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Sympathetic Hyperactivity and Aortic Sympathetic Nerve Sprouting in Patients with Thoracic Aortic Dissection
Clinical Research Sympathetic Hyperactivity and Aortic Sympathetic Nerve Sprouting in Patients with Thoracic Aortic Dissection Hu Zhipeng,1,* Wang Zhiwei,1,* Yu Lilei,2 Zhang Hao,1 Wu Hongbing,1 Ren Zongli,1 Chen Hao,1 and Hu Xiaoping,1 Wuhan, Hubei Province, China
Background: To determine the yet unknown relation between thoracic aortic dissection (TAD) and sympathetic nervous system activity. Methods: Variables such as electrocardiography, blood pressure, respiratory activity, postganglionic muscle sympathetic nerve activity （MSNA）, plasma norepinephrine, tyrosine hydroxylaseepositive nerve fiber density, and growth-associated protein 43epositive nerve fiber density were detected and statistically analyzed. Results: TAD Patients showed a significant lower R-R interval variance and higher blood pressure, heart rate, respiratory rate, MSNA, plasma norepinephrine (reflecting elevated sympathetic nervous system [SNS] activity), higher tyrosine hydroxylase, growth-associated protein 43epositive nerve fiber density (reflecting sympathetic sprouting and innervation) than those of the control group. Conclusions: In TAD patients, both overall and regional aortic SNS activities are elevated.
INTRODUCTION Thoracic aortic dissection (TAD) is a highly lethal vascular disease. In many patients with TAD, the aorta progressively dilates and ultimately ruptures.1 Several risk factors contributing to TAD, such as cocaine abuse, hypertension, sleep apnea syndrome, and pregnancy,2,3 are all in company Funded by: Nature Science Foundation of Hubei province, 2008CHB421 and 2013CKB031. 1 Department of Cardiothoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China. 2 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China.
*Wang Zhiwei and Hu Zhipeng contributed equally to this work. Correspondence to: Wang Zhiwei, MD, Department of Cardiothoracic Surgery, Renmin Hospital of Wuhan University, 99 Ziyang Road, Wuhan, Hubei Province 430060, China; E-mail: wangzhiwp@ gmail.com Ann Vasc Surg 2014; -: 1–6 http://dx.doi.org/10.1016/j.avsg.2013.11.016 Ó 2014 Elsevier Inc. All rights reserved. Manuscript received: April 10, 2013; manuscript accepted: November 25, 2013; published online: ---.
with sympathetic hyperactivity. This raises a possibility that sympathetic nervous system (SNS) activity abnormity also contributes to TAD formation. Cystic medial degeneration, a key feature of TAD, is typically accompanied by elastic fiber fragmentation, loss of smooth muscle cells (SMCs), and accumulation of amorphous extracellular matrix (ECM) in the aortic wall.4e6 Other diseases such as myocardial hypertrophy after congestive heart failure, hypertrophic cardiomyopathy, and ulcerative colitis also experienced pathologic changes of SMC, ECM, and elastic fiber. Especially, these pathologic changes could be regulated by autonomic nervous system activity.7e9 Such studies hint us that SNS activity may be a regulator of both functional and structural properties of the aorta, and furthermore, SNS activity abnormity may affect the pathologic changes of TAD. This possibility has been partly proven by further study. Thoracic sympathectomy, simulating a regional SNS activity reduction, induces structural and biomechanical remodeling of the thoracic aorta.10 But whether SNS activity is abnormal in TAD is still unclear. 1
2 Zhipeng et al.
This study was, therefore, designed to determine the yet unknown relation between TAD and SNS activity.
