| Part I The role of BDNF/trkB-KCC2 signaling in the spinal dorsal horn and remifentanil-induced postoperative hyperalgesia in ratsOpioids are widely used drugs in the clinical treatment of pain.During the operation,opioid receptor agonists(fentanyl,remifentanil,sufentanil,etc.)are the most commonly used analgesic drugs in treating pain.However,in the clinical administration of opioids receptor agonists,hyperalgesia was found in the patients without these drugs medication anymore.The patients who were exposured to opioids during anesthesia always confuse normal stimuli with pain,and can not distinguish light pain from severe pain,which is called a downregulated threshold or opioid-induced hyperalgesia(OIH).Numerous studies have demonstrated that,remifentanil is an opioid receptor agonist characterized by significant postoperative hyperalgesia.Despite of so many advantages of remifentanil as rapid metabolism and elimination,strong and stable analgesic effects,and the effect of lowering blood pressure and heart rate,remifentanil-induced hyperalgesia(RIH)enhanced patients'discomfort,limited postoperative activities and increased the days in hospital.At present,the underlying mechanisms and signling pathways involved in the process of RIH are still enigmatic.In addition,most studies about RIH were performed only for a short time after surgery(e.g.,24h to 48h),and few studies concerned the duration of hyperalgesia.The cotransporter K+-Cl-cotransporter 2(KCC2)plays a critical role in maintaining the stability and dynamic equilibrium of the Cl-concentration inside neurons.The decrease of the expression or activity of KCC2 could result in the accumulation of the Cl-inside cells,and GABAergic disinhibition is caused,leading to the initiation and maintenance of pain.Brain-derived neurotrophic factor(BDNF)could bind with tyrosine receptor kinase B(TrkB),downregulate the expression of KCC2,inhibit the transportation of Cl-and increase the accumulation of intracellar Cl-,which reduces GABAergic inhibition and results in hyperalgesia.But whether BDNF/trkB-KCC2 signaling in the spinal dorsal horn plays a role in the development of RIH in rats is still unreported.In present experiment,we infused remifentanil subcutaneously for 30min(80?g/kg),and made the incision on right feet of rats according to Brennan' method to prepare the rat model of RIH.We investigated the effect of BDNF/trkB-KCC2 signaling in the spinal dorsal horn on the development of RIH by intrathecally injecting TrkB/Fc(the inhibitor of TrkB)and exploring the changes of the expression of BDNF,TrkB and KCC2.Paw withdrawal mechanical threshold(PWMT)and paw withdrawal thermal latency(PWTL)were used to assess pain behavior.Behavior evaluations were recorded at baseline(24 h before incision)and 4 h,in addition to postoperative days 1,2,4,7,10,12,14,21 respectively.The expression of BDNF,trkB and KCC2 in the spinal dorsal horn were explored on postoperative day 1.TrkB/Fc was intrathecally injected at 15min before the operation and the changes of behavior were measured at baseline(24 h before incision)and 4 h,in addition to postoperative days 1,2,4,7,10,12,14,21.The expression of KCC2 in the spinal dorsal horn was explored on postoperative day 1.The results demonstrated:1.RIH could last about two weeks after surgery;2.The protein expression of BDNF and trkB in the spinal dorsal horn after RIH were upregulated,while the KCC2 expression was downregulated;3.The intrathecal administration of TrkB/Fc significantly alleviated RIH and shorten the duration of hyperalgesia,and at the same time,the expression of KCC2 in the spinal dorsal horn was upregulated.In conclusion,this experiment adopts the rat model of RIH to imitate the clinical hyperalgesia by subcutaneous infusion of remifentanil during the process of making incision pain and studied the relationship between BDNF/trkB-KCC2 signaling and RIH.We observed a significant downregulation in KCC2 and a simultaneous upregulation in BDNF and trkB in the spinal dorsal horn following RIH.As the inhibitor of TrkB,TrkB/Fc significantly alleviated RIH and shortened the duration of hyperalgesia by upregulating the expression of KCC2 in the spinal dorsal horn.This study provided new evidence which will help to treat RIH.Part II The effects of a7-nAchRs activation