J Physiol 601.

19 (2023) pp 4251–4262 4251

TECHNIQUE

The jugular venous-to-arterial PCO2 difference during

rebreathing and end-tidal forcing: Relationship with
cerebral perfusion
Jay M. J. R. Carr1 , Trevor A. Day2 , Philip N. Ainslie1 and Ryan L. Hoiland1,3,4,5
1
Centre for Heart, Lung and Vascular Health, University of British Columbia Okanagan, Kelowna, BC, Canada
2
Department of Biology, Faculty of Science and Technology, Mount Royal University, Calgary, AB, Canada
3
Department of Anesthesiology, Pharmacology and Therapeutics, Vancouver General Hospital, University of British Columbia, Vancouver, BC, Canada
4
Department of Cellular and Physiological Sciences, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada
5
International Collaboration on Repair Discoveries, University of British Columbia, Vancouver, BC, Canada

Handling Editors: Harold Schultz & Frank Powell

The Journal of Physiology

The peer review history is available in the Supporting Information section of this article
(https://doi.org/10.1113/JP284449#support-information-section).

Abstract We examined two assumptions of the modified rebreathing technique for the
assessment of the ventilatory central chemoreflex (CCR) and cerebrovascular CO2 reactivity (CVR),
hypothesizing: (1) that rebreathing abolishes the gradient between the partial pressures of arterial

© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society. DOI: 10.1113/JP284449
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4252 J. M. J. R. Carr and others J Physiol 601.19

and brain tissue CO2 [measured via the surrogate jugular venous PCO2 and arterial PCO2 difference
(Pjv-a CO2 )] and (2) rebreathing eliminates the capacity of CVR to influence the Pjv-a CO2 difference,
and thus affect CCR sensitivity. We also evaluated these variables during two separate dynamic
end-tidal forcing (ETF) protocols (termed: ETF-1 and ETF-2), another method of assessing CCR
sensitivity and CVR. Healthy participants were included in the rebreathing (n = 9), ETF-1 (n = 11)
and ETF-2 (n = 10) protocols and underwent radial artery and internal jugular vein (advanced to
jugular bulb) catheterization to collect blood samples. Transcranial Doppler ultrasound was used to
measure middle cerebral artery blood velocity (MCAv). The Pjv-a CO2 difference was not abolished
during rebreathing (6.2 ± 2.6 mmHg; P < 0.001), ETF-1 (9.3 ± 1.5 mmHg; P < 0.001) or ETF-2
(8.6 ± 1.4 mmHg; P < 0.001). The Pjv-a CO2 difference did not change during the rebreathing
protocol (−0.1 ± 1.2 mmHg; P = 0.83), but was reduced during the ETF-1 (−3.9 ± 1.1 mmHg;
P < 0.001) and ETF-2 (−3.4 ± 1.2 mmHg; P = 0.001) protocols. Overall, increases in MCAv were
associated with reductions in the Pjv-a CO2 difference during ETF (−0.095 ± 0.089 mmHg cm−1 s−1 ;
P = 0.001) but not during rebreathing (−0.028 ± 0.045 mmHg · cm−1 · s−1 ; P = 0.067). These
findings suggest that, although the Pjv-a CO2 is not abolished during any chemoreflex assessment
technique, hyperoxic hypercapnic rebreathing is probably more appropriate to assess CCR sensitivity
independent of cerebrovascular reactivity to CO2 .

(Received 24 January 2023; accepted after revision 11 August 2023; first published online 28 August 2023)
Corresponding author R. L. Hoiland: Department of Anesthesiology, Therapeutics and Pharmacology, University of
British Columbia, Vancouver General Hospital, Room 2451, Jim Pattison Pavilion, 2nd Floor, 899 West 12th Avenue,
Vancouver, BC, V5Z 1M9, Canada. Email: ryanleohoiland@gmail.com

Abstract figure legend To address two premises of breathing tests, we assessed cerebral partial pressure of carbon dioxide
(PCO2 ) gradients indexed via veno-arterial PCO2 differences (Pjv-a CO2 , yellow), during modified rebreathing (left side)
and hypercapnic end-tidal forcing (right side) tests. We show that the Pjv-a CO2 difference is related to middle cerebral
artery blood velocity (MCAv) during end-tidal forcing but not rebreathing. During rebreathing, increases in cerebral
blood velocity have a minimal effect on the PJV-a CO2 difference; both the internal jugular venous PCO2 (Pjv CO2 , blue
dashed lines) and arterial PCO2 (PaCO2 , red lines) rise at commensurate rates. By contrast, during end-tidal forcing, at
each hypercapnic stage, the associated increases in cerebral blood velocity and CO2 washout from the brain reduce the
Pjv-a CO2 difference.

Key points
r Modified rebreathing is a technique used to assess the ventilatory central chemoreflex and is based
on the premise that the rebreathing method eliminates the difference between arterial and brain
tissue PCO2 . Therefore, rebreathing is assumed to isolate the ventilatory response to central chemo-
reflex stimulation from the influence of cerebral blood flow.
r We assessed these assumptions by measuring arterial and jugular venous bulb PCO2 and middle
cerebral artery blood velocity during modified rebreathing and compared these data against data
from another test of the ventilatory central chemoreflex using hypercapnic dynamic end-tidal
forcing.
r The difference between arterial and jugular venous bulb PCO2 remained present during both
rebreathing and end-tidal forcing tests, whereas middle cerebral artery blood velocity was
associated with the PCO2 difference during end-tidal forcing but not rebreathing.
r These findings offer substantiating evidence that clarifies and refines the assumptions of modified
rebreathing tests, enhancing interpretation of future findings.

Introduction central chemoreceptor mediated ventilatory sensitivity

to PCO2 (Casey et al., 1987; MacKay et al., 2016; Read,
Rebreathing techniques (e.g. Read, Duffin) are commonly 1967; Read & Leigh, 1967). During rebreathing, arterial
used in research laboratories and classrooms to assess PCO2 (PaCO2 ) equilibrates with alveolar PCO2 (PACO2 )

© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.

