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ORIGINAL ARTICLE
Neural control of arterial pressure variabilityin the neuromuscularly blocked rat
Xiaorui Tang • Tian Hu
Received: 23 March 2011 / Accepted: 30 August 2011 / Published online: 23 September 2011
� Springer-Verlag 2011
Abstract The baroreflexes stabilize moment-to-moment
arterial pressure. Sinoaortic denervation (SAD) of the ba-
roreflexes results in a large increase in arterial pressure
variability (APV) across various species. Due to an
incomplete understanding of the nonlinear interactions
between central and peripheral systems, the major source
of APV remains controversial. While some studies sug-
gested that the variability is endogenous to the central
nervous system (CNS), others argued that peripheral
influences may be the main source. For decades, abnormal
cardiovascular variability has been associated with a
number of cardiovascular diseases including hypertension,
heart failure, and stroke. Delineating mechanisms of the
APV is critical for the improvement of current strategies
that use APV as a clinical tool for the diagnosis and
prognosis of cardiovascular diseases. In this study, with a
unique chronic neuromuscularly blocked (NMB) rat prep-
aration that largely constrains peripheral influences, we
determined the CNS contribution to the post-SAD APV.
First, we confirmed that SAD significantly increased APV
in the NMB rat, then demonstrated that post-SAD gangli-
onic blockade substantially reduced APV, and subsequent
intravenous infusions of phenylephrine and epinephrine (in
presence of ganglionic blockade) only slightly increased
APV. These data suggest that the CNS is an important
source, and skeletal activity, thermal challenges or other
forms of peripherally generated cardiovascular stress are
not required for the post-SAD APV. In addition, we
showed that bilateral aortic denervation produced a larger
increase in APV than bilateral carotid sinus denervation,
suggesting that the aortic baroreflex plays a more dominant
role in the control of APV than the carotid sinus.
Keywords Sinoaortic denervation � Arterial pressure
variability � Heart rate variability � Baroreflex �Neuromuscular block � Ganglionic blockade � Sympathetic
nervous system � Vascular tone
Introduction
For decades, abnormal cardiovascular variability has been
regularly described in, and used as a risk factor for, a
number of cardiovascular diseases including hypertension,
heart failure, myocardial infarction, and stroke (Casolo
et al. 1995; Frattola et al. 1993; Julius and Nesbitt 1996;
Malpas 2002; Pringle et al. 2003). A better understanding
of the factors involved in the generation of arterial pressure
variability (APV) will not only improve current strategies
for the use of APV as a clinical tool in the assessment of
cardiovascular function, but may also shed some light into
the processes involved in the development of cardiovas-
cular pathologies in general.
The arterial baroreflexes have a critical role in regulat-
ing and stabilizing the arterial pressure. Impairment or
interruption of the baroreflexes results in a significant
increase in APV (Dworkin et al. 2000; Martinka et al.
2005; Pringle et al. 2003; Schreihofer and Sved 1994;
Timmers et al. 2004a, b). However, due to complex and
often nonlinear interplays between the central and
Communicated by Susan A. Ward.
X. Tang (&)
Department of Neural and Behavioral Sciences, Pennsylvania
State University College of Medicine, Hershey, PA 17033, USA
e-mail: [email protected]
T. Hu
Department of Public Health, Pennsylvania State University
College of Medicine, Hershey, PA 17033, USA
123
Eur J Appl Physiol (2012) 112:2013–2024
DOI 10.1007/s00421-011-2160-4
peripheral systems, it is unclear whether the APV that
follows baroreflex disfunction arises from the central ner-
vous system (CNS) or from factors extrinsic to the sym-
pathetic nervous system. Some studies (Alper et al. 1987a,
b; Jacob et al. 1989; Trapani et al. 1986) suggested that
APV is produced primarily by influences from the CNS and
secondarily by peripheral non-neurogenic influences.
These conclusions stem from the observation in freely
moving sinoaortically denervated rats that blocking either
sympathetic ganglionic transmission or alpha-adrenergic
receptors substantially attenuated APV, but an additional
blockade of the renin-angiotensin and vasopressin systems
was required to further reduce APV.
In contrast, other studies (Barres et al. 1992; Jacob et al.
1988; Julien et al. 1993) suggested that APV is largely
determined by peripheral non-neurogenic mechanisms and
that the sympathetic nervous system only plays a permis-
sive role by maintaining vascular tone at a normal level.
These conclusions mainly resulted from observations in the
freely moving sinoaortically denervated rat that the low
APV produced by ganglionic blockade was largely restored
by infusion of pressor agents such as phenylephrine or
angiotensin II.
