J Appl Physiol 106: 1564 1573, 2009.

First published February 26, 2009; doi:10.1152/japplphysiol.91590.2008.

Contribution of the carotid body chemoreceptors to eupneic ventilation

in the intact, unanesthetized dog
Gregory M. Blain, Curtis A. Smith, Kathleen S. Henderson, and Jerome A. Dempsey
The John Rankin Laboratory of Pulmonary Medicine, Department of Population Health Sciences, School of Medicine
and Public Health, University of Wisconsin, Madison, Wisconsin
Submitted 11 December 2008; accepted in final form 23 February 2009

Blain GM, Smith CA, Henderson KS, Dempsey JA. Contribution
of the carotid body chemoreceptors to eupneic ventilation in the intact,
unanesthetized dog. J Appl Physiol 106: 1564 1573, 2009. First pub-
lished February 26, 2009; doi:10.1152/japplphysiol.91590.2008.We
used extracorporeal perfusion of the reversibly isolated carotid sinus
region to determine the effects of specific carotid body (CB) chemo-
receptor inhibition on eupneic ventilation (VI) in the resting, awake,
intact dog. Four female spayed dogs were studied during wakefulness
when CB was perfused with 1) normoxic, normocapnic blood; and 2)
rhythm-generating neurons in the pre-Botzinger complex, sug-
gesting a reduced activity of the central respiratory control
system after CBD (22) over and above that resulting from loss
of carotid chemoreceptor afferent input. 3) A significant reduc-
tion of the ventilatory response to focal acidosis with 80% CO2
at multiple raphe sites was found after CBD in the goat (17). 4)
Several studies (2, 20, 26) have shown that, after the initial rise
in eupneic arterial PCO2 (PaCO2) following denervation, PaCO2

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hyperoxic (500 mmHg), hypocapnic (20 mmHg) blood to maxi-
mally inhibit the CB tonic activity. We found that CB perfusion per se
(normoxic-normocapnic) had no effect on VI. CB inhibition caused
marked reductions in VI (60%, range 49 80%) and inspiratory flow
rate (58%, range 44 87%) 24 41 s following the onset of CB
perfusion. Thereafter, a partial compensatory response was observed,
and a steady state in VI was reached after 50 76 s following the onset
of CB perfusion. This steady-state tidal volume-mediated hypoventi-
lation (31%) coincided with a significant reduction in mean dia-
phragm electromyogram (24%) and increase in mean arterial pres-
sure (12 mmHg), which persisted for 725 min until CB perfusion
was stopped, despite a substantial increase in CO2 retention (9 Torr,
arterial PCO2) and systemic respiratory acidosis. We interpret these
data to mean that CB chemoreceptors contribute more than one-half to
the total eupneic drive to breathe in the normoxic, intact, awake
animal. We speculate that this CB contribution consists of both the
normal tonic sensory input from the CB chemoreceptors to medullary
respiratory controllers, as well as a strong modulatory effect on central
chemoreceptor responsiveness to CO2.
control of breathing; eupnea; peripheral chemoreception
SINCE THE LANDMARK FINDINGS of Corneille Heymans and col-
leagues in the 1930s (16), a number of experimental models
have been used in an attempt to determine the role of carotid
body (CB) chemoreflexes in the regulation of breathing. For
example, hypoventilation, which averaged 20% below control,
has been reported in several animal species following CB
denervation (CBD), thereby demonstrating that CB chemore-
ceptor afferents provide a significant tonic excitatory input to
the medullary respiratory neurons during eupnea (3, 13, 18, 26,
31). However, there are major limitations of the CBD model
that severely limit the conclusions that can be drawn from these
studies, because CBD has also been shown to result in several
time-dependent compensatory and adaptive changes that fun-
damentally alter the respiratory control system. For example,
CBD has been shown to cause the following changes: 1) upregu-
lation of aortic chemoreceptors sensitivity (2, 3); 2) marked
reductions in cytochrome oxidase activity in the medullary
Address for reprint requests and other correspondence: G. M. Blain, The
John Rankin Laboratory of Pulmonary Medicine, 1300 Univ. Ave., #4245
MSC, Madison, WI 53706 (e-mail: blain@unice.fr).
falls, and the gain of the ventilatory response to CO2 rises
gradually over days to weeks.
To our knowledge, the role of CB chemoreceptors in eu-
pneic breathing at rest in the intact, unanesthetized preparation
has never been tested. Over the past decade, based on the
pioneering work of Busch et al. (5) in the goat, we have
developed and refined an isolated and perfused CB preparation
in the unanesthetized, intact canine, which reversibly separates
the circulation of the CB chemoreceptors from that of the
systemic circulation (and, therefore, the cerebral circulation
supplying the central chemoreceptors). This preparation allows
precise and independent control of the chemical milieu of these
regions in an intact physiological preparation. Using this tech-
nique, our group showed in intact, unanesthetized dogs, that
inhibition of the CB chemoreceptors via perfusion of the
reversibly isolated carotid sinus region with either hyperoxic or
hypocapnic blood transiently reduced eupneic ventilation to an
average of 29% below control (38). However, the true contri-
bution of CB chemoreceptors to eupneic ventilation cannot be
determined from this study, because the tonic input from these
chemoreceptors may not have been maximally inhibited.
Moreover, the ventilatory response beyond 2 min of inhibi-
tion was not studied, so any further compensation for changes
in PaCO2 and H in the steady-state was not determined. To
overcome these limitations, we combined marked hypocapnia
and hyperoxia to maximally and physiologically inhibit the
isolated CB chemoreceptors over longer durations. With this
approach, combined with an analysis of the time course re-
sponse, we have quantified the CB chemoreceptors maximum
contribution to the eupneic drive to breathe, as well as the
compensatory capability of the central chemoreceptors to re-
spond to systemic hypercapnia in the absence of tonic CB
chemoreceptor input.
METHODS
Studies were performed during wakefulness on four unanesthe-
tized, spayed female, mixed-breed dogs (20 25 kg). The dogs were
trained to lie quietly in an air-conditioned (19 22C), sound-attenu-
ated chamber. Throughout all experiments, the dogs behavior was
monitored by an investigator seated within the chamber and also by
closed-circuit television. The Animal Care and Use Committee of The
University of Wisconsin-Madison approved the surgical and experi-
mental protocols for this study.

