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.     

黑木耳多糖对急性脑缺血/再灌注损伤影响的研究

目的:观察黑木耳多糖(APP)对急性脑缺血大鼠的保护作用并探讨其相关机制。方法:成年雄性SD大鼠给予不同浓度的AAP灌胃20d,每天1次,腹腔注射银杏叶提取物(ginkgo biloba extract,EGb671)作为阳性对照,20d后实施右侧大脑中动脉栓塞(MCAO)建立局灶性脑缺血模型。MCAO60min后复灌,复灌24h后进行Longa神经功能损伤评分,并用2,3,5-氯化三苯基四氮唑(TTC)染色法测定脑梗死面积。复灌48h后用TUNEL免疫组化检测神经元凋亡,测定脑组织线粒体内活性氧簇(ROS)的生成量判断氧化应激水平。结果:黑木耳多糖能降低神经功能损伤评分,减小脑梗死面积,减少神经元凋亡,并且能够使缺血复灌脑组织线粒体ROS生成显著减少。高剂量AAP组的凋亡神经元数量、ROS生成量和阳性对照组相比有显著性差异。结论:黑木耳多糖能够对抗大鼠的局灶性脑缺血损伤,其保护作用和减轻氧化应激水平有关,并优于银杏叶提取物。     

Ghrelin reduces injury of hippocampal neurons in a rat model of cerebral ischemia/reperfusion

Ghrelin, an acyl-peptide gastric hormone and an endogenous ligand for growth hormone secretagogue (GHS) receptor 1a (GHS-R 1a) exerts multiple functions. It has been reported that synthetic GHS-hexarelin reduces injury of cerebral cortex and hippocampus after brain hypoxia-ischemia in neonatal rats. However, the effect of ghrelin in tolerance of the brain tissues to cerebral ischemia/reperfusion (I/R) injury has not been studied. The aim of the present study was to examine whether ghrelin have potential protective effect on hippocampal neurons of rats against I/R injury. I/R injury was induced by a modified four-vessel occlusion model. Ghrelin was administered intraperitoneally after the insult. Histological damage of the neurons was determined with hematoxylin-eosin (H&E) staining and assay of the neuronal apoptosis was performed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). The results showed that I/R decreased the number of surviving neurons and induced apoptosis of the neurons in CA1 area of the hippocampus in rats. In contrast, administration of ghrelin significantly increased the number of surviving neurons and reduced the number of TUNEL-positive apoptotic neurons in the equivalent areas after I/R. In conclusion, the present data provide evidence for the first time that ghrelin can exert a neuroprotective role in vivo in the tolerance of hippocampal neurons to I/R injury, and that the mechanism underlying this effect involves an anti-apoptotic property of ghrelin.     

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。     

Intrinsic and extrinsic erythropoietin enhances neuroprotection against ischemia and reperfusion injury in vitro

This study was designed to investigate the neuroprotective effect of intrinsic and extrinsic erythropoietin (EPO) against hypoxia/ischemia, and determine the optimal time-window with respect to the EPO-induced neuroprotection. Experiments were conducted using primary mixed neuronal/astrocytic cultures and neuron-rich cultures. Hypoxia (2%) induces hypoxia-inducible factor-1alpha (HIF-1alpha) activity followed by strong EPO expression in mixed cultures and weak expression in neuron-rich cultures as documented by both western blot and RT-PCR. Immunoreactive EPO was strongly detected in astrocytes, whereas EPOR was only detected in neurons. Neurons were significantly damaged in neuron-rich cultures but were distinctly rescued in mixed cultures. Application of recombinant human EPO (rhEPO) (0.1 U/mL) within 6 h before or after hypoxia significantly increased neuronal survival compared with no rhEPO treatment. Application of rhEPO after onset of reoxygenation achieved the maximal neuronal protection against ischemia/reperfusion injury (6 h hypoxia followed 24 h reoxygenation). Our results indicate that HIF-1alpha induces EPO gene released by astrocytes and acts as an essential mediator of neuroprotection, prove the protective role of intrinsic astrocytic-neuronal signaling pathway in hypoxic/ischemic injury and demonstrate an optimal therapeutic time-window of extrinsic rhEPO in ischemia/reperfusion injury in vitro. The results point to the potential beneficial effects of HIF-1alpha and EPO for the possible treatment of stroke.     

