Dynamic changes in neuronal autophagy and apoptosis in the ischemic penumbra following permanent ischemic stroke

The temporal dynamics of neuronal autophagy and apoptosis in the ischemic penumbra following stroke remains unclear.Therefore,in this study,we investigated the dynamic changes in autophagy and apoptosis in the penumbra to provide insight into potential therapeutic targets for stroke.An adult Sprague-Dawley rat model of permanent ischemic stroke was prepared by middle cerebral artery occlusion.Neuronal autophagy and apoptosis in the penumbra post-ischemia were evaluated by western blot assay and immunofluorescence staining with antibodies against LC3-Ⅱ and cleaved caspase-3,respectively.Levels of both LC3-Ⅱ and cleaved caspase-3 in the penumbra gradually increased within 5 hours post-ischemia.Thereafter,levels of both proteins declined,especially LC3-Ⅱ.The cerebral infarct volume increased slowly 1–4 hours after ischemia,but subsequently increased rapidly until 5 hours after ischemia.The severity of the neurological deficit was positively correlated with infarct volume.LC3-Ⅱ and cleaved caspase-3 levels were high in the penumbra within 5 hours after ischemia,and after that,levels of these proteins decreased at different rates.LC3-Ⅱ levels were reduced to a very low level,but cleaved caspase-3 levels remained high 72 hours after ischemia.These results indicate that there are temporal differences in the activation status of the autophagic and apoptotic pathways.This suggests that therapeutic targeting of these pathways should take into consideration their unique temporal dynamics.     

应用激光共聚焦显微镜研究脑缺血再灌注后缝隙连接重分布

目的 探讨脑缺血再灌注对脑神经细胞间缝隙连接分布的影响.方法 应用激光共聚焦显微镜技术观察脑缺血再灌注后不同缺血区缝隙连接分布变化,并探讨其可能机制及变化规律.结果 正常组Cx43分布于星形胶质细胞之间及脑毛细血管周围,分布均匀,光斑较小,在毛细血管周围排列紧密;与正常组比较,缺血2h再灌注24h半影区Cx43光斑较大,分散的小光斑相对较少;坏死区Cx43稀疏、散乱.结论 脑缺血再灌注后半暗带区缝隙连接发生重分布,形成了更大的缝隙连接斑,以加强细胞间物质及信号的传递.

Abstract：

Objective To investigate the effect of the redistribution of gap junction after reperfusion subsequent to cerebral ischemia. Methods In the test laser scanning confocal microscope (LSCM) was used to investigate the change of Cx43 distribution, and to study its possible mechanism. Results Compared to the control group, the Cx43 expression formed into bigger plagues and remained linear disposition in the penumbra in 24h of reperfusion after cerebral ischemia and changed sparse and messy in the infarction core of ischemia. Conclusion Gap junctions were redistributed in the penumbra after reperfusion subsequent to cerebral ischemia.     

Expression profiles of microRNAs after focal cerebral ischemia/reperfusion injury in rats

Rat models of focal cerebral ischemia/reperfusion injury were established by occlusion of the middle cerebral artery.Microarray analysis showed that 24 hours after cerebral ischemia,there were nine up-regulated and 27 down-regulated microRNA genes in cortical tissue.Bioinformatic analysis showed that bcl-2 was the target gene of microRNA-384-5p and microRNA-494,and caspase-3 was the target gene of microRNA-129,microRNA-320 and microRNA-326.Real-time PCR and western blot analyses showed that 24 hours after cerebral ischemia,bcl-2 mRNA and protein levels in brain tissue were significantly decreased,while caspase-3 mRNA and protein levels were significantly increased.This suggests that following cerebral ischemia,differentially expressed microRNA-384-5p,microRNA-494,microRNA-320,microRNA-129 and microRNA-326 can regulate bcl-2 and caspase-3 expression in brain tissue.     

Is longer sevoflurane preconditioning neuroprotective in permanent focal cerebral ischemia?

