Download Resistant Hypertension Treatment through Carotid Baroreceptor

ASIA-PACIFIC JOURNAL OF CARDIOLOGY
REVIEW ARTICLE
Resistant Hypertension Treatment through Carotid
Baroreceptor Stimulation
Gino Seravalle1 and Guido Grassi2
Affiliations: 1Cardiology Department, Ospedale San Luca, Istituto Auxologico Italiano, Milan, Italy and 2Clinica Medica, Ospedale San Gerardo, Università
Milano-Bicocca, Monza, Italy
A B S T R A C T
A significant number of hypertensive subjects fail to achieve adequate blood pressure control despite adherence to maximal doses of
several antihypertensive drugs. Electrical stimulation of the carotid sinus is a new and interesting approach to the treatment of resistant
hypertension. The purpose of this paper is to overview the argument starting from the historical physiological background to potential
therapeutical applications through the recent advances in technology regarding electrical baroreceptor stimulators.
Keywords: resistant hypertension, carotid baroreflex, baroreceptor stimulation, sympathetic nervous system, therapy, surgery
Correspondence: Gino Seravalle, Cardiology Department, Ospedale San Luca, Istituto Auxologico Italiano, Via Spagnoletto 3, 20149 Milano,
Italy. Fax: (39)-2-619112906; e-mail: [email protected]
basal level of RSNA and impairing its arterial baroreflex
regulation [12]. A reduction in the pressor influences can be
obtained by denervating the renal arteries through a
percutaneously inserted catheter capable of removing efferent
sympathetic influences, thus lowering overall total-body
norepinephrine spillover, and reducing vasoconstriction in
an a that accounts for a considerable proportion of systemic
vascular resistance [13]. An enhancement of the depressor
influences can be obtained via the sympathoinhibitory effects
of continuous electric stimulation of the carotid baroreceptor
[14]. The renewed interest in electrical stimulation of the
carotid sinus will be the central argument of this review.
INTRODUCTION
Despite growing awareness of the linear relationship
between the level of blood pressure (BP) and the risk of
cardiovascular events [1] and the importance of blood
pressure goals, blood pressure control worldwide remains
poor [2, 3]. Several trials have clearly shown a failure to
achieve BP goals despite receiving more than three antihypertensive drugs [4–7] (Figure 1). A significant number of
hypertensive subjects fail to achieve adequate BP control
despite adherence to maximal doses of several antihypertensive drugs. These patients are regarded as having resistant
hypertension, defined as failure to achieve a BP of ,140/
90 mmHg despite taking three antihypertensive medications,
including a diuretic agent [2, 3, 8, 9]. To this end, the
investigation of new approaches in the drug treatment of
resistant hypertension is increasing with the addition to the
existing multidrug regimen of an antialdosterone agent [10],
thereby more effectively blocking the sodium-retaining
properties of this hormone, or adding an antagonist of
endothelin receptors, which is able to induce a further BP
reduction as a result of its vasodilatory properties [11].
HISTORICAL BACKGROUND
The influence of arterial reflex in BP control has been
known from ancient Rome where it was observed that
manual compression of the arteries anatomically located in
the neck of animals was able to induce sedation. In 1799,
Parry observed for the first time in humans that, along with
bradycardia, carotid compression induced hypotension. At
the beginning of the twentieth century, it was found that
the carotid bifurcation region is particularly sensitive to
pressor and depressor stimuli and has a role in BP control.
Carotid baroreceptors are stretch receptors that transmit
signals to the medullary vasomotor centers through the
carotid nerves. The impulses are processed in the central
nervous system and inhibit the discharge of the sympathetic
nervous system (Figure 2). As the arterial baroreflex
represents a major inhibiting mechanism of the sympathetic
nervous system, it seemed rational to assume that an
alteration in baroreflex activity is implicated in the
pathogenesis of hypertension [15–17].
