cord tissues (see Note 4).
2. If desired, perform therapeutic treatments following the experimental design in animal model of the CNS disease or injury. After the treatments and behavioral studies, sacrifice the animals in control and treatment groups under anesthesia (e.g., ketamine at 100 mg/kg and xylazine at 5 mg/kg), and perfuse via the left ventricle with 100 mL of phosphate-buffered saline (PBS) (see Note 5).
3. Use standardized surgical tools and procedures to collect the brain and spinal cord tissues.
1. Tissue Freezing Medium or TFM (Triangle Biochemical Sciences) (see Note 6).
2. Opimal cutting temperature (OCT) compound cryostat embedding medium (Fisher Scientific) (see Note 7).
3. Cut tissue sections of 5 μm thickness using Reichert-Jung cryostat (Leica) (see Note 8).
4. Superfrost Plus Micro Slides (VWR Scientific Products).
5. 95 % ethanol.
2.4 Special Equipment
2.5 Chemicals and Reagents for TUNEL-nDIFL
6. 10× phosphate-buffered saline (PBS): 100 mM Na2HPO4, 18 mM KH2PO4, 1.37 M NaCl, 27 mM KCl, and pH 7.4 (see Note 9).
1. A battery-operated homogenizer (Kontes Instruments) (see Note 10).
2. A ultraviolet or UV (303 nm) transilluminator.
3. Polaroid film (positive/negative) Type 665.
4. An autoclave for sterilization of the dissection instruments.
5. Reichert-Jung cryostat (Leica) for brain and spinal cord sectioning.
6. Coplin jar.
7. Plastic Coverslip (Promega).
8. OmniSlide Thermal Cycler (Hybaid) for TUNEL reaction.
9. Fluorescence microscope (Olympus Corporation, Japan) equipped with appropriate filters for the fluorescence colors of two fluorescent antibodies (e.g., AMCA and TR, FITC and TR) used in the experiment.
10. Image-Pro Plus software (Media Cybernetics) and ImageJ software (National Institutes of Health, USA).
1. 95 % ethanol.
2. 4 % methanol-free formaldehyde (freshly prepared in PBS).
3. Equilibration buffer (EB): 200 mM K-cacodylate, 25 mM Tris–HCl, pH 6.6, 0.2 mM dithiothreitol (DTT), 0.25 mg/mL bovine serum albumin (BSA), and 2.5 mM CoCl2 (Promega).
4. TUNEL kit (Promega).
5. 10× PCR DIG Labeling mix (2 mM each of dATP, dCTP, dGTP, 1.9 mM dTTP, and 0.1 mM alkali-stable DIG-11dUTP) (Boehringer Mannheim) (see Notes 11 and 12).
6. 20× saline-sodium citrate (SSC) buffer: 3 M NaCl, 0.3 M sodium citrate, pH 7.0.
7. 2 % serum from sheep (lamb), horse, or goat (Sigma) as blocking reagent (see Note 13).
8. Sheep anti-DIG IgG antibody (Fab fragments) conjugated with AMCA (blue), FITC (green), or TR (red) procured from commercial vendor (Roche Molecular Biochemicals).
9. Primary IgG antibodies against NeuN, GFAP, MBP, or OX-42 (BioSource International).
2.6 Chemicals and Reagents for Isolation and Electrophoresis of Genomic DNA from Mixed Culture or Brain and Spinal Cord Tissues
10. Secondary IgG antibody conjugated with a fluorophore (other than that of anti-DIG antibody) procured from commercial vendor (Vector Laboratories).
11. VectaShield Mounting Medium (Vector Laboratories).
1. Cells or tissue homogenization buffer: 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 50 mM EDTA.
2. Cells or tissue homogenate digestion buffer: 10 mM Tris–HCl, pH 8.0, 50 mM NaCl, 10 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), and 250 ng/mL proteinase K (see Note 14).
3. Phenol (which will be used for DNA extraction from cells or tissue digests) for equilibrating with equal volume of 500 mM Tris–HCl, pH 8.0 to avoid partitioning of DNA into the organic phase [15](see Note 15).
4. Mixture of phenol (pH 8.0) and chloroform (1:1, v/v) for DNA extraction (see Note 15).
5. 70 % ethanol.
6. TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) containing RNase A (50 ng/mL) (see Note 16) for dissolving DNA.
7. 6× DNA loading dye: 0.25 % (w/v) bromophenol blue, 0.25 % xylene cyanol, 30 % glycerol in deionized water. DNA loading dye allows the sample to sink visibly into each gel slot and further helps tracking of travel of the DNA samples during gel electrophoresis.
8. Agarose for preparation of 1.8 % agarose gels (see Note 17).
9. 10× TAE buffer: 400 mM Tris–acetate, 10 mM EDTA, pH 8.3.
10. Ethidium bromide or EtBr (1 μg/mL) (see Note 18).
3 Methods
3.1 Processing of CNS Cells in Mixed Culture
Perform all procedures at a laboratory temperature of around 25 °C unless specifically mentioned otherwise.
