| Part oneComparative study of different spinal nerve ligation model of neuropathic pain in miceObjective:To compare efficacy of L4and L5spinal nerve ligation model in mice.Methods:We dissected sciatic nerve structural organization in C57BL/6J mouse. C57BL/6J mice were randomly distributed into three groups:Sham group, L4ligation group, and L5ligation group. The general behavior of each mouse was noted during the entire test period. Paw withdraw mechanical threshold (PWMT) and paw withdraw thermal latency (PWTL) were measured before operation for baseline and in1,3,5,7and14days after operation. On the3rd and7th day after surgery, the L4-5segment of the spinal cord was collected and p-CREB was detected by western blot test.Results:The mouse sciatic nerve was origins predominantly from the L3and L4spinal nerves with a smaller diameter contribution from L5. Foot cupping was found in mice after L4ligation, but not in mice after L5ligation and Sham mice. Hyperalgesia and p-CREB of spinal cord were increased significantly in mice after L4ligation compared with L5ligation.Conclusion:L4spinal nerves ligation model is an ideal spinal nerve ligation mode of neuropathic pain in mice. Part twoInterleukin-17A is both necessary and sufficient for neuropathic painObjective:The study aimed to investigate whether IL-17A of spinal cord involves in neuropathic pain.Methods:The lumbar segments L4-5of the spinal cord were collected in1,3,5,7and14days after SNL. IL-17A and GFAP were measured by western blot test, and double staining for GFAP (green) and IL-17A (red) was confirmed by double-label fluorescent immunohistochemistry. Behavioral tests were measured before SNL for baseline and in1,3,5,7and14day after SNL in Sham group, WT group, and IL-17A knockout (KO) group. On the7th day after operation, the lumbar segments L4-5of the spinal cord from above three groups were removed and measured by western blot test for IL-17A and GFAP. SNL mice (7day after SNL) were randomly divided into2subgroups, which were respectively injected with vehicle (saline) or IL-17A antiserum. Behavior testing was measured before injection and1and2h after the injection. Naive mice were randomly divided into4groups, which were respectively injected (i.t.) with vehicle (saline), rIL-17A at50ng,100ng per mouse, and100ng rIL-17A plus2μg antiserum against IL-17A. Behavioral tests were measured before injection and1and2h after the injection.Results:Compared with the Sham, SNL induced upregulation of IL-17A and GFAPuntil day14after nerve injury. Double immunofluorescence staining indicated that all IL-17A-labeled cells also expressed GFAP. The expression of GFAP and IL-17A were significantly decreased in IL-17A null mice. PWMT and PWTL in the ipsilateral hind paws of WT mice were significantly lower than that of IL-17A KO mice after SNL. IL-17A antiserum significantly improved PWMT and PWTL compared to vehicle injection in SNL mice. Hyperalgesia can be induced by rIL-17A in naive mice, which was reversed by IL-17A antiserum.Conclusion:IL-17A is release by astrocytes in spinal cord after SNL in mice, and contributes to neuropathic pain. Part threeIL-17A contributes to neuropathic pain through CREB activation in spinal neuronsObjective:To investigate whether p-CREB involves in IL-17A pathway in neuropathic pain.Methods:The lumbar segments L4-5of the spinal cord were harvested in1,3,5,7and14days after SNL, and p-CREB were measured by western blot test. Spinal cord sections in7days after SNL were analyzed using double-label fluorescent immunohistochemistry under a fluorescence microscope to confirm whether p-CREB is colocalized with NeuN. The spinal cords (L4-L5segment) were collected in7day after SNL in Sham group, WT group and IL-17A-/-group, and p-CREB were detected by western blot test. Mice (7days after SNL) were divided into2subgroups, which were respectively intrathecally injected with saline and IL-17A antiserum. The spinal cords were collected2h after the injection and p-CREB was measured by western blot test. Naive