MATERIALS AND METHODS Subjects The study protocol was approved by the Medical Ethics Committee of the Renmin Hospital of Wuhan University, and informed consent was obtained from each subject. For comparison 1, 27 patients with TAD (19 men and 8 women) without phenotypic characteristics of any of the known genetic disorders, such as Marfan’s syndrome, were included in the experimental group for detecting overall SNS activity. Control experiments were performed in 14 healthy people (7 men and 7 women), who volunteered to participate in this study. None of the people in the control group was diagnosed for the following diseases or experienced such disease history: hypertension, autonomic nervous functional disorder, rheumatoid diseases, myocardial hypertrophy after any disease, hypertrophic cardiomyopathy, any drug abuse, and sleep apnea syndrome. For comparison 2, full-thickness aortic wall specimens from 19 patients with TAD (14 men and 5 women) in the experiment group of comparison 1 (8 patients with TAD accepted operations without aortic wall resection, so no aorta sample could be obtained from them. Their aorta wall was used to wrap artificial vascular graft and prevent angiorrhea) were obtained and immediately placed in 4% paraformaldehyde for 18e24 hrs. These specimens were then embedded in paraffin, sliced perpendicular to the longitudinal axis of the aorta, and then subjected to H&E or immunohistochemical staining. Abdominal aortic wall specimens from 5 kidney donors (5 men) and 8 thoracic aortic wall specimens from patients (4 men and 4 women) accepted aortic valve replacement surgery were obtained in the control group. Recorded Variables In every subject, electrocardiography, blood pressure, respiratory activity, and postganglionic muscle sympathetic nerve activity (MSNA) were continuously recorded. MSNA was recorded according to the method reported by Furlan et al.6 Plasma Norepinephrine Measurement Venous blood samples for norepinephrine (NE) analysis were collected in ethylene glycol bis(2-
Annals of Vascular Surgery
Table I. Baseline data of study population
Male (n) Female (n) Age (years) Weight (kg) BMI (kg/m2) Hypertension (n) Medication (n) b-Blocker ACEI Nitrate
TAD (n ¼ 27)
CTL (n ¼ 14)
P value
19 8 41 ± 12.66 71 ± 26.44 30±2* 2*
7 7 35 ± 6.18 63 ± 14.56 26 ± 1 0
0.4315 0.4315 0.0415 0.5327 <0.0001 <0.0001
0 0 0
<0.0001 0.0183 <0.0001
25* 4 25*
ACEI, angiotensin-converting enzyme inhibitor; CTL, control group. Values are given as mean ± standard deviation. *P < 0.01.
aminoethyl) tetraacetic acid tubes 30 min after the patients and volunteers woke up in the morning in the recumbent position. Samples were processed immediately in a refrigerated centrifuge. The plasma was stored at 80 C until analysis. Plasma NE was assayed by high-pressure liquid chromatography with electrochemical detection.11 Immunohistochemistry The samples were immunostained for tyrosine hydroxylase (TH, a marker of sympathetic nerve) and for growth-associated protein 43 (GAP43, a marker of active nerve sprouting). Details of the staining techniques have been published previously. Nerve densities were determined by Image-Pro Plus 6.0. Each slide was examined under a microscope to select 3 fields randomly. Nerve density is expressed as the nerve area divided by the total area examined (mm2/mm2). The mean density of nerves in these 3 selected fields was used to represent the nerve density of that slide.12,13 Statistical Analysis Continuous variables are expressed as mean ± standard deviation. The data were analyzed by an unpaired Student’s t-test to determine intergroup differences. Data were analyzed by SPSS 13.0 software. Statistical significance was defined as P < 0.01.
RESULTS Comparison 1: Overall SNS Activity Study Population. Baseline data of the study population in comparison 1 are given in Table I. The TAD group had higher average body mass index (BMI) and higher incidence of hypertension than the
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Table II. Variables indicating overall SNS activity in controls and TAD patients
SBP (mm Hg) DBP (mm Hg) HR (beat/min) RR (cycles/min) RRIV (ms2) MSNA (bursts/min) (bursts/100 beat) PNE (pg/mL)
TAD (n ¼ 27)
CTL (n ¼ 14)
P value
170 ± 43* 120 ± 32* 84 ± 14* 22±3* 1,712 ± 331*
121 71 67 16 2,614
13 12 6 2 499
0.0002 <0.0001 0.0001 <0.0001 <0.0001
17 ± 3.3 25 ± 7.1 251 ± 41
<0.0001 <0.0001 <0.0001
35 ± 4.1* 61 ± 7.7* 369 ± 52*
± ± ± ± ±
DBP, diastolic blood pressure; HR, heart rate; PNE, plasma norepinephrine; RR, respiratory rate; RRIV, R-R interval variance; SBP, systolic blood pressure. *P < 0.01.
