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Phd Dissertation-Aparna Raghavan A Dissertation entitled Neuroprotective Potential of Withania Somnifera in Cerebral Ischemia by Aparna Raghavan Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Medicinal Chemistry Dr. Zahoor Shah, Committee Chair Dr. Hermann von Grafenstein, Committee Member Dr. James T. Slama, Committee Member Dr. Youssef Sari, Committee Member Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo December 2014 Copyright 2014, Aparna Raghavan This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Neuroprotective Potential of Withania Somnifera in Cerebral Ischemia by Aparna Raghavan Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Medicinal Chemistry The University of Toledo December 2014 Withania somnifera (WS), popularly known as ‘Ashwagandha’ has been used for centuries in the treatment of neurological disorders, although its effects on cerebral ischemia are not well understood. We used a combination of in vitro and in vivo methods to examine the neuroprotective properties of an aqueous extract of WS in cerebral ischemia. In a mouse model of permanent middle cerebral artery occlusion (pMCAO), WS (200mg/kg) improved functional recovery and significantly reduced the infarct volume in both pre-treatment and post-treatment paradigms. Upon investigating the protective mechanism/s induced by WS, we found that it upregulated the expression of hemeoxygenase 1 (HO1) and attenuated the expression of the pro-apoptotic proteins poly(ADP-ribose) polymerase-1 (PARP-1) and apoptosis inducing factor (AIF), via the PARP-1-AIF pathway. WS also reduced levels of the negative guidance cue- semaphorin3A (Sema3A), which suggests a potential role in stroke recovery. Overall, the protective effects of WS could, at least in part, be attributed to a combination of its anti- oxidant, anti-apoptotic, and restorative effects. Our results suggest that WS could be a potential prophylactic as well as a therapeutic agent aiding stroke repair. iii Acknowledgements This dissertation is the result of relentless fixation over a plant-Withania somnifera, for a little more than five years. A commitment of this magnitude would not have been possible without the support and understanding of the following people: Dr. Zahoor Shah, my mentor, who has been a source of inspiration, guidance, and patience throughout my research tenure. I owe my next step into independent scientific research, entirely to him. My committee members: Dr. James Slama, Dr. Hermann von Grafenstein, and Dr. Youssef Sari, whose expertise and guidance helped keep the research on track The Dean-Dr. Johnnie Early and Dr. Wayne Hoss, who provided many opportunities to hone my extra-curricular skills and refresh my perspective about research Dr. Shadia Nada, who taught me all that I know about molecular biology My lab members Jatin, Anusha, Qasim, and Kevin, who have been a great source of help My friends Timnit, Divya, Duane, and Chris, with whom I’ve had many intellectual discussions as well as plain banter My husband Bharat, who has overwhelmed me with his support and love during testing times, and my family back home, whose care and blessing didn’t ever feel continents apart iv Contents Abstract iii Acknowledgements iv Contents v List of Figures ix 1 Introduction 1 1.1 What is Cerebral Ischemia………………………………………………… 1 1.2 Current Scenario of Ischemic Stroke Therapy…………………………….. 3 1.3 Withania somnifera: an introduction………………………………………. 6 1.4 The Bioactive Effects of Withania somnifera in CNS Disorders………….. 8 1.4.1WS in Ischemic Stroke………………………………………………... 10 1.5 Cellular Cascades Activated by Ischemic Stroke…………………………. 12 1.5.1 Oxidative stress and Cerebral Ischemia……………………………… 15 1.5.1.1 Hemoxygenase 1 (HO1) for Combating Oxidative Stress……… 16 1.5.2 Apoptosis and Cerebral Ischemia……………………………………. 18 v 1.5.2.1 PARP-1-AIF pathway and Bcl-2 as Targets for Preventing Apoptosis…………………………………………………………….. 20 1.5.3 Repair Mechanisms after Cerebral Ischemia………………………… 21 1.5.3.1 Semaphorin-3A (Sema3A) and the Wnt Pathway as Targets for Promoting Repair after Stroke………………………………….. 23 1.6 Animal Models of Stroke……………………………………………………… 25 1.7 Hypothesis and Specific Aims ………………………………………………… 27 2 Materials and Methods 29 2.1 Withania somnifera Aqueous Root Extract Preparation……………………….. 29 2.2 Rat Pheochromocytoma (PC12) Cell Culture………………………………….. 30 2.3 WSE Treatment Regimen for the in vitro Experiments………………………… 31 2.4 Cell Viability Assays using the MTT Method…………………………………... 31 2.5 Western blotting/Immunoblotting Procedures for the in vitro Study…………… 32 2.5.1 Protein Extraction from PC12 Cells………………………………………... 