The Changes And Effects Of Macrophage Inflammatory Protein-1a (CCL3) And Its Receptor And Glial Cells On Remifentanil-induced Hyperalgesia In Rats

Remifentanil belongs ?-opioid receptor agonist,it has following good characteristics like rapid-onset,short duration,rapid elimination,no cumulative effects,especially the metabolism of remifentanil does not depend on liver and kidney function,so,it is widely used in clinically.However,the phenonmen of remifentanil-induced hyperalgesia(RIH)has been numerous experimental and clinical researches demonstrated,which has allured people's more attention.RIH will limit the usage of remifentanil,so more and more people have interest in exploring what reason could induce RIH and which kind of measures could effective prevent RIH.Recent studies have identified that the glial cells in central nervous system(CNS)play an important role in regulating pain process,it will release a variety of reactive molecules,such as neurotransmitters,nerve growth factor and inflammatory mediators,cytokines and chemokines.In this research,we aimed to explore the changes of chemokine C-C motif ligand 3(CCL3),also called macrophage inflammatory protein-1?(MIP-1?)and its receptor CCR5,belongs to seven-transmembrane domain G protein-coupled receptors(GPCRs)in spinal cord in rats,it will provide a new idea to explain the mechanism of RIH and find a new way to prevent it.Objective To investigate the involvments of CCL3 and CCR5 and changes of glial cells in spinal cord in incisional pain-remifentanil-induced hyperalgesia;To explore the role of CCL3 and CCR5 signaling pathways in RIH after using CCL3 neutralizing antibody and Maraviroc,the CCR5 receptor antagonist by measuring the behavior test.Method 32 male SD rats were distributed into 4 groups(n = 8)randomly,including: group C(control group): Normal saline was infused through caudal vein,normal saline infusion dose was 0.1ml/kg/min;group I(incision pain group): Made a incision pain model according to the method of Brennan with normal saline infusion,normal saline infusion dose was 0.1 ml/kg/min;group R(remifentanil group):Remifentanil was infused through caudal vein,the dose of remifentanil was1.0?g/kg/min;group R+I(remifentanil+incision pain group): Made a incision pain model with remifentanil infusion,the dose of remifentanil was 1.0?g/kg/min.The time of duration was 60 min in four groups.At the time of T0(24h before infusion)and T1(2h after infusion),T2(6h after infusion),T3(24h after infusion),T4(48h after infusion),we measured the PWT(paw withdraw threshold)and PWL(paw thermal latency).After we mesured the last behavioral test,we killed the rats and then took the L4-6 segment for evaluation of the m RNA expression level of CCL3 and CCR5 through using RT-q PCR and total protein expression level of them by using Western blot.And we further tested whether the glial cells in spinal cord been activiated by using immunofluorescence and the location of CCL3 and CCR5 be expressed by using double-staining.80 male SD rats were distributed into 10 groups(n=8)randomly,including,group C(a sham operation group): Normal saline infusion dose was 0.1ml/kg/min through caudal vein,and intrathecally 10?l PBS;group C+Anti-CCL3(a sham operation group): Made a incision pain model according to the method of Brennan with normal saline infusion,the dose of it was 0.1 ml/kg/min,and intrathecally CCL3 neutralizing antibody 20ng/10 ?l;group R: Remifentanil infusion dose was1.0?g/kg/min,and intrathecally CCL3 neutralizing antibody 20ng/10 ?l;group C+M1,M2,M3(a sham operation group): Normal saline infusion dose was 0.1ml/kg/min,and intrathecally Maraviroc 1 pmol/10 ?l,1 pmol/10 ?l,1 pmol/10 ?l;group R+M1,M2,M3: Remifentanil infusion dose was 1.0?g/kg/min,and intrathecally Maraviroc 1pmol/10 ?l,1 pmol/10 ?l,1 pmol/10 ?l.CCL3 neutralizing antibody and Maraviroc were given intrathecally 30 min before after NS or remifentanil infusion.The PWT and PWL were measured at T0?T1?T2?T3 and T4.The time of duration was 60 min in all groups.Result Different from group C,in group I,R and R+I,the PWT and PWL were significantly decreased(P<0.05);CCL3 and CCR5 m RNA and total protein expression were increased,especially in group R+I.And microglia and astrocytes were been activiated in remifentanil group,which reflected that the cells became much larger and denser,and most of them were lacated in spinal dorsal horn.Double-staining showed that microglia and astrocytes could express CCL3 and neuron and microglia could express CCR5.Remifentanil may cause postoperative hypernociception,and CCL3 neutralizingantibody and Maraviroc could improve remifentanil-induced hyperalgesia.Maraviroc not 1pmol but 10 and 100 pmol dosage-dependently attenuated hyperalgesia.Maraviroc dose-dependently plays a preventive role in RIH,furthermore,the dose of 100 pmol exerts a best result.Conclusion The expression of CCL3 and CCR5 were increased in rats spinal cord might be involved in incisional pain–remifentanil induced hyperalgesia.Remifentanil could activiated glial cells in spinal cord,and hyperalgesia was attenuated after CCL3 neutralizing antibody and Maraviroc in a dose-dependent way,which reflected that CCL3 and CCR5 signaling participated in regulating the pain process.