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Alejandra Bosco, Cesar Romero, Alexis Chagovetz, Michael Steele, Kevin Breen, Balamurali Ambati, Monica Vetter; Live imaging of early microglia activation predicts neurodegeneration in chronic glaucoma. Invest. Ophthalmol. Vis. Sci. 2013;54(15):783.
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Microglia activation is the earliest reported retinal change preceding detectable retinal ganglion cell (RGC) degeneration in the DBA/2J (D2) model of chronic glaucoma (Bosco et al, 2011). Here, we asked whether early microglia changes are indicators of later RGC pathology. We addressed this question in the D2 model, in which variable and asynchronous RGC degeneration affects discrete retinal sectors and about half of the optic nerves (ONs). We sought to determine whether early microglia activation positively correlates with the severity of optic neuropathy and/or pattern of retinal degeneration at later stages.
To monitor the progression of microglial changes, we used live laser confocal ophthalmoscopy (Spectralis, Heidelberg Engineering) to image GFP+ microglia in Cx3cr1+/GFP D2 mice (n=30). Each retina was imaged at 2, 3, 4 and 5 months of age (mo) to map sequential changes in the distribution and density of activated microglia in the inner retina, optic disc and nerve head (OD/ONH). At 10 mo, the same eyes were analyzed ex vivo for ON pathology severity and pattern of retinal degeneration. ONs were prepared as semithin plastic cross-sections, stained with PPD, and axonal loss scored in light-microscopy montages. Retinal flatmounts were immunostained for Brn3 or γ-synuclein and p-neurofilament, and the entire inner retina was imaged by confocal microscopy. Last, we compared levels of OD/ONH microgliosis at 2-3mo with severity of ON pathology at 10 mo, and the sectorial distributions of activated microglia at 4-5mo and declining RGCs at 10 mo in the same retinas.
Our live imaging method unambiguously identified activated microglia with large somata and reduced branching, detecting peak OD/ONH microgliosis by 2-3 mo in 81% of the retinas. We found positive correlation between levels of early OD/ONH microgliosis and late ON degeneration, which coincided in 16/18 ONs with severe and moderate pathology, and 5/9 healthy ONs. In the retina, 79% of the areas showing high microglia activation at 4-5 mo, developed RGC somatic and/or axonal degeneration at 10 mo. This colocalization was detected in eyes at all levels of ON pathology (severe, 79±6%, moderate 75±7%, mild/no 83±3%).
These data establish microglia activation as a sensitive and early biomarker of late retinal and ON pathology in D2 glaucoma.
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