1564 8750-7587/09 $8.00 Copyright 2009 the American Physiological Society http://www. jap.org
 EFFECTS OF MAXIMAL CAROTID BODY INHIBITION ON VENTILATION
Chronic Instrumentation
Our preparation required two surgical procedures performed under
general anesthesia and with strict sterile surgical techniques and
appropriate postoperative analgesics and antibiotics. In the first pro-
cedure, dogs were subjected to ovariectomy/hysterectomy, a chronic
indwelling catheter was placed into the abdominal aorta via a branch
of the femoral artery, and bipolar electromyogram recording elec-
trodes were installed into the costal diaphragm. In the second proce-
dure, the left CB was denervated, and the right carotid sinus was
equipped with a vascular occluder and catheter to permit extracorpo-
real perfusion of the reversibly isolated carotid sinus CB. A chronic
indwelling catheter was also placed in the abdominal vena cava via a
branch of the femoral vein. Catheters were tunneled subcutaneously to
the cephalad portion of the dogs back where they were exteriorized.
Dogs recovered for at least 4 days before study. The instrumentation
was protected with a heavy nylon jacket and a padded Elizabethan
collar modified to allow normal eating and drinking.
Carotid Sinus Perfusion
Dogs lay unrestrained on a bed within the sound-attenuated cham-
ber. The extracorporeal circuit was primed with 700 ml of saline,
120 ml of allogenic blood, and 2,500 units of heparin and supple-
mented with 1,000 U/h. PCO2, PO2, and pH in the perfusion circuit
were set by adjustment of the gas concentrations supplying the circuit
and by addition of NaHCO3. The carotid sinus region was perfused at
flow rates 100 ml/min, which raised the pressure in the sinus region
by 10 mmHg above the systemic blood pressure. Before data
acquisition, a 30-min period of normal perfusion of the carotid sinus
region was used to ensure uniformity between systemic and extracor-
poreal circuit blood. Intravenous boluses of NaCN (20 mg/kg) were
used to confirm that 1) the nondenervated CB remained completely
functional before CB perfusion; and 2) there was complete isolation of
the carotid sinus during perfusion and also complete denervation of
the contralateral CB. These techniques have been described in detail
in previous publications (8, 37, 38).
Experimental Setup and Measurements
Ventilation was measured using a tight-fitting muzzle mask con-
nected to a heated pneumotachograph (model 3700, Hans Rudolph,
Kansas City, MO) that was calibrated before each study with four
known flows. Costal diaphragm electromyogram (EMGdi) signals
were amplified, band-pass filtered, rectified, and moving-time aver-
aged (BMA-931; MA-821RSP, CWE). End-tidal PO2 (PETO2) and
PCO2 (PETCO2) were measured using a mass spectrometer (MGA-1100,
Perkin-Elmer, Waltham, MA).
Blood pressure was recorded continuously from the femoral artery.
One-milliliter arterial and perfusion circuit blood samples were
analyzed for pH, PO2, and PCO2 on a blood-gas analyzer (model
ABL-505, Radiometer, Copenhagen, Denmark). The blood-gas ana-
lyzer was validated daily with dog blood tonometered with three
different combinations of PO2 and PCO2, covering the range encoun-
tered in the experiments. Samples were corrected for both body
temperature and systematic errors revealed by tonometry.
Ventilation and blood pressure signals were digitized (128-Hz
sampling frequency) and stored on the hard disk of a PC for subse-
quent analysis. Key signals were also recorded continuously on a
polygraph (AstroMed K2G, West Warwick, RI). All ventilatory data
were analyzed on a breath-by-breath basis by means of custom
analysis software developed in our laboratory.
