Paradoxical respiratory sinus arrhythmia in the anesthetized rat

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Autonomic Neuroscience: Basic and

Paradoxical respiratory sinus arrhythmia in the anesthetized rat

Yu-Chieh Tzeng*, Duncan C. Galletly, Peter D. Larsen

Department of Surgery and Anesthesia, Wellington School of Medicine and Health Sciences, 23A Mein Street, Newtown,

PO Box 7343, Wellington, New Zealand

Received 2 August 2004; received in revised form 10 December 2004; accepted 11 December 2004

Abstract

This study examines respiratory sinus arrhythmia (RSA) in the isoflurane-anesthetized rat. In fifteen female Sprague–Dawley rats, we

recorded continuous ECG and respiratory airflow before and after bilateral vagotomy. RSAwas assessed using power spectral analysis and by

plotting the normalised changes in heart period as a function of the time during the respiratory cycle. Contrary to descriptions of RSA in

conscious rats, we observed in all rats in the current study a dreversedT pattern of RSA in which heart rate decelerated during inspiration.

Elimination of vagal efferent fibres to the heart by vagotomy did not abolish the presence of reversed RSA suggesting that the pattern of heart

period variation is not neural, and may be largely mechanical. Vagotomy altered breathing by increasing respiratory period, tidal volume, and

the time to peak inspiratory flow. These changes did not alter the magnitude of RSA but reduced the latency period between inspiratory onset

and the onset of respiratory related prolongation of heart period. Periods of positive pressure ventilation were associated with reversal of the

inspiratory cardiac-deceleration pattern of RSA to resemble the more widely described pattern of inspiratory cardiac-acceleration. We

conclude that RSA is not a suitable measure of vagal tone during anesthesia in the rat and reiterate the caution that needs to be taken when

working with anesthetized experimental models of cardiac control.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Respiratory sinus arrhythmia; Heart rate variability; Vagotomy; Heart rate; Anesthesia

1. Introduction

In a recent study investigating the role of somatic afferent

nerve stimulation on inspiratory pacemaker activity (Larsen

et al., 2003), we observed that in the rat during spontaneous

breathing isoflurane anesthesia, respiratory sinus arrhythmia

(RSA) was associated with an inspiratory cardio-deceler-

ation, rather than acceleration, as is classically ascribed to

this phenomenon (Ludwig, 1847; Anrep et al., 1936a,b). A

review of the current literature revealed that RSA has been

well described in adult and neonatal humans, baboons, dogs,

rabbits, seals, and cats, but the nature of which is

controversial in rats (Bouairi et al., 2004; Cerutti et al.,

1991; Chess et al., 1975; Galletly and Larsen, 1998;

1566-0702/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.autneu.2004.12.003

* Corresponding author. Tel.: +64 4 385 5888x5152; fax: +64 4 389

5318.

E-mail address: [email protected] (Y.-C. Tzeng).

Japundzic et al., 1990; Neff et al., 2003; Perlini et al.,

1995a,b; Pickering et al., 2002; Rentero et al., 2002). We

found that dreversedT cardio-deceleration pattern of RSA has

previously been described in heart transplant recipients

(Bernardi et al., 1989) in anesthetized humans during

intermittent positive pressure ventilation (Larsen et al.,

1999) and was recently described in urethane anesthetized

rats (Bouairi et al., 2004; Rentero et al., 2002).

The current consensus is that RSA is mediated via

multiple mechanisms. These include (a) the direct inter-

action between central cardiorespiratory centres within the

brainstem, (b) pulmonary reflex pathways, (c) respiratory

gating of central arterial baroreceptor afferent input, (d) an

atrial reflex, and (e) oscillations in PaCO2 and pH in

arterial blood (Daly, 1997). Common to all of these

mechanisms is the phasic withdrawal of vagal tone

(Angelone and Coulter, 1964; Anrep et al., 1936a,b; Daly,

1997; Eckberg, 1983; Hirsch and Bishop, 1981; Levy et

al., 1966). The magnitude of heart period variation during

Clinical 118 (2005) 25–31

Y.-C. Tzeng et al. / Autonomic Neuroscience: Basic and Clinical 118 (2005) 25–3126

RSA is commonly taken as an indirect estimate of the

level of cardiac vagal efferent outflow and has been widely

used in cardiovascular research because computation of

indices, such as the maximum–minimum heart period

intervals or spectral power at the respiratory frequency, can

be readily obtained from non-invasive electrocardiogram

(ECG) recordings. The use of heart rate variability analysis

as a monitor of autonomic balance and dysfunction in

anesthetic practice has also been explored (Baumert et al.,

1995).

Anesthesia is ethically desirable for all experimental

protocols that are deemed to be highly invasive or difficult

to perform in conscious animals, and as such the

anesthetized rat has become a common model used in

cardiovascular research. However, there are limitations

associated with anesthesia because all anesthetic agents

exert effects on cardio-respiratory regulation. Data derived

from anesthetized animals therefore need to be interpreted

cognisant of the possible effects of anesthesia (Bouairi et

al., 2004). If heart rate variability analysis is to be applied

to anesthetized laboratory animals such as the rat, then a

detailed understanding of the altered physiology during

anesthesia must be established. In the current study we

explored features of respiratory sinus arrhythmia in

relation to the respiratory cycle for the isoflurane-

anesthetized rat.

2. Method

2.1. Animal

Experiments were carried out on adult female Sprague–

Dawley rats weighing 300–350 g. Rats were housed in an

institutional containment facility and were fed standard rat

feed and water ad libitum in line with local animal research

guidelines. The experimental protocol was approved by the

Wellington animal ethics committee.

2.2. Surgical preparation

On the day of the experiment, animals were retrieved

from the containment facility and placed in an induction

chamber with 4% isoflurane in oxygen. Once anesthetized,

an endotracheal tube was inserted and anesthesia was

thereafter maintained with isoflurane (2%) in oxygen. A

femoral artery and vein were cannulated (24 gauge IV

cannulae, Becton Dickinson, Utah, USA) for recording of

blood pressure and intravenous drug administration. All

catheters were filled with heparinized saline to prevent

clotting and secured to the surrounding skin flap with

surgical sutures. After cannulation, animals were left

undisturbed for at least 30 min for hemodynamic variables

to reach steady-state. Temperature was measured with

rectal thermo probe and maintained at 37 8C using a

radiant heat source.

2.3. Experimental protocol

After the initial stabilisation period (30 min), we

recorded 15 min of ECG (Grass Instruments P511

amplifier), respiratory activity (Hans Rudolph heated

Pneumotach 8420 A; Vacumed differential pressure trans-

ducer 4500) and blood pressure (Statham P23 ID, Gould,

Cleveland, OH). Arterial blood pressure signals were

passed to a low level DC amplifier (Model 7P122M,

Grass instruments, Quincy, MA, USA). In 15 rats,

bilateral vagotomy was then performed and ECG and

respiratory recordings were repeated for 15 min following

a 30-min stabilisation period. To ensure completeness of

denervation, in six rats we administered high dose

atropine (0.5 mg kg�1) following surgical vagotomy. We

also performed intermittent positive pressure ventilation

(IPPV, 10 min, 15 cm H20, 25 br min�1) to determine the

effects of IPPV on RSA (Dreyfuss et al., 1985; Fukunaga

et al., 1998). A low ventilation rate was chosen to clearly

demarcate cardiorespiratory interactions. Blood pressure

was maintained at levels above 100 mm Hg for all

animals throughout the duration of the study with saline

infusion. All data were sampled at 2000 Hz using a

multifunction I/O DAQ board (PCI-6023E, National

Instruments, TX, USA) and stored on hard-disk for off-

line processing.