Over the past 30 years, our laboratory has developed and
utilized a unique chronic NMB rat preparation (Dworkin
and Dworkin 1990, 1995, 2004; Dworkin et al. 2000a, b;
Tang and Dworkin 2007a, b, 2009) that largely constrains
peripheral influences and significantly limits the complex
interactions between the central and peripheral nervous
systems. As such, this model provides a platform to clarify
the CNS’s contribution to a specific physiological phe-
nomenon because of minimal interference from peripheral
influences. The novelty of this current study resides in the
use of this unique NMB preparation to illustrate the role of
the CNS in generating post-SAD APV. Contrary to the idea
that APV is largely determined by peripheral nonneuro-
genic mechanisms (Barres et al. 1992; Jacob et al. 1988;
Julien et al. 1993), our results suggested that skeletal
activity, thermal challenges or other forms of peripherally
generated cardiovascular stress are not required for the post-
SAD APV. Two aims were addressed in this study. The first
aim was to evaluate the hypothesis that the CNS is a major
contributor to the observed large post-SAD APV. To this
end, we evaluated APV after (1) sino-aortic denervation
(SAD); (2) ganglionic blockade; and (3) ganglionic block-
ade followed by infusion of pressors in SAD and baroreflex-
intact rats. The second aim of this study was to examine the
relative contribution of the aortic and carotid sinus ba-
roreflexes in the control of APV. Therefore, we measured
APV following (1) bilateral aortic denervation followed by
SAD; (2) bilateral carotid sinus denervation followed by
SAD; and (3) unilateral aortic denervation. Finally, to better
understand detail characteristic features of the variability,
APV was analyzed both in the time domain as the standard
deviation (SD) and in the frequency domain with the Fast
Fourier Transform power spectral analysis of the hourly
systolic arterial pressure.
Methods
Sprague–Dawley female (255–390 g) rats were used in the
study. The experimental protocols were supervised and
certified to be in compliance with National Institute of
Health and American Physiology Society guidelines by the
Pennsylvania State University College of Medicine Insti-
tutional Animal Care and Use Committee (IACUC). On
average, each preparation lasted for *10 days, and mul-
tiple protocols were implemented in the same preparation.
Each rat was monitored and attended to around the clock.
The NMB rat was prepared as previously described in
(Dworkin et al. 2000; Tang and Dworkin 2007a). Briefly,
for maintaining the preparation, the following parenteral
solutions were infused continuously: (1) An intra-arterial
solution (0.6 ml/h) consisting of 50 ml H2O, 50 ml 0.5 N
lactated Ringer, 1.25 g oxacillin Na, 2.8 mg a-cobratoxin,
0.3 mg vitamin K (Synkavite), and 20 meq K? (as KCl);
(2) an intra-venous solution (0.6 ml/h) consisting of 50 ml
H2O, 50 ml 0.5 N lactated Ringer and 1.25 g oxacillin Na
for the initial 48 h, then glucose, 8.5% Travasol amino
acid, and intralipid (fat emulsion) were added subsequently
to provide necessary nutrients.
Surgery
The general surgical procedures are provided in detail in
Dworkin et al. (2000) and Tang and Dworkin (2007a).
Briefly, the rat was deeply anesthetized with [1.5% iso-
flurane and monitored to ensure that (1) the electroen-
cephalogram (EEG) was synchronized and dominated by
high-voltage low-frequency activity; (2) mean systolic
arterial pressure was\100 mmHg; (3) heart rate was\420
beats/min; and (4) arterial pressure, heart rate, and EEG did
not respond to surgical manipulation. General surgery
included implantation of a pair of subcutaneous precordial
electrocardiogram (EKG) silver wire electrodes, a transu-
rethral bladder cannula to allow drainage and measurement
of the rate of urine production, a femoral artery cannula for
arterial pressure measurement, and a femoral vein cannula
for recording venous pressure and administering parenteral
solutions. General surgery also included implantations of
EEG electrodes to assure maintenance of adequate levels of
anesthesia during the surgery and the regularity of sleep
and wakefulness cycles throughout the experiment. Core
temperature was servo-regulated at 37�C. Neuromuscular
block was induced with a 75 lg i.v. bolus of a-cobratoxin
2014 Eur J Appl Physiol (2012) 112:2013–2024
123
and maintained by continuous infusion at 250 lg/day. The
rat was ventilated with positive pressure at a rate of 72
breaths/min and at a volume of 200 cc/min. The air mix-
ture consisted of 48.5% O2, 47% N2, 3% CO2, and 1.5%
isoflurane. After the surgery, the rat was allowed to recover
for 2 days during which the isoflurane level was gradually
reduced to 0.5%. During the recovery, adequate level of
anesthesia was maintained by observing that the EEG,
arterial pressure, and heart rate signals were within the
physiological range. If any of these signals indicated signs
of distress during the reduction of anesthesia, the level of
isoflurane was elevated to its previous stage and main-
tained for a longer period of time before the next lowering
attempt. All experimental data used for the final analysis in
this study were acquired at 0.5% isoflurane level.
The aortic and carotid sinus denervation A modification
of Krieger’s sinoaortic denervation method (Krieger 1964)
was used. Briefly, the aortic denervation was achieved by
isolating and resecting the superior cervical ganglion,
superior laryngeal nerve, and aortic depressor nerve
(ADN). The carotid sinus denervation was achieved by
stripping off all connective tissue around the carotid sinus
region and painting the region with a solution of 10%
phenol in ethanol to remove any remaining baroreceptor
afferent fibers. Complete SAD was achieved by bilateral
aortic and carotid sinus denervation. This was confirmed by
the absence of bradycardia in response to a phenylephrine
challenge, which typically increased arterial pressure more
than 30 mmHg.
Experimental protocols
Protocols were arranged into two main categories corre-
sponding to the two major aims of the study: (1) evaluating
the CNS contribution to the post-SAD APV; and (2) evalu-
ating the relative contributions of the aortic and carotid sinus
baroreflexes in stabilizing cardiovascular variability.
