Effects of supplemental oxygen on systemic and cerebral hemodynamics in experimental hypovolemia: Protocol for a randomized, double blinded crossover study.

Supplemental oxygen is widely administered in trauma patients, often leading to hyperoxia. However, the clinical evidence for providing supplemental oxygen in all trauma patients is scarce, and hyperoxia has been found to increase mortality in some patient populations. Hypovolemia is a common finding in trauma patients, which affects many hemodynamic parameters, but little is known about how supplemental oxygen affects systemic and cerebral hemodynamics during hypovolemia. We therefore plan to conduct an experimental, randomized, double blinded crossover study to investigate the effect of 100% oxygen compared to room air delivered by a face mask with reservoir on systemic and cerebral hemodynamics during simulated hypovolemia in the lower body negative pressure model in 15 healthy volunteers. We will measure cardiac output, stroke volume, blood pressure, middle cerebral artery velocity and tolerance to hypovolemia continuously in all subjects at two visits to investigate whether oxygen affects the cardiovascular response to simulated hypovolemia. The effect of oxygen on the outcome variables will be analyzed with mixed linear regression. Trial registration: The study is registered in the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT, registration number 2021-003238-35).

Introduction

Supplemental oxygen is frequently administered in acutely and critically ill patients to avoid arterial hypoxemia and tissue hypoxia [1]. For trauma patients, this is stated in the ATLS (Advanced Trauma Life Support) guidelines: “Supplemental oxygen must be administered to all severely injured trauma patients” [2]. Accordingly, supplemental oxygen is often given to trauma patients, frequently resulting in hyperoxia [3]. However, the clinical evidence for providing supplemental oxygen in all trauma patients is scarce [4] and the liberal use has been largely founded on a presumption that supplemental oxygen is harmless. There is an increasing focus on possible deleterious effects of hyperoxia [1], and a recent retrospective cohort study on trauma patients receiving supplemental oxygen found higher mortality rates in patients with a higher SpO2 [5].

In the initial treatment of trauma patients, detection and treatment of hypovolemia is of paramount importance. The overriding goal for the resuscitation of these patients is to ensure adequate oxygen delivery to the vital organs, which is given by the product of cardiac output and arterial oxygen content. Hypovolemia leads to reduced cardiac filling, stroke volume and cardiac output [6]. Under normal circumstances in unanesthetized humans, this is compensated by an increase in systemic vascular resistance and heart rate to maintain a normal or near-normal mean arterial pressure (MAP). Normobaric hyperoxia induces vasoconstriction and reduced blood flow to several organs in normovolemic healthy volunteers, including the brain, heart and skeletal muscle [7,8]. Accordingly, hyperoxia may lead to an increased tolerance to hypovolemia mediated by vasoconstriction and thereby maintained MAP as well as a potential increase in arterial oxygen content. However, hyperoxia may lead to reduced tolerance to hypovolemia due to reduced cerebral blood flow.

There is a lack of studies investigating the effect of supplemental oxygen on systemic hemodynamics during hypovolemia in a controlled, experimental setting. We therefore plan to conduct an experimental, randomized, double blinded crossover study where healthy subjects will inhale 100% oxygen or room air administered on a face mask with reservoir during simulated hypovolemia in the lower body negative pressure (LBNP) model. LBNP is an experimental model of central hypovolemia where blood is redistributed from the upper to the lower body [9]. While the separated effects of hyperoxia and LBNP on healthy volunteers are described previously [7,9], the potential effects of hyperoxia on the hemodynamic response to LBNP need elucidation. The aim of the present study is therefore to investigate the effect of supplemental oxygen on systemic and cerebral hemodynamics during LBNP.

The primary hypothesis of this study is that supplemental oxygen will induce a different response in cardiac output compared to room air during LBNP. Secondary hypotheses are that supplemental oxygen induces different responses in stroke volume, middle cerebral artery velocity (MCAV) or time to decompensation during LBNP.

Materials and methods

Organization and conduct

The study protocol is written according to the Norwegian Clinical Research Infrastructure Network (NorCRIN) guidelines and registered in the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT, registration number 2021-003238-35). The Norwegian Medical Agency (21/15284-9) and the Regional Ethics Committee (REK South East D, ref. 285164) have assessed and approved the protocol. The original protocol is found in the supporting information file “S1 Protocol” and the spirit checklist in “S1 Checklist”.

The sponsor of this trial is Oslo University Hospital, Norway. Experiments will be conducted at The Section of Vascular Investigations, Oslo University Hospital, Oslo, Norway. We will obtain written informed consent from all subjects before the start of the study.

Design

In this single-center, experimental, randomized, double blinded, crossover trial we will study the effects of supplemental oxygen on systemic and cerebral hemodynamics during simulated hypovolemia in 15 healthy subjects. The schedule of enrolment, interventions, and assessments is shown in , and study design is illustrated in . All subjects will participate on two different visits, with at least one day between each visit. On both visits the subjects will be exposed to LBNP and inhale either 100% oxygen or room air, in a block-randomized order. Except from the inhalation gas, the experiments on Visit 1 and 2 are identical.

