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。     

亚低温对大鼠全脑缺血再灌注损伤后海马CA1区c-Jun蛋白表达的影响

目的研究亚低温对大鼠全脑缺血再灌注损伤后海马CA1区神经元的保护作用,并探讨其可能的机制。方法采用四血管阻断法建立大鼠全脑缺血模型。SD大鼠,随机分为假手术组(SH组)、常温组(IR组)和亚低温组(HIR组)。各组在全脑缺血15min后分别再灌注6h、12h、1d、3d,采用苏木素-伊红(HE)染色观察各时间点海马CA1区细胞形态学变化和TUNEL法检测海马CA1区神经元凋亡,免疫印迹检测c-Jun蛋白表达。结果(1)HE染色结果 IR组和HIR组于全脑缺血再灌注后6h,HE染色未见明显改变,IR组缺血再灌注1d时CA1区出现严重改变,3d时损伤最严重,出现细胞数目减少,细胞胞体缩小、胞核固缩深染,损伤严重,排列紊乱,核膜不清,核仁消失。而HIR组海马存活的锥体细胞数较之IR组12h、1d、3d时间点均明显增加(P<0.05)。(2)TUNEL标记IR组于缺血再灌注后6h在海马CA1区阳性细胞开始增多,缺血再灌注1 d时阳性细胞数最多。而HIR组各时间点阳性细胞数均较IR组明显减少(P<0.01)。(3)免疫印迹结果全脑缺血再灌注后6h c-Jun蛋白在IR组海马CA1区表达开始增加,12h达高峰,持续到3d;HIR组在各时间点的表达均弱于IR组(P<0.01)。结论亚低温通过减少海马CA1区c-Jun的表达,抑制海马CA1区神经元的凋亡,可能是亚低温脑保护作用的机制之一。     

高血压大鼠全脑缺血再灌注后神经细胞DNA损伤修复的研究

目的 从DNA损伤和修复的角度探讨局灶性脑缺血再灌注后神经元凋亡的机制.方法 运用双肾双夹法建立肾血管性高血压大鼠模型,采用四血管法制备高血压大鼠全脑缺血再灌注模型,免疫组化法检测再灌注过程中鼠脑DNA损伤修复,蛋白XRCC1、TUNEL法检测凋亡.结果 与假手术组相比,脑缺血再灌注3h在缺血区皮质和海马CA1区XRCC1表达出现显著减低,这种减低一直持续到缺血再灌注24h(P＜0.05);再灌注24h缺血区皮质和海马CA1区神经细胞发生了凋亡,神经细胞XRCC1的表达强度与凋亡呈负相关.结论 脑缺血再灌注早期神经细胞XRCC1表达的减少可能导致机体对此时出现的DNA单链断裂修复的能力下降,造成了再灌注后期发生了凋亡.     

局灶性脑缺血再灌注大鼠海马CA1区神经元凋亡研究

目的 观察大鼠局灶性脑缺血再灌注(ischemia reperfusion,I/R)损伤后海马CA1区神经元凋亡、TUNEL阳性细胞变化,以及凋亡相关蛋白Bcl-2与Bax蛋白的表达情况.方法 将健康雄性SD (Sprague-Dawley)大鼠随机分为假手术组和I/R组,每组再分为缺血再灌注后3、6、12、24、48、72 h亚组.应用免疫组化方法检测再灌注后不同时间点大鼠海马CA1区神经元凋亡基因Bcl-2和Bax蛋白的表达及Bcl-2/Bax比值变化,采用原位细胞凋亡检测(TUNEL)技术检测凋亡阳性细胞数.结果 各组非缺血侧相应区域神经元胞质中Bcl-2均有微弱表达.I/R组缺血侧海马CA1区于再灌注3 h开始出现Bcl-2和Bax蛋白微弱表达,随再灌注时间延长神经元内Bcl-2表达逐渐增强,再灌注24 h后Bcl-2表达达高峰,假手术组与I/R组比较差异有统计学意义(P<0.05).结论 I/R损伤后海马CA1区神经元不仅存在变性坏死,还存在明显的细胞凋亡且细胞凋亡在大鼠I/R损伤中发挥重要作用;I/R可诱导海马CA1区细胞凋亡基因Bcl-2和Bax蛋白表达,且其表达呈一定规律.     

