Eter Nicole, Engel Daniel R, Meyer Linda, Helb Hans-Martin, Roth Felix, Maurer Juliane, Holz Frank G, Kurts Christian
Department of Ophthalmology, University of Bonn, Bonn, Germany.
Invest Ophthalmol Vis Sci. 2008 Aug;49(8):3649-58. doi: 10.1167/iovs.07-1322. Epub 2008 Mar 3.
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.
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.
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.
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.
研究激光诱导损伤后眼后节中单核吞噬细胞（即树突状细胞[DCs]和巨噬细胞[MPhis]）的体内反应，以更好地理解这些细胞在炎性眼病中的作用。
使用CX(3)CR1(GFP/+)基因敲入小鼠，其中DCs、MPhis和小胶质细胞（microGCs）组成性荧光表达。氩激光凝固后，通过共焦扫描激光检眼镜（cSLO）检查这些报告基因小鼠。通过视网膜铺片和眼冰冻切片的荧光显微镜检查来补充cSLO，以研究细胞形态和位置，并通过多色流式细胞术来确定荧光细胞的数量和类型。
健康报告基因小鼠的视网膜中有大量荧光microGCs。眼底激光损伤后，这些细胞在60分钟后聚集并向损伤部位横向迁移。形状独特的荧光细胞在激光光斑内聚集，并通过流式细胞术和免疫荧光显微镜鉴定为视网膜和脉络膜中的DCs和MPhis。DCs迅速从视网膜中消失，而MPhis停留时间更长。即使在激光损伤后35天仍可检测到脉络膜浸润，特别是在激光强度较高导致的较大光斑中。此外，在脉络膜中检测到非荧光粒细胞。
检眼镜检查、流式细胞术和免疫荧光显微镜的协同使用能够详细剖析单核吞噬细胞对眼底激光损伤的体内反应。视网膜中microGCs的数量增加。DCs和MPhis存在于视网膜和脉络膜浸润中。MPhis和粒细胞在脉络膜浸润中的持续时间比以前认为的更长。