control group. Most of the hypertensive patients accepted two or more medication in the TAD group. Variables Indicating Overall SNS Activity. Almost every hypertensive patient with TAD has been treated with antihypertensive drugs before hospitalization. It is meaningless to compare the blood pressure during hospitalization. So the highest blood pressure that the studied subjects have ever experienced was compared. Patients with TAD exhibited significantly higher systolic and diastolic blood pressure than volunteers. Heart rate and respiratory rate were also significantly higher in subjects of TAD group than control group. R-R interval variance of TAD groups was lower than that of volunteers in the control group. MSNA was significantly higher in patients with TAD compared with in the control group. The plasma NE level of patients with TAD was higher than that of volunteers too. (Table II) Subgroup Analysis for Overall SNS Activity in Patients with TAD. Most of the patients with TAD suffered from hypertension. So the sympathetic hyperactivity and aortic sympathetic nerve sprouting may result from hypertension. To rule out such a possibility, we conducted a subgroup comparison between well-controlled and not well-controlled hypertensive TAD patients included. As it is shown in Table III, although the average heart rate of wellcontrolled hypertensive group was greater than that of not well-controlled hypertensive group, there was not a significant difference of average respiratory rate, average R-R interval variance, average MSNA, and average plasma NE between these 2 subgroups. Comparison 2: Regional Sympathetic Innervation of the Aorta Sympathetic Sprouting and Innervation. Figure 1 shows that significant neural remodeling occurred in the TAD group. Both TH-positive (Fig. 1A, B
and E) and GAP43-positive (Fig. 1C, D and E) nerves were far more abundant in the TAD group than in the control group (P < 0.01). Subgroup Analysis for Regional Sympathetic Innervation in Patients with TAD. We also conducted a subgroup analysis for regional sympathetic innervation in patients with TAD included in comparison 2. As it is shown in Table IV, there was not a significant difference of TH + nerve dense or GAP43 + nerve dense between well-controlled and not wellcontrolled hypertensive subgroup.
DISCUSSION The main finding of our study is that patients with TAD were characterized by an overall enhancement of sympathetic activity compared with healthy controls. In addition, patients with TAD exhibited a sympathetic nerve sprouting in the aortic wall. The observation suggests that sympathetic hyperactivity may be involved in the pathologic process of TAD. Several variables such as blood pressure, heart rate, respiratory rate, R-R interval, MSNA, and plasma NE concentration were used for comparing the overall SNS activity. Although blood pressure, heart rate, respiratory rate, and R-R interval are nonspecific, MSNA and plasma NE are good indicators of overall sympathetic activity.14,15 MSNA is a direct measure of sympathetic nervous outflow.14,16,17 Meanwhile, NE is the transmitter of SNS and is an accurate measure of sympathetic activity. Both MSNA and plasma NE indicated that the overall sympathetic activity was elevated in patients with TAD, suggesting that sympathetic hyperactivity might play an important role in the initiation and progress of TAD. Although plasma NE and MSNA are good markers of overall SNS activity, they cannot reflect
4 Zhipeng et al.
Annals of Vascular Surgery
Table III. Subgroup analysis comparing variables indicating overall SNS activity between well-controlled and not well-controlled hypertensive patients (TAD)
Heart rate (beat/min) Respiratory rate (cycles/min) R-R interval variance (ms2) MSNA (bursts/min) (Bursts/100 beat) Plasma norepinephrine (pg/mL)
Well controlled (n ¼ 8)
Not well controlled (n ¼ 16)
P value
72 ± 8.4 19 ± 3 1,936 ± 395
87 ± 12 18 ± 2.2 1,878 ± 549
0.0046* 0.3624 0.7933
34 ± 6.8 62 ± 8.9 331 ± 67
34 ± 8.2 61 ± 13.1 351 ± 62
1.0000 0.8482 0.4756
*P < 0.01.
Fig. 1. TH-positive sympathetic nerves (A, B, and E) and GAP43-positive sympathetic nerves (C, D, and E) in control group and TAD group (40 objective). * denotes P < 0.01.
the regional SNS activity of a certain organ. To overcome this disadvantage, we detected sympathetic innervation of the aortic wall of patients with TAD. Interestingly, marked sympathetic nerve sprouting was exhibited and provided a strong
evidence to the sympathetic hyperactivity in the aortic wall of patients with TAD. Our finding seems to be inconsistent with some former reports. Angouras DC Reported that the porcine aorta exhibited an aneurysm-like change
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Table IV. Subgroup analysis comparing variables indicating regional sympathetic innervation of the aorta between well-controlled and not well-controlled hypertensive TAD patients
2
2
TH + nerve dense (mm /mm ) GAP43 + nerve dense (mm2/mm2)
Well controlled (n ¼ 14)
Not well controlled (n ¼ 5)
P value
42,931 ± 616 44,811 ± 901
43,209 ± 648 45,121 ± 609
0.4152 0.4891
P < 0.01.
