Green tea polyphenol (−)-epigallocatechin gallate reduces matrix metalloproteinase-9 activity following transient focal cerebral ischemia

Green tea polyphenol (?)-epigallocatechin gallate (EGCG) has been reported to reduce neuronal damage after cerebral ischemic insult. EGCG is known to reduce matrix metalloproteinase (MMP) activity. MMP can play an important role in the pathophysiology of neurological disorders including cerebral ischemia. The purpose of the current study was to investigate whether EGCG shows an inhibitory effect on MMP activity and neural tissue damage following transient focal cerebral ischemia. In the present study, C57BL/6 mice were subjected to 80 min of focal ischemia induced by middle cerebral artery occlusion (MCAO). Animals were killed 24 h after ischemia. EGCG (50 mg/kg) was administered intraperitoneally immediately after ischemia. Gelatin gel zymography showed an increase in the active form of MMP-9 after ischemia. EGCG reduced ischemia-induced up-regulation of the active form of MMP-9. In in situ zymography, EGCG reduced up-regulation of gelatinase activity induced by cerebral ischemia. Co-incubation with EGCG reduced gelatinase activity directly in postischemic brain section. In 2,3,5-triphenyltetrazolium chloride (TTC) assay, brain infarction was remarkable in the middle cerebral artery territory after focal cerebral ischemia. In EGCG-treated mice, infarct volume was significantly reduced compared with vehicle-treated mice. These results demonstrate that EGCG, a green tea polyphenol, may reduce up-regulation of MMP-9 activity and neuronal damage following transient focal cerebral ischemia. In addition to its antioxidant effect, MMP-9 inhibition might be a possible mechanism potentially involved in the neuroprotective effect of a green tea polyphenol, EGCG.     

高压氧对局灶性脑缺血后细胞凋亡的影响及其机制

目的：观察不同时间点高压氧（HBO）治疗对短暂性脑缺血的作用,并探讨其对细胞凋亡的影响。方法：在客观监测局部脑血流的条件下,大鼠经历短暂脑缺血后3h,6h,12h应用HBO治疗,24h后行神经功能评分和梗死体积测定,免疫组化染色各组Bcl-2、Bax、活性Caspase-3、活性Caspase-9以及TUNEL法检测细胞凋亡。结果：缺血后3h HBO治疗减少70％梗死体积,缺血后6hHBO治疗则减少梗死体积约44％,早期应用HBO治疗增加半暗带区细胞内Bcl-2的表达,减少活性Caspase-9和活性Caspase-3以及TUNEL阳性细胞数;缺血后12h应用HBO治疗却恶化神经功能,扩大梗死范围,而对上述凋亡各指标无影响。结论：HBO治疗短暂性局灶脑缺血具有时间窗,应争取在缺血后6h内应用HB0治疗,其早期治疗的神经保护作用与抑制细胞凋亡有关。     

Delayed activin A administration attenuates tissue death after transient focal cerebral ischemia and is associated with decreased stress-responsive kinase activation

Focal cerebral ischemia and reperfusion initiates complex cellular and molecular interactions that lead to either cell repair or destruction. In earlier work, we found that activin A is an early gene response to cerebral ischemia and supports cortical neuron survival in vitro. In this study, the ability of exogenous activin A to attenuate injury from transient middle cerebral artery occlusion was tested in adult mice. Intracerebroventricular administration of activin A prior to middle cerebral artery occlusion reduced infarct volume apparent 1 day after experimental stroke. A single activin A administration at 6 h following ischemia/reperfusion reduced lesion volumes at 1 and 3 days and led to improved neurobehavior. Moreover, activin A treatment spared neurons within the ischemic hemisphere and led to a concomitant reduction in microglial activation. Activation of the stress-responsive kinases p38 and c- jun N-terminal kinase implicated in neuronal apoptosis after stroke was reduced following activin A treatment. Together these findings suggest that activin A promotes tissue survival after focal cerebral ischemia/reperfusion with an extended therapeutic window.     

