| Part oneThe changes of autophagy in the rat spinal dorsal horn after spinal nerve ligationObjective To observe the changes of autophagy in the rat spinal dorsal horn after spinal nerve ligation.Methods Sixty male SD rats (200～220g) were randomly divided into two groups: sham group and L5spinal nerve ligation (SNL) group (n=30each). The rats in the SNL group were underwent L5spinal nerve ligation. The surgical procedure of the rats in the sham group was identical except spinal nerve ligation. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured at1d before SNL (baseline) and at1,3,5,7d after SNL. Six rats in each group were sacrificed after behavioral assessment at1,3,5,7d after SNL, the expression of p62and LC3in the ipsilateral dorsal horn was measured by Western blot. Four rats in the SNL group were sacrificed at7d, the expression of p62/NeuN, p62/GFAP and p62/CD11b in the ipsilateral dorsal horn was examined by double immunofluorescence.Results Compared with the sham group, the PWT was significantly decreased at1d after SNL (P<0.05), the PWL was significantly decreased at3d after SNL (P<0.05), and the expression of p62and LC3was significantly increased (P<0.05). Double immunofluorescence showed that a few p62were colocalized with NeuN and CD1lb in the ipsilateral spinal dorsal horn at7d after SNL.Conclusion Autophagy in the neurons and microglias in the ipsilateral spinal dorsal horn was impaired after SNL, which maybe related to the development of neuropathic pain. Part twoThe effects of autophagy on neuropathic pain and interleukin-ip in the spinal dorsal horn in rats after spinal nerve ligationObjective To investigate the effect of of autophagy on neuropathic pain and interleukin-1β (IL-1β) in the spinal cord in rats after spinal nerve ligation.Methods Sixty male SD rats (200～220g) were randomly divided into four groups: sham group, L5spinal nerve ligation (SNL) group, rapamycin-treatment group (Rap group) and3-methyladenine-treatment group (3-MA group)(n=15each). After intrathecal catheter implantation, the rats in SNL group, Rap group and3-MA group were underwent spinal nerve ligation, the surgical procedure for the sham rats was identical except nerve ligation. Rapamycin (0.1μg) or3-MA(30μg) was injected intrathecally in the Rap group or3-MA group30min after SNL, once daily for7days, the same volume of vehicle(5%DMSO10μl) was administered intrathecally in the sham group and SNL group. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured at1d before SNL(baseline) and at1,3,5,7d after SNL, Rats were sacrificed at7d after SNL after behavioral assessment. The expression of p62and LC3in the ipsilateral dorsal horn was measured by Western blot, ipsilateral L5segment of spinal dorsal horn was examined by transmission electron microscope, and the level of IL-1β was measured by ELISA.Results Compared with the sham group, the PWT and PWL were significantly decreased after SNL, with the LC3-II, p62and IL-1β significantly increased; Compared with the SNL group, the PWT and PWL in the Rap group were significantly increased (P<0.05) with the LC3-II, autophagosomes in microglia significantly increased and the p62, IL-1β significantly decreased (P<0.05), while the PWT and PWL in the3-MA group were significantly decreased (P<0.05) with the LC3-II, autophagosomes in microglia significantly decreased and the p62, IL-1β significantly increased (P<0.05).Conclusion Increased autophagy ameliorated neuropathic pain while decreased autophagy intensified neuropathic pain following SNL, the role may be related to the effcet of autophagy in microglia on IL-1β in the spinal dorsal horn. Part threeThe effects of autophagy on apoptosis of neurons in the spinal cord in rats after spinal nerve ligationObjective To investigate the effect of of autophagy on apoptosis of neurons in the spinal cord in rats after spinal nerve ligation.Methods1) Thirty male SD rats (200-220g) were randomly divided into three groups: sham group, L5spinal nerve ligation (SNL) group and rapamycin-treatment group(Rap group)(n=10each). After intrathecal catheter implantation, the rats in SNL group and Rap group were underwent spinal nerve ligation, the surgical procedure for the sham rats was identical except nerve ligation. Rapamycin(0.1μg) was injected intrathecally in the Rap group30min after SNL, once daily for7days, the same volume of vehicle(5%DMSO) was administered intrathecally in the sham group and SNL group.2) Thirty male SD rats (200-220g) were randomly divided into three groups: sham group, L5spinal nerve ligation (SNL) group and3-methyladenine-treatment group(3-MA group)(n=10each). After intrathecal catheter implantation, the rats in SNL group and3-MA group were underwent spinal nerve ligation, the surgical procedure for the sham rats was identical except nerve ligation.3-MA (30μg) was injected intrathecally in the Rap group30min after SNL, once daily for3days, the same volume of vehicle (5%DMSO) was administered intrathecally in the sham group and SNL group.Rats in part one and part two were sacrificed at7d and3d after SNL, respectively. Ipsilateral L5segment of spinal dorsal horn was examined by transmission electron microscope. The expression of caspase-3and NeuN in the ipsilateral dorsal horn was measured by Western blot.Results In the sham group, neuronal morphology appeared to be normal with healthy looking nuclei, mitochondria, endoplasmic reticulum and lysosomes. In the SNL group, the neurons showed shrunken nuclei, fragmented endoplasmic reticulum, swollen mitochondria with vacuolated and disorganized cristae. Autophagosomes that contained cytoplasmic material or with double-membrane structures were found in the neurons and microglias. In the Rap group, darkened autolysosomes and autophagosomes were frequently observed. Although the neurons displayed swollen mitochondria and expanded endoplasmic reticulum, the neuronal injury seems much less than that in SNL group. By contrast, in the3-MA group, numerous vacuoles and destructive organelles in the cytoplasm were observed with few autophagosomes. In part one, compared with the sham group, the expression of caspase3was significantly increased (P<0.05), with the expression of NeuN was significantly decreased in the SNL group (P<0.05); compared with the SNL group, the expression of caspase3was significantly decreased (P<0.05), with the expression of NeuN was significantly increased (P<0.05) in the Rap group. In part two, the expression of caspase3was significantly increased (P<0.05), while the expression of NeuN was not significantly changed in the SNL group (P>0.05); compared with the SNL group, the expression of caspase3was significantly increased (P<0.05), with the expression of NeuN was significantly decreased (P<0.05) in the3-MA group.Conclusion Increased autophagy ameliorated, while decreased autophagy intensified the damage and apoptosis of the neurons in the ipsilateral spinal dorsal horn after SNL. The effect of autophagy on neuropathic pain may be related to its effcet on the apoptosis of neurons in the spinal dorsal horn. |
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