The carotid body as a putative therapeutic target for the treatment of neurogenic hypertension

Abstract

In the spontaneously hypertensive (SH) rat, hyperoxic inactivation of the carotid body (CB) produces a rapid and pronounced fall in both arterial pressure and renal sympathetic nerve activity (RSA). Here we show that CB de-afferentation through carotid sinus nerve denervation (CSD) reduces the overactive sympathetic activity in SH rats, providing an effective antihypertensive treatment. We demonstrate that CSD lowers RSA chronically and that this is accompanied by a depressor response in SH but not normotensive rats. The drop in blood pressure is not dependent on renal nerve integrity but mechanistically accompanied by a resetting of the RSA-baroreflex function curve, sensitization of the cardiac baroreflex, changes in renal excretory function and reduced T-lymphocyte infiltration. We further show that combined with renal denervation, CSD remains effective, producing a summative response indicative of an independent mechanism. Our findings indicate that CB de-afferentation is an effective means for robust and sustained sympathoinhibition, which could translate to patients with neurogenic hypertension.

ARTICLE

Received 8 Apr 2013 | Accepted 2 Aug 2013 | Published 3 Sep 2013

Fiona D. McBryde1, Ana P. Abdala1, Emma B. Hendy1, Wioletta Pijacka1, Paul Marvar1, Davi J.A. Moraes2, Paul A. Sobotka3 & Julian F.R. Paton1

In the spontaneously hypertensive (SH) rat, hyperoxic inactivation of the carotid body (CB) produces a rapid and pronounced fall in both arterial pressure and renal sympathetic nerve activity (RSA). Here we show that CB de-afferentation through carotid sinus nerve denervation (CSD) reduces the overactive sympathetic activity in SH rats, providing an effective antihypertensive treatment. We demonstrate that CSD lowers RSA chronically and that this is accompanied by a depressor response in SH but not normotensive rats. The drop in blood pressure is not dependent on renal nerve integrity but mechanistically accompanied by a resetting of the RSAbaroreex function curve, sensitization of the cardiac baroreex, changes in renal excretory function and reduced T-lymphocyte inltration. We further show that combined with renal denervation, CSD remains effective, producing a summative response indicative of an independent mechanism. Our ndings indicate that CB de-afferentation is an effective means for robust and sustained sympathoinhibition, which could translate to patients with neurogenic hypertension.

DOI: 10.1038/ncomms3395

The carotid body as a putative therapeutic target for the treatment of neurogenic hypertension

1 School of Physiology and Pharmacology, Bristol Heart Institute, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, England. 2 Department of Physiology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil. 3 Coridea NC1., 134 W 26th Street, Suite 1011, New York, New York 10001, USA. Correspondence and requests for materials should be addressed to J.F.R.P. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 1

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395

The causes of human essential hypertension remain unknown. It is established that in many patients, sympathetic nerve activity increases proportionately as hyper-

tension develops and this may be a causative factor15, but it is unclear what triggers heightened sympathetic trafc (reviewed in ref. 6). Despite good pharmacological control of arterial pressure in most patients with essential hypertension, a signicant proportion remain drug resistant or intolerant to medication7,8, and this has been highlighted as a growing problem9,10. This presents a signicant clinical challenge and provides the motivation to discover novel ways to control arterial pressure.

Recently, the role of the carotid body (CB) chemoreceptors in the pathophysiology of cardiovascular disease is gaining considerable interest1115. The sympathoexcitation in both sleep apnoea and heart failure may originate from enhanced activity of the CB chemoreceptors1620. In both human patients and animal models of hypertension, the CB chemoreex-evoked sympathoexcitatory responses are enhanced15,2123. However, the hypothesis that CB chemoreceptor drive has an important role in the pathogenesis and/or maintenance of high arterial pressure was undetermined until most recently. We found that in spontaneously hypertensive (SH) rats, carotid sinus nerve denervation (CSD) lowered arterial pressure by B17 mm Hg, and that this was well maintained and tolerated11. These data are comparable to those in human hypertensive patients where inactivation of the CB with 100% oxygen reduced both arterial pressure and sympathetic activity24, supporting a causal role for peripheral chemoreceptors in the aetiology of neurogenic hypertension. On the basis of these data, and that CB resection has been performed and well tolerated for the treatment of dyspnoea in B5,600 patients with asthma and chronic obstructive pulmonary disease (reviewed in ref. 14), we recently proposed that CB ablation may be an effective interventional strategy to control blood pressure in drug-resistant hypertensive patients11,14.

To assess the mechanisms underlying the hypotensive effect of CSD, we have made direct long-term simultaneous recordings of blood pressure and renal sympathetic nerve activity (RSA) before and after CSD in conscious SH and Wistar rats. Given the current clinical interest and application of renal denervation (RD) in the treatment of drug-resistant hypertension2529, we have characterized the interaction between CSD and RD in combination to establish the type of interaction: summative, occlusive or facilitatory. Given that T lymphocytes have been shown to contribute to both angiotensin II and neurally mediated chronic hypertension30,31, we have assessed immune function following CSD. Unlike Wistar rats, we nd that the CB of the SH rat has resting tone driving hypertension, which is not dependent on renal nerves, depresses both the cardiac and sympathetic vasomotor baroreex and attenuates the adaptive immune response. We hypothesize that these ndings may translate to patients with neurogenic hypertension.

ResultsTransient inactivation of CB activity with hyperoxia. All numerical data are reported as means.e.m., with statistical signicance (Po0.05) determined using two-way analysis of variance (repeated measures for within-subject comparison), followed with the HolmSidek post hoc test.

In SH rats, exposure to 100% oxygen to inactivate the CB produced falls in both systolic blood pressure (SBP; 12

4 mm Hg, n 5, P 0.047) and RSA ( 2.90.4 mV, n 5,

P 0.019) but no consistent change in the heart rate (HR; Fig. 1).

In stark contrast, pure oxygen was without signicant effect on these variables when delivered to Wistar rats or SH rats after CSD (Supplementary Fig. S1). These data provide evidence that in SH rats, tonic afferent activity emanating from the CB provides a potent drive for neurogenically mediated hypertension.

a b c

Air

RSA

RSA

100% O2

20 V

3s

12

14 SHR

180

*

Mean arterial pressure (mm Hg)

10

SHR

RSA (V)

8

160

6

4

*

2

HYPEROXIA

140

0 1 0 1 2

10 Wistar

120 Wistar

8

100

RSA (V)

6

4

2

HYPEROXIA

HYPEROXIA

80 1 0 1 2Time (min)

0 1 0 1 2

Time (min)

d e

5

0

Wistar

SHR

Mean arterial pressure (mmHg)

Renal sympathetic

activity (%baseline)

20

0

Wistar

SHR

40

5

60

80

10

100

*

15

120

*

20

Figure 1 | The carotid bodies in hypertensive rats are tonically active. Comparing the effect of 100% oxygen to depress tonic CB activity in Wistar(n 6) and SH (n 6) rats on SBP (a) and RSA (b,c). The CB in SH rats, but not Wistar rats, exhibits tonic activity that is functionally relevant and contributes

to basal arterial pressure (d) and sympathetic activity (e). Data are shown as means.e.m. *Po0.05, repeated-measures analysis of variance.

2 NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395 ARTICLE

Peripheral chemoreex sensitivity in Wistar and SH rats. Although previous studies have found increased reex peripheral chemosensitivity in anaesthetized and decerebrate SH versus Wistar rats15, there are no data in conscious animals. Compared with Wistar rats, SH rats showed a more profound peripheral chemoreceptor reex-evoked bradycardia ( 1945 versus 12654 b.p.m., n 6, P 0.046) and RSA excitatory

responses (13.40.8 versus 90.6 mV, n 6, P 0.021), but

not a pressor response (548 versus 4821 mm Hg; P 0.811)

following an intravenous (i.v.) sodium cyanide bolus.

