Very little is known about the activity of the peripheral

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1 Sympathetic Neural Mechanisms in Normal and Hypertensive Pregnancy in Humans John P. Greenwood, MB ChB, PhD; Eleanor M. Scott, BM BS, MD; John B. Stoker, BSc, MB ChB; James J. Walker, MB ChB, MD; David A.S.G. Mary, MB ChB, PhD Background Direct recordings from peripheral sympathetic nerves have shown an increased sympathetic drive in pregnancy-induced hypertension (PIH) and preeclampsia (PE). It is unknown whether sympathetic drive is altered in normal pregnancy, when arterial blood pressure can be normal or relatively low. The aim of this study was to measure and compare peripheral sympathetic discharge, its vasoconstrictor effect and its baroreceptor control, during pregnancy and postpartum in women with normal pregnancy (NP) and PIH and in normotensive nonpregnant (NN) women. Methods and Results Twenty-one women with NP, 18 women with PIH, and 21 NN women had muscle sympathetic nerve activity assessed from multiunit discharges (MSNA) and from single units with defined vasoconstrictor properties (s-msna). The s-msna in NP ( impulses/100 beats) was greater (P 0.05) than in NN women ( impulses/100 beats) despite similar age and body weight but less than in PIH women (P 0.001) ( impulses/100 beats). MSNA followed a similar trend. Cardiac baroreceptor reflex sensitivity (BRS) was impaired in NP and PIH women relative to NN. After delivery, sympathetic activity decreased to values similar to those obtained in NN, and there was an increase in BRS. In women with NP, the decrease in sympathetic output occurred despite an insignificant change in blood pressure. Conclusions Central sympathetic output was increased in women with normal pregnancy and was even greater in the hypertensive pregnant group. The findings suggest that the moderate sympathetic hyperactivity during the latter months of normal pregnancy may help to return the arterial pressure to nonpregnant levels, although when the increase in activity is excessive, hypertension may ensue. (Circulation. 2001;104: ) Key Words: nervous system, autonomic hypertension blood pressure pregnancy Very little is known about the activity of the peripheral sympathetic system in normal pregnancy compared with the nonpregnant state. However, using the technique of microneurography, 1,2 an increase in the mean frequency of bursts representing multiunit discharge of muscle sympathetic nerve activity (MSNA) 3,4 and activity from single units (s-msna) 5 has been shown to occur during the third trimester in patients with preeclampsia (PE) or pregnancy-induced hypertension (PIH) relative to women with normal pregnancy. During the third trimester of normal pregnancy, arterial blood pressure tends to rise toward normal nonpregnant levels, 6,7 but it is unknown whether central sympathetic drive is involved in this process. The use of indirect measures of sympathetic output, such as changes in hemodynamic variables and circulating catecholamines, has yielded conflicting results in both normal and hypertensive pregnancy The present investigation was designed to examine whether central sympathetic vasoconstrictor output to the peripheral vascular bed is altered in normal pregnancy. For this purpose, we studied matched groups of pregnant women prepartum and postpartum with normal pregnancy (NP) and PIH in addition to normotensive nonpregnant (NN) women. Methods Subjects We examined 60 women, ranging in age between 18 and 40 years, between January 1996 and March They comprised 3 agematched groups: 21 NP, 21 NN, and 18 PIH. Only white primigravidae were examined, and they were excluded if there was any evidence of secondary hypertension or chronic disease that may influence the autonomic system, such as diabetes mellitus or neurological dysfunction. Otherwise all patients were included in whom it was possible to identify and record single-unit activity. This represented 60% of the total number of women studied; in the other 40%, it was not possible to obtain a stable recording from a single vasoconstrictor unit. Patients with PIH were recruited shortly after admission. They were accepted as having PIH if their arterial blood pressure was 140/90 mm Hg on at least two separate occasions a minimum of 6 Received June 22, 2001; revision received August 20, 2001; accepted August 20, From the Departments of Cardiology and Obstetrics and Gynecology (J.J.W.), St James s University Hospital, Leeds, UK. Presented at the meetings of the British Hypertension Society, Bristol, United Kingdom, September 15 17, 1997, and the Physiological Society, Prague, Czech Republic, June 20 23, 1998, and published in abstract form (J Hypertens. 1997;15[12 part 1]: and J Physiol [Lond]. 1998;511:11P). Correspondence to Dr J.P. Greenwood, Department of Cardiology, St James s University Hospital, Beckett St, Leeds, LS9 7TF UK. john_greenwood@ hotmail.com 2001 American Heart Association, Inc. Circulation is available at

