irradiation and bone marrow transfer procedure in mice [39]. In this case, the effects of irradiation versus bone marrow transplan- tation were not tested. Nevertheless, it raises the possibility that irradiation may have similar neuroprotective effects in other disease models since early microglial activation has been reported for a number of neurodegenerative diseases [40]. However caution is warranted, since radiation retinopathy associated with adverse effects on retinal vasculature can be a significant complication of irradiating the retina, with few therapeutic options [41,42,43]. It is also possible that irradiation has a direct effect on other retinal cell populations, since there is precedence for irradiation impacting neural tissue. For example, therapeutic irradiation of the brain is used for treating brain tumors, but is known to induce cognitive deficits [44,45]. Some of this may be due to the depletion of dividing neural stem cells and neural precursors in the ventricular zone of some brain regions [44]. This is unlikely to be a factor in this study since there are no dividing neural precursors or stem cells within the retina at this stage, with only microglia and pericytes dividing in the D2 retina [16]. In addition, postmitotic neurons are generally thought to be more radio- resistant [46]. We did not observe any significant acute effects of irradiation on RGC structural integrity (data not shown), suggesting that RGCs are relatively resistant to radiation-induced damage. More subtle effects on neurons have been associated with irradiation. For example in hippocampal pyramidal neurons irradiation caused a loss of expression of some neuronal genes, such as NeuN, within 7 days [47]. In addition, irradiation has been shown to affect behaviorally induced expression of the immediate- early gene Arc (activity-regulated cytoskeleton-associated protein), suggesting alteration of neuronal function [48]. However, we observed normal expression of Brn3 and NeuN, suggesting that RGC gene expression is not negatively affected by irradiation. While these other studies suggest that irradiation causes reduction of neuronal integrity and function, it remains possible that exposure of the retina to high-dose irradiation alters neuronal gene expression, potentially rendering RGCs resistant to subse- quent insult in this glaucoma model. Retinal pericytes, which are associated with the retinal vasculature, also proliferate in the D2 retina [16], so these cells may also be impacted by high-dose irradiation, as suggested by our detection of activated caspases in diminutive cells along vessel walls. In fact, in other studies irradiation has been shown to acutely affect the integrity of the blood–brain barrier and blood- retinal barrier and promote the influx of bone-marrow-derived cells [49,50,51,52], while under normal conditions, bone marrow- derived myeloid cells rarely infiltrate the CNS [53]. Consistent with this, it has been clearly demonstrated in the retina that irradiation triggers recruitment of blood-derived monocytes, as detected by transplantation of GFP-labeled bone marrow cells into mice following whole body irradiation [51,52,54]. This influx of GFP+ cells into the retina was prevented by shielding of the head, demonstrating that irradiation of the retina is required and that normal microglial cell turnover from circulating monocytes is low [52,55]. In contrast, Howell et al. report that irradiation of the head or eyes of the D2 mouse at 2 months of age prevents proinflamma- tory monocytes from entering the optic nerve when analyzed at 10.5 months of age, but prior to detectable neuronal damage, and they provide evidence that these infiltrating monocytes contribute to neuronal decline in D2 glaucoma [26]. Thus, monocyte infiltration may be a critical component of disease in this model. However, it remains to be determined how early irradiation inhibits the invasion of monocytes at much later ages, especially Irradiation Abates Early Microglia Activation when in other models irradiation promotes monocyte invasion [52]. Importantly, bone marrow-derived myeloid cells may have functions or responses that are distinct from those of resident microglia [53,56,57], potentially impacting the course of disease. In our study, Iba1 does not distinguish macrophages from microglia, so it is possible that macrophages are recruited at early stages as well. It remains to be determined whether this contributes to the effects of irradiation in the D2 model of glaucoma. Supporting Information Figure S1 Experimental design. (A) D2 mice received a single high-dose of X-irradiation targeting their eyes and visual pathways at a prepathological age of 4 to 6 weeks of age. Microglial changes following irradiation were examined in a cohort of young mice, while optic neuropathy was measured in aged mice. Identical analysis was performed in age-matched non- irradiated D2 mice as well as non-glaucoma D2G mice. (B) Radiation was confined to the rostral half of the head targeting eyes and visual pathways (orange rectangle). This area was consistently positioned with reference collimated light (white rectangle) and focusing LEDs. The nose and regions behind the ears (5-mm post-lambda, l) were spared, as was the body by shielding with lead plates. (TIF) Figure S2 Intraocular pressure (IOP) elevated with similar patterns in D2 mice from 2 to 8 months of age, regardless of irradiation. A summary of mean IOP per month (and sample size) shows that irradiation did not prevent IOP elevation. (TIF) Figure S3 Irradiation selectively induces apoptosis in retinal microglia. Representative retinal wholemounts from non-irradiated and irradiated D2.CX3CR1gfp/+ mice, where microglia expresses GFP under the control of fractalkine-receptor locus (optic disc position indicated by asterisk). Apoptotic cells were detected with SR-FLIVO, which selectively binds cleaved caspases and emits at 600 nm. (A) Non-irradiated retinas show only very small apoptotic cells positioned along large blood vessels, probably corresponding to pericytes. (B) Irradiated retinas, instead, show numerous and conspicuous apoptotic microglia mostly localized to the optic disc and central retina. Some microglial cells are detectable in the mid-peripheral retina (arrowheads). Notice that, relative to non-irradiated mice, the regularity of microglial cell tiling is lost, and fewer cells with complex branching are present in the central retina. (C, D) Detailed view of the optic disc area (inset in A, B) showing the overlay of FLIVO and GFP highlights the virtual absence of apoptosis in the non-irradiated retina, and the clear overlap of both stainings to cells with activated shape. n = 3 per group. Scale bars, 100 mm (A, B), 10 mm (C, D). (TIF) Figure S4 Irradiation does not induce early gliosis. (A, B) Images of representative radial sections through the central retina and proximal optic nerve from 3 month-old D2 mice detects comparable GFAP expression in astrocytes, but not in Mueller cells, regardless of treatment. Co-immunostaining for Iba1 shows the relative reduction of microglial cells in the irradiated ONH and OL, relative to the non-irradiated sample. (C, D) High- magnification view of astrocytes localized to the OL (insets in A, B) show similar cell size and shape under both experimental conditions, while microglia display morphological signs of PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43602