Protocols for evaluating the CNS contribution
to the post-SAD cardiovascular variabilities
In the baroreflex denervated rats: Baseline ? SAD ?chlorisondamine administration ? phenylephrine and epi-
nephrine infusions Following recovery from the general
surgery, 6 h of baseline data were acquired before SAD.
Twenty-four hours following the SAD, a bolus of the gan-
glionic blocker, chlorisondamine (2.5 mg/kg, i.v.), was given.
Six hours later, phenylephrine and epinephrine were infused
continuously (i.v.) for 6 h each in a random order at a dose that
restored arterial pressure to or above baseline levels, as cus-
tomized for each NMB rat (phenylephrine, 5–73 lg kg-1
min-1; epinephrine, 2.6–36 lg kg-1 min-1). To achieve the
appropriate dose, the infusion rates of phenylephrine and
epinephrine were adjusted from 5 to 33 ll/min and from 3 to
13 ll/min, respectively. The purpose of using two different
pressors, i.e., phenylephrine (an alpha adrenergic agonist) and
epinephrine (an alpha and beta adrenergic agonist), was to
partially control for idiosyncratic effects of the particular
agent on the APV. Saline infusion at a rate of 10 ll/min was
used as a control.
In baroreflex-intact rats: Baseline ? chlorisondamine
administration ? phenylephrine and epinephrine infu-
sions Following recovery from the general surgery, a bolus
of chlorisondamine (2.5 mg/kg, i.v.) was given. Six hours
later, the pressors phenylephrine and epinephrine were
continuously infused (i.v.) for 6 h each in a random order
as described above. Saline infusion at a rate of 10 ll/min
was used as a control.
Protocols for evaluating the relative contributions
of the aortic and carotid sinus baroreflexes
in stabilizing the APV
Bilateral aortic or carotid sinus denervation: Base-
line ? bilateral aortic (or carotid sinus) denerva-
tion ? bilateral carotid (or aortic) denervation, i.e., SAD
Following recovery from the general surgery, bilateral
aortic (or carotid sinus) denervation was performed. This
initial denervation was chosen randomly on either pair of
the baroreceptors (i.e., half of the rats began with the aortic
then the carotid sinus denervation, and another half with
the carotid sinus then the aortic denervation). Twenty-four
hours later, further denervation was conducted on the
remaining pair of the baroreceptors.
Unilateral aortic denervation: Baseline ? left or right
aortic denervation: Following recovery from the general
surgery, aortic denervation was performed on the left aortic
baroreceptors.
Data acquisition and analysis
All experimental signals were continuously acquired 24 h a
day and 7 days a week throughout the entire experiment
using Spike2� and a Power 1401 data acquisition system
(Cambridge Electronic Design, Cambridge, UK). Five
hours of data were analyzed for each experimental condi-
tion in protocols evaluating the CNS contribution to the
post-SAD cardiovascular variability. Ten hours of data
were analyzed for each condition in protocols evaluating
the role of the aortic and carotid sinus baroreflexes in
stabilizing the APV. To eliminate potential transitional
effects of switching from one experimental condition to
another, signals were often acquired more than 5 or 10 h in
an experimental condition, and those from the last 5 or
10 h were analyzed for that condition. Data used for final
Eur J Appl Physiol (2012) 112:2013–2024 2015
123
analysis were always acquired at a 0.5% isoflurane level.
Arterial pressure variability (APV) and heart rate vari-
ability (HRV) were calculated as follows: First, beat-to-
beat systolic arterial pressure and heart rate were generated
from the arterial pressure and EKG waveforms, respec-
tively, using a peak detection algorithm in the Spike2�
software. APV and HRV were calculated in the time
domain as the standard deviations of systolic arterial
pressure and heart rate in each hour; and the standard
deviations across 5 or 10 h were averaged and reported for
each experimental condition. In the frequency domain, Fast
Fourier Transform (0.012 Hz resolution, Hanning window,
no overlapping samples) power spectral analysis was
conducted on systolic arterial pressure for each hour and
the very low-frequency (VLF: 0.01–0.15 Hz) and low-
frequency (LF: 0.15–0.6 Hz) powers were calculated by
integration of the areas in the corresponding frequency
ranges. The VLF and LF powers across 5 or 10 h were
averaged and reported for each experimental condition.
Statistical analysis
A separate analysis of variance (ANOVA) with repeated
measures was used to determine the effect of each exper-
imental intervention on APV, HRV, and the VLF and LF
powers of the systolic arterial pressure spectra; Newman–
Keuls post-hoc comparisons were applied when appropri-
ate. Differences were considered significant at p \ 0.05.
A Student t test was used to evaluate the effect of unilateral
aortic denervation on the APV, and differences were con-
sidered significant at p \ 0.05. All results are presented as
the mean ± SE.
Results
Results are arranged according to the two major aims of
this study: (1) evaluating the CNS contribution to the post-
SAD cardiovascular variability; and (2) evaluating the
relative contributions of the aortic and carotid sinus ba-
roreflexes in stabilizing the APV.
The CNS has a significant contribution to the post-SAD
APV
SAD increased and chlorisondamine decreased APV
in the NMB rat (Fig. 1, N = 9)
Systolic AP following baseline, SAD, chlorisondamine,
phenylephrine, and epinephrine infusion was analyzed using
one-way analyses of variance (ANOVAs) with repeated
measures across stage (Fig. 1B1, N = 9). The main effect
of stage was significant (F(4, 38) = 50.18, p \ 0.05).
