大鼠脑缺血区细胞间粘附分子ICAM-1表达和LFA-1阳性细胞浸润的研究

研究粘附分子和白细胞与脑缺血／再灌流损伤的病理联系,运用原位杂交和免疫组化技术对36只SD大鼠脑缺血区细胞间粘附分子（ICAM－1）表达和淋巴细胞机能相关抗原（LFA－1）阳性细胞浸润进行了观察。结果显示,脑缺血区的毛细胞血管内皮细胞表达ICAM－1 mRNA发生于脑缺血1h,在脑缺血1h／再灌流8h达到高峰。而脑缺血区毛细血管ICAM－1蛋白质的表达则发生于脑缺血1h／再灌流2h,高峰出现于脑缺血1h／再灌流16h,LFA－1阳性细胞在脑缺血区的聚集发生在脑缺血1h,并随再灌流时间延长,其聚集数量逐渐增加。结果提示,脑缺血／再灌流能诱导缺血区的血管内皮细胞表达ICAM－1 mRNA和蛋白质,进而导致白细胞在脑缺血区的浸润,此可能是脑缺血／再灌流损伤的病理机制之一。     

大鼠脑缺血—再灌注损伤细胞间粘附分子—1表达的研究

本文用暂时性脑缺血的大鼠模型对血管内皮细胞间粘附分子１（ＩＣＡＭ１）的表达进行了研究。免疫组化显示,ＩＣＡＭ１的表达于缺血１ｈ再灌６ｈ后明显升高;激光共聚焦扫描显微镜定量检测说明再灌后其表达量比单纯缺血增加了４７％。脑组织过氧化物酶ＭＰＯ（μ／ｇ）含量检测及光镜观察均证实再灌后白细胞在缺血区聚集增多。局部脑组织ＩＬ１含量（ＯＤ值／ｇ）在灌流３ｈ后明显升高。结果说明：①脑缺血再灌注损伤时血管内皮细胞ＩＣＡＭ１的表达为时间依赖性增加;②ＩＣＡＭ１的调节表达与脑损伤时局部细胞因子ＩＬ１的分泌有关;③再灌注后脑缺血区有白细胞的大量聚集;④ＩＣＡＭ１表达量的增加是白细胞在缺血区粘附到血管壁、游出血管损伤周围脑组织的前提条件。     

电针对脑缺血/再灌注模型大鼠双侧脑区IL-1β、ICAM-1表达的影响

目的探讨脑缺血/再灌注损伤大鼠大脑健、患侧白介素-1β(IL-1β)和细胞间粘附分子-1(ICAM-1)的变化趋势及电针对二者的影响。方法采用大脑中动脉线栓法制备大鼠脑缺血/再灌注模型(MCAO)。将大鼠随机分为空白组、模型组和电针组,造模后各组又分为6个时程组(12、24、48、72、96、144 h),每个时程组10只大鼠。取脑组织,制备冰冻切片,免疫组化检测健、患侧脑组织中IL-1β、ICAM-1的表达情况。结果在脑缺血大鼠患侧与健侧脑区,IL-1β和ICAM-1的表达均呈现典型的双峰模式。模型组患侧IL-1β在12 h和48 h到达峰值,健侧在12 h和144 h到达峰值。模型组患侧IL-1β表达在48 h明显高于健侧(P0.05),在96 h和144 h又明显低于健侧(P0.05);电针组患侧IL-1β表达在24 h、48 h和144 h均低于健侧(P0.05)。模型组患侧的ICAM-1在24 h和72 h达到峰值,健侧在24 h和144 h到达峰值;电针组患侧ICAM-1表达在各时间点均低于健侧(P0.05)。结论针刺可能通过降低大鼠患侧脑组织中IL-1β和ICAM-1的表达抑制炎症反应,从而改善脑缺血/再灌注对脑组织的损伤。     

N-乙酰半胱氨酸对大鼠肝脏缺血再灌注损伤NF-κ B和ICAM-1表达的影响

目的：探讨大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达情况及NAC的保护作用机制。方法：45只雄性SD大鼠随机分成三组：假手术组（Sham组,n=5）;缺血再灌注损伤组（I/R组,n=20）缺血60min后分别再灌注1、3、6、12h;N-乙酰半胱氨酸组（NAC组,n=20）：先自阴茎背静脉给大鼠注射溶于生理盐水的NAC,20min后再按I/R组处理。在各规定的再灌注时间点,分别采用western-blot和免疫组化方法测定肝组织中NF-κB和ICAM-1的表达。结果：I/R组和NAC组再灌注1、3、6、12h后,NF-κB的表达均明显高于Sham组（p〈0.01）,于再灌注3h达到高峰;ICAM-1的表达均明显高于Sham组（p〈0.01）,于再灌注6h达到高峰。NAC组再灌注1、3、6h与I/R组相同时间点比较：NF-κB和ICAM-1的表达均低于I/R组（p〈0.05）。NAC组再灌注12h与I/R组相同时间点比较：NF-κB和ICAM-1的表达虽然在数值上有所减少,但统计学上无差异（p〉0.05）。结论：大鼠肝脏缺血再灌注后NF-κB和ICAM-1表达增加,NAC可抑制NF-κB激活,减少ICAM-1表达减轻大鼠肝脏缺血再灌注损伤。     

