| ObjectiveThe MRMT-1 breast cancer cells were injection into thetibial cavity and combine with intraperitoneal injecting morphine to successfully establish the model of bone cancer pain combined with morphine tolerance rat.Tested the change of the pain withdrawal thresholds(PWT)at different time points to clear the time point of tibial cancer pain and bone cancer pain-morphine tolerance.To investigate the effect of electroacupuncture(EA)on pain behavior and the expression of ?-opioid receptor(MOR)and Rab5 positive cells and the coexpression(MOR endocytosis)in locus ceruleus of bone cancer pain combined with morphine tolerance rat.lt is attempted to investigate wheather EA treatment through promoting MOR endocytosis to against the morphine tolerance.MethodsPart 1The24 SD rats were randomly divided into 3 groups:sham operation group(sham BCP),bone cancer pain group(BCP),bone cancer pain combined with morphine tolerance group(BCP-MT).The left tibial bone marrow cavity of rats in the BCP group and BCP-MT group were injected with 3?l MRMT-1 breast cancer cells(3×104)to induce bone cancer pain.The sham BCP group rats were injected with 3?l PBS solution.At the next day of bone cancer pain induced,then BCP-MT group rats were intraperitoneal injecting morphine(q12h)hydrochloride continuous with 11days.Before cancer cells inoculation,on 6 D and 8 D and 10 D after inoculation,onl D and 5 D and 9 D and 11D after morphine injection for 0.5h,we can observe the change of PWT at eight different time points in each group rat.At 21D after inoculation,the pathological changes of tibia of rats in each group were observed by HE stainingand X-ray films of the BCP group rat.Part 2The 50 SD rats were randomly divided into 5 groups:sham BCP,BCP,BCP-MT,BCP-MT+EA,BCP-MT+sham EA.The sham BCP group received sham operation,the BCP group received only tibial cancer pain model,the other three group rats were treated with the model of tibial bone cancer pain combined with morphine tolerance rat.From the next day of bone cancer model established to the end,BCP-MT group rats were given morphine injection(ql2h).Giving respectively electroacupuncture and sham acupuncture treatment,the BCP-MT+EA group and BCP-MT+shamEA group rats at the morphine injection for 0.5 hour later.Then Using 2/100 Hz EA connected with the side point,selected bilateral "Zusanli" and "Kunlun",the intensity of 0.5-1.5mA(0.5mA step,the stimulation of 10min every),treated for 30 mines every day,which had been lasted for 7 days.The sham acupuncture treatment was only given acupunctureto break the skin,and no electricity.Before cancer cells inoculation,on 10D and 21D after inoculation,onl D and 3 D and 5 D and 7D after 0.5 hour of EA treatment,we can observe the changes of the PWT at different time points andthe effect of EA on pain behavior.The locus ceruleus localization and the expression of MOR and Rab5 positive cells and the coexpression(MOR endocytosis)in locus coeruleus in each group rats were observed by brain tissue staining and immunofluorescence staining respectively.Results part 1Before inoculation,there were no statistical differences in PWT among these three group rats(P>0.05).On 10D after cancer cells inoculation,the PWT of rats in BCP group and BCP-MT group were significantly lower than that of the sham BCP group(P<0.01),which lasted for the 21D after inoculation.The day after morphine injection,the PWT of rats in sham BCP group and BCP-MT group were significantly higher than those of the BCP group(P<0.01).On 11D of morphine injection,the PWT of BCP-MT group rats was decreased as the same level as the BCP group(P>0.05),but lower than that of the sham BCP group(P<0.01).X-ray and HE staining showed,the cortical bone of the tibia of rats in the BCP group was discontinuous,and the shadow was enlarged,on the 1/3 tibia of rats in the BCP group and BCP-MT group were visible tumor induced by cancer cells,and the tibial bone marrow cavity with MRMT-1 cancer cells,while the rats in the sham BCP group showed no abnormal change of tibia.part 2 Before inoculation,on 10D and 21D after inoculation,there were no statistical differences in PWT among BCP-MTgroup,BCP-MT+EA group and BCP-MT+shamEAgroup(P>0.05).On1 D and3 D and5 D and 7D of EA treatment,the PWT of rats in BCP-MT+EA group was significantly higher than that of BCP-MT group and BCP-MT+sham EA group(P<0.01),and also was significantly higher than that the same group at the lever of 11D after morphine injection(P<0.01).There was no differences in PWT between BCP-MT group and BCP-MT+sham EA group at each time point(P>0.05).Compared with sham BCP group,the expression of MOR and Rab5 positive cells and the positive ratio of MOR+/Rab5+in locus coeruleus of BCP group,BCP-MT group,BCP-MT+EA group,BCP-MT+shamEA group were decreased(P<0.05orP<0.01).the expression of MOR positive cells between BCP group and Model group(P>0.05).Compared with BCP group,no significant changes in the expression of MORpositive cells(P>0.05),the expression of Rab5 positive cells and the positive ratio of MOR+/Rab5+ in BCP-MT group were decreased(P<0.05).The expression of MOR and Rab5 positive cells and the positive ratio of MOR+/Rab5+ in locus coeruleus of BCP-MT+EA group was each higher than that of BCP-MT group and BCP-MT+shamEAgroup(P<0.01),but there were no statistical differences between the BCP-MTgroup and BCP-MT+shamEAgroup(P>0.05).ConclusionIntraperitoneal injection of morphine for 11 consecutive days can successfully induce the model of morphine tolerance in bone cancer pain rat by MRMT-1 breast cancer cells.The down regulation of MOR expression may be mainly related to the formation of hyperalgesia and Morphine tolerance.EA could improve mechanical pain threshold of bone cancer pain combined with morphine tolerance rat.EA can increase the expression of MOR and Rab5 positive cells,increase the coexpression of MOR and Rab5 in rat locus coeruleus,and then promote the rate of MOR endocytosis,which may be the main factor for the reversal of morphine tolerance. |
PDF Full Text Request