Physiological Reports ISSN 2051-817X

ORIGINAL RESEARCH

The effects of superimposed tilt and lower body negative
pressure on anterior and posterior cerebral circulations
Michael M. Tymko1,2, Caroline A. Rickards3, Rachel J. Skow2,4, Nathan C. Ingram-Cotton2,
Michael K. Howatt2 & Trevor A. Day2
1 Centre for Heart, Lung, and Vascular Health, School of Health and Exercise Science, University of British Columbia, Kelowna, Canada
2 Department of Biology, Faculty of Science and Technology, Mount Royal University, Calgary, Alberta, Canada
3 Institute for Cardiovascular & Metabolic Diseases, University of North Texas Health Science Centre, Fort Worth, Texas
4 Faculty of Physical Education and Recreation, University of Alberta, Edmonton, Alberta, Canada

Keywords
Central hypovolemia, cerebral blood velocity,
head-down tilt, head-up tilt, lower body
negative pressure.
Correspondence
Trevor A. Day, Department of Biology,
Faculty of Science and Technology, Mount
Royal University, 4825 Mt Royal Gate SW,
Calgary, AB, T3E 6K6.
Tel: 403 440 5961
Fax: 403 440 6095
E-mail: tday@mtroyal.ca
Funding Information
Financial support for the initial building of
the apparatus (supplies) was provided by
Mount Royal University, Faculty of Science
and Technology, Laboratory Support Centre,
and most of the funding for data collection
(student salaries) by a Government of Alberta
STEP Grant (MMT and RJS), an NSERC USRA
(MMT), and the MRU Petro-Canada Young
Investigator award (TAD).
Received: 11 August 2016; Accepted: 14
August 2016

Abstract
Steady-state tilt has no effect on cerebrovascular reactivity to increases in the
partial pressure of end-tidal carbon dioxide (PETCO2). However, the anterior
and posterior cerebral circulations may respond differently to a variety of
stimuli that alter central blood volume, including lower body negative pressure (LBNP). Little is known about the superimposed effects of head-up tilt
(HUT; decreased central blood volume and intracranial pressure) and headdown tilt (HDT; increased central blood volume and intracranial pressure),
and LBNP on cerebral blood flow (CBF) responses. We hypothesized that (a)
cerebral blood velocity (CBV; an index of CBF) responses during LBNP would
not change with HUT and HDT, and (b) CBV in the anterior cerebral circulation would decrease to a greater extent compared to posterior CBV during
LBNP when controlling PETCO2. In 13 male participants, we measured CBV
in the anterior (middle cerebral artery, MCAv) and posterior (posterior cerebral artery, PCAv) cerebral circulations using transcranial Doppler ultrasound
during LBNP stress ( 50 mmHg) in three body positions (45°HUT, supine,
45°HDT). PETCO2 was measured continuously and maintained at constant
levels during LBNP through coached breathing. Our main findings were that
(a) steady-state tilt had no effect on CBV responses during LBNP in both the
MCA (P = 0.077) and PCA (P = 0.583), and (b) despite controlling for
PETCO2, both the MCAv and PCAv decreased by the same magnitude during
LBNP in HUT (P = 0.348), supine (P = 0.694), and HDT (P = 0.407). Here,
we demonstrate that there are no differences in anterior and posterior circulations in response to LBNP in different body positions.

doi: 10.14814/phy2.12957
Physiol Rep, 4 (17), 2016, e12957,
doi: 10.14814/phy2.12957

Introduction
Both lower body negative pressure (LBNP) and changes
in body position induce variations in central blood volume (Cooke et al. 2004) and intracranial pressure
(Macias et al. 2015; Rosner and Coley 1986; Schwarz

et al. 2002). Specifically, LBNP and head-up tilt (HUT)
result in a decrease in both central blood volume and
intracranial pressure, while head-down tilt (HDT)
increases both central blood volume and intracranial pressure. Effective physiological compensation to these stressors requires rapid integration of many reflex

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

2016 | Vol. 4 | Iss. 17 | e12957
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M. M. Tymko et al.

Regional Cerebral Blood Velocity During Tilt and LBNP

mechanisms, particularly the cardiovagal and vascular
sympathetic baroreflexes. These reflexes are critical for
rapid regulation of heart rate and vascular resistance in
humans in order to maintain mean arterial pressure
(MAP), and subsequently, cerebral perfusion pressure.
Recent studies have demonstrated that changes in MAP
associated with steady-state tilt and lower body positive
pressure (90°HUT; 90°HDT), are within the autoregulatory capacity of the brain, and thus have no effect on
steady-state CBF (Gelinas et al. 2012; Perry et al. 2013),
nor on cerebrovascular reactivity to increasing and
decreasing partial pressure of arterial CO2 (PaCO2)
(Tymko et al. 2015), likely due to compensatory changes
in intracranial pressure, and thus cerebral perfusion pressure (Macias et al. 2015; Tymko et al. 2015). To date,
however, only one study compared regional CBF regulation during LBNP stress in supine and HUT (Deegan
et al. 2010), and none during HDT positions when central blood volume, intracranial pressure, and MAP are
likely elevated. This might be attributed to the difficulty
in developing an LBNP apparatus that can accommodate
HUT and HDT body positions.
Accumulating evidence supports the notion of differentially regulated blood flow in the anterior and posterior
cerebral circulations. For example, differences in regional
CBF regulation has been demonstrated in response to
changes in PaCO2 (Sato et al. 2012b; Skow et al. 2013; Willie et al. 2012), hyperthermia (Bain et al. 2013), administration of indomethacin (Hoiland et al. 2015), and during
changes in central blood volume such as during tilt (Tymko
et al. 2015), thigh cuff release (Sato et al. 2012a), and
LBNP (Kay and Rickards 2016; Lewis et al. 2015; Ogoh
et al. 2015). However, there is little agreement within the
available literature of regional CBF regulation (i.e., anterior
vs. posterior cerebral circulation comparisons), especially
in terms of orthostatic stress. For example, during reductions in central blood volume (i.e., steady-state tilt, LBNP,
or thigh cuff release), studies have proposed that the anterior cerebral circulation is more responsive compared to
the posterior circulation (Kay and Rickards 2016; Ogoh
et al. 2015; Sato et al. 2012a; Tymko et al. 2015), and viceversa (Lewis et al. 2015), and other studies have suggested
that the anterior and posterior cerebral circulation respond
in the same fashion (Deegan et al. 2010; Lewis et al. 2014).
Collectively, the current evidence on regional differences in
cerebral blood flow suggests that the posterior cerebral circulation is more capable of maintaining a constant supply
of blood flow compared to the anterior cerebral circulation
during central hypovolemia (Kay and Rickards 2016; Ogoh
et al. 2015; Sato et al. 2012a; Tymko et al. 2015). Reasoning for this is unclear, however, from an evolutionary
standpoint, it makes sense that the posterior cerebral circulation be better fit for maintaining blood flow since it

