Evaluating the Optic Nerve Crush Model to Understand the Function of Microglia in Glaucoma Neuroprotection.

Purpose: Microgliosis is a key neuroinflammatory feature in human glaucomatous retinas, believed to contribute to disease progression. This study aims to characterize changes in microglia and intra-retinal axons following optic nerve crush (ONC) and investigate microglial involvement in retinal ganglion cell (RGC) and axonal degeneration. Methods: Using the CD11c.YFP.Venus.Tg mouse line, we tracked microglial activation and assessed the spatiotemporal changes in TUJ1+ intraretinal axons over a 2-week period post-ONC. Microglial function was examined by depleting microglia with the CSF1R inhibitor PLX5622 and using Trem2-deficient mice with dampened microglial activation. Results: Activated microglia accumulated significantly in the retina from day 4 post-ONC, peaking at day 7. Retinal microglia became hypertrophic by day 1 and started proliferating. Axon beading occurred primarily in the peripheral retina by day 2 post-ONC, with more beaded axons appearing along long axonal bundles toward the optic nerve head (ONH) by day 7 and day 14. There was a significant reduction in overall TUJ1 expression and axonal bundle thickness during this period. Despite complete microglial depletion and significantly reduced activation, no differences were observed in the RGC count or the extent of optic nerve damage following ONC. Conclusions: Microglial activation is secondary to axonal injury and plays a bystander role in the ONC model. Robust RGC and axonal degeneration appear unaffected by activated microglia. This finding challenges the utility of the ONC model for evaluating microglia-based glaucoma treatments. Additionally, the study reaffirms the value of combining fluorescent reporter mouse lines with noninvasive ocular imaging for streamlining future research.