2 Greenwood et al Sympathetic Hyperactivity in Normal Pregnancy 2201 TABLE 1. Details of the 3 Groups of Women: NN, NP, and PIH Comparisons (P) Variable NN NP PIH NN vs NP NP vs PIH n Age, y NS NS Gestation, wk NS Weight, kg* NS 0.01 Heart rate, beats/min NS Arterial pressure, mm Hg Mean Systolic NS Diastolic s-msna, impulses/100b MSNA, bursts/100b CVR, units NS 0.05 BRS, ms/mm Hg NS All data were obtained concurrently during nerve recording sessions and are presented as mean SEM. MSNA and s-msna are expressed per 100 cardiac beats (100b). One-way ANOVA for all data except length of gestation (unpaired t test). *P 0.008; P ; P Between group analyses (Newman-Keuls) are shown on the right. hours apart, they were known to have been normotensive before this time, had no albuminuria (a maximum of 300 mg in 24 hours), and had no general systemic abnormality, and they had normal arterial blood pressure by the sixth postpartum week. Subjects with NP were healthy and were attending routine antenatal clinics. The NN subjects were also healthy women (with an arterial blood pressure 130/80 mm Hg) who were recruited from hospital staff or relatives of pregnant women. NP were matched for maternal and gestational age to the group with PIH, and the NN group were age-matched with the other 2 groups. The details of the women are given in Table 1. All normotensive women were studied while receiving no medical therapy other than iron or vitamins. Of the 18 patients with PIH, 9 had recently started oral labetalol as monotherapy to control their hypertension. This was commenced between 12 and 48 hours before the study and did not significantly affect heart rate. General Protocol Each subject provided informed written consent to the investigation, which was performed under the approval of the Leeds Health Authority Ethical Committee. Pregnant women were all in their 3rd trimester and were examined before delivery and then again a minimum of 6 weeks postpartum. The details of the protocol and data analysis have been published previously. 5,14 Briefly, all of the studies were performed under similar conditions between the hours of 9:00 AM and 12:00 PM, and subjects were asked to have had a light breakfast and to empty their bladder before commencing the study. They were also asked to avoid nicotine and caffeine products for 12 hours and alcohol and strenuous exercise for 24 hours before the investigation. Resting blood pressure was measured from the arm using a standard mercury sphygmomanometer. Changes in heart rate and arterial blood pressure were monitored and recorded using a standard ECG and a Finapres device, and blood flow to the muscle of the left calf was obtained using standard strain-gauge plethysmography. Microneurography Postganglionic muscle sympathetic nerve activity was recorded from the right peroneal nerve as previously described. 1,2,5,14 Briefly, the neural signal was amplified ( ), and for the purpose of generating bursts representing multiunit discharge, the signal was filtered (bandwidth of 700 to 2000 Hz) and integrated (time constant 0.1 second). The output of action potentials and bursts from this assembly were passed to a data acquisition system, which system digitized the action potentials at samples/second and other data channels at 2000 samples/second (8 bits). MSNA was differentiated from skin sympathetic activity and afferent activity by previously accepted criteria. 1,2 Single units (s-msna) in the raw action potential neurogram were obtained by adjusting the electrode position. Fast monitor sweep and an online storage oscilloscope were then used to confirm the presence of a single unit by demonstrating consistency in action potential morphology, as previously described. 5,14,15 Only vasoconstrictor units were accepted and examined, the criteria of acceptance being appropriate responses to spontaneous changes in arterial blood pressure, the Valsalva maneuver, and isometric hand-grip exercise. The vasoconstriction function of the activity examined was confirmed by measuring calf vascular resistance (CVR). The Valsalva maneuver was performed by asking the subjects to exhale into a standard mercury manometer, at a pressure of 40 mm Hg for 15 seconds, while a pneumograph was observed to confirm correct performance of the test. The sympathetic activity increased during the latter part of phase II (blood pressure compensation) and phase III (release of strain and fall in blood pressure) and decreased during phase IV (increase and overshoot of blood pressure). An electronic discriminator was used objectively to count the spikes of s-msna and was quantified as mean frequency of impulses per 100 cardiac beats; this avoided any interference by the length of the cardiac cycle. 16 The bursts of MSNA were identified by inspection when the signal-to-noise ratio was 3 and were quantified as mean number of bursts per 100 beats (Figure 1). The variability of measuring both s-msna and MSNA in this laboratory did not exceed 10%. 14 During the fourth phase of the Valsalva maneuver, the slope of the best linear relationship between the systolic blood pressure and its pulse interval (phase 0) or the succeeding one (phase 1) was used as an indicator for baroreceptor reflex sensitivity (BRS). 17 CVR was obtained from the product of mean arterial blood pressure on the mean of at least 3 measurements of calf blood flow during each phase of the test protocol. Statistics One-way ANOVA with Newman-Keuls multiple post-test comparisons were used to compare data between the different groups.

3 2202 Circulation October 30, 2001 Figure 1. Example of a recording from a pregnant woman. Shown are the ECG, respiratory movements (Resp), MSNA (Bursts), Finapres blood pressure (FBP), and action potentials (AP in V) for measuring s-msna. The s-msna is counted from action potentials of the same profile and amplitude (tallest unit in this example) to derive the mean frequency of central vasoconstrictor sympathetic neural discharge. The burst count may include a variable number of units (smaller units in this example) with different profiles. Student s t test for paired variables was used to examine changes in variables after delivery, and t test for unpaired variables was used to examine differences between two groups. The relationship between systolic blood pressure and pulse interval was examined using regression analysis. Values of P 0.05 were considered statistically significant. Data are presented as mean SEM Results The three groups, NN, NP, and PIH, were well matched with the exception of 2 patients in the PIH group who were above average weight. These two patients were not excluded and resulted in a higher (P 0.01) average body weight in the hypertensive pregnant group (Table 1). As expected, the average indices of arterial blood pressure were significantly greater in PIH than in NP and NN groups. Also, as anticipated from the study design, the blood pressure in 3rd trimester NP subjects was only slightly lower than NN, and the resting heart rate was greater in pregnant women compared with NN. Among the patients with PIH, there were no significant differences in heart rate, mean arterial blood pressure, and sympathetic nerve activity between those who received oral labetalol and those who did not. As can be seen in Table 1 and Figure 2, the frequency of s-msna in NP was significantly greater than in NN, with a 2-fold difference. This frequency was even greater in PIH, with nearly a 4-fold difference compared with NP. Similar differences, although of lesser magnitude, were seen in MSNA between the groups. In addition, CVR was found to be greater in PIH than in the NP and NN groups, and the two groups of pregnant women both had a significantly lower BRS than the NN group. Follow-up studies were carried out after delivery in 19 NP and 10 PIH women (Table 2). In both groups, the s-msna (Figure 3) and MSNA decreased back toward normal values. However, arterial blood pressure and CVR decreased significantly and consistently only in the PIH group, whereas BRS increased significantly in both groups after delivery (Table 2). Discussion The present investigation has shown for the first time that normal pregnancy is associated with an increase in resting Figure 2. Resting mean frequency of single-unit muscle sympathetic nerve activity (s-msna) in the 3 groups of subjects, NN, NP, and PIH, expressed as mean (height of columns) and SEM (bars). *P 0.05; P peripheral sympathetic neural discharge having vasoconstrictor properties. The mechanism for this increase could not be explained by changes in body weight or baroreceptor reflex control. This sympathetic hyperactivity occurred to a greater extent in women with PIH than in women with NP. The increase was found both in single-unit activity and in multiunit bursts. The single-unit activity seems to provide a more quantitative estimation of sympathetic discharge than that of multiunit bursts 14,15 and is obtained objectively. Therefore, because the frequency of firing of such units was not affected by other units, it is possible that they reflected the true resting central tone of the peripheral nervous system. 14 We only examined white women because of reported evidence that race can affect responses of MSNA 18 and only primigravidae to avoid the possibility of a confounding effect Figure 3. Resting mean frequency of single-unit muscle sympathetic nerve activity (s-msna) in the two groups of pregnant women (NP and PIH) studied both prepartum and postpartum and the normotensive nonpregnant (NN) group, expressed as mean (height of columns) and SEM (bars). *P 0.001; P

4 Greenwood et al Sympathetic Hyperactivity in Normal Pregnancy 2203 TABLE 2. Data From the Longitudinal Study of Pregnant Women Both Prepartum and Postpartum NP PIH Variable Prepartum Postpartum Prepartum Postpartum Number Weight, kg * Heart rate, beats/min * Arterial pressure, mm Hg Mean * Systolic * Diastolic * s-msna, impulses/ beats MSNA, bursts/100 beats * * CVR, units BRS, ms/mm Hg * All data were obtained concurrently during nerve recording sessions and are presented as mean SEM. Paired Student s t test. *P ; P 0.005; P 0.03; P of multiple pregnancy. 