Early release of cytochrome C and activation of caspase-3 in hyperglycemic rats subjected to transient forebrain ischemia

The mechanisms underlying the aggravating effect of hyperglycemia on brain damage are still elusive. The present study was designed to test our hypothesis that hyperglycemia-mediated damage is caused by mitochondrial dysfunction with mitochondrial release of cytochrome c (cyt c) to the cytoplasm, which leads to activation of caspase-3, the executioner of cell death. We induced 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of recirculation in sham, normoglycemic and hyperglycemic rats. Release of cyt c was observed in the neocortex and CA3 in hyperglycemic rats after only 0.5 h of reperfusion, when no obvious neuronal damage was observed. The release of cyt c persisted after 1 and 3 h of reperfusion. Activation of caspase-3 was observed after 1 and 3 h of recovery in hyperglycemic animals. No cyt c release or caspase-3 activation was observed in sham-operated controls while a mild increase of cyt c was observed in normoglycemic ischemic animals after 1 and 3 h of reperfusion. The findings that there is caspase activation and cyt c relocation support a notion that the biochemical changes that constitute programmed cell death occur after ischemia and contribute, at least in part, to hyperglycemia-aggravated ischemic neuronal death.     

Cloning and characterization of rat caspase-9: implications for a role in mediating caspase-3 activation and hippocampal cell death after transient cerebral ischemia.

Delayed hippocampal neurodegeneration after transient global ischemia is mediated, at least in part, through the activation of terminal caspases, particularly caspase-3, and the subsequent proteolytic degradation of critical cellular proteins. Caspase-3 may be activated by the membrane receptor-initiated caspase-8-dependent extrinsic pathway and the mitochondria-initiated caspase-9-dependent intrinsic pathway; however, the precise role of these deduced apoptosis-signaling pathways in activating caspase-3 in ischemic neurons remains elusive. The authors cloned the caspase-9 gene from the rat brain and investigated its potential role in mediating ischemic neuronal death in a rat model of transient global ischemia. Caspase-9 gene expression and protease activity were extremely low in the adult brain, whereas they were developmentally upregulated in newborn rats, especially at postnatal 12 weeks, a finding consistent with the theory of an essential role for caspase-9 in neuronal apoptosis during brain development. After 15-minute transient global ischemia, caspase-9 was overexpressed and proteolytically activated in the hippocampal CA1 neurons at 8 to 72 hours of reperfusion. The temporal profile of caspase-9 activation coincided with that of cytochrome c release and caspase-3 activation, but preceded CA1 neuronal death. Immunoprecipitation experiments revealed that there was enhanced formation of Apaf-1/caspase-9 complex in the hippocampus 8 and 24 hours after ischemia. Furthermore, intracerebral ventricular infusion of the relatively specific caspase-9 inhibitor N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoro-methylketone before ischemia attenuated caspase-3-like activity and significantly enhanced neuronal survival in the CA1 sector. In contrast, inhibition of caspase-8 activity had no significant effect on caspase-3 activation or neuronal survival. These results suggest that the caspase-9-dependent intrinsic pathway may be the primary mechanism responsible for the activation of caspase-3 in ischemic hippocampal neurons.     

Hyperglycemia enhances DNA fragmentation after transient cerebral ischemia.

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.     

Severe global cerebral ischemia-induced programmed necrosis of hippocampal CA1 neurons in rat is prevented by 3-methyladenine: a widely used inhibitor of autophagy

The true programmed mechanisms of delayed neuronal death induced by global cerebral ischemia/reperfusion injury remain incompletely characterized. Autophagic cell death and programmed necrosis are 2 kinds of programmed cell death distinct from apoptosis. Here, we studied the death mechanisms of hippocampal cornu ammonis 1 neuronal death after a 20-minute severe global ischemia/reperfusion injury in young adult rats and the effects of 3-methyladenine (3-MA), a widely used inhibitor of autophagy. The morphological changes detected by electron microscopy, together with the activation of autophagy, transferase-mediated UTP nick end-labeling-positive neurons, and delayed death, demonstrated that cornu ammonis 1 neuronal death induced in this paradigm was programmed necrosis. No significant activation of caspase-3 after injury was detected by Western blot and immunohistochemistry. Treatment with 3-MA provided time-dependent protection against cornu ammonis 1 neuronal death at 7 days of reperfusion when it was administered before ischemia; administration 60 minutes after reperfusion was not beneficial. The redistribution of the lysosomal enzyme cathepsin B after injury was inhibited by 3-MA administered before ischemia, suggesting that this might be another important mechanism for the protective effect of 3-MA in ischemic neuronal injury.     

Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain.

These studies have addressed the role of caspase-3 activation in neuronal death after cerebral ischemia in different animal models. The authors were unable to show activation of procaspase-3 measured as an induction of DEVDase (Asp-Glu-Val-Asp) activity after focal or transient forebrain ischemia in rats. DEVDase activity could not be induced in the cytosolic fraction of the brain tissue obtained from these animals by exogenous cytochrome c/dATP and Ca2+. However, the addition of granzyme B to these cytosolic fractions resulted in a significant activation of DEVDase, confirming that the conditions were permissive to analyze proteolytic cleavage of the DEVD-AMC (7-amino-4-methyl-coumarin) substrate. Consistent with these findings, zVal-Ala-Asp-fluoromethylketone administered after focal ischemia did not have a neuroprotective effect. In contrast to these findings, a large increase in DEVDase activity was detected in a model of hypoxic-ischemia in postnatal-day-7 rats. Furthermore, in postnatal-day-7 animals treated with MK-801, in which it has been suggested that excessive apoptosis is induced, the authors were unable to detect activation of DEVDase activity but were able to induce it in vitro by the addition of cytochrome c/dATP and Ca2+ to the cytosolic fraction. Analysis of cytochrome c distribution did not provide definitive evidence for selective cytochrome c release in the permanent focal ischemia model, whereas in the transient model a small but consistent amount of cytochrome c was found in the cytosolic fraction. However, in both models the majority of cytochrome c remained associated with the mitochondrial fraction. In conclusion, the authors were unable to substantiate a role of mitochondrially derived cytochrome c and procaspase-3 activation in ischemia-induced cell death in adult brain, but did see a clear induction of caspase-3 in neonatal hypoxia.     

Temporal profile of cytochrome c and caspase-3 immunoreactivities and TUNEL staining after permanent middle cerebral artery occlusion in rats

Abstract

Although apoptotic pathways play important roles in ischemic neuronal injury, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochemical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNED were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reaching a peak at 24 h. Thus, the peak of induction of cytochrome c preceded that of caspase-3, and these two peaks were also precedence of the peak of DNA-fragmentation. Western blot analysis showed cytosolic expression of cytochrome c from mitochondria. This study demonstrated 7. Rapid release of cytochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apoptotic pathways in neuronal death after permanent MCA occlusion of rats. [Neurol Res 2000; 22: 223-228]     

大鼠局灶性脑缺血/再灌注损伤后Caspase-1的表达

目的研究Caspase-1在大鼠脑缺血／再灌注损伤中的作用。方法用Longa法制备大鼠大脑中动脉缺血（2h）／再灌注模型，HE染色观察梗死灶的形成，分别用TUNEL染色及免疫组化技术检测鼠脑缺血中心区及半暗带凋亡细胞与Caspase-1的表达。结果在缺血中心区Caspase-1及凋亡细胞主要见于缺血再灌注损伤早期；在缺血半暗带凋亡细胞与Caspase-1于缺血再灌注损伤早期表达不明显，于缺血再灌注24-48h则明显表达。结论细胞凋亡机制参与了缺血后迟发性神经元死亡，Caspase-1参与了其损伤过程。     

Mu-calpain mediates hippocampal neuron death in rats after lithium-pilocarpine-induced status epilepticus