Experimental Protocol
Each test protocol consisted of a 5-min control period (eupnea),
during which perfusion of the CB was endogenous, i.e., systemic
arterial blood. Two 1-ml blood samples were collected at 3 min for
determination of blood gases and pH control values. Then, CB
J Appl Physiol VOL
1565
perfusion was abruptly switched to the extracorporeal circuit (2 s),
and the ventilatory and EMGdi responses were recorded for at least 7
min before the carotid sinus was abruptly returned to endogenous
perfusion.
The dogs were perfused in random order from the extracorporeal
circuit with blood gases and pH concentrations matching a given
dogs eupneic values (CB normal), or with hyperoxic (500 mmHg)
and hypocapnic (20 mmHg) blood gases (CB inhibited). At least
two 1-ml blood samples were collected at 3 min and 7 min of
perfusion for determination of blood gases and pH. During inhibition
of CB chemoreceptors, inspired O2 fraction was increased to maintain
arterial PO2 at control values.
Speed of Response
We determined the time to the first significant ventilatory response
[breath-by-breath minute ventilation (VI)]. A VI response was con-
sidered significant if it was 3 SDs lower than the mean of the
preceding normocapnic VI. We also quantified 1) the time to VI nadir,
defined as the mean of the lowest VI and its two surrounding breaths;
2) the time required for VI to reach a steady state, defined as the first
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three consecutive breaths within 1 SD of the mean VI from the last
30 s of CB inhibition; and 3) the mean ventilatory values at 5 min
following the beginning of CB inhibition (averaged over a 30-s
period) and from the last 30 s of CB inhibition.
We also determined the time to the first significant response in
mean arterial pressure (MAP). A response in MAP was considered
significant, if it was 3 SDs higher than the mean of the preceding
normocapnic MAP. We also quantified the time required for MAP to
reach a steady state, defined as the first three consecutive beat values
within 1 SD of the mean MAP from the last 30 s of CB inhibition.
Ventilatory and Arterial Blood Variables
Ventilatory data were averaged for the last 2 min of control and CB
perfusion periods to determine the ventilatory response to CB inhibi-
tion. Duplicate arterial blood samples were also obtained during this
2-min period.
Statistics
Normality of the data was confirmed using the Shapiro-Wilk
test. The time-dependent effect of CB inhibition on ventilation (see
Fig. 3) was tested using a one-way repeated-measure ANOVA with
Holm-Sidak post hoc test. Significance of the differences in mean
steady-state data between control and the response to CB perfusion
was determined by means of paired t-tests, with Bonferroni cor-
rection for multiple comparisons. Differences were considered
significant if P 0.05.
RESULTS
Effects of Perfusion Per Se
Consistent with our laboratorys previous studies (37, 38),
CB perfusion with blood gases and pH matched to normal
eupneic systemic arterial values did not significantly affect
ventilation or cardiovascular parameters (Tables 1 and 2).
Response to CB Inhibition: Original Record
Figure 1 is a segment of an original polygraph record
showing the ventilatory, EMGdi, MAP, PETO2, and PETCO2
responses over the first 4 min of perfusion of the isolated CB
with hyperoxic and hypocapnic gases in a representative dog
(dog 2). Note the rapid reduction in tidal volume (VT) and the
amplitude of EMGdi and the development of hypoventilation
(i.e., elevated PETCO2) with the onset of CB perfusion. Also
106 MAY 2009 www.jap.org
1566 EFFECTS OF MAXIMAL CAROTID BODY INHIBITION ON VENTILATION

