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

Abstract

PurposeDespite 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.MethodsJ: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.ResultsJ:DO tissues lacked overt disease. GCC-thickness (62.4 ± 3.7 µm), RGC abundance (3,097 ± 515 BRN3A+ nuclei/mm2; 45,533 ± 9,077 axons), and total inner retinal cell abundance (6,952 ± 810 nuclei/mm2) 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 mm2) and optic nerve (0.09 ± 0.02 mm2) 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.ConclusionsAmongst 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.

Detecting retinal neurodegeneration in people with diabetes: Findings from the UK Biobank

“…A recent meta-analysis on this topic also showed a stronger association with GC-IPL thickness compared to mRNFL thickness between DM patients with no DR and controls [ 25 ]. Our findings, together with prior work from others, suggest that GC-IPL thickness may be a good marker for evaluation of DRN and animal studies suggest that thinning of GC-IPL may represent loss of ganglion cells [ 26 ]. Direct visualization of ganglion cells in-vivo would help further elucidate this.…”

The Retinal Ganglion Cell Response to Blast-Mediated Traumatic Brain Injury Is Genetic Background Dependent

“…While there is a possibility this analysis could introduce variation, previous publications have shown that retinal parameters have high intraocular correlation in J:DO mice. 19 Furthermore, we have shown that BRN3A antibody staining is not significantly different between eyes in C57BL/6J mice. However, we cannot discount the possibility that enucleation of one eye may result in optic nerve and/or RGC damage in the fellow eye, 50 which would have the potential to influence the results presented here.…”

“…J:DO mice lack known overt retinal degenerative mutations, such as rd1 or rd8, 17,18 and have normal appearing retinas. 19 J:DO have approximately 45 million segregating genetic variations and encompass the majority of all existing genetic diversity in laboratory strains of mice. J:DO mice have had multiple generations of opportunity for meiotic recombination, which makes them useful for analysis of complex traits, [20][21][22][23][24] such as the response to blast.…”

AxonDeep: Automated Optic Nerve Axon Segmentation in Mice with Deep Learning

“…Lyst ) with healthy optic nerves ( n =5 nerves); C57BL/6J that had been subjected to either blast-induced traumatic brain injury (TBI blast) within an enclosed chamber or sham treatment (TBI sham; n= 27 nerves); 3, 4, 22 and Diversity Outbred (J:DO) with healthy optic nerves but predicted to exhibit genetic background-dependent natural variability in optic nerve features ( n= 13 nerves). 38, 39 Tissues in the current study were collected from mice contributing to prior publications, 40, 41 however all data reported herein arise from new analyses performed uniquely for this study. All mice used in this study were originally purchased from The Jackson Laboratory (Bar Harbor, ME).…”