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晏义平

摘  要

    谷氨酸神经毒在脑缺血神经元死亡中起重要作用，本研究探索谷氨酸载体在脑缺血及电针对脑缺血性神经元死亡保护中的作用，结果如下：（1）谷氨酸载体摄取抑制剂L-trans-PDC 对脑缺血神经元死亡的影响：侧脑室注射生理盐水组的脑梗塞体积为1.027±0.516 mm³, 侧脑室注射L-trans-PDC 1μg、 2μg 和5μg组的脑梗塞体积分别为1.23±0.3 mm³ 、 1.97±0.451mm³和2.24±0.69mm³ 。与侧脑室注射生理盐水组相比, 侧脑室注射L-trans-PDC 1μg和2μg组的脑梗塞体积有增大趋势，但无显著性差异(P>0.05), 侧脑室注射L-trans-PDC 5μg组的脑梗塞体积显著增大(P<0.05)。结果表明,在脑缺血状态下，抑制谷氨酸载体摄取加重脑缺血神经元死亡。（2）L-trans-PDC对大鼠脑血流量的影响：大鼠侧脑室注射生理盐水、L-trans-PDC 1μg、2μg和5μg后15min，脑血流量无显著变化，脑缺血后10-120min，四组大鼠脑血流量都下降至缺血前基础值的50%左右，与侧脑室注射生理盐水组相比，侧脑室注射L-trans-PDC 1μg、2μg和5μg对正常大鼠以及大鼠脑缺血后脑血流量无显著影响（P>0.05)，表明L-trans-PDC加重脑缺血神经元死亡与脑血流量无关。（3）脑缺血后谷氨酸载体GLAST mRNA表达变化：脑缺血后3小时，大脑皮层缺血边周区GLAST mRNA表达无显著变化，缺血后12小时，表达显著降低（P<0.05)，缺血后24小时，表达回升至假手术组水平，缺血后72小时, 表达显著增加（P<0.05)。（4）GLAST mRNA表达的细胞定位：GLAST mRNA与神经元特异性烯醇化酶（NSE）或GFAP都有表达共存，说明GLAST mRNA在神经元和神经胶质细胞中均有表达，缺血后72小时，缺血边周区的部分神经元和增生的神经胶质细胞中GLAST mRNA表达都增加。（5）谷氨酸载体摄取抑制剂减弱电针对缺血性神经元死亡的保护作用：与单纯缺血组相比，缺血+电针组的脑梗塞体积显著减小(P<0.05)。缺血+L-trans-PDC 1μg 和缺血+电针+L-trans-PDC 1μg组的脑梗塞体积与单纯缺血组之间无显著性差异（P>0.05)。结果表明，抑制谷氨酸载体摄取减弱电针对缺血性神经元死亡的保护作用。（6）电针对大鼠脑内谷氨酸载体GLAST mRNA表达的影响：与假手术组相比，单纯电针组大鼠脑内GLAST mRNA表达无显著性变化，脑缺血后12小时，大脑皮层缺血区边缘锥体细胞层GLAST mRNA表达显著降低（P<0.05)，在缺血后12h+电针组，大脑皮层缺血区边缘锥体细胞层GLAST mRNA表达与缺血后12h组相比，有显著性差异（P<0.05)。结果表明，电针对正常大鼠脑内GLAST mRNA表达无显著影响，但可拮抗脑缺血后12小时缺血边周区GLAST mRNA表达下降。（7）电针对大鼠脑血流量的影响：电针对正常大鼠脑血流量无显著影响，在脑缺血状态下，电针可以改善大鼠脑缺血后的脑血流量，但作用不持久，电针停止后，电针对脑血流量的调节作用消失。（8）电针对脑缺血后神经细胞凋亡的影响：在假手术组和单纯电针组，脑内未见TUNEL染色阳性细胞。与缺血后12h组相比，缺血后12h+电针组梗塞区内TUNEL染色阳性细胞显著减少。结果表明，电针使脑缺血后神经细胞凋亡减少。实验结果提示：（1）谷氨酸载体在限制脑缺血后谷氨酸神经毒中起重要作用，脑缺血后细胞外液谷氨酸浓度升高可能与缺血早期谷氨酸载体GLAST mRNA表达降低，谷氨酸摄取下降有关；脑缺血后72小时，缺血边周区的部分神经元和增生的神经胶质细胞中GLAST mRNA表达都增加，可能为脑缺血后机体的一种代偿机制，以减轻谷氨酸的神经毒作用。（2）电针对缺血性神经元死亡的保护作用可能与电针激活谷氨酸载体功能有关。

关键词：脑缺血 谷氨酸载体 脑血流量 电针 凋亡

Introduction of the Author: Yan Yi-Ping was born on Nov 1.1964. Under the guidance of Prof. Zhang An-Zhong, he was awarded the Dr.degree of philosophy at Shanghai Medical University on July, 1997. Now he continues his research work in the field of neuroscience.

