Abstract
Retinal ganglion cells (RGCs), the exclusive efferent neurons in the retina, represent a heterogenous population of cells. Similar to most projection neurons, they undergo cell death following axotomy proximal to the cell body. Certain subtypes of RGCs display varying responses to axonal injury. For instance, ON-OFF direction selective RGCs (ooDSGCs) are particularly susceptible to damage, whereas intrinsically photosensitive RGCs (ipRGCs) show a notable resilience. Using RNA sequencing and fluorescent in situ hybridization, this study finds that the expression levels of chloride intracellular channel proteins 1 and 4 (Clic1 and Clic4) are significantly elevated in ooDSGCs following axonal injury. To investigate the role of specific genes in RGCs, this study improves the use of adenovirus-associated virus (AAV)-retro vectors to express short hairpin RNA (shRNA). Injecting AAV2-retro into the superior colliculus ensures efficient delivery and expression of shRNA in RGCs. Additionally, by incorporating the nuclear affinitous-histone H2B gene fused with the fluorescent protein mGreenLantern, bright nuclear reporter expression is achieved. This enhancement allows for the precise identification and quantification of individual RGCs in live retinal tissues. Finally, this research shows that simultaneous knock down Clic1 and Clic4, using AAV2-retro, significantly enhances the survival of RGCs following injury. This study combines the use of AAV2-retro vectors for shRNA delivery with real-time fundus imaging, revealing the critical role of CLIC proteins in RGC death. This integrated approach highlights the potential of targeting Clic1 and Clic4 as a therapeutic strategy to promote RGC survival after axonal damage.