Point application with Angong Niuhuang sticker protects hippocampal and cortical neurons in rats with cerebral ischemia

Angong Niuhuang pill, a Chinese materia medica preparation, can improve neurological functions after acute ischemic stroke. Because of its inconvenient application and toxic components(Cinnabaris and Realgar), we used transdermal enhancers to deliver Angong Niuhuang pill by modern technology, which expanded the safe dose range and clinical indications. In this study, Angong Niuhuang stickers administered at different point application doses(1.35, 2.7, and 5.4 g/kg) were administered to the Dazhui(DU14), Qihai(RN6) and Mingmen(DU4) of rats with chronic cerebral ischemia, for 4 weeks. The Morris water maze was used to determine the learning and memory ability of rats. Hematoxylin-eosin staining and Nissl staining were used to observe neuronal damage of the cortex and hippocampal CA1 region in rats with chronic cerebral ischemia. The middle- and high-dose point application of Angong Niuhuang stickers attenuated neuronal damage in the cortex and hippocampal CA1 region, and improved the memory of rats with chronic cerebral ischemia with an efficacy similar to interventions by electroacupuncture at Dazhui(DU14), Qihai(RN6) and Mingmen(DU4). Our experimental findings indicate that point application with Angong Niuhuang stickers can improve cognitive function after chronic cerebral ischemia in rats and is neuroprotective with an equivalent efficacy to acupuncture.     

3-硝基丙酸预处理对大鼠局灶性脑缺血Bcl-2和Bax mRNA表达的影响

目的探讨3-硝基丙酸(3-NPA)预处理对大鼠局灶性脑缺血半暗带Bc l-2和Bax mRNA表达的影响。方法将大鼠腹腔注射3-NPA 20 mg/kg,3 d后制作局灶性脑缺血再灌注模型;采用逆转录聚合酶链反应,观察3-NPA预处理对脑缺血再灌注1 h、6 h、12 h、24 h及48 h额顶部皮质Bc l-2和Bax mRNA表达的影响,并与假手术组和缺血再灌注组比较。结果与假手术组比较,缺血再灌注组和3-NPA预处理组各时间点Bc l-2和Bax mRNA表达极显著增强(均P<0.01);与缺血再灌注组比较,3-NPA预处理组各时间点Bc l-2mRNA表达显著增强(均P<0.05),再灌注12~48 h Bax mRNA的表达显著降低(均P<0.05)。结论增强Bc l-2的表达、抑制Bax的表达,可能是3-NPA预处理抑制细胞凋亡、诱导脑缺血耐受的机制之一。     

雌激素对雄性大鼠缺血性脑损伤的神经保护作用及其可能机制

目的 探讨雌激素对雄性大鼠局灶性脑缺血的神经保护作用及其可能机制。方法 随机将大鼠分为雌激素预处理组、雌激素＋他莫昔芬预处理组和对照组，采用线栓法制作大鼠大脑中动脉闭塞（MCAO）模型，观察缺血后2h和24h脑梗死体积、大鼠死亡率、神经功能缺损程度、神经元凋亡以及Bcl-2、Bax、p53蛋白的表达。结果 缺血后2h和24h，雌激素预处理组较对照组脑梗死体积小、死亡率低、神经功能缺损程度轻、神经元凋亡少，并抑制Bax、p53表达，促进Bcl-2蛋白表达。雌激素受体拮抗剂他莫昔芬可抑制这一作用。结论 雌激素对雄性大鼠缺血性脑损伤具有明显的神经保护作用。对基因和非基因因素的影响是神经保护作用的可能机制。     

Remifentanil postconditioning improves global cerebral ischemia-induced spatial learning and memory deficit in rats via inhibition of neuronal apoptosis through the PI3K signaling pathway