19 The reason for examining women in the 3rd trimester was because during this period in normal pregnancy, the arterial blood pressure and vascular resistance tend to normalize. Because these indices would be similar to a nonpregnant group and also after a normal delivery, significant hemodynamic change would not confound sympathetic measures. To avoid other confounding factors, all studies were undertaken within the same environmental conditions and after a light breakfast with an empty urinary bladder, because visceral distention is known to increase sympathetic activity. 20,21 In addition, the three groups were matched for age, whereas the pregnant groups were also matched for gestational age to avoid any influence on sympathetic output. 22 Two of the hypertensive patients had preconception obesity resulting in a difference in weight between the two pregnant groups. It is known that obesity can affect resting MSNA 23,24 ; however, if the results are analyzed with these 2 subjects excluded, then there is no change in the overall findings. The increase in body weight during pregnancy cannot explain the increased sympathetic activity. The frequency of MSNA bursts has been reported to be greater in subjects with obesity, 23,24 but the weight of such subjects is much greater than in pregnancy and, secondly, our groups (NN and NP) were well matched for body weight. The decrease in sympathetic activity after delivery toward normal values in both the NP and PIH groups, for the same decrease in body weight, is also against body weight being a significant factor. Similarly, the increased sympathetic activity in NP could not be related to baroreceptor reflex compensation, first, because after delivery there was a significant decrease in sympathetic output at a time when there was no significant increase in arterial blood pressure, and, second, because the cardiac baroreceptor reflex sensitivity was reduced in the NP group and recovered after delivery. Using peroneal nerve microneurography, there are reports of an increase in resting peripheral sympathetic activity in women with PIH 3,5 or PE 4 compared with women with NP. In one study, 4 comparisons were available between NP and NN; although no significant differences were found between these two groups, measurements were not made in the same subjects before and after delivery. However, in common with previous studies, 3 5 the present study showed that women with PIH had an increased peripheral sympathetic output relative to women with normal pregnancy. This was additionally confirmed by longitudinal follow-up showing that the sympathetic activity decreased after delivery to values encountered in the NN group. The present findings have important implications. It is possible to suggest that pregnancy per se results in peripheral sympathetic hyperactivity, the mechanism of which involves central factors. As discussed above, the sympathetic hyperactivity was not related to reflex control, race, or constitutional factors, and it could be the mechanism by which the arterial blood pressure and vascular resistance tend to normalize during the third trimester of normal pregnancy. 6,7,19,25 The excessive increase in sympathetic output found in patients with PIH may by implication be involved in the development of their hypertension. From the published evidence, although limited, it is possible to speculate that the increase in central sympathetic activity in pregnancy is related to hormonal changes, of which there are many. Certainly activation of the renin-angiotensin system is known to occur in pregnancy, and angiotensin II has been shown to increase MSNA. 26 Fasting levels of insulin are raised in the 3rd trimester of pregnancy in women who later develop hypertension, 27 and hyperinsulinemia produces sympathetic activation. 28 Reduction in vasopressin levels has also been reported in pregnacy, 29 and infusion of this hormone has been shown to decrease MSNA. 30 More recently it has been shown that MSNA is greater in young women during the midluteal compared with early follicular phase of the menstrual cycle, when estradiol and progesterone are elevated, 31 and in pregnancy these hormones are additionally elevated. Also, there is

5 2204 Circulation October 30, 2001 experimental evidence to suggest that central neurotransmitters in the medulla modulate sympathetic output, eg, nitric oxide, 32 but changes in levels during pregnancy and their implications remain to be determined. Finally, one must also consider the hemodynamic changes associated with pregnancy, in particular the increase in blood volume that may be important in increasing sympathetic output and heart rate through afferent sympathetic fibers. 