Sevoflurane preconditioning has neuroprotective effects in the cerebral ischemia/reperfusion model. However, its influence on permanent cerebral ischemia remains unclear. In the present study, the rats were exposed to sevoflurane for 15, 30, 60, and 120 minutes, followed by induction of perma-nent cerebral ischemia. Results demonstrated that 30-and 60-minute sevoflurane preconditioning significantly reduced the infarct volume at 24 hours after cerebral ischemia, and 60-minute se-voflurane preconditioning additionally reduced the number of TUNEL-and caspase-3-positive cells in the ischemic penumbra. However, 120-minute sevoflurane preconditioning did not show evident neuroprotective effects. Moreover, 60-minute sevoflurane preconditioning significantly attenuated neurological deficits and infarct volume in rats at 4 days after cerebral ischemia. These findings in-dicated that 60-minute sevoflurane preconditioning can induce the best neuroprotective effects in rats with permanent cerebral ischemia through the inhibition of apoptosis.     

Neuroprotective effects of SMAds in a rat model of cerebral ischemia/reperfusion

Previous studies have shown that up-regulation of transforming growth factor β1 results in neuroprotective effects. However, the role of the transforming growth factor β1 downstream molecule, SMAD2/3, following ischemia/reperfusion remains unclear. Here, we investigated the neuroprotective effects of SMAD2/3 by analyzing the relationships between SMAD2/3 expression and cell apoptosis and inflammation in the brain of a rat model of cerebral ischemia/reperfusion. Levels of SMAD2/3 m RNA were up-regulated in the ischemic penumbra 6 hours after cerebral ischemia/reperfusion, reached a peak after 72 hours and were then decreased at 7 days. Phosphorylated SMAD2/3 protein levels at the aforementioned time points were consistent with the m RNA levels. Over-expression of SMAD3 in the brains of the ischemia/reperfusion model rats via delivery of an adeno-associated virus containing the SMAD3 gene could reduce tumor necrosis factor-α and interleukin-1β m RNA levels, down-regulate expression of the pro-apoptotic gene, capase-3, and up-regulate expression of the anti-apoptotic protein, Bcl-2. The SMAD3 protein level was negatively correlated with cell apoptosis. These findings indicate that SMAD3 exhibits neuroprotective effects on the brain after ischemia/reperfusion through anti-inflammatory and anti-apoptotic pathways.     

Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injur y

Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endo-plasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of lfuid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediat-ed dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These ifndings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury.     

Involvement of glucose transporter overexpression in the protection or damage after ischemic stroke

Cerebral ischemia and resultant energy collapse:The ischemic stroke is a complex neurological condition that can be devastating for patients and their families.This disease is the second leading cause of death worldwide and is characterized by a sudden decrease in cerebral blood flow due to major blood vessel blockage.Ischemic stroke generates two damaged zones with distinctive metabolic characteristics.The first is the ischemic core,the region directly irrigated by the occluded artery.A reduction to less than 20%of the baseline blood flow levels defines the area;therefore,depletion of glucose and adenosine triphosphate(ATP)cause an irreversible failure in the energy metabolism that leads to loss of ionic homeostasis,acidosis,and necrosis.The second is the region surrounding the ischemic core,called the penumbra,characterized by significantly depressed tissue perfusion that is barely sufficient to support basal oxygen,glucose,and ATP levels.     

Inhibition of cerebral ischemia/reperfusion injury-induced apoptosis:nicotilforin and JAK2/STAT3 pathway

Nicotilforin is a lfavonoid extracted from Carthamus tinctorius. Previous studies have shown its cerebral protective effect, but the mecha-nism is undeifned. In this study, we aimed to determine whether nicotilforin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway. hTe cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion. Nicotilforin (10 mg/kg) was administered by tail vein injection. Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Bcl-2 and Bax expres-sion levels in ischemic cerebral cortex were examined by immunohistochemial staining. Additionally, p-JAK2, p-STAT3, Bcl-2, Bax, and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay. Nicotilforin altered the shape and structure of injured neurons, decreased the number of apoptotic cells, down-regulates expression of p-JAK2, p-STAT3, caspase-3, and Bax, decreased Bax immunoredactivity, and increased Bcl-2 protein expression and immunoreactivity. hTese results suggest that nicotilforin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.     