Time prole of RSA response to CSD in SH and Wistar rats. Following the surgery-related increase in RSA, there was a prompt and profound reduction in RSA below preoperative baseline after CSD in SH rats ( 1.940.3 mV, 5612%;

n 6, P 0.027; Fig. 2ac). The peak fall occurred 5 days post

CSD and showed no signs of recovery throughout the 3-week experimental period. The fall in RSA was correlated temporally with a fall in the mean arterial pressure of 173 mm Hg from

a baseline of 1377 mm Hg (n 6, P 0.009). Spectral analysis

also revealed a reduction in low-frequency (LF) SBP from3.50.2 to 2.50.2 mm Hg2 (Po0.012; Table 1) suggesting a global reduction in sympathetic trafc. In these animals, we also tested the efcacy of CSD: Fig. 2d shows that the sympathoexcitatory reex response following i.v. sodium cyanide was blunted severely. Despite the substantial fall in basal RSA after CSD, a stressful stimulus (high-frequency (HF) noise) continued to evoke a signicant (P 0.013) sympathoexcitatory response

in SH rats, the magnitude of which was not different from those evoked before CSD (before: 13928%, after: 15313%, n 6,

P 0.529; Fig. 2e). This test served to both validate the viability of

our chronic recordings and show that despite lower resting levels of RSA, alerting/arousal responses remained intact ensuring that fundamental visceral reactions were preserved. In Wistar rats, although there was no signicant change in mean arterial pressure after CSD ( 65 mm Hg, n 6), there was a small

fall in RSA ( 0.90.1 mV, 187%, n 6, P 0.045;

Fig. 2ac), which was much less than that seen in SH rats (Po0.01; Fig. 2ac).

#

a

200

150

RSA (% baseline)

100

*

*

*

*

Wistar

50

** **

SHR

**

**

Carotid sinus denervation

0 5 0 5 10

Time (days)

b

c

0

Day3

Day 10

RSA (% baseline)

RSA

RSA

20

W

40

SHR

**

60

2 s

25 V

80

d

e

Pre-CSD

Post -CSD

Pre-CSD

Post-CSD

RSA

RSA

Na CN

Na CN

50 V

High-frequency noise

20 V

1s

2 s

Figure 2 | Carotid sinus denervation causes substantial renal sympathoinhibition. Time prole of renal sympathetic nerve activity (RSA) response to bilateral carotid sinus nerve denervation (CSD) in SH (n 6) and Wistar (n 6) rats (a). Inset (b) shows the raw activity levels of RSA before

and after CSD in one SH rat sitting quietly. The fall in RSA was B55% relative to baseline in SH rats and signicantly greater than that in Wistar rats (c). Efcacy of CSD is shown in d where the sodium cyanide (i.v.)-evoked RSA excitatory response was greatly attenuated. (e) A CSD SH rat showingthat it is able to generate a robust RSA response to a stressful stimulus. Data are shown as means.e.m. *Po0.05, **Po0.01, between-groups analysis of variance (ANOVA); #Po0.05, repeated-measures ANOVA.

NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 3

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395

Table 1 | Cardiovascular and sympathetic effects of carotid sinus denervation.

MBP (mm Hg)

HR (beats per min)

sBRG(ms mm Hg 1)

RR (breaths per min)

LF:HF (PI) VLF(SBPmm Hg2)

LF (SBPmm Hg2)

LF:HF (SBP)

CSD RDNX (n 7)

Absolute

Baseline 1346 3335 0.660.05 712 0.210.01 5.30.6 3.50.2 0.720.03 CBD 1206**# 3146* 0.970.14**# 642**# 0.220.01 5.80.05 2.50.2* 0.600.06** RDNX 1175*# 3177 1.10.19*# 682 0.230.01 5.70.04 2.40.2 0.520.05* ChangeDiff CBD 143**# 197* 0.300.12**# 71**# 0.0140.009 0.450.28 0.990.16*# 0.130.04**

Diff RDNX 43* 54 0.100.07* 52 0.0120.008 0.360.58 0.040.14 0.070.02

Total fall 173 165 0.440.09 21 0.0250.008 0.400.43 0.520.24 0.200.03 Sham RDNX (n 7)

Absolute

Baseline 1328 3264 0.870.18 765 0.200.01 5.30.6 3.20.3 0.650.03 Sham 1355 3067* 0.810.12 754 0.210.01 5.80.5 3.30.3 0.620.03 RDNX 1229* 3002 0.8210.08 694 0.230.02 5.70.4 2.60.2 0.570.03 ChangeDiff Sham 33 197* 0.060.16 01 0.010.01 0.480.28 0.110.18 0.030.01

Diff RDNX 87* 610 0.0820.09 64 0.010.02 0.360.58 0.630.30 0.040.02 RDNX CSD (n 6)

Absolute

Baseline 1435 3253 0.670.04 864 0.210.01 5.90.3 3.40.2 0.580.01 RDNX 1375*# 3247 0.720.04 884 0.210.01 5.60.1 3.20.2 0.560.01 CBD 1264* 3135* 0.780.04 762** 0.210.01 5.60.3 2.90.1* 0.500.02** ChangeDiff RDNX 62*# 13 0.060.03 21 0.0050.012 0.350.14 0.150.13 0.020.01

Diff CBD 113* 115* 0.060.03 112** 0.0010.007 0.030.21 0.350.02* 0.070.01**

Total fall 172 124 0.120.03 92 0.0050.011 0.330.17 0.500.08 0.090.01 Sham CSD (n 6)

Absolute

Baseline 1288 3299 0.740.07 744 0.190.01 5.90.3 3.50.2 0.650.03 Sham 1329 3235 0.690.06 775 0.200.01 5.50.3 3.10.2 0.650.02 CBD 1229* 3139* 0.850.06 652** 0.200.01 5.70.3 2.90.15 0.590.03* ChangeDiff Sham 42 65 0.050.02 31 0.0040.007 0.390.32 0.350.27 0.010.01

Diff CBD 116* 105* 0.160.04 103** 0.0050.005 0.230.24 0.200.17 0.060.03*

CSD, carotid sinus nerve denervation; HF, high frequency; HR, heart rate; LF, low frequency; MAP, mean arterial pressure; PI, pulse interval; RD, renal denervation; RR, respiratory rate; SBP, systolic blood pressure; sBRG, spontaneous baroreceptor reex gain; VLF, very low frequency.

Sham-operated data are given for each procedure. All data are shown as means.e.m. Statistical analysis is performed using repeated-measures ANOVA*Po0.05.

**Po0.01 (within-subject comparison).

#Po0.05 (between-group comparison).

Baroreex function improvement following CSD. Cardiac baroreceptor reex function curves were constructed recently after CSD11 and showed an increase in sensitivity, which might contribute to the hypotensive effect of CSD; hence, we have analysed this in more detail. Consistent with this was the new nding that spontaneous cardiac baroreex gain increased from 1.160.09 to 1.530.27 b.p.m. mm Hg 1 after CSD

(n 7, Po0.01). We also found an improvement in the

sympathetic baroreex sensitivity after CSD in SH rats (Fig. 3), where the curve was shifted leftwards (n 5, P 0.032) and the

operating point moved to the left and downwards (P 0.026;

Fig. 3). An increase in the sensitivity of the curve was revealed by increases in both the upper and lower curvature parameters (see Supplementary Table S1), which translated into an increase in maximum gain (from 6.81.7 to 9.7

1.9% mm Hg 1, P 0.028) towards that seen in Wistar rats

(13.43.1% mm Hg 1). No signicant changes in cardiac or sympathetic baroreex function were found in Wistar rats post

CSD (Supplementary Fig. S2).

Reduced T-cell inltration into the aorta after CSD and RD. Inammation, in particular the adaptive immune response, has

been shown to have a signicant role in the pathogenesis of hypertension3032. We therefore examined the effects of CSD and RD on the percentage of total aortic leukocytes (CD45 average total cells 1.9 104 per aorta) and

T lymphocytes (CD3 average total cells 2.5 103 per aorta).

