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Neuroprotection by 20(S)Protopanaxadiol in Focal Cerebral Ischemia Mouse Model Wang, Jing 2006

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Neuroprotection by 20(S)ProtopanaxadioI in Focal Cerebral Ischemia Mouse Model B y Jing Wang B.Med . , ChongQing Medical University of China, 1991 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E In T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Surgery) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A Apr i l 2006 . © Jing Wang, 2006 A B S T R A C T 20(S)Protopanaxadiol (aPPD) is a deglycosylation metabolite of ginsenosides. The latter are the major components in ginseng. Previous study in our lab has demonstrated that aPPD i.v. injection at dose 60mg/kg had no noticeable toxicity in animals and was able to cross the blood-brain barrier. Recently, increasing evidence in the literature has reported that aPPD's precursor such as R b l , Rg3 and Rh2, have beneficial effect on the central nervous system. Since aPPD is the common metabolic product of above ginsenosides in the body, the purpose of this study is to investigate the possible protective effect of aPPD upon brain injury after cerebral ischemia. We found that aPPD could protect cultured cortical neurons from N M D A induced excitotoxicity. We have also tested neuropotective actions of aPPD in a mouse ischemia-reperfusion model. Transient focal ischemia was induced by 60min middle cerebral artery occlusion followed by reperfusion in C57 B L / 6 mice. 30mg/kg aPPD i.p. was administered for 7 days from the onset of reperfusion. The outcome of aPPD treatment was assessed by general physiological condition, various behavioral tests and histopathological analysis on day 7 and day 90 post-ischemia. aPPD significantly reduced weight loss, mortality, infarct area and facilitated sensorimotor functional recovery in the early period following focal cerebra ischemia. Moreover, aPPD also improved cognitive deficit o f mice when evaluated post ischemia 90 days. Furthermore, a protein kinase profile has demonstrated that aPPD caused significant elevation o f kinase activity in the ischemic brain, including all the stress-related kinases. In particular, aPPD induced upregulation of p A K T in a neuronal and ischemic specific n fashion. Although outcome of long-term responses to aPPD treatment requires further study, present results provide evidence that aPPD might potentially be a therapeutic agent for preventing brain damage from a stroke. i i i T A B L E O F C O N T E N T S Abstract ii Table of Contents iv List of Tables . vii List of Figures viii Abbreviations x Acknowledgements xii CHAPTER 1: INTRODUCTION.... 1 1.1 S T R O K E 1 1.1.1 Epidemiology and Classification 1 1.1.2 Clinical and Histopathological Features 2 1.1.3 A n Animal Mode l for Transient Focal Cerebral Ischemia 3 1.1.4 Disease Mechanisms in Focal Cerebral Ischemia 4 1.1.4.1 Energy failure, Excitotoxicity and Ionic imbalance 4 1.1.4.2 Oxidative stress .....6 1.1.5 Neuronal Death in Focal Cerebral Ischemia: Apoptosis in Penumbral ..7 1.1.6 . Therapeutic Strategies 10 1.2 G I N G S E N G . ; 11 1.2.1 Chemical structure and metabolism of protopanaxadiol-typQ ginsenosides 12 1.2.2 Beneficial effects of protopanaxadiol-type ginsenosides on C N S 13 1 3 H Y P O T H E S I S A N D S T U D Y A I M 15 iv CHAPTER 2: MATERIAL AND METHODS 16 2.1 IN VITRO S T U D Y 16 2.1.1 Mouse primary cortical neuronal culture 16 2.1.2 Mouse primary cortical neuronal culture treated with N M D A and aPPD 17 2.1.3 L D H assay 17 2.2 IN VIVO S T U D Y 18 2.2.1 Animal 18 2.2.2 Transient focal ischemia 19 2.2.3 Drug treatments ..19 2.2.4 Weight loss and Mortality evaluation. 20 2.2.5 Behavior Tests (post-ischemic 7 days ) 20 2.2.5.1 Four-point scale 21 2.2.5.2 Latency to move 21 2.2.5.3 Latency to fall 21 2.2.6 Behavior Tests (post-ischemic 90 days) 22 2.2.6.1 Water maze 22 2.2.6.2 Context Fear conditioning 23 2.2.7 Histopathology assessment 24 2.3 S T U D Y O F P R O T E I N K I N A S E S 25 2.3.1 Tissue preparation and protein kinase screening 25 2.3.2 Western Blotting for phospho-Akt (pAkt) 27 2.3.3 Immunofluorescence triple-labeling 28 v • 2.3 .4 D N A fragmentation detected by T U N E L 29 2.3.5 Immunofluorescent triple labeling with pAk, Nuclear Dye and T U N E L 30 2.4 STATISTICAL ANALYSIS 31 CHAPTER 3: RESULTS 32 3.1 In Vitro Study 32 3.1.1 aPPD protects primary neuronal culture from N M D A induced excitotoxicity 32 3.2 Mouse t M C A O Model .....33 3.2.1 aPPD reduced animal weight loss and mortality after ischemia 33 3.2.2 aPPD improved functional recovery after ischemia 34 3.2.3 Histological evaluation ....36 3.3 Activity of Kinases 36 3.3.1 aPPD altered kinase activeity after t M C A O 36 3.3.2 aPPD increased expression o f pAkt and enhanced pAkt mainly in neurons 38 3.3.3 aPPD reduced ischemia-induced apoptosis 38 CHAPTER 4: DISCUSSION AND SUMMARY 39 4.1 The Role of aPPD in t M C A O 39 4.2 Conclusion and Future Direction 48 REFERENCE 66 vi L I S T O F T A B L E S Table 3.1. Post-ischemia 7-day behavioral functional test 55 vi i L I S T O F F I G U R E S Figure 1.2.1. aPPD and its precursor (aglycone process) 50 Figure 3.1.1. Protective effects of aPPD against NMD A neurotoxicity in primary neuronal culture 51 Figure 3.2.1. 7-day weight loss after ischemia 52 Figure 3.2.2. 14-day mortality evaluation after ischemia 53 Figure 3.2.3. Post-ischemia 7-day behavioral functional test 54 Figure 3.2.4. Post-ischemic 90 days Water Maze test. 56 Figure 3.2.5. Post-ischemic 90-day Context Fear Conditioning test.. .57 Figure 3.2.6. Short-termhistopathological evaluation 58 Figure 3.2.6. Long-term histopathological evaluation 59 Figure 3.3.1. Protein Kinase Screen Results 60 Figure 3.3.2. Western Blotting analysis of pAkt after tMCAO 61 Figure 3.3.3. Cellular localization of pAkt after ischemia by triple fluorescence immunohistochemistry 62 Figure 3.3.4. aPPD rescued cortical cells from ischemia-induced apoptosis 63 Figure 3.3.5. aPPD rescued primary cortical cultures from NMDA-induced apoptosis ....64 v i i i Figure 4.2. Proposed intracellular signaling involved in aPPD neuroprotective mechanism 65 IX A B B R E V I A T I O N S A I F apoptosis inducing factor Akt (PKB) protein kinase B A M P A a-amino-3-hydroxy-5-methyl-4- isoxazolepropion APaf-1 apoptotic protease activating factor 1 aPPD 20(S)-Protopanaxadiol A T P adenosine triphosphate C A D caspase activated deoxiribonuclease caspase cyteine aspartate-specific proteases C B F cerebral blood flow C C A common carotid artery C N S central nervous system CS conditioned stimulus E A A s excitatory amino acids E C A external carotid artery E R K extracellular signal-related kinases G F A P glial fibrillary acidic protein i.p. intraperitoneally i.v. intravenously IAP inhibitor o f apoptosis I C A internal carotid artery I N K C-Jun NH2-terminal kinase L D H lactate dehydrogenase M A P K mitogen - activated protein kinase M C A middle cerebral artery M C A O middle cerebral artery occlusion mGluR metabotrobic glutamate receptor NeuN neuron specific nuclear protein N M D A N-Methyl-D-Aspartate N O nitric oxide P38 38 K D a subunit of mitogen - activated protein kinase P A R P poly (ADP-ribose) polymerase PI3K phosphatidylinositol 3-kinase P R A S 4 0 proline-rich A K T substrate of 40kDa PTP permeability transition pore R O S reactive oxygen species S D S - P A G E sodium dodecyl sulfate polyacryl amide gel electrophoresis S A P K stress activated protein kinase t M C A O transient middle cerebral artery occlusion T T C 2,3,5 triphenyl tetrazolium chloride T U N E L terminal deoxynucleotidyl transferase mediated dUTP nick end labelin US unconditioned stimulus XI ACKNOWLEDGEMENTS This work was carried out in the Department of Surgery and Brain Research Center, University of British Columbia during the year 2004-2006. I wish to express my deepest gratitude to my principal supervisor Dr. Wi l l i am Jia for introducing me to the field of neuroscience and the fascinating subject - stroke. I w i l l always appreciate the generous gifts of his time, advice, inspiration, and continuous support. I am most grateful to my supervisor and committee members: Dr. Gary Redekop, Dr. Wei Hong Song, and Dr . Y u Tian Wang for their guidance, support and constructive criticism to this study. I would like to express my gratitude to Dr. M a x Cynader for his crucial advice, fruitful discussion, and valuable suggestions. I want to thank PanaGin Pharmaceutials Inc for providing me with ginsenoside products and Kinexus Bioinformatics Corporation for measuring a panel of signaling proteins. I owe a great debt of gratitude to Ivan Cepeda and Gui Qong He, as well as Dr. Wei Hong Song's lab for their help with behavioral tests. I w i l l remember the help from all the members in Brain Research Center. I owe my special thanks to Dong Qiang, L i Pang, Louis Huang, Y u Ping L i , Guo Y u L i u , Xue X i a n Bu , Hang Yan , Y a n Huan Wen, W e i Xiong, A l a n Huang and Shan Shan Zhu for their knowledge in laboratory techniques and practical problem solving capability. Finally, no words are enough to express my appreciation and love for my parents, my husband and my son for their understanding, supporting, and sharing the feelings on the bumpy road of science. x i i C H A P T E R 1 INTRODUCTION 1.1 STROKE A stroke is defined as a sudden loss of brain function caused by a blockage or rupture of a blood vessel to the brain. Cells in the affected region of the brain die because they no longer receive oxygen and nutrients from the blood, leading to the symptoms and disabilities of stroke patients. [1]. 1.1.1 Epidemiology and Classification Stroke remains the third leading cause of death and major cause of disability in industrialized countries. In Canada, it occurs every 10 minutes and approximately 50,000 Canadians suffer stroke each year. It accounts for 8% of hospital bed utilization and the direct cost of taking care of patients (hospital and nursing homes) may be over 4 bill ion dollars annually [2]. In the U S A , stroke killed 283,000 people in 2000 and accounted for about one in every 14 deaths. Three mil l ion Americans are currently permanently disabled because of ischemic stroke, and the direct and indirect cost of stroke in 1998 is estimated at $ 43.4 bi l l ion [3, 4]. The main causes of stroke are ischemia and hemorrhage. They are classified as three major categories: ischemic stroke, subarachnoid hemorrhage and intracerebral hemorrhage [5]. 1 1.1.2 Cl in ica l and Histopathological Features Most strokes (~85%) are ischemic, characterized by inadequate blood flow and death of affected brain regions [6]. Infarction is a histopathological description for the injured regions. According to the size and location of infarction and an evolving neurological deficit, ischemic stroke is characterized into two features: global and focal. Global cerebral ischemia, as occurs during a cardiac arrest, affects the entire brain due to the sudden cessation o f blood flow. Focal ischemia, as occurs in most cases, results from an occlusion of a major cerebral artery by a thrombus or an embolism, which leads to loss of blood flow in a specific region. Thrombotic occlusion of small and large vessels causes approximately 61% of strokes in the anterior, middle, and posterior cerebral vasculature combined. Embolic occlusion results in 15-30% of all strokes [7]. In focal ischemia, the centre or core territory is the region where the blood flow is most severely reduced and where the brain cells rapidly die. Regions adjacent to the core are defined as penumbra, which means a peripheral zone where residual blood flow might transiently sustain tissue viability and cells within the flow-compromised territory might be rescued and resuscitated by restoration of perfusion or other protective therapies. Ischemia core and penumbra can be clearly identified under Positron emission tomography and magnetic resonance imaging [8]. Focal cerebral ischemia can be broadly categorized into two types, permanent or transient focal ischemia followed by reperfusion. In patients suffered from embolic vascular occlusion, spontaneous thrombolysis and recanalization occur at variable times following initial occlusion [9]. Angiographic controlled studies in humans have shown that spontaneous recanalization can occur around 17% of the time within the first 6 to 8 2 hours of stroke and that approximately half of the vessels w i l l reopen in 3 to 4 days [10]. Therefore, damage w i l l be the result of both the ischemia and the consequences of reperfusion. In clinical situation, middle cerebral artery ( M C A ) is by far the largest o f the cerebral arteries and is the vessel most common occlusion in stroke patients [11]. Focal ischemia by middle cerebral artery occlusion ( M C A O ) results in the epicentre of the insult at striatum and complex process of cell death and cell survival in the penumbra [12]. More recent research has focused upon the presence of specific neurological deficits after M C A O and correlation to outcomes and prognosis. 