June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Evaluating the reliability of Brn3a and RBPMS as markers of retinal ganglion cells with age and ocular hypertension-induced disease
Author Affiliations & Notes
  • Miranda Meng
    University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Kimberly Dine
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Brahim Chaqour
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Kenneth S Shindler
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Jipeng Yue
    University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Ahmara Gibbons Ross
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Miranda Meng None; Kimberly Dine None; Brahim Chaqour Gyroscope Therapeutics, Code F (Financial Support); Kenneth Shindler Gyroscope Therapeutics, Code F (Financial Support), Noveome Biotherapeutics, Code F (Financial Support); Jipeng Yue None; Ahmara Ross Gyroscope Therapeutics, Code F (Financial Support), Noveome Biotherapeutics, Code F (Financial Support)
  • Footnotes
    Support  EY019014, EY301163, RWJ-Harold Amos Faculty Development Award, Foundation Fighting Blindness, Research to Prevent Blindness, Paul and Evanina Mackall Foundation Trust, Center for Advanced Retinal and Ocular Therapeutics, and the F. M. Kirby Foundation
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 5461. doi:
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      Miranda Meng, Kimberly Dine, Brahim Chaqour, Kenneth S Shindler, Jipeng Yue, Ahmara Gibbons Ross; Evaluating the reliability of Brn3a and RBPMS as markers of retinal ganglion cells with age and ocular hypertension-induced disease. Invest. Ophthalmol. Vis. Sci. 2023;64(8):5461.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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