3 months after sympathectomy.7 It seems like that sympathetic denervation causes aortic aneurysm-like change. But in fact, sympathetic nerve sprouting and reinnervation will take place after denervation, especially in chronic experimental models. This has been proven in both orthotopic heart transplantation patients and myocardial infarction animal models.18,19 The experiments of Angouras DC did not use sympathetic innervation detection, so we cannot rule out a possibility that the aneurysm-like change resulted from sympathetic reinnervation. The present study is insufficient to explain how SNS activity affects TAD. But former studies have reported that SNS activity could affect several important pathologic processes which are also involved in TAD. For example, in patients with pressureoverload hypertrophy, sympathectomy can blunt myocardial interstitial fibrosis via affect collagen content, MMP Activity, and TIMP Concentration.20 The SNS can also modulate inflammation and remodeling in the hypertensive heart.21 We presume that SNS activity can also affect such pathologic processes during the initiation and progress of TAD. This should be tested by future experiments. Several risk factors of TAD, such as stress, cocaine abuse, intermittent hypoxia and reoxygenation are associated with SNS activity.22e27 So our finding provides a new perspective to understand the mechanism of the promotional effect of these risk factors on TAD. A limitation of this study is abdominal aorta sample was also used as control in comparison 2. How different of sympathetic innervation between thoracic and abdominal aorta in healthy people is unknown, leaving us a risk that the difference of sympathetic innervation may come from an irrelevant control group. Fortunately, 8 thoracic aortic wall specimens from patients accepted aortic valve replacement surgery were also obtained in the control group. The sympathetic innervation of the 8 thoracic aortic wall specimens is roughly similar to that of the 5 abdominal aortic wall specimens. In conclusion, our study shows that in patients with TAD, both overall and regional aortic SNS activities are elevated. Further research is needed to
explore the exact role of sympathetic hyperactivity in the pathologic progress of TAD. REFERENCES 1. Wu D, Shen YH, Russell L, et al. Molecular mechanisms of thoracic aortic dissection. J Surg Res 2013;184:907e24. 2. Greenwood JP, Scott EM, Walker JJ, et al. The magnitude of sympathetic hyperactivity in pregnancy-induced hypertension and preeclampsia. Am J Hypertens 2003;16: 194e9. 3. Paterick TE, Humphries JA, Ammar KA, et al. Aortopathies: etiologies, genetics, differential diagnosis, prognosis and management. Am J Med 2013;126:670e8. 4. Hurley JV. Dissecting aneurysm of the aorta. Histological appearances and an hypothesis of pathogenesis. Australas Ann Med 1959;8:297e306. 5. Ihling C, Szombathy T, Nampoothiri K, et al. Cystic medial degeneration of the aorta is associated with p53 accumulation, Bax upregulation, apoptotic cell death, and cell proliferation. Heart 1999;82:286e93. 6. Isselbacher EM. Thoracic and abdominal aortic aneurysms. Circulation 2005;111:816e28. 7. Esler M, Kaye D. Sympathetic nervous system activation in essential hypertension, cardiac failure and psychosomatic heart disease. J Cardiovasc Pharmacol 2000;35(7 Suppl 4): S1e7. 8. Limbruno U, Strata G, Zucchi R, et al. Altered autonomic cardiac control in hypertrophic cardiomyopathy. Role of outflow tract obstruction and myocardial hypertrophy. Eur Heart J 1998;19:146e53. 9. Furlan R, Ardizzone S, Palazzolo L, et al. Sympathetic overactivity in active ulcerative colitis: effects of clonidine. Am J Physiol Regul Integr Comp Physiol 2006;290:R224e32. 10. Angouras DC, Dosios TJ, Dimitriou CA, et al. Surgical thoracic sympathectomy induces structural and biomechanical remodeling of the thoracic aorta in a porcine model. J Surg Res 2012;172:68e76. 11. May M, Gueler F, Barg-Hock H, et al. Liver afferents contribute to water drinking-induced sympathetic activation in human subjects: a clinical trial. PLoS One 2011;6: e25898. 12. Miyauchi Y, Zhou S, Okuyama Y, et al. Altered atrial electrical restitution and heterogeneous sympathetic hyperinnervation in hearts with chronic left ventricular myocardial infarction: implications for atrial fibrillation. Circulation 2003;108:360e6. 13. Wu X, Jiang H, Yu L, et al. Desipramine pretreatment improves sympathetic remodeling and ventricular fibrillation threshold after myocardial ischemia. J Biomed Biotechnol 2012;2012:732909. 14. Williamson JW, Muzi M, Ebert TJ. Unilateral carotid sinus stimulation and muscle sympathetic nerve activity in man. Med Sci Sports Exerc 1996;28:815e21.
