The aim of this article is to summarize the role of sympathetic nervous system (SNS) in cardiovascular pathophysiology and describe how methods for studying sympathetic activity influence clinical practice. There is no doubt about the great increase in the knowledge on neuroregulation of cardiovascular system during last 3 decades. Methods such as microneurography, direct catecholamine measurements, heart rate variability or baroreflex sensitivity assessment allowed studying pathophysiology of cardiovascular diseases. What is most important, data obtained using those methods changed clinical practice. Some of them were also introduced into clinic as a diagnostic tool.

Studies on SNS changed clinical practice

Cardiovascular diseases are the most frequent causes of morbidity and mortality around the world. However, during last decades, an improvement was made in diagnosis and therapy of cardiovascular diseases, there was still a need for better understanding of their pathophysiology. Among neurohormonal systems, SNS plays a central role in cardiovascular regulation in both health and disease (1 - 5). Activation of SNS can increase peripheral vascular resistance and cardiac output to raise blood pressure. Arteriolar vasoconstriction, as well as sympathetic mediated venoconstriction with consequent central redistribution of blood, both acts to increase blood pressure. Cardiac sympathetic chronotropic and inotropic effects also increase blood pressure, particularly in the setting of increased vascular resistance. Thus, increased sympathetic traffic to the peripheral vasculature and sympathetic discharge to the heart exert complementary effects on blood pressure. Activation of SNS may also contribute to blood pressure levels in the long term by other mechanisms. Effects of sympathetic activation on the kidney, renin-angiotensin system, blood vessel growth and permeability as well as resetting of the arterial baroreflex should be mentioned.

In a few past decades, convincing data were collected to support theory that enhanced sympathetic activity was involved in pathogenesis of many cardiovascular diseases. Numerous data from various observations overwhelmingly attest to the importance of the SNS in essential hypertension, particularly in its early stages (2, 3). Increased sympathetic tone was also proved to promote development and progression of hypertension related complications that led to increased cardiovascular morbidity and mortality. Different techniques were involved to quantify sympathetic cardiovascular effects in humans with essential hypertension. First, tachycardia is the simplest and probably the most reliable marker of sympathetic overactivity in humans with hypertension. An association of tachycardia with higher blood pressure has been found in numerous investigations and both a simultaneous elevation of the heart rate and plasma catecholamines were reported in hypertensives. In some observations, it was described that in normotensive subjects tachycardia might predict development of future hypertension. Also, an association of tachycardia and increased cardiac output was found as a characteristic feature of early stages of essential hypertension. Secondly, important information regarding sympathetic activity in hypertension has come from techniques that assay in a sensitive fashion plasma level of sympathetic neurotransmitter - noradrenaline. Although a number of early comparisons between normotensive and hypertensive individuals led to equivocal results, a meta-analyses of published data did show that essential hypertensive patients displayed greater plasma noradrenaline values than normotensives (6). Thirdly, other biochemical and neurophysiological approaches, such as the noradrenaline radiolabeled technique and the microneurography, have provided further evidence of sympathetic overactivity in hypertensive individuals. In some studies, use of the noradrenaline - radiolabeled tracer, which estimates the secretion of noradrenaline from the sympathetic nerve terminals, confirmed a greater sympathetic activity in young hypertensive subjects as compared to age-matched normotensive individuals (7). An introduction of the microneurography to scientific routine has provided further data that showed an increase of sympathetic drive in essential hypertension. Microneurography was also used to demonstrate sympathetic enhancement during subsequent stages of hypertension. It was confirmed that sympathetic activity might be increased in normotensive subjects with a family history of hypertension and especially in subjects with borderline hypertension as compared to normotensive subjects (8, 9) and might progressively increase. In addition, an increase in sympathetic traffic was presented also in older patients with isolated systolic hypertension (10).

Knowledge regarding enhanced sympathetic activity in patients with essential hypertension has many practical implementations and is currently employed in daily clinical routine. Common use of antiadrenergic agents in therapy of hypertension might be an example.