1.1.3 An Animal Model for Transient Focal Cerebral Ischemia A number of stroke animal models have been developed. Originally described by Koizumi [13] and modified by Longa [14], transient middle cerebral artery occlusion in rodents has been used routinely to mimic transient focal cerebral ischemia in humans. This model is to use an intraluminal nylon fdament, insert into external carotid artery ( E C A ) , and advance along internal carotid artery (ICA) into the circle of Wi l l i s , and then lodge in anterior cerebral artery ( A C A ) / middle cerebral artery ( M C A ) bifurcation. Therefore, middle cerebral artery is occluded at its origin. After occlusion, blood flow can be restored by removing the fdament and transient middle cerebral artery occlusion ( t M C A O ) is established. t M C A O model is non-invasive because it avoids craniotomy and the severity of the insult is controlled by varying the occlusion time. One essential feature of t M C A O model is that ischemic damage, rather paradoxically, progresses for days and even weeks after the restoration of the blood flow. This phenomenon call "reperfusion 3 injury" is well characterized and characteristic for the model. Therefore, t M C A O model is now accepted as the primary model for stroke studies by the fact that clinically the M C A is the primary site of many strokes and reperfusion frequently occurs as the result of recanalization [15, 16]. More recently, t M C A O model is widely accepted for use in mice, particularly because the availability of transgenic and gene knockout mouse strains provides a unique opportunity to evaluate the role of single gene product in the pathophysiology of stroke. 1.1.4 Disease Mechanisms in Focal Cerebral Ischemia Ischemic brain injury results from a complex sequence of pathophysiological events that evolve over time and space. The major pathogenic mechanisms involved in focal cerebral ischemia include energy failure, excitotoxicity, ionic imbalance, and oxidative stress. 1.1.4.1 Energy failure, Excitotoxicity and Ionic imbalance The blockage o f blood flow to the certain vulnerable regions of the brain results in restricting the delivery o f oxygen and glucose and impairing the energy supply required to maintain ionic gradients [17]. When the cerebral blood flow (CBF) does not guarantee adequate perfusion, neurons become depleted of A T P , switch over to anaerobic glycolysis and acidic byproducts of metabolism accumulate. Energy deficits lead to membrane potential lost, neurons and glia depolarized, and ion pump failed. Failure to maintain the ion gradients and depolarization of cell membranes results in excessive extracellular excitatory amino acids (EAAs) , especially glutamate, as a consequence of 4 enhancing efflux and reducing uptake. Glutamate is the most abundant excitatory neurotransmitter in the central nervous system which is stored in the presynaptic vesicles and carries out its activity by stimulating receptors located on the postsynaptic membranes of neurons. There are two classes of glutamate receptors, the ionotropic receptors, which are the ligand-gated ion channels, and the metabotropic glutamate receptors (mGluR), which affect ion-channels by activation of G-proteins and evoke a variety of functions by mediating intracellular signal transduction [18]. Metabotropic glutamate receptors are found pre- and post synaptically and they may modulate the toxicity of ionotropic glutamate receptors, for example during excitotoxicity. There are three subtypes of glutamate ionotropic receptors that are distinctively activated by their selective chemical agonists: A^-methyl-D-aspartate ( N M D A ) , a-amino-3-hydroxy-5-mefhyl-4- isoxazolepropionic acid ( A M P A ) and Kainate [19]. K a i n a t e / A M P A receptors are often referred together as n o n - N M D A receptors [20]. N M D A receptor activation opens C a 2 + channel, allowing C a 2 + influx, and n o n - N M D A receptors activation opens a Na + channel, allowing Na+ influx. One attractive hypothesis, that the ability o f glutamate and related E A A exposure to trigger central neuronal death was first proposed by Olney and colleagues [21]. According to this hypothesis, the early efflux of glutamate occurring immediately after the onset of ischemia is mediated by a calcium-dependent process through activation of voltage dependent calcium channels. Extracellular glutamate overstimulates A M P A , kainate, and N M D A - t y p e receptors and promotes N a + and C a 2 + influx and K + efflux through these receptor-gated ion channels. A s N a + and C a 2 + entry is joined by the influx of CI" and water, marked neuronal cell body swelling and dendrite swelling occur. The calcium-independent influx at later stages is also triggered 5 secondarily by N a + influx through A M P A - , kainate- and N M D A - receptor-gated channels by activation of voltage-gated C a 2 + channels and reverse operation of the N a + / C a 2 + exchanger, thereby again resulting in increased C a 2 + [22]. More recently, attention has been drawn to the possibility that C a 2 + entry leads to variety lethal metabolic derangements [23]. Influx of C a 2 + damages the mitochondria, which further exacerbates energy failure. High intracellular C a 2 + level initiates protease and phospholipase activity that inhibits protein synthesis and degrades cellular membranes. Increased C a 2 + also induces nitric oxide synthase generation and cause oxidative stress. Although the relationship between accumulation of extracellular glutamate and subsequent neuronal cell death is not necessarily direct, the toxic effects of glutamate on central neurons may be mediated by an influx of N a + and C a 2 + into the cell, which initiate a cascade of biochemical changes such as mitochondrial lesions, proteolysis of microfilaments, breakage of membrane phospholipids, formation of free radicals, and cell death [24]. 1.1.4.2 Oxidative stress Oxidative stress is a consequence of a misbalance between the production of oxidants and the ability o f a cell or tissue to defend itself against them. Potential oxidants are free radicals, such as the superoxide and hydroxyl radicals, as well as molecules with a strong oxidative potential, such as hydrogen peroxide, nitric oxide, and peroxynitrite. Among these free radicals, superoxide anion is directly toxic to neurons [25]. After ischemia and particularly reperfusion, energetic and ionic perturbations lead to accelerated formation of free radicals and a wide range of intracellular and extracellular effects, including damage to D N A , proteins and lipids and the formation of post-ischemic 6 inflammatory injury [26]. Limited Oxygen supply reduces electron transport chain of the inner mitochondrial membrane, which causes direct single-electron reduction o f oxygen and formation of superoxide. Calcium accumulation stimulates phospholipase activity, which may generate superoxide or hydroxyl production via arachidonic acid metabolism through the cyclooxygenase pathway or the lipoxygenase pathway. Phospholipid breakdown into arachidonic acid is then further activated by free radicals. Free radicals facilitate mitochondrial transition pore (MTP) formation, leading to mitochondria and D N A damage, and in turn triggering caspase-mediated apoptotic cell death [27, 28]. Nitric oxide (NO) is produced by neuronal constitutive nitric oxide synthetase (NOS) or inducible nitric oxide synthetase (iNOS) in an oxygen-dependent reaction which is activated by Ca 2 + /calmodulin in most neurons and endothelial cells. A n increased production of N O in the brain has been demonstrated during ischemia and both N O S and iNOS are upregulated after focal ischemia [29, 30]. N O combining with superoxide results in peroxynitrite generation, leading to inappropriate protein nitrosylation, l ipid peroxidation and membrane breakdown. N O is able to cause G : C to A : T transitions and to mediate D N A strand breaks[31]. In addition, free radicals induce the formation of inflammatory mediators, which lead to the invasion of blood-born inflammatory cells and post-ischemic injury [32, 33]. 1.1.5 Neuronal Death in Focal Cerebral Ischemia: Apoptosis in Penumbra In recent years it became clear that ischemic neurons die by two different modes: necrosis and apoptosis [34, 35]. Neurons in the central core of the infarction die by necrosis, which is characterized by the sudden failure of cellular energy, and swelling and 7 rupture of the organelles. In contrast, the involvement of apoptotic cell death, particularly of cells within the penumbra, has been observed in a number of reports over the last few years. Apoptosis is an active, energy consuming process of self-destruction in which unnecessary or damaged cells are eliminated [36]. A hallmark distinction between necrotic cell death and apoptosis is that energy and protein synthesis are required for the latter to program an internal self-destruction. This may account for cells in penumbra usually undergoing apoptotic pathway. Linnik et al. (1993) were the first to implicate apoptosis in focal ischemia brain by showing D N A fragmentation on gel electrophoresis [37]. L i et al. (1995) identified morphological features of apoptotic cells in the peripheral part of infarct of rats subjected to t M C A O under electron microscopy [38, 39]. Apoptosis is morphologically characterized by chromatin condensation, cell shrinking, neurite degeneration and D N A fragmentation. D N A fragmentation can be visualized in tissue sections by positive terminal deoxyri-bonucleotidyl transferase (TNT)-mediated dUTP(2'-deoxyuridine-5'-triphosphate)-digoxigenin nick end labeling ( T U N E L ) [40]. Recently, more evidence in molecular signatures of apoptosis and pro- and anti-apoptotic gene localization are documented in experimental focal ischemic models. Cel l death by apoptosis is carried out by several facilitating receptors or factors. These include apoptosis inducing or death receptors (e.g. Apo- l /Fas , Apaf-1), apoptosis initiating factors (AIFs), members of the Bcl-2 and cysteine proteases of the caspase/ calpain family [41, 42]. Caspases, a family of cysteine aspartases constitutively express as zymogens or pro-caspases in adult cells, particularly neurons. Caspases can be divided into two groups: upstream cascade initiators (caspase-8, -9, and -10),.and downstream terminators (caspases-3, -6, and -7). Caspase-1, -2, - 4, -5, -11, and -12 can act as 8 initiators and executioners. Caspase-3 is a predominant protease involved in rapid cleavage of DNA-repair ing enzyme poly (ADP-ribose) polymerase (PARP) , caspase activated DNase ( C A D ) , and D N A damage, leading to apoptosis. Apoptosis is also regulated by proteins of the Bcl -2 family, consisting of antiapoptotic members (such as Bcl-2 itself and B c l - X L ) and pro-apoptotic members (Bax, B i d , Bak, Bad and B c l - X S ) . Although apoptotic cell death is often mediated by a caspase cascade, both extrinsic and intrinsic caspase-dependent cell death pathways, and caspase-independent cell death signaling have been implicated in several paradigms of t M C A O in adult rats and mice [43, 44, 45, 46]. The extrinsic pathway is independent of mitochondria and initiated by receptors binding to ligand, which activates receptor-associated death domain and caspase-8, and cleaves caspase-3 into active proteases. Caspase-8 is also able to activate one of the Bcl -2 family proteins, B i d , and to initiate the mitochondrial pathway of apoptosis [47]. The intrinsic pathway is mitochondrial pathway where various signals can trigger the release of harmful proteins by mitochondria, especially cytochrome C to form apoptosome. For example, Bad, a member of the Bax family, translocates to mitochondria membrane, competes with antiapoptotic members of Bcl -2 family and results in cytochrome c release to the cytosol [48]. When cytochrome C is released from mitochondria, it forms an apoptosome complex [APaf-1 plus pro-caspase 9] in the presence of d A T P , which promotes clipping and activation of caspase 3 [49, 50]. Finally, apoptosis-inducing factor (AIF) translocates to the mitochondria and the nucleus, and produces D N A damage [51]. In addition, activation of other proteases such as calpain, proteasome and serine proteases also leads to the caspase-independent cell death pathway of apoptosis [52, 53]. The genes for caspases as well as genes that suppress (for example, 9 Bcl2) or augment (Bax, p53) cell death are expressed at higher levels and activated in both the early and late stages o f ischemia, and genetic manipulations or drugs that block caspase family members or enhance the actions of B C L 2 demonstrated resistance to ischemic injury, further supporting apoptotic-like pathway following focal cerebral ischemia [54, 55]. 