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Continuous vagal nerve stimulation affects atrial neural remodeling and reduces atrial fibrillation inducibility in rabbits
Cardiovascular Pathology 24 (2015) 395–398
Contents lists available at ScienceDirect
Cardiovascular Pathology
Original Article
Continuous vagal nerve stimulation affects atrial neural remodeling and reduces atrial fibrillation inducibility in rabbits Yuan Yuan, Zhaolei Jiang, Yi He, Fang-Bao Ding, Shi-Ao Ding, Yang Yang, Ju Mei ⁎ Department of Cardiothoracic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
a r t i c l e
i n f o
Article history: Received 20 June 2015 Received in revised form 17 August 2015 Accepted 17 August 2015 Keywords: Vagal nerve stimulation Atrial fibrillation Atrial neural remodeling
a b s t r a c t Background: The effects of continuous vagal nerve stimulation (VNS) on atrial neural remodeling during atrial fibrillation (AF) remain unclear. Objective: To test the hypothesis that VNS affects atrial neural remodeling and reduces AF inducibility. Methods: Twenty rabbits were randomly divided into two groups: rapid atrial pacing (RAP) group and RAP with VNS group. AF inducibility studies and atrial histologic analyses were performed after 4 weeks. Results: Five rabbits of RAP group (5/10) in the RAP group developed sustained AF. None of rabbits in RAP with VNS group had developed AF. The incidence of sustained AF in VNS group was significant lower than that in rapid pacing group (Pb.01). Treatment with VNS resulted in a significant reduction in atrial neural remodeling and AF duration (Pb.01). Conclusions: Atrial neural remodeling plays an important role in the initiation and maintenance of AF. Modulating autonomic nerve function with VNS can contribute to AF control. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
2. Methods
Atrial fibrillation (AF) is one of the most common cardiac arrhythmias. The number of patients with AF in the United States is anticipated to increase to 9.4–11.7 millions in the year 2030, many of whom will have drug-resistant AF [1]. It is highly desirable to develop a less invasive therapy, which can be easily terminated, without causing permanent damage to the autonomic structures, to treat such a large population of patients with AF. Rapid pacing of left atrial can be used in the animal laboratory as a method to induce or maintain sustained AF [2]. Vanoli et al. [3] showed that chronic vagal nerve stimulation (VNS) can prevent ventricular fibrillation and sudden cardiac death in conscious dogs with a healed myocardial infarction. Plenty of studies have shown that VNS might be used to attenuate heart failure development in dogs [4], rats [5], and humans [6–8]. However, no histological data were presented in that article to document the presence of increased sympathetic innervation. The purpose of the present study was to use immunohistochemical techniques to observe atrial sympathetic nerve density in a rabbit model of persistent AF produced by rapid atrial pacing (RAP). The results were set to investigate the potential new therapeutic options for AF that target atrial neural remodeling.
2.1. Animals
Funding: This work is supported by the Project of Science and Technology Committee of Shanghai (11441900200). ⁎ Corresponding author. E-mail address: [email protected] (J. Mei). http://dx.doi.org/10.1016/j.carpath.2015.08.005 1054-8807/© 2015 Elsevier Inc. All rights reserved.
Thirty New Zealand rabbits, either male or female, weighing about 2.0–2.5 kg, were purchased from the Experimental Animal Department, Shanghai Jiaotong University School of Medicine, and received humane care. The experiment was approved by Shanghai Administrative Committee for Laboratory Animals, and the procedures were carried out strictly in compliance with the guide for the care and use of laboratory animals published by the National Institutes of Health in 1996. Thereafter, the animals were randomly divided into two groups: RAP group (Group P) and RAP with VNS group (Group V). 2.2. RAP models of AF Rabbits were anesthetized with intravenous injection of 3% pentobarbital sodium (30 mg/kg) and were ventilated through a cuffed endotracheal tube with a Bird Mark 8 ventilator. The left thoracic cavity was opened via the third intercostal space, and then the heart was exposed by a dilator. A custom-designed set of electrodes, comprising a pair of electrodes with a distal hook for pacing, were sutured to the epicardial surface of the left atrium (LA). The distal ends of these electrode leads were tunneled subcutaneously and exposed on the back and connected to a pacemaker (output of 6 V with 1.0 ms pulse duration; Guangzhou Academy of Sciences, China). The pacemaker was programmed to provide RAP at 900 ppm, and this pacing rate was maintained continuously for 4 weeks. Rabbits in sham control were operated with the identical surgery procedure but RAP.