Prior to the start of the experiment on either visit, the subject will be familiarized to the set-up and rest for 20–30 minutes in the supine position to stabilize hemodynamic parameters before data sampling begins. After a baseline period, a 5 min run-in time for the inhalation gas will follow. The subjects will be exposed to stepwise LBNP starting at 0 mmHg with 10 mmHg increments every 3 minutes until reaching LBNP 80 mmHg or aborting the experiment (see ). As all subjects receive both oxygen and room air, they will act as their own controls due to the crossover design. Both the subjects and the investigators will be blinded to the inhalation gas.

Randomization

At enrolment, subjects will be randomly assigned (block randomization) in a 1:1 ratio to receive oxygen or room air on Visit 1, and the other on Visit 2. To get at balanced design, the subjects will be randomized with permuted blocks of size 4 or 6, using the “blockrand” package [10] in R [11] /Rstudio [12]. The randomization list will be automatically generated by the principal investigator as a.pdf-document and handed to a 3rd party who will prepare hosing for oxygen or room air administration and envelopes for emergency unblinding. Randomization lists will not be available to the investigators collecting data until after end of the study. Each subject will be dispensed blinded study intervention.

Eligibility criteria

Subjects will be recruited according to the inclusion and exclusion criteria given in . In addition, subjects must abstain from caffeine containing products for 6 hours before each visit, nicotine containing products for 12 hours before each visit and strenuous exercise for 3 hours before each visit. Subjects are allowed to have a light meal on the day of the experiment before the experiment begins. Due to potential effects of circadian rhythm on the hemodynamic response to LBNP, we will to the extent possible conduct both visits at a similar time of the day for each subject. However, the evidence supporting the effect of circadian rhythm on the hemodynamic response to LBNP in the literature seems weak [13].

Interventions

Lower body negative pressure

LBNP is a method to simulate central hypovolemia where negative pressure is applied to the body from the waist-down [9] as shown on Fig 3. Thereby, blood is displaced from the central compartment of the upper body to the lower extremities and pelvis. The subject is placed in the supine position in the LBNP chamber which is sealed just above the iliac crest. The model has been used for more than half a century and is considered a safe and useful model for studying hypovolemia in conscious volunteers.

Fig 3

Illustration showing the test subject inside 1) the lower body negative pressure (LBNP) chamber. The chamber is 2) sealed just above the iliac crest and connected to 3) a vacuum pump controlled by 4) a pressure control unit. The applied negative pressure is displayed on 5) a pressure monitor. Measurements such as 6) ECG for heart rate (HR), 7) mean arterial pressure (MAP) and 8) stroke volume (SV) are connected to 9) a data acquisition device and 10) sampled on a laptop continuously. The inhalation gas is administered on 11) a face mask connected to 12) a gas cylinder.

Inhalation gas: Oxygen and room air

At each visit a subject will inhale either 100% oxygen or room air during the entire experiment as shown in Fig 2. The inhalation gas will be administered on a face mask with reservoir from a gas cylinder connected to a flow meter to ensure an output flow of 15 L/min.

Fig 2

Schematic illustration of the study design per visit.

The subject receives either 100% oxygen or room air as inhalation gas on Visit 1, and the other on Visit 2, throughout the entire experiment. Lower body negative pressure (LBNP) is increased stepwise with increments of 10 mmHg every 3 min from 0 mmHg until reaching 80 mmHg or aborting. Inh. gas = inhalation gas, MAP = mean arterial pressure, HR = heart rate.

Administration of normobaric oxygen at 100% is not recommended for >6 h due to formation of reactive oxygen species (ROS) [14] and their possible side-effects, primarily affecting the lungs. During the study, administration of 100% oxygen will in most subjects be limited to approximately 30 min, and never exceed 60 min. In essence, we are not aware of significant medical risks with the short-term use of oxygen in healthy adults. There are no absolute contraindications to normobaric oxygen supplementation [14].