1.1.6 Therapeutic Strategies Although many different therapeutic approaches have been evaluated for possible treatment of stroke patients, unfortunately, there has been a lack of progress for recent two decades. With regard to treatment for ischemic stroke, two major approaches have been developed. The first target is to restore blood flow and establish arterial oxygen and glucose by lysing an intraarterial thrombus after ischemia. In 1996, the first thrombolysis, plasminogen activator (rt-PA) became available but it must be given within the first 3 hours of symptom onset [56]. After this so-called "therapeutic window," the administration of thrombolysis increases the risk of haemorrhagic transformation. In addition, delayed hospital presentation is also precluding its administration. The second major therapeutic approach is to develop neuroprotective agents, which protect ischemic cells, particular in penumbra from progressing death. More than 49 potential neuroptotective agents, which appeared quite effective in animals, have been studied in more than 114 clinical trials in the past years. These include free radical scavengers, excitatory amino acid antagonists, calcium channel blockers and growth factors [57, 58]. However, none of these have proven conclusively to be effective in humans. Why have 10 so many clinical stroke trials failed? The failure probably lies with three reasons: drugs, animal models and preclinical designs. First, because ischemic stroke is a complex event involved overlapping pathways and multiple molecular cascades, single neuroprotective agent intervention w i l l never work and multiagent therapy is needed. Combined thrombolysis-neuroprotective approaches have shown promise in animal studies and are beginning to be investigated in clinical trials. For example, synergistic effects have been demonstrated in animals when thrombolysis is combined with excitatory amino acid antagonists, or other agents [59, 60]. Second, the variable aetiology, pathology, clinical presentation and outcome of stroke patients, coupled with pre-existing disease, can all contribute to variability in outcome [61, 62]. Conversely, animal experiments control or eliminate many of these variables. Finally, the designs established by preclinical study, including drug exposure (dose and subsequent plasma concentration), time window, age and associated illnesses and efficacy evaluation are not integrated and fail to match the complex clinical trial. 1.2 G I N G S E N G Ginseng refers to the root o f several species in the plant genus Panax which is a member of one of the oldest plant families-Araliaceae family [63]. Among them, there are three common species that are most utilized and studied: Panax ginseng (mainly in China and Korea), Panax quinquefolius (in southern Canada and in the United States), and Panax japonicus (in Japan) [64]. In China, Ginseng is a widespread herbal medicine and it has served as an important component of many Chinese prescriptions for thousands of years. Ginseng roots are harvested when the plant is 3-6 years old and then divided to 11 three types based in the different preparation: white ginseng (air drying), red ginseng (steamed), and fresh ginseng. Ginseng root consists o f two major ingredients: crude ginseng saponin and crude ginseng nonsaponin fractions. The principle active ingredients of ginseng are ginseng saponins. In 1963, Shibata S and his colleagues first named ginseng saponins as "ginsenosides" and classified them as two groups, dammarane-type and oleanane-type saponins on the basis of the chemical structures of their aglycones [65, 66]. The dammaranes are further divided into two types according to the number of hydroxyl and glicosidic bond at C-3 and C-6: protopanxadiol type (PPD) and protopanxatriol type (PPT). To date, more than 35 ginsenosides have been isolated from ginseng root and identified chemically. Among them 22 of these ginsenosides are protopanaxadiol type (e.g. R b l , Rb2, Rb3, Rc, Rd , Rg3, Rh2, R s l , and aglycon PPD); and 11 are protopanaxatriol type (e.g. Re, Rf, R g l , Rg2, R h l , and aglycone PPT) [67, 68]. 1.2.1 Chemical structure and metabolism of protopanaxadiol-type ginsenosides The basic structure of protopanaxadiol type ginsenosides consists of a gonane steroid nucleus with 17 carbon atoms arranged in four rings and a modified side-chain at C-20. Although they have the common dammarane skeleton, the characteristic biological responses for each protopanaxadiol are mainly attributed to the differences in the position and number of sugar moieties attached by glicosidic bond at C-3 as well as hydroxyl groups attached at C-20 [69]. The sugar moieties include glucose, maltose, fructose, and saccharose. 20(S) protopanaxadiol (aPPD) is an aglycone metabolite of Rh2. Most ginsenosides that have been isolated are naturally present as enantiomeric mixtures. 12 Ginseng is usually taken by oral administration and the biotransformation of ginsenosides has been reported through human intestinal bacteria [70]. Based on enzyme immunoassay (EIA) and high-performance liquid chromatograph ( H P L C ) , orally administrated red ginseng extracts protopanaxadiol type R b l is not detected in the blood. Human intestinal bacteria assay and H P L C suggest that R b l and Rb2 transformed to Rg3 in the stomach, and then Fusobactrium K-60 , a Rbl-metabolizing bacteria transforms Rg3 to 20(S) protopanaxadiol (aPPD) via Rh2 by bacteria (including Bacteroides sp., Eubacterium sp., and Bifidobacterium sp.) in human intestine [71]. The structure diversities and proposed metabolic pathway of 20(S) protopanaxadiol (aPPD) and other protopanaxadiol-type ginsenosides are illustrated as Figure 1.2.1.. Considering that Ginseng are often orally taken and that ginsenosides are metabolized by intestinal bacteria to monoglucosides and then aglycone products, it is important to investigate the biological activity of the metabolic products, such as aPPD. 1.2.2 Beneficial effects of protopanaxadiol-type ginsenosides on CNS Recently, it has been shown that ginseng and its components, ginsenosides, have a wide range of actions in. the central nervous system. These effects include increased cell survival, extension of neurite growth and protection of neurons from different insults caused cell death both in vivo and in vitro. Here I focus on the recently reported beneficial effects of protopanaxadiol-type ginsenosides in C N S . K i m et al. showed that R b l and Rg3 protected neuronal cultures from glutamate-induced neurotoxicity [72]. L i m et al. reported R b l prevented hippocampal C A 1 neuronal death after forebrain ischemia in gerbils, possibly by scavenging free radicals [73]. Using a spinal neuron model, R b l 13 and R g l proved to be potentially effective therapeutic agents for spinal cord injuries as they protected spinal neurons from excitotoxicity induced by glutamate and kainic acid, and oxidative stress induced by hydrogen peroxide [74]. R b l protects ischemia induced apoptotic neuronal death in vivo and in vitro may be attributed to enhanced expression of B c l - x l , reduced expression of Bad and inhibited activation of caspase-3 [75]. R b l and its metabolized product M l reversed decreases in axonal density and synaptic loss in cerebral cortex and hippocampus of A/3 (25-35)-injected mice [76]. Other studies showed that R b l enhanced long-term potentiation (LTP) in the hippocampal formation and ameliorated cognitive deficits in ischemic and aged rats [77, 78]. Tian et al. demonstrated that Rg3 might be neuroprotective against focal cerebral ischemia in rats through reducing lipid peroxides, scavenging free radicals and improving the energy metabolism [79]. Also, it has been suggested that Rg3 inhibited both N-methyl-D-aspartate ( N M D A ) and n o n - N M D A glutamate receptors, which contribute significantly to most neurological disorders [80, 81]. Rh2 demonstrated neuroprotective effects upon brain injury after transient focal ischemia in rats by reducing infarct area and imbibing inflammatory reactions [82]. Previous study in our lab has proved that aPPD has low toxicity and ability to cross the blood-brain barrier ( B B B ) (data not shown). Since increasing evidence in the literature has demonstrated that precursors of aPPD such as R b l , Rg3 and Rh2, have beneficial effect on the central nervous system, we thought it would be interesting to see whether aPPD also has any neuroprotective effect upon ischemic brain injury. 14 1.2 HYPOTHESIS AND STUDY AIM We hypothesized that aPPD may attenuate brain injury after transient focal cerebral ischemia insult. To test this hypothesis, the first objective o f our studies was to investigate possible neuroprotective effect of aPPD upon N M D A induced excitotoxicity in mouse primary cortical neurons. The second part of our experiments was to assess the neuroprotective actions of aPPD on a transient focal M C A O mouse model. Finally, we sought to understand the mechanism of possible neuroprotective effect of aPPD by measuring levels of various protein kinase activities. 15 C H A P T E R 2 MATERIAL AND METHODS 2.1 IN VITRO STUDY 2.1.1 Mouse primary cortical neuronal culture Pregnant C57 B L / 6 male mice from Charles River Laboratories (Laval, Canada) were sacrificed according to the guidelines of Institutional Animal Care and Use Committee ( I A C U C ) . Primary cerebral cortical cultures were prepared from 15-day fetal mice using minor modification of an established technique [83]. The mouse head was open from the base of the skull to the mid-eye area. The brain was released from the skull cavity, and transferred to 4°C Hanks Balanced Salts ( G I B C O B R L , Grand Island, N Y ) containing l O M m Hepes (Sigma, Saint Louis, M O ) , P H 7.4, in a 60 mm Petri dish. Meninges were removed and frontal cortex was dissected under microscope. Tissue was transferred to 0.25% trypsin ( G I B C O B R L , Grand Island, N Y ) and digested at 37°C for 15 min. Tissue was then resuspended in D M E M ( G I B C O B R L , Grand Island, N Y ) plus 10% fetal bovine serum ( G I B C O B R L , Grand Island, N Y ) and triturated 4-6 times through the fire-polished tip. The supernatant was centrifuged at 200g for 45 seconds. Ce l l pellet was resuspended in Neurobasal, ( G I B C O B R L , Grand Island, N Y ) , B 2 7 ( G I B C O B R L , Grand Island, N Y ) , 2 M m L-glutamine (Sigma, Saint Louis, M O ) and seeded at a density of 2 x 10 5 /well onto poly-D-lysine (Sigma, Saint Louis, M O ) coated 24-well plates. These cultures were maintained in serum-free Neurobasal-B27 medium and one-half of the 16 medium was replaced on day 3 or 4 by equal volume of fresh medium. Gl ia l cell growth at five days is reduced to less than 0.5% for a nearly pure neuronal population [84]. 