用逆转录——多聚酶链反应检测局灶脑缺血及再灌注后c—fo…

本研究应用逆转录－多聚酶链反应方法检测大鼠局灶脑缺血模型中即早基因ｃ－ｆｏｓ和ｃ－ｊｕｎ的表达。结果发现缺血１５ｍｉｎ时可见到ｃ－ｆｏｓ和ｃ－ｊｕｎ ｍＲＮＡ表达缺血３０ｍｉｎ时引起轻微左侧局灶脑缺血改变，可诱导左侧局灶脑缺血区ｃ－ｆｏｓ和ｃ－ｍＲＮＡ广泛的表达；缺血９０ｍｉｎ后，导致大面积局灶脑缺血改变，诱导上述两种基因在同侧缺血区与同侧非大脑中动脉供血区的海马中表达。后者有相当轻的缺血症状。再     

大鼠局灶性脑缺血时细胞凋亡与c-fos表达

目的：研究凋亡细胞在脑缺血及再灌注后的时空表达方式及原癌基因ｃ－ｆｏｓ的表达，以期探讨脑缺血时ｃ－ｆｏｓ表达与细胞凋亡的关系。方法：用线栓法建立局灶性脑缺血再灌注模型，ＤＮＡ缺口末端标记法原位检测细胞凋亡，免疫组化检测ｃ－ｆｏｓ的表达。结果：缺血侧鼠脑凋亡细胞数随再灌注时间延长而增多，凋亡细胞主要分布于额顶叶皮层与纹状体，以梗死灶边缘区密度最高。正常组、假手术组未见ｃ－ｆｏｓ表达，缺血及再灌注各组     

大鼠局灶性脑缺血再灌注时c-fos、bcl-2蛋白的表达

目的：为探讨细胞凋亡在脑缺血再灌注时脑损伤过程中的作用。方法：采用免疫组化法检测大鼠脑缺血再液注不同时间内ｃ－ｆｏｓ、ｂｃｌ－２蛋白表达的水平。结果：①脑缺血再灌注时在缺血侧皮层和基底节区可见ｃ－ｆｏｓ阳性表达,并于缺血 ３０ｍｉｎ再灌注１ｈ时阳性表达达高峰;②脑缺血再灌注时缺血侧皮层和基底节区均有ｂｃｌ－２阳性表达,于缺血２ｈ再灌注３ｈ时阳性表达达高峰。结论：ｃ－ｆｏｓ和ｂｃｌ－２参与大鼠脑缺血再灌注时缺血性细胞损伤的发生。     

局灶性脑缺血大鼠海马bcl-2 mRNA、Bax mRNA表达水平的改变

局灶性脑缺血大鼠海马ｂｃｌ－２ｍＲＮＡ、ＢａｘｍＲＮＡ表达水平的改变张苏明王伟选用局灶性脑缺血模型，观察缺血及再灌注期大鼠海马细胞程序性死亡（ＰＣＤ）调控基因ｂｃｌ－２ｍＲＮＡ、ＢａｘｍＲＮＡ表达水平的变化，以期了解缺血与神经元ＰＣＤ二者的关系。材料...     

脑缺血选择性海马CA1区神经元损害的实验研究

采用Ｐｕｌｓｉｎｅｌｉ－Ｂｒｉｅｒｌｅｙ４血管阻塞脑缺血模型观察了大鼠全脑缺血２０ｍｉｎ再灌流８ｈ，ｃ－ｆｏｓ基因表达及再灌流７ｄ海马ＣＡ１区迟发性神经元损害。在缺血再灌流早期（８ｈ）海马ＣＡ１区极少ｃ－ｆｏｓ表达，而齿状回、海马ＣＡ３区、杏仁核大量ｃ－ｆｏｓ表达。缺血再灌流晚期（７ｄ）镀银染色显示海马ＣＡ１区神经元及其突触终末带呈黑色溃变相，而齿状回、海马ＣＡ３区、杏仁核呈金黄色正常相。相邻切片ＨＥ染色示缺血组海马ＣＡ１区核完整的锥体细胞数（５±２．６个／２００μｍ）与对照组（４０±２．９个／μｍ）比较差异有显著意义（Ｐ＜０．０１）。脑缺血诱导的ｃ－ｆｏｓ基因表达对于缺血易损海马ＣＡ１区迟发性神经元坏死可能起直接的调控作用。     