mice were randomly divided into4groups, which were respectively injected (i.t.) with vehicle, rIL-17A at50ng,100ng per mouse, and100ng rIL-17A plus2μg antiserum against IL-17A. The spinal cords were collected2h after the injection and p-CREB was measured by western blot test. Cultured spinal neuron were randomly divided into3groups, which were respectively administrated with saline, rIL-17A at10ng/ml, or100ng/ml for15min. Cultured spinal neuron after treatment were immunofluorescence stained of p-CREB. Cultured spinal neuron were randomly divided into3groups, which were respectively cultured with vehicle, rIL-17A at100ng/ml, and100ng/ml rIL-17A plus2μg antiserum for15min. Cultured spinal neuron after treatment were collected and the expressions of p-CREB and NeuN were detected by immunofluorescence labeling.Results:SNL induced upregulation of p-CREB compared with the sham group until day14after nerve injury. Double staining demonstrated that p-CREB and NeuN colocalize in neurons of spinal cord. SNL-induced phosphorylation of CREB was reversed by specific IL-17A knockdown and IL-17A antiserum. rIL-17A at100ng/mouse significantly increased pCREB levels compared to saline control, which was reversed by IL-17A antiserum. CREB phosphorylation was increased by rIL-17A in vivo, which was decreased by anti-IL-17A mAb.Conclusion:IL-17A contributes to neuropathic pain through phosphorylation of CREB. Part fourIL-17A maintains neuropathic pain through activation of CaMKⅡ/CREB signaling in spinal neuronsObjective:To determine whether p-CaMKII involves in IL-17A/CREB pathway in neuropathic pain.Methods:The lumbar segments L4-5of the spinal cords were collected in7day after SNL in Sham group, WT group and IL-17A KO group and p-CaMKII were detected by western blot analysis. Mice (7day after SNL) were divided randomly into2subgroups, which were respectively injected with vehicle and IL-17A antiserum. The spinal cords (L4-L5segment) were collected2h after the injection and p-CaMKⅡ was measured by western blot test. Naive mice were randomly divided into4groups, which were respectively administrated (i.t.) with vehicle, rIL-17A at50ng,100ng per mouse, and100ng rIL-17A plus2μg antiserum against IL-17A. The spinal cords were collected2h after the injection and p-CaMKII was measured by western blot test. Mice (7day after SNL) were randomly vehicle divided into2subgroups, which were respectively administrated (i.t.) with10% DMSO,45nmol KN93. Behavioral tests were measured before injection and1and2h after the injection. The spinal cords were harvested2h after the injection, and p-CREB and p-CaMKⅡ were measured by western blot test. Naive mice were randomly divided into4groups, which were respectively injected with10%DMSO, rIL-17A at100ng per mouse, and100ng rIL-17A plus45nmol KN93(pre-treated30min) against p-CaMKII. Behavioral tests were measured before injection and1and2h after the injection. The spinal cords were harvested2h after the injection, and p-CREB and p-CaMKII were measured by western blot test. Cultured spinal neuron were randomly divided into3groups, which were respectively treated administrated with10%DMSO, rIL-17A at100ng/ml, and100ng/ml rIL-17A plus45nmol KN93(pre-treated30min) for15min. Cultured spinal neurons after treatment were collected and the expressions of p-CREB were detected by immunofluorescence labeling.Results:SNL-induced phosphorylation of CaMKII was reversed by specific IL-17A knockdown and IL-17A antiserum. rIL-17A at100ng/mouse significantly increased p-CaMKII levels compared to saline control, which was reversed by IL-17A antiserum. SNL-induced hyperalgesia, phosphorylation of CaMKII and CREB was reversed by KN93. rIL-17A at100ng/mouse significantly increased hyperalgesia, p-CaMKII and p-CREB levels, which was reversed by KN93. CREB phosphorylation was increased by rIL-17A in vivo, which was decreased by KN93.Conclusion:IL-17A contributes to neuropathic pain through activation of CaMKII/CREB signaling... |
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