Retinal Ganglion Cell Quantification for Evaluating Neurodegeneration in Mouse Models of Multiple Sclerosis​.

Quantification of retinal ganglion cell (RGC) loss provides a sensitive and reproducible measure of neurodegeneration in mouse models of neurological  disease and injury. In experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of multiple sclerosis, inflammation of the optic nerve can lead to optic neuritis, axonal injury, and progressive RGC loss. Therefore, quantification of RGC survival provides a valuable readout of inflammation-mediated neurodegeneration and can be used to evaluate disease severity and neuroprotective interventions. Here, we describe a protocol for retinal dissection, flatmount preparation, immunostaining, and quantification of RGCs in the mouse retina. Using whole-mount preparations, this method preserves the structure and spatial organization of the retina, which allows visualization and quantification of RGCs using markers such as Brn3a. This protocol enables reliable identification and quantification of Brn3a-positive RGCs. When combined with complementary neuronal and stress markers, changes in labeling intensity and cellular morphology can help identify stressed or injured RGCs and distinguish reduced marker expression from overt cell loss. Representative results demonstrate that the protocol detects both early RGC stress and overt RGC loss in EAE mice. This approach provides a robust and scalable framework for evaluating inflammation-driven neuronal injury and assessing neuroprotective strategies in preclinical studies.