神经生长因子对大鼠前脑缺血再灌注后细胞凋亡及FaS蛋白表达的影响

目的：研究神经生长因子（NGF）对大鼠前脑缺血再灌注后海马CA1区Fas蛋白表达和细胞凋亡的影响。方法：夹闭大鼠双侧颈总动脉造成前脑缺血，30min后松夹再灌注，NGF组和生理盐水组于再灌注开始时分别肌肉注射NGF30μg·mL^-1和生理盐水0．1mL，应用免疫组化法和TUNEL法检测各组大鼠海马CA1区Fas蛋白表达和细胞凋亡。结果：再灌注后6和24h，NGF组Fas蛋白平均吸光度值小于生理盐水组（P〈0．001和P〈0．05）。再灌注后48h两组比较差异无统计学意义（P〉0．5）。再灌注后6、24和48h，NGF组TUNEL阳性细胞率均低于生理盐水组，差异有统计学意义（P〈0．005）。结论：NGF可以减少大鼠脑缺血再灌注后海马CA1区Fas蛋白表达，抑制细胞凋亡，从而发挥其神经保护作用。     

亚低温对缺血性神经元凋亡、细胞色素C释放的影响

通过大鼠短暂全脑缺血模型来探讨亚低温对大鼠脑缺血后细胞色素C(CytochromeC ,CytC)释放及缺血性神经元凋亡的影响 ,揭示亚低温的部分神经保护机制。用原位细胞凋亡检测法 (TUNEL染色 )检测及电镜观察脑缺血后大鼠脑海马CA1区神经元凋亡发生情况 ;免疫组织化学法测定脑缺血后大鼠脑海马区神经元中细胞色素C释放情况。结果显示 :①低温缺血组海马CA1区凋亡神经元数明显少于常温缺血组 (P <0 .0 1) ;②低温缺血3h组海马CA1区神经元CytC阳性表达低于常温缺血 3h组 (P <0 .0 1)。据此认为 ,全脑缺血后的迟发性神经元死亡很可能经由凋亡途径 ,而CytC激活、释放是缺血性神经元凋亡的一个关键事件。亚低温可抑制CytC的释放 ,推测经此途径减少缺血性神经元凋亡而发挥一定的神经保护作用。     

亚低温对缺血性神经元凋亡、细胞色素C释放的影响

通过大鼠短暂全脑缺血模型来探讨亚低温对大鼠脑缺血后细胞色素c(Cytoehrome c,CytC)释放及缺血性神经元凋亡的影响,揭示亚低温的部分神经保护机制.用原位细胞凋亡检测法(TUNEL染色)检测及电镜观察脑缺血后大鼠脑海马CAi区神经元凋亡发生情况;免疫组织化学法测定脑缺血后大鼠脑海马区神经元中细胞色素C释放情况.结果显示:①低温缺血组海马CAi区凋亡神经元数明显少于常温缺血组(P<0.01);②低温缺血3 h组海马CA1区神经元CytC阳性表达低于常温缺血3 h组(P<0.01).据此认为,全脑缺血后的迟发性神经元死亡很可能经由凋亡途径,而CytC激活、释放是缺血性神经元凋亡的一个关键事件.亚低温可抑制CytC的释放.推测经此途径减少缺血性神经元凋亡而发挥一定的神经保护作用.     

阿魏酸钠对脑缺血大鼠海马CA1区神经细胞凋亡的影响

为了探讨脑缺血迟发性神经细胞死亡与细胞凋亡的关系以及阿魏酸钠的抗凋亡作用,利用大鼠全脑缺血及再灌流模型,TUNEL法原位标记DNA片段,观察用阿魏酸钠后海马CA_1区神经细胞凋亡改变.结果发现,脑缺血10min再灌流3d后海马CA_1区存在较明显的神经细胞凋亡现象,凋亡细胞分布与光镜下神经细胞迟发性死亡的病理改变相对应,缺血前用药组神经细胞凋亡数目较对照组明显减少,神经细胞迟发性改变减轻.而缺血后用药组未见显效.结果提示,脑缺血海马CA_1区神经细胞迟发性死亡可能是通过细胞凋亡完成,阿魏酸钠对脑缺血再灌流引起的海马CA_1区神经细胞凋亡具有一定的防止作用.     

