| Objective: Remifentanil is an ultra-short-acting ?-opioid receptor agonist.With little risk of delayed postoperative recovery or respiratory depression,remifentanil has been widely used in clinical practice.However,comparing with other opioids,remifentanil is more likely to induce hyperalgesia.Remifentanil-induced hyperalgesia?RIH?occurs very soon?< 60 min?after remifentanil administration and contributes to an increase rate of postoperative pain,which is a thought-provoking issue.The mammalian TRP channels are tetrameric,nonselective cation permeable pores,which can be divided into six TRP channel protein families: TRPC,TRPM,TRPV,TRPP,TRPML and TRPA.Transient receptor potential subtype V1?TRPV1?is voltage-gated calcium channel.TRPV1 plays an important role in nociception and neuroinflammation,as it can be activated by multiple modulated mediators,such as low p H??5.9?,heat stimulation?>42??,endorphin,endogenous lipid and capsaicin.In recent years,abundant researches show us that microglial has significances to immunoregulation,as well as neuropathic pain.Microglial can modulate neuroimmunology and synaptic transmission,making it an important cell type in pain.However,rarely research reports microglial associated with RIH.We aim to establish animal models of RIH,observe the changes of TRPV1 and microglia,and investigate the mechanisms of RIH.In addition,TRPV1 inhibitor is administrated intrathecally to prevent RIH,providing theoretic foundation of clinical anesthesia and analgesia.Methods: 1.40 SD male rats were divided into 4 groups?n=10?randomly: control group: It was infused with normal saline via caudal vein;incision group: Made a Brennan incision model and infused with normal saline via caudal vein;Remifentanil group: it was infused with remifentanil via caudal vein;Remifentanil plus incision group: Made a Brennan incision model and infused with remifentanil via caudal vein.The saline infusion rate was 0.1 m L·kg-1·min-1,and remifentanil infusion rate was 1.2 ?g·kg-1·min-1,and the infusion time was 60 min.The thermal hyperalgesia was measured respectively in five time points 1 day before surgery,2h,6h,24 h,48h after surgery,using hot plate measurement.When the time reaches 30 seconds,we stop the test to avoid the experimental animal tissue damage.And after the end of the behavioral measurement,lumbar L46 dorsal root ganglion and spinal cord was taken from the sacrificed rat.Changes of TRPV1 expression in dorsal root ganglion and spinal cord were tested by Western blot;Elisa and Western blot technique was used to determine the expression of TNF-?in rat spinal cord;The expression of TRPV1 in dorsal root ganglion and the changes of the number and quantity of microglia in spinal cord were determined by immunofluorescence assay.2.60 SD male rats were divided into 6 groups?n=10?randomly: control group: It was infused with normal saline via caudal vein,The saline infusion rate was 0.1 m L·kg-1·min-1,and the infusion time was 60 min,intrathecally injection DMSO 10?l;control plus CPZ group: It was infused with normal saline via caudal vein,The saline infusion rate was 0.1 m L·kg-1·min-1,and the infusion time was 60 min,intrathecally injection TRPV1 antagonist 16?g/10 ?l;Remifentanil plus incision group: It was infused with remifentanil via caudal vein,the remifentanil infusion rate was 1.2 ?g·kg-1·min-1,and the infusion time was 60min;R+I+CPZ3 group: It was infused with remifentanil via caudal vein,the remifentanil infusion rate was 1.2 ?g·kg-1·min-1,and the infusion time was 60 min,intrathecally injection TRPV1 antagonist 16?g/10 ?l;CPZ1 group: It was infused with remifentanil via caudal vein,the remifentanil infusion rate was 1.2 ?g·kg-1·min-1,and the infusion time was 60 min,intrathecally injection TRPV1 antagonist 2?g/10 ?l;CPZ2 group: It was infused with remifentanil via caudal vein,the remifentanil infusion rate was 1.2 ?g·kg-1·min-1,and the infusion time was 60 min,intrathecally injection TRPV1 antagonist 8?g/10 ?l.Using hot plate measured thermal hyperalgesia.Results: 1.The results of PWL showed that control group,incision group,remifentanil group,remifentanil plus incision group are no statistical difference in the 24 h before surgery P>0.05.After infusion remifentanil 2-48 h,the control group are no statistical difference in the different time points P>0.05;compared with the control group,the incision and remifentanil group is lower with statistical difference P<0.01;compared with the other three group,the remifentanil plus incision group is the lowest P<0.01.2.Immunofluorescence results showed: The expression of TRPV1 in rat dorsal root ganglia was increased in R group and I group compared with C group;Compared with R group and I group,the RI group was significantly increased,the difference was statistically significant P<0.01;TRPV1 is mainly expressed in small diameter neurons,which plays an important role in remifentanil hyperalgesia.Compared with the normal control group,the expression of microglia in spinal cord tissue of remifentanil group was significantly higher than that of the control group,the difference was statistically significant P<0.01.There was no significant difference in the expression of microglia in the spinal cord of remifentanil group compared with incision pain group;Compared with remifentanil group,the expression of microglia in RI group was significantly increased and the difference was statistically significant P<0.01.It is suggested that the release of inflammatory mediators after the activation of microglia in the spinal cord tissue of remifentanil induced hyperalgesia in rats.At the same time,the release of inflammatory factors can promote the activation of microglia,the production of long-time pretension and central sensitization.3.Western Blot results showed that: compared with C group,R group,I group,the expression of TRPV1 protein in RI group was increased in dorsal root ganglion and spinal dorsal horn,with statistical significance P<0.01;Compared with I group or R group,the expression of TRPV1 in RI group was increased P<0.01;but there was no significant difference between R group and I group P>0.05.4.Western blot and ELISA indicated that the remifentanil group compared with normal control group,significantly increased the expression of TNF-?protein in rat spinal cord tissue,the difference was statistically significant P<0.01;There was no significant difference in the expression of TNF-? protein in the spinal cord of rats in I group and R group,the difference was not statistically significant P>0.05;Compared with other groups,the expression of TNF-? was significantly increased in the spinal cord of remifentanil + incisional pain group,and the difference was statistically significant P<0.01.5.The behavior test was measured after intrathecal administration of saline or TRPV1 antagonist.The results showed that the C group of PWL was not statistically significant P>0.05;The difference of C+CPZ group was not statistically significant suggests that intrathecal injection of TRPV1 antagonist did not affect the normal pain threshold in rats.The PWL threshold of R+I group and RI+CPZ group was significantly lower than that of C group P<0.01;Compared with R+I group,the PWL level of RI+CPZ group increased,the difference was statistically significant P<0.01.Different doses of TRPV1 specific antagonists were given.Compared with RI group,CPZ1,CPZ 2 and CPZ 3 groups can improve the thermal pain threshold,the difference has a unified meaning P<0.01.These results suggest that TRPV1 antagonists can effectively improve hyperalgesia and dose dependently.Conclusion: 1.When Remifentanil infused 1.2 ?g·kg-1·min-1,60 min can induce hyperalgesia.The mechanism is associated with the upregulation of TRPV1 both in the dorsal root ganglia and spinal cord.2.Up regulation of TRPV1 can activate microglia and promote the release of TNF-,increase pain and induce hyperalgesia.3.TRPV1 antagonist can partly improve remifentanil induced hyperalgesia,and dose dependently.There is hope to become a potential therapy to prevent the hyperalgesia induced by remifentanil. |
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