A partial depletion of the microglia influences the phenotype and survival of the NIRG cells.

<p>The dose of clodronate-liposomes was titered-down so that nearly one-half of the microglia survived at 1 day after treatment. Retinas were obtained 1 or 7 days after treatment with IL6 alone (control) or IL6 with clodronate-liposomes (treated). Vertical sections of the retina were for Sox9 (red) and transitin (green; <b>a</b>–<b>c</b>, <b>j</b> and <b>k</b>), or DRAQ5 (red) and CD45 (green; <b>d</b>–<b>f</b>). The histogram in <b>g</b> illustrates the mean (±SD) illustrates the mean (±SD) number of NIRG cells in the IPL and INL at 1 and 7 days after treatment. The histogram in <b>h</b> illustrates the mean (±SD) number of microglia in the retina at 1 and 7 days after treatment. The plot in <b>i</b> combines the data from the histograms in <b>g</b> and <b>h</b> to demonstrate the depletion of microglia and NIRG cells over time following treatment with IL6/clodronate-liposomes. Panels <b>j</b> and <b>k</b> include representative high-magnification images of representative NIRG cells within the IPL of control (IL6 alone) or treated (IL6+ clodronate-liposomes) retinas at 1 day after treatment. Images were obtained using confocal (<b>a</b>–<b>c</b>, <b>j</b> and <b>k</b>) and widefield (<b>d</b>–<b>f</b>) microscopy. Images of control and treated retinas were obtained using identical microscope, camera settings, and post-acquisition processing settings. Arrows indicate NIRG cells, hollow arrows indicate the somata of microglia, and arrow-heads indicate the nuclei of Müller glia. The scale bar (50 µm) in panel <b>c</b> applies to <b>a</b>–<b>c</b>, and the bar (10 µm) in <b>k</b> applies to <b>j</b> and <b>k</b>. Significance of difference was determined by using a one-way ANOVA (p<0.0001). Significance of difference between control and treated groups (*p<0.0001), and between treatment time-points (bracket and p-values) was determined by using a post-hoc two-tailed Student’s t-test. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.