J Physiol 601.19 The venous–arterial CO2 difference during rebreathing
(Laszlo et al., 1971; Read & Leigh, 1967) and rises at a rate
commensurate to whole body metabolic CO2 production.
This increase in PaCO2 elevates brain tissue PCO2 (Pbt CO2 ),
which stimulates the central chemoreceptors and evokes
an increase in ventilation (i.e. central ventilatory chemo-
reflex, CCR) (Ainslie & Duffin, 2009; Guyenet, 2014).
As described by Duffin (1990, 2011), a fundamental
premise of the hyperoxic hypercapnic rebreathing test
is that the initial elevation in the fraction of inspired
CO2 (FICO2 , typically 0.07 initially held in the rebreathing
circuit) and subsequent coupling of the rise in PaCO2 to the
rate of whole body metabolic CO2 production minimizes
the brain tissue-to-arterial PCO2 gradient (Pbt-a CO2 ), such
that its subsequent effects on CCR sensitivity are negligible
(Duffin, 1990, 2011; Read, 1967; Read & Leigh, 1967). This
premise is commonly expressed as, the Pbt-a CO2 gradient
is abolished by rebreathing (Boulet et al., 2016; MacKay
et al., 2016). This assumption may have arisen through
misinterpretation of comments by Read and Leigh (1967),
where they suggest that the PCO2 gradient across the brain
is suddenly reduced following the initial step of PaCO2
upon initiation of rebreathing. Yet, to our knowledge,
no experimental data have been reported in support or
contradiction of this assumption.
An adjunct to this assumption is that rebreathing
precludes any influence of cerebral blood flow (CBF)
on the Pbt-a CO2 gradient, and, therefore, the measured
ventilatory response to increases in PaCO2 . Outside
the context of rebreathing, increases in PaCO2 mediate
elevations in CBF, which offsets the minimization of the
Pbt-a CO2 gradient that would otherwise occur (Neubauer
& Santiago, 1985), therefore offsetting the elevation
of Pbt CO2 , via CO2 washout (Carr et al., 2023). The
magnitude of change in CBF for a given CO2 stimulus,
termed cerebrovascular CO2 reactivity (CVR) (Hoiland
et al., 2019), is therefore a contributing determinant of
the PCO2 stimulus for central ventilatory chemoreceptors
(Ainslie & Duffin, 2009; Carr, Caldwell, Ainslie, 2021),
as well as the subsequent ventilatory response (Xie et al.,
2006). During rebreathing, however, increased CBF via
CVR mechanisms could not further reduce the Pbt-a CO2
gradient if it were already abolished. Indeed, marked
(∼30%) pharmacological reductions in CVR do not
0 Jay Carr is a Postdoctoral Fellow at the University of British Columbia, Okanagan with Professor Phil Ainslie. His research
and academic interests include cerebral vascular function, integrative physiology particularly in the control of ventilation with
cerebrovascular reactivity and respiratory physiology, adaptation to resistance exercise, as well as philosophy and the history of
science. He spends most of his time climbing and exploring the Okanagan mountain, as well as biking and running.
© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.
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4253
influence CCR assessed with rebreathing (Fan et al.,
2010), whereas this influence is present using steady-state
methods (Xie et al., 2006), presumably as a result of
the persistent and changing gradient across the stages of
the CVR test. Nonetheless, no experimental data have
reported Pbt-a CO2 gradients during rebreathing in support
of this assumption.
Thus, the present study aimed to quantify the
arterial-to-jugular venous PCO2 (Pjv CO2 ) difference
(Pjv-a CO2 ) (a proxy for Pbt-a CO2 gradient) (Bradley et al.,
1965) and its association with middle cerebral artery
blood velocity (MCAv; a proxy for CBF) during hyperoxic
hypercapnic rebreathing. Moreover, as prior work has
demonstrated the presence of a Pjv-a CO2 difference with
other hypercapnic tests that are commonly employed to
assess ventilatory chemoreflex control (e.g. steady-state
methods) (Hoiland et al., 2015; Peebles et al., 2007; Xie
et al., 2006), we also quantified the Pjv-a CO2 difference
and its association with MCAv during a hypercapnic
end-tidal forcing (ETF) test. We hypothesized that during
rebreathing: (1) PaCO2 and Pjv CO2 would not equilibrate,
such that Pjv CO2 would remain higher than PaCO2 , but
that (2) the Pjv-a CO2 difference would not be associated
with MCAv responses to hypercapnia. We further hypo-
thesized that, during ETF (3), the change in the Pjv-a CO2
difference would be greater than during rebreathing; and
(4), increases in MCAv would be related to a decrease in
the Pjv-a CO2 difference.
Methods
Ethical approval
The experiments were approved by the University
of British Columbia Clinical Research Ethics Board
(H11-03287; H16-01028; H18-01764), harmonized with
the Mount Royal University Human Research Ethics
Board (Protocol 103288), and conformed to the Canadian
Government Tri-Council Policy Statement on Research
with Human Participants, which is consistent with the
Declaration of Helsinki, except for registry in a database.
Participants provided written and verbal informed
consent in advance.

