Irradiation Abates Early Microglia Activation Figure 3. Irradiation reduced early microglia activation within the central retina and proximal optic nerve compartments. (A) Quantitative RT-PCR analysis of Iba1 transcript in a sample including the central 100 mm of retina, ONH, OL and proximal 200 mm of myelinated optic nerve, collected from non-irradiated D2 and non-glaucoma D2G, and irradiated D2 mice (n = 10 per group and age). 7 days post- Figure 2. Irradiation reduces microgliosis in the unmyelinated optic nerve. Quantification of the proportion of cycling microglial cells (PCNA and Iba1-positive) per optic nerve compartment in non- irradiated and irradiated D2 mice (n = 6 samples per timepoint and experimental group). The statistical significance indicated for each compartment and timepoint is relative to non-irradiated controls. (A) In non-irradiated D2 mice aged 4 to 6 weeks, the proportion of proliferating microglia is ,50% within the OL, and ,25% within the ONH and ON. At 1 day and 7 days after irradiation, numbers of cycling microglia are reduced in all optic nerve portions, with maximal reduction within the OL (p,0.001), and with significant effect in the ONH and proximal ON (p,0.01). 30 days post-treatment the unmyelinated ONH and OL compartments have recovered only half of its original proliferating microglia, whereas the proximal ON has been repopulated. (B) Total number of Iba1-positive microglia (per area) within each optic nerve compartment. The density of total microglia demonstrates that irradiation has an acute and specific effect on cycling microglia resident within the OL (p,0.01). doi:10.1371/journal.pone.0043602.g002 expression was observed in numerous microglial cells in non- irradiated D2 retinas (Fig. 3C, C’; pseudocolored, with red representing the highest levels of expression), and this is clearly reduced in age-matched tissue after irradiation (Fig. 3D, E; pseudocolored). Our findings indicate that irradiation suppresses the intense microglia activation, characteristic of the early D2 retina. Irradiation did not induce early gliosis Glaucoma-like pathology in the D2 retina is also associated with significant astrocyte and Mueller cell reactive gliosis, which is evident by upregulation of GFAP expression [16]. To assess whether irradiation induces gliosis at 3 months of age, we generated radial sections through the central retina and its proximal optic nerve regions and immunostained them for GFAP. Non-irradiated and irradiated D2 mice showed similar levels and PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43602