Figure 1.
iNOS immunolabeling of amoeboid microglia in retinas of quail embryos at 8 and 9 days of incubation.
Confocal micrographs of QH1 (green) and anti-iNOS (red) double-immunolabeled amoeboid microglial cells in whole-mounted retinas (WM) and retinal cross-sections (CS) of quail embryos at 8 (E8, A, B) and 9 days of incubation (E9, C, D). Cell nuclei in retinal cross sections are stained with Hoechst (blue). All QH1-positive amoeboid microglial cells in E8 and E9 embryo retinas are located in the nerve fiber layer (NFL) and are also immunolabeled with the anti-iNOS antibody. Anti-iNOS labeling of microglial cells is similar to QH1 labeling but with some differences. Thus, QH1 labels the entire microglial cell profile, including lamellipodia, whereas anti-iNOS labeling is exclusively cytoplasmic in distinct zones of the soma and cell processes. OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar, 50 µm for A and C; 60 µm for B and D.
Figure 2.
Representative confocal images of single immunolabeling (IL) for iNOS (A) and its negative control, omitting the primary antibody (B) from whole-mounted retinas of quail embryos at 8 days of incubation. Note that the single iNOS labeling is similar to the iNOS labeling in double immunolabeled specimens shown in figure 1A, whereas no labeling is seen when anti-iNOS was omitted. Scale bar, 25 µm.
Figure 3.
Anti-iNOS immunofluorescence intensity in microglial cells of quail embryo retinas from 8 days of incubation to adulthood.
The histogram represents anti-iNOS fluorescence intensities per microglial cell obtained in the nerve fiber layer (NFL), inner plexiform layer (IPL) and outer plexiform layer (OPL) on confocal micrographs of double QH1/anti-iNOS immunolabeled whole-mounted retinas of quail embryos at 8 days of incubation (E8), E9, E14, and of 4-day-old (P4) and adult quails. Pixel intensities ranged from 0 (the darkest shade) to 255 (the lightest shade). Data are expressed as means ± SEM (n = 30 cells for each age and retinal layer). Asterisks indicate significant differences (* p<0.05 and ** p<0.001, one-way ANOVA followed by Tukey test for multiple comparisons). Note that iNOS immunofluorescence intensities of microglial cells in the NFL are higher than in the IPL and OPL and show the highest value at E8, decreasing until P4 and then increasing in adulthood. In the IPL and OPL, the highest fluorescence intensity is observed at E14, with a significant decrease until adulthood. No data are shown for iNOS-immunofluorescence intensity in the IPL and OPL at E8 and E9 because no microglial cells were present in these layers at these ages.
Figure 4.
Pecten-adhered vitreal macrophages are also positive for iNOS immunostaining.
Confocal image of a QH1 (green) and anti-iNOS (red) double-immunostained cross section of the pecten from a quail embryo at 8 days of incubation. Note that QH1-positive vitreal macrophages adhered to the pecten surface are strongly iNOS-positive, whereas endothelial cells of blood vessels (arrows) within the pecten show strong QH1 labeling but are weakly immunostained with anti-iNOS. Scale bar, 25 µm.
Figure 5.
Decreased iNOS immunolabeling in ramifying microglial cells at the end of embryonic development of the quail retina.
Representative confocal images of QH1 (green) and anti-iNOS (red) double-immunolabeled microglial cells in a whole-mounted retina (WM, A-C) and a retinal cross section (CS, D) of quail embryos at 14 days of incubation (E14). Cell nuclei in the retinal cross section are stained with Hoechst (blue). In the nerve fiber layer (NFL), microglial cells have an amoeboid appearance with strong iNOS immunolabeling (A, D). However, in the inner plexiform layer (IPL) and outer plexiform layer (OPL), ramifying microglial cells have a weaker iNOS labeling (B-D), suggesting that iNOS expression becomes downregulated as amoeboid microglia differentiate into ramified microglia. INL: inner nuclear layer; GCL: ganglion cell layer. Scale bar, 50 µm for A-C; 60 µm for D.
Figure 6.
Weak iNOS immunolabeling of ramified microglia in the developing quail retina during the first post-hatching week.
Representative confocal images of QH1 (green) and anti-iNOS (red) double-immunolabeled microglial cells in a whole-mounted retina (WM, A-C) and retinal cross sections (CS, D) from 4-day-old quails (P4). Hoechst staining was used to visualize nuclei (blue) in the retinal cross section. Microglial cells at this developmental stage are mainly located in the nerve fiber layer (NFL, A), inner plexiform layer (IPL, B), and outer plexiform layer (OPL, C) and show a more profuse ramification and a similar anti-iNOS immunostaining in comparison to ramifying microglial cells in retinas from embryos at 14 days of incubation. During the first post-hatching week, some microglial cells can be seen in the inner nuclear layer (INL), apparently traversing this layer towards the OPL (D). GCL: ganglion cell layer. Scale bar, 50 µm for A-C; 65 µm for D.
Figure 7.
iNOS immunolabeling of ramified microglial cells in the adult quail retina is weak in the plexiform layers and stronger in the nerve fiber layer.
Representative confocal images of a QH1 (green) and anti-iNOS (red) double-immunolabeled adult whole-mounted retina showing mature ramified microglial cells in the nerve fiber layer (NFL, A), inner plexiform layer (IPL, B), and outer plexiform layer (OPL, C). In the NFL, microglial cell processes are parallel to the course of ganglion cell axon fascicles (not shown) and show stronger iNOS immunolabeling in comparison to ramified microglial cells in the IPL and OPL, whose cell processes are oriented in all directions. Microglial iNOS immunostaining in the IPL and OPL is similar to that in retinas from quail embryos at 14 days of incubation and from 4-day-old quails. Scale bar, 50 µm.