1. In one set, grow the cells, and subsequently treat on sterile glass cover slips inserted within 6-well plates [57]. Process and save cells (from control and all treatment groups) for conducting TUNEL-n-DIFL method [15, 57].
2. In another set, grow and subsequently treat the cells in the 6-well plates (without sterile glass cover). Process and save cells (from control and all treatment groups) for conducting DNA laddering assay [15].
3.2
Processing of Brain or Spinal Cord Tissue Sections
3. Use the CNS cells grown and subsequently treated on sterile glass cover slips inserted within 6-well plates. Gently spin (at 150× g for 10 min) the plates to settle down less adherent apoptotic cells on cover slips. Fix the cells with 95 % ethanol for 10 min and wash twice with 1× PBS.
4. Further fix the cells with 4 % methanol-free formaldehyde in PBS for 15 min, and wash twice with 1× PBS.
1. After surgical collection, immediately freeze a same small amount (at least 0.5 g) of the same area of brain or spinal cord tissue from each animal in the TFM (Triangle Biochemical Sciences).
2. Coat the brain or spinal cord tissue with the OCT further to avoid disintegration during sectioning. Snap-freeze in liquid nitrogen for 1 min, and store all frozen tissue samples at -80 °C until ready for sectioning.
3. Also, collect and save a same small amount (at least 1.0 g) of the same area of brain or spinal cord tissue from each animal in the 1.5 mL Eppendorf tube. Store all tissue samples at -80 °C until ready for isolation of genomic DNA.
4. Use the frozen tissue samples to cut 4 μm sections on a cryostat (Cryocut 1800, Reichert-Jung, Leica), and thaw mount onto the Superfrost Plus Micro Slides (VWR). Take two tissue sections on one slide (see Note 19).
5. Place slides on a slightly warm surface for approximately 1 min to dry the sections.
6. Immerse the slide in 95 % ethanol in a Coplin jar for 10 min.
7. Wash the slide twice in PBS in a Coplin jar for 5 min to remove all traces of OCT.
8. Place the slides in 4 % methanol-free formaldehyde (freshly prepared in PBS) in a Coplin jar for 15 min.
9. Rinse twice in PBS for 5 min each.
3.3 TUNEL
1. Cover the cells or tissue sections with 50 μL of EB (Equilibration Buffer, Promega TUNEL kit). Place ½ piece of Plastic Coverslip (Promega TUNEL kit) to ensure even spreading and prevent evaporation of the reagent. Equilibrate for 5–10 min (see Note 20).
2. While the cells or tissue sections are equilibrating, prepare sufficient TdT reaction mix to use 25 μL/slide. Prepare 25 μL of TdT reaction mix by adding 21.5 μL of EB, 2.5 μL o nucleotide mix (PCR DIG Labeling mix, Boehringer Mannheim), and 1 μL of TdT (TUNEL kit, Promega). Keep the TdT reaction mix on ice.
f
3.4 DIFL
3. Remove the Plastic Coverslip and excess liquid from the slides. Blot around the equilibrated areas with tissue paper (see Note 21), but do not allow the cells or tissue sections to dry out.
4. Immediately, add 25 μL of TdT reaction mix to the tissue sections on each slide. Cover with ½ piece of Plastic Coverslip to ensure even spreading of the reagent and carefully remove any air bubbles.
5. Incubate the slides at 37 °C for 1 h in the humid OmniSlide Thermal Cycler (Hybaid, UK).
6. Dilute 20× SSC (Promega TUNEL kit) to 40 mL of 2× SSC with deionized water.
7. Remove the Plastic Coverslip and terminate the DIG Labeling reaction by immersing the slides in 2× SSC for 15 min.
8. Wash the slides in PBS for 5 min. Repeat two times for a total of three washes to remove unincorporated alkali-stable DIG-dUTP.
1. To reduce nonspecific background fluorescence (following treatments with antibodies), block the cells or tissue sections on each slide with 200 μL of a mixture of two sera (2 % each of the two sera) in PBS for 30 min.
2. Remove sera by tender tapping of the slide on a paper towel. Add 200 μL of primary IgG antibody targeted to specific CNS cell marker (e.g., NeuN, GFAP, MBP, or OX-42) with a desired dilution (1:100) in blocking solution to the cells or tissue sections and incubate at room temperature for 1 h.
3. Rinse the slide for 5 min in PBS. Repeat.
4. Now is the time to add fluorescent antibodies (see Note 22). Treat the tissue sections with 200 μL of a mixture of sheep antiDIG antibody (Fab fragments) conjugated with AMCA, FITC, or TR (1:50) and a secondary antibody conjugated with a fluorophore (1:100), which is different from that of anti-DIG antibody, in blocking solution for 30 min in the dark.
