Ischemia-induced cleavage of OPA1 at S1 site aggravates mitochondrial fragmentation and reperfusion injury in neurons

Neuronal mitochondrial dynamics are disturbed after ischemic stroke. Optic atrophy 1 (OPA1) and its GTPase activity are involved in maintaining mitochondrial cristae and inner membrane fusion. This study aimed to explore the role of OMA1-mediated OPA1 cleavage (S1-OPA1) in neurons exposed to cerebral ischemia and reperfusion. After oxygen-glucose deprivation (OGD) for 60 min, we found that mitochondrial fragmentation occurred successively in the axon and soma of neurons, accompanied by an increase in S1-OPA1. In addition, S1-OPA1 overexpression significantly aggravated mitochondrial damage in neurons exposed to OGD for 60 min and 24 h after OGD/R, characterized by mitochondrial fragmentation, decreased mitochondrial membrane potential, mitochondrial cristae ultrastructural damage, increased superoxide production, decreased ATP production and increased mitochondrial apoptosis, which was inhibited by the lysine 301 to alanine mutation (K301A). Furthermore, we performed neuron-specific overexpression of S1-OPA1 in the cerebral cortex around ischemia of middle cerebral artery occlusion/reperfusion (MCAO/R) mice. The results further demonstrated in vivo that S1-OPA1 exacerbated neuronal mitochondrial ultrastructural destruction and injury induced by cerebral ischemia-reperfusion, while S1-OPA1-K301 overexpression had no effect. In conclusion, ischemia induced neuronal OMA1-mediated cleavage of OPA1 at the S1 site. S1-OPA1 aggravated neuronal mitochondrial fragmentation and damage in a GTPase-dependent manner, and participated in neuronal ischemia-reperfusion injury.Subject terms: Stroke, Cell death in the nervous system     

Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain

Transient focal cerebral ischemia leads to extensive excitotoxic neuronal damage in rat cerebral cortex. Efficient reuptake of the released glutamate is essential for preventing glutamate receptor over-stimulation and neuronal death. Present study evaluated the expression of the glial (GLT-1 and GLAST) and neuronal (EAAC1) subtypes of glutamate transporters after transient middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Between 24h to 72h of reperfusion after transient MCAO, GLT-1 and EAAC1 protein levels decreased significantly (by 36% to 56%, p < 0.05) in the ipsilateral cortex compared with the contralateral cortex or sham control. GLT-1 and EAAC1 mRNA expression also decreased in the ipsilateral cortex of ischemic rats at both 24h and 72h of reperfusion, compared with the contralateral cortex or sham control. Glutamate transporter down-regulation may disrupt the normal clearance of the synaptically-released glutamate and may contribute to the ischemic neuronal death.     

大鼠局灶性脑缺血后不同时间点磷酸化Rb蛋白与凋亡神经元的共定位研究

目的探讨大鼠局灶性脑缺血后磷酸化Rb蛋白(p-Rb,ser 795)的表达定位与神经元凋亡的时空关系。方法制备大鼠大脑中动脉梗塞(MCAO)模型,分为假手术对照组、缺血1h再灌注12h,1d,3d,7d组。利用TUNEL法检测缺血周边区细胞凋亡情况;TUNEL与p-Rb荧光双标观察神经元凋亡与p-Rb表达、定位的关系。结果缺血半暗带内大部分TUNEL阳性细胞为神经元;大鼠MCAO再灌注12h和1d,TUNEL与p-Rb分别以重叠和镶嵌的方式共定位;再灌注3d,7d发生p-Rb核浆转移的神经元与TUNEL染色细胞仍然分别维持在高水平,但是两者却没有明显的共定位关系。结论 p-Rb可能参与短暂局灶脑缺血后神经元早期凋亡过程,间接或者不参与神经元晚期凋亡过程。     

大鼠小脑缺血再灌注损伤的细胞凋亡及尼莫通的保护作用

采用四血管闭塞法制作全脑缺血再灌动物模型, 再灌后４８ 小时取小脑, 石蜡包埋切片。应用末端转移酶介导的缺口末端标记法原位检测到小脑皮质及小脑核有阳性反应的凋亡细胞, 表明细胞凋亡是迟发性神经元损伤的主要形式。缺血前３０ 分钟给以尼莫通能有效地减少细胞凋亡, 尼莫通对小脑缺血再灌注损伤有显著性保护作用     

