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 ramifications of stroke damage commonly include paralysis, vision problems, and memory loss (American Heart Association, About Stroke, 2014 http://www.strokeassociation.org/STROKEORG/AboutStroke/About-
Stroke_UCM_308529_SubHomePage.jsp).
Stroke is the fourth leading cause of death and the leading cause of long-term disability in the United States (Go, Mozaffarian et al. 2014). Each year, about 795,000 people
1 experience new or recurrent strokes in the United States, which translates to someone having a stroke every 40 seconds. It is predicted that by 2030, there will be a 20.5% increase in stroke prevalence from what was observed in 2012. The direct and indirect costs of stroke care amounted to $36.5 billion in 2010 (Go, Mozaffarian et al. 2014).
Apart from the economic costs of stroke therapy, there is tremendous societal burden on patients and caregivers due to the long-term disability associated with it. It is therefore important to be aware of the risk factors and warning signs of stroke. Common risk factors of stroke include high blood pressure, diabetes, smoking, heart disorders, high cholesterol, physical inactivity, and chronic kidney disease (Go, Mozaffarian et al. 2014).
Figure 1-1: Schematic description of ischemic stroke and hemorrhagic stroke
The left panel depicts ischemic stroke, which occurs due to a blood clot lodged in a cerebral artery, and the right panel shows a stroke that occurs due to rupture of a cerebral blood vessel. Both types of stroke result in loss of blood supply and subsequent cell death in areas of the brain (Picture source: National Stroke Association www.stroke.org)
2 Ischemic strokes account for 87% of all stroke cases followed by 10% intracerebral hemorrhage and 3% subarachnoid hemorrhage. Therefore, cerebral ischemia formed the focus of our study. Ischemic strokes can been further classified as embolic and thrombotic strokes (American Heart Association; About Stroke, 2014). Embolic strokes result from blood clots that develop in other locations—generally the heart and the large arteries in the neck and upper chest—that enter the brain via the circulatory system and cause an obstruction in the smaller arteries. Thrombotic strokes occur when a clot develops within blood vessels supplying the brain; this happens sometimes due to the accumulation of fatty deposits in the lumen of the artery.
The staggering mortality, debilitating outcomes and the exorbitant costs associated with this disease have spurred extensive drug development and rehabilitative efforts. Research is also ongoing for the development of preventive strategies that reduce the probability of stroke occurrence in susceptible individuals. The following section focuses on the current scenario of stroke therapy, particularly that of cerebral ischemia, and its future scope.
1.2 Current Scenario of Ischemic Stroke Therapy
Presently, there is only one FDA-approved therapy for stroke—recombinant tissue plasminogen activator (rtPA) (Zivin 2009). It is a serine protease that is endogenously present on endothelial cells; it causes thrombolysis by catalyzing the conversion of plasminogen to plasmin, an enzyme important for clot dissolution. It is produced using recombinant DNA technology and administered to patients in the acute stage of stroke.
At the time of approval, tPA carried a highly restrictive time window of administration within three hours following onset of stroke symptoms. This was an impractical goal to
3 achieve and resulted in many patients being excluded from its use (Zivin 2009).
However, recent clinical trials suggest that although the three-hour time window is optimal for reversing stroke damage, drug administration as late as six hours following stroke onset could improve functional outcomes (group, Sandercock et al. 2012).
However, despite the extension of the time window of action, tPA therapy is still riddled with issues of time, cost, hemorrhagic adverse effects, and other exclusion criteria like age and hypertension (Zivin 2009). This prevents a huge subset of stroke patients from gaining effective stroke therapy.
Therefore, there is a dire need to develop an effective therapeutic agent for stroke.
However, this is becoming an increasingly daunting goal due to the extensive clinical failure of neuroprotective agents that were successful in various pre-clinical studies. This bleak translational power of drug candidates for stroke has led to the reassessment of pre- clinical testing paradigms. The poor performance of drugs at the clinical level could be attributed to reasons like:
(a) Heavy reliance at the pre-clinical level on drug administration within three hours after stroke: A mixture of prophylactic treatment and several doses of a drug after stroke would give a better indicator of clinical success (Gladstone, Black et al. 2002)
(b) Use of histological outcomes as the prime indicator of success at the pre-clinical level, whereas clinical success depends on improvement in functional recovery
(Gladstone, Black et al. 2002)
(c) Extrapolating the efficacy of the drug based on performance in a single animal model of ischemic stroke: The conventional view that the transient model of stroke is the
4 clinically relevant one has been debunked (Hossmann 2012) and researchers now favor the permanent model of focal ischemia (discussed in detail in Section 1.6). The Stroke
Therapy Academic Industry Roundtable (STAIR) recommends that drugs be evaluated in both permanent and transient models in both rodent and primate models before advancing them to clinical trials (Stroke Therapy Academic Industry 1999).
(d) Drugs targeted at a single molecular target: Considering the complex cascade of events that ensue after stroke (Section 1.5), it is impractical to adopt a ‘magic bullet’ approach to stroke therapy. Multiple drugs or even single drugs acting at multiple targets are preferred candidates (Gladstone, Black et al. 2002).
The wealth of information gleaned from the pitfalls encountered in clinical trials has reoriented pre-clinical stroke research. Natural products are being increasingly tapped into in search of potential leads (Newman and Cragg 2007). They provide unique chemical diversity that could be either manipulated to design novel structures or used directly as drugs against targets of choice. Natural products drug discovery has been substantially simplified with the advent of sophisticated analytical techniques and screening procedures (Rishton 2008). The inherent complexity of natural products makes them amenable to polypharmacology—a new paradigm in drug discovery which transforms the traditional ‘one drug one target’ view to ‘one drug multiple targets’
(Reddy and Zhang 2013). This has the advantage of modulating multiple targets at once, which is important in the rapidly changing cellular milieu observed in stroke. Crude plant extracts are gaining popularity due to polypharmacology exhibited by their constituents; several of them are in the pre-clinical pipeline for stroke therapy.
5 1.3 Withania somnifera: an introduction
Withania somnifera (WS), also commonly known as ‘Ashwagandha’ or Winter cherry is a shrub that has been used for centuries to treat a host of ailments in the ayurvedic as well as indigenous systems of medicine as an aphrodisiac, nerve-tonic, anti-inflammatory and anti-cancer agent, and considered to have therapeutic potential in many cognitive and neurological disorders (Kulkarni and Dhir 2008). It belongs to the family solanaceae, which is commonly known as the nightshade family. Its etymology traces back to the
Latin name for sleep-inducing—somnifera.
The major constituents of this plant include steroidal lactones and alkaloids; these structures are together called withanolides and are responsible for most of its biological effects. These withanolides have an ergostane scaffold with a C9-side chain in the form of a lactone ring (Elsakka, Grigorescu et al. 1990), generally attached to the ‘D’ ring of the steroid (Figure 1-2). Some of the withanolides exist as glycosides formed with the hydroxyl group on the ‘A’ ring (e.g. withanoside-IV) or with the lactone (e.g. sitoindoside). Due to the structural resemblance of withanolides to the ginsenosides present in Panax ginseng, Ashwagandha is also known as the Indian Ginseng (Grandhi,
Mujumdar et al. 1994). Twelve alkaloids, 35 withanolides, and some sitoindosides have been isolated from WS and tested for their bioactive effects in various disorders.
Amongst the bioactive alkaloids, withanine is found to be the most important; the other isolated alkaloids include somniferine, somnine, cuscohydrine, tropine, and anaferine
(Kulkarni and Dhir 2008). The most researched withanolide is withaferin A, which has been found to inhibit angiogenesis and therefore protective in many types of cancers
(Szarc vel Szic, Op de Beeck et al. 2014). However, this property is undesirable in a drug
6 candidate for stroke (discussed in Section 1.5.3), although its anti-inflammatory properties might be helpful in treating the microglial inflammatory sequelae following stroke (Min, Choi et al. 2011). Withanolide A, withanoside-IV, and some sitoindosides are amongst the individual components being pursued for their effects on nervous system disorders (Zhao, Nakamura et al. 2002, Kulkarni and Dhir 2008).
Figure 1-2: Structures of some of the withanolides isolated from Withania somnifera
7 Withanolides are steroidal lactones with an ergostane skeleton and the lactone ring attached to the D ring of the steroid. Withanosides have the glycosidic linkage on the A ring, whereas sitoindosides could have them either on the lactone or both the lactone and the A ring.
1.4 The Bioactive Effects of WS in CNS Disorders
WS has been found to have an effect in most disorders affecting the CNS, particularly those that cause neurodegeneration. Most studies utilize aqueous or hydroalcoholic extracts of WS roots, although the leaves (Kataria, Wadhwa et al. 2012) and fruits (Alam,
Hossain et al. 2012) have bioactive constituents too.
Withanolides possess acetylcholinesterase and butyrylcholinesterase activity—a property, which in addition to its calcium antagonistic effects makes it an ideal candidate for
Alzheimer’s disease (AD) therapy (Choudhary, Nawaz et al. 2005). WS components have been able to reverse cognitive deficits and the reduction in cholinergic markers in an ibotenic acid model of AD (Glotter 1991). The loss of synapses and neurite atrophy characteristic of AD and other dementias were reversed by withanoside-IV in an animal model of AD (Kuboyama, Tohda et al. 2006). This model also demonstrated the functional improvement in cognitive function along with a plausible molecular mechanism for the effect—increase in neurite outgrowth and induction of synaptogenesis and synaptic integration. Additionally, withanolide A has also been shown to improve regeneration of both axons and dendrites and synaptic reconstruction in an Aβ (25-35) mouse model of AD (Kuboyama, Tohda et al. 2005). Oxidative stress is often implicated
8 in AD-induced neurodegeneration; WS has also been shown to combat AD-related oxidative stress in an in vitro model of Aβ toxicity (Kumar, Seal et al. 2010).
WS extract (100 mg/kg; p.o.) improved locomotor activity and physiological abnormalities observed in an MPTP-induced mouse model of Parkinson’s disease (PD)
(RajaSankar, Manivasagam et al. 2009). The authors proposed that WS-mediated increase in the anti-oxidants—glutathione and glutathione peroxidase levels as well as reduction in lipid peroxidation could have led to the observed effect. Similarly, in a 6- hydroxy dopamine model of PD, WS (100 mg/kg, 200 mg/kg, 300 mg/kg) prevented oxidative stress damage by inducing the expression of the anti-oxidant panel: glutathione peroxidase, superoxide dismutase, and catalase in addition to improving dopamine function (Ahmad, Saleem et al. 2005). The oxidative damage and mitochondrial dysfunction prevalent in Huntington’s disease was also reversed by chronic administration of WS in rats (Kumar and Kumar 2009).
In addition to neurodegenerative conditions, WS also exerts palliative effects in neuropsychiatric conditions like epilepsy and anxiety due to its ability to increase levels of the inhibitory neurotransmitter gamma amino butyric acid (GABA) in the brain
(Kulkarni and Dhir 2008). WS is also widely known as an anti-stress agent, and shown to reduce markers of stress in various models of stress including foot-shock and the forced swimming tests (Dhuley 1998). Sitoindosides have been heavily studied for this effect; the mechanism is attributed to reduction of lipid peroxidation and DNA oxidation and increased levels of anti-oxidants (Bhattacharya, Ghosal et al. 2001). WS also possesses anti-inflammatory properties owing to its inhibition of inflammatory cytokines like tumor necrosis factor-alpha (TNF-alpha), IL-1beta and transcription factors like nuclear factor
9 kappa-light-chain-enhancer of activated B cells (NF-kappaB) (Singh, Aggarwal et al.
2007); it is being considered as a treatment option in inflammatory conditions like arthritis.
1.4.1 WS in Ischemic Stroke
Considering the recurrent role of the anti-oxidant effects of WS in neurodegenerative conditions, it is a logical extension that these properties would be useful in ischemic stroke, wherein oxidative stress is a major causative factor of cellular damage (Starkov,
Chinopoulos et al. 2004). Few studies have evaluated the effects of WS in cerebral ischemia; however, in contrast to our study, they were conducted in rats, used different stroke models, involved only WS pre-treatment, and failed to establish a molecular mechanism of action. In a rat model of transient middle cerebral artery occlusion, WS
(500mg/kg for 7 days) was found to significantly decrease the DNA fragmentation in the ischemic cerebral cortex as compared to that of control animals (Adams, Yang et al.
2002). Since DNA fragmentation is a hallmark of apoptosis, this points to a putative anti- apoptotic effect. In another study employing the same surgical procedure in rats, a 30-day pre-treatment with a hydroalcoholic extract of WS (1g/kg) significantly decreased the malondialdehyde (MDA) levels as compared to vehicle-treated rats. MDA being an oxidative stress marker, the authors suggested that its significant decrease in the WS treated group of animals could involve an increased anti-oxidant defense (Chaudhary,
Sharma et al. 2003).
Thus, the paucity of data on WS in other models of stroke, the effect of post-treatment, and the detailed molecular mechanism of its effects led us to devise the current study.
10 Based on WS’s mechanisms in other disorders and ischemia (Figure 1-3), its anti- oxidative, regenerative and anti-apoptotic properties seem to be prime candidates. Some other relevant mechanisms include its anti-inflammatory and anti-excitotoxic effects and glutamate modulation (Chaudhary, Sharma et al. 2003, Parihar and Hemnani 2003).
Figure 1-3: Schematic of WS-mediated mechanisms in CNS disorders
WS exhibits anti-oxidative (increase in levels of anti-oxidants glutathione (GSH), superoxide dismutase (SOD) and catalase), anti-inflammatory (reduction in levels of
TNF-alpha and NF-kappaB) and other effects such as inhibition of acetyl- (AChE) and butyrylcholinesterase (BChE) activities, and calcium antagonism. It also increases GABA levels, induces neurite regeneration and has anti-apoptotic properties. (Misc: miscellaneous; the upward and downward arrows stand for increase and decrease, respectively).
11 1.5 Cellular Cascades Activated by Ischemic Stroke
Ischemic stroke activates complex cellular mechanisms that function simultaneously and culminate in cell death. These mechanisms include but are not limited to: energy failure, excitotoxicity, accumulation of reactive oxygen species (ROS) and their damage, programmed cell death, and inflammatory processes (Culmsee and Krieglstein 2007).
When the cerebral blood flow dips down to 20-30 ml/min/100 g from the normal flow of
45-50 ml/min/100 g, energy disturbances begin to manifest (Dearden 1985); at levels lower than 10 ml/min, cells undergo anoxic depolarization—a condition that results from the malfunctioning of the Na+/K+ ATPase channel. The lack of oxygen and glucose causes a rundown of the ATP stores, which depletes the channel’s capacity to maintain the concentration gradients of Na+ and K+, leading to the accumulation of K+ in the extracellular space and the influx of Na+ and water (Balestrino 1995). This causes the cell-swelling, particularly in astrocytes, and compression of capillaries causing a ‘no- reflow’ phenomenon which prevents reperfusion for a certain time even after removal of occlusion (Rezkalla and Kloner 2002).
The high intracellular levels of sodium changes the concentration gradient required for the Na+/Ca2+ exchanger (NCX)-mediated Ca2+ extrusion, thereby causing intracellular accumulation of Ca2+(Blaustein and Lederer 1999). Increased levels of calcium is a key mediator of the deleterious effects of stroke damage (Culmsee and Krieglstein 2007).
Apart from the failure of NCX, Ca2+ influx can be caused by several other mechanisms.
The membrane depolarization caused by the disturbances of Na+ and K+ concentration gradients also leads to excess glutamate release, which is an excitatory neurotransmitter that acts via receptors— α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
12 (AMPA) and N-Methyl-D-aspartic acid (NMDA). This causes activation of voltage- dependent calcium channels leading to calcium influx (Rothman and Olney 1986).
Ischemia is also characterized by the reduced reuptake of glutamate from the synaptic space, which prolongs their receptor mediated activation (Rossi, Oshima et al. 2000); moreover, the excess calcium itself triggers further release of glutamate, thus setting up a positive feedback loop that amplifies the damaging effects of glutamate—a condition known as excitotoxicity. Neuronal death from excitotoxicity mainly results from Ca2+ overload, mitochondrial dysfunction, free radical generation, and secondary excitotoxicity (Rothman and Olney 1986). Clinical trials in the past have focused on drugs blocking the NMDA receptor, but have failed mainly because the excitotoxic damage is too rapid to salvage in a clinic, and that the long-term inhibition of NMDA might actually hinder neuronal survival (Ikonomidou and Turski 2002).
Calcium influx during ischemia can also be triggered by acid-sensing ion channels
(ASIC), which are activated by the low pH conditions that exist during ischemic conditions (Culmsee and Krieglstein 2007). Mitochondria are important organelles that maintain calcium homeostasis by sequestering the excess calcium (Duchen 2000); however this mechanism is overwhelmed in ischemia and leads to mitochondrial dysfunction (Lin and Beal 2006). This leads to failure of oxidative phosphorylation causing the release of free radicals. Additionally, ischemia causes a drop in the redox potential of mitochondria, which also generates ROS via the electron transport chain
(ETC). Overproduction of ROS further disrupts the ETC, hampers the calcium buffering capacity and metabolic function culminating in more ROS generation. ROS and other
13 reactive species cause oxidative modification of essential proteins, lipids, and DNA, which eventually results in cell death (Piantadosi and Zhang 1996, Lin and Beal 2006).
In addition to cell death that results from ROS damage and mitochondrial dysfunction, cell death can also be caused by other cues. Once again, calcium overload can act as a trigger by activating calpains, which are calcium-dependent proteases that cleave the cytoplasmic protein called the BH3-interacting domain death agonist (Bid). The truncated form of Bid called tBid acts as a common gateway for death signals to trigger apoptosis or programmed cell death (Culmsee and Krieglstein 2007). This protein (tBid) translocates to the mitochondrial membrane and interacts with pro-apoptotic protein Bax, which is usually inhibited by the anti-apoptotic members of the B‐cell leukaemia/lymphoma 2 (Bcl‐2) family proteins: Bcl-2 or Bcl‐xL. The levels of these protective proteins run low during ischemia, thereby potentiating the effects of tBid and
Bax. They form dimers that open the mitochondrial transition permeability pores, releasing apoptotic mediators like cytochrome c and apoptosis inducing factor (AIF), which induce caspase-dependent and independent apoptosis, respectively (Culmsee and
Krieglstein 2007).
Another contributor to ischemic damage is the inflammatory process initiated by leukocyte and macrophage infiltration, cytokines, adhesion molecules, prostanoids and nitric oxide, which eventually contributes to expansion of the infarct (Huang, Upadhyay et al. 2006). Inhibitors of inflammatory enzymes like cyclooxygenases and nitric oxide synthases could provide protection (Iadecola and Alexander 2001).
14 The next three sub-sections will focus in detail on the mechanisms relevant to the present study. It will also include an introduction of the proteins tested in the study and how they relate to the mechanisms discussed.