Newman–Keuls post hoc test showed that, following SAD,
systolic AP was significantly increased from the baseline
(from 133 ± 4.9 to 151 ± 4.17 mmHg; p \ 0.05 vs. base-
line); the subsequent application of chlorisondamine
decreased systolic AP from 151 ± 4.17 to 67 ± 1.98
mmHg (p \ 0.05 vs. every other experimental stages). In the
presence of chlorisondamine, phenylephrine, and epineph-
rine infusion both returned systolic AP to the baseline control
level, from 67 ± 1.98 to 138 ± 7.72 mmHg for phenyl-
ephrine (p \ 0.05 vs. the chlorisondamine stage); and from
67 ± 1.98 to 153 ± 5.26 mmHg for epinephrine (p \ 0.05
vs. the chlorisondamine stage).
APV was analyzed using one-way repeated measures of
ANOVAs across stage (Fig. 1B2, N = 9). The main effect
of stage was significant (F(4, 38) = 84.31, p \ 0.05).
Newman–Keuls post hoc test showed that, following SAD,
APV was significantly increased from the baseline (from
8.3 ± 1.4 to 25 ± 1.98 mmHg; p \ 0.05 vs. baseline); the
subsequent application of chlorisondamine decreased APV
from 25 ± 1.98 to 2.44 ± 0.36 mmHg (p \ 0.05 vs. every
other experimental stages). In the presence of chlorisond-
amine, phenylephrine, and epinephrine infusion both only
slightly increased APV, from 2.44 ± 0.36 to 7.18 ± 0.47
mmHg for phenylephrine (p \ 0.05 vs. the chlorisond-
amine stage); and from 2.44 ± 0.36 to 7.46 ± 0.71 mmHg
for epinephrine (p \ 0.05 vs. the chlorisondamine stage).
These data suggest that the CNS has a significant contri-
bution to the post-SAD APV. In corollary, these data also
suggest that the baroreflex is frequently engaged to restrain
moment-to-moment APV.
VLF power was analyzed using one-way repeated mea-
sures of ANOVAs across stage (Fig. 1B3, N = 9). The main
effect of stage was significant (F(4, 38) = 41.97, p \ 0.05).
Newman–Keuls post hoc test showed that, following SAD,
VLF was significantly increased from the baseline (from
13.23 ± 2.07 to 30.89 ± 2.38 mmHg/HHz; p \ 0.05 vs.
baseline); the subsequent application of chlorisondamine
decreased VLF power from 30.89 ± 2.38 to 3.77 ± 0.51
mmHg/HHz (p \ 0.05 vs. every other experimental stages).
In the presence of chlorisondamine, phenylephrine and epi-
nephrine infusions both slightly increased VLF power, from
3.77 ± 0.51 to 11 ± 0.99 mmHg/HHz for phenylephrine
(p \ 0.05 vs. the chlorisondamine stage); and 3.77 ± 0.51
to 9.87 ± 0.85 mmHg/HHz for epinephrine (p \ 0.05 vs.
the chlorisondamine stage). VLF was identified as the prin-
cipal frequency region of the baroreflex system (Dworkin
et al. 2000). The VLF changes in response to each experi-
mental intervention corresponded well with the APV chan-
ges, further suggesting that a substantial proportion of the
APV likely arises from the CNS, and the baroreflex is fre-
quently engaged to restrain moment-to-moment APV.
LF power was analyzed using one-way repeated mea-
sures of ANOVAs across stage (Fig. 1B4, N = 9). The
2016 Eur J Appl Physiol (2012) 112:2013–2024
123
Fig. 1 Effects of sinoaortic denervation (SAD), chlorisondamine
administration, intravenous infusions of phenylephrine, and epineph-
rine on the arterial pressure (AP) and arterial pressure variability
(APV) in NMB rats. a Shows representative traces of AP (A1) and
systolic AP (A2) and a frequency histogram of the systolic AP (A3)
from each experimental procedure. b Summarizes the results of
systolic AP (B1), APV (B2), very low-frequency (VLF: 0.01–0.15 Hz,
B3) and low-frequency (LF: 0.15–0.6 Hz, B4) powers of the systolic
AP spectra at baseline, following sinoaortic denervation, chlorisondamine
administration, and intravenous infusions of phenylephrine and
epinephrine. While SAD significantly increased APV and VLF
power, chlorisondamine decreased the values of both variables to
below their corresponding baseline levels; subsequent phenylephrine
and epinephrine infusions only slightly increased APV. APV was
calculated as standard deviation of the hourly systolic AP. Data are
mean ± SEM. Error bars SEM. *p \ 0.05. ##Statistically significant
difference from every other experimental stages
Eur J Appl Physiol (2012) 112:2013–2024 2017
123
main effect of stage was significant (F(4, 38) = 20.63,
p \ 0.05). Newman–Keuls post hoc test showed that, fol-
lowing SAD, LF power was decreased from the baseline
(from 12.17 ± 1.68 to 6.34 ± 0.43 mmHg/HHz; p \ 0.05
vs. baseline); the subsequent application of chlorisond-
amine further decreased LF power from 6.34 ± 0.43 to
1.48 ± 0.11 mmHg/HHz (p \ 0.05 vs. every other
experimental stages). In the presence of chlorisondamine,
the phenylephrine and epinephrine infusion both slightly
increased LF power, from 1.48 ± 0.11 to 4.98 ± 0.59
mmHg/HHz for phenylephrine (p \ 0.05 vs. the chloris-
ondamine stage), and 1.48 ± 0.11 to 4.76 ± 0.45 mmHg/
HHz for epinephrine (p \ 0.05 vs. the chlorisondamine
stage). These data suggest that LF is the resonant (i.e.,
oscillation) region of the baroreflexes (Dworkin et al.
