Chapter 5: Discussion Chapter Discussion Department of Pharmacology, YLL School of Medicine 135 Chapter 5: Discussion During ischemia, the disruption of cerebral blood flow deprives brain cells of oxygen, leading to a reduction in energy production associated with building-up toxic metabolites such as glutamate, inflammatory mediators, free radicals that could ultimately trigger ischemic cascade. As mentioned in section 2.1.3, over production of free radicals during ischemia results in oxidative stress. Several possible mechanisms have been postulated for the generation of ROS during ischemia (Loh et al, 2006a). The inherent biochemical and physiological characteristics of brain, including high poly-unsaturated fatty acid and the energy requirements, make it particularly vulnerable to the oxidative stress. Oxidative stress does not play in isolation, but participates in the complex interplay between excitotoxicity, apoptosis, inflammation and so forth. Low antioxidant defense mechanisms and high oxygen consumption again subjects the brain to be more vulnerable to the oxidative stress insult. Therefore, modulation of oxidative stress could be a potential therapeutic approach for various neurodegenerative diseases. Mitochondria are the primary cellular source of ROS as they generate huge numbers of oxidation-reduction reactions and they consume massive amounts of oxygen. Mitochondrial dysfunction is also well-documented in the ischemic cascade. Thus, mitochondria are both the initiator and target of oxidative stress. Mitochondria damage leads to cell death given the role of mitochondria in the energy metabolism, calcium homeostasis and programmed cell death. Therefore, prevention of mitochondrial damage could confer a protective mechanism to ischemic stroke. Department of Pharmacology, YLL School of Medicine 136 Chapter 5: Discussion 5.1 Discussion on experiment I Left MCAO resulted in infarction spanning left cortical area and corpus striatum and severe neurological deficit. Oral administration of pHL could ameliorate both signs. Tissue infarction involving death of essentially all cells within the core infarct area is a common pathology outcome of stroke insult and an important determinant for the long term neurological disorder. In this study, infarction at the area in left cortical area and corpus striatum was consistently resulted by left MCAO, as shown by TTC staining. Study also demonstrated that rats suffering from left MCAO had the severe impairment of motor function as measured by neurological deficit grading system. Under the treatment of pHL, the infarct volume was significantly reduced. This phenomenon indicates the lesser histological damage, possibly within the area of penumbra was resulted in the stroke group treated with pHL as simplistic view of ischemic penumbra suggests cell death in this area undergo apoptosis which could be possibly inhibited but not necrosis cell death in core infarct. Neurological impairment was slightly alleviated by pHL day after MCAO. Significant difference of functional outcome was observed between vehicle group and stroke group treated with pHL days after MCAO, as measured by neurological deficit grading system. As ample evidences from 15O (Furlan et al, 1996; Heiss et al, 1998) and 18F-fluoromisonidazole (Read et al, 2000; Markus et al, 2004) PET studies indicate that survival of the tissue in penumbra has benefit on subsequent neurological recovery. A positive correlation does exist with improvement of month recovery scores and the survival of penumbra, leading to the hypothesis that survival of penumbra influences late recovery, possibly through allowing the subsequent Department of Pharmacology, YLL School of Medicine 137 Chapter 5: Discussion peri-infarct neuronal reorganization (Moustafa and Baron, 2008). With this rationale, pHL is suggested to have the effect of preventing the damage within the affected area, particularly through the protection of ischemic penumbra. Oxidative stress is associated with MCAO as shown by reduced plasma total antioxidant concentration and enhanced cerebral DNA oxidative damage. pHL treatment was shown to ameliorate oxidative stress in MCAO-induced rats. Endogenous antioxidant system under the influence of pHL was also investigated. Plasma was selected as the sample for this experiment as it contains many non-enzymatic antioxidants and antioxidant enzymes. The major antioxidant defenses in plasma include ascorbate, protein thiols, bilirubin, urate and α-tocopherol (Nicholas, 1993). Plasma contains also preventive antioxidants such as caeruloplasmin and transferring. Ironscavenging proteins present in plasma can contribute to the total antioxidant capacity because they scavenge the iron availability (Stocks, 1974). The total antioxidant concentration decreased significantly after MCAO. This result is in consistent with the work done previously by Antonio et al (2000), which showed that mean plasma levels of nonenzymatic antioxidants and antioxidant enzymes activities were lower in patients than in healthy controls (Antonio, 2000). Sun et al (2005) also showed that rat undergone myocardial infarction had lower level of antioxidant enzyme activities in hearts (Sun et al, 2005). The low level of antioxidant capacity in stroke groups suggests that much of the antioxidant molecules have been used up and react with ROS that over produced during ischemia. Under the pHL treatment, both in healthy or stroke group, the plasma Department of Pharmacology, YLL School of Medicine 138 Chapter 5: Discussion antioxidant concentration was greatly enhanced. This implies that pHL may have enhancing effects on endogenous antioxidants with or without oxidative stress condition, thereby confer protective barrier against stroke. Single strand break and base modifications characteristic of oxidative injury to DNA can be detected after permanent brain ischemia. Free radicals cause also