Outcome measures

During each visit we will measure heart rate with a three-lead ECG (Powerlab; ADInstruments, Dunedin, New Zealand). MAP and cardiac stroke volume will be measured with the volume-clamp method on the third finger of the left hand (Nexfin; Edwards Lifesciences corp., CA, USA) and by suprasternal Doppler ultrasound (SD-50 (SD-50; Vingmed Ultrasound, Horten, Norway). Cardiac output is calculated as the product of stroke volume from the Doppler ultrasound and heart rate from the ECG. Middle cerebral artery velocity (MCAV) will be measured using triplex ultrasound (GE E95; General Electric/ Vingmed, Horten, Norway) as a surrogate for cerebral blood flow. Arterial pulse oximetry will be obtained (Masimo Radical 7; Maximo corp., CA, USA) in addition to cerebral oxygen saturation by near infrared spectroscopy (Invos 5100C cerebral/somatic oximeter; Somanetics, Troy, MI, USA). We will use laser Doppler flowmetry to measure acral skin blood flow (PeriFlux 4001 Master; Perimed AB, Järfälla, Sweden), and volumetric capnography to measure respiratory frequency and end-tidal CO2 (Medlab CAP 10; Medlab GmbH, Stutensee, Germany). Tolerance to hypovolemia will be estimated as time from the start of LBNP 0 to hemodynamic decompensation, where decompensation is defined by Stop-criteria. All data will be sampled continuously and stored on the hospital’s secured server. Our primary and secondary objectives with corresponding endpoints are shown in .

Discontinuation of study

LBNP is released after 3 min at LBNP 80 mmHg or sooner by occurrence of any of the stop-criteria in given in . For safety reasons, an envelope containing a paper stating the given inhalation gas will we present at all visits for the purpose of emergency unblinding due to medical considerations.

Sample size

The estimated effect of LBNP on cardiac output with its standard deviation was estimated from the raw data from a previous study [15]. A change of 15% in cardiac output is often used as a threshold when evaluating interventions to increase cardiac output [16]. We assume a mean cardiac output of 4.85 ± 1.08 L/min at baseline, and a change of -0.489 L/min for each LBNP-level (Δ-20 mmHg/level). Error within subjects is assumed independent between LBNP-levels with SD 0.385 L/min. Assuming that a 15% reduction in cardiac output during oxygen inhalation compared to air is significant, this would give an increased reduction (interaction effect) of 0.18 L/min per LBNP level. If assuming a SD of 0.18 L/min for this interaction effect, including 15 subjects would give a 1−β = 0.87 to detect this effect with α = 0.05, based on simulations.

Trial oversight

This study will be monitored by the Clinical Trials Unit (CTU) at Oslo University Hospital to ensure all procedures follow Good clinical practice (GCP) guidelines. Adverse events (AEs) and serious adverse advents (SAEs) will be collected from the start of the experiment on Visit 1 and until the end of Visit 2. All SAEs will be recorded and reported to the sponsor or designee immediately. The investigator will submit any updated SAE data to the sponsor within 24 hours. Fatal or life threatening suspected unexpected adverse reactions (SUSARs) will be reported to The Norwegian Medicines Agency within 7 days, and other SUSARs within 15 days.

Statistical methods

The effect of oxygen on cardiac output will be analyzed in a mixed linear regression model to account for repeated measurements within subjects. The effect of oxygen on MCAV and cardiac stroke volume will be analyzed in a similar fashion. LBNP-tolerance (time to decompensation) will be analyzed in a mixed proportional hazards model. No interim analysis will be performed.

Discussion

There are few experimental studies investigating the effect of supplemental oxygen on systemic hemodynamics during simulated hypovolemia. This is unfortunate since trauma patients often receive supplemental oxygen and may suffer from hypovolemia. To our knowledge, only one study has previously exposed healthy volunteers to LBNP and 100% oxygen while measuring systemic hemodynamics [17]. They found no difference in hemodynamic response to LBNP between 100% oxygen and room air. A limitation to this study was that the authors only applied one level of LBNP, which was also low to moderate (-40 mmHg). In our planned study we will use graded LBNP from 0 to -80 mmHg to induce a greater span of hypovolemia and also estimate cerebral blood flow. We hope that our results can contribute to the understanding of the effect of oxygen on systemic and cerebral hemodynamics during hypovolemia.

When designing this study, we had to weigh the duration of each LBNP-level against the desire to reach a sufficiently high (negative) level of LBNP. By increasing the duration of each LBNP-level, we could potentially increase the number of decompensations at the cost of fewer observations at high LBNP-levels. Based on the decompensation rate in prior work [15,18], we believe that the present LBNP protocol will be able to reveal an effect of oxygen on time to decompensation, i.e. LBNP tolerance. We also believe that the MAP stop-criterion of 25% below baseline values is suitable to detect hemodynamic decompensation, as the change relative to the individual subject’s habitual blood pressure is considered. Also, a MAP reduction to less than 75% of baseline values largely coincides with a substantial reduction in systolic blood pressure using an absolute threshold of e.g. 80 mmHg.

There are a few considerations regarding the validity of the suprasternal Doppler ultrasound which is used to measure our main outcome variable. The velocity profile in the ascending aorta is rectangular and preserved for the first 3 cm distal to the aortic orifice, even if the aortic diameter changes [19]. Consequently, slight changes in sample volume location in either lateral or caudal direction will have minor influence on the obtained velocity. In addition, since the suprasternal ultrasound probe is pointed in a craniocaudal direction, a theoretical caudal displacement of the heart with LBNP should have negligible influence on the angle of insonation and hence the obtained velocity.