Prospects for the future: the role of free radicals in the treatment of stroke

Studies using animal models of stroke have demonstrated that free radicals are highly reactive molecules generated predominantly during cellular respiration and normal metabolism. Imbalance between cellular production of free radicals and the ability of cells to defend against them is referred to as oxidative stress. After ischemic brain damage introduced by ischemic stroke or reperfusion, production of reactive oxygen species may increase, sometimes drastically, leading to tissue damage via several different cellular molecular pathways. The damage can become more widespread due to weakened cellular antioxidant defense systems after ischemic stroke. These experimental findings have important implications for the treatment of human cerebral ischemia. Agents directed at eliminating oxygen radicals must be administered before, or in the early stages of, reperfusion after ischemia. The therapeutic window seems to be narrow and limited to, at most, a few hours. Future research may clarify the current hypothesis that the accuracy of gene expression could account for the recovery of cellular function after ischemic stroke. This may open the window to the future use of drug combinations that may be rationally administered sequentially. If the phenomenon of ischemic tolerance plays a role in this concept is still a matter of debate.     

Transient focal cerebral ischemia/reperfusion induces early and chronic axonal changes in rats: its importance for the risk of Alzheimer's disease

The dementia of Alzheimer's type and brain ischemia are known to increase at comparable rates with age. Recent advances suggest that cerebral ischemia may contribute to the pathogenesis of Alzheimer's disease (AD), however, the neuropathological relationship between these two disorders is largely unclear. It has been demonstrated that axonopathy, mainly manifesting as impairment of axonal transport and swelling of the axon and varicosity, is a prominent feature in AD and may play an important role in the neuropathological mechanisms in AD. In this study, we investigated the early and chronic changes of the axons of neurons in the different brain areas (cortex, hippocampus and striatum) using in vivo tracing technique and grading analysis method in a rat model of transient focal cerebral ischemia/reperfusion (middle cerebral artery occlusion, MCAO). In addition, the relationship between the changes of axons and the expression of β-amyloid 42 (Aβ42) and hyperphosphorylated Tau, which have been considered as the key neuropathological processes of AD, was analyzed by combining tracing technique with immunohistochemistry or western blotting. Subsequently, we found that transient cerebral ischemia/reperfusion produced obvious swelling of the axons and varicosities, from 6 hours after transient cerebral ischemia/reperfusion even up to 4 weeks. We could not observe Aβ plaques or overexpression of Aβ42 in the ischemic brain areas, however, the site-specific hyperphosphorylated Tau could be detected in the ischemic cortex. These results suggest that transient cerebral ischemia/reperfusion induce early and chronic axonal changes, which may be an important mechanism affecting the clinical outcome and possibly contributing to the development of AD after stroke.     

Neuroprotective Effects of Ischemic Preconditioning in Brain Mitochondria Following Cerebral Ischemia

Numerous studies support the hypothesis that reperfusion following cerebral ischemia contributes substantially to ischemic injury and that mitochondrial dysfunction plays a central role. Defining the mechanisms by which mitochondrial dysfunction occurs may be important for the development of new therapies against delayed neuronal cell death. Ischemic preconditioning (IP) increases an organ's resistance to ischemic injury. There are two windows for IPC, one that requires several hours to develop and another one with a rapid setting (rapid window). However, the rapid window only provides neuroprotection for few days. We have recently determined that this lack of chronic protection by the rapid window was due to lack of protection against mitochondrial dysfunction.     

Role of Hydrogen Sulfide in Early Blood-Brain Barrier Disruption following Transient Focal Cerebral Ischemia

We determined the role of endogenous hydrogen sulfide (H₂S) in cerebral vasodilation/hyperemia and early BBB disruption following ischemic stroke. A cranial window was prepared over the left frontal, parietal and temporal cortex in mice. Transient focal cerebral Ischemia was induced by directly ligating the middle cerebral artery (MCA) for two hours. Regional vascular response and cerebral blood flow (CBF) during ischemia and reperfusion were measured in real time. Early BBB disruption was assessed by Evans Blue (EB) and sodium fluorescein (Na-F) extravasation at 3 hours of reperfusion. Topical treatment with DL-propargylglycine (PAG, an inhibitor for cystathionine γ-lyase (CSE)) and aspartate (ASP, inhibitor for cysteine aminotransferase/3-mercaptopyruvate sulfurtransferase (CAT/3-MST)), but not O-(Carboxymethyl)hydroxylamine hemihydrochloride (CHH, an inhibitor for cystathionine β-synthase (CBS)), abolished postischemic cerebral vasodilation/hyperemia and prevented EB and Na-F extravasation. CSE knockout (CSE^-/-) reduced postischemic cerebral vasodilation/hyperemia but only inhibited Na-F extravasation. An upregulated CBS was found in cerebral cortex of CSE^-/- mice. Topical treatment with CHH didn’t further alter postischemic cerebral vasodilation/hyperemia, but prevented EB extravasation in CSE^-/- mice. In addition, L-cysteine-induced hydrogen sulfide (H₂S) production similarly increased in ischemic side cerebral cortex of control and CSE^-/- mice. Our findings suggest that endogenous production of H₂S by CSE and CAT/3-MST during reperfusion may be involved in postischemic cerebral vasodilation/hyperemia and play an important role in early BBB disruption following transient focal cerebral ischemia.     

