Towards live assessment of human leukocyte dynamics in retinal disease with fluorescein-labeled peripheral blood mononuclear cells

Abstract

Purpose: Based on an abundance of animal model data, the pathogenesis of several retinal diseases like diabetic retinopathy (DR) is linked to inflammatory processes such as leukostasis, which is an early and important trigger of vascular hyperpermeability and damage. However, in humans, the dynamics and precise contribution of leukocytes to these retinal diseases and the potential therapeutic benefit of modulating these cells are still unclear. In this study we developed optimal conditions for the passive labeling of human leukocytes with sodium fluorescein, with the near term aim of autologous administration and monitoring of these cells in patients.

Methods: Individual human blood samples were centrifuged to isolate peripheral blood mononuclear cells (PBMCs) and then the optimal concentration, time, media and temperature for robust and safe labeling of cells with sodium fluorescein were determined. Fluorescence intensity, percent labeled cells, and percent cell survival were monitored by flow cytometry. PBMCs and different leukocyte subpopulations were also assessed for signs of cell activation. Finally, the optimized human leukocyte labeling conditions were used to label rat and mouse PBMCs and the ability to detect leukostasis in vivo was assessed using a scanning laser ophthalmoscope (SLO).

Results: Incubation of human PBMCs with 0.1% fluorescein for 2 minutes at room temperature, followed by storage at 4°C for up to 4 hours, led to robust cellular fluorescence without affecting cell viability. Labeling and further storage of cells for up to 24 hours at 37°C had no detrimental effect on survival and did not trigger cell activation. Using a SLO with rat PBMCs labeled using the optimized human cell conditions cells were readily recognized as single fluorescent dots moving in the retinal vessels and in VEGF-A injected rat eyes and in a mouse model of DR, leukostasis could be readily detected.

Conclusions: These findings provide the labeling conditions and initial safety assurances that will help enable imaging of autologous human leukocyte dynamics. The eventual aim will be to correlate leukocytes and their subpopulation numbers and dynamics with local retinal abnormalities. Moreover, in the future, the ability to monitor retinal leukocytes could prove invaluable in patient diagnosis, and the ability to monitor the therapeutic activity of drugs in clinical development.

Keywords: 552 imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • 688 retina • 529 flow cytometry