人参皂甙Rg1对脑缺血再灌注大鼠脑组织Bax和Bcl-2蛋白表达的影响

目的探讨人参皂甙Rg1对脑缺血再灌注大鼠脑组织Bcl-2和Bax表达的影响及其意义。方法分别给大鼠腹腔注射人参皂甙Rg1 10、20、40 mg/kg,采用大脑中动脉闭塞方法建立脑缺血再灌注模型,观察大鼠脑缺血再灌注后不同时间段（2 h、24 h）神经功能缺损评分;应用免疫组化法检测脑组织缺血再灌注24h后Bcl-2、Bax的表达。结果人参皂甙Rg1组大鼠脑缺血后各时间点神经功能缺损评分显著低于单纯缺血再灌注组（P〈0.05）;与单纯缺血再灌注组相比,人参皂甙Rg1各组Bcl-2表达显著增高,Bax表达显著降低,Bcl-2/Bax比值显著上调（P〈0.05）。结论人参皂甙Rg1防治大鼠脑缺血再灌注损伤的机制可能与促进脑组织Bcl-2表达、抑制Bax表达有关,且以高剂量效果较好。     

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。     

大鼠局灶性脑缺血后一氧化氮合酶基因表达的变化

目的:观察大鼠局灶性脑缺血后3种类型一氧化氮合酶(NOS)mRNA表达的变化.方法:大鼠随机分为正常对照组、缺血后2、6、12、24 h组,以逆转录-聚合酶链反应(RT-PCR)法分别检测缺血脑组织NOS基因表达的变化.结果:脑缺血后eNOS、nNOSmRNA表达增强,分别于2、6 h达高峰;iNOS mRNA表达亦增高,但在缺血后12 h达高峰.结论:大鼠脑缺血早期eNOS和nNOS占主要地位,缺血后期iNOS占主要地位.     

当归对大鼠缺血性脑损伤后Flt-1、Flk-1 mRNA表达的影响

目的：研究大鼠缺血性脑损伤后不同时间点Flt-1、Flk-1 mRNA的表达及当归对其表达的影响。方法：雄性Wistax大鼠,随机分为缺血损伤组和当归治疗组。采用线栓法制作大鼠短暂性大脑中动脉阻断（MCAO）与再灌模型。治疗组腹腔注射当归注射液（剂量5g／kg）。36只大鼠（每组各18只）在脑缺血／再灌后1d、3d、7d神经行为学评分完成后被处死,取大脑行氯化三苯四唑（TTC）染色以测脑梗死比;另取72只大鼠（每组各36只）在脑缺血／再灌后3h、6h、12h、1d、3d、7d分别被处死,应用半定量逆转录聚合酶链反应（RT-PCR）技术检测缺血侧Flt-1、Flk-1 mRNA的表达。结果：在同时间点神经功能缺损评分比较,缺血损伤组明显高于当归治疗组（P〈0．05）;在同时间点当归治疗组梗塞比明显小于缺血损伤组（P〈0．01）。RT-PCR检测表明,缺血损伤组Flt-1、Flk-1 mRNA在缺血／再灌后3h即开始表达增强,于3d达高峰,后逐渐降低;当归治疗组Flt-1、Flk-1 mRNA表达比缺血损伤组明显增加,于3d达到高峰后缓慢降低至第7d仍保持较高水平。缺血损伤组和当归治疗组中Flt-1 mRNA与Flk-1 mRNA的表达呈正相关性,相关系数为r=0．957（P〈0．01）。结论：当归可增强缺血性脑损伤后Flt-1、Flk-1 mRNA表达。Flt-1、Flk-1 mRNA的表达紧密相关。     

N-乙酰半胱氨酸对大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达的影响

目的:探讨大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达情况及NAC的保护作用机制.方法:45只雄性SD大鼠随机分成三组:假手术组(Sham组,n=5);缺血再灌注损伤组(I/R 组,n=20)缺血60min后分别再灌注1、3、6、12h;N-乙酰半胱氨酸组(NAC组,n=20):先自阴茎背静脉给大鼠注射溶于生理盐水的NAC,20min后再按I/R组处理.在各规定的再灌注时间点,分别采用western-blot和免疫组化方法测定肝组织中NF-κB和ICAM-1的表达.结果:I/R组和NAC组再灌注1、3、6、12h后,NF-k B的表达均明显高于Sham组(p＜0.01),于再灌注3h达到高峰;ICAM-1的表达均明显高于Sham组(p＜0.01),于再灌注6h达到高峰.NAC组再灌注1、3、6h与VR组相同时间点比较:NF-k B和ICAM-1的表达均低于I/R组(p＜0.05).NAC组再灌注12h与I/R组相同时间点比较:NF-K B和ICAM一1的表达虽然在数值上有所减少,但统计学上无差异(p＞0.05).结论:大鼠肝脏缺血再灌注后NF-κB和ICAM-1表达增加,NAC可抑制NF-k B激活,减少ICAM-1表达减轻大鼠肝脏缺血再灌注损伤.     