the DNA strand break with a less extent by nitric oxide. Nitric oxide is capable of causing base modifications such as nitration or deamination (Love S, 1999). With many base modifications produced, the major base modifications that result from the reaction between peroxynitrite and DNA are the conversion of guanine to form 8-nitroguanine and the deamination of guanine to form xanthine (Love S, 1999). Level of DNA oxidative damage was elevaluated as one of the marker of oxidative stress. GC/MS has of late been widely used to quantify DNA damage and the level of oxidative stress due to its ability to identify a wide range of DNA base product in a single analysis. For the DNA oxidative damage analysis, direct damage by ROS that can affect purine, pyrimidine bases and/or deoxyribose sugar was focused (Halliwell, 4th Edition). From this study, it is showed that DNA oxidative damage was increased after MCAO, suggesting the increase free radical activity, as well as reciprocal decreased endogenous antioxidant activity contributes to DNA oxidative damage after stroke insult. Stroke rats treated with pHL not only had high endogenous antioxidant concentration but also had significant reduction on DNA oxidative damage. Therefore, it is proposed that the in vivo therapeutic Department of Pharmacology, YLL School of Medicine 139 Chapter 5: Discussion effects of pHL might be strongly related to its antioxidant effects by enhancing the endogenous antioxidant capacity and also alleviating the oxidative stress. TUNEL assay and Immunohistochemical staining of apoptotic-related proteins showing the presence of these proteins only in the infarct area after MCAO indicates the apoptosis involvement during ischemia injury. Significant reduction of green fluorescence and immunoreactivity of pro-apoptotic proteins could be observed in stroke rats with pHL treatment. Experimental animal models of global ischemia and focal ischemia have suggested the involvement of cell survival/death signaling pathway in the pathogenesis of cerebral ischemia (Sugawara et al, 2004). Morphological and biochemical evidences of apoptosis have also well documented in experimental animal models of cerebral ischemic injury (Charriaut-Marlangeu et al, 1996; Li et al, 1997; Fujimura et al, 1998). It is clear that both necrosis and apoptosis contribute to the cerebral ischemic injury, the relative contribution of each form varies substantially with the experimental model, brain cell type, and age of the experimental animal (Starkov et al, 2004). A simplistic view introduced in chapter 2, in focal ischemia, there is rarely complete blockade of blood flow to the affected region because plethora of collateral vessels provides some flow to the area, making the affected region a core infarct area and an ischemic penumbra. The cells in the core infarct are destined to die by necrosis while those in ischemic penumbra still receive collateral flow that sufficient to prevent cell Department of Pharmacology, YLL School of Medicine 140 Chapter 5: Discussion death by necrosis, but apoptosis. There is indeed a clear indication of initiation of apoptosis pathway in the core infarct, such as cytoplasmic and nuclear condensations and activation of specific caspase cascade (Benchoua et al, 2001). However, complete morphological changes of apoptosis are not observed at the end stages of infarction. The abortion of apoptosis in the core infarct might be attributed by the severe impairment of energy level that might cause a shift towards secondary necrosis from apoptosis (Nicotera et al, 1998). In contrast, many studies have demonstrated that cells in ischemic penumbra undergo delayed cell death by apoptosis due to the allowance given by the residual blood supply to maintain a low level of ATP. On the other hand, activated calpains and caspases could eventually cleave and inactivate the ion pumps. This situation could lead to the disruption of ion homeostasis and switch the apoptosis signaling to necrosis (Schwab et al, 2002). Therefore, the cell death in the ischemic area is rather a complex phenomenon. Since the necrosis is beyond the therapeutic reach, the rational treatment of stroke has likewise focused on protecting the cells from undergoing apoptosis at the early stage of stroke pathology. In past, direct treatment of oxidants like hydrogen peroxide was thought to induce exclusively necrosis. However, more recent studies have shown that lower doses of free radicals can trigger apoptosis as well. Reciprocally, broad-spectrum of anti-apoptotic proteins such as BCL-2 have been ascribed as an antioxidant function by maintaining cells in a more reduced state by scavenging ROS either directly or by up-regulating other ROS scavengers such as thiol compounds and antioxidant enzymes, further indicating that ROS generation is one of the crucial initiation step for apoptotic event (Chandra et al, Department of Pharmacology, YLL School of Medicine 141 Chapter 5: Discussion 2000). Recently, it has also been shown that oxidative stress can lead to apoptosis by inducing mitochondria permeability transition pore (MPTP) opening (Galindo et al, 2003). In addition, ROS can also lead to oxidation of the anionic phospholipids cardiolipin to facilitate cytochrome c release from mitochondria (Halliwell, 4th Edition). Since ample evidences demonstrate the link between oxidative stress and apoptosis, we would like to investigate whether pHL with antioxidant properties ameliorates the level of apoptosis during stroke. The TUNEL system is designed for detection of nuclear DNA fragmentation, an important biochemical hallmark of apoptosis in many cell types. It has been suggested that caspase may be the key executioner of nuclear degradation in ischemic neuron and contributes to the DNA fragmentation, possibly by activation of caspase-activated deoxyribonuclease (CAD) (Nakka et al, 2008). Results obtained from TUNEL assay showed