Knockdown of glucose-6-phosphate dehydrogenase (G6PD) following cerebral ischemic reperfusion: the pros and cons

NADPH derived from glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, has been implicated not only to promote reduced glutathione (GSH) but also enhance oxidative stress in specific cellular conditions. In this study, the effects of G6PD antisense oligodeoxynucleotides (AS-ODNs) was examined on the CA1 pyramidal neurons following transient cerebral ischemia. Specifically knockdown of G6PD protein expression in hippocampus CA1 subregion at early reperfusion period (1-24 h) with a strategy to pre-treated G6PD AS-ODNs significantly reduced G6PD activity and NADPH level, an effect correlated with attenuation of NADPH oxidase activation and superoxide anion production. Concomitantly, pre-treatment of G6PD AS-ODNs markedly reduced oxidative DNA damage and the delayed neuronal cell death in rat hippocampal CA1 region induced by global cerebral ischemia. By contrast, knockdown of G6PD protein at late reperfusion period (48-96 h) increased oxidative DNA damage and exacerbated the ischemia-induced neuronal cell death in hippocampal CA1 region, an effect associated with reduced NADPH level and GSH/GSSG ratio. These findings indicate that G6PD not only plays a role in oxidative neuronal damage but also a neuroprotective role during different ischemic reperfusion period. Therefore, G6PD mediated oxidative response and redox regulation in the hippocampal CA1 act as the two sides of the same coin and may represent two potential applications of G6PD during different stage of cerebral ischemic reperfusion.     

Involvement of inhibition of nucleus GAPDH over-expression in erythropoietin's reduction of neuronal apoptosis induced by brain ischemia/reperfusion in rats

To study whether recombinant human erythropoietin (rhEPO) reduces neuronal apoptosis through inhibiting over-expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nucleus induced by brain ischemia/reperfusion in rats, 48 adult Sprague-Dawley rats were randomly divided into 3 groups: sham, saline and EPO groups. Animal models of brain ischemia/reperfusion were established by middle cerebral artery occlusion in rats. The effects of EPO on the sizes of ischemia tissue were observed by TTC staining. The over-expression of GAPDH in nucleus was detected by Hoechst-33258 and anti-GAPDH antibody double staining. The neuronal apoptosis in penumbral was detected by Nissl's staining and Hoechst-33258 immunofluorescence, respectively. The results showed that rhEPO treatment (3 000 U/kg, three times daily, i.p.) apparently reduced the sizes of infarct brain tissue in ischemia/reperfusion rats. rhEPO inhibited over-expression of GAPDH in nucleus of apoptotic neurons. In the meantime rhEPO decreased the number of apoptotic neurons in ischemia/reperfusion rats. These results suggest that rhEPO may induced reduction of neuronal apoptosis in penumbra may be through inhibiting over-expression of GAPDH in nucleus of apoptotic neurons induced by ischemia/reperfusion. Reduction of GAPDH over-expression in nucleus may play a pivotal role in EPO inhibiting neuronal apoptosis in cerebral ischemia/reperfusion rats, providing experimental evidence for EPO neuro-protecting effects against ischemia/reperfusion.     

The Involvement of Upregulation and Translocation of Phospho-Rb in Early Neuronal Apoptosis Following Focal Cerebral Ischemia in Rats

The aim of this study was to investigate the temporal and spatial relationship between phospho-Rb (ser 795) and neuronal apoptotic death in rats subjected to transient focal cerebral ischemia. We found increased phosphorylation of Rb and translocation from neuronal nucleus to cytoplasm in the penumbra zone at 12 h, 1 day, 3 days and 7 days after middle cerebral artery occlusion (MCAO)/reperfusion, compared with sham-operated controls. At 12 h and 1 day, phospho-Rb appeared to be colocalizated with TUNEL staining in neurons, but staining was not colocalizated at 3 days and 7 days. These results demonstrated that cytoplasmic translocation of phospho-Rb from nucleus of neurons occurs in potential apoptotic neurons in the early stages of ischemia/reperfusion, suggesting that the Rb pathway may only be involved in early neuronal apoptosis and may be not an apoptotic signal in the late stages of transient cerebral ischemia. Ying Yu and Xiang Luo contributed equally to this work.     

Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract: Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16–72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.     

Cdk5与大鼠脑缺血再灌注损伤时细胞凋亡的关系

目的利用大鼠可逆性大脑中动脉栓塞模型（MCAO）,研究周期素依赖性蛋白激酶5（cyclin dependent kinase5,Cdk5）对视网膜母细胞瘤蛋白（Rb）的磷酸化作用与细胞凋亡的关系。方法线栓法制作大鼠MCAO模型,随机分为再灌注0、3、6、9、24h组。通过免疫组化染色观察缺血侧大脑Cdk5和磷酸化Rb的数量和变化,TUNEL标记法检测凋亡神经元数量和变化,Western blot检测不同再灌注时间Cdk5蛋白水平的表达。结果缺血侧cdk5随再灌注时间增加而增加,24h达到高峰;再灌注6h出现磷酸化的视网膜母细胞瘤蛋白（pRb）,并随再灌注时间增加而增加,24h达到高峰。缺血侧凋亡阳性细胞变化趋势与Cdk5基本一致。缺血侧Cdk5蛋白的表达随再灌注时间增加而增加,缺血对侧无明显变化。结论大鼠局限性脑缺血再灌注损伤可以诱导Cdk5数量和激酶活性增加,通过底物磷酸化作用引起神经细胞凋亡。     

神经途径在肢体缺血预处理抗脑缺血/再灌注损伤中的作用

目的：探讨神经途径在肢体缺血预处理（limbi schemic preconditioning,LIP）抗脑缺血／再灌注损伤中的作用。方法：脑缺血采用四血管闭塞模型,重复短暂夹闭放松大鼠双侧股动脉3次作为LIP。将凝闭椎动脉的大鼠随机分为sham组、脑缺血组、股神经切断＋脑缺血组、LIP＋脑缺血组、股神经切断＋LIP＋脑缺血组。于Sham手术和脑缺血后7d处死大鼠,硫堇染色观察海马CA1区锥体神经元迟发性死亡的变化。于Sham手术和脑缺血后6h心脏灌注固定大鼠,免疫组化法测定海马CAI区c-Fos表达的变化。结果：硫堇染色结果显示,与sham组比较。脑缺血组和股神经切断＋脑缺血组大鼠海马CAI区均有明显组织损伤。LIP＋脑缺血组CAI区无明显细胞缺失,神经元密度明显高于脑缺血组（P〈0．01）。而股神经切断＋LIP＋脑缺血组大鼠海马CA1区明显损伤,锥体细胞缺失较多,与LIP＋脑缺血组组比较,神经元密度显著降低（P〈O．01）,提示LIP前切断双侧股神经取消了LIP抗脑缺血／再灌注损伤作用。c—Fos免疫组化染色结果显示,Sham组海马CAI区未见明显的c-Fos蛋白表达。脑缺血组海马CAI区偶见c—Fm的阳性表达。LIP＋脑缺血组c—Fos表达增强,数量增加,与Sham组和脑缺血组比较。c-Fos阳性细胞数和光密度均明显升高（P〈0．01）。而股神经切断＋LIP＋脑缺血组c-Fos表达明显减少,仅见少量弱阳性e-Fos表达。结论：LIP可通过神经途径发挥抗脑缺血／再灌注损伤作用,而LIP诱导c—Fos表达增加可能是LIP诱导脑缺血耐受神经途径的一个环节。     

Oxysophoridine Protects Against Focal Cerebral Ischemic Injury by Inhibiting Oxidative Stress and Apoptosis in Mice

Our previous studies have demonstrated that oxysophoridine (OSR) has protective effects on cerebral neurons damage in vitro induced by oxygen and glucose deprivation. In this study, we further investigated whether OSR could reduce ischemic cerebral injury in vivo and its possible mechanism. Male Institute of cancer research mice were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days, then subjected to brain ischemia induced by the model of middle cerebral artery occlusion. After reperfusion, neurological scores and infarct volume were estimated. Morphological examination of tissues was performed. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Oxidative stress levels were assessed by measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. The expression of various apoptotic markers as Caspase-3, Bax and Bcl-2 were investigated by immunohistochemistry and Western-blot analysis. OSR pretreatment groups significantly reduced infract volume and neurological deficit scores. OSR decreased the percentage of apoptotic neurons, relieved neuronal morphological damage. Moreover, OSR markedly decreased MDA content, and increased SOD, GSH-Px activities. Administration of OSR (250 mg/kg) significantly suppressed overexpression of Caspase-3 and Bax, and increased Bcl-2 expression. These findings indicate that OSR has a protective effect on focal cerebral ischemic injury through antioxidant and anti-apoptotic mechanisms.     

