| Background:Opioids are the most prescribed and effective analgesics.Therapeutic effects of morphine and other opioids are limited by side effects: nausea,vomiting,abdominal pain,and constipation,collectively known as opioid-induced intestinal dysfunction.Current studies have shown that the interstitial cells of Cajal(ICCs)in the gastrointestinal tract are pacemaker cells of the gastrointestinal tract.Their differentiation and maturation have important effects on gastrointestinal function;and intestinal dysfunction caused by opioids.Whether it is related to gastrointestinal ICCs and its specific mechanism of action remains unclear.Previous studies have shown that the number of proximal colonic ICCs in morphine or combined with low-dose naloxone in rabbits is reduced and the morphology is affected.Destruction,c-kit mRNA and protein expression were significantly reduced,while low-dose naloxone could reverse the above performance;its research on electrophysiological aspects of ICCs has not been studied by other scholars.The study(basic study)on the electrophysiological effects of morphine or combined with low-dose naloxone on rabbit intestinal ICCs and(clinical studies)morphine or combined with low-dose naloxone for postoperative analgesia in patients with lower extremity fractures observed on the stomach A combination of effects of intestinal function was explored to explore possible mechanisms of morphine epidural infusion for gastrointestinal function.Objective:The aim of this study was to investigate the following questions: 1,culture and identification of rabbit intestinal ICCs cells in vitro;2,Effects of morphine or combinedlow dose naloxone on electrophysiology of rabbit intestinal ICCs cells and its possible mechanism;3,in order to exclude the influence of abdominal surgery such as gastrointestinal tract on the observation index,elective lower extremity orthopedic surgery using epidural infusion of morphine or combined with low-dose naloxone analgesia,using patient subjective questionnaire and MRI objective detection A combination of methods to observe its effect on gastrointestinal function and explore its impact on gastrointestinal function;4,the above related exploration and elaboration for perioperative postoperative analgesia and clinical pain treatment to prevent opioid side effects Better strategies and further research on the mechanism of opioids affecting gastrointestinal function and its ICC cells provide important reference information.Methods:Part ?:rabbits were randomly divided into three groups: morphine group(M group),morphine combined with low dose naloxone group(MN group)and saline group(NS group),with 6 rabbits in each group.One day after successful epidural puncture,CBI epidural analgesia pump was connected and infused continuously for 5days.Carry out the following studies:1,in vitro culture: one day after the drug was stopped,air 20 ml was injected into the ear margin vein and put to death.Immediately,the colon tissue from the cecum to the rectum was taken from the lower cecum.The colon tissue was washed and rinsed and fixed to the bottom of the petri dish.Then placed in a petri dish containing calcium-free Hank's solution,the cells were separated with type ? collagenase by the conventional method of stripping the mucosa layer under the anatomical microscope and cutting into small fragments.The isolated cells were inoculated into the SMGM medium and cultured in the incubator.After one day of culture,the cells were replaced with the SMGM medium containing SCF factor;2,Immunologic identification of ICC: after 72 hours of cell culture,immunofluorescence was used to identify the cells and inverted culture was carried out in a petri dish.Therabbit SA1022-antibody(Wuhan Baude Bioengineering Co.,Ltd.)was incubated overnight at 37 ? and washed 2min × 3 times by PBS.Anti-rabbit anti-IgG antibody(),PBS flushing by Wuhan Baude Bioengineering Co.,Ltd.,DAB staining,slight restaining of hematoxylin,transparent xylene carbonate,neutral gum seal film),dripping plus 1 / 200 diluted biotinylated goat anti-rabbit antibody(Wuhan Baude Bioengineering Co.,Ltd.),PBS).The cells with brown staining were positive cells;3,Laser confocal fluorescence labeling and microscope observation of ICC: the culture medium solution was diluted to 4 ? mol / L working solution(avoiding light)on the day of experiment.Take out the culture dish for 72 hours,remove the culture medium,rinse with the above-mentioned configuration liquid,rinse twice,place