A Research For The Mechanism In The Modulation Of Left Posterior Insular Cortex For Atrial Fibrillation With A Novel Cardiac Stereotactic Electrophysiological Epicardial Mapping System

Atrial fibrillation(AF)is the most common arrhythmia in neurology and neurosurgery department.AF has also been found the most common arrhythmia among the patients who caught insular stroke and with no organic cardiac disease.However the mechanism of the sort of cortex-driven AF is obscure at present for a shortage of the relevant study.Insular cortex(IC)has a close relationship with autonomic nerve system(ANS).It has been reported that the stimulation of left IC may cause the variation of electrocardiogram(ECG).In addition,IC has brain lateralization that left IC is responsible for the modulation of parasympathetic nervous system.When activating the left posteror insular(LPIC)with electrical stimulation,BP and HR was declined.With the development of imaging technology recent years,the documents of functional magnetic resonance imaging(fMRI)has also indicated the control of parasympathetic nervous system by LPIC.On the other hand,parasympathetic nervous system is a very important factors to influence AF.Thus wheather LPIC as the upper stream of ANS could inervate AF through the modulation for parasympathetic nervous system or other path,which have not been investigated yet.In view of the interdiscipline of the research,we need to deal with two main problems.Firstly,we should select a suitable AF in vivo model to preserve the intact nervous system in order to the study of the dynamic regulation for AF by IC.Secondly,we need to combine the experimental method of neuroscience and the evaluating method of cardiac electrophysiology in AF.However,the model for the study is scarity.Therefore,we invented a cardiac stereotactic electrophysiology epicardial mapping system(CREAMS)served as a useful tool for the electrophysiological evaluation in AF.In addition,we used CREAMS to creat two practicable models.We will demonstrate these through tree parts as follows: 1.The development of CREAMS:The system induced the concept into the multiple mapping about the operational principle of cerebral stereotaxis to fulfill the function of taking the electrode to the site we needed to map.According to the characteristic of cardiac morphology and physiology,we invented two devices,an all-around rotating and positioning electrode holder and cardiac repository,to build up a artificial three dimension coordinate system and use two set of coordinations(GPC and LEPC)to fulfill the mapping for multiple sites.2.Validity test for CREAMS:(1)The measurement of cardiac electrophysiology: CREAMS was used to successfully record unipolar electrograms and to measure ERPs and WOV at HRA,RA,LRA,LAA,LA and JLA-LPV.(2)Test for the positioning function: Corresponding coordinates were noted at the 6 sites and all 6 sites were relocated in terms of coordinates.Five consecutive unipolar electrograms were selected and the average of maximum and minimum peak value,amplitude,area under curve,the maximum ascending velocity of voltage(dMax/dt),and the minimum descending velocity of voltage(dMin/dt)were obtained.Comparison of the graphic parameters of the unipolar electrograms allowed for examination of the accuracy of positioning.The results has no statistical significant.This part tested with CREAMS the validity of the measurable capacity of electrophysiological parameters and the positioning function.2.Practicalbility evaluation for CREAMS(1)To build up a focal atrial fibrillation triggers model with high-frequency stimulation(HFS)in JLA-LPV and make a electrophysiological evaluation with CREAMS:after JLA-LPV HFS 1.5h(frequency 40 Hz,pacing 2×threshold,duration 2ms),ERPs and WOV of 6 measured sites(HRA?RA?LR?LAA?LA?JLA-LPV)was shortened(P<0.05),comparing before JLA-LPV HFS.The dERP and ?WOV were calculated offline.dERP got increased(P<0.01)and ?WOV became enlarged(P<0.05).AF-CL was decreased(P<0.01)and AF-D increased(P<0.001).All of these results were shown that acute electric remodeling in atrium was found by JLA-LPV HFS and increased spatial heterogeneity to facilitate AF happening.(2)CREAMS for electrophysiological evaluation of drug-induced AF in rat in vivo with LL-VNS: All the 30 adult healthy SD rats were randomly assigned to three groups: blank control group,ACh-CaCl2 control group and low-level vagus nerve stimulation(LL-VNS)group.All chests were opened through a median sternotomy at the fourth intercostal space or the intercostal at the cardiac point of maximal impulse to achieve maximum exposure of heart.Drug induced AF model was build up in vivo in rat with ACh-CaCl2 injection of caudal vein(the solution recipe: 60 ? g/ml ACh:10 mg/ml CaCl2:1ml/kg)and make a electrophysiological evaluation with CREAMS.Compared with control group,ACh-CaCl2 group significantly increased electric remodeling in atria and facilitate AF happening(P<0.05).LL-VNS group,comparing ACh-CaCl2 group,ERP was significantly reduced(P<0.05).dERP,WOV and ?WOV were decreased obviously(P<0.05),which turned out that LL-VNS was able to depress AF by reversing the electric remodeling in atria.All above,CREAMS successfully made an atrial electrophysiological evaluation in two of the AF in vivo models.It is the first to use dERP and ?WOV to evaluate the atrial electrophysiological properties in rat AF in vivo model,which makes the evaluation more