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Y. Yuan et al. / Cardiovascular Pathology 24 (2015) 395–398
2.3. VNS For VNS implantation in Group V, a pair of Teflon-coated stainless steel wires (UL1330; Triumph Cable Co, Ltd, Dongguan, China) was looped around the left vagal nerve in the neck. This was connected to the output terminals of the stimulator (BL-420S Data Acquisition and Analysis System; Chengdu TME Technology Co, Ltd) through the same subcutaneous tunnel with the pacing leads, which provides stimulation over a range of frequencies (0.1–100 Hz), strengths (1–10 V), and pulse widths (0.001–10 s). In the present study, the vagal nerve was stimulated with rectangular pulses of 40 ms duration at 1 Hz, 2 V.
was significant lower than those in Group P (Pb .01). VNS treatment resulted in a significant reduction in average AF duration from 1589± 225 s in Group P to 305±54 s in Group V (Pb.001). 3.2. Immunohistochemical studies The nerves immunopositive to GAP43 and TH in the atrium of Group P were significantly higher than that of Group V (Figs. 1 and 2). In Group P, the nerve densities of GAP43 and TH are 6701±1385 μm 2/mm2 and 3750±865 μm 2/mm 2, respectively. In comparison, VNS had reduced the nerve density of GAP43 (839±455 μm 2/mm 2, Pb .001) and TH (494±285 μm 2/mm2, Pb .001).
2.4. ECG recordings 4. Discussion Electrocardiogram (ECG) was recorded before and after the pacing. During the period of pacing, ECG was measured every day to ensure that the pacemakers were working properly. 2.5. AF inducibility studies At the end of the experiment, all rabbits were anesthetized and ventilated. The chest was opened with a midline sternotomy. A pericardial cradle was created at one LA site and one right atrium site. AF inducibility was assessed by single extrastimulus pacing and an atrial burst pacing protocol. Signals were sampled at 2 kHz and stored with the UnEmap system (University of Auckland, New Zealand). A total of 16 burst stimulations were performed for each animal, with each atrial site receiving eight burst pacings (4 for 6 s and 4 for 12 s) at a cycle length of 50 ms and a stimulus output of 0.5 V plus twice the diastolic threshold. AF was considered sustained if the induced episode lasted 30 min. If persistent AF was achieved at any point in the protocol, further testing was not performed, and the longest AF duration was taken as 1800 s. 2.6. Immunohistochemical studies Tissues were obtained from LA at the end of AF inducibility study. The whole procedure was performed in cold conditions. Anti-growthassociated protein 43 (GAP43) antibody and anti-tyrosine hydroxylase (TH) antibody were used for immunohistochemical staining. Nerve densities were determined by a computer-assisted image analysis system (Image-Pro Plus 6.0). Each slide was examined under a microscope with 20× objectives, and three fields were selected randomly with the highest density of nerves. The computer automatically detected the stained nerves in these fields by the brown-stained color and then calculated the area occupied by the nerves in each field. The nerve density was the nerve area divided by the total area examined. The mean density of nerves in these three selected fields was used to represent the nerve density of that slide. 2.7. Statistical analysis All offline measurements were made by investigators blinded to the treatment. Quantitative data were presented as mean±S.D. Statistical product and service solutions (SPSS) 22.0 software package (IBM SPSS Inc, Chicago, USA) was used for statistical analysis. The chi-square test was used to compare categorical variables between the two groups. The Student’s t test was used to compare continuous variables between the two groups. A value of Pb .05 was regarded as statistically significant. 3. Results 3.1. AF inducibility Only rabbits in Group P developed sustained AF (5 of 10). There was no sustained AF in Group V. The incidence of sustained AF in Group V