Neuroprotective effects of atorvastatin against cerebral ischemia/reperfusion injury through the inhibition of endoplasmic reticulum stress

Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize that the regulatory mechanisms are mediated by the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α in the endoplasmic reticulum stress signaling pathway. To verify this hypothesis, we occluded the middle cerebral artery in rats to establish focal cerebral ischemia/reperfusion model. Results showed that the expression levels of protein kinase-like endoplasmic reticulum kinase and caspase-3, as well as the phosphorylation of eukaryotic initiation factor 2α, were increased after ischemia/reperfusion. Administration of atorvastatin decreased the expression of protein kinase-like endoplasmic reticulum kinase, caspase-3 and phosphorylated eukaryotic initiation factor 2α, reduced the infarct volume and improved ultrastructure in the rat brain. After salubrinal, the specific inhibitor of phosphorylated eukaryotic initiation factor 2α was given into the rats intragastrically, the expression levels of caspase-3 and phosphorylated eukaryotic initiation factor 2α in the were decreased, a reduction of the infarct volume and less ultrastructural damage were observed than the untreated, ischemic brain. However, salubrinal had no impact on the expression of protein kinase-like endoplasmic reticulum kinase. Experimental findings indicate that atorvastatin inhibits endoplasmic reticulum stress and exerts neuroprotective effects. The underlying mechanisms of attenuating ischemia/reperfusion injury are associated with the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/caspase-3 pathway.     

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.     

Caspase-dependent and caspase-independent signalling of apoptosis in the penumbra following middle cerebral artery occlusion in the adult rat

Transient focal ischaemia by middle cerebral artery occlusion (MCAO) may produce cell death, but the mechanisms leading to cell death differ in the infarct core and in the penumbra, the immediate zone surrounding the infarct core. In the present study, transient focal ischaemia to adult rats was produced by intraluminal occlusion of the middle cerebral artery for 1 h followed by 0 h (n=6), 1 h (n=10), 4 h (n=8), 6 h (n=2) and 12 h (n=3) of reperfusion. The present model of ischaemia causes a large cortico-striatal infarct extending through the mediolateral cortex and dorsolateral striatum at 12 h. The expression and subcellular distribution of several proteins involved in apoptosis have been examined in the penumbra and in the infarct core by using combined methods of immunohistochemistry, cell subfractionation and Western blotting. Transient focal ischaemia by MCAO results in activation of complex signal pathways for cell death in the penumbra. Increased expression of Bcl-2 and Bax, but not of Bcl-x, occurs in the penumbra at the time when Bax translocates from the cytosol to the mitochondria, cytochrome c is released to the cytoplasm and active caspase-3 is expressed. Bax translocation, cytochrome c release and active caspase-3 are observed at 4 h, but not at 1 h, following reperfusion, and together indicate activation of the caspase-dependent pathway of apoptosis in the penumbra. In contrast, reduced Bax expression but not Bax translocation and cytochrome c release occurs in the infarct core, thus suggesting apoptosis signals restricted to the penumbra. In addition, increased expression of an apoptosis-inducing factor in the cytoplasm and nuclei of selected cells shows, for the first time, activation of the caspase-independent mitochondrial pathway in the penumbra following transient focal ischaemia and reperfusion.     