2.1.2 Mouse primary cortical neuronal culture treated with NMDA and aPPD Primary cortical neuronal culture was maintained in B27/Neurobasal medium and used on 9-10 days in vitro (DIV). The day before experiment, a half of the medium was replaced by fresh one. aPPD, which was kindly provided by Pegasus Pharmaceutical Inc. (Vancouver, B C ) was stored in 100% ethanol at concentration of 50mg/ml (108mM). Before each experiment, stock solutions of aPPD, N-Methyl-D-Aspartate ( N M D A ) (Sigma, Saint Louis, M O ) and glycine (Sigma, Saint Louis, M O ) were diluted in fresh medium to final concentrations of aPPD (2.5uM, 5uM, l O u M and 20uM), N M D A lOOuM, and glycine 20um.The cultures were then changed to a medium containing lOOuM N M D A plus 20uM glycine for 60 min in the absence and presence of aPPD of 2.5um, 5 u M , l O u M , and 2 0 u M concentrations. Negative control was cells without N M D A or aPPD treatment but exposed to sham washes. After 60-min N M D A exposure, cells were changed back to previous medium and incubated at 37°C, 5% C 0 2 incubator overnight. 2.1.3 L D H assay Cel l death was determined 24h after N M D A exposure using lactate dehydrogenase (LDH) assay (Roche Cytotoxicity Detection Ki t , L D H ) . L D H is a stable cytoplasmic enzyme and is released to culture supernatant when the cell dies or when the plasma membrane is damaged. Released L D H reduces N A D + to N A D H + and H+, then 2 H are transferred to tetrazolium salt INT (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-17 phenyltetrazolium chloride) to form a formazan dye. Therefore, the amount of formazan formed in the supernatant directly correlates to the amount of cell death. Briefly, the medium was collected and prepared cell-free by centrifugation. A lOOul aliquot was pipetted to a 96-well plate and mixed with lOOul of LDH reagent. Spontaneous LDH release from sister cultures without any treatment but just washed with fresh medium were used as the low control. The high controls were obtained by adding 3% Tritonx-100 to untreated cultures to lyse 100% cells (maximum LDH release). The change of absorbance per minute was determined with a Multiskan plate reader (Labsystems, Helsinki, Finland) measured at 500 nm. Results from at least three separate experiments were averaged. Percentage of cytotoxicity was calculated by the following equation: Cytotoxicity (%) = 100 x (exp.value-low control) / (high control-low control) 2.2 IN VIVO STUDY 2.2.1 Animal A total of 130 adult male C57BL/6 mice, with body weights 23-28 g, were obtained from Charles River Laboratories (Laval, Canada). The mice were housed in the animal facility of University of British Columbia and were bred (12h dark-light cycle) at least one week before being used for experiments. All experiments were performed in a dark cycle of the mice. The experimental protocol (A 03-0151) was approved by the Animal Care and Use Committee of University of British Columbia. 18 2.2.2 Transient focal ischemia Transient focal cerebral ischemia was induced by using an intraluminal fdament to occlude middle cerebral artery as previously described [85]. Each mouse was weighed and anesthetized through a cylindrical face mask with 3% isoflurane in a mixture of 70%N2O and 30%O2 administered through a Harvard rodent ventilator. A heating pad was used to maintain rectal temperatures between 37°C to 37.5°C during the surgery. The surgery was performed under a Zeiss operating microscope (Cal Zeiss A G , Gottingen, Germany). Neck incision was made and the left common carotid artery ( C C A ) and external carotid artery ( E C A ) were exposed. A 5-0 nylon monofilament (Syneture, Japan), blunted at the tip in a flame, was inserted into left E C A , advanced along left internal carotid artery ( ICA) into the circle of Wil l i s , and lodged in left anterior cerebral artery ( A C A ) and middle cerebral artery ( M C A ) bifurcation. Therefore, middle cerebral artery was occluded at its origin. Then the wound was closed and the animal was allowed to awaken. After lhour occlusion, the mouse was reanesthetized and reperfusion was established by withdrawal of the fdament. Neurological deficit examination (Four-point scale) was performed before reperfusion to ensure the occlusion occurred. M i c e without observable neurological deficit were excluded from further study. 2.2.3 Drug treatments aPPD was kindly provided by Pegasus Pharmaceutical Inc. (Vancouver, B C ) . aPPD was dissolved in Caster O i l ( B A S F , Germany)/ 100% Ethanol solution at concentration of 60mg/ml. Before experiment, aPPD was diluted in 0.9% saline and its working concentration is 30mg/kg. M i c e subjected to transient focal ischemia were injected aPPD 19 intraperitoneally at the onset of reperfusion and given daily for 7 days. In the vehicle control group, the vehicle of the same volume was administered instead. The choice of the dose, duration, time and the route of aPPD administration were based on the results of preliminary work. I found by those experiments that 30mg/kg aPPD reduced infarct area when evaluated 24h after ischemia (aPPD n=21 vs. vehicle n=14, P=0.059) and no difference was noticed between i.p. and i.v injections. 2.2.4 Weight loss and Mortality evaluation Weight loss after surgery is an important measure to evaluate the general physiological condition of experimental mice. Mouse body weight (aPPD treated n=9; vehicle treated n=7) was monitored daily before ischemia and in 7days after ischemia. Weight loss was expressed as the percentage of mean weight subtracted by the mean weight before surgery day. Mortality was compared between aPPD-treated mice (n=20) and vehicle treated mice (n=20) for the first 14 days after ischemia since there was no death occurred after day 14 post-ischemia. 2.2.5 Behavior Tests (post-ischemic 7 days ) A s a part of short-term functional recovery evaluation, sensorimotor behavioral tests were performed in 9 aPPD-treated and 7 vehicle-control mice for 7 days after t M C A O . 20 2.2.5.1 Four-point scale A neurological score was assigned to each animal before and after surgery for 7 days. The scale was used to quantify sensorimotor deficit which was characterized by contralateral hemiparalysis and hemiparesis and described previously [86]. Each mouse was assigned a score of 0-4, where 0 represented no observable neurological deficit; 1, failure to extend the right forelimb or torso turning to the right side when held by tail; 2, circling to the right; 3, unable to bear weight on right side; 4, no spontaneous locomotor activity. 2.2.5.2 Latency to move This test is used to assess motor deficit after ischemia. The animal was placed on a flat surface, and the time for the animal to move 1 body length was recorded. Two trials were performed before ischemia and every day after ischemia for 7 days and average was taken. 2.2.5.3 Latency to fall This experiment was performed before ischemia and every day after ischemia for 7 days. The animal was placed at the center of a horizontal wooden pole (2 cm in diameter) that was 75 cm above the ground. Two trials were performed per day and an average was taken. Latency to fall was recorded as the duration Of animal stayed on the pole (maximum of 60 seconds). The latency to fall is attributed to deficiency in locomotor balance that most likely resulted from damage to the frontal cortex during M C A O . 21 2.2.6 Behavior Tests (post-ischemic 90 days) A s a long-term functional recovery evaluation, cognitive function was assessed in 6 normal mice, 10 aPPD treated, and 9 vehicle-control mice post-ischemic 90 days. 2.2.6.1 Water maze The Morris water maze is used to test spatial learning and memory in rodents [87]. Briefly, the mouse swims in a round pool with water to reach a submerged platform, which is the only place to escape from the water and localization of the hidden platform can be performed and learned only by using distal room cues. A 1.5m diameter and 2 ft deep round pool was fdled with water at temperature 24 ± 2C°. The pool was divided into four quadrants of equal surface area. The starting locations were called north, south, east, and west, and were located at equal distances on the pool rim. A 10cm diameter round platform was covered with a fibrous mesh. There were five platform positions in the pool and at least three cues placed on the walls around the pool. The visible platform task was started in the first three days with 5 trials (5 swims) per day. This is followed by 5 days hidden platform training with 6 trials per day. On day 3 and day 5, there was an additional 60 - second probe trial without the platform. The trials were separated from each other by a period of 30 min and the start point was changed after each trail. In visible platform task, the mice (aPPD, n = 10; vehicle, n = 9; normal, n=6) were trained to locate a cued, flagged platform in clear water. After each trial the location of the platform was changed to a different quadrant of the pool. In the following 5 days, the cued platform was switched with a submerged position and the pool was filled with water that is colored white with non-toxic paint. The swim-pattern of the mice to find the 22 platform was monitored by a video camera connected to a computer through an image analyzer (HVS2020 video tracking system, H V S Image, San Diego). Once the mouse either found the platform or was manually guided there, it was allowed to stay at the platform for 10 seconds, and then it was lifted off the platform and dried with tissues. Latency (time to reach the platform) and path (length the mouse swam to find the platform) were used to assess memory function. On the 3rd and the 5th day, 60-second probe trial without platform was used to further assess how the animals remembered the location of the platform by percentage of time in the target quadrant of the pool (QUAD%sc) . Swimming speed (path / latency) was evaluated during the first three training days to assess the motor activity of mice in water-maze task. 2.2.6.2 Context Fear conditioning Fear is an emotion associated with learning and memory. Based on Pavlovian classical conditioning, fear conditioning test is widely used to evaluate a form of learning in which fear is associated a particular neutral context (e.g., a room) or neutral stimulus (e.g., a tone) with an aversive stimulus (e.g., a shock,). Eventually, the neutral stimulus alone can elicit the state of fear [88, 89, 90, 91, 92, 93, 94]. For context fear conditioning test, the neutral context is the "conditioned stimulus" (CS), the aversive stimulus is the "unconditioned stimulus" (US), and the fear is the "conditioned response" (CR) [95]. The procedure was done as described previously [96, 97]. Briefly, mice (aPPD, n = 10; vehicle, n = 9; normal, n=6) were placed within the conditioning chamber for 3 min to allow them to develop a representation of the context via exploration before the onset of a single U S (1 s/1 m A foot shock) for 2 seconds. Elicited movement and jump 23 were observed for each mouse due to the intensities of electric shock. After the shock, they then were left in the chamber for additional 2 min (immediate freezing) and returned to its home cages. Conditioning was tested 24 h after training for 4 min in the same conditioning chamber. Freezing response was monitored by using a computerized system (FreezeFrame Actimetrics, Wilmette, IL), which combined infrared beams and video tracking systems to trace freezing behavior, such as minute movements of grooming, sniffing, turning and rearing. Fear was assessed by measuring freezing behavior and presenting as freezing time. 2.2.7 Histopathology assessment Terminal histopathology was assessed at the 7th day (14 mice, aPPD n=9, vehicle n=7) and by the end o f 3rd month (19 mice, aPPD n=10, vehicle n=9) after ischemia. Infarct volume was determined by staining with 2, 3, 5-triphenyltetrazolium chloride (TTC) (Sigma, Saint Louis, M O ) according to Bedersen et al. (1986). T T C , a marker for mitochondrial enzyme activity, is widely used to assess infarct area o f cerebral ischemia in vivo. This dye is reduced to a brick-red-colored precipitate in the presence of the co-enzyme N A D H . Dying cells lose their ability to retain N A D