Trial status

The study is planned to enroll test subjects from December 2021 to June 2022.

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22 Feb 2022

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Reviewer #1: General concerns with the protocol:

Based upon the text in lines 66-70, I presumed that the primary variable of interest was tolerance to LBNP. However, Table 3 indicates that this variable is a secondary observation, with differences in the change in cardiac output between trials being the primary observation. This is perplexing given the absence of a physiologically-sound rationale for the mechanisms by which hyperoxia would alter cardiac output during LBNP. Cardiac output changes during LBNP are primarily mediated by reductions in venous return, coupled with baroreflex-driven withdrawal of cardiac vagal tone (affecting heart rate) and accompanying increases in cardiac sympathetic activity (affecting heart rate and contractility). Given this, it is not clear how hyperoxia would alter variables influencing cardiac output during LBNP. A mechanistic rationale should be proposed justifying the primary outcome variable.

I struggle with the assumption that the diameter of the aorta for cardiac output calculations does not change throughout LBNP. There are two possible reasons why that diameter may in fact change. 1) It is highly unlikely that the location of the ultrasound beam (ideally the aortic root, which is not verified and thus unknown) would remain stable throughout LBNP. Possible factors that could influence the location of this beam include subtle angle changes of the probe associated with user “variability”, gradual position changes as the participant is pulled into the LBNP chamber, and/or possible changes in the position of the heart during LBNP. 2) Prior work has shown that low levels of LBNP (e.g., 40 mmHg) change the diameter of the ascending aorta (see PMID 7776239). Given that it is unlikely that the ultrasound beam is consistently focused on the aortic root, there is a high likelihood that LBNP itself is reducing the diameter of the assessed area. Both issues are critical given the substantial error that small differences in aortic diameter have on calculations of stroke volume, and thus cardiac output. As an example, a 10% error in the diameter of the aorta (e.g., 18 mm actual diameter rather than a proposed 20 mm fixed diameter) would result in an ~1 l/min error in cardiac output at a heart rate of 60 bpm. These two potential sources of error for the primary variable are very concerning.

Given the author’s prior work showing that approximately 50-75% of participants can tolerate 4.5 min of 80 mmHg LBNP, for the LBNP tolerance question it is unclear why LBNP does not continue until pre-syncope for all participants. If only 25% of the proposed 15 participants (e.g., ~4 participants) achieve pre-syncope at 80 mmHg, it is unlikely that an effect of hyperoxia on LBNP tolerance will be identified.

Specific recommendations:

Introduction:

Please include a hypothesis statement to inform the reader what the authors are proposing will occur during the hyperoxia trial.

Table 1: The MAP and HR “stop-criteria” are of concern for the LBNP tolerance question. In reviewing the authors’ prior work (and associated figures), I didn’t see any evidence of MAP being 75% below pre-LBNP baseline in the individuals who stopped LBNP prior to 80 mmHg. If someone has a blood pressure of 120/80 (mean = 93 mmHg, depending on the formula used), then a blood pressure of 100/55 during LBNP would equal a 75% reduction from pre-LBNP baseline, thus stopping the LBNP trial. In the hundreds of LBNP trials that I’ve conducted, I can’t think of a single occurrence when someone became pre-syncopal at such a high (relatively speaking) blood pressure. Consider using a more accepted blood pressure threshold for pre-syncope such as a sustained systolic blood pressure below 80 mmHg (see PMID: 25071587). I am also perplexed by the HR statement of “to less than 75% baseline values”. Though HR often decreases during LBNP at pre-syncope, HR values less than 75% below baseline almost never occurs (e.g., resting HR of 60 bpm so a HR of 45 bpm would be a stopping criterion).

Line 134-135: Given the impact of meals on gut blood flow, the authors should ensure similar meals are consumed for both trials.

Line 198-200: I believe there is a mistake in this sentence given that LBNP stages will be 10 mmHg every 3 min, as conveyed in figure 2. Also, please provide a citation supporting the statement that cardiac output decreases linearly at a rate of 0.489 L/min for each 20 mmHg LBNP; do we know that this is a linear response (e.g., the same reduction in cardiac output between 0 and 20 mmHg LBNP as between 60 and 80 mmHg LBNP)? Clearly, adding 20 mmHg LBNP from 0 mmHg would be less of a cardiovascular stress relative to adding 20 mmHg LBNP from 60 mmHg LBNP (e.g., from 60 to 80 mmHg).

Line 201: I am confused regarding the statement of a 15% reduction in oxygen in a protocol where 100% oxygen is administered.

Line 219: Wouldn’t LBNP tolerance be assessed between trials via a paired T-test?

Reviewer #2: Manuscript Number: PONE-D-21-37763

Full Title: Effects of supplemental oxygen on systemic and cerebral hemodynamics in experimental hypovolemia: Protocol for a randomized, double blinded crossover study

Thank you for the privilege in reviewing this protocol for the above study. The reviewer works as an academic anaesthetist in a university teaching hospital, with clinical and research interests in an anaesthesia for complex cardiac surgery and liver transplantation. This reviewer has no conflicts of interests.