Apart of hypertension, sympathetic overactivity has been implicated in the pathogenesis of other diseases as metabolic disorders, coronary artery disease, cardiac arrhythmias or heart failure. Studies using microneurography have consistently shown increased muscle sympathetic nerve activity in obese subjects. Earlier data based on plasma catecholamine measurements and whole-body and regional noradrenaline release revealed inconsistient results (11, 12). However, recent observations suggest that obesity in humans is associated with increased sympathetic outflow and that body fat is a major determinant of sympathetic neural discharge. Moreover, sympathetic overactivity is also involved in pathogenesis of metabolic syndrome. Primarily enhanced sympathetic drive can produce vasoconstriction, diminish the regional blood flow and tissue glucose delivery, and thus generate insulin resistance, a key phenomenon in pathogenesis of many metabolic and cardiovascular disorders. Some other mechanisms of sympathetic influence on insulin resistance are also described. Activation of adrenergic peripheral ß-receptors changes proportion between slow and fast twitch muscle fibers and decreases number of small blood vessels in the skeletal muscles (13).

Increased sympathetic drive in hypertensive subjects may be independently implicated in atherosclerotic vascular disease, especially coronary artery disease and associated fatal cardiovascular events. Although coronary artery disease has a multifactorial origin sympathetic overdrive can be crucial for its development. Increased sympathetic activity induces vascular and cardiac hypertrophy, produces coronary vasoconstriction and increases cardiac oxygen consumption. Procoagulative state, activation of platelets, increased hematocrit level and endothelial dysfunction are also well recognized results of sympathetic overactivity (13). In addition an imbalance between sympathetic and parasympathetic system as well as sympathetically mediated disturbances in electrolytes may exert proarrhytmic action and induce life frightening arrhythmias. Another evidence for a role of sympathetic overactivity in ischemic heart disease is the efficacy of pharmacological ß-blockade in decreasing cardiac death in patients after myocardial infarction (14). Numerous studies clearly showed that sympathetic activity was more pronounced after myocardial infarction than after unstable angina, while sympathetic activity in patients with stable angina did not differ from that in control subjects (15). This observation may at least in part explain why patients with both myocardial infarction and unstable ischemic syndromes are at increased risk of sudden cardiac death.

There is also a growing body of evidence that elevated sympathetic activity plays an important role in the pathophysiology of congestive heart failure. Early studies has shown an increased plasma noradrenalin concentration and total, cardiac and renal noradrenaline spillover in patients with congestive heart failure (16). Later studies demonstrated that prognosis in cardiac failure was directly linked to the level of activation of the SNS and most strongly with that in the high sympathetic outflow to the heart (17, 18). Finally, one of the major advances in cardiology of the past years has been the successful introduction of ß-adrenergic drugs to the therapy of cardiac failure, which has substantially improved the clinical outcome in patients with congestive heart failure.


METHODS OF EVALUATION OF THE SYMPATHETIC NERVOUS SYSTEM
Measurements of urine and plasma noradrenaline

Traditionally, activity of the SNS was assessed using measurements of urine noradrenaline and adrenaline or their precursors and metabolites. However, this "static" approach cannot provide reliable assessment of short-term changes in sympathetic activity and, therefore, has been replaced by measurement of plasma noradrenaline concentration. These measurements provide useful information, but also have significant limitations (19). First, circulating noradrenaline represents only a small fraction (5 - 10%) of the amount of neurotransmitter secreted from nerve terminals. Second, plasma levels of noradrenaline are influenced, in addition to the level of sympathetic neural outflow, by prejunctional modulation of neurotransmitter release, as well as the clearance, metabolism and uptake of noradrenaline from the circulation. Thus, plasma measurements do not allow discrimination between central (increased secretion) and peripheral (reduced clearance) mechanisms of inreased levels of the neurotransmitter (5). Third, the use of plasma noradrenaline is based on the assumption that these measurements reflect "overall" sympathetic activity. Contrary to this assumption, there are profound regional differences in the activity and control of sympathetic function. Furthermore, the reproducibility and sensitivity of plasma noradrenaline values are lower than those of microneurographic recordings (20).

