Table 1.
List of antibodies and lectins used in western blotting and immunofluorescence.
Table 2.
List of Qiagen Quantitect Primer assays used in RT-qPCR.
Fig 1.
Norgestrel-supplemented diet significantly protects against photoreceptor cell death out to P40 in the rd10 retina.
(A) Apoptosis of retinal cells was detected by terminal dUTP nick end-labeling (TUNEL; green & greyscale) of DNA strand breaks in cell nuclei. Norgestrel treatment (NORG diet) decreased TUNEL-positive staining in the central P15 and P20 retina compared to control mice (Control diet). N = 3 mice per group. Hoechst (blue) staining reveals the cell layers in the retina. Scale bar 50μm. (B) Quantification of outer nuclear layer (ONL) thickness in the central and peripheral retina. N = 3 mice per group, n = 20 sections per mouse. Results are presented as mean ± SEM (t-test, *p<0.05, **p<0.01, ***p<0.005).
Fig 2.
Norgestrel-supplemented diet significantly preserves retinal function in the rd10 retina.
(A) Representative scotopic ERG waveforms performed on (i) P20 and (ii) P30-31 rd10 mice on a control or Norgestrel-supplemented diet or C57 mice indicating increased neurotransmission in Norgestrel-fed rd10 mice at both time-points. (B) Light intensity-response curves of (i) P20 and (ii) P31 rd10 mice on a control (circles) or Norgestrel-supplemented diet (squares), showing statistically significant differences between treatments, in both a- and b-wave amplitudes. N = 4 mice per group, n = 3–10 recordings per mouse. Results are presented as mean ± SEM (MANOVA, Bonferroni post-hoc test, *p<0.05, **p<0.001).
Fig 3.
Norgestrel preserves rod and cone outer segment morphology in the rd10 retina.
Confocal microscopic images of (A) rod (rhodopsin; red) and (B) cone outer segments (PNA; green) in the central retina from P15-P40 of control and Norgestrel-fed rd10 mice. Outer segments are shorter and sparser in the control diet retina from P15 for rods (A) and from P20 for cones (B). N = 3 mice per group. Hoechst (blue) staining reveals the cell layers in the retina. Scale bar 50μm. (C) Quantification of rod and cone outer segment thickness in the central retina in control vs. Norgestrel-fed rd10 mice. N = 3 mice per group, n = 8 sections per mouse. Results are presented as mean ± SEM (t-test, *p<0.05, ****p<0.0001).
Fig 4.
Norgestrel dampens pro-inflammatory microglial activity in vivo and decreases DAMP release from photoreceptors.
(A) Confocal microscopic images of microglia (Iba1; red) and activated microglia (CD68; green) in the central retina from P15-P25 control and Norgestrel-fed rd10 mice. Less microglia including CD68+ microglia were observed at P15 and P20 in Norgestrel-fed mice. Hoechst (blue) staining reveals the cell layers in the retina. Scale bar 50μm. N = 3 mice per group, n = 8 sections per mouse. (B & C) Quantification of microglial number and CD68-positivity confirmed (B) significantly less microglia at P15 and P20 and (C) significantly less CD68-positivity at P20 with Norgestrel-supplementation. (D) RT-qPCR analysis of mRNA encoding (i) pro-inflammatory (M1) and (ii) & (iii) anti-inflammatory (M2) microglial markers in whole retina from P20-P30 control and Norgestrel-fed rd10 mice. Norgestrel significantly reduced pro-inflammatory and increased anti-inflammatory markers in the retina. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. (E) RT-qPCR analysis of mRNA encoding danger associated molecular patterns (DAMP), (i) high motility box group 1 (HMGB1) and (ii) interleukin-1α (IL-1α) in whole retina from P20-P30 control and Norgestrel-fed rd10 mice. Norgestrel significantly reduced DAMP expression at P20 and P25 in Norgestrel-fed mice. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. Results are presented as mean ± SEM (t-test, *p<0.05, ***p<0.005, ****p<0.0001).
Fig 5.
Norgestrel significantly abrogates rd10 microglial-induced 661W cell death.
(A) Conditioned media was collected from primary microglial cells cultured with (CM + NORG), or without (CM) 20 μM Norgestrel over 24 h. 661W cells were cultured over a further 24h in the absence (Control) or presence of conditioned media (CM, CM + NORG) and percentage cell death was assessed by the MTS assay. Exposing 661Ws to CM from rd10 microglia resulted in increased neuronal cell death whereas pre-treatment of rd10 microglia with Norgestrel significantly reduced cell death. N = 6 biological replicates, n = 4 technical replicates per group. (One way ANOVA followed by Tukey’s post-hoc test,). (B) Representative fluorescent microscopic image of primary rd10 microglial cells (Iba1; red) in culture with 661W cells (Cone arrestin; green). Hoechst (blue) staining reveals cell nuclei. Scale bar 30μm. (C) 661W cells were cultured in the presence of C57 or rd10 primary retinal microglia. Apoptosis of 661W cells as detected by TUNEL of DNA strand breaks in cell nuclei revealed significantly more 661W cell death when cultured with rd10 microglia. (D) 661W cells were cultured in (i) the presence of (healthy 661W) or (ii) absence of (serum-starved 661W) serum over 3 h. These cells were simultaneously treated with either 20 μM Norgestrel (661W NORG) or vehicle control (661W). The treated 661W cells were then placed in culture with microglia that had been treated with (Microglia NORG) or without (Microglia) 20 μM Norgestrel over 24h. Co-cultures were incubated for a further 24h. Apoptosis of 661W cells was detected by TUNEL of DNA strand breaks in cell nuclei. All Norgestrel-treated co-cultures had significantly less TUNEL-positive 661W cells than equivalent vehicle controls. Hoechst (blue) staining reveals the cell nuclei. Scale bars 50μm. (E) Quantification of TUNEL-positive 661W cells cultured in (i) the presence of (healthy 661W) or (ii) the absence of (serum-starved 661W) serum and following Norgestrel or vehicle DMSO treatment and co-incubation with primary rd10 microglial cells pre-treated with Norgestrel or vehicle DMSO. N = 8 mice for primary culture, n = 6 technical replicates per group. Results are presented as mean ± SEM (t-test, *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).
