Comparing the Reliability of Manual Retinal Ganglion Cell Counting using Brn3a and RBPMS Labeling

Abstract

Purpose : Retinal ganglion cell (RGC) identification and counting provide the basis for determining cell survival in research on optic neuropathies. Manual counting of micrographs is a commonly used method, but its reliability can be affected by multiple factors. This study examines the reliability of manual RGC counting using two RGC-selective markers, Brn3a and RBPMS.

Methods : Retinal flat mounts from C57BL/6 mice were immunostained with Brn3a or RBPMS antibodies and imaged using fluorescent microscopy at 40x. Representative images were selected, reflecting the level of clarity and brightness typical of RGCs stained with either marker. Images were mirrored vertically and horizontally, yielding 3 copies x 5 images/marker x 2 markers = 30 total images. Reliability was tested by comparing cell counts by 4 individuals masked to the experimental conditions using the intraclass correlation coefficient.

Results : Each individual counter demonstrated significantly higher variation when counting RGCs labeled with RBPMS compared to Brn3a. From ICC analysis, Brn3a labeling provided good interperson reliability (0.866; 95% CI: 0.7894-0.9429) and excellent intraperson reliability (0.970; 95% CI: 0.9401-0.9995). RBPMS counts had poor interperson reliability (0.110; 95% CI: -0.027-0.2479) and moderate intraperson reliability (0.657; 95% CI: 0.3141-0.9995).

Conclusions : RGC labeling with Brn3a, a nuclear marker, provided more consistent cell counts than labeling with the cytoplasmic marker RBPMS. This presents an advantage for Brn3a in minimizing the variability and subjective bias associated with manual counting.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.