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supplies blood to brain regions responsible for crucial
homoeostatic functions such as the medulla oblongata,
cerebellum, hypothalamus, thalamus, and brainstem (Tatu
et al. 1996).
A physiological response that commonly occurs during
LBNP is involuntary hyperventilation, resulting in a
reduction in PaCO2. Blood flow through the cerebrovasculature is highly sensitive to changes in PaCO2. During
hypercapnia (high PaCO2), blood flow through the brain
vasculature increases due to downstream vasodilatation of
arterioles, while during hypocapnia (low PaCO2), blood
flow through the brain vasculature decreases due to arteriolar vasoconstriction (Kety and Schmidt 1948; Willie
et al. 2014; Wolff et al. 1930). This physiological response
aids in the tight regulation of substrate delivery, metabolic waste wash-out, and acid–base balance, which is
particularly important for breathing stability (Ainslie and
Duffin 2009). However, changes in PaCO2 can be a
potential confound when quantifying the CBF responses
to physiological perturbations such as orthostatic stress,
as baroreflex-associated changes in ventilation can acutely
alter PaCO2, and thus CBF (Brunner et al. 1982; Heymans and Bouckaert 1930). Although some recent work
has controlled PETCO2, a surrogate of PaCO2, while
investigating CBF responses to tilt-table testing and LBNP
(Gelinas et al. 2012; Lewis et al. 2014, 2015), it is surprising that relatively few studies investigating cerebrovascular
responses to LBNP have instituted PaCO2 control (Brown
et al. 2003; Cencetti et al. 1997; Levine et al. 1994; Ogoh
et al. 2015). In this study, we attempted to control this
PaCO2 confounding variable.
The primary purpose of this study was to investigate the
regional cerebrovascular responses to simultaneous changes
in body position and LBNP stress. Although it is well established that body position results in changes in central blood
volume and intracranial pressure (e.g., both decrease with
HUT and increase with HDT) (Cooke et al. 2004; Macias
et al. 2015; Rosner and Coley 1986), less is known about
whether body position has an effect on cerebrovascular regulation during LBNP, particularly in the position of HDT
where central blood volume and intracranial pressure are
increased. Additionally, it is currently unknown whether
alterations in intracranial pressure can affect the cerebral
blood flow response to LBNP-induced central hypovolemia, furthermore, the differences between the anterior
and posterior circulations under these conditions remains
unclear (Deegan et al. 2010; Kay and Rickards 2016; Lewis
et al. 2014, 2015; Ogoh et al. 2015). Using a novel, purpose-built experimental apparatus, we tested the hypotheses that (a) anterior and posterior cerebral blood velocity
responses during lower body negative pressure would not
be altered with head-up tilt and head-down tilt, and (b)
cerebral blood velocity in the anterior cerebral circulation

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

M. M. Tymko et al.

would decrease to a greater extent compared to the posterior cerebral circulation during lower body negative pressure, when controlling for the partial pressure of arterial
carbon dioxide (via end-tidal carbon dioxide).

Regional Cerebral Blood Velocity During Tilt and LBNP

cardiovascular response in each body position, while
allowing all participants to withstand at least 5 min of
LBNP in each body position (particularly in HUT).

Respiratory and cardiovascular measures

Materials and Methods
Ethical approval
All experimental procedures and protocols were reviewed
and approved by the Mount Royal University Human
Research Ethics Board (Ethics ID 2011-91Sa) and conformed to the Declaration of Helsinki and the Canadian
Government Tri-Council Policy Statement on research
ethics (TCPS2). All participants provided written
informed consent prior to participation in this study.

Participants
All experiments were conducted at Mount Royal University (Calgary, AB). Recruited participants (n = 14) were
required to complete a health history questionnaire to
ensure normal pulmonary, cardiovascular, and cerebrovascular health. Participants were male, between the
ages of 18–40 years, had a body mass index <30 kg/m2,
and were normotensive (systolic blood pressure
123.0  3.2, diastolic blood pressure 60.8  3.6). Female
participants were excluded due to difficulty creating an
airtight seal due to the large size of the LBNP kayak skirt
(see below). Participants were also nonsmokers, had no
reported previous history of respiratory, cardiovascular,
cerebrovascular diseases, and were not taking any medications. Participants were asked to refrain from vigorous
physical activity, alcohol consumption, and caffeine for at
least 12 h prior to experimentation.

Instrumentation
Upon arrival on the experimental day, participants placed
a kayak skirt, which was integrated to the lid of the LBNP
chamber, around their waist and positioned themselves
within the LBNP chamber in the upright (i.e., 90°HUT)
position. The kayak skirt was secured to the inside of the
lid of the LBNP chamber (see Fig. 1). The LBNP chamber
was then moved into the supine position, where it was
confirmed that the participant had the kayak skirt positioned at the level of the iliac crest, and a stretchable
waist belt was wrapped around the kayak skirt to ensure
a tight seal of the LBNP chamber. The LBNP vacuum was
turned on briefly to 50 mmHg in order to familiarize
the participant with the LBNP stress, and to ensure that
adequate LBNP could be achieved. The moderate level of
LBNP was chosen (i.e., 50 mmHg) in order to elicit a

All respiratory and cardiovascular measurements were collected at 200 Hz using an analog-to-digital converter
(Powerlab/16SP ML880; ADInstruments; Colorado Springs,
CO) and analyzed offline using commercially available
software (ADI LabChart Pro 7.2; ADInstruments, Colorado
Springs, CO). Participants breathed through a mouthpiece
(with nose clip), where expired CO2 was sampled and
measured in percent (ADI ML206; ADInstruments). The
gas analyzer was calibrated with known gas concentrations
prior to each test. The partial pressure of end-tidal CO2
(PETCO2; mmHg) was calculated using peak analysis and
corrected for body temperature and pressure saturated
(BTPS) using the daily atmospheric pressure.
Participants were instrumented with electrocardiogram
(ECG) electrodes in lead II configuration in conjunction
with a bioamp (ADI ML132; ADInstruments) to derive
instantaneous heart rate from the R-R interval of the
ECG. Beat-by-beat arterial blood pressure, cardiac output
(CO), and stroke volume (SV), were measured using finger photoplethysmography (Finometer Pro, Finapres
Medical Systems, Amsterdam, NL). Prior to baseline data
collection, the Finometer was calibrated using the returnto-flow function, and blood pressure accuracy was confirmed with manual sphygmomanometer. Mean arterial
pressure (MAP) was calculated using the area under the
curve of the arterial pressure envelope tracing.

Intracranial blood velocity
Cerebral blood velocity (CBV) in the right middle cerebral
artery, (MCA) and left posterior cerebral artery, (PCA)
were measured using a 2-MHz pulsed TCD ultrasound
system (PMD150B, Spencer Technologies, Redmond, WA)
using insonation techniques described in Willie et al.
(2011), and identical to previous studies in our laboratory
(Skow et al. 2013; Tymko et al. 2015). The same experienced sonographers (MMT and TAD) insonated and confirmed the MCA and PCA for all participants. Mean CBV
was calculated from the envelope of the velocity tracings
for both MCA and PCA. Cerebrovascular resistance (CVR)
was calculated by dividing MAP by mean CBV.

