| Background:Atrial fibrillation(AF)has gradually become a kind of cardiovascular epidemics,and is also one of the most common clinical arrhythmia,which has high morbidity?fatality and invalidism rate.The disease itself and its complications have a serious threat to people's health.A number of studies suggest that autonomic nervous tension imbalance causes cardiac electric-anatomical remodeling,which plays an important role in the occurrence and maintaining of atrial fibrillation.Spinal cord stimulation treatment can pass to specific spinal cord segmental distribution of low energy,high frequency electrical stimulation,regulating the balance of the body's autonomic nervous activity and the autonomic nervous tension.Prompting intervention of autonomic nervous system activity and tension can be treatment of atrial fibrillation,and there are methods for the treatment of atrial fibrillation targets in autonomic nerve,but with some limitations.By intervening in the external autonomic nervous manner,releasing energy for electrical stimulation to the external autonomic nerve's specific spinal cord segmental to treat atrial fibrillation has great potential and prospect.Objectives:The purpose of this study were to establish the beagle atrial fibrillation model and give Spinal cord stimulation treatment;to investigate the feasibility,methodology and the curative effect of spinal cord stimulation treatment for regulating cardiac autonomic nervous tension and explore its possible mechanism and molecular mechanism.The study will provide a scientific basis for new strategy to explore the treatment of atrial fibrillation and new therapeutic targets.Method:1?Select 18 adult beagle,male and female informal,and randomly divide into blank control group(Ctrl group,6),atrial fibrillation model without the spinal cord stimulation group(AF group,6),atrial fibrillation model in the spinal cord stimulation group(SCS group,6).(1)Use SelectSecure system of Medtronic company developed,implant the most fine bipolar solid electrode wire-3830 active fixed electrode,connect to the special atrial fibrillation model pacemakers and establish atrial pacing system.Then adopt AOO pacemaker mode of rapid atrial pacing method to establish atrial fibrillation model.Do standard body lead electrocardiogram(ECG)preoperative and postoperative weekly,at the same time using implantable ECG monitor pass Reveal LINQ AF tracker real-time tracking.(2)In the SCS group,with the aid of mature technique of lumbar puncture,puncture the experimental dogs L2,L3 lumbar vertebra clearance to less than 45 degrees,then implant 8 standard spacing contact percutaneous puncture electrode wires and connect the spinal cord stimulator to establish the spinal cord electrical stimulation system.During the period of spinal cord stimulation,use of implantable ECG monitor pass Reveal LINQ tracker real-time monitoring load during the treatment of atrial fibrillation.(3)For AF group of experimental dog,make ultrasonic cardiogram preoperative,between atrial fibrillation model successfully established and executed;For SCS group of experimental dog,make ultrasonic cardiogram preoperative,when atrial fibrillation model successfully established,when spinal cord electrical stimulation achieved after the treatment course,before executed;For Blank group experimental dogs,make ultrasonic cardiogram for the same period,measurement the left,right atrium end systolic atrium area at the apical four-chamber view.(4)Use of CARTO system of 3D measurement,The intracardiac electrophysiology information combined with heart cavity space anatomical structure to measure the cardiac electrophysiological changes of three groups of experimental dogs in the test.In the measuring process,successful puncture the right femoral vein innovatively in the case of no X ray,then place the PentaRay standard test tube with high precision through sheath tube,and build the right atrium and inferior vena cava on the 3D model;Do a transverse incision of about 3 cm long in the chest on the left side of the first four to the fifth floor and exposure the heart in assisted chest brace,then longitudinal shear the pericardium happy package to expose left aurcle.Look straight into the tip of ear and puncture,place the PentaRay standard test tube with high precision through sheath tube,rapidly build 3D models of left atrium,anterior chamber of the pulmonary vein and left aurcle,and electrical physiological indexes of standard test.