沙鼠脑缺血再灌注后海马中c—fos蛋白表达及纳洛酮 …

目的 观察沙鼠脑缺血再灌注后纳洛酮对海马神经元ｃ－ｆｏｓ蛋白表达及神经元形态改变的影响。方法 采用沙鼠急性全脑缺血模型,用免疫组织化学及组织化学方法观察海马ｃ－ｆｏｓ蛋白表达及细胞形态改变。结果 纳洛酮能明显加强急性全脑缺血沙鼠海马各区ｃ－ｆｏｓ蛋白的表达,ＣＡ１区尤为明显。同时纳洛酮能明显改善缺血后ＣＡ１区神经元细胞的变性坏死。结论 纳洛酮对缺血后海马神经元细胞的保护作用可能与加强ｃ－ｆｏｓ蛋白     

大鼠脑缺血再灌注后脑组织C—fos原癌基因表达水平的变化

本文采用核酸分子斑点杂交技术制作脑缺血模型，观察脑缺血１０分钟及再灌注后不同时间脑组织Ｃ－ｇｏｓ原癌基因的变化。结果发现：与对照组比较，脑缺血１０分钟，Ｃ－ｆｏｓ ｍＲ－ＮＡ即增加。缺血后再灌注４５分钟Ｃ－ｆｏｓｍＲＮＡ达到峰值，１５０分钟降至对照组水平，提示脑缺血后再灌注可诱导脑组织Ｃ－ｆｏｓ原部基因－过性增高。     

脑缺血再灌注后脑组织c-fos基因表达与丹参的影响

采用线栓法制成大鼠大脑中动脉缺血再灌注模型，用地高辛精标记ｃ－ｆｏｓ探针进行原位杂交。结果示缺血再灌注鼠栓塞侧皮层及海马ｃ－ｆｏｓ基因表达显著增多，图像分析灰阶值为１１８．６±５．１，对侧为１５９．６±３．１（Ｐ＜０．００１）。丹参组栓塞侧皮层及海马ｃ－ｆｏｓ基因表达亦增多，灰阶为１３５．００±２．０５，对侧为１６７．００±２．００（Ｐ＜０．００１）。丹参组与缺血再灌注组比较，栓塞侧丹参组ｃ－ｆｏｓ基因表达显著低于缺血再灌组（Ｐ＜０．０５），而两组栓塞对侧比较无显著差异。本实验表明，脑缺血再灌注后脑组织ｃ－ｆｏｓ基因表达显著增多，丹参能部分抑制缺血后ｃ－ｆｏｓ基因的表达，这可能是其治疗缺血性脑血管病的机理之一。     

大鼠脑缺血再灌注后脑组织C－fos原癌基因表达水平的变化

本文采用核酸分子斑点杂交技术制作脑缺血模型，观察脑缺血１０分钟及再灌注后不同时间脑组织Ｃ－ｆｏｓ原癌基因表达的变化，结果发现：与对照组比较，脑缺血１０分钟，Ｃ－ｆｏｓｍＲ－ＮＡ即增加（Ｐ＜０．０５）。缺血后再灌注４５分钟Ｃ－ｆｏｓｍＲＮＡ达到峰值（Ｐ＜０．０１），１５０分钟降至对照组水平，提示脑缺血后再灌注可诱导脑组织Ｃ－ｆｏｓ原癌基因一过性增高     

实验性局灶性脑血及再灌流后AP—1复合物的结合活性测定

本研究应用凝胶迁移法检测局灶脑缺血区中Ｆｏｓ与Ｊｕｎ基因的蛋白产物形成异源二聚体复合物－转录因子ＡＰ－（ａｃｔｉｖａｔｏｒ ｐｒｏｔｅｉｎ－１）的ＤＮＡ结合活性。缺血３０ｍｉｎ或９０ｍｉｎ伴随再灌流４ｈ，结果表明ＡＰ－１结合活性增加。提高的ＤＮＡ结合活性持续２４ｈ。以上结果说明提高转录因子ＡＰ－１的表达水平在缺血病变的基因调节改变中起一定作用。     

脑缺血再灌注损伤时 c-fos、c-jun 的表达和细胞凋亡

目的　研究脑缺血再灌注大鼠神经细胞凋亡和原癌基因c fos、c jun表达。 方法　 8周龄健康雄性Wistar大鼠 2 4只 ,随机分为缺血再灌注组、假手术组和对照组 ,每组各 8只。制作大鼠大脑中动脉栓塞 (MCAO)再灌注模型 ,缺血 4h再灌注 2h后断头处死 ,TUNEL法检测神经细胞凋亡 ,免疫组织化学法检测神经细胞c fos、c jun蛋白的表达。结果　缺血再灌注组细胞凋亡率、平均吸光度及c fos、c jun阳性细胞率、平均吸光度均高于假手术组和对照组 (P <0 .0 5 )。结论　脑缺血再灌注损伤可诱导c fos、c jun蛋白的表达和细胞凋亡 ;脑缺血再灌注大鼠神经功能评分与c fos、c jun蛋白的表达和细胞凋亡呈正相关。     

