| Part 1 The relationship between different degree of compression of sciatic nerve and neuropathic painObjective Through the quantitative classification of ligation intensity of sciatic nerve in rats,on the one hand,we can explore whether the translocation associated membrane protein 1(TRAM1)can be used as a biological index of basic research of neuropathic pain;on the other hand,we can explore whether we can establish different degrees of sciatic nerve chronic constriction injury(CCI)model,and provide data support for quantitative and fixed ligation intensity modeling.Method 30 male rats(weight 200-250 g)were divided into blank group(naive group,N group,n=8),sham operation group(sham group,S group,n=8)and nerve ligation group(CCI group,n=14)by random number table method.All rats were screened for pain behavior before operation,then 5 rats in each group were randomly selected for pain behavior test.According to classic CCI modeling methods(when the sciatic nerve is ligated,on the surface of the sciatic nerve under a 40-fold microscope,the blood vessels in the epineurium were only compressed but the blood flow was not interrupted.Occasionally,a slight twitch was observed in the surrounding muscles),the CCI group using dissecting microscope,biological signal collecting system and drawing force transducer to detect the nerve ligation intensity in the process of modeling,the neuropathic pain model was established.S group did not ligate the sciatic nerve,and the other operations were the same as CCI group,N group were done nothing.On the 1st,3rd,5th,7th,10th,14th,21st and 28th day after operation,the posture,gait,mechanical pain threshold and thermal pain threshold were observed,and the behavior and pain threshold of rats before and after ligation were compared.At the same time point,we observed the morphological changes of the epineural vessels and nerves in each group,and explored the ligation intensity under the classic CCI model.2.102 male rats(weight 200-250 g)were divided into blank group(naive group,N group,n=17),sham operation group(sham group,S group,n=17)and nerve ligation group(CCI group,n=68).nerve ligation group was divided into four subgroups according to different ligation intensity:0 g(CCI-A group,n=11),1 g(CCI-B group,n=11),2 g(CCI-C group,n=11)and 3 g(CCI-D group,n=35)by random number table method.All rats were screened for pain behavior before operation,then 5 rats in each group were randomly selected for pain behavior test.After anesthesia,the CCI model of different degree of sciatic nerve ligation was established by using anatomical microscope,biological signal collecting system and drawing force transducer to monitor the nerve ligation intensity during the modeling process.S group did not ligate the sciatic nerve,and the other operations were the same as CCI group,N group were done nothing.On the 1st,3rd,5th,7th,10th,14th,21st and 28th day after operation,the posture,gait,mechanical pain threshold and thermal pain threshold were observed,and the behavior and pain threshold of rats before and after ligation were compared;At the same time point,the rats in each group were taken to observe the morphological changes of the epineural vessels and nerves;The expression of IL-6、p-NF-κB、p-ERK、caspase 3 and TRAM1 in spinal cord and dorsal root ganglion was detected by Western blot.The activation of astrocytes and microglia in spinal cord was analyzed by tissue immunofluorescence.Result 1.Using biological signal collecting system and drawing force transducer,it was found that the ligation strength of sciatic nerve in traditional model was 3 g.2.The expression of TRAM1 increases with ligation intensity of sciatic nerve.3.All the subgroups of CCI induced the changes of behavior and pain threshold,and the evaluation showed that all the subgroups were successfully modeled.The compression degree of sciatic nerve,the change of behavior and pain threshold,the distortion of adventitial vessels and the formation of new blood vessels,the expression of inflammatory factors IL-6,p-NF-κB,p-ERK and Caspase-3 in spinal cord,the activation of astrocytes and microglia in spinal cord all increased with the increase of sciatic nerve ligation.4.Using biological signal collecting system and drawing force transducer,we found that when the ligation strength of sciatic nerve in CCI model was 3 g,the changes of behavior and pain threshold,expression of inflammatory factors in spinal cord,activation of astrocytes and microglia in spinal cord were significant.Conclusion The expression of TRAM 1 was positively correlated with the degree of compression of the sciatic nerve,it can exist as a biomarker in basic experimental research to some extent.It is preliminarily believed that the establishment of neuropathic pain models with different nerve compression in rats is successful,which is helpful for the study of the mechanism of neuropathic pain with different compression and the ligation force of sciatic nerve in CCI model could be fixed to 3 g.Part 2 Study on the strength of sciatic nerve ligation in CCI modelObjective By quantifying the ligation force of the traditional CCI model,the stability of the fixed force modeling model and the expression of TRAM1 in the traditional modeling group and the fixed force modeling group are preliminarily evaluated,and whether the fixed force modeling can improve the stability of the CCI model to a certain extent and further verify whether TRAM1 can be used as biomarker for basic research of neuropathic pain.Method 40 male rats(weight 200-250 g)were divided into blank group(Naive group,N Group,n=10),sham operated group(Sham group,S group,n=10),traditional model group(CCI group,n=10)and fixed dynamics model group(CCI-D group,n=10)by random number table method.All rats were screened for pain behavior before operation,and 5 rats in each group were randomly selected for pain behavior test.After anesthesia,the traditional model group(CCI group)used the traditional CCI model method(when the sciatic nerve is ligated,on the surface of the sciatic nerve under a 40-fold microscope,the blood vessels in the epineurium were only compressed but the blood flow was not interrupted.Occasionally,a slight twitch was observed in the surrounding muscles)to establish the animal model of neuropathic pain,and the fixed dynamics model group(CCI-D group)used the high-power dissecting microscope,biological signal collecting