Mechanisms of cell in following brain ischemia and reconstruction of disturbed function by neural graft

本实验采用大鼠大脑中动脉缺血再灌注模型，探讨缺血性细胞死亡的机制，并尝试通过神经移植重建受损的脑功能。1. mu-钙蛋白酶的分布、激活和神经死亡我们制备了一种抗mu-钙蛋白酶N端乙酰化肽段的抗体，该抗体识别mu-钙蛋白酶N端缺失前的酶原。用该抗体检测发现，在CNS和PNS神经元中，μ-calpain酶原均大量分布于细胞核，少量分布于细胞质，除红细胞外，其它细胞均无分布。在缺血/再灌注的极早期（0- 30 min），N端肽段缺失，酶原转化为活性形式（钙蛋白酶激活），MAP 2免疫反应消失，细胞骨架损伤，嗜银暗神经元出现。经pSV.SPORT1载体转导MAP 2C cDNA的COS 7细胞，其MAP 2-和微管蛋白表达阳性，对无糖/无氧环境产生耐受性，不嗜银。2.血脑屏障功能障碍再灌注后2 ~ 3 h，可见星形胶质细胞和血脑屏障损伤。检测到IgG、补体因子（如C3 b）等渗出，提示脑免疫活性和细胞毒作用增强。地塞米松治疗可抑制IgG的外渗，并使后者的梗死范围缩小。3.神经移植重建受损的脑功能在梗死纹状体内移植胎儿纹状体细胞可减少安非他明引起的旋转次数。采用罗丹明荧光染色法检测移植物。移植细胞在施加电刺激（串刺激）后增加宿主纹状体或皮质中的Fura-2荧光，其指示[Ca^++]_i，表明来自宿主脑的功能输入到移植物。这可能是神经移植后功能改善的部分原因。

Using model rats with ischemia/reperfusion in the middle cerebral artery, we investigated the mechanism of ischemic cell death and tried to reconstruct disturbed brain function by neural graft.1.Distribution of mu-calpain, its activation and neroral deathWe raised an antibody against acetylated N-terminal peptides of mu-calpain 80 KD.This antibody recognized mu-calpain proenzyme before deletion of the N-terminal. Using this antibody we found that mu-calpain proenzyme distributed very heavily in the nucleus and in lesser amount in the cytoplasm of neurons in both CNS and PNS.It does'nt distribute in any other cells except red blood cells. In the very early stage (0-30min) of the ischmia/reperfusion, the N-terminal peptides were deleted, proenzyme was transformed to an active form (calpain activation), MAP2 immunoreaction was disappeared, cytoskeleton damage was detected, and argyrophilic dark neurons were detected. COS7 cells that were transduced MAP2C cDNA by pSV.SPORT1 vecter bore MAP2-and tubulin positive processes, became resistant to glucose/oxygen-free situation, and were never argyrophilic.2.Dysfunction of the BBBTwo to three hours after reperfusion, damages in astrocytes and BBB were detected. The extravasation of IgG,complement factor (such as C3b) etc.was detected, suggesting the elevation of brain immune activity and cytotoxicity. Treatment with dexamethasone suppressed the extravasation of IgG and made the latter infarction smaller.3.Reconstruction of disturbed brain function by neural graftFetal striatal cell grafts in infarct striatum reduced the number of amphetamine-induced rotations. The grafts were detected by preloaded rhodamine fluorescene. The graft cells increased fura-2 fluorescene that indicated [Ca^<++>]_i after application of electrical stimuli (train stimuli) in the host striatum or cortex, suggesting the functional inputs to the graft from the host brain. This may in part underlie the functional amelioration following neural graft.