In Vivo Visualization of Intravascular Patrolling Immune Cells in the Primate Eye

Insight into the immune status of the living eye is essential as we seek to understand ocular disease and develop new treatments. The nonhuman primate (NHP) is the gold standard preclinical model for therapeutic development in ophthalmology, owing to the similar visual system and immune landscape in the NHP relative to the human. Here, we demonstrate the utility of phase-contrast adaptive optics scanning light ophthalmoscope (AOSLO) to visualize immune cell dynamics on the cellular scale, label-free in the NHP. Phase-contrast AOSLO was used to image preselected areas of retinal vasculature in five NHP eyes. Images were registered to correct for eye motion, temporally averaged, and analyzed for immune cell activity. Cell counts, dimensions, velocities, and frequency per vessel were determined manually and compared between retinal arterioles and venules. Based on cell appearance and circularity index, cells were divided into three morphologies: ovoid, semicircular, and flattened. Immune cells were observed migrating along vascular endothelium with and against blood flow. Cell velocity did not significantly differ between morphology or vessel type and was independent of blow flood. Venules had a significantly higher cell frequency than arterioles. A higher proportion of cells resembled "flattened" morphology in arterioles. Based on cell speeds, morphologies, and behaviors, we identified these cells as nonclassical patrolling monocytes (NCPMs). Phase-contrast AOSLO has the potential to reveal the once hidden behaviors of single immune cells in retinal circulation and can do so without the requirement of added contrast agents that may disrupt immune cell behavior.