(5)Executed experimental dogs.Collect blood samples before executed and centrifuge and store for inspection.After executed experimental dogs,take out heart,separate and collect left atrium.Respectively carry on light microscopy,electron microscopy and subsequent immunohistochemical,left atrial tissue transcriptome of related research.2?Using label free quantitative proteomic technology to analysis the differences in the amount of protein expression of experimental canine serum between AF group,SCS group and control group.Through bioinformatics analysis,clustering analysis GO analysis and protein interaction analysis were carried out on the differentially expressed proteins.Selection for the difference proteins between three groups,and verify related protein expression in the experimental canine myocardial tissue volume by using ELISA,early screening biological marker protein which has a guiding significance and can play a key role in the process of SCS intervention AF.3?For AF group,the SCS group and control group experimental canine atrial muscle tissue,using the transcriptome sequencing method,through the analysis of gene expression level,genetic screening with expression differences between the groups and pick out the differences between genes,and do the GO analysis,KEGG pathway annotations and enrichment analysis on the differentially expressed proteins through the application of bioinformatics analysis,find out the differences gene significantly enriched GO and KEGG entries.Quantitative analysis using the qRT-PCR to distinguish gene,and using Western blot method to verify the relevant purpose protein expression in the experimental canine myocardial tissue.Result:1.(1)15 dogs completed the model and the other 3 dogs died of 18 experimental dogs.In establishing the atrial fibrillation model,the total success rate of atrial fibrillation model was 100%.The time to establish a successful atrial fibrillation was 10.63±2.13(weeks);When the atrial fibrillation model is successful,atrial stimulation frequency of high frequency atrial fibrillation model pacemaker is 588.75±11.26(times/min)in AOO mode.The atrial fibrillation load of AF group and SCS group was recorded dynamically using the implanted ECG monitor-Reveal LINQ.To monitor the occurrence of atrial fibrillation and the change of load in atrial fibrillation after SCS treatment,the atrial fibrillation load of experimental dogs in SCS group was significantly lower than that in AF group(7.34±2.34%vs.46.43±5.16%,P<0.001).(2)Using GE Vivid q Ultrasonic Cardiogram imaging instrument,the probe transmitting frequency of 2.5MHZ was performed on the experimental dogs for echocardiography.After the success of the atrial fibrillation model,the area of left atrium was significantly enlarged before modeling(8.20±0.83cm2 vs.3.80±0.08cm2,p<0.05);the right atrium area was larger than that before modeling(4.52±0.44cm2 vs.2.75±0.96cm2,p<0.001).The comparison of the area between the left atrium and the right atrium between the three groups,the left atrium area of the SCS group was smaller than the AF group(5.04±0.89 cm2 vs.8.20±0.83cm2,P<0.05),the right atrium area was smaller than AF group(4.20±0.13 cm2 vs.4.52±0.44cm2,p>0.05);the left atrial area of AF group was larger than that of control group(8.20±0.83 cm2 vs.3.72±0.15cm2,p<0.05);the right atrium area is larger than before modeling(4.52±0.44cm2 VS 2.78±0.18cm2,p<0.05);the left atrium area and the right atrium area of SCS group were both larger than that of control group((5.04±0.89cm2 VS 3.72±0.15cm2,p<0.05;4.20±0.13cm2 VS 2.78±0.18cm2,p<0.05).(3)Compare the atrial effective refractory period of three groups of experimental dogs in electrophysiological test.The AF group compared with the control group,the left atrium effective refractory period was shorter(101.50±6.16ms vs.116.88±6.85ms,P<0.05),the right atrium effective refractory period was shorter(96.38±11.54ms vs.117.13±10.69ms,P<0.05);The SCS group was compared with the AF group,the left atrium effective refractory period was extended,(107.34±5.87ms vs.101.50±6.16ms,P>0.05),the difference was not statistically significant;the right atrium effective refractory period was extended(101.75±10.26ms vs.96.38±11.54ms,P>0.05),the difference was not statistically significant;The SCS group compared to the Control group,the left atrium effective refractory period was shorter(107.34±5.87ms vs.116.8±6.85ms,P<0.05),the right atrium effective refractory period was shorter(101.75±10.26ms vs.117.13±10.69ms,P<0.05);(1)The results of the left atrial myocardial cells in light microscopy were indicated that on the HE staining section,the myocardial cell structure of Control group(blank group)was complete,arranged neatly,and the nuclei are clear,the rule web filled with the entire cardiomyocytes;the shapes of fibroblasts in mesenchyme is regulation and their quantity is moderate.In AF group,myocardial celsl enlarged,arrange disordered,nucleus sized irregular and appeared abnormal changes,some cells appeared dissolve necrosis,vacuoles degeneration and granular degeneration with inflammatory cells infiltration and myocardial fibrosis.In the SCS group,the myocardial cells were intact,slightly disordered,and the nuclei had partial heterotypic changes,and the cytolytic necrosis was reduced,and some myocardial fibrosis changed,slightly less than the AF group.In the left atrial myocardial cell electron microscopy,the left atrial myocardial fibers of the control group was ordered and the mitochondrial size was uniform,the chromatin structure is normal in the nuclear membrane.Left atrium muscle cell ultrastructure of AF group was obviously abnormal,myofibril section appeared serious degradation,mitochondria vacuoles degeneration,serious karyopyknosis with more cells apoptosis,more