Ambulatory Neuroproprioceptive Facilitation and Inhibition Physical Therapy Improves Clinical Outcomes in Multiple Sclerosis and Modulates Serum Level of Neuroactive Steroids: A Two-Arm Parallel-Group Exploratory Trial

Round 1

Reviewer 1 Report

The authors have certainly improved the manuscript following the revision. However, this reviewer still has a point of contention or misunderstanding with how participants were selected.

In the response to my initial comments, the authors clarified how study participants were selected by neurological profile, this is a very appropriate selection criteria.

My major concern is the fact that the authors claim that those study participants were "without severe cardiovascular dysfunction". However, all of the serum was collected from those that were recipients of CF-LVADs. By definition, a patient receiving an LVAD HAS a significant cardiac dysfunction. This discrepancy must be addressed. Furthermore, serum profiles between non-cardiac dysfunction patients and those with cardiac dysfunction are likely to be substantially different at basal.

Participants were recruited from the database of the Centre for diagnosis and 121 Treatment of Multiple Sclerosis, Kralovske Vinohrady University Hospital in 122 Prague. For the inclusion into the study they were selected by a neurologist based 123 on their anamnesis and clinical assessment (not on any serum analysis): both 124 genders, adults, diagnosed with definite multiple sclerosis (37) of moderate to 125 severe disease severity [Expanded Disability Status Scale (EDSS) (38) ranging from 126 3 to 7.5], and without severe orthopaedic or cardiovascular dysfunction or presence of another neurological disorder.

Author Response

Responses to the reviewers.
Reviewer 1:
The authors have certainly improved the manuscript following the revision.
However, this reviewer still has a point of contention or misunderstanding with
how participants were selected.
In the response to my initial comments, the authors clarified how study
participants were selected by neurological profile, this is a very appropriate
selection criteria.
My major concern is the fact that the authors claim that those study participants
were "without severe cardiovascular dysfunction". However, all of the serum
was collected from those that were recipients of CF-LVADs. By definition, a
patient receiving an LVAD HAS a significant cardiac dysfunction. This
discrepancy must be addressed. Furthermore, serum profiles between noncardiac
dysfunction patients and those with cardiac dysfunction are likely to be
substantially different at basal.
I appreciate your comment because the sentence in the paragraph Primary
Outcomes Measures was a mistake that we failed to notice . Our study did not
include any patients with CF-LVADs (Continuous-Flow Left Ventricular Assist
Devices) or LVAD (Left Ventricular Assist Device). Therefore the sentence “The
levels of serum hormones were assessed in continuous-flow left ventricular
assist device (CF-LVAD) recipients.” was deleted.
In this context, there is not any discrepancy in the description of inclusion
criteria now.
Participants were recruited from the database of the Centre for diagnosis and
121 Treatment of Multiple Sclerosis, Kralovske Vinohrady University Hospital in
122 Prague. For the inclusion into the study they were selected by a neurologist
based 123 on their anamnesis and clinical assessment (not on any serum
analysis): both 124 genders, adults, diagnosed with definite multiple sclerosis
(37) of moderate to 125 severe disease severity [Expanded Disability Status
Scale (EDSS) (38) ranging from 126 3 to 7.5], and without severe orthopaedic --
or cardiovascular dysfunction or presence of another neurological disorder.

Author Response File: Author Response.docx

Reviewer 2 Report

This is a report of an interesting study, with some clear strengths: the aim to examine the effects of two forms of physical therapy on levels of corticosteroids and neurosteroids and clinical outcomes in multiple sclerosis has significant value. The approach and methodology are sound, even though groups were unbalanced on some key demographic variables which complicated interpretation of the findings, and is a major weakness of the study. Some changes are recommended below to improve the manuscript.

• It is not clear from the title or the abstract that this is a study in patients with multiple sclerosis. Please consider adding to title: “…improves clinical outcomes in multiple sclerosis and….”; in addition, consider adding to abstract in line 20: “…on clinical outcomes in patients with multiple sclerosis….”;

• Add to line 22: “44 patients with multiple sclerosis…”.

• Abstract: please clarify the first sentence in the results section that there is a trend decrease in 7-oxo-DHEA. It is difficult to match this up with the results: is this referring to both groups? Because there is a significant effect of group according to results, in that the VRL group had significant decrease in this steroid.

• Line 67: please add in multiple sclerosis after “…a part of rehabilitation…”

• Participants: the groups differ on important characteristics which could have impacted the results: gender distribution, age, type of MS, disease duration. Were groups not balanced on these factors at the time of randomization? It appears that perhaps due to the drop-out after randomization, the groups were left un-balanced on these characteristics.

• Results: The results are at times difficult to follow and interpret. Please define any abbreviations in the text in the Methods section or in the Results section, rather than only in the Figure legends. E.g. MPAT; TUG.

• Reference #36 and #54 are the same.

Author Response

This is a report of an interesting study, with some clear strengths: the aim to examine the effects of two forms of physical therapy on levels of corticosteroids and neurosteroids and clinical outcomes in multiple sclerosis has significant value. The approach and methodology are sound, even though groups were unbalanced on some key demographic variables which complicated interpretation of the findings, and is a major weakness of the study. Some changes are recommended below to improve the manuscript.