Value of plasma catecholamines measurement is increased if it is combined with assessment of responses to adrenergic antagonists and agonists. Using this approach, it has been shown that mildly hypertensive individuals had elevated plasma noradrenaline levels, augmented decreases in vascular resistance in response to a-adrenergic blockade, and no increase in alpha-receptor sensitivity as estimated by responses to noradrenaline (21). This study demonstrated augmented sympathetic vasoconstrictor activity in young mildly hypertensive humans, suggesting that increased sympathetic vasoconstriction results from enhanced sympathetic neural release of noradrenaline, and not from augmented a-adrenergic response to the neurotransmitter.

Noradrenaline spillover rate measurements

The noradrenaline radiolabeled method is based on intravenous infusion of small amounts of tritiated noradrenaline, which allows tissue clearance of this substance to be subtracted from plasma noradrenaline values and to make the remainder a marker of the neurotransmitter "spillover" from the neuroeffector junctions. This "spillover" in steady-state conditions mirrors the secretion of noradrenaline from the sympathetic nerve terminals. The noradrenaline "spillover" technique avoids the confounding influence of neurotransmitter clearance and permits assessment of noradrenaline release from specific target organs (22). Hypertension, in particular "early" hypertension, may be characterized by increased sympathetic traffic not only to the heart and blood vessels, but also to the kidneys. Using measurements of noradrenaline spillover, Esler et al. (23) found that noradrenaline release was elevated in hypertensive patients, particularly in young hypertensives, and that the increased spillover occurred mainly from the heart and kidneys.

Using jugular vein noradrenaline spillover measurements, Ferrier et al. (24) have reported that higher sympathetic activity in hypertension may be explained by increased cerebral noradrenaline release, mostly from subcortical forebrain regions. The same group of investigators subsequently reported that subcortical noradrenaline release was linked with both total body noradrenaline spillover as well as renal noradrenaline spillover (25). Since the forebrain is involved in the emotional responses (especially the defense reaction) it has been suggested that increased noradrenaline spillover from certain subcortical regions may represent a neurochemical manifestation of stress.

Quantitative assessment of tritiated noradrenaline uptake from plasma demonstrated impairment of noradrenaline transporter function in essential hypertension (26). The potential role of impaired neuronal noradrenaline reuptake can be directly assessed by infusion of the noradrenaline transport inhibitor desipramine (27). Finally, noradrenaline stores in the human heart could be estimated by quantifying the processing inside sympathetic nerves of tritiated noradrenaline to its intraneuronal metabolite, dihydroxyphenylglycol (DHPG), coupled with measurement of DHPG in coronary sinus plasma (28, 29).

Microneurography

Direct intraneural recordings using microneurography provide a moment-to-moment measure of central sympathetic neural outflow independent of the influence of the neuro-effector junction. This technique involves the recording of multiunit sympathetic nerve discharge from a peripheral nerve, usually the peroneal nerve (30, 31). Sympathetic nerve activity is recorded using tungsten microelectrodes (shaft diameter 200 µm, tapering to an uninsulated tip of 1 - 5 µm) inserted selectively into muscle or skin fascicles. Recently, micronuerographic approach allowed also quantification of single-fibre muscle sympathetic nerve traffic (32, 33).

Microneurography permits separate recordings of sympathetic nerve activity to muscle (MSNA) vessels or skin (SSNA). MSNA reflects the vasoconstrictor signal to the skeletal muscle vasculature, is acutely sensitive to blood pressure changes, and is closely regulated by the arterial and cardiopulmonary baroreflexes. SSNA is not altered by either arterial or cardiopulmonary baroreflexes. At rest, in a room temperature environment, SSNA reflects vasomotor neural traffic to skin blood vessels with little if any sudomotor activities present (34). MSNA and SSNA differ markedly with regard to morphology (Fig. 1). SSNA bursts are broad based and may extend over several cardiac cycles. The duration of each MSNA burst is limited by the cardiac cycle.