5. Rinse twice in PBS for 5 min each.
6. Rinse once in distilled water for 3 min.
7. Add just one drop of VectaShield Mounting Medium (Vector Laboratories), and keep the sample covered with a glass coverslip. Transfer slides to a slide box to keep them in the dark before imaging.
8. Immediately examine the slide at 400× under fluorescence microscope equipped with epi-illumination and appropriate optical bandpass filters (see Note 23). Use Image Pro Plus 3.0 software (Media Cybernetics) to capture and save double immunofluorescent images of the cells or tissue sections (see Note 24).
3.5 Statistical Analysis of TUNEL-nDIFL
3.6 Internucleosomal DNA Fragmentation (DNA Laddering) Assay
1. Perform quantitative analysis of mean fluorescence intensity (MFI) of images from the TUNEL-n-DIFL using ImageJ software (National Institutes of Health, USA) following our previous report [21].
2. Obtain images of fluorescent colors of TUNEL and NeuN for detection and estimation of apoptosis in neurons in a CNS disease; for example, rotenone-induced apoptosis in neurons in rat model of PD (Fig. 1). Similarly, obtain images of fluorescent colors of TUNEL and NeuN, GFAP, or OX-42 for in situ detection and estimation of apoptosis in neurons, astrocytes, and microglia, respectively, in a CNS disease; for example, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced apoptosis in neurons, astrocytes, and microglia in mouse model of PD (Fig. 2). TUNEL-n-DIFL method is equally useful for in situ detection and estimation of apoptosis specifically in the neurons in a CNS injury; for example, apoptosis in neurons in rat model of SCI (Fig. 3).
3. For quantitative results, convert the collaged four images of TUNEL and NeuN (for example) staining into 8-bit format and subtract background. Set an intensity threshold and keep it constant for all images analyzed.
4. Outline NeuN positive cells using an outlining tool in any neuronal staining panel of the collage, and move outline over to respective TUNEL staining panel of collage to delineate neurons.
5. Measure the MFI of TUNEL staining in these outlined areas as fluorescence intensity in arbitrary units. Calculate the MFI per unit area by dividing the MFI units by area of outlined neurons and represent data as arbitrary units ± standard error of the mean (SEM).
6. Express data as mean ± SEM of separate experiments (n ≥ 6). Consider differences significant at p ≤ 0.05.
1. Homogenize cells, brain tissue, or spinal cord tissue in the homogenization buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 50 mM EDTA).
2. Digest the homogenate in the digestion buffer (10 mM Tris–HCl, pH 8.0, 50 mM NaCl, 10 mM EDTA, 0.5 % SDS, 250 ng/mL proteinase K) at 37 °C for 1 day.
3. Extract twice with a mixture of phenol (pH 8.0) and chloroform (1:1, v/v) and once with chloroform only (see Note 25).
4. Add two volumes of absolute ethanol to the aqueous phase to precipitate DNA (see Note 26), and centrifuge to obtain DNA pellet. Wash DNA pellet twice with 70 % ethanol and air-dry.
Fig. 1 Use of TUNEL-n-DIFL method for in situ detection and estimation of apoptosis (TUNEL-positive) specifically in neurons (NeuN positive) in a rat model of PD. This study was aimed at finding pathology in spinal cord (SC) in cervical and lumbar SC areas in this animal model of PD. (a) Representative photomicrographs of staining for TUNEL (red) and NeuN (green) in coronal sections (5 μm) of cervical and lumbar SC areas. No TUNEL-positive (apoptotic) neurons were identified in the sections from control animals (see Merge, green); however, apoptosis in many neurons occurred in dorsal horn and ventral horn regions of the cervical and lumbar SC sections from rotenone-induced PD animals (see Merge, yellow resulting from mixing of red and green), clearly indicating that rotenone-induced pathogenesis in the SC of the animals. Images were taken at 200× magnification. (b) Estimation of amount of apoptosis in neurons in the SC by analysis of mean fluorescence intensity (MFI) per unit area of TUNEL-positive neurons. Data showed significant differences in the arbitrary units between samples from control animals and rotenone-induced PD animals (n ≥ 4, *p ≤ 0.05). (Reproduced from ref. 21 with permission from Elsevier)
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14‒16. The great disaster which befell Amaziah at the hands of Joash king of Israel and which is about to be narrated in verses 17‒24 seemed to require some heinous transgression for its cause. This the Chronicler supplies in the assertion that, after the defeat of Edom, Amaziah actually brought back Edomite images and set them up in Jerusalem for worship (verses 14‒16): a truly horrible result of a victory which had resulted from obedience to Jehovah’s word by His prophet!
¹⁴Now it came to pass, after that Amaziah was come from the slaughter of the Edomites, that he brought the gods of the children of Seir, and set them up to be his gods, and bowed down himself before them, and burned incense unto them.
14. bowed down ... and burned incense] The tenses in the Hebrew are imperfects and imply that this became Amaziah’s practice. The act was according to a policy frequently pursued in ancient times. Solomon affords an instance of it (1 Kings xi. 7).