Upregulation of EMMPRIN after permanent focal cerebral ischemia

Elevated activities of matrix metalloproteinases (MMPs) following ischemic stroke have been shown to mediate ischemic injury as well as neurovascular remodeling. The extracellular MMP inducer (EMMPRIN) is a 58-kDa cell surface glycoprotein, which has been known to play a key regulatory role for MMP activities. The roles of EMMPRIN in stroke injury are not clearly understood. In this study, we investigated changes of EMMPRIN in a mouse model of permanent focal cerebral ischemia, and examined potential association between EMMPRIN and MMP-9 expression. Adult male CD-1 mice were subjected to permanent focal ischemia by intraluminal occlusion of the left middle cerebral artery (MCAO) under anesthesia. EMMPRIN expression was markedly upregulated in the peri-infarct area at 2-7 days after ischemia compared to the contralateral non-ischemic hemisphere by Western blot analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals co-localized with vwF-positive endothelial cells and GFAP-positive peri-vascular astrocytes. In contrast, EMMPRIN signal did not co-localize with NeuN-positive neurons, or MPO-positive neutrophils. Dual fluorescent staining revealed that EMMPRIN co-localized with MMP-9. Our data also demonstrated that increased EMMPRIN expression correlated with increased MMP-9 levels in a temporal manner. In summary, we report for the first time that EMMPRIN expression was significantly increased in a mouse model of permanent focal cerebral ischemia. The spatial and temporal association between increased EMMPRIN expression and elevated MMP-9 levels suggest that EMMPRIN may modulate MMP-9 activity, and participate in neurovascular remodeling after ischemic stroke.     

MMP-9 Inhibition: a Therapeutic Strategy in Ischemic Stroke

Ischemic stroke is a leading cause of disability worldwide. In cerebral ischemia there is an enhanced expression of matrix metallo-proteinase-9 (MMP-9), which has been associated with various complications including excitotoxicity, neuronal damage, apoptosis, blood–brain barrier (BBB) opening leading to cerebral edema, and hemorrhagic transformation. Moreover, the tissue plasminogen activator (tPA), which is the only US-FDA approved treatment of ischemic stroke, has a brief 3 to 4 h time window and it has been proposed that detrimental effects of tPA beyond the 3 h since the onset of stroke are derived from its ability to activate MMP-9 that in turn contributes to the breakdown of BBB. Therefore, the available literature suggests that MMP-9 inhibition can be of therapeutic importance in ischemic stroke. Hence, combination therapies of MMP-9 inhibitor along with tPA can be beneficial in ischemic stroke. In this review we will discuss the current status of various strategies which have shown neuroprotection and extension of thrombolytic window by directly or indirectly inhibiting MMP-9 activity. In the introductory part of the review, we briefly provide an overview on ischemic stroke, commonly used models of ischemic stroke and a role of MMP-9 in ischemia. In next part, the literature is organized as various approaches which have proven neuroprotective effects through direct or indirect decrease in MMP-9 activity, namely, using biotherapeutics, involving MMP-9 gene inhibition using viral vectors; using endogenous inhibitor of MMP-9, repurposing of old drugs such as minocycline, new chemical entities like DP-b99, and finally other approaches like therapeutic hypothermia.     

A Type-II Positive Allosteric Modulator of α7 nAChRs Reduces Brain Injury and Improves Neurological Function after Focal Cerebral Ischemia in Rats

In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.     

