Stimulation of Synthesis and Lysis of Extracellular Matrix Proteins in Fibrosis Associated with Lymphedema

Round 1

Reviewer 1 Report

The study is interesting, but the clumsy structure and poor English are major flaws that should be addressed

Furthermore, the following issues should be addressed with a reply point-by-point.

• Extensive syntax and grammar editing should be accomplished. I suggest a careful revision of the entire manuscript to improve readability.

• After reading the aim of this study, I would suggest to include the keyword “Lymphedema” and “fibrosis” in the title.  It is appropriate and should be done. Moreover, they are hot topic and would give more visibility to your manuscript, if accepted. The title could be totally changed, if deemed appropriate.

Materials and methods

• How many patients were enrolled?

• The patient were classified as stage II. Which classification method was used?

• The patients were affected with unilateral or mixed bilateral and unilateral lymphedema?

• Page 2 “Other causes of fibrosis….. “ are exclusion criteria? If yes they should be also mentioned in the appropriate section

• The paragraph “development” is the description of a case. This should be avoided. Instead, the treatment protocol of this study should be described thoroughly

• Replace “statistical treatment” with “statistical analysis”

• The statistical analysis was repeated two times. Please remove the second one.

• Paragraph “treatment"        It is right to mention the original treatment (i.e. cervical, supraclavicular), but the lower limb application should be clearly described in this section.

• Page 4. “The bar chart in the figure shows the significant increase in the relative area of collagen III in the different regions of the dermis after treatment”. Please mention the number of the figure

Table 1.

• Please clarify the meaning of letters a and b. Please add a column in this table to show the p-values.
• Please revise the rows and columns / number and words to improve ease of reading.

Discussion

• The bibliography should be expanded in order to sustain this section. I recommend to expand the bibliography as follows:

 1.      Page 8: “The literature offers few data on the histological evaluation of lymphedema but re-ports important inflammatory processes in filariasis, which should be evaluated better in cases of primary lymphedema. Different physiopathological processes may be involved in the development of lymphedema. However, stimulation of the lymphatic system seems to be the main common pathway of treatment and reversal in such cases.” Please add references

2.     page 9: “The dynamics of the mobilization of macromolecules from the ECM to the blood circulation almost mandatorily passes through the lymphatic system. This system is the functional reserve of the venous system and edema occurs when this reserve is sur-passed. Thus, it is a problem involving microcirculation and both arterial and venous circulation can interfere in these dynamics.”

Please support this paragraph discussing the followings

Cigna E, et al. Lymphatico-venous anastomosis in chronic ulcer with venous insufficiency: A case report. Microsurgery. 2021 Sep;41(6):574-578. doi: 10.1002/micr.30753.

Franzeck UK, et al. Microangiopathy of cutaneous blood and lymphatic capillaries in chronic venous insufficiency (CVI). Yale J Biol Med. 1993 Jan-Feb;66(1):37-46. 

3) page 9 “It is difficult to treat and cure fibrosis when there is a failure of the lymphatic sys-tem. To date, there is no cure, but improvement can be achieved, as we have demon-strated in our publications.”

Please add the reference of your publications

• Page 8: “The present study was conducted to confirm clinical findings in the treatment of lymphedema, with the achievement of normality or near normality in all clinical stages, including clinical stage III”. Please clarify this sentence. An inclusion criteria was stage II lymphedema

• Page 8. Please clarify and revise the syntax of the following paragraph to improve readability

The treatment employed in the present study interferes in the physiopathology of such cases, mobilizing macromole-cules through the lymphatic system. Thus, the therapeutic strategy in the reversal of various causes of fibrotic processes involves interference in the physiopathology of each disease.

Conclusions

• Please revise the syntax of this section to improve readability

Author Response

Attached reply reviewer 1.

Author Response File: Author Response.doc

Reviewer 2 Report

The Authors conducted a clinical trial analysing the changes in collagen and elastic fibers and thickness of the epidermidis and dermis in histological samples taken from the skin of one patient before and after intensive treatment of stage II primary lower limb lymphedema using Godoy Method.

The study revealed significant changes in all fields evaluated: a reduction in the thickness of the epidermidis and dermis, an increase in elastin, a reduction in type I collagen and an increase in type III collagen.

The topic of the study is very interesting and timely. The physiopathological mechanism of lymphedema inevitably lead to an irreversible subversion of the structure of the lymphatic vessels and the activation of adipogenetic and inflammatory process that induce an increase of the skin thickness and fibrotic tissues. As in all diseases, lymphedema’s treatment is highly personalized on the single patient and on alterations and characteristics of his lymphatic system. In the Literature, few studies offer histological data in patients with lymphedema, in particular before and after conservative therapies. It is of crucial importance to know the impact of these therapies to improve the management and control the clinical course of this chronic disease.

Anyway, the manuscript needs major revisions before it can be considered for publication.

COMMENTS:

I think the title “Stimulation of synthesis and lysis of extracellular matrix proteins” is not appropriate for this study that appears to be a case report.

The language is poor throughout the text and the manuscript needs to be edited by a professional language editing service or by an English native speaker.

In the Abstract,

• In the Background:

Since the authors analyse the modification of fibrotic tissue in patients affected by primary lymphedema, the background should be focused on the features and pathological process of this disease.