There is an association between abnormal autonomic innervation and AF in both animal models and humans. The abnormal autonomic innervation may be important in the mechanisms of AF [9,10]. Jayachandran et al. [11] used [C-11] hydroxyephedrine to label sympathetic nerve terminal in dogs with pacing-induced AF and documented heterogeneously increased atrial sympathetic innervation. The increased sympathetic nerve densities were later confirmed by immunohistochemical staining using antibody against TH in dogs with pacing-induced AF [12]. Atrial nerve sprouting and sympathetic hyperinnervation also occur after ventricular myocardial infraction and are associated with increased incidence and duration of AF [13]. Consistent with these results, atrial sympathetic nerve densities are also significantly increased in patients with chronic AF [14]. Olgin et al. [15] reported that sympathetic atrial denervation by phenol creates heterogeneous autonomic innervation, facilitating sustained AF. The present study extended their observations by documenting sympathetic hyperinnervation in the atrial using immunohistochemical techniques. GAP43, a protein expressed in the growth cones of sprouting axons [16], is a marker for nerve spouting. A robust increase of GAP43-positive nerves in rabbits of Group P suggests that nerve sprouting is responsible for the sympathetic hyperinnervation during pacing-induced AF. One possible cause of nerve sprouting in this model is the electrical current, which has been used to induce nerve sprouting in the brain and in the kindling model of seizure disorder [17]. However, we do not have sufficient data from this study to test that hypothesis. It is also unclear whether neural remodeling is causally related to the pathogenesis of AF. Adrenergic stimulation in the electrically remodeled myocardium increases significant electrophysiological changes and may be proarrhythmic [18,19]. Sympathetic nerve sprouting and hyperinnervation may strengthen this interaction and contribute to the generation and maintenance of AF. Although RAP resulted in an increase in AF duration, treatment with VNS prevented this change. This effect is consistent with the immunohistochemical finding of increased nerve sprouting and sympathetic hyperinnervation in the Group P that was markedly attenuated with VNS. It is highly desirable to develop a neuromodulation method targeted for sympathetic hyperinnervation in AF. VNS can be used in the animal laboratory as a method to induce or maintain sustained AF [20]. Recently, Shen et al. [21] showed that, in ambulatory dogs, continuous left low-level VNS effectively suppresses left stellate ganglion nerve activity and significantly reduces the frequency of paroxysmal atrial tachyarrhythmia in paroxysmal AF dog model. Significant neural remodeling of left stellate ganglion was evident. Kusunose et al. [22] found that VNS improved LA function and volumes and suppressed LA fibrosis in the dog highrate ventricular pacing model. The neural remodeling findings of atrium after VNS were never documented before. In the present study, we evaluated the effects of VNS treatment on AF inducibility in the rabbit AF model induced by RAP. Our findings show that
Y. Yuan et al. / Cardiovascular Pathology 24 (2015) 395–398
397
Fig. 1. GAP43 staining of cardiac nerve (brown twigs) in RAP group (A) could be found significantly higher than that in VNS group (C) at low magnification. In RAP group, GAP43-positive nerves could confluent into a large area (B), which could hardly be observed in VNS group (D) [(A) 20×, (B) 40×, (C) 20×, and (D) 40×].
treatment of RAP rabbits with VNS resulted in a marked reduction in AF duration, but the AF inducibility was still significantly greater than that in controls. These findings were associated with less atrial nerve sprouting and sympathetic hyperinnervation in Group V, implicating a possible mechanism that may contribute to a better preservation of AF inducibility.
5. Study limitations Our main limitation was that we did not perform functional electrophysiological measurements. Therefore, the refractory period and the dispersion of refractoriness are not available for comparison with the magnitude of nerve sprouting.
Fig. 2. TH staining of cardiac nerve (brown twigs) could be could be found significantly increased in RAP group (A), which was much weaker or negative in VNS group (C). (B) and (D) show the TH staining at high magnification [(A) 20×, (B) 40×, (C) 20×, and (D) 40×].
