PONE-D-21-20626A systematic review of in vivo animal stretching regimens on inflammation and its relevance to yoga researchPLOS ONE

Dear Dr. Munoz Vergara,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: No

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: No

Reviewer #4: N/A

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for this excellent manuscript. Please consider the following minor editing/improvement suggestions:

170 Table 3: Both of the first two stretching terms include “movement applied by an external force to increase range of motion” within their definitions. Would it be possible to specify more clearly the differences between these terms in this respect; i.e when such an externally induced tissue stretch will be counted as ‘active’ and when as ‘passive’?

170 Table 3: The inclusion of the term ‘In vivo model of an acute nerve stretch injury’ in the left column could possibly be seen as a contradiction to the previously specified inclusion of studies evaluation “muscles, skin and connective tissue” only. Since the nerve bundles evaluated are often part of a larger ‘muscle’ or ‘connective tissue’, their inclusion could therefore be possibly justified based on this or similar reasoning. Please clarify your reasons for inclusion.

644 I suggest including a brief discussion of potential age effects within this larger ‘DISCUSSION’ section. You Already ADDRESSED THIS ASPECT IN LINES 294-297. At least in humans, there are recent indications that in older individuals the stretching effect may primarily target different tissues than in younger ones (see e.g. doi: 10.1249/MSS.0000000000002360). If verified, this could possible have some interesting implications for yoga practice.

Above all: my congratulations to this very good review study and manuscript.

Reviewer #2: The present study aimed to inform a basic biological framework for designing and interpreting clinical studies that evaluate the impact of yogic stretching on inflammation and health. This is an interesting study, however, I have some major concerns in procedures.

1. First, the novelty of the results/analysis is poor.

2. I wonder if this manuscript is not aligned with guidelines or poor description, although the authors cited [de Vries, RBM, Evidence-based Preclinical Medicine, 2015], the guidelines of the Systematic Review Center for Laboratory Animal Experimentation. The authors can follow PRISMA and SYRCLE. That is through inclusion/exclusion criteria, search strategy, data extraction, and risk of bias.

3. The inclusion/exclusion should be defined more clearly, based on PICO even if systematic review for animal models. Is the exclusion criteria only “all original peer-reviewed articles”? For example, lung stretch? Vascular stretch? Meanwhile, the authors showed some reason for exclusion in Fig 1.

4. I am not sure of the validity of the search strategy. (1) The therapeutic stretching technique is sometimes included in mobilization, manipulation, manual therapy, and so on. Why didn’t the authors include the term? (2) Why were the injurious stretching techniques different from delayed onset muscle soreness model, or eccentric muscle contraction model? Table 3 is not enough to show that. (3) Can the authors justify the species as only rat or mouse, not other animals in Table 1? Meanwhile, the results included the articles in rabbit. (4) Why the term for yoga is used “AND search” in supplement 1? The treatment should be stretching. (5) Didn’t the authors communicate with the librarian? (6) Didn’t the authors submit PROSPERO before this study? (7) The periods of search should be updated whenever.

5. The variables for data extraction were shown. However, the procedure of actual data extraction is not shown. Multiple inflammatory outcomes? Multiple time points? Mean and SD? How about discrepancies between reviewers?

6. Could the authors justify the modified checklist for risk of bias? SYRCLE's risk of bias tool for animal studies is also published based on Cochrane recommendations.

7. This manuscript of systematic review is in vivo animal stretching studies, not yoga research. The title would cause misunderstanding.

Reviewer #3: The authors describe a systematic review of reasonable quality on animal models for stretching. As a great fan of systematic reviews on animal models, I was very enthusiastic about this paper upon reading the title in the review request. Upon reviewing the paper however, my enthusiasm progressively decreased. The methods leave room for improvement (see below), there is relatively little real data synthesis (the authors repeat the findings from the included papers but hardly add to them except for several suggestive vote counting exercises), referencing is tremendously liberal, and some of the conclusions are extremely far-fetched.

As I appreciate the effort that has gone in to collecting the data, I do provide a full review report, hoping that with a thorough rewrite the paper may become acceptable for publication. The data are certainly interesting.

Major issues:

The relevance of the animal models to yoga remains completely unclear. The animal models have very little resemblance to voluntary conscious stretching up to a certain “sweet spot”. The face, concept and predictive validity of the animal models for yoga is not at all discussed. Without a comparison with human data, you cannot state anything on the topic (other than that it inspired the review perhaps). I suggest the authors read into the topics of animal model validation and animal-to-human translation, and either leave the yoga aspect out of the paper, or include human data for comparison (which could make this paper my favorite read of the year, and I do dare to say that in January).

Study quality scores are highly disputable. They are not very relevant, high scores can go together with low risk of bias and vice versa. Please skip these, and provide table 2 totals by column instead of by row, this is far more interesting.

