Effect of the Broad-Spectrum Caspase Inhibitor Q-VD-Oph on Neurodegeneration
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Effect of the Broad-Spectrum Caspase Inhibitor Q-VD-OPh on Neurodegeneration and Neuroinflammation of Sarin exposed mice A thesis submitted in partial fulfillment Of the requirements for the degree of Master of Science By Ekta J Shah B.Pharmacy, Mumbai University, 2011 2014 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL July 14, 2014 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Ekta J Shah ENTITLED Effect of the Broad-Spectrum Caspase Inhibitor Q-VD-OPh on Neurodegeneration and Neuroinflammation of Sarin exposed mice BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. David R Cool, Ph.D. Thesis Director Norma Adragna, Ph.D., Interim Chair Department of Pharmacology and Toxicology Committee on Final Examination David R Cool, Ph.D. James B. Lucot, Ph.D. Courtney E.W. Sulentic, Ph.D. Robert E. W. Fyffe, Ph.D. Vice President for Research and Dean of the Graduate School ii Abstract Shah, Ekta J, M.S. Department of Pharmacology and Toxicology, Wright State University, 2014. Effect of Q-VD-OPh on Neurodegeneration and Neuroinflammation of Sarin Exposed Mice Organophosphates (OP) cause CNS hyperstimulation resulting in seizures, convulsions and brain damage. Sarin is a toxic nerve agent used in Syria, the Gulf War and in terrorist attacks in Japan. Exposure to sarin is associated with presence of dead neurons, activation of astrocytes, microglia and neuroinflammation. Since, cell death and neuroinflammation are found in sarin exposed animals, we evaluated the efficacy of Q-VD-OPh, a broad spectrum caspase inhibitor that prevents cell death and has anti-inflammatory properties. Currently, treatment for sarin exposures is effective only if administered within 30-40 minutes of exposure. In this study, adult female C57BL/6 mice were subjected to an injection of 1.5 mg/kg CBDP before a 0.04mg/kg injection of sarin followed by 20 mg/kg injection of Q-VD-OPh 30 minutes later. Mice were sacrificed at two and fourteen day time points followed by cytokine analysis of amygdala and hippocampus using Bio-Plex 200 Pro Mouse Cytokine Grp I Panel (23-Plex) kit. We found a significant increase in 13 out of 23 examined cytokines in amygdala after 2 days of sarin exposure. Also, sarin elicits a strong response in amygdala and hippocampus. After treatment with Q-VD-OPh, the cytokines did not change appreciably between 2 and 14 days in the sarin/Q-VD-OPh treated animals. Q-VE-OPh, a negative control for Q-VD-OPh was used and it showed no iii significant change at the two or fourteen day time points in amygdala and hippocampus. To detect DNA fragmentation, 12μm thick sections were TUNEL-stained using NeuroTacs Apoptosis kit. Q-VD-OPh effect on cholinesterase activity was detected by ChE assay. Frontal cortex was used to analyse apoptosis markers and for cholinesterase activity. TUNEL staining was increased in sarin treated sections but was seen to be less in sections treated with Q-VD-OPh at 2 days. At 14 days, there was not much difference seen. Also, Q-VE-OPh showed intense staining as compared to Q-VD-OPh. ChE assay showed that Q-VD-OPh did not appear to have an inhibitory effect in mice treated with it and sarin. These trends may be indicative of Q-VD-OPh being effective in attenuating neurodegeneration and neuroinflammation in sarin exposed mice. iv TABLE OF CONTENTS 1. INTRODUCTION………………………………………………………….. 1 Global Effect of Sarin exposure……………………………………... 3 Sarin: Structure, Properties and Pharmacokinetics………………….. 5 Sarin: Mechanism of Action……………………………………..….. 6 Signs, Symptoms and Chronic effects of Sarin exposure………….... 9 Neurodegeneration and Neuroinflammation of sarin exposure……... 12 Cell death……………………………………………………………. 13 Neuroinflammation…………………………………………………. 19 Current treatment for OP exposure…………………………………. 20 Q-VD-OPh: Broad Spectrum Caspase Inhibitor……………….…… 22 FMK versus Q-VD-OPh as caspase inhibitors…………………….. 23 Q-VE-OPh: Negative Control……………………………………… 24 2. HYPOTHESIS AND SPECIFIC AIMS…………………………................. 26 3. MATERIALS AND METHODS………………………………....…..…..... 27 Use of CBDP……………………………….……………………….... 28 Brain Sectioning and Tissue Collection………………........................ 30 Brain regions of interest…………………………………………….... 30 Cytokine Analysis……………………………………………………. 31 v TUNEL Staining……………………………………………............... 32 Statistical Analysis…………………………………………………… 33 4. RESULTS………………………………………………………………….. 34 5. DISCUSSION…………………………………………………………........ 64 6. CONCLUSION…………………………………………………………….. 70 7. APPENDICES……………………………………………………………… 72 8. REFERENCES……………………………………………………………... 83 vi LIST OF FIGURES 1. General Structure of Nerve Agents & Insecticides…………………. 2 2. G and V series nerve agents.………………………………………….. 