irradiation there are comparable levels of Iba1 expression as in age- matched non-irradiated D2 or D2G samples. 30 to 45 days after irradiation Iba1 expression is significantly lower than the elevated levels of expression seen in non-irradiated D2 tissue at this age (p,0.01), and is comparable to levels of Iba1 expression in age-matched non- glaucoma D2G mice. (B, C) Representative retinal wholemounts from non-irradiated and irradiated D2 mice at 3 months of age show clustering of large, activated microglia with high Iba1 expression within the central retina and optic disc (yellow asterisk) in non-irradiated conditions. In age-matched irradiated mice few high-Iba1 expressing microglia are detectable, and display little clustering. (D, E) In non- irradiated D2 retinas, Iba1 expression (pseudocolor code indicated as inset bar) is highly upregulated in the microglial cells concentrated at the optic disc, while only few cells show high expression levels in untreated D2. (B’–E’) High magnification views of framed areas shown in B–E. Scale bars, 100 mm. doi:10.1371/journal.pone.0043602.g003 patterns of GFAP expression, confined in the retina to the NFL where astrocytes reside, and spanning the proximal optic nerve segments (Fig. S4). Irradiation reduces RGC axon degeneration After 9 months of age, optic nerves from non-irradiated D2 mice showed significant structural degeneration, including an abundance of dystrophic axons with expanded diameters and compacted myelin sheaths, dense and dark axonal profiles with multilayered myelin sheaths, and enlarged glial cell somata, indicative of gliosis (Fig. 4A, C), consistent with previous reports [32]. In contrast, irradiated mice showed relatively normal optic nerves with few degenerating axonal profiles and generally intact myelin sheaths (Fig. 4B, D). Since D2 mice typically show significant axon loss by 10 to 12 months of age we quantified axons to assess whether irradiation had a protective effect. We found that irradiated D2 mice had significantly larger axon numbers as compared to untreated D2 mice, indicating reduction of RGC axonal degeneration (Fig. 4E). Another feature of axonal decline in the D2 mouse is progressive loss of anterograde axonal transport from the retina to the superior colliculus (SC), which is severely compromised by 11–12 months of age [31]. To assess whether this is improved by irradiation, we performed intravitreal injection of cholera toxin b- subunit (CTB) in live mice, which is actively taken up by RGCs and transported by their axons to the superficial layers of the SC. Coronal sections of 11-month old SC of non-irradiated D2 mice reveal almost complete loss of CTB label along the medial-lateral extent of the SC, throughout its entire length (Fig. 4F, H). In contrast, irradiated 12-month old mice showed persistent CTB label throughout the entire SC (Fig. 4G, I). We quantified this effect across multiple animals by calculating the percentage of animals with intact anterograde axonal transport, as defined by $70% CTB signal density, and found a significant increase in irradiated D2 mice compared to untreated animals (Fig. 4J). Thus, axonal transport function is also robustly preserved in D2 mice by high-dose head irradiation. Previous reports have demonstrated significant decline in retrograde transport of FluoroGold tracer in advance of significant RGC death (Buckingham, et al., 2008). Significantly, irradiation did not have a direct toxic effect on cells in the ganglion cell layer since the NeuN+ cell numbers did not differ between irradiated and non-irradiated control (Fig. 4M). Irradiated mice injected with FluoroGold demonstrated no significant improvement of retro- grade transport of the tracer as compared to non-irradiated mice, although there was a trend towards increased transport (Fig. 4M). Irradiation improves retinal ganglion cell integrity. Since RGC optic axon structure and transport Irradiation Abates Early Microglia Activation Figure 4. Head irradiation protects D2 mice from optic nerve degeneration. (A, B) Optic nerve cross-sections stained with PPD, from na¨ıve (11 mo old) and irradiated (12 mo old) D2 mice. Scale bar, 250 mm. (C, D) Higher magnification view of the same nerves. Scale bar, 10 mm. (C) Non-irradiated D2 nerves show abundant degenerating axon profiles with dark, condensed axoplasm (arrowheads) and disorganized myelin sheaths (arrows), as well as extensive gliosis. (D) Irradiated D2 nerves mostly show healthy axons with clear axoplasm and uniform myelin sheaths, and rare degenerating profiles (arrows). (E) Total optic axon counts from irradiated D2 nerves (n = 29; 12 mo old) show a significant increase in mean axon number compared to non-irradiated D2 mice (n = 16; 10 and 11 mo old; p,0.01). (F–J) The anterograde transport of CTB by RGC axons from the retina to the superior colliculus (SC) was measured in non-irradiated (n = 16; 10 and 11 mo old) and irradiated D2 mice (n = 17; 12 mo old). (F) Non-irradiated D2 mice (11 mo old) showed severe loss of CTB signal, as seen in coronal section of the SC (dorsal, d; lateral, l). Scale bar, 500 mm. (G) Irradiated D2 mice (12 mo old) showed persistent CTB transport to the superficial layers of PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43602