摘要
目的:探讨经侧脑室给予蛇毒型神经生长因子(vNGF)对脑缺血再灌注损伤后大鼠内源性神经前体细胞增殖与迁移的影响。
方法:取健康清洁级雄性Wistar大鼠,按随机分组原则分为2天组(N=30)、7天组(N=30)、14天组(N=30)。每个时间点再分为五个亚组vNGF 25u、vNGF 50u、vNGF 100u、生理盐水对照组、以及假手术组,每个亚组6只大鼠。生理盐水对照组和vNGF组大鼠采用大脑中动脉线栓法建立局灶性脑缺血一再灌注损伤模型,术后各时间点vNGF亚组在规定的时间段每日采用改良的侧脑室置管术法给予相应剂量蛇毒型神经生长因子,生理盐水对照组给予等渗盐水。在术后每个时间点对脑缺血再灌注损伤后大鼠神经功能恢复的程度进行神经功能评分。采用免疫组织化学法测定术后第2天、第7天以及第14天缺血皮质周围和海马CA3/DG区神经前体细胞标志物Doublecortin(DCX)阳性细胞数以及细胞信号调节激酶(ERK)阳性细胞数
结果:1、与对照组相比,经侧脑室给予蛇毒型神经生长因子各组第2天开始,大鼠神经功能就显著恢复。各组比较经分析,差异有统计学意义(P<0.05)。显示了外源性补充蛇毒型神经生长因子的明显正性干预作用。假手术组大鼠无缺血再灌注损伤。2、DCX免疫组织化学结果显示:经侧脑室给予vNGF各亚组DCX阳性细胞在缺血皮质周围和海马CA3/DG区第2天就开始增加,第7天达到高峰,第14天有所下降。与相应时间点对照组相比,呈明显增高趋势。各时间点中vNGF各亚组以vNGF50u表达最多呈现出vNGF干预的量效关系;其中以第7天vNGF 50uDCX阳性细胞数目最高,与第2、第14天vNGF 50u相比,差异明显(P<0.03)呈现出vNGF干预的时效关系。3、ERK免疫组织化学结果显示:经侧脑室给予vNGF各亚组ERK阳性细胞在缺血皮质周围和海马CA3/DG区第2天表达为高峰,第7天明显下降,第14天恢复基线水平。与相应时间点对照组相比,呈明显下降趋势。对照组中ERK表达水平,初步体现了ERK的表达趋势。各时间点中vNGF各亚组以vNGF50u表达最少,以第7天vNGF 50u为著,呈现出vNGF负性干预的量效关系。
结论:1、脑缺血再灌注损伤可以诱导神经前体细胞在缺血皮质周围及海马CA3/DG增殖,同时还可以激活ERK信号转导途径。2、经侧脑室给予蛇毒型神经生长因子可以有效逆转脑缺血再灌注损伤后神经生长因子匮乏局面,并促进缺血皮质周围及海马CA3和齿状回神经前体细胞增殖与迁移。3、ERK阳性细胞在各组间的表达差异,提示了NGF可以负性调节ERK而发挥促进血管再生和神经发生的作用。4、ERK与DCX阳性细胞在相同时间点相同部位相同干预措施的情况下,表达的明显差异,提示ERK可能对DCX增殖和迁移有一定影响。
Objective:To investigate the effect of intracerebroventricular administra tion of vevom nerve growth factor on proliferation and migration of endogenous neural progenitor cells in rats with cerebral ischemia/reperfusion injury.
Methods:Wistar rats were randomly divided into three groups:2 days group, 7 days group and 14 days group (n= 30 in each group), then each group randomly assigned to vNGF 25u, vNGF 50u, vNGF 1OOu, sham-operation, and ischemia/reperfusion control subgroups (n=6 in each subgroup). After that a focal cerebral ischemia/reperfusion model was induced by middle cerebral artery intraluminal suture occlusion (MCAO) method in the I/R control and vNGF subgroups. Normal saline or Different concentrations vNGF was administered via intracerebroventricular to the I/R control or vNGF subgroups rats per day according to the corresponding time points. After cerebral ischemia /reperfusion injury was established, the degree of neurological functional recovery of rats was assessed by longa score, and the numbers of positive cells of DCX+ cells and ERK+ cells in the Peri-ischemic cortex and Hippocampus CA3/DG were assessed by immunohistochemical method in the corresponding time points.
Result:1, As compared with control subgroups, snake venom nerve growth factor subgroups which was administered via intracerebroventricular, the neurological function began to recover in the first two days. Each subgroups by statistical analysis, the statistically difference was significant (P<0.05), which showed its positive intervention effects markedly. However, there were no ischemia/reperfusion injury in Sham-operated subgroups.The results of DCX immuno-histochemistry showed that:the numbers of DCX positive cells in Peri-ischemic cortex and Hippocampus CA3/DG of snake venom nerve growth factor subgroups began to increase at 2 days after the procedures, reach the peak at 7 days, the first decline in 14 days. As compared with the I/R control subgroups, the trend was markedly higher. The vNGF50u subgroups expressed much more DCX+ cells than other subgoups at the corresponding time points, which showed its positive intervention of dose-response effects markedly; Among them, the numbers of DCX positive cells vNGF 50u subgroup in the 7th day were significantly higher than those vNGF 50u subgroups in 2 day or 14 day, which showed its positive intervention of aging-response effects markedly. The differences aresignificant (P<0.03).3、The results of ERK immunohisto-chemistry showed that:the numbers of ERK positive cells in Peri-ischemic cortex and Hippocampus CA3/DG of snake venom nerve growth factor subgroups reach the peak at 2 days after the procedures, began to decline at 7 days, then restored baseline levels at 14 days. As compared with the I/R control subgroups, snake venom nerve growth factor subgroups showed a clear down-ward trend. The vNGF50u subgroups expressed much less ERK positive cells than other subgoups at the corresponding time points, Especially at the 7days, which showed its negative intervention of dose-response effects markedly.
Conclusions:1, The cerebral ischemia/repufusion injury can induce proliferation and migration of endogenous neural progenitor cells in the Peri-ischemic cortex and Hippocampus CA3/DG.2, Exogenous snake venom nerve growth factor can effectively compensate for the insufficient of endogenous nerve growth factor after cerebral ischemia/repufusion injury, as well as facilitate proliferation and migration of endogenous neural progenitor cells in the Peri-ischemic cortex and Hippocampus CA3/DG.3,The significant differences expression levels of ERK-positive cells among the three groups, suggesting that NGF may play a negative regulator in ERK, which provide a possible theoretical source for NGF on vascular regeneration and the role of neurogenesis.4,The significant differences expression of ERK and DCX-positive cells in the Peri-ischemic cortex and Hippocampus CA3/DG suggesting that there maybe had an association between ERK and the prolife-ration and migration of the DCX after reperfusion.
引文
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