Early Reduction of Microglia Activation by Irradiation in a Model of Chronic Glaucoma Alejandra Bosco1, Samuel D. Crish2¤, Michael R. Steele1, Cesar O. Romero1, Denise M. Inman3¤, Philip J. Horner3, David J. Calkins2, Monica L. Vetter1* 1 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah, United States of America, 2 Department of Ophthalmology and Visual Sciences, Vanderbilt University, Nashville, Tennessee, United States of America, 3 Department of Neurosurgery, University of Washington, Seattle, Washington, United States of America Abstract Glaucoma is a neurodegenerative disease that results in the progressive decline and ultimate death of retinal ganglion cells (RGCs). While multiple risk factors are associated with glaucoma, the mechanisms leading to onset and progression of the disease remain unknown. Molecular analysis in various glaucoma models has revealed involvement of non-neuronal cell populations, including astrocytes, Mueller glia and microglia, at early stages of glaucoma. High-dose irradiation was reported to have a significant long-term protective effect in the DBA/2J (D2) mouse model of glaucoma, although the cellular and molecular basis for this effect remains unclear. In particular, the acute effects of irradiation on specific cell populations, including non-neuronal cells, in the D2 retina and nerve have not been assessed. Here we report that irradiation induces transient reduction in proliferating microglia within the optic nerve head and glial lamina within the first week post-irradiation. This was accompanied by reduced microglial activation, with no effect on astrocyte gliosis in those regions. At later stages we confirm that early high-dose irradiation of the mouse head results in improvement of axonal structural integrity and anterograde transport function, without reduction of intraocular pressure. Thus reduced microglial activation induced by irradiation at early stages is associated with reduced optic nerve and retinal neurodegeneration in the D2 mouse model of glaucoma. Citation: Bosco A, Crish SD, Steele MR, Romero CO, Inman DM, et al. (2012) Early Reduction of Microglia Activation by Irradiation in a Model of Chronic Glaucoma. PLoS ONE 7(8): e43602. doi:10.1371/journal.pone.0043602 Editor: Andreas Ohlmann, University of Regensburg, Germany Received June 6, 2012; Accepted July 23, 2012; Published August 30, 2012 Copyright: 2012 Bosco et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funded by a generous grant from the Melza M. and Frank Theodore Barr Foundation through the Glaucoma Research Foundation (www.glaucoma. org/) (Catalyst for a Cure to MLV, PJH and DJC) and NIH grant # R01EY02878 to MLV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: monica@neuro.utah.edu ¤ Current address: Northeast Ohio Medical University, Rootstown, Ohio, United States of America Introduction Glaucoma is a neurodegenerative disease that destroys vision through progressive degeneration of the optic nerve and decline and death of retinal ganglion cells (RGCs). This disease affects roughly 70 million people, and is a leading cause of non-curable vision loss [1]. Multiple risk factors contribute to glaucoma, including race, age and elevated intraocular pressure [2,3]. However, how these factors influence neuronal loss in glaucoma is complex. In human patients, as well as in experimental animal models, there is evidence that age- and IOP-related RGC axon damage first occurs in the optic nerve head (ONH), where optic axons exit the eye [4]. But degenerative changes also affect other RGC compartments, localized to the retina, nerve and brain, in an asynchronous manner, impacting cell somata, dendrites and their synapses, myelinated axons, and axonal terminations [5,6,7]. Analysis of molecular and cellular changes in various animal models of glaucoma have suggested involvement of diverse non- neuronal cell populations, including astrocytes, Mueller glia and microglia, although the relative contributions of these glial cells to RGC degeneration or protection remain unclear [8]. Microglia are resident immune cells of the central nervous system that have been implicated in neurodegenerative diseases, including glauco- ma [8,9]. These glial cells respond to neuronal stress or injury with changes in distribution and cell activation, which involves simplification of cell morphology, expression of various growth factors and cytokines, and in some cases migration, proliferation or phagocytic activity [9,10]. Notably, microglia reside in proximity to all RGC compartments impacted in glaucoma including dendrites and synapses, cell somata, and axons [11]. In human glaucoma, activated microglia have been found clustered in the region of the ONH, which is the site of initial axonal injury [12]. In the DBA/2J mouse (D2), which is an established model of inherited pigmentary glaucoma [13], microglia become activated prior to evidence of RGC structural decline [11]. Spatially, activated microglia first localize to the ONH and lamina around unmyelinated optic axons [11], the presumed sites of initiation of optic neuropathy in D2 mice [14,15]. At later stages microglia activation spreads to the peripheral inner retina [11], and this is associated with slow microglial proliferation that doubles retinal microglia numbers from 4 to 10 months of age [16]. Consistent with involvement of microglia in glaucoma, microarray analysis of the D2 mouse has shown that there are changes in gene expression within the retina as well as the ONH that suggest an innate PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43602