The brainstem has also been identied as a site of increased inammatory molecules during hypertension33,34; therefore, we have examined the percentage of CD45 CD3 cells

(average total cells 3.6 102 per brainstem) within this tissue.

As shown in Fig. 3b, compared with the surgical shams there were no signicant differences in the percentage of aortic CD45 cells following CSD or RD procedures. However,

when we examined the number of aortic inltrates of CD3 cells, there was a signicant decrease in both CSD

(Fig. 3c,d; Po0.01) and RD groups (Fig. 3cd; Po0.01). Similarly, in single-cell suspensions of whole-brainstem homegenates, a signicant reduction in percentage of CD3 cells was deter

mined following RD (Supplementary Fig. S3; Po0.05) and a trend, which did not reach statistical signicance, for a reduction in CD3 cells in the CSD group. These data suggest that

both CSD and RD procedures can reduce T-cell-mediated inltration of tissue that has been associated with many forms of hypertension.

4 NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395 ARTICLE

a b

250 SHR

7 days post CSD

200

RD

100

%CD45+ cells %CD45+CD3+ cells

SNA (%)

c

100

80

60

40

20

20

15

10

5

150

0 SS CSD

RD

0 SS CSD

*P<0.05

*P<0.05

*

50

Control *

0

100 120 140 160

Mean arterial pressure (mm Hg)

d

SS RD CSD

21.9%

7.2%

12.6%

CD3+

CD45+

Figure 3 | Effects on the sympathetic baroreex and vascular inltration. (a) The renal sympathetic activity baroreex function curve is rightwards shifted over lower pressure ranges after carotid sinus nerve denervation (CSD) in SH rats (n 6) but no changes were observed in Wistar rats

(n 6, Supplementary Fig. S3). Percent total leukocytes (CD45 cells) in the aorta of surgical sham (SS), RD and CSD animals (b). There was reduced

vascular T-cell inltration following RD and CSD in the SH rats as the percentage of T lymphocytes (CD45 CD3 cells) in the aorta of RD and

CSD animals was less relative to SS (n 6 and 7 per group; c). Representative ow cytometry contour plot showing T lymphocytes (CD45 CD3 ) in

aortic samples of each rat group (d). All data are shown as means.e.m. *Po0.05, analysis of variance.

Renal excretory function after CSD. Given the alterations in arterial pressure and RSA, we looked to see whether renal function was altered. Despite our observed reduction in arterial pressure, SH rats showed an increase in 24-h urine volume at 12 days following CSD (from 4.250.28 to 5.570.32 ml/24 h/100 g body weight; Po0.05), which was sustained at days 21 and 28.

This was accompanied by an increase in creatinine clearance (from 0.940.03 to 1.150.07 ml min 1; Po0.01), indicating an increase in glomerular ltration rate. The total protein in urine decreased signicantly (from 10.51.0 to 8.70.8 mg ml 1;

Po0.05), albuminuria was lower after CSD but not signicant (from 0.570.05 to 0.450.05 mg ml 1; P 0.067), although

water intake also increased signicantly (from 101.2 to12.81.6 ml/24 h/100g body weight; Po0.01). For comparison, Wistar rats had a lower total protein and albumin level in their urine compared with SH rats before denervation: 9.750.84 versus 10.490.77 mg ml 1 and 0.400.06 versus 0.57

0.05 mg ml 1, respectively. The water intake and creatinine clearance was similar between Wistar and SH rats before denervation: 30.812.13 versus 30.492.13 ml/24 h and 0.830.07 versus 0.940.03 ml min 1, respectively. Taken together, these data indicate that the progression of glomerular kidney damage/

leakage in SH rats may have been retarded by CSD.

Time to sympathoinhibition after CSD in SH rats. In conscious SH rats, it was not possible to assess the precise time to onset of the depressor and sympathoinhibition following CSD because of the side effects of surgery and anaesthesia. To this end, we recorded internal cervical (iCSN) and lumbar sympathetic

post-ganglionic nerves simultaneously before and after CSD in in situ-unanesthetized decerebrated SH rats. Figure 4 shows that by 40 min, there was already a signicant decrease in sympathetic nerve activity in all outows and that this was due, in part, to a decrease in the amplitude of respiratory-modulated discharge (Po0.05; Fig. 4b). The peak response was achieved at 60 min post CSD, as activity levels at 75 or 90 min were not different from the level recorded at 40 min post CSD but were different from their own baseline (both Po0.01; Fig. 4c).

Consistent with the reduction of respiratory-sympathetic modulation, we found a decrease in HF SBP in conscious SH rats after CSD (from 4.520.3 to 3.90.3 mm Hg2; n 7,

Po0.05).

Comparing uni- versus bilateral CSD in SH rats. We wished to test any difference in the sympathoexcitatory drive from the left versus the right CB as well as their individual responses. Figure 5 shows the time prole of the response in SBP following unilateral and subsequent bilateral CSD. Unilateral CSD, whether targeting the left (n 4) or right side (n 4) produced no change in SBP

(Supplementary Table S2). The fall in SBP achieved when bilateral CSD was staggered in time by 15 days (that is, denervating left then right CB, or denervating right then left CB) was no different from the response when both the CBs were denervated at the same time ( 162 versus 173 mmHg, respectively,

P 0.831). In our in situ preparation, which allowed high tem

poral resolution of changes in sympathetic activity (see above), unilateral CSD did not produce a measureable change in any of the recorded variables.

NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 5

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395

a

Pre-CSD Post-CSD

7.5 mm Hg

10 V

10 V

10 V

10 V

PP

iCSN

iCSN

iCSN

iCSN

iCSN

ISN

ISN

ISN ISN

iCSN

ISN

ISN

PN

PN

PP 7.5 mm Hg

10 V

10 V

50 V

0.5 s

PN

PN

PN PN

50 V

0.5 s

b

Pre-I burst

10 V

10 V

0.5 s 0.5 s

c

80 10 20 18

14 12 10

8 6 4 2 0

16

70 60 50 40 30 20 10

PP (mm Hg)

TH (mm Hg)

9 8 7 6 5 4 3

Respiratory-related burst

(V) iCSN

Respiratory-related burst

(V) iSN

********

* *

** ** **

** ** *****

20 18

14 12 10

8 6 4 2 0

16

CSD

2 CSD CSD

CBD

SHR CSD (n=7) Wistar CSD (n=5) SHR SHAM (n=5)

0 0 20 40 60 80Time (min) Time (min) Time (min) Time (min)

20 0 20 40 60 80 100

1 0 20 0 20 40 60 80 100

20 0 20 40 60 80 100

20

100

Figure 4 | Immediate effects of carotid sinus denervation. The time course of the fall in sympathetic nerve activity (raw and integrated) after carotid sinus nerve denervation (CSD) was compared for the internal cervical (iCSN) and lumbar (lSN) sympathetic post-ganglionic nerves in situ. By 40 min, there was a signicant decrease in the sympathetic nerve activity in both outows (a). Note the decrease in the amplitude of respiratory-modulated discharge (Po0.05; b). The peak response was achieved at 60 min post CSD as activity levels at 75 or 90 min were not different from the level recorded at 40 min post CSD but were different from their own baseline (c). Data are shown as means.e.m. *Po0.05, **Po0.01, ***Po0.001, repeated-measures analysis of variance; nZ5. PN, phrenic nerve; PP, perfusion pressure (aortic); Pre-I, pre-inspiratory; TH, TraubeHering arterial pressure waves.

