CD74 as a microglial activation marker in the pathogenesis of retinal vasoregression in a transgenic rat model

Aims: Vasoregression is a hallmark of the early phase of diabetic retinopathy (DR). Our previous study has demonstrated in a transgenic rat model – the polycystic kidney disease (PKD) model – retinal vasoregression which is similar to DR. Microarray analysis in this model indicated a major upregulation of CD74. Immunofluorescence (IF) staining exhibited that CD74 is expressed by microglia – resident macrophage in retina and central nervous system. In the present study, we aimed to establish CD74 as microglial activation marker in the pathogenesis of vasoregression in PKD model.

Materials and methods: Sprague-Dawley (SD) rats were subjected to generate transgenic rat (TGR) with overexpression of polycystin-2. 3 months old PKD (PKD 3mo) and SD (SD 3mo) rats were studied in this research (n=5 in each group). IF staining of CD74 was performed to identify the morphology of positive cells in different layers of retina. Triple IF staining of CD74, CD11b (marker of microglia) and lectin was conducted to investigate the colocalization of CD74 with CD11b and quantification of microglia in different layers of retina. The ratio of colocalization (the amount ratio of CD74-positive cells to CD11b-positive cells) represents how many microglia express CD74. Quantitation of CD11b and CD74 positive cells was performed by confocal microscope (Leica TCS SP2, Heidelberg, Germany) (ten fields in each layer of retina, randomly).

Results: There were five layers of CD74-positive cells in PKD group with different morphology under microscope: superficial1 (S1) layer and superficial2 (S2) layer (ramified shape with foot processes), intermediate (M) layer and deep1 (D1) layer (ramified shape with longer foot processes), deep2 (D2) layer (oval or round shape with short processes, activated state). CD74-positive cells were absent in D1 and D2 layers in SD group. 91.1±8.1% CD74-positive cells co-expressed CD11b and showed similar morphology of microglia, which indicated CD74 maybe a potent marker of microglia. Microglia was detected exactly on the site of acellular capillaries in deep vasculature. Quantification of CD74- or CD11b-positive cells demonstrated that the amounts of microglia increased significantly in each layer in PKD group, compared with SD group and this differences were more obvious in CD74 staining, especially in deep layer (S1: 12.7±5.3 vs. 3.3±0.5, S2: 9.9±5.3 vs. 1.3±1.0, M: 7.2±4.0 vs. 0.8±1.0, D1: 23.5±6.7 vs. 0, D2: 20±19.8 vs. 0; PKD vs. SD). The ratio of colocalization in PKD group achieved peak value in D1 and D2 layer (S1: 18.7±10.2%, S2: 26.3±17.9%, M: 26.0±12.2%, D1: 76.1±13.4%, D2: 72.8±19.8%).

Conclusion: Microglial activation is indicated by CD74 expression in the transgenic model of vasoregression, in particular in the deep vasculature where vasoregression starts. These also are suggestive of an active role of microglial activation in the pathogenesis of vasoregression and the important role of glial-neuronal-endothelial interaction in retinopathy.