Status epilepticus (SE) is a severe clinical manifestation of epilepsy which causes brain damage. The pathological process and underlying mechanisms involved in the programmed cell death (PCD) are still not fully clear. In the current study, rats were induced SE by lithium–pilocarpine administration. Our data showed hippocampal neurons death appeared at 6 h after SE and sustained for 7 days. By blotting the activation of μ-calpain and its specific cleavage of nonerythroid -spectrin (SpII) (145 kDa) was evident at 1 and 3 days after SE, which coincided with Bid activation, apoptosis inducing factor (AIF) translocation and cytochrome c release from mitochondria, whereas, activated caspase-3 and caspase-3-specific fragments of SpII (120 kDa) predominantly appeared at 5 and 7 days after SE. Moreover, MDL-28170, a calpain inhibitor, partially rescued the neuron death and attenuated the expression of activated μ-calpain, cleavage of Bid (15 kDa), AIF translocation and cytochrome c release. Taken together, our study indicated that μ-calpain mediated hippocampal neuron PCD is prior to caspase-3 activation. It functioned via translocation of Bid, AIF and cytochrome c release.     

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.     

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK‐3β) and cAMP response element‐binding protein (CREB) through phosphatidylinositol 3‐kinase (PI3‐K)‐dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four‐vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK‐3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3‐K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK‐3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3‐K, compromised the neuroprotective effect of resveratrol and decreased the level of p‐Akt, p‐GSK‐3β and p‐CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro‐survival states of Akt, GSK‐3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3‐K/Akt signaling pathway, subsequently downregulating expression of GSK‐3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.     

Overexpression of UCP2 protects thalamic neurons following global ischemia in the mouse.

Uncoupling protein 2 (UCP2) is upregulated in the brain after sublethal ischemia, and overexpression of UCP2 is neuroprotective in several models of neurodegenerative disease. We investigated if increased levels of UCP2 diminished neuronal damage after global brain ischemia by subjecting mice overexpressing UCP2 (UCP2/3tg) and wild-type littermates (wt) to a 12-min global ischemia. The histopathological outcome in the cortex, hippocampus, striatum, and thalamus was evaluated at 4 days of recovery, allowing maturation of the selective neuronal death. Global ischemia led to extensive cell death in the striatum, thalamus, and in the CA1 and CA2, and less-pronounced cell death in the CA3 and dentate gyrus (DG) hippocampal subfields. Histologic damage was significantly lower in the ventral posterolateral VPL and medial VPM thalamic nuclei in UCP2/3tg animals compared with wt. These thalamic regions showed a larger increase in UCP2 expression in UCP2/3tg compared with wt animals relative to the nonprotected DG. In the other regions studied, the histologic damage was lower or equal in UCP2/3tg animals compared with wt. Consequently, neuroprotection in the thalamus correlated with a high expression of UCP2, which is neuroprotective in a number of models of neurodegenerative diseases.     

2ME2对全脑缺血大鼠模型HIF-1α及凋亡相关基因的作用

目的 探讨2-甲氧基雌二醇(2ME2)对全脑缺血大鼠缺氧诱导因子-1α(HIF-1α)及凋亡相关基因的作用. 方法 将成年雄性SD大鼠168只按随机数字表法分为伞脑缺血组(n=84)和全脑缺血2ME2干预组(n=841),然后按再灌注时间不同又分为6h、12h、24h、48h、72h、5d和7d7个亚组.采用改良的Pu刘lsineli 4-VO法制作全脑缺血大鼠模型.应用Nissl染色进行海马神经元计数,免疫组化及RT-PCR技术进行HIF-1α、caspase-3蛋白及RTP801 mRNA表达检测. 结果 全脑缺血2ME2干预组48h至7d亚组神经元计数分别为37.09±3.52、26.93±3.10、22.22±3.091、6.98±3.07.与全脑缺血组相应时间点亚组相比明显增加,差异均有统计学意义(P＜0.05).全脑缺血2ME2干预组48h至7d亚组HIF-1α、caspase-3蛋白表达阳性细胞计数分别为11.47±1.98、20.27±2.07、3.12±0.89、1.07±0.83和12.39±1.67、20.65±2.01、15.6l±1-26、6.57±1.12,与全脑缺血组相应时间点亚组相比明显减少,差异均有统计学意义(P＜0.05).全脑缺血2ME2干预组12h至5d亚组RTP801mRNA表达吸光度比值分别为0.750±0.078、1.008±0.090、0.717±0.072、0.431±0.047、0.231±0.028,与全脑缺血组相应时间点亚组相比明显减低,差异均有统计学意义(P＜0.05). 结论 在全脑缺血急性期,2ME2抑制HIF-1α及凋亡相关基因RTP801、caspase-3的表达,具有一定的神经保护作用.     

