Association between non-acute traumatic injury (TI) and heart rate variability (HRV) in adults: A systematic review protocol.

Heart Rate Variability (HRV) is an indirect measure of autonomic function. Attenuated HRV is linked to worsening health outcomes including Major Adverse Cardiovascular Events (MACE). The relationship between traumatic injury (TI) and HRV has been limitedly studied. This research protocol has been designed to conduct a systematic review of the existing evidence on the association between non-acute TI and HRV in adults. Four electronic bibliographic databases (Web of Science, CINAHL, Medline, and Scopus) will be searched. The studies on non-acute (>7 days post injury) TI and HRV in adults will be included, followed by title-abstract screening by two reviewers independently. The quality and risk of bias of the included studies will be assessed using Axis and a six-item Risk of Bias Assessment tool for of Non-randomized Studies (RoBANS) respectively. Grading of Recommendations Assessment, Development and Evaluation (GRADE) will assess the quality of evidence. The extracted data will be synthesized using narrative syntheses and a Forest plot with or without meta-analysis- whichever permitted by the pooled data. This will be the first systematic review to examine the relationship between generalized TI and HRV in adults. Trial registration: (PROPSERO registration number: CRD: CRD42021298530) https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021298530.

Introduction

Traumatic injury (TI) is a leading cause of death and long-term disability [1]. It is typically defined as any physical injury of sudden onset and severity requiring immediate medical care [2]. In total, 38 different injury mechanisms have been defined [3]. Among these road traffic accidents, falls and sport injuries tend be predominant among civilian populations [3] whereas some occupational population such as law enforcement officers have been reported to have four times higher risk of mortality than civilians as a result of traumatic injuries during assaults [4]. Similarly, in deployed military populations, there is a greater predominance of gunshots and blast, but this depends on the conflict and environment [5].

Traumatic injury initiates an immediate autonomic response typified by an increase in sympathetic activation and gradual withdrawal of parasympathetic tone [6]. This autonomic response can be measured non-invasively using the concept of Heart Rate Variability (HRV)- which measures the fluctuations in cardiac inter-beat intervals in response to competing sympathetic nervous system and parasympathetic nervous system tones [6,7]. This can be examined in greater detail using time domain, frequency domain and non-linear methods, with each offering their own unique insight into autonomic balance [8].

Owing to HRV’s ability to indicate somatic tissue damage before the onset of pain and the injury [9], HRV has also been proposed as a new vital sign and triage tool in trauma settings [10-12]. In addition to being an objective marker of autonomic function [13], HRV has also been shown to be a robust marker of injury severity and as a predictor of morbidity and mortality in patients with severe traumatic injuries to head, neck, torso, abdomen [14], burns [15], and Traumatic Brain Injury (TBIs) [16]. With this prognostic and diagnostic value, HRV has emerged as an increasingly useful measure of physical fitness and the identification of overtraining. Heart rate variability is a robust cardiovascular marker and reduced HRV has been strongly linked to major adverse cardiovascular events (MACE) and all-cause mortality [17].

In the last two decades, research into the impact of TI on HRV has been largely focused on acute trauma and during hospital admission [18-21]. While recent systematic reviews have summarized the relationship between HRV and depression [22], Post Traumatic Stress Disorder (PTSD) [23], and selected TBI [24] and spinal cord injury [25]; the current knowledge gap is in non-acute TI and HRV with the need to include multiple injury types. To date, no such systematic review has been undertaken. This systematic review is essential considering the recent conflicts in Iraq and Afghanistan which have brought the societal and long-term physical and psychological impact of TI among military conflicts into sharp focus. In a recent study of combat veterans, suffering previous TI in Afghanistan, it was shown that TI and its severity was independently associated with an increased cardiovascular risk [26]. Given the utility of HRV as a global non-invasive CVD risk its relationship to non-acute TI warrants further research interest.

This protocol for systematic review aims to address this research gap by reviewing the existing evidence base relating to generalized non-acute TI (excluding selected TBI, spinal cord, haemorrhage, PTSD and depression) [22-25] and HRV. This should lead to greater understanding of the longer-term impact of TI on autonomic function and the potential for preventive measures to moderate this pathway. The review question is: What is the association between non-acute traumatic injury and heart rate variability in adults with unselected TI versus uninjured controls? The objectives of this systematic review are to: examine the association between traumatic injury and heart rate variability, report which indices (whether time, frequency, non-linear) and injuries have been mostly reported in the literature and identify any patterns of confounders (age and time from injury) in relation to TI and HRV.