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15. Narkiewicz K, Winnicki M, Schroeder K, et al. Relationship between muscle sympathetic nerve activity and diurnal blood pressure profile. Hypertension 2002;39: 168e72. 16. Snitker S, Pratley RE, Nicolson M, et al. Relationship between muscle sympathetic nerve activity and plasma leptin concentration. Obes Res 1997;5:338e40. 17. Snitker S, Tataranni PA, Ravussin E. Respiratory quotient is inversely associated with muscle sympathetic nerve activity. J Clin Endocrinol Metab 1998;83:3977e9. 18. Kim DT, Luthringer DJ, Lai AC, et al. Sympathetic nerve sprouting after orthotopic heart transplantation. J Heart Lung Transplant 2004;23:1349e58. 19. Oh YS, Jong AY, Kim DT, et al. Spatial distribution of nerve sprouting after myocardial infarction in mice. Heart Rhythm 2006;3:728e36. 20. Perlini S, Palladini G, Ferrero I, et al. Sympathectomy or doxazosin, but not propranolol, blunt myocardial interstitial fibrosis in pressure-overload hypertrophy. Hypertension 2005;46:1213e8. 21. Levick SP, Murray DB, Janicki JS, et al. Sympathetic nervous system modulation of inflammation and
Annals of Vascular Surgery
22.
23.
24.
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27.
remodeling in the hypertensive heart. Hypertension 2010;55:270e6. Liao M, Liu Z, Bao J, et al. A proteomic study of the aortic media in human thoracic aortic dissection: implication for oxidative stress. J Thorac Cardiovasc Surg 2008;136: 65e72. 71-2. Palmiere C, Burkhardt S, Staub C, et al. Thoracic aortic dissection associated with cocaine abuse. Forensic Sci Int 2004;141:137e42. Naito R, Sakakura K, Kasai T, et al. Aortic dissection is associated with intermittent hypoxia and re-oxygenation. Heart Vessels 2012;27:265e70. Wilkinson DJ, Thompson JM, Lambert GW, et al. Sympathetic activity in patients with panic disorder at rest, under laboratory mental stress, and during panic attacks. Arch Gen Psychiatry 1998;55:511e20. Macedo TR, Ribeiro CA, Morgadinho T, et al. Influence of concurrent heroin and cocaine abuse on the adrenergic and serotonergic systems in man. Ann N Y Acad Sci 1998;844:208e13. Narkiewicz K, Somers VK. Sympathetic nerve activity in obstructive sleep apnoea. Acta Physiol Scand 2003;177:385e90.
Background: To determine the yet unknown relation between thoracic aortic dissection (TAD) and sympathetic nervous system activity. Methods: Variables such as electrocardiography, blood pressure, respiratory activity, postganglionic muscle sympathetic nerve activity （MSNA）, plasma norepinephrine, tyrosine hydroxylaseepositive nerve fiber density, and growth-associated protein 43epositive nerve fiber density were detected and statistically analyzed. Results: TAD Patients showed a significant lower R-R interval variance and higher blood pressure, heart rate, respiratory rate, MSNA, plasma norepinephrine (reflecting elevated sympathetic nervous system [SNS] activity), higher tyrosine hydroxylase, growth-associated protein 43epositive nerve fiber density (reflecting sympathetic sprouting and innervation) than those of the control group. Conclusions: In TAD patients, both overall and regional aortic SNS activities are elevated.
INTRODUCTION Thoracic aortic dissection (TAD) is a highly lethal vascular disease. In many patients with TAD, the aorta progressively dilates and ultimately ruptures.1 Several risk factors contributing to TAD, such as cocaine abuse, hypertension, sleep apnea syndrome, and pregnancy,2,3 are all in company Funded by: Nature Science Foundation of Hubei province, 2008CHB421 and 2013CKB031. 1 Department of Cardiothoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China. 2 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China.
