MTOR signaling pathway of spinal cord is involved in peripheral nerve injury-induced hyperalgesia in rats

Abstract

To investigate whether mammalian target of rapamycin (mTOR) signaling pathway is involved in peripheral nerve injury-induced hyperalgesia through activation of spinal dorsal astrocytes in rats. Methods: A total of 30 male Sprague-Dawley (SD) rats were randomly divided into 6 groups (n=5): the 1 day group (D1 group), the 4 days group (D4 group), the 7 days group (D7 group), the 14 days group (D14 group), the normal group and the sham group. The sciatic nerve chronic constriction injury (CCI) model was established in the D1, D4, D7 and D14 group. The normal group received no treatment while the sham group was only exposed the sciatic nerve. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at the 1st, 4th, 7th, and 14th day after CCI in the different groups. Lumbar spinal cord were harvested on the 1st, 4th, 7th and 14th day in the D1, D4, D7, D14 group correspondingly, which were harvested on the 14th day in the normal group and the sham group. Distribution of mTOR in rat spinal cord was assessed by immunohistochemistry. The expressions of mTOR mRNA and protein in the spinal cord in different groups were determined by real-time PCR and Western blotting, respectively. Another 30 male intrathecal catheterized SD rats were randomly divided into 6 groups (n=5): a blank group, a CCI group, a CCI+early rapamycin (RAPA) group, a CCI+early dimethylsulfoxide (DMSO) group, a CCI+ later RAPA group, and a CCI+later DMSO group. The blank group didn't received any treatment; The CCI group was carried out the treatment of CCI model in the left hind limbs. 10 μL of 1% RAPA was given to the CCI+early RAPA group intrathecally at 4 hours after CCI for 3 days; the CCI+later RAPA group were treated with the same dose of RAPA on the 7th days after CCI for 3 days; the CCI+early DMSO group and the CCI+later DMSO group were injected with the same volume of 4% DMSO at the corresponding time as controls. The PWTL and PWMT were measured before and after intrathecal catheterization, and every other day after CCI. The lumbar spinal cords were selected and the expression of glial fibrillary acidic protein (GFAP) in spinal dorsal horn were examined by immunohistochemistry in the 14th day after CCI. Results: The immunohistochemistry positive particles of mTOR were widely distributed in the cytoplasm of the normal spinal neurons. Compared with the base line, the PWMT in the D14 group on the 1st, 4th, 7th and 14th day after CCI were significantly lower, and the PWTL on the 4th, 7th and 14th day after CCI were also significantly lower (P<0.05 or P<0.01). The expressions of mTOR mRNA and protein in the CCI groups (D1, D4, D7 and D14 group) were significantly increased than those in the normal group (P<0.05 or P<0.01). Compared with the CCI+early DMSO group, the PWMT and PWTL in the CCI+early RAPA group were obviously increased on 4th, 6th, 8th, 10th, 12th or 14th day after CCI (P<0.05 or P<0.01); compared with the CCI+later DMSO group, the PWMT and PWTL in the CCI+later RAPA group were also significantly increased at the 8th, 10th or 14th day after CCI (P<0.01 or P<0.05). The GFAP immunohistochemistry positive area and absorbance value in the dorsal horn of the lumbar spinal cord in the CCI rats were decreased in the CCI+early RAPA group compared with the CCI+early DMSO group (P<0.05 or P<0.01), and which were also decreased in the CCI+later RAPA group compared with the CCI+later DMSO group (P<0.05 or P<0.01). Conclusion: mTOR signaling pathway may be involved in hyperalgesia induced by peripheral nerve injury via spinal astrocyte activation in the dorsal horn of the spinal cord.