Definitions for active and passive stretching overlap. This needs to be clarified. Without clarification figure 2A is meaningless.

Table 4 and 5 are not internally consistent and need harmonization. An example: “no mention of randomisation” for Best et al., but not for the other non-randomising studies. Also, why the bold? Can the name of the second column be changed to “study aim as described in the paper” (or something similar)? Is there a reason to the bold text? I find it confusing. If there is a consistent decision making process behind the inconsistencies, please describe it somewhere beforehand.

In the results section, there is quite a bit of phrasing that encourages vote counting (e.g. “of these [six] studies, five found …”. If there are 6 studies, a meta-analysis would be interesting. Without effect sizes, 5 out of 6 sounds informative but is not (which makes the phrasing misleading).

The authors refer to an evidence gap in human literature. I think the human literature needs to be further described (what were the comparators in the 25 randomised control studies included in the systematic review that is reference 73? and in the 301 randomized controlled trials of yoga summarized in reference 121? Is there really nothing that can be analysed there?). I also think resolving this human evidence gap should be prioritized over further animal studies because a. humans can give informed consent, and b. the value of the animal models has not been proven. Remove all suggestions for future animal experiments from the manuscript.

Minor comments:

L30: were studies <1900 not eligible or is this the general database cut-off?

L70-71: if the authors want to state that inflammation is evolutionary preserved, this needs a specific reference. I think it will be difficult to find one, as the immune system varies substantially between species and we do not fully understand how.

L76: “biological framework”? Please explain or leave out.

L91: delete “structure and”, and change “guidelines” into “format” or “template”. Also, why was the protocol not posted?

L101 and further: why are the species not included in all searches? Why are some species searched for and others not? Why were the available animal filters not used? (doi: 10.1258/la.2010.009117 and 10.1258/la.2011.011087, and for future work 10.1177/00236772211045485)

L108: delete “different” (current phrasing suggests that you only included studies that compared multiple stretching techniques, and excluded stretching vs. control only)

L122: did these reviewers evaluate all retrieved studies independently or did both do half the set? Was this the same for all phases?

L137: Why not the SYRCLE Risk of Bias tool?

L158: change “included” into “described”. For most animal studies a power analysis is performed, as it is a mandatory part of the ethics review process.

L363: I think you mean “either” where you write “both”.

L403: Please change “one” into “four”. (Yin-yoga is gaining popularity.)

L625 and elsewhere: please delete figure 4. It has little to do with your evidence which focussed on in vivo (in rodents) only.

Paper flow chart: please move the box with the duplicates to where it belongs (they go out, not through)

Reviewer #4: The authors have done great job in preparing and writing this systematic review article, including 1,411 English articles between 1900 and 2020 as per their ‘predefined criteria’.

This systematic review summarizes the findings from 28 experimentally controlled animal studies (in vivo) conducted in last 20 to 25 years, employing mechanical stretching forces on the musculoskeletal and integumentary (i.e., skin and surrounding connective tissue) systems to evaluate its potential impact on inflammatory processes, and its relevance to yoga research.

It may be an effective and useful approach for studying the effect of yogic stretching on inflammation and health as well as future translational research.

There are some queries/suggestions which may be addressed by the authors and may add more value to this article:

1. The main concern is that yoga includes “active” stretching exercises (asana) in humans, whereas authors have used 25 (out of total 28) animal studies using “passive” stretching exercises. How it can be justified for this systematic review where theses “passive” animal studies are being tried to relate with “active” yoga stretching exercises (asana)?

The authors try to explain this phenomenon using concept of ‘stretching’ as per sports medicine terminology, that does not appear much convincing, scientifically.

2. The animals used in these studies were primarily rats or, mice. Again, how this stretching exercises in tiny rats/mice can be compared with human yoga stretching exercises (asana)?

3. Why authors have included the studies using stretching of skin of lower back (3 studies out of total 28) to understand the related pathophysiology. The studies using skin may be removed from the list/ table 5.

4. In the animal stretching (under anesthesia or, without anesthesia) vs human yoga stretching (asana), how the role of cognition and stress can be ignored?

The stress during these stretching exercises is different in humans vs tiny animals (rats/mice).

5. Page 4, line 53: Can we delete the word, “therapeutic” from here. As the yoga etc is also useful to maintain the fitness in healthy participants.

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Reviewer #1: Yes: Rober Schleip, Dr. biol. hum.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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A systematic review of in vivo stretching regimens on inflammation and its relevance to translational yoga research

PONE-D-21-20626R1

Dear Dr. Munoz Vergara,

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Additional Editor Comments (optional):

Dear authors

Thank you very much for your detailed response, in which you successfully addressed all points addressed in the review process.

Reviewers' comments: