| Part1.Evaluation of the brain functional activities in rats various location-endometriosis pain modelObjective:Endometriosis(EM)is a common gynecological disease in women of reproductive age.These patients in approximately 80%suffer the various degree pain.This study will investigate synergistically the mechanism of the higher-position central sensitization and offer a pre-clinical experiment evidence for treatment of various location-EM patients with pain.Methods:Twenty Sprague-Dawley rats were induced three types EM including abdominal EM(n=5),gastrocnemius EM(n=5)and ovary EM group(n=5)and one sham control group(n=5).All groups were measured the pain sensitization by hotplate test,then scanned by the functional magnetic resonance imaging(fMRI).The resting-state fMRI(rs-fMRI)date was analyzed using regional homogeneity(ReHo)approach to find out the abnormal functional activity brain regions.Nissl staining method observed the state of neurons in aberrant ReHo signal brain regions.Results:Rats with EM pain sensitization were increased in abdominal EM and gastrocnemius EM than ovary EM group and sham control.The ReHo value is decreased in gastrocnemius EM in right thalamus and left olfactory tubercle compared with other three groups.The number of neurons was decreased;cavitation around nucleus,and pyknotic homogenous nuclei.Nissl bodies were stained deeply,and the shape was irregular in gastrocnemius EM by Nissl staining in right thalamus.In left olfactory tubercle,there was no significant difference in 4 groups.Conclusions:The thalamus may be the potential key brain region for the central sensitization mechanism of various location-EM pain.The oxidative activation may be weakened in thalamus in gastrocnemius EM group with more severe pain.This finding could support for future research on the imageology and pathology of various location-EM pain.Part2.Study on the molecular mechanism of abnormal activation brain regions in pain rat model of endometriosisObjective:Endometriosis is a common gynecological disease among women of childbearing age,with 70-80%of patients suffering different degrees of pain.Currently,the mechanism of endometriosis-associated pain is unclear,and central sensitization may be the cause of long-term pain or pain recurrence,but most studies on endometriosis pain are currently limited to behavioral or morphological observations,which do not provide sufficient evidence of molecular mechanisms.Therefore,the objective of this study is to further investigate the changes in abnormal areas brains for central sensitization,explore the molecular mechanism of central sensitization of endometriosis-related pain,solve the problem at the source,and provide a reliable experimental basis for clinical treatment.Methods:40 Sprague-Dawley female rats were induced in endometriosis model,behavioral assessment selected 28 endometriosis-related pain model rats as experimental group.20 Sprague Dawley rats were performed sham operation as the control group.All rats were scanned the whole brain by fMRI.ReHo method was performed to found abnormal activated brain regions associated for central sensitization.Nissl’s staining was observed the state of nerve cells in target regions and mRNA sequencing was explored difference cxpression genes in target brain regions.Real time quantitative PCR was verified the correlated significantly differences expression genes.Bioinformatics methods analyzed the functions and pathways of proteins related to the mechanism of central sensitization in endometriosis-related pain.The expression of key proteins TRPV1 and NMDRA in target brain regions were observed by immunohistochemical staining.Results:The pain threshold of the endometriosis-related pain group was significantly lower than that of the control group(P<0.001),The ReHo signals in the left anterior cingulate cortex 2(Cg2),left and right thalamus(Tha),hippocampal CAI and right brainstem(BS)of the pain group were significantly enhanced compared with the control group(P<0.005)by the fMRI scan of the whole brain.Nissl’s staining showed that there were deeper nissl bodies,abnormal morphology,and apoptotic neurons around them in the brain regions of anterior cingulate cortex,thalamus and hippocampus with enhanced ReHo signal,.The cell nuclei of the neurons were concentrated and deformed,the cytoplasm was stained deeply,and the volume of the neurons around the apoptotic cells was compensatory increased compared with that of the control group.mRNA sequencing results showed that in the three target brain regions test 139.101 and 66 difference expression genes respectively.The top significant abnormally difference expressed genes were mostly involved in the protein synthesis process required by REDOX reaction.GO and KEGG analysis revealed that the difference expressed genes were mainly involved in the calcium signaling pathway,axon orientation,focal adhesion and pi3k-akt signaling pathway.The key proteins TRPV1 and NMDAR were strongly positive expression in the target brain regions through immunohistochemistry.Conclusion:The key brain areas for central sensitization in endometriosis-related pain may occur in the anterior cingulate cortex,hippocampus and thalamus.The level of blood and oxygen balance in related brain regions might be regulated mainly by the plasticity of nerve cells and synapses.The molecular mechanism may be involved in the regulation of the PI3K/Akt signaling pathway,the transient receptor potential pathway mediated via calcium ions,and the regulation of synaptic plasticity mediated by local adhesion kinases.Part3.The activation of NLRP3 in inflammatosomes of endogeneic pain rats was reduced by tDCS to relieve central sensitized painObjective:Pain,abnormal menstruation and infertility are still major symptoms of endometriosis.About 70 to 80 percent of adolescents and adult women with endometriosis suffer from pain.The mechanism of pain is still unclear,which may be related to the inflammation and pain mediators by the implantation of ectopic lesions outside uterine,leading to the local related nerve dysfunction,and thus causing central sensitization.The balance of estrogen receptor expression is regulated by inflammasome pathways mediated.NLRP3 inflammasome plays an important role in the development of the endometriosis-related pain.Transcranial direct current stimulation(tDCS)is a noninvasive treatment of brain stimulation.In this study,we investigated whether tDCS could relieve central sensitized pain by reducing the activation of NLRP3 inflammasome in endometriosis rats.Methods:The 28 pain rats screened by behavioral assessment were randomly divided into two groups,14 experimental rats were treated with tDCS,and 14 control rats were treated with sham treatment.Rs-fMRI was used to scan the whole brain of the two groups.ReHo was used to analyze the abnormal activation signals in the different brain areas.Transmission electron microscopy further to observe the ultrastructural changes of nerve cells in the different brain areas of the treated and sham-treated rats.Immunohistochemistry was used to detect the differences in estrogen levels and inflammasome-related proteins in the brain areas with abnormal activation signals between the two groups.unpaired t tests were used to compare the differences between the groups,P<0.05 was considered statistically significant.Results:The hot plate test results show the pain threshold value of the two groups before treatment were 10.86±0.71s and 11.32 ±0.78s,P>0.05.After tDCS,pain threshold of control group was 11.64±0.80s,and the pain threshold value of tDCS group was 27.07±1.66s,P<0.05,which indicated the tDCS group rats ease pain sensitization degree.Rs-fMRI results showed that ReHo signaling in tDCS rats t decreased in the anterior cingulate cortex(left),hippocampal CA1(left),thalamus(left),and primary motor cortex(right)regions,compared with the control group.Transmission electron microscopy was used to further observe the brain areas with weakened ReHo signal,and it was found that the number of neurons in the endometriosis-related pain control group was relatively increased,the volume of intracellular mitochondria compensatory increased,and even some of them showed swelling,while the mitochondria volume of tDCS group reversibly returned to normal size.In the control group,the lamellar structure of myelin sheath was loosely arranged,and even lamellar detachment and morphological changes were observed.However,the myelin lamellar structure of the tDCS treatment group was closely and neatly arranged.Immunohistochemical results showed that estrogen receptor(ER)was positively expressed in the normal endometrium,eutopic endometrium and ectopic cyst epithelium of both groups.Caspase-1,NLRP3 and IL-1 β were negatively expressed in the normal reference endometrium,positively expressed in the eutopic endometrium of both groups,positively expressed in the ectopic cyst tissues of control group,and weakly positive or negative in the ectopic cyst tissues of the tDCS treatment group.Caspase-1,IL-1 β and NLRP3 were positively expressed in the hippocampal neurons of the control group,while most of the neurons in the tDCS treatment group were negatively expressed,and the h-score difference was statistically significant.Conclusion:In this study,it was found that tDCS could reduce the activation of NLRP3 inflammasome and the release of IL-1 β,further to relieve the central sensitized pain of endometriosis through the verification of subjective indicators of ethology,and objective indicators of imaging and histomorphology. |
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