Global cerebral ischemia followed by reperfusion, which leads to extensive neuronal damage, particularly the neurons in the hippocampal CA1 region. Apoptosis is one of the major mechanisms that lead to neuronal death after cerebral ischemia and reperfusion. The neuroprotective effects of remifentanil preconditioning against cerebral ischemia/reperfusion injury have been recently reported. Here we investigated whether remifentanil postconditioning exerts neuroprotective effects against global cerebral ischemia/reperfusion injury in rats and its potential mechanisms. Global cerebral ischemia was performed via 10 min of four-vessel occlusion. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells and expression of Bcl-2 and Bax in the hippocampal CA1 region were assessed after reperfusion. Morris water maze task was used to quantify spatial learning and memory deficits after reperfusion. We found remifentanil postconditioning markedly improved the spatial learning and memory as well as attenuated neuronal apoptosis in hippocampus caused by cerebral ischemia/reperfusion injury. In addition, remifentanil postconditioning enhanced the expression of anti-apoptotic gene Bcl-2 while suppressed the expression of pro-apoptotic gene Bax in hippocampal CA1 region. However, the neuroprotective effects of remifentanil postconditioning were abolished by pretreatment of the PI3K inhibitor LY294002. The results suggest that remifentanil postconditioning exhibits neuroprotective effects against global cerebral ischemia/reperfusion injury in rats, and its mechanisms might involve inhibition of neuronal apoptosis through the PI3K pathway.     

小檗碱对小鼠全脑缺血后神经元凋亡相关基因的影响

目的 探讨小檗碱对小鼠全脑缺血后神经元凋亡相关基因的影响,以了解小檗碱保护脑缺血的机制,为其开发利用提供理论依据。方法 利用改良的Pulsinelli-Brierley4血管闭塞法制成小鼠全脑缺血再灌注动物模型。小檗碱用量为1mg/kg,于术前30min,术后每日1次,腹腔注射。免疫组织化学技术检测凋亡相关基因Bcl-2,Bax蛋白的表达。结果 正常组海马区未见Bcl-2或Bax蛋白表达;缺血组再灌注6h海马CA3区可见Bcl-2阳性细胞,24h达到高峰,48h开始下降;小檗碱治疗组再灌注24h、48h及168hBcl-2阳性细胞明显减少（P＜0．01）。缺血组再灌注6h海马CA1区可见Bax阳性细胞;48h达高峰;168h明显下降;小檗碱组再灌注24h,48h及168hBax阳性细胞数明显减少（P＜0．01）。结论 小檗碱可以增加小鼠全脑缺血后海马CA3区bcl-2基因的表达,降低CA1区Bax基因的表达,从而减少凋亡的发生,可能为其保护脑缺血的机制之一。     

脑源性神经营养因子对脑梗死大鼠神经细胞凋亡及Bcl-2、Bax蛋白表达的影响

目的 探讨侧脑室内立体定向注射脑源性神经营养因子(BDNF)对脑梗死大鼠神经细胞凋亡及Bcl-2、Bax蛋白表达的影响.方法 32只SD大鼠成功制作大脑中动脉闭塞模型,随机分为BDNF组(n=16)和对照组(n=16),在两组大鼠侧脑室内分别注射10μ1 BDNF溶液(0.5 μmol)和10μl磷酸盐缓冲液(PBS液).注射2周后,2组大鼠分别行神经功能严重性评分(mNSS)及梗死面积测定,应用免疫组化染色、Western blot分析脑组织Bcl-2、Bax蛋白的表达,Tunel检测细胞凋亡.结果 与对照组比较,BDNF组脑梗死面积缩小,神经细胞凋亡数少,Bax蛋白表达低,Bcl-2蛋白表达高,短期内神经功能恢复好,差异均具有统计学意义(P＜0.05).结论 BDNF可能通过调节Bcl-2、Bax蛋白表达,减少神经细胞凋亡,改善脑缺血大鼠的功能.     