Table 1. Mean steady-state values for blood gas, ventilatory, and EMGdi variables
f, breaths/
Dog No. PaCO2, Torr PaO2, Torr pHa HCO3, meq/l VI, l/min VT, liter min TI, s TE , s EMGdi, AU

Control
1 46.42.9 92.63.2 7.300.02 22.11.4 3.270.59 0.230.05 14.84.2 1.560.26 2.941.13 0.350.33
2 36.01.3 104.16.7 7.350.01 19.40.2 5.050.40 0.310.03 16.50.5 1.240.13 2.430.08 0.580.07
3 48.42.7 87.05.3 7.330.02 24.81.7 5.330.79 0.420.19 16.06.5 2.421.55 1.900.50
4 41.11.0 99.69.8 7.340.02 21.40.6 3.850.46 0.260.02 14.21.9 1.550.09 2.790.49 0.460.28
MeanSD 43.05.6 95.87.6 7.330.02 21.92.2 4.380.98 0.310.08 15.41.1 1.690.51 2.520.46 0.460.12
CB perfused normoxic-normocapnic
1 43.90.1 97.31.9 7.290.03 20.41.3 3.900.50 0.260.03 15.13.6 1.600.07 2.600.96 0.340.06
2 38.81.8 94.70.7 7.330.03 19.90.3 4.560.18 0.280.02 16.51.9 1.230.18 2.460.24 0.540.01
3 48.51.9 91.42.7 7.310.01 23.61.5 5.131.34 0.530.16 9.70.3 3.701.60 2.551.38
4 40.70.8 101.04.8 7.330.02 20.60.4 3.780.76 0.270.02 13.31.8 1.690.05 3.380.81 0.470.25
MeanSD 43.04.2 96.14.1 7.320.02 21.11.7 4.340.63 0.340.13 13.72.9 2.061.11 2.750.43 0.450.10
CB perfused hyperoxic-hypocapnic
1 60.43.2 91.24.7 7.240.02 24.40.41 1.700.56 0.180.03 10.13.9 1.640.27 5.362.58 0.230.03

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2 45.53.2 100.45.0 7.280.02 20.524.4 3.771.70 0.270.03 14.13.9 1.380.27 2.902.58 0.470.07
3 57.12.2 89.35.1 7.290.02 26.11.83 4.460.49 0.220.08 18.47.5 2.462.06 1.600.39
4 47.71.2 97.53.4 7.270.02 20.42.10 2.520.73 0.260.02 11.63.5 2.000.67 3.470.96 0.370.17
MeanSD 52.77.2* 94.65.2 7.270.02* 22.92.9 3.111.24* 0.260.04* 13.63.6 1.870.47 3.331.56 0.360.12*
Values are means SD. CB, carotid body; PaCO2, arterial PCO2; PaO2, arterial PO2; pHa, arterial pH; HCO3, HCO3 concentration; VI, ventilation; VT, tidal
volume; f, breathing frequency; TI, inspiratory time; TE, expiratory time; EMGdi, moving-time-averaged electromyogram of the costal diaphragm; AU, arbitrary
units. Blood gases during CB perfusion are from the last (steady-state) blood sample. Note that 1) control refers to the condition following the second surgery
(denervation of one CB; see METHODS); 2) for CB, perfused hyperoxic-hypocapnic inspired O2 fraction was increased to maintain PaO2 at control values.
Significant difference from *control values and CB normal values, P 0.05.