Methods

This protocol has been designed using the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) protocol guidelines and checklist [27] (S1 Table). The protocol has been registered on PROSPERO- International Prospective Register of Systematic Reviews (CRD: CRD42021298530) and is available at: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=298530. The research question has been designed using the Population, Exposure, Comparator and Outcome (PECO) framework [28].

The results will be reported in the subsequent systematic review following the PRISMA 2020 guidelines [29]. Any changes to the protocol will be noted and explained there.

Eligibility criteria

Population

We aim to include studies with adult human participants aged 18 or above with no restriction on gender and occupation. The rationale is to include a broad spectrum of participants (civilian and military) [3-5]. Studies with participants who sustained chronic TI (>7 days post injury) and post initial hospital discharge will be included instead of acute injuries which led to death upon hospital admission. We selected the cut-off point of >7 days post injury based on a previous review on mild TBI [30] and to mitigate the impact of early physiological impact of acute trauma on HRV [18-21]. Any studies with animals or children and adolescents aged <18 years will be excluded.

Exposure

For this systematic review, traumatic injuries are defined as any physical injuries or trauma such as injuries resulting from: burn, fall, explosion, blast, gunshot, amputation and accidents. Studies reporting traumatic brain injury (TBI), spinal cord injury, haemorrhage and psychological trauma such as PTSD, depression, and anxiety will be excluded [22-25].

Comparator

Only studies with healthy controls (who didn’t sustain any traumatic injuries) will be considered for inclusion.

Outcome

Any measure of HRV- includes and not limited to: time domain (root mean square of successive differences between normal heartbeats (RMSSD), standard deviation of the average normal-to-normal (NN) intervals (SDANN), standard deviation of NN intervals (SDNN), frequency domain (low frequency power (LF), high frequency power (HF), total power (TP), LF/HF ratio) and non-linear measures for heart rate complexity (HRC) (e.g. entropy, SD1, SD2) will be considered as the primary outcomes in this study [8]. Studies measuring HRV either as a primary or secondary outcome will be eligible for inclusion. This study will include heart rate, type and mechanism of TI and time from TI as the secondary outcomes: There will be no restrictions on date, and geographical area. The inclusion and exclusion criteria are based on previous studies on the topic [3,4,18-25] and are listed in Table 1.

Table 1

The inclusion and exclusion criteria.

	Inclusion	Exclusion
Population	Young/middle/older adults from both sexes, aged >18, no history of cardiovascular disease	Animals, children, or adolescents aged <18, history of cardiovascular disease
Exposure	any physical trauma sustained (>7 days post injury) suchas gunshot wounds, amputation, limb loss, burn and fall etc.	Studies which involve PTSD, depression, anxiety, head/brain injury, traumatic brain injury, spinal cord injury and haemorrhage.
Comparison	Controls with no traumatic injury.	No control
Outcome	Any index of Heart Rate Variability (HRV)	No HRV measure reported as a primary of secondary outcome.
Setting	Any	-
Study Design	Observational, cohort, cross-sectional, prospective, case control	Systematic reviews, reviews
Language	English	Other than English
Date	Any	-
Publication status	Published research papers	In-press, grey literature, conference proceedings, meeting abstracts, case reports, case series, opinion/editorial, in-vitro and animal study.

Information sources

Cochrane library and PROSPERO registry were searched to see if a systematic review had already been undertaken. An initial scoping search was conducted to identify the relevant key terms in the study area and identify the gap. The preliminary search strategy has been developed around exposure and outcome by the authors with the help of an experienced librarian. The title and abstract fields of four electronic bibliographic databases (Medline, Web of Science, CINAHL and Scopus) will be searched with the search strategy (adapted for each database). MeSH terms and CINAHL subject headings will be added for “wounds and injury” and “heart rate variability” wherever applicable.

Search strategy

Following is the master search strategy for Web of Science.

Exposure: trauma* OR wound* OR "blast" OR explosion* OR trauma N3 injur* OR “burn*”.

Outcome: "heart rate variability" OR "HRV" OR "heart rate variation*" OR “heart rate complexity” OR "SDNN" OR "RMSSD" OR "autonomic function*" OR "autonomic reactivity" OR "HR-variability" OR “autonomic regulation” OR “autonomic activity".

The reference lists of included studies will also be scanned to supplement the searches and ensure the inclusion of important data sources which might have been missed in our search.

Study records

Data management

The records will be exported to Mendeley Desktop (version 1.19.8) and maintained in Mendeley throughout the review. This will be followed by de-duplication which will be supplemented with manual screening and elimination of duplicates by both reviewers (RM and CJB).