大鼠脑缺血再灌注后Caspase-3、Bcl-2和Bax的表达

目的探讨大鼠脑缺血再灌注后caspase-3、Bcl-2和Bax在脑皮质神经元中的表达。方法将动物随机分为假手术组及缺血组,参照zea longa线栓法建立大鼠左侧大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)局灶性脑缺血再灌注模型,各组大鼠分别在左侧MCAO2h再灌注不同时间点断头取脑,脑皮质神经元中caspase-3、Bcl-2和Bax的表达通过免疫组化法来测定。结果缺血组大鼠脑皮质caspase-3的表达较假手术组显著增强(P<0.01),缺血组大鼠脑皮质Bcl-2的表达较假手术组显著增强(P<0.01),缺血组大鼠脑皮质Bax的表达较假手术组显著增强(P<0.01)。结论短暂性脑缺血再灌注上调脑皮质神经元中caspase-3和Bax的表达促细胞凋亡,上调脑皮质神经元中Bcl-2的表达抗细胞凋亡。     

针刺预处理对短暂性脑缺血再灌注损伤海马神经元的保护

The present study established a model of brain ischemia in aged rats using four-vessel occlusion.We observed hippocampal CA1 neuronal apoptosis and apoptosis-mediated protease caspase-3 expression following preconditioning of electroacupuncture at Baihui(GV 20).Our results showed that the number of hippocampal CA1 normal neurons was decreased,and degenerated neurons were increased 12 hours to 3 days following cerebral ischemia/reperfusion.The number of hippocampal CA1 apoptotic neurons and caspase-3-positive neurons in rats with cerebral ischemia/reperfusion injury was significantly decreased following acupuncture preconditioning.Acupuncture preconditioning protects aged rats against ischemia/reperfusion injury by regulating caspase-3 protein expression.     

香港远志提取物对局灶性脑缺血大鼠脑保护作用与作用机制

目的探讨香港远志提取物对局灶性脑缺血大鼠脑保护作用与机制。方法线栓法制备SD大鼠大脑中动脉闭塞(MCAO)模型,随机分4组:假手术组、模型组及治疗A、B组。治疗组按设定方式给药,假手术组、模型组给1%吐温溶液。观察术后24h神经功能缺损评分(NBDS)、脑梗死体积(IV)、血清神经元烯醇化酶(NSE)及神经元凋亡、Bcl-2、Bax表达。结果与假手术组比较,模型组和治疗组NBDS、IV、NSE、神经元凋亡及Bcl-2、Bax显著增高、Bcl-2/Bax显著降低(P<0.05)。与模型组比较,治疗组NBDS、IV、NSE及神经元凋亡、Bax显著降低、Bcl-2、Bcl-2/Bax显著增高(P<0.05),治疗组间数据亦有显著差异(P<0.05)。结论香港远志提取物预处理,对局灶性脑缺血大鼠脑神经有保护作用,机制可能是促进Bcl-2、抑制Bax表达,上调Bcl-2/Bax比值,阻止神经元凋亡。     

奥扎格雷钠对大鼠局灶性脑缺血血小板内皮细胞黏附分子-1、Bcl-2、Bax表达的影响

目的 观察大鼠局灶性脑缺血后脑组织中血小板内皮细胞黏附分子-1(PECAM-1、CD31)、Bcl-2、Bax表达的改变及奥扎格雷钠对其表达的影响. 方法 采用线栓法制作大鼠大脑中动脉局灶性脑缺血模型,制模成功大鼠随机分为奥扎格雷钠组和生理盐水组,脑组织切片免疫组化染色检测不同时间点PECAM-1、Bcl-2、Bax在各组大鼠脑组织中的表达变化. 结果大鼠大脑中动脉闭塞后脑组织PECAM-1、Bcl-2、Bax的表达明显增高(均P＜0.01).奥扎格雷钠组缺血12h、24h、36h表达PECAM-1较生理盐水组增高,缺血12h、24h 表达Bcl-2较生理盐水组增高,缺血24h、36h表达Bax较生理盐水组增高,均P＜0.01. 结论 脑组织表达的PECAM-1、Bcl-2、Bax分别参与了脑缺血不同时期的病理生理作用;奥扎格雷钠可促进PECAM-1和Bcl-2的表达,抑制Bax的表达.     