*Wang Zhiwei and Hu Zhipeng contributed equally to this work. Correspondence to: Wang Zhiwei, MD, Department of Cardiothoracic Surgery, Renmin Hospital of Wuhan University, 99 Ziyang Road, Wuhan, Hubei Province 430060, China; E-mail: wangzhiwp@ gmail.com Ann Vasc Surg 2014; -: 1–6 http://dx.doi.org/10.1016/j.avsg.2013.11.016 Ó 2014 Elsevier Inc. All rights reserved. Manuscript received: April 10, 2013; manuscript accepted: November 25, 2013; published online: ---.
with sympathetic hyperactivity. This raises a possibility that sympathetic nervous system (SNS) activity abnormity also contributes to TAD formation. Cystic medial degeneration, a key feature of TAD, is typically accompanied by elastic fiber fragmentation, loss of smooth muscle cells (SMCs), and accumulation of amorphous extracellular matrix (ECM) in the aortic wall.4e6 Other diseases such as myocardial hypertrophy after congestive heart failure, hypertrophic cardiomyopathy, and ulcerative colitis also experienced pathologic changes of SMC, ECM, and elastic fiber. Especially, these pathologic changes could be regulated by autonomic nervous system activity.7e9 Such studies hint us that SNS activity may be a regulator of both functional and structural properties of the aorta, and furthermore, SNS activity abnormity may affect the pathologic changes of TAD. This possibility has been partly proven by further study. Thoracic sympathectomy, simulating a regional SNS activity reduction, induces structural and biomechanical remodeling of the thoracic aorta.10 But whether SNS activity is abnormal in TAD is still unclear. 1
2 Zhipeng et al.
This study was, therefore, designed to determine the yet unknown relation between TAD and SNS activity.
MATERIALS AND METHODS Subjects The study protocol was approved by the Medical Ethics Committee of the Renmin Hospital of Wuhan University, and informed consent was obtained from each subject. For comparison 1, 27 patients with TAD (19 men and 8 women) without phenotypic characteristics of any of the known genetic disorders, such as Marfan’s syndrome, were included in the experimental group for detecting overall SNS activity. Control experiments were performed in 14 healthy people (7 men and 7 women), who volunteered to participate in this study. None of the people in the control group was diagnosed for the following diseases or experienced such disease history: hypertension, autonomic nervous functional disorder, rheumatoid diseases, myocardial hypertrophy after any disease, hypertrophic cardiomyopathy, any drug abuse, and sleep apnea syndrome. For comparison 2, full-thickness aortic wall specimens from 19 patients with TAD (14 men and 5 women) in the experiment group of comparison 1 (8 patients with TAD accepted operations without aortic wall resection, so no aorta sample could be obtained from them. Their aorta wall was used to wrap artificial vascular graft and prevent angiorrhea) were obtained and immediately placed in 4% paraformaldehyde for 18e24 hrs. These specimens were then embedded in paraffin, sliced perpendicular to the longitudinal axis of the aorta, and then subjected to H&E or immunohistochemical staining. Abdominal aortic wall specimens from 5 kidney donors (5 men) and 8 thoracic aortic wall specimens from patients (4 men and 4 women) accepted aortic valve replacement surgery were obtained in the control group. Recorded Variables In every subject, electrocardiography, blood pressure, respiratory activity, and postganglionic muscle sympathetic nerve activity (MSNA) were continuously recorded. MSNA was recorded according to the method reported by Furlan et al.6 Plasma Norepinephrine Measurement Venous blood samples for norepinephrine (NE) analysis were collected in ethylene glycol bis(2-
Annals of Vascular Surgery
Table I. Baseline data of study population
Male (n) Female (n) Age (years) Weight (kg) BMI (kg/m2) Hypertension (n) Medication (n) b-Blocker ACEI Nitrate
TAD (n ¼ 27)
CTL (n ¼ 14)
P value
19 8 41 ± 12.66 71 ± 26.44 30±2* 2*
7 7 35 ± 6.18 63 ± 14.56 26 ± 1 0
0.4315 0.4315 0.0415 0.5327 <0.0001 <0.0001
0 0 0
<0.0001 0.0183 <0.0001
25* 4 25*
ACEI, angiotensin-converting enzyme inhibitor; CTL, control group. Values are given as mean ± standard deviation. *P < 0.01.
