Wnt5a Is Involved In The Study Of The Mechanism Of Chronic Pain After Thoracotomy

Background and ObjectiveRegarding to post-surgical pain,the incidence of chronic post-thoracotomy pain(CPTP)is second only to that caused by amputation and the underlying mechanism remains elusive.The emerging role of Wnts has been confirmed in the pathogenesis of neuropathic and inflammatory pain,both of which are known components of CPTP.Wnts are involved in inflammation regulation as showed in inflammatory diseases and immune cells in vitro,while neuroinflammation plays a key role in peripheral and central sensitization.So,we initially investigated whether Wnts were involved in the development of CPTP in a previously established rat CPTP model.Then,we focused on peripheral and central inflammation existed in CPTP,and their regulation by Wnts.MethodsThe rat CPTP model was built according to Buvanendran.The rats were divided into three groups,naive,sham and model group.The mechanical withdrawal threshold(MWT),cold allodynia threshold,and the qulitative hyperalgesia profile(QHP)were used to assess pain behavior.Both spinal cord dorsal horn(SDH)and dorsal root ganglion(DRG)RNA on postoperative day(POD)21 in the rat CPTP model were extracted for RT-PCR for the changed Wnts-related signals including Wnt ligands,membrane receptors,intracellular kinases and inflammatory ones.Immunoflurosence was used for their location in SDH and DRG on POD 21.Western blot was further used to confirm the results showed by RT-PCR.Furthermore,the selected pro-inflammatory factors in serum were continually monitored by ELISA.Then early intrathecal inhibition(from POD 7 to 9)of the increased Wnts was used to see the analgesia effects,also the regulation on peripheral and central nervous system inflammatory mediators,and on systemic inflammation.ResultsRats in the model group on POD 21 would be divided into two pain states according to the MWT,that is CPTP group(MWT?4 g)and non-CPTP(MWT>4 g).The QHP in the two groups on the same day was significant different from POD 5 to 21.Compared with the sham and non-CPTP group,only Wnt5b mRNA in the CPTP group were upregulated in the SDH,while the mRNA levels of Wnt5a,Wnt5b,Wnt11,Ror2 and PKC?2 were increased in the DRG.Immunoflurosence showed that both Wnt3a and Wnt5a were mainly located in neurons,while the increased Wnt3a and Wnt5a in the CPTP group were mainly expressed in axons in the SDH,and in cytoplasm in the DRG.Western blot showed that proteins of Wnt3a,Wnt5a,?-catenin,GFAP and TLR4 in the SDH and DRG were all upregulated in the CPTP group.Besides,proteins of IL-1? and IL-6 in the SDH,and IL-1?,NF-?B in the DRG were all increased in the CPTP group.Proteins of IL-1? and IL-6 in serum were increased from POD 7 to 21.Furthermore,intrathecal inhibition of Wnt5a by Box5 but not Wnts by IWP-2 increased the MWT from POD 9 to 21,ameliorated the QHP on POD 21,down-regulated expression of TLR4,and inactivated astrocytes in the SDH and DRG.What's more,the increased IL-1? was also decreased in both DRG and serum by Box5 but not IWP-2.ConclusionsThe above results suggest suppression of Wnt5a,but not Wnts alleviates the development of CPTP,possibly by regulating the activated central and peripheral nervous system inflammatory response.In addition,the increased TLR4 in the SDH and DRG could also be regulated by Wnt5a.Background and ObjectiveAs well-characterized ligands involved in inflammation and tissue regeneration,Wnts are emerging as promising targets in pain pathogenesis.The first part of our study showed that intrathecal inhibition of Wnt5a,but not Wnts,relieves chronic post-thoracotomy pain(CPTP)via anti-inflammation effects in rats.Concerning Wnt5a takes effects mainly through activating the non-canonical Wnt pathways,we aimed to further explore their activation and regulation by Wnt5a in the thoracic dorsal root ganglion(DRG)in CPTP development.In addition,both nerve regeneration and sympathetic sprouting play key roles in pain persistence,and their modulation by Wnt5a has been verified in vitro,thus we tested their expressions and regulation by Wnt5a in CPTP.Furthermore,the time-dependent changes of the above proteins were detected,and also their regulation by Wnt5a in different periods.MethodsThe distribution of Wnt5a in the small size neurons of the DRG was located by immunoflurosence.Proteins of Wnt5a ligand,Ror2 receptor,intracellular signals like pCaMKII,CaMKII,pERK/ERK and RhoA,GAP43(nerve regeneration marker),TH(sympathetic sprouting marker)and GFAP(astrocytes marker)collected on postoperative day(POD)7,14 and 21 were detected by Western blot.Intrathecal inhibition of Wnt5a by its specific antagonist Box5 was injected preoperatively(POD-1,0,1),acute postoperatively(POD 7,8,9),and chronic postoperatively(POD 14,15,16)respectively to see its effects on pain threshold.Furthermore,changes of the above protein levels were tested following the three interventions on POD 21.ResultsIncreased protein levels of Wnt5a,Ror2 and GFAP were found in the thoracic DRG from POD 7 to 21.The activated non-canonical Wnt pathways members like pCaMKII,CaMKII,pERK/ERK,RhoA,GAP43 and TH were detected from POD 14 to 21.Furthermore,preoperative and acute postoperative blockade of Wnt5a significantly increased the pain threshold;while chronic postoperative intervention alleviated pain,the analgesic response was not as effective as that conducted preoperatively and acute postoperatively.Additionally,preoperative and acute postoperative Wnt5a blockade obviously reversed CPTP-induced activation of Wnt5a,Ror2,non-canonical Wnt pathways,nerve regeneration,sympathetic sprouting and astrocytes.While chronic postoperative Wnt5a blockade only down-regulated the increased nerve regeneration.ConclusionsThese results suggest that the analgesic effects of early blockade of Wnt5a was better than intervention in chronic postoperative period.The increased Wnt5a in acute postoperative period in the DRG promotes the development of CPTP by activating Ror2 receptor,non-canonical Wnt pathways,nerve regeneration,and sympathetic sprouting.While in the persistence stage of CPTP,Wnt5a upregulation only stimulate nerve regeneration.Therapeutic intervention by targeting Wnt5a may present an effective strategy for preventing and treating early CPTP.