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R. M. Sappington, L. Holmgren, S. M. Sims; Interleukin-6 Receptor Expression as a Function of Microglial Activation in the DBA/2 Mouse. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5219.
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Microglial interleukin-6 (IL-6) has been shown in vitro to inhibit pressure-induced apoptosis of retinal ganglion cells (RGCs). Here we examine the relationship between microglial activation and IL-6 receptor (IL-6R) expression in the nerve fiber and ganglion cell layers of retina from DBA/2 mice.
Cell type-specific expression of IL-6R and microglial activation were assessed in wholemount retina from DBA/2 mice (ages 3 and 10 months), using immunohistochemistry against IL-6R, the RGC marker SMI-31 and the microglia marker Iba-1. IL-6R expression was analyzed by measuring label intensity in digital confocal images. We quantified hypertrophy, ramification and cell density as indicators of microglial activation. Hypertrophy was quantified as the area of cell soma. We quantified ramification as the number of intersections between microglial processes and a 10µm grid mask. Density was quantified by counts of Iba-1-positive cells. Correlations between microglial activation and IL-6R expression were assessed by various statistical analyses, including Mann-Whitney U test, analysis of variance, pearson product moment correlation and regression analysis.
Compared to 3 month mice, the number of process intersections decreased by 42% (p < 0.01), while the density of microglia increased by 14% (p < 0.05) at 10 months. Soma area did not differ significantly between 3 and 10 months (p > 0.05). IL-6R co-localized with RGCs in both 3 month and 10 month retina. However, IL-6R also co-localized with microglia at 10 months. At 10 months, IL-6R intensity decreased by 43%, as compared to 3 months (p < 0.01). Correlation analysis revealed that at 10 months, the number of process intersections correlated strongly with IL-6 intensity (correlation co-efficient = -0.623, p < 0.01). Regression analysis (r2 = 0.37) revealed an inverse, linear relationship between the number of process intersections and IL-6R intensity (F = 24.1, p < 0.01). IL-6R intensity did not correlate statistically with any other parameter of microglia activation we measured in either age group.
Our data suggest that IL-6R expression changes with age in the DBA/2 and that this change is accompanied by changes in microglial reactivity. Furthermore, IL-6R expression is inversely proportional to microglia ramification, where increased IL-6R expression occurs in the presence of microglia with more ameboid versus ramified morphology.
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