脑缺血后大鼠神经元和星形胶质细胞P21cip1表达的研究

目的研究大鼠局灶性脑缺血再灌注损伤后细胞周期蛋白依赖性激酶抑制因子P21cip1在神经元和星形胶质细胞的表达。方法建立大鼠大脑中动脉阻塞(MCAo)再灌注模型,应用流式细胞术检测各组MCAo再灌注后不同时期神经元和星形胶质细胞中的P21cip1的表达。结果缺血侧皮层区星形胶质细胞和神经元中的P21cip1的表达在再灌注3d、7d、14d后表达下调,与假手术组比较有显著性差异(P<0.05);神经元中的P21cip1的表达和星形胶质细胞中的P21cip1的表达无显著性差异(P>0.05)。结论局灶性脑缺血再灌注损伤后,缺血侧皮层区星形胶质细胞和神经元的p21cip1表达下调。     

Atorvastatin Attenuates Ischemia/Reperfusion-Induced Hippocampal Neurons Injury Via Akt-nNOS-JNK Signaling Pathway

Ischemia-induced brain damage leads to apoptosis like delayed neuronal death in selectively vulnerable regions, which could further result in irreversible damages. Previous studies have demonstrated that neurons in the CA1 area of hippocampus are particularly sensitive to ischemic damage. Atorvastatin (ATV) has been reported to attenuate cognitive deficits after stroke, but precise mechanism for neuroprotection remains unknown. Therefore, the aims of this study were to investigate the neuroprotective mechanisms of ATV against ischemic brain injury induced by cerebral ischemia reperfusion. In this study, four-vessel occlusion model was established in rats with cerebral ischemia. Rats were divided into five groups: sham group, I/R group, I/R+ATV group, I/R+ATV+LY, and I/R+SP600125 group. Cresyl violet staining was carried out to examine the neuronal death of hippocampal CA1 region. Immunoblotting was used to detect the expression of the related proteins. Results showed that ATV significantly protected hippocampal CA1 pyramidal neurons against cerebral I/R. ATV could increase the phosphorylation of protein kinase B (Akt1) and nNOS, diminished the phosphorylation of JNK3 and c-Jun, and further inhibited the activation of caspase-3. Whereas, all of the aforementioned effects of ATV were reversed by LY294002 (an inhibitor of Akt1). Furthermore, pretreatment with SP600125 (an inhibitor of JNK) diminished the phosphorylation of JNK3 and c-Jun, and further inhibited the activation of caspase-3 after cerebral I/R. Taken together, our results implied that Akt-mediated phosphorylation of nNOS is involved in the neuroprotection of ATV against ischemic brain injury via suppressing JNK3 signaling pathway that provide a new experimental foundation for stroke therapy.     

Aminoguanidine Administration Ameliorates Hippocampal Damage After Middle Cerebral Artery Occlusion in Rat

The effects of a selective inducible nitric oxide synthase inhibitor aminoguanidine (AG) on neuronal cells survival in hippocampal CA1 region after middle cerebral artery occlusion (MCAO) were examined. Transient focal cerebral ischemia was induced in rats by 60 or 90 min of MCAO, followed by 7 days of reperfusion. AG treatment (150 mg/kg i.p.) significantly reduced total infarct volumes: by 70% after 90 min MCAO and by 95% after 60 min MCAO, compared with saline-treated ischemic group. The number of degenerating neurons in hippocampal CA1 region was also markedly lower in aminoguanidine-treated ischemic groups compared to ischemic groups without AG-treatment. The number of iNOS-positive cells significantly increased in the hippocampal CA1 region of ischemic animals, whereas it was reduced in AG-treated rats. Our findings demonstrate that aminoguanidine decreases ischemic brain damage and improves neurological recovery after transient focal ischemia induced by MCAO.     