1.5.1 Oxidative stress and Cerebral Ischemia
Free radicals are continually produced as a result of oxidative phosphorylation in the mitochondria; they serve important functions when present in physiological concentrations (Valko, Leibfritz et al. 2007). Their levels are kept in check by a host of enzymatic anti-oxidants like glutathione peroxidase (GPx), catalase, superoxide dismutase (SOD), and peroxiredoxins. This process is also aided by the free-radical scavenging activity of non-enzymatic components like glutathione, ascorbic acid, pyruvate, α-tocopherol etc. (Yu 1994). When the oxidant/anti-oxidant balance is tipped in the favor of formation of free radicals and reduction in anti-oxidant response as seen in ischemic stroke (Saito, Maier et al. 2005), it leads to oxidative stress (Halliwell 1999).
This effect is extremely pronounced in neurons due to their high oxygen consumption and metabolic demand and inherently low anti-oxidant defense (Cooper and Kristal
1997).
Excitotoxicity is one of the prime contributors of free radicals (Bondy and LeBel 1993).
ROS and reactive nitrogen species (RNS) are responsible for the generation of free radicals. ROS include superoxide anion radical (O2•-), hydroxyl radical (OH•) and some precursors to radicals like hydrogen peroxide (H2O2) and singlet oxygen (O2*). H2O2 can form the OH• radical either in the presence of superoxide radical (Haber-Weiss reaction)
(Koppenol 2001) or in the presence of transition metal ions (Fenton reaction)
15 (Winterbourn 1995). Conversely, H2O2 could be converted to water in the presence of anti-oxidant enzymes like GPx and catalase (Droge 2002). RNS are comprised of reactive species derived from nitric oxide like NO•, which can combine with superoxide anion
- and H2O2 to form peroxynitrite anion (ONOO ) and OH•. Thus, the ROS generated from the ETC during mitochondrial dysfunction (Section 1.5) can combine with NO to form peroxynitrite, whose downstream events include DNA, protein and lipid damage and cell death (Iadecola 1997).
In addition to ETC and excitotoxicity-mediated production of ROS and RNS, excess calcium accumulation also contributes to it by the activation of phospholipases, cyclooxygenases and neuronal nitric oxide synthase (nNOS) (Kristian and Siesjo 1998).
Oxidative stress leads to cell death due to free-radical mediated irreversible oxidative modification of membrane lipids, cytoprotective proteins and DNA fragmentation (Ryter,
Kim et al. 2007).
Oxidative stress is so crucial to ischemic cell death that drugs acting as free-radical scavengers or inducers of endogenous anti-oxidant enzymes are ideal candidates for stroke therapy (Slemmer, Shacka et al. 2008).
1.5.1.1 Hemoxygenase 1 (HO1) as a Target for Combating Oxidative Stress
HO catalyzes the cleavage of pro-oxidant heme to form iron, carbon monoxide (CO), and biliverdin, which is reduced into bilirubin (BR). Two isoforms of HO have been distinguished and well studied (Shibahara, Muller et al. 1985, Dore 2002). HO1, the first to be isolated, is an inducible enzyme barely detectable in the brain under normal conditions, but many reports have shown that it is significantly increased after stress
16 conditions. HO1 is considered to be a heat shock protein that can be induced in a cell- specific manner by multiple stress factors like hyperthermia (Ewing, Haber et al. 1992),
Alzheimer’s disease (Smith, Kutty et al. 1994) and ischemia (Takeda, Kimpara et al.
1996). The infarct damage after stroke was significantly reduced by increasing HO1 activity in neurons (Panahian, Yoshiura et al. 1999). Moreover, HO1 knockout mice showed higher infarct volumes compared to wild type mice (Shah, Nada et al. 2011).
There are abundant heme-containing enzymes in mitochondria and endoplasmic reticulum (such as myoglobin, catalase, GPx, SOD, nitric oxide synthases, etc.) that undergo turnover during oxidative stress. HO1 is primarily responsible in neutralizing the free heme released from these hemoproteins and preventing them from causing oxidative stress (Bidmon, Emde et al. 2001). The salutary effects of HO1 stem not only from the degradation of heme but also from the protective effects of its reaction products. HO1 is the prime source of endogenous CO, which at physiological concentrations is known promote endothelial cell survival partly by mimicking the vasodilatory and proangiogenic effects of NO (Dore 2002, Dulak, Deshane et al. 2008). Additionally, CO is also known to have anti-apoptotic and anti-inflammatory effects (Otterbein, Bach et al. 2000, Al-
Owais, Scragg et al. 2012). Moreover, the antioxidative effect of HO1 is supplemented also by the free-radical scavenging properties of biliverdin and its reduced form, BR
(Dore 2002). Thus, the conglomerate anti-oxidant, angiogenic, anti-inflammatory and anti-apoptotic effects make HO1 a valuable cyto-protective enzyme.
17 1.5.2 Apoptosis and Cerebral Ischemia
Minutes after ischemic stroke, the precipitous reduction in ATP levels cause anoxic depolarization, excitotoxic damage and necrotic cell death. This forms the ischemic core wherein there is no perfusion. The core is surrounded by an edge of tissue with below- normal perfusion, and which is metabolically impeded yet viable known as the penumbra.
The penumbral region predominantly undergoes a form of delayed cell death known as apoptosis and presents a practical window for the action of neuroprotective drugs to prevent further cell death and infarct expansion (Broughton, Reutens et al. 2009).
Apoptotic pathways could either be intrinsic or extrinsic. Intrinsic pathways are activated by internal events/triggers. High intracellular Ca2+ levels act as one of the key initiators of the intrinsic pathway. Ca2+ activates calpains, which cause the truncation and the subsequent translocation of tBid to the mitochondrial membrane (Section 1.5). The tBid then induces conformational changes in proapoptotic proteins like Bak, Bax, Bad, and
Bcl-XS and forms heterodimers with them. Barring neutralization by anti-apoptotic members of the Bcl-2 family, these complexes open the mitochondrial transition pores and release groups of sequestered apoptogenic proteins like cytochrome c,
Smac/DIABLO and HtrA2/Omi although data is scarce on the latter two proteins
(Broughton, Reutens et al. 2009). What then follows is an apoptotic cascade that requires the activation of caspases. Upon release into the cytoplasm, cytochrome c binds to the apoptotic protein-activating factor-1 (Apaf-1) and procaspase-9 and forms an
‘apoptosome’. This process requires the consumption of ATP and acts as the activating step for caspase-9, which subsequently activates caspase-3 (Broughton, Reutens et al.
2009). Caspase-3 is the downstream mediator of caspase-mediated apoptosis; its levels
18 are elevated in ischemia (Rami, Sims et al. 2003), and its genetic abrogation or pharmacological inhibition has a neuroprotective effect in stroke (Endres, Namura et al.
1998, Le, Wu et al. 2002).
The mitochondrial transition pores also release apoptotic mediators like AIF, endonuclease G and Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP3), of which, AIF is likely the most investigated. Under conditions of energy depletion rampant in ischemia, the energy intensive caspase activation may be substituted with the AIF- mediated apoptotic pathway. This is resistant to inhibition by caspase inhibitors, thereby substantiating its caspase-independent role (Broughton, Reutens et al. 2009). Ischemia is known to trigger the translocation of AIF to the nucleus wherein it initiates large-scale
DNA fragmentation signifying the final steps of apoptosis. It was found that low levels of
AIF attenuated ischemic neuronal death and that the inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1) prevented the nuclear translocation of AIF and the resultant cell death (Culmsee, Zhu et al. 2005).
Finally, apoptosis can also be initiated by external signals via the extrinsic mechanism.
They function by the means of ligands activating death receptors on plasma membranes.
The death receptors comprise of the tumor necrosis factor receptor (TNFR) superfamily and include TNFR-1, Fas, and p75NTR as its members. In one such mechanism, the ligand for the Fas receptor-FasL binds to its receptor and activates a cytoplasmic protein Fas- associated death domain protein (FADD). This initiates an apoptotic cascade that involves procaspase-8 and the eventual activation of caspase-3 (Broughton, Reutens et al.
2009).
19
1.5.2.1 PARP-1-AIF pathway and Bcl-2 as Targets for Preventing Apoptosis
PARP-1 is a 116-kDa nuclear enzyme involved in DNA–base-excision-repair and modification of proteins by poly-ADP-ribosylation. DNA damage enhances the activity of this enzyme, which under physiological circumstances, controls cell repair, DNA transcription and replication and protein function by means of adding poly(ADP-ribose)
(PAR) polymers onto its substrates. However, excessive DNA damage causes the generation of large branch-chained PAR polymers, which signal cell death (Hong,
Dawson et al. 2004).
PARP-1 has been implicated in ischemic pathophysiology and animals with abrogated
PARP-1 gene show lower infarct volumes (Goto, Xue et al. 2002). Many mechanisms have been postulated to explain the cell death mediated by PARP-1: peroxynitrite induced mitochondrial damage, PARP-1 mediated NAD+ consumption and subsequent energy depletion and necrosis, and PAR-initiated death signals. However, none of these have been able to sufficiently explain all the features of PARP-1 mediated cell death.
There is growing evidence for the involvement of another mechanism—PARP-1 mediated AIF release.
AIF is a mitochondrial protein structurally similar to flavoproteins. It is thought to have oxidoreductase activity, although it is not required for its apoptotic effect. PARP-1 activators cause the translocation of AIF from the mitochondria to the nucleus resulting in nuclear condensation and cell death—a process that is not perturbed by caspase inhibitors. However, PARP-1 inhibitors prevent AIF translocation and antibodies to AIF
20 prevent PARP-1 dependent cell death program, indicating that AIF is an essential mediator of PARP-1 mediated cell death. Agents that inhibit AIF translocation might thus have great therapeutic potential (Hong, Dawson et al. 2004).
Bcl-2 is an antiapoptotic member of the Bcl-2 family that comprises of both pro- and anti-apoptotic proteins. The interaction between these members in the outer mitochondrial space is important to the apoptotic fate of the cell. The anti-apoptotic members (Bcl-2 and Bcl-xL) reside in the outer mitochondrial membrane and prevent apoptosis by maintaining mitochondrial redox potential, preventing the release of apoptotic factors and maintaining the integrity of the transition pores (Broughton,
Reutens et al. 2009). Bcl-2 and Bcl-xL upregulation has neuroprotective effects
(Martinou, Dubois-Dauphin et al. 1994, Wiessner, Allegrini et al. 1999), whereas Bcl-2 knockout mice show higher infarct size (Hata, Gillardon et al. 1999).
1.5.3 Repair Mechanisms after Cerebral Ischemia
Following ischemic stroke, the nervous system spurs a plethora of mechanisms that together reconstruct severed networks with the aim of eliciting functional recovery. These processes are active under normal conditions and are amplified under pathological conditions like stroke. They include neurogenesis, angiogenesis, and synaptic plasticity
(Font, Arboix et al. 2010).
Neurogenesis or the formation of new neurons occurs mainly in the subgranular zone of dentate gyrus, subventricular zone of some cortical areas, substantia nigra and perinfarcted areas. The process begins with the proliferation of neuronal precursor cells
(NPC) in these areas, their migration to the injured site, differentiation into mature
21 neurons, and final functional integration into the circuit. Although stroke is a potent inducer of neurogenesis, not many precursor cells survive to differentiate into mature neurons. Therefore, exogenous application of neural stem cells has gained interest as a therapy to increase the number of surviving, functional neurons and has indeed met with moderate success (Meamar, Dehghani et al. 2013).
Blood vessels play an important role in the providing a migratory route for the NPCs; they also provide the support required for their survival, thus functioning as a neurovascular unit. Hence, angiogenesis or the formation of new blood vessels is vital for neurogenesis, and pro-angiogenic factors like vascular endothelial growth factor (VEGF) promote both angiogenesis and neurogenesis. VEGF administration and modulation of its receptor-VEGFR is known to stimulate the recovery process; targeting this system has tremendous recovery potential (Ruiz de Almodovar, Lambrechts et al. 2009).
The final functional recovery after stroke involves the integration of the neurovascular niche to reestablish the network. This is achieved by inducing synaptogenesis and activating silent synapses, thus reversing the excitotoxicity-inflicted damage to synaptic connections (Font, Arboix et al. 2010).
The repair processes stimulated after ischemia face a major obstacle to their progress— the negative environment established by the overexpression of molecules that impede axonal sprouting. These inhibitory molecules belong to three classes: myelin associated proteins like NogoA and myelin-associated glycoprotein; extracellular matrix proteins like chondroitin sulfate proteoglycans and tenascin; developmentally associated axonal
22 guidance molecules like ephrin A & B and semaphorins (Carmichael 2008). Thus, successful recovery can only result after the inhibitory environment is subdued.
1.5.3.1 Semaphorin-3A (Sema3A) and the Wnt Pathway as Targets for Promoting
Repair after Stroke
Sema3A is an important member of the semaphorin family of negative guidance cues known to inhibit axonal growth and cause growth cone collapse. Its actions are mediated by neuropilin-1 (NRP-1), a member of the NRP family of receptors that function as co- receptors with plexins and VEGFRs to facilitate signal transduction (Hou, Keklikian et al.
2008). Both Sema3A and NRP-1 levels were found to be elevated for a long time after ischemia, preventing neurons from integrating into the circuit and thwarting any functional recovery (Hou, Keklikian et al. 2008). Therefore, surmounting this barrier could be a viable target for stroke recovery (Pekcec, Yigitkanli et al. 2013).
Another interesting facet of Sema3A action is the selective inhibition of VEGF-mediated angiogenesis acting via NRP1, which is common to both Sema3A and VEGF. Thus, inhibition of Sema3A could also have the potential consequence of removing its anti- angiogenic influence, thereby indirectly promoting repair (Acevedo, Barillas et al. 2008).
The Wnt signaling pathway influences the NPC proliferation and differentiation steps of neurogenesis during embryonic development, and is a key mediator of adult hippocampal neurogenesis (Mu, Lee et al. 2010). Wnts are secreted proteins pivotal to the development of the central nervous system; their roles as diverse as specifying cell polarity, neuronal migration, axon guidance, and synaptogenesis to name a few. Wnts are highly conserved and function by activating many signaling cascades via its receptors: transmembrane
23 frizzled receptors (Fzd), tyrosine kinases and, the recently discovered, insulin-like growth factor 1 (IGF1) (Rosso and Inestrosa 2013). They function via two distinct pathways:
Wnt/β-catenin or the canonical pathway and the β-catenin independent or non-canonical pathway (Oliva, Vargas et al. 2013). The canonical Wnt/β-catenin pathway is most studied in terms of both cortical and hippocampal neurogenesis, cell differentiation, proliferation, and synaptic plasticity (Hirabayashi, Itoh et al. 2004, Toledo, Colombres et al. 2008). Moreover, its activation is associated with improved neurogenesis after focal cerebral ischemia (Shruster, Ben-Zur et al. 2012).
The pathway involves transcription of Wnt target genes via stabilization of β-catenin. In the absence of Wnt, the kinases casein kinase-1α (CK-1α) and glycogen synthase kinase-
3β (GSK-3β) sequentially phosphorylate and signal β-catenin for ubiquitination and subsequent degradation by the proteasome. The downstream events of Wnt signaling phosphorylate and inactivate GSK-3β, thereby preventing β-catenin degradation, which is now free to translocate to the nucleus and commence transcription (Oliva, Vargas et al.
2013).
The non-canonical Wnt pathway plays a role in cell movement, tissue separation, cardiogenesis and myogenesis amongst many others (Veeman, Axelrod et al. 2003). It functions either by activating C-jun N-terminal kinase (JNK) to maintain tissue polarity or by triggering the intracellular calcium release and concomitant activation of calcium dependent kinases like Ca2+-calmodulin-dependent protein kinase II (CaMKII), calcineurin, and protein kinase-C (PKC). The kinases JNK and CaMKII have both been implicated in the ischemic pathophysiology; their inhibition is linked to improved outcomes after stroke (Benakis, Bonny et al. 2010, Coultrap, Vest et al. 2011). The
24 collapsin response mediator protein-2 (CRMP-2), a regulator of neuronal plasticity and neurite outgrowth, is a common substrate for both JNK and CaMKII. Its phosphorylation at Thr555 by these kinases (Schlessinger, Hall et al. 2009) (Arimura, Menager et al.
2005) (Hou, Jiang et al. 2009) inhibits axonal growth. The apparent deleterious effects of the activation of the non-canonical pathway in stroke is further supported by the evidence to suggest that both these pathways might serve opposite functions (Veeman, Axelrod et al. 2003).
1.6 Animal Models of Stroke
Having discussed the pathophysiology of stroke, it is important to understand how stroke is modeled in animals, in order to assess the effectiveness of a drug.
Stroke models can be broadly classified into two groups: global ischemia and focal ischemia. Global ischemia is characterized by the cessation of cerebral blood flow (CBF) throughout the brain. When it is carried out by the bilateral occlusion of the common carotid artery, it is called complete global ischemia. Sometimes, only unilateral occlusion is conducted, which leads to a less severe reduction in CBF called incomplete global ischemia. Gerbils are the animals of choice for this model (Graham, McCullough et al.
2004).
Focal ischemia models involve blood flow reduction in a restricted area of the brain supplied by one of the major cerebral arteries. Since most strokes occur due to the occlusion of a cerebral artery in the brain, this model is the most studied (Liu and
McCullough 2011). Focal models usually involve occlusion of the middle cerebral artery
(MCA) and are classified into transient middle cerebral artery occlusion (tMCAO) and
25 permanent occlusion models (pMCAO). The intraluminal suture method is one of the most common ways to induce tMCAO. A surgical filament is introduced through the internal carotid artery (ICA) to occlude the origin of the MCA. The removal of the suture restores perfusion and the occlusion is therefore transient (Canazza, Minati et al. 2014).
This model is often characterized by larger lesions, higher neurological deficits and mortality rate. Once thought to be the clinically relevant model, recent failures at the clinical trials have raised serious questions about the predictive capacity of this model
(Hossmann 2012).
On the other hand, the permanent model (pMCAO) involves irreversible occlusion by means of clipping or coagulating the MCA. This model has relatively higher survival rates and produces consistent infarcts restricted to the cortical region. This makes it amenable to conduct long-term functional outcome studies. Recently, this has become the model of choice for testing neuroprotective agents in the pre-clinical level considering that agents tested solely in transient models have failed at the clinic so far (Hossmann
2012). It is suggested that pMCAO is the more stringent model because it usually takes a higher dose of the drug candidate to show efficacy in this model. Therefore, it is believed that compounds effective in this model have better chances of clinical success (Richard
Green, Odergren et al. 2003).
In addition to the selection of the appropriate model, it is also imperative to choose outcomes relevant to our end goal. In this regard, a high premium is placed on assessing functional recovery after surgery. It is also important to consider that stroke is a sexually dimorphic disease that often affects the elderly and presents with various comorbidities at
26 the clinic (Liu and McCullough 2011). A model that could account for these variables would provide answers that closely reflect the situation at the clinic.
