Oxidative stress and neuronal death/survival signaling in cerebral ischemia

It has been demonstrated by numerous studies that apoptotic cell death pathways are implicated in ischemic cerebral injury in ischemia models in vivo. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides oxygen as a substrate for numerous enzymatic oxidation reactions and for mitochondrial oxidative phosphorylation to produce adenosine triphosphate. Oxygen radicals, the products of these biochemical and physiological reactions, are known to damage cellular lipids, proteins, and nucleic acids and to initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways could provide novel therapeutic strategies in clinical stroke.     

Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of reperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG® in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 90 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated short-term ischemia and reperfusion which also reduced the ischemic reperfusion injury as evidenced by better recovery of left ventricular performance in the preconditioned myocardium. The results of this study indicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by myocardial adaptation to ischemia.     

Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this is also true for other animals, an isolated perfused mouse heart was subjected to 30 min of ischemia followed by 2 h of reperfusion. Experiments were terminated before ischemia (baseline), after ischemia, and at 30, 60, 90 and 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The results of our study revealed that apoptotic cells appear only after 60 min of reperfusion as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). Since our previous studies showed a role of glutathione peroxidase (GSHPx) in apoptotic cell death, we performed identical experiments using isolated hearts from GSHPx-l knockout mice and transgenic mice overexpressing GSHPx-l. GSHPx-l knockout mice showed evidence of apoptotic cell death even after 30 min of reperfusion. Significant number of apoptotic cells were found in the cardiomyocytes as compared to non-transgenic control animals. To the contrary, very few apoptotic cells were found in the hearts of the transgenic mice overexpressing GSHPx-l. Hearts of GSHPx-l knockout mice were more susceptible to ischemia/reperfusion injury while transgenic mice overexpressing GSHPx- 1 were less susceptible to ischemia reperfusion injury compared to non-transgenic control animals. The results of this study clearly demonstrate a role of GSHPx in ischemia/reperfusion-induced apoptosis in mouse heart.     

Influence of Pigment Epithelium-Derived Factor on Outcome after Striatal Cerebral Ischemia in the Mouse

We here suggest that pigment epithelium-derived factor (PEDF) does not have an effect on lesion size, behavioral outcome, cell proliferation, or cell death after striatal ischemia in the mouse. PEDF is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. It influences self-renewal of neural stem cells and proliferation of microglia. We investigated whether intraventricular infusion of PEDF reduces infarct size and cell death, ameliorates behavioral outcome, and influences cell proliferation in the one-hour middle cerebral artery occlusion (MCAO) mouse model of focal cerebral ischemia. C57Bl6/N mice were implanted with PEDF or artificial cerebrospinal fluid (control) osmotic pumps and subjected to 60-minute MCAO 48 hours after pump implantation. They received daily BrdU injections for 7 days after MCAO in order to investigate cell proliferation. Infarct volumes were determined 24 hours after reperfusion using magnetic resonance imaging. We removed the pumps on day 5 and performed behavioral testing between day 7 and 21. Immunohistochemical staining was performed to determine the effect of PEDF on cell proliferation and cell death. Our model produced an ischemic injury confined solely to striatal damage. We detected no reduction in infarct sizes and cell death in PEDF- vs. CSF-infused MCAO mice. Behavioral outcome and cell proliferation did not differ between the groups. However, we cannot exclude that PEDF might work under different conditions in stroke. Further studies will elucidate the effect of PEDF treatment on cell proliferation and behavioral outcome in moderate to severe ischemic injury in the brain.     

Effects of L-carnitine and its derivatives on postischemic cardiac function,ventricular fibrillation and necrotic and apoptotic cardiomyocyte death in isolated rat hearts

