Figure 2
From: Sectoral activation of glia in an inducible mouse model of autosomal dominant retinitis pigmentosa
Microglia in the I307N Rho mouse migrate to the outer retina, change to an ameboid morphology, and expand in number after induction. I307N Rho mice were exposed to 20,000 lx of light for thirty minutes and eyes were enucleated before and between two hours and three days thereafter for preparation of PFA-fixed retinal flat-mounts and subsequent IF-staining for CD45 and DAPI. (a) Full-thickness images were captured in the area of light damage and Z-projections were created with ImageJ to depict CD45-positive cells (green) in the outer (OPL and ONL) or inner retina (IPL and GCL). Microglia responded by progressively changing from a ramified to an ameboid configuration. Isolated phagocytic events were observed at twelve hours and one day (white arrows). CD45-positive monocytic cells were seen in the inner retina on day three (red arrow). Original magnification = 10x. (b) Microglia were counted in the inner and outer retina in two separate images for a given animal and summed. The bar graph represents the mean count ± s.e.m. (n = 3 per time point). Microglia distribution changed as early as two hours after light damage. One-way ANOVA followed by Dunnett’s multiple comparisons tests was performed to compare the inner retina, outer retina, and total counts to baseline. Normality was tested with the Shapiro–Wilk test of residuals. Statistical symbols: black circle = inner retina; white circle = outer retina; * = total count. One-symbol = p < 0.05; Two-symbols = p < 0.01; Three-symbols = p < 0.001; Four-symbols = p < 0.0001. (c) 3D reconstructions (CD45 = green; ONL = blue; INL = purple) of isolated microglia (original magnification = 100x) at the interface of the OPL and ONL were created with ImageJ. Microglia dendrites rapidly infiltrated the ONL and began to retract as early as two hours and twelve hours, respectively. A frank ameboid morphology is achieved by day three. A 125 µm segment of the SD-OCT B-scan is provided to show the development of hyper-reflectivity alongside the microglia morphological changes (ONL: blue bar; INL: purple bar). The choroid and nerve-fiber layer (NFL) are positioned superiorly and inferiorly, respectively. Statistical data can be viewed in Supplementary Table S3.