葛根素对大鼠全脑缺血再灌注后学习记忆障碍保护作用及其机制的研究

目的 探讨葛根素对大鼠全脑缺血再灌注后学习记忆能力的影响及其机制。方法 采用四血管阻断法建立SD大鼠全脑缺血再灌注损伤模型，暗回避反应法测定学习记忆能力，并应用免疫组织化学法、原位末端标记法，检测大鼠全脑缺血再灌注海马CA，区的bcl-2阳性细胞数、凋亡细胞数的动态变化。结果 (1)与再灌注组相比，葛根素组大鼠潜伏期明显延长；(2)脑缺血再灌注后，海马CA1区bcl-2蛋白的表达随再灌注时间不同而变化，缺血20min后再灌注24h达高峰，葛根素组bcl2蛋白的表达于相应的时间点明显增多；(3)脑缺血再灌注后海马CA1区神经元凋亡损伤在再灌注72h损伤最重，葛根素组可减少相应时点神经细胞凋亡数。结论 葛根素对全脑缺血再灌注后大鼠学习记忆能力具有明显的改善作用，其作用机制可能与通过上调bcl-2基因表达从而抑制或延迟脑缺血再灌注后细胞凋亡有关。     

大鼠局灶性脑缺血再灌注后海马Bcl-2、Bax蛋白的表达

目的 探讨大鼠局灶性脑缺血再灌注后Bcl-2、Bax蛋白在海马表达的变化.方法 线栓法制作大鼠局灶性脑缺血再灌注模型,应用免疫组化染色检测Bcl-2、Bax蛋白表达,应用TUNEL法检测海马区细胞凋亡.结果 缺血再灌注2h后海马神经元Bcl-2、Bax蛋白开始表达,Bcl-2蛋白12h达高峰,Bax蛋白12h～24h达高峰,之后开始下降.再灌注2h后海马凋亡细胞开始表达,随着再灌注时间的延长,其表达不断增加.Bcl-2/ Bax的比率在再灌注开始时升高,再灌注12h达高峰,随后开始下降.结论 凋亡是脑缺血再灌注损伤的重要形式之一,Bcl-2/ Bax的改变与缺血再灌注后海马的神经元存亡有关,缺血再灌注可导致海马神经元凋亡.     

亚低温对大鼠短暂全脑缺血后神经元凋亡的影响

目的 探讨亚低温对大鼠脑缺血后神经元凋亡的影响，揭示亚低温的部分神经保护机制。方法 采用“双侧颈总动脉阻断＋全身低血压”方法来建立大鼠短暂性全脑缺血模型。用神经元尼氏体亚甲兰特殊染色法观察大鼠脑缺血后海马CA1区神经元损害情况；原位细胞凋亡检测法（TUNEL染色）及电镜观察脑缺血后CA1区神经元凋亡情况。结果 与假手术组、低温缺血组相比，常温缺血组海马CA1区神经元缺失明显（P＜0．01）。常温及低温缺血组海马CA1区均存在神经元凋亡，但低温缺血组海马CA1区凋亡神经元数明显少于缺血组（P＜0．01）。结论 经“双侧颈总动脉阻断＋全身低血压”方法建立的大鼠短暂全脑缺血模型证实了亚低温的脑保护作用。全脑缺血后的迟发性神经元死亡很可能经由凋亡途径，而亚低温可通过抑制缺血性神经元凋亡而发挥一定的神经保护作用。     

Effects of berberine on hippocampal neuronal damage and matrix metalloproteinase-9 activity following transient global cerebral ischemia