脑缺血耐受与细胞凋亡及p53、p21、Bax表达关系的实验研究

目的　探讨大鼠局灶性缺血预处理的脑保护作用与细胞凋亡及凋亡相关因子p5 3、p2 1和Bax的关系。方法　采用线栓法阻塞大鼠大脑中动脉造成脑缺血预处理和致死性缺血模型 ,图像分析测算相对梗死体积 ,使用TUNEL染色标记神经细胞凋亡 ,免疫组化染色观察p5 3、p2 1和Bax蛋白表达。结果　与致死缺血组比较 ,缺血预处理组梗死体积减少 4 6 % (P <0 0 5 ) ,半暗区TUNEL阳性细胞数和p5 3阳性细胞数均明显降低 (均P <0 0 5 ) ,p2 1和Bax阳性细胞数无显著变化 (均P >0 0 5 )。结论　缺血预处理可能通过抑制严重缺血后p5 3表达 ,减轻神经细胞凋亡 ,发挥脑保护作用。p2 1和Bax在脑缺血耐受形成中可能不起重要作用。     

沙土鼠脑缺血后海马CA1区神经细胞凋亡、相关基因表达及亚低温的干预作用

目的 研究沙土鼠脑缺血后海马CA1区神经细胞凋亡、相关基因表达及亚低温的干预作用. 方法 72只沙土鼠采用随机数字表法分为假手术组(SH)、低温假手术组(HSH)、常温再灌注组(IR)和低温再灌注组(HIR).采用双侧颈总动脉阻断5 min制作脑缺血再灌注损伤模型,各组依术后处死动物时间的不同再分为1、3、7d亚组(n=6),在预定时间点行开阔法迷宫检查、TUNEL法检测海马CA1区神经细胞的凋亡、免疫组化检测肿瘤抑制基因p53、核因子-kB的表达情况.结果 常温状态下脑缺血5 min可诱导沙土鼠1、3、7d的探索活动增加(P<0.051.亚低温状态下仅缺血再灌注后1 d探索活动增加(P<0.05);TUNEL与免疫组化染色显示海马CA1区神经细胞凋亡数量及p53蛋白和NF-KB表达增加,亚低温对以上过程有明显抑制作用(P均<0.05). 结论 海马CA1区p53蛋白和NF-KB表达增加可能是沙土鼠脑缺血5 min神经元凋亡的机制之一,亚低温脑保护机制可能与其对此过程的抑制作用有关.     

钙拮抗剂对大鼠缺血性脑损伤后Bcl-2和Bax基因表达的作用

目的探讨大鼠实验性脑缺血后海马组织Bcl-2和Bax基因的表达及尼莫地平预处理对Bcl-2和Bax基因表达的影响。方法采用线栓法建立大鼠脑缺血模型；尼莫地平预处理；逆转录聚合酶链式反应(RT-PCR)法测定其海马组织中Bcl-2和Bax mRNA。结果大鼠大脑中动脉阻断(MCAO)后，海马组织Bcl-2和Bax基因均被诱导表达，Bcl-2表达量持续升高，而Bax表达量在脑缺血24h达高峰，随后逐渐下降。在脑缺血后6h和24h，经尼莫地平预处理7d的大鼠，海马组织Bcl-2基因的表达水平较未经预处理的大鼠明显上调，而Bax基因表达水平则较未经预处理的大鼠明显下调。结论钙拮抗剂尼莫地平能有效地调控大鼠缺血性脑损伤后海马组织Bcl-2和Bax基因表达的水平，为干预脑卒中后基因表达提供依据。     

p53 potentiates hippocampal neuronal death caused by global ischemia.