H and are delineated as pale areas within the red stained viable brain tissue. Briefly, brains were removed from the skull, cut into seven 1-mm coronal sections, stained with 2% T T C (dissolved in 0.9% saline) at 37°C for 20 min and stored in 10% formalin. Sections were measured with an image analysis system (Image J 1.32, Bethesda, M D ) . For the total ipsilateral infarct volume, the ipsilateral infarction area obtained from each section was multiplied by the section thickness and each sectional volume was summed up to the total volume. To 24 exclude the influence of edema, the infarct size was calculated as the percentage of ipsilateral hemisphere volume [98, 99]: % Infarct volume =100% x [total ipsilateral infarct volume/total ipsilateral hemisphere volume] 2.3 STUDY OF PROTEIN KINASES 2.3.1 Tissue preparation and protein kinase screening To investigate changes in protein kinase activities after M C A O , tissue samples were collected from animals treated with different conditions. Animals were divided into five groups (n=4/group). Group 1: Mice subjected to l h M C A O then sacrificed immediately (occlusion phase); Group 2: mice had l h M C A O and allowed for lhr. reperfusion before being sacrificed (reperfusion phase); Group 3: mice were given aPPD i.p. at the onset of reperfusion and sacrificed l h after reperfusion; Group 4: As group 3 but vehicle treated; Group 5: Sham operated animals underwent same surgery but without filament insertion were sacrificed 2 hour after surgery. A l l mice were sacrificed with an i.p. injection of 300ul euthanyl (Sigma, Saint Louis, MO) and cortical tissue of ipsilateral hemispheres was collected from the penumbra region and immediately immersed in a lysis buffer ( 20 mM MOPS; pH 7.0, 2 m M EGTA; 5 m M EDTA; 30 m M sodium fluoride; 40 m M 0-glycerophosphate, pH 7.2; 20 m M sodium pyrophosphate; 1 m M sodium ortho vanadate; 1 mM phenylmethylsulfonylfluoride; 3 m M benzamidine; 5 uM pepstatin A ; 10 /xM leupeptin; 0.5% Nonidet P-40). 1 g wet weight of the chopped brain tissue was homogenized in 4 ml of lysis buffer. Homogenization was performed on ice with 10 strokes of a glass dounce and then subjected to ultracentrifugation for 30 min at 100,000 25 x g at 4°C. The supernatant was collected as whole-cell fraction and sent to Kinexus Bioinformatics Corporation for measuring a panel of signaling proteins. Upon receipt of tissue samples, Kinexus uses m i n i - S D S - P A G E for immunoblotting analysis with as little as 350 pg of tissue lysate protein. Standardized conditions and procedures in the preparation and operation of S D S - P A G E are rigidly followed. Essentially all of tissue lysate sample is applied in one lane of the S D S - P A G E gel. Molecular weight marker proteins are deposited on both side of the S D S - P A G E gel to ensure accurate assignment of molecular masses of lysate proteins. To permit maximum resolution of target proteins, S D S - P A G E is performed until after lower molecular weight proteins have eluted from the gel and the gels are formulated with a lower ratio of bisacrylamide to acrylamide. After electrophoretic transfer of proteins from the S D S - P A G E gel onto a nitrocellulose membrane, the membrane is probed for protein with Ponceau stain. If insufficient protein is detected, , a multiblotting apparatus that features 20 separate slot chambers was used for incubation with a panel of 25 or more primary antibodies. N o more than 3 antibodies are incubated in the same lane to minimize the risk of a cross-reacting band co-migrating with a target protein band. The antibody mixes have been very carefully optimized in at least 20 model systems to avoid overlap of any immunoreactive proteins. After incubation with the primary antibodies at 4°C overnight and then for 30 minutes at room temperature with a panel of secondary antibodies, the immunoblot is incubated with enhanced chemoluminescence ECL-plus reagent. This ensures high sensitivity detection of sub-nanogram levels of the target proteins. The E C L signal produced on the immunoblot is detected using a Bio-Rad FluorS M a x Multi-Imager with a 16-bit camera and able to quantitate the levels of immuno-reactivity over a 2000-fold range of linearity. 26 The reproducibility of this analysis ranges from 80 to 99% for similar samples. The reports of Kinetworks™ immunoblot include the locations of the immunoreactive target bands which permit their accurate identification, the images of the immunoblots both with and without all o f the lane and band numbers indicated, and the quantification of all detected proteins. 2.3.2 Western Blotting for phospho-Akt (pAkt) After l h M C A O and l h reperfusion, mouse ipsilateral and contralateral cortical tissue (4 mice in each group) of the peripheral region of the M C A territory (ischemic penumbra) were dissected and immediately immersed in the lysis buffer. Homogenization and ultracentrifugation were performed as described above. The resulting supernatant fraction was removed and immediately measured for the protein concentration with a modified D C Protein Assay ( B I O - R A D , Mississauga, ON) . After mixing the protein sample with loading buffer (62.5mMTris-HCl, pH6.8; 2% SDS; 10%glycerol, 5 0 m M DTT; 0.01% (W/V) bromo-phenol blue), the samples were boiled for 5 min to denature.the protein. Equal amount of protein (50-100ug/lane) from each sample was loaded to 10% SDS-polyaccarylamide gel for electrophoresis in MiniProtean ( B I O - R A D , Mississauga, ON)apparatus, run under 150V for about 40 min, followed by electrophoretically transferring to P V D F membrane (Amersham Pharmacia Biotech, Quebec, C A ) under 20V for about 30 min. The membranes were blocked in phosphate buffered saline (PBS) with 5% non-fat milk ( B I O - R A D , Mississauga, ON) for l h at room temperature, and probed with primary antibodies at 4°C overnight. The primary antibodies were diluted in PBS with 1%) Bovine serum albumin (BSA) . Antibodies used included rabbit polyclonal 27 antibody against phospho-Akt (serine-473, pAkt , 1:1000), rabbit polyclonal antibody against Akt (1:1000) (Cell Signaling, Beverly, M A , U S A ) , mouse monoclonal anti-actin antibody (Sigma, St. Louis, M O ) (1:5000). After washed with PBS/0 .05% Tween 20 (PBST) for 3 times, the membrane was incubated with 1:2000 horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (PerkinElmer Life Sciences, Boston M A ) for l h at room temperature. Western blots were detected by enhanced chemiluminescence reaction assay (PerkinElmer Life Sciences, Boston M A ) and visualized under Kodak image system (Kodak, U S A ) . Data were analyzed with Kodak I D 3.5 software. 2.3.3 Immunofluorescence triple-labeling One hour after onset of the reperfusion, mice (aPPD n=4, vehicle n=4) were sacrificed with an i.p. injection o f 300ul euthanyl. The mice were perfused through heart with 30-50ml 0 .1M P B S , followed by 30ml 4% paraformaldehyde (PFA) (Sigma, Saint Louis, M O ) . The brains were dehydrolyzed by 20% sucrose at 4°C overnight and then imbedded in O.C.T. (optimal cutting temperature) compound (Tissue-Tek®; Torrance, U S A ) . The mould was wrapped with parafilm and foil, and placed in a -80 C freezer until sectioning. Tissue blocks were cut into coronal sections with a L E I C A Cryostat ( C M 3050 S) (Meyer Instruments Inc.; Houston, T X , U S A ) at 10-14um thickness, mounted on glass slides, and then air-dried overnight (O/N) at room temperature (RT). The slides were washed 3 times in P B S and the sections were outlined on the glass slides with a Pap pen (Vector Laboratory; Burlingame, C A , U S A ) . For immunofluoresence staining, the slides were placed in a moist chamber and incubated with 10% goat serum ( C H E M I C O N International; Temecula, C A , U S A ) for 1 hour at RT to block non-specific binding. The 28 primary antibodies against phospho-Akt (pAkt) (Cell Signaling, # 3787) or neuron-specific nuclear protein (NeuN) (Chemicon International, Temecula, M A B 3 7 7 ) was applied with 1:200 dilution in 10% goat serum and incubated O / N at 4°C. Negative controls without adding primary antibody was also performed. The next day, the slides were washed with 0.1% PBS/Tween-20 for 5 minutes x 5 times. The secondary antibodies, which were Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 546 goat anti-mouse IgG (Molecular Probes Burlingto, ON) at 1:1000 dilution in 10% goat serum, were applied for 30 minutes at RT. The slides were washed with 0.1% PBS/Tween-20 5 times (5min/wash). Subsequently, the slides were, covered with V E C T A S H I E L D mounting medium (Vector Laboratories) with Hoechst 33342 (Sigma). The slides were visualized under fluorescent microscope (Zeiss, Oberkochen, Germany) and images were taken by Northern Eclipse software (Germany). Immunostaining after a preabsorption procedure or without a primary antibody showed weak or no immunoreactivity, suggesting the specificity of the antibody to the antigen. 2.3.4 DNA fragmentation detected by T U N E L 24h after ischemia, mice were sacrificed (aPPD n=4; vehicle n=4) and the brains were dissected for D N A fragmentation detection by terminal deoxynucleotidyl transferase-mediated uridine 5_-triphosphate-biotin nick end labeling ( T U N E L ) using a fluorescent In Situ Ce l l death Detection K i t , (Roche, Germany). This assay used an optimized terminal transferase (TdT) to label free 3 ' O H ends in genomic D N A with fluorescein-dUTP or T M R - d U T P . The procedure involved: cryosecting mouse brain as described above; fixing the brain slides by 4% P F A ; permeabilizing slides in 0.1% TritonX-100 and 29 0.175 mol /L sodium acetate for 15 minutes; and then washing in P B S 3 times (5min/wash). The slides were incubated in T U N E L solution (10% T U N E L enzyme and 90% T U N E L label) at 37°G for l h in dark. After washed with 0.1% PBS/Tween-20 for 5 times (5min/wash), the slides were covered with V E C T A S H I E L D mounting medium (Vector Laboratories), with 2 u M Hoechst 33342 (Sigma), and were visualized under fluorescent microscope (Zeiss, Germany). 2.3.5 Immunofluorescent triple labeling with pAkt, Nuclear Dye and T U N E L Mouse, primary cortical neuronal cultures were treated with N M D A and aPPD as described above. 24h after N M D A exposure, cells were washed with 1ml P B S per well and then fixed in 1 m l 4% P F A on ice. After washed 3 times in P B S , cells were incubated with primary antibody for pAkt (IHC special, Cel l Signaling, Beverly, M A , U S A ) with 1:200 dilution in 10% goat serum for 2 hours at RT. Cells were washed with 0.1% PBS/Tween-20 for 5 minutes x 5 times and.incubated with 1:1000 Alexa Fluor 546 goat anti-Rabbit IgG (Molecular Probes) for 30 minutes at RT. Fol lowing immunofluorescent staining of pAkt, the cell cultures were further processed with Fluorescein In Situ Ce l l death Detection K i t (Roche) for T U N E L staining as described above. Subsequently, cells were covered with V E C T A S H I E L D mounting medium with 2 u M Hoechst 33342 (Sigma), and images were taken under fluorescent microscope. 30 2.4 STATISTICAL ANALYSIS Comparisons between two groups were achieved with two-tailed Student-/- test. Repeated measures A N O V A was used to correct for multiple comparisons in Weight loss, Four-point scale, Latency to move, and Latency to fall test after one-tailed Student-/- test. Mortality was analyzed with Kaplan-Meier survival analysis. Fear conditioning was analyzed with Kruskal-Wallis One-way A N O V A on ranks followed by post hoc analysis with Dunnett's method. Comparisons among multiple groups such as Water Maze results were analyzed with two-way A N O V A with posthoc Bonferroni comparisons. A l l data were expressed as mean ± S E M and analyzed by using Graphpad Prism version 4.0 (Graphpad software, San Diego, C A , U S A ) . P O . 0 5 was considered statistically significant. 