system and drawing force transducer to fix the ligation force to 3 g for the sciatic nerve ligation,the same method as CCI group;In the S group,except that the sciatic nerve was not ligated,the other operations were the same as CCI group;No operation in N group.On the 1st,3rd,5th,7th,10th,14th,21st and 28th day after operation,the rats in each group were observed in posture and gait,and the mechanical pain threshold and thermal pain threshold were observed and compared;The expression of IL-6,TRAM-1 and p-ERK in spinal cord and DGR were detected by Western blot and ELISA,and the stability of pain behavior and related factors expression in traditional CCI model and fixed dynamics model were compared and analyzed.Result 1.Traditional CCI model and fixed dynamics model both induce neuropathic pain,and the evaluation showed that the two groups were successfully modeled,but the pain behavior and expression of IL-6 and p-ERK in spinal cord and DGR were more stable in fixed dynamics model.2.The expression of TRAM 1 was more stable in the fixed force group.Conclusion The behavioral and pain threshold changes of rats,the expression of IL-6 and p-ERK in spinal cord and DGR are relatively stable in fixed force modeling group,which is helpful to improve the stability of CCI modeling to a certain extent.The stable expression of TRAM 1 in the fixed force model further confirmed that it can exist as a biomarker for basic research of neuropathic pain.Part 3 The relationship between TRAM1 and neuropathic painObjective Through the establishment of a CCI model with a fixed ligation strength of 3 g,the correlation between TRAM1 and neuropathic pain and its synthesis site were studied,and the possible mechanism of TRAM1 involved in the development of neuropathic pain was explored.Method 104 male rats(weight 200-250 g)were divided into blank group(naive group,N group,n=11),sham operation group(sham group,S group,n=11)and experimental group(CCI group,n=82)by random number table method.All rats were screened for pain behavior before operation,and 5 rats in each group were randomly selected for pain behavior test.After anesthesia,the CCI group used the high-power dissecting microscope,biological signal collecting system and drawing force transducer to fix the ligation force to 3 g for the sciatic nerve ligation.S group did not ligate the sciatic nerve,and the other operations were the same as CCI group,N group were done nothing.On the 1st,3rd,5th,7th,10th,14th,21st and 28th day after operation,the posture,gait,mechanical pain threshold and thermal pain threshold were observed,and the behavior and pain threshold of rats before and after ligation were compared.Materials were taken according to the same time point.Western blot was used to detect the expression of TRAM1 and p-ERK in spinal cord and DGR,and immunofluorescence was used to analyze the expression and distribution of TRAM1.At the peak of expression,rats in each group were injected with TAK-242(TLR4 inhibitor),BAY11-7082(IKBα/NF-KB inhibitor)and TRAM1-siRNA,and the changes of pain behavior were observed.The expression of TRAM1 and p-NF-KB were detected,the correlation between TRAM1 and pain signaling pathway molecules was analyzed,and the possible mechanism of TRAM 1 mediated neuropathic pain was explored..Result 1.The expression of TRAM1 increased in spinal cord and DRG of CCI model rats,and the peak appeared on the 7th day after operation,which was positively correlated with the degree of pain.2.The synthesis of TRAM1 is mainly located in the neurons of Spinal dorsal horn and DRG.3.After intrathecal injection of TAK-242,BAY 11-7082 and TRAM1-siRNA,the pain threshold increased,and the expression of TRAM1 and p-NF-κB in spinal dorsal horn and DRG decreased significantly,which was negatively correlated with the degree of pain.Conclusion TRAM1 is involved in the development of neuropathic pain,its synthesis is mainly located in the neurons of Spinal dorsal horn and DRG,and may participate in the production and regulation of neuropathic pain through TLR4/p-NF-κB pathway.Part 4:Expression and clinical significance of TRAM1 in patients with carpal tunnel syndromeObjective To study the expression of TRAM1 in different types of patients with carpal tunnel syndrome and the effect of dexmedetomidine on the expression of TRAM1 in patients with carpal tunnel syndrome,and to explore whether TRAM1 can be used as a biological index to guide the clinical diagnosis and treatment of neuropathic pain diseases to some extent.Methods 50 patients with carpal tunnel syndrome and 10 healthy volunteers were selected.According to Gu Yudong’s typing method,50 patients with carpal tunnel syndrome diagnosed clinically and electrophysiologically were divided into light,mediun and heavy.10 cases in group A(light),20 cases in group B(middle),20 cases in group C(heavy),10 healthy in group N.The levels of IL-6 and TRAM1 were measured before treatment.Group A was treated with wrist brake and physical therapy;group B was divided into group B1(ropivacaine)and group B2(ropivacaine+dexmedetomidine),group C was divided into group C1(ropivacaine)and group C2(ropivacaine+dexmedetomidine).After nerve block anesthesia,the patients in four groups were treated with neurolysis of median nerve.The plasma levels of inflammatory factors(IL-6)and TRAM1 were measured at 6 h,12 h and 24 h after injection.Results The concentrations of IL-6 and TRAM1 in the patients with carpal tunnel syndrome were higher than those in the healthy patients,and the concentrations of IL-6 and TRAM 1 in the patients with carpal tunnel syndrome increased as the severity increased(group C>group B>group A>group N);the pain in the patients with carpal tunnel syndrome was relieved after the treatment of nerve block and neurolysis of median nerve,and the levels of IL-6 and TRAM1 were decreased as the time decreased(T6 h>T12 h>T24 h);the therapeutic effect of dexmedetomidine group was better than that of ropivacaine group alone.Conclusion The expression of TRAM 1 in different types of patients with carpal tunnel syndrome is positively correlated with the degree of pain,and has a grade trend.Dexmedetomidine can reduce the expression of TRAM 1 in the plasma of patients with carpal tunnel syndrome.Therefore,TRAM 1 can be used as a biological index to guide the clinical diagnosis and treatment of neuropathic pain. |
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