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Y. Yuan et al. / Cardiovascular Pathology 24 (2015) 395–398
The second limitation of this study was that we were unable to perform histological inflammation or neurotransmitters analysis in the LA to enable the mechanistic insight into VNS effects. 6. Conclusions Significant nerve sprouting and sympathetic hyperinnervation are present in a rabbit model of persistent AF produced by RAP. VNS treatment results in a marked attenuation of atrial neural remodeling by suppressing the sympathetic innervation, with reduction AF inducibility. Modulating autonomic nerve function such as VNS can contribute to AF control.
References [1] Miyasaka Y, Barnes ME, Gersh BJ, Cha SS, Bailey KR, Abhayaratna WP, et al. Secular trends in incidence of atrial fibrillation in Olmsted County, Minnesota, 1980 to 2000, and implications on the projections for future prevalence. Circulation 2006; 114:119–25. [2] Goldberger AL, Pavelec RS. Vagally-mediated atrial fibrillation in dogs: conversion with bretylium tosylate. Int J Cardiol 1986;13:47–55. [3] Vanoli E, De Ferrari GM, Stramba-Badiale M, Hull SS, Foreman RD, Schwartz PJ. Vagal stimulation and prevention of sudden death in conscious dogs with a healed myocardial infarction. Circ Res 1991;68:1471–81. [4] Zhang Y, Popovic ZB, Bibevski S, Fakhry I, Sica DA, Van Wagoner DR, et al. Chronic vagus nerve stimulation improves autonomic control and attenuates systemic inflammation and heart failure progression in a canine high-rate pacing model. Circ Heart Fail 2009;2:692–9. [5] Li M, Zheng C, Sato T, Kawada T, Sugimachi M, Sunagawa K. Vagal nerve stimulation markedly improves long-term survival after chronic heart failure in rats. Circulation 2004;109:120–4. [6] De Ferrari GM, Crijns HJ, Borggrefe M, Milasinovic G, Smid J, Zabel M, et al. Chronic vagus nerve stimulation: a new and promising therapeutic approach for chronic heart failure. Eur Heart J 2011;32:847–55. [7] De Ferrari GM, Sanzo A, Schwartz PJ. Chronic vagal stimulation in patients with congestive heart failure. Conf Proc IEEE Eng Med Biol Soc 2009;2009:2037–9.
[8] De Ferrari GM, Schwartz PJ. Vagus nerve stimulation: from pre-clinical to clinical application: challenges and future directions. Heart Fail Rev 2011;16:195–203. [9] Arora R. Recent insights into the role of the autonomic nervous system in the creation of substrate for atrial fibrillation: implications for therapies targeting the atrial autonomic nervous system. Circ Arrhythm Electrophysiol 2012;5:850–9. [10] Volders PG. Novel insights into the role of the sympathetic nervous system in cardiac arrhythmogenesis. Heart Rhythm 2010;7:1900–6. [11] Jayachandran JV, Sih HJ, Winkle W, Zipes DP, Hutchins GD, Olgin JE. Atrial fibrillation produced by prolonged rapid atrial pacing is associated with heterogeneous changes in atrial sympathetic innervation. Circulation 2000;101:1185–91. [12] Chang CM, Wu TJ, Zhou S, Doshi RN, Lee MH, Ohara T, et al. Nerve sprouting and sympathetic hyperinnervation in a canine model of atrial fibrillation produced by prolonged right atrial pacing. Circulation 2001;103:22–5. [13] Miyauchi Y, Zhou S, Okuyama Y, Miyauchi M, Hayashi H, Hamabe A, et al. Altered atrial electrical restitution and heterogeneous sympathetic hyperinnervation in hearts with chronic left ventricular myocardial infarction: implications for atrial fibrillation. Circulation 2003;108:360–6. [14] Nguyen BL, Fishbein MC, Chen LS, Chen PS, Masroor S. Histopathological substrate for chronic atrial fibrillation in humans. Heart Rhythm 2009;6:454–60. [15] Olgin JE, Sih HJ, Hanish S, Jayachandran JV, Wu J, Zheng QH, et al. Heterogeneous atrial denervation creates substrate for sustained atrial fibrillation. Circulation 1998;98:2608–14. [16] Meiri KF, Pfenninger KH, Willard MB. Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones. Proc Natl Acad Sci U S A 1986;83:3537–41. [17] Sutula T, He XX, Cavazos J, Scott G. Synaptic reorganization in the hippocampus induced by abnormal functional activity. Science 1988;239:1147–50. [18] Cao JM, Chen LS, KenKnight BH, Ohara T, Lee MH, Tsai J, et al. Nerve sprouting and sudden cardiac death. Circ Res 2000;86:816–21. [19] Cao JM, Fishbein MC, Han JB, Lai WW, Lai AC, Wu TJ, et al. Relationship between regional cardiac hyperinnervation and ventricular arrhythmia. Circulation 2000; 101:1960–9. [20] Wang Z, Page P, Nattel S. Mechanism of flecainide’s antiarrhythmic action in experimental atrial fibrillation. Circ Res 1992;71:271–87. [21] Shen MJ, Shinohara T, Park HW, Frick K, Ice DS, Choi EK, et al. Continuous lowlevel vagus nerve stimulation reduces stellate ganglion nerve activity and paroxysmal atrial tachyarrhythmias in ambulatory canines. Circulation 2011;123: 2204–12. [22] Kusunose K, Zhang Y, Mazgalev TN, Van Wagoner DR, Thomas JD, Popovic ZB. Impact of vagal nerve stimulation on left atrial structure and function in a canine high-rate pacing model. Circ Heart Fail 2014;7:320–6.
Contents lists available at ScienceDirect
Cardiovascular Pathology
Original Article
Continuous vagal nerve stimulation affects atrial neural remodeling and reduces atrial fibrillation inducibility in rabbits Yuan Yuan, Zhaolei Jiang, Yi He, Fang-Bao Ding, Shi-Ao Ding, Yang Yang, Ju Mei ⁎ Department of Cardiothoracic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
a r t i c l e
i n f o
Article history: Received 20 June 2015 Received in revised form 17 August 2015 Accepted 17 August 2015 Keywords: Vagal nerve stimulation Atrial fibrillation Atrial neural remodeling
a b s t r a c t Background: The effects of continuous vagal nerve stimulation (VNS) on atrial neural remodeling during atrial fibrillation (AF) remain unclear. Objective: To test the hypothesis that VNS affects atrial neural remodeling and reduces AF inducibility. Methods: Twenty rabbits were randomly divided into two groups: rapid atrial pacing (RAP) group and RAP with VNS group. AF inducibility studies and atrial histologic analyses were performed after 4 weeks. Results: Five rabbits of RAP group (5/10) in the RAP group developed sustained AF. None of rabbits in RAP with VNS group had developed AF. The incidence of sustained AF in VNS group was significant lower than that in rapid pacing group (Pb.01). Treatment with VNS resulted in a significant reduction in atrial neural remodeling and AF duration (Pb.01). Conclusions: Atrial neural remodeling plays an important role in the initiation and maintenance of AF. Modulating autonomic nerve function with VNS can contribute to AF control. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
2. Methods
Atrial fibrillation (AF) is one of the most common cardiac arrhythmias. The number of patients with AF in the United States is anticipated to increase to 9.4–11.7 millions in the year 2030, many of whom will have drug-resistant AF [1]. It is highly desirable to develop a less invasive therapy, which can be easily terminated, without causing permanent damage to the autonomic structures, to treat such a large population of patients with AF. Rapid pacing of left atrial can be used in the animal laboratory as a method to induce or maintain sustained AF [2]. Vanoli et al. [3] showed that chronic vagal nerve stimulation (VNS) can prevent ventricular fibrillation and sudden cardiac death in conscious dogs with a healed myocardial infarction. Plenty of studies have shown that VNS might be used to attenuate heart failure development in dogs [4], rats [5], and humans [6–8]. However, no histological data were presented in that article to document the presence of increased sympathetic innervation. The purpose of the present study was to use immunohistochemical techniques to observe atrial sympathetic nerve density in a rabbit model of persistent AF produced by rapid atrial pacing (RAP). The results were set to investigate the potential new therapeutic options for AF that target atrial neural remodeling.