2000a). When denervation eliminated the negative feed-
back, it also eliminated the oscillation in this closed-loop
negative feedback system, which resulted in a power
decrease in the LF band of the APV spectrum.
Chlorisondamine, phenylephrine, and epinephrine
infusions produced similar effects in baroreflex-intact NMB
rats as in SAD NMB rats (Fig. 2)
Systolic AP, APV, VLF, and LF powers following base-
line, chlorisondamine, phenylephrine, and epinephrine
infusion in the baroreflex-intact and SAD rat were analyzed
separately using repeated measures of two-way ANOVAs
varying group and stage. There was a significant two-way
interaction varying group and stage in systolic AP, APV,
VLF, and LF (systolic AP, F(3, 33) = 3.82, p \ 0.05,
Fig. 2a; APV, F(3, 33) = 32.93, p \ 0.05, Fig. 2b; VLF, F
(3, 33) = 11.69, p \ 0.05, Fig. 2c; and LF, F(3,
33) = 9.76, p \ 0.05, Fig. 2d). Newman–Keuls post hoc
test showed that, similarly, in both SAD and baroreflex-
intact NMB rats, chlorisondamine significantly decreased
systolic AP, APV, VLF, and LF powers compared with the
pre-chlorisondamine baseline condition (p \ 0.05 vs. every
other experimental stages); the subsequent phenylephrine
and epinephrine infusion (in presence of the chlorisond-
amine) both returned systolic AP to the baseline condition
and increased APV, VLF, and LF power compared with the
chlorisondamine condition (p \ 0.05 vs. the chlorisond-
amine stage). Between the SAD and the baroreflex-intact
rats, the SAD rats had a higher systolic AP (Fig. 2a), APV
(Fig. 2b), and VLF power (Fig. 2c), but a lower LF power
(Fig. 2d) than the baroreflex-intact rats during the pre-
chlorisondamine baseline stage (p \ 0.05, for all vari-
ables). Note that, the baroreflexes were intact for the ‘Intact
Rats,’ and completely denervated for the ‘SAD Rats’ in the
baseline stage in Fig. 2. Interestingly, epinephrine infusion
produced a larger increase in APV, VLF power, and LF
power in the baroreflex-intact rats than in the SAD rats; the
significance of this increase, if any, has yet to be
determined.
Independent of prior status of the baroreflex system (i.e.,
SAD or intact), a bolus application of chlorisondamine
substantially decreased APV and VLF in both SAD and
baroreflex-intact NMB rats, suggesting that, by interrupting
the baroreflex efferent pathway, chlorisondamine blocked
the CNS contribution to the APV. Moreover, in presence of
the chlorisondamine, subsequent phenylephrine and epi-
nephrine infusions both returned systolic AP to or above
the baseline levels in both SAD and intact rats (Fig. 2a,
p \ 0.05). This restoration of systolic AP significantly
increased APV (Fig. 2b), VLF (Fig. 2c), and LF powers
(Fig. 2d) in both SAD and intact NMB rats, suggesting that
the APV is not only modulated by the CNS, but also by the
peripheral system.
The aortic baroreflex has a more dominant role
in the control of APV than the carotid sinus baroreflex
The aortic denervation produced a larger increase in APV
than the carotid sinus denervation in the NMB rat (Fig. 3,
N = 6 in each group)
APV, VLF power, and LF power following baseline,
bilateral aortic, or carotid sinus denervation and complete
SAD were analyzed using separate two-way analyses of
variance (ANOVAs) with repeated measures varying group
and stage. There was a significant two-way interaction
varying group and stage in APV and VLF (APV, F(2,
20) = 5.09, p \ 0.05, Fig. 3a; and VLF, F(2, 20) = 3.63,
p \ 0.05, Fig. 3b). Newman–Keuls post hoc test showed
that, for the APV (Fig. 3a), it was increased from the
baseline 7.02 ± 0.66 to 17.27 ± 0.82 mmHg following
bilateral aortic denervation (p \ 0.05 vs. baseline) and
further increased to 24.02 ± 2.07 mmHg following the
subsequent complete SAD (p \ 0.05 vs. baseline and
bilateral aortic denervation); the APV was increased from
the baseline 7.31 ± 0.96 to 12.14 ± 1.83 mmHg following
bilateral carotid sinus denervation (p \ 0.05 vs. baseline),
and further increased to 27.29 ± 1.12 mmHg following the
subsequent complete SAD (p \ 0.05 vs. baseline and
bilateral carotid sinus denervation). For the VLF power
(Fig. 3b), it was increased from the baseline 12.01 ± 1.5 to
24.69 ± 1.73 mmHg/HHz (p \ 0.05 vs. baseline) follow-
ing bilateral aortic denervation, and further increased to
30.63 ± 1.53 mmHg/HHz following the subsequent com-
plete SAD (p \ 0.05 vs. baseline and bilateral aortic
denervation); the VLF power was increased from
11.21 ± 1.31 to 19.12 ± 3.49 mmHg/HHz following
bilateral carotid sinus denervation (p \ 0.05 vs. baseline),
and further increased to 34.38 ± 1.85 mmHg/HHz fol-
lowing the subsequent complete SAD (p \ 0.05 vs.