Integrated tilt-table LBNP chamber
Orthostatic stress was elicited using a custom-built integrated tilt-table LBNP apparatus (designed and built by
the first author, MMT; see Fig. 1). Participants were

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

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M. M. Tymko et al.

Regional Cerebral Blood Velocity During Tilt and LBNP

Figure 1. Schematic of a participant positioned in 45°HDT in the custom-built integrated tilt and LBNP apparatus. The participant was
instrumented with a mouthpiece and nose clip (to sample PETCO2), Finometer (for monitoring beat-by-beat blood pressure), ECG leads (for
monitoring HR), and TCD (for monitoring MCAv and PCAv). The LBNP chamber was secured to a custom-built frame on an axle, and was
connected to an industrial shop-vacuum to generate negative pressure, and a digital manometer was used to monitor pressure within the
chamber. The participant was secured inside the chamber with ankle restraints, and they were sealed within the chamber by means of a kayak
spray skirt. ECG, electrocardiogram; HR, heart rate; LBNP, lower body negative pressure; PCA, posterior cerebral artery.

placed in a rectangular LBNP chamber fastened onto a
custom-built tilt-table that was suspended on a steel
frame by an axle. Ankles were secured using restraints
(Teeter Hang-ups, Tacoma, WA) that were attached to a
bar installed inside the LBNP chamber. This LBNP apparatus has the unique ability to place participants in varying body positions, from 90°HUT to 90°HDT. The LBNP
chamber was sealed at the participant’s iliac crest by use
of a kayak spray skirt secured to the LBNP chamber.
Pressure across the wall of the box was generated using
an industrial 6-horsepower vacuum, and measured using

2016 | Vol. 4 | Iss. 17 | e12957
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a digital manometer (DigiMano 1000, 200–200IN, Netech
Corporation, Farmingdale, NY). The magnitude of negative pressure was manipulated using a 120-volt input/140volt output variable transformer (Powerstat transformer,
The Superior Electric Co, Bristol, CT).

Experimental protocol
To ensure that experimentally induced changes in blood
volume distribution were representative of a true change
from baseline, participants were instructed to lie motionless

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the American Physiological Society and The Physiological Society.

M. M. Tymko et al.

and breathe normally for 20 min before LBNP was applied
in each body position (HDT, supine, HUT) (Goswami
et al. 2008; Levine et al. 1994). The protocol consisted of
an initial 5-min quiet baseline period in the supine position, after which the pressure inside the LBNP chamber
was immediately lowered to 50 mmHg for 10 min or
until (a) the participant voluntarily terminated the test due
to the onset of subjective symptoms (e.g., tunnel vision,
nausea, dizziness, or discomfort) or (b) the participant
reached presyncope, which was identified in real time by
the investigators by rapid onset of bradycardia, and/or a
30% reduction of systolic blood pressure from baseline
values.
To ensure that any changes in CBV were not due to
the effects of changing PaCO2 (Brown et al. 2003; Gelinas
et al. 2012), the investigators coached each participant to
breathe at their resting ventilatory rate and depth in
order to maintain PETCO2 levels at baseline values
during LBNP in each position. Immediately after LBNP
termination, the participant completed a 15-min recovery
period, and then repeated the same protocol (20-min
baseline, LBNP, and recovery period) in the remaining
two randomized tilt positions (45°HDT or 45°HUT).
Experimentation in the supine position was always conducted first.

Data analysis
For each protocol, baseline measurements were averaged
over 2 min immediately prior to the start of the LBNP.
As participants reached presyncope (defined above) at different time points (notably in 45°HUT, n = 8), data
within each body position trial were normalized within
individual by taking a 30 sec average of all outcome variables immediately prior to 20%, 40%, 60%, 80%, and
100% of the total LBNP protocol duration for that participant; this approach has been used previously (Cooke
et al. 2009; Levine et al. 1994; Soller et al. 2008).
To compare differences in respiratory, cardiovascular,
and cerebrovascular responses to the LBNP, two-factor
(Factor A: body position, three levels; Factor B: % of
LBNP protocol, five levels) repeated measures analysis of
variances (RM ANOVAs) were used. To compare data
between the MCA and PCA (absolute and relative data),
a two-factor (Factor A: vessel, two levels; Factor B: % of
LBNP protocol, five levels) RM ANOVA was used. To
compare the differences in protocol time between body
positions, a one-way RM ANOVA was used. Participants
who did not reach presyncope during LBNP were designated a 100% LBNP value of 600 sec (10 min). When
significant F-ratios were detected, post hoc comparisons
were made using Tukey’s post hoc test for pair-wise comparisons. All statistical analyses were performed using

Regional Cerebral Blood Velocity During Tilt and LBNP

SigmaStat V11.5 (Systat, Chicago, IL). All data in figures
are expressed as mean values  SEM. Statistical significance was defined with a critical a < 0.05.

Results
Participants
Fourteen participants were recruited for this study, but
one participant was excluded from data analysis due to
extreme intolerance to LBNP. Thirteen males
(mean  SEM) aged 24.2  1.5 years were subsequently
included in the data analysis (height, 178.5  0.7 cm;
weight, 84.6  2.7 kg; BMI, 26.5  0.8 kg/m2. The MCA
was insonated at mean depth of 52.4  1.4 mm and the
PCA at a mean depth of 62.4  1.5 mm.
During LBNP, some participants reached presyncope
with application of LBNP in the supine position (n = 3)
and in the 45°HUT position (n = 8). In contrast, no participants included in the mean data analysis reached
presyncope in the 45°HDT position. The average protocol
time for all participants in the 45°HUT position was
467.5  39.5 sec, which was shorter than the protocol
time in supine position (565.5  23.1 sec; P = 0.014),
and 45°HDT (600  0 sec; P < 0.001). There was no statistical difference in protocol time between supine and
45°HDT (P = 0.534).

Baseline cardiovascular, respiratory, and
cerebrovascular data
Cardiovascular responses during baseline are presented in
Table 1. At baseline, SV was lower during 45°HUT by
13.9  3.8% and 13.3  4.7% compared to supine
(P = 0.001) and 45°HDT (P = 0.006) positions, respectively. To compensate for these differences in SV, heart rate
(HR) was higher in the 45°HUT position by 11.1  1.6%
and 14.8  2.3% compared to supine (P = 0.016) and
45°HDT (P = 0.001), respectively, and there was subsequently no difference in CO across body positions
(P = 0.294). MAP was higher during 45°HDT by
10.0  3.7% compared to supine (P = 0.003), but there
were no differences in MAP between supine and 45°HUT
(P = 0.370), and 45°HUT and 45°HDT (P = 0.089).
Cerebrovascular and respiratory responses during baseline are presented in Table 2. No differences were found in
MCAv (P = 0.055), PCAv (P = 0.676), or PCAv CVR
(P = 0.118), across all body positions at baseline. However,
MCAv CVR was greater in 45°HUT compared to supine
(P = 0.017), but there was no difference detected between
45°HUT and 45°HDT (P = 0.860) nor between supine and
45°HDT (P = 0.052). No differences were found in baseline PETCO2 between body positions (P = 0.757).