Fig. 1. Recordings of skin and muscle sympathetic nerve activity in a normal subject. Duration of each muscle sympathetic nerve activity burst is limited by the cardiac cycle; skin sympathetic nerve activity bursts are broad based and may extend over several cardiac cycles. Both Recordings were performed in the young patient with essential hypertension.

Measurement of sympathetic nerve activity from peripheral nerves in humans has been shown to be safe, accurate, quantifiable and reproducible (35). Also important is that simultaneous measurements of sympathetic nerve activity from different limbs show identical profiles in terms of burst frequency and morphology. Thus, recordings in one limb can be reliably assumed to reflect recordings of sympathetic nerve activity to the muscle vascular bed throughout the body (36).

The neural signals are amplified, filtered, rectified, and integrated to obtain a voltage display of sympathetic nerve activity. Sympathetic bursts are identified by a careful visual inspection of the voltage neurogram or by dedicated software. Muscle sympathetic nerve activity can be expressed as bursts per minute and burst per 100 heart beats, which allows comparison of sympathetic discharge between individuals (Fig. 2). The amplitude of each burst can also be determined and sympathetic activity may be calculated as bursts/minute multiplied by mean burst amplitude and expressed as units/minute. Measurements of nerve activity at baseline before each intervention are expressed as 100%. Changes in integrated MSNA allow evaluation of within subject changes in sympathetic traffic in response to different stressors during the same recording session.

Fig. 2. Recordings of muscle sympathetic nerve activity illustrating low (top) and high (bottom) activity. Recordings were performed in the young patient before and after 1minute apnea.

The introduction of microneurography has enabled a direct evaluation of the reflex sympathetic neural response to chemoreflex stimulation. These studies have documented that the peripheral and central chemoreflexes have powerful effects on sympathetic activity in both health and disease and may contribute importantly to disease pathophysiology, particularly in conditions such as hypertension (37), obstructive sleep apnea (38) and heart failure (39).

Although introduced into scientific practice methods of SNS evaluation described above are not commonly used in the clinic. Limitations and disadvantages of the various techniques has been reviewed in greater details elsewhere (19). However two of the methods: analysis of heart rate variability (HRV) and baroreflex sensitivity (BRS) were recommended as the diagnostic tools and can be found in clinical guidelines as basic assessment methods.

Heart rate variability

For more than 20 years spectral analysis of heart rate variability was used to assess autonomic control of the heart (40). Assessment of heart rate variability (HRV) is based on the analysis of consecutive sinus rhythm R-R intervals and may provide quantitative information about the modulation of cardiac vagal and sympathetic nerve activities. HRV measurements can be derived from short term (2 to 5 minutes) or long-term ECG recordings (24 to 48 hours). It can be quantified in a number of ways but techniques of conventional time domain (statistical and geometrical) and frequency domain measurements (power spectral density) remain predominantly utilized. Recently (41), analysis of heart rate dynamics by methods based on non-linear system theory has been introduced, which may be an alternative way for studying the abnormalities in heart rate.

In normal humans, short term RR interval variability occurs predominantly at a low frequency (0.04 to 0.14 Hz) and a high frequency (±0.25 Hz, synchronous with the respiratory frequency) (Fig. 3). The respiratory-related HF component is attributed mainly to vagal mechanisms. By contrast, different hypotheses have been proposed for the LF oscillation of RR interval variability. In several studies, LF component was not related to rates of noradrenaline spillover from the heart and or muscle sympathetic nerve traffic (19). Thus, while the LF/HF ratio may be considered as a marker of sympatho-vagal balance, it is unjustified to consider the low frequency power as a surrogate measure of sympathetic nerve firing.

Fig. 3. Spectral analysis of simultaneous recordings of RR variability in a patient with heart failure (low) and in a control subject (high). There is a relative predominance of the LF component over the HF component of RR interval in the patient with heart failure.

HRV as an independent cardiovascular risk factor

The closely monitored elderly population from the Framingham Heart Study was assessed using HRV calculated from 2-hour ambulatory ECG recordings. It was found that HRV was significantly associated with all-cause mortality and provided additional assessment of cardiovascular risk regardless traditional cardiovascular risk factors. A later study showed that HRV was also an independent risk factor in the healthy cohort of the Framingham study (42).

