Study On The Mechanism Of Central Nervous System Ror2 And Astrocyte Polarization In Chronic Pain After Thoracotom

Part ⅠRor2 in spinal cord dorsal horn mediates chronic post-thoracotomy pain by inducing the transformation of A1/A2 reactive astrocytes in ratsBackground and ObjectiveChronic post-thoracotomy pain(CPTP)can seriously affect a patient’s postoperative recovery and quality of life.The underlying mechanism of CPTP remains elusive.Ror2 plays an important role in neuronal development,neuronal plasticity,and neuropathic pain,and it is thought to be a crucial regulator in acute and chronic pain.Astrocytes activity may contribute to the pathogenesis of chronic pain.Reactive astrocytes can be divided into neurotoxic A1 astrocytes and neuroprotective A2 astrocytes,but little is known about the potential functions of the A1/A2 astrocytes in CPTP development.In our previous pilot study,we found that Ror2 and GFAP protein levels increased in thoracic dorsal root ganglia from postoperative day(POD)7 to POD 21 in rats with CPTP.We have investigated the changes in Ror2,c-JUN,C3aR and astrocyte activation in the spinal cord during the development of CPTP.We further explored the role of Ror2 in modulating A1/A2 astrocyte transformation in CPTP.MethodsAccording to Buvanendron’s method,the CPTP model was established on rats.Mechanical hyperalgesia and cold hyperalgesia of the rat back skin were measured during the day.Behavioral assays were performed on the day before thoracotomy(baseline)and then 1,3,5,7,10,14,18 and 21 days after thoracotomy.The rats were divided into Naive group,Sham group,CPTP group and non-CPTP group.The ipsilateral thoracic fourth and fifth SDHs were collected for western blot and immunofluorescence of GFAP,S100A10,C3,Ror2,c-JUN and C3aR on POD 7,POD 14 and POD 21.AAV-GFAP-Mir30-Ror2 for inhibiting the expression of Ror2 in spinal cord astrocytes and control AAV-GFAP-NC under control of the astrocyte specific promoter GFAP were injected intrathecally 21 days prior to thoracotomy to explore the effects of Ror2 on pain behaviors,the expression of GFAP,S100A10,C3,Ror2,c-JUN and C3aR and in regulating A1/A2 reactive astrocyte transformation in CPTP.ResultsThoracotomy induced mechanical hyperalgesia and cold allodynia in rats.Compared with the sham group,the expressions of GFAP,C3,Ror2,c-JUN and C3aR increased and the expression of S100A10 decreased in SDH with CPTP from POD 7 to POD 21,and there was an increase in the levels of GFAP,C3,Ror2,c-JUN and C3aR and a decrease in the level of S100A10 in the CPTP group relative to the Naive,Sham,and non-CPTP groups on POD 21.Astrocytes were activated and mainly expressed as the A1 phenotype in the SDH after CPTP.Knockdown of the spinal astrocyte Ror2 before thoracotomy alleviated thoracotomy-induced mechanical hyperalgesia and cold allodynia,reverted the A1/A2 ratio of reactive astrocytes and downregulated the expression of c-JUN and C3aR in rats with CPTP.ConclusionsThe expressions of Ror2,c-JUN,and C3aR were increased and the astrocytes were activated and mainly expressed as the A1 phenotype in the SDH of the rats with CPTP.Ror2 in the spinal cord astrocytes mediates the transformation of A1/A2 reactive astrocytes via regulating the expressions of the c-JUN and C3aR in CPTP.Part ⅡRor2 in anterior cingulate cortex mediates chronic post-thoracotomy pain by regulating the transformation of A1/A2 reactive astrocytes and the expression of CCL2 and CXCL1 in ratsBackground and ObjectiveChronic post-thoracotomy pain(CPTP)is related to inflammatory pain,neuropathic pain,peripheral sensitization and central sensitization,but its exact pathogenesis is still unclear,so there is no effective prevention and treatment measures at present.In the inflammatory pain model and neuropathic pain model,the expression of GFAP in anterior cingulate cortex(ACC)increased.Inhibiting the activation of astrocytes in ACC can alleviate the behaviors associated with chronic pain.Chemokines CCL2 and CXCL1 secreted by astrocytes are involved in the occurrence and development of chronic pain by regulating the excitability of peripheral and spinal sensory neurons and mediating the interaction between neurons and glia.The results of our first part show that Ror2 in spinal cord dorsal horn(SDH)mediates CPTP by inducing the transformation of A1/A2 reactive astrocytes in rats.This study intends to establish a CPTP rat model to explore the roles of Ror2,the transformation of reactive astrocytes and the expression of CCL2 and CXCL1 in ACC during the development of CPTP.MethodsThe CPTP model was established according to Buvanendron’s method.The pain behaviors of rats were evaluated by mechanical withdrawal threshold(MWT)and cold allodynia threshold one day before thoracotomy as baseline value and 1,3,5,7,10,14,18 and 21 days after thoracotomy.The rats were divided into Naive group,Sham group,CPTP group and non-CPTP group.The expressions of GFAP,S100A10,C3,Ror2,CCL2 and CXCL1 were detected by western blot in ACC collected on postoperative day(POD)7,POD 14 and POD 21 respectively.S100A10,C3,Ror2,CCL2 and CXCL1 and GFAP were co-stained with immunofluorescence to observe their expressions in ACC astrocytes.21 days before thoracotomy,adeno-associated virus AAV-GFAP-Mir30-Ror2 for knocking down the expression of Ror2 in astrocytes and blank control virus AAV-GFAP-NC were injected intrathecally to explore the effects of Ror2 in ACC on pain behaviors,the expressions of GFAP,S100A10,C3,Ror2,CCL2 and CXCL1 and the transformation of reactive astrocytes during the development of CPTP.ResultsCompared with the sham group,the expressions of GFAP,C3,Ror2,CCL2 and CXCL1 in ACC of rats with CPTP significantly increased from POD 7 to POD 21,while the expressions of S100A10 decreased from POD 7 to POD 21.The expressions of GFAP,C3,Ror2,CCL2 and CXCL1 in ACC of CPTP increased compared with Naive group,Sham group and non-CPTP group on POD 21,while the expression of S100A10 decreased compared with Naive group,Sham group and non-CPTP group.Immunofluorescence showed that the expressions of C3,Ror2,CCL2 and CXCL1 increased and the expression of S100A10 decreased in ACC astrocytes with CPTP.Knocking down the expression of Ror2 in ACC astrocytes before thoracotomy can alleviate mechanical hyperalgesia and cold hyperalgesia induced by CPTP,and significantly reduce the incidence of CPTP.Compared with AAV-GFAP-NC-CPTP on POD 21,the expressions of C3,Ror2,CCL2 and CXCL1 were significantly down-regulated,while the expression of S100A10 was upregulated in ACC on POD 7,POD 14 and POD 21 of AAV-GFAP-Mir30-Ror2-CPTP group.ConclusionsThe expressions of Ror2,CCL2 and CXCL1 were up-regulated,and the reactive astrocytes were mainly expressed in the A1 phenotype in ACC of CPTP.Ror2 in ACC may mediate the development of CPTP by regulating the transformation of A1/A2 reactive astrocytes and the expressions of CCL2 and CXCL1.