33 2.5.2 Protein Quantification using Bradford Assay……………………………… 33 2.5.3 Western blotting…………………………………………………………… 34 2.6 Animal Studies………………………………………………………………….. 37 2.7 Permanent Middle Cerebral Artery Occlusion (pMCAO)……………………… 37 2.8 Drug Dosing Regimen………………………………………………………….. 38 2.9 Locomotor Activity……………………………………………………………… 39 vi 2.10 Neurological Deficits Analyses………………………………………………… 40 2.11 Infarct Volume Analyses………………………………………………………. 40 2.12 Immunohistochemistry Procedures…………………………………………….. 42 2.12.1 Perfusion of Mice Brains and their Isolation……………………………… 42 2.12.2 Tissue Sectioning…………………………………………………………. 42 2.12.3 TUNEL Assay…………………………………………………………….. 43 2.12.4 Immunohistochemical Analysis………………………………………….. 44 2.13 Western blotting Procedures for the in vivo Study…………………………….. 45 2.14 HPLC Fingerprinting of the Withania somnifera Extract……………………… 46 2.15 Statistical Analyses……………………………………………………………. 47 3 Results 49 3.1 WSE Protects PC12 Cells from H2O2-induced Oxidative Damage…………… 49 3.2 WSE Induces the Expression of HO1 when Exposed to H2O2………………… 51 3.3 WSE Pre-treatment Attenuated Infarct Volume and Improved Functional Outcomes when Mice were subjected to pMCAO……………………………… 53 3.4 Mice Subjected to pMCAO and Post-treated with WSE had Attenuated Infarct Volume and Improved Functional Outcomes…………………………………… 56 3.5 WSE Treatment did not have a Considerable Effect on the Neurological Deficits Suffered by Mice Subjected to pMCAO………………………………………… 59 vii 3.6 WSE Pre-treated Mice Show Higher Levels HO1 in Mice Brain Cortices............ 61 3.7 WSE Pre-treated Mice show Lower Expression of Pro-apoptotic Proteins-PARP-1 and AIF………………………………………………………………………….. 63 3.8 WSE Pre-treatment Prevents the Nuclear Translocation of AIF under Ischemic Conditions………………………………………………………………………. 65 3.9 WSE Pre-treatment does not Significantly Alter Bcl-2 Levels in Mice that Underwent pMCAO…………………………………………………………….. 68 3.10 WSE Pre-treatment Attenuates the Expression of Sema3A in Mice Brains Following Ischemic Stroke……………………………………………………… 70 3.11 WSE Pre-treatment does not Substantially Affect Wnt Expression…………… 72 3.12 WSE Pre-treatment does not Perturb the Wnt Signaling Pathways……………. 74 3.13: HPLC Fingerprinting of WSE and Determining Abundance of Two Bioactive Molecules-Withanoside-IV and Withanolide A………………………………… 76 4 Discussion 78 5 Conclusion and Future Directions 91 References 93 viii List of Figures 1-1 Schematic description of ischemic stroke and hemorrhagic stroke……………... 2 1-2 Structures of some of the withanolides isolated from Withania somnifera……… 7 1-3 Schematic of WS-mediated mechanisms in CNS disorders……………………… 11 2-1 Drug dosage regimen for pre-treatment and post-treatment studies……………… 39 3-1 The effect of WSE on PC12 cells when subjected to H2O2-mediated oxidative stress……………………………………………………. 50 3-2 The effect of WSE on HO1 expression levels in PC12 cells subjected to oxidative stress………………………………………………………………………………... 52 3-3a,b Cortical infarct volume in WSE pre-treated mice subjected to pMCAO……….. 54 3-3c Comparison of the locomotor activity in WSE pre-treated and vehicle treated mice……………………………………………………………………….. 55 3-4a,b Cortical infarct volume in mice treated with WSE after pMCAO……………… 57 3-4c Comparison of the locomotor activity in WSE post-treated and vehicle treated mice………………………………………………………………. 58 3-5 NDS measurement in WSE pre- and post-treated mice on day 7 after pMCAO….. 60 ix 3-6a,b HO1 expression in mice pre-treated with WSE (200 mg/kg)………………….. 62 3-7a-c PARP-1 and AIF expression levels in mice pre-treated with WSE (200 mg/kg)……………………………………………………….. 64 3-8 Sub-cellular localization of AIF in WSE pre-treated mice subjected to pMCAO… 67 3-9 Bcl-2 expression in mice pre-treated with WSE (200 mg/kg)…………………….. 69 3-10 Sema3A levels in mice pre-treated with WSE (200 mg/kg)……………………… 71 3-11 Wnt1 expression in WSE pre-treated mice……………………………………….. 73 3-12a,b Expression levels of GSK3-β and CRMP2 in WSE pre-treated mice…………. 75 3-13a,b HPLC fingerprint of WSE and its composition in terms of markers-withanoside-IV and withanolide A…………………………………… 77 x Chapter 1 Introduction 1.1 What is Cerebral Ischemia? Brain stroke is a cerebrovascular disease characterized by the loss of blood supply to regions of the brain. This occurs either due to the obstruction of the blood vessel as a result of a clot (ischemic stroke/cerebral ischemia) or its rupture that prevents blood flow (hemorrhagic stroke) (Figure 1-1). The brain is uniquely sensitive to the rundown of oxygen and glucose stores; this manifests as large-scale cell damage and demise followed by loss of function in areas of the brain perfused by the compromised artery/s. The functional
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