Irradiation Abates Early Microglia Activation Figure 5. Irradiation reduces RGC decline and microglia activation within the retina. Confocal images of triple-immunostained (Brn3, pNF and Iba1) flat-mounted retinas from non-irradiated (11 mo old) and irradiated (12 mo old) D2 mice (n = 8 retinas per group). Representative fields (,1.660.9 mm2) show matching position (optic disc at left of asterisks) and a projection of the NFL and GCL, from central to peripheral eccentricities. (A–B) Most of the non-irradiated retina displays low numbers of RGCs expressing Brn3, whereas irradiated mice maintain a higher density of brightly labeled RGCs. (C, D) Co-immunostaining for pNF in the same non-irradiated retina shows a reduced number of optic axons, thinner axonal bundles and defasciculated axons that show beaded profiles in their entire length. In the irradiated retina, RGC axons are organized in bundles that show a higher and uniform pNF content, and some fragmentation mostly limited to the peripheral area. Only non-irradiated retinas show abundant RGCs with somadendritic accumulation of pNF (arrowheads). (E, F) Overlay of Brn3 and pNF staining reveals the colocalization of declining axons and sparse Brn3-positive RGCs in the non-irradiated D2 retina, in contrast to healthier, fasciculated axons in the irradiated mice. (G, H) Reflecting the abnormalities shown by the RGC markers, microglia (Iba1) had altered tiling around RGC axons and variable, increased density in non-irradiated retinas. Activated microglia, recognizable by their enlarged somata and reduced branching, were abundant in the non-irradiated retina, and infrequent in the irradiated ones. (G’, H’) High magnification views of framed areas shown in G, H. Scale bars, 100 mm. doi:10.1371/journal.pone.0043602.g005 PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43602