亚低温对大鼠局灶性脑缺血后细胞凋亡及Caspase-3的影响

目的 探讨亚低温对脑缺血损伤的保护作用。方法 用原位末端标记（TUNEL）和原位杂交技术分别观察亚低温组、常温组脑缺血不同时间点神经细胞凋亡的变化及Caspase-3的表达。结果 （1）常温组脑缺血后凋亡神经细胞主要分布于缺血周围区，随着时间的延长凋亡细胞数逐渐增加，至12h达高峰，24h后开始下降，7d时仍高于假手术组；（2）亚低温组脑缺血后，凋亡神经细胞也主要位于缺血周围区，数量相对较少，其变化规律与常温组相似，同一时间点相比较，亚低温组均显著低于常温组；（3）常温组脑缺血2h后，神经细胞Caspase-3开始表达，并随着时间的延长而增强，24h达高峰，其后逐渐下降，至7d略高于假手术组；（4）亚低温组脑缺血后，神经细胞Caspase-3的表达也主要位于缺血周围区，其变化规律与常温组相似，同一时间点相比较，亚低温组均显著低于常温组。结论 脑缺血后，缺血周围区神经细胞的凋亡是一个动态的渐进过程，Caspase-3基因在介导脑缺血损伤神经元凋亡过程中起关键作用。亚低温对短暂性脑缺血后的神经元凋亡有明显的抑制作用，亚低温可能通过Caspase-3途径抵抗脑缺血损伤。     

Hyperglycemic exacerbation of neuronal damage following forebrain ischemia: microglial, astrocytic and endothelial alterations

We undertook a detailed characterization of the cellular responses to acute global cerebral ischemia complicated by hyperglycemia. Anesthetized, physiologically monitored male Wistar rats received 12.5 min of global forebrain ischemia by bilateral common carotid artery occlusions plus hemorrhagic hypotension to 45 mm Hg. Cranial temperature was maintained at normothermic levels. Hyperglycemic animals received dextrose (2.5 ml of a 25% solution, intraperitoneally) prior to ischemia; this doubled the mean plasma glucose concentration to 296 mg/100 ml. At 3 days (n = 10) or 24 h (n = 4) after ischemia, brains were perfusion-fixed and paraffin-embedded for light microscopic histopathology and for the histochemical visualization of activated microglia and the immunocytochemical visualization of glial fibrillary acid protein. Normal-neuron counts in the vulnerable hippocampal CA1 sector of hyperglycemic-ischemic (HI) rats were reduced to one-third the number observed in normoglycemic-ischemic (NI) animals. Ischemic cell counts in the striatum were increased fivefold or more in HI compared to NI rats, and normal small-neuron counts were reduced by two-thirds. The neocortex and striatum of NI rats showed only mild damage, while the majority of HI rats had extensive lesions, and several showed large cortical, striatal or thalamic infarcts. In addition, widespread cortical ischemic neuronal changes were evident in HI animals. No endothelial alterations were present in NI rats. By contrast, HI rats showed prominent peri- and intravascular polymorphonuclear and monocytic accumulation evident at 24 h; frequent white cell thrombi in pial arterioles on day 3; and thickening of vascular endothelium, with foci of parenchymal rarefaction or microinfarction adjacent to occluded vessels. Prominent microglial activation, often along the course of penetrating blood vessels, was common in the striatum and neocortex of HI animals but was much less extensive in the NI group. Activated microglia in HI rats were typically hypertrophic and amoeboid. These results suggest that the detrimental influence of hyperglycemia in ischemia is initially mediated by an action on vascular endothelium, which in turn leads to widespread foci of infarction and neuronal loss. Received: 18 March 1998 / Revised, accepted: 2 June 1998     

Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra

Focal ischemia by middle cerebral artery occlusion (MCAO) results in necrosis at the infarct core and activation of complex signal pathways for cell death and cell survival in the penumbra. Recent studies have shown activation of the extrinsic and intrinsic pathways of caspase-mediated cell death, as well as activation of the caspase-independent signaling pathway of apoptosis in several paradigms of focal cerebral ischemia by transient MCAO to adult rats and mice. The extrinsic pathway (cell-death receptor pathway) is initiated by activation of the Fas receptor after binding to the Fas ligand (Fas-L); increased Fas and Fas-L expression has been shown following focal ischemia. Moreover, focal ischemia is greatly reduced in mice expressing mutated (nonfunctional) Fas. Increased expression of caspase-1, -3, -8, and -9, and of cleaved caspase-8, has been observed in the penumbra. Activation of the intrinsic (mitochondrial) pathway following focal ischemia is triggered by Bax translocation to and competition with Bcl-2 and other members of the Bcl-2 family in the mitochondria membrane that is followed by cytochrome c release to the cytosol. Bcl-2 over-expression reduces infarct size. Cytochrome c binds to Apaf-1 and dATP and recruits and cleaves pro-caspase-9 in the apoptosome. Both caspase-8 and caspase-9 activate caspase-3, among other caspases, which in turn cleave several crucial substrates, including the DNA-repairing enzyme poly(ADP-ribose) polymerase (PARP), into fragments of 89 and 28 kDa. Inhibition of caspase-3 reduces the infarct size, further supporting caspase-3 activation following transient MCAO. In addition, caspase-8 cleaves Bid, the truncated form of which has the capacity to translocate to the mitochondria and induce cytochrome c release. The volume of brain infarct is greatly reduced in Bid-deficient mice, thus indicating activation of the mitochondrial pathway by cell-death receptors following focal ischemia. Recent studies have shown the mitochondrial release of other factors; Smac/DIABLO (Smac: second mitochondrial activator of caspases: DIABLO: direct IAP binding protein with low pI) binds to and neutralizes the effects of the X-linked inhibitor of apoptosis (XIAP). Finally, apoptosis-inducing factor (AIF) translocates to the mitochondria and the nucleus following focal ischemia and produces peripheral chromatin condensation and large-scale DNA strands, thus leading to the caspase-independent cell death pathway of apoptosis. Delineation of the pro-apoptotic and pro-survival signals in the penumbra may not only increase understanding of the process but also help to rationalize strategies geared to reducing brain damage targeted at the periphery of the infarct core.     

蛋白激酶抑制剂H-7降低局灶性脑缺血半暗带和核心区半胱氨酸蛋白酶的活化

目的 研究蛋白激酶抑制剂H-7对局灶性脑缺血半暗带和核心区半胱氨酸蛋白酶Calpain和Caspase-3活性的影响.方法 采用动脉腔内插线法建立大鼠局灶性脑缺血模型,在缺血前15min经脑室给予H-7(125μg/大鼠),测定缺血1h再灌注23h(R23h)时,半暗带和核心区Calpain和Caspase-3的活性、Calpastatin和微管相关蛋白-2(MAP-2)的含量及梗死体积.结果 H-7明显降低R23h时半暗带和核心区μ-和m-Calpain及Caspase-3的活性,升高核心区Calpastatin的含量及半暗带和核心区MAP-2的含量,缩小梗死体积.结论 H-7通过抑制半暗带和核心区Calpain和Caspase-3的活性,降低局灶性脑缺血损伤.