note that, as CB inhibition was maintained, the VT and EMGdi
amplitude tended to increase slowly over time; however, a
substantial hypoventilation remained throughout the 4-min
period of CB perfusion.
Steady-State Ventilatory Response to CB Inhibition
We performed a total of 27 CB inhibition experiments in
four dogs. The mean values for blood gases and ventilatory
Table 2. Mean steady-state values
for cardiovascular variables
Dog No. MAP, mmHg
Control
1 101.66.8
2 92.73.6
3 105.98.3
4 98.45.7
MeanSD 99.75.6
CB perfused normoxic-normocapnic
1 102.82.9
2 99.87.8
3 103.93.6
4 90.09.6
MeanSD 99.16.3
CB perfused hyperoxic-hypocapnic
1 117.77.7
2 108.57.0
3 111.010.4
4 108.61.2
MeanSD 111.59.2*
Values are means SD. MAP, mean arterial pressure; HR, heart rate.
Significant difference from *control values and CB normal values, P 0.05.
J Appl Physiol VOL
variables obtained during control and steady-state periods of
CB inhibition are summarized in Table 1.
Representative breath-by-breath time course of response of
VI and PETCO2 are shown in Fig. 2 for each animal. In all four
animals, VI fell abruptly, and PETCO2 rose with the onset of CB
inhibition; shortly thereafter, VI rose slightly and reached a
steady-state plateau over the remaining several minutes of each
trial. In the steady state, PaCO2 (superimposed on the figure)
rose to 714 Torr greater than control and was accompanied by
a significant respiratory acidosis. EMGdi fell 24% (range 19
34%), mean inspiratory flow rate (VT/TI, where TI is inspira-
tory time) was reduced by 30% (range 22 49%), and VI
HR, beats/min decreased 31% (range 16 48%) below control. This reduction
in steady-state VI was due almost entirely to a reduced VT, with
only one of the four animals showing a significant decrease in
78.7113 breathing frequency as well as in VT (see Table 1).
77.011.6
67.33.4
58.34.8 Time Course and Magnitude of the Ventilatory Depression
70.39.5 via CB Inhibition
Figure 3 divides the time course of the ventilatory response
68.19.0 with CB inhibition into five segments.
78.110.7 Time to first ventilatory depression. The time to the first
65.12.3
57.84.2 detectable breath with a reduction in VI and VT/TI to more than
67.38.4 3 SDs below the control mean averaged 8 s (range, 4 12 s)
from the onset of CB hyperoxic hypocapnia.
Nadir of the ventilatory response. Thirty seconds (range
84.310.5
78.411.9
24 41 s) following the onset of CB inhibition, VI and VT/TI
69.97.3 were reduced to an average of 60% (range 49 80%) and 58%
53.40.7 (range 44 87%) of control, respectively. These nadir values
71.513.4 were averaged over three breaths (1318 s), during which time
the reduced VI and VT/TI remained unchanged. Thereafter, VI
and VT/TI began to increase systematically, presumably repre-
106 MAY 2009 www.jap.org
 EFFECTS OF MAXIMAL CAROTID BODY INHIBITION ON VENTILATION
Fig. 1. Polygraph record of a representative trial of carotid body (CB) inhibition (dog 2). Perfusion begins at time 0 (solid vertical line). VI, ventilation; EMGdi,
moving-time-averaged electromyogram of the costal diaphragm; VT, tidal volume; BP, blood pressure; PETCO2, end-tidal PCO2; PETO2, end-tidal PO2; au, arbitrary
units. Interruption in the BP trace is due to blood sampling. Note that the immediate hypoventilation and PETCO2 increase with CB inhibition persists for the
duration of the trial.
senting a compensatory response to the initial hypoventilation
and systemic CO2 retention.
Compensatory response to steady state. From their nadir,
both VI and VT/TI rose over the subsequent 30 s to reach a
new steady state, with values that averaged 31% (range 16
48%) and 30% (range 22 49%) less than control, respectively
(see Fig. 3 and Table 1).
Individual mean values for VI and VT/TI are shown after 5
min of continued CB inhibition in each animal, as well as for
the average maximum duration of CB inhibition, which varied
from 7 to 10 min across the four animals. The constancy of these
values in all animals in all trials between 1 and 10 min of CB
inhibition demonstrates that the compensatory increases of VI
and VT/TI were essentially complete within 30 s following the
nadir of hypoventilation achieved via CB inhibition.
Off-transient. Due to normal behavioral patterns (stretching,
licking, etc.), technically acceptable ventilatory and cardiovascu-
lar data following the abrupt cessation of CB inhibition were not
available in all dogs. In two dogs (dogs 1 and 3) in which
acceptable off-transient data were available, cessation of CB
inhibition was accompanied by a brief VI overshoot, followed by
a return to control values within 27 s (range 24 29 s; Fig. 4).
Note, in this example, that the CB inhibition-induced hypoventi-
lation was maintained throughout 25 min of CB inhibition.
Cardiovascular Response to CB Inhibition
MAP and heart rate changes in response to CB inhibition are
shown in Fig. 5 and Table 2. MAP began to rise significantly
above 3 SD of control (13.7, range 9 18 mmHg) 32 s on
average (range 22 45 s) after the onset of CB inhibition. A
steady-state response, with MAP increased by 12 mmHg
(range 517 mmHg) greater than control, was achieved at 52
s (range 3292 s) following the onset of CB inhibition and
J Appl Physiol VOL
1567
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remained elevated throughout the duration of CB inhibition.
Heart rate did not change systematically from control over the
time course of CB inhibition. In the two dogs (dogs 1 and 3) in
which acceptable off-transient data were available, cessation of
CB inhibition was accompanied by a return of MAP to control
values within 30 s (range 26 34 s).
DISCUSSION
Our study shows that physiological inhibition of the carotid
chemoreceptor via hyperoxic-hypocapnic perfusion in intact,
unanesthetized dogs significantly and markedly inhibits eu-
pneic EMGdi, VT, and VI. The maximal ventilatory depression
that was observed consistently within 30 s of the onset of
isolated CB inhibition suggests that more than one-half of the
eupneic drive to breathe in the normoxic, intact animal is
attributable to sensory tonic input from the carotid chemore-
ceptor. Longer periods of isolated CB inhibition were accom-
panied by marked hypoventilation and systemic respiratory
acidosis for which there was significant but incomplete venti-
latory compensation. This limited compensatory response was
attributed to a secondary inhibitory effect on medullary che-
moresponsiveness (to brain hypercapnia) caused by the ab-
sence of CB sensory input.
Significance/Limitations of the Unanesthetized, Intact,
Carotid Sinus-Perfused Model
We believe that the present study, utilizing the intact, un-
anesthetized, carotid sinus-perfused dog model to isolate the
CB chemoreceptors from the systemic circulation and central
chemoreceptors is an important advance over the CBD model.
Indeed, this model, with intact but reversibly isolated CB,
preserves the tonic CB sensory input, thereby avoiding 1) any
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1568 EFFECTS OF MAXIMAL CAROTID BODY INHIBITION ON VENTILATION

