In vivo tracking of hematopoietic cells in the retina of chimeric mice with a scanning laser ophthalmoscope

Abstract

We examine the effect of bone marrow transplantation (BMT) on retinal cell turnover by performing simultaneous cell tracking of native microglia and engrafting donor bone marrow-derived cell (BMDC) populations in the retinae of live mice using a custom-built multi-color confocal scanning laser ophthalmoscope (SLO) specifically developed for murine retinal imaging. CX₃CR₁^GFP/+ mice whose retinal microglia express the green fluorescent protein (GFP) were exposed to a lethal dose of gamma radiation and subsequently rescued with bone marrow cells from universal DsRed donor mice. Over a time course of four months after the irradiation and BMT, progressive loss of GFP⁺ microglia was accompanied by delayed engraftment of DsRed⁺ BMDC. Morphologic examination revealed that the remaining GFP⁺ microglia were ramified, while engrafting DsRed⁺ cells exhibited both ramification and dendriform shape. Leukocyte endothelial interaction, normally absent in healthy retinal vasculature, was observed even after three months, indicating sustained inflammation long after the radiation exposure. Fluorescein angiography demonstrated that the blood-retina barrier is compromised early after irradiation. In vivo imaging provides a powerful means to study dynamic cellular processes over a broad range of timescales from seconds to months that have previously not been accessible by ex vivo analysis.

Keywords: :

• scanning laser ophthalmoscope
• retinal imaging
• microglia
• turnover
• bone marrow derived cells
• engraftment
• irradiation
• blood-retina barrier

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This research was funded by the Department of Defense, project FA9550–10–1-0537. The authors thank Dr. Mark Cronin-Golomb of Tufts University for the generous loan of a laser beam propagation analyzer and Dr. Daniela Krause for help with the bone marrow transplantation. We thank Drs. Francesca Ferrara, Stefania Lymperi, Daniela Krause and Andrew Catic for help with gamma irradiations.