视神经顺行压力梯度变化对视网膜神经节细胞轴浆流运输的影响

Our previous study found that the elevated pressure difference along the optic nerve is correlated with glaucomatous optic neuropathy. Either reduced intracranial pressure or increased intraocular pressure can elevate pressure difference along the optic nerve. Then, why the elevation of pressure difference along the optic nerve will induce glaucomatous optic neuropathy? Through mathematical deduction, we found that elevated pressure difference along the optic nerve either by reducing intracranial pressure or increasing intraocular pressure will affect the orthograde and retrograde axonal transport, and cause the depletion of mitochondria inside the axons and lack of neurotrophins, which may lead to death of RGCs. A review of available data has proved axonal transport in ganglion cell axons can be blocked in experimental ocular hypertension. However, there is no direct experimental evidence to elucidate the effect of low CSFP on axonal transport. To test this hypothesis, we propose to artificially decrease CSFP and increase IOP of the rat to elevate the pressure difference along the optic nerve, and observe the change of axonal transport, the motor molecules involved in it and its energy supplier-mitochondria, and investigate the difference of the change of the axonal transport caused by different reasons which formed elevated pressure difference along the optic nerve. Our research may provide a new theory of the pathogenesis of the glaucomatous optic damage, and open up new avenues of therapeutic approach based on the mechanism of optic neuropathy.

前期研究发现视神经顺行压力梯度增大与青光眼视神经损害相关。眼内压增高或颅内压降低均可导致视神经顺行压力梯度增加。那么，这种视神经顺行压力梯度增加为什么会造成视神经损害呢？通过数学推理，我们提出不论低颅压还是高眼压所造成的视神经顺行压力梯度增加均会影响视神经正逆向轴浆流运输，导致线粒体流空缺失及神经营养因子缺乏，而发生视网膜神经节细胞凋亡。既往研究已证实了单纯高眼压对视神经轴索轴浆流运输的影响，而单纯低颅压对轴浆流运输的影响尚无直接实验证据支持。本课题拟以大鼠为研究对象，通过降低其颅内压和增加其眼内压增大视神经顺行压力梯度，观察轴浆流运输和参与轴浆流运输的动力蛋白及其主要能量来源线粒体的变化，观察比较不同成因视神经顺行压力梯度增大致视神经轴索轴浆流运输损害的异同，以期证实上述假说。该课题有望为青光眼视神经损害机制提供新的理论基础，并在该理论的指导下，建立基于损害机制的青光眼治疗策略。

前期研究发现视神经顺行压力梯度增大与青光眼视神经损害相关。眼内压增高或颅内压降低均可导致视神经顺行压力梯度增加。既往研究已证实单纯高眼压可影响视神经轴索轴浆流运输，而单纯低颅压对轴浆流运输的影响尚无直接实验证据支持。本课题基于此现状，通过试验研究，首先通过脑脊液引流成功建立了大鼠低颅压模型，模型建立后，通过核磁共振成像检测和脑组织切片HE染色检查均证实该模型对脑组织未造成损伤改变。在建立动物模型的基础上，通过荧光金上丘逆行标记的方法发现低颅压和高眼压同样导致了视网膜神经节细胞逆向轴浆流运输障碍，通过罗丹明染料玻璃体腔注射的方法发现低颅压和高眼压同样导致了视网膜神经节细胞正向轴浆流运输的障碍。在此基础上，进一步通过免疫组织化学染色和Western plot的方法研究证实，高眼压和低颅压可导致视网膜神经节细胞轴浆流运输动力蛋白dynein和kinesin表达变化，在一定程度上分析了高眼压和低颅压影响视网膜神经节细胞轴浆流运输的机制。本课题进一步通过TUNEL凋亡检测和HE染色发现，低颅压虽然影响了轴浆流运输，但尚未造成视网膜神经节细胞损伤。通过电镜扫观察了高眼压和低颅压导致的视神经轴索形态学及轴索内显微结构的改变，发现低颅压和高眼压均导致视神经轴突肿胀，低颅压组可见神经髓鞘的结构被破坏，肿胀的神经纤维内可见线粒体，及其他电子致密度高的小体，线粒体面积稍变大，变短，变宽。免疫组织化学染色和Western plot检测显示与正常对照组相比，低颅压组线粒体自噬相关蛋白PINK1，Parkin，和LAMP1随时间表达升高，而LC3B-II/I仅在术后第0天表达明显升高。本课题首次通过试验研究证实了低颅压可以和高眼压一样导致视网膜神经节细胞轴浆流运输的障碍，进而导致压力相关性视神经损害，并进一步探讨了低颅压导致视神经损伤的可能病理机制，为青光眼类视神经损害机制提供了新的理论基础，为青光眼的治疗的发展提供新的理论基础和可能靶点。