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Fig. 2. PETCO2 (left) and VI (right) responses from one representative trial of CB inhibition from each dog: dog 1 (A and B), dog 2 (C and D), dog 3 (E and F),
and dog 4 (G and H). Data are breath by breath and normalized to control (endogenous CB perfusion; i.e., CB not inhibited). CB inhibition commenced at time
0 (vertical line). Numbers at arrow indicate arterial PCO2 (Torr) and pH measured at that time. Note that the hypoventilation is maintained throughout CB
inhibition, despite marked CO2 retention and arterial acidosis. , Change.

potential compensatory changes in the central respiratory con-
troller or in central and/or non-CB peripheral chemoreceptor
sensitivities resulting from CBD (2, 20, 22, 26); and 2) any
changes in baseline ventilation or acid-base status, as is com-
monly observed following bilateral CBD (14, 31) (also see
Introduction). Moreover, our study provides unique data in the
unanesthetized dog, in which both chemoreflexes, pulmonary
J Appl Physiol VOL
mechanoreflexes, and baroreflexes were not obtunded, as is
known to occur with anesthesia (10, 15, 29). Finally, the
isolated, blood-perfused CB preparation permits reversible
physiological inhibition of the CB chemoreceptor, thereby
allowing analysis of the time course of the cardiorespiratory
response to sustained inhibition of carotid chemoreceptor
sensory input in the absence, as well as in the presence, of
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 EFFECTS OF MAXIMAL CAROTID BODY INHIBITION ON VENTILATION 1569
ing three animals (14, 9.5, and 6.6 Torr), which showed
no significant alteration in eupneic PaCO2 following unilateral
CBD. Taken together, these findings suggest that animals with
one intact CB have a ventilatory control system that responds
similarly to that of the intact animal.
To produce retrograde flow through the carotid sinus region
sufficient to isolate the CB from systemic blood, perfusion
pressure was slightly higher (510 mmHg) than systemic
arterial pressure. However, data from the present study using
normoxic and normocapnic control perfusions, as well as a
previous study using increases in blood pressure limited to the
carotid sinus in the awake dog (33), showed that pressure
increases of this magnitude had no significant ventilatory or
cardiovascular effects. Consequently, we do not think that the
imposed slight increase in carotid sinus pressure is a significant
limitation to this study.
Significant sex differences in peripheral and central chemo-
sensitivities could be a potential limitation to our use of spayed
female dogs in the present study. However, our interpretation

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of the human literature is that the preponderance of evidence,
when scaled correctly for body mass and/or vital capacity,
supports a lack of sex difference in ventilatory responses to
hypoxia or hypercapnia (19, 28, 41, 45). In studies that do find
a difference, women tend to have a lower apneic threshold for
CO2 (i.e., a wider CO2 reserve) (46) or an increased ventilatory
response to hypoxia (1) relative to men, although one paper
reports an increased peripheral response to CO2 in men vs.
women based on single-breath CO2 tests (35). The limited
Fig. 3. Bar graph of the time course of VI (top) and inspiratory flow rate [ratio
of tidal volume to inspiratory time (VT/TI), bottom] during CB inhibition. Data
are normalized to control (%control, where control is normal, endogenous CB
perfusion; i.e., CB not inhibited) and presented as means SD. Control is
arbitrarily set to zero to more clearly indicate the direction of change. For each
dog, the first error bar (without associated bar) indicates the SD of the control
values. 3 SD, the first VI response 3 SDs below the control mean; Nadir,
the mean of the lowest VI and its two surrounding breaths; Steady State, the
mean values of the first 3 consecutive breaths within 1 SD of the mean VI from
the last 30 s of the experiment (7 min of perfusion); 5, the mean ventilatory
values of the last 30 s of the 5th min of CB inhibition; End, the mean
ventilatory values from the last 30 s of each experiment, regardless of duration.
Note that the nadir ventilatory response to CB inhibition averaged 60% below
control, and the maintained steady-state response was 38% below control.
Significant difference from *control values and nadir values, P 0.05.

secondary changes in systemic arterial blood gases (see

below).
Our method of CB perfusion does require unilateral CBD on
the nonperfused side. The potential effects on the central
ventilatory control system following denervation are thus a
concern. There is evidence using this preparation in both the
goat (5) and the dog (8, 38) showing that unilateral CBD has no
consistent effects on ventilation during control, air-breathing
conditions, or on the ventilatory response to hypoxia or CO2. In
contrast, Pan et al. (26) showed in the goat that unilateral CBD
resulted in a transient (i.e., over 7 days) but significant hy- Fig. 4. Example of PETCO2 (A) and VI (B) responses to prolonged (25 min) CB
poventilation during room-air breathing, yet the ventilatory inhibition in dog 2 (same dog shown in Fig. 1). Data are breath by breath and
response to intravenous injections of sodium cyanide was not normalized to control (endogenous CB perfusion; i.e., CB not inhibited). Mean
affected. We also found high PaCO2 values (48.5 1.9 Torr) in PETCO2 and VI during control were 43.1 Torr and 5.51 l/min, respectively. CB
one dog (dog 3) during eupneic control in the present study, inhibition commenced at time 0 (first vertical line) and stopped after 25 min of
CB inhibition (second vertical line). Note that the hypoventilation is main-
even though inhibition of the remaining CB in this dog elicited tained throughout CB inhibition, despite marked CO2 retention, and, after a
a further marked increase in PaCO2 (8.7 Torr) in the steady brief overshoot, it returned to control values within 30 s upon a return to
state, which was similar to the increase observed in the remain- endogenous CB perfusion.

