Biological Correlations and Confounding Variables for Quantification of Retinal Ganglion Cells Based on Optical Coherence Tomography using Diversity Outbred Mice

Purpose Despite popularity of optical coherence tomography (OCT) in glaucoma studies, it’s unclear how well OCT-derived metrics compare to traditional measures of retinal ganglion cell (RGC) abundance. Here, Diversity Outbred (J:DO) mice are used to directly compare ganglion cell complex (GCC)-thickness measured by OCT to metrics of retinal anatomy measured ex vivo with retinal wholemounts or optic nerve cross sections. Methods J:DO mice ( n = 48) underwent OCT and fundoscopic exams, with GCC-thickness measured using automated segmentation. Following euthanasia, RGC axons were quantified from para-phenylenediamine-stained optic nerve cross sections and RGC somas from BRN3A-immunolabeled retinal wholemounts with total cellularity assessed by TO-PRO or hematoxylin nuclear staining. Results J:DO tissues lacked overt disease. GCC-thickness (62.4 ± 3.7 µm), RGC abundance (3,097 ± 515 BRN3A + nuclei/mm 2 ; 45,533 ± 9,077 axons), and total inner retinal cell abundance (6,952 ± 810 nuclei/mm 2 ) varied broadly. GCC-thickness correlated significantly to RGC somal density (r = 0.46) and axon number (r = 0.49), whereas total inner retinal cellularity did not. Retinal area (20.3 ± 2.4 mm 2 ) and optic nerve (0.09 ± 0.02 mm 2 ) cross-sectional area varied widely. Sex did not significantly influence any of these metrics. In bilateral comparisons, GCC-thickness (r = 0.89), inner retinal cellularity (r = 0.47), and RGC axon abundance (r = 0.72) all correlated significantly. Conclusions Amongst outbred mice with widely variable phenotypes, OCT-derived measurements of GCC thickness correlate significantly to RGC abundance and axon number. The extensive phenotypic variability exhibited by J:DO mice make them a powerful resource for studies of retinal anatomy using quantitative genetics.