immune response [17,18,19]. Similar changes in gene expression have been reported in microarray studies of the retina following acute IOP elevation in various species [20,21,22,23]. Furthermore, early and chronic deactivation of microglia by administration of minocycline results in improved RGC axonal integrity and transport function [24]. Thus microglia activation represents a significant and early component of pathology in glaucoma. High-dose radiation treatment of the entire animal with syngeneic bone marrow transfer (BMT) had an unexpected and remarkably profound protective effect on glaucomatous damage in the D2 mouse [25]. Neither iris disease nor IOP elevation were affected by treatment, but there was dramatic improvement in axon preservation out to 14 months of age, when most D2 mice normally show severe optic nerve pathology. In addition, there was protection against RGC somal loss and excavation of the ONH. The protective effect was also observed with irradiation of the head or eye without BMT [26]. In these studies irradiation was performed from 5 to 8 weeks of age, which is just prior to significant microglial activation in the D2 mouse [11]. Thus, it is possible that treatment had an effect on microglia with the potential to impact the subsequent course of degeneration. Here we tested the effects of high-dose irradiation of the head region alone on D2 mice and found transient reduction in proliferating microglia within the ONH and glial lamina, with maximal effect within the first week post-irradiation. This was accompanied by reduced microglial activation within the central retina and proximal optic nerve compartments, with no effect on astrocyte gliosis in those regions. We also found significant improvement in axonal survival and anterograde transport without reduction of IOP, confirming that bone marrow transfer is not required to abate degeneration. Thus, one consequence of high-dose irradiation is to acutely reduce the levels of microglia activation in this glaucoma model. Materials and Methods Ethics Statement This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Utah (Animal Welfare Assurance Number: A3031-01). All procedures were performed under avertin anesthesia, and every effort was made to minimize suffering. Animals Adult DBA/2J mice (Jackson Laboratory (JAX), Bar Harbor, ME; stock # 000671) served as an experimental model of inherited pigmentary glaucoma, and its DBA/2J-Gpnmb+/Sj substrain (stock # 007048) as non-glaucoma control [27,28]. These strains are referred to as D2 and D2G mice, respectively. Mice were bred and aged at the University of Utah in a pathogen- free barrier facility under a 12 hr light/dark cycle, and every 4 generations breeders purchased from JAX were introduced to avoid genetic drift. Mice under anesthesia were identified with ear tattoos (Harvard Apparatus, Holliston, MA). D2.CX3CR1gfp/+ mice, where microglia (and other myeloid cells) specifically express GFP under the control of fractalkine receptor locus [29], were generated after backcrossing B6.129P-CX3CR1tm1Litt/J mice (JAX stock #005582) onto the D2 background for over 10 generations. All animal maintenance and procedures were approved by the University of Utah Institutional Animal Care and Use Committee, and followed the Statement for the Use of Animals in Ophthalmic Irradiation Abates Early Microglia Activation and Vision Research from the Association for Research in Vision and Ophthalmology (ARVO). Head irradiation Four to six week-old female and male D2 mice had the rostral half of their heads exposed to a single high dose of electromagnetic radiation (8 Gy or 800 rads, 1 Gy/min) delivered by a biological X-ray generator (X-Rad 320, Precision X-Ray, North Branford, CT). To restrict irradiation to the eyes and visual pathways, each mouse was positioned inside a 50 mL conical tube with its muzzle exposed through the truncated end of the tube, while the rest of the body was shielded with two 6 mm-thick lead plates. Irradiation targeted an area delimited by the base of the ears and the beginning of the eyes, sparing the nose. The head region and the depths targeted were maintained constant across animals with the aid of reference collimated light and focusing LEDs. During the procedure, mice were anesthetized via intraperitoneal Avertin injection, and kept warm over isothermal pads (Braintree Scientific, Braintree, MA). Intraocular pressure (IOP) measurement IOP was measured monthly from 2 to 8 months of age in both irradiated and untreated D2 mice using the TonoLab (Colonial Medical Supply, Franconia, NH), as previously described [11]. Each data point represents individual (per eye) monthly, mean IOP values (20 readings). Mean value per age and experimental group were tabulated, as well as sample size. Optic nerve histology and analysis Axon counts were obtained in optic nerve cross-sections (2 mm- thick) from 9–12 month-old irradiated and control D2 mice. Optic nerves 2 mm proximal to the globe were stained with paraphen- ylenediamine (PPD) and counterstained with toluidine blue then prepared for plastic embedding and semithin sectioning, as previously described [30,31,32]. Using differential interference contrast optics at 1006magnification, cross-sections of optic nerve were photographed en montage using an Olympus Provis AX70 microscope. Custom algorithms were used to identify and count axons across the entire cross-section, as previously described [33]. Anterograde transport analysis To measure the preservation of RGC axonal transport from the retina to the superior colliculus (SC), cholera toxin b-subunit (CTB) transport was assayed in 10–12 month-old irradiated and non-irradiated mice as recently described [31]. Briefly, 1 mL of 1% CTB conjugated to Alexa Fluor 488 nm (Molecular Probes, Eugene, OR) in sterile PBS was injected intravitreally, using a 33 G needle attached to a 25 mL Hamilton syringe (Hamilton Co., Reno, NV). After 2 days, serial coronal brain sections (50 mm) were collected from mice prepared by transcardial perfusion with 4% paraformaldehyde in PBS. CTB signal was imaged throughout alternate SC sections to reconstruct the total retinotopic map in the superficial layers. CTB signal was normalized to the background of the deeper, non-retinorecipient layers and label and density was calculated and plotted across the total SC. CTB density is represented colorimetrically, and the fraction of intact transport map is defined as the percent area with CTB signal $70%. Retrograde Transport Analysis To measure retrograde transport of cargo from superior colliculus to retina within the retinal ganglion cell axons, a small burr hole was drilled through the skull above the superior PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43602