Selection process

At the initial screening stage, the titles and abstract will be scanned by two reviewers (RM and CJB) independently. A supplementary selection criterion (S2 Table) will also be used at the initial stage to screen studies. Studies meeting the eligibility criteria will be retrieved for the full text. Full text articles will be evaluated by two independent reviewers (RM and CJB) using the inclusion and exclusion criteria. In cases of disagreement, a third reviewer (AK) will be invited to decide, followed by documenting the number of included and excluded studies. The search and screening results will be documented and displayed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram [29].

Data collection process

A data-extraction form (S3 Table) has been developed by RM and customized for this systematic review using the guidelines set by Centre for reviews and dissemination [31]. In case of absence, the data as reported by the authors in the studies will be extracted. The second reviewer (CJB) will cross-check the extraction performed by the lead reviewer (RM). The data-extraction form may also be continually developed as it will be piloted with two studies initially to test its reliability and comprehensiveness to extract data. Once completed, the data extraction forms will be cross reviewed by both reviewers (RM and CJB) to discuss discrepancies (if any). Any differences observed at this stage by the two reviewers will be resolved by inviting the third reviewer (AK). All data will be stored on Microsoft Excel and managed by the lead reviewer (RM). The corresponding authors will be contacted to obtain any missing data. If not available, the study will be excluded.

Data items

For each article, following data (not limited to) will be extracted by the lead reviewer (RM): date of publication, authors, title, setting, country, study design, sample size, participants’ gender and age, exposure and outcome variables, type of traumatic injury, injury-severity measure, time from injury, and measure of HRV, and heart rate. Wherever appropriate, means, standard deviation, p-values, correlation coefficients, confidence intervals and any other statistical findings will be extracted.

Outcomes and prioritization

Any standardised index of HRV reported in the studies will be prioritised to be included as a primary outcome whereas heart rate will be reported as the secondary outcome. Injury-related characteristics such as time from injury and type of injury will be prioritised to be included in the narrative synthesis.

Risk of bias in individual studies

Both reviewers (RM and CJB) will perform the critical appraisal of included studies using the Axis quality appraisal tool. The Axis checklist has 20 questions with response of either a yes (1) or a no (0) [32]. Study quality will be further categorized as low (<10), moderate (10–15) and high (>15) as previously used [33]. The final quality assessment score of each study will be documented and displayed on the quality appraisal table. Any disagreements at the quality assessment stage will be resolved by the third reviewer (AK).

Since non-randomized studies will be included in the review, the Risk of Bias (RoB) will be assessed using the six-item Risk of Bias Assessment tool for of Non-randomized Studies (RoBANS) [34]. The following six domains will be assessed in the included studies: the selection of participants, confounding variables, the measurement of exposure, the blinding of the outcome assessments, incomplete outcome data, and selective outcome reporting [34]. Studies scoring 0, 0–2, >2 will be rated as having low, moderate and high risk of bias, respectively as previously defined [33]. Two reviewers (RM and CJB) will independently evaluate the included studies for RoB against the RoBANS criteria. Disagreements will be resolved by discussion with the third reviewer (AK).

Data synthesis

To detect and test the heterogeneity across studies, the Forest plot will be visually inspected followed by statistical tests- Chi-squared test and I2 statistic [35] using Review Manager (RevMan 5.4.1) [36]. Outcome of the heterogeneity tests will inform the data-synthesis approach.

Given the methodological (different measurement tools/software used for ECG-recording and HRV analysis) and statistical heterogeneity (coefficient correlations, t-scores, odds ratio and means/medians etc), approach to data synthesis will be informed by the heterogeneity score. If heterogeneity is greater than 75%, it is likely that a narrative synthesis will be performed by tabulating the extracted data as suggested in the guidelines by Centre for reviews and dissemination [31] with a forest plot using Review Manager (RevMan 5.4.1) [36] without meta-analysis. However, if the characteristics of three or more studies are homogenous, a meta-analysis of the pooled data will be performed in RevMan using random-effects approach [37]. Mean difference (MD) or standardized mean difference (SMD) will be generated if the outcome of interest in the included study is given in the same or different outcome measures on a continuous scale [38], respectively.

Meta-bias

For publication bias, the funnel plot will be visually inspected for asymmetry. If the number of studies included in the meta-analysis is less than ten, the Egger’s test will not be used as suggested [39].

Confidence in cumulative evidence

The quality of evidence will be assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE) by two reviewers (RM and CJB). GRADE is a system to rank the quality of evidence in a systematic review and make recommendations [40]. Using the online tool, GRADEproGDT [41], each study will be ranked for quality of evidence into 4 ranks: very low, low, moderate, and high. GRADE evidence profile and summary of evidence will be reported in the subsequent systematic review.

Discussion

Traumatic injury is one of the leading causes of death in adults aged <40 years [1]. The reported adverse health consequences of injuries sustained during recent conflicts in Afghanistan and Iraq have brought the impact of TI into greater clinical focus [26].