Changes in the Extracellular Concentrations of Amino Acids in the Rat Striatum During Transient Focal Cerebral Ischemia

Abstract: Although considerable evidence supports a role for amino acids in transient global cerebral ischemia and permanent focal cerebral ischemia, effects of transient focal cerebral ischemia on the extracellular concentrations of amino acids have not been reported. Accordingly, our study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, GABA, taurine, glutamine, alanine, and phosphoethanolamine in the striatum of transient focal cerebral ischemia, as evidence to support their pathogenic roles. Focal ischemia was induced using the middle cerebral artery occlusion model, with no need for craniotomy. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. One hour of middle cerebral artery occlusion followed by recirculation caused neuronal damage that was common in the frontoparietal cortex and the lateral segment of the caudate nucleus. During 1 h of ischemia, the largest increase occurred for GABA and moderate increases were observed for taurine, glutamate, and aspartate. Alanine, which is a nonneuroactive amino acid, increased little. After recirculation, the levels of glutamate and aspartate reverted to normal baseline values right after reperfusion. Despite these rapid normalizations, neuronal damage occurred. Therefore, uptake of excitatory amino acids can still be restored after 1 h of middle cerebral artery occlusion, and tissue damage occurs even though high extracellular levels of glutamate are not maintained.     

Neuroprotective Role of Nanoencapsulated Quercetin in Combating Ischemia-Reperfusion Induced Neuronal Damage in Young and Aged Rats

Cerebral stroke is the leading cause of death and permanent disability among elderly people. In both humans and animals, cerebral ischemia damages the nerve cells in vulnerable regions of the brain, viz., hippocampus, cerebral cortex, cerebellum, and hypothalamus. The present study was conducted to evaluate the therapeutic efficacy of nanoencapsulated quercetin (QC) in combating ischemia-reperfusion-induced neuronal damage in young and aged Swiss Albino rats. Cerebral ischemia was induced by occlusion of the common carotid arteries of both young and aged rats followed by reperfusion. Nanoencapsulated quercetin (2.7 mg/kg b wt) was administered to both groups of animals via oral gavage two hours prior to ischemic insults as well as post-operation till day 3. Cerebral ischemia and 30 min consecutive reperfusion caused a substantial increase in lipid peroxidation, decreased antioxidant enzyme activities and tissue osmolality in different brain regions of both groups of animals. It also decreased mitochondrial membrane microviscosity and increased reactive oxygen species (ROS) generation in different brain regions of young and aged rats. Among the brain regions studied, the hippocampus appeared to be the worst affected region showing increased upregulation of iNOS and caspase-3 activity with decreased neuronal count in the CA1 and CA3 subfields of both young and aged rats. Furthermore, three days of continuous reperfusion after ischemia caused massive damage to neuronal cells. However, it was observed that oral treatment of nanoencapsulated quercetin (2.7 mg/kg b wt) resulted in downregulation of iNOS and caspase-3 activities and improved neuronal count in the hippocampal subfields even 3 days after reperfusion. Moreover, the nanoformulation imparted a significant level of protection in the antioxidant status in different brain regions, thus contributing to a better understanding of the given pathophysiological processes causing ischemic neuronal damage.     

肢体缺血预处理减轻大鼠海马缺血/再灌注损伤

目的:探讨肢体缺血预处理(LIP)对大鼠全脑缺血/再灌注损伤的影响.方法: 36只大鼠椎动脉凝闭后随机分为假手术(Control)组、脑缺血组、肢体缺血组、LIP 0 d组(LIP后即刻行脑缺血)、LIP 1 d组(LIP后1 d行脑缺血)和LIP 2 d组(LIP后2 d行脑缺血).重复夹闭大鼠双侧股动脉3次(每次10 min,间隔10 min)作为LIP,夹闭颈总动脉进行全脑缺血8 min后再灌注.硫堇染色观察海马CA1区组织学分级及锥体神经元密度以判断海马损伤程度.结果:脑缺血组海马CA1区锥体神经元损伤严重,与Control组比较,组织学分级明显升高,神经元密度明显降低(P<0.01).LIP 0 d组海马CA1区神经元损伤较脑缺血组明显减轻,组织学分级明显降低,神经元密度明显升高(P<0.01).而LIP 1 d组和LIP 2 d组大鼠海马CA1区锥体细胞缺失较多,仍有明显的组织损伤.结论:LIP可减轻随后立即发生的脑缺血/再灌注损伤,但对间隔1 d后的脑缺血/再灌注损伤无显著对抗作用.     