2.4. Graphical analysis

From the respiratory flow signal, we determined the

time of all inspiratory onsets (I) and from the recorded

ECG we determined the time of each R wave peak. For

each data set, we constructed the averaged respiratory

waveform by signal averaging all breaths over the

recording period, and the bRSA curveQ by plotting, for

each R wave after inspiratory onset, the normalised

preceding RR-interval (duration of the preceding heart

periods divided by the mean RR-interval determined from

a window ten beats on either side of the interval under

examination) as a function of time following inspiratory

onset. A ten beat moving averager was chosen to

adequately remove beat to beat fluctuations. The RSA

curve essentially demonstrates the acceleration or deceler-

ation of heart rate during the respiratory cycle (Larsen et

al., 1999). A mean estimate of RSA magnitude (Rmax) in

the time domain was obtained by calculating the maximum

within breath difference of the RR-interval times from the

RSA curve. Tidal volume was measured by integrating the

respiratory waveform. The II-interval denotes the respira-

tory period.

2.5. Spectral analysis

In addition to the RSA curve, spectral analysis was also

used to quantify RSA magnitude. The RR-interval time

series was manually checked for the presence of artefacts

Fig. 1. Stylistic representation of signal averaged respiratory flow and the

RSA curve showing changes in normalised RR-interval throughout the

respiratory cycle. Time of inspiratory onset (t=0 s), and times of peak

inspiratory flow (a), expiratory onset (b), and peak expiratory flow (c) were

determined from the averaged respiratory flow signal. From the RSA curve,

Y.-C. Tzeng et al. / Autonomic Neuroscience: Basic and Clinical 118 (2005) 25–31 27

and spuriously detected or missed R waves were corrected

by linear interpolation or deletion. The resulting RR-

interval time series were then re-sampled at 10 Hz to

provide equidistant data points. The data set for spectral

analysis was obtained from each recording epoch by

applying a series of windows of length n=1024 points,

shifted by 512 points across the epoch. For each data set, a

Hanning window was applied prior to Fast-Fourier Trans-

form analysis. The average heart rate variability (HRV)

power spectrum from each window was obtained. We

defined the respiratory component of the power spectrum

as the centre frequency corresponding to the respiratory

frequency peak. The magnitude of RSA was quantified by

estimating the mean amplitude of the spectral peak at the

respiratory frequency, calculated by the formula (Hayano et

al., 1994),

HFamp ms=Hz1=2�¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2� power ms2=Hzð Þ�½

p�

All data analysis was performed on a Macintosh G4

computer using custom written software in LabView7

(National Instruments, TX, USA).

2.6. Statistical analysis

Results are expressed as meanFS.E. The effect of

vagotomy was determined with a paired t-test. Correlations

were obtained using correlation Z-test. Significant differ-

ences were accepted at Pb0.05. All statistical analysis was

performed using StatView 5 (SAS institute).

we determined, from inspiratory onset (t=0 s), latency of RSA (d), time to

maximum RSA (e), time to end of RSA (f) as well as the deceleration

period (g) and acceleration period (h) of RSA. The maximum within breath

difference in RR-interval was taken as an estimate of RSA magnitude

(Rmax).

3. Results

3.1. RSA during anesthesia (n=15)

In all rats we observed a paradoxical reversed RSA

with transient prolongation of the RR-interval at variable

latencies following inspiratory onset. A stylistic repre-

sentation showing principal features and definition of

associated nomenclature of the RSA curve and respira-

tion waveform is shown in Fig. 1, with the individual

animal data shown in Fig. 2. The mean magnitude of

RSA as estimated by Rmax was unrelated to the mean

RR-interval (164 (3) ms), mean II-interval (866 (37) ms),

or to mean tidal volume (3.5 (0.5) ml) both between and

within animals.

3.2. Effect of vagotomy and atropine (n=15)

Vagotomy was associated with significant increases in

II-interval, tidal volume, time of peak expiratory flow

and a significant reduction in time of maximum

inspiratory flow (Table 1) but did not alter RR-interval

(Table 2). Vagotomy did not alter the time of maximum

RSA, time to end RSA or the magnitude of RSA as

estimated by HFamp and Rmax, but was associated with a

significant reduction in RSA latency as well as an

increase in deceleration period of RSA (Table 2). The

acceleration period of RSA was unchanged by vagotomy

(Table 2).