Stem Cell Treatment After Cerebral Ischemia Regulates the Gene Expression of Apoptotic Molecules

Evidence suggests that apoptosis contributes significantly to cell death after cerebral ischemia. Our recent studies that utilized human umbilical cord blood-derived mesenchymal stem cells (hUCBSCs) demonstrated the potential of hUCBSCs to inhibit neuronal apoptosis in a rat model of CNS injury. Therefore, we hypothesize that intravenous administration of hUCBSCs after focal cerebral ischemia would reduce brain damage by inhibiting apoptosis and downregulating the upregulated apoptotic pathway molecules. Male Sprague–Dawley rats were obtained and randomly assigned to various groups. After the animals reached a desired weight, they were subjected to a 2 h middle cerebral artery occlusion (MCAO) procedure followed by 7 days of reperfusion. The hUCBSCs were obtained, cultured, and intravenously injected (0.25 × 10⁶ cells or 1 × 10⁶ cells) via the tail vein to separate groups of animals 24 h post-MCAO procedure. We performed various techniques including PCR microarray, hematoxylin and eosin, and TUNEL staining in addition to immunoblot and immunofluorescence analysis in order to investigate the effect of our treatment on regulation of apoptosis after focal cerebral ischemia. Most of the apoptotic pathway molecules which were upregulated after focal cerebral ischemia were downregulated after hUCBSCs treatment. Further, the staining techniques revealed a prominent reduction in brain damage and the extent of apoptosis at even the lowest dose of hUCBSCs tested in the present study. In conclusion, our treatment with hUCBSCs after cerebral ischemia in the rodent reduces brain damage by inhibiting apoptosis and downregulating the apoptotic pathway molecules.     

Protection by 3'-methoxypuerarin of rat hippocampal neurons against ischemia/reperfusion injury

3'-Methoxypuerarin (3'-MOP) is an isoflavone extracted from radix puerariae. The aim of this study was to investigate the role and the mechanism of 3'-MOP in the protection of hippocampal neurons against cerebral ischemia/reperfusion (I/R) injury in rats. I/R injury was induced by a modified four-vessel occlusion model. Rats were randomly divided into an I/R group, an I/R + 3'-MOP group and a control group. Histological changes in the neurons of the hippocampal CA1 region were observed with hematoxylin and eosine (H&E) staining. The apoptotic neurons in the hippocampal CA1 area were counted with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The results showed that compared with the I/R group, 3'-MOP increased the number of surviving neurons in the hippocampal CA1 region (P < 0.001) and markedly reduced the number of apoptotic pyramidal neurons (P < 0.001) after I/R injury. In conclusion, 3'-MOP can protect hippocampal neurons against I/R injury by inhibiting apoptosis.     

The relationship between erythropoietin pretreatment with blood-brain barrier and lipid peroxidation after ischemia/reperfusion in rats

Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including ischemia and some neurodegenerative disorders. In recent years, erythropoietin (EPO) has been shown to exert neuroprotection in many pathological conditions including ischemia in the brain. This study aimed to investigate the effects of EPO on BBB integrity, infarct size and lipid peroxidation following global brain ischemia/reperfusion in rats. Wistar male rats were divided into four groups (each group n=8); Group I; control group (sham-operated), Group II; ischemia/reperfusion group, Group III; EPO treated group (24 h before decapitation--000 U/kg r-Hu EPO i.p.), Group IV; EPO+ ischemia/reperfusion group (24 h before ischemia/reperfusion--3000 U/kg r-Hu EPO i.p.). Global brain ischemia was produced by the combination of bilateral common carotid arteries occlusion and hemorrhagic hypotension. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage was observed for BBB integrity. Infarct size was calculated based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Lipid peroxidation in the brain tissue was determined as the concentration of thiobarbituric acid-reactive substances (TBARS) for each group. Ischemic insult caused bilateral and regional BBB breakdown (hippocampus, cortex, corpus striatum, midbrain, brain stem and thalamus). EPO pretreatment reduced BBB disruption, infarct size and lipid peroxide levels in brain tissue with 20 min ischemia and 20 min reperfusion. These results suggest that EPO plays an important role in protecting against brain ischemia/reperfusion through inhibiting lipid peroxidation and decreasing BBB disruption.     

