Sonic hedgehog signaling in spinal cord contributes to morphine-induced hyperalgesia and tolerance through upregulating brain-derived neurotrophic factor expression

Figures & data

Figure 1 Effects of cyclopamine on morphine-induced hyperalgesia and tolerance.

Notes: The MPE% (refers to tolerance) decreased after repeated morphine injection (A and B). Coadministration of morphine with cyclopamine (10 mg/kg) on days 1, 2, and 3 significantly delayed MPE% decrease (A). However, coadministration of morphine with cyclopamine on days 5, 6, and 7 showed no effect on the reduction of MPE% (B). The thermal withdrawal latency (refers to hyperalgesia) of mice was significantly decreased after chronic morphine treatment (C and D). Coadministration of morphine with cyclopamine on days 1, 2, and 3 significantly delayed or attenuated hyperalgesia (C), but coadministration of morphine with cyclopamine on days 5, 6, and 7 showed no effect on hyperalgesia induced by chronic morphine treatment. *P<0.05, **P<0.01 versus sham group; ^#P<0.05, ^##P<0.01 versus Mor group. Ten mice were included in each group.
Abbreviations: i.p., intraperitoneally; MPE%, percentage of maximal possible effect; Mor, morphine; CLP, cyclopamine; DMSO, dimethyl sulfoxide.

Figure 2 Knockdown of Shh in spinal cord prevents the reduction of chronic morphine treatment-induced MPE%.

Notes: (A) Effects of Shh targeting siRNA (1 μg i.t., daily for 3 consecutive days) on the expression of Shh in naïve mice (n=6 in each group). Whole spinal cord tissues were collected 2 hours after the last injection. (B) Knockdown of Shh significantly prevented the reduction of chronic morphine treatment-induced MPE% (n=10 in each group). (C) Effects of Shh targeting siRNA (1 μg i.t., daily for 3 consecutive days) on the expression of Shh in morphine-treated mice (n=6 in each group). Whole spinal cord tissues were collected at day 4 after the first morphine injection. *P<0.05, **P<0.01 versus con-siRNA+Mor group.
Abbreviations: MPE%, percentage of maximal possible effect; siRNA, small interfering RNA; i.t., intrathecally; Mor, morphine; con-siRNA, control-siRNA.

Figure 3 Shh signaling inhibition with cyclopamine suppressed morphine treatment-induced upregulation of c-fos and CGRP in the spinal cord of mice.

Notes: (A) Examples of immunofluorescence images and data summary (n=6 in each group) showing the expression of c-fos and CGRP in the dorsal horn of the spinal cord. Magnification: 200×. (B) Examples of Western blot analysis and data summary (n=6 in each group) showing the expression of c-fos and CGRP in the spinal cord. Samples were collected at day 4 after the first morphine injection. Cyclopamine (10 mg/kg, i.p.) was coadministrated with morphine on days 1, 2, and 3. *P<0.05, **P<0.01 versus sham group. ^##P<0.01 versus Mor group.
Abbreviations: i.p., intraperitoneally; Mor, morphine; CLP, cyclopamine.

Figure 4 Expression and activation of Shh signaling after chronic morphine treatment.

Notes: (A) Examples of Western blot analysis and data summary (n=6 in each group) showing a time-dependent increased expression of Shh, Ptch1, and Smo in the SC of the mice. (B) Examples of Western blot analysis and data summary (n=6 in each group) showing a time-dependent increased expression of Gli1 in nuclear extract of the SC of the mice. (C) Immunofluorescence staining shows the expression and location of Shh in the dorsal horn of the SC (n=4 in each group) (bar: 50 μm). (D) Examples of Western blot analysis and data summary showing a time-dependent increased expression of Shh and Gli1 in DRG (n=6 in each group). (E) Immunofluorescence staining shows the expression of Shh in DRG (n=6 in each group) (bar: 250 μm). (F) ELISA showing the concentration of Shh in DRG and SC (n=6 in each group). For immunofluorescence staining, samples were collected on day 7 after the first morphine injection. *P<0.05, **P<0.01 versus sham group.
Abbreviations: SC, spinal cord; DRG, dorsal root ganglion; i.t., intrathecally; Mor, morphine; ELISA, enzyme linked immunosorbent assay.

Figure 5 Shh signaling contributed to tolerance and MIH through regulating BDNF expression.

Notes: (A) Expression of BDNF increased in a time-dependent manner after chronic morphine treatment. Inhibiting Shh signaling by cyclopamine (B) and Shh-target siRNA (C) significantly suppressed upregulation of BDNF. Tissues were collected on day 4 after morphine injection (B and C). (D) Activating Shh signaling by SAG injection (i.p.) in naïve mice significantly increased BDNF expression, and pretreatment with cyclopamine effectively prevented the upregulation of BDNF induced by SAG (E). Tissues were collected at 2 hours after SAG injection (D and E). Six mice were included in each group (A–E). BDNF inhibitor K252 and anti-BDNF antibody significantly reversed SAG-induced thermal hyperalgesia in naïve mice (F). BDNF inhibitor K252 effectively suppressed and delayed chronic morphine treatment-induced MPE decrease (G) and thermal hyperalgesia (H). BDNF (green) were colocalized with Shh (I, left) and Gli1 (I, right) in the spinal cord (n=4, bar: 50 μm). Insets are magnified areas. Yellow represents colocalization. Tissues were collected on day 7 after the first morphine injection. Ten mice were included in each group (F–H). *P<0.05, **P<0.01 versus sham group (A–C) or DMSO group (D–F). ^#P<0.05, ^##P<0.01 versus Mor group (B, C, G, and H) or SAG group (E and F).
Abbreviations: MIH, morphine-induced hyperalgesia; BDNF, brain-derived neurotrophic factor; siRNA, small interfering RNA; i.p., intraperitoneally; i.t., intrathecally; Mor, morphine; CLP, cyclopamine; DMSO, dimethyl sulfoxide; SAG, smoothened agonist; MPE, maximal possible effect.