Fig. 2 shows the diversity of RSA curves in relation to

the respiratory waveform for all preparations before and

after vagotomy. Prominent features of the RSA curve, as

indicated on representative waveforms include (i) prolonged

cardio-deceleration plateaus and double RSA peaks (Fig.

2a), (ii) biphasic RSA curves with an initial cardio-

acceleration (Fig. 2b), (iii) period of dovershootT followingthe end of RSA (Fig. 2c).

Deceleration plateaus and biphasic RSA curves were

observed both before and after vagotomy, however double

RSA peaks and dovershootsT were observed exclusively in

the vagotomized state. RSA latency varied between

preparations with the majority showing cardio-deceleration

Fig. 2. RSA curves and signal averaged respiratory waveforms superimposed for all rats during anesthesia (a–o), before (left panel) and after vagotomy (right

panel). Details of how these curves are created are given in the methods. In the majority of the RSA curves, both before and after vagotomy, cardiac-

deceleration starts during inspiratory onset, although the latency to the onset of deceleration is highly variable. In some cases, such as (i) the latency is

prolonged, and deceleration begins during the expiratory phase. Specific features on RSA curves are highlighted by the horizontal bar and arrows. In (a),

horizontal bar highlights the period of cardio-deceleration plateau while the arrows point to the presence of double RSA peaks. In (b), arrows point to an initial

period of cardio acceleration preceding onset of cardio-deceleration period on the RSA curve. In (c), the arrows point to dovershootT cardio-accelerationfollowing the end of RSA.

Y.-C. Tzeng et al. / Autonomic Neuroscience: Basic and Clinical 118 (2005) 25–3128

occurring during inspiration, although in some prepara-

tions, such as the example in Fig. 2i, cardio-deceleration

was observed during expiration.

Table 1

Effect of vagotomy on respiration

Variable Before

vagotomy

After

vagotomy

P

II-interval (ms) 866 (37) 1806 (86) b0.01

Tidal volume (ml) 3.5 (0.5) 5.8 (0.9) b0.05

Time to peak inspiratory flow (ms) 203 (8) 127 (4) b0.01

Time to peak expiratory flow (ms) 368 (8) 429 (21) b0.05

Time to expiratory onset (ms) 300 (7) 325 (16) NS

All values are reported as mean (SE). Respiratory period, II-interval. Time to

peak inspiratory flow, peak expiratory flow, and expiratory onset are given

as values relative to inspiratory onset. Comparisons were made using the

paired t-test. Pb0.05 denotes statistical significance; NS, non-significant.

In Fig. 3, the RR-interval time series and power spectrum

of a representative example show the cardio-deceleration

pattern of RSA that clearly persists following vagotomy.

To ensure complete vagotomy had been achieved, we

administered atropine in six rats post vagotomy and

observed no change in the pattern of RSA (Fig. 3).

3.3. Effect of IPPV (n=6)

A representative example of RSA during IPPV is shown

in Fig. 3. We found that mechanical ventilation caused the

RSA curve to reverse from the inspiratory deceleration

pattern to the classically described inspiratory acceleration

pattern. This reversal was observed in both the RR-interval

time series showing a reversal in heart interval fluctuations

and in the RSA curve. IPPV under the conditions of the

Table 2

Effect of vagotomy on RR-interval and RSA curve

Variable Before

vagotomy

After

vagotomy

P

RR-interval (ms) 164 (3) 166 (4.3) NS

HFamp (ms/Hz1/2) 1.00 (0.10) 0.80 (0.09) NS

Rmax (ms) 28 (2) 29 (3) NS

RSA latency (ms) 183 (21) 124 (9) b0.05

Time of maximum RSA (ms) 390 (20) 418 (26) NS

Time of end RSA (ms) 727 (30) 811 (50) NS

Deceleration period (ms) 207 (16) 293 (29) b0.05

Acceleration period (ms) 336 (33) 393 (41) NS

All values are expressed as mean (SE). Heart period, RR-interval.