the fluo-4AM with 5 ? M in the 5%C02 incubator at 37 ?,incubate for 5 minutes,then rinse twice after taking out the culture medium,and put it at room temperature for 10 minutes.The cell temperature was the same as that of room temperature,and the cell stability was restored.Place the petri dish on the laser confocal microscope platform,select a well-stained field of view of the cells under the microscope,each petri dish At random,20 cells were scanned and recorded,and the length of each cell was measured by META analysis software.The 200ms/ images of 10 ICC-like cells were scanned at random.The intracellular relative fluorescence intensity of ICC-like cells represented the concentration of calcium ion,and the intracellular calcium fluorescence intensity was obtained at each time point;4,ICC cells cultured for 72 hours were flushed with normal saline without calcium for 3 times at room temperature,and the glass slides were poured down to the bottom of the irrigation tank on the microscope platform for1~3ml/min.The perfusion temperature was set between 30 ? and 32 ?.The membrane potential was clamped at-60 mV with a glass electrode with a resistance of2? 5m ? and a whole cell clamp mode.The current was recorded and analyzed by EPC-10 patch clamp amplifier.Part ?: Application of random double-blind study design,and 30 patients whounderwent elective lower extremity fracture internal fixation were enrolled.All patients underwent spinal anesthesia.Randomized results were concealed in opaque envelopes.Postoperative analgesia was randomized into 3 groups: ropivacaine(group L),ropivacaine + morphine(LM group),ropivacaine + morphine + Naloxone group(LMN group),continuous postoperative analgesia for 5 days.Unlimited gender,age20 to 60 years old,ASAI-II,expected hospital stay for at least 7 days.The intraoperative blood pressure,heart rate,duration of surgery,blood loss,and intraoperative fluid volume were recorded.VAS scores were measured on day 1,3,and 5 postoperatively at rest(VAS rest)and activity(VAS dynamics).Patients were monitored for side effects such as nausea,vomiting,itching,sedation,exercise or sensory block,hypotension,bradycardia,and respiratory depression.The patient's daily independent gastrointestinal function questionnaire(BFI,GSRS,PAC-SYM,BSFS)was collected and analyzed accordingly;intestinal MRI was performed 1 day and 5 days postoperative analgesia and the results were analyzed.Results:Part ?: 1,It is feasible to culture ICCs cells in vitro;the C-kit protein of ICC cells cultured in vitro in the saline and morphine combined with low-dose naloxone group is positive,and the cells and cells are stained obviously.The cells expressed are specific to ICC cells.Morphological characteristics: The cell body is fusiform or triangular,with large nucleus and few cytoplasm,which are connected to adjacent cell processes and cell bodies in a network.However,C-kit protein expression in ICC cells in morphine group is significantly less,and the morphology is affected.A large degree of damage,a significant reduction in the number;2,The intracellular calcium fluorescence value of ICC cells in the naloxone group of normal saline group and morphine group was not significantly changed,while the intracellular calcium ion fluorescence value of ICC in morphine group was significantly lower than that ofnormal saline group and morphine combined with low dose naloxone group.(P<0.05);there was no significant change in the length of ICC-like cells in the naloxone group in the saline group and the morphine group,but the length of ICC-like cells in the morphine group was significantly shorter than that in the saline group and the morphine combined low-dose naloxone group(P<0.05);3,The pacing current amplitudes of single ICC in the saline group and morphine combined low-dose naloxone group were:(19.85±3.75)pA,(19.53±3.81)pA,and the amplitude of single ICC pacing current in the morphine group.