缝隙连接参与蛛网膜下腔出血后脑微循环功能障碍的机制研究

Delayed ischemic neurological deficit (DIND) is the leading cause of high fatality and disability rate after spontaneous subarachnoid hemorrhage (SAH). The pathophysiological mechanism of DIND remains poorly understood. Currently, cerebral microcirculation dysfunction is considered to be the most important factor in causing DIND. Physiologically, the tissue blood flow perfusion is maintained by microcirculation with two important regulation mechanisms, conducted vasodilation (CVD) and feedback. CVD refers to that after ischemia and hypoxia induce precapillary arterioles and capillaries vasodilation, the signal of vasodilation is conducted along the wall of upstream vessel, causing feed arteries of microcirculation dilates and the tissue blood flow is therefore increased. Feedback refers to that smooth muscle cell (SMC) contraction can induced endothelial cell hyperpolarization by increasing intracellular IP3 and Ca2+ . The hyperpolarization can spread to SMC and in turm inhibit the contraction of SMC, to prevent the sustained vasoconstriction. Gap junctions play a key role in these two regulation mechanisms. After SAH, arteriolar vasospasm, formation of microthrombus, ischemia and hypoxia in tissue occur in parenchymal cerebral, demonstrated the dysfunction of CVD and feedback in microcirculation. We hypothesize that CVD and feedback can not be effective in conduction and diffusion of vasodilation signal would be caused by GJ remodeling after SAH. This subject will focus on this idea, to further clarify the mechanism of cerebral microcirculation dysfunction and pathogenesis of DIND after SAH and provide a new target and treatment method.

迟发性缺血性神经功能障碍（DIND）是蛛网膜下腔出血(SAH)后致死致残的主要原因，目前认为脑微循环功能紊乱是引起DIND的重要因素。微循环通过两个重要的调节机制来维持组织的血流灌注，一是传导性舒张（CVD)：局部缺血缺氧等刺激毛细血管和微动脉舒张，舒张信号还能沿上游血管传导，引起供应微循环的小动脉舒张,从而改善局部血流量；二是负反馈：当微血管平滑肌细胞收缩的同时，通过IP3、Ca2+升高等引起内皮细胞超极化，反馈性抑制平滑肌细胞收缩，避免持续收缩导致的血流量减少。缝隙连接在上述两种机制中发挥了关键作用。SAH后脑微动脉痉挛、微血栓形成，组织缺血缺氧，表明微循环的调节机制失效，我们推测可能系缝隙连接功能发生了紊乱，导致CVD和负反馈中的舒张信号不能有效传导和扩散，继而引起脑微动脉持续痉挛。本课题将就此设想展开研究，明确SAH后脑微循环紊乱和DIND的发病机制，并为其治疗提供新的靶点和方法。

脑血管痉挛是神经科学急待解决的难题，其发病机制未明，治疗也无特效方法。我们前期研究表明缝隙连接参与脑血管痉挛（CVS）的形成，尤其是缝隙连接蛋白43(Cx43)和缝隙连接蛋白40（Cx40）,其机制可能是通过调节缝隙连接蛋白的表达来影响细胞间信号传导的通透性和传导性，继而引起血管收缩与舒张信号平衡的紊乱，导致CVS的发生。本课题在前期研究的基础上，对Cx43和Cx40参与CVS的机理进行深入探讨，研究内容包括：①探讨Cx43在蛛网膜下腔出血(SAH)后CVS的调节机制； ②探讨Cx40在SAH后CVS的调节机制；③SAH大鼠血浆小细胞外囊泡（sEVs）的差异miRNA的初步探索。本研究得出以下结果：①SAH后Cx43的表达上调，且Cx43的的改变可能是由PKC途径介导的；②SAH后可通过NO-cGMP-PKG通路上调Cx40的表达缓解CVS；③通过下一代测序技术发现：SAH大鼠与sham大鼠的血浆sEVs之间存在明显的差异miRNA，具有统计学意义的有142个，其中SAH较sham组上调的有73个，下调的有69个(P<0.05; fold change≥2)。以上结果揭示Cx43的PKC途径及Cx40的NO-cGMP-PKG通路是其参与脑血管痉挛的重要病理形式，特异性降低Cx43的表达或者提高Cx40的表达能够对脑血管痉挛的病理改变产生明确影响，进一步研究Cx43、Cx40和差异miRNA的作用机制，可能是一种治疗和预防脑血管痉挛的新靶点。

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