that green fluorescence could only detect in infarct zone, indicating that apoptosis occurs only at the affected region. In addition, strongest nuclear green fluorescence was observed in infarct zone of stroke-induced rats, which is correlated with the results obtained from other analysis in this study. Lesser nuclear green fluorescence was observed in the stroke rats treated with pHL. Together with its ability of reducing infarct volume, neurological impairment and DNA oxidative damage, result obtained from TUNEL assay further indicates the therapeutic potential of pHL in the middle cerebral artery occluded Wistar rats, most probably through its antioxidant and anti-apoptosis properties. Department of Pharmacology, YLL School of Medicine 142 Chapter 5: Discussion Although TUNEL assay has been widely accepted for the detection of apoptosis, it is vulnerable to false-positives in the form of non-apoptotic necrotic cells (Iijima T, 2006). Therefore, it is always recommended to confirm the diagnosis with additional techniques such as study based on the criteria of morphological changes of apoptosis. In this experiment, existence of apoptosis was confirmed through immunohistochemical staining via probing the apoptotic-related proteins. Generally, apoptosis can be classified as intrinsic pathway and extrinsic pathway. There are considerable cross talks between both pathways. Subsequently, they converge to result ultimately in cellular morphological and biochemical alterations characteristic of apoptosis (Ashe et al, 2003). For Fas, it is involved in the extrinsic pathway of apoptosis (Ashe et al, 2003). In healthy individuals, Fas ligand (FasL) and Fas are mainly restricted to the immuno tissues and to sites of immune privilege such as the eye and the testes where they can trigger apoptosis of invading immune cells. The function of this death inducing receptor in the healthy brain is not so clear though it was suggested that it has a role of maintenance of immune privilege through FasL binding in brain (Mehmet, 2001). In particular, it is well documented that up-regulation of Fas is involved in ischemic injury in adult brain, which has also been shown in this experiment. According to the Mehmet’s report (2001), Fas knockout mice have a reduction of 54% of infarct area when the mice were subjected to MCAO, indicating the important role of Fas in pathophysiological progression of stroke. Department of Pharmacology, YLL School of Medicine 143 Chapter 5: Discussion Central to intrinsic pathway are proteins in the Bcl-2 family. The Bcl-2 family can be subdivided into pro- and anti-apoptotic proteins. Bax belongs to the pro-apoptotic protein while Bcl-2 and Bcl-xL belong to anti-apoptotic proteins (Love, 2003). Both Bcl-2 and Bcl-xL are localized at the mitochondrial outer membrane and to the endoplasmic reticulum and perinuclear membrane (Nakka et al, 2008). Bcl-2 is expressed at high level during the development and down-regulated after birth in the CNS. In peripheral nervous system, Bcl-2 expression is maintained throughout the life and its deletion causes profound loss of peripheral motor, sensory and sympathetic neurons after birth (Merry and Korsmeyer, 1997). Bcl-xL is expressed throughout life as well (Gonzalez-Garcia et al, 1995), indicating crucial role of Bcl-2 and Bcl-xL in neuronal survival. Bcl-2 and Bcl-xL could also inhibit pro-apoptotic Bcl-2 family through heterodimerization such as Bax (Nakka et al, 2008). Furthermore, Bcl-2 inhibits apoptosis through increasing mitochondrial redox capacity and increase resistance for the formation of mitochondrial transition permeability pore opening caused by the Ca2+ and oxidative stress (Starkov et al, 2004). Consistent with the previous works done (Ferrer et al, 2003; Sairanen et al, 2001), immunohistochemical staining results showed the presence of pro-apoptotic proteins BAX and FAS only in the infarct area after MCAO but undetectable in the healthy individuals. Highest immunoreactivity of pro-apoptotic proteins in vehicle group is correlated with observation of strongest nuclear green fluorescence in vehicle. This indicates the up-regulation of pro-apoptotic proteins and thereby intense cell death with dramatic morphological changes via both intrinsic (BAX upregulation) and extrinsic Department of Pharmacology, YLL School of Medicine 144 Chapter 5: Discussion In this experiment, we identified the protective effect of pHL on mitochondria with several limitations needed to be noted. Firstly, effects of pHL on reduction of ROS generation and ATP biosynthesis were observed in isolated mitochondria. Further experiments are suggested to be carried out to identify if pHL has the similar effects in vivo particularly in MCAO model. Secondly, the hypothesis of mild uncoupling effects on pHL needs further evaluation. JC-1 assay could be carried out to identify if pHL lowers the mitochondrial membrane potential which is an important indicator for the reduction in ROS generation and mild uncoupling effect. Lastly, in addition to the GSH determination, experiments should be carried out to elucidate the redox state of mitochondria by measuring the ratio of GSH and GSSG. In experiment II, results obtained from in vitro studies showed that pHL could suppress mitochondrial ROS generation in both physiological and oxidative stress condition. pHL inhibits ATP biosynthesis and possible confer the effect of metabolic arrest or uncoupling effects to the isolated mitochondria. In left MCAO model, reduction in mitochondrial respiration and RCR value from left cortical mitochondria indicates the mitochondrial dysfunction is resulted during MCAO. Cerebral ischemia caused by MCAO could also offset the GSH level in mitochondria. In vivo study leads us to conclude that pHL could enhance mitochondrial respiration and protect mitochondria from being damage during brain ischemia as shown by mitochondrial RCR value. In addition, it