Increased intranuclear matrix metalloproteinase activity in neurons interferes with oxidative DNA repair in focal cerebral ischemia

Increased matrix metalloproteinase (MMP) activity is implicated in proteolysis of extracellular matrix in ischemic stroke. We recently observed intranuclear MMP activity in ischemic brain neurons at early reperfusion, suggesting a possible role in nuclear matrix proteolysis. Nuclear proteins, poly-ADP-ribose polymerase-1 (PARP-1) and X-ray cross-complementary factor 1 (XRCC1), as well as DNA repair enzymes, are important in DNA fragmentation and cell apoptosis. We hypothesized that intranuclear MMP activity facilitates oxidative injury in neurons during early ischemic insult by cleaving PARP-1 and XRCC1, interfering with DNA repair. We induced a 90-min middle cerebral artery occlusion in rats. Increase activity of MMP-2 and -9, detected in the ischemic neuronal nuclei at 3 h, was associated with DNA fragmentation at 24 and 48 h reperfusion. The intranuclear MMPs cleaved PARP-1. Treatment of the rats with a broad-spectrum MMP inhibitor, BB1101, significantly attenuated ischemia-induced PARP-1 cleavage, increasing its activity. Degradation of XRCC1 caused by ischemic insult in rat brain was also significantly attenuated by BB1101. We found elevation of oxidized DNA, apurinic/apyrimidinic sites, and 8-hydroxy-2'-deoxyguanosine, in ischemic brain cells at 3 h reperfusion. BB1101 markedly attenuated the early increase of oxidized DNA. Using tissue from stroke patients, we found increased intranuclear MMP expression. Our data suggest that intranuclear MMP activity cleaves PARP-1 and XRCC1, interfering with oxidative DNA repair. This novel role for MMPs could contribute to neuronal apoptosis in ischemic injuries.     

Reversibility of Nimodipine Binding to Brain in Transient Cerebral Ischemia

Using autoradiography, we have measured the in vivo binding of [3H]nimodipine to brain in a rat model of reversible cerebral ischemia. Ischemia was induced by simultaneous occlusion of the middle cerebral artery (MCA) and ipsilateral common carotid artery by microaneurysm clips. Rats were studied after 15 min of ischemia (ischemic group) or after 45 min of reperfusion following 15 min of ischemia (reperfused group). Regional cerebral blood flow (CBF) was determined autoradiographically using [14C]iodoantipyrine in both ischemic (n = 6) and reperfused (n = 6) groups. During ischemia blood flow in the territory of the MCA was depressed and recovered to normal only in the distal territory of the MCA following reperfusion. [3H]Nimodipine binding in the ischemic group (n = 12) was elevated in ischemic brain regions and declined significantly (p < 0.01) in these regions in the reperfused group (n = 11). The ratio of the volume of cortex showing increased binding to the total volume of the forebrain was 0.113 +/- 0.025 (mean +/- SD) in the ischemic group and declined to 0.080 +/- 0.027 following reperfusion (p < 0.005). In general, infarct was only observed in regions showing persistent elevation of nimodipine binding following reperfusion as determined by histology performed in a separate group of rats (n = 8) after 24 h of reperfusion. We conclude that increased nimodipine binding to ischemic tissue is initially reversible with prompt reestablishment of CBF and is a sensitive indicator of early and reversible ischemia-induced cerebral dysfunction.     

The characteristics of therapeutic effect of pinocembrin in transient global brain ischemia/reperfusion rats

AimsThe therapeutic effect of pinocembrin, together with the therapeutic time window, in the transient global cerebral ischemia/reperfusion (I/R) rats was investigated.Main methodsAdult male Sprague–Dawley rats were subjected to global cerebral ischemia for 20 min by four-vessel occlusion. Pinocembrin (1 and 5 mg/kg) was administrated intravenously 30 min before ischemia and 30 min, 2 h, 6 h after reperfusion, respectively. Neurological scores, brain edema and histological examination by Nissl staining were employed to assess the neuronal injury after ischemia and the neuroprotection by pinocembrin. The activities of superoxide dismutase (SOD), myeloperoxidase (MPO) and the content of malondialdehyde (MDA) in brain tissue were tested by colorimetric assays. Alterations of neurotransmitters were determined by a high performance liquid chromatography–electrochemical method.Key findingsPinocembrin significantly ameliorated neurological deficits and brain edema, and alleviated the degree of hippocampal neuronal loss at 24 h after global cerebral I/R with a broad therapeutic time window. It was found that treatment with pinocembrin reduced the compensatory increase of SOD activity and decreased the MDA level and MPO activity in a dose-dependent manner. The metabolic balance between excitatory and inhibitory amino acids was modulated by pinocembrin treatment.SignificanceThese findings suggest that pinocembrin provides neuroprotection against global cerebral ischemic injury with a wide therapeutic time window, which may be attributed to its antioxidative, antiinflammatory and antiexcitotoxic effects.     

