Department of Surgical Sciences, School of Veterinary Medicine.
Department of Ophthalmology & Visual Sciences, School of Medicine and Public Health.
Mol Vis. 2021 Nov 19;27:608-621. eCollection 2021.
The purpose of this study was to identify a robust, representative region of interest (ROI) for studies of retinal ganglion cell (RGC) soma loss in feline congenital glaucoma (FCG), a spontaneous, large-eyed glaucoma model.
Seven FCG and three wild-type (wt) eyes were collected from 10 adult cats of both sexes. Eyes enucleated postmortem were immediately fixed overnight in 4% paraformaldehyde and then stored in 0.1 M PBS at 4 °C. The retinas were wholemounted, Nissl stained with cresyl violet, and imaged using light microscopy. Somas of RGCs were manually identified according to long-established morphological criteria and quantified using a semiautomated method; their coordinates were used to create density maps and plots of the retinal topography. The RGC axon counts for the corresponding eyes were obtained from glutaraldehyde-fixed, resin-embedded optic nerve cross-sections stained with 0.1% p-phenylenediamine (PPD) using a semiautomated counting method. Correlations between total optic nerve axons and RGC soma counts were assessed by linear regression. A k-means cluster algorithm was used to identify a retinal ROI, with further definition using a probability density algorithm.
Interindividual variability in RGC total soma counts was more pronounced in FCG cats (mean = 83,244, range: 0-155,074) than in wt cats (mean = 117,045, range: 97,373-132,972). In general, RGC soma counts were lower in FCG cats than they were in wt cats. RGC axon counts in the optic nerve cross-sections were lower than, but strongly correlated to, the total RGC soma count across all cats (in wt and FCG retinas; R = 0.88) and solely FCG eyes (R = 0.92). The k-means cluster algorithm indicated a region of the greatest mean difference between the normal wt retinas and FCG-affected retinas within the temporal retina, incorporating the region of the area centralis.
As in other species, RGC soma count and topography are heterogeneous between individual cats, but we identified an ROI in the temporal retina for future studies of RGC soma loss or preservation in a large-eyed model of congenital glaucoma. Many of the methods refined and established to facilitate studies in this FCG model will be broadly applicable to studies in other large-eyed models.
本研究旨在确定一个稳健、有代表性的兴趣区域（ROI），用于研究猫先天性青光眼（FCG）中视网膜神经节细胞（RGC）体丢失的情况，FCG 是一种自发性的、大眼青光眼模型。
从 10 只成年公猫和母猫中收集了 7 只 FCG 和 3 只野生型（wt）眼睛。死后眼球立即在 4%多聚甲醛中固定过夜，然后储存在 0.1 M PBS 中于 4°C。视网膜全铺片，经 cresyl violet 尼氏染色，用光学显微镜成像。根据长期确立的形态学标准，手动识别 RGC 体，并使用半自动方法进行定量；它们的坐标用于创建视网膜地形图的密度图和图。相应眼睛的 RGC 轴突计数是通过戊二醛固定、树脂包埋的视神经横截面获得的，这些横截面用 0.1%对苯二胺（PPD）染色，使用半自动计数方法。通过线性回归评估总视神经轴突与 RGC 体细胞计数之间的相关性。使用 k-均值聚类算法识别视网膜 ROI，然后使用概率密度算法进一步定义。
FCG 猫（平均值= 83244，范围：0-155074）的 RGC 总体细胞计数的个体间变异性比 wt 猫（平均值= 117045，范围：97373-132972）更为明显。一般来说，FCG 猫的 RGC 体细胞计数低于 wt 猫。视神经横截面中的 RGC 轴突计数低于但与所有猫（wt 和 FCG 视网膜；R = 0.88）和仅 FCG 眼（R = 0.92）的总 RGC 体细胞计数密切相关。k-均值聚类算法表明，在颞视网膜中存在一个区域，该区域在正常 wt 视网膜和受 FCG 影响的视网膜之间的平均差异最大，包含中央凹区域。
与其他物种一样，个体猫之间的 RGC 体细胞计数和拓扑结构存在异质性，但我们在颞视网膜中确定了一个 ROI，用于研究大眼先天性青光眼模型中 RGC 体细胞丢失或保存。为促进 FCG 模型中研究而改进和建立的许多方法将广泛适用于其他大眼模型的研究。