33 Indeed, in accordance with our present findings, a recent study of heart rate variability performed in similar subjects to this study 34 suggested that normal pregnancy had increased sympathetic modulation compared with the nonpregnant state, and that this increase was even greater in hypertensive pregnancy. In summary, the present investigation has shown that pregnancy is associated with peripheral sympathetic hyperactivity. In women with PIH, this hyperactivity is extreme, but in both situations the levels fall to normal after delivery. There were indications that the mechanisms of this sympathetic hyperactivity involved central factors. Acknowledgments This work was funded by the British Heart Foundation (Grant No. FS/97085). The authors thank J. Bannister and J. Corrigan for technical assistance. References 1. Vallbo AB, Hagbarth KE, Torebjörk HE, et al. Somatosensory, proprioceptive and sympathetic activity in human peripheral nerves. Physiol Rev. 1979;59: Wallin BG. Assessment of sympathetic mechanisms for recordings of postganglionic efferent nerve traffic. In: Hainsworth R, Mark AL, eds. Cardiovascular Reflex Control in Health and Disease. Philadelphia, Pa: WB Saunders; 1993: Sander K, Hansen J, Sander M, et al. Sympathetic overactivity in pregnancy induced hypertension. Circulation. 1995;92(suppl I): Schobel HP, Fischer T, Heuszer K, et al. Preeclampsia: a state of sympathetic overactivity. 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Plasma noradrenaline and adrenaline levels in normal pregnancy and in pregnancyinduced hypertension. Br J Obstet Gynaecol. 1982;89: Ekholm EM, Tahvanainen KU, Metsala T. Heart rate and blood pressure variabilities are increased in pregnancy-induced hypertension. Am J Obstet Gynecol. 1997;177: Eneroth-Grimfors E, Westgren M, Ericson M, et al. Autonomic cardiovascular control in normal and pre-eclamptic pregnancy. Acta Obstet Gynecol Scand. 1994;73: Greenwood JP, Stoker JB, Mary DASG. Single-unit sympathetic discharge: quantitative assessment in human hypertensive disease. Circulation. 1999;100: Macefield VG, Wallin BG, Vallbo AB. The discharge behaviour of single vasoconstrictor motoneurones in human muscle nerves. J Physiol (Lond). 1994;481: Sundlöf G, Wallin BG. The variability of muscle nerve sympathetic activity in resting recumbent man. J Physiol (Lond). 1977;272: Ekholm EM, Vesalainen RK, Tahvanainen KU, et al. Valsalva manoeuvre can be used to study baroreflex sensitivity in pregnancy. Eur J Obstet Gynecol Reprod Biol. 1998;76: Calhoun DA, Mutinga ML, Collins AS, et al. Normotensive blacks have heightened sympathetic response to cold pressor test. Hypertension. 1993; 22: Broughton Pipkin F. The hypertensive disorders of pregnancy. Br Med J. 1995;311: Fagius H, Karhuvaara S. Sympathetic activity and blood pressure increases with bladder distension in humans. Hypertension. 1989;14: Cox HS, Kaye DM, Thompson JM, et al. Regional sympathetic nervous activation after a large meal in humans. Clin Sci (Colch). 1995;89: Ng AV, Callister R, Johnson DG, et al. Age and gender influence muscle sympathetic nerve activity at rest in healthy humans. Hypertension. 1993; 21: Andersson B, Elam M, Wallin BG, et al. Effect of energy-restricted diet on sympathetic muscle nerve activity in obese women. Hypertension. 1991;18: Scherrer U, Randin D, Tappy L, et al. Body fat and sympathetic nerve activity in healthy subjects. Circulation. 1994;89: Teghini L, Livi R, Parretti E, et al. Maternal 24-hour blood pressure and heart rate changes in normal pregnancy. Am J Hypertens. 2000;13:231A. 26. Matsukawa T, Gotoh E, Minamisawa K, et al. Effects of intravenous infusions of angiotensin II on muscle sympathetic nerve activity in humans. Am J Physiol. 1991;261:R690 R Solomon CG, Carroll JS, Okamura K, et al. Higher cholesterol and insulin levels in pregnancy are associated with increased risk for pregnancyinduced hypertension. Am J Hypertens. 1999;12: Anderson EA, Hoffman RP, Balon TW, et al. Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans. J Clin Invest. 1991;87: van der Post JA, van Buul BJ, Hart AA, et al. Vasopressin and oxytocin levels during normal pregnancy: effects of chronic dietary sodium restriction. J Endocrinol. 1997;152: Floras JS, Aylward PE, Abboud FM, et al. Inhibition of muscle sympathetic nerve activity in humans by arginine vasopressin. Hypertension. 1987;10: Minson CT, Halliwill JR, Young TM, et al. Influence of the menstrual cycle on sympathetic activity, baroreflex sensitivity, and vascular transduction in young women. Circulation. 2000;101: Zanzinger J, Czachurski J, Seller H. Inhibition of basal and reflexmediated sympathetic activity in the RVLM by nitric oxide. Am J Physiol. 1995;268:R958 R Bishop VS, Lombardi F, Malliani A, et al. Reflex sympathetic tachycardia during intravenous infusions in chronic spinal cats. Am J Physiol. 1976;230: Yang CC, Chao TC, Kuo TB, et al. Preeclamptic pregnancy is associated with increased sympathetic and decreased parasympathetic control of HR. Am J Physiol. 2000;278:H1269 H1273.