Glutathione monoethylester prevents mitochondrial glutathione depletion during focal cerebral ischemia

Glutathione is a central component in the antioxidant defences of cells. We have recently reported an early and selective loss of total (reduced plus oxidised) glutathione from mitochondria isolated from rat brain following occlusion of the middle cerebral artery. This mitochondrial glutathione depletion showed an apparent association with the tissue damage that developed during subsequent reperfusion, suggesting that it could be an important determinant of susceptibility to cell loss. In the present study, we have investigated whether in vivo treatment with glutathione ethyl ester can modulate mitochondrial glutathione in the brain and whether this treatment can influence the response to focal ischemia. In further support of our previous findings, middle cerebral artery occlusion caused a duration-dependent partial loss of mitochondrial glutathione. Bilateral injections of glutathione ethyl ester immediately prior to induction of unilateral focal ischemia resulted in a substantial increase in glutathione in mitochondria from the striatum of both the non-ischemic hemisphere (190% of saline-treated controls) and the ischemic hemisphere (240% of controls) at 2h after arterial occlusion. Total tissue glutathione was not affected by the ester treatment at this time. A smaller increase in mitochondrial glutathione was observed at 3h of occlusion in the non-ischemic striatum following ester treatment but at this time point glutathione was not significantly altered in mitochondria from the ischemic hemisphere. Pre-ischemic treatment with glutathione ester did not significantly change the volume of tissue infarction assessed at 48 h following ischemia for 2 or 3h. These studies demonstrate that glutathione ethyl ester is a highly effective modulator of the mitochondrial glutathione pool in the intact brain and provides a useful means for further investigating the role of this antioxidant in the development of tissue damage in ischemia and other brain disorders.     

Neuroprotective effects of VEGF administration after focal cerebral ischemia/reperfusion: dose response and time window

Administration of vascular endothelial growth factor (VEGF) has been shown to increase cerebral blood flow and reduce neurological damage after experimental ischemic brain injury. The purpose of this study was to examine the optimal dose and time window for the neuroprotective effect of VEGF when administrated after focal ischemia/reperfusion injury in rabbits. Focal cerebral ischemia/reperfusion was induced by the middle cerebral artery occlusion (MCAO) method. In a dose response experiment, low (1.25 ng/μL), middle (2.5 ng/μL) and high (5.0 ng/μL) doses of VEGF were administered 2h after MCAO by the route of perifocal region. The VEGF at a dose of middle (2.5 ng/μL) displayed excellent effects on neuroprotective efficacy for focal cerebral ischemia/reperfusion injury. In another experiment, 2.5 ng/μL VEGF was administered at times varying from 2 to 8h after MCAO. Infarct volume, water content and neurological deficits were significantly reduced when VEGF was given at 2 and 3h after injury. The protective effect was less when the same dose was given at the later times. Thus, the present findings indicated that VEGF reduced ischemic neuronal danger with a therapeutic time window within the first 3h of transient MCAO and may be useful in the treatment of acute ischemic stroke in humans.     

Delayed treatment with a novel neurotrophic compound reduces behavioral deficits in rabbit ischemic stroke

Acute ischemic stroke is a major risk for morbidity and mortality in our aging population. Currently only one drug, the thrombolytic tissue plasminogen activator, is approved by the US Food and Drug Administration to treat stroke. Therefore, there is a need to develop new drugs that promote neuronal survival following stroke. We have synthesized a novel neuroprotective molecule called CNB-001 (a pyrazole derivative of curcumin) that has neurotrophic activity, enhances memory, and blocks cell death in multiple toxicity assays related to ischemic stroke. In this study, we tested the efficacy of CNB-001 in a rigorous rabbit ischemic stroke model and determined the molecular basis of its in vivo activity. CNB-001 has substantial beneficial properties in an in vitro ischemia assay and improves the behavioral outcome of rabbit ischemic stroke even when administered 1?h after the insult, a therapeutic window in this model comparable to tissue plasminogen activator. In addition, we elucidated the protein kinase pathways involved in neuroprotection. CNB-001 maintains the calcium-calmodulin-dependent kinase signaling pathways associated with neurotrophic growth factors that are critical for the maintenance of neuronal function. On the basis of its in vivo efficacy and novel mode of action, we conclude that CNB-001 has a great potential for the treatment of ischemic stroke as well as other CNS pathologies.     