¹⁵Wherefore
the anger of the L was kindled against Amaziah, and he sent unto him a prophet, which said unto him, Why hast thou sought after the gods of the people, which have not delivered their own people out of thine hand?
15. which have not delivered] Such deliverance being in popular thought the proof of a deity’s power; compare Isaiah xxxvi. 18 ff., xxxvii. 18 ff.
¹⁶And it came to pass, as he talked with him, that the king said unto him, Have we made thee of the king’s counsel? forbear; why shouldest thou be smitten? Then the prophet forbare, and said, I know that God hath determined to destroy thee, because thou hast done this, and hast not hearkened unto my counsel.
16. of the king’s counsel] Literally, “counsellor to the king.”
hath determined] Literally, “hath counselled” (with a play on the king’s word).
17‒24 (= 2 Kings xiv 8‒14).
A C J .
The overwhelming defeat of Amaziah by Joash of Israel, involving the destruction of part of the defences of Jerusalem and the plundering of the Temple, must have been an affair of the highest importance in Judean history. The relative weakness of Judah compared with Israel is still less apparent in Chronicles than in Kings. For a discussion of the evidence see Cook in Encyclopedia Britannica, s.v. Jews, p. 379.
¹⁷Then Amaziah king of Judah took advice, and sent to Joash, the son of Jehoahaz the son of Jehu, king of Israel, saying, Come, let us look one another in the face.
17. took advice] Took counsel, presumably (according to the Chronicler’s narrative) with a view to demanding satisfaction from Joash for the ravages of the Israelite mercenaries (verse 13). The sequel suggests that Joash refused to give satisfaction.
let us look one another in the face] The proposal may be either to fight or (better) to discuss Amaziah’s claims, the two kings meeting as equals. The latter is probably the right alternative, for the answer of Joash draws a scoffing parallel between Amaziah’s proposition and a thorn’s proposal of alliance with a cedar. Had Amaziah’s words been a challenge to fight, Joash’s answer might rather have taken the form of the parable in Judges ix. 15, “The thorn said, Fire shall come out of the thorn and devour the cedars of Lebanon,” etc.
¹⁸And Joash king of Israel sent to Amaziah, king of Judah, saying, The thistle¹ that was in Lebanon sent to the cedar that was in Lebanon, saying, Give thy daughter to my son to wife: and there passed by a wild beast that was in Lebanon, and trode down the thistle.
¹ Or, thorn
18. the thistle] margin, thorn; compare Proverbs xxvi. 9 (same Hebrew word).
¹⁹Thou sayest, Lo, thou hast smitten Edom; and thine heart lifteth thee up to boast: abide now at home; why shouldest thou meddle to thy hurt¹ , that thou shouldest fall, even thou, and Judah with thee?
¹ Or, provoke calamity
19. Thou sayest] i.e. to thyself.
meddle to thy hurt] margin, provoke calamity, i.e. by making claims which he could not enforce.
²⁰But Amaziah would not hear; for it was of God, that he might deliver them into the hand of their enemies, because they had sought after the gods of Edom.
20. for it was of God] Not in Kings. This turn is characteristic of the Chronicler; compare x. 15, xxii. 7.
²¹So Joash king of Israel went up; and he and Amaziah king of Judah looked one another in the face at Beth-shemesh, which belongeth to Judah.
21. he and Amaziah ... looked one another in the face] The historian by a kind of irony takes up Amaziah’s phrase (verse 17) and gives it a fresh application. Compare the double application (by a similar irony) of the phrase, “lift up the head” in Genesis xl. 13, 19.
at Beth-shemesh] Compare 1 Chronicles vi. 59 [44, Hebrew] (note).
²²And Judah was put to the worse before Israel; and they fled every man to his tent.
22. to his tent] Compare vii. 10 (note).
²³And Joash king of Israel took Amaziah king of Judah, the son of Joash the son of Jehoahaz, at Beth-shemesh, and brought him to Jerusalem, and brake down the wall of Jerusalem from the gate of Ephraim unto the corner gate¹ , four hundred cubits.
¹ So in 2 Kings xiv. 13. The text has, the gate that looketh.
23. the son of Jehoahaz] i.e. the son of Ahaziah, Jehoahaz and Ahaziah being varying forms of the same name; compare xxi. 17 (note).
brake down the wall] Rather, made a breach (or breaches) in the wall. The same verb is used in Nehemiah i. 3 (“broken down”) and Nehemiah iv. 7 (“the breaches”).
the gate of Ephraim] Its precise position is not known, but it was no doubt in the north or north-west wall of the city, on the road to Ephraim. Compare Nehemiah viii. 16.
the corner gate] Hebrew text doubtful, but LXX. ἕως
Compare xxvi. 9; Jeremiah xxxi. 38; Zechariah xiv. 10. Most probably this gate also was near the north-west angle of the walls, but nothing certain is known of its position.
four hundred cubits] About 600 feet according to the ancient cubit, and 700 according to the later standard; compare iii. 3 (note).