C—fos反义寡脱氧核苷酸部分阻断MCAO大鼠缺血侧皮质内BDNF的表达

用c fos反义寡脱氧核苷酸侧脑室微量注射和细胞免疫化学等技术和方法 ,探讨大鼠局灶性脑缺血(MCAO)模型中 ,即早反应基因c fos表达与脑源性神经营养因子 (BDNF)表达的关系。结果表明 ,局灶性脑缺血再灌注可引起c fos和BDNF在缺血侧皮质的大量表达。侧脑室微量注射c fos反义寡脱氧核苷酸后 ,脑内BDNF的部分表达明显被阻断 ,脑缺血损伤加重。提示脑缺血损伤后 ,脑内BDNF的表达对脑缺血再灌注损伤起一定的保护作用 ;脑缺血后BDNF的表达可能部分通过c fos调控。     

胰岛素对局灶脑缺血c-jun基因表达的影响

目的观察不同剂量胰岛素对高血压大鼠局灶缺血脑组织ｃｊｕｎ基因表达的影响。方法以肾血管性高血压大鼠（ＲＨＲ）复制大脑中动脉闭塞（ＭＣＡＯ）模型，采用原位杂交技术检测不同剂量胰岛素组和对照组ＭＣＡＯ后３ｈ脑组织ｃｊｕｎ基因的表达。结果与对照组相比，在缺血侧广泛的大脑皮层，低剂量胰岛素组ｃｊｕｎｍＲＡＮ有统计学意义的增加（Ｐ＜００５），而较高剂量胰岛素组ｃｊｕｎｍＲＮＡ增加更为显著（Ｐ＜００１）。结论胰岛素促进缺血脑组织ｃｊｕｎ基因表达可能是其神经保护作用机制之一，而且这种作用呈剂量依赖性。     

脑缺血再灌注后脑组织c—fos基因表达与东菱克栓酶的影响

本实验采用线栓法制成大鼠大脑中动脉缺血再灌注模型,用地高辛精标记的c—fos探针进行原位杂交。结果示缺血再灌注组栓塞侧皮层及海马c—fos基因表达显著增多,图像分析灰阶值为118.6±5.1,对侧为159.6±3.1(P<0.001),东菱克栓酶组栓塞侧皮层及海马c—fos基因表达亦增多,灰阶为131.0±4.5,对侧为164.6±4.0(P<0.001)。克栓酶组与缺血再灌注组比较,栓塞侧东菱克栓酶组c—fos基因表达显示低于缺血再灌组(P<0.05),而两组栓塞对侧比较无显著差异。本实验表明,脑缺血再灌注后脑组织的c—fos基因表达显著增多,东菱克栓酶能抑制缺血后c—fos基因的表达,这可能是其治疗缺血性脑血管病的机理之一。     

Large-conductance Ca2+-activated K+ channel involvement in suppression of cerebral ischemia/reperfusion injury after electroacupuncture atShuigou (GV26) acupoint in rats

Excess activation and expression of large-conductance Ca2+-activated K+ channels (BKCa channels) may be an important mechanism for delayed neuronal death after cerebral ischemia/reperfusion injury. Electroacupuncture can regulate BKCa channels after cerebral ischemia/reperfusion injury, but the precise mechanism remains unclear. In this study, we established a rat model of cerebral ischemia/reperfusion injury. Model rats received electroacupuncture of 1 mA and 2 Hz atShuigou (GV26) for 10 minutes, once every 12 hours for a total of six times in 72 hours. We found that in cerebral ischemia/reperfusion injury rats, ischemic changes in the cerebral cortex were mitigated after electroacupuncture. Moreover, BKCa channel protein and mRNA expression were reduced in the cerebral cortex and neurological function noticeably improved. These changes did not occur after electroacupuncture at a non-acupoint (5 mm lateral to the left side of Shuigou). Thus, our ifndings indicate that electroacupuncture atShuigou improves neurological function in rats following cerebral ischemia/reperfu-sion injury, and may be associated with down-regulation of BKCa channel protein and mRNA expression. Additionally, our results suggest that theShuigou acupoint has functional speciifcity.     

