Visualizing immune cell dynamics in the living non-human primate eye following retinal surgery

Abstract

Purpose : The retina mounts an immune response to foreign pathogens, injury, disease, and therapeutic interventions that may be helpful or harmful. Our understanding of these processes is limited by the paucity of imaging modalities that allow us to visualize single immune cells in the living eye. This study uses adaptive optics scanning laser ophthalmoscopy (AOSLO) with phase contrast to observe the evolution of immune activity at the cellular scale in the living retina of non-human primates (NHP) after surgical removal of the inner limiting membrane (ILM). We perform label-free, non-invasive monitoring of immune cell activity at the same retinal locations over the course of injury and recovery.

Methods : Retinal imaging was performed pre and post-surgery in 3 eyes from 2 male Maccaca fascicularis using AOSLO and clinical imaging. In each eye, an ILM peel (~ 19mm²) centered on the fovea was performed by a vitreoretinal surgeon. AOSLO data were collected using offset aperture imaging (790nm, 2.6 mW/mm², 10 Airy Disc Diameter (ADD) detection pinhole offset to 10 ADD). 3-6-minute videos were recorded at 60 locations (1.6^o x 1.6^o) in the vascular layer pre and 1-5 weeks post-ILM peel. Fundus imaging, SLO, and OCT were collected 2 to 3 weeks post-surgery.

Results : We observed a dramatic 17 fold increase in motile cells in the retinal parenchyma 1-2 weeks post-surgery (from pre-surgical levels of 21 ± 30 cells per mm², mean ± SD, to 350 ± 200 cells per mm²). Motile processes were also observed consistent with immune cell activity. Putative immune cells ranged from 5.7-8.4 μm in diameter. The number of moving cells decreased to 140 ± 70 cells per mm² by five weeks post-surgery. Static microstructural changes in the tissue were also observed, resolving over a similar timescale. Despite changes in density of motile cells in the parenchyma, there were no significant changes in the speed of cells crawling in vessels. Intravascular cells crawled at 14-25 μm/min, consistent with the behavior of patrolling monocytes.

Conclusions : This study reveals parenchymal immune cell dynamics for the first time label-free in NHP. These findings demonstrate the utility of offset aperture AOSLO to monitor immune cell activity in the central nervous system, offering valuable insights for both clinical and research applications when assessing diseases, surgical procedures, and next-generation treatments like gene and cell therapies.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.