全脑缺血/再灌注大鼠海马PPARγ mRNA表达变化

目的 观察大鼠全脑缺血/再灌注损伤后海马PPARγ mRNA表达的动态变化.方法 采用夹闭双侧颈总动脉,颈总静脉抽血后回输建立大鼠全脑缺血/再灌注损伤模型.Morris水迷宫检测大鼠空间定向能力变化,HE染色观察海马病理组织学改变及RT-PCR法检测缺血再灌注后不同时间点PPARγ mRNA的表达变化.结果 全脑缺血/再灌注损伤导致大鼠空间学习及记忆能力明显下降,海马神经元出现明显的核固缩和细胞丢失.PPARγ mRNA的表达先升高后降低,以缺血/再灌注后48 h表达水平最高,30 d后接近正常水平.结论 全脑缺血/再灌注损伤大鼠海马组织中PPARγ mRNA的表达,在再灌注30 d内明显增加,表达高峰在48 h.     

大鼠脑缺血再灌注血管壁NOS和ICAM-1的表达

一氧化氮（NO）和一氧化氮合酶（NOS）与脑血管功能有重要关系,细胞间粘附分子1（ICAM－1）可由脑缺血／再灌注诱导产生并与脑组织损伤密切相关,本实验用免疫组织化学和NADPH－d酶组织化学方法,观察了SD大鼠实验性脑缺血再灌注内皮细胞ICAM－1和NOS的表达,结果显示正常对照组大鼠脑血管ICAM－1免疫组织化学显色为阴性或弱阳性反应,再灌注2h,ICAM－1阳性反应明显增强,与对照组相比,P＜0.01。随再灌注至16h,ICAM－1表达增加近一倍。脑缺血1h缺血侧侧脑血管壁开始出现NOS的阳性表达,与对照组相比,P＜0.01,再灌注2h,NOS表达最多,随后逐渐下降,结果提示脑缺血再灌注与ICAM－1和NOS表达升高有关。     

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.     

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。     

脑缺血损伤时脑片细胞内Ca2+及脂质过氧化物的测定

用插细法制作局灶性脑缺血／再灌损伤模型,激光共聚焦扫描显微镜观察活体脑片细胞内Ｃａ＾２＋的分布及脂质过氧化物水平的动态变化,结果表明：缺血１ｈ时只有纹状体Ｃａ＾２＋含量增加,而缺血４ｈ时梗塞侧皮质、纹状体区域的Ｃａ＾２＋与脂质过氧化物含量均进一步增高,且与缺血时间相关,而与再灌持续时间无明显关系;轻度缺血／再灌损伤时细胞内脂质过氧化物增加的发生较增钙反应为早;纹状体对缺血／再灌损伤比皮质敏感。     

AMD3100对局灶脑缺血/再灌注大鼠大脑皮质血管再生的影响

目的探讨AMD3100阻断SDF-1/CXCR4轴后,对局灶脑缺血/再灌注大鼠缺血半暗带血管再生的影响。方法将SD大鼠随机分为假手术组(S组)、模型组(IR组)、AMD3100组(IRA组)、生理盐水组(IRN组)。采用线栓法制备大鼠局灶脑缺血/再灌注模型,缺血2h后将IR、IRA和IRN组分为再灌注12h,1、3和7d四个亚组。HE染色观察局灶脑缺血/再灌注后大脑皮质病理变化。免疫组化法检测CD31在缺血半暗带表达。荧光定量PCR检测外周血中AC133mRNA表达。结果与IRN组比较,IRA 12h外周血中AC133mRNA显著升高,第1d升高达峰值(P0.01),IRA 3dAC133mRNA表达比IRA1d显著减少(P0.05);与IRN组比较,IRA组CD31阳性血管密度在第1d无显著变化(P0.05),第3和7d血管密度显著减少(P0.01);IRA 7d梗死区由大量坏死神经细胞和泡沫细胞填充,坏死较严重。结论持续注射AMD3100能动员干/祖细胞快速进入外周血,但可能抑制局灶脑缺血/再灌注大鼠缺血半暗带血管再生,加重梗死区坏死。     