More intriguing is the interest in invasive procedures
capable of reducing the pressor or increasing the depressor
influences that physiologically modulate BP. Long-term
control of arterial pressure has been attributed to the kidney
because of its ability to couple regulation of blood volume
with the maintenance of sodium and water balance by the
mechanisms of pressure natriuresis and diuresis. There is
evidence that an important cause of the defect in renal
excretory function in hypertension is an increase in renal
sympathetic nerve activity (RSNA) resulting from the direct
action of angiotensin II on brain stem nuclei, increasing the
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Figure 1. Effects of antihypertensive drug treatment on systolic blood pressure (SBP) in trials on essential hypertensive (left) and diabetic hypertensive (right)
patients. Values at trial entry (B) and during treatment (T) are shown for each trial. Dashed horizontal lines refer to goal BP values indicated by international
guidelines to be achieved during treatment. Modified with permission from [4]
Figure 2. Scheme illustrating the main reflex mechanisms that, through central integration, are responsible for the cardiovascular homeostasis. NTS, nucleus
tracti solitari; CNS, central nervous system; Ach, acetylcholine; NE, norepinephrine; GI, gastrointestinal
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Carotid baroreceptor stimulation in resistant hypertension
included: (1) paralysis of the hypoglossal nerve with
dysarthria and hemiatrophy of the tongue; (2) blurring of
vision and dizziness in the standing position; (3) perioperative death attributed to the fact that the surgical approach to
the carotid baroreceptor area was in its infancy. Technical
difficulties included: (1) current spread around the inserted
electrode with discomfort and pain; (2) difficulties in
communication between the external antenna and the
inserted receiver; (3) nerve damage; and (4) stimulation of
the nearby chemoreceptor afferents leading to sympathoexcitation. The above-mentioned disadvantages have been overcome by recent advances in technology.
BAROREFLEX CARDIOVASCULAR CONTROL
As BP increases, baroreceptor triggering leads to diminished sympathetic outflow to the heart, kidneys, and
peripheral vasculature, as well as heightened parasympathetic
tone in the heart. The result is a fall in peripheral vascular
resistance, heart rate, stroke volume, and BP. The decrease in
renal sympathetic tone reduces the activity of the renin–
angiotensin–aldosterone system (RAAS), with a resultant
decrease in renal salt and water retention as well as
diminished angiotensin II-mediated vasoconstriction [15–
19]. Decreased arginine vasopressin secretion is also observed
during increased baroreceptor activity, contributing to reduce
systemic vasoconstriction and renal water retention [15–18].
Despite the early successes with an implantable carotid
simulator system, the appeal of a surgical approach to
hypertension declined as the armamentarium of effective
antihypertensive drugs increased and because of the invasiveness of carotid baroreceptor therapy. Although recent decades
have seen a dramatic improvement in the treatment of
hypertension, we remain limited in our ability to treat the
most resistant patients effectively.
It is well established that the arterial baroreflex buffers
short-term fluctuations in BP, whereas arterial baroreceptors
are rapidly reset in response to sustained BP elevations [16,
17].
BARORECEPTOR ACTIVATION
The concept of carotid baroreceptor activation is actually
based on the idea that increased nerve traffic from the carotid
sinus to brainstem cardiovascular centers would result in a
sustained fall in BP. This continuous neural signal evokes a
subsequent decrease in BP and heart rate. The manipulation
of BP levels by electrical stimulation of the carotid
baroreceptors could ‘‘re-reset’’ the baroreflexes, and thus
hypertension could be controlled more easily.
NEW APPEAL OF CAROTID STIMULATION
In the last two or three decades, significant evidence has
accumulated suggesting a causative or accessory role of
sympathetic nerve activity in the pathogenesis of essential
hypertension [28–31]. Findings indicate that a sympathoexcitatory state is especially pronounced in individuals in whom
BP control is particularly difficult, as in isolated systolic
hypertension [32], in hypertension associated with obesity
[33, 34] and obstructive sleep apnea [35], and in subjects with
an altered dipping profile [36] (Figure 3). These studies
suggest that suppression of sympathetic activity should be
one of the major goals of hypertensive therapy [30]. In the
setting of progressively lowering BP targets needed to prevent
end-organ disease and continued refinements in implantable
medical technology, this has led to a renewal of interest in
carotid sinus stimulation. The use of electrical carotid
baroreceptor stimulation is also supported by the evidence
that, in hypertension, the baroreflex undergoes a ‘‘resetting’’
that avoids baroreceptor saturation, thus preserving the reflex
ability to cause vasodilation and decrease BP in response to an
increase in its activity [15].