1.7 Hypothesis and Specific Aims
Before undertaking any scientific research it is essential to know the status quo of the field of chosen study. This is basically the null hypothesis or the default position that the study needs to test and, in the end, conclude whether or not the findings of the study are sufficient to reject the hypothesis. The null hypothesis for our study is:
An aqueous extract of Withania somnifera roots has the same effect as that of a placebo
when tested in experimental models of ischemic stroke
Based on a thorough understanding of the mechanistic and logistic underpinnings of ischemic stroke, we formulated a set of aims to test the hypothesis:
Aim 1: To determine the effects of WS extract in an in vitro model of oxidative stress
We will use the H2O2-model of oxidative stress as a preliminary screen for WS’s neuroprotective actions.
Aim 2: To determine stroke damage in mice pre-treated with WS extract and subjected to pMCAO This is meant to evaluate the potential of WS to be used as a prophylactic treatment for ischemic stroke. We will test mice for functional recovery in terms of locomotor activity and neurological deficits.
27 Aim 3: To determine stroke damage in mice subjected to pMCAO and post-treated with WS extract
We will evaluate the therapeutic potential of the extract in stroke by administering it as a post-treatment; Functional outcomes will also be assessed.
Aim 4: To delineate the molecular mechanisms of action of WS that could explain its observed effects in the aims 1-3 We will evaluate WS’s effects on known markers of oxidative stress, apoptotic cell death and recovery mechanisms in ischemic stroke. Targeting multiple cellular mechanisms is a desired property in a neuroprotective agent.
Aim 5: To establish a molecular fingerprint of the WS extract
We will use HPLC to analyze the extract and create a fingerprint that can be used for comparison purposes in future studies. We will also test for quantities of known bioactive constituents of WS: withanoside-IV and withanolide A.
28 Chapter 2
Materials and Methods
2.1 WS Aqueous Root Extract Preparation
Ashwangandha herbal supplement caplets were purchased from Himalaya pure herbs
(Himalaya Healthcare, Texas, USA). It is a commercially available supplement containing a mixture of a supercritical carbon dioxide extract (10 mg) and standardized root extract of organic ashwagandha root (Withania somnifera Dunal) (280 mg) and organic ashwagandha root powder (380 mg) per caplet as per the label claim. The product was subjected to chromatographic fingerprinting at the site of manufacture to ensure herbal potency.
We prepared the extract from the caplets using a procedure slightly modified from Kumar et al (Kumar, Seal et al. 2010). The caplets were crushed to a coarse powder, weighed, and infused in freshly boiled deionized water (1:50 w/v) for 25 min. The infusion was left to cool to room temperature and centrifuged at 5000 rpm for 20 min. The supernatant was re-centrifuged at 5000 rpm for 15 min. The pellet was discarded, and the supernatant was subjected to a wash with chloroform or methylene chloride (10-12% of the volume of supernatant) to minimize the interference from oily substances that make the final extract
29 sticky and difficult to handle. The chloroform/methylene chloride layer was discarded, and the aqueous layer was concentrated in a rotary vacuum evaporator. This step also aided evaporation of traces of organic solvent present, which would otherwise interfere with the freeze-drying process. The concentrated aqueous fraction was then freeze-dried, upon which a hygroscopic, yellowish-brown powder was obtained. Accurately weighed freeze-dried aliquots of the powder were reconstituted in physiological saline (0.9%
Sodium chloride) or distilled water. Aliquots were stored in light-resistant containers at -
20 °C until further use. This aqueous extract shall henceforth be called WSE (Withania somnifera extract) throughout the text.
2.2 Rat Pheochromocytoma (PC12) Cell Culture
PC12 cells were purchased from American Type Culture Collection (ATCC, Manassas,
VA) and cultured in RPMI 1640 medium (Mediatech, Inc, Manassas, VA) supplemented with 10% horse serum (Life Technologies, Carlsbad, CA), 5% fetal bovine serum
(HyClone, Thermo scientific, Waltham, MA, USA), penicillin/streptomycin (100 units/mL; Fisher, Hanover Park, IL), amphotericin B/fungizone (Fisher, Hanover Park,
IL, USA), and 1 mM L-glutamine (Fisher). Cells were grown on 60-mm tissue culture plates pre-coated with poly-L-lysine (50 µg/ml; Sigma-Aldrich, St. Louis, MO, USA) and incubated under humidified atmosphere of 5% CO2, 95% air at 37 °C. The medium was replaced with fresh medium once in two days and cells were split in a ratio of 1:2 when
80% confluent. Trypsin (0.05%; HyClone, Thermo scientific) was used to disengage cells and 1X phosphate buffered saline (1X PBS) was used to wash cells during the splitting procedure.
30 2.3 WSE Treatment Regimen for the in vitro Experiments
The in vitro experiments were designed to study the effect of WSE when PC12 cells were subjected to hydrogen peroxide (H2O2) mediated oxidative stress. A combination of cell viability assays and Western blotting procedures was used to test this effect. In case of the cell viability assay, PC12 cells were pre-treated for 24 h with different concentrations
(25, 50, 100 and 200 µg/ml) of WSE or vehicle (0.9% NaCl). For the Western blotting study, the best WSE concentration from the cell viability assay was used, which in our case was 100 µg/ml. At the end of the treatment period, cells were challenged with H2O2
(100 µM) for additional 24 h.
2.4 Cell Viability Assays using the MTT Method
PC12 cells were counted using the trypan blue exclusion test. Trypan blue dye enters and selectively stains dead cells because they have leaky cell membranes. This test therefore excludes dead cells and helps ensure the integrity of the cells being tested. Viable PC12 cells were plated at a density of 0.12 x 106 per well in 24 well plates (pre-coated with poly-L-lysine) and incubated for 24 h to allow for attachment of cells to the surface of the plate. Following this, cells were treated to WSE/vehicle as mentioned in the treatment protocol in Section 2.3. Control cells received no treatment. At the end of the oxidative stress insult, cells were assessed for their viability using the MTT assay kit (Promega,
Madison, WI, USA). MTT or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is a yellow-colored tetrazolium dye, which when reduced, forms a purple formazan product. Metabolically active cells have functional NAD(P)H-dependent oxidoreductase enzymes that carry out this reduction reaction. Thus, under defined
31 conditions, the colorimetrically determined absorbance at a wavelength where the purple dye shows maximum absorbance would be proportionate to the viability of cells being tested.
For the experimental procedure, cells were treated with the tetrazolium dye reagent (25
µl/well) and incubated at 37 °C for 2 h followed by addition of the stop solution (125
µl/well) to terminate the reaction and solubilize the formazan. Following incubation for 1 h, the reaction mixture from each well was transferred to appropriately labeled microcentrifuge tubes and centrifuged at 14,000 rpm for 1 min. The supernatant was pipetted into 96-well plates for absorbance measurement at 570 nm using Synergy Hybrid plate reader (BioTek, Winooski, VT, USA). Within an experiment, each treatment was done in triplicate, and each experiment was conducted three times with separate batches of cell cultures. The plating cell density was optimized so that the absorbance stays within the linear range.
2.5 Western blotting/Immunoblotting Procedures for the in vitro Study
For this study, only a single concentration of WSE (100 µg/ml) was tested for its ability to modulate the expression of the anti-oxidant enzyme HO1. PC12 cells were plated at a density of 3.2 x 106 onto 60 mm petri dishes, followed by the drug and oxidative stressor treatment outlined in Section 2.3. Upon completion of this step, several preparative procedures were done to obtain cellular protein for the immunoblotting experiment. They are listed below in the order in which they were carried out:
32 2.5.1 Protein Extraction from PC12 Cells
The cells were prepared for protein extraction by removing their growth medium and gently washing them with 1X PBS. The cells were then lysed using 1X RIPA buffer containing 20mM Tris HCL (pH 7.5), 150 mM Nacl, 1 mM Na2EDTA, 1 mM EGTA, 1%
NP-40, and 1% sodium deoxycholate. Prior to lysis, the RIPA buffer was supplemented with protease inhibitors (1:100 dilution; Protease inhibitor cocktail kit, Thermo scientific), phosphatase inhibitors (0.1 mM sodium pyrophosphate, 50 mM sodium fluoride, 10 mM sodium vanadate), 1 mM dithiothreitol (DTT), and 1 mM phenylmethanesulfonylfluoride (PMSF). Each plate received about 250-300 µL of the lysis buffer so prepared, and placed on ice for a minute to ensure distribution of the buffer. The cells were then scraped from the plate using a cell scraper and the suspension so obtained was rapidly transferred to microcentrifuge tubes and placed on ice for 1 h to ensure complete lysis. During this time, the tubes were mildly vortexed every 15 min to facilitate homogenization. At the end of lysis procedure, the suspension was centrifuged at 12000 rpm for 12 min at 4°C. The supernatant containing the whole cellular protein was collected in separate microcentrifuge tubes and stored at -20 °C. Repeated thawing- freezing cycles of protein samples was avoided.
2.5.2 Protein Quantification using Bradford Assay
The Bradford assay is a colorimetric method used to quantify proteins. It is based on the absorbance shift of the dye Coomassie Blue, which forms a blue colored complex when bound to amino acids of the protein. This complex has maximum absorbance at 595 nm.
33 Therefore, the absorbance at this wavelength is proportional to the concentration of protein in the test solution, within the linear range of this assay.
We used bovine serum albumin fraction V (BSA; Research Products International, IL,
USA) to construct the standard curve. A 1 mg/ml stock solution of BSA was prepared in deionized water and diluted to obtain concentrations ranging from 50-800 µg/ml, which is within the linear range of the assay. The assay was performed in a 96-well plate.
Standards and samples were loaded in triplicate to minimize error. The protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) was diluted with deionized water in the ratio of 1:4 and added to each well (200 µl) using a repeater pipette. The blank wells had the assay reagent and deionized water instead of the protein. The absorbance was measured immediately at 595 nm using Synergy Hybrid plate reader (BioTek). The optical density value of each sample was subtracted from the blank values to obtain the true sample value. A standard curve was plotted using optical density values of BSA versus the known concentration of BSA. The unknown concentrations of the samples were estimated by interpolating the optical density values using the equation obtained from the standard graph. A fresh standard curve was constructed for each experiment.
2.5.3 Western blotting
Western blotting involves the separation of proteins on a polyacrylamide gel based on their molecular weight followed by their transfer to a membrane, which could be probed with antibodies against proteins of interest.
The first step prior to electrophoretic separation involves sample preparation. Based on the protein concentrations obtained in Section 2.5.2, protein samples were added to
34 sample buffer (25% v/v mercaptoethanol, 5% v/v glycerol, 5% w/v SDS and 0.625 M
Tris chloride pH 6.8, 5 mg bromophenol blue) to obtain the desired final concentration.
Bromophenol blue was added to help track the movement of samples across the gel. The higher density of glycerol ensured that the samples remained in their wells instead of floating up. Once suspended in the sample buffer, protein samples were mixed thoroughly and heated at 95 °C to facilitate denaturation of proteins.
Equivalent amounts of protein samples thus prepared (20-25 µg) were loaded on wells of polyacrylamide gels (10-12%, depending on the molecular weight of the protein of interest). A molecular weight marker (Thermo scientific) was loaded onto a separate well to locate the protein band of interest. The apparatus was placed in a reservoir containing
1X Tris-glycine running buffer (3.03 g Tris base, 14.4 g glycine and 1 g SDS) and electrophoresis was commenced at a voltage of 75 V, and incrementally increased as required during the procedure. Both the gel running buffer and the sample buffer contain sodium dodecyl sulfate (SDS), which denatures proteins and gives them a uniform negative charge. Therefore, under the influence of electric current, proteins migrate solely on the basis of their molecular weight and not their charge. Low molecular weight proteins migrate the farthest from their origin (bottom of the gel) whereas higher molecular weight proteins migrate relatively less and stay close to the origin (top of the gel). At the end of the electrophoresis, gels were sandwiched with pretreated polyvinylidene fluoride (PVDF) membranes (Bio-Rad) and set up in a transfer chamber containing 1X transfer buffer (3.03 g Tris base, 14.4 g glycine) supplemented with 10-
20% methanol. This process was carried out at 115 V for 1.5-2 h. The electric current
35 causes the movement of proteins from the gel to the membrane in their original configuration.
The membrane containing the transferred proteins was then blocked using 5% non-fat dry milk (Lab Scientific, Inc, Livingston, NJ) for 1 h at room temperature (RT). This was done to prevent non-specific binding of antibodies in the subsequent steps. Following blocking, membranes were incubated with the primary antibodies- rabbit anti-HO1 dilution 1:1000 (Assay Designs, Ann Arbor, MI, USA) and rabbit anti-GAPDH dilution
1:2000 (Millipore, Billerica, MA, USA) overnight at 4 °C. The antibody incubation was carried out in polyethylene pouches secured to a rotary shaker to enhance thorough distribution.
Following the primary antibody incubation, membranes were washed thrice with PBS-T
(1X PBS, 0.1 % Tween-20) buffer, with each wash lasting for 15 min. The membranes were then incubated with the horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (1:2000; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. After the incubation, membranes were once again rinsed thrice with
PBS-T buffer. The protein bands were then visualized by chemiluminescence reaction produced by the interaction of HRP enzyme with the added substrate. The chemiluminescence detection reagent consists of hydrogen peroxide and luminol, the breakdown of the former by the peroxidase enzyme immobilized on the membrane causes the oxidation of the latter. The product emits light, which can be detected using an X-ray film or a CCD (charged-couple device) camera system. In our in vitro experiments, the
X-ray technique was used for detection. Membranes were incubated with a mixture containing equal volumes of the chemiluminescence reagents (Super Signal West Pico 36 reagent kit, Thermo scientific) for 3 min to facilitate the reaction. They were then immediately developed using an X-ray autoradiography cassette (Fisher). The bands were quantified using Image J software provided by the National Institutes of Health and
Adobe photoshop. The quantification process involved subtracting the background and de-speckling of images. The bands were normalized to those of the loading control-
GAPDH, to account for any gel loading errors.
2.6 Animal Studies
All the animal experiments were carried out in concordance with the protocol approved by the University of Toledo Health Science Campus Institutional Animal Care and Use
Committee. The guidelines prescribed by the National Institutes of Health were adhered to throughout the study. C57BL/6 male mice (5–10 weeks old; 20–25 grams) were procured from Charles River Laboratories, Wilmington, MA, USA and were housed at
22±1°C with a 12 h:12 h light/dark cycle with water and food available ad libitum.
2.7 Permanent Middle Cerebral Artery Occlusion (pMCAO)
This model of focal ischemia involves the permanent occlusion of the distal portion of the middle cerebral artery, thereby cutting off the blood supply to the regions supplied by this artery.
The surgical procedure was carried out as described by Zeylanov et al (Zeynalov, Shah et al. 2009). Mice were anesthetized using 5% isoflurane (Baxter Healthcare, Deerfield, IL,
USA) in oxygen in the induction chamber, and maintained at 2% isoflurane throughout the surgery with the use of a nasal cone. Their rectal temperature was continuously
37 monitored with a rectal probe and maintained at 37.0±0.5°C during the surgery with a heating blanket. With the aid of a surgical microscope, a 10 mm vertical skin incision was made between the right eye and ear; the area was scrubbed using 70% isopropyl alcohol and iodine prior to the incision. The temporal bone was then exposed by moving the temporal muscle aside, and the middle cerebral artery was located beneath it. A 2 mm burr hole was drilled through the skull to expose the distal portion of the middle cerebral artery. A bipolar coagulator was used to cauterize this portion of the artery, thus minimizing blood loss. Upon confirmation of complete cessation of blood supply at the occlusion site, the wound was sutured and the animals were left to recover in a temperature regulated recovery chamber before moving them to their home cages.
2.8 Drug Dosing Regimen
We tested two treatment paradigms in the animal studies: pre-treatment and post- treatment. In the pre-treatment regimen, animals were dosed with WSE (200 mg/kg, p.o) or Vehicle (distilled water) daily for seven days, and then subjected to pMCAO on day seven (first day counted as day zero) (Figure 2-1). Following a survival period of another seven days, during which they were evaluated for behavioral parameters, mice were sacrificed on day 14 (i.e, seven days after pMCAO). In the post-treatment regimen, WS
(200 mg/kg, p.o) was administered four hours immediately after surgery followed by daily doses for seven days, during which they were evaluated based on behavioral parameters (Figure 2-1). Mice were sacrificed seven days after surgery.
38
Figure 2-1: Drug dosage regimen for pre-treatment and post-treatment studies
The upper panel (a) shows the schedule for pre-treatment, whereas the lower panel (b) depicts the post-treatment schedule. Each perpendicular arrow represents one dose of
WSE/vehicle.
2.9 Locomotor Activity
Locomotor activity is one of the behavioral tests used to evaluate animals on the recovery of their motor skills after surgery. In this test, animals were placed on a horizontal rod
(Columbus instruments, OH, USA) that was made to rotate at 1 r.p.m. with an acceleration rate of 1 r.p.m. every ten seconds, and the time taken for the animal to fall down was noted. Each animal was tested three times per trial. All the animals were trained on the rotarod assembly prior to surgery. The duration for which each animal was able to stay on the accelerating rod was recorded as the latency to fall and registered manually. Baseline locomotor activity was recorded on the day of surgery, and the effect
39 of drug-treatment was tested on the first and seventh day post-surgery. The automated nature of this test gives an objective indicator of the locomotor activity of animals being tested.
2.10 Neurological Deficits Analyses
Mice were assessed on a detailed 28-point neurological scoring system (Clark, Gunion-
Rinker et al. 1998) in which the animals were scored based on seven parameters encompassing their sensory and motor deficits. These included their body symmetry, gait, circling & gripping behavior, and whisker response, with a higher number being associated with a higher deficit. Each parameter had a scoring range from zero to four, thus making 28 the maximum deficit score attainable. A trained experimenter blinded to the identity of the animals scored the animals for their deficits and tabulated the total score. NDS was evaluated on the day of sacrifice for both treatment regimens.
2.11 Infarct Volume Analyses
Animals from both treatment groups were euthanized seven days after pMCAO using carbon dioxide. Brains were dissected out and sliced into five 2 mm-thick coronal sections before incubating in 1% triphenyltetrazolium chloride (TTC) (SigmaAldrich) in
1X Phosphate buffered saline (PBS) solution for 20 min at 37°C. Due to the high oxidoreductase activity in live tissue, the tetrazolium dye stains it reddish pink
(mechanism similar to the MTT assay), while leaving the dead tissue unstained or whitish in color. This unstained region is considered the infarcted region for quantification
40 purposes. The stained brain sections were stored in 4% formaldehyde solution overnight to fix the sections and make them amenable to handling for quantification. Both rostral and caudal surfaces of brain sections were photographed, and analyzed for infarct areas using Image J software. The infarct area was estimated from five slices of each brain, measuring rostral and caudal surfaces of each individual slice in conjunction with the thickness of each slice to obtain the volume and expressed as a percentage of the volume of the contralateral hemisphere. These estimates were corrected for swelling in the ipsilateral hemisphere by using the difference between the volumes of normal tissue
(non-infarcted) in the two hemispheres to calculate the actual infarct. This is best understood by the equation outlined by Swanson et al (Swanson, Morton et al. 1990):
� − � % � = 100× ! ! �!