31 C H A P T E R 3 RESULTS 3.1 IN VITRO STUDY 3.1.1 aPPD protects primary neuronal culture from N M D A induced excitotoxicity In vitro ischemic model was utilized to study the effect of aPPD upon N M D A induced excitotoxicity. Primary neurons of 9-10 D I V C57 B L / 6 mice were exposed to lOOuM N M D A for 60 minutes. N M D A induced neurotoxicity in the absence or presence of 20uM aPPD. Sister cultures (DIV 9) were exposed to sham washes., Immediately after the exposure, neuronal cell bodies started swollen, neurites appeared fragmented, and by the next day, a significant cell death was seen (Figure 3.1.1.). These morphological changes were confirmed by levels of L D H activity in the bathing medium 24h after N M D A exposure. L D H release from the damaged cells was used as a marker of cell death and the data were expressed as the averages of 6 individual experiments. The dose-response relationship o f aPPD against N M D A neurotoxicity was obtained and the aPPD concentrations with significantly protective effect were l O u M and 20uM with percentages of cell death 13 ± 0.05% and 18 ± 0.06%, respectively. This is in contrast with the neuronal cultures treated with N M D A in the absence of aPPD (31 ± 0.06% cell death; p<0.05) (Figure 3.1.1.). 32 3.2 MOUSE tMCAO MODEL 3.2.1 aPPD reduced animal weight loss and mortality after ischemia Animal body weight was monitored after ischemia to compare general physiological condition of mice between aPPD and vehicle treated groups. Dai ly body weight loss was expressed as percentage of the mean weight before surgery day subtracted by the mean weight after surgery. aPPD treated group demonstrated less weight loss after surgery. The percentage of weight loss was statistically significant in aPPD treated group on day 1 ( aPPD 9.75 ± 1.49%; vehicle 16.8 ± 1.62%; p<0.05), day 2 ( aPPD 15.3 ± 1.34%; vehicle 25.9 ± 2.13%; p<0.05), day 3 ( aPPD 18.9 ± 2.5%; vehicle 29.5 ± 2.83%; p<0.05), day 4 (aPPD 20.2 ± 2.78%; vehicle 28.4 ± 3.29%; p<0.05) and day 7 ( aPPD 19.9 ± 2.93%; vehicle 30.5 ± 3.72%; p<0.05) compared with the vehicle control group by one-tail Mest for each testing day (Figure 3.2.1.). Because experiments conducted over a series of days, repeated measures A N O V A was used to reveal overall effects of treatment and days for the entire testing period. A significant change in aPPD treatment [F(l,112) = 35.17; PO.0001] and in performance across days [F(7,112) = 23.61; PO.0001] but with negative geotaxis of interaction between treatment and day [F(7,112) = 0.9; P=0.509] was demonstrated. The mortality of aPPD and vehicle control mice (n=20 for each group) was measured with Kaplain-Meir analysis for the period of 14 days post- ischemia. This is because that the most animal death of both groups occurred within 3-6 days after ischemia and no more death occurred after day 14 post-ischemia in both groups. aPPD significantly reduced the mortality compared to vehicle group.(Figure 3.2.2.). B y 14 days after 33 ischemia, the mortality rate for aPPD treated group was 40% while it was 70% in the vehicle control group (P=0.0320). 3.2.2 aPPD improved functional recovery after ischemia The most striking functional effect after stroke is frequently the impairment of movement and motor control. Therefore, it is important to show that aPPD can improve ischemia-caused deficiency in neurological and motor functions. Although sensorimotor recovery occurred in both groups, motor deficits in vehicle-treated group seemed more seriously than aPPD treated group. Four-point scale assay was utilized to determine the neurological deficits after ischemia. It is apparent that animals in aPPD treated group recovered much faster with significantly lower scores starting from day 2 post-ischemia compared to the vehicle control group. The scores maintained constantly for the rest of days during the test, indicating aPPD treated group had a faster and stable neurological recovery after the t M C A O (see Table 3.1). Repeated measures A N O V A again revealed overall significant effects of aPPD treatment [F(l,112) = 35.14; PO.0001] , effect of day on recovery of function [F(7,112) = 19.53; PO.0001] but no significant interaction between treatment and day [F(7,112) - 1.21; P=0.3016]. Similarly, effects o f aPPD treatment in latency to move was significantly demonstrated in group performance [F(l,112) = 10.64; PO.0015] , day [F(7,112) = 5.88; P<0.0001] but no significant interaction between treatment and day [F(7,112) = 0.51; P=0.8281]. Independent analyses at each day revealed that the latency of aPPD treated mice significantly less than vehicle mice in the first three days post ischemia. 34 The latency to fall in vehicle-treated animal group was the worst on the first day post-ischemia reflecting the severe locomoter deficit due to ischemic brain damage. The performance o f the animals improved in the following 3 days and reached the plateau on day 4. Again, animals.treated with aPPD could stay on the role much longer on the first day after t M C A O . The latency to fall was significantly longer than the vehicle control group in the first three days. There was significantly effect of aPPD treatment on post-ischemic performance in latency to fall [F(l,112) = 16.94; PO.0001] , day [F(7,112) = 4.88; PO.0001] and without significant interaction between treatment and day [F(7,112) = 0.82;P=0.5733]. The long-term cognitive impairment after t M C A O was assessed again in survived animals three months later. In water maze test, animals in vehicle treated group had longer escape latency and path lengths compared to normal animals and aPPD-treated animals when performed invisible platform or probe trial task (Figure 3.2.4.). However, there were no significant differences between aPPD (n=10) and vehicle treatment groups (n=9) or between ischemic mice and non-ischemia (n=6) control in escape latency, path lengths, and percentage of time in the target quadrant (Figure 3.2.4.). Only effect of trial day on path [F(4,80) = 3.60; P = 0.0095] among groups was demonstrated significantly different by two-way repeated-measures A N O V A analysis. Although swimming speed was significantly different between vehicle and non-ischemia control on day 2 visible platform task, there were no differences in swimming speed among three groups on the third training day. Abi l i ty of learning and memory in animals with ischemia was also examined using context fear conditioning test. On the first day, electric foot shock was paired with 35 conditioning chamber to elicit freezing behavior in animals. On the next day, post-shock freezing behavior was seen in both groups as a result of the process of learning and memory. The aPPD-treated mice (n=10) performed equally wel l as non-ischemic normal animals. However, the vehicle-treated group (n=9) had significantly less freezing behavior compared to both normal and aPPD treated mice (3.23 ± 2.95 vs. aPPD 16.9 ± 9.29; P<0.05 and vs. normal control 19.05 ± 11.7, n=6, p<0.01), indicating more severe learning deficits after ischemia without aPPD treatment (Figure 3.2.5.). 3.2.3 Histological evaluation Mice (aPPD n=9, vehicle n=7) were subjected to l h ischemia and following reperfusion. On the 7 t h day after ischemia, histological damage in the brain was determined by T T C staining (Figure 3.2.6.). aPPD treated animals had significantly reduced cerebral injury in both the cortex and striatum in the ischemic side of the hemisphere. (aPPD 14.44 ± 5.26% vs vehicle 44.14 ± 6.58%; p=0.0031; two-tailed unpaired Mest). N o histological evaluation was performed by 3 month post ischemia as gliosis, cavitation and atrophy were formed in the brains of both groups, which was impossible to measure quantitatively (Figure 3.2.7.). 3.3 ACTIVITY OF KINASES 3.3.1 aPPD altered kinase activeity after t M C A O A panel of proteins elevated their phosphorylation levels during ischemia but declined 1 hr after reperfusion was obtained in the penumbra cortical area of ischemic hemisphere 36 (Figure 3.3.1). aPPD seemed to sustain the activity of most of these phosphoproteins at the level of ischemia occlusion phase during ischemia reperfusion period. These included M A P kinase cascade, such as extracellular signal-regulated kinase 1 and 2 (ERK1/2) cascade, which preferentially regulates cell growth and differentiation, as well as the c-Jun N-terminal kinase ( JNK) and p38 M A P K cascades, which function mainly in stress responses like inflammation and apoptosis. The significance of aPPD induced activation of stress kinases during reperfusion phase remains to be studied (Figure 3.3.1.). Among many proteins that changed their phosphorylation status after treated with aPPD, phosphorylated Ak t is one of the most significantly increased kinases, accompanied by the increasing phosphorylation level o f its substrate P R A S 4 0 . Results of western blotting demonstrated that levels of p A K T (Serine-473) were significantly different between aPPD (n=4) and vehicle (n=4) treated groups (aPPD 2,32 ± 0.02 %, vehicle 0.79 ± 0.03 %; p=0.017) while total amount of Ak t was similar among aPPD-, vehicle-treated and sham-operated animals. Little activated Ak t was seen in the sham-operated brain tissue (n=4). Most interestingly, while the level of pAkt markedly increased in aPPD-treated penumbra regions of the ischemic hemisphere, there was no elevation of pAkt in the contralateral non-ischemic hemisphere. Results of the western blots were quantified using densitometry. The intensity of each band was normalized with the corresponding intensity of actin (Figure 3.3.2.). 37 3.3.2 aPPD increased expression of pAkt and enhanced pAkt mainly in neurons Triple fluorescent labeling of pAkt, N e u N and nuclear dye Hoechst was performed in tissue sections to support the results of Western blottings. The enhanced Akt phosphorylation was localized in the discrete cortical areas adjacent to the M C A territory of aPPD-treated mouse brain. There was no pAkt expression in the contralateral non-ischemic hemisphere. In particular, N e u N staining showed that the pAkt positive cells were mainly neurons i n the brains o f aPPD-treated mice (Figure 3.3.3.). 3.3.3 aPPD reduced ischemia-induced apoptosis T U N E L staining was performed to assess ischemia-induced apoptotic cell death 24 hours after t M C A O (n=4/group). aPPD markedly decreased the number of apoptotic cells in the ischemic caudate putamen and the cortex 24h after reperfusion, whereas, many TUNEL-posi t ive cells were apparent in the vehicle treated group with morphologically shrunken, condensed nuclei (Figure 3.3.4.). Finally, to demonstrate that elevated pAkt levels could protect neuronal cells from apoptosis, double immunofluorescence staining for pAkt and T U N E L were performed in NMDA-treated primary neuronal cultures. Reduced number of TUNEL-pos i t ive cells was observed in aPPD-treated cultures 24h after exposed to N M D A . In addition, phosphor-Akt-positive cells were not colocalized with TUNEL-pos i t ive cells, suggesting that expression of pAkt might prevent neurons from NMDA- induced apoptosis (Figure 3.3.5.). 