Abstract
Purpose :
Retinal ganglion cells (RGCs) deliver visual information to the brain but are lost in neurodegenerative ocular disease. Reliable markers that identify the entire population of RGCs are necessary to accurately quantify RGC loss in models of optic neuropathy and neuroprotective treatments. This study compared two markers for RGCs, Brn3a and RBPMS, in age and a chronic ocular hypertension model of disease.
Methods :
RGC quantification from immunofluorescent staining and RT-qPCR was performed on retinas from 3 week (N=6 mice) and 6 month-old (N=8) wild-type C57BL/6 mice. RNA was isolated from the retina of one eye of each mouse for RT-qPCR, and the retina of the contralateral eye was flat mounted for immunofluorescent staining to determine the effect of age on Brn3a and RBPMS gene and protein expression. Elevated IOP was induced by intravitreal microbead injection into one eye/mouse (N=4 mice), and RT-qPCR was used to compare retinal gene expression 6 months later between eyes with elevated IOP and contralateral control eyes.
Results :
RBPMS stains a larger population of RGCs than Brn3a in all samples. At 6 months, the total number of RBPMS+ and Brn3a+ RGCs decreased compared to 3 weeks (by 22.7±8.3% and 19.7±3.0% respectively), but no significant decrease in fold change of gene expression was observed for either marker (p<0.05), reflecting a potential increase in RBPMS and Brn3a protein degradation with age. The percent of RBPMS+ cells that did not exhibit Brn3a staining (Brn3a-/RPBMS+) in retinal flat mounts also decreased with age: at 3 weeks, there were 9.78±2.9% Brn3a-/RBPMS+ cells, which decreased to 6.44±0.50% at 6 months. There is a significant difference in the number of RBPMS+ compared to Brn3a+ RGCs at 3 weeks, but not at 6 months. With elevated IOP, Brn3a gene expression increased (227±19% fold change from the control), whereas RBPMS showed no significant change.
Conclusions :
Results suggest that RBPMS marks more RGCs than Brn3a, and is a more consistent marker, when considering changes induced by age and disease. However, with age, the difference in total RGC count stained using Brn3a and RBPMS decreases, so the marker chosen may be less of a concern at older ages.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.