(6.56±4.22)pA;compared with the saline group and morphine combined with low-dose naloxone group,the clamp current of the morphine group was significantly lower(P<0.05);while the saline group was compared with the morphine combined with low-dose naloxone.There was essentially no change in the two groups(P>0.05).The pacing frequency distribution of the low-dose naloxone ICC in saline group and morphine was(22.5±2.15)times/min,(21.5±2.92)times/min,while the pacing frequency of ICC in morphine group was(10.25±2.35).Times/min,compared with the saline group and morphine combined with low-dose naloxone group,the ICC pacing frequency of the morphine group was significantly lower(P<0.05);while the saline group was compared with the morphine combined with low-dose naloxone,two There was essentially no change in the group(P>0.05).Part ?:1,There were no significant differences in age,gender,BMI,and ASA grades between the three groups.There was no significant difference in anesthesia time,operative time,fluid volume,blood loss,and urine volume between the three groups.Postoperative analgesia with ropivacaine The amount of L group was higher than that of LM group and LMN group,the difference was statistically significant(p<0.05).There was no significant difference in the total amount of morphine administration between the three groups(P>0.05).;2,During the whole postoperative period,all patients had sufficient analgesic effect.There was no significant difference between the three groupsat the resting VAS pain score from day 1 to day 5(P>0.05).The L group and the LM group were in the group.There was no difference in VAS score from day 1 to day 5(P>0.05).The VAS score of LMN group was significantly lower than that of L group and LM group(p<0.05);3,No side effects such as sedation,exercise or sensory block,hypotension,bradycardia and respiratory depression were observed in the three groups.L group and LMN group were not found to have nausea,vomiting,or itchy skin;group L had vomiting during treatment(2%(20%),nausea in 4 cases(40%),and pruritus in 4cases(40%),compared with group L,The LMN group was significantly elevated,and the difference was statistically significant(P<0.05);4,Baseline-adjusted BFI symptom scores(first day and fifth)in the ropivacaine + morphine group were significantly increased by 460% compared with the ropivacaine group and the ropivacaine +morphine + naloxone group.The difference was statistically significant(P<0.001);baseline corrected BFI symptom scores in the ropivacaine group and ropivacaine +morphine + naloxone group did not change significantly,respectively,increased by 40%,45% compared with baseline(P>0.05);three groups of GSRS total scores(average scores of the first day and the fifth total score),ropivacaine + morphine group and ropivacaine group and ropivacaine + morphine + nanolo In the ketone group,the total score was statistically significant(F=29.72;P<0.001),and the overall difference between the five items was statistically significant(F=15.54;P<0.001);and between the total score and the project.The difference was statistically significant(F = 12.48;P <0.001);there was no significant change in the ropivacaine group compared with the ropivacaine + morphine + naloxone group.The difference between the ropivacaine +morphine group and the ropivacaine group and the ropivacaine + morphine + naloxone group was statistically significant(P<0.001),ropivacaine group and Luo There was no significant difference between the group of pikaine+morphine+naloxone(P>0.05).The patient evaluation of the constipation symptom questionnaire(PAC-SYM)was significantly higher in the LM group than in the L group and the LMN group.Thesignificance of learning(F=24.42;P<0.001);the difference of PAC-SYM score between LM group and postoperative treatment and treatment day was statistically significant(F=3.87;P=0.01),in the fourth treatment Days(P < 0.001)and day 5(P < 0.001)were significantly different from the first day.In addition,the PAC-SYM score during the LM group treatment was between Day 2 and Day 4(2% decrease from baseline on Day2,17% increase from baseline on Day 4;P=0.002)and Day 2 and Significant differences were detected between day 5(2% decrease from baseline on day 2,and 19%increase from baseline on day 5,P < 0.001).In the PAC-SYM scores of L group and LMN group,the difference of baseline correction values ??between different days was not detected(P>0.05);the frequency of defecation was significantly lower in LM group than L group and LMN group,and the frequency of defecation was significantly lower.Statistical significance(F=7.87;P=0.005);the difference between the LM group treatment and treatment date defecation frequency,the difference was statistically significant(F=8.11;P<0.001);data analysis showed defecation frequency LM group and L group,Compared with the LMN group,the number of bowel movements decreased by 87 on the second day,and the difference was statistically significant(87reductions in bowel movements,P < 0.001).The difference of fecal shape scores between LM group and L group