38 C H A P T E R 4 DISCUSSION AND S U M M A R Y 4.1 THE ROLE OF aPPD IN tMCAO The present study was to investigate the neuroprotective effect of aPPD, a common metabolic product of ginsenosides. The beneficial effect of aPPD was first shown by results from in vitro experiments. Excitatory amino acid ( E A A ) neurotoxicity (excitotoxitotoxicity) has been proposed to contribute to neuronal loss in cerebral ischemia, probably through a large influx of calcium upon activation of glutamate iontropic receptors [100, 101,102]. Among iontropic receptors, N M D A receptor is one of the major glutamate receptor subtypes contributing to intracellular calcium overloading [103]. N M D A receptor is activated by N M D A (or glutamate) and glycine [104]. Since activation of iontropic glutamate receptors is sufficient to produce most of the E A A neurotoxicity [105, 106, 107], we used N M D A receptor-mediated neuronal death as our in vitro stroke model to investigate neuroprotective effect of aPPD. A n important observation was that aPPD protected primary cultured cortical neurons from N M D A induced excitotoxicity. The protective effect of aPPD was dose-dependent. Compared to NMDA-treated only cultures (31% cell death), treatment with 20uM aPPD reduced cell death to 13%. It was impossible to obtain more than 30-40% cell ki l l ing in N M D A treated neuronal cultures under our culture condition. Partially because L D H release occurs when the plasma membrane is damaged, L D H release may be more sensitive to necrotic cell death than to apoptotic cell death. Furthermore, neurons were cultured in a serum-free medium supplemented with Neurobasal - B27, which contains antioxidants 39 [108]. Because oxygen free-radicals are involved in cell damage associated with downstream production of N M D A receptor activation [109, 110]. Thus, cell cultures raised in B27 supplement medium might resist to N M D A neurotoxicity. Nevertheless, treatment with aPPD still showed significant decrease in cell death compared with the control. To simulate the most frequently occurred clinical stroke, mouse transient focal cerebral ischemia ( t M C A O ) was utilized in the present study. M C A O is one of the most common causes of focal stroke in humans [111]. Moreover, intraluminal filament occlusion technique is able to induce a transient period of ischemia followed by restoration of blood flow. Therefore, this model has become the model of choice for approximating the pathology and symptoms of human stroke [112]. Because of the above similarities to clinical stroke, relatively noninvasive manner, intraluminal filament M C A occlusion model has been modified for use in mice since most of transgenic and knockout animals are mice [113]. However, it is noteworthy that it was generally believed that the mouse model is significantly inconsistent in infarct size [114, 115]. I conducted a preliminary experiment to improve the consistency of this model. Three factors may affect the reproducibility of this model: 1) mouse weight, 2) the diameter of nylon filament round tip, and 3) the duration of occlusion [116, 117]. According to Tureyen et al, for weighing 22 to 24 g C57BL/6 mice, sutures of 180 pm in diameter should reliably produce occlusion and consistent infarct size. Before surgery, the 5-0 suture was flame-blunt and measured under microscope to ensure the tip diameter was with 0.18-0.185pm. Because previous studies suggested that SI hour occlusion leads to reproducible infarcts of the M C A territory [118], the duration of 60 minutes of M C A O in C 5 7 B L / 6 mice was 40 chosen in our experiment and the mice weight were strictly controlled (25±2g). Our Preliminary experiment not only demonstrated consistent infarction volumes when evaluated 24h after ischemia (Mean ± S E M ; aPPD 38.19 ± 3.752%, n=21; vehicle 49.57 ± 4.371, n=14). We also found 30mg/kg might be an effective dose of aPPD and no difference was noticed between i.p. and i.v. injectionsl hour after onset of reperfusion (aPPD n=21 vs. vehicle n=14; P=0.059). In addition, previous study in our lab has demonstrated that aPPD i.v. injection at a dose of 60mg/kg has no noticeable toxicity in animals. Recent evidence suggests that ischemic damage is a dynamic process, in which neurons continue to die over a long period of time after initiating ischemic injury [119]. Moreover, in humans, functional outcome as a direct consequence of stroke is a major end point in clinical trials [120.121]. Recently, the Stroke Therapy Academic Industry Roundtable (STAIR) group pointed out that compounds progress to clinical trial should combine infarct volume measures with functional tests, including short-term and long-term assessment [122]. Thus, the neuroprotective effects of aPPD were evaluated by two periods: short-term outcome (7 day post-ischemia) and long-term outcome (90 days post-ischemia) with histopathology analysis and functional behavior outcomes. It is noteworthy that during the first 7days post-ischemia, aPPD prevented ischemia-caused weight loss in the treated mice compared to vehicle treatment (Figure 3.2.). Weight loss continued to progress even after 7 days post-ischemia in vehicle treated mice but not aPPD-treated ones. Body weight reflects general physiological condition of the animals and closely correlates with post-operational mortality. In contrast, survival rate significantly increased in aPPD-treated group, especially, after 7 day post ischemia. 41 In mice, unilateral occlusion of the M C A caused infarction lies more in subcortical and posterior cortical regions of the affected hemisphere. The ipsilateral lateral septum, thalamus, and hippocampus-structures are also involved [123]. The most striking functional effect after stroke is frequently the impairment of movement and motor control. As evidenced by Figure 3.4., the recovery of locomotion was significantly earlier in aPPD group within 3 days post- ischemia, demonstrated by a shorter average latency to move. Sensory-motor coordination also significantly improved in aPPD treated mice during the firs 3 days, evidenced by longer latency to fall from the role. Four-point scale was used to determine the severity of stroke quantified by sensorimotor deficit scores [124]. Less neurological deficits were present in aPPD group compared to the vehicle group from post-ischemic day 2 up to 7 days. Although it has been reported that Four-point scale can detect sensorimotor deficits up to 30 and 60 days post ischemia [125], it is noteworthy that there was a recovery of function over time in both groups. The rapid spontaneous functional recovery in sensorimotor deficit is consistent with previous studies, which suggest that mice are able to rapidly develop functional compensation for damaged brain regions [126, 127, 128, 129]. In addition, many factors could contribute to results of locomotion and sensorimotor behavior tests, including familiarization/enrichment as a result of daily testing. These may explain no apparent difference between aPPD and vehicle control groups in latency to move and latency to fall tests observed at the later days post ischemia. However, clinical studies have shown that early neurological functional deficits are major predictors of stroke outcome [130]. Therefore, significant neurological improvement by aPPD treatment in the early stage of post-ischemia in mice renders further study for its potential clinical application. 42 Since cognitive deficits have frequently been reported in patients after ischemic stroke [131], we assessed the effect of aPPD on learning and memory function 90 days after t M C A O . Learning and memory are complex phenomenon requiring the coordinated interaction of multiple brain structures. Previous experimental studies supported the view that hippocampal damage is not solely responsible for ischemia-induced learning and memory deficits. Rather, they suggest that both the intra- and extra-hippocampal damage contribute to the pattern of memory impairments observed following ischemia [132, 133, 134]. Focal ischemia by M C A occlusion leads to cell death in the striatum and various cortical areas, as well as secondary neuronal damage in remote brain regions, such as the thalamus. A l l o f these regions are involved in learning and memory [135, 136, 137]. Water maze was used to evaluate the ability of spatial learning and memory function 90 days after the initial t M C A O . Although vehicle-treated animals had a longer average latency and path-length in getting to the invisible platform, there was no statistical difference among the three groups. To our current knowledge, no study in mice has examined spatial learning deficit using water maze 90 days after M C A O . Due to high mortality (40% to 80%) [138, 139], long-term cognitive outcome after t M C A O in mice was rare. Recently, Winter et al reported that mice, subjected to 30 min M C A occlusion followed by reperfusion, had no obvious deficits in spatial learning in water maze when evaluated 42 days after t M C A O [140]. The lack of a water-maze deficit might be attributed to several factors: l .The performance was occurred in the later phase of post-ischemic recovery period and the mice had developed functional compensation to solve the navigation problem. 2. Training paradigm (six trials on one day, 30-s trials intertribal interval) might not very sensitive to hippocampal damage. Since there was no difference 43 between the vehicle-treated ischemic animals and the non-ischemic normal controls, the results of water maze test in the present study cannot be interpreted as a failure of aPPD in protecting related brain regions. In contextual fear conditioning test, the mice learn to associate a conditioned stimulus (CS; test chamber) with a U S (footshock) [141]. After the pairing of C S and U S , a robust associative memory of the C S - U S is formed such that the CS alone can elicit a fear response (e.g., freezing). Freezing is a widely used measure of conditioned fear defined as remaining motionless [142, 143, 144]. The amygdala is a critical region for fear conditioning by receiving input through its lateral nucleus from cortical areas and the thalamus and sending output via its central nucleus to a variety of brain regions that are known to mediate fear responses, such as the hypothalamus and hippocampus. Fear responses are determined by the integrity of the above amygdale pathway [145, 146]. aPPD treated mice demonstrated stronger fear responses than the vehicle group.(Figure 3.5.) Because the amygdale complex is involved in t M C A O damage, stronger fear displayed in aPPD-treated animals may reflect less severity of the brain injury or better preserved amygdale complex. The strong neuroprotective effect of aPPD was proved by histological assessment 7 day post ischemia. aPPD treated mice demonstrated significant reduced infarct area compared to the vehicle group by T T C staining (p<0.01).(figure 3.6.) T T C and crystal violet (CV) stainings are two routinely used methods for determination of infarct volumes and areas after T M C A O in previous studies. T T C staining method, introduced by Jestaedt et al. was first used to detect myocardial infarct [147]. Compared to C V staining, T T C staining is quicker, easier and more reproducible. More importantly, T T C staining 44 accurately measures infarct area or volumes without significant difference from C V and hematoxylin-eosin (HE) staining [148, 149]. However, T T C staining is only capable of detecting short-term brain infarction but not for delineating long-term brain damage. B y 3-month post ischemia, the ischemic hemisphere showed atrophy and cavitation, which cannot be delineated by T T C staining. Nevertheless, results presented here still clearly demonstrate that aPPD, at least during short-term post ischemia period was protective against experimental stroke in vivo. Molecular biology and biochemistry study were conducted to investigate possible mechanisms of aPPD induced neuroprotection. A t least three mechanisms have been previously proposed for neuroprotection actions of ginsenosides against cerebral ischemia: 1) reducing excitotoxicity by blocking N M D A receptor activity [150], 2) scavenging free radicals [151, 152], and 3) inhibiting inflammatory reactions [153]. Although other mechanisms may also be involved [154, 155], effects of ginsenoside on intracellular signaling pathway have never been studied in ischemia models. We started the investigation by profiling the kinase activity in animals with three conditions, a) occlusion one hour, b) occlusion one hour followed by one reperfusion, and c) b) plus aPPD i.p. injection by the end of occlusion. It seems that treatment with aPPD resulted in an activation of all stress-related kinases such as M A P kinase /ERK-l / -2 and J N K , as well as p38 phosphorylation state. Furthermore, aPPD treatment was associated with a marked increase in phosphorylated Ak t and its substrate P R A S 4 0 levels in ischemic brain tissue. In non-aPPD-treated mice, phosphorylation of Ak t was enhanced in cortex 1 hour after occlusion but declined after reperfusion whereas treatment with aPPD maintained the high level of activated Ak t in penumbra area during reperfusion phase. This is in 45 agreement with other reports showing ischemia induced transient increasing of phosphorylation A k t in penumbra and sustaining Ak t activation promoted neuronal cells survival [156, 157]. Neurons whose survival is promoted by A k t activity are healthy and appear functional [158]. The serine-threonine kinase Akt , also known as protein kinase B , is activated by a mechanism involving PI3-K, phosphorylation Ak t on