The goal of lowering BP in humans by activating carotid
baroreceptors is not a new approach. The first studies in the
late 1950s performed in experimental animals [19–21] showed
that a significant reduction in BP was achieved in normotensive dogs for periods of up to 90 min following direct
electrical stimulation of the carotid sinus nerve. A consistent
BP reduction lasting for the whole year of evaluation was
observed by Schwartz et al [22] in studies performed both in
normotensive and hypertensive dogs.
The hemodynamic effects of bilateral electrical stimulation
of the carotid sinus nerves in humans have been evaluated in
several studies. In the study by Tuckman et al [23], the acute
carotid stimulation of supine hypertensive patients reduced
cardiac output by 11%, peripheral resistance by 10%, mean
arterial pressure by 21%, and heart rate by 16%. Similar
results were also reported in other studies [24–26].
In the first years of the millennium, studies by Lohmeier et
al [37, 38] and Barrett et al [39] showed that, in animals with
angiotensin II-induced acute and chronic hypertension, this
treatment produced a decrease in renal sympathetic nerve
activity in animals with an intact baroreflex but not in animals
that had undergone sinoaortic denervation. These data
suggest that the baroreflex is important in chronic hypertension and that renal sympathoinhibition, with a resultant
increase in natriuresis, may be the mechanism by which the
baroreflex participates in long-term BP control [18, 40]. A
further step was evaluation of the effect of long-term
stimulation on the baroreflex using electrodes implanted
around both carotid sinuses of dogs [38]. Baroreflex
activation for 7 days produced a rapid and sustained reduction
in heart rate and BP as well a significant reduction in plasma
Another important issue is whether unilateral or bilateral
baroreceptor activation is required to obtain the optimal
response. Parsonnet et al [27] have noted that, although
maximum BP reduction was obtained by simultaneous
bilateral application, this response was similar to that
obtained by stimulation of the more responsive of the two
carotid sinus nerves. In contrast, Schwartz et al [22]
demonstrated that simultaneous bilateral stimulation is much
better than the stimulation of either nerve, and usually more
effective than the summation of the unilateral stimulation.
However, the use of these early devices was not devoid of
adverse events and technical difficulties. Adverse events
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Figure 3. Behavior of muscle sympathetic nerve traffic (MSNA), expressed as burst incidence corrected for heart rate (bs/100hb) in moderate (MHT) and
severe (SHT) hypertension (left), in obese subjects (O) and obese subjects with sleep apnea syndrome (OSAS) (middle), and in non-dipper (NDHT) and
reverse dipper (RDHT) hypertensive patients (right). In each panel, data referring to normotensive control subjects (NT) are shown. Data are represented as
mean¡SEM. Symbols refer to the statistical significance between data obtained in control subjects vs other conditions. Modified with permission from [32, 33,
35, 36]
norepinephrine levels, but plasma renin levels did not increase,
suggesting that baroreflex-mediated suppression of renin
activity played a role in the sustained hypotensive response.
More recently, the same group evaluated the effects of
prolonged electrical carotid baroreflex stimulation in dogs
with obesity-induced hypertension, showing a significant
reduction in mean arterial pressure and plasma catecholamines
without a concomitant increase in plasma renin activity [40].
Thus, in obesity, baroreflex activation can suppress the
endogenous activation of the sympathetic nervous system and
reduce high BP. These data also support the hypothesis that
baroreflex-mediated suppression of renal sympathetic nerve
activity and renin secretion is an important mechanism by
which the carotid stimulation exerts its antihypertensive effect.
stimulation showing a voltage-dependent reduction in BP
levels.
To date, several studies are under way with a Rheos device
for chronic electrical stimulation of the carotid sinus (CVRx,
MN, USA): the European and US Feasibility studies and the
Rheos Pivotal trial. In the Device Based Therapy of
Hypertension (DEBuT-HT) study, 16 patients completed the
2-year follow-up [43]. Both systolic and diastolic BP were
significantly lowered by 35¡8 mmHg and 24¡6 mmHg,
respectively. These reductions were comparable to those
observed at 3 and 12 months after activation of the device.
