Baez Hector C, LaPorta Jennifer M, Walker Amber D, Fischer William S, Hollar Rachel, Patterson Sara, DiLoreto David A, Gullapalli Vamsi, McGregor Juliette E
Department of Biomedical Engineering, University of Rochester, Rochester, NY.
Center for Visual Science, University of Rochester, Rochester, NY.
bioRxiv. 2025 Mar 7:2024.06.02.597041. doi: 10.1101/2024.06.02.597041.
Adaptive Optics Scanning Light Ophthalmoscopy (AOSLO) paired with intravitreal injection of a viral vector coding for the calcium indicator GCaMP has enabled visualization of neuronal activity in retinal ganglion cells (RGCs) at single cell resolution in the living eye. However, the inner limiting membrane (ILM) restricts viral transduction to the fovea in humans and non-human primates (NHP), hindering both therapeutic intervention and physiological study of the retina. To address this, we explored peeling the ILM before intravitreal injection to expand calcium imaging beyond the fovea in the living primate eye.
Five eyes from Macaca fascicularis (age 3-10; n=3; 2 males, 1 female) underwent vitrectomy and ILM peel centered on the fovea prior to intravitreal delivery of 7m8:SNCG:GCaMP8s. RGC responses to visual flicker were evaluated using AOSLO calcium imaging 1-6 months post intravitreal injection.
Calcium activity was observed in RGCs throughout the ILM peeled area in all eyes, representing a mean 8-fold increase in accessible recording area relative to a representative control eye. RGC responses in the ILM peeled and control eyes were comparable and showed no significant decrease over the 6 months following the procedure. In addition, we demonstrated that activity can be recorded directly from the retinal nerve fiber layer.
Peeling the ILM is a viable strategy to expand viral access to the GCL for gene therapies in NHP. Overall, this approach has potential to advance visual neuroscience, including pre-clinical evaluation of retinal function, detection of vision loss, and assessment of therapeutic interventions.
自适应光学扫描光检眼镜（AOSLO）与玻璃体内注射编码钙指示剂GCaMP的病毒载体相结合，能够在活体眼中以单细胞分辨率可视化视网膜神经节细胞（RGC）的神经元活动。然而，内界膜（ILM）限制了病毒在人类和非人灵长类动物（NHP）中的转导仅局限于中央凹，这阻碍了视网膜的治疗干预和生理学研究。为了解决这一问题，我们探索了在玻璃体内注射前剥离ILM，以在活体灵长类动物眼中将钙成像扩展到中央凹以外的区域。
对5只食蟹猴的眼睛（年龄3 - 10岁；n = 3；2只雄性，1只雌性）进行玻璃体切除术，并在向玻璃体腔内注射7m8:SNCG:GCaMP8s之前，以中央凹为中心剥离ILM。在玻璃体内注射后1 - 6个月，使用AOSLO钙成像评估RGC对视觉闪烁的反应。
在所有眼睛的整个ILM剥离区域的RGC中均观察到钙活性，相对于一只代表性的对照眼，可记录区域平均增加了8倍。ILM剥离眼和对照眼中的RGC反应相当，并且在手术后的6个月内没有显著下降。此外，我们证明可以直接从视网膜神经纤维层记录活动。
剥离ILM是一种可行的策略，可扩大病毒进入NHP中神经节细胞层进行基因治疗的途径。总体而言，这种方法有潜力推动视觉神经科学的发展，包括视网膜功能的临床前评估、视力丧失的检测以及治疗干预的评估。