aminoethyl) tetraacetic acid tubes 30 min after the patients and volunteers woke up in the morning in the recumbent position. Samples were processed immediately in a refrigerated centrifuge. The plasma was stored at 80 C until analysis. Plasma NE was assayed by high-pressure liquid chromatography with electrochemical detection.11 Immunohistochemistry The samples were immunostained for tyrosine hydroxylase (TH, a marker of sympathetic nerve) and for growth-associated protein 43 (GAP43, a marker of active nerve sprouting). Details of the staining techniques have been published previously. Nerve densities were determined by Image-Pro Plus 6.0. Each slide was examined under a microscope to select 3 fields randomly. Nerve density is expressed as the nerve area divided by the total area examined (mm2/mm2). The mean density of nerves in these 3 selected fields was used to represent the nerve density of that slide.12,13 Statistical Analysis Continuous variables are expressed as mean ± standard deviation. The data were analyzed by an unpaired Student’s t-test to determine intergroup differences. Data were analyzed by SPSS 13.0 software. Statistical significance was defined as P < 0.01.
RESULTS Comparison 1: Overall SNS Activity Study Population. Baseline data of the study population in comparison 1 are given in Table I. The TAD group had higher average body mass index (BMI) and higher incidence of hypertension than the
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Table II. Variables indicating overall SNS activity in controls and TAD patients
SBP (mm Hg) DBP (mm Hg) HR (beat/min) RR (cycles/min) RRIV (ms2) MSNA (bursts/min) (bursts/100 beat) PNE (pg/mL)
TAD (n ¼ 27)
CTL (n ¼ 14)
P value
170 ± 43* 120 ± 32* 84 ± 14* 22±3* 1,712 ± 331*
121 71 67 16 2,614
13 12 6 2 499
0.0002 <0.0001 0.0001 <0.0001 <0.0001
17 ± 3.3 25 ± 7.1 251 ± 41
<0.0001 <0.0001 <0.0001
35 ± 4.1* 61 ± 7.7* 369 ± 52*
± ± ± ± ±
DBP, diastolic blood pressure; HR, heart rate; PNE, plasma norepinephrine; RR, respiratory rate; RRIV, R-R interval variance; SBP, systolic blood pressure. *P < 0.01.
control group. Most of the hypertensive patients accepted two or more medication in the TAD group. Variables Indicating Overall SNS Activity. Almost every hypertensive patient with TAD has been treated with antihypertensive drugs before hospitalization. It is meaningless to compare the blood pressure during hospitalization. So the highest blood pressure that the studied subjects have ever experienced was compared. Patients with TAD exhibited significantly higher systolic and diastolic blood pressure than volunteers. Heart rate and respiratory rate were also significantly higher in subjects of TAD group than control group. R-R interval variance of TAD groups was lower than that of volunteers in the control group. MSNA was significantly higher in patients with TAD compared with in the control group. The plasma NE level of patients with TAD was higher than that of volunteers too. (Table II) Subgroup Analysis for Overall SNS Activity in Patients with TAD. Most of the patients with TAD suffered from hypertension. So the sympathetic hyperactivity and aortic sympathetic nerve sprouting may result from hypertension. To rule out such a possibility, we conducted a subgroup comparison between well-controlled and not well-controlled hypertensive TAD patients included. As it is shown in Table III, although the average heart rate of wellcontrolled hypertensive group was greater than that of not well-controlled hypertensive group, there was not a significant difference of average respiratory rate, average R-R interval variance, average MSNA, and average plasma NE between these 2 subgroups. Comparison 2: Regional Sympathetic Innervation of the Aorta Sympathetic Sprouting and Innervation. Figure 1 shows that significant neural remodeling occurred in the TAD group. Both TH-positive (Fig. 1A, B
and E) and GAP43-positive (Fig. 1C, D and E) nerves were far more abundant in the TAD group than in the control group (P < 0.01). Subgroup Analysis for Regional Sympathetic Innervation in Patients with TAD. We also conducted a subgroup analysis for regional sympathetic innervation in patients with TAD included in comparison 2. As it is shown in Table IV, there was not a significant difference of TH + nerve dense or GAP43 + nerve dense between well-controlled and not wellcontrolled hypertensive subgroup.