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

2016 | Vol. 4 | Iss. 17 | e12957
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M. M. Tymko et al.

Regional Cerebral Blood Velocity During Tilt and LBNP

Table 1. Cardiovascular data during baseline and LBNP in each body position.
Baseline

20%

40%

HR (bpm)
87.4  3.3*,§,†
97.3  3.5*,§,†
45°HUT
73.3  2.9§,†
Supine
65.0  2.3
74.6  2.7*
80.3  2.2*
45°HDT
62.2  2.5
73.7  2.0*
74.7  2.0*
Body position: P < 0.001; % Protocol: P < 0.001; Interaction: P < 0.001
SV (mL)
45°HUT
96.8  6.1
69.0  3.7*
68.1  2.2*
Supine
108.1  4.5†
76.6  5.3*,†
72.8  3.0*,†
‡
45°HDT
106.5  3.3
70.5  2.5*
73.3  2.6*,§
Body position: P < 0.001; % Protocol: P < 0.001; Interaction: P = 0.001
CO(L/min)
45°HUT
6.9  0.3
5.9  0.3*,§
6.1  0.3*,§
Supine
6.9  0.3
5.6  0.3*
5.8  0.2*
45°HDT
6.6  0.3
5.2  0.2*
5.5  0.3*
Body position: P < 0.001; % Protocol: P < 0.001; Interaction: P = 0.059
MAP (mmHg)
45°HUT
82.2  3.3
81.7  3.2
82.2  2.4
Supine
79.0  3.8
79.2  2.5
80.8  2.6
45°HDT
87.4  2.3‡
82.8  1.8
84.3  1.8
Body position: P = 0.009; % Protocol: P = 0.008; Interaction: P < 0.001.

60%

80%

100%

103.5  3.4*,§,†
80.6  2.5*
74.4  2.1*

107.9  0.3*,§,†
82.9  1.9*,‡
73.0  2.2*

106.8  4.8*,§,†
82.2  3.6*,‡
74.1  2.4*

63.3  4.0*
73.5  3.2*,†
73.4  2.7*,§

62.8  4.0*
71.5  3.8*,†
75.6  3.1*,§

58.2  4.1*
68.9  3.3*,†
75.0  2.2*,§

6.4  0.3*,§
5.9  0.2*
5.4  0.3*

6.6  0.3*,§
5.9  0.3*
5.5  0.3*

5.9  0.3*,§
5.6  0.3*
5.6  0.3*

82.2  2.4
80.8  2.3
85.3  1.9

79.9  2.5
79.5  2.3
84.6  1.8

73.4  4.1*
75.1  4.5
86.8  1.8‡,†

P-values for main effects and interactions are displayed underneath each variable.
HR, heart rate; SV, stroke volume; CO, cardiac output; MAP, mean arterial pressure; HUT, head-up tilt.
*
P < 0.05, versus baseline.
†
P < 0.05, 45° HUT versus supine.
§
P < 0.05, 45°HUT versus 45°HDT.
‡
P < 0.05, Supine versus 45°HDT.

Effects of body position on cardiovascular
variables during LBNP
Table 1 illustrates cardiovascular responses during LBNP
in each body position. In all body positions, HR was higher
throughout the LBNP protocol compared to baseline
(P < 0.05). In the 45°HUT position, HR was greater compared to supine and 45°HDT throughout the entire LBNP
protocol (P < 0.05). In the supine position, HR was greater
compared to 45°HDT position at the 80% (P = 0.004) and
100% (P = 0.021) of the LBNP protocol. As expected, SV
was lower during LBNP in all body positions compared to
baseline (P < 0.05). SV in the supine and 45°HDT position
was greater compared to 45°HUT throughout the entire
LBNP protocol (P < 0.05), with the exception of the 20%
LBNP stage, where no difference existed between 45°HUT
and 45°HDT (P = 0.876). Similar to SV, CO was lower
during LBNP in all body positions compared to baseline
(P < 0.05), but CO was greater in the 45°HUT position
compared to 45°HDT (main effect, P < 0.001). MAP did
not change from baseline nor between body positions at
any level of LBNP (P > 0.05), except at 100% of the LBNP
protocol where MAP was lower compared to baseline in
the 45°HUT position (P < 0.001), which was likely related

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to the large number of participants that reached presyncope
in 45°HUT (see above). Also, at 100% of LBNP, MAP was
higher in the 45°HDT position compared to supine
(P < 0.001) and 45°HUT (P < 0.001).

Effects of body position on cerebrovascular
responses during LBNP
Table 2 and Figure 2 illustrate the cerebrovascular and
respiratory responses during LBNP in each body position
for all participants. MCAv was lower during LBNP compared to baseline in all three body positions (P < 0.001),
however, there was no difference in MCAv during LBNP
between body positions (main effect: P = 0.077). Similarly, during all LBNP stages in the supine position, PCAv
was lower compared to baseline (P < 0.05). PCAv was
lower compared to baseline during 45°HDT and LBNP
(P < 0.05), with the exception of the 20% level of LBNP
where it was the same as baseline (P = 0.095). In the
45°HUT position, PCAv was lower compared to baseline
only during 60% (P = 0.049), 80% (P < 0.001), and
100% (P < 0.001) of maximal LBNP; at 100% of LBNP
in the 45°HUT position, PCAv was lower compared to
45°HDT (P = 0.037). There was no difference in PCAv

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

M. M. Tymko et al.

Regional Cerebral Blood Velocity During Tilt and LBNP

Table 2. Cerebrovascular and respiratory data during baseline and LBNP in each body position.
Baseline

