Long-Term Effects Of Atrial Ganglionated Plexi Ablation On Function And Structure Of Sinoatrial And Atrioventricular Node In Canine

Autonomic innervation of the heart involves both the extrinsic and the intrinsic cardiac autonomic nervous system (ANS). The The intrinsic cardiac autonomic nervous system (ICANS) is consisted of ganglionated plexi (GP) concentrated within epicardial fat pads, nerve fibers connecting GP, ganglia located in the ligament of Marshall, and axons. Neurons in the GP send out postganglionic fiber to dominate the heart. GP are particularly well innervated with both adrenergic and vagal nerve endings. Anterior right ganglionated plexi (ARGP) which is located just anterior and inferior right superior pulmonary vein, in proximity to the junction of right superior pulmonary vein and right atria, and inferior right ganglionated plexi (IRGP) which is situated at the junction of inferior vein cava and both atria, dominate sinoatrial node (SAN) and atrioventricular node (AVN) functions, respectively, as well as their adjacent atrium muscles.GP ablation may affect ANS function and inhibited AF inducibility. However, the denervation effect of GP ablation may destroy intrinsic cardiac ANS and has potential effects on the function of SAN and AVN. Therefore, the safety of GP ablation, particularly the effect of GP ablation on pacing function of SAN and conduction function of AVN, is gradually drawing attention. Now, the acute effect of GP ablation on the function of SAN and AVN has no final conclusion. There is no evidence about the long-term effect on the function and structure of SAN and AVN.On these grounds, we ablated the ARGP and IRGP by anatomic location and high frequency stimulation and detected the changes of the electrophysiology function of SAN and AVN before and after ablation immediately, one month, six months, twelve months. We also detected the long-term changes of the histological characteristics as well as connexin (Cx) 43 and Cx 45 expression in SAN and AVN after ablation of ARGP and IRGP so as to investigate the possible mechanism of these changes. The aim of this study is to investigate the acute and long-term safety of GP ablation and provide the theoretic evidence for clinic application of GP ablation.Part Ⅰ:Long-term Effects of Atrial Ganglionated Plexi Ablation on Electrophysiology Function of Sinoatrial and Atrioventricular Node in CanineObjective:Ganglionated plexi (GP) ablation is an important therapy for atrial fibrillation (AF). Acute and long-term effects of GP ablation on the electrophysiology function of sinoatrial node (SAN) and atrioventricular node (AVN) remain unclear. This study is to investigate the acute and long-term effects of ablation of cardiac anterior right GP (ARGP) and inferior right GP (IRGP) on electrophysiology function of SAN and AVN in canine. The aim is to investigate the safety of GP ablation and provide the theoretic evidence for clinic application of GP ablation.Methods:Twenty-four dogs were anesthetized with Na-pentobarbital and were ventilated by a positive pressure respirator. Two multipolar electrode catheters were positioned at right atrium and His bundle through right internal jugular vein and femoral vein with indication of intracardiac electrogram. Continuous surface electrogram and intracardiac tracings were recorded. The functions of SAN and AVN were detected with a Electrophysiology Management System.1. The SAN function was evaluated by resting heart rate (HR), sinus node recovery time (SNRT) and corrected sinus node recovery time (cSNRT) at four levels of decremental pacing cycle length (PCL):330,300,280, and 250 ms. The pulse width of pacing was 1.5 ms, and the strength was twice the pacing threshold. The duration of each episode of pacing lasted for 2 min and was temporarily stopped for another 2 min before the next pacing period. The SNRT is defined by the duration from the last paced atrial complex to the following electrogram of spontaneous sinus beat, and the cSNRT is equal to the difference between SNRT and sinus cycle before pacing.2. The AVN function was evaluated by:(1) AVN effective refractory period (ERP); (2) atria-His (AH) interval at four levels of decremental pacing cycle length (PCL):330,300,280, and 250 ms; (3) average ventricular rate (VR) during AF (the average duration of cycle lengths over 10 ventricular beats during AF induced by right atrium burst pacing [S1S1=100 ms]); (4) the PCLs at which Wenckebach atrioventricular block (AVB) and 2:1 AVB occurred during incremental pacing.After all parameters were detected, thoracotomy was performed through the right fourth intercostal space and heart was exposed. Right atrium, superior and inferior vena cava, right pulmonary vein were identified. The locations of ARGP and IRGP:(1) anatomic location:ARGP was located just anterior and inferior right superior pulmonary vein, in proximity to the junction of right superior pulmonary vein and right atria; IRGP was situated at the junction of inferior vein cava and both atria, (2) high-frequency stimulation:Vagus response (progressive slowing of sinus HR or atrioventricular conduction block) was observed during high-frequency stimulation (20 Hz,0.1 ms,0.6-4.5 V) with a Multifunctional Electrophysiology Stimulator to the two GPs. ARGP and IRGP were ablated. After successful ablation immediately, the functions of SAN and AVN were evaluated again. Twenty-four dogs were randomly divided into three groups. The SAN and AVN functions were re-evaluated at one, six, and twelve months after ablation.Date analysis:The changes of electrophysiology parameters of SAN and AVN before and after GP ablation were analyzed. Pair f-test was used for comparison of electrophysiological parameters between pre-ablation and immediately after ablation in twenty-two dogs. ANOVA (Tukey’s HSD and Dunnett’s T3) was used for comparison of electrophysiological parameters in three groups before and after ablation.Results:At the end of the study, twenty-two dogs were