2018 Eur J Appl Physiol (2012) 112:2013–2024
123
baseline and bilateral aortic denervation). These data sug-
gest that while both aortic and carotic sinus baroreflexes
are important in stabilizing APV, the aortic baroreflex has a
more dominant role than the carotid sinus baroreflex. These
data also suggest that the effect of complete SAD on APV,
VLF, and LF variables is independent of the sequence of
the denervation.
A two-way analysis of variance with repeated measures
revealed a significant difference on main effect of stage in
LF (F(2, 20) = 21.33, p \ 0.05). Newman–Keuls post hoc
test indicated that, when combined, baseline LF was higher
in baseline than in the partial denervation (i.e., AD or CSD)
stage (Fig. 3c).
Single-side aortic denervation did not elicit a significant
increase in APV in the NMB rat (Fig. 4, N = 6)
Following unilateral (left or right only) aortic denervation
in the NMB rat, APV increased from 7.2 ± 1.03 to
8.87 ± 1.47 mmHg and VLF power from 9.54 ± 0.95 to
12.81 ± 1.89 mmHg/HHz. LF power decreased from
11.06 ± 2.04 to 8.58 ± 1.48 mmHg/HHz (N = 6 for all;
Fig. 2 Chlorisondamine administration, intravenous infusions of
phenylephrine, and epinephrine produced similar effects on the
systolic arterial pressure (AP), arterial pressure variability (APV),
VLF and LF powers of the systolic AP spectra in both SAD and
baroreflex-intact NMB rats. a The systolic AP. In both SAD and
baroreflex-intact rats, chlorisondamine significantly decreased sys-
tolic AP and subsequent infusions of phenylephrine and epinephrine
both returned systolic AP to or above the corresponding baseline
levels. b The APV. In both SAD and baroreflex-intact rats, chlorisond-
amine significantly decreased the APV and subsequent infusions of
phenylephrine and epinephrine both increased APV to the baroreflex-
intact baseline variability level. c VLF power of the systolic AP
spectrum. In both SAD and baroreflex-intact rats, chlorisondamine
significantly decreased the VLF power and subsequent infusions of
phenylephrine and epinephrine both increased the VLF power to the
baroreflex-intact baseline level. d LF power of the systolic AP
spectrum. In both SAD and baroreflex-intact rats, chlorisondamine
significantly decreased LF power and subsequent infusions of phenyl-
ephrine and epinephrine both increased the power to the SAD level.
Moreover, the SAD rat had a higher systolic AP, larger APV, and VLF,
but a lower LF power than the baroreflex-intact rat during the baseline
stage. In the baseline stage, the baroreflexes were completely dener-
vated for the ‘SAD Rats’ and un-manipulated for the ‘Intact Rats’. APV
was defined as standard deviation of the hourly systolic AP. Data are
mean ± SEM. Error bars SEM. *p \ 0.05. ##Statistically significant
difference from every other experimental stage
Eur J Appl Physiol (2012) 112:2013–2024 2019
123
Fig. 4). None of the above variables were significantly
altered by unilateral aortic denervation. Taken together
with the results shown in Fig. 3, these data suggest that
with the carotid sinuses intact, only a single aortic limb was
required to stabilize the pressure.
SAD had no effect on the HRV (Fig. 5, N = 9)
In the NMB rat, baseline values of heart rate and HRV
were 373 ± 13 and 12 ± 1 beats/min, respectively. Fol-
lowing SAD, neither heart rate nor HRV was significantly
altered (p [ 0.05; Fig. 5). The different outcomes of SAD
on the APV and HRV suggest that different mechanisms
are involved in producing and stabilizing heart rate and
arterial pressure variability.