Edaravone ameliorates oxidative stress associated cholinergic dysfunction and limits apoptotic response following focal cerebral ischemia in rat

Stroke is a life-threatening disease with major cause of mortality and morbidity worldwide. The neuronal damage following cerebral ischemia is a serious risk to stroke patients. Oxidative stress and apoptotic damage play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. The objective of this study was to test the hypothesis that administration of edaravone (Edv) maintains antioxidant status in brain, improves the cholinergic dysfunction and suppresses the progression of apoptosis response in rat. To test this hypothesis, male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) of 2 h followed by reperfusion for 22 h. Edv was administered (10 mg/kg bwt) intraperitoneally 30 min before the onset of ischemia and 1 h after reperfusion. After reperfusion, rats were tested for neurobehavioral activities and were sacrificed for the infarct volume, estimation of oxidative damage markers. Edv treatment significantly reduced ischemic lesion volume, improved neurological deficits, contended oxidative loads, and suppressed apoptotic damage. In conclusion, treatment with Edv ameliorated the neurological and histological outcomes with elevated endogenous anti-oxidants status as well as reduced induction of apoptotic responses in MCA occluded rat. We theorized that Edv is among the pharmacological agents that reduce free radicals and its associated cholinergic dysfunction and apoptotic damage and have been found to limit the extent of brain damage following stroke.     

大鼠局灶性脑皮质缺血/再灌注对脑神经元损伤作用及瘦素受体表达的改变

目的:研究脑缺血/再灌注(I/R)损伤后瘦素受体(OB-R)表达的变化情况.方法:雄性成年Wistar大鼠20只,随机分成4组:假手术24 h、72 h对照组及I/R 24 h、72 h实验组.线栓法制备大鼠局灶性脑皮质I/R损伤模型,在脑I/R后相应时间点分别处死大鼠,采用免疫组织化学、免疫电镜方法观察大脑皮质OB-R的表达,在光镜及电镜下观察神经元损伤改变.结果:左顶叶皮质锥体细胞、血管内皮、脉络丛发现有OB-R阳性表达;与假手术对照组相比,I/R 24 h(I/R早期)锥体细胞OB-R免疫反应阳性细胞表达减少(P＜0.05),I/R 72 h(I/R晚期)锥体细胞OB-R免疫反应阳性细胞减少更明显(P＜0.001);光镜及电镜对缺血中心区神经元的观察均显示I/R晚期的神经元损伤明显重于早期.结论:脑I/R损伤后早期神经元损害和迟发性神经元损害均伴随有OB-R的表达减少,且迟发性神经元损害表达减少更明显,因此在脑梗塞的防治中有必要对瘦素及其OB-R的作用进一步研究.     

Phosphorylation of JNK Increases in the Cortex of Rat Subjected to Diabetic Cerebral Ischemia

Previous studies have demonstrated that the c-Jun N-terminal kinase (JNK) pathway plays an important role in inducing neuronal apoptosis following cerebral ischemic injury. JNK signaling pathway in activated during cerebral ischemic injury. It participates in ischemia-induced neuronal apoptosis. However, whether JNK signaling is involved in the process of neuronal apoptosis of diabetes-induced cerebral ischemia is largely unknown. This study was undertaken to evaluate the influence of cerebral ischemia–reperfusion injury on phosphorylation of JNK in diabetic rats. Twenty-four adult streptozotocin induced diabetic and 24 adult non-diabetic rats were randomly subjected to 15 min of forebrain ischemia followed by reperfusion for 0, 1, 3, and 6 h. Sixteen sham-operated diabetic and non-diabetic rats were used as controls. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Protein expression of phospho-JNK was examined by immunohistochemistry and Western blot. The numbers of TUNEL-positive cells and phospho-JNK protein expression in the cerebral cortices after 1, 3 and 6 h reperfusion was significantly higher in diabetic rats compared to non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). Western blot analysis showed significantly higher phospho-JNK protein expression in the cerebral cortices of the diabetic rats after 1 and 3 h reperfusion than that was presented in non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). These findings suggest that increased phosphorylation of JNK may be associated with diabetes-enhanced ischemic brain damage.