The results of several recent original publications have highlighted a plausible association between combat-related TI injury and subclinical cardiovascular risk [26,33,42]. The majority of this research has focussed on more well-established markers of cardiovascular risk such as heart rate, obesity, blood pressure, glucose and lipids [26]. The examination of the impact of TI on HRV has the potential to bridge the research gap. Heart rate variability is a unique non-invasive marker of autonomic balance that has been strongly linked to adverse health outcomes including MACE [17] across a broad spectrum of patient populations [14-16]. While the effects of acute TI on HRV have been studied [18-21], there appears to be a paucity of research on the longer terms impacts of non-acute TI and HRV, in particular beyond that of mild traumatic brain injury [24] and spinal cord injury [25]. Examination of the longer-term effects of TI on HRV has the potential to enhance existing research knowledge gaps and offer mechanistic insight to the reported elevation of cardiovascular risk with TI.

Addressing this research gap, this paper presents the protocol for the systematic review of literature on the association between non-acute TI and HRV. This systematic review is timely considering the recent trend of examining the long-term impact of TI in vulnerable groups such as military veterans.

This systematic review protocol offers several strengths. Firstly, it has been registered in the PROSPERO database to ensure a transparent conduct of the systematic review. Secondly, the literature searches conducted in the 4 databases will allow a comprehensive search. Thirdly, the rigour and the quality of the included studies will be assessed using validated critical appraisal and risk of bias tools along with GRADE, independently by two reviewers. Lastly, to our knowledge, this will be the protocol of the first systematic review which examines the association between a diverse spectrum of non-acute TIs and HRV in adults. Upon completion, the systematic review will be submitted to a peer-reviewed journal. However, some limitations are also anticipated such as heterogeneity in HRV data acquisition and analysis across the studies as found in the preliminary searches.

Nevertheless, the importance of this systematic review is not lessened by these limitations given the transparent conduct and rigorous evaluation of the included studies using validated tools. The outcomes of this systematic review may have implications to inform trauma care practice and intervention development for civilians and vulnerable populations such as military personnel and law enforcement officers.

This is the S1 Table PRISMA-P (Preferred Reporting Items for Systematic review and Meta-Analysis Protocols) 2015 checklist.

(DOCX)

Click here for additional data file.

This is the S2 Table the supplementary study selection criteria.

(DOCX)

Click here for additional data file.

This is the S3 Table data extraction form.

(DOCX)

Click here for additional data file.

9 Jun 2022

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The present study protocol is interesting but there still some improvements that can be made before further consideration.

For instance, some methodological aspects need more rational and support such as, the characteristics of the target population in study, the inclusion/exclusion criteria, and the development of some preliminar results and discussion to stretch the importance of such study.

Please consider the comments made by the reviewers.

Thank you

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The present study is of interest to investigate the association between non-acute traumatic injury (TI) and heart rate variability (HRV) in adults, throughout a systematic review protocol.

Despite the interesting work, I strongly suggest following the comments to improve the quality of the manuscript.

1. Line 137 and 138 Population: "We aim to include studies with adult human participants aged 18 or above with no restriction on gender and occupation." Please explain why?

2. Line 139-140: "Studies with participants who sustained chronic TIs (>7 days post injury) and post initial hospital discharge will be included instead of acute injuries which led to death upon hospital admission." Please explain why?

3. Table1. Authors should explain why this inclusion/exclusion criterias were chosen across the text. Please use valid references to support your rationale.

4. Why authors decided to use RoB for the risk of bias analyses? Please clarify.

5. Despiste being this a "systematic review protocol" I think that will be very interesting to presente some "preliminar results" accompanied with some "preliminar discussion" (more robust) around the topic here presented. As it stands it is not clear how your questions/hypothesis will be really answered.

Reviewer #2: I carefully studied the file. The importance of the work in the introduction should be written thoroughly with up-to-date sources. Also it should be mentioned relevant studies in the introduction. The article is also in the discussion section Too short and too weak. It is not possible to be published in this way. It should be discussed with the articles included in the study. This section must be developed.

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Reviewer #1: Yes: Júlio Alejandro Henriques da Costa

Reviewer #2: No

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17 Jun 2022

Also attached as the 'Response to reviewers'.

PONE-D-22-12255: Association between non-acute traumatic injury (TI) and heart rate variability (HRV) in adults: a systematic review protocol

We would like to thank the academic editor and the reviewers for providing insightful feedback on our manuscript which has improved our paper. The suggestions made by the academic editor and the reviewers have been incorporated into the manuscript and are highlighted. Please see below, in blue, for a point-by-point response to the reviewers’ comments.