More than 75% of these patients showed a reduction of
.20 mmHg in systolic BP, and 31% showed BP control. In
the European/US study [44], the Rheos device applied to 16
resistant hypertensive patients was able to induce statistically
significant reductions in BP values at 3 months accompanied
by reduction in left ventricular mass index (–24.1¡18.7 g/
m2), septal wall thickness (–1.3¡1.8 mm), and left ventricular
posterior wall thickness (–1.4¡1.1 mm). These results were
also accompanied by a reduction in the number of
antihypertensive drugs. A study of 12 patients with resistant
hypertension fitted with the Rheos device did not show an
impairment in renal function after a 1-year follow-up [45]. In
this study, glomerular filtration rate was unchanged, while
the effective plasma flow tended to decrease, thus resulting in
a significant rise in filtration fraction. A recent study of 12
resistant hypertensive patients by Heusser et al [46] showed
that acute electric baroreflex stimulation decreased systolic BP
by 32¡10 mmHg, and this was correlated with a reduction in
muscle sympathetic nerve activity, heart rate, and plasma
renin concentration. This result is relevant because it shows
that the surgical procedure by which the stimulating device is
bilaterally implanted does not impair, through scarring,
inflammation, or direct baroreceptor damage, the ability of
the reflex to exert its sympathetic and vasomotor modulation.
As far as human studies are concerned, carotid baroreceptor
stimulation was first studied in the 1960s. After less than
optimal results resulting from technical problems and
symptoms related to local tissue and nerve stimulation
(dysphagia, coughing, gagging, hyperpnea, dyspnea), the
development of systems that permitted radiofrequency
adjustment of implanted devices allowed tailored stimulation
of parameters to individual patients. Thanks to these
innovations, Tuckman et al [23] implanted bilateral carotid
sinus nerve stimulators, permitting fine regulation of the
stimulus in achieving BP reduction and avoiding undesirable
effects over a period of 2–18 months. Peters et al [24] used a
device that matched stimulator frequency to patient heart rate,
based on the hypothesis that heart rate elevations signaled
increases in sympathetic tone that would need to be answered
with greater activation of the baroreflex to achieve BP control.
Patients fitted with these stimulators were able to increase
their heart rate and BP with exercise, thus avoiding the
inability to increase sympathetic tone during physiologic
demands as seen with previous devices. Schmidli et al [41, 42]
reported results on the acute and chronic effects of carotid
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Carotid baroreceptor stimulation in resistant hypertension
14. Mohaupt MG, Schmidli J, Luft FC. Management of uncontrollable
hypertension with a carotid sinus stimulation device. Hypertension. 2007;
50:825–828.
15. Mancia G, Ludbrook J, Ferrari A, et al. Baroreceptor reflexes in human
hypertension. Circ Res. 1978;43:170–177.
16. Folkow B. Physiological aspects of primary hypertension. Physiol Rev.
1982;62:347–404.
17. Mancia G, Mark AL. Arterial baroreflexes in humans. In: Shepherd JT,
Abboud EM, eds. Handbook of Physiology, Section 2: The Cardiovascular
System. Bethesda, MD: American Physiological Society; 1983:755–793.
18. Lohmeier TE, Hildebrandt DA, Warren S, et al. Recent insights into the
interactions between the baroreflex and the kidneys in hypertension. Am J
Physiol Regul Integr Comp Physiol. 2005;288:R828–R836.
19. McCubbin JW, Green JH, Page IH. Baroreceptor function in chronic renal
hypertension. Circ Res. 1956;4:205–210.
20. Werner HR. The frequency-dependent nature of blood pressure
regulation by the carotid sinus studied with an electrical analog. Circ
Res. 1958;6:35–40.
21. Bilgutay A, Lillehei W. Treatment of hypertension with an implantable
electronic device. JAMA. 1965;191:113–117.
22. Schwartz S, Griffith L, Neistadt A, et al. Chronic carotid sinus nerve
stimulation in the treatment of essential hypertension. Am J Surg. 1967;
114:5–15.
23. Tuckman J, Reich T, Lyon A, et al. Electrical stimulation of the sinus
nerves in hypertensive patients-clinical evaluation and physiological
studies. Hypertension. 1967;194S:23–38.