HRV in sudden cardiac death (SCD) and coronary heart disease risk assessment

HRV analysis was found useful in risk stratification for SCD in patients with heart failure. High LF values obtained during controlled breathing were found predictive of sudden cardiac death (43). HRV analysis was also classified as recommendation Class I A for risk assessment by the Task Force on Sudden Cardiac Death of European Society of Cardiology (44). It was also found that low HRV predicts risk in coronary heart disease (45).

HRV in diabetes

Diabetic autonomic neuropathy is one of major complication of diabetes contributing significantly to the morbidity and mortality of the disease. Although traditional measures and symptoms of autonomic function like resting tachycardia, exercise intolerance, orthostatic hypotension, constipation, gastroparesis, erectile dysfunction, sudomotor dysfunction, impaired neurovascular function or hypoglycemic autonomic failure are able to document the presence of neuropathy, usually they are abnormal when there is severe clinical symptomatology. Thus by the time changes in function are evident, the natural course of autonomic neuropathy is well established. HRV analysis determines the relative powers of the sympathetic and parasympathetic activities, is a very sensitive and early measure of autonomic neuropathy, and allows monitoring of disease progression. American Diabetes Association recommends HRV analysis as a part of the diagnosis of autonomic neuropathy (46).

Baroreflex sensitivity

Baroreflex sensitivity measurement is based on the principle that the increase in blood pressure stimulates baroreceptors of the carotid sinus and aortic arch and results in the activation of vagal fibers. As a result decrease in heart rate occurs. Sensitivity of baroreflex is defined as proportion of heart rate decrease due to blood pressure increase. Impaired baroreflex sensitivity is characterized by decreased HVR and increased BP variability. As a result, normal buffering of BP increases by HR decreases is lost.

In last decades neck suction (47) or pharmacological stimulation (48) were used to activate baroreceptors and evoke heart rate changes. Advances in beat-by-beat blood pressure (BP) monitoring methods have now made possible noninvasive estimation of baroreflex sensitivity from the RR interval changes associated with spontaneous fluctuations in BP. This new methodology offers clear advantages over traditional techniques of assessing baroreflex control. Noninvasive estimates of baroreflex sensitivity are obtained from beat-to-beat BP and heart rate recordings by one of two methods to extract concordantly changing systolic BP (SBP) and RR interval. Power spectral analysis provides a baroreflex estimate based on the RR interval changes associated with rhythmic BP oscillations over a range of frequencies reported to be associated with baroreflex function (Fig. 4). Second, recently developed method extracts covarying pressure and RR interval based on the magnitude of the changes occurring across sequential beats. In this technique, beat-by-beat BP and RR interval recordings are scanned for sequences in which SBP and RR interval concurrently increase or decrease for at least three consecutive beats. Baroreflex sensitivity is then assessed from the relationship between SBP and RR interval across these sequences (Fig. 4). Positive correlation was reported between baroreflex sensitivity estimates obtained by the frequency-domain-based and sequence method and from pharmacological manipulations of BP and RR interval (49).

Fig. 4. Two methods of baroreflex sensitivity analysis-spectral (A) and sequence (B). Recordings were made in the young patient with high-normal blood pressure.

Spontaneous baroreflex sensitivity is a very important marker for risk stratification particularly in patients who suffered from myocardial infarction (50 - 53). A low BRS in patients with ischemic heart disease and impaired left ventricular function is the important prognostic parameter (54, 55). The Task Force on Sudden Cardiac Death of European Society of Cardiology also classified BRS analysis as a recommendation Class I A for cardiovascular risk assessment (44).


CONCLUSIONS
This brief review indicates that SNS is involved in pathophysiology of many cardiovascular disorders and points out an importance of its investigation for both clinical and experimental research. Many aspects of the role of sympathetic system are still controversial or remain a matter of debate. However, wider implementation of objective methods like microneurography, may contribute to better understanding of the role of SNS in cardiovascular disease and translate into better patient care.


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