4254 J. M. J. R. Carr and others
Study overview
We assessed nine young healthy participants (eight males
and one female; age: 29 ± 8 years; body mass index: 24.9
± 3.8 kg m–2 ) during a modified rebreathing protocol
(hyperoxic and hypercapnic with prior hyperventilation).
We also conducted retrospective analyses of two pre-
viously published data sets that included arterial and
jugular bulb blood sampling during hypercapnia using
ETF (Carr et al., 2023; Hoiland et al., 2022).
Instrumentation and measurements
During rebreathing and ETF, participants rested in the
supine position and were instrumented with internal
jugular vein and radial artery catheters as previously
described (Carr et al., 2023; Hoiland et al., 2022).
Following catheterization, the remainder of participant
instrumentation was completed and participants rested
for 20 min.
Cardiorespiratory measurements. Cardiorespiratory
variables were sampled continuously at 1 kHz via
an analogue-to-digital converter (Powerlab, 16/30;
ADInstruments, Colorado Springs, CO, USA) and
archived for offline analysis, including: heart rate
(HR; ADI bioamp ML132), radial artery and inter-
nal jugular venous pressure (Truwave Transducer;
Edwards Life Sciences, Irvine, CA, USA), CO2 and
O2 concentrations sampled at the mouth and recorded as
a percentage (%) using a calibrated gas analyser (model
ML206; ADInstruments), respiratory flow via calibrated
pneumotachograph (MLT1000L; ADInstruments) and
spirometer (FE141; ADInstruments) connected with
bacteriological filter, and partial pressures of end-tidal
CO2 (PETCO2 ) and O2 (PETO2 ) were the calculated
from raw percentage (%) gas channels in BTPS (i.e.
body temperature and pressure, saturated). All data
were collected and archived using LabChart, version 7
(ADInstruments).
Cerebrovascular measurements. A 2 MHz Transcranial
Doppler ultrasound (TCD; Spencer Technologies, Seattle,
WA, USA) was utilized to measure MCAv (right side),
with the probe secured via headpiece (model M600
bilateral head frame; Spencer Technologies). Technique
standardization guidelines were observed (Willie et al.,
2011). Mean MCAv was calculated from the raw TCD
signal in LabChart.
Blood sampling and analyses. We collected ∼1.0 mL
of radial arterial and jugular venous blood into
pre-heparinized syringes (SafePICO; Radiometer,
Copenhagen, Denmark) for the immediate analysis
© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.
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J Physiol 601.19
of the partial pressure of arterial oxygen (PaO2 ), PaCO2
and arterial oxygen saturation (SaO2 ) (ABL90 FLEX;
Radiometer).
Rebreathing protocol
Following 2 min at eupnoeic baseline (e.g. PETCO2 ≈
40 mmHg, PETO2 ≈ 100 mmHg), participants were
coached to hyperventilate to ∼18 mmHg PETCO2
for 5 min (Boulet et al., 2016; Casey et al., 1987).
Participants then performed a maximal expiration
during which the inspiratory port was switched to
a 5 L bag containing 7% CO2 and 93% O2 using a
three-way valve. Maximal expiration was followed by
three deep breaths to equilibrate with the rebreathing
circuit. Thereafter, participants were instructed to breathe
normally. Rebreathing ended following attainment of
either 60 mmHg PETCO2 (n = 7) or maximum tolerance
(∼55 mmHg PETCO2 ; n = 2) (Duffin, 1990, 2011).
Our target timepoints for data acquisition and blood
sampling were poikilocapnic baseline, hyperventilation
(PaCO2 ≈ 25 and then 18 mmHg) and during rebreathing
when PETCO2 was 35, 40, 45, 50, 55 and/or 60 mmHg. Blood
pressure and MCAv data were extracted as 30 s means
centred around each PETCO2 timepoint.
End-tidal forcing studies
Data from two previously published ETF protocols were
used (Carr et al., 2023; Hoiland et al., 2022). We controlled
PETO2 and PETCO2 with a portable dynamic ETF system.
Alterations in PaCO2 were calculated individually from
resting eupnoeic breathing end-tidal values. Our ETF
system controls end-tidal gases through wide ranges of
PETCO2 and PETO2 independent of ventilation (Hoiland
et al., 2015; Howe et al., 2020; Tymko et al., 2016).
(1) ETF protocol 1: In 11 participants (11 males; age:
28 ± 7 years; body mass index = 23 ± 2 kg m–2 ),
end-tidal gases were controlled at eupnoeic levels
(e.g. PETCO2 = 40 mmHg and PETO2 = 100 mmHg
for a 2 min baseline period. Thereafter, PETCO2 was
elevated in three sequential stepwise increments to
+3, +6 and +9 mmHg PETCO2 above baseline. Each
stage lasted 5 min following the attainment of steady
end-tidal gases (three breaths within ±1 mmHg of
target PETCO2 ) (Carr, Caldwell, Carter et al., 2021).
(2) ETF protocol 2: In 10 participants (10 males; age:
26 ± 5 years; body mass index = 24 ± 3 kg m–2 ),
end-tidal gases were controlled at eupnoeic levels (e.g.
PETCO2 = 40 mmHg and PETO2 =100 mmHg for a 2 min
baseline period. Two sequential stepwise increments
in PETCO2 of +4.5 and +9 mmHg were performed.
Each stage of elevated PETCO2 lasted 5 min following
the attainment of steady-state.
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J Physiol 601.19 The venous–arterial CO2 difference during rebreathing 4255

Table 1. Cardiovascular and blood gas variables during rebreathing

Hyperventilation Rebreathing

Baseline 25 mmHg 18 mmHg 35 mmHg 40 mmHg 45 mmHg 50 mmHg 55 mmHg 60 mmHg

HR (beats min–1 ) 58 ± 8 78 ± 13 84 ± 13 51 ± 22 56 ± 8 56 ± 9 60 ± 10 68 ± 14 53 ± 34
MAP (mmHg) 112 ± 5 108 ± 6 111 ± 8 115 ± 7 117 ± 9 121 ± 9 123 ± 8 129 ± 7 133 ± 8
IJV pressure 15 ± 4 13 ± 3 12 ± 3 14 ± 7 17 ± 5 17 ± 5 18 ± 6 20 ± 6 19 ± 8
(mmHg)
CPP (mmHg) 97 ± 6 95 ± 7 99 ± 8 99 ± 8 100 ± 9 103 ± 10 105 ± 9 109 ± 9 113 ± 8
Arterial pH 7.42 ± 0.02 7.59 ± 0.03 7.60 ± 0.06 7.45 ± 0.02 7.40 ± 0.02 7.37 ± 0.02 7.3 ± 0.02 7.30 ± 0.01 7.28 ± 0.02
Venous pH 7.37 ± 0.02 7.45 ± 0.04 7.47 ± 0.06 7.41 ± 0.02 7.37 ± 0.02 7.33 ± 0.01 7.31 ± 0.01 7.28 ± 0.01 7.26 ± 0.01
SaO2 (%) 97 ± 2 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0.0 100 ± 0 100 ± 0 100 ± 0
Sjv O2 (%) 65 ± 3 42 ± 9 51 ± 20 76 ± 4 79 ± 2 84 ± 1 88 ± 1 91 ± 1 92 ± 1
Blood samples drawn at target partial pressure of arterial carbon dioxide.
Abbreviations: CPP, cerebral perfusion pressure; HR, heart rate; IJV, internal jugular vein; MAP, mean arterial pressure; SaO2 , arterial
saturation of oxygen; Sjv O2 , jugular venous saturation of oxygen. Data are mean ± standard deviation (SD) (n = 9).

