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Gareth R. Howell, Gregory L. Sousa, Margaret M. Ryan, Danilo G. Macalinao, Vittorio Porciatti, Simon W. John; Radiation Prevents Early Signs Of Glaucomatous Injury In DBA/2J Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2605.
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A one-time treatment of gamma radiation, combined with a syngeneic transfer of bone marrow, robustly protects DBA/2J mice from glaucomatous retinal ganglion cell (RGC) loss. However, it has not been previously determined whether the protection prevents very early stages of glaucoma such as focal damage to RGC axons, loss of axon transport and pattern electroretinogram (PERG), reactive astrocytosis, and microglial activation. Therefore, to better understand the mechanism(s) of radiation-based neuroprotection, we have comprehensively assessed early glaucomatous events in irradiated DBA/2J mice.
DBA/2J and control mice (D2-Gpnmb+) were irradiated at 2-3 months of age and glaucomatous events assessed at 9-12 mos. Focal damage to RGC axons at the ONH was determined using irradiated DBA/2J-Thy1(CFP) mice, a substrain of DBA/2J mice that have RGCs labeled with cyan fluorescent protein. Anterograde axon transport studies were performed using fluorescently labeled cholera toxin B subunit. To determine the extent of reactive astrocytosis and microglia activation we used a combination of Affymetrix gene expression profiling and immunofluorescence. For each experiment, at least 6-10 eyes in each treatment group were assessed.
Irradiated DBA/2J have a significantly better axon transport (same as controls in >90% of eyes, 9 mos) and PERG responses (AVE±SD, 7.2 uV±2.4, 9 mos) than non-treated DBA/2J mice (2.2 uV±0.85, P<0.001). Irradiated mice are very similar to age-matched D2-Gpnmb+ mice. Additionally, irradiated mice have no detectable focal damage to RGC axons at the ONH. While a robust accumulation of phagocytosing microglia were present in the ONH of untreated DBA/2J eyes prior to significant RGC loss, these cells were not found in aged-matched treated eyes. This result is supported by gene expression profiling which shows the microglia markers Aif1 and Cd68 are not significantly elevated in the optic nerve head compared to controls (P>0.05). Finally, key markers of reactive astrocytosis, such as Gfap, Stat3 and Vim, that increase during early stages of glaucoma, are not significantly elevated in irradiated DBA/2J mice (P>0.05).
Together, our data shows that radiation robustly protects almost all DBA/2J eyes from even early stages of glaucoma. While further work is required to understand the mechanism of protection, this work provides a promising avenue to both understand the pathogenesis of and develop improved treatments for human glaucoma.
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