Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States.
Invest Ophthalmol Vis Sci. 2024 Oct 1;65(12):18. doi: 10.1167/iovs.65.12.18.
The long-term evaluation of RPE65 gene augmentation initiated in middle-aged RPE65 mutant dogs previously uncovered notable inter-animal and intra-retinal variations in treatment efficacy. The study aims to gain deeper insights into the status of mutant retinas and assess the treatment impact.
Immunohistochemistry utilizing cell-specific markers and reverse transcription-quantitative PCR (RT-qPCR) analysis were conducted on archival retinal sections from normal and RPE65 mutant dogs.
Untreated middle-aged mutant retinas exhibited marked downregulation in the majority of 20 examined genes associated with key retinal pathways. These changes were accompanied by a moderate increase in microglia numbers, altered expression patterns of glial-neuronal transmitter recycling proteins, and gliotic responses in Müller glia. Analysis of advanced-aged mutant dogs revealed mild outer nuclear layer loss in the treated eye compared to moderate loss in the corresponding retinal regions of the untreated control eye. However, persistent Müller glial stress response along with photoreceptor synapse loss were evident in both treated and untreated eyes. Photoreceptor synaptic remodeling, infrequent in treated regions, was observed in all untreated advanced-aged retinas, accompanied by a progressive increase in microglial cells indicative of ongoing inflammation. Interestingly, about half of the examined genes showed similar expression levels between treated and untreated advanced-aged mutant retinas, with some reaching normal levels.
Gene expression data suggest a shift from pro-degenerative mechanisms in middle-aged mutant retinas to more compensatory mechanisms in preserved retinal regions at advanced stages, despite ongoing degeneration. Such shift, potentially attributed to a number of surviving resilient cells, may influence disease patterns and treatment outcomes.
先前对中年 RPE65 突变犬进行的 RPE65 基因增强治疗的长期评估发现，治疗效果在动物间和视网膜内存在显著差异。本研究旨在更深入地了解突变视网膜的状况，并评估治疗的影响。
利用细胞特异性标志物对正常和 RPE65 突变犬的存档视网膜切片进行免疫组织化学和逆转录定量 PCR（RT-qPCR）分析。
未经治疗的中年突变视网膜中，与关键视网膜途径相关的 20 个检测基因中有大多数出现明显下调。这些变化伴随着小胶质细胞数量的适度增加、神经胶质-神经元递质再循环蛋白表达模式的改变以及 Müller 胶质细胞的胶质反应。对老年突变犬的分析显示，与未经治疗的对照眼相应视网膜区域的中度损失相比，治疗眼的外核层损失较轻。然而，在治疗眼和未经治疗眼中都可以观察到持续的 Müller 胶质细胞应激反应以及光感受器突触的丧失。在所有未经治疗的老年视网膜中都观察到光感受器突触重塑，在治疗区域中很少见，同时伴随着小胶质细胞的数量呈进行性增加，表明存在持续的炎症。有趣的是，大约一半的检测基因在治疗和未经治疗的老年突变视网膜之间表现出相似的表达水平，其中一些达到正常水平。
基因表达数据表明，从中年突变视网膜中的促退行性机制向高级阶段保留的视网膜区域中的更补偿性机制转变，尽管仍存在退行性变。这种转变可能归因于许多存活的有弹性的细胞，可能会影响疾病模式和治疗结果。