Fig 6.
Norgestrel reduces pro-inflammatory and promotes anti-inflammatory phenotypes in rd10 microglia.
(A) RT-qPCR analysis detected similar levels of mRNAs encoding progesterone receptor membrane complexes 1 and 2 (PGRMC1, PGRMC2) and all three membrane progesterone receptor isoforms, α, β and γ (mPRα, mPRβ, mPRγ) in rd10 retinal microglial cells. Classical progesterone receptor (PR A/B) was not expressed. Actin, HPRT and GAPDH served as reference genes. (B) Immunolabeling confirmed the presence of these progesterone receptors in primary rd10 microglial cells. Hoechst (blue) staining reveals the cell nuclei. Scale bar represents 10μm. (C) Fluorescent microscopic images of primary rd10 microglia immunolabeled for (C) (i) markers of activation (CD68) and pro-inflammation (M1) (iNOS) and (C) (ii) anti-inflammatory activation (M2) (MRC1); after treatment with 20 μM Norgestrel over 24h. Norgestrel reduced pro-inflammatory and increased anti-inflammatory markers in rd10 microglia. Scale bar 10μm. (D) Quantification of fluorescence intensity of (D) (i) CD68 and iNOS and (D) (ii) MRC1 in primary rd10 microglial cultures following Norgestrel treatment over 24h. N = 8 mice, n = 4 technical replicates per group. (E) Nitrite concentration in the media of rd10 microglia in vitro treated with Norgestrel or vehicle over 24hr. Norgestrel significantly reduced nitrite release from rd10 microglia. N = 8 biological replicates, n = 2 technical replicates per sample. Results are presented as mean ± SEM (t-test, *p<0.05, ***p<0.005, ****p<0.001).
Fig 7.
Norgestrel reduces inflammation in 661Ws and the rd10 retina at P15 and P20.
(A) 661W cells were serum-starved and treated with 20μM Norgestrel or the equivalent DMSO control (Control) over 24h. RT-qPCR analysis detected a significant decrease in mRNA levels of macrophage inflammatory protein 1α and 1β (MIP-1α, MIP-1β), monocyte chemoattractant proteins 1 and 3 (MCP1, MCP3) and interleukin 6 (IL-6) in serum-starved cells treated with Norgestrel. N = 6 biological replicates, n = 3 technical replicates per sample. (B-C) Retinal cytokine mRNA levels were measured by RT-qPCR analysis, in P15-30 rd10 mice. Relative (B) chemokine (MIP-1α, MIP-1β, MCP1 and MCP3) and (C) pro-inflammatory cytokine (tumour necrosis factor α (TNFα), interleukin 1β (IL-1β) and interleukin 6 (IL-6)) mRNA expression levels in retinas of rd10 mice supplemented with Norgestrel. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. Results are presented as mean ± SEM (t-test, *p<0.05, **p<0.01, ****p<0.001).
Fig 8.
Norgestrel upregulates fractalkine-CX3CR1 signaling in the rd10 retina.
(A) 661W cells were serum-starved and treated with 20 μM Norgestrel (Norgestrel) or the equivalent DMSO control (Control) over 6 and 24h. RT-qPCR analysis detected a significant increase in mRNA levels of fractalkine in serum-starved cells treated with Norgestrel. N = 6 biological replicates, n = 3 technical replicates per sample. (B) Norgestrel-supplementation in rd10 mice induced a significant increase in fractalkine and CX3CR1 mRNA expression in P20-P30 retinas compared to control. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. (C) Western blot for fractalkine at P20 and P25 confirmed an increase in full-length fractalkine (100kDa; red box) at both time-points with Norgestrel treatment. Total protein stain shows similar loading between samples. Blots are representative of 6 biological replicates per group. (D) Fluorescent microscopic images of fractalkine (green) in the central retina from P15-40 of control and Norgestrel-fed rd10 mice. Fractalkine staining is evident in the outer nuclear layer (ONL) of the retina in Norgestrel-supplemented mice. Hoechst (blue) staining reveals the cell layers in the retina. Scale bar 50μm. N = 3 mice per group, n = 3 sections per mouse. (E) 661W cells were cultured in the absence of serum (serum-starved 661W) over 3 h. 661W cells were then placed in culture with microglia that had been treated with (Microglia Fract.) or without (Microglia) 1.2μg/ml recombinant full-length fractalkine over 24h. Co-cultures were incubated for a further 24h. Apoptosis of 661W cells was detected by TUNEL of DNA strand breaks in cell nuclei. Co-cultures with fractalkine-treated microglia had less TUNEL-positive 661W cells than equivalent vehicle control. Scale bar 30μm. (F) Quantification of TUNEL-positive 661W cells cultured in the absence of serum and following co-incubation with primary rd10 microglial cells pre-treated with recombinant full-length fractalkine or vehicle. N = 9 mice for primary culture, n = 5 technical replicates per group. Results are presented as mean ± SEM (t-test, *p<0.05, ***p<0.005, ****p<0.001).