threonine 308, and on serine 473 [159, 160]. Ak t activation is known for its pro-survival function through initiating various downstream pathways to inhibit apoptotic cell death [161, 162, 163, 164]. Recently, apoptotic cell death has been implicated in cerebral ischemia and mitochondria-dependent apoptosis have been thoroughly studied in transient focal cerebral ischemia in rodents [165, 166, 167]. The role of pAkt in modulating of mitochondrial-dependent survival pathway may be through its ability to phosphorylate, and thus inactivate Bad on serine 136 [168]. Subsequently, Bad is prevented of disassociation from its binding protein 14-3-3, translocates to the outer membrane of the mitochondria and inactivates B c l - X L b y dimerizing [169, 170].The antiapoptotic effect of B c l - x L is through conserving mitochondrial membrane potential, avoiding the permeability transition pore (PTP) formation and ultimately obviating the release of cytochrome c [171, 172]. Blocking the release of cytochrome c prevents the activation of downstream caspases-9 and -3, and D N A fragmentation and results in cell survival [173, 174, 175, 176]. Upregulating pAkt seems crucial for aPPD's acute neuroprotective action since in the N M D A in vitro model; all pAkt positive cells were not T U N E L positive, proving a close reversed association between pAkt and apoptosis. Furthermore, we have shown that aPPD induced increase in pAkt is associated with an increased phosphorylation of its substrate protein P R A S 40. P R A S 40 phosphorylation causes the 46 formation of a dimer between P R A S 4 0 and 14-3-3 protein. In transient cerebral focal ischemia, overexpression of phosphorylated P R A S 4 0 was found to attenuate apoptosis [177, 178]. Most recently, it has been further shown that oxidative damage during focal cerebral ischemia may be the cause of reduced levels of phosphorylated PARS40 in the brain [179]. While detailed downstream effect of activated Ak t in aPPD treated neurons remains unclear, a recent study has shown that one of the aPPD's precursors, R b l upregulates B c l - X L and protects ischemic neurons in vivo and in vitro [180]. Our data point toward a role of phosphatidyl inositol-3 kinase (PI3K)/Akt signaling in acute aPPD-induced neuroprotection, while ERK-1 / -2 and/or JNK/p38 might more specifically be involved in aPPD's long-term effects. E R K 1 / E R K 2 activation, which preferentially regulates cell growth and differentiation, has been implicated against apoptotic cell death by inactivating B A D at the Ser-112 [181, 182]. c-Jun N-terminal kinase ( INK) and p38 M A P K cascades function mainly in stress responses like inflammation and apoptosis.[183] aPPD seemed to remain the activity of above stress-related kinases at the levels of ischemia phase when administrated at the end of ischemia period. In ischemic preconditioning, both of E R K - 1 / - 2 and p38 has been implicated in ischemic tolerance, induced by short-lasting nonlethal episodes of global ischemia and cortical spreading depression [184, 185]. These two conditions are known to attenuate brain injury evoked by subsequent longer-lasting ischemic episodes [186]. It w i l l be interesting to understand whether the elevated activity o f the stress pathways by aPPD plays any role in its neuroprotection. Nevertheless, the present findings support the notion that the protective mechanism of aPPD may involve PI3k-Akt pathway. 47 4.2 CONCLUSION AND FUTURE DIRECTION A s describing in the previous chapters, we showed aPPD protects against ischemia-like injury in neuronal cultures and experimental stroke in vivo. Those results can be summarized as follows: 1. aPPD, when administered 7 days from the onset of reperfusion has demonstrated neuroprotective effects upon a mouse t M C A O model. 2. aPPD may protect penumbra neuronal death through increased activation of A K T and other protein kinases. 3. The above results suggest that aPPD might potentially be a therapeutic agent for preventing brain damage after stroke. At present we have no direct evidence about the exact mechanism how aPPD activate Akt after ischemia. We hypothesize that the molecular mechanism of aPPD mediated neuroprotective effects through the modulation of P I 3 K - A K T - B a d pathway, which upregulates B c l - X L and opposes apoptosis. Figure 4.2. is a diagram of hypothesized mechanism which may be involved in the action of aPPD based on our current results. These results raise a possibility that aPPD would constitute a therapeutic strategy. In order to support our hypothesis, future study should investigate the pAkt upstream PI3k and downstream Bad or other substrates. For example, by intracerebroventricular infusion PI3k inhibitor LY294002 before ischemia, we can clarify whether aPPD-induced pAkt is associated with PI3k pathway. Moreover, we can detect B c l - X L m R N A or B c l - X L protein expression after ischemia to elucidate i f aPPD can upregulate B c l - X L as its precursor R b l . 48 Although the present results have provide evidence for a role o f aPPD in improvement of learning and memory after stroke, further studies are required to determine i f the present results generalize to different behavioral tasks that involve different anatomical areas. Battery behavioral tests, such as the cylinder test and the corner test which have been reported to be more sensitive for long-term continued sensorimotor deficits assessments [187], may be needed to tease out further the potential effects of confounding factors on the performance of locomotion and sensorimotor behavior tests. Several different tasks including passive avoidance, spontaneous alternation in a T maze or the radial arm maze may be used to assess the effect of aPPD on the long-term cognitive recovery. Finally, the accurate histological outcome of long-term responses to aPPD treatment requires further study. 49 3-0-[y?-D-glucopyranosy!-(l—2)-/8-D-gluco pyranosyl]-20-O-[^-D-glucopyranosyi(l—6) -£-D-glucopyranosyl]-20(S)-protopanaxadiol 3-Or[j8-D-glucopyranosyl-(l—-2)-/S-D-gluco pyianosyl]-20(5')-protopanaxadiol (3-O-^-D-g]ucopyranosyl-20(5r) -protopanaxadiol) (20(S)-protopanaxadiol) Figure 1.2.1. aPPD and its precursor (aglycone process). A D Control iPPU il'I'H >I'PD il'PD MOTTO 2.5tdi 5uil KluM 20nM Figure 3.1.1. Protective effects of aPPD against N M D A neurotoxicity in primary neuronal culture. Micrographs (Phase-contrast) of sister cultures taken 24h after a 60-min lOOuM N M D A exposure were represented as: A , normal neurons without N M D A insult; B , treatment neurons in the presence of lOOum N M D A and 20uM aPPD; C, N M D A with l O u M aPPD; D , N M D A with 5uM aPPD; E , N M D A with 2.5uM aPPD; F, control neurons with N M D A exposure; G , Histogram represents L D H released and dose-response neuroprotctive effect of aPPD. Scale bar, lOOum (* P<0.05). 51 1 I 1 1 1 1 1 I dayi day2 day3 day4 day5 day6 day? Figure 3.2.1. 7-day weight loss after ischemia. aPPD treated group (n=9) demonstrated significantly less body weight lost on day l , 2, 3, 4 and 7 compared to vehicle group (n= 7). (* p<0.05; ** P< 0.01) 52 0.0 2.5 5.0 7.5 10.0 12.5 15.0 Days Elapsed Figure 3.2.2. 14-day mortality evaluation after ischemia. aPPD treatment (n significantly reduced mortality compared to vehicle treatment (n=20). (* p<0.05) 3.5-1 3.0-2.5-£ 2.0-O 1.0-0.5-o.o-L Four-Point Scale M i l l ! • a P P D • Vehicle i i 1 i i 1 i 1d 2d 3 d 4d 5 d Sd 7 d Latency to Move ! I I al 'PD "di d2 d3 d4 d5 d6 dT 65-, 60-55-50-T3 45-40-c 35-o o 30-4> CO 25-20-15-10-5-o-l Latency to Fall z I •• j 1 m —i 1 1 — i 1 1 r— d1 d2 d3 d4 d5 d6 d7 Figure 3.2.3. Post-ischemia 7-day behavioral functional tests. aPPD treated group (n=9) vs. vehicle group (n=7) in the 7 days following ischemia. (*p<0.05; **P< 0.01) 54 Behivioral Test Four-point Scale Latency to move Latency to Fal l Day! aPPD(9) 2.22 ±0.66 5.33 ±0.86* 35.7 ±23.9* Vehicle(7) 2.57±0.78 6.28±0.48 13.1 ±9.37 Day2 aPPD(9) Vehicle(7) 1.88 ±0.60* 2.71 ±0.75 5.0 ±1.22* 6.14 ±0.89 •45:7 ±21.8** 21 ±9.57 Day3 aPPD(9) Vehicle(7) 1.55 ±0.72* 2.57 ±0.97 5.33 ±0.86* 6.28 ±0,75 57.2±8.33* 33.1 ±25.3 Day4 aPPD(9) 1.66±0.70* . 5.22 + 1.39 52.7±14.3 Vehicle(7) 2.57 ±0:78 5.71 ±1.11 40.4±25.7 Day5 aPPD(9) 1.44 ±0.52* 5.0 ±0.86 55 ± 15 Vehicle(7) 2.57 ±1.13 5.71 ±0.75 43.7±27.8 Day6 aPPD(9) 1.44±0.52* 5.11 ±1.45 46.4 ±21.5 Vehicle(7) 2.42 ±0.97 5.57 ±1.61 38 ±28.0 Day7 aPPD(9) Vehicle(7) 1.22 ±0.44* 1.85 ±0.69 4.88 ±1.53 5.28 ±1.11 53.6±19 43.7± 27.8 Table 3.1. Post-ischemia 7-day behavioral functional tests. Data were expressed as Mean ± S E M which compared the difference between aPPD and vehicle treated mice with Mest for each testing day. (*p<0.05; **P< 0.01) 55 Figure 3.2.4. Post-ischemic 90 days Water Maze test. aPPD n=10; vehicle n=9; no-ischemic normal mice n=6. (*p<0.05) 56 Context Fear Conditioning aPPD Vehicle Normal Figure 3.2.5. Post-ischemic 90-day Context Fear Condi t ioning test. aPPD n= vehicle n=9; no-ischemic normal mice n=6. (*p<0.05; **P< 0.01) aPPD Vehicle Figure 3.2.6. Short-term histopathological evaluation. Representative coronal sections were from aPPD treated mice (A) and vehicle treated mice (B), sacrificed on the 7th day after ischemia and stained by T T C . Percentage infarct size (C) indicated that aPPD significantly reduced brain injury (** P<0.01) vs. vehicle treated mice on the post-ischemia 7 day. 58 B Figure 3.2.7. Long-term histopathological evaluation (A) Dorsal and sagittal surface profiles of mouse brain post ischemic 90 days. Instead of infarction, cavitation and atrophy were formed on the T T C stained coronal brain sections (B). 59 =IF2a[S51] PKCg[T514i Adducin gamma [Sc tope] PKACb[S338| P R A S « [ T 2 * i PKCb2 JT641] ERCflUSYtffl] Addu-^ in dftia [S72G] JNK[TlB3/Y1S]p;i T3u[S7ig PAK t'2'3 [SI l«BM1«154| JNK[T183/Y1«](p46) F-arriBQfYtaq AJd[S473]H R b [ S 6 1 p !M1[S2S1 Bmm -200.0 I I ~ QiaPPD • 1hrocclusion+1hr reperfusion • 1 hr Occlusion 0.0 200.0 400.0 G0O.0 600.0 10CO.0 Changes in phosphorylation i%of Sham! 1200.O 140O.il Figure 3.3.1. Protein Kinase Screen Results. 60 Vechicle aPPD Sham pAkt Akt ^-actin B 2.5 5 I. 5 0. 5 - 0 v c h i c l i aPPD sham aPPD Vechicle C O N T R A IPSI C O N T R A IPSI pAkt /J-actin Figure 3.3.2. Western Blot t ing analysis of p A k t after t M C A O . (A) 1 hour after reperfusion, the band of pAkt was evident at 60 kDa and increased in the cortex adjacent to M C A territory of aPPD treated brain. (B) Histogram was expressed as percentage of mean control (P-actin). (C) aPPD and vehicle treated contralateral side and ipsilateral side pAkt expression. (n=4;*P < 0.05). 61 aPPD Vehicle IPSI CONTRA IPSI Figure 3.3.3. Cellular localization of pAkt after ischemia by triple labeling fluorescent immunohistochemistry. Representative photomicrographs showed, 1 hour after reperfusion, aPPD and vehicle treated brain. pAkt positive cells were observed in cortical areas adjacent to M C A territory. pAkt expression was intensely increased in aPPD treated ipsilateral hemisphere compared with the same region of vehicle treated. N o pAkt expression in the non-ischemic hemisphere. Triple labeling of pAkt, N e u N and Hoechst immunohistochemistry demonstrated that pAkt-positive cells were primarily neurons. (A), pAkt (green); (B), N e u N (red); (C), Hoechst (blue), (D), pAkt/NeuN/Hoe. Scale bar, 50um. 62 aPPD V e h i c l e Figure 3.3.4. aPPD rescued cortical cells from ischemia-induced apoptosis. 24 hours after t M C A O , T U N E L positive cells were in the cerebral cortex and caudate putamen. Representative photomicrographs revealed more TUNEL-pos i t ive cells were in the vehicle treated group with shrunken, condensed nucleus. ( A ) , T U N E L (green); (B), Hoechst (blue); (C), TUNEL/Hoechs t . Scale bar, 50pm. 63 NMDA+aPPD NMDA D Figure 3.3.5. aPPD rescued primary cortical cultures from NMDA-induced apoptosis. Primary neuronal cultures taken 24h after a 60-min 1 OOuM N M D A exposure along with 20um aPPD and absence of aPPD (control) were stained for: (A) pAkt , red; (B) T U N E L , green; (C) Hoechst, blue; (D) p A k t / T U N E L / H o e . Noted phosphor-Akt-positive cells had no colocalization with TUNEL-pos i t ive staining. 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