Title of the Dr. Degree’Thesis: Roles of glutamate transporter in the pathogenesis of cerebral ischemia and the protective effects of electro-acupuncture on ischemic neuronal damage

Key Words: glutamate transporter, cerebral ischemia, neurotoxicity

Abstract: Neurotoxcity of glutamate plays an important role in ischemic neuronal death. In this study, we investigated the roles of glutamate tramsporter in the pathogenesis of cerebral ischemia and in the protective effects of EA on ischemic neuronal damage. The results were as follows: (1) Effect of L-trasns-PDC, a glutamate transporter uptake Inhibitor, on ischemic neuronal death: The infarction volume was 1.027±0.516 mm³ in the ischemia+saline group. In the rats pretreated with L-trans-PDC 1μg or 2μg, There was a trend toward an incresed infarction volume (1.23±0.3mm³ and 1.97±0.451mm³, respectively, P>0.05 Vs the ischemia+saline group). L-trans-PDC at a dose of 5μg significantly increased the infarction volume to 2.24±0.89mm³ (P<0.05 Vs the ischemia+saline group). The results showed that L-trans-PDC dose-dependently increased the infarction volume induced by thrombotic focal cerebral ischemia. (2) Effects of L-trans-PDC on cerebral blood flow (CBF) in rats: CBF did not change significantly at 15min after injection(icv) of 5μl saline, L-trans-PDC 1μg, 2μg or 5μg. Following ischemic insult, CBF decreased to 50% of  baseline from10min to 120min postischemia, respectively. There were no significant differences in CBF among four groups. The results showed that injection(icv) of L-trans-PDC 1μg, 2μg or 5μg did not significantly affect CBF in rats with or without brain ischemia. This indicated that the aggravating effect of L-trans-PDC on infarction volume was not related to CBF. (3) Change of  glutamate transporter GLAST mRNA expression in the brain following focal ischemia: GLAST mRNA expression did not significantly change at 3h postischemia, decreased obviously at 12h postischemia ischemia (P<0.05 Vs sham-operated group ), recovered to the level of the sham-operated group at 24h postischemia and significantly increased at 72h following focal ischemia (P<0.05 Vs sham-operated group ). (4) Cellular localization of GLAST mRNA expression: Double staining showed that GLAST mRNA co-expressed with neuron specific enolase(NSE) or GFAP. The expression of GLAST mRNA at 72h after ischemia increased in some neurons and proliferated glial cells in the area penumbra of cerebral cortex. (5) Inhibition of glutamate uptake decreases the protective effects of EA on ischemic neuronal damage: Compared with the control ischemia group, the infarction volume did not significantly increase in the ischemia+ L-trans-PDC 1μg group, decreased significantly in the ischemia+EA group. There was no difference between the control ischemia group and the ischemia+EA+L-trans-PDC 1μg group. The results indicated that inhibition of glutamate transporter uptake attenuated the protective effects of EA on ischemic neuronal damage. (6) Effects of EA on GLAST mRNA expression in rat brain: Compared with the sham-operated group, the expression of GLAST mRNA expression in brain did not change significantly in EA group. In the 12h postischemia group, the expression of GLAST mRNA expression in pyramidal cell layer of cerebral cortex below the infarcted area decreased significantly (P<0.05 Vs the sham-operated group). In the 12h postischemia+EA group, the expression of GLAST mRNA expression in pyramidal cell layer of cerebral cortex below the infarcted area was higher than that in the 12h postischemia group(P<0.05 ). The results showed that EA did not affect the GLAST mRNA expression in normal rat brain, but increased the GLAST mRNA expression in the penumbra area following focal ischemia. (7) Effects of EA on CBF in rats: EA did not significantly affect CBF in normal rats, but improved CBF in the ischemic rats. The effects of EA on CBF was transient. After the cease of EA treatment, the effect of EA on CBF disappeared. (8) Effects of EA on apoptotic neuronal death induced by focal ischemia: In the sham-operated group and the EA group, TUNEL positive stained cell was not detected in the brain. Comparing with 12h postischemia group, the number of TUNEL positive stained cells significantly decreased in the 12h postischemia+EA group. The results showed that EA reduced apoptotic neuronal death induced by focal ischemia. The results of this study suggest that (1) Glutamate transporter plays an important role in limiting glutamate neurotoxicity following cerebral ischemia. Decrease of GLAST mRNA expression during the early period following cerebral ischemia may be related to the elevation of extracellular glutamate concentration. The increase of GLAST mRNA expression in the penumbra area at 72h postischemia possibly represent a protective mechanism for increasing extracellular glutamate uptake and limiting glutamate neurotoxicity. (2) The protective effects of EA on ischemic neuronal damage may be related to the increase of glutamate uptake induced by EA.