24. Peters T, Koralewski H, Zerbst F. Search for optimal frequencies and
amplitudes of therapeutic electrical carotid sinus nerve stimulation by
application of the evolutionary strategy. Artif Organs. 1989;13:133–143.
25. Peters T, Koralewski H, Zerbst F. The principle of electrical carotid sinus
nerve stimulation: a nerve pacemaker system for angina pectoris and
hypertension therapy. Ann Biomed Eng. 1980;8:445–458.
26. Wallin BG, Sundlof G, Delius W. The effect of carotid sinus nerve
stimulation on muscle and skin nerve sympathetic activity in man. Pflugers
Arch. 1975;358:101–110.
27. Parsonnet V, Myers GH, Holcomb WG, et al. Radio-frequency stimulation
of the carotid baroreceptors in the treatment of hypertension. Surg Forum.
1966;17:125–127.
28. Grassi G. Assessment of sympathetic cardiovascular drive in human
hypertension: achievements and perspectives. Hypertension. 2009;54:690–
697.
29. Esler M. The sympathetic system and hypertension. Am J Hypertens. 2000;
13:99S–105S.
30. Mancia G, Grassi G, Giannattasio C, et al. Sympathetic activation in the
pathogenesis of hypertension and progressive organ damage.
Hypertension. 1999;34:724–728.
31. Anderson EA, Sinkey CA, Lawton WJ, et al. Elevated sympathetic nerve
activity in borderline hypertensive humans: evidence from direct
intraneural recordings. Hypertension. 1989;14:177–183.
32. Grassi G, Seravalle G, Bertinieri G, et al. Sympathetic and reflex
alterations in systo-diastolic and systolic hypertension of the elderly. J
Hypertens. 2000;18:587–593.
33. Grassi G, Seravalle G, Dell’Oro R, et al. Adrenergic and reflex
abnormalities in obesity-related hypertension. Hypertension. 2000;36:
538–542.
34. Grassi G, Dell’Oro R, Quarti Trevano F, et al. Neuroadrenergic and reflex
abnormalities in patients with metabolic syndrome. Diabetologia. 2005;48:
1359–1365.
35. Grassi G, Facchini A, Quarti Trevano F, et al Obstructive sleep apneadependent and -independent adrenergic activation in obesity.
Hypertension.2005;46:321–325.
36. Grassi G, Seravalle G, Quarti Trevano F, et al. Adrenergic, metabolic, and
reflex abnormalities in reverse and extreme dipper hypertensives.
Hypertension. 2008;52:925–931.
37. Lohmeier TE, Loheimer JR, Haque A, et al. Baroreflexes prevent neurally
induced sodium retention in angiotensin hypertension. Am J Physiol Regul
Integr Comp Physiol. 2000;279:R1437–R1448.
38. Lohmeier TE, Irwin E, Rossing M, et al. Prolonged activation of the
baroreflex produces sustained hypotension. Hypertension. 2004;4:306–311.
CONCLUSIONS
Although intriguing, the results obtained with carotid
baroreceptor stimulation for the treatment of resistant
hypertension leave a number of clinically relevant questions.
First, are the favorable effects of this intervention maintained
over time? Is there any evidence of a time-dependent
baroreflex desensitization to the electrical stimuli? What
about the long-term safety of the procedure? How much can
the baroreflex function be quantitatively altered by the
surgical procedure? What about the battery9s half-life and
the necessity of several changes after implantation? Taking
into account these problems, what is the economic impact of
this procedure in the treatment of resistant hypertension?
Taken together, these questions demonstrate that much
needs to be learned about this procedure, which nevertheless
opens new options for the treatment of resistant hypertension.
Disclosure: The author declares no conflict of interest.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
MacMahon S, Peto R, Cutler J, et al. Blood pressure, stroke, and coronary
heart disease. Part 1. Prolonged difference in blood pressure: prospective
observational studies corrected for the regression dilution bias. Lancet.
1990;335:765–774.
Chobanian AV, Bakris GL, Black HR, et al. The seventh report of the
Joint National Committee on Prevention, Detection, Evaluation and
Treatment of High Blood Pressure: the JNC-7 report. JAMA. 2003;289:
2560–2572.
The Task Force for the Management of Arterial Hypertension of the
European Society of Hypertension (ESH) and of the European Society of
Cardiology (ESC). 2007 guidelines for the management of arterial
hypertension. J Hypertens. 2007;25:1105–1187.