Berberine, an isoquinoline alkaloid with a long history of use in Chinese medicine, has several important pharmacological effects. Several studies have revealed that berberine has neuroprotective and neuropsychiatric effects. However, there are few reports regarding the protective effect of berberine against neuronal damage following transient global cerebral ischemia. In this study, mice were subjected to 20 min of global brain ischemia and sacrificed 72 hr later. Berberine was administered for 7 days prior to ischemia and daily until sacrifice. Mice treated with berberine showed reduced matrix metalloproteinase-9 (MMP-9) activity. Berberine inhibited gelatinase activity directly in in situ zymography and reduced neuronal damage following global ischemia. Laminin expression and NeuN expression were markedly reduced in CA1 and CA2 areas after ischemia, and berberine reduced the laminin degradation and neuronal loss. In the TUNEL assay, damaged neurons were also apparent in the CA1 and CA2 areas, and berberine reduced TUNEL-positive cells. These data demonstrate that berberine, a plant alkaloid, may protect from hippocampal neuronal damage following transient global ischemia by reducing MMP-9 activity.     

Tea polyphenols increase X-ray repair cross-complementing protein 1 and apurinic/apyrimidinic endonuclease/redox factor-1 expression in the hippocampus of rats during cerebral ischemia/reperfusion injury

Recent studies have shown that tea polyphenols can cross the blood-brain barrier, inhibit apoptosis and play a neuroprotective role against cerebral ischemia. Furthermore, tea polyphenols can decrease DNA damage caused by free radicals. We hypothesized that tea polyphenols repair DNA damage and inhibit neuronal apoptosis during global cerebral ischemia/reperfusion. To test this hypothesis, we employed a rat model of global cerebral ischemia/reperfusion. We demonstrated that intraperitoneal injection of tea polyphenols immediately after reperfusion significantly reduced apoptosis in the hippocampal CA1 region; this effect started 6 hours following reperfusion. Immunohistochemical staining showed that tea polyphenols could reverse the ischemia/reperfusion-induced reduction in the expression of DNA repair proteins, X-ray repair cross-complementing protein 1 and apurinic/apyrimidinic endonuclease/redox factor-1 starting at 2 hours. Both effects lasted at least 72 hours. These experimental findings suggest that tea polyphenols promote DNA damage repair and protect against apoptosis in the brain.     

Relationship between transmembrane signal transduction pathway and DNA repair and the mechanism after global cerebral ischemia-reperfusion in rats

Objective

To investigate the protein levels of phospho-ERK and phospho-APE/Ref-1 in hippocampal neurons after global cerebral ischemia reperfusion in rats, and observe the relationship between transmembrane signal transduction and repair of DNA damage. The role of ERK signal transduction pathway following global cerebral ischemia reperfusion in rats is further discussed.

Methods

Ninety healthy male SD rats were divided into 3 groups randomly: Sham group (S group), Ischemia reperfusion group (IR group) and Pd98059 pretreatment/ischemia reperfusion group (PD group). Global cerebral ischemia reperfusion model was established by four-vessel occlusion (4-VO) method, and reperfusion was performed 5 minutes following ischemia. Protein levels of phospho-ERK and phospho-APE/Ref-1 were detected using immunohistochemical method at 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after reperfusion, and neuron apoptosis was observed by HE and TUNEL staining.

Results

In CA1 region of IR group, TUNEL positive cells began to appear at 6 h after IR, and reached the apex during 24 h to 48 h. However, TUNEL positive was most strongly exhibited in PD group. In IR group, phospho-ERK was obviously detected in CA3 region at 2 h after IR, and its level was gradually decreased from 6 h until totally absent at 48 h. Besides, phospho-ERK expression in PD group was weaker than that in IR group. For phospho-APE/Ref-1, its expression began to appear in CA1 region in IR group at 2 h after IR, with no obvious changes during 2 h to 12 h. Phospho-APE/Ref-1 expression began to decrease at 24 h and this decrease continued thereafter. Expression level of phospho-APE/Ref-1 in PD group was lower than that in IR group. Results showed the concurrence of decreased phospho-ERK expression level and increased neuron apoptosis after cerebral ischemia reperfusion, the former of which was consistent with the decrease of phospho-APE/Ref-1 expression. Also, the greater the inhibition of ERK phosphorylation was, the greater decrease of APE/Ref-1 expression occurred.

