Glaucomatous Retinal Ganglion Cell (RGC) Loss: Density Distribution and Relationship to RGC Size in a Mouse Model of Glaucoma

Abstract

Abstract: : Purpose: To evaluate the relationship between retinal ganglion cell (RGC) size, density distribution and survival in senescent DBA2/NNia mice. To determine whether nearest neighbor distance (NND) can be used to quantify RGC loss in these animals. Method: 15-month-old DBA2/NNia mice (n=8, mixed gender) were used. RGCs were labeled retrogradely with Fluorogold from the superior colliculus. Animals were sacrificed 7 days later; retinas were flat-mounted and imaged in their entirety using a microscope equipped with an automated stage. Digital frame images were used to construct a map of the retinal wholemounts and automatically count all RGCs. RGC density for each frame image was calculated for a sub-set of retinas (n=5). In addition, RGC size in pixels and NND in pixels were determined for the rest of the retinas (n=7). Results: RGCs in 15-month-old DBA2/NNia animals ranged from 22,330 to 92,150 per retina and RGC frame density ranged from 500 to 11,000 RGC/mm². Mean RGC area ranged from 14.26 to 24.48 pixels and showed good linear correlation (R²= 0.8466) with the total number of RGCs per retina. The mean NND of retinas ranged from 10.18 to 13.60 pixels and showed an excellent linear correlation (R²= 0.9773) to their total RGC count. For distribution analysis, NND values (a close approximation of the focal density for each RGC in a given retina) were divided into 42 bins (1-41, >41 pixels). The distribution of NND in each retina was bimodal with the >41 bin increasing in retinas with RGC loss. Conclusion: Cell size is linearly correlated to the total RGC count in 15-month-old DBA2/NNia mice. Retinas with higher RGC counts had a larger mean cell size indicating preferential loss of large RGCs or reduction in size of surviving cells. Mean NND for all RGCs in a retina is correlated to the total RGC number and is a better measure than RGC frame density for evaluating RGC loss. The bimodal NND distribution of RGCs and the finding that loss of RGCs correlates with a shift/amplitude change in both modes suggests two different patterns of RGC loss caused by different pathological processes.

Keywords: ganglion cells • retina • degenerations/dystrophies