Where % � = Percentage of the cortex in the ipsilateral hemisphere that is infarcted
�! = Volume of the normal matter in the cortex of the contralateral (control) hemisphere
�! = Volume of the normal matter in the cortex of the ipsilateral (lesioned) hemisphere
This original equation by Swanson et al was meant for infarct calculation within any structure in the brain, and was adapted to measure cortical infarct in our case as the permanent occlusion model produces infarcts restricted to the cortex.
41 2.12 Immunohistochemistry Procedures
Proteins exert varied, often opposing effects, depending on the cellular sub-compartment they are expressed in or translocated to. We used immunohistochemistry to detect the cellular localization, and to thereby infer the actions of the proteins under study. A series of procedures were used to detect these proteins. A detailed account of each procedure is given below:
2.12.1 Perfusion of Mice Brains and their Isolation
A separate cohort of mice that received pre-treatment with WSE (200mg/kg, p.o) or vehicle (distilled water) was used for this experiment. At the end of the survival period of seven days, mice were anesthetized using sodium pentobarbital (50-65 mg/kg, i.p) and transcardially perfused with physiological saline (0.9% NaCl) followed by 4% buffered paraformaldehyde using a peristaltic pump. The perfusion with saline ensured the drainage of blood, which would otherwise interfere with the immunohistochemical detection. Paraformaldehyde fixes the soft brain tissue, preparing it for further processing. The perfusion was discontinued upon the first signs of the rigidity in the tail of the mouse. Brains were then dissected from the skulls and post-fixed for 24 h in 4% paraformaldehyde. The isolated brains were stored at -80°C.
2.12.2 Tissue Sectioning
The frozen brains were sectioned using a cryostat. About 6-7 micron thick sections were obtained and deposited onto positively charged slides (Superfrost Plus, VWR, Radnor,
PA, USA) that adsorb thin tissue sections by means of electrostatic attraction. Slides were
42 immediately stored at -20 °C to prevent dehydration. These sections were then used for the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and the immunostaining methods.
2.12.3 TUNEL Assay
TUNEL assay is a standard method of detecting apoptosis, which involves large scale
DNA fragmentation. The principle of this assay is to label the nick ends (3′-OH) of fragmented DNA with a fluorescein-12-dUTP, which is catalyzed by the enzyme- terminal deoxynucleotidyl transferase (TdT). The labeled DNA can then be detected using fluorescence microscopy. Here, we used TUNEL assay to delineate the infarcted region in our tissue sections so that we could explore the border of the injury site for protein expression.
Thawed tissue sections were fixed using 4% paraformaldehyde in 1X PBS for 15 min at
RT. The slides were then washed thrice in 1X PBS and then treated with proteinase K (20
µg/ml; 100 µl per section) for 20 min. Proteinase K aids permeabilization of the tissues.
At the end of the permeabilization step, sections were washed once again with 1X PBS.
The TUNEL procedure was carried out as per the manufacturer’s protocol (DeadEnd™
Fluorometric TUNEL System, Promega, Madison, WI, USA). The reaction mixture (100
µl) was composed of 98 µl of equilibration buffer, 1 µl of biotinylated nucleotide mix, and 1 µl of recombinant TdT enzyme. Each section was first treated with just the equilibration buffer for 10 min. The excess solution was blotted out, and the sections were treated with 100 µl of the TdT reaction mixture under dark conditions for 60 min at
37 °C. All the subsequent steps including the ones in the next section (Section 2.12.4)
43 were also carried out in the dark to avoid bleaching of the fluorescent dye. The reaction was ended by immersing the slides containing the sections in 2X SSC (saline sodium citrate) for 15 min. This was followed by two PBS washes after which the slides were further processed for immunohistochemical analysis.
2.12.4 Immunohistochemical Analysis
Following the TUNEL procedure, slides were blocked with 3% BSA in 1X PBS for 1 h at
RT. About 100 µl BSA was added to each section; this step served to block the non- specific binding sites on the sections. The slides were then incubated with the primary antibody- polyclonal rabbit Anti-AIF at a dilution of 1:100(Abcam, USA, Cambridge,
MA, USA), overnight at 4 °C. Even distribution of the antibody solution was ensured by placing parafilm (Pechiney Plastic Packaging, WI) over the sections. After the overnight incubation, the parafilm was gently removed and sections washed with PBS thrice for 10 min each. The sections were then incubated with the secondary antibody- anti-rabbit IgG
(tagged with fluorescent Texas Red) at a dilution of 1:800 (Jackson ImmunoResearch) for
2 h at RT. Following another set of washings, slides were mounted with 4′,6-dimidino-2- phenylindole (DAPI) to stain nuclei and covered with coverslips. The gap between the slide and the coverslip was sealed to prevent the sections from drying. The slides were then photomicrographed using a fluorescence microscope (Nikon Eclipse Ti microscope,
Nikon, Japan). Around 10-15 captures were obtained per animal and three animals were used per treatment group.
44 2.13 Western blotting Procedures for the in vivo Study
The procedures differ from those described in Section 2.5 primarily in the tissue preparation and protein extraction method and the antibodies used. Therefore, only procedural details unique to the animal studies are given here. The Western blotting was carried out only in animals subjected to a) pre-treatment with WSE, b) vehicle treatment, and c) no surgery/sham
Mice were allowed to survive for seven days after pMCAO, at the end of which they were sacrificed using carbon dioxide. Their brains were then isolated and their lesioned cortices carefully dissected out. The tissue was weighed and homogenized using a tissue homogenizer. All procedures were carried out on ice to minimize protein degradation.
Thereafter, cytoplasmic and nuclear fractions were obtained from the tissue using buffer
A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5% NP40 1 mM EDTA; pH 7.9) and buffer B (5 mM HEPES, 1.5 mM MgCl2, 1 mM EDTA, 25 % glycerol, 420 mM NaCl; pH 7.9) respectively. Protease and phosphatase inhibitors as well as DTT and PMSF were added freshly to both buffers at the final concentration mentioned in Section 2.5.1. The cytoplasmic fraction was obtained first by incubating the homogenized tissue with buffer
A and spinning the suspension at 5000 rpm for 10 min. The supernatant was collected as the cytoplasmic fraction and the pellet was treated with buffer B. The nuclear fraction was obtained by centrifuging the suspension at 10,000 rpm for 10 min. Both the fractions were stored at -20 °C until further use.
The protein extraction procedure was identical to that in Section 2.5.2. The electrophoresis procedure also involved the same steps except that the gels for analyzing cytoplasmic proteins had a lower protein load (20-25 µg) as compared to the nuclear
45 protein gels (40 µg). The primary antibodies used for the animal studies were: rabbit anti-
PARP-1 dilution 1:1000 (Cell signaling, Danvers, MA, USA); rabbit anti-AIF (Cell signaling) dilution 1:1000, rabbit anti-Sema3A dilution 1:1000 (Abgent, San Diego, CA), rabbit anti-HO1 dilution 1:1000 (Assay Designs, Ann Arbor, MI), mouse anti-GSK3 α/β dilution 1:1000 (ThermoScientific), rabbit anti-phospho-GSK3 α/β (Ser21/9) dilution
1:1000 (ThermoScientific), rabbit anti-Wnt1 dilution 1:1000 (Abcam), rabbit anti-
CRMP2 dilution 1:50,000 (Millipore, Billerica, MA), rabbit anti-phospho-CRMP2
(Thr555) dilution 1:1000, rabbit-anti-Histone H3 dilution 1:3000 (ThermoScientific,
Waltham, MA, USA), rabbit anti-actin dilution 1:2000 (SigmaAldrich), and rabbit anti-
GAPDH dilution 1:2000 (Millipore). The secondary antibodies were: goat anti-rabbit and goat anti-mouse dilution 1:2000-1:5000 (Jackson ImmunoResearch Laboratories, West
Grove, PA).
The bands were photographed using the ChemiDoc molecular imager (Bio-Rad). The analysis was done using the volume tool of the ChemiDoc. The densitometric values were normalized with respect to the values of histone H3/GAPDH immunoreactivity to correct for any loading and transfer differences between samples.
2.14 High-performance Liquid Chromatography (HPLC) Fingerprinting of the
Withania somnifera Extract
An HPLC system (Waters Alliance 2695 separation module, Milford, MA) equipped with a Phenomenex C18 (25 cm x 4.6 mm I.D., 5 µm particles) and photodiode array (Waters
2998) detector was used for analysis. We selected withanoside IV and withanolide A— both components of the extract— as our reference compounds, as they are reported to be
46 highly bioactive in neurological disorders (Kuboyama, Tohda et al. 2005, Kuboyama,
Tohda et al. 2006). Withanoside IV (ChromaDex, Irvine, CA) and withanolide A
(ChromaDex) were analyzed using aqueous mobile phase containing 1mM KH2PO4 in a gradient combination with acetonitrile, pumped at a flow rate of 1.5 ml/min. The column temperature was maintained at 27˚C. The HPLC gradient program (run time in minutes/% aqueous phase) was set as follows: 0/95%, 18/55%, 25/30%, 30/30%,
30.01/95%, and 32/95%. Different calibration standards of withanoside IV and withanolide A were prepared in 14% v/v acetonitrile solution. For the calibration curve, each standard was analyzed in triplicate and the average peak area was plotted against concentration. WSE was tested for its withanoside IV and withanolide A content by intrapolating the peak areas on their respective calibration curves. Results were expressed as percentage by weight of the reference compounds present in the aqueous extract
(WSE) as well as the original caplet. This was calculated based on the weights of the powdered caplet and the freeze-dried aqueous extract. The intra- and inter-assay precisions of withanoside IV and withanolide A expressed as the relative standard deviations did not exceed 2%. The limit of detection and limit of quantification of withanoside IV was found to be 37.4 ng/ml and 124.8 ng/ml, respectively. For withanolide A, the limit of detection and limit of quantification was found to be 42.7 ng/ml and 142.3 ng/ml, respectively.
2.15 Statistical Analyses
The experimental results are expressed as the mean ± SEM and are accompanied by the number of observations. One-way ANOVA, followed by Tukey’s post-hoc test was used
47 to determine significant differences between groups in the infarct volume and behavioral studies. Student’s unpaired t test was used to compare1) control and H2O2 group, 2) H2O2 and each drug-treated group, 3) sham and vehicle group, and 4) vehicle and each drug- treated group in the in vitro experiments and immunoblot analyses. A value of p < 0.05 was considered to be statistically significant.
48 Chapter 3
Results
3.1 WSE Protects PC12 Cells from H2O2-induced Oxidative Damage
Oxidative stress is one of the predominant pathological features of ischemia (Chen, Yang et al. 2011). H2O2 releases highly reactive hydroxyl radicals in the presence of trace metal ions like Fe2+ via the Fenton reaction. These radicals cause irreversible damage to cellular structures, thereby causing their demise. This in vitro model of oxidative stress is a highly convenient, reproducible model to conduct a preliminary screen for neuroprotective drugs. The MTT assay gives a reliable indicator of the metabolically active cells present in the cell population.
In our experiment, we pre-treated PC12 cells with different concentrations of WSE (25-
200 µg/ml) followed by the oxidative insult by H2O2 (100 µM). The cell viability as determined by the MTT assay is shown in Figure 3-1. Cells subjected to H2O2 show drastically reduced viability when compared to control cells that received no treatment
(50% reduction; statistically significant with p= 0.0002). At lower concentrations of WSE
(25, 50 µg/ml), cells showed no considerable improvement in viability when compared with cells that were treated with H2O2 alone. However, we saw a spike in cell viability at
49 higher doses of WSE (100 and 200 µg/ml), with the 100 µg/ml showing the highest increase (about 40%). The results were statistically significant (p= 0.03 and 0.01 respectively) at both these concentrations. The decline at WSE 200 µg/ml wasn’t significant when compared to the WSE 100 µg/ml group. This experiment shows that
WSE has potential in reversing the oxidative damage induced at the cellular level.
Fig 3-1: The effect of WSE on PC12 cells when subjected to H2O2-mediated oxidative stress
PC12 cells were pre-treated with WSE (25, 50, 100 and 200 µg/ml) or vehicle (0.9%
NaCl) for 24 h and then treated with H2O2 (100 µM) for 24 h. WSE (100 µg/ml) showed the highest efficacy in improving cell viability when compared to the H2O2 group.
50 Results are expressed as a percentage of control cells, with each value corresponding to the mean with the standard error (SEM) obtained from three readings. This graph shows the representative data obtained from three individual experiments using three separate batches of cell cultures. *p<0.05 vs. H2O2 group; #p<0.05 vs. control
3.2 WSE Induces the Expression of HO1 when Exposed to H2O2
In the experiment previously discussed (Figure 3-1), we found that WSE rescued PC12 cells from oxidative damage. We sought to investigate whether WSE possessed anti- oxidant properties, which could have been responsible for the observed protection from oxidative stress. WS extracts have been previously reported to possess anti-oxidant activity (Bhattacharya, Ghosal et al. 2001), however its effects on HO1 have not yet been established.
We selected the best working concentration of WSE (100 µg/ml) obtained from results in
Section 3.1 to study HO1 expression. PC12 cells were subjected to the same treatment regimen as outlined in Section 3.1. Additionally, a separate group of cells received treatment with WSE (100 µg/ml) alone to provide an indication of its effects on HO1 expression in absence of a stressor. Whole cell lysates from PC12 cells were immunoblotted for HO1 levels (Figure 3-2). Control cells showed marginal expression of
HO1, which increased with cells that were treated with WSE alone. HO1 levels were relatively lower in the H2O2 group, although higher than that in the control cells. This decline was completely reversed in the group that received both WSE and H2O2 treatment. The induction in HO1 expression brought about by WSE, though not
51 statistically significant, provided a plausible link to its observed protective effects under oxidative stress.
The values obtained from quantifying the pixels associated with each band were normalized with their respective GAPDH bands. GAPDH is a housekeeping gene, which stably expresses high amounts of protein and serves as a loading control to account for gel loading or transfer errors.
1.5
ns 1.0 ns
0.5
ratio HO-1/GAPDH 0.0 2 2 WS O O H 2 2 Control WS + H
Figure 3-2: The effect of WSE on HO1 expression levels in PC12 cells subjected to oxidative stress
PC12 cells were pre-treated with WSE (100 µg/ml) or vehicle (0.9% NaCl) for 24 h and then treated with H2O2 (100 µM) for 24 h. A separate group of cells received treatment
52 with WSE only. Upon Western blotting, we found that WSE induced HO1 expression both in the presence and absence of H2O2, whereas the H2O2-treated group showed relatively low HO1 levels. The changes in expression were however statistically insignificant (ns). The results are expressed as a ratio of HO1 to GAPDH bands for each group. This is a representative blot of three such experiments. Each value is the mean with the standard error (SEM) associated with three readings.
3.3 WSE Pre-treatment Attenuated Infarct Volume and Improved Functional Outcomes when Mice were subjected to pMCAO
The results from the in vitro experiments prompted us to evaluate the effects of WSE in an in vivo model of ischemic stroke. Focal models of ischemia are the most commonly used for pre-clinical testing. We chose the permanent focal occlusion model as it produces consistent, defined infarcts restricted to the cortical region. Also, due to its inherently low mortality, it allows for a longer survival period, which is beneficial for studying long-term outcomes. The dose of WSE for this study was selected on the basis of literature reports (Kulkarni and Dhir 2008, Kumar and Kumar 2009) and a pilot experiment we conducted with a 100 mg/kg dose which failed to provide significant results. Mice were pre-treated with WSE (200 mg/kg p.o.) or vehicle (distilled water) daily for seven days followed by pMCAO. After a survival period of seven days mice were sacrificed, brains dissected and stained with TTC to calculate infarct volumes
(Figure 3-3a). Mice were also evaluated for their locomotor activity in the interim survival period. Mice pre-treated with WSE showed significantly lower (p= 0.01) cortical infarct volumes (about 30% reduction) when compared to the vehicle treated ones (Figure 3-3b). Similarly, drug-treated mice performed better in the rotarod test for
53 locomotor activity (Figure 3-3c). This effect was significant only at the 24 h time-point
(p= 0.01) following pMCAO (about 60% increase when compared to vehicle), and not on
the day of sacrifice (day 7). Although, WSE treated mice still had higher motor function
(higher latency to fall) than the vehicle controls on day 7.
Our results indicate that WSE pre-treatment has favorable histological and functional
outcomes following stroke damage.
a)
b)
Figure 3-3a and b: Cortical infarct volume in WSE pre-treated mice subjected to
pMCAO
54 Mice pre-treated with WSE (200 mg/kg, p.o.) or vehicle (distilled water) daily for seven
days were subjected to pMCAO and sacrificed after a seven-day survival. (a)
Representative TTC-stained brain sections for infarct volume calculation. (b) WSE
treated mice (n=12) exhibited significantly lower infarct volume (p= 0.01) as compared
to vehicle treated mice (n=10). Values are expressed as mean+SEM. *p<0.05 vs. vehicle
c)
Figure 3-3c: Comparison of the locomotor activity in WSE pre-treated and vehicle treated mice
The same cohort of mice mentioned in Figure 3.3a,b was used for this study. Locomotor
activity was measured at 24 h and 7d time-points post-surgery. The values are expressed
as a percentage of their respective baseline values taken prior to surgery. A higher latency
to fall indicates better locomotor activity. WSE pre-treated mice exhibited a significantly
higher locomotor activity (p= 0.01) as compared to the vehicle-treated group at the 24 h
55 time-point, whereas the relative improvement in locomotor function was not statistically significant 7 days post-surgery. Values are expressed as mean+SEM *p<0.05 vs. vehicle
3.4 Mice Subjected to pMCAO and Post-treated with WSE had Attenuated Infarct Volume and Improved Functional Outcomes
The promising results from the WSE pre-treatment study encouraged us to evaluate its effects when given as a post-stroke treatment. Although WSE, being a crude extract, is highly likely to be given as a preventive herbal supplement, the post-treatment study was designed to appraise whether the extract constituents had therapeutic potential. This could be helpful for future studies involving individual components of the extract. We conducted the post-treatment study in the same permanent occlusion model and using the same dose of WSE (200 mg/kg) as that of the pre-treatment study.