Table 2. Cardiovascular and blood gas variables during end-tidal forcing

ETF-1 (n = 11) ETF-2 (n = 10)

Baseline +3 +6 +9 Baseline +4.5 +9

HR (beats min–1 ) 63 ± 10 68 ± 11 72 ± 11 78 ± 13 67 ± 10 76 ± 13 75 ± 16
MAP (mmHg) 98 ± 5 100 ± 5 103 ± 6 108 ± 8 86 ± 7 90 ± 8 97 ± 9
IJV pressure (mmHg) 7 ± 2 7 ± 2 7 ± 2 8 ± 2 8 ± 2 9 ± 3 12 ± 4
CPP (mmHg) 91 ± 5 93 ± 5 96 ± 6 100 ± 8 78 ± 6 81 ± 8 85 ± 10
Arterial pH 7.40 ± 0.02 7.37 ± 0.02 7.36 ± 0.01 7.34 ± 0.01 7.38 ± 0.02 7.35 ± 0.02 7.32 ± 0.02
Venous pH 7.35 ± 0.01 7.34 ± 0.01 7.33 ± 0.01 7.31 ± 0.01 7.34 ± 0.01 7.32 ± 0.01 7.29 ± 0.01
SaO2 (%) 98 ± 0 98 ± 0 97 ± 0 97 ± 0 95 ± 2 95 ± 2 95 ± 2
Sjv O2 (%) 67 ± 5 72 ± 5 77 ± 5 81 ± 3 69 ± 3 75 ± 3 81 ± 2
Abbreviations: ETF-1, end-tidal forcing protocol one; ETF-2, end-tidal forcing protocol two; CPP, cerebral perfusion pressure; HR, heart
rate; IJV, internal jugular vein; MAP, mean arterial pressure; SaO2 , arterial saturation of oxygen; Sjv O2 , jugular venous saturation of
oxygen. Data are the mean ± SD.

For both ETF protocols, cerebrovascular,
haemodynamic, cardiorespiratory variables and blood
samples were collected within the last 1−2 min of each
stage following attainment of physiological steady-state
(Carr, Caldwell, Carter et al., 2021).
Statistical analyses
The sample for this study is one of convenience; therefore,
no a priori power calculation was performed. The
Pjv-a CO2 difference at eupnoeic PCO2 and change in
the Pjv-a CO2 difference with hypercapnia were compared
between the rebreathing and ETF tests using one-way
analysis of variance. When significant F-ratios were
detected, post hoc tests were conducted and corrected for
multiple comparisons using a Dunnett’s test. Whether a
significant change in the Pjv-a CO2 gradient occurred
with hypercapnia was assessed with a two-tailed
one-sample t test. Comparison of the influence of MCAv
on Pjv-a CO2 between rebreathing and ETF tests was
assessed using linear mixed effects modelling with a
compound symmetry covariance structure. Condition
(i.e. rebreathing/ETF) and MCAv were included as fixed
effects and participant ID was included as a random
effect. For all statistical testing, an alpha of P < 0.05 was
considered statistically significant.
Results
Ancillary data pertaining to cardiorespiratory metrics are
presented in Table 1 for rebreathing and Table 2 for the
ETF protocols.
End-tidal, arterial and jugular venous PCO2
During rebreathing the difference between PETCO2 and
PaCO2 was minimal (Fig. 1). The end-tidal to arterial PCO2
gradient was 0.1 ± 2.0 mmHg at a PaCO2 of 40 mmHg,

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4256 J. M. J. R. Carr and others J Physiol 601.19

