Pathomorphological Features of Diabetic Myopathy in Comorbid Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review this interesting manuscript. The topic is potentially relevant, and the authors provide extensive morphological observations; however, in its current form, the manuscript has several important methodological, interpretative, and presentation-related limitations that should be carefully addressed before it can be considered for publication:

1. The sample size is small. The study includes only 28 rats in total, with 7 animals per group, which limits the robustness of the findings and makes the results more vulnerable to biological variability.
2. There is an important inconsistency in the description of the animals. The abstract refers to male rats, whereas the Methods section reports female rats. This discrepancy is not trivial, as sex may influence both metabolic and stress responses.
3. The experimental model is highly specific and has limited translational value. The study focuses on the masseter muscle in a streptozotocin-induced diabetes model combined with chronic immobilization stress, which may not adequately reflect the broader clinical spectrum of diabetic myopathy in humans.
4. The study is largely morphological and lacks functional validation. Although the structural damage is extensively documented, no functional endpoints are provided, such as muscle strength, contractile performance, electrophysiological testing, or behavioral outcomes.
5. Some biological conclusions appear overstated. Terms such as ferroptosis, necroptosis, and partial denervation are used rather assertively, although these mechanisms are inferred mainly from morphological and ultrastructural findings without specific molecular confirmation.
6. The statistical analysis is relatively basic for the complexity of the dataset. Given the number of groups and the large number of morphological and ultrastructural parameters assessed, the analytical approach appears rather simple and does not clearly address multiplicity.
7. The timing of tissue collection is not fully linear across groups. Samples were collected at day 56 in Groups I, II, and IV, but at day 42 from the onset of chronic immobilization stress in Group III, which makes comparisons less straightforward.
8. Overall, the study remains descriptive rather than truly mechanistic. It documents damage convincingly, but it does not fully clarify the molecular causal pathways underlying the observed changes.
9. The title and overall framing appear broader than the actual scope of the work. The manuscript refers to diabetic myopathy in comorbid conditions, whereas the comorbidity investigated is limited to one highly specific and artificial condition, namely chronic immobilization stress.
10. The concept of stress is somewhat oversimplified. In this study, stress essentially corresponds to repeated immobilization, which combines physical restraint, neuroendocrine stress, and muscle disuse. Therefore, the conclusions may reflect immobilization/disuse effects as much as stress itself.
11. There is a potential risk of observational bias in the morphological assessment. Much of the study relies on histology and electron microscopy, and it is not clearly stated whether the evaluations were performed in a blinded manner.
12. Immunohistochemistry is mentioned among the methods, but its contribution to the reported results is unclear and appears marginal compared with the histological, ultrastructural, and morphometric analyses.
13. The terminology is at times too assertive. Expressions such as partial denervation, ferroptosis, necroptosis, and axonopathy would require stronger mechanistic or molecular support to be used with such confidence.
14. The manuscript has several formal and editorial weaknesses. These include the inconsistency regarding the sex of the animals, uneven English style, and sections that still appear insufficiently refined.
15. The study does not include models more closely related to type 2 diabetes. Since streptozotocin primarily reflects an insulin-deficient condition, the relevance of the findings to the more common clinical setting of type 2 diabetes remains limited.
16. No corrective or reversibility strategy was tested. The manuscript documents the lesions but does not explore whether the observed changes can be prevented, attenuated, or reversed.
17. In the combined group, the causal role of each factor remains difficult to disentangle. The worsening may result from several overlapping mechanisms, including hyperglycemia, hypercortisolemia, disuse, and microvascular injury.
18. No direct quantitative markers of inflammation or oxidative stress were measured, despite the extensive discussion of ROS-related and inflammatory mechanisms.
19. The study relies mainly on a final time point. As a consequence, it provides limited information on the temporal evolution of the lesions.
20. No dose-response analysis of immobilization stress is provided. Only one stress protocol was used, so it remains unclear whether the damage progresses according to stress duration or intensity.
21. The anatomical generalizability is limited. The masseter muscle has specific structural and functional features, and the conclusions cannot automatically be extended to skeletal muscle as a whole.
22. Metabolic characterization is incomplete. Blood glucose, HbA1c, and cortisol were measured, but a broader metabolic profile would have strengthened the link between systemic status and tissue damage.
23. At several points, the discussion risks circular reasoning: morphological findings are observed and then assigned to specific biological pathways mainly on the basis of the literature, without direct demonstration in the present model.
24. The reproducibility of the morphometric analyses is insufficiently documented. No clear information is provided regarding intra-observer or inter-observer variability.
25. The manuscript presents many results, but the hierarchy of endpoints is not clearly defined. It is therefore difficult to distinguish the main outcomes from the exploratory observations.
26. The work resembles a rich morphological atlas more than a rigorous translational or mechanistic study. While the descriptive component is substantial, the overall scientific solidity and clinical relevance remain limited.