Calpain inhibitor A-558693 in experimental focal cerebral ischemia in rats

OBJECTIVES: Calpains are intracellular proteases, which are activated in various cerebral injuries. We studied the expression of mu-calpain in a model of focal cerebral ischemia/reperfusion and the efficacy of the calpain inhibitor A-558693. METHODS: A transient occlusion of the middle cerebral artery was produced in male Wistar rats by using the suture model with 3 hours of ischemia and 24 hours of reperfusion. Six animals were given the calpain inhibitor and six animals were treated with placebo. The infarct size was determined by the loss of the calpain substrate microtubule-associated protein-2 (MAP-2) immunohistochemistry using volumetry in serial slices of the brains. Furthermore mu-calpain positive-stained cells were detected by immunohistochemistry and western blotting. RESULTS: In placebo-treated animals the mu-calpain expression was significantly increased in the ischemic hemisphere compared with the contralateral non-ischemic hemisphere (88.6 versus 10.5% in the basal ganglia, 60.7 versus 10.7% in the cortex, p < 0.001, respectively) with a subsequent loss its substrate MAP-2. However, the use of the calpain inhibitor A-558693 did not significantly change the mu-calpain expression, nor significantly reduce the infarct volume. DISCUSSION: The present data indicate that mu-calpain proteolysis plays an important role in the chain of events following cerebral ischemia. However, the calpain inhibitor A-558693 failed to prevent these changes.     

D1和D2受体拮抗剂对乙灶性脑缺血梗塞体积及脑血流的影响

目的 研究多巴胺（ＤＡ）Ｄ１受体拮抗剂ＳＣＨ－２３３９０和Ｄ２受体拮抗剂Ｅｔｉｃｌｏｐｒｉｄｅ对可逆性乙灶性脑缺血梗塞体积及皮层半暗带脑血流的影响。方法 采用激光多普勒脑血流计测量大鼠可逆性乙灶性脑缺血各时相皮层半暗带脑血流,并于缺血后２４小时断头取脑切片,ＴＴＣ染色,计算机图样分析系统测量脑梗塞体积。结果 Ｄ１受体拮抗剂ＳＣＨ－２３３９０可明显缩小局灶性脑缺血梗塞体积,改善缺血期各时相皮层半暗带     

2-（2-苯并呋喃基）-2-咪唑啉对大鼠局灶性脑缺血再灌注损伤的初步观察

目的：初步观察咪唑啉I2受体的高选择性配体2-（2-苯并呋喃基）-2-咪唑啉（2-BFI）对大鼠局灶性脑缺血再灌注损伤的影响。方法：建立SD大鼠大脑中动脉局灶性脑缺血模型。在脑缺血后即通过大鼠尾静脉给予生理盐水、2-BFI或咪唑克生。通过2,3,5-三苯基氯化四唑染色检测梗死体积并评价大鼠的神经功能缺损。通过免疫组化方法检测caspase-3蛋白和原位细胞凋亡法检测缺血半暗带中凋亡神经细胞数。结果：在局灶性脑缺血24h后,2-BFI和咪唑克生都能够显著提高神经功能评分。给予2-BFI或咪唑克生都可以显著降低梗死体积、减少caspase-3阳性细胞数（P〈0．01）和凋亡细胞数（P〈0．05）。结论：2-BFI和咪唑克生对局灶性脑缺血再灌注损伤大鼠有神经保护作用,为脑卒中的治疗提供了新的治疗方法。     

淀粉前体蛋白基因的Swedish突变对光化学法所致脑缺血的影响

目的观察淀粉前体蛋白基因的Swedish突变(APP/SWE)对光化学法所致脑缺血的影响。方法在APP/SWE转基因鼠与非转基因鼠应用光化学法诱导脑梗塞形成,以激光多普勒流量计测定缺血半暗带区脑血流变化,以TTC方法测定脑梗塞体积。结果(1)在APP/SWE转基因鼠组和非转基因鼠组之间缺血半暗带区脑血流在各个时间点均无显著差异,从照射6 min开始,两组缺血半暗带区脑血流较基线显著降低。(2)缺血后2 d APP/SWE转基因鼠组总梗塞体积、皮层梗塞体积及脑水肿指数均显著小于非转基因鼠组。(3)缺血后7d两组总梗塞体积、皮层梗塞体积、海马梗塞体积及脑水肿指数均无显著差异。结论APP/SWE在光化学法所致脑缺血最初30 min对缺血程度没有影响,在缺血后2d可起到神经保护作用,减小梗塞体积,在缺血后7d神经保护作用消失。其机制可能与APP/SWE所致脑血管舒张功能障碍,限制自由基扩散有关。     