−0.3 ± 1.9 mmHg at 45 mmHg PaCO2 , 0.4 ± 2.1 mmHg
at 50 mmHg PaCO2 , 0.4 ± 2.1 mmHg at 50 mmHg PaCO2 ,
0.4 ± 2.1 mmHg at 55 mmHg and −0.3 ± 1.4 mmHg at
60 mmHg PaCO2 . The end-tidal to arterial PCO2 gradients
were comparably minimal with end-tidal forcing. For the
ETF-1 protocol, the end-tidal to arterial PCO2 gradient was
0.7 ± 0.7 mmHg at 40 mmHg PaCO2 , 1.2 ± 0.9 mmHg
at 43 mmHg PaCO2 , 1.0 ± 1.1 mmHg at 46 mmHg
PaCO2 and 1.3 ± 0.7 mmHg at 49 mmHg PaCO2 . For the
ETF-2 protocol, the end-tidal to arterial PCO2 gradient was
−1.2 ± 1.5 mmHg at 40 mmHg PaCO2 , −1.1 ± 0.9 mmHg
at 44.5 mmHg PaCO2 and −0.9 ± 1.1 mmHg at 49 mmHg
PaCO2 .
After participants began breathing on the rebreathing
circuit, PaCO2 increased by 23.2 ± 2.3 mmHg from
36.2 ± 2.6 to 59.4 ± 2.9 mmHg (P < 0.001 for all
stages compared to the first stage) (Fig. 1A). At all stages
of the rebreathing protocol, Pjv CO2 was higher than
PaCO2 (P < 0.001 for all stages). In the first end-tidal
forcing protocol, PaCO2 increased by 8.1 ± 0.7 mmHg
from 41.9 ± 2.2 to 50.0 ± 2.2 mmHg (P < 0.001 for all
stages compared to the first stage) (Fig. 1B). At all stages,
Pjv CO2 was higher than PaCO2 (P < 0.001 for all stages). In
the second end-tidal forcing protocol, PaCO2 increased by
8.6 ± 1.1 mmHg from 43.2 ± 3.4 to 51.8 ± 2.9 mmHg
(P < 0.001 for all stages compared to the first stage)
(Fig. 1B). At all stages, Pjv CO2 was higher than PaCO2
(P < 0.001 for all stages). The increase in PaCO2 was not
different between the two end-tidal forcing protocols, and
so the data were combined (P = 0.33).
Arterial to jugular venous difference in PCO2
Individual data for the Pjv-a CO2 difference during
rebreathing and ETF protocols are depicted in Fig. 2A.
We compared Pjv-a CO2 between the rebreathing and ETF
protocols at a discrete time point matched for PaCO2 noted
in Fig. 2B, where PaCO2 was not different between either
ETF protocol 1 (41.9 ± 2.2 mmHg; P = 0.59) or ETF
protocol 2 (43.2 ± 3.4 mmHg; P = 0.19) compared to
the rebreathing protocol (42.3 ± 2.3 mmHg). There was
a significant Pjv-a CO2 difference for each protocol at a
PaCO2 of ∼40 mmHg (i.e. baseline; P < 0.001, for each
protocol) (Fig. 2C). The Pjv-a CO2 difference was greater
at the beginning of the ETF protocol 1 (9.3 ± 1.5 mmHg;
P = 0.002) and protocol 2 (8.6 ± 1.4 mmHg; P = 0.016)
compared to rebreathing (6.2 ± 2.6 mmHg) at a matched
PaCO2 value of ∼40 mmHg (either following switching to
the rebreathing circuit or commencing ETF) (Fig. 2C).
To facilitate the assessment differences in the change
of Pjv-a CO2 across each test, we compared the change
in the Pjv-a CO2 difference between discrete timepoints
where PaCO2 could be matched between tests (i.e. between
∼40–50 mmHg). The range of data assessed is noted
in Fig. 2B, where the change in PaCO2 was not different
between either ETF protocol 1 (+8.2 ± 0.6 mmHg;
P = 0.97) or ETF protocol 2 (+8.6 ± 1.1 mmHg;
P = 0.53) compared to the rebreathing protocol (+9.5
± 1.1 mmHg). For the change in Pjv-a CO2 , there was a
significant PaCO2 × test protocol interaction (P < 0.001).
There was no change in Pjv-a CO2 across this range of PaCO2
during rebreathing (−0.03 ± 0.12 mmHg · mmHg−1 ;
P = 0.52), whereas Pjv-a CO2 was reduced across both the
ETF-1 (−0.47 ± 0.18 mmHg · mmHg−1 ; P < 0.001) and
ETF-2 tests (−0.35 ± 0.17 mmHg · mmHg−1 ; P < 0.001)
(Fig. 2D). On simply assessing the change in Pjv-a CO2
when PaCO2 was raised from ∼40 mmHg to ∼50 mmHg
(i.e. no intermediate data points), there was similarly
a significant change in the Pjv-a CO2 difference during
the ETF protocols (P < 0.001, for both), but not the

Figure 1. PCO2 responses with rebreathing and end-tidal forcing

The changes in end-tidal PCO2 (PETCO2 ; dark red), arterial PCO2 (PaCO2 ; blue) and jugular venous PCO2 (Pjv CO2 ; yellow)
are depicted for the rebreathing protocol (A) (n = 9) and two end-tidal forcing protocols (B), with end-tidal forcing
protocol 1 on the left (n = 11) and end-tidal forcing protocol 2 on the right (n = 10). A dagger symbol (†) indicates a
difference between Pjv CO2 and PaCO2 (yellow indicating Pjv CO2 is higher), whereas an asterisk symbol (∗ ) indicates a
significant increase in PaCO2 from the 40 mmHg (rebreathe) or baseline (BL; end-tidal forcing) stages (blue indicating
PaCO2 ). Grey boxes indicate the time at which the participant was on either the rebreathing circuit or end-tidal
forcing breathing apparatus. Data are presented as the mean ± SD. Comparisons were made with a one-way
analysis of variance, with a Dunnett’s test used to correct for multiple comparisons for post hoc tests. [Colour
figure can be viewed at wileyonlinelibrary.com]
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 14697793, 2023, 19, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP284449 by Universidad Nacional Autonoma De Mexico, Wiley Online Library on [19/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
J Physiol 601.19 The venous–arterial CO2 difference during rebreathing 4257

rebreathing protocol (P = 0.83) (Fig. 2E). Therefore, there
was a greater reduction in Pjv-a CO2 in ETF protocol 1
(−3.86 ± 1.1 mmHg; P < 0.001) and ETF protocol 2
(−3.42 ± 1.2 mmHg; P < 0.001) compared to rebreathing
(−0.1 ± 1.2 mmHg) (Fig. 2E).
Cerebral blood velocity and Pjv-a CO2
MCAv during each protocol is presented in Fig. 3A. The
increase in MCAv during rebreathing was not associated
with a reduction in the Pjv-a CO2 difference (P = 0.069).
To compare the relationship between MCAv and the
Pjv-a CO2 difference with rebreathing and ETF, the data
from the two ETF protocols were combined. There was
a significant main effect of MCAv (P < 0.001) and
protocol (rebreathing vs. ETF; P = 0.004) on the Pjv-a CO2
difference. There was also a significant MCAv × test
protocol interaction (P = 0.001), whereby there was a
significant reduction in Pjv-a CO2 per cm s−1 increase in
MCAv in the ETF protocols (−0.095 ± 0.089 mmHg ·
cm s−1 ; P = 0.001) but not in the rebreathing protocol
(−0.028 ± 0.045 mmHg · cm · s−1 ; P = 0.067)
(Fig. 3B). This result was not altered when the analysis
of the rebreathing data was restricted to the changes in
MCAv and Pjv-a CO2 that occurred in the same range of
PaCO2 employed in the ETF studies (i.e. 40−50mmHg)
(−0.01 ± 0.08 mmHg · cm · s−1 ; P = 0.82).
Discussion
We assessed the Pjv-a CO2 difference and its association
with MCAv during hyperoxic hypercapnic rebreathing
and hypercapnic ETF. The main findings are that during
rebreathing: (1) PaCO2 and Pjv CO2 did not equilibrate, with