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1570 EFFECTS OF MAXIMAL CAROTID BODY INHIBITION ON VENTILATION

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Fig. 5. Mean arterial pressure (MAP; left) and heart rate (HR; right) responses from one representative trial of CB inhibition from each dog: dog 1 (A and B),
dog 2 (C and D), dog 3 (E and F), and dog 4 (G and H). Data are beat by beat and normalized to control (endogenous CB perfusion; i.e., CB not inhibited).
CB inhibition commenced at time 0 (solid line). Discontinuities in the data are due to blood sampling, which interrupted pressure measurement. Note the
persistent increase in MAP during CB inhibition. bpm, Beats per minute.

animal literature (again, when scaled correctly) is generally in
agreement with that of the human literature in that there are
either no differences in the ventilatory responses to hypoxia
and hypercapnia (34, 42) or slightly increased ventilatory
responses to hypoxia in women (23, 42). It is important to
recall that, in the present study, using spayed female dogs,
ovarian hormone levels would be low and cycling absent, so
J Appl Physiol VOL
our dogs would be most analogous to postmenopausal
women. Even if modest non-hormone-mediated sex differ-
ences in peripheral and central chemosensitivities do exist,
we think it unlikely that there would be qualitative effects
on our results. Large differences would, of course, be a
greater concern, but, to our knowledge, there is no evidence
for such differences.
106 MAY 2009 www.jap.org
 EFFECTS OF MAXIMAL CAROTID BODY INHIBITION ON VENTILATION
Carotid Chemoreceptor Contributions to Eupneic Drive
to Breathe in Normoxia
Our estimates of the carotid chemoreceptor contribution to
eupneic drive to breathe are at least about twofold greater than
previously reported. Prior estimates of this contribution include
the following: 1) the 10% reduction in VT and VI achieved
transiently in humans via a few breaths of a hyperoxic inspirate (7,
11, 12); 2) the 28% reduction in VI achieved transiently in
unanesthetized dogs during 100% O2 breathing (6); and 3) the
20% reduction in VI several days after chemoreceptor denerva-
tion in dogs, goats, or ponies (3, 14, 26, 31). Previously, we used
extracorporal perfusion of the isolated CB to inhibit the chemo-
receptor with either hyperoxic or hypocapnic blood (38). With
either of these perfusates, we observed a maximum 29% reduc-
tion in VI after 30 s of perfusion. In the present study, our time
course analysis revealed again a maximum ventilatory depression
30 s following the onset of CB inhibition, but the magnitude
was much greater, i.e., 60% (range 49 80%) below normocap-
nic control conditions. It is important to emphasize that this nadir
of the ventilatory response did not occur for only a single breath,
but was sustained over several breaths and was reproducible upon
repeat trials of CB inhibition in the same animal. We propose that
the greater ventilatory depression obtained in the present study is
due to the combined effects of hyperoxia and hypocapnia, which
likely provided a greater inhibition of carotid sinus nerve activity
than hyperoxia or hypocapnia alone. Furthermore, in contrast to
the reported response to CBD, the response to acute physiological
inhibition of the intact CB in the present study was likely influ-
enced little, if at all, by upregulation of other sources of peripheral
chemoreception or compensatory alterations in the sensitivity of
medullary respiratory control mechanisms (see also Significance/
Limitations of the Unanesthetized, Intact, Carotid Sinus-Perfused
Model above).
Accordingly, we propose that the present data support the
idea that a contribution of the normoxic carotid chemoreceptor
contributes more than one-half of the total drive to eupnea in
normoxia. Given that the nadir of ventilation was not reached
until after 30 s of CB inhibition, it is likely that at least some
limited level of brain acidosis coincided with and, therefore,
limited the reduction of respiratory motor output achievable via
CB inhibition per se (also see DISCUSSION below, regarding
compensation). So, if anything, we may have underestimated
the contribution of the CB chemoreceptors.
Modulatory Effects of Carotid Chemoreceptor
on the Response to Systemic Hypercapnia
After the initial 30 s of marked ventilatory inhibition in re-
sponse to CB inhibition, EMGdi, VT/TI, and VI rose again and
plateaued over the remaining several minutes of CB perfusion at
an average of 24 31% below control values (see Figs. 2 and 3).
The timing of this secondary rise in VI coincides with the venti-
latory response time of 28 30 s for central chemoreceptors
observed following a step increase in inspired CO2 fraction in the
awake dog, whose intact CB was perfused with normoxic, nor-
mocapnic blood (37). The level of CO2 retention presently