and LMN group was statistically significant(F=56.34,P<0.001).Data analysis showed that the fecal morphology of all days during the treatment of LM group compared with L group and LMN group.The scores were significantly lower(the scores on Day 1 to 5 were reduced by 37%,52%,64%,40%,73%,respectively,P < 0.005);5,LM group compared with L group,LMN group(MRI scan results on the first and fifth days),the total volume of intestinal gas volume increased significantly,the difference was statistically significant(F=9.68,P=0.002),in Gas accumulation in the cecum/ascending colon and transverse colon was also significantly increased,and the difference was statistically significant(F=4.89,P<0.001).During the treatment of LM group,the volume of cecum/ascending colon wassignificantly increased from the first day to the fifth day.(41%,P=0.005),the transverse colon increased significantly(20%,P=0.005),and the total colon also increased(16%,P=0.008),the difference was statistically significant.In the L group and the LMN group,the volume of the cecum/ascending colon was significantly increased on the first day(14%,P=0.03),and there was no significant difference between the other sites and the number of treatment days(P>0.05).Conclusion:1,the reliability of in vitro culture of ICCs cells is exact;2,ISC cells cultured in vitro in NS group and MN group have their unique morphological characteristics: the cell body is fusiform or triangular,with large nuclei and few cytoplasm,and adjacent cell protrusions and cell bodies are connected to each other in a network;The morphology of ICC cells was greatly destroyed and the number was significantly reduced.The intracellular calcium fluorescence values of ICC cells in NS group and MN group did not change significantly,while the intracellular calcium fluorescence values ??in group I ICC were significantly lower than those in NS group and MN group.The length of ICC-like cells in NS group and MN group did not change significantly,while the length of ICC-like cells in group M was significantly shorter than that in NS group and MN group.The amplitude of pacing current of single ICC in group M was significantly lower than that in NS group and MN group.The frequency of pacing current was significantly lower than that of NS group and MN group,while the amplitude and frequency of pacing current in NS group were basically unchanged from those in MN group.3,epidural infusion of morphine caused the expression of ICC in the proximal colon tissue of rabbits,the ICCs-like cells decreased the calcium current,and the pacing current of ICCs-like cells decreased,which may lead to the pacing function of proximal colonic ICCs-like cells./Attenuation and cell signal/information transmission have certain obstacles,and then opioid-induced intestinal dysfunction syndrome;NS groupand MN group have basically no change,combined with low-dose naloxone can reverse morphine to ICCs-like The above effects caused by the cells.4,Based on this,we hypothesized that morphine may cause intestinal dysfunction by blocking or obstructing the expression of ICCs,pacing current and signal transmission through opioid receptors acting on pacemaker cells of rabbit intestinal ICCs.5,L group,LM group and LMN group had good postoperative analgesia effect.The LMN group had better analgesic effect after the first,third and fifth days of activity,and reduced the adverse reactions of morphine.6,through the MRI on the objective measurement of the patient's intestinal volume,to assess the impact of morphine on the function of the bowel function is feasible;7,LMN group enhanced analgesic effect,the gastrointestinal tract BFI,GSRS,PAC-SYM,BSFS score and MRI detection of intestinal volume were more clinically significant than the LM group,while reducing the amount of local anesthetic.Through a two-part study,we speculate that the possible mechanism is that morphine mainly acts on opioid receptors expressed by ICCs in pacemaker cells of the gastrointestinal tract,by blocking the expression of ICCs,pacing current block,signal/Obstruction of information transmission leads to intestinal dysfunction;application of the receptor antagonist naloxone can reverse the above effects of morphine.It provides an important reference for providing better strategies for perioperative analgesia and clinical pain management as well as further research on the mechanism of opioids affecting gastrointestinal function and its ICC cells. |
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