Figure 2. Jugular venous-to-arterial CO2 differences during rebreathing and end-tidal forcing
Rebreathing data are represented by magenta colour, with the two end-tidal forcing tests (ETF-1 and ETF-2) denoted
by gold and cyan colours, respectively. A, individual Pjv-a CO2 difference data during each test. B, corresponding
mean ± SD. The dashed line rectangle in (B) indicates the discrete points used to derive the data in (C) and (E), with
the entire range of these discrete data points included in (D). The selection of data from a PaCO2 of 40–50 mmHg
was selected because this magnitude change in PaCO2 could be matched between groups (see text). C, Pjv-a CO2
difference at ∼40 mmHg PaCO2 (i.e. eupnoeic PCO2 ) for the ETF and rebreathing protocols. D, individual participant
changes in Pjv-a CO2 from ∼40 mmHg PaCO2 to ∼50 mmHg PaCO2 . For each test, the black line represents the
overall linear mixed model regression. To better visualize the changes in Pjv-a CO2 for each participant, the change
in Pjv-a CO2 difference from 40 to 50 mmHg PaCO2 is depocyed in (E) for the rebreathe and ETF protocols. Summary
data represent the mean ± SD, with individual data overlaid in (C) and (E). An asterisk (∗ ) in (C) and (E) indicates
a significant difference from zero (one-sample t test). Sample sizes are n = 9 for rebreathing, n = 11 for ETF-1
and n = 10 for ETF-2. For (C) and (E), two-tailed one sample t tests were used to determine whether the Pjv-a CO2
difference (or Pjv-a CO2 ) was different from zero. A one-way analysis of variance was used to compare the Pjv-a CO2
difference (or Pjv-a CO2 ) between the rebreathe, ETF-1 and ETF-2 protocols, with a Dunnett’s test used to correct
for multiple comparisons for post hoc testing. In (D), the association between PaCO2 and Pjv-a CO2 was compared
between rebreathing and the ETF tests using linear mixed effects modelling with a compound symmetry covariance
structure. For the individual data, the female participant is depicted as a diamond symbol. [Colour figure can be
viewed at wileyonlinelibrary.com]

© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.
 14697793, 2023, 19, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP284449 by Universidad Nacional Autonoma De Mexico, Wiley Online Library on [19/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
4258 J. M. J. R. Carr and others J Physiol 601.19

the Pjv-a CO2 difference constant throughout the test, and
(2) changes in the Pjv-a CO2 difference were not associated
with MCAv responses. We also show that during ETF:
(1) the change in the Pjv-a CO2 difference was greater than
during rebreathing, that is, the Pjv-a CO2 difference was
reduced across the ETF protocol with increases in PaCO2 ,
and (2) changes in the Pjv-a CO2 difference were associated
with changes in MCAv responses. We place these results
into the context of prior work and provide fundamental
propositions on how the findings of the present study may
inform the use of hyperoxic hypercapnic rebreathing as a
method to test central ventilatory chemoreflex sensitivity.
Keir et al. (2020) suggested that with rebreathing a
Pbt-a CO2 gradient of only 0.7 mmHg should be expected
between the lung and central compartments as a resultof
the time required for circulation of blood. In their semi-
nal study, Read and Leigh (1967) reported theoretical pre-
dictions that rebreathing reduces the Pbt-a CO2 gradient
to near zero. However, they also compared their pre-
dictions against experimental animal data during hyper-
toxic rebreathing (Mithoefer and Kazemi, 1963; Read
& Leigh, 1967) and found that the measured Pjv-a CO2
difference was greater than the predicted Pbt-a CO2
gradient by ∼2–4 mmHg Pbt-a CO2 (Mithoefer & Kazemi,
1963; Read & Leigh, 1967). Mithoefer and Kazemi (1963)
demonstrated that, across 15 min of rebreathing in
dogs, Pjv CO2 (and thus assumed Pbt CO2 ) was always
higher than PaCO2 . Read and Leigh (1967) attributed
the discrepancy between their predictions and Mithoefer
and Kazemi’s (1963) experimental data to either: (1)
the experimental conditions differing from the model
assumptions; (2) inaccurate normative baseline values in
the model; or (3) the assumptions of the model over-
simplifying the reality of the relationship between PaCO2 ,
Pbt CO2 and CBF. Given the maximum PaCO2 achieved
during rebreathing by Mithoefer and Kazemi (1963) was
∼136 mm Hg, which is well above that achievable in
conscious humans, it could be expected that CBF would
have plateaued at maximal levels (Harper & Glass, 1965)
and the Pjv-a CO2 difference would have been reduced to
the greatest possible extent.
For the present study, within a physiological and
tolerable range of PaCO2 , we experimentally demonstrated
that the assumption that rebreathing abolishes the
gradient between PaCO2 and Pbt CO2 (Pjv CO2 ) is not
accurate. Nonetheless, during rebreathing: (1) the gradient
magnitude is consistently maintained (i.e. unchanged)
and (2) the relationship between MCAv and Pbt-a CO2
(Pjv-a CO2 ) is attenuated. The implications of these data for
interpretation of CCR sensitivity are summarized below.
Rebreathing does not fully abolish the Pbt-a CO2
gradient
During rebreathing, the difference between Pjv CO2 (a
proxy for Pbt CO2 ) (Bradley et al., 1965) and PaCO2 is not
abolished (Fig. 2A), probably as a result of continuous
cerebral metabolic CO2 production (Carr, Caldwell,
Ainslie, 2021). Even when PaCO2 reaches ∼60 mmHg,
a typical end-point for rebreathing tests, the Pjv-a CO2
ranged from 3.9 to 9.4 mmHg in our participants (Fig. 2A).
Rebreathing reduces the influence of cerebrovascular
reactivity on the Pbt-a CO2 gradient compared to
end-tidal forcing
Rebreathing attenuates the capacity of CVR to reduce
the Pjv-a CO2 difference (Fig. 3B). The commonly asserted