The study aimed to examine whether L-carnitine and its derivatives, acetyl-L-carnitine and propionyl-L-carnitine, were equally effective and able to improve postischemic cardiac function, reduce the incidence of reperfusion-induced ventricular fibrillation, infarct size, and apoptotic cell death in ischemic/reperfused isolated rat hearts. There are several studies indicating that L-carnitine, a naturally occurring amino acid and an essential cofactor, can improve mechanical function and substrate metabolism not only in hypertrophied or failing myocardium but also in ischemic/reperfused hearts. The effects of L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine, on the recovery of heart function, incidence of reperfusion-induced ventricular fibrillation (VF), infarct size, and apoptotic cell death after 30 min ischemia followed by 120 min reperfusion were studied in isolated working rat hearts. Hearts were perfused with various concentrations of L-carnitine (0.5 and 5 mM), acetyl-L-carnitine (0.5 and 5 mM), and propionyl-L-carnitine (0.05, 0.5, and 5 mM), respectively, for 10 min before the induction of ischemia. Postischemic recovery of CF, AF, and LVDP was significantly improved in all groups perfused with 5 mM of L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine. Significant postischemic ventricular recovery was noticed in the hearts perfused with 0.5 mM of propionyl-L-carnitine, but not with the same concentration of L-carnitine or L-acetyl carnitine. The incidence of reperfusion VF was reduced from its control value of 90 to 10% (p < 0.05) in hearts perfused with 5 mM of propionyl-L-carnitine only. Other doses of various carnitines failed to reduce the incidence of VF. The protection in CF, AF, LVDP, and VF reflected in a reduction in infarct size and apoptotic cell death in hearts treated with various concentrations of carnitine derivatives. The difference between effectiveness of various carnitines on the recovery of postischemic myocardium may be explained by different membrane permeability properties of carnitine and its derivatives.     

Biochemical and Molecular Characteristics of the Brain with Developing Cerebral Infarction

1. We review the biochemical and molecular changes in brain with developing cerebral infarction, based on recent findings in experimental focal cerebral ischemia.2. Occlusion of a cerebral artery produces focal ischemia with a gradual decline of blood flow, differentiating a severely ischemic core where infarct develops rapidly and an area peripheral to the core where the blood flow reduction is moderate (called penumbra). Neuronal injury in the penumbra is essentially reversible but only for several hours. The penumbra area tolerates a longer duration of ischemia than the core and may be salvageable by pharmacological agents such as glutamate antagonists or prompt reperfusion.3. Upon reperfusion, brain cells alter their genomic properties so that protein synthesis becomes restricted to a small number of proteins such as stress proteins. Induction of the stress response is considered to be a rescue program to help to mitigate neuronal injury and to endow the cells with resistance to subsequent ischemic stress. The challenge now is to determine how the neuroprotection conferred by prior sublethal ischemia is achieved so that rational strategies can be developed to detect and manipulate gene expression in brain cells vulnerable to ischemia.4. Expansion of infarction may be caused by an apoptotic mechanism. Investigation of apoptosis may also help in designing novel molecular strategies to prevent ischemic cell death.5. Ischemia/reperfusion injury is accompanied by inflammatory reactions induced by neutrophils and monocytes/macrophages infiltrated and accumulated in ischemic areas. When the role of the inflammatory/immune systems in ischemic brain injury is revealed, new therapeutic targets and agents will emerge to complement and synergize with pharmacological intervention directed against glutamate and Ca²⁺ neurotoxicity.     

Cellular neuroprotective mechanisms in cerebral ischemia: Bcl-2 family proteins and protection of mitochondrial function

Mitochondria are central to brain cell response to ischemia, with critical roles in generation of ATP, production of free radicals, and regulation of apoptotic cell death. Changes in the permeability of the outer mitochondrial membrane to regulators of apoptosis can control ischemic cell death and this permeability is directly controlled by the Bcl-2 family of proteins. The Bcl-2 family regulate apoptosis by several mechanisms including affecting the formation of apoptotic protein-conducting pores in the outer mitochondrial membrane. The anti-apoptotic protein Bcl-2 improves neuron survival following various insults, and is protective even when administered after stroke onset in a rat model of focal ischemia. Despite intense study, the precise molecular mechanisms underlying protection by the anti-apoptotic members of the Bcl-2 family are not completely understood. This review focuses on the mechanisms by which Bcl-2 family members control the permeability of the mitochondrial membrane and influence other aspects of mitochondrial function after brain ischemia, concluding with discussion of the potential use of Bcl-2 for the treatment of cerebral ischemia.     

