| Remifentanil is a new type of ultra-short-actingμ-opioid receptor agonist commonly used in clinic.It can be rapidly hydrolyzed by non-specific esterase in tissues and blood,so it is safe and reliable,with quick onset,good controllability and efficacy.It plays an important role in clinical analgesia,and patients have a lower risk of respiratory depression and delayed resuscitation in clinical applications.,but it may induce hyperalgesia which limit its use.Studies have shown that Neuroligin-1/Neurexin-1αregulates synaptic neurotransmitter release and postsynaptic membrane receptor function,participates in synapse formation and plasticity changes,and plays an important role in learning,memory and mechanism of pain.Neuroligin-1/Neurexin-1αregulates the activation of AMPA receptor function by mediating the formation of glutamatergic excitatory synapses,thereby maintaining synaptic efficacy and information transmission.Previous studies by our research group have showed that the expression level and transport of AMPA receptor-expressing membrane protein containing GluR1 in remifentanil induced hyperalgesia（RIH）rats’spinal cord neurons increased.Therefore,it is speculated that Neuroligin-1/Nerexin-1αis likely to affect the formation of excitatory synapses by regulating membrane translocation of AMPA receptors,and thus participates in the activation of AMPA receptors in remifentanil hyperalgesia.This study was designed to investigate the mechanism of action of Neuroligin-1/Nerexin-1αin a rat model of remifentanil-induced postincisional hyperalgesia.Objective To detect the influence of Neuroligin-1 and Neurexin-1αon RIH by analyzing the expression of Neuroligin-1 and Neurexin-1αin spinal dorsal horn in rats,and evaluate its effect.MethodsI:32 healthy male rats,weighing 250g,aged 2-3 months,were randomly divided into 4 groups using a method of random number table.In Group C,saline(0.1 ml kg-1min-1,1h)was infused;In Group R,remifentanil(1μg·kg-1·min-1,1 h)was infused;In Group I,Intravenously infuse saline(1.0μg·kg-1·min-1,1 h)through the tail vein catheter and create a plantar incision pain model simultaneously;In Group R+I,Intravenously infuse remifentanil(1.0μg·kg-1·min-1,1 h)through the tail vein catheter and create a plantar incision pain model simultaneously.Hyperalgesia was measured by paw withdrawal threshold（PWT）and paw withdrawal latency（PWL）24 hours before anesthesia and 2 hours,6 hours,1 day,2 days,3 days,5 days and 7 days after anesthesia.The rats were sacrificed by means of spinal cord dislocation after the measurement of pain behavioral 2 days after anesthesia,then collect spinal dorsal horn and determined the expression level of Neuroligin-1,Neurexin-1α,mGluA1,tGluA1 and m/tGluA1.II:55 healthy male rats were randomly divided into 5 groups（n=11）:Group C:Intravenously infuse saline（1.0μg·kg-1·min-1,1 h）through the tail vein catheter.Group saline+Neuroligin-1-shRNA（C+NL1-shRNA）:transfection by intrathecal injection of Neuroligin-1-shRNA（10μL;3×108 transducing units[TU]/mL）,and 2weeks after successful transfection,saline（0.1 ml·kg-1·min-1,1 h）was infused;incisional pain+remifentanil group（IR group）:Intravenously infuse remifentanil（1.0μg·kg-1·min-1,1 h）through the tail vein catheter and create a plantar incision pain model simultaneously;incision pain+remifentanil+Negative control-shRNA group（IR+NC-shRNA group）:intrathecal injection of NC-shRNA（10μL;3×108transducing units[TU]/mL）,then establish a model of incisional pain and infusion of remifentanil（1.0μg·kg-1·min-1,1h）;incision pain+remifentanil+Neuroligin-1-shRNA group（IR+NL1-shRNA group）:Intrathecal injection of Neuroligin-1-shRNA（10μL;3×108 transducing units[TU]/mL）,then establish a model of incisional pain and infusion of remifentanil（1.0μg·kg-1·min-1,1h）.PWT PWL were measured 24 hours before anesthesia and 2 hours,6 hours,1 day,2 days,3days,5 days and 7 days after anesthesia.The rats were sacrificed by means of spinal cord dislocation after the measurement of pain behavioral 48h after anesthesia,then the rats were sacrificed and the spinal dorsal horn tissues were taken.The expression levels of Neuroligin-1,mGluA1,tGluA1 and m/tGluA1 in the spinal dorsal horn were determined by Western blot.The rats were sacrificed after the measurement of pain behavioral 48h after anesthesia,then the rats were sacrificed by means of spinal cord dislocation and the spinal dorsal horn tissues were taken.GolgiStain was performed to analyse dendritic spine.ResultsI:PWT was significantly decreased and PWL was significantly shortened,the expression level of Neuroligin-1 and Neurexin-1αwere increased in Groups R,I and R+I compared with Group C,（P<0.05）.Compared with Group I and Group R,respectively,PWT was declined and PWL was shortened significantly,and the expression of Neuroligin-1 and Neurexin-1αwere increased in Group R+I（P<0.05）.II:PWL and PWT were significantly down-regulated in the IR group,IR+NL1-shRNA group and IR+NC-shRNA group compared with the control group,（P<0.05）.Compared with the IR group,the PWL and PWT in the IR+NL1-shRNA group were significantly increased（P<0.05）;However,compared with group C,there was no significant difference in PWL and PWT between C+NL1-shRNA group（P>0.05）.There was no significant differences between PWL and PWT in IR group and IR+NC-shRNA group at every test time（P>0.05）.The expression of Neuroligin-1 in the spinal dorsal horn of rats after transfection with NL1-shRNA was significantly decreased（P<0.05）.And the expression of Neuroligin-1 in the spinal dorsal horn of rats after transfection with NC-shRNA was not significantly changed（P>0.05）.Compared with the control group,the expression of mGluA1 in the spinal dorsal horn of the IR group and the IR+NL1-shRNA group was higher than that of the control group（P<0.05）,the expression of tGluA1 was increased,and the ratio of m/t GluA1 was increased（P<0.05）;The AMPA receptor of mGluA1 in the spinal dorsal horn of rats in the IR+N1-shRNA group was decreased compared with the IR group（P<0.05）,the expression of tGluA1 was decreased,and the ratio of m/t GluA1 was down-regulated（P<0.05）.The number of dendritic spines in the spinal dorsal horn of SD rats in the IR group was significantly higher than that in the control group（P<0.05）,while the length of dendritic spines was significantly longer than the control group（P<0.05）.The number of dendritic spines in the spinal dorsal horn of rats of IR+Neuroligin-1-shRNA group was significantly lower than that in the IR group（P<0.05）,and the length of dendritic spines was significantly shortened（P<0.05）.Conclusion Postincisional hyperalgesia can be exacerbate by Remifentanil,and the increased Neuroligin-1 and Neurexin-1αexpression in rats spinal dorsal horn is involved in remifentanil-induced hyperalgesia.Inhibition of spinal cord level of Neuroligin-1 has an antagonistic effect on RIH,which may be through down-regulation of AMPA expression in the GluA1 subunit of rat spinal dorsal horn,affecting receptor synthesis and transport to achieve its anti-RIH effect.RIH may be due to increased neuroligin-1 synthesis in the spinal dorsal horn,which in turn regulates the remodeling of dendritic spine structures,ultimately leading to the formation of RIH. |
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