Figure 3. The association between intracranial blood velocity and jugular venous-to-arterial CO2
differences during rebreathing and end-tidal forcing
A, middle cerebral artery blood velocity (MCAv) response to increased PaCO2 for the rebreathing and ETF protocols.
B, relationship between MCAv and the Pjv-a CO2 difference for the rebreathing and ETF protocols for a PaCO2 of
40–60 mmHg, as indicated by the callout area in (A). The black line represents the overall linear mixed model
regression for the two ETF trials combined, whereas the magenta line represents the overall linear mixed model
regression line for the rebreathing protocol. In (B), the influence of MCAv on Pjv-a CO2 was compared between
rebreathing and the ETF tests using linear mixed effects modelling with a compound symmetry covariance structure.
In (A), sample sizes are n = 9 for rebreathing, n = 11 for ETF-1 and n = 10 for ETF-2 and, in (B), the sample size is
n = 21 for the ETF data, which are combined for analysis. In (B), the female participant is depicted as a diamond
symbol. [Colour figure can be viewed at wileyonlinelibrary.com]

© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.

J Physiol 601.19 The venous–arterial CO2 difference during rebreathing
assumption that rebreathing eliminates the capacity of
CVR to reduce the Pjv-a CO2 appears to be correct
(Fig. 2D) and supports previous work (Fan et al., 2010);
however, this probably represents an oversimplification.
For example, between-individual differences in the rate
of rise of PaCO2 , Pjv CO2 and/or CBF could produce some
variation in the stability of Pjv-a CO2 across the course of a
rebreathing protocol (Fig. 2A).
Methodological considerations
Throughout the course of rebreathing, Pjv-a CO2 is
somewhat variable between individuals (Fig. 2A),
although the mean Pjv-a CO2 is stable and the change
in Pjv-a CO2 from the beginning of rebreathing is
minimal across individuals (Fig. 2D). By contrast,
during ETF, the within-individuals Pjv-a CO2 appears
less variable than with rebreathing, but the mean
Pjv-a CO2 decreases at each stage, probably as a result
of the influence of CVR. This produces a relatively
predictable stage-wise decrease in Pjv-a CO2 because of
the steady-state nature of each PaCO2 stage (Caldwell
et al., 2021). This difference between methods could be
the result of normal between-individual physiological
variability in the rate of rise of the relevant parameters
(PaCO2 , Pjv CO2 and/or CBF), or the result of the dynamic
nature of the PaCO2 ramp during rebreathing increasing the
technical variability involved in simultaneous arterial and
jugular venous blood draws. Furthermore, although
we did not detect a significant influence of MCAv
on the Pjv-a CO2 difference with rebreathing (−0.028
± 0.045 mmHg · cm · s−1 ; P = 0.067) (Fig. 2E), we
acknowledge the potential for a type II error given our
limited sample size (n = 9). Finally, TCD measures of
MCAv represent a surrogate of CBF, which underestimates
changes in CBF that occur with the magnitude of hyper-
capnia employed herein (+10–20 mmHg) (Ainslie &
Hoiland, 2014; Coverdale et al., 2014; Verbree et al.,
2014). The influence of this limitation on the results of
the present study is probably minimal given that the
primary comparisons were made on data collected at
similar PaCO2 values, and thus differential responses
of MCA diameter to changes in PaCO2 other than
that pertaining to individual variability, would not be
expected. Therefore, although the influence of CBF on
the Pjv-a CO2 difference is certainly less with rebreathing
compared to ETF, we cannot definitively conclude that
CVR has no influence on the Pbt-a CO2 gradient during
rebreathing. Nevertheless, compared to ETF, the Pjv-a CO2
difference during rebreathing is relatively consistent
across the rising stages of PaCO2 and considerably less
influenced by CVR (Fig. 3B). Incidentally, we cannot
exclude the possibility of hyperoxia-mediated cerebral
vasoconstriction during rebreathing (Armstead, 1999;
Damato et al., 2020; Kety & Schmidt, 1948). However, such
© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.
14697793, 2023, 19, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP284449 by Universidad Nacional Autonoma De Mexico, Wiley Online Library on [19/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
4259
changes would probably result from hyperoxia-induced
hyperventilation causing hypocapnia, with the reduction
in PaCO2 explaining the majority of the effect on CBF
in studies assessing hyperoxia-associated cerebral vaso-
constriction (Brugniaux et al., 2018). There may be some
influence of reactive oxygen species on the cerebral
vasculature (Faraci, 2006) during hyperoxia (Mattos et al.,
2019, 2020) independent of PaCO2 . If hyperoxia-mediated
constriction of the MCA were to occur, this would lead
to an overestimation of CBF during the later stages
of the rebreathing test. Similarly, hyperoxia-mediated
vasoconstriction would also lead to a lower CBF response
compared to when the test was conducted under normoxic
conditions. This relative attenuation of the possible CBF
increase would result in less CO2 washout and thereby
a greater Pjv-a CO2 difference. If this is the case, then
a smaller Pjv-a CO2 difference may be observed during
normoxic rebreathing; however, a normoxic test would
also trigger peripheral chemoreceptor-driven ventilatory
responses, and, in such a test, the Pjv-a CO2 difference
would be of less significance. We utilized hyperoxia in
one protocol but not the other because the hyperoxic
rebreathe and the normoxic hypercapnic ETF protocols
are the archetypical tests of central chemoreceptor
sensitivity and cerebrovascular reactivity, respectively.
The participants in the present study comprised
eight males and one female. Although it is important to
address the potential for sex to have a role as a biological
factor in influencing the relationship between MCAv
and Pjv-a CO2 , there is no obvious difference between the
female and male participants in our study (the female
participant is denoted by a diamond symbol in Figs 2
and 3). Pertaining specifically to cerebrovascular CO2
reactivity, studies conducted in the late 1990s observed a
higher cerebrovascular CO2 reactivity in premenopausal
women compared to males (Kastrup et al., 1997, 1999;
Matteis et al., 1998). These studies used TCD to measure
MCAv and did not standardize testing to a specific phase
of the menstrual cycle. By contrast, more recent work has
demonstrated that, when females are tested in the early
follicular phase (Peltonen et al., 2015; Skinner et al., 2023;
Willie et al., 2012) or ovulatory phase (Skinner et al.,
2023), their hypercapnic CO2 reactivity does not differ
from males, with additional studies demonstrating that
hypercapnic CO2 reactivity does not change across the
menstrual cycle (Peltonen et al., 2016; Skinner et al., 2023).
Therefore, there is currently no clear evidence as to the
likelihood that biological sex influences the relationship
between CBF (e.g. MCAv) and Pjv-a CO2 .
The cardinal implication for these findings concerns the
measurement of ventilatory responses to increases in PaCO2
in light of the relationship between CBF and the Pbt-a CO2
gradient. However, the rebreathing and ETF samples for
the present study were used out of convenience; the
ETF protocols were iso-oxic, whereas the rebreathing

