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. 2023 Mar 15;17:1120400. doi: 10.3389/fncel.2023.1120400

FIGURE 6.

FIGURE 6

Reduction of microglial reactive oxygen/nitrogen species (RONS) and cell death by iNOS inhibition. iNOS inhibition by L-NMMA greatly decreases both microglial production of RONS (A) and cell death (B,D) in LPS-treated retina explants. It also increases the percentage of Islet 1-positive cells (C). (A) Quantification of the flow cytometry analysis of RONS production by microglia in LPS-treated (LPS, grey bar) versus LPS plus L-NMMA-double treated (LPS + LNMMA, striped grey bar) retina explants from E8 quail embryos cultured for 12 h in vitro (E8 + 12 hiv). Bars represent the percentages (means ± SEM) of microglial cells (QH1-positive) producing ROS (C-DCF-positive) from the whole microglia population. The percentage of RONS-producing microglial cells is significantly higher in LPS-treated than in LPS + LNMMA explants (**P < 0.01, Student’s t-test; n = 6 for each condition). (B) Percentages (means ± SEM) of annexin V-positive cells in E8 + 12 hiv retina explants as determined by flow cytometry analysis in LPS-treated (LPS, grey bar), LPS plus L-NMMA-double-treated (LPS + LNMMA, striped grey bar), non-treated (NT, white bar), and L-NMMA-treated (LNMMA, striped white bar) explants (n = 12 for LPS and NT and n = 6 for LPS + LNMMA and LNMMA conditions). The percentage of annexin V-positive cells are significantly higher in LPS-treated explants, while the differences between LPS plus L-NMMA-double treated, non-treated and L-NMMA-treated explants are not significant (***P < 0.001, one-way ANOVA followed by Tukey test for multiple comparisons). (C) Percentages (means ± SEM) of Islet 1-positive cells as determined by flow cytometry analysis in LPS-treated (LPS, grey bar) and LPS plus L-NMMA-double treated (LPS + LNMMA, striped grey bar) E8 + 24 hiv retina explants (n = 11 for each condition). The percentage of Islet 1-positive cells is significantly lower in LPS-treated than LPS plus L-NMMA-double treated explants (**P < 0.01, Student’s t-test). (D) Relative areas of TUNEL-positive profiles in the ganglion cell layer (GCL) of E8 + 24 hiv retina explants as determined in two experiments comparing LPS-treated explants (LPS, grey bars) versus LPS plus L-NMMA double-treated (LPS + LNMMA, striped grey bar) explants. Data are presented as means ± SEM of percentages of TUNEL-positive pixels as measured in three microscopic fields of 250 μm × 250 μm in the ganglion cell layer (GCL) of whole-mounted explants (n = 8 for each condition). The relative area of TUNEL-positive profiles is significantly higher in LPS-treated than in LPS plus L-NMMA double-treated explants (**P < 0.01, Student’s t-test).