In summary, this research is unique, commendable, and addresses a clinically important and relevant question. Moreover, this research could provide critical pilot data for the design of larger clinical trial for trauma patients. The safety aspects for the healthy volunteers have been carefully considered and the primary and secondary end points are valid. The study design is thorough. This is an excellent study and the authors are to be congratulated.

Minor points

1. Please comment on the preop fluids allowances and ensure this is built into the protocol and is consistent for all experiments. The authors state that patients may have a lights meal before, but volaemic status is arguably more important to control for, as this could confound the results. Please consider.

2. As a secondary outcome, please consider adding in the measurements of venous blood gases (baseline, mid experiment, end). Translating the changes in biochemical outcomes e.g., base deficit, lactate, hemoglobin, hematocrit, may further strengthen the scientific integrity of this excellent study. A venous blood gas imposes minimal additional risks and there would be little reason to think that these participants would not consent for this small additional step.

3. As an exploratory outcome, please consider whether funding allows for the assessment of markers of endothelial/glycocalyx function (e.g., heparin sulphate, syndecan 1 etc.). This will be an exploratory outcome but may inform the design of a larger clinical trial, especially if a signal is seen between the groups. If the samples are frozen and even analyzed at a later date, this would provide further valuable information about strategies to protect the endothelium during major trauma.

4. Will all experiments be undertaken at the same time of the day? If not, could the hydration status from overnight fasting impact on any of the outcomes, especially if the cross over is such that first experiment for a given participant is conducted in the morning and the second experiment for the same participant in the afternoon. Please comment.

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Reviewer #1: No

Reviewer #2: Yes: Laurence Weinberg

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7 Apr 2022

Dear Editor,

Many thanks for the constructive and positive feedback on our manuscript. Please see the revised version addressing all reviewers’ concerns. Below is a list commenting on all reviewers’ concerns and describing the actions we have taken to improve the manuscript. Changes in the manuscript are highlighted with red color.

Reviewer #1:

Based upon the text in lines 66-70, I presumed that the primary variable of interest was tolerance to LBNP. However, Table 3 indicates that this variable is a secondary observation, with differences in the change in cardiac output between trials being the primary observation. This is perplexing given the absence of a physiologically-sound rationale for the mechanisms by which hyperoxia would alter cardiac output during LBNP. Cardiac output changes during LBNP are primarily mediated by reductions in venous return, coupled with baroreflex-driven withdrawal of cardiac vagal tone (affecting heart rate) and accompanying increases in cardiac sympathetic activity (affecting heart rate and contractility). Given this, it is not clear how hyperoxia would alter variables influencing cardiac output during LBNP. A mechanistic rationale should be proposed justifying the primary outcome variable.

We completely agree with the reviewer regarding the general mechanisms behind changes in cardiac output during LBNP alone. While we also agree that any effect of hyperoxia on the tolerance to hypovolemia is of interest, we believe that the general hemodynamic effects of hyperoxia during LBNP still remain to be sufficiently elucidated, as stated in lines 71-72, before tolerance can be studied more closely. Only one study has investigated the effect of hyperoxia on cardiac output during LBNP and they only applied one level of 40 mmHg of LBNP (https://doi.org/10.1177/147323000803600203). For this reason, effects on changes in cardiac output is reflected in the primary hypothesis of the present study as we believe changes in cardiac output is the better parameter to describe the degree of hypovolemia. We agree with the reviewer that it is not clear how hyperoxia would alter variables influencing cardiac output during LBNP, which is why we will perform the present study. The mechanistic rationale is given by the finding that cardiac output is reduced with hyperoxia during normovolemia in healthy volunteers (lines 64-66). Further, hyperoxia leads to an increase in systemic vascular resistance and accompanying reductions in heart rate and cardiac output, although the mechanisms are not fully understood (https://doi.org/10.1152/ajpregu.00165.2017). We have added a sentence (line 77-79) to clarify the rationale behind the study interventions. Therefore, we think it is of interest to investigate whether hyperoxia changes the cardiovascular response to LBNP with cardiac output as the primary outcome variable.

I struggle with the assumption that the diameter of the aorta for cardiac output calculations does not change throughout LBNP. There are two possible reasons why that diameter may in fact change. 1) It is highly unlikely that the location of the ultrasound beam (ideally the aortic root, which is not verified and thus unknown) would remain stable throughout LBNP. Possible factors that could influence the location of this beam include subtle angle changes of the probe associated with user “variability”, gradual position changes as the participant is pulled into the LBNP chamber, and/or possible changes in the position of the heart during LBNP. 2) Prior work has shown that low levels of LBNP (e.g., 40 mmHg) change the diameter of the ascending aorta (see PMID 7776239). Given that it is unlikely that the ultrasound beam is consistently focused on the aortic root, there is a high likelihood that LBNP itself is reducing the diameter of the assessed area. Both issues are critical given the substantial error that small differences in aortic diameter have on calculations of stroke volume, and thus cardiac output. As an example, a 10% error in the diameter of the aorta (e.g., 18 mm actual diameter rather than a proposed 20 mm fixed diameter) would result in an ~1 l/min error in cardiac output at a heart rate of 60 bpm. These two potential sources of error for the primary variable are very concerning.