2.1. Animals
Funding: This work is supported by the Project of Science and Technology Committee of Shanghai (11441900200). ⁎ Corresponding author. E-mail address: [email protected] (J. Mei). http://dx.doi.org/10.1016/j.carpath.2015.08.005 1054-8807/© 2015 Elsevier Inc. All rights reserved.
Thirty New Zealand rabbits, either male or female, weighing about 2.0–2.5 kg, were purchased from the Experimental Animal Department, Shanghai Jiaotong University School of Medicine, and received humane care. The experiment was approved by Shanghai Administrative Committee for Laboratory Animals, and the procedures were carried out strictly in compliance with the guide for the care and use of laboratory animals published by the National Institutes of Health in 1996. Thereafter, the animals were randomly divided into two groups: RAP group (Group P) and RAP with VNS group (Group V). 2.2. RAP models of AF Rabbits were anesthetized with intravenous injection of 3% pentobarbital sodium (30 mg/kg) and were ventilated through a cuffed endotracheal tube with a Bird Mark 8 ventilator. The left thoracic cavity was opened via the third intercostal space, and then the heart was exposed by a dilator. A custom-designed set of electrodes, comprising a pair of electrodes with a distal hook for pacing, were sutured to the epicardial surface of the left atrium (LA). The distal ends of these electrode leads were tunneled subcutaneously and exposed on the back and connected to a pacemaker (output of 6 V with 1.0 ms pulse duration; Guangzhou Academy of Sciences, China). The pacemaker was programmed to provide RAP at 900 ppm, and this pacing rate was maintained continuously for 4 weeks. Rabbits in sham control were operated with the identical surgery procedure but RAP.
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Y. Yuan et al. / Cardiovascular Pathology 24 (2015) 395–398
2.3. VNS For VNS implantation in Group V, a pair of Teflon-coated stainless steel wires (UL1330; Triumph Cable Co, Ltd, Dongguan, China) was looped around the left vagal nerve in the neck. This was connected to the output terminals of the stimulator (BL-420S Data Acquisition and Analysis System; Chengdu TME Technology Co, Ltd) through the same subcutaneous tunnel with the pacing leads, which provides stimulation over a range of frequencies (0.1–100 Hz), strengths (1–10 V), and pulse widths (0.001–10 s). In the present study, the vagal nerve was stimulated with rectangular pulses of 40 ms duration at 1 Hz, 2 V.
was significant lower than those in Group P (Pb .01). VNS treatment resulted in a significant reduction in average AF duration from 1589± 225 s in Group P to 305±54 s in Group V (Pb.001). 3.2. Immunohistochemical studies The nerves immunopositive to GAP43 and TH in the atrium of Group P were significantly higher than that of Group V (Figs. 1 and 2). In Group P, the nerve densities of GAP43 and TH are 6701±1385 μm 2/mm2 and 3750±865 μm 2/mm 2, respectively. In comparison, VNS had reduced the nerve density of GAP43 (839±455 μm 2/mm 2, Pb .001) and TH (494±285 μm 2/mm2, Pb .001).