Mancia G, Grassi G. Systolic and diastolic blood pressure control in
antihypertensive drug trials. J Hypertens. 2002;20:1461–1464.
Zanchetti A, Grassi G, Mancia G. When should antihypertensive drug
treatment be initiated and to what levels should systolic blood pressure
be lowered? A critical reappraisal. J Hypertens. 2009;27:923–934.
Zanchetti A, Mancia G, Black HR, et al. Facts and fallacies of blood
pressure control in recent trials: implications in the management of
patients with hypertension. J Hypertens. 2009;27:673–679.
Struijker-Boudier HA, Ambrosioni E, Holzgreve H, et al. The need for
combination antihypertensive therapy to reach target blood pressures:
what has been learned from clinical practice and morbidity-mortality
trials? Int J Clin Pract. 2007;61:1592–1602.
Calhoun DA, Jones D, Textor S, et al. Resistant hypertension: diagnosis,
evaluation, and treatment: a scientific statement from the American Heart
Association Professional Education Committee of the Council for High
Blood Pressure Research. Hypertension. 2008;51:1403–1419.
Sarafidis PA, Bakris GL. Resistant hypertension: an overview of
evaluation and treatment. J Am Coll Cardiol. 2008;52:1749–1757.
Chapman N, Dobson J, Wilson S, et al. For the Anglo-Scandinavian
Cardiac Outcomes Trial Investigators. Effect of spironolactone on blood
pressure in subjects with resistant hypertension. Hypertension. 2007;49:
839–845.
Weber MA, Black H, Bakris G, et al. A selective endothelin-receptor
antagonist to reduce blood pressure in patients with treatment-resistant
hypertension: a randomized double-blind placebo-blind placebo-controlled trial. Lancet. 2009;374:1423–1431.
DiBona GF. Sympathetic nervous system and the kidney in hypertension.
Curr Opin Nephrol Hypertens. 2002;11:197–200.
Krum H, Schlaich M, Whitbourn R, et al. Catheter-based renal
sympathetic denervation for resistant hypertension: a multicentre safety
and proof-of-principle cohort study. Lancet. 2009;373:1275–1281.
www.slm-asia.com
5
APJOC 2010; 000:(000). Month 2010
Asia-Pacific Journal of Cardiology
39. Barrett CJ, Guild S, Ramchandra J, et al Baroreceptor denervation prevents
sympathoinhibition during angiotensin II-induced hypertension.
Hypertension. 2005;46:1–5.
40. Lohmeier TE, Dwyer TM, Irwin ED, et al. Prolonged activation of the
baroreflex abolishes obesity-induced hypertension. Hypertension. 2007;49:
1307–1314.
41. Schmidli J, Mohaupt M, Savolainen H, et al. Response to acute electrical
activation of the carotid baroreflex is maintained in drug refractory
hypertension. J Hypertens. 2005;23(Suppl 2):S6.
42. Schmidli J, Savolainen H, Eckstein F, et al. Acute device-based blood
pressure reduction: electrical activation of the carotid baroreflex in
patients undergoing elective carotid surgery. Vascular. 2007;15:63–69.
43. Scheffers I, Schmidli J, Kroon AA, et al. Sustained blood pressure
reduction by baroreflex hypertension therapy with a chronically
implanted system: 2-years data from the Rheos DEBUT-HT study in
patients with resistant hypertension. J Hypertens. 2008;26(Suppl 1):S19.
44. De Leeuw P, Gangahar D, Bach D, et al. Left ventricular reverse
remodeling with chronic treatment of resistant hypertension using an
implantable device: results from European and United States trials of the
Rheos baroreflex hypertension therapy system. J Hypertens. 2008;26(Suppl
1):S471.
45. Scheffers I, Kroon AA, de Leeuw P. Renal hemodynamics during chronic
therapy with electrical stimulation of the carotid sinus in humans. J
Hypertens. 2008;26(Suppl 1):S465.
46. Heusser K, Tank J, Engeli S, et al. Carotid baroreceptor stimulation,
sympathetic activity, baroreflex function, and blood pressure in
hypertensive patients. Hypertension. 2010;55:619–626.
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