DLK/JNK/c-JUN信号通路在间接性外伤性视神经病中的作用机制研究

Indirect traumatic visual neuropathy (ITON) is caused by the transmitted forces secondary to head trauma without direct disruption of normal tissue structure, which leads to partial or full vision loss. However, pathobiology and molecular mechanism of ITON is still not clear. Clinical therapies including steroids and surgical decompression or combination of both approaches to reduce nerve swelling and release nerve from compression have failed to show benefits as compared to no treatment. In our impact acceleration (IA) mouse model which mimics these human concussive injury scenarios for the study of traumatic axonal injury (TAI), visual system is one of the major systems involved in TAI. Our previous experiments confirmed that retinal ganglion cell (RGC) loss have been found in this model. Gallyas silver staining presented axonal degeneration occurred in optic nerve, optic tract and superior colliculus under IA injury. Similarly, concomitant with axon degeneration, there is neuroinflammation, i.e. activation of microglia, involved in the same locations. We assessed the complete ON by APP immunostaining and CTB transport visualized with clarity. It was confirmed that one third segment of the nerve from chiasm was the initial part of TAI occurred at the optic nerve by both of the above methods. In addition, activation of DLK/JNK/c-JNU pathway in RGC of ITON after IA injury could be confirmed by increase of pathway related protein expression. Here in this study, we investigate pathological changes of ITON in mice after pharmacologically blocking DLK/JNK/c-JNU pathway with DLK inhibitor sunitinib or JNK inhibitor SP600125. We detect the expression of DLK、p-MKK4、MKK4、p-MKK7、MKK7、p-JNK、JNK and p-c-JUN in the whole retina by western blot and immunohistochemical(IHC) staining at 1, 3 and 7 days after IA injury. We count the number of survival RGCs and p-c-JUN（+）RGC s by γ-synuclein and p-c-JUN double labelled immunofluorescence(IF) staining of whole mount retina at 1 day, 2 weeks and 4 weeks after IA injury. We study axonal degeneration with Gallyas silver staining and neuroinflammatory responses with IBA1 staining labelling microglia in visual pathway at 7 days after IA injury. In addition, we explore the permeability of blood brain barrier, the integrity of optic nerve and axon degeneration by APP and mouse IgG double labelled IF staining of optic nerve at 4 and 24 hours after IA injury. We research the axonal transport of optic nerve at 2 days after IA injury by intravitreal injection of CTB488 into the animal eyes at 2 hours after IA injury and processing optic nerves through clarity techniques. Then we observe the myelinated axon in semithin optic nerve sections processed for toluidine blue staining at 2 and 4 weeks after IA injury. Through these researches, we can further illuminate the role of DLK/JNK/c-JUN pathway in ITON and the effect of pharmacologically blocking this pathway to RGC survival and axon degeneration, and also provide more directions and targets for the treatment of ITON.

间接性外伤性视神经病(ITON)继发于头部创伤，造成部分或完全视力丧失，病理及分子机制不清，且无有效治疗手段。加速撞击(IA)模型最大程度模拟ITON的情况，我们前期试验证实IA损伤导致视网膜神经节细胞(RGC)减少，视觉通路的轴突变性和神经炎症发生，同时DLK/JNK/c-JUN通路被激活。因此本研究通过药物阻断该通路，western blot和免疫染色观察视网膜DLK、p-MKK4/7、p-JNK和p-c-JUN表达，γ-synuclein和p-c-JUN双染观察RGC数量，银染和IBA1染色脑切片观察视觉通路轴突变性及神经炎症，APP及鼠IgG双染视神经和clarity处理CTB标记的视神经观察血脑屏障通透性、轴突完整性及轴突转运，视神经半薄切片甲苯胺蓝染色观察有髓神经轴突变化，明确ITON中DLK/JNK/c-JUN通路对RGC和轴突损伤的影响及作用机制，为该病治疗提供有效的靶点。

间接性创伤性视神经病变（ITON）是由无穿透性头部外伤引起的视神经退化疾病，由冲击力传导所致，对正常组织结构无直接破坏作用。患者通常是 30 多岁的年轻男性，在很多情况下 ITON 会被其他严重脑外伤症状所掩盖，所以常常被忽视。ITON 所致的视力丧失可以是暂时的或是永久的。现有治疗手段主要目的是减轻神经肿胀、缓解神经纤维受压，未见明显获益。ITON 的病理及分子生物学机制尚不清楚，因此有必要对其进行更深一步的研究从而找到有效的治疗靶点。.. ITON 病例多为钝器伤，加速撞击（IA）的小鼠模型能够很好的模拟人类发生 ITON 的情境，我们既往的研究亦证实 IA 模型可以有效地进行创伤性轴突损伤（TAI）的研究，且结果提示 IA 模型中存在神经轴突变性、视网膜神经节细胞（RGC）的减少和神经炎症。DLK/MKK4/JNK/c-JUN 通路在 RGC 的存活和功能方面起重要作用，我们的前期试验也证实了 IA 所致的 ITON 模型中上述通路激活，参与 ITON 中 RGC 的损伤，由此，针对该通路的抑制作用很有可能成为神经保护性治疗策略的新方向。. 本研究应用DLK或JNK抑制剂对DLK/JNK/c-JUN通路进行了有效阻断，减轻了视觉通路轴突变性和炎症反应并提高了RGC的存活率，为 ITON 的治疗提供了新的干预方向。