²⁴And he took all the gold and silver, and all the vessels that were found in the house of God with Obed-edom, and the treasures of the king’s house, the hostages also, and returned to Samaria.
24. And he took] The verb is missing in Chronicles, and is supplied from Kings.
with Obed-edom] i.e. with the family of Obed-edom which (1 Chronicles xxvi. 4‒8, 15) served as doorkeepers in the House of God. The words are an addition of the Chronicler.
25‒28 (= 2 Kings xiv 17‒20).
T E A .
²⁵And Amaziah the son of Joash king of Judah lived after the death of Joash son of Jehoahaz king of Israel fifteen years. ²⁶Now the rest of the acts of Amaziah, first and last, behold, are they not written in the book of the kings of Judah and Israel? ²⁷Now from the time that Amaziah did turn away from following the L they made a conspiracy against him in Jerusalem; and he fled to Lachish: but they sent after him to Lachish, and slew him there.
27. from the time] The Chronicler characteristically connects the conspiracy with Amaziah’s apostasy; in Kings the only fact of the conspiracy is stated. a conspiracy] Athaliah, Joash, Amaziah each fell one after the other before a conspiracy. Jehoiada’s example had far-reaching results.
to Lachish] Perhaps he was trying to reach Egypt.
²⁸And they brought him upon horses, and buried him with his fathers in the city of Judah¹ . ¹ In 2 Kings xiv. 20, the city of David.
28. upon horses] Render, upon the horses; i.e. upon the horses of some of his pursuers.
the city of Judah] Read, with the margin, the Versions and 2 Kings, the city of David.
C XXVI.
1‒4 (= 2 Kings xiv 21, 22, xv 2, 3).
U ’ R .
¹And all the people of Judah took Uzziah¹ , who was sixteen years old, and made him king in the room of his father Amaziah.
¹ In 2 Kings xiv. 21, Azariah.
1. all the people of Judah] Popular choice does not seem to have determined the succession to the throne, except when the reigning king had perished by a violent or untimely death, compare xxii. 1.
Uzziah] Called “Azariah” in 1 Chronicles iii. 12 and in 2 Kings (eight times), but “Uzziah” in 2 Kings xv. 13, 32, 34; Isaiah i. 1, vi. 1; Hosea i. 1; Amos i. 1; Zechariah xiv. 5. The two forms of the name when written in Hebrew consonants closely resemble each other; moreover the meanings of the two are similar, “Jah is my strength” and “Jah hath given help.” Perhaps the king bore both names; compare “Abram” and “Abraham”—“Eliakim” and “Jehoiakim” (xxxvi. 4).
²He built Eloth, and restored it to Judah, after that the king slept with his fathers.
2. Eloth] So spelt in viii. 17 (= 1 Kings ix. 26), but “Elath” in Deuteronomy ii. 8; 2 Kings xiv. 22. In 2 Kings xvi. 6 the two forms are found side by side in one verse.
after that the king, etc.] The meaning seems to be it was after king Amaziah slept with his fathers that Uzziah his son restored Elath to Judah; and it is a natural inference that Uzziah was ruling in Jerusalem for some while before the death of Amaziah at Lachish left him sole and undisputed king. A considerable time may have elapsed between Amaziah’s flight and his capture as related in xxv. 27. Yet this is not very likely, and from the position of the present verse in Kings it would seem as though the statement in its original context should be interpreted thus: “he, Amaziah, built Eloth,” etc.; and the king referred to in the clause “after that the king slept with his fathers” is probably Jeroboam king of Israel (so Barnes on 2 Kings xiv. 22).
³Sixteen
years old was Uzziah when he began to reign; and he reigned fifty and two years in Jerusalem: and his mother’s name was Jechiliah of Jerusalem.
3. Jechiliah] so the Kethīb; the Ḳerī Jecoliah agrees with the parallel passage of Kings.
⁴And he did that which was right in the eyes of the L , according to all that his father Amaziah
had done.
4. his father Amaziah] This verse suits its original context in Kings, for Kings records nothing against Amaziah; it is out of place in Chronicles, for according to xxv 14 Amaziah was an idolater
5‒10 (not in Kings).
T P U .
5‒10. It is probable that the Chronicler had old and genuine tradition to rely on for the account which he here gives of Uzziah’s prosperity—his wars against neighbouring tribes (verses 6‒8), and his building activity (verses 8‒10). Doubtless in the earlier years of Uzziah’s reign Judah was still suffering from the effects of the defeat inflicted by Joash of Israel. But the general accuracy of the picture of the reign is assured by such facts as (1) the stout resistance offered by Jerusalem to the Assyrians in Hezekiah’s time as contrasted with its capture by the Israelites in Amaziah’s reign (xxv. 23); (2) the frequency of metaphors from building implements and materials in the pages of the prophets of this period (e.g. Amos vii. 7 ff.); (3) the commercial activity and luxury of Jerusalem in the reign of Uzziah’s successor Ahaz—witness the writings of Isaiah, passim.