SBP response to combined CSD and RD. Given the current interest in RD2529, the relative potency and interaction (occlusive, summative and facilitatory) of RD versus CSD and was deemed scientically and clinically important. All sham-operated SH rats showed a time-related increase in SBP (44 and 32 mm Hg for sham CSDRD and sham RDCSD, respectively; Figs 6,7). The efcacy of RD was conrmed by measuring renal noradrenaline content after RD (with or without concurrent CSD), which showed a signicant reduction compared with sham controls (RD alone: 456; RD with CSD: 319; sham: 44354 pg per 100 mg protein; n 18, Po0.01).

The data reported herein are all expressed relative to the preoperative control level.

Figure 6 shows the time prole of the SBP response to RD followed by CSD (Fig. 6a) and CSD followed by RD (Fig. 6b). In SH rats, performing RD rst produced a fall of SBP of

92 mm Hg from a baseline of 1665 mm Hg (n 6,

Po0.013). A further fall of 123 mm Hg ensued after

subsequent CSD (Po0.001) and showed no recovery over the observational period. In SH rats undergoing the CSDRD surgical sequence, basal SBP was 1608 mm Hg, CSD alone evoked a prompt fall of 142 mm Hg (n 7, Po0.001) with a further

fall of 62 mm Hg (Po0.001) after RD; no recovery was

observed over the time course of the study. The total fall

( 172 mm Hg) was not different from that measured with the

reversed sequence of RDCSD ( 203 mm Hg; P 0.363). These

data are summarized in Fig. 7 and show an additive interaction between RD and CSD that this is similar irrespective of the order in which the surgical procedures are carried out in SH rats. CSD, but not RD, produced signicant bradycardia (P 0.018) and a small

reduction in respiratory frequency (Po0.01) in SH rats (Table 1). Table 1 documents spectral frequency analysis of both pulse interval and SBP after RDCSD and CSDRD. RD alone resulted in an increase in spontaneous baroreex gain ( 1.730.4 b.p.m.

mm Hg 1 mmHg; Po0.01; Table 1) and decrease in CD3 cells

(Fig. 3bd; Po0.01). Figure 6c demonstrates that the reex bradycardia and pressor responses to sodium cyanide (i.v.), used to activate peripheral chemoreceptors in conscious SH rats, were abolished after bilateral CSD.

DiscussionOur data demonstrate that the CB in the conscious SH rat is constitutively active and provides a tonic prohypertensive drive. Mechanistically, the antihypertensive effects of CSD in SH rats include a substantial reduction in sympathetic nerve discharge including that to the kidney, involving a reduction of respiratory modulation; a resetting of the sympathetic baroreex over lower

6 NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395 ARTICLE

a

Unilateral CSD

Bilateral

CSD

180

SBP (mm Hg)

**

160

140

5 0 5 10 15 20 25 30 35 Time (days)

b + right + left

5

Sham Left Right

bilateral

0

SBP (mm Hg)

5

Sham

Unilateral

Bilateral

10

15

20

** ** **

Figure 5 | Unilateral carotid sinus denervation does not lower blood pressure in the rat. Unilateral carotid sinus nerve denervation (CSD) was ineffective at lowering SBP in SH rats whether the left or right CB was removed (n 8; a). Irrespective of whether the left or right CB was removed rst, there was no

difference in the fall in SBP observed on removal of the contralateral CB (b). Note that time-staggered unilateral CB denervation does not affect the magnitude of the fall in SBP, which is the same as that seen when both left and right CB are denervated simultaneously (see Figs 2,3). Data are shown as means.e.m. **Po0.01, within-group analysis of variance.

a

RD/sham +CSD

b

180

180

CSD/sham

+RD

*

160

*

**

160

SBP (mm Hg)

SBP (mm Hg)

*

## *

140

140

RD-CBD

Sham-CBD

CBD-RD Sham-RD

120 7 0 7 14 21 28

Time (days)

120 7 0 7 14 21 28

Time (days)

c

Pre-CSD Post-CSD

200

175

SBP

(mm Hg)

100

150

125

Heart rate

(b.p.m.)

400

300

200

100

10 s

0

Na CN

Na CN

Figure 6 | Carotid sinus and RD interaction time course. Time prole of the SBP response to (a) renal nerve denervation (RD) followed by carotid sinus nerve denervation (CSD; n 6) and (b) CSD followed by RD resection in SH rats (n 7). Note the time-dependent increase in SBP in shams

(n 6 and 7, respectively). The completeness of the CSD is shown by the absence of a reex pressor and bradycardic response following i.v. injections of

sodium cyanide (NaCN; c). Data are shown as means.e.m., repeated-measures analysis of variance (ANOVA): *Po0.05, **Po0.01; between-group ANOVA, ##Po0.01.

NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 7

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395

10

5

Sham Sham

0

SBP (mm Hg)

RD

5

RD

CSD

CSD

10

*

*

15

CSD

RD

**

*

NS

20

*

25

*

NS

#

Figure 7 | Carotid sinus and RD interaction summary. Group data show the interaction between carotid sinus nerve denervation (CSD; n 6) and

renal nerve denervation (RD; n 7) in SH rats. There was an additive effect

of CSD and RD, irrespective of the order in which the intervention was made. The combined intervention produced a greater fall in SBP than each RD or CSD alone. Data are shown as means.e.m., repeated-measures analysis of variance (ANOVA): *Po0.05, **Po0.01; between-group

ANOVA, #Po0.05.

arterial pressures; an improvement in spontaneous cardiac baroreex gain; possible changes in renal excretory function; and a reduction in vascular T-cell inltration. Unilateral CSD, whether on the left or right side, was ineffective in reducing arterial pressure but subsequent resection of the contralateral side remained an effective antihypertensive therapy. In conscious SH rats, we show that CSD is more potent as an antihypertensive intervention than RD but that the renal nerves are not necessary for the hypotensive effect of CSD. Combining RD with CSD, or reversing the sequence (CSD then RD), gives an additive response that is similar in magnitude, irrespective of the order in which the surgical procedures are performed.

RD in young SH rats prevents the full development of hypertension35,36. The fall in arterial pressure by B9 mm Hg we report herein following RD in Wistar rats is consistent with previous studies in SpragueDawley rats37 but not with Wistar Kyoto rats, where arterial pressure was unaltered35,38. Previous reports in SH rats demonstrated larger falls in arterial pressure after RD (B19 mm Hg (refs 36,38)) relative to our results (B9 mm Hg). The difference may reect the use of tail cuff/ indwelling catheters rather than radio-telemetry and between-group versus within-animal analyses. Despite these quantitative differences, renal nerves (afferent and/or efferent) have a tonic role in regulating arterial pressure in some strains of normotensive and SH rats, as reviewed recently39.

In conscious SH rats, our nding that the CB chemoreex sensitivity is elevated relative to the Wistar animal is consistent with prior data from animals and humans15,2123. For the rst time, we describe heightened CB tonicity in SH rats as revealed by a depressor and sympathoinhibitory response to hyperoxia in SH but not Wistar rats. This suggests that the CB has elevated activity in the SH rat providing excitatory drive to the generation of sympathetic vasomotor tone that in turn contributes to the hypertension, as recently described in hypertensive human patients24. It may also explain why CSD is effective in producing

a long-term antihypertensive response. The tonic activity in SH rats may result from increased expression of ASIC3 and TASK1 two acid-sensing non-voltage-gated channels15. Others suggest that the balance between carbon monoxide and hydrogen sulphide within the CB and the activity of HIF-1a versus HIF-2a have a role40. Animal models of chronic heart failure have revealed that CB chemoreceptor activity is augmented by angiotensin II receptors41, impaired nitric oxide synthase activity20, reduced CB blood ow42, enhanced adenosine monophosphate-activated protein kinase43 and inammation44,45. Heightened sympathetic drive to the arterioles of the CB itself may also contribute to its hyperactivity because of hypoperfusion (via vasoconstriction and vascular remodelling46) and inammation.