Discussion
In the present study we first confirmed that SAD produced
a substantial increase in APV in the NMB rat. We then
demonstrated that ganglionic blockade reduced the mean
systolic pressure and APV in both SAD and baroreflex-
intact NMB rats, and following ganglionic blockade,
Fig. 3 While bilateral aortic and carotid sinus denervation signifi-
cantly increased APV and VLF, bilateral aortic denervation produced
a larger increase in APV than carotid sinus denervation. a, b and
c depicted the effects of different denervation (i.e., AD, CSD, or
SAD) on APV, VLF, and LF, respectively. Bilateral aortic and carotid
sinus denervation both significantly increased APV and VLF power
compared with the baroreflex-intact baseline (p \ 0.05 vs. baseline,
for the APV and the VLF power); the subsequent SAD further
increased APV and VLF. (p \ 0.05 vs. baseline and AD or CSD
denervation stages). Bilateral aortic denervation produced a larger
increase in APV and VLF than carotid sinus denervation (p \ 0.05,
aortic vs. carotid sinus denervation). Data are mean ± SEM. Errorbars SEM. *p \ 0.05. APV was calculated as standard deviation of
the hourly systolic arterial pressure. VLF and LF: the very low-
frequency (VLF: 0.01–0.15 Hz) and the low-frequency (LF:
0.15–0.6 Hz) power of the systolic arterial pressure spectra, respec-
tively. AD aortic denervation, CSD carotid sinus denervation, SADsinoaortic denervation
Fig. 4 Unilateral aortic denervation had no effect on the APV. From
top to bottom, APV was characterized as the standard deviation of the
hourly systolic arterial pressure. The VLF and LF were the very low-
frequency (VLF: 0.01–0.15 Hz) and the low-frequency (LF:
0.15–0.6 Hz) power of the hourly systolic arterial pressure spectra,
respectively. Data are mean ± SEM. Error bars SEM. *p \ 0.05
2020 Eur J Appl Physiol (2012) 112:2013–2024
123
pressor infusions restored mean systolic pressure to base-
line levels, but only slightly increased APV in both SAD
and baroreflex-intact NMB rats. These results suggest that
the CNS is an important source for the large APV fol-
lowing the SAD. However, pressor infusions following
chlorisondamine application also increased APV, indicat-
ing that the peripheral input also plays a role. The
peripheral input, however, is not necessarily required for
the manifestation of the large post-SAD APV, because
APV was substantially increased following SAD in the
NMB rat, in which the peripheral variance is largely
constrained.
We have also shown that (1) while bilateral aortic and
carotid sinus denervation both significantly increased APV,
the bilateral aortic denervation produced a larger increase
in APV than the bilateral carotid sinus denervation; and (2)
unilateral (left or right) aortic denervation alone had no
significant effect on the APV. These data suggest that the
aortic baroreflex has a more dominant role in stabilizing
moment-to-moment APV compared with the carotid sinus.
In addition, these data also suggest that, with a pair of
intact carotid sinus baroreflexes, a single aortic limb was
sufficient to stabilize the pressure.
Last, we showed denervation had no effect on HRV.
Different effects of baroreflex denervation on APV and
HRV suggest that different mechanisms are involved in
producing and stabilizing these variabilities.
The CNS is a major source of post-SAD APV
The NMB rat provides an ideal setting to analyze the
sources of APV: First, it is an extremely stable preparation.
We (Dworkin et al. 2000a) have shown previously that
baseline drift for arterial pressure for a typical NMB rat is
?0.01 mmHg/h (see bottom panel in Fig. 1 of Dworkin
et al. 2000a). Thus, for a typical 10-day experiment per-
formed in the current study, only 2–3 mmHg increase is
expected in arterial pressure over the entire experimental
duration. Also, APV was demonstrated to be consistent
over a 10-day period (bottom panel of Fig. 1 in Dworkin
et al. 2000a). Second, the NMB rat is largely free of
peripheral interference. Specifically, (1) chronic neuro-
muscular block removes the influence of skeletal muscle
activity on the regional blood flow and thereby regional
APV; (2) controlled ventilation at a precise rate and vol-
ume circumvents the influence of respiratory variations on
APV; (3) servo-regulated core temperature removes the
influence of temperature fluctuations on the APV; and (4)
experiments are conducted in a controlled, sound-isolated
environment which eliminates the presence of other
external stimuli. Finally, data in this study were acquired at
0.5% isoflurane level. It is well recognized that, at high
doses (3.0 MAC & 4.5%, Eger et al. 2003), isoflurane
produces direct cardiac depression, affecting both systolic
and diastolic function. However, no differences were found
in systolic and diastolic functions of rats between the low-
dose (\1 MAC) and isoflurane-free control conditions
(Skeehan et al. 1995). We concluded from over 30 years of
experience with the NMB rat that 0.5% isoflurane is a
comfortable analgesia level that minimally alters auto-
nomic cardiovascular function. At 0.5% isoflurane level,
the cardiovascular parameters (heart rate, blood pressure,
and even the post-SAD APV, etc.,) in NMB rats are well
within the range of those in freely moving rats, suggesting
that the NMB rat highly emulates the condition of freely
moving rats in the cardiovascular aspect.
Given the above, the evidence of a substantial increase
in APV following SAD in the NMB rat (Fig. 1) strongly
suggests that the CNS is an important source of the post-
SAD APV. The large increase in post-SAD APV also infers
that the intact baroreflex is frequently engaged to restrain
moment-to-moment APV.
The conclusion that the CNS is an important source of
post-SAD APV is further strengthened by the observation
that restoration of systolic pressure with pressor infusions
only slightly increased the APV levels in both SAD and
baroreflex-intact NMB rats. Chlorisondamine reduced APV
significantly, but also induced a significant drop in mean
Fig. 5 Sinoaortic denervation (SAD) had no effect on both the heart rate (a) and the heart rate variability (HRV) (b). Data are mean ± SEM.
Error bars SEM. *p \ 0.05
Eur J Appl Physiol (2012) 112:2013–2024 2021
123
systolic pressure, and thus may have limited the range in
which arterial pressure could fluctuate. The fact that res-
toration of systolic pressure only slightly increased the
APV and VLF levels in both SAD and baroreflex-intact
NMB rats strongly supported the hypothesis that the CNS
is an important source of the post-SAD APV.
