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. 2019 Dec 26;17(2):722–742. doi: 10.1007/s13311-019-00800-w

Fig. 4.

Fig. 4

The role of the chronic i.t. morphine exposure-induced upregulation of HMGB1 in the spinal dorsal horn in the development of analgesic tolerance and hyperalgesia. (a) Diagram of the timeline of this experiment. (b) Repeated i.t. injections of morphine led to a significant reduction in morphine’s maximal possible analgesic effect (% MPAE). # P < 0.05; ## P < 0.01 vs. day one. Data are presented as the mean ± SEM and were analyzed by two-way ANOVA. Intrathecal coadministration of morphine (Mor) plus glycyrrhizin (GL), an inhibitor of HMGB1, dose-dependently prevented the decrease in the MPAE. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. morphine plus vehicle (Veh) group (one-way ANOVA). (c, d) Chronic i.t. morphine exposure resulted in decreased paw withdrawal threshold (PWT) (c) and paw withdrawal latency (PWL) (d) in the left hind paw. ## P < 0.01 vs. baseline. Repeated intrathecal coinjections of morphine plus GL prevented the reduction in PWT and PWL after morphine withdrawal. * P < 0.05; ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT and PWL were not changed by repeated i.t. injections of GL alone. (e) Repeated i.t. injections of morphine plus vehicle (Veh: transfection regent) or morphine plus scramble (sc) RNA resulted in significant reductions in the MPAE at day 5 and day 7. ## P < 0.01 vs. day one (two-way ANOVA). This effect was clearly prevented by the coadministration of morphine with HMGB1 siRNA intrathecally. ** P < 0.01; *** P < 0.001 vs. morphine plus vehicle or morphine plus HMGB1 scRNA (one-way ANOVA). (f, g) Repeated i.t. injections of morphine plus vehicle or morphine plus HMGB1 scRNA led to significant decreases in PWT (f) and PWL (g) in the left hind paw, but these reductions were prevented by i.t. coinjections of morphine plus HMGB1 siRNA. # P < 0.05, ## P < 0.01 vs. baseline; * P < 0.05, ** P < 0.01 vs. morphine plus vehicle or morphine plus HMGB1 scRNA (Student’s t-test). The basal tail-flick response, PWT, and PWL were not changed by repeated i.t. injections of HMGB1 siRNA or transfection regent alone. (h, i) Repeated i.t. coinjections of morphine plus vehicle or morphine plus HMGB1 scRNA led to significantly increased expression of the HMGB1 protein (h) and HMGB1 mRNA (i) in the spinal dorsal horn. These effects were inhibited by i.t. coadministration of morphine with HMGB1 siRNA. * P < 0.05, ** P < 0.01 vs. vehicle group; ### P < 0.001 vs. morphine plus vehicle or morphine plus scramble RNA (one-way ANOVA).