Treatment commenced after the pMCAO procedure with the first dose of WSE/vehicle given 4 h after the onset of occlusion. We set a time lag for the drug administration to mimic clinical settings, where there are often time delays between onset of stroke symptoms and onset of treatment. The first dose was then supplemented with daily doses of WSE/vehicle for seven days, after which mice were euthanized for histological analysis of their brains. Mice were also tested for their locomotor activity 24 h and seven days following pMCAO. We found that WSE post-treatment caused a significant reduction (p= 0.009) in the infarct volume (about 35% reduction) when compared to vehicle controls (Figure 3-4 a,b). WSE treated mice also had significantly better motor function at the 24 h time-point (about 70% increase compared to vehicle; p= 0.006), and a favorable trend at the 7-day time-point (Figure 3-4c).
56 Our post-treatment results with WSE cemented its neuroprotective effects in our stroke
model.
a)
b)
Figure 3-4a and b: Cortical infarct volume in mice treated with WSE after pMCAO
Mice were subjected to pMCAO and dosed with WSE (200 mg/kg, p.o.) or vehicle 4 h
after occlusion followed by daily doses for seven days. (a) Representative TTC stained
brain sections of the treatment groups (b) Quantification of infarct volume revealed that
the WSE post-treatment group (n=10) had significantly lower infarct volume (p= 0.009)
57 as compared to the vehicle-treated group (n=10). Values are expressed as mean+SEM.
*p<0.05 vs. vehicle c)
Figure 3-4c: Comparison of the locomotor activity in WSE post-treated and vehicle treated mice
The same cohort of mice used for infarct volume calculations mentioned in Figure 3.4 a,b
was used to study the locomotor activity. Mice were tested at 24 h and on day 7 after the
surgery. The results were similar to that obtained in the pre-treatment study; WSE post-
treatment showed a higher increase at the 24 h time-point only (about 70% increase; p=
0.006). The motor function exhibited by the WSE group was higher than the vehicle-
treated group on day 7, but not statistically significant. Values are expressed as
mean+SEM. *p<0.05 vs. vehicle
58
3.5 WSE Treatment did not have a Considerable Effect on the Neurological Deficits Suffered by Mice Subjected to pMCAO
Mice belonging to the pre-treatment and post-treatment groups were graded on their sensory and motor deficits on the day of sacrifice. In this manual scoring system, an observer blinded to the treatment groups, scored mice on seven parameters: gait, body symmetry, climbing, circling behavior, front limb symmetry, compulsory circling, and whisker response. The aggregate score was expressed as the neurological deficit score
(NDS).
Upon comparing the NDS of different treatment groups, we found that there was no substantial change observed either in WSE pre- or post-treatment when compared to the vehicle controls (Figure 3-5). This is in concordance with WSE’s lack of a significant effect on the locomotor activity 7 days after surgery. Thus, WSE was only able to improve functional outcomes in the acute phase of our model of ischemic stroke.
59
Figure 3-5: NDS measurement in WSE pre- and post-treated mice on day 7 after pMCAO
On the day of sacrifice, mice belonging to the treatment groups mentioned in Sections 3.3 and 3.4 were graded manually on their sensory and motor deficits, together constituting the NDS. On a scale of 0-28, with higher scores indicating higher deficits, the drug- treated groups (both pre- and post-treatment) did not perform considerably better than the vehicle treated group. Values are expressed as mean+SEM
60
3.6 WSE Pre-treated Mice Show Higher Levels of HO1 in Mice Brain Cortices
In the course of the infarct damage and functional outcome studies, we found that both pre- and post-treatment with WSE (200 mg/kg) have therapeutic potential in ischemic stroke. In order to understand the molecular underpinnings behind the observed effects, we conducted a series of experiments to study the cellular mechanisms of action. We explored the literature on the mechanisms of WS in other central nervous system disorders with similar pathophysiology to ischemia, and found that its anti-oxidant activity was a common theme in its reported mechanisms. This and the encouraging results in the in vitro experiment measuring HO1 expression levels (Section 3.2) prompted us to confirm whether or not HO1 could be responsible for some of the observed neuroprotective actions of WSE. We used immunoblotting to compare expression levels of HO1 following the WSE pre-treatment regimen in mice. A separate cohort of mice was pre-treated with WSE (200 mg/kg, p.o.) or vehicle for seven days, followed by pMCAO and a survival period of seven days. Mice were sacrificed, and their ipsilateral cortices were dissected out for the protein expression study. A separate group of mice (sham) received no treatment and underwent all procedures except the occlusion of the MCA. When the cytoplasmic fractions of the protein from the treatment groups were probed for HO1 levels, we found that sham mice expressed little to no HO1 (Figure
3-6a), consistent with the stress-inducible nature of the protein. Vehicle treated mice showed marginally higher expression as compared to sham, whereas WSE pre-treated mice showed a dramatic increase in HO1 levels—almost five times that of the vehicle group (Figure 3-6b). The WSE-mediated HO1 increase was found to be statistically significant with respect to the vehicle group (p= 0.001).
61
Figure 3-6a & b: HO1 expression in mice pre-treated with WSE (200 mg/kg)
Mice were exposed to WSE (200 mg/kg) pre-treatment regimen outlined in Section 3.6, and probed for HO1 expression levels in their ipsilateral cortices. (a) Representative immunoblots of the cytoplasmic fractions revealed low expression levels in the sham group. There was a slight increase in HO1 levels in the vehicle treated group, and a substantial induction in the WSE pre-treated group (b) The increase in WSE-mediated
HO1 expression was found to be statistically significant (p= 0.001) when corrected for
GAPDH levels. Values are expressed as mean+SEM (n=3 for each treatment group).
*p<0.05 vs. vehicle
62 3.7 WSE Pre-treated Mice show Lower Expression of Pro-apoptotic Proteins-PARP-
1 and AIF
Upon confirming WSE-mediated HO1 induction under ischemic conditions, we sought to test whether WSE could prevent the cell death associated with ischemic damage.
Prevention of neuronal cell death is the primary requirement of neuroprotective therapy.
In order to investigate whether WSE fulfilled this condition, we focused on the cell death mechanisms that are predominant during ischemia. Necrotic death occurs within minutes to hours following stroke, a time-window that is often impractical for clinical use.
Apoptosis, on the other hand, is the dominant mechanism during the delayed phase of cell death, and presents an avenue for therapeutic intervention and salvage of valuable brain tissue (Broughton, Reutens et al. 2009). It is known that apoptotic cell death functions both with and without the involvement of caspases, with the PARP-1-AIF pathway constituting a key mechanism of the latter type (Hong, Dawson et al. 2004). We tested whether WSE could modulate the levels of either protein, to influence this pathway of apoptosis. The treatment groups used for this study were the same as those mentioned in
Section 3.6. We used immunoblotting to probe for levels of PARP-1 and AIF in the nuclear fractions of brain cortices. We found a significant increase in both PARP-1 and
AIF expression levels in vehicle treated mice when compared to sham mice (Figure 3-7a- c; p= 0.03 and 0.04 respectively). On the other hand, WSE pre-treatment reinstated the nuclear AIF levels back to sham levels (Figure 3-7a & c) and reduced PARP-1 expression even below the observed sham levels (Figure 3-7a & b). The reduction in the expression of both proteins was found to be statistically significant with respect to the vehicle treated
63 group (p= 0.004 and 0.01 for PARP-1 and AIF respectively), when corrected for histone levels.
Figure 3-7a-c: PARP-1 and AIF expression levels in mice pre-treated with WSE
(200 mg/kg)
64 Mice were exposed to WSE (200 mg/kg) pre-treatment regimen outlined in Section 3.6, and probed for PARP-1 and AIF expression levels in their ipsilateral cortices. (a)
Representative immunoblots of the nuclear fractions show higher expression levels of both PARP-1 and AIF in vehicle treated mice as compared to sham, and lower levels in
WSE pre-treated group as compared to the vehicle group. (b and c) The increase in the expression levels found in the vehicle treated group as well as the decrease in the WSE pre-treatment group were found to be statistically significant with respect to the sham and the vehicle group, respectively (p= 0.004 for PARP-1 and 0.01 for AIF). The values are expressed as mean+SEM (n=3 for each treatment group). *p<0.05 vs. vehicle group;
#p<0.05 vs. sham group
3.8 WSE Pre-treatment Prevents the Nuclear Translocation of AIF under Ischemic Conditions
The previous experiment revealed that one of the modes of WSE’s action involves reduction of nuclear expression of PARP-1 and AIF. This pathway of caspase- independent apoptosis is activated by the PARP-1-mediated release of AIF from the mitochondria, and its subsequent translocation to the nucleus to convey the death signal
(Li, Klaus et al. 2010). In order to confirm that WSE prevents this mechanism of cell death, it is therefore important to verify the effect of WSE on the nuclear translocation of
AIF.
We used immunohistochemical methods to visualize the sub-cellular localization of AIF, and thus confirm the mechanistic consequence of WSE pre-treatment. A separate cohort of mice was randomly divided into different treatment groups (sham, vehicle and WSE
65 pre-treatment 200 mg/kg; n=3 each). The treatment regimen was the same as outlined in
Section 3.7. Brain sections obtained from mice were triple stained for their nuclei,
TUNEL positive region, and AIF expression. The TUNEL assay was carried out to demarcate the infarct region (dense TUNEL expression), so that we could scan for AIF expression in the border of the injury site (sparse TUNEL expression). We found that the sham group expressed little to no AIF in their cortices (Figure 3-8). On the other hand, there was a remarkable increase in AIF expression in the peri-infarct region of vehicle treated mice. Moreover, AIF was predominantly localized in the nuclei of the TUNEL positive cells. On the contrary, in WS treated mice, AIF was found to be predominantly extra-nuclear. These results corroborate the immunoblot findings seen in Section 3.7, confirming that WSE plausibly acts by preventing PARP-1-mediated AIF release and translocation, thereby thwarting the cell-death signaling cascade.
66
Figure 3-8: Sub-cellular localization of AIF in WSE pre-treated mice subjected to pMCAO
The TUNEL assay was used to delineate the ischemic region (green) to study apoptotic death (10X). The white boxes denote the area chosen to study the AIF translocation.
Sham brains show little or no TUNEL or AIF expression (100X). AIF was predominantly nuclear in the cortices of vehicle treated mice (overlay panel). On the other hand, WS pre-treated mice showed mainly extra-nuclear localization of AIF, confirming that it prevents the translocation of AIF to the nucleus under ischemic conditions. (Blue: DAPI for nuclei; Green: TUNEL positive cells, Red: AIF; n=3 for each treatment group)
67 3.9 WSE Pre-treatment does not Significantly Alter Bcl-2 Levels in Mice that Underwent pMCAO
We confirmed the involvement of WSE pre-treatment in preventing PARP-1-AIF mediated caspase-independent apoptosis. To investigate its participation in apoptosis involving caspase activation, we assessed for levels of Bcl-2—an anti-apoptotic protein which prevents mitochondrial release of cytochrome c and subsequent caspase activation
(MacManus and Linnik 1997).
The same cohort of mice used for the immunoblot studies in Sections 3.6 and 3.7 were utilized for this study. When we probed the cytoplasmic fractions for Bcl-2 levels (Figure
3-9a), we found a decrease in the vehicle-treated mice when compared to the sham group
(decrease not significant; Figure 3-9b). This is consistent with the notion that ischemia overwhelms the cellular anti-apoptotic defense systems (Broughton, Reutens et al. 2009).
Although we did see an increase in Bcl-2 expression in the WSE pre-treated group when compared to vehicle, there was high intra-group variability for this increase to be of statistical significance. This study, therefore, does not attach confidence to the involvement of WSE in the Bcl-2-mediated prevention of caspase activation.
68
Figure 3-9a & b: Bcl-2 expression in mice pre-treated with WSE (200 mg/kg)
Mice pre-treated with WSE (200 mg/kg) and processed as mentioned in Section 3.6 were probed for Bcl-2 levels in the cytoplasmic fractions. (a) Representative blot with Bcl-2 expression. (b) Both the decrease in the vehicle group and increase in WSE group were found to statistically insignificant when corrected for actin levels. The values are expressed as mean+SEM (n=3 for each treatment group; ns= not significant)
69 3.10 WSE Pre-treatment Attenuates the Expression of Sema3A in Mice Brains Following Ischemic Stroke
The in vivo results we have shown until now focused on the mechanisms that ensue during the acute-to-intermediate phase of an ischemic insult. We were curious if WSE’s effects could extend to the restorative or rehabilitative phase which begins several days following ischemia and extends to weeks or even months after onset (Gladstone, Black et al. 2002). Being a protracted phase, the restorative process offers the advantage of a wider window for drug intervention (Font, Arboix et al. 2010). Hence, this phase is more flexible to manipulation of drug doses and frequency, and less sensitive to their deleterious effects. The repair mechanisms involve the functioning of two processes in tandem: spurring the trophic cues that aid in reestablishing severed neuronal networks and overcoming the negative guidance cues that inhibit this reorganization. Sema3A is an example of one such negative guidance cue that poses an inhibitory environment to the regrowth of axons (Joyal, Sitaras et al. 2011). We wanted to evaluate WSE pre-treated mice for their cortical Sema3A levels and see how they compared to that in the vehicle and sham groups. Immunoblots showed that the levels of Sema3A increased in the vehicle treated group when compared to sham (Figure 3-10a), although the change wasn’t significant (Figure 3-10b). However, the levels of Sema3A plummeted in the WSE pre- treated group, and this effect was significant with respect to the vehicle group (p= 0.03).
These results show that WSE could have a role to play in stroke recovery by impeding the Sema3A-mediated inhibitory signals.
70
Figure 3-10a & b: Sema3A levels in mice pre-treated with WSE (200 mg/kg)
Mice pre-treated with WSE (200 mg/kg) and processed as mentioned in Section 3.6 were assessed for Sema3A expression levels. (a) Immunoblotting of the cytoplasmic fractions revealed higher levels of Sema3A in the vehicle group as compared to sham, whereas there was substantial reduction in the WSE pre-treated group (b) The decrease in the
WSE group was statistically significant with respect to vehicle (p= 0.03) when corrected for GAPDH levels, and not significant when the vehicle group was compared to sham.
The values are expressed as mean+SEM (n=3 for each treatment group).
71 3.11 WSE Pre-treatment does not Substantially Affect Wnt Expression
In light of the possibility of WSE’s involvement in the recovery processes following ischemic stroke (Section 3.10), we sought to investigate whether it could induce neurogenesis or the formation of new neurons. Neurogenesis, along with angiogenesis
(formation of new blood vessels) and neuroplasticity, are the key trophic mechanisms underplay during the recovery phase following a stroke attack (Font, Arboix et al. 2010).
The Wnt family of secreted proteins activates diverse signaling mechanisms modulating axon guidance, cell polarity, neuronal migration, and neurogenesis to name a few (Rosso and Inestrosa 2013). They function via two distinct pathways— the β-catenin or the canonical pathway and the β-catenin independent or non-canonical pathway (Oliva,
Vargas et al. 2013), the former being one of the prime pathways underlying adult neurogenesis (Hirabayashi, Itoh et al. 2004).
We assessed Wnt1 expression levels in our WSE pre-treatment as a preliminary indicator of the involvement of its pathways. Upon immunoblotting for Wnt1 levels in the cytoplasmic fractions of mice brain cortices (sham, vehicle and WSE), we found a marginal, non-significant increase in expression in the vehicle group when compared to sham (Figure 3-11a & b). The Wnt1 levels in the WSE group were virtually unaltered with respect to the vehicle group. This led us to believe that Wnt ligand modulation wasn’t likely to be one of the mechanisms responsible for its protective effects.
72
Figure 3-11: Wnt1 expression in WSE pre-treated mice
Mice pre-treated with WSE (200 mg/kg) and processed as outlined in Section 3.6 were probed for Wnt1 expression in their cytoplasmic fractions. (a) We found slightly increased Wnt1 expression levels in the vehicle group as compared to sham; however,
WSE pre-treated mice exhibited virtually no change in Wnt1 expression with respect to the vehicle group (b) None of the changes observed between treatment groups (vehicle vs. sham and WSE vs vehicle) were statistically significant. The values are expressed as mean+SEM (n=3 for each treatment group; ns= not significant)
73 3.12 WSE Pre-treatment does not Perturb the Wnt Signaling Pathways
Once we established that WSE does not significantly alter Wnt1 expression levels, we checked for levels of the downstream mediators of both canonical and non-canonical pathways. This was done in order to sufficiently explore the downstream events of Wnt signaling instead of concluding its non-involvement based on Wnt1 expression levels alone. Additionally, the markers involved in Wnt signaling function in diverse cellular mechanisms, some of which are not under the direct control of Wnt ligands (Kim and
Snider 2011). We tested for levels of key markers of Wnt pathway activation: GSK-3β and CRMP2, with the former being the prime mediator of the canonical pathway and the latter a downstream target of mediators of the non-canonical pathway— JNK and
CaMKII.
Once again, we used immunoblotting to assess the levels of these markers. Since the phosphorylation status of the effectors/mediators of this pathway is crucial to understanding their effects, we tested both the phosphorylated and the total levels of
GSK3-β and CRMP2. We found a gradual decline in the phospho-GSK3-β (p-GSK3-β
Ser 21/9) levels from sham to vehicle to the WSE pre-treated group (Figure 3-12a) We compared the ratios of p-GSK3-β /totalGSK3-β levels and found that WSE-mediated decrease in p-GSK3-β expression was not statistically significant (Figure 3-12b).
Similarly, the p-CRMP2 (Thr555)/total CRMP2 ratios weren’t significantly different amongst either treatment groups (Figure 3-12a & b). These results suggest that WSE pre- treatment doesn’t substantially perturb either of the Wnt pathway mediators, also extending to any Wnt-independent effects they may have.
74
Figure 3-12a & b: Expression levels of GSK3-β and CRMP2 in WSE pre-treated mice
Mice pre-treated with WSE (200 mg/kg) and processed as outlined in Section 3.6 were probed for phosphorylated and total expression levels of GSK3-β and CRMP2 in the cytoplasmic fractions. (a) We saw a gradual decline in p-GSK3-β (Ser 21/9) levels in the vehicle and WSE pre-treated groups as compared to sham. The WSE pre-treated group also exhibited low p-CRMP2 (Thr555) levels as compared to both vehicle and sham groups. (b) When the ratios of the phosphorylated/total expression of both these markers were compared across treatment groups, we found no significant alteration in the WSE pre-treated group as compared to the vehicle group. The values are expressed as mean+SEM (n=3 for each treatment group; ns= not significant)
75 3.13: HPLC Fingerprinting of WSE and Determining Abundance of Two Bioactive Molecules-Withanoside-IV and Withanolide A
This experiment was done so as to obtain a fingerprint of the extract (WSE) we have used throughout this study. Also, we wanted to probe WSE for its content of reported bioactive constituents. We chose withanoside-IV and withanolide A as the bioactive markers as they have been shown to have neuroprotective properties in neurological disorders with similar pathophysiological features to ischemic stroke (Zhao, Nakamura et al. 2002,
Kuboyama, Tohda et al. 2006). WSE prepared as mentioned in Section 2.1 was subjected to an HPLC analysis using a gradient elution method with the mobile phase consisting of aqueous buffer (1mM KH2PO4) and an increasing gradient of acetonitrile. The assay method was found to be linear in the range of 1.25-12.5 µg/ml with a correlation coefficient greater than 0.9984. The retention times of withanoside IV and withanolide A
(λ = 227 nm) were found to be 14.571 and 19.867 minutes (Figure 3-13a), respectively.