• In the Objective:

The aim of the study is not clear. Patients with primary lymphedema could be undergone to several treatment, such as conservative or surgical therapies. Authors should indicate which type of treatment they refer to.

• In the Methods, the authors should provide more information:
  • Specify the number of patients enrolled. Was it a sequential enrolling or did they adopt inclusion/exclusion criteria??
  • Duration of the study and period of enrolment.
  • Which variables were included?

• In the Results:
  • Some patient’ features such as stage of lymphedema, unilateral or bilateral lymphedema, BMI and comorbidities.

• The conclusion is not appropriated for a study including only one patient with 10 histological fields as a study group and should be

In the Main text:

In the “Introduction”:

• For a more comprehensive reading of the manuscript, the authors should spend more words on the pathophysiological process of fibrotic tissue’s formation in the lymphedematous limb, the modality of treatment and the description of the Godoy technique used in the study, citing the relevant reference.
• Authors stated that: “In recent years, novel concepts have emerged in the treatment of lymphedema, with the proposal of the normalization or near normalization of the affected limb in all clinical stages, including elephantiasis, and this treatment modality has been adapted to the clinical reversal of fibrosis”. This sentence is not clear. In which terms do they refer to the “normalization of the affected limb”?
• The aim of the study is not clear. Patients with primary lymphedema could be undergone to several treatment, such as conservative or surgical therapies. Authors should indicate which type of treatment they refer to.

In the “Methods”

• How many patients were enrolled? Authors should specify the enrolment of only one patient.
• In which part of the lower limb were biopsies performed?
• Provide a more adequate description of the methods and timing of the conservative treatment.
• Authors stated that “Inclusion criterion. Stage II lower limb lymphedema with intense fibrosis and absence of the Godet sign.” Which clinical classification do the authors refer to? Please, cite the relevant reference.
• Inclusion and exclusion criteria should be reviewed. They should underline that the study analyses only one case of primary lymphedema. How many patients were excluded?
• Authors reported in the section “Development” that “After the clinical normalization of the fibrosis and the return of elasticity, a second biopsy was performed 0.5 cm parallel and lateral to the first incision (Figure 2).” Which parameters were considered to define the level of normalization of the fibrosis and tissue’s elasticity?

Any way this paragraph contains methods and results and is reported in a form that confuses the normal reading of the manuscript. Authors should decide whether remove it or submit the study as case report.

• Why do authors choose the variant of lymphatic therapy using cervical stimulation and not the lower limb manual lymph drainage using hands according to Godoy method?

In the “Results”

• “The bar chart in the figure shows the significant increase in the relative area of collagen III in the different regions of the dermis after treatment.” Indicate the number of the Figure which is referred to.
• Information about the maintenance of treatment should be given.

In the Discussion:

• “The present study was conducted to confirm clinical findings in the treatment of lymphedema, with the achievement of normality or near normality in all clinical stages, including clinical stage III (elephantiasis).”

“We are able to bring the affected limb to within or close to normality and without fibrosis.”

These sentences should be modified because the study analyses sample of only one patient with lower limb primary lymphedema stage II. Authors cannot confirm the clinical reversal of fibrosis through specific physiological stimulation of the lymphatic system in all stages of lymphedema. Moreover, these results should be reanalysed in a more numerous study group and compared with a control group to obtain a statistical value.

In the Conclusion:

• The present conclusion is not appropriated for a study including only one patient and should be The reproducibility, safety and efficacy of this approach needs further studies to be proven, particularly in the short- and long-term follow up. As extensively described, lymphedema is a chronic, progressive and worsening disease which necessitates chronic therapies.

Author Response

Attache reply reviewer 2.

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

Dear Authors, the following comments of the first round of review  (answered in the response letter) were not addressed in the text.

Please modify the text accordingly

• How many patients were enrolled?

Reply: This is the first in a series of 10 patients.

• The patient were classified as stage II. Which classification method was used?

Reply: The one used by the International Society of Lymphology.

I could not find any answer to the following comments of the first round of review

• Extensive syntax and grammar editing should be accomplished. I suggest a careful revision of the entire manuscript to improve readability.

Conclusions

• Please revise the syntax of this section to improve readability

2nd round of review

The structure of the manuscript is not conventional. It is a mix between a case report and an original article. Please modify the structure and subtitle according to instructions for authors.

Please have the revised manuscript reviewed and edited by a native English speaker with editing experience before resubmission.

"The selection of samples was random" The authors should clarify if random is referred to any part of the body or specific ones

Author Response

Reply

2nd round of review-Reviewer 1

The structure of the manuscript is not conventional. It is a mix between a case report and an original article. Please modify the structure and subtitle according to instructions for authors.

 Reply: Are 10 histological fields evaluation, I believe the layout of the article is correct because it is easy and compressible for readers.

Please have the revised manuscript reviewed and edited by a native English speaker with editing experience before resubmission.

 Reply: Its ok, send MDPI service.

"The selection of samples was random" The authors should clarify if random is referred to any part of the body or specific ones

Reply: Explain in the text.

Thanks

Author Response File: Author Response.doc

Reviewer 2 Report

The authors improved the quality of their work. I feel that the manuscript is worthy of publication in the present form. 

Author Response

Attached revision II.

Thanks

Author Response File: Author Response.doc