2 3. Structure of Sarin……………………………………………………... 5 4. Mechanism of action of Sarin………………………………………… 8 5. Primary and secondary targets of organophosphates…………………. 10 6. Signs and Symptoms of Sarin exposure……………………………… 11 7. Apoptosis Vs Necrosis………………………………………………... 15 8. Caspase activation model in mitochondria………………………….... 16 9. Pathways of Apoptosis……………………………………………….. 18 10. Nerve Agent Antidote Kit (NAAK)…..……………………………. 22 11. Structure of Q-VD-OPh and Q-VE-OPh………………………………… 25 12. Experimental Design…………………………………………………. 27 13. Coronal section of brain region………………………………………. 34 vii 14. Diagram showing the effect of CBDP on cytokines at 2 and 14 day time points in amygdala and hippocampus…………………… 36 15. Diagram showing the effect of sarin exposure on cytokines in amygdala and hippocampus at 2 and 14 day time point…………….. 42 16. Diagram showing effect of Q-VD-OPh on sarin induced cytokines in amygdala nad hippocampus at 2 and 14 day time points……………. 43 17. TUNEL staining on dentate gyrus region of brain at day 2…………. 59 18. TUNEL staining on dentate gyrus region of brain at 14 days………. 60 19. AChE activity in frontal cortex………………………………………. 63 viii LIST OF TABLES 1. History of Sarin……………………………………………………….. 4 2. Treatment Groups for the Project……………………………………... 29 3. Experimental Substances with dose and route of administration……... 29 4. Effect of CBDP (without sarin) on Cytokines at 2 Days in Amygdala.. 37 5. Efect of CBDP (without sarin) on Cytokines at 14 Days in Amygdala. 38 6. Effect of CBDP (without sarin) on Cytokines at 2 Days in Hippocampus……………………………………………………….. 39 7. Effect of CBDP (without sarin) on Cytokines at 14 Days in Hippocampus………………………………………………………….. 40 8. Sarin Effect on Cytokines at 2 Days in Amygdala…………………… 44 9. Sarin Effect on Cytokines at 14 Days in Amygdala………………….. 45 10. Effect of Sarin on Cytokines at 2 Days in Hippocampus…………….. 46 11. Effect of Sarin on Cytokines at 14 Days in Hippocampus…………… 47 12. Sarin effect at 2 days and 14 days in Amygdala……………………… 48 13. Sarin effect at 2 days and 14 days in Hippocampus………………….. 49 14. Effect of Q-VD-OPh (with sarin) Cytokines at 2 Days in Amygdala….. 50 15. Effect of Q-VD-OPh (with sarin) on Cytokines at 14 Days in Amygdala… 51 16. Effect of Q-VD-OPh (with sarin) on Cytokines at 2 Days in ix Hippocampus……………………………………………………….. 52 17. Effect of Q-VD-OPh (with sarin) on Cytokines at 14 Days in Hippocampus……………………………………………………………… 53 18. Effect of Q-VE-OPh (with sarin) on Cytokines at 2 Days in Amygdala….. 54 19. Effect of Q-VE-OPh (with sarin) on Cytokines at 2 Days in Hippocampus 55 20. Effect of Q-VE-OPh (with sarin) on Cytokines at 14 Days in Amygdala… 56 21. Effect of Q-VE-OPh (with sarin) on Cytokines at 14 Days in Hippocampus…………………………………………………………….. 57 x ACKNOWLEDGEMENTS The completion of this thesis is the result of the support, guidance and encouragement of several people. I would like to express my deepest appreciation to Dr David R Cool for guiding, supporting, motivating, helping and believing in me for the completion of this thesis and related project work. I would also like to thank committee members Dr James Lucot and Dr Courtney Sulentic for their support. In addition, I would like to thank William C Grunwald and Teressa Garrett for providing appropriate training that helped me coordinate my project especially in completion of experiments. Furthermore, I would like to thank all my friends for their support and motivation. Anall, Richa, Sourav, Sarfaraz, Shreyansh and Pratik-you all made a family away from family, a home away from home. I will be grateful forever for your love, care and concern. Last, but certainly not least, I owe my deepest gratitude to my family especially my grandma, parents, uncle-aunt, two brothers, two sisters and three little angels in the form of niece and nephews. You all gave me an opportunity of an education from the best country and institution and helped me throughout my life. I could not have made it this far without all of your support and motivation xi Introduction Chemical warfare agents (CWA) are chemical substances with toxic properties that can kill, injure or incapacitate an enemy and associated military operations (Chauhan et al., 2008). Most widely known compounds of this category are nerve agents, a class of compounds developed as chemical warfare weapons. They are extremely potent inhibitors of the enzyme acetylcholinesterase, a key regulator of cholinergic neurotransmission (Geoghegan & Tong, 2006). So far, the best-known nerve agents are organophosphate (OP) compounds that share similarity in chemical structure to organophosphate insecticides. Nerve agents can be inhaled or absorbed through skin and the time of onset of intoxication can vary with the dose and route of exposure. Onset of hyper secretion, loss of consciousness,