力学敏感通道Piezo1介导青光眼视神经筛板区星形胶质细胞骨架解聚的机制研究

The main course of glaucomatous neuropathy is owing to the pathological raising intraocular pressure (IOP). It is not confirmed that how IOP rising leads to the optic nerve damage and which is the initial pathological change in glaucoma. In our research, we have discovered that withdrawing of astrocytes processes in lamina cribrosa is the initial lesion of glaucoma. Lamina cribosa astrocyte (LCA) is the initial effector of raising IOP. Mechanosensitive channel Piezo1 is a key channel in LCA to receive and transduct the damage effects of raising IOP. Expression and opening of Piezo1 will induce Ca2+ influx as IOP rising. Transcription factor YAP/TAZ will be activated. As a result, cytoskeleton of LCA will depolymerize, and then withdrawing processes of LCA will happen. Many experimental techniques will be employed in this study, including duel-photon techniques, atomic force microscopy，patch clamp and so on. GCaMP protein gene will be introduced into glial cells with GFAP positive expression in the rat’s eyes. With these GCaMP+-GFAP+ rats，we will induce glaucoma model with beads injection into anterior chamber. With these experiments, we will observe many effects of LCA with pressure rising, including the change of expression and activation of Piezo1, the relationship between Piezo1 opening and cytoskeleton depolymerization, the activation of Pizeo1 affected by cytoskeleton depolymerization factors. We will try to reveal the rule of changes of expression and activation of Piezo1 in the course of IOP rising. And then will will try to explore the feature of molecules of astrocytes glial processes rollback. We expect to help the diagnosis of glaucoma at early stage and to find new targets for glaucoma therapy.

病理性高眼压是青光眼视神经损害的主要原因，目前对高眼压致青光眼视神经损害的机制和初始病变特征认识不清。我们发现高眼压致筛板区星形胶质细胞（LCA）的突起回退是青光眼初始病变，LCA是高眼压的初始效应器。力学敏感通道Piezo1是LCA感受和传导高眼压损害效应的重要通道。我们推测：高眼压下Piezo1通道表达改变和开放促使Ca2+内流，触发转录因子YAP/TAZ活化，LCA骨架解聚因子活化致骨架解聚和突起回退。本研究拟采用双光子、原子力显微镜、膜片钳等技术，用GCaMP基因在体标记的高眼压大鼠模型，分析高眼压作用下Piezo1在LCA的表达和活性变化，及其与LCA骨架解聚的关系。本研究可望阐明高眼压导致LCA骨架解聚的分子机制，为临床青光眼早期诊断和治疗开辟新的途径。

青光眼是研究星形胶质细胞（AC）微环境变化的适合模型。青光眼的疾病进程与眼内压升高密切相关，其病变的可能机制包括神经营养障碍、兴奋性毒性、氧化应激以及免疫性系统的紊乱等。青光眼病变过程中，早期眼压升高，筛板区无髓鞘的神经节细胞在压力作用下以囊泡形式释放谷氨酸激活AC，导致AC 的活化、增生，而AC 胞膜上谷氨酸转运体表达下降，组织间隙游离谷氨酸浓度升高，神经节细胞轴突谷氨酸受体表达升高，上述改变共同造成神经节细胞轴突损伤，轴突损伤导致的轴浆运输障碍进一步加速了神经节细胞胞体的变性和细胞凋亡。因此，AC 主导的视网膜视神经谷氨酸微环境变化在青光眼发展早期具有重要作用。采用以星形胶质细胞活化为早期事件的青光眼大鼠作为研究对象，通过前房内注射微磁珠建立Long-Evans 大鼠青光眼模型，以正常Long-Evans 大鼠（未作前房注射）作为对照，采用视网膜免疫荧光染色、 qRT-PCR、谷氨酸含量测定等方法研究青光眼模型大鼠视神经区AC 及兴奋性氨基酸代谢变化及使用嗅鞘细胞（OECs） 移植的作用。结果发现：前房内注射微磁珠能够稳定、持久维持高眼压，可造成大鼠视神经胶质增生，神经元轴突凋亡，视功能减退，能模拟青光眼的病理生理特点。移植OECs 到视乳头旁血管间的视网膜下腔后，青光眼模型大鼠的视敏度以及视功能得到改善，视神经区的胶质增生得到显著抑制，视神经轴突得到保护。同时，离体研究发现：200mM 谷氨酸能够诱导AC 凋亡，可作为AC 凋亡的离体模型；而加入OECs 共培养，能通过激发AC 代谢微环境中的谷氨酸。在体研究发现：在青光眼模型的病变过程中，视神经区谷氨酸代谢进行性下降，导致谷氨酸在该区域堆积。移植的OECs 可通过调节AC 活化，增加视神经区域谷氨酸代谢，降低谷氨酸含量，保护视神经轴突。上述结果提示OECs通过调控AC，降低微环境中的谷氨酸，保护神经节细胞轴突，从而延缓青光眼模型中视神经的退变。