DISCUSSION The main finding of our study is that patients with TAD were characterized by an overall enhancement of sympathetic activity compared with healthy controls. In addition, patients with TAD exhibited a sympathetic nerve sprouting in the aortic wall. The observation suggests that sympathetic hyperactivity may be involved in the pathologic process of TAD. Several variables such as blood pressure, heart rate, respiratory rate, R-R interval, MSNA, and plasma NE concentration were used for comparing the overall SNS activity. Although blood pressure, heart rate, respiratory rate, and R-R interval are nonspecific, MSNA and plasma NE are good indicators of overall sympathetic activity.14,15 MSNA is a direct measure of sympathetic nervous outflow.14,16,17 Meanwhile, NE is the transmitter of SNS and is an accurate measure of sympathetic activity. Both MSNA and plasma NE indicated that the overall sympathetic activity was elevated in patients with TAD, suggesting that sympathetic hyperactivity might play an important role in the initiation and progress of TAD. Although plasma NE and MSNA are good markers of overall SNS activity, they cannot reflect
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Annals of Vascular Surgery
Table III. Subgroup analysis comparing variables indicating overall SNS activity between well-controlled and not well-controlled hypertensive patients (TAD)
Heart rate (beat/min) Respiratory rate (cycles/min) R-R interval variance (ms2) MSNA (bursts/min) (Bursts/100 beat) Plasma norepinephrine (pg/mL)
Well controlled (n ¼ 8)
Not well controlled (n ¼ 16)
P value
72 ± 8.4 19 ± 3 1,936 ± 395
87 ± 12 18 ± 2.2 1,878 ± 549
0.0046* 0.3624 0.7933
34 ± 6.8 62 ± 8.9 331 ± 67
34 ± 8.2 61 ± 13.1 351 ± 62
1.0000 0.8482 0.4756
*P < 0.01.
Fig. 1. TH-positive sympathetic nerves (A, B, and E) and GAP43-positive sympathetic nerves (C, D, and E) in control group and TAD group (40 objective). * denotes P < 0.01.
the regional SNS activity of a certain organ. To overcome this disadvantage, we detected sympathetic innervation of the aortic wall of patients with TAD. Interestingly, marked sympathetic nerve sprouting was exhibited and provided a strong
evidence to the sympathetic hyperactivity in the aortic wall of patients with TAD. Our finding seems to be inconsistent with some former reports. Angouras DC Reported that the porcine aorta exhibited an aneurysm-like change
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2014
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Table IV. Subgroup analysis comparing variables indicating regional sympathetic innervation of the aorta between well-controlled and not well-controlled hypertensive TAD patients
2
2
TH + nerve dense (mm /mm ) GAP43 + nerve dense (mm2/mm2)
Well controlled (n ¼ 14)
Not well controlled (n ¼ 5)
P value
42,931 ± 616 44,811 ± 901
43,209 ± 648 45,121 ± 609
0.4152 0.4891
P < 0.01.
3 months after sympathectomy.7 It seems like that sympathetic denervation causes aortic aneurysm-like change. But in fact, sympathetic nerve sprouting and reinnervation will take place after denervation, especially in chronic experimental models. This has been proven in both orthotopic heart transplantation patients and myocardial infarction animal models.18,19 The experiments of Angouras DC did not use sympathetic innervation detection, so we cannot rule out a possibility that the aneurysm-like change resulted from sympathetic reinnervation. The present study is insufficient to explain how SNS activity affects TAD. But former studies have reported that SNS activity could affect several important pathologic processes which are also involved in TAD. For example, in patients with pressureoverload hypertrophy, sympathectomy can blunt myocardial interstitial fibrosis via affect collagen content, MMP Activity, and TIMP Concentration.20 The SNS can also modulate inflammation and remodeling in the hypertensive heart.21 We presume that SNS activity can also affect such pathologic processes during the initiation and progress of TAD. This should be tested by future experiments. Several risk factors of TAD, such as stress, cocaine abuse, intermittent hypoxia and reoxygenation are associated with SNS activity.22e27 So our finding provides a new perspective to understand the mechanism of the promotional effect of these risk factors on TAD. A limitation of this study is abdominal aorta sample was also used as control in comparison 2. How different of sympathetic innervation between thoracic and abdominal aorta in healthy people is unknown, leaving us a risk that the difference of sympathetic innervation may come from an irrelevant control group. Fortunately, 8 thoracic aortic wall specimens from patients accepted aortic valve replacement surgery were also obtained in the control group. The sympathetic innervation of the 8 thoracic aortic wall specimens is roughly similar to that of the 5 abdominal aortic wall specimens. In conclusion, our study shows that in patients with TAD, both overall and regional aortic SNS activities are elevated. Further research is needed to
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