20%

40%

MCAv (cm/sec)
45°HUT
55.2  3.0
51.2  3.1*
50.8  3.0*
Supine
59.8  2.0
54.4  2.0*
55.4  2.6*
45°HDT
59.3  2.3
54.3  2.8*
54.6  3.0*
Body Position: P = 0.077; % Protocol: P < 0.001; Interaction: P = 0.053
MCAv CVR (mmHg/cm/sec)
1.67  0.11†,*
1.79  0.11†,*
45°HUT
1.55  0.10†
Supine
1.36  0.09
1.50  0.09*
1.49  0.08*
45°HDT
1.51  0.08
1.60  0.12*
1.61  0.11*
Body Position: P = 0.040; % Protocol: P < 0.001; Interaction: P = 0.970
PCAv (cm/sec)
45°HUT
31.7  2.9
29.7  2.9
29.8  3.0
Supine
32.9  2.1
29.8  1.9*
30.5  2.0*
45°HDT
32.6  2.4
30.5  2.4
29.9  2.3*
Body Position: P = 0.583; % Protocol: P < 0.001; Interaction: P = 0.046
PCAv CVR (mmHg/cm/sec)
45°HUT
2.99  0.28
3.12  0.33*
3.16  0.32*
Supine
2.59  0.24
2.84  0.24*
2.85  0.25*
45°HDT
2.92  0.25
2.98  0.27*
3.08  0.26*
Body Position: P = 0.183; % Protocol: P < 0.001; Interaction: P < 0.542
PETCO2 (mmHg)
45°HUT
31.5  0.9
30.3  0.7
30.1  0.9
Supine
31.8  1.1
30.0  1.1*
30.2  1.1
45°HDT
31.6  0.9
30.4  1.1
30.1  1.2
Body Position: P = 0.219; % Protocol: P < 0.001; Interaction: P < 0.034

60%

80%

100%

49.9  2.6*
55.8  2.3*
54.3  2.4*

48.5  3.0*
52.6  2.1*
54.1  2.9*

45.2  3.2*
49.9  1.6*
53.6  2.6*

1.70  0.10†,*
1.50  0.09*
1.62  0.10*

1.72  0.10†,*
1.56  0.10*
1.62  0.09*

1.69  0.11†,*
1.52  0.10*
1.65  0.08*

29.4  2.7*
30.6  2.2*
30.0  2.2*

28.5  2.7*
29.2  2.0*
30.0  2.2*

26.3  2.5*
27.9  1.9*
30.0  2.2*,§

3.13  0.30*
2.87  0.25*
3.07  0.25*

3.13  0.29*
2.92  0.24*
3.04  0.23*

3.09  0.31*
2.85  0.24*
3.12  0.24*

29.9  1.0*
30.2  1.0
30.9  1.0

29.2  1.0*
30.3  1.1
30.9  1.0§

28.1  0.9*
29.7  1.2*,†
30.3  1.2§

MCAv, middle cerebral artery velocity; MCAv CVR, middle cerebral artery cerebrovascular resistance; PCAv, posterior cerebral artery velocity;
PCA CVR, posterior cerebral artery cerebrovascular resistance, PETCO2, partial pressure of end-tidal carbon dioxide, HUT, head-up tilt; LBNP,
lower body negative pressure; PCA, posterior cerebral artery.
*
P < 0.05, versus baseline.
†
P < 0.05, 45°HUT versus supine.
§
P < 0.05, 45°HUT versus 45°HDT.
‡
P < 0.05, Supine versus 45°HDT. P-values for main effects and interactions are displayed underneath each variable.

during LBNP between body positions (main effect:
P = 0.583). MCA CVR and PCA CVR were higher during
LBNP compared to baseline in each body position
(P < 0.001). Additionally, MCA CVR was greater in
45°HUT compared to supine (main effect: P = 0.040).

Anterior and posterior cerebral circulation
responses during LBNP
MCAv and PCAv at baseline and during LBNP for each
body position are illustrated in Figure 2. As expected,
MCAv was greater than PCAv during LBNP in each body
position (main effect, P < 0.001 for each body position;
Fig. 2 panels A–C). In 45°HUT, MCAv was lower at
100% of LBNP protocol, compared to 20%, 40%, 60%,
and 80% of LBNP protocol. In addition, there was a main
effect for percent of LBNP protocol across the two vessels
(P < 0.001), and there was an interaction effect for the

cerebral vessel (MCAv and PCAv) and percent of LBNP
(P < 0.001). In contrast, in the 45°HUT, PCAv was lower
at 100% of LBNP protocol compared to 20%
(P = 0.012), 40% (P = 0.012), and 60% (P = 0.030) of
LBNP protocol. In both supine and 45°HDT positions,
no differences were found during LBNP for both MCAv
and PCAv. When normalizing CBV to baseline values
during LBNP (i.e., percent change from baseline), MCAv
and PCAv decreased from baseline throughout LBNP in
each body position (P < 0.001), and there was no difference in relative changes from baseline during LBNP
between the MCA and PCA in 45°HUT (main effect:
P = 0.100), supine (main effect: P = 0.529), or 45°HDT
(main effect: P = 0.407) (Fig. 2, panels D–F). Additionally, the relative decrease in CBV in the MCA and PCA
was greater at 100% LBNP compared to 20%, 40%, 60%,
and 80% LBNP in 45°HUT (P < 0.001), and compared
to 40% and 60% LBNP in supine (P < 0.01).

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

2016 | Vol. 4 | Iss. 17 | e12957
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M. M. Tymko et al.

Regional Cerebral Blood Velocity During Tilt and LBNP

45 HUT

MCAv and PCAv (cm/sec)

A

70
60

*

Supine

B

*

*

50

*

**
*

40
30

60

*

10

*
***

D

MCAv and PCAv % change

*

*

60

*

50

50

40

40

20

*

*

*

*

20

E

*

*

*

–10

–5
**
*

0
*

–10

*

*
*

***
*

–5

–15

–20

–20

–20
BL 20% 40% 60% 80% 100%

G

H

34

34

30

32
*

***
*

28
26

*

*

*

*

*

*

*

BL 20% 40% 60% 80% 100%
I

34
*

*

NSD

32

30

30

28

28
26

26
BL 20% 40% 60% 80% 100%

*

–25

–25
BL 20% 40% 60% 80% 100%

*

*

–10

–15

32

*

5

–15

–25

*

F

0

*

*

BL 20% 40% 60% 80% 100%

5

0

*

10
BL 20% 40% 60% 80% 100%

5

*

30
*

10

BL 20% 40% 60% 80% 100%

PETCO2 (mmHg)

*

*

30
*

20

–5

70

70
*

45 HDT

C

BL 20% 40% 60% 80% 100%

BL 20% 40% 60% 80% 100%

Figure 2. Absolute and relative MCAv and PCAv responses to LBNP in each body position. (▲) represents MCAv data, (Δ) represents PCAv
data, (•) represents PETCO2. Absolute MCAv and PCAv during baseline (BL) and at 20%, 40%, 60%, 80%, and 100% of the LBNP protocol in
45°HUT (Panel A), supine (Panel B), and 45°HDT (Panel C) positions. Relative MCAv and PCAv during BL and at 20%, 40%, 60%, 80%, and
100% of the LBNP protocol in 45°HUT (Panel D), supine (Panel E), and 45°HDT (Panel F) positions. PETCO2 during BL and at 20%, 40%, 60%,
80%, and 100% of the LBNP protocol in 45°HUT (Panel G), supine (Panel H), and 45°HDT (Panel I) positions. Mean data  SEM is represented
at each data point. No statistical comparisons between BL and percent of LBNP protocol are shown in the figure, these comparisons can be
found in Table 2. Brackets between MCAv and PCAv data represents main effect (P < 0.05) between the two vessels. *P < 0.05, between
baseline and percent of the LBNP protocol. **P < 0.05, between 100% of LBNP protocol and 20%, 40%, 60% of LBNP protocol. ***P < 0.05,
between 100% of LBNP protocol and 40%, and 60% of LBNP protocol. NSD, no significant differences detected; HUT, head-up tilt; LBNP,
lower body negative pressure; PCA, posterior cerebral artery.