厄贝沙坦对大鼠局灶性脑缺血再灌注后炎症反应的影响

目的观察厄贝沙坦对大鼠局灶性脑缺血再灌注后脑内及外周炎症反应的影响。方法采用改良Longa方法制备大鼠大脑中动脉阻塞(middle cerebralartery occlusion,MCAO)模型,于缺血90min再灌注后24h和72h进行梗死体积的测量,采用免疫组化和ELISA方法测量脑内和外周血的粘附分子。结果厄贝沙坦可以显著减少局灶性脑缺血再灌注后24h和72h的梗死体积(均P<0.01),改善神经功能(均P<0.01);降低脑内ICAM-1、VCAM-1的表达及其外周血浆中可溶性的形式sICAM-1、sVCAM-1蛋白的水平(均P<0.05)。结论厄贝沙坦可以降低粘附分子的表达,减少梗死体积,改善神经功能,对脑缺血再灌注起保护作用。     

实验性局灶性脑缺血及再灌流后AP-1复合物的结合活性测定

本研究应用凝胶迁移法检测局灶脑缺血区中Ｆｏｓ与Ｊｕｎ基因的蛋白产物形成异源二聚体复合物—转录因子ＡＰ１（ａｃｔｉｖａｔｏｒｐｒｏｔｅｉｎ１）的ＤＮＡ结合活性。缺血３０ｍｉｎ或９０ｍｉｎ伴随再灌流４ｈ，结果表明ＡＰ１结合活性增加。提高的ＤＮＡ结合活性持续２４ｈ。以上结果说明提高转录因子ＡＰ１的表达水平在缺血病变的基因调节改变中起一定作用     

大鼠局灶性脑缺血再灌注后bcl-2蛋白在大脑皮质的表达

目的观察bcl-2蛋白在大鼠局灶性脑缺血再灌注(FCIR)后表达的变化。方法大脑中动脉内线栓法(MCAO)建立缺血再灌注(IR)模型,用免疫组化(SP)法观察bcl-2蛋白不同时间的表达。HE染色观察各个时间点细胞形态学变化。结果脑缺血2h再灌注2hbcl-2表达升高(P<0.01),IR6h达到高峰,IR12h开始下降。结论随着脑缺血再灌注时间延长脑损伤加重,bcl-2蛋白表达减少,bcl-2蛋白表达增加对细胞存亡有重要意义。     

Nimodipine attenuates both increase in cytosolic free calcium and histologic damage following focal cerebral ischemia and reperfusion in cats

To clarify the mechanism of its effect on ischemic stroke, we investigated the effect of nimodipine, a dihydropyridine calcium antagonist, on changes in cytosolic free calcium, cortical blood flow, and histologic changes following focal cerebral ischemia and reperfusion in 14 cats. Using indo-1, a fluorescent intracellular Ca2+ indicator, we simultaneously measured changes in the Ca2+ signal ratio (400:506 nm), reduced nicotinamide adenine dinucleotide fluorescence (464 nm), and reflectance (340 nm) during an ultraviolet excitation (340 nm) directly from the cat cortex in vivo. In six cats treated with vehicle only, the calcium signal ratio increased from 5 minutes after middle cerebral artery occlusion to 30 minutes into reperfusion. The elevation of cytosolic free calcium was significantly attenuated by nimodipine, which was administered by intravenous infusion in eight cats starting 5 minutes after occlusion. Nimodipine had no effect on cortical blood flow during ischemia but induced a hyperperfused state following reperfusion. Nimodipine did not modify changes in the mitochondrial oxidation-reduction state. Nimodipine proved to have beneficial effects on recovery of the electroencephalogram following reperfusion as well as on the extent of focal histologic damage. Our results suggest that nimodipine, when administered during the early stage of focal ischemia, can favorably modify the outcome of stroke by reducing the Ca2+ entry during both the ischemic and reperfusion periods.     

Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats

While the immunosuppressant tacrolimus (FK506) is known to be neuroprotective following cerebral ischemia, the mechanisms underlying its neuroprotective properties are not fully understood. To determine the mode of action by which tacrolimus ameliorates neurodegeneration after transient focal ischemia, we therefore evaluated the effect of tacrolimus on DNA damage, release of cytochrome c, activation of microglia and infiltration of neutrophils following a 60-min occlusion of the middle cerebral artery (MCA) in rats. In this model, cortical brain damage gradually expanded until 24 h after reperfusion, whereas brain damage in the caudate putamen was fully developed within 5 h. Tacrolimus (1 mg/kg) administered immediately after MCA occlusion significantly reduced ischemic damage in the cerebral cortex, but not in the caudate putamen. Tacrolimus decreased both apoptotic and necrotic cell death at 24 h and reduced the number of cytochrome c immunoreactive cells at 8 h after reperfusion in the ischemic penumbra in the cerebral cortex. In contrast, tacrolimus did not show significant neuroprotection for necrotic cell death and reduction of cytochrome c immunoreactive cells in the caudate putamen. Tacrolimus also significantly decreased microglial activation at 8 h and inflammatory markers (cytokine-induced neutrophil chemoattractant and myeloperoxidase [MPO] activity) at 24 h after reperfusion in the ischemic cortex but not in the caudate putamen. These results collectively suggest that tacrolimus ameliorates the gradually expanded brain damage by inhibiting both apoptotic and necrotic cell death, as well as suppressing inflammatory reactions.