Because of the complications of anaesthesia and surgical recovery, we were unable to determine the precise time-dependent effects following CSD in conscious SH rats. Using our in situ rat preparation, a fall in perfusion pressure and sympathetic activity occurred at 40 min post CSD indicating a relatively rapid response. Presumably, 40 min permits a reconguration of the cardio-respiratory brainstem network in the in situ rat, as reected by a reduction in both respiratory-sympathetic coupling and the magnitude of the TraubeHering wave amplitude. Consistent with reduced respiratory-sympathetic coupling, we saw a decrease in HF SBP spectra in conscious SH rats after CSD. Given that respiratory-sympathetic coupling contributes signicantly to vascular resistance and arterial pressure development the SH rat47, this could be a major mechanism for the CSD-evoked antihypertensive effect.

It was evident that removal of a single CB, whether the left or right, in vivo or in situ, was ineffective in lowering arterial pressure in SH rats, although there was a brief reduction in respiratory frequency, perhaps indicative of a transient effect. Given that CB afferents terminate in the commissural nucleus tractus solitarious (NTS) with axons traversing the midline providing ample innervation of contralateral targets48, it perhaps is not surprising that unilateral CSD was not effective long-term.

On subsequent removal of the second CB from a SH rat with chronic, unilateral CSD, arterial pressure fell precipitously; the level reached was not different from that recorded following a procedure where both CBs were removed at the same time. This suggests that any compensation occurring after loss of a single CB does not affect the efcacy of response following contralateral CB denervation. Whether these ndings relate to humans with hypertension is unknown but provides two predictions. The rst is that unilateral CSD may be effective transiently in lowering arterial pressure and compensated for with time. Second, subsequent CB resection following a unilateral procedure will cause a potent hypotensive effect. For safety reasons, a clinical study should start with unilateral CB ablation; thus, knowing that a hypotensive effect persists following staggered bilateral CSD (that is, not ameliorated by a prior unilateral intervention) is an important preclinical information.

Previously, we have shown that CSD in young, prehypertensive SH rats prevented the full manifestation of hypertension and lowered arterial pressure in adult SH rats11. Using both power spectral analysis and hexamethonium to block ganglionic transmission, we provided indirect evidence that the hypotension was caused, in part, by a generalized reduction in sympathetic activity11. Here we demonstrate for the rst time that the CSD-induced hypotension was accompanied with a substantial reduction in RSA that fell with a similar time course. Despite this robust reduction of B55% in RSA, we showed that animals were not left sympathetically incompetent, and, importantly, could respond vigorously to a stressor stimulus. Given that lumbar and cervical sympathetic outows also fell (as shown acutely), we suspect that there is widespread sympathoinhibition following

8 NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395 ARTICLE

CSD and that it is not conned to the kidneys. This is supported by a chronic reduction in LF spectra of SBP. This has important clinical implications regarding the applicability of CSD beyond hypertension to conditions such as heart failure, sleep apnoea and insulin resistance, where there is excessive and pathological sympathetic activity14.

The mechanisms for blood pressure reduction following CSD are likely to be numerous and operate over different time scales giving a persistent effect. Our hyperoxia data clearly shows that removal of CB tone in SH rats can, at least acutely, provide sympathoinhibition. This may suggest a hypoxic drive within the CB of the SH rat, which might be the stimulus for the increased density of small vessels in the CB of SH versus normotensive rats46. However, we do not rule out non-hypoxic intra-CB drives to explain the tonicity within the SH rat CB. Other mechanisms by which CSD lowers blood pressure include an improvement in both cardiac and sympathetic vasomotor limbs of the baroreex after CSD11. As our method of CSD removes innervation of the carotid sinus baroreceptors, the improvement in baroreceptor reex function must be of aortic baroreceptor origin. On the basis of this observation, it is expected that selective removal of the CB, sparing carotid sinus baroreceptors, would be predicted to enhance their gain also and produce a greater antihypertensive effect. The known antagonistic interaction between chemo- and baro-afferents both centrally (at a neuronal level within nucleus tractus solitarii49) and of their respective reex responses50 could explain this benecial result as enhanced baroreex function can contribute to the long-term lowering of blood pressure and sympathetic activity in dogs51 and humans52. We do not rule out improved compliance of the aorta, central plasticity and/or improved cardiac vagal transmission to explain better baroreex function after CSD in SH rats. The nding of reduced iCSN activity may improve the brainstem blood ow; given that the SH rat brainstem is hypoperfused, improving ow may contribute to reducing arterial pressure53. Our observation of increased urine production and glomerular ltration rate in the face of decreased systemic arterial pressure indicates that CSD may be associated with active improvements in renal excretory function. This may be mediated, at least in part, directly through the substantial inhibition of renal sympathetic activity and may contribute to the hypotensive effect of CSD through uid excretion. The reduced percentage of vascular and brainstem inltrates of T lymphocytes is unlikely to contribute to the prompt fall in the arterial pressure because of its rapid time course but may assist in the long-term maintenance of the hypotensive effect of CSD especially because T lymphocytes have been shown to contribute to both angiotensin II and neurally mediated chronic hypertension54,55. We speculate that the sympathoinhibitory effect of CSD contributes to reduced T-cell inltration in the aorta and brainstem described herein, which could improve vascular compliance including the aortic baroreceptor sensitivity and improved baroreex transmission centrally33,34. The location of the vascular immune cell inltrates is unknown but we speculate based on previous studies56 that these cells predominantly accumulate in the adventitia and perivascular adipose tissue of blood vessels. Given the sympathetic innervation of immune system organs such as the spleen, thymus and bone marrow26, we suggest that reductions in sympathetic activity may be the trigger for the anti-inammatory response, thereby breaking this positive (and pathological) feedback loop6.

One successful new antihypertensive interventional approach is RD, which appears to be a safe treatment in some humans with drug-resistant hypertension2529. In one study, blood pressure was lowered by 410 mm Hg with a high success rate (84%), although all patients remained hypertensive and their antihypertensive medication was maintained25. In contrast, an 84% success rate was

not conrmed by other groups who reported no overall change in the arterial pressure, although some individuals did respond27 or around a 4050% blood pressure response rate26,28,29. Given these ndings, and the recent success of unilateral CB resection in a heart failure patient57, CB ablation to control arterial pressure in drug-resistant hypertensive patients could be considered. Thus, scenarios are imagined that include patients in which RD has not lowered arterial pressure to target levels; not circumvented prescribed and poorly tolerated medication; and failed and not been possible because of renal artery stenosis or bifurcation. Thus, it becomes critical to study any interaction on arterial pressure following combined RD and CSD as well as CSD alone procedures.

The nding that unilateral CB resection in a heart failure patient was effective in correcting partially both cardiac autonomic balance and cardiac baroreex gain57, yet ineffective in altering these variables in SH rats is curious. Clearly, heart failure and hypertension have distinct pathologies, which could explain this difference. Equally, there may be differences in CB signalling mechanisms between SH rats and humans that could contribute to response differences between unilateral versus bilateral CB resection. This remains an open question.

In conclusion, there is an additive antihypotensive interaction between CSD and RD, which is unaffected by either the order or timing of the procedures. Bilateral CSD provides a potent and persistent antihypertensive response in SH rats accompanied by substantial sympathoinhibition in multiple outows, improved baroreex and renal function, and reduced T-lymphocyte inltration in the aorta and brainstem. Whether CSD offers therapeutic benet to drug-resistant/intolerant human patients with sympathetically mediated diseases is currently undergoing clinical trial.

Methods

Renal and CB surgery. Animal experiments were conducted in accordance with the UK Animals (Scientic Procedures) Act 1986 and associated guidelines and approved by the University of Bristol Ethics Committee. Adult male (12 weeks;n 38) SHR and Wistar rats (12 weeks; n 6) bred in the University of Bristol

Animal Services Unit were used. For all surgeries, rats were anaesthetized with ketamine (60 mg kg 1; intramuscular) and medetomidine (250 mg kg 1, intramuscular), and aseptic techniques were used.

