| Objective:mTOR( mammalian target of rapamycin)is mostly known for its role in nonneural cells proliferation, neuronal development, synaptic plasticity regulation and learning and memory formation. But, it is not well understood how mTOR works in the pathgenesis of neuropathic pain. To interpret the role of mTOR in neuropathic pain, the change of mTOR expression in spinal cord of rats with neuropathic pain induced by chronic constriction injury to the sciatic nerve, and the association between mTOR and astrocyte activation in spinal cord dorsal horn will be observed.Method:30 male and healthy SD rats were randomly divided into 6 groups with 5 rats each. Basic mechanical and thermal pain threshold were tested before building CCI model. CCI model was established in 20 rats. Five rats were selected as sham-operation group. Five rats were selected as control group. CCI rats were killed on post-operative day of 1 (group D1), 4(group D4), 7 (group D7) and 14(group D14) after pain threshold test respectively; rats in sham-operation group and control group were killed on 14th day. Lumbar spinal cords of rats in all groups were dissected out. The localization of mTOR was demonstrated by immuo-histochemical assay. The expression of mRNA level of mTOR was detected by relative real-time fluorescent quantitative PCR assay. The expression of protein level of mTOR was detected by Western blot method.Another 30 male and healthy SD rats were randomly divided into 6 groups with 5 rats each. Basic mechanical and thermal pain threshold were tested before the experiment. Five rats were selected as control group. CCI model was established in 25 rats. Five rats were selected as CCI group without any treatment after CCI. At 4 hours after CCI, five rats were given rapamycin(10μg)10μl+NS10μl intrathecal injections once a day for 3 days, and five rats were given the vehiculum of the same volume and concentration. On 7th day after CCI, five rats were given rapamycin (10μg)10μl+NS10μl intrathecal injections once a day for 3 days, and five rats were given the vehiculum of the same volume and concentration. Mechanical and thermal pain threshold were tested on 2th, 4th,6th,8th,10th,12th,14th day after CCI. The rats in all groups were killed on 14th day. Lumbar segment of spinal cords was removed for determination of glial fibrillary acidic protein (GFAP) in spinal cord by immuo-histochemical assay.Result:1. Hyperalgesia in CCI rats: Mechanical hyperalgensis emerged on 1th day after CCI. Thermal hyperalgensis emerged on 4 th day after CCI(P 2. Localization of mTOR: In Lumbar segment of spinal cords of rats in control group, mTOR positive particles were presented in neuronal cytoplasm widespreadly.3. mRNA expression of mTOR in CCI rats: The mean mTOR mRNA content was significantly higher in the CCI groups than that in the control group(P < 0.01). The mRNA expression of mTOR in 1D, D4, D7, D14 groups were 5.254, 7.547, 15.192 and 17.432 folds that of the control group, respectively. No significant difference was observed in the mean mTOR mRNA content between the control group and the sham-operation group(P>0.05).4. Protein expression of mTOR in CCI rats: The relative protein content of mTOR was higher in the CCI groups than that in the control group significantly (P<0.05 or P<0.01). There was no significant difference of mTOR protein content between the control group and the sham-operation group(P>0.05).5. Hyperalgesia in CCI rats after rapamycin treatment : CCI group and two vehiculum treated groups developed mechanical and thermal hyperalgesia from 4th day to 14th day after CCI, no significant difference in mechanical and thermal pain threshold between those three groups. Intrathecal injection of rapamycin at 4 hours after CCI, attenuated CCI-induced mechanical and thermal hyperalgesia from 4th day to 14th day significantly (P < 0.01 or P < 0.05). Intrathecal injection of rapamycin on 7th day after CCI, attenuated CCI-induced mechanical and thermal hyperalgesia from 8th day to 14th day significantly (P<0.01 or P< 0.05).6. Expression of spinal GFAP in CCI rats after rapamycin treatment : Compared with the control group, the sum area of GFAP positive and the mean density of GFAP positive area in the left dorsal horn were greatly increased in CCI group and two vehiculum treated groups(P<0.01). These two indexes in rapamycin treated groups were lower than that in CCI group with out any treatment(P< 0.01).Conclusion1. The mRNA and the protein expression of mTOR was greatly increased in CCI rats. mTOR signal pathway may play a significant role in the pathgenesis of neuropathic pain in rats induced by chronic constriction injury to the sciatic nerve.2. Intrathecal rapamycin treatment significantly attenuated CCI-induced hyperalgesia and inhibited the activation of astrocyte. mTOR signal pathway may be involved in the astrocyte activation in spinal dorsal horn after periphery nerve injury. |
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