Magnitude of RSA is estimated from the mean amplitude of high frequency

RR-interval oscillations (HFamp) and the magnitude of maximum RR-

interval change from the RSA curve (Rmax). RSA latency, time to maximum

and end RSA are mean intervals relative to inspiratory onset. Comparisons

were made using the paired t-test. Pb0.05 denotes statistical significance;

NS, non-significant.

Y.-C. Tzeng et al. / Autonomic Neuroscience: Basic and Clinical 118 (2005) 25–31 29

current study was associated with a shorter RSA latency

(106 (13) ms) than during spontaneous breathing. Spectral

peak corresponding to the ventilation frequency was seen

for all animals.

4. Discussion

In the present study we examined the nature of RSA in

the anesthetized rat in relation to the respiratory cycle. We

found that, contrary to descriptions of RSA in conscious

Fig. 3. Representative RR-interval, RSA curve and heart rate variability power spe

vagotomy and atropine with intermittent positive pressure ventilation (IPPV). Disti

interval power spectra at the respiratory frequency indicate the presence of RSA

RSA is present in the absence of vagal innervation to the heart, but is reversed d

rats, in which RR-interval decreased with inspiration and

increased during expiration (Bouairi et al., 2004; Neff et al.,

2003), all rats in the current study showed a dreversedTpattern of RSA in which cardio-deceleration, usually

beginning in inspiration, was observed, similar to that

described in human heart transplant recipients (Bernardi et

al., 1989) and urethane anesthetized rats (Bouairi et al.,

2004). We observed that the magnitude and direction of the

RR-interval change of this RSA pattern was unaffected by

surgical vagotomy but could be reversed with positive

pressure mechanical ventilation.

To our knowledge, the inspiratory cardio-deceleration

pattern of RSA in the anesthetized rat has not been

adequately explained. Bouairi and co-workers observed that

RSA was dependant on the type of anesthetic agent used.

Ketamine–xylazine anaesthesia was associated with a

reduction in magnitude of RSA although cardiac-acceler-

ation was still observed during inspiration. Pentobarbital

anesthesia abolished RSA altogether and urethane anaes-

thesia was associated with inspiratory cardio-deceleration

pattern of RSA. The changes associated with ketamine–

xylazine and pentobarbital sodium were attributed to central

depression of cardiac parasympathetic output, while no

mechanism for reversal of RSA during urethane anesthesia

was proposed (Bouairi et al., 2004).

The results of this present study demonstrate that the

mechanism of reversed RSA during isoflurane anaesthesia is

not vagally mediated. Isoflurane increases steady-state heart

rate, decreases heart rate variability, central respiratory

ctra during (a) anesthesia, (b) vagotomy, (c) vagotomy and atropine, and (d)

nct oscillations in the RR-interval time series and a spectral peak on the RR-

during anesthesia. RSA curves show that the cardio-deceleration pattern of

uring IPPV.

Y.-C. Tzeng et al. / Autonomic Neuroscience: Basic and Clinical 118 (2005) 25–3130

drive, and like urethane, decreases the sensitivity of the

baroreceptor reflex loop (Lee et al., 2002). In the present

study, during isoflurane anesthesia there was no demon-

strable efferent vagal-cardiac nerve activity evidenced by

vagotomy producing no significant change in heart rate or

RSA. Moreover, the persistence of cardio-deceleration RSA

following vagotomy and high dose atropine demonstrate

that the mechanism of this reversed RSA is not mediated by

changes in vagal tone.

It has previously been shown that, in addition to neural–

humoral control of heart rate, the heart possesses an intrinsic

mechanism of rate regulation (Pathak, 1973; Keatinge,

1959; Lange et al., 1966; Vick, 1963; Oosting et al., 1997).