evaluated during the study (eight dogs each in one-month and six-months groups; six dogs in twelve-months group).1. The changes of SAN electrophysiology function:after GP ablation immediately (n= 22), resting HR was increased (164±10 vs 170±9 bpm, P= 0.029), SNRT and cSNRT at different PCLs were shortened. One months after ablation (n= 8), resting HR, SNRT and cSNRT were reverted to pre-ablation level. The SAN function had no significant changes at six months (n= 8) and twelve months (n= 6) after ablation compared with pre-ablation.2. The changes of AVN electrophysiology functions:AVN ERP was prolonged (108±10 vs 125±13 ms, P=0.000), PCLs as Wenckebach AVB (175±10 vs 189± 16 ms, P= 0.001) and 2:1 AVB (157±12 vs 171±15 ms, P=0.004) occurred were prolonged, the VR during AF (279±28 vs 265±30 bpm, P=0.11) and AH intervals at different PCLs had no significant changes. One months after ablation, AVN ERP was reverted to pre-ablation level. However, the VR during AF (242±27 bpm) was decreased (P= 0.044), AH intervals at short PCLs (280ms:87±11 vs 104±12 ms, P= 0.029; 250ms:94±12 vs 110±12 ms, P=0.035) were prolonged. The PCLs as Wenckebach AVB (180±11 vs 200±13 ms, P=0.01) and 2:1 AVB (159±14 vs 180 ±13 ms, P=0.015) occurred were still prolonged. Six months after ablation, all above parameters were reverted to pre-ablation level. All parameters had no significant changes before and twelve months after ablation.Conclusion:After ARGP and IRGP ablation immediately, the SAN function was strengthened with increasing resting HR and shortened SNRT and cSNRT. One month after ablation, the SAN function was reverted to pre-ablation level. The AVN conduction function was decreased at one month and was reverted to pre-ablation level at six months after ablation. Ablation of ARGP and IRGP has short-term effects on function and structure of SAN and AVN rather than long-term effects, which suggests that ablation of ARGP and IRGP is safe.Part II:Long-term effects of atrial ganglionated plexi ablation on structure of sinoatrial and atrioventricular node in canineObjective:The long-term changes of histological characteristics and connexin (Cx) expression in SAN and AVN with denervation after GP ablation remain unclear. This study is to investigate the long-term effects of ablation of cardiac ARGP and IRGP on histological characteristics and connexin expression of SAN and AVN in canine, and to investigate the relationship between connexin expression and electrophysiologic function changes. The changes of electrophysiologic function were tried to be explained by connexin expression. The results of this study would provide the theoretic evidence for clinic safe application of GP ablation.Methods:Besides GP ablation group (24 dogs),8 dogs were selected into sham-operated group (control group) in which SAN and AVN functions were also observed to elinminate the effect of electrophysiologic study program. We only performed thoracotomy and did not ablate GP in sham-operated group. After electrophysiologic functions were detected, SAN and AVN specimens were cut from dogs in three subgroups of operated group and the specimens were cut directly from 8 dogs in sham-operated group after the electrophysioligic function was observed and the chest was opened. The histological characteristics of SAN and AVN were observed under light microscope after HE staining. Western-blot was not performed to quantify protein in this study because SAN and AVN were surrounded closely or mixed by near tissues and the boundaries were only identified under microscope. Cx43 and Cx45 expression were detected by immunofluorescence in this study. The expression level was represented with mean optical density (MOD). Date analysis:ANOVA (Tukey’s HSD and Dunnett’s T3) was used for comparison of Cx43 and Cx45 expression in SAN and AVN among sham-operated group and different periods in operated group after GP ablation.Results:1. The changes of histological characteristics of SAN and AVN:(1) SAN:the histological characteristics of SAN was compact with conglobate or bunchy cells in the central zone and infiltrated by fatty tissue. The SAN artery was observed in every specimen. Compared with the sham group, there were no obvious differences in the quantity and arrangement of cells at one, six, and twelve months after ablation.(2) AVN:AVN was located at the junction of interatrial septum and interventricular septum, and much fatty tissue infiltrated into the AVN connecting to the central fibrous body (cavity was formed during frozen section preparation). The histological characteristics of AVN after GP ablation were in accordance with those before ablation.2. The changes connexin expression of SAN and AVN:(1) SAN:One month after ablation, Cx43 (0.14±0.04 vs 0.52±0.04, P=0.000) and Cx45 (1.28±0.29 vs 3.17±0.40, P=0.000) expression in SAN were downregulated compared with those in sham-operated group. Cx43 and Cx45 expression had no statistical difference among the levels at six months (Cx43:0.51± 0.03,P= 0.865; Cx45:2.87±1.42, P= 0.179)and twelve months (Cx43:0.48±0.03, P= 0.268; Cx45:3.08±0.20, P=0.933) after ablation and the level in sham-operated group.(2) AVN:One month after ablation, Cx43 (0.35±0.07 vs 1.09±0.17, P=0.000) and Cx45 (2.11±0.42 vs 4.32±0.40, P=0.000) expression in AVN were also downregulated compared with those in sham-operated group. Cx43 and Cx45 expression had no statistical difference among the levels at six months (Cx43:0.94± 0.33, P=0.787; Cx45:4.43±0.62, P=0.969) and twelve months (Cx43:1.16±0.13, P= 0.939; Cx45:4.29±0.45, P=0.999) after ablation and the level in sham-operated group.Conclusion:One month after ARGP and IRGP ablation, Cx43 and Cx45 expression in SAN and AVN were downregulated and reverted to normal level at six months after ablation. The atrioventricular conduction dysfunction might be related to the downregulation of Cx43 and Cx45 expression in AVN. ARGP and IRGP ablation had no long-term effects on the structure and connexin expression in SAN and AVN.