Decreased expression of vesicular GABA transporter, but not vesicular glutamate, acetylcholine and monoamine transporters in rat brain following focal ischemia

In nerve terminals, vesicular transporters pack neurotransmitters into synaptic vesicles, which is an essential prerequisite for transmitter release. To date, three distinct families of vesicular transporters have been identified which are specific for (a) excitatory amino acids (glutamate and aspartate), (b) inhibitory amino acids (GABA and glycine) and (c) acetylcholine and monoamines. The present study evaluated the effect of transient focal cerebral ischemia on the expression of these vesicular transporters in adult rat brain. Ischemia was induced by a 1 h transient middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. At various reperfusion periods (3-72 h), mRNA levels of the vesicular transporters were estimated in the contralateral and the ipsilateral cerebral cortex by real-time PCR analysis. Following transient focal ischemia, mRNA expression of the vesicular GABA transporter (VGAT) decreased significantly by 3 h of reperfusion and remained at a significantly lower level than sham until at least 72 h of reperfusion. Western blotting showed a significant decrease in the VGAT immunoreactive protein levels in the ipsilateral cortex of rats subjected to focal ischemia and 24 h reperfusion. Immunohistochemistry demonstrated many VGAT immunopositive puncta in the contralateral cortex, which were significantly decreased in the ipsilateral cortex at 24 h reperfusion. Focal ischemia had no effect on the mRNA levels of the vesicular transporters specific for glutamate/aspartate, acetylcholine and monoamines at either 6 h or 24 h of reperfusion.     

Immunohistochemical and in situ hybridization studies on glycine transporter 1 after transient ischemia in the rat forebrain

Glycine is a critical factor in ischemia as reduced astrocytic and increased extracellular glycine levels aggravate the neurotoxic effect of glutamate and consequently, increase the extent of brain damage. Extracellular levels of glycine are primarily regulated by the plasma membrane glycine transporter 1. In the present study, we examined the effects of transient ischemia (1 h occlusion of the middle cerebral artery; followed by 0 h, 0.5 h, 1 h, 2 h, 4 h, 24 h or 48 h reperfusion) on immunoreactivity and mRNA expression of glycine transporter 1 in the rat forebrain. In control animals, glycine transporter 1-immunoreactivity was strong in diencephalic and certain telencephalic structures, moderate in the globus pallidus, and rather low in the cortex and striatum. In situ hybridization studies revealed a similar distribution pattern of glycine transporter 1 mRNA expression. One hour occlusion of the middle cerebral artery resulted in a significant decrease in ipsilateral glycine transporter 1-immunoreactivity and mRNA expression in a circumscribed region of the preoptic/hypothalamic area; both the immunoreactivity and mRNA exhibited further reductions with increasing reperfusion time. In contrast, the cerebral cortex and the globus pallidus showed an increase of glycine transporter 1-immunoreactivity after 0.5 h reperfusion; the elevation proved to be transient in the somatosensory cortex and remained sustained in the globus pallidus after longer reperfusion times. Western blot analysis of globus pallidus samples from the ipsilateral side confirmed higher glycine transporter 1 protein levels. These results suggest an elevated expression of the transporter protein facilitating the glial uptake of glycine from the extracellular space. However, glycine transporter 1 mRNA expression was not significantly different in the penumbra regions from the corresponding contralateral sites of the injury. Together, these findings indicate that post-translational mechanisms are of primary importance in elevating glycine transporter 1 protein levels following transient ischemia.     

Dynamic Expression Pattern of Neuro-oncological Ventral Antigen 1 (Nova1) in the Rat Brain after Focal Cerebral Ischemia/Reperfusion Insults

The present study aimed to evaluate the expression of neuro-oncological ventral antigen 1 (Nova1) in cerebral ischemia/reperfusion (I/R) insults by immunohistochemistry. The focal cerebral I/R model was induced by right middle cerebral artery occlusion (MCAO) for 120 min followed by 1 day, 7 days, and 14 days of reperfusion in Sprague-Dawley (SD) rats. The results showed that Nova1 was expressed in nearly the whole brain, although with higher density in hippocampus, hypothalamus, cingulate cortex, and medial habenular nucleus. The immunoreactivity of Nova1 neurons was increased dramatically, especially on both sides of the hippocampal CA₁ region, after 1 day of reperfusion. A strong response occurred at the ipsilateral CA₁ region between 1 day and 7 days of reperfusion. Likewise, strong compensatory responses of Nova1 expression were observed on the contralateral side of the striate cortex, dentate gyrus, and hypothalamus. Interestingly, more Nova1 neurons were observed to translocate to the dendrites and growth cones of the axons in the hypothalamus on the ischemic side after 7 days of reperfusion. In conclusion, our data suggest that Nova1 might mediate neuronal responsiveness, and its expression might positively correlate with neural repair after I/R insults in the rat brain.