This was in accordance with their relative polarities, with withanoside-IV being the more polar molecule. The areas of the peaks corresponding to withanoside-IV and withanolide
A were intrapolated on the calibration curves to obtain their respective concentration in the extract. The results were expressed as the percentage by weight of the marker compounds in the extract (WSE) and the starting material (Ashwagandha caplets). We found that withanoside-IV was the more abundant of the two, with a content of 0.07% in the extract—twice the amount present in the starting material (Figure 3-13b). The content of withanolide A in the extract was found to be 0.016%, which was about one and a half times its content in the caplets. The representative chromatogram (Figure 3-13a) could be used as a map for verification and comparison purposes for future extraction procedures.
76
Figure 3-13a & b: HPLC fingerprint of WSE and its composition in terms of markers-withanoside-IV and withanolide A
WSE was subjected to an HPLC analysis using an aqueous buffered mobile phase (1mM
KH2PO4) with increasing gradients of acetonitrile. The HPLC gradient program (run time in minutes/% aqueous phase) was set as follows: 0/95%, 18/55%, 25/30%, 30/30%,
30.01/95%, and 32/95%. (a) The chromatogram shows the retention time in minutes on the X axis and the absorbance units (AU) on the Y axis. Withanoside-IV and withanolide
A were eluted at retention times 14.5 and 19.8 min respectively. (b) Withanoside-IV was found to be more abundant (0.07%) in the extract than withanolide A (0.016%). Both markers were present at a higher concentration in the extract as compared to the starting material. Results are expressed as mean percentage by weight along with their standard deviation based on three recordings.
77 Chapter 4
Discussion
The results of this study suggest a strong therapeutic potential of WSE when given either as a preventive measure, or as a treatment post-stroke. Therefore, we can conclusively reject the null hypothesis postulated in Section 1.7. We used a combination of in vitro and in vivo experiments to test this hypothesis and graded the extract on a combination of histological, functional and molecular outcomes. Our initial studies were conducted on the rat pheochromocytoma cell line (PC12), which has been widely used with success, to predict neuroprotective effects of drugs using in vitro models of ischemic stroke (Hillion,
Takahashi et al. 2005, Tabakman, Jiang et al. 2005). In addition to the convenience and reproducibility of this particular cell line, in vitro studies as such, have the added advantage of quick results, relatively lower costs, and the avoidance of ethical issues surrounding animal usage for preliminary studies. This way, only those candidates that succeed in the preliminary screen are advanced to the more intensive in vivo level.
We chose to evaluate an aqueous extract of WS root because of its preponderance to exhibit neuroprotective properties in neurological disease models with pathophysiologies similar to cerebral ischemia (Kumar, Harris et al. 2012, Pingali, Pilli et al. 2014).
78 We subjected PC12 cells to H2O2-induced oxidative stress, as oxidative damage is central to the devastating effects of ischemic stroke (Love 1999). The brain is metabolically very active and is the highest consumer of oxygen; this makes it susceptible to the negative effects of the disruption of oxygen handling systems and attendant metabolic pathways
(Mariani, Polidori et al. 2005). We found a 50% reduction in cell viability in cells treated with H2O2 alone, thereby confirming the validity of our model. We demonstrated that
WSE protects PC12 cells from oxidative damage at the higher range of test concentrations (100 µg/ml, 200 µg/ml) with the effect being highest at the 100 µg/ml concentration (Figure 3-1). This led us to speculate whether WSE possessed constituents that could act as free-radical scavengers and/or act on components of the anti-oxidant defense system. A thorough literature review on the anti-oxidative effects of WS revealed that its constituents possess free-radical scavenging activity and could modulate the expression of endogenous anti-oxidant enzymes like glutathione peroxidase, catalase, and superoxide dismutase (Bhattacharya, Ghosal et al. 2001, Alam, Hossain et al. 2011). We sought to explore the anti-oxidant potential of WSE by testing its effects on another important anti-oxidant enzyme—HO1. We chose HO1 as one of our molecular targets not only due to its multifaceted actions in addition to the anti-oxidative effects (Section
1.5.1.1), but also because it is a well-validated target in ischemic stroke (Nimura,
Weinstein et al. 1996). In fact, HO1 null mice show significantly higher infarct volumes and neurological deficits when compared to their wild type counterparts (Shah, Nada et al. 2011). In our immunoblotting studies in PC12 cells, we used the same pre-treatment regimen used for the cell viability assay. We tested the best working concentration of
WSE (100 µg/ml) and found that WSE induced the expression of HO1 both in isolation
79 as well as in the presence of H2O2, as compared to cells exposed to H2O2 alone, which showed relatively lower expression levels (Figure 3-2). The cells that received no treatment showed little to no HO1 expression, which is consistent with the stress- inducible nature of the enzyme. One might reason that the increase in HO1 expression in cells treated with WSE alone could mean that WSE acts as a stressor itself. We believe that WSE has a preconditioning effect which, by itself acting as a mild stressor triggers the physiological stress-response system, thus fortifying the body’s defense for a future insult (ischemic stroke, in our case). The fact that WS significantly improved cell viability at the same concentration it causes HO1 upregulation suggests that it has no cytotoxicity at that concentration. Although, there was a lack of statistical significance to our findings in this experiment, it gave an indication of the HO1 modulating propensity of the extract, subject to further verification in vivo.
We based our in vivo studies on the pMCAO model due to the following reasons: It has inherently low mortality rate and produces reproducible infarcts restricted to the cortical region (Bacigaluppi, Comi et al. 2010), without damaging deeper structures—both desirable aspects in a pre-clinical animal model. Also recently, based on the abysmal performance of drug candidates in clinical trials, the conventional view that tMCAO is necessarily the clinical relevant model has been debunked (Hossmann 2012). In fact, pMCAO constitutes a much more rigorous model with compounds showing activity in this model more likely to be successful at the clinical level (Richard Green, Odergren et al. 2003). We chose the dose of the extract based on studies conducted on its effects in animal models of other neurodegenerative disorders like Parkinson’s and Huntington’s
80 disease (Kulkarni and Dhir 2008, Kumar and Kumar 2009). Appropriate dose adjustments were made according to the surface area when converting rat doses to those for mice (Freireich, Gehan et al. 1966). We conducted a pilot study using the 100 mg/kg dose of WSE, but found no significant results (unpublished data); hence, we increased the dose to 200 mg/kg. Since multicomponent herbal supplements are likely to be taken orally, we chose the oral route of administration for WSE. We found that both pre- and post-treatment with WSE caused a significant reduction in the infarct volume (between
30-35% reduction as compared to the vehicle group). Since infarct volume measurement is the prime histological parameter for pre-clinical stroke studies, our results suggest that
WSE is a viable candidate for stroke therapy. The infarct volume measurements took into consideration any overestimation resulting from brain swelling (Section 2.11), thereby minimizing artifacts in the results obtained.
According to the STAIR guidelines, it is essential to evaluate neuroprotective candidates on their ability to improve functional outcomes in addition to histological parameters
(Stroke Therapy Academic Industry 1999). Therefore, we assessed the effect of WSE on the locomotor activity and neurological deficits post-surgery. Our results show that both
WSE pre- and post-treatment improve the locomotor recovery in mice to a greater extent than control mice (vehicle), the effect being significant 24 h after surgery. However, neither of the treatments had a substantial effect on improving the neurological deficits after surgery. There are two plausible explanations for the lack of significant locomotor recovery at the later time point (7 d) and the absence of an effect on the neurological deficits. Behavioral outcome studies have inherently low statistical power— more so, for subjective tests like NDS —and their results do not necessarily correlate with histological
81 outcomes. In such a case, we believe that an increase in the sample size of each treatment group would provide an indicator of the true trend of the parameter. The other reason could be that the dose of WSE used in this study is effective in improving only short-time functional outcomes, and a higher dose might be needed to extend the effect. Thus, we found significant improvement in infarct damage and short-term functional recovery with a promising trend for a long-term effect. To the best of our knowledge, this is the first study to report the therapeutic feasibility of WSE when given as a post-treatment. Based on its performance in the post-treatment paradigm, we envisage the use of WSE as an adjuvant to rt-PA at the clinic, thereby serving to extend its therapeutic window well beyond three hours.
Few studies have reported the effects of Withania somnifera in rat models of transient stroke, but they haven’t elucidated the molecular mechanism of action beyond speculating that it could involve combating oxidative stress and apoptosis (Adams, Yang et al. 2002, Chaudhary, Sharma et al. 2003). Our in vitro results suggested a possible involvement of HO1 in the observed effects of WSE. In order to confirm this theory in vivo, we probed for HO1 expression levels in mice pre-treated with WSE (200 mg/kg).
We found that indeed, WSE pre-treated mice exhibited higher levels of HO1 in their cortices, and this effect was statistically significant when compared to HO1 levels in the vehicle group (Figure 3-6). Consistent with the stress-inducible nature of HO1, sham mice expressed little to no protein. These results cement the proposition that WSE- mediated HO1 upregulation is responsible for at least part of the observed protective effects. These effects could be mediated via the anti-oxidant, anti-inflammatory, anti-
82 apoptotic, and/or the pro-angiogenic properties elicited by HO1 and its reaction by- products (Otterbein, Bach et al. 2000, Dore 2002, Al-Owais, Scragg et al. 2012).
Since prevention of cell death is the ideal outcome of any neuroprotective therapy, our next step was to assess whether WSE could protect from the widespread cell death resulting from ischemia. As discussed previously (Section 1.5.2), ischemic cell death could be a result of both necrosis and apoptosis (Nakka, Gusain et al. 2008). However, necrotic death is often rapid, and contributes to the ischemic core, which is practically beyond the point of rescue due to the considerable lag time between the onset of symptoms and clinical intervention. On the other hand, apoptosis is a delayed process predominantly responsible for the expansion of the lesion by signaling cell death in the salvageable penumbral tissue (Charriaut-Marlangue, Margaill et al. 1996). Prevention of apoptotic cell death is therefore the strategy with the best chance of success for a neuroprotective agent.
Apoptosis could result from the functioning of either extrinsic or intrinsic pathways
(Section 1.5.2). Within the intrinsic pathway, in addition to the widely studied mechanisms that require the activation of caspases, there is growing evidence for the contribution of caspase-independent mechanisms (Broughton, Reutens et al. 2009). As with many cellular cascades rampant in ischemia, there is sexual dimorphism in the apoptotic pathways with the caspase-independent pathway being the dominant player in males and the females showing primarily caspase-dependent cell death (Liu and
McCullough 2011).
The PARP-1-AIF pathway is a key mediator of apoptosis not involving caspases (Hong,
Dawson et al. 2004). AIF is known to translocate from the mitochondria to the nucleus
83 and induce DNA fragmentation within hours of a focal ischemic insult (Plesnila, Zhu et al. 2004). This translocation process is resistant to the pharmacological inhibition of caspases or gene knockouts of caspase-9, further clarifying its caspase-independent role
(Zhu, Qiu et al. 2003). While many triggers exist for this translocation including proteases like calpains and cathepsins, PARP-1 is the most relevant cue to focal ischemia
(Yu, Wang et al. 2002, Chaitanya and Babu 2008). Although its primary function is DNA repair, PARP-1 activation is associated with cell death under ischemic conditions. Free radicals and fragmented DNA—both characteristic features of the ischemic pathophysiology— trigger the activation of PARP-1. PARP-1 activation depletes NAD+ stores in cells that are already energy compromised, thus aggravating the insult (Goto,
Xue et al. 2002). Mitochondria sense the low NAD+ levels and signal the release of AIF, thus providing a molecular link between PARP-1 activation and AIF release (Yu, Wang et al. 2002). Additionally, PARP-1 null mice show lower infarct volumes, and pharmacological inhibition of this enzyme is touted to be a viable strategy for stroke therapy (Eliasson, Sampei et al. 1997). Our results showing reduced nuclear levels of both PARP-1 and AIF in WSE pre-treated mice thus suggest that WSE could prevent the deleterious effects of both these apoptotic mediators. This combined with the immunohistochemical evidence that WSE pre-treatment prevents the nuclear translocation (Figure 3-8) makes the prevention of PARP-1-mediated AIF release a likely mechanism of WSE’s action. However, the individual cytoprotective effects resulting from lower levels of either AIF or PARP-1—i.e., independent of their interaction— cannot be ruled out. For example, in addition to apoptosis, PARP-1 could initiate necrotic death of its own accord (Ying 2013); thus, WSE-mediated reduction in the expression of
84 PARP-1 could be significant despite the relatively low relevance of necrosis to our model.
Although caspase-independent apoptosis is the predominant mode of cell-death following ischemic stroke in males, we were interested to see if WSE had an influence at all on caspase-dependent mechanisms. These mechanisms are initiated by noxious stimuli causing the proliferation and the dimerization of pro-apoptotic proteins in the peri- mitochondrial space. The detrimental effects of the pro-apoptotic proteins like tBid, Bax and Bad are often neutralized by the anti-apoptotic members of the Bcl-2 family
(Broughton, Reutens et al. 2009). Bcl-2 forms homodimers or heterodimers with other protective members of its family (e.g. Bcl-xL), which in totality negates the effect of the apoptotic dimers, thereby preventing cytochrome c release and the subsequent caspase activation (MacManus and Linnik 1997).
Thus, Bcl-2 upregulation is thus a useful indicator of the status of the caspase-dependent apoptotic processes. In our case, we noticed a dip in the levels of Bcl-2 expression in the vehicle control and a slight increase in the WSE group; however, neither result was significant to warrant its role in the protective mechanism of WSE. These results taken together strongly suggest that WSE primarily influences the caspase-independent mechanism of cell death, particularly that involving PARP-1 and AIF.
As discussed previously (Section 1.5.1.1), the induction of HO1 could have anti- apoptotic effects by virtue of its enzymatic by-products: biliverdin, bilirubin, and CO.
Also, it is possible that the anti-oxidant effect by itself could have contributed to the prevention of apoptosis of cells in the penumbral region (Mattson, Culmsee et al. 2000).
Likewise, the attenuated apoptosis could have also bolstered the anti-oxidant response by
85 simply preserving cells that will express cytoprotective proteins like HO1. Thus, it is reasonable to suggest the involvement of crosstalk between the anti-oxidant and anti- apoptotic mechanisms exhibited by WSE, although a mechanistic link between the proteins involved in the pathways (HO1 and PARP-1/AIF) is yet to be established.
Following the bout of encouraging results on the neuroprotective properties of WSE pre- treatment, we shifted our focus to mechanisms downstream of the ischemic cascade. The endogenous repair mechanisms kick-start within a few days following an ischemic insult and last for weeks, even months thereafter (Gladstone, Black et al. 2002). Our aim was to test whether WSE’s ameliorative effects could extend to the reparative phase of stroke, which would widen its influence on the ischemic sequelae. Recovery from stroke involves the collaborative functioning of three processes within the neurovascular niche: neurogenesis, angiogenesis, and neuroplasticity (Font, Arboix et al. 2010). However, these processes are impeded by the presence of an inhibitory microenvironment comprised of negative or chemorepulsive guidance cues that prevent axonal sprouting
(Hou, Keklikian et al. 2008, Soleman, Filippov et al. 2013). Therefore, drugs surmounting the inhibitory effects and/or inducing the trophic effects would be ideal candidates for promoting stroke repair.
Sema3A is an important member of the semaphorins class of negative guidance cues known to inhibit axonal growth and cause growth cone collapse (Kolodkin, Matthes et al.
1993). Additionally, Sema3A could hamper angiogenesis, a crucial component of the neurorestorative phase (Font, Arboix et al. 2010). It does so by antagonizing the pro-
86 angiogenic effects of VEGF by competing with it for occupation of the same receptor
(Acevedo, Barillas et al. 2008) (Joyal, Sitaras et al. 2011)
When we immunoblotted for Sema3A in our mice brain cortices we found that the vehicle group exhibited elevated levels of the protein (Figure 3-10), consistent with reported literature (Hou, Keklikian et al. 2008, Pekcec, Yigitkanli et al. 2013). We found that WSE pre-treatment nearly abrogated the expression of Sema3A suggesting that it could pose a relatively less inhibitory environment to the repair process and possibly have an indirect pro-angiogenic role. Our deductions based on the lowered levels of
Sema3A are opposite to that of Nada et al, 2014 (Nada, Tulsulkar et al. 2014) who argue that a surge in cyclic nucleotide concentration might have caused a shift from repulsive nature of Sema3A to attraction, therefore making higher levels of Sema3A beneficial to the regenerative process. However, it is known that a combination of various factors influences the global levels of cyclic nucleotides, not just the microenvironment in question (Song, Ming et al. 1998). Moreover, our conclusion that low levels of Sema3A is beneficial for repair is substantiated by two points of evidence: HO1 is known to induce the expression of VEGF (Dulak, Deshane et al. 2008). Sema3A is a competitive antagonist of VEGF (Suchting, Bicknell et al. 2006); therefore, lower levels of Sema3A would have the net effect of promoting the pro-angiogenic effects of VEGF, while reducing the axonal growth inhibitory effects.
WSE’s potential to curb the abovementioned inhibitory cues spurred interest in supplementary pathways that aid neuronal repair. Neurogenesis, or the formation of neurons is one such crucial mechanism of repair. It is known that neurogenesis hinges on angiogenesis for its successful execution (Font, Arboix et al. 2010); the possible pro-
87 angiogenic effects of WSE therefore provided a just rationale to test for neurogenesis.
Neurogenesis is an active process in the adult nervous system; its activity is enhanced under pathological conditions like ischemia, which threaten to compromise the functioning of the system. It is a multi-step process involving proliferation of precursor cells, migration of neuroblasts to the injured site, differentiation into neurons, and finally, integration into the neuronal circuit (Ohab, Fleming et al. 2006). This process in under the control of many cellular cascades including the Wnt (Hirabayashi, Itoh et al. 2004), sonic hedgehog (Ho and Scott 2002), fibroblast growth factor (Galvez-Contreras,
Gonzalez-Castaneda et al. 2012), and the notch signaling pathways (Shimojo, Ohtsuka et al. 2011).
As described in Section 1.5.3.1, the Wnt pathway functions via two mechanisms: canonical and non-canonical. The canonical pathway is dependent on the stabilization and subsequent translocation of beta-catenin to cause transcription of Wnt target genes that control the neurogenesis process (Oliva, Vargas et al. 2013). The upregulation of
Wnt has been shown to improve neurogenesis after focal cerebral ischemia, and primarily involves beta-catenin signaling (Toledo, Colombres et al. 2008, Shruster, Ben-Zur et al.
