Abstract
Purpose: :
Due to their limited numbers, dendritic cells (DC) have been difficult to study for phenotype and function in retina. Previously, we found that they responded to minor retinal injury, this response will be used to facilitate the study of their frequency, morphology, origin, and function.
Methods: :
Chimeric B6 mice [CD11c-DTR Ly5.2 recipients of Ly5.1 bone marrow (BM), and vice versa] were used to analyze the origin of the retinal GFP+ cells. Chimeric and control mice received an ONC to one eye and later depleted of retinal GFP+ cells by injecting diphtheria toxin (DTx) into the anterior chamber (AC). An ONC is used as a stimulus to increase the number of GFP+ cells in the retina. Retinal whole mounts were harvested at specified times, stained for DC and microglia (MG), and analyzed by confocal microscopy.
Results: :
Analysis of retinas from Ly5.1 recipients of CD11c-DTR BM, in the absence of injury, shows a decrease in GFP+ cells relative to normal CD11c-DTR retina. This suggests minimal turnover from the grafted cells up to six months, even though the mice were irradiated. Retinas from Ly5.1 recipients of CD11c-DTR BM that received an ONC 10 days prior to harvesting had many donor GFP+ cells in the crushed eyes around the optic disc and in the periphery. These GFP+ cells appear to be derived from circulating precursors. The persistence of retinal GFP+ cells in CD11c-DTR recipients of Ly5.1 BM up to 7 months pointed to a precursor that must have come from a non-circulating source such as MG or local progenitor cells. The number of MG was found to be unchanged after the retinas of these mice were repeatedly depleted of GFP+ cells by DTx injections. Thus, MG are unlikely to be major contributors of GFP+ cells. The injection of DTx not only led to the depletion of GFP+ cells; it also seemed to deplete local progenitor cells, as no GFP+ cells were found after the retinas had received an additional stimulus to recruit more GFP+ cells. In non-chimeric CD11c-DTR mice, which have all possible precursors, the number of MG did not show a reduction after the retinas were repeatedly stimulated and depleted of GFP+ cells with DTx. These results suggest that MG do not contribute to the GFP+ cells.
Conclusions: :
The use of normal and chimeric mice led us to the conclusion that these GFP+ cells were derived in part from a local progenitor cell population.
Keywords: microglia • retina • immunohistochemistry