It is not clear from the title or the abstract that this is a study in patients with multiple sclerosis. Please consider adding to title: “…improves clinical outcomes in multiple sclerosis and….”; in addition, consider adding to abstract in line 20: “…on clinical outcomes in patients with multiple sclerosis….”;

We added the information that study was realised on people with multiple sclerosis in the title and the abstract.

Add to line 22: “44 patients with multiple sclerosis…”.

Added.

Abstract: please clarify the first sentence in the results section that there is a trend decrease in 7-oxo-DHEA. It is difficult to match this up with the results: is this referring to both groups? Because there is a significant effect of group according to results, in that the VRL group had significant decrease in this steroid.

The paragraph was reformulated to be clear that the trend is in both groups, with a higher effect of Vojta’s reflex locomotion.

Line 76: please add in multiple sclerosis after “…a part of rehabilitation…”

Added.

Participants: the groups differ on important characteristics which could have impacted the results: gender distribution, age, type of MS, disease duration. Were groups not balanced on these factors at the time of randomization? It appears that perhaps due to the drop-out after randomization, the groups were left un-balanced on these characteristics.

Thank you for this comment. We added following explanation into the discussion “The next limitation is non-uniform distribution of participants within groups that could be caused by the drop-out after randomization. Although all participants fulfilled inclusion criteria and were divided into groups independently, they differed at some baseline characteristics, clinical outcomes and the level of neurohormones. On the other hand, this study brings information about personalized rehabilitation in ‘real-life’ settings.”

Results: The results are at times difficult to follow and interpret. Please define any abbreviations in the text in the Methods section or in the Results section, rather than only in the Figure legends. E.g. MPAT; TUG.

We went through the text and explained all the abbreviations when first used in the text. In the results, we tried not to use abbreviations where it was possible. We believe that now the text is better understandable.

Reference #36 and #54 are the same.

Thank you for this warning. We deleted the duplicity reference and renumbered citation in the whole text.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for the clarification and revision on the methods and inclusion criteria. I have no further concerns or comments.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This paper describes a study assessing the effects of two therapies for multiple sclerosis on functional and endocrine measures immediately after therapy and after a delay/washout period.  Although the research question is interesting and important, there are some suggestions provided as follows about the study design and analysis and write-up of this work.

1. In the Introduction, please provide more details about previous studies that justify the present one about hormones role in multiple sclerosis as well as in therapy.
2. What was the authors’ hypothesis? 
3. Were several assays run for all the different biochemical measures, and if so can the inter- and intra-assay variances be provided?  Were measurements done in duplicate?
4. A major point is not taking into consideration major variables that influence endocrine measures, and some functional measures as well.  It seems problematic to have included men and women together (albeit, there were much fewer men in study).  Additionally, the range in age from 19-71 is huge especially when considering endpoints that are age-related.  Also, how would length of time since multiple sclerosis symptoms or diagnosis influence results?  People were treated with corticosteroids (currently or in the past?), which would influence results. 
5. The description of the Results was confusing and lacking critical details. 

• There were no inferential statistics results provided; only providing p values is insufficient and makes it nearly impossible to understand and interpret these results. Similarly, it really is not that informative to describe specific p values when they are all under 0.05.  Please also do not include he p values in the Abstract.
• Consider how you would best tell your reader what the important results are graphically.

1. The Discussion could be more focused on the results of the present study in the context of the existing literature and hypotheses of the authors.  The order of paragraph here (and in the Introduction) could be improved so that they follow from the preceding point. 
2. In general, the paper needs to be thoroughly edited, especially by a native English speaker.  Please try to minimize the number of abbreviations that are used and do not have to be to make the paper easier to read and understand.

Reviewer 2 Report

In the present study, Angel and colleagues attempt to link physical therapy regimes to the production of neuroactive steroids. They suggest their data on 44 patients with MS demonstrate shows a decrease in DHEA following physical therapy intervention and improvements in symptomatic scoring.

The idea of modulating serum levels of neuroactive steroids through exercise or physical therapy is very attractive as it does not require pharmaceutical intervention and could help lessen pharmacological burden on patients with MS. However, in the present study the authors have not fully illustrated a substantial link between physical therapy and appreciable changes in serum DHEA, let alone other neuroactive substrates or modulatory compounds including serum cytokines. Furthermore, while they do show changes in pathological score at the M1 vs. M2 points, those changes are almost whole lost by examination M3, bringing into question the sustainability of physical therapy on both physiological parameters as well as observed disease improvement.

The authors nicely discuss the need for continued therapy and that acute therapy is likely not sufficient for improved MS outcome. 

However, my major point of contentions with the study are with respect to serum mediators as listed below.

1) The authors state in the methods that all participants were selected due to limited co-morbidities including no cardiovascular disease. However, all serum values were obtained form patients with CF-LVADs? Please explain.

2) The authors should include serum concentrations of other neuroactive mediators and markers of MS, such as serum neurofilament light, TNFa, IL1beta, neutralizing antibodies against IFNb, and analysis of OCB and other markers in CSF would be ideal (this reviewer realizes this may be beyond the scope of the present study).

Additionally, there are multiple minor grammatical and punctuation errors throughout the text. The manuscript should be reviewed by a native English speaker.