Furthermore, the result that chlorisondamine produced
similar effects on APV, VLF, and LF in the SAD and
baroreflex-intact NMB rat provided additional evidence
suggesting that the CNS is an important source for the post-
SAD APV. If the major source of post-SAD APV were in
the peripheral system, chlorisondamine application should
have produced different effects in the baroreflex-intact and
SAD rat. Chlorisondamine should have substantially
increased the APV in baroreflex-intact rats (particularly
when the systolic pressure was restored with the phenyl-
ephrine and epinephrine infusion) and had no substantial
effect on the post-SAD APV in the SAD rat. By blocking
the neural transmission in the efferent pathway of the
baroreflex system, chlorisondamine disrupts the baroreflex
control of the APV, which should result in a large increase
in the APV in baroreflex-intact rats. Conversely, since the
baroreflex system is already interrupted in the SAD rat,
further disruption of the system with chlorisondamine
should not further influence the APV (given that the major
source is at the periphery). Thus, the findings that chlo-
risondamine had similar effects on APV, VLF, and LF in
the SAD and baroreflex-intact NMB rat further suggest that
the CNS is an important source for the post-SAD APV.
Certainly, following chlorisondamine, the slight
increase of APV produced by phenylephrine and epi-
nephrine infusions in both SAD and baroreflex-intact rats
also suggests that peripheral input plays a role in the post-
SAD APV. Obviously, this input is not necessarily required
for the manifestation of the large post-SAD APV.
Aortic baroreflex has a more dominant role in control
of APV than the carotid baroreflex
In agreement with findings of Van Vliet et al. (1999), we
found that the aortic baroreflex has a more dominant role in
controlling APV than the carotid sinus baroreflex, because
aortic denervation produced a larger increase in APV than
the carotid sinus denervation. In support, Sanders et al.
(1988) showed that stimulation of the aortic baroreflexes,
but not carotid sinus reflexes, produced a substantial and
sustained inhibition of muscle sympathetic nervous activity
in humans. Prior studies (Dampney et al. 1971; Donald and
Edis 1971; Pelletier et al. 1972; Pickering et al. 2008;
Sanders et al. 1988) have also shown that the cardiac
baroreflex is critically dependent upon the aortic afferents
with relatively little contribution from the carotid sinus. All
together, these results indicate that the aortic baroreflex has
a more dominant role than the carotid baroreflex in overall
blood pressure regulation.
In the present study we reported that HRV was not sig-
nificantly altered by either partial or complete SAD. This is
similar to what was observed in previous reports (Alper et al.
1987a, b; Mancia et al. 1999; Tang and Dworkin 2009;
Trapani et al. 1986), indicating that HRV has no relevant role
in APV. The different effects of denervation on the HRV and
APV suggest that the stabilization of heart rate and arterial
pressure variability is controlled by different mechanisms.
The similar HRV observed between the denervation and
baseline stages suggests that HRV is unlikely to be the source
of the post-SAD APV. In support of these conclusions,
Ferrari et al. (Ferrari et al. 1987) showed that administration
of atropine to freely moving rats with intact baroreceptors
significantly reduced HRV while increasing APV. This
indicates that the vagally mediated fluctuations in heart rate
are probably triggered by the arterial baroreflexes, instead of
being the source of APV, and reflect the ability of the baro-
reflex system to buffer arterial pressure changes through
opposing changes in cardiac output.
Power spectral analysis of the APV
In this study, we found that a partial denervation (the aortic
or carotid sinus denervation) increased and a complete
SAD further increased VLF power of the APV spectra.
These results are consistent with the findings of our prior
study (Dworkin et al. 2000b) and also findings of others
(Burgess et al. 1997; Cerutti et al. 1994; Jacob et al. 1995;
Julien et al. 2003; Ramanathan et al. 1994). In a previous
study (Dworkin et al. 2000b), using electrical stimulation
of the aortic depressor nerve in NMB rats, we identified a
very low-frequency (VLF) band (0.01–0.2 Hz) as the
principal frequency region of the baroreflexes. Thus, when
the baroreflex was intact, the APV was well constrained by
the negative feedback mechanism of the baroreflex in the
VLF region. However, when the constraint was removed
step-by-step via a partial and then a complete baroreflex
denervation, the VLF power increased progressively. In
addition, in this study, we found that, in contrast to the
VLF, LF power was decreased in the APV spectra after the
denervation, which is, again, consistent with our prior
findings. Previously, we (Dworkin et al. 2000b) measured
an overall baroreflex transportation lag of *1.05 s in the
NMB rat, which determined a low-frequency (LF) band of
0.2–0.6 Hz as the resonant (i.e., oscillation) region of the
baroreflexes. SAD interrupted the baroreflex system, thus
removed the oscillation in this closed loop negative feed-
back system, and resulted in a power decrease in the LF
band of the APV spectrum.
In conclusion, the present study suggests that (1) the
CNS has a significant contribution to the increased APV
2022 Eur J Appl Physiol (2012) 112:2013–2024
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observed following baroreflex denervation. Although
peripheral interference contributes, it is not necessarily
essential. (2) Compared with the carotid baroreflexes, the
aortic baroreflexes have a more dominant role in stabilizing
moment-to-moment APV. With the carotid sinuses intact,
only a single aortic limb was required to stabilize the
pressure.
Acknowledgments The authors would like to thank Dr. Sean
Stocker for his input for the manuscript, Dr. A. R. Travagli for his
significant editing of the manuscript, and Dr. Christopher Freet and
Mr. James Stoner for their help in fine tuning the manuscript.
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