Department of Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania, United States.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States.
Invest Ophthalmol Vis Sci. 2024 Apr 1;65(4):19. doi: 10.1167/iovs.65.4.19.
Retinal ganglion cell (RGC) loss provides the basis for diagnosis and stage determination of many optic neuropathies, and quantification of RGC survival is a critical outcome measure in models of optic neuropathy. This study examines the accuracy of manual RGC counting using two selective markers, Brn3a and RBPMS.
Retinal flat mounts from 1- to 18-month-old C57BL/6 mice, and from mice after microbead (MB)-induced intraocular pressure (IOP) elevation, are immunostained with Brn3a and/or RBPMS antibodies. Four individuals masked to the experimental conditions manually counted labeled RGCs in three copies of five images, and inter- and intra-person reliability was evaluated by the intraclass correlation coefficient (ICC).
A larger population (approximately 10% higher) of RGCs are labeled with RBPMS than Brn3a antibody up to 6 months of age, but differences decrease to approximately 1% at older ages. Both RGC-labeled populations significantly decrease with age. MB-induced IOP elevation is associated with a significant decrease of both Brn3a- and RBPMS-positive RGCs. Notably, RGC labeling with Brn3a provides more consistent cell counts than RBPMS in interpersonal (ICC = 0.87 to 0.11, respectively) and intra-personal reliability (ICC = 0.97 to 0.66, respectively).
Brn3a and RBPMS markers are independently capable of detecting significant decreases of RGC number with age and in response to IOP elevation despite RPBMS detecting a larger number of RGCs up to 6 months of age. Brn3a labeling is less prone to manual cell counting variability than RBPMS labeling. Overall, either marker can be used as a single marker to detect significant changes in RGC survival, each offering distinct advantages.
视网膜神经节细胞（RGC）的损失为许多视神经病变的诊断和分期提供了依据，而 RGC 存活的定量是视神经病变模型中的一个关键的结果衡量指标。本研究使用两种选择性标记物 Brn3a 和 RBPMS 来检查手动 RGC 计数的准确性。
使用 Brn3a 和/或 RBPMS 抗体对 1 至 18 个月大的 C57BL/6 小鼠以及微珠（MB）诱导的眼内压（IOP）升高后的小鼠的视网膜平面进行免疫染色。四名对实验条件不知情的人员对五张图片的三张进行了标记 RGC 的手动计数，并通过组内相关系数（ICC）评估了个体间和个体内的可靠性。
在 6 个月之前，用 RBPMS 标记的 RGC 数量比 Brn3a 抗体标记的 RGC 数量多约 10%，但在老年时差异减少到约 1%。两种 RGC 标记的群体都随着年龄的增长而显著减少。MB 诱导的 IOP 升高与 Brn3a 和 RBPMS 阳性 RGC 的显著减少有关。值得注意的是，Brn3a 标记的 RGC 计数比 RBPMS 标记的 RGC 计数更一致，个体间的 ICC 分别为 0.87 至 0.11，个体内的 ICC 分别为 0.97 至 0.66。
尽管 RBPMS 在 6 个月之前可以检测到更多的 RGC，但 Brn3a 和 RBPMS 标记物都能够独立地检测到年龄增长和 IOP 升高导致的 RGC 数量的显著减少。Brn3a 标记比 RBPMS 标记更不容易受到手动细胞计数变异性的影响。总的来说，两种标记物都可以用作单一标记物来检测 RGC 存活的显著变化，每种标记物都有其独特的优势。