We attempted to control PETCO2 to baseline levels by
coaching the participants’ ventilation during LBNP (see
Fig. 2, panels G–I). In the 45°HDT position, there was no
difference in PETCO2 throughout LBNP compared with
baseline among all participants (n = 13). In contrast,
PETCO2 was slightly lower compared to baseline in the
supine posture at 20% (P = 0.023) and 100% (P = 0.004)
of the LBNP protocol, and during 60% (P = 0.045), 80%

2016 | Vol. 4 | Iss. 17 | e12957
Page 8

(P < 0.001), and 100% (P < 0.001) of LBNP protocol in
45°HUT. Since we were unsuccessful at controlling
PETCO2 in 45°HUT and supine position, we analyzed
data from a subset of participants where PETCO2 was successfully controlled in 45°HUT (n = 8; P = 0.063) and
supine positions (n = 7; P = 0.101) (see Fig. 3). As previously described, we were successful at controlling PETCO2
in 45°HDT using all participants (n = 13).

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

M. M. Tymko et al.

Regional Cerebral Blood Velocity During Tilt and LBNP

significantly lower compared to baseline at 80%
(P = 0.002) and 100% (P < 0.001) LBNP. Also, MCAv
during 100% LBNP was lower compared to 20%
(P = 0.007), 40% (P = 0.009), and 60% (P = 0.005)
LBNP. In the supine position, MCAv was lower compared
to baseline at 20% (P < 0.001), 80% (P = 0.008), and
100% (P ≤ 0.001) LBNP, and was lower during 100%
LBNP compared to 40% (P = 0.024) and 60%
(P = 0.001) LBNP. In 45°HUT, PCAv was only different

Anterior and posterior cerebral circulation
responses during LBNP with no change in
PETCO2
Figure 3 illustrates the cerebrovascular responses to LBNP
in participants who did not have a significant change in
PETCO2 during the LBNP protocol. The data in Figure 3
for 45°HDT (panels C, F, and I) are the same as in Figure 2 (n = 13). In the 45°HUT position, MCAv was

MCAv and PCAv (cm/sec)

A

45 HUT (n = 8)

70
*

60

Supine (n = 7)

B
**
*
*

70
60

*

*

60
50

40

40

40

30

30
*

20

*

20

20

E

0

0

–5

–5

0
*

*

****
*

–5
–10

–15

–15

–15

–20

–20

–20
BL 20% 40% 60% 80% 100%

H
NSD

*

*

36

*

*

*

*

*

BL 20% 40% 60% 80% 100%

NSD

36

34

34

32

32

32

30

30

30

28

28

28

NSD

26

26
BL 20% 40% 60% 80% 100%

*

I

34

26

*

–25

–25
BL 20% 40% 60% 80% 100%

36

*

5

–10

G

*

BL 20% 40% 60% 80% 100%

–10

–25

*

F

5
**
*

*

10
BL 20% 40% 60% 80% 100%

5
*

*

30

*

10

10
D

MCAv and PCAv % change

70
***
*

50

50

BL 20% 40% 60% 80% 100%

PETCO2 (mmHg)

45 HDT (n = 13)

C

BL 20% 40% 60% 80% 100%

BL 20% 40% 60% 80% 100%

Figure 3. Absolute MCAv and PCAv responses to LBNP in each body position when PETCO2 was appropriately controlled. (▲) represents MCAv
data, (Δ) represents PCAv data, (•) represents PETCO2. Absolute MCAv and PCAv during baseline (BL) and at 20%, 40%, 60%, 80%, and
100% of LBNP protocol in 45°HUT (n = 8; Panel A), supine (n = 7; Panel B), and 45°HDT (n = 13; Panel C). Relative MCAv and PCAv during BL
and at 20%, 40%, 60%, 80%, and 100% of LBNP protocol in 45°HUT (n = 8; Panel D), supine (n = 7; Panel E), and 45°HDT (n = 13;
Panel F). PETCO2 during BL and at 20%, 40%, 60%, 80%, and 100% of LBNP protocol in 45°HUT (n = 8; Panel G), supine (n = 7; Panel H),
and 45° HDT (n = 13; Panel I). Mean data  SEM is represented at each data point. Brackets between MCAv and PCAv data represent main
effect (P < 0.05) between the two vessels. *P < 0.05, between baseline and percent of LBNP protocol. **P < 0.05, between 100% of LBNP
protocol and 20%, 40%, 60% of LBNP protocol. ***P < 0.05, between 100% of LBNP protocol and 40%, and 60% of LBNP protocol.
****P < 0.05, between 100% of LBNP protocol and 60% of LBNP protocol. NSD, no significant differences detected; LBNP, lower body
negative pressure; PCA, posterior cerebral artery.

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

2016 | Vol. 4 | Iss. 17 | e12957
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M. M. Tymko et al.

Regional Cerebral Blood Velocity During Tilt and LBNP

between baseline and 100% LBNP (P = 0.006), while in
the supine position, PCAv was different between baseline
and 20% (P = 0.042), and 100% (P = 0.025) LBNP.
When normalizing CBV to baseline values during LBNP
(i.e., % change from baseline), we found that CBV in
both the MCA and PCA was lower during 80%
(P = 0.013) and 100% (P < 0.001) LBNP compared to
baseline, and lower during 100% LBNP compared to 20%
(P = 0.031), 40% (P = 0.025), and 60% (P = 0.018) in
45°HUT. In the supine position, relative CBV in both the
MCA and PCA was lower during 20% (P = 0.011), 80%
(P = 0.001), and 100% (P < 0.001) LBNP compared to
baseline, and lower during 100% LBNP compared to 60%
(P = 0.037).
When controlling for changes in PETCO2, no differences were found between the relative reductions in
MCAv and PCAv during LBNP in 45°HUT (P = 0.348),
supine (P = 0.694), and 45°HDT (P = 0.407) (Fig. 3,
panels D–F), consistent with the responses in the uncontrolled condition (Fig. 2, panels D–F).