Perspectives on reperfusion-induced damage in rodent models of experimental focal ischemia and role of gamma-protein kinase C

Ischemic stroke represents the leading cause of death and disability among elderly people. Most stroke survivors are left with lifelong disability. With the exception of tissue-type plasminogen activator (t-PA), no effective therapy exists for the management of acute stroke. Understanding the role of various extrinsic and intrinsic pathogenic factors of ischemic damage represents a prime objective of ongoing stroke research. An important variable affecting stroke outcome is the presence or absence of reperfusion (recanalization of the occluded vessel) following an ischemic event. It appears that early reperfusion after a stroke is beneficial and capable of reversing the majority of ischemic dysfunctions. However, in some instances, late reperfusion may contrarily trigger deleterious processes and lead to more ischemic damage. Examples of ischemia/reperfusion damage using an experimental model of focal ischemia in rodents are provided, along with evidence that the brain-enriched gamma-isoform of protein kinase C may represent an important mediator of reperfusion-induced brain injury in mutant mice.     

Human-Derived Physiological Heat Shock Protein 27 Complex Protects Brain after Focal Cerebral Ischemia in Mice

Although challenging, neuroprotective therapies for ischemic stroke remain an interesting strategy for countering ischemic injury and suppressing brain tissue damage. Among potential neuroprotective molecules, heat shock protein 27 (HSP27) is a strong cell death suppressor. To assess the neuroprotective effects of HSP27 in a mouse model of transient middle cerebral artery occlusion, we purified a “physiological” HSP27 (hHSP27) from normal human lymphocytes. hHSP27 differed from recombinant HSP27 in that it formed dimeric, tetrameric, and multimeric complexes, was phosphorylated, and contained small amounts of αβ-crystallin and HSP20. Mice received intravenous injections of hHSP27 following focal cerebral ischemia. Infarct volume, neurological deficit scores, physiological parameters, and immunohistochemical analyses were evaluated 24 h after reperfusion. Intravenous injections of hHSP27 1 h after reperfusion significantly reduced infarct size and improved neurological deficits. Injected hHSP27 was localized in neurons on the ischemic side of the brain. hHSP27 suppressed neuronal cell death resulting from cytochrome c-mediated caspase activation, oxidative stress, and inflammatory responses. Recombinant HSP27 (rHSP27), which was artificially expressed and purified from Escherichia coli, and dephosphorylated hHSP27 did not have brain protective effects, suggesting that the phosphorylation of hHSP27 may be important for neuroprotection after ischemic insults. The present study suggests that hHSP27 with posttranslational modifications provided neuroprotection against ischemia/reperfusion injury and that the protection was mediated through the inhibition of apoptosis, oxidative stress, and inflammation. Intravenously injected human HSP27 should be explored for the treatment of acute ischemic strokes.     

Post-ischemic apoptotic death of rat neonatal cardiomyocytes

Despite the clinical importance of cardiomyocyte death following ischemia and reperfusion, little is known about the nature of the process. In primary rat neonatal cardiomyocyte cultures, cell death was induced by ischemia (deprivation of oxygen, serum and glucose) and reperfusion. We report here that ischemia induced primarily necrosis, whereas subsequent reperfusion induced apoptosis. Apoptosis of rat neonatal cardiomyocytes could not be prevented by protein synthesis inhibitors, suggesting that molecular components of the apoptotic pathway pre-exist in these cells. IGFs and calpain inhibitors had no effect on necrotic death during ischemia, but they significantly reduced apoptotic death during reperfusion. These results support the concept that inhibition of post-ischemic apoptotic death in the myocardium may provide a valuable new therapeutic strategy for the treatment of acute myocardial ischemia.     

大鼠局灶性脑缺血再灌注中nNOS来源的NO对细胞凋亡的影响

目的探讨大鼠局灶性脑缺血再灌注早期nNOS来源的NO对细胞凋亡的影响.方法闭塞大鼠左侧大脑中动脉造成局灶性脑缺血模型,给予选择性nNOS抑制剂-7硝基吲唑,应用原位末端标记法及流式细胞术检测缺血2h再灌注6h细胞凋亡的变化.结果 50mg/kg、25mg/kg剂量的7硝基吲唑可使1、NO含量显著降低.2、NT阳性细胞荧光强度及阳性细胞百分比显著减少.3、TUNEL阳性细胞明显减少.4、细胞凋亡百分率降低,AP峰降低.结论 nNOS来源的NO参与介导脑缺血再灌注早期的细胞凋亡.     

Chloride channel inhibition prevents ROS-dependent apoptosis induced by ischemia-reperfusion in mouse cardiomyocytes.

