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

¹ Department of Ophthalmology, University of Bonn Medical Center, Ernst-Abbe-Str. 2, Bonn, 53127, Germany
² Institute of Molecular Medicine and Experimental Immunology, University of Bonn Medical Center, Bonn, Germany
³ Department of Ophthalmology, University of Bonn Medical Center, Bonn, Germany

^* To whom correspondence should be addressed. E-mail: eter{at}uni-bonn.de.

	Abstract

Purpose: We investigated the in vivo response of mononuclear phagocytes, i.e. dendritic cells (DCs) and macrophages (Ms), in the posterior eye segment after laser-induced injury, to better understand the role of these cells in inflammatory eye disease. Methods: We used CX3CR1GFP/+ knockin mice, in which DCs, Ms and microglia cells (µGCs) 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 flat-mounts and eye cryosections to study cell morphology and location, and by multicolor flow-cytometry to determine cell numbers and identity. Results: The retina of healthy reporter mice featured abundant fluorescent µGCs. After laser injury to the fundus, these cells accumulated and migrated laterally towards injury after 60 minutes. Distinctly shaped fluorescent cells accumulated within laser spots, and were identified by flow-cytometry and immunofluorescence microscopy as DCs and Ms in retina and choroid. DCs rapidly disappeared from the retina, whereas Ms stayed longer. Choroidal infiltrates were detectable even 35 days after laser injury, in particular in larger spots resulting from higher laser intensity. In addition, non-fluorescent 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. µGC numbers increased in the retina. DCs and Ms were present in the retina and choroid infiltrate. Ms and granulocytes persisted in the choroid infiltrate longer than previously thought.

Key Words: laser, immunoregulation, knockout animals, scanning laser ophthalmoscopy, macrophages

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