Discussion
The primary purpose of this study was to investigate the
regional cerebrovascular responses to simultaneous body
position and LBNP stress. We previously demonstrated
that steady-state tilt (HUT and HDT) has no effect on
cerebrovascular reactivity to PaCO2 (Tymko et al. 2015).
In addition, recent studies explored regional differences
between the anterior and posterior circulations during
LBNP (Deegan et al. 2010; Kay and Rickards 2016; Lewis
et al. 2014, 2015; Ogoh et al. 2015), but there are no
studies that have investigated regional CBF responses during LBNP and steady-state tilt, both of which alter central
blood volume and intracranial pressure. Our main findings were that (a) steady-state tilt (i.e., 45°HUT, supine,
and 45°HDT) had no effect on the CBV responses during
LBNP in both the MCA and PCA, and (b) despite controlling for PETCO2, both the MCA and PCA CBV
decreased during LBNP in each body position, and the
observed decrease in CBV for the MCA and PCA was the
same between 45°HUT, supine, and 45°HDT.

The physiological effects of steady-state tilt
A change in body position has a profound effect on cardiovascular physiology. When moving from supine into
the upright position (e.g., HUT), gravity draws blood
down into the lower extremities causing a decrease in
central blood volume and intracranial pressure; the opposite occurs for the HDT position (Bundgaard-Nielsen
et al. 2009; Macias et al. 2015; Murrell et al. 2011; Rosner
and Coley 1986; Schwarz et al. 2002). In order to

2016 | Vol. 4 | Iss. 17 | e12957
Page 10

compensate for changes in body position, baroreceptors
(e.g., aortic arch and carotid sinus) detect changes in
MAP, and alter HR and vascular resistance in order to
maintain CO, MAP, and thus, cerebral perfusion pressure
(Cooke et al. 2004; Wehrwein and Joyner 2013).
Between the three body positions tested in this study
(45°HUT, supine, and 45°DT), CO was similar, but the
components that contribute to CO (HR and SV) were
different between body positions. In the 45°HUT position, where a large opposing hydrostatic pressure gradient
was present due to the effects of gravity, venous return
was reduced resulting in a decrease in SV. In order to
maintain CO and MAP, HR consequently increased (refer
to Table 1). At baseline, there were no differences in
MAP between supine and 45°HUT, but as expected, MAP
increased with 45°HDT compared to supine, likely as a
result of increased thoracic blood volume (Tymko et al.
2015). Interestingly, the HDT-associated increases in thoracic blood volume was not reflected in changes in SV or
CO at baseline, meaning that the observed increase in
MAP was likely due to increases in peripheral resistance.
We attempted to control PETCO2 by coaching participants’ breathing pattern throughout LBNP to baseline
levels, as changes in PETCO2 (reflective of changes in
PaCO2) can alter the resistance of downstream cerebral
arterioles, drastically changing CBV (Kety and Schmidt
1948; Wolff et al. 1930). Fortunately, PETCO2 did not
change between body positions at baseline, so any body
position related changes in MAP appear to be countered
with changes in intracranial pressure (Tymko et al. 2015).

Cerebrovascular responses to steady-state
tilt and LBNP
There is limited literature on the effects of body position
on CBF responses during LBNP (Deegan et al. 2010). In
a previous study, no differences were reported between
CBV in the MCA and volumetric blood flow in the vertebral artery, which is located proximal to the PCA, during
combined HUT and LBNP, but no responses were measured during HDT (Deegan et al. 2010). Another study
compared regional differences in CBF by measuring volumetric flow within the internal carotid and vertebral
arteries during thigh cuff release in supine and HUT positions (Sato et al. 2012a). Interestingly, these investigators
report that body position (supine vs. HUT) elicited a differential CBF response, where internal carotid artery
blood flow was reduced during HUT compared to supine.
In contrast, when measuring CBV responses during
steady-state tilt and LBNP, we found that body position
had no effect on MCAv and PCAv during LBNP in HUT,
or during the relatively unexplored body position of
HDT. This discrepancy with previous work could be due

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the American Physiological Society and The Physiological Society.

M. M. Tymko et al.

to several reasons. First, we measured regional cerebral
blood velocity using TCD rather than volumetric flow,
opposite to the previous report that used thigh cuff
release (Sato et al. 2012a). As such, it is possible that
there are differences in cerebral blood flow regulatory
mechanisms between intra- and extracranial arteries, and
between the anterior and posterior intracranial circulations. The validity of using TCD for assessing flow
responses has been debated, as CBV is only indicative of
CBF if the diameter remains unchanged within the insonated conduit cerebral arteries (i.e., MCA and PCA; see
further discussion below) (Aaslid et al. 1982; Giller 2003).
Second, another potential reason for the observed differences between experimental studies is that the stimulus
for central hypovolemia was different; thigh cuff release
combined with HUT (Sato et al. 2012a) versus LBNP and
HUT (Deegan et al. 2010). Thigh cuff release results in a
rapid, transient drop in central blood volume and MAP
(i.e., within 1 min), while LBNP progressively decreases
central blood volume, which results in little to no change
in MAP until presyncope is reached. This means that
potentially, central hypovolemia in-and-of itself does not
elicit body positional related changes in CBF, and that a
decrease in MAP (i.e., hypotension) is what governs the
observed regional differences in cerebral blood flow (Sato
et al. 2012a). In the 45°HUT position, we observed a
decrease in MAP at 100% LBNP, probably due to the
high volume of participants that reached presyncope.
However, no differences between MCAv and PCAv were
observed at this time period, and this is likely because the
observed hypotension was not to the same extent that is
typically seen with thigh cuff release (i.e., transient drop
in MAP by ~15–20 mmHg). To date, there is only one
study that has measured regional cerebral blood flow
responses to LBNP until presyncope, while controlling for
PaCO2 (Lewis et al. 2015). It was found that at the point
of presyncope (characterized by hypotension), the posterior cerebral circulation (vertebral artery) was better at
maintaining blood flow compared to the anterior cerebral
circulation (internal carotid artery). Our results contrast
these previous findings, however, the aim of our experiment was not to reach presyncope in each body position,
and it is possible that the degree of hypotension reached
in our study during LBNP (particularly in HUT) was not
significant enough to elicit a differential regional cerebrovascular response.

Regional cerebral blood velocity during
steady-state tilt and LBNP
Historically, when investigating the cerebrovascular
response to specific stimuli, the MCA was the vessel of
interest, primarily because it is relatively easy to insonate

