Figure 1.
Olig2 is expressed by Nkx2.2/Sox9-positive NIRG cells.
Vertical sections of the retina were labeled with antibodies to Oilg2 (green) and Nkx2.2 (red) (a–c) or Olig2 (green) and Sox9 (red) (d–f). Arrows indicate the nuclei of cells labeled for Olig2 and Nkx2.2 or Olig2 and Sox9, arrow-heads indicate the nuclei of Sox9-positive Müller glia, and small double-arrows indicate cells in the GCL that are labeled for Sox9 or Nkx2.2 alone. The scale bar (50 µm) in panel f applies to a–f. Abbreviations: INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.
Figure 2.
IGF1 stimulates NIRG cells to up-regulate Nkx2.2, but not Sox2.
Retinas were obtained from eyes that received 2 consecutive daily injections vehicle (control) or IGF1 (treated). Vertical sections of the retina were labeled with antibodies to Sox2 (green; a and b) and Nkx2.2 (red; c and d). Images of control and treated retinas were obtained using identical microscope, camera and post-acquisition processing settings. Arrows indicate the nuclei of NIRG cells that are labeled for Sox2 and Nkx2.2, arrow-head indicate nuclei of Müller glia, and small double-arrows indicate the nuclei of cholinergic amacrine cells. The histograms in panel e illustrate the mean (±SD) pixel intensity, area and density sum for Nkx2.2-immunofluorescence per cells. Image Pro 6.2 was used to measure total area for pixel intensities >68 (0 = black, 255 = saturated green), average pixel intensity and the density sum, as described in the Methods. Immunofluorescence for Nkx2.2 was measured in individual nuclei for ≥50 cells per individual, per experimental condition. Significance of difference between control and IGF1-treated retinas was determined by using a two-tailed Student’s t-test. The scale bar (50 µm) in panel d applies to a–d. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.
Figure 3.
IGF1 causes a transient reactive phenotype and accumulation of NIRG cells in the retina.
Whole-mount preparations of control and treated retinas were labeled with antibodies to Sox2 (red), Sox9 (magenta), and transitin (green) (a–c). Vertical sections of the retina were labeled with antibodies to transitin (e–i). Micrographs were obtained from projections of optical sections collected through the IPL (a–c) or wide-field microscopy of retinal sections (e–i). Images of control and treated retinas were obtained using identical microscope, camera settings, and post-acquisition processing settings. Histograms illustrate the mean (± SD) number of NIRG cells within the IPL, INL and the entire retina (d). Significance of difference among groups was determined by using a one-way ANOVA (p<0.0001). Significance of difference between control (time 0) and treatment groups (days 1, 2, 3 and 7 after IGF1-treatment) (*p<0.05, **p<0.001, ***p<0.0001), and between treatment groups at different time points (bracket and p-values) was determined by using a two-tailed post-hoc Student’s t-test. The scale bar (50 µm) in panel c applies to a–c, and the panel in i applies to e–i. Abbreviations: INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.
Figure 4.
IGF1-treatment causes the sustained reactivity of microglia within the retina.
Retinal sections were labeled with antibodies to CD45 (a–d). Arrows indicate the somata of microglia within the retina and arrow-heads indicate CD45-positive processes and/or cells at the ILM. Images of control and treated retinas were obtained using identical microscope, camera settings, and post-acquisition processing settings. The scale bar (50 µm) in panel a applies to a–d. The histograms in panels e–g illustrate levels of CD45-immunfluorescence in retinas at 2, 3 and 7 days after IGF1-treatment. ImagePro 6.2 was used to measure total area (e) for pixel intensities >72 for CD45 (0 = black, 255 = saturated), mean pixel intensity (f) above threshold, and the density sum (g; total values of pixels within thresholded objects). Measurements of CD45-immunofluoresence included all layers across 640 µm of linear retina (12 µm-thick section). The plot in panel h illustrates the mean (±SD) number of microglial somata (CD45+ DRAQ5 or ToPro3) per 19,800 µm2 of retina (n = 5 for each time point). Significance of difference among groups were determined by using a one-way ANOVA (p<0.0001). Significant of different between control and treated groups (*p<0.05, **p<0.001, ***p<0.0001), and between different treatment time-points (brackets and p-values) was determine by using a two-tailed post-hoc Student’s t-test. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.