肢体缺血预处理减少大鼠全脑缺血再灌注诱导的海马CA1区锥体神经元凋亡

探探讨肢体缺血预处理(limb ischemic preconditioning,LIP)对大鼠全脑缺血再灌注后海马CA1区锥体细胞凋亡的影响。46只大鼠椎动脉凝闭后分为假手术组、肢体缺血组、脑缺血组、LIP组。重复夹闭大鼠双侧股动脉3次(每次10min,间隔10min)作为LIP,之后立即夹闭双侧颈总动脉进行全脑缺血8min后再灌注。DNA凝胶电泳、TUNEL和吖啶橙/溴乙锭(AO/EB)双染技术从生化和形态学方面观察海马神经元凋亡的情况。凝胶电泳显示,脑缺血组出现了凋亡特征性DNA梯状条带,而LIP组无上述条带出现。与脑缺血组比较,LIP可明显减少海马CAI区TUNEL阳性神经元数(17.8±5.8vs 69.8±12,P<0.01)。AO/EB染色也显示LIP可明显减少脑缺血再灌注引起的神经元凋亡。以上结果提示,LIP可抑制脑缺血再灌注后海马神经元的凋亡,进而减轻脑缺血再灌注损伤,提供脑保护作用。     

20-羟基蜕皮甾酮对大鼠全脑缺血再灌注后海马神经元损伤和炎症反应的影响

目的:观察20-羟基蜕皮甾酮对全脑缺血再灌注后SD大鼠海马神经元和认知功能的保护作用,并探讨其相关机制。方法:采用四血管闭塞法建立SD大鼠全脑缺血再灌注模型,脑电图和脑组织Nissl染色评估模型的可靠性。将实验动物分为假手术组,缺血再灌注组和缺血再灌注+20-羟基蜕皮甾酮组。TUNEL染色观察海马神经元凋亡,Morris水迷宫实验评价大鼠的认知功能,酶联免疫法测定缺血再灌注后3-24小时大鼠血清中白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α)的浓度。结果:全脑缺血再灌注后大鼠海马神经元凋亡率从4.50±1.90%上升至72.90±8.40%(p0.01),给予20和40 mg/kg 20-羟基蜕皮甾酮干预,大鼠海马神经元凋亡率分别下降至51.40±8.60%(p0.05)和42.70±6.80%(p0.01)。与假手术组相比,全脑缺血再灌注后大鼠在Morris水迷宫定位航行试验中逃避潜伏期明显延长(p0.01),在空间探索试验中目标象限停留时间和穿越目标象限次数明显减少(p0.01),而20-羟基蜕皮甾酮显著抑制上述变化,改善大鼠的认知功能。缺血再灌注后3-24小时,大鼠血清中IL-1β和TNFα浓度较假手术组显著升高,20-羟基蜕皮甾酮能抑制上述各时间点大鼠血清中IL-1β和TNFα浓度的升高。结论:20-羟基蜕皮甾酮对全脑缺血再灌注后大鼠海马神经元和认知功能有显著保护作用,抑制缺血再灌注后的炎症反应是其保护机制之一。     

大鼠脑缺血再灌注后神经元和星形胶质细胞凋亡的比较研究

目的比较研究大鼠局灶性脑缺血再灌注后神经元和星形胶质细胞的凋亡规律。方法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,在缺血再灌注后1、3、7、14d断头取脑,应用流式细胞分选技术和原位末端标记法分别检测各组MCAO后不同时期神经元和星形胶质细胞凋亡情况。结果局灶性脑缺血再灌注后,海马区星形胶质细胞凋亡数量超过神经元,其凋亡以再灌注3d最为显著,而神经元则以7d最为显著;而皮层区神经元凋亡数量超过星形胶质细胞,两种细胞凋亡均在再灌注后7d达高峰。结论脑缺血再灌注后,皮层和海马区的神经元及星形胶质细胞均可发生凋亡,海马区星形胶质细胞比皮层区更易凋亡,而皮层区神经元比海马区更易凋亡。     