Comments on the Quality of English Language

The English language requires substantial revision throughout the manuscript. There are numerous grammatical errors, inconsistencies in tense and style, awkward phrasing, and several sentences that are difficult to follow. In addition, the overall presentation would benefit from careful editing by a fluent English speaker or a professional language editing service in order to improve clarity, precision, and readability.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 

In my opinion, the article submitted for review “Pathomorphological Features of Diabetic Myopathy in Comorbid Conditions” raises an interesting and important topic, but requires significant corrections:

Generally, please standardize the group nomenclature throughout the text and figures. Either groups I, II, II, etc., group 1,2 etc., or control, SIDM, etc.

My main concern regarding the submitted article concerns the research methods. The stains used by the authors are standard in histology, but for precise analysis of blood vessels, histological stains are insufficient. Especially in the case of capillaries, immunohistochemical reactions for the presence of vascular endothelial markers, such as the CD31 protein, also known as PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1), should be performed.

L: 131-152 Incorrect conclusion. According to Figure 1a, in terms of blood glucose levels, group III differs significantly from group IV (control) – likely p<0.05 – no explanation '*'. All abbreviations and symbols used must be explained in the figure descriptions. Please clarify how the muscles were sliced, in what orientation relative to the muscle fibers – along the fibers or across the fibers? What muscle cross-sections were used for which analyses? How many areas were analyzed in each preparation?

L: 132-133 Please clarify: muscle samples ‘will be fixed’ or ‘were they fixed’?

L: 154-155 Question: Were muscle fragments for electron microscopy fixed only in osmium tetroxide or also in glutaraldehyde?

L: 176 The presentation of the data requires changes. Although very popular, the mean ± SD notation is inappropriate. Arithmetic mean is a measure of location, and standard deviation is a measure of variation. They measure completely different characteristics of a sample and there is no point in linking them with the ± sign. The proper notation is mean ± SEM (or just SE) or possibly without the ± sign.

L: 181-182 “In Group III, carbohydrate metabolism parameters tended to increase but did not differ significantly from control values (Figure 1a–b)” – wrong conclusion. According to Fig. 1a, in the case of blood glucose levels, group III differs significantly from group IV (control) – likely p<0.05 – no explanation '*, **, ***'.

Fig. 1, 3 All abbreviations and symbols used must be explained in the Figure descriptions.

Fig. 2. Please provide a figure showing the stained capillaries and the walls and lumen of the capillaries, as well as a figure on the basis of which the authors analyzed the number of capillaries - as mentioned by the authors in the "Material and Methods" chapter L: 151-152. Please also explain the inconsistency of  (L: 205-206) 'Group 1 (SIDM + CIS, a–c, d), Group 2 (SIDM, d–f)' what does Fig 2D actually represent - group I or II muscle?

L: 226-226 ‘In contrast, the cross-sectional area of venules increased compared to controls due to lumen dilation’ – Comment: According to Table 1, statistically significant differences are only found in the ‘Luminal Area’.

