the SC. (H, I) Reconstruction of the retinotopic SC map, showing the density of CTB signal (red, green and blue indicate 100, 50 and 0% density, respectively). Dotted lines indicate location of sections shown in F and G; rostral, r; lateral, l. (H) Non-irradiated D2 mice showed near total CTB depletion. (I) Irradiated D2 mice showed almost complete CTB label and a normal retinotopic pattern. (J) Irradiation significantly protected axonal anterograde transport in D2 optic nerves (p,0.05). K) Flat-mount of an irradiated D2 retina at 9 mo of age showing FG labeled cells (red). L) Most NeuN+ RGCs (green) in this retina are retrogradely labeled with FG (red). Scale bar, 100 mm. M) Quantitative stereology of both NeuN+ RGCs and FG+/NeuN+ RGCs in flat-mounted retinas. Non-irradiated retinas (dark bars, n = 8; 9–10 mo old) and irradiated retinas (yellow bars, n = 8; 9–10 mo old) showed no differences in the total number of NeuN+ RGCs per retina between treatments, but slightly more retrogradely labeled FG+ RGCs in the irradiated retinas. Error bars = SEM. doi:10.1371/journal.pone.0043602.g004 function were preserved by irradiation, we investigated whether there is also preservation of intraretinal RGC compartments. To identify and assess the health status of RGCs, we collected retinas from 12 month-old mice that had received high-dose head irradiation at 4 to 6 weeks of age and, and compared these to non- irradiated D2 controls. We performed immunostaining in whole retinas with a Brn3 antibody that recognizes all isoforms of Brn3, a POU homeodomain transcription factor that identifies the majority of adult mouse RGCs. Brn3 expression is known to downregulate in aged D2 retinas prior to RGC loss (Buckingham et al., 2008; Soto et al., 2008). Confocal images of retinal flat- mounts showed that irradiated mice displayed a higher density of RGC somata expressing Brn3 compared to non-irradiated mice, as well as relatively more intense levels of Brn3 expression (Fig. 5A, B). Half of the non-irradiated D2 retinas analyzed (4/8 retinas) showed almost complete or severe loss of Brn3 immunostaining throughout the entire GCL, while only one irradiated retina (1/8 retinas) showed a significant portion of the retina with reduced Brn3, with none showing the most severe loss. In the same tissue we performed co-immunostaining for phosphorylated neurofilament (pNF), which is normally localized to optic axons, but aberrantly accumulates within the somata, dendrites and intraretinal axon segment of declining RGCs in D2 mice [15]. We found that in non-irradiated D2 retinas, there were reduced numbers of pNF-labeled axons overall, as well as beading or fragmentation of the remaining axons (Fig. 5C). In contrast, RGCs in irradiated mice showed uniform levels of pNF expression along their intraretinal axons, although axonal beading was present in the far peripheral retina (Fig. 5D). Somal and dendrite accumulation of pNF immunostaining was detected in most non- irradiated D2 retinas, particularly in RGCs localized to the mid- and far-peripheral retina (Fig. 5C, arrowheads), and in some cases somal pNF was observed within every declining RGC in the far- periphery (4/8 retinas). However, irradiated retinas had very few RGCs with somal pNF build up, with only low levels of accumulation (Fig. 5D, arrowhead; 7/8 retinas). The merged view of the Brn3 and pNF immunostainings reveals that in the non-irradiated retina there is colocalization of dystrophic axons to areas with reduced density of Brn3-positive cells, while these areas are reduced, but not completely absent, in retinas from irradiated mice (Fig. 5E, F). Since microglia activation is associated with neuron decline and degeneration in glaucoma and other neurodegenerative condi- tions, we immunostained the same tissue for Iba1 to assess the distribution and presence of activated microglia, identifiable by enlarged somata, thick processes and upregulation of Iba1 expression. We observed higher density of Iba1-positive microglia in non-irradiated D2 retinas (Fig. 5G), in comparison to irradiated Irradiation Abates Early Microglia Activation ones (Fig. 5H). Moreover, non-irradiated retinas showed a more irregular tiling of microglia resident in the NFL and GCL (Fig. 5G, H). The presence of activated microglia was noticeably reduced in irradiated retinas, presumably due to reduced RGC decline and degeneration. Most cells showed complex, slender processes and small somata (Fig. 5H, H’), versus the conspicuous soma and processes of activated microglia in non-irradiated retinas (Fig. 5G, G’). Thus, head irradiation results in overall improvement of RGC integrity at 12 months of age, when the RGC somal loss by apoptosis is quite apparent in a majority of D2 mice [13,34,35,36]. The late effect includes preservation of Brn3 expression and reduction of pNF buildup, in conjunction with reduced microglia activation. Discussion Here we examined the acute effects of high-dose irradiation on microglia in the D2 mouse glaucoma model. We found that irradiation causes an immediate reduction in proliferating microglia in the optic nerve head and glial lamina region, as well as a sustained reduction in the levels of microglia activation in the central retina, optic nerve head and laminar region. We also confirmed that irradiation of the head alone reduces RGC degeneration, improving both the structural integrity of the axons as well as anterograde axonal transport at later stages. While the effects of high dose irradiation on neuronal tissue are complex, these findings suggest that a component of the response to irradiation involves changes in microglia. High-dose irradiation has previously been shown to have significant effects on microglia, particularly in the young brain where proliferating microglia are more abundant than in the adult. For example, following high-dose irradiation of rats at P9 or P21, apoptotic microglia were evident by six hours, with a significant decrease in microglia cell numbers seven days later [37]. In the D2 retina, it has previously been shown that the major proliferating cell population is microglia [16], consistent with our observation that these cells are vulnerable to irradiation. Previously we reported microgliosis, as well as clustering and activation of microglial cells in the inner central retina and unmyelinated optic nerve regions (ONH and OL) in D2 mice at early ages [11]. In this study we found evidence for proliferating microglia concentrated in the OL by 1 mo of age, which were depleted by high-dose irradiation. In some cases, whole brain irradiation has been associated with increased microglia activation coupled with secondary astrocyte gliosis [38], although this was not observed here. In the D2 retina, the effect may target proliferating microglia, which are selectively increased in this disease model. In this study, irradiation was performed in young D2 animals, from 4 to 6 weeks of age, which is prior to the onset of detectable neurodegeneration of RGCs. Since microglia activation can be detrimental to neurons [9], it is possible that the protective effect of irradiation is due to selective depletion of an activated, prolifer- ating population of microglia, resulting in less neuronal damage. In support of this idea we have found that other interventions that reduce the levels of microglia activation, such as long-term minocycline treatment, can improve RGC axon integrity and axon transport function [24]. High dose irradiation with bone marrow transfer has also been shown to be neuroprotective in another neurodegenerative disease, Sandhoff’s disease, which is an inherited lysosomal storage disorder causing acute and rapid neuronal decline usually leading to early childhood death [39]. Interestingly, neurodegeneration in this disease is preceded by extensive microglia activation in both human cases as well as in a mouse model of the disease, which was prevented by the PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43602