Abstract：

Objective To investigate the effects of protein kinase inhibitor H-7 on the activation of calpain and caspase-3 in penumbra and core after focal cerebral ischemia in rats. Methods Rats received 1h focal cerebral ischemia by intraluminal filament. H-7 ( 125 μg/rat) was administered intracerebroventricularly 15 min before ischemia. The activities of calpain and caspase-3, the levels of calpastatin and microtubule-associated protein-2 ( MAP-2 ) , and the infarct volume were assessed by casein zymography,fluorometry, Western blot analysis,and staining the brain sections with 2,3 ,5-tripheny-ltetrazolium chlorides,respectively. Results Compared with ischemic control, H-7 markedly reduced the activities of μ-and m-calpain, and caspase-3 , increased the levels of MAP-2 in penumbra and core, and enhanced the levels of calpastatin in core. Moreover, animals treated with H-7 showed a significant reduction in infarct volume. Conclusions These data demonstrate the protection of H-7 against focal cerebral ischemia through inhibiting the activation of calpain and caspase-3.     

胰岛素对大鼠脑缺血再灌注后Caspase-3表达及细胞凋亡的影响

目的探讨胰岛素对大鼠脑缺血再灌注后Caspase-3表达及细胞凋亡的影响。方法将动物随机分为假手术组、缺血组及干预组,参照zea longa线栓法建立大鼠左侧大脑中动脉闭塞（mid-dle cerebral artery occlusion,MCAO）再灌注模型,干预组大鼠在脑缺血即刻给予胰岛素及葡萄糖腹腔注射,分别在左侧MCAO2h再灌注不同时间点断头取脑,脑皮质神经元Caspase-3的表达通过免疫组化法来测定,并采用TUNEL法原位标记DNA片段,检测TUNEL阳性细胞的变化。结果缺血组大鼠脑皮质Caspase-3的表达较假手术组显著增强（P〈0.01）,TUNEL阳性细胞数较假手术组显著增多（P〈0.01）;给予胰岛素处理后,Caspase-3的表达较缺血组明显减弱（P〈0.01）,TUNEL阳性细胞数较缺血组明显减少（P〈0.01）,但两者均显著高于假手术组（P〈0.01）。结论短暂的脑缺血再灌注可导致脑皮质神经元Caspase-3的表达增加,促进细胞凋亡,胰岛素可下调脑皮质神经元Caspase-3的表达,发挥神经保护作用。     

Kainic acid dose affects delayed cell death mechanism after status epilepticus

Kainic acid (KA)-induced status epilepticus (SE) produces hippocampal neuronal death, which varies from necrosis to apoptosis or programmed cell death (PCD). We examined whether the type of neuronal death was dependent on KA dose. Adult rats were induced SE by intraperitoneal injection of KA at 9 mg/kg (K9) or 12 mg/kg (K12). Hippocampal neuronal death was assessed by TUNEL staining, electron microscopy, and Western blotting of caspase-3 on days 1, 3 and 7 after SE induction. K12 rats showed higher a mortality rate and shorter latency to the onset of SE when compared with K9 rats. In both groups, acidophilic and pyknotic neurons were evident in CA1 at 24h after SE and neuronal loss developed from day 3. The degenerated neurons became TUNEL-positive on days 3 and 7 in K9 rats but not in K12 rats. Caspase-3 activation was detected on days 3 and 7 in K9 rats but was undetectable in K12 rats. Ultrastructural study revealed shrunken neurons exhibiting pyknotic nuclei containing small and dispersed chromatin clumps 24h after SE in CA1. No cells exhibited apoptosis. On days 3 and 7, the degenerated neurons were necrotic with high electron density and small chromatin clumps. There were no ultrastructural differences between the K9 and K12 groups. These results revealed that differences in KA dose affected the delayed cell death (3 and 7 days after SE); however, no effect was seen on the early cell death (24h after SE). Moderate-dose KA induced necrosis, while low-dose KA induced PCD.     

Temporal profile of cytochrome c and caspase-3 immunoreactivities and TUNEL staining after permanent middle cerebral artery occlusion in rats

Although apoptotic pathways play important roles in ischemic neuronal injury, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochemical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL) were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reaching a peak at 24 h. Thus, the peak of induction of cytochrome c preceded that of caspase-3, and these two peaks were also precedence of the peak of DNA-fragmentation. Western blot analysis showed cytosolic expression of cytochrome c from mitochondria. This study demonstrated 1. Rapid release of cytochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apoptotic pathways in neuronal death after permanent MCA occlusion of rats.