L: 226-227 “Microvessel remodeling led to the formation of arteriovenous anastomoses (Figure 2c, f)” - Where do these conclusions come from? In both Fig. 2c and 2F, the authors did not mark arteriovenous anastomoses.

L: 229-231 It is difficult to comment on the results presented in this section, as none of the figures provided by the authors demonstrate precise capillary differentiation in muscle cross-sections. To accurately determine the number of capillaries (as mentioned earlier in the review), more detailed immunohistochemical analyses are necessary.

Please explain L: 28 male or L: 98 female?

L: 31 What immunohistochemical methods were used? Where is their description and figures?

Please avoid citing tables or figures in the 'Discussion' section (L: 464).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for your revised manuscript and for addressing several of the previous comments. The revised version is improved in some important formal and methodological aspects, including correction of the inconsistency regarding the sex of the animals, removal of the unsupported reference to immunohistochemistry, and clearer reporting of some morphometric and statistical procedures. These revisions are appreciated.

However, the main critical issue remains unresolved. In its current form, the manuscript is still fundamentally a descriptive morphological study, whereas parts of the text continue to present the work in a more mechanistic and pathophysiological way than the data actually support. The study convincingly documents structural damage of the masseter muscle, microcirculation, and neuromuscular junctions under the modeled conditions, but it does not provide functional validation or direct molecular evidence sufficient to support several of the stronger mechanistic interpretations.

For this reason, the manuscript can only be considered further if the authors are willing to substantially revise its conceptual framing and conclusions. In particular, the paper should be clearly repositioned as a morphological and morphometric characterization of tissue damage, rather than as a mechanistic demonstration of specific biological pathways.

The following points should be addressed very carefully:

1. The overall framing of the manuscript should be downgraded from mechanistic/pathophysiological claims to a descriptive morphological study. The title, abstract, discussion, and conclusions should consistently reflect what the data truly demonstrate: structural alterations and their distribution/severity across groups.
2. Terms such as “ferroptosis”, “necroptosis”, “partial denervation”, and “axonopathy” should be used with much greater caution. Without direct molecular or functional confirmation, these should be presented as morphologically suggestive features rather than definitive mechanistic conclusions.
3. The conclusions must be substantially softened. At present, several statements still go beyond the evidence provided. The manuscript supports the observation of damage patterns, not a definitive mechanistic explanation of the underlying biological processes.
4. The descriptive nature of the study should be explicitly acknowledged as a limitation in both the Discussion and the Conclusions. The lack of functional validation and the absence of specific molecular markers should not be deferred only to future work; they should be clearly recognized as current limitations of this manuscript.
5. The translational implications should be moderated. The experimental model remains highly specific, and the findings should not be overextended beyond the actual scope of the study.
6. The Discussion should be revised to avoid overinterpretation. At several points, morphological findings are linked to specific molecular pathways mainly through literature-based reasoning rather than direct demonstration in the present model. This distinction must be made explicit.
7. The manuscript would benefit from a clearer hierarchy between observed findings and interpretative hypotheses. Readers should always be able to distinguish what was directly shown from what is inferred.
8. The English language and overall presentation still require further refinement to improve precision and avoid overly assertive wording.

In summary, the revised manuscript is improved, but the core issue remains the mismatch between the descriptive nature of the evidence and the strength of the mechanistic claims. If the authors are willing to fully realign the manuscript with the actual level of evidence, the paper may still be reconsidered. Otherwise, in its present conceptual form, the manuscript remains difficult to support for publication.

Comments on the Quality of English Language

The English language requires substantial revision throughout the manuscript. There are numerous grammatical errors, inconsistencies in tense and style, awkward phrasing, and several sentences that are difficult to follow. In addition, the overall presentation would benefit from careful editing by a fluent English speaker or a professional language editing service in order to improve clarity, precision, and readability.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In my opinion, the work submitted for review may be accepted for publication in the form presented.

Author Response

Thank you for the reviewer's suggestions.