Abstract
Purpose: :
To characterize the neovascular response of CCR2 -/- mice in the oxygen induced retinopathy mouse model and measure macrophage infiltration and cytokine levels during the establishment and regression of pre-retinal neovascularization.
Methods: :
Pre-retinal neovascularization was assessed by enumerating endothelial cell nuclei in paraffin embedded sections from age-matched wild-type and CCR2 -/- mice. FITC dextran (2x10^6 MW) perfusion as well as Isolectin B4 staining of retinal flatmounts was also performed. Flow cytometry for macrophages was performed in samples collected from age-matched animals at time points following hyperoxic exposure. Retinal extracts were analyzed by multiplex bead array for inflammatory cytokine levels.
Results: :
CCR2 -/- show a more slightly robust neovascular response in the OIR model than wild-type mice of the same background at postnatal day 17, 44.5 nuclei per section vs. 35.9 nuclei per section, but the differences were not statistically significant (n=3). Flow cytometry of retinal cells from CCR2 -/- mice and WT during the OIR model showed increased levels of macrophages in the period immediately following return to room air. VEGF levels in the retinas of CCR2 -/- mice are markedly increased after the return to room-air, 22.5 ng/ml vs. 9.4 ng/ml for WT mice (p<0.05). Levels for IL-18, M-CSF, and bFGF were also significantly altered over the course of neovascular establishment and regression in the OIR model.
Conclusions: :
Interestingly, CCR2 -/- mice seem to share a similar phenotype in this model as MCP-1 -/- mice; MCP-1 is the cognate receptor for CCR2. MCP-1 -/- have previously been reported to have an unchanged level of neovascularization at day 17, but a delayed regression of pathologic retinal vasculature (Davies, et al, IOVS, 2008). Unlike the reduction in macrophages in that study, our data indicate that mice deficient in the receptor CCR2 have significant levels of macrophages in the retina during the OIR model. This provocative finding hints at the existence of an alternate receptor for MCP-1 that may be signaling macrophage response in these animals and modulating their neovascular response. Disregulation of bFGF, VEGF, M-CSF, and IL-18 may contribute to the differential progression observed between MCP-1 -/- and CCR2 -/- mice in the OIR model.
Keywords: diabetic retinopathy • inflammation • cytokines/chemokines