2.4. ECG recordings 4. Discussion Electrocardiogram (ECG) was recorded before and after the pacing. During the period of pacing, ECG was measured every day to ensure that the pacemakers were working properly. 2.5. AF inducibility studies At the end of the experiment, all rabbits were anesthetized and ventilated. The chest was opened with a midline sternotomy. A pericardial cradle was created at one LA site and one right atrium site. AF inducibility was assessed by single extrastimulus pacing and an atrial burst pacing protocol. Signals were sampled at 2 kHz and stored with the UnEmap system (University of Auckland, New Zealand). A total of 16 burst stimulations were performed for each animal, with each atrial site receiving eight burst pacings (4 for 6 s and 4 for 12 s) at a cycle length of 50 ms and a stimulus output of 0.5 V plus twice the diastolic threshold. AF was considered sustained if the induced episode lasted 30 min. If persistent AF was achieved at any point in the protocol, further testing was not performed, and the longest AF duration was taken as 1800 s. 2.6. Immunohistochemical studies Tissues were obtained from LA at the end of AF inducibility study. The whole procedure was performed in cold conditions. Anti-growthassociated protein 43 (GAP43) antibody and anti-tyrosine hydroxylase (TH) antibody were used for immunohistochemical staining. Nerve densities were determined by a computer-assisted image analysis system (Image-Pro Plus 6.0). Each slide was examined under a microscope with 20× objectives, and three fields were selected randomly with the highest density of nerves. The computer automatically detected the stained nerves in these fields by the brown-stained color and then calculated the area occupied by the nerves in each field. The nerve density was the nerve area divided by the total area examined. The mean density of nerves in these three selected fields was used to represent the nerve density of that slide. 2.7. Statistical analysis All offline measurements were made by investigators blinded to the treatment. Quantitative data were presented as mean±S.D. Statistical product and service solutions (SPSS) 22.0 software package (IBM SPSS Inc, Chicago, USA) was used for statistical analysis. The chi-square test was used to compare categorical variables between the two groups. The Student’s t test was used to compare continuous variables between the two groups. A value of Pb .05 was regarded as statistically significant. 3. Results 3.1. AF inducibility Only rabbits in Group P developed sustained AF (5 of 10). There was no sustained AF in Group V. The incidence of sustained AF in Group V
There is an association between abnormal autonomic innervation and AF in both animal models and humans. The abnormal autonomic innervation may be important in the mechanisms of AF [9,10]. Jayachandran et al. [11] used [C-11] hydroxyephedrine to label sympathetic nerve terminal in dogs with pacing-induced AF and documented heterogeneously increased atrial sympathetic innervation. The increased sympathetic nerve densities were later confirmed by immunohistochemical staining using antibody against TH in dogs with pacing-induced AF [12]. Atrial nerve sprouting and sympathetic hyperinnervation also occur after ventricular myocardial infraction and are associated with increased incidence and duration of AF [13]. Consistent with these results, atrial sympathetic nerve densities are also significantly increased in patients with chronic AF [14]. Olgin et al. [15] reported that sympathetic atrial denervation by phenol creates heterogeneous autonomic innervation, facilitating sustained AF. The present study extended their observations by documenting sympathetic hyperinnervation in the atrial using immunohistochemical techniques. GAP43, a protein expressed in the growth cones of sprouting axons [16], is a marker for nerve spouting. A robust increase of GAP43-positive nerves in rabbits of Group P suggests that nerve sprouting is responsible for the sympathetic hyperinnervation during pacing-induced AF. One possible cause of nerve sprouting in this model is the electrical current, which has been used to induce nerve sprouting in the brain and in the kindling model of seizure disorder [17]. However, we do not have sufficient data from this study to test that hypothesis. It is also unclear whether neural remodeling is causally related to the pathogenesis of AF. Adrenergic stimulation in the electrically remodeled myocardium increases significant electrophysiological changes and may be proarrhythmic [18,19]. Sympathetic nerve sprouting and hyperinnervation may strengthen this interaction and contribute to the generation and maintenance of AF. Although RAP resulted in an increase in AF duration, treatment with VNS prevented this change. This effect is consistent with the immunohistochemical finding of increased nerve sprouting and sympathetic hyperinnervation in the Group P that was markedly attenuated with VNS. It is highly desirable to develop a neuromodulation method targeted for sympathetic hyperinnervation in AF. VNS can be used in the animal laboratory as a method to induce or maintain sustained AF [20]. Recently, Shen et al. [21] showed that, in ambulatory dogs, continuous left low-level VNS effectively suppresses left stellate ganglion nerve activity and significantly reduces the frequency of paroxysmal atrial tachyarrhythmia in paroxysmal AF dog model. Significant neural remodeling of left stellate ganglion was evident. Kusunose et al. [22] found that VNS improved LA function and volumes and suppressed LA fibrosis in the dog highrate ventricular pacing model. The neural remodeling findings of atrium after VNS were never documented before. In the present study, we evaluated the effects of VNS treatment on AF inducibility in the rabbit AF model induced by RAP. Our findings show that
Y. Yuan et al. / Cardiovascular Pathology 24 (2015) 395–398
397
Fig. 1. GAP43 staining of cardiac nerve (brown twigs) in RAP group (A) could be found significantly higher than that in VNS group (C) at low magnification. In RAP group, GAP43-positive nerves could confluent into a large area (B), which could hardly be observed in VNS group (D) [(A) 20×, (B) 40×, (C) 20×, and (D) 40×].
