In vivo visualization of dendritic cells, macrophages, and microglial cells responding to laser-induced damage in the fundus of the eye.
OBJECTIVE
To study the in vivo response of mononuclear phagocytes (i.e., dendritic cells [DCs] and macrophages [MPhis]) 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, MPhis, and microglia cells (microGCs) 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 microGCs. 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 MPhis in the retina and choroid. The DCs rapidly disappeared from the retina, whereas the MPhis 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 microGCs increased in the retina. DCs and MPhis were present in the retina and choroid infiltrate. MPhis and granulocytes persisted in the choroid infiltrate longer than previously thought.
Eter
Nicole
N
Department of Ophthalmology, University of Bonn, Bonn, Germany. eter@uni-bonn.de
Engel
Daniel R
DR
Meyer
Linda
L
Helb
Hans-Martin
HM
Roth
Felix
F
Maurer
Juliane
J
Holz
Frank G
FG
Kurts
Christian
C
D000818:Animals; D000073883:CX3C Chemokine Receptor 1; D002465:Cell Movement; D002829:Choroid / Q000293:injuries* / Q000473:pathology; D003713:Dendritic Cells / Q000473:pathology*; D005131:Eye Injuries / Q000473:pathology*; D005434:Flow Cytometry; D005654:Fundus Oculi; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054018:Lasers, Excimer / Q000009:adverse effects*; D008264:Macrophages / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000473:pathology*; D008856:Microscopy, Fluorescence; D009887:Ophthalmoscopy; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000293:injuries* / Q000473:pathology; D013047:Specific Pathogen-Free Organisms
D000073883:CX3C Chemokine Receptor 1; C471885:Cx3cr1 protein, mouse; D019707:Receptors, Chemokine; D049452:Green Fluorescent Proteins