Figure 8.
iNOS gene is expressed in the quail retina during embryonic and post-hatching development and adulthood.
A. Representative gel from three independent experiments of agarose gel electrophoresis of iNOS mRNA by RT-PCR of cDNAs prepared from quail retinas of different embryonic (E8, E9, E14 and E16) and post-hatching (P4 and adulthood) ages. iNOS RT-PCR products at each age are referred to the corresponding 18S rRNA bands. Expression of the iNOS gene is observed in retinas at all tested ages (embryonic, postnatal and adulthood). B. Quantitative analysis of iNOS mRNA expression in quail retinas of different ages, as shown by RT-PCR of six RT-PCR experiments. The histogram represents changes in the iNOS mRNA levels (mean ± SEM) at the different ages with respect to E8. No significant differences are observed between the different ages (one-way ANOVA followed by Tukey test for multiple comparisons).
Figure 9.
In vitro LPS treatment of quail embryo retina explants induces morphological changes in amoeboid microglia compatible with activation.
A, B. Representative images of a pair of QH1 immunolabeled retina explants from a quail embryo at 8 days of incubation cultured for 24 hrs in vitro (E8+24hiv) in medium containing LPS (LPS, A) and in LPS-free medium (CTRL, B). QH1-positive microglial cells in the LPS-treated explant are more rounded than those in the control explant. C. QH1 immunolabeled non-cultured retina explant from a quail embryo at 9 days of incubation (E9), showing the typical polarized morphology of amoeboid microglia migrating tangentially in the nerve fiber layer, with elongated cell body, polarized cell processes, and broad lamellipodia. Note that the microglial cell morphology is similar in the control E8+24hiv retina explant (B) to that in the non-cultured E9 retina explant (C), although lamellipodia are noticeably less abundant in the former. D-F. Morphometric analysis of cell area (D), elongation index (E), and cell rounding index (F) for microglial cells in LPS-treated E8+24hiv (red bars), non-treated control E8+24hiv (blue bars), and non-cultured E9 (green bars) retina explants. Data are expressed as means ± SEM (n = 30 for each). Asterisks indicate significant differences (* p<0.05 and ** p<0.001, one-way ANOVA followed by Tukey test for multiple comparisons). Scale bar, 50 µm.
Figure 10.
LPS treatment markedly increases the lysosomal compartment in amoeboid microglia of quail embryo retina explants.
Confocal images of QH1 (green) and LEP100 (specific lysosomal membrane marker, red) double-immunolabeled amoeboid microglial cells in retina explants from quail embryos at 8 days of incubation cultured for 24 hrs in vitro (E8+24hiv) in medium containing LPS (LPS, A, A′) and in LPS-free medium (CTRL, B, B′). Double-immunolabeled microglial cells in non-cultured retina explants from quail embryos at 9 days of incubation (E9) are shown in C and C′. The lysosomal compartment is clearly larger in the LPS-treated E8+24hiv retina explant than in control E8+24hiv and non-cultured E9 retina explants. D. Morphometric analysis shows that the relative area of the microglial cell lysosomal compartment is significantly higher in LPS-treated E8+24hiv retina explants (red bar) than in non-treated control E8+24hiv (blue bar) or non-cultured E9 (green bar) retina explants. Data are expressed as means ± SEM (n = 45 for each). Asterisks indicate significant differences (* p<0.05 and ** p<0.001, one-way ANOVA followed by Tukey test for multiple comparisons). Scale bar, 50 µm for A-C; 23 µm for A′-C′.
Figure 11.
iNOS gene expression is upregulated in LPS-treated quail embryo retina explants cultured for 12 hrs in vitro (hiv).
A, B. Confocal images of QH1 (green) and anti-iNOS (red) double immunostained microglial cells in non-treated control (CTRL, A) and LPS-treated (LPS, B) retina explants from quail embryos at 8 days of incubation cultured for 12 hiv (E8+12hiv). The iNOS labeling is higher in LPS-treated versus control explants. C. Western blot analysis results, representative of three independent experiments, for iNOS protein levels in non-treated control (CTRL) and LPS-treated (LPS) E8+12hiv retina explants. The 135 kDa band, which corresponds to the iNOS protein, is noticeably more intense in LPS-treated versus control explants. β-tubulin antibody was used as a loading control. D. Representative gel of three independent experiments on agarose gel electrophoresis of iNOS mRNA, showing a more intense band in LPS-treated explants (LPS) than in control explants (CTRL). iNOS amplification products are referred to the corresponding 18S rRNA bands. E. Quantitative analysis of iNOS mRNA expression by real-time PCR. The histogram represents changes in the iNOS mRNA levels of LPS-treated E8+12hiv retina explants with respect to non-treated explants (mean ± SEM) obtained from three real-time PCR experiments. iNOS mRNA levels are more than two-fold higher in LPS-treated explants than non-treated explants. Scale bar, 25 µm.
Figure 12.
Microglial cells produce NO in quail embryo retina explants from quail embryos at 8 days of incubation cultured for 12 hrs in vitro (E8+12hiv).
Representative images of QH1 (green) immunolabeled amoeboid microglia in non-treated control (CTRL, A) and LPS-treated (LPS, B) E8+12hiv retina explants cultured in the presence of DAR-4M AM (DAR, red), a reliable fluorescent indicator of subcellular NO production by iNOS. All microglial cells are specifically labeled with DAR in the LPS-treated and control explants. An entirely DAR-fluorescent cell body is observed in some cells (right inserts), whereas other cell bodies contain several distinct DAR-fluorescent masses of variable size (left inserts). Scale bar, 50 µm; 20 µm for inserts.