Academic Editor:

Dear authors,

The present study protocol is interesting but there still some improvements that can be made before further consideration. For instance, some methodological aspects need more rational and support such as, the characteristics of the target population in study, the inclusion/exclusion criteria, and the development of some preliminary results and discussion to stretch the importance of such study.

Please consider the comments made by the reviewers.

Response: Thank you for the feedback. Previously, for brevity, some methodological aspects were only briefly discussed. However, in the light of your helpful comments, the above suggestions have now been incorporated into our revised manuscript and are tracked.

Reviewer #1: The present study is of interest to investigate the association between non-acute traumatic injury (TI) and heart rate variability (HRV) in adults, throughout a systematic review protocol.

Response: Thank you!

Despite the interesting work, I strongly suggest following the comments to improve the quality of the manuscript.

1. Line 137 and 138 Population: "We aim to include studies with adult human participants aged 18 or above with no restriction on gender and occupation." Please explain why?

Response: Thanks for this comment. This sentence is worded in this way to emphasise that our review is intended to be as inclusive as possible in terms of incorporating both men and women as well as a broad spectrum of participants (e.g. civilian and military). The following changes have been made (line 141-142).

“The rationale is to include a broad spectrum of participants (civilian and military) [3, 4, 5].”

Please note that the line numbers originally quoted by the reviewers have changed due to some addition in the revised manuscript.

2. Line 139-140: "Studies with participants who sustained chronic TIs (>7 days post injury) and post initial hospital discharge will be included instead of acute injuries which led to death upon hospital admission." Please explain why?

Response: Apologies for the confusion. We agree that this needed more explanation. Setting the background, the following lines have now been added in the introduction section:

(Line 84-85): In the last two decades, research into the impact of TI on HRV has been largely focused on acute trauma and during hospital admission [14, 15, 16, 17].

(Line 88-90): (the current knowledge gap is in non-acute TI and HRV with the need to include multiple injury types. To date, no such systematic review has been undertaken)”.

-and under the heading “Eligibility criteria”:

(Line 145-147): We selected the cut-off point of >7 days post injury based on a previous review on mild TBI [29] and to mitigate the impact of early physiological impact of acute trauma on HRV [18, 19, 20, 21].

In sum, the rationale is that previous systematic reviews have looked at the relationship between HRV and acute trauma (selected TIs such as spinal cord, traumatic brain injury). To address this research gap, our systematic review will examine the association between non-acute (or generalised) TI and HRV in adults.

Seven new references have been added (see below). Therefore, the in-text citations and reference list have been updated (tracked).

• Batchinsky AI, Wolf SE, Molter N, Kuusela T, Jones JA, Moraru C, Boehme M, Williams K, Bielke P, Wade C, Holcomb JB, Cancio LC. Assessment of cardiovascular regulation after burns by nonlinear analysis of the electrocardiogram. J Burn Care Res. 2008;29(1):56-63.

• Colombo J, Shoemaker WC, Belzberg H, Hatzakis G, Fathizadeh P, Demetriades D. Noninvasive monitoring of the autonomic nervous system and hemodynamics of patients with blunt and penetrating trauma. J Trauma Acute Care Surg. 2008;65(6):1364-73.

• Riordan Jr WP, Norris PR, Jenkins JM, Morris Jr JA. Early loss of heart rate complexity predicts mortality regardless of mechanism, anatomic location, or severity of injury in 2178 trauma patients. J Surg Res. 2009;156(2):283-9.

• Mowery NT, Morris Jr JA, Jenkins JM, Ozdas A, Norris P. Core temperature variation is associated with heart rate variability independent of cardiac index: a study of 278 trauma patients. J Crit Care 2011;26(5):534-e9.

• Buker DB, Oyarce CC, Plaza RS. Effects of spinal cord injury in heart rate variability after acute and chronic exercise: a systematic review. Top Spinal Cord Inj. 2018;24(2):167-76.

• Krainin BM, Forsten RD, Kotwal RS, Lutz RH, Guskiewicz KM. Mild traumatic brain injury literature review and proposed changes to classification. J Spec Oper Med. 2011;11(3):38-47.

• Bhatnagar V, Richard E, Melcer T, Walker J, Galarneau M. Retrospective study of cardiovascular disease risk factors among a cohort of combat veterans with lower limb amputation. Vasc Health Risk Manag. 2019;15:409.

3. Table1. Authors should explain why this inclusion/exclusion criteria were chosen across the text. Please use valid references to support your rationale.