secondary lysosomes,obvious swelling of the collapse of the endoplasmic reticulum and some muscle,disorder and chaos of muscle fibers.the muscle cell ultrastructure abnormalities of the SCS group,myofibril section appeared a certain degree of degradation,but still can see most of the myofibril order,some mitochondria cristae decreased density with chromatin and nuclear membrane expansion and deformation of nucleus pycnosis,swollen endoplasmic reticulum,increase of glycogen,still can see the compact density and muscle fibers.2.Using label free quantitative proteomic technology,there were 349 quantifiable proteins in the three groups of Experimental dogs.Among these proteins,there were 18 differentially expressed proteins in AF group and Control group,16 in SCS group and Control group and 23 in SCS group and AF group.Through bioinformatics analysis,we analyzed the differentially expressed proteins with clustering analysis,GO analysis,protein interaction analysis.We applied ELISA to verify the differentially expressed proteins KNGI?CFP?C1R?SERPINA4,the result shows that these 4 differential proteins are different between groups.In SCS group,the expression quantity of KNG1 was higher than AF group and Control group;CFP in AF group was higher than Control group and SCS group,there was no difference in Control group and SCS group;The expression of C1R showed that AF group was higher than SCS group and Control group,there was no marked difference between Ctrl group and AF group;for SERPINA4,it was significantly higher in SCS group than Ctrl and AF group.3.We applied transcriptome sequencing approach to analyze the overall gene expression level for dogs canine atrial muscle tissue in AF,SCS and Ctrl groups,to screen out genes that have differentially expressed between groups,selected the differential genes,the up-regulated genes were MG21,MTMR2,EPHX2,INPP5,AMACR,ACOX2,IP6K3,ITPKC and PTGS2.The down-regulated were COL21 A1,COL6A6,CRY2,SPP1,TFRC,SLC8A3,PER1,KCNJ8,MYLK3.Clustering differentially expressed genes by clustering analysis and analysing differential genes by GO analysis..Differentially expressed genes were annotated and categorized by Pathway in the KEGG database.According to the functional analysis of Pathway in KEGG database of differentially expressed genes,we can predict the differentially expressed genes related signaling pathway,which may be related to the Peroxisome and Primary pile acid biosythesis pathway.Quantitative analysis of the selected differential genes by real-time fluorescence quantitative nucleic acid amplification detection system(qRT-PCR)suggested that the expression trends of the differential genes were approximately the same as the sequencing results.Western blot method was used to verify the expression of the target protein in the canine myocardial tissue.In the Ctrl group,INPP5B expression was significantly down-regulated,KCNJ8 expression was small,MGST and IP6K3 substantially up-regulated;In the AF group,INPP5B and KNJ8 expression was significantly up-regulated,while MGST and IP6K3 expression was not notable;INPP5B and MGST expression was significantly down-regulated in SCS group,KNJ8 and IP6K3 expression was up-regulated.Immunohistochemical staining was performed with KCNJ8 for atrial muscle,the results showed high expression of KCNJ8 in AF group,but not obvious in Ctrl and SCS group.Conclusion:1.The use of rapid atrial pacing can successfully establish a stable model of atrial fibrillation.The method using the SelectSecure system and high 3830 atrial pacing electrode implanted can accurately implant the electrode and improve the success rate of electrode implantation in the experimental dog.Real-time dynamic monitoring of atrial fibrillation load with Reveal LINQ can enhance the efficiency of experimental monitoring.2.Experimental treatment of atrial fibrillation dogs by using SCS is safe,feasible and effective,which can slow down and improve the remodeling of the left atrium caused by atrial fibrillation.3.The use of Label free quantitative proteomics preliminary screening has instructive meaning in the process of intervention by SCS.The biomarker protein KNG1?CFP?C1R?SERPINA4,which play a key role,are expected to obtain more sensitive and specific protein biomolecules for further investigation and to provide dynamic monitoring of prognosis for further work on clinical intervention,become the basis for the exploration of new targets.4.Through the use of transcriptome sequencing,we screened out that there were multiple genes that were up-regulated and down-regulated during the Intervention of AF experimental dogs by SCS method,which may be related to the curative effect of SCS on AF,they may lay the foundation for further in-depth study,with guiding significance and provide more new entry points.5.In the SCS treatment of AF experimental dogs in the effective mechanism,Peroxisome and Primary bile acid biosynthesis signaling pathway may play an important role in the regulation of related signal networks,which may provide new ideas for the further research of mechanism. |
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