Conclusion

Activation of ERK signal transduction pathway increased the expression of phospho-APE/Ref-1, and thus faciliated the repair of DNA damage. So, activation of ERK signal transduction pathway may protect neurons from apoptosis after cerebral ischemia reperfusion.     

大鼠脑缺血再灌流后海马氨基酸水平变化与神经元损害的关系探讨

目的探讨脑缺血再灌流后海马氨基酸递质变化与神经元损害的关系。方法建立大鼠前脑缺血再灌流模型,测定海马ＣＡ１区和ＣＡ３／齿状回区游离氨基酸含量,观察阻断隔－海马通路对海马神经元损害和氨基酸水平的影响。结果（１）海马结构中仅ＣＡ１区神经元明显损害,但ＣＡ１区和ＣＡ３／齿状回区的Ｇｌｕ、Ａｓｐ和ＧＡＢＡ含量无差异。（２）阻断隔－海马通路可明显减轻海马神经元损害,但对海马氨基酸水平变化无影响。结论脑缺血再灌流后,氨基酸递质水平的异常变化不是海马ＣＡ１区神经元选择性易损的唯一决定因素,隔－海马通路末梢释放的神经递质也参与海马神经元损害过程。     

大鼠局灶性脑缺血再灌注后磷酸化细胞外信号调节激酶表达的变化

目的研究细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)在局灶性脑缺血再灌注中的作用。方法制备大鼠局灶性脑缺血再灌注模型,据Longa's的5级标准评分法进行神经功能评分,用免疫组织化学方法检测磷酸化ERK的表达,TUNEL染色检测神经元凋亡。结果在假手术组未检测到磷酸化的ERK表达,再灌注1h开始检测到阳性表达,再灌注4h达到峰值,12h下降;假手术组高倍镜视野仅见个别凋亡细胞,阴性对照片未检出阳性细胞,再灌注1h阳性细胞增加不明显,4h开始有明显的阳性细胞增加,24h细胞凋亡达到高峰,48h下降;磷酸化ERK阳性细胞计数与凋亡细胞数的相关分析显示相关系数(r)为-0.036,P=0.863,无统计学意义。结论局灶性脑缺血再灌注后磷酸化ERK表达增加,但其表达可能与神经元凋亡无关。     

缺血预处理后Fas蛋白表达与迟发性神经元凋亡的关系初步探讨

目的动态观察缺血预处理后大鼠大脑皮层和海马CA1区神经元凋亡与Fas蛋白表达变化情况,初步探讨缺血预处理后Fas蛋白表达与迟发性神经元凋亡的关系。方法四血管阻断法复制全脑缺血模型,动物随机分为非缺血对照组、预处理对照组、缺血预处理组和缺血组。采用尼氏和TUNEL染色法观察皮层及海马CA1区神经元存活数和凋亡细胞数,免疫组化方法检测Fas蛋白在缺血预处理后表达变化情况。结果缺血组缺血6h在皮质及海马CA1区Fas阳性表达细胞计数升高,12h达高峰;缺血预处理组缺血12h阳性细胞计数升高,24h达高峰。缺血组缺血6h出现凋亡细胞,48h凋亡细胞数达到高峰;缺血预处理组凋亡细胞数较缺血组明显减少。缺血组缺血7d神经元数明显减少,12周时神经元大量减少;缺血预处理组缺血7d时神经元数无明显变化,但12周时神经元同样大量减少。结论全脑缺血可能通过诱导Fas蛋白的表达增多,启动细胞凋亡,导致缺血后神经元凋亡的发生;缺血预处理虽可延缓缺血后神经元的凋亡,但无法提供真正的长时期的神经元保护作用,其有限的保护作用可能是通过延缓Fas蛋白的表达而减缓了神经元凋亡的进程。