Response: Thank you for raising this. In our introduction, we have presented the background and rational of our target population as well as highlighting some of the key research gaps with reference to our targeted population with the inclusion of up-to-date references. These references have now been cited in line 165-166:

The inclusion and exclusion criteria are based on previous studies on the topic [3, 4, 18-25]

4. Why authors decided to use RoB for the risk of bias analyses? Please clarify.

Response: Thank you for highlighting this. We decided to use RoBANS for assessing RoB due to its validity and suitability for non-randomised and observational studies. The following lines (215-220) have now been added to the manuscript to clarify this point:

Since non-randomized studies will be included in the review, the Risk of Bias (RoB) will be assessed using the six-item Risk of Bias Assessment tool for of Non-randomized Studies (RoBANS) [33]. The following six domains will be assessed in the included studies: the selection of participants, confounding variables, the measurement of exposure, the blinding of the outcome assessments, incomplete outcome data, and selective outcome reporting [33].

5. Despite being this a "systematic review protocol" I think that will be very interesting to present some "preliminary results" accompanied with some "preliminary discussion" (more robust) around the topic here presented. As it stands it is not clear how your questions/hypothesis will be really answered.

Response: Thanks for this suggestion. Our preliminary searches indicate a rather small body of evidence on the topic. Sharing the preliminary results may be premature at the moment. However, the anticipated limitations of the included studies (e.g. heterogeneity in terms of HRV acquisition and analysis) have been discussed in “discussion”. Similarly, the anticipated data analysis approaches have been described under “data synthesis”. Furthermore, the importance of our work in the light of current research has also been discussed (please see our response to Reviewer 2’s 2nd comment).

Reviewer #2: I carefully studied the file. The importance of the work in the introduction should be written thoroughly with up-to-date sources. Also, it should be mentioned relevant studies in the introduction.

Response: Thanks for this suggestion. In the Introduction, the existing literature has been reviewed briefly from old to new studies. Addressing Reviewer 1’s comment as well, a few more studies have also been added to clarify the inclusion and exclusion criteria (Introduction section: line 84-90). References have now been cited in lines 99, 142, 146-147, 153, 166, 264). To our knowledge, all sources are up-to-date.

The article is also in the discussion section Too short and too weak. It is not possible to be published in this way. It should be discussed with the articles included in the study. This section must be developed.

Response: Thank you for highlighting this. Considering Reviewer 1’s comment 5 as well, the discussion section has now been rewritten to include the importance, strengths, and limitation of the work in the light of current research as follows (Lines 258-295):

Traumatic injury is one of the leading causes of death in adults aged <40 years [1]. The reported adverse health consequences of injuries sustained during recent conflicts in Afghanistan and Iraq have brought the impact of TI into greater clinical focus [26].

The results of several recent original publications have highlighted a plausible associated between combat-related TI injury and subclinical cardiovascular risk [26, 32, 41]. The majority of this research has focussed on more well-established markers of cardiovascular risk such as heart rate, obesity, blood pressure, glucose and lipids [26]. The examination of the impact of TI on HRV has the potential to bridge the research gap. Heart rate variability is a unique non-invasive marker of autonomic balance that has been strongly linked to adverse health outcomes including MACE [17] across a broad spectrum of patient populations [14, 15, 16]. While the effects of acute TI on HRV have been studied [18, 19, 20, 21], there appears to be a paucity of research on the longer terms impacts of non-acute TI and HRV, in particular beyond that of mild traumatic brain injury [24] and spinal cord injury [25]. Examination of the longer-term effects of TI on HRV has the potential to enhance existing research knowledge gaps and offer mechanistic insight to the reported elevation of cardiovascular risk with TI.

Addressing this research gap, this paper presents the protocol for the systematic review of literature on the association between non-acute TIs and HRV. This systematic review is timely considering the recent trend of examining the long-term impact of TIs in vulnerable groups such as military veterans.

This systematic review protocol offers several strengths. Firstly, it has been registered in the PROSPERO database to ensure a transparent conduct of the systematic review. Secondly, the literature searches conducted in the 4 databases will allow a comprehensive search. Thirdly, the rigour and the quality of the included studies will be assessed using validated critical appraisal and risk of bias tools along with GRADE, independently by two reviewers. Lastly, to our knowledge, this will be the protocol of the first systematic review which examines the association between a diverse spectrum of non-acute TIs and HRV in adults. Upon completion, the systematic review will be submitted to a peer-reviewed journal. However, some limitations are also anticipated such as heterogeneity in HRV data acquisition and analysis across the studies as found in the preliminary searches.

Nevertheless, the importance of this systematic review is not lessened by these limitations given the transparent conduct and rigorous evaluation of the included studies using validated tools. The outcomes of this systematic review may have implications to inform trauma care practice and intervention development for civilians and vulnerable populations such as military personnel and law enforcement officers.