comprehensive and reliable.CREAMS could serve as a useful tools to support the following study.After solving the main problems about model and method of electrophysiological evaluation for AF,we utilized the multiple experimental method,including cardiac electrophysiology,neuroelectric physiology,morphology and immunohistochemistry,to investigate the relationship between LPIC and AF and try to disclose the possible path between them.We discussed that with the following two parts: 1.The effect of the variation of LPIC function for AFWe useed DBS and chemical genetics technique to manipulate the function of LPIC and observed the influence for atrial electrophysiological properties and AF.In the DBS experiment,we randomized 72 SD adult rats into 6 group that are baseline,DBS,Sham,VNSt,aatenolol and atropine.We observed the influence of the LPIC activating for the atrial electrophysiological effect and AF caused by JLA-LPV HFS.We observed that LPIC activating influenced the atrial electrophysiological properties and AF.Then we compared with each groups to find out the possible path about the regulation for AF causing by LPIC.In the chemical genetics experiment,likewise,we randomized 72 SD adult rats into 6 group that are HM4 Di,mCherry and the rest groups are the same as previous.We injected chemical genetics virus(AAV2/8-hSyn-DIO-HM4Di-mCherry)into LPIC.Then after 4 weeks,we started the cardiac electrophysiology in vivo experiment.Before the cardiac electrophysiology in vivo experiment,we identified the activity of the chemical genetics virus with patch clamp technique and AAV2/8-hSyn-DIO –mCherry was used as the control virus in the experiment.The results were as follows:(1)The effect of atrial electrophysiological properties and AF regulated by activating LPIC with DBS:Compared with Sham,DBS were able to increase dERP(P<0.05)and enlarge ?WOV(P<0.05)further,which identified that LPIC excitement could change the atrial electrophysiological properties further causing by JLA-LPV HFS,which leaded to aggravate atrial electric-remodeling and facilitate AF.In addition,AF-D was increased(P<0.05)and AF-CL was decreased(P<0.05),which turned out that LPIC participated in happening and maintainnence of AF.(2)The possible regulating path of the regulation of AF by LPIC excitement: atenolol blockading the activity of sympathetic nerve system seemed not to change the electrophysiological effect from DBS.Whereas,VNSt and atropine seemed to offset the effect.Moreover,DBS leaded to the decrement of BP in the process of the experiment(comparing with Sham,P<0.01).All of the evidences indicated that the excitement of LPIC was able to regulate AF through modulating parasympathetic tone.(3)The effect of atrial electrophysiological properties and AF regulated by deactivating LPIC with chemical genetics virus(HM4Di): after providing CNO to deactivate LPIC,we found that the atrial electrophysiological properties also be changed(dERP increased,?WOV enlarged,AF-D lengthened and AF-CL decreased,all P<0.05)to deteriorate atrial electrical-remodeling by JLA-LPV HFS and facilitate AF happening and maintenance.(4)The possible regulating path of the regulation of AF by LPIC deactivation: atenolol could counteract the effect of atrial electrical-remodeling exerting by HM4 Di and depress AF happening and maintenance.VNSt could not offset the effect causing by HM4 Di.Moreover,HM4 Di also leaded to the increment of BP in the process of the experiment(comparing with Sham,P <0.01).All of the evidences indicate that the deactivation of LPIC will regulate AF through modulating sympathetic tone.These shows that the variation of the function of LPIC is able to control atrial electrophysiologial properties and facilitatle AF.AF can be regulated through parasympathetic path in the condition of the excitement of LPIC and sympathetic path in LPIC deactivation.2 The efferents connections of the LPIC:We injected an anterograde tracer Phaseolusvulgaris-leukoagglutinin(PHA-L)into LPIC with iontophoretical method.14 days after injection,the rats were perfused,brains were cut into slices and make an immumohistochemical staining.To observe brain areas and nucleus containing by PHA-L with microscope.As a result,we found that PHA-L labeled fibers and terminals were mainly found in the following: ipsilateral prelimbic cortex(PrL),BLA,mediodorsal thalamic nucleus(MD),central medial thalamic nucleus(CM),parvicellular part(VPPC),lateral hypothalamic area(LH),periaqueductal gray(PAG);contralateral LPIC,nucleus of the solitary tract(Sol)and the dorsal motor nucleus of the vagus nerve(DMVN).From all of these,LPIC connection was so complicated for projecting towards many brain areas and nucleus.In particularly,we found LPIC connected with Sol,DMVN and hypothalamic area.The former two nucleus has close relationship with the regulation for cardiovascular activity,which may be responsible for parasympathetic effect.The latter area was responsible for modulating the hypothalamic-pituitary-adrenal to exert sympathetic effect.All of these morphological evidences make us easilier understand the electrophysiological phenomenon of the modulation of AF by LPIC.In summary,all of our experiment showed that:(1)CREAMS system was a effective and convinient tool to evaluate AF electrophysiology;(2)the variation of LPIC function can inervate AF through autonomic nerve system(ANS);(3)We observed some possible path participating in AF regulation.