Figure 1. Irradiation results in depletion of proliferative microglia within the proximal optic nerve regions. (A) Iba1- positive cells with and without nuclear expression of the proliferative marker (PCNA) were detected in radial sections along the ONH, OL and proximal ON (delineated) shown in a single 1 mm optical slice. (B) Overlay of Iba1 and PCNA, plus single-channel view for PCNA. Scale bar represents 10 mm. The colocalization of Iba1 and PCNA to individual cells was confirmed by tracing their linear intensity profiles in single optical slices (between the arrows). Cycling microglial cells were positively identified by the nuclear localization of PCNA expression within the cytoplasmic Iba1 expression (graph). (C, E) Non-irradiated D2 mice show concentration of proliferating microglia within the unmyelinated ONH and OL, shown in a maximal Z-projection. Cycling microglia have a green or white nucleus in the overlay view of Iba1 and PCNA stainings, which is shown as single PCNA-channel for clarity. (D, F) 7 days (d7) after irradiation microglia proliferation appears negligible along the proximal nerve, as shown in a maximal Z-projection. Scale bars, 100 mm. doi:10.1371/journal.pone.0043602.g001 whether this was due to induction of apoptosis we delivered SR- FLIVO probe to 2–3 month-old D2.CX3CR1gfp/+ mice by tail vein injection 8 hours post-irradiation, then sacrificed animals 8 hours later for retina dissection and analysis. We detected apoptotic microglia in irradiated mice, which were mostly Irradiation Abates Early Microglia Activation concentrated in the central retina, while apoptotic microglia were absent in non-irradiated controls (Fig. S3). To quantify the effect of radiation on proliferating microglia, we counted the percent of Iba1+ PCNA+ microglia localized to the ONH, OL and proximal ON in untreated D2 animals, and at 1, 7 and 30 days post-irradiation. In non-irradiated 4 to 6 week-old D2 mice we found proliferating microglia in all three regions, but detect the highest proportion in the lamina (OL), representing half of the microglia population in this region (Fig. 2A). At both 1 and 7 days post-irradiation, proliferating microglia were dramatically reduced in the ONH, OL and ON, with some recovery in the ONH by 7 days. By 30 days post-irradiation the proportion of cycling microglia within both the ONH and OL had recovered to nearly half of their original numbers, while the repopulation within the first 250 mm of ON was complete (Fig. 2A). When we assessed changes in overall density of microglia, we found a general reduction in all compartments at 1 day post-irradiation, but most significantly in the OL, consistent with the high proportion of proliferating microglia in that region (Fig. 2B). Microglial cell density was restored to levels approaching non-irradiated D2 mice by 7 days post-irradiation and was maintained out through 30 days. Thus, we conclude that irradiation has an acute and significant effect by reducing the presence of proliferating microglia. Irradiation results in reduced levels of microglia activation Since the numbers of proliferating microglia were acutely reduced by high dose irradiation, we sought to investigate whether irradiation also had an acute effect on the level of microglial activation, as well as an impact on the peak activation detectable by 3 months of age in the central retina and ONH of D2 mice [11]. To quantify changes in Iba1 mRNA expression 7 days and 30 to 45 days post-irradiation, we performed quantitative RT- PCR on mRNA isolated from microdissected samples that included the central 100 mm of central retina, the unmyelinated optic nerve regions and the first ,200 mm of myelinated optic nerve. We compared these samples to age-matched control samples from non-irradiated D2 and D2G retinas. After 7 days, irradiation did not alter the levels of Iba1 expression, which are low at this age and were comparable between the three groups analyzed. Normally there is an increase in microglia activation in the central retina and proximal nerve region by 3 months of age [11], which is also observed here as indicated by an elevation of Iba1 expression (Fig. 3A). In irradiated D2 tissue at this age, which is 30 to 45 days after irradiation, we found a significant reduction in Iba1 expression down to levels corresponding to those in tissue from non-glaucoma D2G mice (Fig. 3A), suggesting that irradiation prevents the increase in microglia activation. We conclude that, after high dose irradiation, the microglia localized to the central retina and proximal nerve region show sustained reduced levels of activation, comparable to non-glaucoma controls. The striking finding that irradiation lowers the levels of Iba1 mRNA expression to control levels by 3 months of age was confirmed by immunostaining retinal wholemounts from non- irradiated and irradiated D2 mice for Iba1. To assess microglia activation, microglia localized to the central retina and optic disc were imaged at high resolution. We found that non-irradiated D2 retinas show clustering of microglia with large soma size and reduced branching within the central retina, demonstrating cell activation (Fig. 3B, B’). In contrast, irradiated retinas show microglia with small somata and thinner branches, and therefore with reduced levels of activation (Fig. 3D, D’). High levels of Iba1 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43602