We agree with the reviewer and are aware of the possible theoretical limitations of suprasternal Doppler ultrasound to measure cardiac stroke volume. However, for the use of suprasternal Doppler, we refer to PMID 2287179. The velocity profile in the ascending aorta has been shown to be preserved for the first 3 cm distal to the aortic orifice, even if the aortic diameter changes. Further, the velocity profile in the LVOT is rectangular, and not parabolic. Therefore, minor changes in location of the Doppler sample volume both in depth and distance from the aortic wall, will have minor influence on the Doppler measurements. The angle of insonation may change slightly if the heart is displaced laterally, but caudal displacement during LBNP will probably not have a large influence on this angle of insonation as the suprasternal acoustic window gives a very craniocaudal direction of the ultrasound beam. We have addressed this on line 255-262 in the revised manuscript.

In addition to the maintenance of velocity despite aortic diameter changes (as stated above), the reference provided by the reviewer on aortic diameter during LBNP refers to a reduction in pulse area, and not area per se. In this reference, the maximal aortic diameter decreases, but the minimal diameter (at start of systole) increases with LBNP. The diastolic area, which is the area at the beginning of systolic anterior flow thus increases with LBNP, and these effects will therefore to some extent cancel each other out.

Even if changes in aortic diameter and angle would cause a bias in the suprasternal Doppler measurements, we believe one can assume that this effect is comparable between the two visits. As each subject will act as its own control, there is little reason to suspect that these possible sources of error will affect the estimated effect of the intervention between the two visits (within subjects). Also, other non-invasive methods to estimate cardiac stroke volume have their own limitations, with pulse wave analysis (PWA) among others being sensitive to changes in systemic vascular resistance (https://doi.org/10.1016/j.bja.2020.09.049), which does change during LBNP. That being said, we also estimate cardiac stroke volume with PWA in addition to suprasternal Doppler ultrasound in our lab, and plan to present the agreement between these methods in a separate manuscript.

Given the author’s prior work showing that approximately 50-75% of participants can tolerate 4.5 min of 80 mmHg LBNP, for the LBNP tolerance question it is unclear why LBNP does not continue until pre-syncope for all participants. If only 25% of the proposed 15 participants (e.g., ~4 participants) achieve pre-syncope at 80 mmHg, it is unlikely that an effect of hyperoxia on LBNP tolerance will be identified.

In our latest study using LBNP (https://doi.org/10.1007/s00421-021-04693-6) 11 of 16 (69%) subjects tolerated and finished 6 min of 60 mmHg of LBNP, and only 2 of 16 (13%) completed 80 mmHg of LBNP. We therefore believe that the planned LBNP protocol is suitable to detect potential differences between visits due to hyperoxia. When designing the study, we had to balance the time at each LBNP-level with the desire to reach a sufficiently high (negative) level of LBNP. By increasing the time at each LBNP-level, we could potentially increase the number of decompensations at the cost of fewer observations at high LBNP-levels. We have addressed this on line 245-250 in the revised manuscript.

Specific recommendations:

Introduction:

Please include a hypothesis statement to inform the reader what the authors are proposing will occur during the hyperoxia trial.

We have added a hypothesis statement at the end of the Introduction section (line 81-84).

Table 1: The MAP and HR “stop-criteria” are of concern for the LBNP tolerance question. In reviewing the authors’ prior work (and associated figures), I didn’t see any evidence of MAP being 75% below pre-LBNP baseline in the individuals who stopped LBNP prior to 80 mmHg. If someone has a blood pressure of 120/80 (mean = 93 mmHg, depending on the formula used), then a blood pressure of 100/55 during LBNP would equal a 75% reduction from pre-LBNP baseline, thus stopping the LBNP trial. In the hundreds of LBNP trials that I’ve conducted, I can’t think of a single occurrence when someone became pre-syncopal at such a high (relatively speaking) blood pressure. Consider using a more accepted blood pressure threshold for pre-syncope such as a sustained systolic blood pressure below 80 mmHg (see PMID: 25071587). I am also perplexed by the HR statement of “to less than 75% baseline values”. Though HR often decreases during LBNP at pre-syncope, HR values less than 75% below baseline almost never occurs (e.g., resting HR of 60 bpm so a HR of 45 bpm would be a stopping criterion).

