CXCL12/CXCR4 Signaling Activation In Spinal Cord Contributes To The Pathogenesis Of Postsurgical Pain In Rats

BackgroundPostoperative pain often brought great suffering to the patients.Despite increased basic and clinical research have improved understanding of its pathologic mechanisms,optimal postsurgical pain therapy still remains a challenge for physicians.Previous studies have demonstrated that the surgery-induced upregulation of inflammatory mediators,including TNF-? and IL-1?,contributes to postoperative pain.Recent studies have found that chemokine CXCL12 and its receptor CXCR4-mediated signaling pathways are involved in multiple types of chronic pain.However,whether the CXCL12/ CXCR4 signal pathway involved in postoperative pain has not been determined to date.Compared with inflammatory and neuropathic pain,surgical pain is a unique acute pain state in which there are various central sensitization mechanisms,in particular in the spinal cord.Thus although a number of studies have demonstrated the CXCL12/CXCR4 signaling contributes to various types of chronic pain,whether it also plays a role in postsurgical pain process still need to preciously study.ObjectiveIn this study,the expression of CXCL12 and CXCR4 in the spinal cord was observed by using plantar incision model in rats.And the role of CXCL12/CXCR4 signaling activation in postoperative pain was proved.Finally,the signal pathway of plantar incision-induced upregulation of CXCL12 and the downstream molecules of CXCR4 mediated postsurgical pain were further determined.Methods 1 Expression of CXCL12/CXCR4 in the spinal cord of rats after plantar incision and the distribution of cell typesMale SD rats were randomly divided into experimental group(PI group)and control group(Sham group),the paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)were measured continuously from 1 day before operation to the 7th day after operation.The expression of CXCL12,CXCR4,p-p65,p-ERK and p-Akt in the spinal dorsal horn was detected by Western-blot.The cell types of CXCL12 and CXCR4 expressed in the spinal cord were observed by immunofluorescence double staining.2 The role of blocking CXCL12/CXCR4 signaling in postoperative painMale SD rats were randomly divided into 6 groups: Sham group,PI + Saline group,PI + AMD 5 ?g group,PI + AMD 10 ?g group,PI + AMD 20 ?g group and AMD 20 ?g group.The rats were treated with pre-intrathecal injection,then PWT and PWL were measured after operation.The expression of p-ERK and p-Akt was detected by Western Blotting.Another group of male SD rats were randomly divided into experimental group(PI + CXCL12 antibody 4 ?g group)and control group(PI + IgG 4 ?g group).Intrathecal injection was initiated 30 minutes before surgery,PWT and PWL were measured continuously at 30 min,2 h,4 h,8 h,16 h,24 h after PI.3 Regulation mechanism of up-regulation of CXCL12 expression in spinal cord of rats after PIMale SD rats were randomly divided into 4 groups: Sham + Vehicle group,PI + Vehicle group,PI + PDTC 0.5 ?g group and Sham + PDTC 0.5 ?g group.The rats were treated with pre-intrathecal injection,then PWT and PWL were measured after operation.The expression of p-p65 and CXCL12 was detected by Western Blot.4 Downstream pathway of CXCL12/CXCR4 signaling mediated postsurgical painThe first two steps of this section have been completed by Part 1 and Part 2,and this section will proceed to the 3rd Step.Male SD rats were randomly divided into 2 groups: PI + Vehicle group,PI + PD98059 10 ?g group.The rats were treated with pre-intrathecal injection,then PWT and PWL were measured after PI.Results 1 Plantar incision induced pain-related hypersensitivity and upregulation of CXCL12 and CXCR4 in spinal cordPlantar incision(PI)produced a rapid mechanical allodynia and thermal hyperalgesia.Pain-related behavioral test revealed a clear reduction of PWT and PWL,which started at 1 h after PI and persistent to the fifth day after surgery.The western blotting data showed PI induced robust increased expressions of CXCL12 and CXCR4 in spinal cord.The results of immuohistochemistry also showed PI induced a significant increased expression of CXCL12 and CXCR4 in spinal dorsal horn.Double immunofluorescence staining revealed that the up-regulated CXCL12 primarily co-localized with the neuronal marker NeuN and the astrocytic marker GFAP,but not with the microglia marker OX42,whereas CXCR4 was exclusively co-localized with neuronal marker NeuN.2 The role of spinal CXCL12/CXCR4 signaling in the induction of postoperative painPrior to administration of AMD3100,a specific antagonist of CXCR4,or CXCL12 neutralizing antibody intrathecally attenuated PI-induced mechanical allodynia and thermal hyperalgesia.Compared with vehicle(i.t.10 ?l saline)group,AMD3100 treatment(30 min before PI and daily for 5 days)dose-dependently increased PWT and PWL in ipsilateral,but not contralateral,hind paw.The results showed that a single dose of CXCL12 neutralizing antibody i.t.resulted in a significant increase of PWT and PWL following PI.Compared to control group,the statistical difference of PWT and PWL started at 30 min after surgery,and maintained to the 16 h of post-surgery.3 Nuclear factor kappa B(NF-?B)activation mediates the plantar incision-induced CXCL12 upregulation in spinal cordThe results of western blot showed that the expression of phosphorylated p65 was significantly increased started at 2 h and persistent to the 3rd day after surgery.The total NF-?B p65 protein in spinal cord was not changed following PI.The PI-induced upregulation of CXCL12 in spinal dorsal horn was inhibited following i.t.administration of PDTC(a specific inhibitor of NF-?B activation,500 ng/10 ?l).Behavior data analysis showed that i.t.administration of PDTC attenuated PI-induced mechanical allodynia and thermal hyperalgesia.4 Downstream pathway of CXCL12/CXCR4 signaling mediated postsurgical painPI also resulted in an increased phosphorylation of ERK1/2 and Akt in spinal cord.Results from western blot showed prior to i.t.administration of AMD3100 significantly reduced the phosphorylation level of PI-induced ERK1/2,but not Akt,in spinal dorsal horn.Behavioral data showed prior to i.t.PD098059(10 ?g/10 ?l,30 min before surgery and daily for 5 days)significantly prevented the PI-induced reduction of PWT and PWLConclusionOur results reveal that plantar incision-induced NF-?B activation mediates the upregulation of CXCL12 in spinal dorsal horn.The increased release of CXCL12 via acting on CXCR4 evoked ERK signal activation contributes to the generation of postsurgical pain.