Apoptosis of cardiomyocytes following ischemia and Apoptosis of cardiomyocytes following ischemia and known about the mechanism by which it is induced. Recently, essential roles of a Cl- channel whose activity triggers the apoptotic volume decrease and of reactive oxygen species (ROS) in activation of this channel have been identified in mitochondrion-mediated apoptosis. Therefore, in this study, involvement of Cl- channels and ROS in apoptosis was studied in primary mouse cardiomyocyte cultures subjected to ischemia-reperfusion. Apoptotic cell death as measured by caspase-3 activation, chromatin condensation, DNA laddering, and cell viability reduction was observed tens of hours after reperfusion but never immediately after ischemia. A non-selective Cl-channel blocker (DIDS or NPPB) rescued cells from apoptotic death when applied during the reperfusion, but not ischemia, period. Another blocker relatively specific to the volume-sensitive outwardly rectifying (VSOR) Cl-channel (phloretin) was also effective in protecting ischemic cardiomyocytes from apoptosis induced by reperfusion. A profound increase in intracellular ROS was detected in cardiomyocytes during the reperfusion, but not ischemia, period. Scavengers for ROS, H2O2 and superoxide all inhibited apoptosis induced by ischemia-reperfusion. Thus, it is concluded that the mechanism by which cardiomyocyte apoptosis is induced by ischemia-reperfusion involves VSOR Cl- channel activity and intracellular ROS production.     

Placental growth factor signaling regulates isoform splicing of vascular endothelial growth factor A in the control of lung cancer cell metastasis

The omega-3 fatty acid, alpha linolenic acid (ALA) found in plant-derived foods induces significant cardiovascular benefits when ingested. ALA may be cardioprotective during ischemia; however, the mechanism(s) responsible for this effect is unknown. Isolated adult rat cardiomyocytes were exposed to medium containing ALA for 24 h and then exposed to non-ischemic (control), simulated ischemia (ISCH), or simulated ischemia/reperfusion (IR) conditions. Cardiomyocyte phospholipids were extracted and analyzed by an HPLC/electrospray ionization tandem mass spectrometry system. Pre-treatment of cells with ALA resulted in a significant incorporation of ALA within cardiomyocyte phosphatidylcholine. Cell death, DNA fragmentation and caspase-3 activity increased during ischemia and ischemia/reperfusion. Two pro-apoptotic oxidized phosphatidylcholine (OxPC) species, 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) were significantly increased during both ischemia and ischemia/reperfusion. Pre-treatment of the cells with ALA resulted in a significant reduction in cell death during ischemia and ischemia/reperfusion challenge. Apoptosis was also inhibited during ischemia and ischemia/reperfusion as shown by reduced DNA fragmentation and decreased caspase activation. ALA pre-treatment significantly decreased the production of POVPC and PGPC during ischemia and ischemia/reperfusion. ALA pre-treatment also significantly increased in resting Ca²⁺ during ischemia or ischemia/reperfusion but did not improve Ca²⁺ transients. ALA protects the cardiomyocyte from apoptotic cell death during simulated ISCH and IR by inhibiting the production of specific pro-apoptotic OxPC species. OxPCs represent a viable interventional target to protect the heart during ischemic challenge.     

Heat shock protein 27 as a neuroprotective biomarker and a suitable target for stem cell therapy and pharmacotherapy in ischemic stroke

Ischemic stroke is a major common cause of death and long‐term disability worldwide. Several pathophysiological events including excitotoxicity, oxidative/nitrative stress, inflammation, and apoptosis are involved in ischemic injuries. Recently, the molecular mechanisms involved in cerebral ischemia through a focus on a member of small heat shock proteins family, Hsp27, has been developed. Notably, following exposure to ischemia, Hsp27 expression in the brain could be increased rather than the normal condition and it may play an important role in neuroprotection after ischemic stroke. The neuroprotection effects of Hsp27 may arise from its anti‐oxidant, anti‐inflammatory, anti‐apoptotic, and chaperonic properties. Moreover, some therapeutic strategies such as stem cell therapy and pharmacotherapy have been developed with Hsp27 targeting. In this review, we describe the function and structure of Hsp27 and its possible role in neuroprotection after ischemic stroke. Finally, we present current studies in stroke therapy, which focused on Hsp27 targeting.     