For CSD, the CB was approached via an anterior midline neck incision and the carotid sinus nerve branches were sectioned11. Sham-operated rats underwent the same surgical procedures to expose the CB but the carotid sinus nerves were left intact. Unilateral CSD rats (SH, n 8) were randomized to have either their left

(n 4) or right (n 4) carotid sinus denervated rst, followed by contralateral

denervation 14 days later. On denervating the second CB, a transient apnoea occurred in all SH rats with some requiring resuscitation. This apnoea was not observed in Wistar rats. No post-surgical mortalities occurred, suggesting that there were no lethal apnoeic episodes post CSD and all rats gained weight normally. To assess the efcacy of CSD, arterial pressure was recorded (and from this, HR and respiratory frequency were derived) during an injection of sodium cyanide (NaCN, 100 ml 0.04% i.v.) 12 weeks post CSD.

Bilateral RD was achieved via a retroperitoneal incision to allow retraction of each kidney and exposure of the renal artery. The nerves and adventitia were stripped from the renal artery and renal plexus, which were then painted with a dilute (10% in ethanol) phenol solution. Sham-operated rats had both kidneys exposed via the retroperitoneal incision and the renal nerves visualized but left intact.

Renal sympathetic activity response to CSD. In rats (SHR, n 6; Wistar, n 6)

following implantation of the blood pressure catheter into the abdominal aorta and closure of the midline incision33, the right kidney was exposed via a retroperitoneal incision. Using a ne round-tipped glass hook, the renal nerve was freed from the surrounding connective tissue and a small patch of paralm was slipped underneath to prevent uid ingress. The nerve was passed over the electrodes from a sympathetic nerve activity telemeter (Model TR46SP, Telemetry Research Ltd., Auckland, New Zealand), carefully xed in place using sutures, and then isolated from surrounding tissue with silicone elastomer (Kwik-Sil, WPI Inc.). A polyurethane catheter (0.033 in (B0.84 mm) outer diameter (OD); 0.014 in (B0.36 mm) inner diameter (ID); Micro-Renathane, Braintree Scientic) was implanted into the left femoral vein, connected to a transcutaneous port between

NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 9

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395

the scapulae and ushed every 23 days with saline-containing heparin(100 U ml 1) and penicillin G (2,000 U ml 1) solution11. On days 2 and 8, the

venous lines were connected and solutions of phenylephrine (0.1 mg ml 1) and sodium nitroprusside (0.1 mg ml 1) were infused i.v. to obtain ramp increases in arterial pressure of 12 mm Hg s 1 between B60 and 180 mm Hg. These data were then used to generate 5-parameter sigmoidal regression baroreex function curves. A brief sodium cyanide bolus (NaCN, 100 ml 0.04% i.v.) was given to stimulate peripheral chemoreceptors. The zero level of renal sympathetic activity (RSA) was assessed after ganglionic blockade using hexamethonium tartrate(100 ml, 10 mg ml 1 i.v.). To assess any tonic activity generated by the CB, hyperoxia was used to silence the CBs by briey placing their cages into a plexiglass box connected to a 100% oxygen source on day 1. Arterial pressure was

recorded 24 h per day, whereas RSA was recorded for 60 min at approximately the

same time each day when animals were observed to be resting quietly.

Arterial pressure and RSA signals were sampled at 500 Hz using an analogue-digital data acquisition card (PCI 6024E National Instruments, Austin, Texas) and continuously displayed by a data acquisition programme (Universal Acquisition 11, University of Auckland, Auckland, New Zealand). HR was derived from the arterial pressure waveform. The RSA signal was amplied, ltered between 50 and5,000 Hz, full-wave rectied and integrated using a low-pass lter with a 20-ms time constant. At least 7 days were allowed for recovery before a 3-day baseline period was recorded. Arterial pressure and HR were recorded for 24 h a day. To avoid movement artefact, RSA was recorded for an hour at the same time each day, whereas the rats undisturbed in their home cage were observed to be quiescent. On days 2 and 7, rats were exposed to a brief burst of HF noise, which produces a

stress increase in RSA. The magnitude of this response was used to check that the RSA signal remained viable and responsive throughout the experiment.

Renal excretory function. Rats were housed in metabolic cages with free access to water and food for 24 h. Blood was withdrawn from the tail vein. Plasma and urine creatinine were measured by improved Jaffe method with the commercially available QuantiCrom Creatinine Assay Kit (DICT-500, Universal Biologicals, Cambridge, UK)58. Creatinine clearance was estimated as (urinary creatinine ((mmol l 1) volume urine produce in 24 h (ml))/(plasma creatinine

(mmol l 1) 1,440 (min)). Urinary albumin was measured by the improved

bromocresol green (BCG) method59 with the commercially available Albumin Assay (Randox Laboratories Ltd, Crumlin, UK) and the total protein was measured by the Lowry method60 with the DC Protein Assay Kit (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). Urinary sodium was measured using an ion-selective electrode (Cole-Parmer, London,UK). Renal excretory function was assessed over a 24-h period before (day 2) and after (day 28) CSD (n 6) or sham (n 6) surgery.

Renal noradrenaline content. The left kidneys were crushed in liquid nitrogen and homogenized in an extraction buffer (0.01 M HCl, 1 mM EDTA, 4 mM sodium metabisulphite). Samples were centrifuged (8,000 r.p.m. for 30 min) and the supernatant protein concentration was determined by DC Protein Assay Kit (Bio-Rad Laboratories Ltd, No 500-0112). Tissue noradrenaline content was assessed by enzyme-linked immunosorbent assay (Alpco Diagonstics, USA) and normalized to protein concentration.

FACS analysis of cellular inammation. Analysis of T cells in vascular homogenates of the aorta and brainstem was performed using uorescence-activated cell sorting (FACS). To analyse leukocytes in the aorta and brain, tissue was digested using collagenase type IX (125 m ml 1); collagenase type IS (450 U ml 1) and hyaluronidase IS (60 U ml 1) dissolved in 20 mM HEPESPBS buffer for 30 min at 37 C, during continuous agitation. The dissolved tissue was then passed through a 70-m sterile lter (Falcon, BD Biosciences), yielding a single-cell suspension. An additional step was applied for brain tissue using a 30/70% percoll gradient to separate out the mononuclear cell layer. All cells were then washed twice with FACS buffer (0.5% BSA in PBS) then counted, stained and analysed using multi-colour ow cytometry61. Antibodies (BD Biosciences) used for staining were as follows: V450 anti-CD45 and PE anti-CD3. After immunostaining, cells were resuspended in FACS buffer (0.5% BSA in PBS) and analysed immediately on a LSR-II ow cytometer with DIVA software (Becton Dickinson). Data were analysed with FlowJo software (Tree Star Inc., Ashland, Oregon, USA).

In situ working heartbrainstem preparation. As described originally62, animals were anaesthetized deeply (halothane), heparinized (1,000 units, intraperitoneal), bisected below the diaphragm and decerebrated pre-collicularly. Anaesthesia was terminated. Preparations were transferred to a recording chamber and a double lumen cannula was placed into the descending aorta for retrograde perfusion with a Ringer solution containing in mM NaCl (125), NaHCO3 (24), KCl (3), CaCl2(2.5), MgSO4 (1.25), KH2PO4 (1.25), and D-glucose (10). Ficoll (molecular weight: 20,000; 1.25%) was added as an oncotic agent and the perfusion solution was saturated with 95% O2-5% CO2 (pH, 7.357.4; osmolality 2905 mosmkg

H2O 1 at 31 C). Perfusion pressure was maintained between 60 and 80 mm Hg with additions of arginine vasopressin (200400 pM, Sigma). A muscle relaxant, vecuronium bromide (4 mg ml 1; Organon Teknica, Cambridge, UK) was added to the perfusate. Recordings from the phrenic, lumbar sympathetic nerves and iCSN

were made simultaneously using custom bipolar glass suction electrodes. Signals were amplied ( 20k), ltered (603,000 Hz), rectied and integrated (50 ms time

constant), and the noise level (obtained at the end of the experiment) was subtracted. Phrenic-triggered averaging of integrated sympathetic activity (over 10 phrenic cycles) allowed peak-to-trough amplitudes of the respiratory-related sympathetic burst to be measured. The TraubeHering waves were also analysed using phrenic-triggered averaging.

