colliculus in anesthetized mice; FluoroGold was injected (1 mL, bilaterally) as described (Buckingham, et al., 2008). The holes were plugged with Gelfoam (Pfizer) soaked in 5% FluoroGold. Three days later, retinas were collected from mice after transcardial perfusion with 0.1M PBS followed by 4% paraformaldehyde in PBS, dissected out, immunolabeled with NeuN (1:500, Millipore, Temecula, CA) and FluoroGold (1:500, Millipore), mounted flat with gelvitol, then coverslipped. Unbiased stereological analysis of NeuN and FG-positive cell number was undertaken in whole- mount retinas using the optical fractionator module within StereoInvestigator software (MicroBrightfield, Middlebury, VT). Retinal immunofluorescence, microscopy and analysis Processing, immunostaining and imaging of retinal whole- mounts and cryosections (30 mm-thick) including the ONH, OL and proximal ON regions were performed as previously described [11]. We used polyclonal antibodies against Brn3 to identify RGCs, which recognize its three isoforms, (1:50, C-13; Santa Cruz, Santa Cruz, CA), Iba1 to identify microglia (1:1,000; Wako, Richmond, VA), and monoclonal antibodies against GFAP to label astrocytes (1:1,000; Sigma, St. Louis, MO), and against the 68 and 200 kDa isoforms of phosphorylated neurofilament to identify optic axons (1:50, clone 2f11; Dako, Carpinteria, CA). An antibody recognizing PCNA was used to identify mitotic cells (1:500, clone PC10; Dako, Carpinteria, CA), which required prior antigen unmasking with a solution of 0.18 mM citric acid, 77 mM sodium citrate, pH 6 for 15 min at 90uC. Immunostaining of retinal wholemounts and sections consisted of a 3-day incubation of three different primary antibodies at 4uC, followed by 2 hr incubation in Alexa Fluor-conjugated secondary antibodies (1:400, Molecular Probes). Confocal images were acquired with identical settings for each marker to allow comparison across samples (A1 and NIS-Elements 3.1, Nikon, Melville, NY), and are published with minimal adjustment of brightness (Photoshop CS3, Adobe Systems, San Jose, CA). Each flat mounted retina was imaged by acquiring 625 XY images throughout inner 25–35 mm of the retina (0.7 mm-step). For analysis, these images were stitched to produce a high-resolution image (0.41 mm/pixel) of the retinal surface, and published as maximal intensity projections that span the nerve fiber layer (NFL) and ganglion cell layer (GCL). To represent variations in relative intensity of protein levels, some published images were pseudocolored as indicated by a color code bar. Confocal images of cryosections of the central retina and proximal optic nerve regions show 20-mm thick maximal intensity projections. Microglia cell counting Total and proliferating microglial cells were counted in images of radial sections along the central retina and proximal optic nerve from irradiated D2 mice (1, 7 and 30 days post-treatment) and aged-matched untreated D2 mice. Cells were counted within areas localized to the head, laminar and myelinated optic nerve regions, defined as ONH (150 mm below NFL-plane), OL (100 mm from choroidal plane), and ON (250 mm from OL). Briefly, total microglia numbers were obtained from counting Iba1-positive cells with an identifiable Hoechst-stained nucleus (total microglia) within each nerve region. Proliferating microglia were recognized as cells with cytosolic Iba1 and nuclear PCNA, unambiguously identified by tracing their linear density profile across the cell soma, in single optical slice views. Cell counts in each compart- ment were normalized to represent percentage of proliferating 2 cells and cells per mm . Irradiation Abates Early Microglia Activation Detection of apoptotic microglia To detect apoptosis in microglial cells, avoiding artifacts derived from euthanasia and histology, we used D2.CX3CR1gfp/+ mice, in which microglia express GFP, and delivered SR-FLIVO, a red- fluorescent tracer that selectively forms covalent bonds with active caspases in vivo (Immunochemistry Technologies LLC, Blooming- ton, MN). Following manufacturer’s protocol, 100 mL FLIVO was administered to 2–3 month-old mice via tail vein injection 8 hr after irradiation and 8 hr before sacrifice, which was replicated in non-irradiated control mice. Retinas were dissected and flat mounted fresh, then fixed for 30 min and counterstained with Hoechst 33258 (Invitrogen, Carlsbad, CA). Confocal images were collected for each entire retina as described above. Quantitative real time PCR analysis Levels of Iba1 mRNA expression were measured in a sample that included the central 1-mm of retina, with its ONH and OL, using techniques and primers previously described [11]. Iba1 expression was assessed 1 week and 4 to 6 weeks after irradiation, and compared to untreated age-matched D2 and D2G samples (n = 10 per timepoint and strain). Data is presented as mean fold change normalized to b-actin expression within each sample. Statistical analysis Statistical significance was determined using two-way analysis of variance, followed by two-tailed Student’s t-test (GraphPad, La Jolla, CA). Statistical significance is indicated by asterisks, p,0.05 (*) and p,0.01 (**). Sample sizes are also reported in Figure legends. Results High dose head irradiation alters early microgliosis One effect of high-dose irradiation is to kill dividing cells, so we hypothesized that irradiation may be affecting a proliferative glial cell population in the retina, optic nerve head (ONH), optic lamina (OL), or optic nerve (ON). Previous analysis has shown that microglia represent the only significant dividing cell population in the D2 retina, doubling their numbers from 4 to 10 months of age [16]. Furthermore, we previously showed that microglial cell density increased in the central inner retina and ONH in D2 mice by 3 months of age versus D2G controls, suggesting increased proliferation [11]. To directly assess whether microglia are proliferating at 4 to 6 weeks of age (the time of irradiation) in the central retina, ONH, OL and/or proximal ON, radial retinal sections were double-immunostained for Iba1 and PCNA to identify and quantify dividing microglial cells within each region (Fig. 1A, B). Numerous PCNA+/Iba1+ cells were detected in the ONH, OL and ON from non-irradiated D2 animals, suggesting the presence of proliferating microglia (Fig. 1C, E). Few PCNA+/ Iba1+ cells localized to the peripheral retina (data not shown), consistent with previous analysis [16]. To assess the effects of irradiation on microglia in the eye, we selected mice from 4 to 6 weeks of age, shielded the body with lead plates, and delivered a single high dose of X-irradiation (8 Gy) to the rostral half of the head spanning the eye region and visual pathway (see Methods and Fig. S1). IOP was measured monthly from 2 to 8 months of age in both non-irradiated and irradiated mice. The irradiated cohort of D2 mice still showed the expected IOP elevation (.20 mm Hg) by 7–8 months (Fig. S2), consistent with previous reports [25,26]. We then assessed the effects of irradiation on proliferating microglia and found that in irradiated D2 mice, the presence of PCNA+/Iba1+ cells was dramatically reduced one week post-irradiation (Fig. 1D, F). To determine PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43602