⁵And he set himself to seek God in the days of Zechariah, who had understanding¹ in the vision² of God: and as long as he sought the L , God made him to prosper.
¹ Or, gave instruction.
² Hebrew the seeing Many ancient authorities have, the fear
5. Zechariah] Nothing is known (apart from this passage) of this Zechariah.
who had understanding] margin, “who gave instruction Hebrew mēbhīn, a word applied to a leader of song (1 Chronicles xv. 22, “skilful”; 1 Chronicles xxv. 7, “cunning”; 1 Chronicles xxv. verse 8, “teacher”).
in the vision of God] Read, in the fear of God (so LXX., Targum Peshitṭa), making a slight correction of the Hebrew text.
⁶And he went forth and warred against the Philistines, and brake down the wall of Gath, and the wall of Jabneh, and the wall of Ashdod; and he built cities in the country of Ashdod, and among the Philistines.
6. the Philistines] Compare xvii. 11, xxi. 16, xxviii. 18; 2 Kings xviii. 8; 1 Maccabees v. 66‒68, xiv. 34.
brake down the wall] See note on xxv. 23.
Jabneh] Mentioned only here in the Old Testament, but probably to be identified with “Jabneel” (Joshua xv. 11). At a later date it was called “Jamnia” (2 Maccabees xii. 8), and, after the fall of Jerusalem in 70 . ., it became for a while the chief centre of Jewish intellectual and religious activities. Its ruins are to be seen about 10 miles south of Jaffa (Joppa) on the coast. The modern Yebna is a few miles inland. Bädeker, Palestine⁵, p. 122.
Ashdod] compare 1 Samuel v. 1 ff.; Isaiah xx. 1; Zephaniah ii. 4; Nehemiah iv. 7, xxiii. 23; Acts viii, 40 (Ἄζωτος). Ashdod (modern Esdūd) was situated between Gaza and Joppa, some three miles from the sea.
in the country of Ashdod] (literally “in Ashdod”). Perhaps the name has been repeated through an early scribal error and we should read simply “and built cities among the Philistines.”
⁷And God helped him against the Philistines, and against the Arabians that dwelt in Gurbaal, and the Meunim.
7. against the Philistines, and against the Arabians] “Conditions in the comparatively small and half-desert land of Judah depended essentially on its relations with the Edomite and Arabian tribes on the
south-east and with the Philistines on the west”: note how this comes out in the traditions of the period as narrated in Chronicles Jehoshaphat dominated both Philistines and Arabians (Edomites) (xvii. 11); but Libnah (near Lachish) and Edom revolted successfully against his son Jehoram (xxi. 10). After the reigns of Ahaziah and Joash, Amaziah found himself able to assail Edom and gained a great victory (xxv. 11, 12). Later in his reign Amaziah suffered a crushing defeat at the hands of Joash of Israel, and it is reasonable to suppose that Edom would seize the opportunity to reassert its independence, though Chronicles is silent on the point. It is therefore in harmony with the sequence of events as narrated by the Chronicler, when in the present verse (compare verse 2) we are told that Amaziah’s successor, Uzziah, reestablished the Judean power over Edom, and that later, against Ahaz, Edom and the Philistine cities gained the upper hand (xxviii. 17, 18).
Gur-baal] An unidentified place; a “Gur” is mentioned in 2 Kings ix. 27. A slight correction of the Hebrew would give “in Gerar (compare Genesis xx. 1) and against the Meunim.”
Meunim] compare xx. 1 (note).
⁸And the Ammonites gave gifts to Uzziah: and his name spread abroad even to the entering in of Egypt; for he waxed exceeding strong.
8. gave gifts] i.e. tribute. Compare 1 Chronicles xviii. 2 (note).
⁹Moreover Uzziah built towers in Jerusalem at the corner gate, and at the valley gate, and at the turning of the wall, and fortified them.
9. towers in Jerusalem, etc.] The Chronicler is evidently fond of recording such traditions; compare xxxii. 30, xxxiii. 14; and the Introduction § 7, p. xlviii.
the corner gate] At the north-west corner of the walls. Compare xxv. 23 (note).
the valley gate] Nehemiah ii. 13, iii. 13. Probably near the southwest corner of the walls.
the turning of the wall] Mentioned Nehemiah iii. 19, 24. See G. A. Smith, Jerusalem, II. 120.
¹⁰And he built towers in the wilderness, and hewed out many cisterns, for he had much cattle; in the lowland also, and in the plain¹ : and he had husbandmen and vinedressers in the mountains and in the fruitful fields² ; for he loved husbandry.
¹ Or, table land. ² Or, Carmel See 1 Samuel xxv. 2.