2012). Stabilization of beta-catenin is achieved by phophorylating and inactivating GSK-
3β (Oliva, Vargas et al. 2013); therefore, the phosphorylation status of GSK-3β serves as a marker of canonical pathway activation. GSK-3β plays a vital role in neural development pathways, several not under the direct influence of the Wnt pathway (Hur and Zhou 2010, Wu and Pan 2010). When we probed for Wnt and GSK-3β in our WSE pre-treatment group, we failed to see a marked change in Wnt expression levels in animals treated with WS as compared to vehicle controls (Figure 3-11). Similarly, no
88 significant changes were seen in the phosphorylated form of GSK-3β (Ser21/9) (Figure
3-12), thereby making it unlikely that WSE activated the canonical pathway or modulated
GSK-3β phosphorylation to mediate its protective effects.
In light of evidence suggesting that the non-canonical Wnt pathway might function antagonistic to the canonical pathway (Veeman, Axelrod et al. 2003, Grumolato, Liu et al. 2010), we sought to verify if WSE modulated proteins involved in that pathway.
The general functions of the non-canonical pathway involve cell movement, tissue separation, cardiogenesis and myogenesis, to name a few (Veeman, Axelrod et al. 2003).
The signaling mechanism comprises of activation of two kinases: JNK and calcium- dependent kinases (CaMKII, calcineurin, Protein kinase C etc.). That the pathway activates JNK and CaMKII kinases and that both of them have been implicated in mediating some of the deleterious effects of ischemia (Repici, Centeno et al. 2007, Lu,
Harris et al. 2013) lends further credence to the non-canonical pathway functioning opposite to the protective canonical pathway in our settings.
We chose to study the phosphorylation levels of CRMP2 (Thr555), as it is a common phosphorylation target of both JNK and CaMKII, causing axonal destruction and growth cone collapse as a result (Arimura, Menager et al. 2005, Hou, Jiang et al. 2009,
Schlessinger, Hall et al. 2009). In our study, we did not observe a significant alteration in the phosphorylation levels of CRMP2 in both the vehicle and the WSE-treated groups
(Figure 3-12). Therefore, it is reasonable to conclude that WSE doesn’t induce the supposed harmful effects of the non-canonical signaling mechanism in our model of ischemia. In short, WS does not considerably perturb either the canonical or the non- canonical pathway, to mediate its observed protective effects.
89 Having elucidated WSE’s mechanisms of action, we proceeded to obtain a fingerprint of the crude extract for establishing a molecular template with which to compare for future experiments and also to gain a general understanding about its constituents. HPLC is a highly sensitive and commonly used analytical method for multi-component mixtures. It can be used for quantification of specific components when used with the appropriate standards. We quantified levels of withanoside-IV and withanolide A in the extract due to their high bioactivity in disease models with pathologies similar to ischemic stroke
(Kuboyama, Tohda et al. 2005, Kuboyama, Tohda et al. 2006). They have been shown to improve neurite outgrowth, synaptic integration and reverse cognitive impairment in animal models—properties that could assist with stroke rehabilitation.
We found that both these compounds were present at a higher concentration in the extract than the starting material (Ashwagandha caplets) (Figure 3-13), with withanoside-IV being the more abundant one (0.07%). While it is tempting to attribute some of the neuroprotective effects of WSE to either of these marker compounds, one cannot rule out the contribution of synergistic effects or that of other individual components we haven’t probed for in this study.
90 Chapter 5
Conclusion and Future Directions
The results of this study demonstrate the neuroprotective and potential neuroreparatory potential of WSE using a combination of in vitro and in vivo models of ischemic stroke.
Both pre- and post-treatment regimens were effective in improving infarct damage and short-term functional outcomes. We scanned well-validated, disease-relevant markers to identify targets responsible for WSE’s actions. We found that WSE pre-treatment increased HO1 expression and attenuated the PARP-1-AIF pathway of caspase- independent apoptosis suggesting that its anti-oxidant and anti-apoptotic effects could have been responsible for part of its protective effects. It is worth noting that both these effects could be interlinked, with a bidirectional cause and effect relationship between them. We also found that WSE reduced levels of the negative guidance cue-Sema3A, which could aid the repair process following stroke. We were not able to associate the
Wnt pathway of neurogenesis or Bcl-2 modulation with the observed protective effects. It is hoped that inconclusive results such as these would refine future target elucidation efforts for this extract.
91 This study unearthed many important findings pertaining to WSE’s therapeutic potential in stroke. Nevertheless, it has some limitations: The constituents of the extract haven’t been completely identified, and would make for an interesting future study. The reason for the lack of WSE’s influence on long-term functional outcomes wasn’t unequivocally established; dosage and frequency modulation could clarify the issue. Repair mechanisms including alternative neurogenesis pathways need to be thoroughly explored before ruling out their effect. Lastly, extracts obtained using different solvents could be compared to uncover new activities and constituents.
We believe that there is considerable scope to explore the beneficial effects of WSE in ischemic injury. Some of our future endeavors in this direction include testing individual components of the extract and studying other cellular pathways modulated by this fascinating herb.
92
References
Acevedo, L. M., S. Barillas, S. M. Weis, J. R. Gothert and D. A. Cheresh (2008). "Semaphorin 3A suppresses VEGF-mediated angiogenesis yet acts as a vascular permeability factor." Blood 111(5): 2674-2680.
Adams, J. D., Jr., J. Yang, L. C. Mishra and B. B. Singh (2002). "Effects of ashwagandha in a rat model of stroke." Altern Ther Health Med 8(5): 18-19.
Ahmad, M., S. Saleem, A. S. Ahmad, M. A. Ansari, S. Yousuf, M. N. Hoda and F. Islam (2005). "Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats." Hum Exp Toxicol 24(3): 137-147.
Al-Owais, M. M., J. L. Scragg, M. L. Dallas, H. E. Boycott, P. Warburton, A. Chakrabarty, J. P. Boyle and C. Peers (2012). "Carbon monoxide mediates the anti- apoptotic effects of heme oxygenase-1 in medulloblastoma DAOY cells via K+ channel inhibition." J Biol Chem 287(29): 24754-24764.
Alam, N., M. Hossain, M. I. Khalil, M. Moniruzzaman, S. A. Sulaiman and S. H. Gan (2011). "High catechin concentrations detected in Withania somnifera (ashwagandha) by high performance liquid chromatography analysis." BMC Complement Altern Med 11: 65.
Alam, N., M. Hossain, M. A. Mottalib, S. A. Sulaiman, S. H. Gan and M. I. Khalil (2012). "Methanolic extracts of Withania somnifera leaves, fruits and roots possess antioxidant properties and antibacterial activities." BMC Complement Altern Med 12: 175.
Arimura, N., C. Menager, Y. Kawano, T. Yoshimura, S. Kawabata, A. Hattori, Y. Fukata, M. Amano, Y. Goshima, M. Inagaki, N. Morone, J. Usukura and K. Kaibuchi (2005). "Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones." Mol Cell Biol 25(22): 9973-9984.
Bacigaluppi, M., G. Comi and D. M. Hermann (2010). "Animal models of ischemic stroke. Part two: modeling cerebral ischemia." Open Neurol J 4: 34-38.
Balestrino, M. (1995). "Pathophysiology of anoxic depolarization: new findings and a working hypothesis." J Neurosci Methods 59(1): 99-103.
93 Benakis, C., C. Bonny and L. Hirt (2010). "JNK inhibition and inflammation after cerebral ischemia." Brain Behav Immun 24(5): 800-811.
Bhattacharya, A., S. Ghosal and S. K. Bhattacharya (2001). "Anti-oxidant effect of Withania somnifera glycowithanolides in chronic footshock stress-induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum." J Ethnopharmacol 74(1): 1-6.
Bidmon, H. J., B. Emde, E. Oermann, R. Kubitz, O. W. Witte and K. Zilles (2001). "Heme oxygenase-1 (HSP-32) and heme oxygenase-2 induction in neurons and glial cells of cerebral regions and its relation to iron accumulation after focal cortical photothrombosis." Exp Neurol 168(1): 1-22.
Blaustein, M. P. and W. J. Lederer (1999). "Sodium/calcium exchange: its physiological implications." Physiol Rev 79(3): 763-854.
Bondy, S. C. and C. P. LeBel (1993). "The relationship between excitotoxicity and oxidative stress in the central nervous system." Free Radic Biol Med 14(6): 633-642.
Broughton, B. R., D. C. Reutens and C. G. Sobey (2009). "Apoptotic mechanisms after cerebral ischemia." Stroke 40(5): e331-339.
Canazza, A., L. Minati, C. Boffano, E. Parati and S. Binks (2014). "Experimental models of brain ischemia: a review of techniques, magnetic resonance imaging, and investigational cell-based therapies." Front Neurol 5: 19.
Carmichael, S. T. (2008). "Themes and strategies for studying the biology of stroke recovery in the poststroke epoch." Stroke 39(4): 1380-1388.
Chaitanya, G. V. and P. P. Babu (2008). "Multiple apoptogenic proteins are involved in the nuclear translocation of Apoptosis Inducing Factor during transient focal cerebral ischemia in rat." Brain Res 1246: 178-190.
Charriaut-Marlangue, C., I. Margaill, A. Represa, T. Popovici, M. Plotkine and Y. Ben- Ari (1996). "Apoptosis and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis." J Cereb Blood Flow Metab 16(2): 186-194.
Chaudhary, G., U. Sharma, N. R. Jagannathan and Y. K. Gupta (2003). "Evaluation of Withania somnifera in a middle cerebral artery occlusion model of stroke in rats." Clin Exp Pharmacol Physiol 30(5-6): 399-404.
Chen, S. D., D. I. Yang, T. K. Lin, F. Z. Shaw, C. W. Liou and Y. C. Chuang (2011). "Roles of oxidative stress, apoptosis, PGC-1alpha and mitochondrial biogenesis in cerebral ischemia." Int J Mol Sci 12(10): 7199-7215.
94 Choudhary, M. I., S. A. Nawaz, Z. ul-Haq, M. A. Lodhi, M. N. Ghayur, S. Jalil, N. Riaz, S. Yousuf, A. Malik, A. H. Gilani and A. ur-Rahman (2005). "Withanolides, a new class of natural cholinesterase inhibitors with calcium antagonistic properties." Biochem Biophys Res Commun 334(1): 276-287.
Clark, W., L. Gunion-Rinker, N. Lessov and K. Hazel (1998). "Citicoline treatment for experimental intracerebral hemorrhage in mice." Stroke 29(10): 2136-2140.
Cooper, A. J. and B. S. Kristal (1997). "Multiple roles of glutathione in the central nervous system." Biol Chem 378(8): 793-802.
Coultrap, S. J., R. S. Vest, N. M. Ashpole, A. Hudmon and K. U. Bayer (2011). "CaMKII in cerebral ischemia." Acta Pharmacol Sin 32(7): 861-872.
Culmsee, C. and J. Krieglstein (2007). "Ischaemic brain damage after stroke: new insights into efficient therapeutic strategies. International Symposium on Neurodegeneration and Neuroprotection." EMBO Rep 8(2): 129-133.
Culmsee, C., C. Zhu, S. Landshamer, B. Becattini, E. Wagner, M. Pellecchia, K. Blomgren and N. Plesnila (2005). "Apoptosis-inducing factor triggered by poly(ADP- ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia." J Neurosci 25(44): 10262-10272.
Dearden, N. M. (1985). "Ischaemic brain." Lancet 2(8449): 255-259.
Dhuley, J. N. (1998). "Effect of ashwagandha on lipid peroxidation in stress-induced animals." J Ethnopharmacol 60(2): 173-178.
Dore, S. (2002). "Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer's disease." Free Radic Biol Med 32(12): 1276- 1282.
Droge, W. (2002). "Free radicals in the physiological control of cell function." Physiol Rev 82(1): 47-95.
Duchen, M. R. (2000). "Mitochondria and calcium: from cell signalling to cell death." J Physiol 529 Pt 1: 57-68.
Dulak, J., J. Deshane, A. Jozkowicz and A. Agarwal (2008). "Heme oxygenase-1 and carbon monoxide in vascular pathobiology: focus on angiogenesis." Circulation 117(2): 231-241.
Eliasson, M. J., K. Sampei, A. S. Mandir, P. D. Hurn, R. J. Traystman, J. Bao, A. Pieper, Z. Q. Wang, T. M. Dawson, S. H. Snyder and V. L. Dawson (1997). "Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia." Nat Med 3(10): 1089-1095.
95
Elsakka, M., E. Grigorescu, U. Stanescu, U. Stanescu and V. Dorneanu (1990). "New data referring to chemistry of Withania somnifera species." Rev Med Chir Soc Med Nat Iasi 94(2): 385-387.
Endres, M., S. Namura, M. Shimizu-Sasamata, C. Waeber, L. Zhang, T. Gomez-Isla, B. T. Hyman and M. A. Moskowitz (1998). "Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family." J Cereb Blood Flow Metab 18(3): 238-247.
Ewing, J. F., S. N. Haber and M. D. Maines (1992). "Normal and heat-induced patterns of expression of heme oxygenase-1 (HSP32) in rat brain: hyperthermia causes rapid induction of mRNA and protein." J Neurochem 58(3): 1140-1149.
Font, M. A., A. Arboix and J. Krupinski (2010). "Angiogenesis, neurogenesis and neuroplasticity in ischemic stroke." Curr Cardiol Rev 6(3): 238-244.
Freireich, E. J., E. A. Gehan, D. P. Rall, L. H. Schmidt and H. E. Skipper (1966). "Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man." Cancer Chemother Rep 50(4): 219-244.
Galvez-Contreras, A. Y., R. E. Gonzalez-Castaneda, S. Luquin and O. Gonzalez-Perez (2012). "Role of fibroblast growth factor receptors in astrocytic stem cells." Curr Signal Transduct Ther 7(1): 81-86.
Gladstone, D. J., S. E. Black, A. M. Hakim, Heart and R. Stroke Foundation of Ontario Centre of Excellence in Stroke (2002). "Toward wisdom from failure: lessons from neuroprotective stroke trials and new therapeutic directions." Stroke 33(8): 2123-2136.
Glotter, E. (1991). "Withanolides and related ergostane-type steroids." Nat Prod Rep 8(4): 415-440.
Go, A. S., D. Mozaffarian, V. L. Roger, E. J. Benjamin, J. D. Berry, M. J. Blaha, S. Dai, E. S. Ford, C. S. Fox, S. Franco, H. J. Fullerton, C. Gillespie, S. M. Hailpern, J. A. Heit, V. J. Howard, M. D. Huffman, S. E. Judd, B. M. Kissela, S. J. Kittner, D. T. Lackland, J. H. Lichtman, L. D. Lisabeth, R. H. Mackey, D. J. Magid, G. M. Marcus, A. Marelli, D. B. Matchar, D. K. McGuire, E. R. Mohler, 3rd, C. S. Moy, M. E. Mussolino, R. W. Neumar, G. Nichol, D. K. Pandey, N. P. Paynter, M. J. Reeves, P. D. Sorlie, J. Stein, A. Towfighi, T. N. Turan, S. S. Virani, N. D. Wong, D. Woo, M. B. Turner, C. American Heart Association Statistics and S. Stroke Statistics (2014). "Heart disease and stroke statistics--2014 update: a report from the American Heart Association." Circulation 129(3): e28-e292.
Goto, S., R. Xue, N. Sugo, M. Sawada, K. K. Blizzard, M. F. Poitras, D. C. Johns, T. M. Dawson, V. L. Dawson, B. J. Crain, R. J. Traystman, S. Mori and P. D. Hurn (2002).
96 "Poly(ADP-ribose) polymerase impairs early and long-term experimental stroke recovery." Stroke 33(4): 1101-1106.
Graham, S. M., L. D. McCullough and S. J. Murphy (2004). "Animal models of ischemic stroke: balancing experimental aims and animal care." Comp Med 54(5): 486-496.
Grandhi, A., A. M. Mujumdar and B. Patwardhan (1994). "A comparative pharmacological investigation of Ashwagandha and Ginseng." J Ethnopharmacol 44(3): 131-135. group, I. S. T. c., P. Sandercock, J. M. Wardlaw, R. I. Lindley, M. Dennis, G. Cohen, G. Murray, K. Innes, G. Venables, A. Czlonkowska, A. Kobayashi, S. Ricci, V. Murray, E. Berge, K. B. Slot, G. J. Hankey, M. Correia, A. Peeters, K. Matz, P. Lyrer, G. Gubitz, S. J. Phillips and A. Arauz (2012). "The benefits and harms of intravenous thrombolysis with recombinant tissue plasminogen activator within 6 h of acute ischaemic stroke (the third international stroke trial [IST-3]): a randomised controlled trial." Lancet 379(9834): 2352-2363.
Grumolato, L., G. Liu, P. Mong, R. Mudbhary, R. Biswas, R. Arroyave, S. Vijayakumar, A. N. Economides and S. A. Aaronson (2010). "Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors." Genes Dev 24(22): 2517-2530.
Halliwell, B. (1999). "Antioxidant defence mechanisms: from the beginning to the end (of the beginning)." Free Radic Res 31(4): 261-272.
Hata, R., F. Gillardon, T. M. Michaelidis and K. A. Hossmann (1999). "Targeted disruption of the bcl-2 gene in mice exacerbates focal ischemic brain injury." Metab Brain Dis 14(2): 117-124.
Hillion, J. A., K. Takahashi, D. Maric, C. Ruetzler, J. L. Barker and J. M. Hallenbeck (2005). "Development of an ischemic tolerance model in a PC12 cell line." J Cereb Blood Flow Metab 25(2): 154-162.
Hirabayashi, Y., Y. Itoh, H. Tabata, K. Nakajima, T. Akiyama, N. Masuyama and Y. Gotoh (2004). "The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells." Development 131(12): 2791-2801.
Ho, K. S. and M. P. Scott (2002). "Sonic hedgehog in the nervous system: functions, modifications and mechanisms." Curr Opin Neurobiol 12(1): 57-63.
Hong, S. J., T. M. Dawson and V. L. Dawson (2004). "Nuclear and mitochondrial conversations in cell death: PARP-1 and AIF signaling." Trends Pharmacol Sci 25(5): 259-264.
97 Hossmann, K. A. (2012). "The two pathophysiologies of focal brain ischemia: implications for translational stroke research." J Cereb Blood Flow Metab 32(7): 1310- 1316.
Hou, S. T., S. X. Jiang, A. Aylsworth, G. Ferguson, J. Slinn, H. Hu, T. Leung, J. Kappler and K. Kaibuchi (2009). "CaMKII phosphorylates collapsin response mediator protein 2 and modulates axonal damage during glutamate excitotoxicity." J Neurochem 111(3): 870-881.
Hou, S. T., A. Keklikian, J. Slinn, M. O'Hare, S. X. Jiang and A. Aylsworth (2008). "Sustained up-regulation of semaphorin 3A, Neuropilin1, and doublecortin expression in ischemic mouse brain during long-term recovery." Biochem Biophys Res Commun 367(1): 109-115.