Although p53 controls cell death after various stresses, its role in neuronal death after brain ischemia is poorly understood. To address this issue, we subjected p53-deficient (p53-/- and p53+/-) mice (backcrossed for 12 generations with C57BL/6 mice) and wild-type mice (p53+/+) to transient global ischemia by the three-vessel occlusion method. Despite similar severity of ischemia, as shown by anoxic depolarization and cortical blood flow, neuronal death in the hippocampal cornus ammonis (CA)1 region was much more extensive in p53+/+ than in p53-/- mice (surviving neuronal count, 9.3%+/-3.0% versus 61.3%+/-34.0% of nonischemic p53+/+ controls, respectively, P<0.0037). In p53+/- mice, a similar trend was also observed, though not statistically significant (43.5% of nonischemic p53+/+ controls). In p53+/+ mice, p53-like immunoreactivity in hippocampal CA1 neurons was enhanced at 12 h after ischemia, and messenger ribonucleic acid for Bax, a direct downstream target of p53, was also increased. These results indicate that p53 potentiates ischemic neuronal death in vivo and suggest that this molecule could be a therapeutic target in neuronal death after cerebral ischemia.     

人尿激肽原酶对急性局灶性脑缺血再灌注损伤大鼠细胞凋亡的影响

目的 研究人尿激肽原酶(HUK)对大鼠急性局灶性脑缺血再灌注(FCIR)损伤后细胞凋亡数量及B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2联合X蛋白(Bax)蛋白表达的影响.方法 84只雄性SD大鼠分为假手术组(12只)、脑缺血再灌注(IR)组(36只)、HUK处理组(36只),IR组和HUK处理组剩余大鼠又按照再灌注时间6 h、12 h、24 h、72 h、168 h分为5个亚组(均为6只).建立大鼠大脑中动脉FCIR模型.假手术组、IR组及HUK处理组中各取6只SD大鼠用于测定梗死体积,其余大鼠用于观察神经功能缺陷评分、TIJNEL法及免疫组化检测凋亡细胞数量及凋亡蛋白Bcl-2、Bax的表达.结果 HUK处理组神经功能缺陷评分、梗死灶体积、除168 h亚组外的各时 间点的凋亡细胞数及Bax蛋白表达均显著少于IR组(P＜0.05),除168 h亚组外的各时间点的Bcl-2蛋白表达均显著高于IR组(P＜0.05).结论 HUK对FCIR后的脑组织起保护作用,其机制可能为损伤后3 d内通过上调Bcl-2、下调Bax蛋白表达来抑制细胞凋亡.     

阻断缝隙连接对局灶性脑缺血再灌注后海马迟发性神经元死亡及Bax表达的影响

目的 探讨阻断缝隙连接对大鼠局灶性脑缺血再灌注后海马迟发性神经无死亡(delayed neu-ronal death,DND)及Bax表达的影响.方法 术前2 h左侧脑室注射缝隙连接阻断剂甘珀酸(carbenoxolone,CBX),颈内动脉插线法制备大鼠大脑中动脉缺血模型,采用TUNEL及免疫荧光技术,观察3 d后海马DND及Bax表达水平的变化.结果 缺血再灌注生理盐水有45%的大鼠出现海马DND;用CBX后仅30%的大鼠出现海马DND,机率明显减小(P<0.01);与假手术组比较,缺血再灌注中CBX组Bax的表达水平明显增高,但低于缺血再灌注生理盐水(P<0.01).结论 缝隙连接与局灶性脑缺血再灌注引起的海马DND有密切关系,其原因可能与缺血再灌注后凋亡启动信号由缺血再灌注区通过缝隙连接向远隔部位播散有关.Bax参与了海马神经元凋亡的调节.     