treatment of RAP rabbits with VNS resulted in a marked reduction in AF duration, but the AF inducibility was still significantly greater than that in controls. These findings were associated with less atrial nerve sprouting and sympathetic hyperinnervation in Group V, implicating a possible mechanism that may contribute to a better preservation of AF inducibility.
5. Study limitations Our main limitation was that we did not perform functional electrophysiological measurements. Therefore, the refractory period and the dispersion of refractoriness are not available for comparison with the magnitude of nerve sprouting.
Fig. 2. TH staining of cardiac nerve (brown twigs) could be could be found significantly increased in RAP group (A), which was much weaker or negative in VNS group (C). (B) and (D) show the TH staining at high magnification [(A) 20×, (B) 40×, (C) 20×, and (D) 40×].
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Y. Yuan et al. / Cardiovascular Pathology 24 (2015) 395–398
The second limitation of this study was that we were unable to perform histological inflammation or neurotransmitters analysis in the LA to enable the mechanistic insight into VNS effects. 6. Conclusions Significant nerve sprouting and sympathetic hyperinnervation are present in a rabbit model of persistent AF produced by RAP. VNS treatment results in a marked attenuation of atrial neural remodeling by suppressing the sympathetic innervation, with reduction AF inducibility. Modulating autonomic nerve function such as VNS can contribute to AF control.
References [1] Miyasaka Y, Barnes ME, Gersh BJ, Cha SS, Bailey KR, Abhayaratna WP, et al. Secular trends in incidence of atrial fibrillation in Olmsted County, Minnesota, 1980 to 2000, and implications on the projections for future prevalence. Circulation 2006; 114:119–25. [2] Goldberger AL, Pavelec RS. Vagally-mediated atrial fibrillation in dogs: conversion with bretylium tosylate. Int J Cardiol 1986;13:47–55. [3] Vanoli E, De Ferrari GM, Stramba-Badiale M, Hull SS, Foreman RD, Schwartz PJ. Vagal stimulation and prevention of sudden death in conscious dogs with a healed myocardial infarction. Circ Res 1991;68:1471–81. [4] Zhang Y, Popovic ZB, Bibevski S, Fakhry I, Sica DA, Van Wagoner DR, et al. Chronic vagus nerve stimulation improves autonomic control and attenuates systemic inflammation and heart failure progression in a canine high-rate pacing model. Circ Heart Fail 2009;2:692–9. [5] Li M, Zheng C, Sato T, Kawada T, Sugimachi M, Sunagawa K. Vagal nerve stimulation markedly improves long-term survival after chronic heart failure in rats. Circulation 2004;109:120–4. [6] De Ferrari GM, Crijns HJ, Borggrefe M, Milasinovic G, Smid J, Zabel M, et al. Chronic vagus nerve stimulation: a new and promising therapeutic approach for chronic heart failure. Eur Heart J 2011;32:847–55. [7] De Ferrari GM, Sanzo A, Schwartz PJ. Chronic vagal stimulation in patients with congestive heart failure. Conf Proc IEEE Eng Med Biol Soc 2009;2009:2037–9.
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