Huang, J., U. M. Upadhyay and R. J. Tamargo (2006). "Inflammation in stroke and focal cerebral ischemia." Surg Neurol 66(3): 232-245.
Hur, E. M. and F. Q. Zhou (2010). "GSK3 signalling in neural development." Nat Rev Neurosci 11(8): 539-551.
Iadecola, C. (1997). "Bright and dark sides of nitric oxide in ischemic brain injury." Trends Neurosci 20(3): 132-139.
Iadecola, C. and M. Alexander (2001). "Cerebral ischemia and inflammation." Curr Opin Neurol 14(1): 89-94.
Ikonomidou, C. and L. Turski (2002). "Why did NMDA receptor antagonists fail clinical trials for stroke and traumatic brain injury?" Lancet Neurol 1(6): 383-386.
Joyal, J. S., N. Sitaras, F. Binet, J. C. Rivera, A. Stahl, K. Zaniolo, Z. Shao, A. Polosa, T. Zhu, D. Hamel, M. Djavari, D. Kunik, J. C. Honore, E. Picard, A. Zabeida, D. R. Varma, G. Hickson, J. Mancini, M. Klagsbrun, S. Costantino, C. Beausejour, P. Lachapelle, L. E. Smith, S. Chemtob and P. Sapieha (2011). "Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A." Blood 117(22): 6024-6035.
Kataria, H., R. Wadhwa, S. C. Kaul and G. Kaur (2012). "Water extract from the leaves of Withania somnifera protect RA differentiated C6 and IMR-32 cells against glutamate- induced excitotoxicity." PLoS One 7(5): e37080.
Kim, W. Y. and W. D. Snider (2011). "Functions of GSK-3 Signaling in Development of the Nervous System." Front Mol Neurosci 4: 44.
Kolodkin, A. L., D. J. Matthes and C. S. Goodman (1993). "The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules." Cell 75(7): 1389-1399.
98 Koppenol, W. H. (2001). "The Haber-Weiss cycle--70 years later." Redox Rep 6(4): 229- 234.
Kristian, T. and B. K. Siesjo (1998). "Calcium in ischemic cell death." Stroke 29(3): 705- 718. Kuboyama, T., C. Tohda and K. Komatsu (2005). "Neuritic regeneration and synaptic reconstruction induced by withanolide A." Br J Pharmacol 144(7): 961-971.
Kuboyama, T., C. Tohda and K. Komatsu (2006). "Withanoside IV and its active metabolite, sominone, attenuate Abeta(25-35)-induced neurodegeneration." Eur J Neurosci 23(6): 1417-1426.
Kulkarni, S. K. and A. Dhir (2008). "Withania somnifera: an Indian ginseng." Prog Neuropsychopharmacol Biol Psychiatry 32(5): 1093-1105.
Kumar, P. and A. Kumar (2009). "Possible neuroprotective effect of Withania somnifera root extract against 3-nitropropionic acid-induced behavioral, biochemical, and mitochondrial dysfunction in an animal model of Huntington's disease." J Med Food 12(3): 591-600.
Kumar, S., R. J. Harris, C. J. Seal and E. J. Okello (2012). "An aqueous extract of Withania somnifera root inhibits amyloid beta fibril formation in vitro." Phytother Res 26(1): 113-117.
Kumar, S., C. J. Seal, M. J. Howes, G. C. Kite and E. J. Okello (2010). "In vitro protective effects of Withania somnifera (L.) dunal root extract against hydrogen peroxide and beta-amyloid(1-42)-induced cytotoxicity in differentiated PC12 cells." Phytother Res 24(10): 1567-1574.
Le, D. A., Y. Wu, Z. Huang, K. Matsushita, N. Plesnila, J. C. Augustinack, B. T. Hyman, J. Yuan, K. Kuida, R. A. Flavell and M. A. Moskowitz (2002). "Caspase activation and neuroprotection in caspase-3- deficient mice after in vivo cerebral ischemia and in vitro oxygen glucose deprivation." Proc Natl Acad Sci U S A 99(23): 15188-15193.
Li, X., J. A. Klaus, J. Zhang, Z. Xu, K. K. Kibler, S. A. Andrabi, K. Rao, Z. J. Yang, T. M. Dawson, V. L. Dawson and R. C. Koehler (2010). "Contributions of poly(ADP- ribose) polymerase-1 and -2 to nuclear translocation of apoptosis-inducing factor and injury from focal cerebral ischemia." J Neurochem 113(4): 1012-1022.
Lin, M. T. and M. F. Beal (2006). "Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases." Nature 443(7113): 787-795.
Liu, F. and L. D. McCullough (2011). "Middle cerebral artery occlusion model in rodents: methods and potential pitfalls." J Biomed Biotechnol 2011: 464701.
Love, S. (1999). "Oxidative stress in brain ischemia." Brain Pathol 9(1): 119-131.
99
Lu, Q., V. A. Harris, X. Sun, Y. Hou and S. M. Black (2013). "Ca(2)(+)/calmodulin- dependent protein kinase II contributes to hypoxic ischemic cell death in neonatal hippocampal slice cultures." PLoS One 8(8): e70750.
MacManus, J. P. and M. D. Linnik (1997). "Gene expression induced by cerebral ischemia: an apoptotic perspective." J Cereb Blood Flow Metab 17(8): 815-832.
Mariani, E., M. C. Polidori, A. Cherubini and P. Mecocci (2005). "Oxidative stress in brain aging, neurodegenerative and vascular diseases: an overview." J Chromatogr B Analyt Technol Biomed Life Sci 827(1): 65-75.
Martinou, J. C., M. Dubois-Dauphin, J. K. Staple, I. Rodriguez, H. Frankowski, M. Missotten, P. Albertini, D. Talabot, S. Catsicas, C. Pietra and et al. (1994). "Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia." Neuron 13(4): 1017-1030.
Mattson, M. P., C. Culmsee and Z. F. Yu (2000). "Apoptotic and antiapoptotic mechanisms in stroke." Cell Tissue Res 301(1): 173-187.
Meamar, R., L. Dehghani, M. Ghasemi, F. Khorvash and V. Shaygannejad (2013). "Stem cell therapy in stroke: a review literature." Int J Prev Med 4(Suppl 2): S139-146.
Min, K. J., K. Choi and T. K. Kwon (2011). "Withaferin A down-regulates lipopolysaccharide-induced cyclooxygenase-2 expression and PGE2 production through the inhibition of STAT1/3 activation in microglial cells." Int Immunopharmacol 11(8): 1137-1142.
Mu, Y., S. W. Lee and F. H. Gage (2010). "Signaling in adult neurogenesis." Curr Opin Neurobiol 20(4): 416-423.
Nada, S. E., J. Tulsulkar and Z. A. Shah (2014). "Heme oxygenase 1-mediated neurogenesis is enhanced by Ginkgo biloba (EGb 761(R)) after permanent ischemic stroke in mice." Mol Neurobiol 49(2): 945-956.
Nakka, V. P., A. Gusain, S. L. Mehta and R. Raghubir (2008). "Molecular mechanisms of apoptosis in cerebral ischemia: multiple neuroprotective opportunities." Mol Neurobiol 37(1): 7-38.
Newman, D. J. and G. M. Cragg (2007). "Natural products as sources of new drugs over the last 25 years." J Nat Prod 70(3): 461-477.
Nimura, T., P. R. Weinstein, S. M. Massa, S. Panter and F. R. Sharp (1996). "Heme oxygenase-1 (HO-1) protein induction in rat brain following focal ischemia." Brain Res Mol Brain Res 37(1-2): 201-208.
100 Ohab, J. J., S. Fleming, A. Blesch and S. T. Carmichael (2006). "A neurovascular niche for neurogenesis after stroke." J Neurosci 26(50): 13007-13016.
Oliva, C. A., J. Y. Vargas and N. C. Inestrosa (2013). "Wnts in adult brain: from synaptic plasticity to cognitive deficiencies." Front Cell Neurosci 7: 224.
Otterbein, L. E., F. H. Bach, J. Alam, M. Soares, H. Tao Lu, M. Wysk, R. J. Davis, R. A. Flavell and A. M. Choi (2000). "Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway." Nat Med 6(4): 422-428.
Panahian, N., M. Yoshiura and M. D. Maines (1999). "Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice." J Neurochem 72(3): 1187-1203.
Parihar, M. S. and T. Hemnani (2003). "Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity." J Biosci 28(1): 121- 128.
Pekcec, A., K. Yigitkanli, J. E. Jung, S. Pallast, C. Xing, A. Antipenko, M. Minchenko, D. B. Nikolov, T. R. Holman, E. H. Lo and K. van Leyen (2013). "Following experimental stroke, the recovering brain is vulnerable to lipoxygenase-dependent semaphorin signaling." FASEB J 27(2): 437-445.
Piantadosi, C. A. and J. Zhang (1996). "Mitochondrial generation of reactive oxygen species after brain ischemia in the rat." Stroke 27(2): 327-331; discussion 332.
Pingali, U., R. Pilli and N. Fatima (2014). "Effect of standardized aqueous extract of Withania somnifera on tests of cognitive and psychomotor performance in healthy human participants." Pharmacognosy Res 6(1): 12-18.
Plesnila, N., C. Zhu, C. Culmsee, M. Groger, M. A. Moskowitz and K. Blomgren (2004). "Nuclear translocation of apoptosis-inducing factor after focal cerebral ischemia." J Cereb Blood Flow Metab 24(4): 458-466.
RajaSankar, S., T. Manivasagam, V. Sankar, S. Prakash, R. Muthusamy, A. Krishnamurti and S. Surendran (2009). "Withania somnifera root extract improves catecholamines and physiological abnormalities seen in a Parkinson's disease model mouse." J Ethnopharmacol 125(3): 369-373.
Rami, A., J. Sims, G. Botez and J. Winckler (2003). "Spatial resolution of phospholipid scramblase 1 (PLSCR1), caspase-3 activation and DNA-fragmentation in the human hippocampus after cerebral ischemia." Neurochem Int 43(1): 79-87.
Reddy, A. S. and S. Zhang (2013). "Polypharmacology: drug discovery for the future." Expert Rev Clin Pharmacol 6(1): 41-47.
101 Repici, M., C. Centeno, S. Tomasi, G. Forloni, C. Bonny, A. Vercelli and T. Borsello (2007). "Time-course of c-Jun N-terminal kinase activation after cerebral ischemia and effect of D-JNKI1 on c-Jun and caspase-3 activation." Neuroscience 150(1): 40-49.
Rezkalla, S. H. and R. A. Kloner (2002). "No-reflow phenomenon." Circulation 105(5): 656-662. Richard Green, A., T. Odergren and T. Ashwood (2003). "Animal models of stroke: do they have value for discovering neuroprotective agents?" Trends Pharmacol Sci 24(8): 402-408.
Rishton, G. M. (2008). "Natural products as a robust source of new drugs and drug leads: past successes and present day issues." Am J Cardiol 101(10A): 43D-49D.
Rossi, D. J., T. Oshima and D. Attwell (2000). "Glutamate release in severe brain ischaemia is mainly by reversed uptake." Nature 403(6767): 316-321.
Rosso, S. B. and N. C. Inestrosa (2013). "WNT signaling in neuronal maturation and synaptogenesis." Front Cell Neurosci 7: 103.
Rothman, S. M. and J. W. Olney (1986). "Glutamate and the pathophysiology of hypoxic--ischemic brain damage." Ann Neurol 19(2): 105-111.
Ruiz de Almodovar, C., D. Lambrechts, M. Mazzone and P. Carmeliet (2009). "Role and therapeutic potential of VEGF in the nervous system." Physiol Rev 89(2): 607-648.
Ryter, S. W., H. P. Kim, A. Hoetzel, J. W. Park, K. Nakahira, X. Wang and A. M. Choi (2007). "Mechanisms of cell death in oxidative stress." Antioxid Redox Signal 9(1): 49- 89.
Saito, A., C. M. Maier, P. Narasimhan, T. Nishi, Y. S. Song, F. Yu, J. Liu, Y. S. Lee, C. Nito, H. Kamada, R. L. Dodd, L. B. Hsieh, B. Hassid, E. E. Kim, M. Gonzalez and P. H. Chan (2005). "Oxidative stress and neuronal death/survival signaling in cerebral ischemia." Mol Neurobiol 31(1-3): 105-116.
Schlessinger, K., A. Hall and N. Tolwinski (2009). "Wnt signaling pathways meet Rho GTPases." Genes Dev 23(3): 265-277.
Shah, Z. A., S. E. Nada and S. Dore (2011). "Heme oxygenase 1, beneficial role in permanent ischemic stroke and in Gingko biloba (EGb 761) neuroprotection." Neuroscience 180: 248-255.
Shibahara, S., R. Muller, H. Taguchi and T. Yoshida (1985). "Cloning and expression of cDNA for rat heme oxygenase." Proc Natl Acad Sci U S A 82(23): 7865-7869.
Shimojo, H., T. Ohtsuka and R. Kageyama (2011). "Dynamic expression of notch signaling genes in neural stem/progenitor cells." Front Neurosci 5: 78.
102
Shruster, A., T. Ben-Zur, E. Melamed and D. Offen (2012). "Wnt signaling enhances neurogenesis and improves neurological function after focal ischemic injury." PLoS One 7(7): e40843.
Singh, D., A. Aggarwal, R. Maurya and S. Naik (2007). "Withania somnifera inhibits NF-kappaB and AP-1 transcription factors in human peripheral blood and synovial fluid mononuclear cells." Phytother Res 21(10): 905-913.
Slemmer, J. E., J. J. Shacka, M. I. Sweeney and J. T. Weber (2008). "Antioxidants and free radical scavengers for the treatment of stroke, traumatic brain injury and aging." Curr Med Chem 15(4): 404-414.
Smith, M. A., R. K. Kutty, P. L. Richey, S. D. Yan, D. Stern, G. J. Chader, B. Wiggert, R. B. Petersen and G. Perry (1994). "Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer's disease." Am J Pathol 145(1): 42-47.
Soleman, S., M. A. Filippov, A. Dityatev and J. W. Fawcett (2013). "Targeting the neural extracellular matrix in neurological disorders." Neuroscience 253: 194-213.
Song, H., G. Ming, Z. He, M. Lehmann, L. McKerracher, M. Tessier-Lavigne and M. Poo (1998). "Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides." Science 281(5382): 1515-1518.
Starkov, A. A., C. Chinopoulos and G. Fiskum (2004). "Mitochondrial calcium and oxidative stress as mediators of ischemic brain injury." Cell Calcium 36(3-4): 257-264.
Stroke Therapy Academic Industry, R. (1999). "Recommendations for standards regarding preclinical neuroprotective and restorative drug development." Stroke 30(12): 2752-2758.
Suchting, S., R. Bicknell and A. Eichmann (2006). "Neuronal clues to vascular guidance." Exp Cell Res 312(5): 668-675.
Swanson, R. A., M. T. Morton, G. Tsao-Wu, R. A. Savalos, C. Davidson and F. R. Sharp (1990). "A semiautomated method for measuring brain infarct volume." J Cereb Blood Flow Metab 10(2): 290-293.
Szarc vel Szic, K., K. Op de Beeck, D. Ratman, A. Wouters, I. M. Beck, K. Declerck, K. Heyninck, E. Fransen, M. Bracke, K. De Bosscher, F. Lardon, G. Van Camp and W. Vanden Berghe (2014). "Pharmacological levels of Withaferin A (Withania somnifera) trigger clinically relevant anticancer effects specific to triple negative breast cancer cells." PLoS One 9(2): e87850.
103 Tabakman, R., H. Jiang, I. Shahar, H. Arien-Zakay, R. A. Levine and P. Lazarovici (2005). "Neuroprotection by NGF in the PC12 in vitro OGD model: involvement of mitogen-activated protein kinases and gene expression." Ann N Y Acad Sci 1053: 84-96.
Takeda, A., T. Kimpara, H. Onodera, Y. Itoyama, S. Shibahara and K. Kogure (1996). "Regional difference in induction of heme oxygenase-1 protein following rat transient forebrain ischemia." Neurosci Lett 205(3): 169-172.
Toledo, E. M., M. Colombres and N. C. Inestrosa (2008). "Wnt signaling in neuroprotection and stem cell differentiation." Prog Neurobiol 86(3): 281-296.
Valko, M., D. Leibfritz, J. Moncol, M. T. Cronin, M. Mazur and J. Telser (2007). "Free radicals and antioxidants in normal physiological functions and human disease." Int J Biochem Cell Biol 39(1): 44-84.
Veeman, M. T., J. D. Axelrod and R. T. Moon (2003). "A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling." Dev Cell 5(3): 367-377.
Wiessner, C., P. R. Allegrini, K. Rupalla, D. Sauer, T. Oltersdorf, A. L. McGregor, S. Bischoff, B. W. Bottiger and H. van der Putten (1999). "Neuron-specific transgene expression of Bcl-XL but not Bcl-2 genes reduced lesion size after permanent middle cerebral artery occlusion in mice." Neurosci Lett 268(3): 119-122.
Winterbourn, C. C. (1995). "Toxicity of iron and hydrogen peroxide: the Fenton reaction." Toxicol Lett 82-83: 969-974.
Wu, D. and W. Pan (2010). "GSK3: a multifaceted kinase in Wnt signaling." Trends Biochem Sci 35(3): 161-168.
Ying, W. (2013). "Roles of NAD (+) , PARP-1, and Sirtuins in Cell Death, Ischemic Brain Injury, and Synchrotron Radiation X-Ray-Induced Tissue Injury." Scientifica (Cairo) 2013: 691251.
Yu, B. P. (1994). "Cellular defenses against damage from reactive oxygen species." Physiol Rev 74(1): 139-162.
Yu, S. W., H. Wang, M. F. Poitras, C. Coombs, W. J. Bowers, H. J. Federoff, G. G. Poirier, T. M. Dawson and V. L. Dawson (2002). "Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor." Science 297(5579): 259-263.
Zeynalov, E., Z. A. Shah, R. C. Li and S. Dore (2009). "Heme oxygenase 1 is associated with ischemic preconditioning-induced protection against brain ischemia." Neurobiol Dis 35(2): 264-269.
104 Zhao, J., N. Nakamura, M. Hattori, T. Kuboyama, C. Tohda and K. Komatsu (2002). "Withanolide derivatives from the roots of Withania somnifera and their neurite outgrowth activities." Chem Pharm Bull (Tokyo) 50(6): 760-765.
Zhu, C., L. Qiu, X. Wang, U. Hallin, C. Cande, G. Kroemer, H. Hagberg and K. Blomgren (2003). "Involvement of apoptosis-inducing factor in neuronal death after hypoxia-ischemia in the neonatal rat brain." J Neurochem 86(2): 306-317.
Zivin, J. A. (2009). "Acute stroke therapy with tissue plasminogen activator (tPA) since it was approved by the U.S. Food and Drug Administration (FDA)." Ann Neurol 66(1): 6- 10.
105