As previously stated, this study was primarily designed to investigate the hemodynamic response to ongoing hypovolemia, and not hemodynamics at the point of cardiovascular decompensation. Neither was our previous work designed to do so and therefore the figures do not show MAP values at the point of termination of LBNP. In fact, in our previous work, the LBNP-level where decompensation occurred were removed from the analyses (as explained in the manuscripts), as this represents a physiological condition completely different from that of compensated hypovolemia. While the reduction in blood pressure (120/80 to 100/55) suggested by the reviewer represents a 25% reduction in MAP, it also represents an increase in pulse pressure (from 40 to 45), which does not typically occur with LBNP. For the systolic pressure of 100 mmHg presented, one would therefore typically see a higher diastolic pressure than 55 and thereby a higher MAP.

The range of normal systolic blood pressure (SBP) in men and women from the age 18-50 is quite broad with the 5th percentile being 95 mmHg and the 95th percentile being 160 mmHg (https://doi.org/10.1038/jhh.2013.85). A reduction to <80 mmHg as suggested by the reviewer, may thus represent a reduction of 15 mmHg for some and 80 mmHg for others. Due to the large intraindividual variation in absolute blood pressure, we therefore chose a stop criterion based on a relative reduction, with a 25% reduction in MAP being a substantial reduction.

Lastly, we find it hard to ethically defend a greater reduction in MAP of more than 25% without aborting the trial. This is also why we included the stop-criteria of 25% reduction in HR, as this consistently indicates decompensation. In essence, our experience is that a reduction of SBP to <80 mmHg in most cases coincides with a reduction of MAP of >25%, but based on the above, we believe a reduction of 25% is appropriate. See line 250-254 in the revised manuscript.

Line 134-135: Given the impact of meals on gut blood flow, the authors should ensure similar meals are consumed for both trials.

We agree and believe “light meal” as stated in the manuscript is sufficient to provide similar amount of food intake for both visits.

Line 198-200: I believe there is a mistake in this sentence given that LBNP stages will be 10 mmHg every 3 min, as conveyed in figure 2. Also, please provide a citation supporting the statement that cardiac output decreases linearly at a rate of 0.489 L/min for each 20 mmHg LBNP; do we know that this is a linear response (e.g., the same reduction in cardiac output between 0 and 20 mmHg LBNP as between 60 and 80 mmHg LBNP)? Clearly, adding 20 mmHg LBNP from 0 mmHg would be less of a cardiovascular stress relative to adding 20 mmHg LBNP from 60 mmHg LBNP (e.g., from 60 to 80 mmHg).

As with all statistical models, power and sample size calculations always assume some simplifications. The assumption of the stated decrease in cardiac output is, as stated in the original manuscript, from the raw data for https://doi.org/10.1371/journal.pone.0219154, designated as reference 15. We have clarified this on line 205 in the revised manuscript. The assumption of a linear relationship between LBNP-level and cardiac output seems reasonable given the estimates presented in the figure below (panel bottom right). Although the cardiac stress may be less with a change from 0 to 20 mmHg vs. a change from 60 to 80 mmHg, the relative reduction in cardiac output seems similar, allowing the assumption of a linear relationship. From 0 to 20 mmHg, the reduction in stroke volume seems less than at higher LBNP, but so is the increase in heart rate. After multiplication, this results in a similar change in cardiac output.

Line 201: I am confused regarding the statement of a 15% reduction in oxygen in a protocol where 100% oxygen is administered.

We apologize for the spelling mistake, which has been corrected.

Line 219: Wouldn’t LBNP tolerance be assessed between trials via a paired T-test?

Time to decompensation between groups could be compared in a paired t-test, but we plan to compare these in a mixed proportional hazards model (mixed Cox regression). This has been specified in the revised manuscript on line 229-230. As previously stated by the reviewer, we will not expect all subjects to decompensate, and these observations will thus be censored at the end of the protocol. To apply a paired t-test, these observations would have to be designated as missing or be given a fixed maximal value. A Cox-regression would however handle these observations as censored. In essence, we believe survival analyses (time-to event) such as Cox regression is well suited to describe time to decompensation.

Reviewer #2:

Minor points

1. Please comment on the preop fluids allowances and ensure this is built into the protocol and is consistent for all experiments. The authors state that patients may have a lights meal before, but volaemic status is arguably more important to control for, as this could confound the results. Please consider.

We agree that baseline volaemic status is important and will generally consider healthy volunteers as euvolemic after a light meal. We will also strive to conduct both visits within a subject on the same time of day.

2. As a secondary outcome, please consider adding in the measurements of venous blood gases (baseline, mid experiment, end). Translating the changes in biochemical outcomes e.g., base deficit, lactate, hemoglobin, hematocrit, may further strengthen the scientific integrity of this excellent study. A venous blood gas imposes minimal additional risks and there would be little reason to think that these participants would not consent for this small additional step.