Figure 5.
NIRG cells and microglia continue to proliferate for several days after exposure to IGF1.
Vertical sections of the retina were labeled for Sox9 (red; b–e), BrdU (green; b–g), Nkx2.2 (magenta; g), and RCA1 (red; g). (a) Retinas were obtained from eyes that received injections of IGF1 at P4 and P5, and a single dose of BrdU was applied 4 hrs before harvesting at P6 (c), P7 (d), P8 (e), P9 and P10 (not shown). Alternatively, eyes received injections of IGF1+BrdU at P4 and P5, BrdU alone at P6, P7 and P8, and retinas harvested at P12 (f–h). Wide-field microscopy (b–e) and confocal microscopy were used to obtain images (f and g). The scale bar (50 µm) in panel g applies to b–g. Arrows indicate NIRG cells labeled for BrdU and Sox9 (b–e) or BrdU and Nkx2.2 (f and g), hollow arrows indicate Nkx2.2-positive NIRG cells that are BrdU-negative, arrow-heads indicate BrdU-labeled RCA1-positive microglia, hollow arrow-heads indicate BrdU-negative RCA1-positive microglia, and small double arrows indicate BrdU-labeled cells that are negative for RCA1 and Nkx2.2 (f and g). Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.
Figure 6.
NIRG cells and microglia transiently accumulate in the retina following NMDA-induced excitotoxic damage.
Retinas were obtained from eyes treated with NMDA at P7 and harvested at P8 (day 1), P10 (day 3), P14 (day 7) and P21 (day 14). Vertical sections of the retina were labeled with antibodies to Nkx2.2 (green) and Sox9 (red) (a–e), or CD45 (green) and ToPro3 (red) (i–m). Arrows indicate the nuclei of NIRG cells that are labeled for Nkx2.2 and Sox9 (a–e) or CD45-positive microglia (i–m). Plots illustrate the mean (±SD) number of NIRG cells (f–h) or microglia (n) per 19,800 µm2. Significance of difference among groups was determined by using a one-way ANOVA (p<0.0001). Significance of difference between control and treated groups (*p<0.05, **p<0.001, ***p<0.0001), and between treatment time-points (brackets and p-values) was determined by using a post-hoc two-tailed Student’s t-test. The scale bar (50 µm) in panel e applies to a–e, and the bar in m applies to i–m. Abbreviations: ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.
Figure 7.
NIRG cells do not survive when the microglia have been selectively ablated by the combination of IL6 and clodronate-liposomes.
The dose of clodronate-liposomes was titered-down to the minimum dose required to eliminate >90% of the microglia at 1 days after treatment. Vertical sections of the retina were labeled for CD45 (a–c), RCA1 (d and e), CD45 and DRAQ5 (f), and transitin and Sox9 (g). Retinas were obtained from eyes one day after treatment with IL6 alone (a and d), clodronate liposomes alone (b) or IL6 with clodronate liposomes (c and e–g). Arrows indicate NIRG cells in the IPL, hollow arrow-heads indicate the somata of microglia, arrow-heads indicate the nuclei of Müller glia, and small double-arrows indicate monocytes in the choroid. The scale bar (50 µm) in panel f applies to a–c and f, the bar in e applies to d and e, and the bar in g applies to g alone. The histograms in panels h and i, and the plot in j, illustrate the mean (±SD) number of microglia in the retina (i and j) or NIRG cells (h and j) in the IPL and INL at 1 and 7 days after treatment with IL6 alone (control) and IL6 with clodronate-liposomes. Significance of difference was determined by using a one-way ANOVA (p<0.0001). Significance of difference between control and treated groups (*p<0.001, **p<0.0001), and between treatment time-points (brackets and p-values) was determined by using a post-hoc two-tailed Student’s t-test. Abbreviations: RPE – retinal pigmented epithelium, ONL – outer nuclear layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer.
