SV2A配体抑制CD8＋T淋巴细胞脱颗粒对急性脑缺血神经保护作用研究

An accumulating body of evidence from experimental studies supports a definite neurotoxic role of CD8＋ T lymphocytes and a potential neuroprotective effect of SV2A ligands in cerebral ischemia-reperfusion (I/R) injury, but the neuroprotective mechanism of SV2A remains enigmatic. Recently, we found that the anticonvulsant levetiracetam showed an attenuating effect on degranulation of CD8+ T lymphocytes and SV2A exist in CD8+ T lymphocytes. Thus, we hypothesize that attenuating effect on degranulation of CD8+ T lymphocytes might underlie the neuroprotective mechanism of SV2A ligands for cerebral I/R injury. In the current study, we will observe the neuroprotective effect of three kinds of SV2A ligands (levetiracetam, brivaracetam and seletracetam) under different dosage and course of treatment. The attenuating effect of SV2A ligands on degranulation of intracephalic CD8+ T lymphocytes after cerebral I/R will be measured using the the flow cytometry analysis after isolation of intracephalic lymphocytes. The relationship between attenuating effect on degranulation of CD8+ T lymphocytes and neuroprotective effect of SV2A ligand will be investigated using mice with CD8+ T lymphocytes mutation and mice with perforin mutation. As other candidate neuroprotective strategy of SV2A ligand, the selective modulation of N-type calcium channel and the blockage of Fas/FasL pathway are compared with the attenuating effect on degranulation by preconditioning of SNX-111 ( Ziconotide, a selective blocker of N type calcium channel) and by mice with FasL mutation, respectively. Taken together, our aims are to verify the neuroprotective effect of SV2A ligands in cerebral ischemia-reperfusion (I/R) injury and to elucidate the exact role of attenuating effect on degranulation of CD8+ T lymphocytes in neuroprotective mechanism of SV2A ligand after cerebral I/R injury. The conclusion of this study might contribute to open new perspectives for the immunoloregulation treatment of acute cerebral ischemia.

我们新近发现：CD8＋T淋巴细胞（CD8T）中存在突触囊泡蛋白2A（SV2A），而SV2A配体左乙拉西坦能够抑制CD8T脱颗粒。CD8T是脑缺血-再灌注（I/P）后重要神经毒性细胞，脱颗粒是其主要毒性机制，因而SV2A配体抑制CD8T脱颗粒可能有助于I/P后脑保护。基于上述假设，本项目首先采用小鼠脑I/P模型确证SV2A配体的缺血性神经保护作用（预实验已获肯定结果）；然后采用脑组织内淋巴细胞分离技术和各种流式细胞仪分析技术，检测SV2A配体对缺血脑组织中CD8T脱颗粒的抑制作用；进而采用CD8T基因缺陷小鼠和穿孔素基因缺陷小鼠研究抑制CD8T脱颗粒与SV2A配体缺血脑保护的关系；最后通过与其他候选机制（Fas/FasL通路阻滞、N型钙通道阻滞）进行对比，明确抑制CD8T脱颗粒是否为主要神经保护机制。鉴于SV2A配体的临床实用性，本项目将为探索治疗急性脑梗死可行的新方法提供重要实验依据。

该研究对sv2a配体对急性局灶性脑缺血再灌注小鼠的神经保护作用及其机制进行了研究。研究提示了穿孔素在小鼠缺血再灌注急性期的神经损伤中介导毒性作用。t提示了SV2A 配体（LEV、Brivaracetam和 Seletracetam）对小鼠实验性急性局灶性脑缺血具有神经保护作用，SV2A 配体（LEV、Brivaracetam和 Seletracetam）抑制小鼠缺血再灌注后脑组织中CD8+T淋巴细胞内穿孔素的释放。为了检验穿孔素途径是否为SV2A 配体对小鼠缺血再灌注后的神经损伤的唯一途径，我们对N 型钙离子通道阻滞、Fas/FasL 通路阻滞、CD8＋T 淋巴细胞脱颗粒抑制等候选机制分别进行了检验，结果表明： SV2A 配体对穿孔素基因敲除小鼠缺血再灌注后的神经损伤仍有一定的保护作用，这种保护作用被证实与小鼠缺血-再灌注后脑内 CD8＋T 淋巴细胞脱颗粒的抑制有关，但未被证实与Fas/FasL 通路阻滞和 N 型钙通道阻滞有关。