10. the wilderness] i.e. the southern pasture land of Judah. Compare Psalms lxv. 12. Fortified towers have always proved effective for controlling the Bedouin and keeping the desert roads open.
in the lowland also, and in the plain] For the “lowland” (Hebrew Shephēlah) see i. 15 (note). The “plain” (margin table land; Hebrew Mishōr) is the name of the high pasture lands east of Jordan; apparently the part occupied by the Ammonites whom Uzziah had subdued is meant here. (For a different view see Smith, Jerusalem, II. 119, note.)
11‒15 (no parallel in Kings). U ’ A .
¹¹Moreover Uzziah had an army of fighting men, that went out to war by bands, according
to the number of their reckoning made by Jeiel the scribe and Maaseiah the officer, under the hand of Hananiah, one of the king’s captains.
¹²The whole number of the heads of fathers’ houses, even the mighty men of valour, was two thousand and six hundred. ¹³And under their hand was a trained army ¹ , three hundred thousand and seven thousand and five hundred, that made war with mighty power, to help the king against the enemy.
¹ Or, the power of an army.
13. three hundred thousand and seven thousand and five hundred] Compare xxv. 5 (Amaziah’s army), and the notes on xiv. 8 and xvii. 14 (the forces of Asa and of Jehoshaphat).
¹⁴And Uzziah prepared for them, even for all the host, shields, and spears, and helmets, and coats of mail, and bows, and stones for slinging.
14. stones for slinging] Such stones needed to be carefully chosen, for they had to be smooth and of a suitable size, compare 1 Samuel xvii. 40. Bows and slings appear to have been favourite weapons in Benjamin, compare 1 Chronicles xii. 2; Judges xx. 16.
¹⁵And he made in Jerusalem engines, invented by cunning men, to be on the towers and upon the battlements¹ , to shoot arrows and great stones withal. And his name spread
far abroad; for he was marvellously helped, till he was strong.
¹ Or, corner towers.
15. engines] Doubtless contrivances similar to the Roman catapulta and balista. It is questionable whether such engines of war were really in use as early as the time of Uzziah, at least among the Israelites (see Smith, Jerusalem, ii. 121, 122; and the Encyclopedia Biblia s.v. siege, especially col. 4510). The next reference to similar instruments of war is in 1 Maccabees vi. 51, 52.
helped] compare verse 7.
16‒20 (not in Kings). U ’ P .
16‒20. Uzziah died from leprosy, as is related in verses 21‒23 (= 2 Kings xv. 5‒7). That terrible disease was always regarded as a manifestation of Divine anger against the sufferer (compare Numbers xii. 9 ff.; 2 Kings v. 27), but no special cause is assigned in Kings why the disaster befell Uzziah. In the present verses an adequate reason is brought forward—Uzziah, blinded by the pride of his success, infringed the privileges of the priesthood and was guilty of sacrilege. The motive for some such tale is so strong and the actual sin alleged so akin to the Chronicler’s prejudices that it may well be that the tale originated with him or his immediate circle. Yet it is possible that there may be behind the present form of the tale a valid tradition of a dispute at this period between the hierarchy and the authority of the king.
¹⁶But when he was strong, his heart was lifted up so that he did corruptly¹ , and he trespassed against the L his God; for he went into the
temple of the L to burn incense upon the altar of incense.
¹ Or, to his destruction.
16. did corruptly] Compare xxvii. 2.
he trespassed] compare xii. 2; Joshua vii. 1, xxii. 16. The Hebrew word implies presumptuous dealing with holy things.
the altar of incense] Compare Exodus xxx. 1‒10. Not only the altar, but the incense itself was “most holy”; Exodus xxx. verses 34‒38.
¹⁷And Azariah the priest went in after him, and with him fourscore priests of the L , that were valiant men:
17. Azariah the priest] i.e. the high-priest (verse 20). He cannot be identified with any priest in the list given 1 Chronicles vi. 4‒15 (v. 30‒41, Hebrew).
¹⁸and they withstood Uzziah the king, and said unto him, It pertaineth not unto thee, Uzziah, to burn incense unto the L , but to the priests the sons of Aaron, that are consecrated to burn incense: go out of the sanctuary; for thou hast trespassed; neither shall it be for thine honour from the L God.
¹⁹Then Uzziah was wroth; and he had a censer in his hand to burn incense; and while he was wroth with the priests, the leprosy
brake forth¹ in his forehead before the priests in the house of the L , beside the altar of incense.
¹ Hebrew rose (as the sun)
18. the priests the sons of Aaron] Compare xiii. 10, 11 and Numbers xvi. 40.
neither shall it be for thine honour] A euphemism covering a threat of danger and disgrace.
²⁰And Azariah the chief priest, and all the priests, looked upon him, and, behold, he was leprous in his forehead, and they thrust him out quickly from thence; yea, himself hasted also to go out, because the L had smitten him.
20. the L had smitten him] So 2 Kings xv. 5.
21‒23 (= 2 Kings xv. 5‒7).
T E U .
