In vivo visualization of dendritic cells, macrophages, and microglial cells responding to laser-induced damage in the fundus of the eye

PURPOSE. To study the in vivo response of mononuclear phagocytes (i.e., dendritic cells [DCs] and macrophages [M Phi s]) in the posterior eye segment after laser-induced injury, and to gain a better understanding of the role of these cells in inflammatory eye disease. METHODS. CX(3)CR1(GFP/+) knockin mice were used, in which DCs, M Phi s, and microglia cells (mu GCs) are constitutively fluorescent. These reporter mice were examined by a confocal scanning laser ophthalmoscope (cSLO) after argon laser coagulation. cSLO was complemented by fluorescence microscopy of retinal flatmounts and eye cryosections, to study cell morphology and location, and by multicolor flow cytometry, to determine the number and identity of the fluorescent cells. RESULTS. The retina of healthy reporter mice featured abundant fluorescent mu GCs. After laser injury to the fundus, these cells accumulated and migrated laterally toward injury after 60 minutes. Distinctly shaped fluorescent cells accumulated within laser spots and were identified by flow cytometry and immunofluorescence microscopy as DCs and M Phi s in the retina and choroid. The DCs rapidly disappeared from the retina, whereas the M Phi s stayed longer. Choroidal infiltrates were detectable even 35 days after laser injury, in particular in larger spots resulting from higher laser intensity. In addition, nonfluorescent granulocytes were detected in the choroid. CONCLUSIONS. The synergistic use of ophthalmoscopy, flow cytometry, and immunofluorescence microscopy allows detailed dissection of the in vivo response of mononuclear phagocytes to laser injury of the fundus. The number of mu GCs increased in the retina. DCs and M Phi s were present in the retina and choroid infiltrate. M Phi s and granulocytes persisted in the choroid infiltrate longer than previously thought.