balances the GSH pool in the mitochondria so that GSH concentration is kept within the physiological range. Department of Pharmacology, YLL School of Medicine 158 Chapter 5: Discussion 5.3 Discussion on experiment III Leonurine as one of the key compounds found in HL could also significantly reduce the infarct volume and neurological deficit resulted from MCAO. Similarly, the effect of Leonurine is believed to act through antioxidant effects. Similar to the previous studies, left MCAO model consistently caused the infarction at the area in left cortical area and corpus striatum shown by TTC staining. Rats suffering from MCAO would have severe neurological deficit score throughout the entire study. Treatment of Leonurine at 60mg/kg/day could significantly reduce the infarct volume and alleviate the neurological impairment, indicating the lesser histological damage was observed as compared to the vehicle group. This has prompted us to further the study to evaluate the possible therapeutic mechanisms of Leonurine. Leonurine is one of the active compounds of pHL which has been shown to act as antioxidant for stroke treatment as discussed in previous experiments, therefore question for next is if Leonurine as a newly synthesized compound acts also an antioxidant for stroke treatment. In this study, one week pre-treatment was selected as it was found that one week of pre-treatment conferred similar protective effects as two weeks of pretreatment of Leonurine. SOD dismutates superoxide to hydrogen peroxide and oxygen. Hydrogen peroxide has modest oxidative potential and freely crosses cell membranes. Through iron-catalyzed reaction, H2O2 can be converted to much more reactive radical, OH·. Therefore elimination of hydrogen peroxide is equally important in alleviating oxidative stress. Department of Pharmacology, YLL School of Medicine 159 Chapter 5: Discussion Catalase and glutathione Peroxidase (GPx) serve this purpose. GPx was targeted in this study as in brain, the level of GPx is seven-fold greater than catalase. In addition, glutathione peroxidase is present in the cytosol while catalase locates mainly in peroxisomes. GPx converts hydrogen peroxide to water by oxidizing reduced glutathione (GSH) to oxidized glutathione (GSSG) (Warner et al, 2004). The reduced activities of SOD and GPx in vehicle group imply that severe oxidative stress occurred during permanent MCAO as much of the antioxidant molecules have been used up to react with overproduced free radicals. Leonurine treatment significantly restored the suppressed activities of SOD and GPx in stroke group to the level almost comparable to sham group, indicating that Leonurine has enhancing effects on endogenous antioxidant enzymes and therefore confers a protection against cerebral ischemia. Next, level of Melondialdehyde (MDA), a byproduct of oxidation of polyunsaturated fatty acid was evaluated as one of the marker of oxidative stress. This study showed that MDA level was increased after MCAO, further confirmed the involvement of increased free radical activity and reciprocal reduced endogenous antioxidant during cerebral ischemia. The obtained results are consistent with previous works done (Wang et al, 2007; Thiyagarajan and Sharma SS, 2004; Love S, 1999). Leonurine treatment was found not only enhance the level of SOD and GPx but also decrease the level of MDA. Therefore, it is proposed that in vivo therapeutic effect of Leonurine is strongly related to antioxidant effect by enhancing the endogenous antioxidant capacity and therefore alleviates the oxidative stress during ischemic stroke. Department of Pharmacology, YLL School of Medicine 160 Chapter 5: Discussion Similarly, mitochondrial ROS generation and ATP biosynthesis in vitro were reduced by Leonurine. In addition to the energy state and ion homeostasis, mitochondrial function is disrupted by ROS during neurodegenerative diseases (Brad et al, 2009). Modulation of mitochondrial function by antioxidant would be ideal therapy for neurodegenerative diseases. Dysfunctional mitochondria will produce more ROS, and a feed-forward loop is set up whereby ROS-mediated oxidative damage to mitochondria favors more ROS generation, resulting in a vicious cycle (Szeto HH, 2006). This phenomenon was shown in this study, that as compared to the control group, H2O2-treated mitochondria produced much more free radicals. This study demonstrated for the first time that mitochondrial ROS production was reduced by Leonurine in both physiological and H2O2- treated condition, in a dose dependent manner. The reduction of physiological mitochondrial ROS generation indicates that pretreatment with Leonurine could provide a protective barrier to oxidative stress. A reduction of mitochondrial ROS production or mitochondrial oxidative stress is believed to break the vicious cycle of mitochondrial ROS generation and therefore slow down the progression of ischemic stroke. Several enzymes of ETC were shown to be inhibited by ROS. In addition, single strand breaks DNA generated by ROS activate poly ADP-ribose polymerase (PARP) for DNA repair. Activated PARP cleaves NAD+ into nicotinamide and ADP-ribose, resulting in NAD+ deficiency. ROS could also attack the mitochondrial membrane directly to cause the membrane damage. Together with the possibility of the reversal action of F0F1ATPase under stress condition as discussed in experiment II, these factors lead to Department of Pharmacology, YLL School of Medicine 161 Chapter 5: Discussion depletion of ATP (Erdelyi et al, 2005). In this study, ATP biosynthesis was shown to be suppressed as mitochondria were treated with H2O2. In both physiological and H2O2– treated condition, ATP biosynthesis was further suppressed by Leonurine dosedependently, suggesting that Leonurine might induce mitochondrial metabolic arrest, reducing ATP production to the minimal. It has been reported that animal undergoing hibernation had