²¹And Uzziah the king was a leper unto the day of his death, and dwelt in a several house¹ , being a leper; for he was cut off from the house of the L : and Jotham his son was over the king’s house, judging the people of the land.
¹ Or, lazar house.
21. a several house] i.e. separate, special; compare Numbers xxviii. 13; Matthew xxv. 15. The same Hebrew word is used in Psalms lxxxviii. 5, “free (Revised Version ‘cast off’) among the dead.”
cut off] The same Hebrew word is translated in the same way in Isaiah liii. 8.
²²Now the rest of the acts of Uzziah, first and last, did Isaiah the prophet, the son of Amoz, write.
22. did Isaiah ... write] This statement is not in Kings. Uzziah is mentioned in Isaiah vi. 1, and this fact may be all that lies behind the present statement. It is utterly improbable that the reference is to some writing of Isaiah which has not been preserved. Possibly some section of the midrashic Book of the Kings of Judah and Israel is meant, presuming that such a work was known to the Chronicler actually or by tradition (see Introduction § 5, pp. xxxii, xxxvi).
²³So Uzziah slept with his fathers; and they buried him with his fathers in the field of burial which belonged to the kings; for they said, He is a leper: and Jotham his son reigned in his stead.
23. the field of burial] i.e. not actually in the tombs of the kings, lest they should be defiled, but in ground adjoining the royal tombs. Kings has simply “in the city of David.” Compare xxi. 20, xxiv. 25, xxviii. 27.
C XXVII.
1‒6 (compare 2 Kings xv. 32‒35).
J S .
¹Jotham was twenty and five years old when he began to reign; and he reigned sixteen years in Jerusalem: and his mother’s name was Jerushah the daughter of Zadok.
1. he reigned sixteen years] The years during which he acted as regent in place of his father (see above xxvi. 21) are included in the sixteen. Jotham’s independent reign was probably very brief.
²And he did that which was right in the eyes of the L , according to all that his father Uzziah had done: howbeit he entered not into the temple of the L . And the people did yet corruptly.
2. according to all that ... howbeit he entered not into the temple of the L ] i.e. he imitated Uzziah in all his virtues, but not in his sin against the ritual of the Temple (xxvi. 16 ff.). The clause howbeit, etc., is not in Kings, since Kings makes no reference to Uzziah’s transgression.
did yet corruptly] In Kings, “Howbeit the high places were not taken away; the people still sacrificed and burned incense in the high places.”
³He built the upper gate of the house of the L , and on the wall of Ophel he built much.
3. the upper gate] Compare the note on xxiii. 20.
and on the wall of Ophel he built much] The statement is made only in Chronicles Like similar notices of building activity, etc.—a subject of great interest to the Chronicler—it may possibly have some basis in fact; compare xxvi. 9 f., xxxii. 30, xxxiii. 14.
Ophel] compare xxxiii. 14; Nehemiah iii. 26, 27. It was a southern spur of the Temple Hill. Bädeker, Palestine⁵, p. 31; and Smith, Jerusalem, i. 152 ff.
⁴Moreover he built cities in the hill country of Judah, and in the forests he built castles and towers.
4. castles] compare xvii. 12 (note).
⁵He fought also with the king of the children of Ammon, and prevailed against them. And the children of Ammon gave him the same year an hundred talents of silver, and ten thousand measures ¹ of wheat, and ten thousand of barley. So much did the children of Ammon render unto him, in the second year also, and in the third.
¹ Hebrew cors
5. the children of Ammon] Compare xx. 1 ff., xxvi. 8.
an hundred talents of silver] Compare 2 Kings xxiii. 33.
measures] Hebrew kōrīm. A kōr (= a ḥōmer, Ezekiel xlv. 14, Revised Version) was a dry measure holding about 11 bushels.
⁶So Jotham became mighty, because he ordered his ways before the L his God.
6. became mighty] The same Hebrew word as in i. 1 (see note).
7‒9 (= 2 Kings xv. 36‒38).
T S J ’ R .
⁷Now the rest of the acts of Jotham, and all his wars, and his ways, behold, they are written in the book of the kings of Israel and Judah. ⁸He was five and twenty years old when he began to reign, and reigned sixteen years in Jerusalem. ⁹And Jotham slept with his fathers, and they buried him in the city of David: and Ahaz his son reigned in his stead.
7. all his wars] Only a war with Ammon is mentioned above, but according to 2 Kings xv. 37 the Syro-Ephraimite war also began in Jotham’s reign. The notices in Kings and Chronicles may be regarded as supplementary. Ammon was a natural ally of the Syrians, and perhaps the wording of verse 5 (end) hints that after the third year Ammon was able to refuse to pay tribute. The information of Chronicles is therefore plausible; but it is curious that Chronicles preserves the one incident and Kings the other. The point is highly significant. Not only does it illustrate very forcibly the comparative independence of the Chronicler’s narrative, which is so marked a feature in these later reigns; but also it adds to the evidence in favour of the view that the Chronicler had traditions before him other