大蒜素对全脑缺血再灌注大鼠脑保护机制的研究

目的：通过大蒜素预处理,观察全脑缺血再灌注大鼠海马区ICAM-1 的表达,从而探讨大蒜素的脑保护机制。方法：雄性 Wistar 大鼠30 只,随机分为5 组：假手术组、缺血再灌注组、缺血再灌注+ 大蒜素10、20、30 mg/kg 组。采用四血管闭塞法制备大 鼠全脑缺血再灌注模型,于再灌注24 h 取出海马,硫堇染色观察海马组织的形态学改变,免疫组织化学染色测定海马CA1 区 ICAM-1 免疫反应阳性细胞面积和积分光密度值。结果：通过给予大鼠全脑缺血8 min 再灌注24 h处理,海马CA1 区组织形态学 改变显著,神经元密度明显降低;ICAM-1的表达显著增加。静脉给予大蒜素可使缺血再灌注海马组织形态学改变明显改善,存活 神经元数目增加,ICAM-1 表达显著较少。结论：大蒜素可以通过减少ICAM-1 的表达抑制全脑缺血再灌注后的炎症损失从而发 挥脑保护作用。     

K252a suppresses neuronal cells apoptosis through inhibiting the translocation of Bax to mitochondria induced by the MLK3/JNK signaling after transient global brain ischemia in rat hippocampal CA1 subregion

It is demonstrated that the c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Our previous studies have suggested that K252a can obviously inhibit JNK activation induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. Here, we further discussed the potential mechanism of ischemic brain injury induced by the activation of JNK after 15?min of transient global cerebral ischemia. As a result, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and 14-3-3 protein (a cytoplasmic anchor of Bax) induced by the activation of JNK, K252a decreased the release of Bax from Bcl-2/Bax and 14-3-3/Bax dimers, further attenuating the translocation of Bax from cytosol to mitochondria and the release of cytochrome c induced by ischemia/reperfusion, which related to mitochondria-mediated apoptosis. Importantly, pre-infusion of K2525a 20?min before ischemia showed neuroprotective effect against neuronal cells apoptosis. These findings imply that K252a induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 subregion via inhibiting the mitochondrial apoptosis pathway induced by JNK activation.     

Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats

Recent evidence indicates that cell-cycle regulating proteins are involved in apoptotic process in post-mitotic neurons. In this study, we examined cell-cycle regulators for G1/S cell-cycle progression after a transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion. In the cerebral frontoparietal cortex, we observed a marked induction of Cyclin D1 (a coactivator of Cdks), and proliferating cell nuclear antigen (PCNA), together with upregulated Cdk kinase activities. This process is accompanied with multiple phosphorylation of retinoblastoma (Rb) protein at Cdk phosphorylation sites in neurons from the ischemic cortex. We further examined DNA synthesis by the incorporation of BrdU, a nucleotide analog that incorporates into newly synthesized DNA. Within 24-h of reperfusion after 60-min occlusion, substantial BrdU-positive neurons were observed in the ischemic cortex. Inhibition of Cdk4 activity during this ischemia/reperfusion is highly neuroprotective. These results suggest that ischemia/reperfusion cerebral damage induces signalings at the G1/S cell-cycle transition, and may constitute a critical step in the neuronal apoptotic pathway in ischemia/reperfusion induced neuronal damage.     

大豆异黄酮基于RhoA/ROCK2信号通路改善MCAO大鼠神经功能损伤

目的:探讨大豆异黄酮对脑缺血再灌注大鼠RhoA/ROCK2信号通路介导的氧化应激反应和神经元凋亡的影响。方法:60只SD大鼠随机分为3组,对照组、模型组、大豆异黄酮组。连续给药7天后,给药剂量200 mg/kg。应用中动脉栓塞再灌注模型致大鼠缺血损伤。24 h后评价大鼠神经功能,TTC染色检测脑梗死体积,试剂盒检测脑中氧化因子含量,免疫组化检测神经元损伤,Western Blotting检测RhoA/ROCK2相关蛋白含量。结果:与对照组比较,模型组大鼠神经功能评分降低(P0.05),脑梗死体积增加(P0.05),氧化因子含量增加(P0.05),神经元凋亡显著(P0.05),RhoA/ROCK2蛋白表达增加(P0.05)。与模型组相比,大豆异黄酮升高了大鼠神经功能评分(P0.05),减少的脑梗死体积(P0.05),降低脑中氧化因子含量(P0.05),抑制了神经元凋亡(P0.05),抑制了RhoA/ROCK2蛋白表达(P0.05)。结论:大豆异黄酮可以缓解脑缺血再灌注损伤介导的氧化应激及细胞凋亡,进而减轻神经功能障碍,其机制可能与抑制RhoA/ROCK2信号通路相关。     