4260 J. M. J. R. Carr and others
protocol was hyperoxic and, as such, the ventilatory
responses are not directly comparable. Nonetheless, when
conducting a study aiming to assess central chemo-
receptor sensitivity, the research question influences
which method (rebreathing vs. ETF) is most appropriate
to assess central chemoreceptor sensitivity. Indeed, if a
researcher is interested in the interaction between CBF
and measured ventilatory CO2 sensitivity, then ETF may
be ideal; however, if they are interested in assessing
CCR sensitivity independent of potentially confounding
changes in CBF, so that the central CO2 stimulus is better
reflected by assessments of PaCO2 (or PETCO2 ), a rebreathing
test is ideal (Ainslie & Duffin, 2009).
Conclusions
In summary, our data demonstrate that researchers should
be aware that the Pjv-a CO2 difference (and thus Pbt-a CO2
gradient) is not abolished with hyperoxic hypercapnic
rebreathing, as previously assumed. There remains a
physiological precedent for changes in CBF or Pbt CO2
(as a result of changes in metabolic CO2 production) to
influence this gradient across the course of a rebreathing
test. Nonetheless, marked reductions in CVR (e.g. ∼30%
decrease) do not influence CCR sensitivity assessed with
a hyperoxic hypercapnic rebreathing test (Fan et al.,
2010). Regardless, our data demonstrate that the Pjv-a CO2
gradient is not abolished during rebreathing, but remains
relatively consistent across the time-course of the test
(i.e. the increases in both Pjv CO2 and PaCO2 are parallel).
Therefore, a superimposed hyperoxic and incremental
hypercapnic rebreathing test apparently remains the most
appropriate available technique to assess the central
chemoreflex ventilatory response in a manner that is
independent of the influence of cerebral perfusion on the
arterial to jugular venous PCO2 difference.
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Additional information
Data availability statement
The de-identified, numerical data can be made available to any
qualified researcher upon reasonable request.
Competing interests
The authors declare that they have no competing interests.
Author contributions
T.A.D. and P.N.A. conceived the original study design. J.M.J.R.C.
and R.L.H conceived the retrospective hypotheses and data
analyses. J.M.J.R.C. and R.L.H analysed and interpreted the data
and drafted the article. All authors critically reviewed the article.
All authors approved the final version of this article and agree to
be accountable for all aspects of the work. All persons designated
as authors qualify for authorship, and all those who qualify for
authorship are listed.
Funding
This work was funded by Natural Science and Engineering
Research Council of Canada to PNA and TAD. PNA was
supported by a Canada Research Chair.
Acknowledgements
We thank Drs. David MacLeod, Kurt Smith, Michael Tymko,
Nia Lewis, Glen Foster, Joe Donnelly and Chris Willie for their
dedicated and exceptional contributions to data collection for
the rebreathing protocol.
Keywords
cerebral blood flow, chemoreceptor, chemoreflex, end-tidal
forcing, rebreathing, ventilation
Supporting information
Additional supporting information can be found online in the
Supporting Information section at the end of the HTML view of
the article. Supporting information files available:
Statistical Summary Document
Peer Review History

Translational perspective
Modified rebreathing is a widely used assessment of central chemoreflex ventilatory sensitivity. Two assumptions
are that rebreathing: (1) abolishes the difference between arterial and brain tissue PCO2 and (2) isolates central
chemoreflex ventilatory sensitivity from the influence of cerebral blood flow. The data from the present study
demonstrated that the first assumption was not accurate because the difference between arterial and brain tissue
PCO2 remained at ∼6 mmHg throughout rebreathing. However, we also found a reduction in the relationship
between the PCO2 difference and a surrogate of cerebral blood flow during rebreathing compared to during a
steady-state test using computerized control of blood gases. These findings refine the existing assumptions for
modified rebreathing, such that we now know that the difference between arterial and brain tissue PCO2 is not
abolished, but that the method does indeed reduce the influence of blood flow on said difference. These findings
also add further nuance to the premises in that (1) the PCO2 difference is steady across the course of a rebreathing
test when interpreted using a mean value (individually, there is some variation) and (2) although the relationship
between cerebral blood flow (indexed via intracranial blood velocity) and the PCO2 difference was reduced in our
data, we cannot discount the possibility that a relationship does exist. As such, although modified rebreathing
tests remain the most appropriate available method to assess the ventilatory central chemoreflex independent of
the influence of cerebral blood flow, application of this and other methods still requires careful consideration of
the research question at hand.

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