Quantitative neuroproteomics of an in vivo rodent model of focal cerebral ischemia/reperfusion injury reveals a temporal regulation of novel pathophysiological molecular markers

Cerebral ischemia or stroke, an acute neurological injury lacking an effective therapy, is the second leading cause of death globally. The unmet need in stroke research is to identify viable targets and to understand their interplay during the temporal evolution of ischemia/reperfusion (I/R) injury. Here we report a temporal signature of the ischemic hemisphere revealed by the isobaric tag for relative and absolute quantification (iTRAQ)-based 2D-LC-MS/MS strategy in an in vivo middle cerebral artery occlusion (MCAO) model of focal cerebral I/R injury. To recapitulate clinical stroke, two hours of MCAO was followed by 0, 4, and 24 h of reperfusion to capture ischemia with an acute and subacute durations of reperfusion injury. The subsequent iTRAQ experiment identified 2242 proteins from the ischemic hemisphere with <1.0% false discovery rate. Data mining revealed that (1) about 2.7% of detected proteins were temporally perturbed having an involvement in the energy metabolism (Pygb, Atp5b), glutamate excitotoxicity (Slc1a3, Glud1), neuro-inflammation (Tf, C3, Alb), and cerebral plasticity (Gfap, Vim, Gap43); (2) astrocytes participated actively in the neurometabolic coupling underlining the importance of a cerebro-protective rather than a neuro-protective approach; and (3) hyper-acute yet progressive opening of the blood brain barrier (BBB), accompanied by stimulation of an innate immune response and late activation of a regenerative response, which provides an extended therapeutic window for intervention. Several regulated proteins (Caskin1, Shank3, Kpnb1, Uchl1, Mtap6, Epb4.1l1, Apba1, and Ube1x) novel in the context of stroke were also discovered. In conclusion, our result supports a dynamic multitarget therapy rather than the traditional approach of a unilateral and sustained modulation of a single target to address the phasic regulation of an ischemic proteome.     

Bid-mediated mitochondrial pathway is critical to ischemic neuronal apoptosis and focal cerebral ischemia

We have investigated the role of the BH3-only pro-death Bcl-2 family protein, Bid, in ischemic neuronal death in a murine focal cerebral ischemia model. Wild-type and bid-deficient mice of inbred C57BL/6 background were subjected to 90-min ischemia induced by left middle cerebral artery occlusion followed by 72-h reperfusion. The volume of ischemic infarct was significantly smaller in the bid-deficient brains than in the wild-type brains, suggesting that Bid participated in the ischemic neuronal death. Indeed, following the ischemic treatment there was a significant reduction of apoptosis in the ischemic areas, particularly in the inner infarct border zone (the penumbra), of the bid-deficient brains. In addition, activation of Bid in the wild-type brains could be readily detected at approximately 3 h after ischemia, as evidenced by its proteolytic cleavage and translocation to the mitochondria as determined using Western blot analysis and immunofluorescence staining. Correspondingly, mitochondrial release of cytochrome c could be detected around the same time Bid was cleaved in the wild-type brains. However, no significant cytochrome c release was detected in the bid-deficient brains until 24 h later. This suggests that, although the mitochondrial apoptosis pathway might be activated by multiple mechanisms during focal cerebral ischemia, Bid is critical to its early activation. This notion was further supported by the finding that caspase-3 activation was severely impaired in the bid-deficient brains, whereas activation of caspase-8 was much less affected. Taken together, these data suggest that Bid is activated early in neuronal ischemia in a caspase-8-dependent fashion and that Bid is perhaps one of the earliest and most potent activators of the mitochondrial apoptosis pathway. Thus, the role of Bid in the induction of ischemic neuronal death may render this molecule an attractive target for future therapeutic intervention.     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.