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. Author manuscript; available in PMC: 2016 Aug 4.
Published in final edited form as: Nat Neurosci. 2013 Jan 6;16(2):183–192. doi: 10.1038/nn.3295

Figure 8. Activation of two separate signaling pathways is necessary for morphine-induced hyperalgesia.

Figure 8

a–c. Effect of intrathecal injections of low doses of (+)NL (5 ng) on morphine-induced pain hypersensitivity, tolerance and microglia activation: a. Thermal withdrawal threshold prior to morphine or saline injections in(+)NL or vehicle treated rats ((+)NL, n = 8; morphine+(+)NL, n = 7; morphine, n = 8; *P < 0.05); b. Thermal pain threshold 1 h after treatment. c. CD11b immunostaining in rat SDH following 5 days of intrathecal morphine or morphine+(+)NL (scale bar, 30 μm) and quantification (CTR: n = 28 sections; MS: n = 34 sections; morphine+(+)NL: n = 34 sections; ***P < 0.001). d. LI EGABA following 3 h in vitro with (−)NL alone (n = 6 cells) vs morphine+(−)NL (n = 6; U: 14; P > 0.05) and with (+)NL (n = 6) vs morphine + (+)NL (n = 8, U: 26; P > 0.05). e. Mechanical withdrawal threshold 5 h after intrathecal injections of microglia cultures treated with morphine+(−)NL (n = 8; H: 12.97, *P < 0.05), morphine+(+)NL (n = 5; *P < 0.05) vs morphine alone (n = 7). f. Western blot analysis of P2X4R protein expression in microglia cultures following treatment with morphine (100 nM, n = 10 trials, H: 26.49, ***P < 0.001), morphine+(−)NL (P > 0.05), morphine+(+)NL (n = 5, ***P < 0.001), (−)NL (n = 3, P > 0.05), (+)NL (n = 6, P > 0.05) vs saline (n = 10). g. ATP-evoked rise in intracellular [Ca2+] in cultured microglia treated with morphine (100 nM, n = 54 cells, H: 67.98, ***P < 0.001), morphine+(−)NL (n = 38, P > 0.05), morphine+(+)NL (n = 40,***P < 0.001), (−)NL (n = 15, P > 0.05), (+)NL (n = 15, P > 0.05), or saline (n = 35). h. Western blot of P2X4R protein from spinal cords isolated from rats treated for 5 days with intrathecal saline, morphine, morphine+(−)NL, morphine+(+)NL. i. ELISA-based measurement of BDNF release from microglia treated with morphine+(−)NL (n = 5 trials, H: 14.29, ***P < 0.001), morphine+(+)NL (n = 5,***P < 0.001), vs morphine (n = 14). j. ELISA-based measurement of BDNF release from microglia treated with morphine (n = 4 trials, H: 15.84, ***P < 0.001), morphine+LPS-RS 1 ng/ml (n = 4,***P < 0.001), morphine+LPS-RS 10 ng/ml (n = 4,***P < 0.001), morphine+LPS-RS 100 ng/ml (n = 4,***P < 0.001), LPS-RS 100 ng/ml (n = 4,***P < 0.001) vs control (n = 4). k. Effect of morphine (escalating doses from 10 to 40 mg/kg intraperitoneally twice a day for 7 days) on mechanical sensitivity of TLR4 deficient C3H/HeJ mice (n = 11) vs the wild type C3H/HeOuJ (n = 8). Mechanical sensitivity index was calculated as (10-PWs)/PWs (10 = stimulations with 3.41 g calibrated filament; PWs = paw withdrawals). No differences were observed in the development of mechanical allodynia between mice groups (U: 30; P > 0.05). All threshold values in the figure are normalized to the baseline. Abb.: PWT = paw withdrawal threshold; CTR = control; MS = morphine sulphate; (−)NL = (−)-naloxone; (+)NL = (+)-naloxone; LPS-RS = lipopolysaccharide from Rhodobacter sphaeroides; i.u.: intensity units; error bars = s.e.m.