Baez Hector C, LaPorta Jennifer M, Walker Amber D, Fischer William S, Hollar Rachel, Patterson Sara S, DiLoreto David A, Gullapalli Vamsi, McGregor Juliette E
Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States.
Center for Visual Science, University of Rochester, Rochester, NY, United States.
Invest Ophthalmol Vis Sci. 2025 Sep 2;66(12):25. doi: 10.1167/iovs.66.12.25.
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, hindering both therapeutic intervention and physiological study of the retina. To address this issue, we explored peeling the ILM before intravitreal injection to expand calcium imaging beyond the fovea in the living primate eye.
Five eyes from three Macaca fascicularis (aged 3-10 years; 2 males, 1 female) that were immune suppressed with cyclosporine, underwent vitrectomy and ILM peel centered on the fovea prior to intravitreal delivery of 7m8:SNCG:GCaMP8. RGC responses to visual flicker were evaluated using AOSLO calcium imaging 1 to 6 months after intravitreal injection.
Calcium activity was observed in RGCs throughout the ILM peeled area in all eyes, representing a mean eight-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 month period after 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 RGCs for gene therapy, and when paired with GCaMP imaging has the potential to advance visual neuroscience, preclinical 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）限制了病毒转导在人类和非人类灵长类动物中仅限于中央凹，这阻碍了视网膜的治疗干预和生理学研究。为了解决这个问题，我们探索了在玻璃体内注射前剥离ILM，以将钙成像扩展到活体灵长类动物眼的中央凹以外。
对三只食蟹猴（年龄3 - 10岁；2只雄性，1只雌性）的五只眼睛用环孢素进行免疫抑制，在玻璃体内注射7m8:SNCG:GCaMP8之前，进行玻璃体切除术并以中央凹为中心剥离ILM。在玻璃体内注射后1至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八倍。ILM剥离眼和对照眼中RGC的反应相当，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，可扩大病毒对RGC的感染以进行基因治疗，并且与GCaMP成像相结合有潜力推动视觉神经科学、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。
