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M. R. Steele, A. Bosco, L. Luo, B. K. Ambati, M. L. Vetter; Characterization of GFP-expressing Microglia in DBA/2J-Cx3cr1 +/GFP Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2771.
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The goal was to determine whether a DBA/2J mouse strain with the Cx3cr1 promoter linked to green fluorescent protein (GFP) reporter is an appropriate tool for visualizing retinal microglia in the DBA/2J mouse both in vivo and ex vivo. We propose to perform in vivo imaging of GFP-labeled microglia and correlate this with detailed morphometry analysis of retinal GFP+-microglial cells from the same tissue fixed at single time points and compare this with known microglial markers.
B6.129P-CX3cr1tm1Litt/J mice, homozygous for the Cx3cr1GFP targeted mutation, were backcrossed with DBA/2J mice to produce a DBA/2J sub-strain of Cx3cr1+/GFP mice. Live mice (1, 3, 7, 11 months; n=10 each) were imaged with a confocal scanning laser ophthalmoscope (Spectralis HRA+OCT) to determine distribution, numbers and size of GFP+-expressing cells localized in the retinal vitreal surface. These mice had their retinas isolated and immunostained as whole mounts for Iba1, CD11b, Cx3cr1 as markers of microglia and for PCNA to mark dividing cells. GFP+-cells that were localized to the inner layers and optic nerve head were imaged using confocal microscopy to characterize their number, morphology and distribution. Data from live and histological analysis was compared.
DBA/2J-Cx3cr1+/GFP offspring are viable, fertile, and physically and behaviorally normal. Retinal microglia somata were readily imaged in vivo at multiple ages, with reliable reporting of relative somal size (an indirect readout for activation) and distribution of microglia when compared with postmortem analysis of fixed tissue. In fixed tissue, retinal microglial activation, monitored by Iba1 expression levels, correlated with morphological changes revealed by GFP-expressing cells, including increases soma size and process diameter and decreased branching complexity. Clustering of larger microglial cells was detectable in the central retina and/or peripheral sectors starting at 3 months of age, both in fixed and live retinas, which escalated with age.
DBA/2J-Cx3cr1+/GFP sub-strain provides a new tool for observation and profiling of microglial changes in single time points and throughout life, which may enable us to evaluate the still unclear contribution of microglial cells to the neurodegeneration of the retinal ganglion cell (RGC) population in DBA/2J mice.
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