We agree that biochemical analyses, both “traditional” tests such as those from venous blood gas, or more specific analyses for glycocalyx degradation (below) or possibly tests of oxidative stress, would be of interest. In this study, however, our resources are unfortunately limited to focus on the hemodynamic response. We will keep the suggestions in mind for future studies.

3. As an exploratory outcome, please consider whether funding allows for the assessment of markers of endothelial/glycocalyx function (e.g., heparin sulphate, syndecan 1 etc.). This will be an exploratory outcome but may inform the design of a larger clinical trial, especially if a signal is seen between the groups. If the samples are frozen and even analyzed at a later date, this would provide further valuable information about strategies to protect the endothelium during major trauma.

Please see our response above. Depending on the results of the present study, we hope to address these questions in future studies.

4. Will all experiments be undertaken at the same time of the day? If not, could the hydration status from overnight fasting impact on any of the outcomes, especially if the cross over is such that first experiment for a given participant is conducted in the morning and the second experiment for the same participant in the afternoon. Please comment.

As stated above, we will strive to conduct the experiments at the same time of day for each subject. The subjects are told to have “a light meal” before both visits and we will therefore consider them as euvolemic. To account for circadian variations, we will to the extent possible attempt to perform the visits at a similar time of the day for each subject as specified on line 141-144. However, the evidence supporting the effect of circadian rhythm on the hemodynamic response to LBNP in the literature seems weak. To our knowledge, only one study (https://doi.org/10.1152/jappl.1994.76.6.2602) have investigated how time of day affect the response to LBNP. Gillen et al state that preliminary experiments showed a smaller decrease in stroke volume when LBNP was applied in the morning compared with the afternoon, but they do not present data supporting the statement. Also, they show in a subgroup of three subjects that LBNP 40 mmHg in the morning induces a similar decrease in stroke volume as LBNP 30 mmHg in the afternoon (morning ΔSV = 52±3 mL, afternoon ΔSV = 60±6 mL).

Submitted filename: Response to Reviewers.docx

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14 Jun 2022

Effects of supplemental oxygen on systemic and cerebral hemodynamics in experimental hypovolemia: Protocol for a randomized, double blinded crossover study

PONE-D-21-37763R1

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Despite the authors' replies, several methodological concerns remain that I believe will adversely affect both the publishability and associated conclusions of this work. That said, I recognize that this is not the ideal forum to debate those concerns.

Reviewer #2: I am satisfied that the detailed responses to the Reviewers have been adequately addressed. The responses to both reviewers have been suitably responded to.

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16 Jun 2022

PONE-D-21-37763R1

Effects of supplemental oxygen on systemic and cerebral hemodynamics in experimental hypovolemia: Protocol for a randomized, double blinded crossover study

Dear Dr. Lindvåg Lie:

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Table 1

Stop-criteria.

Stop-criteria

Symptoms or signs of impending circulatory collapse

• Symptoms of pre-syncope

1. Light-headedness

2. Nausea

3. Sweating

• Occurrence of hemodynamic thresholds preceding circulatory collapse (determined from measurements at baseline)

1. MAP-reduction to less than 75% of baseline values (measured at normovolemia) for >3 s

2. HR-reduction to less than 75% baseline values (measured at normovolemia) for >3 s

Subject request for reasons other than above

MAP = mean arterial pressure, HR = heart rate.

Table 2

Inclusion and exclusion criteria.

Inclusion criteria

Age ≥ 18 and < 50 years at the time of signing the consent

Overtly healthy as determined by medical evaluation including medical history, heart and lung auscultation, focused cardiac ultrasound and measurement of cardiac conduction times

Woman of childbearing potential (WOCBP) must 1) use adequate birth control* or 2) have a negative pregnancy test less than 14 days before visit

Capable of giving a signed informed consent

Exclusion criteria

Any medical condition limiting physical exertional capacity or requiring regular medication (allergy and contraceptives excepted)

Pregnancy

Breastfeeding

History of syncope (syncope of presumed vasovagal nature with known precipitating factor excepted)

Any known cardiac arrhythmia

Any drug (contraceptives excepted) used on a regular basis for a chronic condition (allergy excepted)

*See “S1 Protocol” for specific requirements to adequate birth control.

Table 3

Primary and secondary objectives and endpoints.

Objectives	Endpoints
Primary
Study the effect of supplemental oxygen on cardiac output during LBNP	Difference in the change in cardiac output between oxygen and room air during LBNP
Secondary
Study the effect of supplemental oxygen on cardiac stroke volume during LBNP	Difference in the change in cardiac stroke volume between oxygen and room air during LBNP
Study the effect of supplemental oxygen on MCAV during LBNP	Difference in the change in MCAV between oxygen and room air during LBNP
Study the effect of supplemental oxygen on time to hemodynamic decompensation during LBNP	Difference in time to decompensation between oxygen and room air during LBNP

LBNP = lower body negative pressure, MCAV = middle cerebral artery velocity.