Abstract
Purpose :
Non-human primates (NHPs) are critical to the pre-clinical development of human therapies for eye disease due to similarities in ocular anatomy, physiology, and immune function. The retinal immune response to injury, disease, and therapeutics is currently poorly understood. We employ phase contrast adaptive optics scanning light ophthalmoscopy (AOSLO) to visualize in vivo immune activity in the NHP retina on a cellular scale.
Methods :
Offset aperture AOSLO recordings focused on retinal vascular layers were collected over six minute periods (4 Airy Disc (AD) pinhole, Offset laterally ~10 AD, 3mW, 1.5° x 1.5°, 730 nm pulsed laser) from five eyes of three NHPs over nine sessions. Imaging was performed in intact eyes and in the hour following high intensity ultrafast laser exposure (26.6 - 32.5 J/cm2, 730nm) to focally ablate the photoreceptor layer (PRL). Retinal videos were registered to correct for eye motion, averaged, and reviewed to identify putative immune cells. Counts and speeds of visible cells were determined manually. Metrics were computed in one eye, pre- and post-PRL ablation, by averaging data from 9-15 retinal locations 1.2 - 6.5° from laser exposure.
Results :
In all imaging sessions, putative systemic immune cells were identified flowing within and adhering to the inner wall of retinal vessels. Immune cell behaviors characteristic of the extravasation cascade such as capture, rolling, and crawling were observed. Immune cell diameters were ~9 - 12 µm, consistent with literature, and 107 cells were observed in total. In one intact eye, an average of 5.5 ± 6 S.D. cells/mm of retinal vasculature was observed with crawling speeds ranging from 0.17 to 0.32 μm/s. In the first hour following PR ablation in the same eye, an average of 7.1 ± 8 S.D. cells/mm was observed with crawling speeds of 0.11 to 0.30 μm/s. Averaging all sessions and locations in this eye, immune cells were 5 fold more numerous in venules than arterioles (X2 (1, N = 25)=32.9, p < .0001). Vascular perfusion remained uninterrupted throughout imaging.
Conclusions :
Immune cell activity within retinal vasculature can be visualized label free in living NHPs. This proof of concept study demonstrates that offset aperture AOSLO may serve to be a useful tool to assess the engagement of the systemic immune system in disease models and next-generation therapeutic interventions in NHP.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.