Fig. 7

CCL2-CCR2 mediated signaling recruits monocytes but does not limit the rate of retinal thinning. a, b Cytokine array screening of retinal homogenates of dark-reared Arr1^−/− and WT mice before (0 h) and after 12 h of light exposure. There were few light-dependent changes overall, other than a striking increase in CCL2 expression in Arr1^−/− retinas. Representative results from 8 blots, 4 from each genotype. c ELISA analysis of dark-reared Arr1^−/− and WT mice revealed that CCL2 levels rose rapidly after 12 h of light exposure, peaking at 18 h (n = 3–5 mice per time point). d IHC of retinal sections of dark-reared (0 h) and light-exposed (18 h) Arr1^−/− mice showed early CCL2 expression (green) in Müller glial cells (red). e Expression of fluorescently tagged CCL2 (green) was observed abundantly throughout the retina at 24 h but not at 48 h, consistent with the previous IHC and ELISA data. f In PDGFRa-Cre⁺ mice, the LoxP sites flanking CCL2 were excised only in Müller glia cells, preventing these cells from producing CCL2, and resulting in an almost complete absence of CCL2 in the retina of Cre⁺ mice at 24 h. Only punctate staining was observed in circulating and infiltrating monocytes, which did not lose their ability to produce CCL2. Representative images from 2 to 3 sections from 2 mice per strain per time point. These data demonstrate that Müller glia cells are the primary producers of CCL2 in the retina. g Knocking out CCR2, the primary receptor for CCL2, resulted in an almost complete loss of recruited CD45^high cells. h There was a significant loss of both CD45^high Ly6C^high and CD45^high Ly6C^low subpopulations (n = 4–6 retinas per strain per time point). i, j In vivo OCT B-scans were used to measure the thickness of the outer and inner retinal layers during degeneration. i Blocking CCL2-CCR2 signaling did not affect the extent of outer retinal thinning in CCR2-RFP^rfp/rfp knockout mice (compare Arr1^−/− (blue), Arr1^−/− CCR2-RFP^+/rfp (black), and Arr1^−/− CCR2-RFP^rfp/rfp (brown)), by using an intravitreal injection of CCL2 neutralizing antibody (compare saline (light blue) to anti-CCL2 (green)), or with a conditional CCL2 knockout in Müller glia (compare Arr1^−/− CCL2-RFP^lox/lox PDGFRa-Cre⁻ (purple) to Arr1^−/− CCL2-RFP^lox/lox PDGFRa-Cre⁺ (orange)). j No appreciable change was detected in the thickness of the inner plexiform layer (IPL) in any of the mice, confirming that retinal degeneration was limited to the photoreceptor layer (n = 3–4 retinas from 2 mice per strain per time point). Error bars represent SEM; **p < 0.01, ***p < 0.001; ONL outer nuclear layer, INL inner nuclear layer, RGC retinal ganglion cell layer. Scale bar is 20 μm in d–f