Regional Cerebral Blood Velocity During Tilt and LBNP

with TCD, and it supplies a large region of the brain with
blood and nutrients, so it is a sound “representation” of
CBF (Schoning et al. 1994). However, recent evidence
suggests that the anterior and posterior cerebral circulations respond differently to physiological stimuli, notably
to changes in PaCO2 (Sato et al. 2012b; Skow et al. 2013;
Willie et al. 2012) and to LBNP (Kay and Rickards 2016;
Lewis et al. 2015; Ogoh et al. 2015). In response to
LBNP, recent work suggests that blood flow within the
posterior cerebral circulation is better preserved compared
to the anterior cerebral circulation (Kay and Rickards
2016; Ogoh et al. 2015). In contrast, it has also been
found that the posterior cerebral artery is more reactive
to hypotension compared to the anterior cerebral artery
(Lewis et al. 2015).
Previous studies demonstrated that CBF declines with
LBNP, but unfortunately, PaCO2 is rarely controlled during these studies and is often overlooked as a potent
vasoconstrictor stimulus under these conditions (Brown
et al. 2003; Levine et al. 1994; Ogoh et al. 2015; Rickards
2015). The advantage of this study is that we assessed
the cerebrovascular responses to LBNP while simultaneously controlling for PETCO2 (a surrogate for PaCO2; see
Fig. 3). Our data show that CBV in the MCA and PCA
still decrease from baseline during LBNP despite controlling PETCO2, in fact, CBV responds similarly to LBNP
between our overall mean data (Fig. 2) and PETCO2-controlled data (Fig. 3). Although we did not control
PETCO2 statistically in all of our participants (n = 13),
our data suggest that the small decreases in PETCO2
observed during LBNP (~2 mmHg in supine, ~3 mmHg
in HUT) was not great enough to elicit a significant differential response between the two groups (i.e., Fig. 2 vs.
3). In addition, there was no difference in the relative
decrease in CBV between the MCA and PCA, supporting
the conclusion that velocity in the intracranial cerebral
vessels respond in a similar fashion to submaximal
LBNP. Previous literature has shown that the anterior
and posterior cerebral circulations respond similarly during LBNP stress (Deegan et al. 2010; Lewis et al. 2014),
while others have shown that there might be regional
differences in CBF responses to LBNP (Kay and Rickards
2016; Lewis et al. 2015; Ogoh et al. 2015) and thigh cuff
release (Sato et al. 2012a). Interestingly, these studies
used both TCD and duplex Doppler ultrasound measurement techniques, suggesting that the discrepancy may be
due to other differences in methodology, such as the
magnitude of central hypovolemic stress (maximal vs.
submaximal). Nevertheless, our data add to the growing
literature exploring regional CBF responses during central hypovolemia, and add a novel component by highlighting CBV measurements in both HUT and HDT
positions.

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

2016 | Vol. 4 | Iss. 17 | e12957
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M. M. Tymko et al.

Regional Cerebral Blood Velocity During Tilt and LBNP

Methodological considerations
Despite providing valuable mechanistic insight, our study
had several limitations. The stimulus of central hypovolemia induced by LBNP was different between body positions, as illustrated by the differential cardiovascular
responses to LBNP between HDT, supine, and HUT (see
Table 1). However, in each body position, a significant
increase in heart rate and reduction in stroke volume
occurred during LBNP, indicating that central blood volume was decreasing in each of the three body positions, in
fact, one participant was excluded from data analysis for
being extremely LBNP intolerant, and reached presyncope
in HDT. However, central blood volume was not directly
measured, so the difference in LBNP stimulus between the
body positions cannot be accurately quantified. In addition
to central blood volume not being directly measured,
intracranial pressure was also not measured. However, it is
known that intracranial pressure is altered during changes
in body position (Macias et al. 2015; Rosner and Coley
1986; Schwarz et al. 2002). For example, previous reports
suggest that for every 10 degrees of HUT, intracranial pressure decreases by 1 mmHg (Rosner and Coley 1986). Additionally, LBNP may mildly reduce intracranial pressure
(Macias et al. 2015), but this is based on intraocular pressure, a surrogate for intracranial pressure. To date, no studies have explored the effects of LBNP on intracranial
pressure measured directly across the skull.
As previously mentioned, in terms of measuring CBF,
we assessed CBV via TCD as a surrogate for CBF, with
the assumption that the cross-sectional area of the insonated vessel does not change. Despite previous demonstrations that MCA diameter does not change during
alterations in PaCO2 and mild LBNP challenges (Serrador
et al. 2000), there is still debate. More recent studies suggest that hypo- and hypercapnia may in fact elicit changes
in MCA diameter (Ainslie and Hoiland 2014; Coverdale
et al. 2014; Verbree et al. 2014). At present, measures of
extracranial arteries (e.g., internal carotid and vertebral
arteries) can provide the most reliable noninvasive measures of global cerebral volumetric inflow (Hoiland et al.
2015; Willie et al. 2012). However, it would have been
very difficult to perform these types of measurements due
to the positioning of our participants. Participants were
suspended within the tilt-LBNP apparatus, approximately,
a meter above the ground, which would make it difficult
getting reliable measurements, as ultrasound measures of
the internal carotid and vertebral cerebral arteries would
require the sonographer to consistently insonate the vessel
at the same angle, in each body position.
Lastly, although mean PETCO2 changed only very little
from baseline during LBNP (~2 mmHg in supine, and
~3 mmHg in HUT), we were unable to successfully coach

2016 | Vol. 4 | Iss. 17 | e12957
Page 12

PETCO2 levels to remain at baseline values in several of
our participants during LBNP in the 45°HUT and supine
position due to the participants hyperventilating during
LBNP. To investigate the effects of LBNP on regional
CBV independent of changes in PaCO2, we performed
analysis on a subset of participants (45°HUT, n = 8;
supine, n = 7) that had no statistical change in PETCO2
during the LBNP protocol. Although the number of participants included in analysis decreased, we still achieved
adequate statistical power (>0.80) for all CBV comparisons, however, we lacked statistical power for PETCO2 in
45°HUT (0.413), and supine (0.313), due to the smaller
sample size (Fig. 3). Despite this, we demonstrated that
the CBV responses to LBNP were similar regardless of
PaCO2 control. A future approach could be to include
end-tidal gas control using an end-tidal forcing system.

Perspectives and significance
We measured the regional cerebrovascular responses to
50 mmHg of steady-state LBNP in three different body
positions, which were used as modalities to alter central
blood volume and intracranial pressure. We found that (a)
body position had no effect on CBV responses during
LBNP within both the MCA and PCA, and (b) despite controlling for PETCO2, both the MCA and PCA CBV
decreased during LBNP in all body positions, and (c) the
observed decrease in MCAv and PCAv was the same in
45°HUT, supine, and 45°HDT. Our novel tilt-table LBNP
apparatus allowed us to conduct the first study that measured regional CBV during LBNP in both HUT and HDT
positions. Our results demonstrate that body positional
changes in central blood volume and intracranial pressure
were not substantial enough to alter CBV responses to
LBNP, illustrating the regulatory capacity regulatory capacity of the brain. Moreover, in contrast to some previous literature, we found that there are no regional differences in
cerebral blood flow between anterior and posterior cerebral
circulations during central hypovolemia, despite the differences in brain regions (e.g., frontal lobe vs. brainstem) to
which the anterior and posterior cerebral circulations provide oxygen and nutrients.

Acknowledgments
We gratefully acknowledge the time and effort of our
research participants. We thank Tatania Karaman, M.Sc.
(http://www.tatianakaraman.com) for her illustration of
the integrated tilt-table LBNP apparatus in Figure 1.

Conflict of Interest
None declared.

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.

M. M. Tymko et al.

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