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Response: Thank you for this comment. To best of our knowledge, the manuscript has been written and revised according to the PLoS one’s style requirements including those for file naming. Please see revised lines 432-434. We have also followed the Study Protocol template available at PloS One website:

https://storage.googleapis.com/plos-published-prod/c9fb/Study%20Protocol%20Article%20Template.pdf?X-Goog-Algorithm=GOOG4-RSA-SHA256&X-Goog-Credential=wombat-sa%40plos-prod.iam.gserviceaccount.com%2F20220615%2Fauto%2Fstorage%2Fgoog4_request&X-Goog-Date=20220615T101150Z&X-Goog-Expires=86400&X-Goog-SignedHeaders=host&X-Goog-Signature=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

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"We would like to acknowledge Bournemouth University and the ADVANCE charity, UK for jointly funding the PhD studentship of RM at Bournemouth University"

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The authors received no specific funding for this work"

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: Thank you for highlighting this. Please change the funding statement (line 32-33) to the following on my behalf:

This project is a part of RM’s Ph.D. studentship- jointly funded by Bournemouth University and the ADVANCE charity, UK.

The Acknowledgment statement has now been revised (line 303-304)

We would like to acknowledge Mr. Caspian Dugdale, Academic Liaison Librarian at Bournemouth University, for helping us with building the search strategy.

Submitted filename: Response to reviewers.docx

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10 Jul 2022

23 Jul 2022

The Response to Reviewers is also attached as a document.

PONE-D-22-12255R1: Association between non-acute traumatic injury (TI) and heart rate variability (HRV) in adults: a systematic review protocol

We would like to thank the academic editor and the reviewers for providing insightful feedback on our manuscript once again. The suggestions made by the academic editor and the reviewers have been incorporated into the manuscript and are highlighted. Please see below, in blue, for a point-by-point response to the reviewers’ comments.

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.

Response: Thank you for this comment. The reference list has now been proofread and revised according to the format available under PloS One’s reference section: https://www.nlm.nih.gov/bsd/uniform_requirements.html.

Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

Response: Thank you for highlighting this. Below are the reference numbers 27 and 29 which have now been updated in the manuscript for the PRISMA-P checklist 2015, and 2020 guidelines, respectively:

27. Moher D, Shamseer L, Clarke M, Ghersi D, Liberati A, Petticrew M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015;4(1):1-9.

29. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. Syst rev. 2021;10(1):1-1.

Owing to the addition of one new reference (29), the reference list has been updated accordingly (please see the tracked changes). Following are the updated reference numbers for which a few changes have been made in the reference list related to et al. and formatting:

• 1, 2, 6,11, 13, 18, 26, 27, 29, 31, 34, 35, 37, 40, 41

Please note a few changes made in the manuscript as a result of proofreading:

• Line 69- ….which measures the fluctuations….

• Line 70-71: Deletion of abbreviations SNS, PNS

• Line 91-92- ..long-term physical and psychological impact…

• Line 110: addition of word ‘protocol’.

• Line 116-117: The results will be reported in the subsequent systematic review following the PRISMA 2020 guidelines [29].

• Line 132: Deletion of abbreviation SCI

• Line 151: Table 1: Deletion of repeated words history of

• Line 425-426: S1 Table. This is the S1 table PRISMA-P (Preferred Reporting Items for Systematic review and Meta-Analysis Protocols) 2015 checklist.

If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: Thanks for this suggestion. I can confirm that a retracted article was not cited and was removed from the reference list. We do not aim to cite any retracted article. The manuscript attached contains the complete and revised reference list.

Additional Editor Comments:

Dear authors,

The authors have addressed all comments of both reviewers. Nonetheless, reviewer 1 suggests that an explanation of the procedures to collect HRV should be added.

Response: Thank you. Please see our response to reviewer 1’s comment.

In addition, I found out that some methods need an update according to the most recent PRISMA 2020 guidelines, namely:

-the reference of the PRISMA 2020 update should be used;

Response: Thank you for drawing our attention to this. However, we would like highlight that the PRISMA 2020 checklist refers to the reporting of a systematic review whereas the PRISMA-P 2015 checklist is used to report a systematic review protocol- as also suggested in the recent PRISMA 2020 guidelines:

Furthermore, PRISMA 2020 is not intended to inform the reporting of systematic review protocols, for which a separate statement is available (PRISMA for Protocols (PRISMA-P) 2015 statement 47,48). (Page et al. 2021).

Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. Syst rev. 2021;10(1):1-1.

