PD-1及其配体调控小胶质细胞活化在青光眼视网膜神经节细胞损伤中的免疫机制研究

As the second leading cause of blindness, glaucoma is a neurodegenerative disease of the optic nerve, characterized by the progressive loss of retinal ganglion cells (RGCs). Neuroinflammation is an important process in glaucoma, which is regarded as a key research direction in this area. Previous studies have shown that retinal microglia plays an important role in neuroinflammation by abnormal activation and mutual regulation with neurons and other immune cells. It has also been verified in spinal cord injury model that PD-1 could regulate the activation type of microglia. We previously identified the constitutive expression of PD-1 in the central nerve system and further demonstrated its important role in developmental and traumatic death of RGC, which is a novel mechanism of RGC loss. In this proposal, we will investigate the immune mechanism of PD-1 and its ligands PDL-1/PDL-2 in the regulation of microglia activation in RGC loss in glaucoma by deletion of PD-1 or PDL-1/PDL-2 genes. We will evaluate therapeutic effects of PD-1 agonist or antagonist antibody in CX3CR1 GFP/+ transgenic mice by using Scanning Laser Ophthalmoscopy (SLO) autofluorescence technique to visualize microglia activation and distribution in vivo. The purpose of this study is to find a breakthrough point in the neuroimmune mechanism of RGC loss in glaucoma and provide novel therapeutic targets for optic nerve protection.

青光眼是世界第二大致盲眼病，是以视网膜神经节细胞(RGC)损伤为特征的神经退行性病变。青光眼的神经免疫机制是近几年研究的热点，研究显示在青光眼中视网膜小胶质细胞异常活化，并与其它免疫细胞和神经细胞互相调节，在神经免疫反应中起至关重要的作用，而在脊髓神经损伤模型中已证实PD-1可调控小胶质细胞的活化类型。我们前期的研究率先揭示了PD-1在中枢神经系统有表达，并在发育期RGC凋亡及外伤性RGC损伤中均起重要作用，从而揭示了RGC损伤的新机制。本课题拟在青光眼模型中借助基因敲除鼠研究PD-1及其配体PDL-1/PDL-2调控视网膜小胶质活化类型在RGC损伤中的免疫机制，借助CX3CR1GFP/+转基因鼠和SLO技术活体检测小胶质细胞的活化，并评估调控PD-1信号和小胶质细胞活化类型在青光眼中的治疗作用。本课题旨在为青光眼神经免疫机制研究找到新的突破点，为青光眼视神经保护治疗提供新靶点。

青光眼是以视网膜神经节细胞损伤为特征的视神经退行性病变，其中细胞凋亡、小胶质细胞活化和炎症与视网膜神经节细胞的死亡有关。我们通过对双敲除PDL-1/PDL-2基因小鼠的体内外研究发现，双敲除PDL-1/PDL-2基因可使小鼠视网膜神经节细胞数目增多，减少视网膜小胶质细胞数目并促进其向M2型分化，STAT6 通路激活也增加，但对视网膜的组织结构和形态几乎没有影响，视觉功能也几乎没有差异。此后我们研究了双敲除PDL-1/PDL-2基因在磁珠诱导的慢性高眼压小鼠模型中是否具有视网膜神经节细胞保护作用。利用磁珠诱导小鼠慢性高眼压。通过小鼠视网膜westernblot 和免疫荧光染色实验评估视网膜神经节细胞的凋亡和小胶质细胞的活化，探究小胶质细胞活化相关通路STAT1 和STAT6 的磷酸化。发现PDL-1/PDL-2双基因敲除在小鼠慢性高眼压模型中一定程度上保护了视网膜神经节细胞的凋亡。阻断PD-1 通路还增加了抑炎性M2型小胶质细胞的活化并增强了其相关通路STAT6的磷酸化。即在磁珠诱导的小鼠COHT模型中，阻断PD-1通路对RGCs具有保护作用。