metabolic depression on mitochondrial ATP biosynthesis (Boutilier and St Pierre, 2002) and it is known to be cytoprotective against various hypoxic and ischemic insults, enabling the organism to survive under deprived conditions over a period of time (Roth and Nystul, 2005). Slight increase of state respiration in isolated mitochondria and enhanced state respiration in vivo were observed under Leonurine treatment. Mitochondrial respiration, particularly the state respiration is important for determining the coupling state of oxidative phosphorylation in the mitochondria, thus regulating the level of mitochondrial metabolic efficiency. The RCR (state respiration rate / state respiration rate) indicates the tightness of the coupling respiration and phosphorylation. In isolated mitochondria, significant changes of oxygen consumption upon Leonurine treatment was not observed. Although treatment of Leonurine had a little impact on reducing RCR value, dose-dependently, it did not reach statistical significant. This is to confirm that reduction of ATP by Leonurine was not resulted from the impairment of mitochondrial ETC that among the treatment group and control groups, that mitochondria had the same functional coupling. Department of Pharmacology, YLL School of Medicine 162 Chapter 5: Discussion However, in vivo experiment, Leonurine treatment could enhance mitochondrial oxygen consumption in sham group with no change of RCR value, suggesting that pharmacological preconditioning by Leonurine could enhance the function of mitochondria. In addition, left cortical mitochondrial oxygen consumption (state respiration) was greatly suppressed hours after MCAO. In consistent with previous work (Wang et al, 2007), the lowest RCR obtained from vehicle group indicates that mitochondrial dysfunction was resulted after permanent MCAO. Leonurine could rescue the mitochondria by improving the oxygen consumption to the level that is almost comparable to sham untreated group. The RCR value from stroke Leonurine treated group was also increased, suggesting that Leonurine could prevent the mitochondrial dysfunction from ischemic insult. Mitochondrial GSH pool was balanced by Leonurine treatment in vivo. Normal cells maintain a specific redox homeostasis such that the redox couples such as NAD+/NADH, GSSG/GSH are maintained within a physiological range for oxidation and reduction. Therefore, any of these redox proteins moves out of physiological range will bring the biological adverse effect to the system. As discussed in experiment II, GSH is synthesized in the cytosol and transported actively into the mitochondria (Coll et al, 2004). In many cells, mitochondrial GSH level is much more important than the cytoplasmic pool in maintaining cell viability. Meredith and Reed (1982) found that depletion of both cytoplasmic and mitochondrial GSH greatly increased cell death than Department of Pharmacology, YLL School of Medicine 163 Chapter 5: Discussion those with depletion of cytoplasmic GSH alone. In contrast to the reduction of total GSH concentration found by previous works (Anderson and Sims, 2002; Madrigal et al, 2001), mitochondrial GSH level was increased in the vehicle group. The drastic increase of the GSH level observed is believed to be resulted from the active transport from cytosol into mitochondria. Studies from Shen and coworkers (2005) suggested that under the conditions where the reduction state of glutathione is high, mitochondrial membrane would be hyperpolarized and superoxide production would increase. Therefore, the increased of GSH level in vehicle group might cause adverse effect to the system by indirectly increasing the ROS production. Under the treatment of Leonurine, GSH level was brought back to the level that is not close to the sham group, indicating the ability of Leonurine to maintain the appropriate level of GSH pool in the mitochondria. To the best of our knowledge, this is the first demonstration of the therapeutic potential of Leonurine on permanent MCAO. Pretreatment of Leonurine at 60mg/kg/day for one week could protect rats from stroke insult by permanent MCAO. In vivo experiments, Leonurine pretreatment could reduce infarct volume and improve neurological deficit in stroke groups. Leonurine protects the cells from neuronal injury at least through the intervention with oxidative stress, as seen from the increased activities of antioxidant enzymes SOD and GPx, and decreased level from lipid peroxidation marker MDA. Further study on possible protective effects of Leonurine on mitochondria showed that Leonurine could modulate mitochondrial function by inhibiting ROS production, ATP biosynthesis, indicating a possible metabolic arrest conferred. Animal studies also demonstrated that the mitochondrial dysfunction was rescued by Leonurine treatment. In Department of Pharmacology, YLL School of Medicine 164 Chapter 5: Discussion addition, mitochondria GSH level could be balanced by Leonurine. Therefore, Leonurine has neuroprotective effects and carries a therapeutic potential for treatment of stroke. Department of Pharmacology, YLL School of Medicine 165 Chapter 5: Discussion 5.4 General discussion Ischemic stroke is the leading cause of death in the developed and developing countries while a large number of survivors suffer from debilitating consequences including permanent disabilities. Neuronal injury from ischemic stroke results from the interaction of complex pathophysiological processes such as excitotoxicity, depolarization, apoptosis and inflammation. The current available only therapy is tPA with therapeutic window of within hours after stroke onset, yet it is limited to a portion of patients, therefore, a neuroprotective or therapeutic agent with extended therapeutic window and minimized side effect is urgently needed. For many years, ischemic brain damage was regarded as a passive outcome resulted from the reduction of oxygen