Cardiovascular responses to CSD and RD. Radio-telemeters (PA-C40, Data Sciences Inc., St Paul, Minnesota, USA) were implanted as described before33,34. After 7 full days of recovery, 4 days of baseline arterial pressure was recorded before the rst surgical intervention (All SH rats: CSD, n 7; shamCSD n 7;

RD, n 6; shamRD n 6). Twenty-one days after CSD/shamCSD, RD surgery

was performed; conversely, 14 days after RD/shamRD, CSD surgery was performed. Arterial pressure was recorded for 5 min every hour, 24 h per day using Hey Presto software, and HR and respiratory rate were derived from the arterial pressure waveform33. The data shown for cardiovascular parameters and ventilatory frequency represent daily averages from these 5-min periods. On the nal day of the experiment, rats were euthanized (sodium pentobarbital40 mg kg 1, intraperitoneal) and the kidneys were extracted, weighed and frozen in liquid nitrogen for a noradrenaline assay. The aorta was removed for FACS and analysis of cellular inammation (see below).

Power spectral analysis. Power spectral density was computed using purpose-written software33. The following frequencies were calculated in normalized units: o0.27 Hz (very low frequency), 0.270.75 Hz (LF) and 0.753.3 Hz (HF). The ratio of the LF to the HF component was used as an indicator of cardiac sympathoparasympathetic balance. Spontaneous baroreex gain was computed and respiratory rate was inferred from the peaks of respiratory modulation of the systolic pressure frequency spectrum.

Data analysis. From the arterial pressure waveform, values for systolic, diastolic, mean pulse pressure, HR and respiratory rate were derived. RSA is expressed as a percentage, where 100% is the average integrated value across the baseline period. Values are presented as means.e.m. except where stated. Data were compared between baseline and CSD, and where applicable, RD time points, by repeated measures two-way analysis of variance, were followed where appropriate by the HolmSidek post hoc multiple comparison test. Differences within or between groups with P-values of o0.05 were considered signicant.

References

1. Anderson, E. A., Sinkey, C. A., Lawton, W. J. & Mark, A. L. Elevated sympathetic nerve activity in borderline hypertensive humans. Evidence from direct intraneural recordings. Hypertension 14, 177183 (1989).

2. Esler, M., Rumantir, M., Kaye, D. & Lambert, G. The sympathetic neurobiology of essential hypertension: disparate inuences of obesity, stress, and noradrenaline transporter dysfunction? Am. J. Hypertens. 14, 139S146S (2001).

3. Grassi, G. Sympathetic and baroreex function in hypertension: implications for current and new drugs. Curr. Pharm. Des. 10, 35793589 (2004).

4. Grassi, G. Counteracting the sympathetic nervous system in essential hypertension. Curr. Opin. Nephrol. Hypertens. 13, 513519 (2004).

5. Smith, P. A., Graham, L. N., Mackintosh, A. F., Stoker, J. B. & Mary, D. A. Relationship between central sympathetic activity and stages of human hypertension. Am. J. Hypertens. 17, 217222 (2004).

6. Fisher, J. P. & Paton, J. F. The sympathetic nervous system and blood pressure in humans: implications for hypertension. J. Hum. Hypertens. 26, 463475 (2012).

7. Falaschetti, E., Chaudhury, M., Mindell, J. & Poulter, N. Continued improvement in hypertension management in England: results from the Health Survey for England. Hypertension 53, 480486 (2006).

8. Plump, A. Accelerating the pulse of cardiovascular R&D. Nat. Rev. Drug. Discov. 9, 823824 (2010).

9. Myat, A., Redwood, S. R., Qureshi, A. C., Spertus, J. A. & Williams, B. Resistant hypertension. BMJ 345, e7473 (2012).

10. Pimenta, E. & Calhoun, D. A. Resistant hypertension: incidence, prevalence, and prognosis. Circulation 125, 15941596 (2012).

11. Abdala, A. P. et al. Hypertension is critically dependent on the carotid body input in the spontaneously hypertensive rat. J. Physiol. 590, 42694277 (2012).

12. Hering, D. et al. Tonic chemoreex activation contributes to the elevated muscle sympathetic nerve activity in patients with chronic renal failure.J. Hypertens. 25, 157161 (2007).13. Narkiewicz, K. et al. Contribution of tonic chemoreex activation to sympathetic activity and blood pressure in patients with obstructive sleep apnea. Circulation 97, 943945 (1998).

14. Paton, J. F. et al. The carotid body as a therapeutic target for the treatment of sympathetically mediated diseases. Hypertension 61, 513 (2013).

10 NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3395 ARTICLE

15. Tan, Z. Y. et al. Chemoreceptor hypersensitivity, sympathetic excitation, and overexpression of ASIC and TASK channels before the onset of hypertension in SHR. Circ. Res. 106, 536545 (2010).

16. Chua, T. P. et al. Clinical characteristics of chronic heart failure patients with an augmented peripheral chemoreex. Eur. Heart. J. 18, 480486 (1997).

17. Narkiewicz, K. et al. Selective potentiation of peripheral chemoreex sensitivity in obstructive sleep apnea. Circulation 99, 11831189 (1999).

18. Ponikowski, P. et al. Peripheral chemoreceptor hypersensitivity: an ominous sign in patients with chronic heart failure. Circulation 104, 544549 (2001).

19. Sun, S. Y., Wang, W., Zucker, I. H. & Schultz, H. D. Enhanced peripheral chemoreex function in conscious rabbits with pacing-induced heart failure.J. Appl. Physiol. 86, 12641272 (1999).20. Sun, S. Y., Wang, W., Zucker, I. H. & Schultz, H. D. Enhanced activity of carotid body chemoreceptors in rabbits with heart failure: role of nitric oxide.J. Appl. Physiol. 86, 12731282 (1999).21. Przybylski, J. Do arterial chemoreceptors play a role in the pathogenesis of hypertension? Med. Hypotheses. 7, 127131 (1981).

22. Somers, V. K., Mark, A. L. & Abboud, F. M. Potentiation of sympathetic nerve responses to hypoxia in borderline hypertensive subjects. Hypertension 11, 608612 (1988).

23. Trzebski, A., Tal, M., Zoltowski, M. & Przybylski, J. Increased sensitivity of the arterial chemoreceptor drive in young men with mild hypertension. Cardiovasc. Res. 16, 163172 (1982).

24. Sinski, M. et al. Tonic activity of carotid body chemoreceptors contributes to the increased sympathetic drive in essential hypertension. Hypertens. Res. 35, 487491 (2012).

25. Esler, M. D. et al. Renal sympathetic denervation in patients with treatment-resistant hypertension (The Symplicity HTN-2 Trial): a randomised controlled trial. Lancet 376, 19031909 (2010).

26. Hart, E. C. et al. Translational examination of changes in baroreceptor reex function after bilateral renal denervation in hypertensive rats and humans. Hypertension 62, 533541 (2013).

27. Brinkmann, J. et al. Catheter-based renal nerve ablation and centrally generated sympathetic activity in difcult-to-control hypertensive patients: prospective case series. Hypertension 60, 14851490 (2012).

28. Prochnau, D. et al. Catheter-based renal denervation for drug-resistant hypertension by using a standard electrophysiology catheter. Eurointervention 7, 10771080 (2012).

29. Vase, H. et al. Catheter-based renal denervation for treatment of resistant hypertension. Dan. Med. J. 59, A4439 (2012).