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     

Glycine attenuates cerebral ischemia/reperfusion injury by inhibiting neuronal apoptosis in mice

Glycine is a cytoprotector to protect cells against ischemic damage by counteracting neuronal depolarization. However, whether it can directly inhibit neuronal apoptosis is unknown. In this study, we demonstrated that glycine could attenuate ischemia/reperfusion (I/R) induced cerebral infarction and improved neurological outcomes in mice. The protective effect of glycine was associated with reduction of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) positive cells, deactivation of phosphor-JNK, inhibition of caspase-3 cleavage, down-regulation of FasL/Fas, and up-regulation of bcl-2 and bcl-2/bax in the mouse I/R penumbra. The beneficial effect of glycine against oxygen and glucose deprivation (OGD) induced injury was also confirmed in SH-SY5Y cells as well as in primary cultured neurons, which was significantly dampened by knockdown of glycine receptor α1 (GlyR α1) with siRNA transfection or by preventing glycine binding with glycine receptor using a specific antibody against glycine receptor. These results suggest that glycine antagonize cerebral I/R induced injury by inhibiting apoptosis in mice. Glycine could block both extrinsic and intrinsic apoptotic pathways for which GlyR may be required.     

Butylphthalide Suppresses Neuronal Cells Apoptosis and Inhibits JNK–Caspase3 Signaling Pathway After Brain Ischemia /Reperfusion in Rats

Although Butylphthalide (BP) has protective effects that reduce ischemia-induced brain damage and neuronal cell death, little is known about the precise mechanisms occurring during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to investigate the neuroprotective mechanisms of BP against ischemic brain injury induced by cerebral I/R through inhibition of the c-Jun N-terminal kinase (JNK)–Caspase3 signaling pathway. BP in distilled non-genetically modified Soybean oil was administered intragastrically three times a day at a dosage of 15 mg/(kg day) beginning at 20 min after I/R in Sprague–Dawley rats. Immunohistochemical staining and Western blotting were performed to examine the expression of related proteins, and TUNEL-staining was used to detect the percentage of neuronal apoptosis in the hippocampal CA1 region. The results showed that BP could significantly protect neurons against cerebral I/R-induced damage. Furthermore, the expression of p-JNK, p-Bcl2, p–c-Jun, FasL, and cleaved-caspase3 was also decreased in the rats treated with BP. In summary, our results imply that BP could remarkably improve the survival of CA1 pyramidal neurons in I/R-induced brain injury and inhibit the JNK–Caspase3 signaling pathway.     

脑缺血和缺血再灌注大鼠皮层神经元自噬的动态变化

目的：探讨脑缺血和缺血/再灌注不同时间大鼠大脑皮层神经元自噬的变化。方法：健康雄性SD大鼠60只,随机分为：假手术（Sham）组（n=10）,脑缺血和缺血/再灌注模型组（n=50）.模型组分别在缺血30min、2h,缺血2h再灌注1h、6h、24h五个时间点,随机抽取10只大鼠,测定脑梗死体积和脑含水量,同时采用Western印迹法测定各组大鼠大脑皮层中微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）的水平,透射电镜检测大脑皮层神经细胞自噬情况。结果：脑缺血30min时LC3-Ⅱ/Ⅰ比值未见明显上升,缺血2h时LC3-Ⅱ/Ⅰ比值开始升高,明显高于Sham组（P<0.01）;缺血/再灌注1h、6h时LC3-Ⅱ/Ⅰ比值虽较缺血2h组有所下降,但仍明显高于Sham组（P<0.05）;缺血/再灌注24h时LC3Ⅱ/Ⅰ比值达高峰,明显高于Sham组（P<0.01）。透射电镜观察进一步证实该现象。缺血/再灌注6h和24h时大鼠脑梗死体积明显增加,与Sham组比较有统计学差异（P<0.01）。缺血/再灌注24h大鼠脑组织含水量明显增加,明显高于Sham组（P<0.05）。HE染色显示：仅在缺血/再灌注24h组大鼠皮层见组织水肿、疏松,部分细胞变性、凋亡,海马区见大量神经元细胞核皱缩、深染呈变性凋亡状。结论：局灶性脑缺血和缺血/再灌注模型中大脑皮层缺血2 h神经元自噬即明显激活,缺血/再灌注1 h、6 h自噬均持续增高,缺血/再灌注24 h自噬达高峰。