Figure 8.
A partial depletion of the microglia influences the phenotype and survival of the NIRG cells.
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; a–c, j and k), or DRAQ5 (red) and CD45 (green; d–f). The histogram in g 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 h illustrates the mean (±SD) number of microglia in the retina at 1 and 7 days after treatment. The plot in i combines the data from the histograms in g and h to demonstrate the depletion of microglia and NIRG cells over time following treatment with IL6/clodronate-liposomes. Panels j and k 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 (a–c, j and k) and widefield (d–f) 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 c applies to a–c, and the bar (10 µm) in k applies to j and k. 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.
Figure 9.
IL6 stimulates the rapid migration of microglia to vitread surface of the retinas.
Retinas were obtained from eyes injected with vehicle (control; a, g–i) or IL6 at 1 (b), 3 (c), 4 (d, j–l) and 6 (e) hours after treatment. Whole-mount preparations of the retina were labeled with DRAQ5 (magenta; g–i) and antibodies to CD45 (a–e and g–l). The scale bar (50 µm) in panel e applies to a–e, and the bar in l applies to g–l. The histogram in panel f illustrates the mean (±SD) area for CD45-immunofluorescence above threshold, per field of view (42,400 µm2), at 1, 3, 4 and 6 hours after treatment. Image Pro 6.2 was used to measure the total area for pixel intensities >68 (0 = black, 255 = saturated green) per field of view, as described in the Methods. 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, NFL – nerve fiber layer.
Figure 10.
Clodronate-DiI-liposomes accumulate, along with the microglia, at the vitread surface of the retina, whereas the NIRG cells appear unaffected by short-term exposure to IL6 and liposomes.
Vertical sections of the retina were labeled with DRAQ5 (red, a–c; magenta, d–g) and antibodies to CD45 (green, a–g), Sox9 (red, h and i), and transitin (green, h and i). The red fluorescent puncta in panels d–g are DiI-labeled liposomes. Images were obtained by using wide-field (a–c, inset in d, h and i) or confocal (d–g) microscopy. Images were obtained from eyes that were treated with saline (a), clodronate-DiI-liposomes (b and h), or the combination of clodronate-DiI-liposomes and IL6 (c–g and i). Arrows indicate the somata of microglia or NIRG cells, hollow arrows indicate DiI-liposomes, and hollow arrow-heads indicate the nuclei of Müller glia. The scale bar (50 µm) in panel d applies to d alone, the bar in e applies to a–c and e, the bar in i applies to h and I, and the bar (10 µm) in the inset in panel d applies to the inset alone. Abbreviations: ONL – outer nuclear layer, OPL- outer plexiform layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer, NFL – nerve fiber layer, ILM – inner limiting membrane.
Figure 11.
IGF1-treatment reduced the survival of colchicine-damaged ganglion cells.
Retinas were obtained from eyes that were injected with saline (a) and colchicine (b) 10 days after treatment. Whole-mount preparations of the retina were labeled with antibodies to Brn3a to identify the nuclei of ganglion cells. Panel c is a histogram illustrating the mean number of Brn3a-positive ganglion cells per 70,000 µm2 in central and peripheral regions of the retina. Significance (p-values) of difference between numbers of Brn3a-positive ganglion cells in control and treated retinas was determined by using a two-tailed Student’s t-test. The scale bar (50 µm) in panel b applies to panels a and b.
Figure 12.
Levels of pro-inflammatory cytokines fluctuate in retinas treated with IGF1.
qRT-PCR was used to measure levels of mRNA in retinas treated with IGF1 at 1 and 3 days after treatment. qRT-PCR was used to measure retinal levels of IL1β, IL6, TNFα and ADAM17. Significance (*p<0.01) of difference between mRNA levels in control and IGF1-treated retinas was determined by using a two-tailed Student’s t-test.