30. Harrison, D. G. et al. Inammation, immunity, and hypertension. Hypertension 57, 132140 (2011).

31. Muller, D. N., Kvakan, H. & Luft, F. C. Immune-related effects in hypertension and target-organ damage. Curr. Opin. Nephrol. Hypertens. 20, 113117 (2011).

32. Zubcevic, J., Waki, H., Raizada, M. K. & Paton, J. F. Autonomic-immune-vascular interaction: an emerging concept for neurogenic hypertension. Hypertension 57, 10261033 (2011).

33. Waki, H. ,K., Li, B.-H., Kasparov, S., Murphy, D. & Paton, J. F. R. Over expression of junctional adhesion molecule -1 in nucleus tractus solitarii is prohypertensive in the rat. Hypertension 49, 13211327 (2007).

34. Xu, H. et al. Upregulation of junctional adhesion molecule-1 is associated with experimental and clinical hypertension. Cardiovasc. Res. 96, 552560 (2012).

35. Kline, R. L., Stuart, P. J. & Mercer, P. F. Effect of renal denervation on arterial pressure and renal norepinephrine concentration in Wistar-Kyoto and spontaneously hypertensive rats. Can. J. Physiol. Pharmacol. 58, 13841388 (1980).

36. Winternitz, S. R., Katholi, R. E. & Oparil, S. Role of the renal sympathetic nerves in the development and maintenance of hypertension in the spontaneously hypertensive rat. J. Clin. Invest. 66, 971978 (1980).

37. Jacob, F., Ariza, P. & Osborn, J. W. Renal denervation chronically lowers arterial pressure independent of dietary sodium intake in normal rats. Am.J. Physiol. Heart. Circ. Physiol. 284, H2302H2310 (2003).38. Norman, Jr. R. A. & Dzielak, D. J. Role of renal nerves in onset and maintenance of spontaneous hypertension. Am. J. Physiol. 243, H284H288 (1982).

39. DiBona, G. F. & Esler, M. Translational medicine: the antihypertensive effect of renal denervation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298, R245R253 (2010).

40. Prabhakar, N. R. & Semenza, G. L. Gaseous messengers in oxygen sensing.J. Mol. Med. 90, 265272 (2012).41. Schultz, H. D. Angiotensin and carotid body chemoreception in heart failure. Curr. Opin. Pharmacol. 11, 144149 (2011).

42. Ding, Y., Li, Y. L. & Schultz, H. D. Role of blood ow in carotid body chemoreex function in heart failure. J. Physiol. 589, 245258 (2011).

43. Wyatt, C. N. et al. Key roles for AMP-activated protein kinase in the function of the carotid body? Adv. Exp. Med. Biol. 605, 6368 (2008).

44. Ackland, G. L., Kazymov, V., Marina, N., Singer, M. & Gourine, A. V. Peripheral neural detection of danger-associated and pathogen-associated molecular patterns. Crit. Care. Med. 41, e85e92 (2013).

45. Lam, S. Y. et al. Chronic intermittent hypoxia induces local inammation of the rat carotid body via functional upregulation of proinammatory cytokine pathways. Histochem. Cell. Biol. 137, 303317 (2011).

46. Clarke, J., de Burgh Daly, M. & Ead, H. in Vascular Analysis Of The Carotid Body In The Spontaneously Hypertensive Rat. (eds Data, P.) 38 (Plenum Press, 1993).

47. Simms, A. E., Paton, J. F., Pickering, A. E. & Allen, A. M. Amplied respiratory-sympathetic coupling in the spontaneously hypertensive rat: does it contribute to hypertension? J. Physiol. 587, 597610 (2009).

48. Finley, J. C. & Katz, D. M. The central organization of carotid body afferent projections to the brainstem of the rat. Brain. Res. 572, 108116 (1992).

49. Paton, J. F., Deuchars, J., Li, Y. W. & Kasparov, S. Properties of solitary tract neurones responding to peripheral arterial chemoreceptors. Neuroscience 105, 231248 (2001).

50. Somers, V. K., Mark, A. L. & Abboud, F. M. Interaction of baroreceptor and chemoreceptor reex control of sympathetic nerve activity in normal humans.J. Clin. Invest. 87, 19531957 (1991).51. Lohmeier, T. E. & Iliescu, R. Chronic lowering of blood pressure by carotid baroreex activation: mechanisms and potential for hypertension therapy. Hypertension 57, 880886 (2011).

52. Heusser, K. et al. Carotid baroreceptor stimulation, sympathetic activity, baroreex function, and blood pressure in hypertensive patients. Hypertension 55, 619626 (2010).

53. Cates, M. J., Steed, P. W., Abdala, A. P., Langton, P. D. & Paton, J. F. Elevated vertebrobasilar artery resistance in neonatal spontaneously hypertensive rats.J. Appl. Physiol. 111, 149156 (2011).54. Marvar, P. J., Lob, H., Vinh, A., Zarreen, F. & Harrison, D. G. The central nervous system and inammation in hypertension. Curr. Opin. Pharmacol. 11, 156161 (2011).

55. Marvar, P. J. et al. Central and peripheral mechanisms of T-lymphocyte activation and vascular inammation produced by angiotensin II-induced hypertension. Circ. Res. 107, 263270 (2010).

56. Elenkov, I. J., Wilder, R. L., Chrousos, G. P. & Vizi, E. S. The sympathetic nerve: an integrative interface between two supersystems: the brain and the immune system. Pharmacol. Rev. 52, 595638 (2000).

57. Niewinski, P. et al. Carotid body removal for treatment of chronic systolic heart failure. Int. J. Cardiol. doi: 10.1016/j.ijcard.2013.03.011 (2013).

58. Cornock, R., Langley-Evans, S. C., Mobasheri, A. & McMullen, S. The impact of maternal protein restriction during rat pregnancy upon renal expression of angiotensin receptors and vasopressin-related aquaporins. Reprod. Biol. Endocrinol. 8, 105 (2010).

59. Langley, S. C., Seakins, M., Grimble, R. F. & Jackson, A. A. The acute phase response of adult rats is altered by in utero exposure to maternal low protein diets. J. Nutr. 124, 15881596 (1994).

60. Loria, A. et al. Sex and age differences of renal function in rats with reduced ANG II activity during the nephrogenic period. Am. J. Physiol. Renal. Physiol. 293, F506F510 (2007).

61. Guzik, T. J. et al. Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction. J. Exp. Med. 204, 24492460 (2007).

62. Paton, J. F. A working heart-brainstem preparation of the mouse. J. Neurosci. Methods 65, 6368 (1996).

Acknowledgements

This study was supported by the British Heart Foundation (JFRP) and a gift from Cibiem NC1 to JFRP. We acknowledge the assistance of Dr. Andrew Herman and the University of Bristol Faculty of Medical and Veterinary Sciences Flow Cytometry Facility.

Author contributions

F.D.M. performed the majority of experiments and analysis assisted by A.P.A. and E.B.H. W.P. performed the renal function and noradrenaline assays. P.M. performed FACS analysis. D.J.A.M. contributed to all data in Fig. 6. P.A.S. provided intellectual input. J.F.R.P. designed/conceived the experiments, provided funding, wrote the manuscript with F.D.M. and P.M.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Web End =http://www.nature.com/ http://www.nature.com/naturecommunications

Web End =naturecommunications

Competing nancial interests: J.F.R.P. is a consultant for Cibiem and P.A.S. is the Chief Medical Ofcer for Cibiem. The remaining authors declare no competing nancial interests.

Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions/

Web End =reprintsandpermissions/

How to cite this article: McBryde, F. D. et al. The carotid body as a putative therapeutic target for the treatment of neurogenic hypertension. Nat. Commun. 4:2395 doi: 10.1038/ ncomms3395 (2013).

NATURE COMMUNICATIONS | 4:2395 | DOI: 10.1038/ncomms3395 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 11

& 2013 Macmillan Publishers Limited. All rights reserved.