achieved in the steady state also approximates that obtained 1 day
to many years in humans after CB resection in patients with CB
tumor (9, 43) or chronic obstructive pulmonary disease (44), in the
chronically CB denervated dog (31), pony (2), goat (26), rabbit
(4), or rat (25).
J Appl Physiol VOL
1571
It is somewhat surprising that these steady-state levels of
systemic CO2 retention and brain acidosis do not yield greater
compensatory ventilatory responses. Studies in intact animals
using ventricular-cisternal perfusion of brain extracellular fluid
(27, 36) or of inhaled CO2 with the isolated CB maintained
normocapnic (37), claim that the great majority of the ventilatory
response to systemic hypercapnia is attributable to the central
chemoreceptors. One explanation for the limited responsiveness
of the central chemoreceptors observed in the present study may
be that central chemoreceptor responsiveness to brain CO2 is
critically dependent on the level of afferent input reaching che-
mosensitive neurons, including that from carotid chemoreceptors,
via pathways through the nucleus of the tractus solitarius. In
support of this postulate, Takakura et al. (40) showed that CB
stimulation via cyanide or hypoxia activated CO2-sensitive neu-
rons in the retrotrapezoid nucleus in anesthetized rats, and that
these responses were prevented via CBD. Furthermore, Hodges
et al. (17) observed that CBD caused a significant depression of
the ventilatory response in awake goats to focal acidosis in the
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raphe nucleus. A more direct test of this proposed dependence of
central chemoreceptor responsiveness on CB chemoreceptor input
might be accomplished in our intact, awake preparation by super-
imposing progressive, systemic hypercapnia on a normal vs.
maximally inhibited CB.
CB Chemoreceptor Inhibition, Systemic Hypercapnia,
and Blood Pressure
Numerous studies have reported that normoxic hypercapnia
elicits an increase in MAP via sympathoexcitation in both healthy
humans (30, 39) and intact dogs (21, 32), which is likely due to
CO2 stimulating both the CB and central chemoreceptors. Our
findings during inhibition of the tonic input from the CB suggest,
however, that a signal of central origin is the main cause of this
CO2-induced increase in MAP. This agrees with findings of
Oikawa et al. (24), who demonstrated that, in conscious rats at
rest, normoxic hypercapnia induced significant increases in MAP
and renal sympathetic nerve activity, and that this response was
not attenuated by CBD (7 mmHg in both conditions).
Although the hypercapnia-induced sympathoexcitation
should result in a positive cardiac chronotropic response, we
observed no change in heart rate during CB inhibition. We
postulate that a baroreflex-induced vagal stimulation, in re-
sponse to MAP increase, counteracted the sympathoexcitation.
This hypothesis agrees with previous findings in intact rats
(24), which showed a vagally induced bradycardia with nor-
moxic hypercapnia, whereas intravenous administration of at-
ropine resulted in an increase in heart rate.
Conclusions
In conclusion, our findings in an intact and unanesthetized
preparation showed that CB chemoreceptors provide a major
excitatory input to medullary respiratory neurons, thereby contrib-
uting more than one-half of the drive to eupnea in normoxia.
Furthermore, we speculate that the contribution of CB input to
eupneic breathing is not limited to a direct modulation of the
ventilatory controller. This tonic input may also exert a strong
modulatory effect on central CO2/H chemoreceptors and, there-
fore, the ventilatory response to systemic hypercapnia/acidosis.
There would be major implications for our understanding of the
chemical control of breathing if future studies confirm this spec-
106 MAY 2009 www.jap.org
1572 EFFECTS OF MAXIMAL CAROTID BODY INHIBITION ON VENTILATION
ulation. 1) Central and peripheral chemoreceptors do not act
independently of one another. 2) It follows that interaction of
central and peripheral chemosensory inputs to the ventilatory
control system would not be simply additive but would likely be
more complex than currently appreciated. 3) If, as suggested by
Guyenet and colleagues (15a, 40), the chemosensitive neurons of
the RTN also possess integrating properties for a variety of
sensory inputs (e.g., pulmonary stretch receptors, baroreceptors,
central locomotor areas), then there is potential for complex
interactions between chemosensory and other nonchemosensory
reflexes.
GRANTS
This material is based on work supported, in part, by grants to C. A. Smith
and J. A. Dempsey from the National Heart, Lung, and Blood Institute of the
National Institutes of Health. G. M. Blain was supported by a postdoctoral
fellowship from the American Heart Association.
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