on the duration of RIH and BDNF/trkB-KCC2 signaling in the spinal dorsal horn of rats with RIHBoth central and periphery immune cells play important roles in the occurrence and maintenance of hyperalgesia,and they participated in the development of central sensitization.As the immune cells in CNS,microglia activation may release proinflammatory cytokines and regulate downstream signal transduction pathways,which was recognized as the important factors of the occurrence and maintenance of hyperalgesia.Therefore,inhibition of the downstream signaling pathways of microglia could be invoked as a direction for the treatment of pain.The a7nAChRs were richly expressed in CNS,especially on microglia.And the a7nAChRs activation could significantly reduce proinflammatory cytokines released.It has been demonstrated that the activation of spinal a7nAChRs could produce significant antinociception effect in various pain models.Previous studies of our team showed that microglia in the spinal dorsal horn of rats with RIH actived significantly,and the activation of spinal a7nAChRs could alleviate RIH by inhibiting microglia and proinflammatory cytokines derived from microglia.Studies demonstrated that inhabitation of microglia activation could downregulate the expression of BDNF in the spinal dorsal horn and inhibit its binding with tyrosine receptor kinase B(TrkB).In this process,the expression of KCC2 increased,the Cl-was transported out of the cell and the effect of GABAAR was enhanced.As a result,inflammatory reaction and central sensitivity were alleviated.The results in the first part of the present study showed that,BDNF/trkB-KCC2 signaling in the spinal dorsal horn play a role in the development of RIH,but whether spinal a7nAChRs activation could alleviated RIH by inhibit BDNF/trkB-KCC2 signaling remain unclear.RIH is a continuous process and may last for several days or longer time,which is bad for the recovery of patients,especially those who need functional activities after surgery.However,the effect of spinal a7nAChRs activation on the duration of RIH is still unreported.Therefore,this part of the experiment study on the effects of a7nAChRs type II positive allosteric modulators(PAMs)PNU-120596 on the duration of RIH and BDNF/trkB-KCC2 signaling in the spinal dorsal horn.1.PNU-120596 was intrathecally injected at 15min before the operation and the changes of behavior were measured at baseline(24 h before incision)and 4 h,in addition to postoperative days 1,2,4,7,10,12,14,21.The effect of a7nAChRs activation on duration of RIH was investigated.2.We investigated the effect of a7nAChRs activation on BDNF/trkB-KCC2 signaling in the spinal.dorsal horn by intrathecally injecting PNU-120596 at 15min before the operation and exploring the expression of BDNF,TrkB and KCC2 in the spinal dorsal horn at baseline(24 h before incision)and 4 h,in addition to postoperative days 1,2,4,7 and 14.3.The inhibitor of KCC2 VU0240551 was intrathecally injected at 15min before the operation and the changes of behavior were measured at baseline(24 h before incision)and 4 h,in addition to postoperative days 1,2,4,7,10,12,14,21.And the role of KCC2 in the anti-hyperalgesia effect of a7nAChRs activation was evaluated.The results demonstrated:1.The activation of spinal a7nAChRs could significantly alleviated RIH and shorten the duration of RIH.2.The intrathecal injection of PNU-120596 could lead to a persistent downregulation of BDNF/trkB and a sustained upregulation of KCC2 in the spinal dorsal horn of RIH rats at all time points of evaluation after surgery.And the time courses of all the protein expression changes were significantly shortened,which concurred with the complete recovery of nociceptive thresholds.3.VU0240551 could significantly inhibit the anti-hyperalgesia effect of PNU-120596.In summary,this study illustrated the effects of activation of spinal a7nAChRs on the duration of remifentanil-induced hyperalgesia and BDNF/trkB-KCC2 signaling in the spinal dorsal horn.Our results showed that spinal a7nAChRs activation significantly alleviated RIH and shortened the duration of RIH by inhibiting BDNF/trkB-KCC2 signaling in the spinal dorsal horn.This result provides a theoretical and experimental basis of looking for new rational treatment of RIH. |
PDF Full Text Request