Fig 1: Chronic morphine exposure activated microglia via the TLR3-TRIF signaling pathway in rats(A) Representative TLR3 and TRIF western blot and quantification results in rat spinal dorsal horn. n = 6 rats/group, ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(B) Representative TLR3 immunostaining and quantification results on day 7 in spinal cord segments from rats treated with twice-daily intrathecal saline or morphine injections. n = 5 slices/5 rats/group, ∗∗p < 0.01, non-parametric test (Mann-Whitney test). Scale bar: 100 μm.(C) Double immunostaining of TLR3 with NeuN, GFAP, and IBA1 on day 7 in spinal cord segments from rats treated with twice-daily intrathecal morphine injections. Scale bars: 50 μm.(D) Representative TLR3, TRIF, and IBA1 western blot and quantification results on day 7 in spinal dorsal horn of rats treated with twice-daily intrathecal morphine (10 μg) or morphine plus TLR3 inhibitor (100 μg) injections. n = 6 rats/group, ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(E) Co-injection of TLR3 siRNA (2 μg) blocked morphine-induced (10 μg) increases in TLR3, TRIF, and IBA1 on day 7 in spinal cords of mice treated with twice-daily intrathecal morphine injections. ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(F) Co-injection of TRIF siRNA (400 μg) blocked morphine-induced (10 μg) increases in TRIF, IL-6, and IBA1 on day 7 in spinal cords of mice treated with twice-daily intrathecal morphine injections. ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(G) Co-injection of GW4869 (1 μg) blocked morphine-induced (10 μg) increases in TLR3, TRIF, and IL-6 on day 7 in spinal cords of mice treated with twice-daily intrathecal morphine injections. ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(H) Representative TLR3, TRIF, and IL-6 western blot and quantification results on day 7 in spinal cord segments of rats treated with twice-daily intrathecal saline or morphine injections after intrathecal LV-GFP or LV-ADAR1 injection. ∗∗p < 0.01, non-parametric test (Mann-Whitney test). Data are shown as the mean ± SEM.
Fig 2: Inhibiting spinal TLR3-TRIF signaling prevented the development of morphine-induced tolerance and hyperalgesia in rats(A) Co-administration of intrathecal morphine and TLR3 inhibitor (10 and 100 μg) attenuated morphine tolerance on days 4–7 as evidenced by the measurement of tail-flick latency. n = 6 rats/group. Morphine plus TLR3 inhibitor (10 μg) vs. morphine plus vehicle, ∗∗p < 0.01; morphine plus TLR3 inhibitor (100 μg) vs. morphine plus vehicle, #p < 0.05, ##p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test.(B) Right shift in the dose-response curve for morphine was prevented by TLR3 inhibitor (10 and 100 μg) in the tail-flick test.(C and D) Co-administration of TLR3 inhibitor (10 and 100 μg) blocked morphine-induced decreases in (C) left hind paw withdrawal threshold and (D) latency on day 7. n = 6 rats/group. ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(E) Co-administration of TLR3 inhibitor (100 μg) beginning on day 7 reversed morphine-induced reductions in latency on days 9 and 11. n = 6 rats/group. ∗∗p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test.(F and G) Co-administration of TLR3 inhibitor (100 μg) beginning on day 7 reversed the reductions in (F) left hind paw withdrawal threshold and (G) latency on day 11. ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(H) Co-administration of intrathecal morphine and poly (I:C) (10 μg) accelerated the development of morphine tolerance. n = 6 rats/group, ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test.(I) Co-administration of intrathecal morphine and TLR3 siRNA (2 μg) attenuated morphine tolerance on days 5 and 7. n = 6 rats/group, ∗∗p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test.(J and K) Co-administration of TLR3 siRNA (2 μg) blocked morphine-induced decreases in (J) left hind paw withdrawal threshold and (K) latency on day 7. n = 6 rats/group. ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(L) Co-administration of intrathecal morphine and TRIF siRNA (400 μg) attenuated morphine tolerance on days 5 and 7. n = 6 rats/group, ∗∗p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test.(M and N) Co-administration of intrathecal morphine and TRIF siRNA (400 μg) decreased left hind paw withdrawal threshold and latency on day 7. n = 6 rats/group. ∗∗p < 0.01, non-parametric test (Mann-Whitney test). Data are shown as the mean ± SEM.
Fig 3: Morphine-Exo dsRNA mediated spinal microglial activation via the TLR3-TRIF signaling pathways in vitro(A) Double dsRNA immunostaining with NeuN in cultured primary spinal neurons. Scale bars: 50 μm.(B) Representative ADAR1 western blot and quantification results in cultured primary spinal neurons. n = 4 biological replicates, ∗p < 0.05, non-parametric test (Mann-Whitney test).(C) Quantification of dsRNA immunostaining in cultured primary spinal neurons. n = 5 wells/group, ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(D) Detection of dsRNA using ELISA in Exos isolated from conditioned media from cultured primary spinal neurons. n = 5 biological replicates, ∗∗p < 0.01, non-parametric test (Mann-Whitney test).(E) Representative IBA1, TLR3, TRIF, and IL-6 western blot and quantification results in cultured primary spinal microglia. n = 4 biological replicates, ∗∗p < 0.01, non-parametric test (Mann-Whitney test). Data are shown as the mean ± SEM.
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