and glucose supply to allow adequate energy production for cell survival. With current ample experimental evidences, we understand that much of the active processes mediate the ischemic injury, particularly with the involvement of generation of highly reactive free radical species that lead to cellular injury. Experimental observations and preliminary data collected from clinical studies suggest that cellular damage and cell death by oxidative injury may be preventable by the early administration of antioxidants. Research using animal models has also shown that pharmacological intervention in oxidative stress could be a potential candidate to reduce the substantial brain damage and neurological dysfunction resulted from ischemic stroke. Over many years of stroke investigation, different models of ischemic injury have been established. The patterns of cellular changes that result in the cell death are different in all Department of Pharmacology, YLL School of Medicine 166 Chapter 5: Discussion types of models, depending on the severity of ischemia, whether the occlusion is permanent or transient, vessels to be occluded, duration of the ischemia and the specific properties of the affected cells. In this project, rodent model of permanent focal ischemia by left middle cerebral artery occlusion was employed (MCAO). MCAO was done by transcranial method which involves the opening of skull and by electro-cauterization to occlude the cerebral artery. Left MCAO model established in this project has consistently produced results of infarct volume of about 25% spanning the region of ipsilateral cortex and striatum. In addition, animal suffering from MCAO would have severe neurological impairment with the score of about measured by neurological deficit grading system. Throughout the studies, oxidative stress was showed to be associated with permanent focal ischemia as the rats suffering from MCAO had reduction of total plasma antioxidant concentration level, cortical SOD and GPx activities, and enhanced cortical MDA level. This is accompanied with the increased level of apoptosis as shown by TUNEL staining. Strong immunoreactivities of pro-apoptotic proteins BAX and FAS were also detected. Both results indicate that apoptotic cells are scattered throughout the ischemic territory. In addition, left cortical mitochondria isolated from MCAO-induced rat had lowered respiration rate, indicating the mitochondrial dysfunction is involved in the stroke induced by MCAO. As discussed above, transcranial procedure opens the skull, thus this method affects the intracranial pressure and may reduce the edema that an intact skull would otherwise cause. Alternative less invasive noncraniectomy models are the suture model (Belayev et al, 1996), thrombus model (Overgaard et al, 1992), endothelin model (Callaway et al, Department of Pharmacology, YLL School of Medicine 167 Chapter 5: Discussion 1999) and photothrombotic model (Cai et al, 1998). The occlusion of the MCA in these models is caused by filaments, heterologous or autologous clots, or endothelin administrated via the common carotid artery. For photothrombotic model, the photosensitive dye excited by light at a specific wavelength injures the endothelium of blood vessels, leading to a nidus for thrombosis. This project was started by clarifying the therapeutic potential of pHL on MCAO-induced Wistar rats. The aspects of antioxidant and apoptosis were emphasized. Pretreatment with vehicle (water), or pHL was applied for 14 days prior to the induction of stroke, followed by another days of post-operation treatment before the rats were sacrificed for sample collection. The pre-surgery treatment was included in the experiment so as to determine if pHL was advantageous when delivered prophylatically before stroke onset in addition to their effects on stroke treatment. As pHL has antioxidant properties as discussed in session 2.3, when it is administered prophylatically, it scavenge and prevent free radical generation. During the onset of stroke, it could reach the optimal concentration to prevent the damage caused by oxidative stress. In other words, if pHL was administered only after stroke onset, free radical mediated damaging effect could be preceded before the drug has reached the site of injury and the drug might not be available in sufficient concentration at time (Gupta et al, 2003). In addition, inclusion of sham operated pHL treated group is to identify its possible protective effect in healthy individual. This project aims to promote pHL as an “antioxidant supplement”. Results obtained from this study showed that pHL serve as Department of Pharmacology, YLL School of Medicine 168 Chapter 5: Discussion antioxidant supplement could enhance plasma antioxidant concentration so to confer a protective barrier to diseases related to oxidative stress. Under physiological condition, it helps in preventing the ROS generation from mitochondria, it also enhances mitochondrial respiration so to protect mitochondrial from being dysfunction upon ischemic challenge. The pilot study on pHL (experiment I) showed that treatment of pHL could statistically reduce the infarct volume and neurological deficit score in animal subjected to MCAO. Therapeutic effect of pHL is believed to act through antioxidant effects by the observation of reduction in DNA oxidative damage and improvement of total plasma antioxidant concentration. pHL might interfere with apoptosis signaling after stroke onset as treatment of pHL is associated with the reduction of TUNEL apoptotic green fluorescence, increase of anti-apoptotic immunoreactivities and reduction of proapoptotic immunoreactivities. From the first part of study, pHL was shown to confer neuroprotective effects to the ischemic stroke by reducing oxidative stress and interfering the apoptotic cascade. While necrosis results from the passive accumulation of deleterious changes that disrupt