A Single Immediate Use of the Cathodal Transcranial Direct Current Stimulation Induces Neuroprotection of Hippocampal Region Against Global Cerebral Ischemia

ObjectivesGlobal cerebral ischemia (CI) causes severe neuronal injury, mainly in the hippocampal CA1 region. This study aimed to investigate an immediate using transcranial direct current stimulation (tDCS) in reducing neuronal injury induced by CI.Materials and methodsThe 32 Wistar male rats were randomly divided into four groups (n=8 per group). In the ischemia group (I), CI was induced via the 4-vessel occlusion model. In the sham group (Sh), rats did not receive any intervention. In the ischemia+cathodal group (I+c/tDCS), the cathodal current was applied during CI. In the ischemia+anodal group (I+a/tDCS), the anodal current was applied. The current intensity of 400 μA was applied for 15-min during the ischemia. Hippocampal tissue was used to assess levels of NMDAR, IL-1β, TNF-α, MDA, SOD, NOS, and apoptosis markers. Histological assessment and TUNEL staining were performed in CA1 hippocampal region.ResultsThe c/tDCS significantly decreased the levels of IL-1β and TNF-α than the I and a/tDCS groups. The c/tDCS significantly reduced MDA and NOS levels, while increasing the level of SOD than the I and a/tDCS. The c/tDCS caused a significant decrease in NMDAR level than the a/tDCS. Using c/tDCS significantly reduced the Bax and Caspase-3 expressions, while increasing the Bcl-2 expression than the I group. In the c/tDCS group, DNA fragmentation and neuronal death were significantly lower than the I and a/tDCS groups.ConclusionUsing cathodal a direct current could attenuate primary pathophysiological pathways induced by CI, and it eventually reduced neurons death and apoptosis in the CA1 hippocampal region.     

Enhanced expression of p53 in reactive astrocytes following transient focal ischemia

The present study used immunohistochemistry to investigate p53 expression in rat brain following transient occlusion of the middle cerebral artery. In the control group, no p53-immunoreactive cells were found in any region of the central nervous system. P53 expression in reactive astrocytes was not obvious in the forebrain one day or three days following ischemic insults. Seven days following ischemic injury, increased expression of p53 was clearly detectable in reactive astrocytes in affected cortical regions, such as forelimb area, hindlimb area, and parietal cortex. At seven days of recirculation, there was also a significant increase in the number of p53-immunoreactive neurons in the cerebral cortex, striatum, and hippocampal CA1-3 regions. Although the present study has not addressed multiple mechanisms contributing to cell death following ischemic injury, the first demonstration of a significant increase in p53 expression in glial cells may prove useful for future investigations of the pathophysiology of ischemia.     

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.     

Electroacupuncture protected pyramidal cells in hippocampal CA1 region of vascular dementia rats by inhibiting the expression of p53 and Noxa

Aim: Clinically electroacupuncture (EA) is proved an effective therapy for vascular dementia (VD), but their mechanisms remain uncertain. The aim of this study was to determine whether EA protects pyramidal cells from apoptosis in hippocampus of a VD rat model by inhibiting the expression of p53 and Noxa. Methods: One month after a VD animal model was established by bilateral occlusion of common carotid arteries, EA treatment was given at “Baihui” (DU20), “Dazhui” (DU14), and “Shenshu” (BL23). The learning and memory ability was assessed by Morris water maze. Neuronal apoptosis in hippocampus was evaluated with hematoxylin–eosin (HE) staining, and the expression of p53 and Noxa was analyzed by confocal laser scanning microscope with immunofluorescence staining. Results: Expressions of p53 and Noxa in the EA group and sham‐operated group were less than in the VD model group (P < 0.01), and the expression of p53 was positively correlated to expression of Noxa in hippocampus of VD rats (r = 0.918, P < 0.01). EA treatment could reduce the amount of apoptotic neurons in hippocampal CA1 area of rats with VD. The average latency in the Morris water maze test was significantly shorter, and escape strategies improved from edge and random searches to more linear swim pathway in the EA group compared with the VD model group (P < 0.01). Conclusions: The increasing expressions of p53 and Noxa play important roles in the pathogenesis of VD. EA improves learning and memory ability and protects pyramidal cells from apoptosis by blocking expression of p53 and Noxa in the hippocampal CA1 region of VD rats. These results suggest a novel mechanism of EA treatment to VD.