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Low Dose Nerve Agent Sarin Causes Dilated Cardiomyopathy and Autonomic Imbalance in Mice

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Low Dose Nerve Agent Sarin Causes Dilated Cardiomyopathy and Autonomic Imbalance in Mice Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2011 Low Dose Nerve Agent Sarin Causes Dilated Cardiomyopathy and Autonomic Imbalance in Mice Swapnil Vijay Shewale Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Pharmacology, Toxicology and Environmental Health Commons Repository Citation Shewale, Swapnil Vijay, "Low Dose Nerve Agent Sarin Causes Dilated Cardiomyopathy and Autonomic Imbalance in Mice" (2011). Browse all Theses and Dissertations. 1059. https://corescholar.libraries.wright.edu/etd_all/1059 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. LOW DOSE NERVE AGENT SARIN CAUSES DILATED CARDIOMYOPATHY AND AUTONOMIC IMBALANCE IN MICE A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By SWAPNIL VIJAY SHEWALE B.Pharmacy, University of Pune, 2007 2011 Wright State University WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES AUGUST-20-2010 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Swapnil Vijay Shewale ENTITLED “Low Dose Nerve Agent Sarin Causes Dilated Cardiomyopathy and Autonomic Imbalance In Mice” BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science Dr. Mariana Morris, Ph.D. Thesis Director Dr. Mariana Morris, Ph.D. Chair, Department of Pharmacology and Toxicology Committee on Final Examination Dr. Mariana Morris, Ph.D. Dr. James Lucot, Ph.D. Dr. Mark Anstadt, M.D. Dr. Andrew Hsu, Ph.D. Dean, Graduate School ABSTRACT Shewale, Swapnil Vijay, M.S., Pharmacology/Toxicology Masters Program, Department of Pharmacology and Toxicology, Wright State University, 2011. Low Dose Nerve Agent Sarin Causes Dilated Cardio myopathy and Autonomic Imbalance Sarin, a lethal chemical nerve agent, may be a causative fa ctor in multifactorial syndrome implicated in the Gulf War and Tokyo terrorist attacks. While high dose results in seizure and death, low dose exposure may lead to autonomic imbalance and chronic cardiac pathologies. In the present study, echocardiography and electrocardiography were used to examine the late onset effects of a low dose sarin on cardiac structure and function in mice. Stress responsiveness of the hypothalamic pituitary adrenal (HPA) axis was also tested. Findings demonstrate changes consistent with a dilated cardiomyopathy, including left ventricular dilatation, reduced contractility, and altered electrophysiological and inotropic responses to β adrenergic stimulation. Results also indicate altered stress responsiveness of HPA indicating autonomic imbalance . The role of low-dose sarin/organophosphate exposure needs to be considered in military and civilian population who suffer from autonomic imbalance and/or cardiomyopathies of indeterminate origin. iii Table of Contents I. Introduction ..................................................................................................... 13 I.1. Sarin: Structure, Function and Mechanism of Action ....................................... 13 I.2. Mechanism of Action of OP’s ............................................................................ 14 II. Background ..................................................................................................... 16 III. Health effects of Sarin and current treatment ................................................. 17 III.1 Symptoms seen in Gulf war veterans . ........................................................... 17 III.2. Cardiovascular Effects ...................................................................................... 18 III.3. Current therapy for acute nerve agent / OP-poisoning .................................. 19 IV. Problem Statement, Hypothesis and Specific Aims…………………………………………22 IV.1. Problem Statement ......................................................................................... 20 IV.2. Hypothesis: ...................................................................................................... 20 IV.3 Specific Aims ..................................................................................................... 20 V. Methods ......................................................................................................... 21 V.1. Animal Protocol ................................................................................................ 21 V.2. Sarin Exposure .................................................................................................. 21 V.3. Cholinesterase Assay ........................................................................................ 22 V.4. Transthoracic Echocardiography ...................................................................... 22 V.5. Electrocardiography (EKG) Methods ................................................................ 24 V.6. Body Weights .................................................................................................... 24 V.7. Urine Collection ................................................................................................ 24 V.8. Cage Change Induced Stress Experiment ......................................................... 25 V.9. ANP and BNP Staining and Quantification ....................................................... 25 V.10. H&E Staining ................................................................................................... 25 iv V.11. Cell Size Quantification ................................................................................... 26 V.12. In situ hybridization (ISH) for TH mRNA ......................................................... 27 V.13. Spot Urine Collection and Cage Change Stress Experiment: ......................... 28 V.14. Corticosterone Radioimmunoassay (RIA) ....................................................... 25 V.15. Protein Assay: ................................................................................................. 29 V.16. Creatinine assay: EIA ...................................................................................... 29 V.17. Statistical Analysis ........................................................................................ 29 VI.Equations and Derived Parameters ................................................................. 30 VI.1. ECHO Derived Parameters ............................................................................... 30 VI.2. Electrocardiography Derived Parameters ....................................................... 32 VII. Figures and Results ....................................................................................... 34 VII.1. Time course of sarin induced LV dilation ....................................................... 36 VII.2. Dobutamine stress test: Impaired stress response in sarin mice .................. 39 VII.3. Electrocardiography: ST depression and pro-arrhythmic phenotype in sarin mice.......................................................................................................................... 42 VII.4. Histological and Immunohistochemical evidence of cardiac remodeling ..... 43 VII.5. Evidence for autonomic imbalance: Stress induced Hypothalamic–pituitary– adrenal (HPA)-axis dysregulation ............................................................................ 43 VIII. Discussion .................................................................................................... 45 VIII.1. Sarin induced dilated cardiomyopathy ......................................................... 45 VIII.2. Sarin induced autonomic dysfunction: .......................................................... 48 IX. Conclusion ..................................................................................................... 50 XI. Bibliography................................................................................................... 51 v List of Figures Figure Page Figure 1: General Chemical Structure of Nerve Agents ............................................ 13 Figure 2: Structure and active center of AChE .......................................................... 15 Figure 3: Demolition of bunkers at Khamisiyah, 4 March 1991 and Plume Models for the Demolition of Bunkers at Khamisiyah, 12 March 1991 ................................ 16 Figure 4 :In-situ Hybridization .................................................................................... 27 Figure 5: Representative Area Measurement Method for 2D Echocardiograms ........ 31 Figure 6:Representative Measurement Methods for 2D M-mode Echocardiograms .. 31 Figure 7: Example Pulsed Wave Doppler Image Showing Eight Full Sinus Rhythms31 Figure 8: Pulsed Wave Doppler Method of Quantification ....................................... 31 Figure 9: Representative EKG Trace with Software Markings .................................. 33 Figure 10: EKG Trace Demonstrating S-T peak and Tpeak-Tend Calculations using QATN interval marked using software ............................................................... 33 PAGE Figure 11: Blood ChE activity was determined 24 hrs following the second sarin injection. .............................................................................................................. 34 vi Figure Page Figure 12: Sarin causes progressive cardiac
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