the cell function to loss in viability, apoptosis is a programmed cell death which could be possibly inhibited along the signaling pathway. Caspase plays a key role in executing apoptosis. Inhibiting apoptosis by caspase inhibitor was suggested to be a possible approach for stroke therapy. However, such approach is controversial as caspase inhibitors might inhibit other signaling pathway that needed for survival in a non-specific Department of Pharmacology, YLL School of Medicine 169 Chapter 5: Discussion manner. It might also shift the injured nonfunctional cells to necrosis by giving the apoptotic pathway a pause, leading to spreading of inflammation and death of adjacent neurons. Therefore, re-evaluation of appropriate anti-apoptosis approach for stroke therapy is taking place. Oxidative stress is a complex interplay that involves not only apoptosis, but other damaging signaling such as inflammation, excitotoxicity and mitochondrial dysfunction. Oxidative stress mediated apoptosis during ischemic cascade provide a new insight for the stroke therapy that with the administration of antioxidant, oxidative stress would be alleviated and therefore apoptosis pathway could be prevented. Therefore, pharmacological intervention and modification is current believed to be a promising avenue for stroke therapy. Mitochondria are the important regulator of cellular oxidative stress as they generate ROS as byproducts of oxygen consumption in ETC. They are also the targets of ROS. Mitochondrial dysfunction has been well documented in the ischemic cascade of stroke. Therefore, mitochondria protection could be a key role in the stroke therapy. As from experiment I, we demonstrated that pHL reduce oxidative stress, we also showed the reduction of BAX immunoreactivity and induction of BCL-2, BCL-XL immunoreactivities in pHL treated stroke operated group, which these proteins are closely related to the mitochondria associated intrinsic pathway of apoptosis. Possible role of pHL in mitochondrial ROS generation and mitochondrial function were evaluated in next set of experiments. Department of Pharmacology, YLL School of Medicine 170 Chapter 5: Discussion In experiment II, to elucidate the effect of pHL on modulation of mitochondrial function (objective 2), isolation of cortical mitochondria from Wistar rats was carried out. In isolated cortical mitochondria, basal level of ROS generation and ATP biosynthesis upon challenged by succinate (complex II substrate) were observed, indicating the initiation of ETC. ROS generation is considered as the byproducts of oxygen consumption. In addition, upon stimulated by succinate in the presence of ADP (state respiration), mitochondrial respiration was observed as indicated by the increase of oxygen consumption. When the isolated mitochondria were treated with pHL, a lowered ROS generation level was observed in a dose dependent manner, suggesting that pHL could inhibit the endogenous production of ROS from mitochondria, thereby preventing the contribution of mitochondria to oxidative stress under any circumstances. This is accompanied with the lowered ATP biosynthesis. Possible toxic effect of pHL that might lead to reduction in ATP biosynthesis is excluded, as subsequent experiment on mitochondrial respiration showed that pHL did not cause any significant changes of mitochondrial RCR value, indicating the normal function of mitochondria was observed under the treatment of pHL. Therefore, pHL might have effect of metabolic arrest to the mitochondria, leading to the cytoprotective barrier to the mitochondria in the case of stress condition, such as ischemia. However, this is yet to be confirmed. Although RCR value did not change significantly by pHL treatment, a slight reduction in RCR value was observed upon treatment of pHL in a dose-dependent manner. This is due to the slight increase of state respiration. This observation leads us to suggest that pHL might have a mild uncoupling effect to the Department of Pharmacology, YLL School of Medicine 171 Chapter 5: Discussion mitochondria respiration. Mild uncoupling has been shown to be a cytoprotective strategy, especially in brain, under the condition of oxidative stress (Brookes PS, 2006). In order to induce oxidative stress, isolated mitochondria were challenged with 1mM of H2O2. A greater extent of ROS generation was observed in mitochondria treated with H2O2. It was reported that during ischemic cascade, Ca2+ induces the superoxide generation in the mitochondrial respiratory chain by flavin mononucleotide (FMN) group of complex I and ubiquinone-cytochrome b-c1 of complex III (Christophe and Nicolas, 2006). In addition, ATP biosynthesis was suppressed by H2O2, reflecting the phenomenon mitochondria are the both contributors and targets of free radicals during oxidative stress. In the presence of pHL, both mitochondrial ROS generation and ATP biosynthesis are suppressed, indicating that effects of pHL could be executed in both physiological and pathological conditions. As discussed above, mitochondrial dysfunction was observed hours after the MCAO. Treatment of pHL could enhance mitochondrial respiration, particularly state respiration in both healthy and MCAO-induced rats, indicating that pHL could protect mitochondria from being dysfunctional, which could protect the cell from undergoing apoptosis and challenging with free radicals. GSH level of mitochondria was also balanced by treatment of pHL as shown by the similar level of GSH observed in pHL treated stroke operated group with sham group. Department of Pharmacology, YLL School of Medicine 172 Chapter 5: Discussion In the third experiment, pretreatment of Leonurine for days prior to MCAO was applied to the animals. One day after MCAO, animal was sacrificed to assess the effect of Leonurine. Leonurine treatment could reduce infarct volume and improve neurological function. It exerts its effect at least through