This is based upon studies in isolated hearts demonstrating

that when the sino-atrial node is physically distended, the

rate of sinus depolarisation may either increase, or decrease,

depending on the initial heart rate and the degree of stretch

applied (Pathak, 1973). Such a dmechanical hypothesisT hasalready been suggested to contribute to RSA in the

conscious rat (Perlini et al., 1995a) and the anesthetized

rabbit (Perlini et al., 1995b). Therefore it is possible to

speculate that the reversed pattern of RSA observed in this

study is due to the fluctuations in atrial pressure, secondary

to respiration, exerting tensile forces on the atrial myocar-

dium to cause phasic fluctuations in RR-interval.

Our observation that reverse RSA reverts to the classical

pattern of inspiratory cardio-acceleration during positive

pressure ventilation appears consistent with a mechanical

hypothesis. Positive pressure ventilation leads to a reversal

of intrathoracic pressure, and therefore the transmural

forces acting on the atrial myocardium will be reversed.

However, another possible explanation for our observations

is that the increase in intrathoracic pressure during the

inspiratory phase of ventilation activates sympatho-sympa-

thetic excitatory pathways through sympathetic afferents

and that this reflex activity may account for the reversal of

RSA during IPPV (Malliani and Montano, 2002). It is also

interesting to note that Yli-Hankala et al. (1991) have

previously demonstrated that the normal pattern of RSA

seen in anaesthetised spontaneously breathing human

subjects is reversed by the application of IPPV, although

the mechanism for this has not been identified. The

changes in RSA that occur during IPPV clearly warrant

further investigation.

The current study shows that the morphology of the RSA

curve and RSA latency varies considerably between

preparations and may change following vagotomy, possibly

as a result of changes to respiratory pattern. Abolition of

pulmonary stretch afferents with vagotomy resulted in

marked decreases in mean II-interval, time interval to peak

inspiratory flow as well as an increase in tidal volume.

These alterations in respiratory pattern did not alter the

absolute magnitude of RSA as measured by Rmax or HFamp,

but were associated with a reduction in RSA latency. One

possible explanation is that the rate of maximal change in

myocardial stretch occurs comparatively earlier following

vagotomy, although this requires experimental validation. In

addition a number of other observations require further

investigation. For example, it is currently unclear why RSA

curve morphology and latency exhibit such widespread

variation with inspiratory cardio-deceleration RSA occur-

ring soon after inspiratory onset in some animals while, in

others, the onset of RSA occurred considerably later, in

some cases during the expiratory phase. This is in contrast

to IPPV where the latency of RSA was minimal. It is also

presently unclear why the magnitude of RSA remains

unchanged following vagotomy despite significant changes

in respiratory flow pattern.

The physiological role of RSA also remains uncertain.

An emerging hypothesis is that RSA functions to

optimise pulmonary gas exchange on the premise that

more heart beats occur during inspiration when alveolar

ventilation is maximal, while fewer heart beats occur

during expiration, when alveolar ventilation is minimal,

thereby minimising ventilation–perfusion mismatch and

conserving energy (Hayano and Yasuma, 2003). If this is

true, then reversal or diminution of RSA during anes-

thesia may result in increased physiological dead space

and intrapulmonary shunt, exacerbating overall increase in

ventilation–perfusion mismatch and therefore contributing

to the deleterious effects of anesthesia on cardiopulmo-

nary function.

In summary, the current study shows that the pattern of

cardio-acceleration/deceleration during RSA in the sponta-

neously breathing rat under isoflurane–oxygen anesthesia is

reversed. This pattern of RSA is not mediated by vagal-

cardiac nerve activity and we believe should not be used as

a measure of vagal tone. Because the use of spectral or time

domain measures of RSA alone as an indicator of RSA

magnitude ignores the possible change to RSA polarity, we

recommend that future research in HRV should always

involve the direct inspection of R–R interval time series

fluctuations. The current study reiterates the caution that

needs to be taken when interpreting physiology from

anesthetized rat models.

Acknowledgements

This study was supported by grants from the Wellington

Medical Research Foundation.

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