While in this instance, we aim to keep the PRISMA-P checklist 2015 in the manuscript (systematic review protocol), the subsequent systematic review will be reported according to the recent PRISMA 2020 guidelines (Please see lines 116-117).

-the PRISMA 2020 checklist should be followed in the exact same order, same sections and topics (as an example, the assessment of methodological quality should be removed. Instead, the Study risk of bias assessment should be added).

Thank you

Response: Thanks for raising this point. Since there is no significant difference in headings between the 2015 and 2020 checklists, the following section headings have now been renamed/updated (please see the tracked changes). The old sub-heading ‘assessment of heterogeneity’ has now been merged into ‘synthesis methods’ as per the order and headings of the PRISMA 2020 checklist:

• Information sources (line 153)

• Selection process (line 176)

• Data items (line 187)

• Study risk of bias assessment (line 206)

• Synthesis methods (line 227)

• Certainty assessment (line 244)

Please note that the section “Eligibility criteria” (line 119) has been moved up following the checklist order.

Review Comments to the Author:

Reviewer #1: The authors did a good job of reviewing the manuscript and answering all the revisions made.

Response: Thank you.

However, I think the paper could be more enriched if the authors could mention (briefly) how HRV can be measured in this type of population (civilian and military). We can use the same procedures?

Response: We appreciate your feedback. However, we would like to point out that this will be a systematic review and not a primary study. We do not aim to measure HRV in participants but present the results of previously reported work. However, we have cited references that provide a more in-depth review of HRV measurements and their interpretation. A detailed discussion on HRV measurement methods may be beyond the scope of this protocol. Nonetheless, we aim to address this in greater detail in the subsequent systematic review where it would be more relevant in the context of results.

Reviewer #2: I examined the Response "the Reviewer file". All corrections are made by the authors. Thank you so much for their effort. My decision is "accept"

Response: Thank you!

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

9 Aug 2022

11 Aug 2022

Response to Reviewers is also attached as a separate file.

PONE-D-22-12255R2: Association between non-acute traumatic injury (TI) and heart rate variability (HRV) in adults: a systematic review protocol

We would like to thank the academic editor and the reviewers for their feedback. The suggestions made by the academic editor have been incorporated into the manuscript and are highlighted.

Please see below, in blue, for a point-by-point response to the reviewers’ comments.

ACADEMIC EDITOR:

Dear authors,

Congratulations! Both reviewers already accept the work in the current form while I still have a minor revision to suggest.

Response: Thank you!

Considering that this is a protocol for a systematic review that follows PRISMA-P 2015 (Moher, D., Shamseer, L., Clarke, M., Ghersi, D., Liberati, A., Petticrew, M., Shekelle, P., Stewart, L. A., & PRISMA-P Group (2015). Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Systematic reviews, 4(1), 1. https://doi.org/10.1186/2046-4053-4-1) as you stated in the manuscript, I would like to suggest that authors also follow the same checklist item recommended in the reference.

For better clarity, after the section "Search strategy" you should present "Study records" in which you will include "data management" + "selection process" + "data collection process". After "Data items", it is suggested adding "Outcomes and

prioritization". "Study risk of bias assessment" should be replaced by "Risk of bias in individual studies". "Synthesis methods" should be replaced by "Data synthesis" as before. Then, you can add "Meta-bias(es)". Finally, instead of "Certainty assessment" used in the PRISMA 2020, it is suggest to replace by "Confidence in cumulative evidence".

I hope the authors can understand my suggestions.

After this step and if the authors follow these suggestions.

Thank you

Response: Thanks for raising this point. The revised manuscript now contains the suggested headings according to the PRISMA-P 2015 checklist. The following headings have been updated; line numbers are given in brackets.

Study records (174), Data management (175), Data collection process (190), Outcomes and prioritization (210), Risk of bias in individual studies (215), Data synthesis (233), Meta-bias (250), Confidence in cumulative evidence (254).

Please note a few changes made in the manuscript as a result of change in the headings:

• Lines 176-179 moved into the section of Data management

• Section on Outcomes and Prioritization added.

• Lines 191-202 moved into Data Collection Process

• Lines 251-253 moved into Meta-bias

• Reference numbers updated in the reference list and the text [35-39]

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

15 Aug 2022

Association between non-acute traumatic injury (TI) and heart rate variability (HRV) in adults: a systematic review protocol

PONE-D-22-12255R3

Dear Dr. Maqsood,

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Additional Editor Comments (optional):

Dear authors,

Congratulations on your work.

My opinion is to accept.

Best regards

Reviewers' comments:

18 Aug 2022

PONE-D-22-12255R3

Association between non-acute traumatic injury (TI) and heart rate variability (HRV) in adults: a systematic review protocol

Dear Dr. Maqsood:

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