antioxidant effects as shown by its ability in reduction of MDA level and improve the cortical SOD and GPx levels. To higher degree of similarity, Leonurine showed much profound protective effect to isolated mitochondria as compared to pHL, with wider therapeutic range as compared to pHL, again confirm that Leonurine is one of the active ingredients in pHL. In vivo experiments also showed that Leonurine could enhance state respiration in mitochondria isolated from rats undergone MCAO. Mitochondrial GSH level could also be balanced by the treatment of Leonurine. To conclude the observed findings, pHL confers neuroprotective effects and therapeutic effects to ischemic stroke via few parameters: reduction in infarct volume, improvement of neurological deficit score, increase of plasma total antioxidant concentration, reduction of DNA oxidative damage, reduction of mitochondrial ROS generation, inhibition of ATP biosynthesis, improvement of oxygen consumption and balancing the mitochondrial GSH pool. Leonurine is believed to be one of the major active ingredients of pHL as it carries the similar but more enhanced therapeutic effects as compared to pHL. Department of Pharmacology, YLL School of Medicine 173 [...]... potential of Leonurine on permanent MCAO Pretreatment of Leonurine at 60mg/kg/day for one week could protect rats from stroke insult by permanent MCAO In vivo experiments, Leonurine pretreatment could reduce infarct volume and improve neurological deficit in stroke groups Leonurine protects the cells from neuronal injury at least through the intervention with oxidative stress, as seen from the increased... enzymes SOD and GPx, and decreased level from lipid peroxidation marker MDA Further study on possible protective effects of Leonurine on mitochondria showed that Leonurine could modulate mitochondrial function by inhibiting ROS production, ATP biosynthesis, indicating a possible metabolic arrest conferred Animal studies also demonstrated that the mitochondrial dysfunction was rescued by Leonurine treatment... on mitochondrial ATP biosynthesis (Boutilier and St Pierre, 2002) and it is known to be cytoprotective against various hypoxic and ischemic insults, enabling the organism to survive under deprived conditions over a period of time (Roth and Nystul, 20 05) Slight increase of state 4 respiration in isolated mitochondria and enhanced state 3 respiration in vivo were observed under Leonurine treatment Mitochondrial. .. The RCR value from stroke Leonurine treated group was also increased, suggesting that Leonurine could prevent the mitochondrial dysfunction from ischemic insult Mitochondrial GSH pool was balanced by Leonurine treatment in vivo Normal cells maintain a specific redox homeostasis such that the redox couples such as NAD+/NADH, GSSG/GSH are maintained within a physiological range for oxidation and reduction... that pHL could enhance mitochondrial respiration and protect mitochondria from being damage during brain ischemia as shown by mitochondrial RCR value In addition, it balances the GSH pool in the mitochondria so that GSH concentration is kept within the physiological range Department of Pharmacology, YLL School of Medicine 158 Chapter 5: Discussion 5. 3 Discussion on experiment III Leonurine as one of the... volume and neurological deficit resulted from MCAO Similarly, the effect of Leonurine is believed to act through antioxidant effects Similar to the previous studies, left MCAO model consistently caused the infarction at the area in left cortical area and corpus striatum shown by TTC staining Rats suffering from MCAO would have severe neurological deficit score throughout the entire study Treatment of Leonurine. .. School of Medicine 164 Chapter 5: Discussion addition, mitochondria GSH level could be balanced by Leonurine Therefore, Leonurine has neuroprotective effects and carries a therapeutic potential for treatment of stroke Department of Pharmacology, YLL School of Medicine 1 65 Chapter 5: Discussion 5. 4 General discussion Ischemic stroke is the leading cause of death in the developed and developing countries while... potential and apoptotic cell death by accumulation of extracellular glutamate through excitotoxicity Hence, in addition through enhancing the mitochondrial oxygen consumption, pHL might prevent the further decrease of membrane potential resulted from excitotoxicity so that confer the neuroprotective effect MCAO-induced rats had enhanced level of mitochondrial GSH and pHL administration balanced the mitochondrial. .. physical injury (Roth and Nystul, 20 05) Department of Pharmacology, YLL School of Medicine 150 Chapter 5: Discussion According to Geng and Su (1991), Chinese herbs are generally classified into four categories: “Yang-invigorating”, “Yin-invigorating”, “Qi-invigorating” and “Bloodenriching”, with “Qi-invigorating” and “Blood-enriching” belong to Yang and Yin family While maintaining the balance of Yang and. .. across the inner mitochondrial membrane to dissipate the electrochemical proton Department of Pharmacology, YLL School of Medicine 152 Chapter 5: Discussion gradient (Skulachev VP, 1997) Diminished brain damage resulted from experimental stroke and traumatic brain injury was observed in mice over-expressing human UCP-2 (Mattiasson et al, 2003) UCP-2 is suggested to play a role in neuroprotection by inhibiting . impairment and DNA oxidative damage, result obtained from TUNEL assay further indicates the therapeutic potential of pHL in the middle cerebral artery occluded Wistar rats, most probably through. Chapter 5: Discussion Department of Pharmacology, YLL School of Medicine 153 gradient (Skulachev VP, 1997). Diminished brain damage resulted from experimental stroke and traumatic brain injury. oxygen deprivation or physical injury (Roth and Nystul, 20 05) . Chapter 5: Discussion Department of Pharmacology, YLL School of Medicine 151 According to Geng and Su (1991), Chinese herbs