Fig 1: Changes in mRNA expression of pro-inflammatory cytokines in response to ocular hypertension and SNC-121 treatment. Intraocular pressure (IOP) was raised by 2.0 M hypertonic saline injection followed by d-opioid receptor agonist (1 mg/kg; i. p injections) treatment for 7 days, once a day. The retinas were collected at day 7, post hypertonic saline injection and mRNA was analyzed for IL-1ß (A), TNF-a (B), FAS (C), IL-6 (D), LIF (E), and IFN-? (F) using cDNA that was synthesized from 1 µg total RNA. The relative changes in mRNA levels were measured by quantitative RT-PCR (qRT-PCR) using primers specific for each gene as indicated in Table 1. The qRT-PCR data was normalized using ß-actin gene expression as an internal control. The relative expression was calculated based on the comparative threshold cycle (2-??Ct) method. Bar graph represents mean data ±SEM. OH Eyes; ocular hypertensive eyes; OH Eyes + SNC-121, ocular hypertensive eyes with SNC-121 treatment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n = 4–9. In this experiment “n” represent a biological replicate.
Fig 2: In adult mice, social isolation and decreased PDE11A4 signaling upregulate markers associated with neuroinflammation. Previous studies comparing expression of PDE11A and the cytokine interleukin-6 (IL-6) between mouse strains determined lower expression of PDE11A4 in ventral hippocampal membranes (VHM) correlated with higher expression of IL-6 in ventral hippocampal the soluble fractions (VHS) (Pathak et al., 2017). (A) Similarly, western blots of ventral hippocampal fractions from adult group housed mice (GH) versus adult mice single-housed for 1 h (SH), 1 day (SD), 1 week (SW), or 1 month (SM) show that social isolation does not significantly affect IL-6 expression in the membrane compartment of the VHIPP (n = 7–8/group/sex; effect of group x sex: F (4,55) = 1.58, P = 0.192), (B) but does increase IL-6 expression in the soluble fraction, albeit at different time points for each sex (n = 7–8/group/sex; effect of group x sex: F (4,51) = 6.52, p < 0.001; Post hoc: GH-M vs SM-M and SW-M, P = 0.046-0.025 and GH-F vs SH-F, P = 0.062; however, paired t-test GH-F vs SH-F: t (6) = 4.404, FDR-P = 0.02). Consistent with our previous work (Pathak et al., 2017), (C) expression of VHM PDE11A4 in isolated mice correlated negatively with VHS IL-6 (r = -0.456, p < 0.001). To determine if a loss of PDE11A4 signaling is sufficient to increase soluble IL-6, we compared IL-6 expression in VHIPP fractions from Pde11a WT vs KO mice (n = 8-9F, 2M/genotype). (D) Genetic deletion of PDE11A did not alter IL-6 expression in the membrane compartment (n = 10/genotype, t (8) = 1.45, p = 0.186) but (E) did increase IL-6 expression in the soluble fraction with an effect size comparable to that observed in the isolated mice (n = 10/genotype, t (8) = 2.033, p = 0.076). Next, we determined a potential source of this increased IL-6. (G) Immunofluorescence for a marker of microglia shows that, (H) relative to Pde11a WT mice, Pde11a KO mice exhibit more microglia in the VHIPP (n = 5/genotype, t (8) = -2.88, p = 0.02) (I) and higher levels of IBA-1 expression per microglia (n = 5/genotype, t (8) = -3.03, p = 0.02). (J) In contrast, we see no change in the number of astrocytes within the VHIPP of Pde11a KO mice (n = 4M and 5F/genotype; effect of genotype: t (16) = 0.17, P = 0.867) (K) nor in the level of GFAP expressed by each astrocyte (t (16) = 0.59, P = 0.566). Post Hoc *vs GH, P = 0.046-0.025; #vs GH, FDR-P = 0.02. Data are plotted as means ± SEMs. Brightness and contrast of images adjusted for graphical clarity.
Fig 3: Effects of d-opioid receptor agonist (SNC-121) treatment on interleukin-1ß (IL-1ß) (A) and interleukin-6 (IL-6) (B) production in ocular hypertensive animal. Animals were euthanized and eyes were enucleated at day 7, post hypertonic saline injections. Retina cryosections were immunostained using a selective anti-IL-1ß or anti-IL-6 antibody. The nuclei were counterstained with DAPI. Red color indicates staining for IL-1ß and IL-6 and blue for the nuclei. There was no positive immunostaining when primary antibodies were omitted (not shown). Data shown here is a representation of at least four independent experiments. We have used four animals in each treatment group. OH Eyes; ocular hypertensive eyes; OH Eyes + SNC-121, ocular hypertensive eyes with SNC-121 treatment, bar size 20 µm.
Fig 4: Inhibitory effects of Stattic, a selective STAT3 inhibitor, on the mRNA expression of IL-1ß (A), TNF-a (B), IL-6 (C), and IFN-? (D). The retina from normal, ocular hypertensive, and Stattic treated ocular hypertensive animals were collected at day 7, post hypertonic saline injection. Complementary DNA was synthesized from 1 µg total RNA extracted from retina. The relative changes in mRNA levels were measured by quantitative RT-PCR (qRT-PCR) using primers specific for each gene as indicated in Table 1. The qRT-PCR data was normalized using ß-actin gene expression as an internal control. The relative expression was calculated based on the comparative threshold cycle (2-??Ct) method. Bar graph represents mean data ±SEM. OH Eyes; ocular hypertensive eyes; OH Eyes + Stattic, ocular hypertensive eyes with Stattic treatment. *p < 0.05; **p < 0.01; ***p < 0.001; n = 6–9. In this experiment “n” represent a biological replicate.
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