Erythropoietin-induced neurovascular protection, angiogenesis, and cerebral blood flow restoration after focal ischemia in mice.

Restoration of local blood supply in the post-ischemic brain plays a critical role in tissue repair and functional recovery. The present investigation explored beneficial effects of recombinant human erythropoietin (rhEPO) on vascular endothelial cell survival, angiogenesis, and restoration of local cerebral blood flow (LCBF) after permanent focal cerebral ischemia in adult mice. Saline or rhEPO (5,000 U/kg, intraperitoneal) was administered 30 mins before ischemia and once daily after ischemic stroke. Immunohistochemistry showed an enhancing effect of rhEPO on expression of EPO receptor (EPOR) of endothelial cells in the penumbra region 3 to 21 days after the ischemic insult. The treatment with rhEPO decreased ischemia-induced cell death and infarct volume 3 days after stroke. Specifically, rhEPO reduced the number of terminal deoxynucleotidyl transferase biotin-dUPT nick end labeling- and caspase-3-positive endothelial cells in the penumbra region. Colocalization of the vessel marker glucose transporter-1 (Glut-1) and cell proliferation marker 5-bromo-2'-deoxyuridine indicated enhanced angiogenic activity in rhEPO-treated mice 7 to 21 days after stroke. Western blot showed upregulation of the expression of angiogenic factors Tie-2, Angiopoietin-2, and vascular endothelial growth factor in rhEPO-treated animals. Local cerebral blood flow was measured by laser scanning imaging 3 to 21 days after stroke. At 14 days, LCBF in the penumbra was recovered to preischemia levels in rhEPO-treated mice but not in control mice. Our data suggest that rhEPO treatment upregulates the EPOR level in vascular endothelial cells, confers neurovascular protection, and enhances angiogenesis. We further show a promoting effect of rhEPO on LCBF recovery in the ischemic brain. These rhEPO-induced effects may contribute to therapeutic benefits in the treatment of ischemic stroke.     

Inhibition of caspase-3 activation and apoptosis is involved in 3-nitropropionic acid-induced ischemic tolerance to transient focal cerebral ischemia in rats

Our aim was to investigate the involvement of caspase-3 activation and apoptotic cell death in mitochondrial toxin 3-nitropropionic acid (3-NPA)-induced ischemic tolerance to transient focal cerebral ischemia in rats. Rats were administrated either vehicle control or 3-NPA ip doses of 20 mg/kg. Three days later, rats were exposed to 2 h of middle cerebral artery occlusion, followed by 24 h of reperfusion. Infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after reperfusion. We measured neural cell apoptosis in the cerebral ischemic penumbra by terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and flow cytometry (FCM). Cleavage of the fluorogenic substrate zDEVD-afc was used to assay caspase-3 activity. Compared with the vehicle-injected group, pretreatment with 3-NPA reduced the infarct volume by 22.3% and decreased the number of TUNEL-positive neural cells and apoptotic percentages by 47% (p < 0.05) and 43.9% (p < 0.01), respectively. In terms of caspase-3 activity in ischemic penumbral tissues, the 3-NPA-pretreated group showed 13.9% (p < 0.05) less caspase-3 activity than the control group. The development of 3-NPA-induced ischemic tolerance in brain may be related to decreases in caspase-3 activation, which leads to decreased neural cell apoptosis.