亚低温联合镁剂对脑缺血再灌注大鼠保护作用的研究

目的:rt-PA溶栓为缺血性卒中最有效的治疗方法,脑血流再通后挽救濒临死亡的神经细胞同时,也可能发生更为严重而持久的脑缺血再灌注损伤。本研究探讨联合应用局部亚低温(32-35℃)及硫酸镁对局灶性脑缺血再灌注大鼠的保护作用及其可能机制。方法:通过线栓法建立大鼠大脑中动脉阻塞(MCAO)及再通模型,将50只雄性Wistar大鼠随机分为假手术组、常温组、亚低温组、硫酸镁组、亚低温+硫酸镁组,每组10例,采用Longa神经功能评分、TTC染色、干湿重法、TUNEL技术,检测和比较各组脑缺血再灌注后大鼠的神经功能、脑梗死体积、脑组织含水量及凋亡细胞数。结果:与常温组相比,亚低温组与亚低温+硫酸镁组的梗死体积、神经功能评分、脑组织含水量、凋亡细胞数均明显降低,差异有显著意义(P0.05);而与亚低温组相比,亚低温+硫酸镁组局灶脑缺血大鼠的脑梗死体积、神经功能评分、脑组织含水量、凋亡细胞数均显著减少,差异有显著意义(P0.05)。结论:与单独应用亚低温相比,局部亚低温与硫酸镁联合应用,对局灶性脑缺血再灌注大鼠可发挥更有效的脑保护作用。其机制可能与抑制脑缺血再灌注后凋亡及减轻脑水肿有关。二者联用可能为缺血性卒中患者提供一种减轻溶栓后再灌注损伤的有效脑保护方法。     

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion

Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its anti-apoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.     

Protective Effects of Spatholobi Caulis Extract on Neuronal Damage and Focal Ischemic Stroke/Reperfusion Injury

Neuronal apoptotic cell death plays an important role in many neurological disorders, including Alzheimer’s disease, Parkinson’s disease, and ischemic stroke. Spatholobi Caulis (SC) has been widely used in traditional herbal medicine for the treatment of cancer, inflammation, viral infection, and anemia. However, the protective effects of SC extract (SCE) against apoptotic cell death in the brain have not been reported. We investigated the protective effects of SCE against neuronal injury etoposide-induced neurotoxicity and in rats subjected to focal transient ischemic stroke middle cerebral artery occlusion (MCAO) for 45 min, followed by 7 days of reperfusion. The in vitro study demonstrated that SCE protected cells against etoposide-induced cell viability loss in SH-SY5Y cells. Apoptotic phenotypes, such as cleaved PARP and caspase-3, and oxidative stress in etoposide-treated cells were ameliorated by SCE treatment. In MCAO-reperfusion injury, SCE promoted neuronal survival and level of brain-derived neurotrophic factor (BDNF) by reducing glial activation, oxidative stress, and apoptosis in the ipsilateral cortex. These results indicated that SCE exerted protective effects under etoposide treatment and in a MCAO-reperfusion model by reducing JNK and p38 MAPK activation. This study presents the first evidence that SCE has therapeutic potential for the treatment of ischemic stroke or neurological disorder-related cell death.     

Characterization of neuronal cell death in normal and diabetic rats following exprimental focal cerebral ischemia.

We have studied the forms of cell death following ischemia/reperfusion, and the influence of diabetes mellitus (DM) as an additional factor. Based on the models of diabetes and middle cerebral artery occlusion (MCAO), characteristics of cell death after ischemia/reperfusion were evaluated synthetically by different methods: pathology, FCM, TUNEL and DNA agarose electrophoresis. The results showed that the occurrence of cerebral injury after ischemia/reperfusion was accompanied by cell necrosis and cell apoptosis. Cell apoptosis was mainly located in the ischemic penumbral (IP) zone around the densely ischemic focus. The ischemic core was characterized by cell necrosis. At the same time, the results showed that the process of ischemic cerebral injury worsened by DM was related to inducing cell apoptosis in IP and mid zone. In conclusion, there existed not only cell apoptosis but cell necrosis in brain damage following focal cerebral ischemia/reperfusion and showed a close, internal relationship between them. Brain damage following cerebral ischemia/reperfusion was worsened distinctly under diabetic conditions.