Expression Of Cellular Biomarkers In Subretinal Fibrosis In The Mouse Laser-induced CNV Model

Abstract

Purpose: : Subretinal fibrosis leading to disciform scar formation is a serious companion pathology associated with choroidal neovascular disease that leads to loss of vision. To better understand subretinal fibrosis, key cellular and protein biomarkers involved in the epithelial to mesenchymal cell transition(EMT), and the fibrotic process have been examined in the mouse laser induced choroidal neovascular, CNV, model by immunofluorescence labeling.

Methods: : Cell biomarker expression was examined in CNV lasered eyes from 4 month old C57BL/6J mice at 3, 7 and 30 days post laser. Cell markers included Iba1, for microglia/macrophages, RPE65 for retinal pigmented epithelium, GFAP for activated Muller glial cells, vimentin and α-smooth muscle actin (αSMA) for mesenchymal cells, Griffonia simplicifolia- iso-B4 Lectin, for endothelial cells and matrix proteins collagen I and IV. Antibody labeling was detected with a sets of secondary antibodies conjugated to fluorophores and DAPI nuclear counter stain.

Results: : Laser ablation in the mouse CNV model resulted in distinct patterns of biomarker expression in retina and choroid at all times points. At 3 days, RPE65 expression was absent at the lesion site with the death of RPE. At 30days, the RPE and RPE65 expression begin to appear in spindle shaped cells to cover the fibrotic wound. Microglial/ macrophages appeared at 3 days but had different shapes in the retina and choroid. Activated Muller glial cells and vimentin were up regulated in area of retinal damage but were not observed in the sub-retinal fibrotic lesion. Vimentin positive cells (myofibroblasts), endothelial cells and collagen IV were observed in the subretinal fibrotic lesion at all times.

Conclusions: : Retinal and choroidal CNV lesions shared common biomarker expression in different cells. Vimentin positive, activated Muller cells were observed only in the retinal lesion. Vimentin positive macrophages and endothelial cells are the main cellular component of the fibrotic scar. Collagen IV and Iba-1-positive cells have been observed in SRF samples from AMD patients (data not shown).

Keywords: EMT (epithelial mesenchymal transition) • age-related macular degeneration • pathology: experimental