Non-Healing Wound Model in Diabetic C57BL/6 Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study establishes and validates a streptozotocin-induced model of diabetic non-healing wounds in C57BL/6 mice, supported by comprehensive histological, immunofluorescence, and morphometric analyses. The topic is clinically relevant, the experimental design is rigorous, and the data validate the suitability of this model for preclinical investigations. The manuscript can be improved by addressing the following points:

1. Please specify the age of the experimental mice, the precise dimensions of the excisional wound, the blood glucose cutoff value for defining diabetes, and the duration of established hyperglycemia prior to wounding. The sample size (n) for each experimental group and time point should also be clearly reported.
2. Elaborate on the mechanisms by which diabetes impairs wound contraction (e.g., myofibroblast dysfunction) and early angiogenesis (e.g., high glucose-induced endothelial injury). A brief comparison between the current model and the authors’ previously reported ischemic wound model is recommended, highlighting the main improvements and limitations.
3. Provide detailed information on antibody incubation conditions and imaging parameters for CD31 and CK14 immunofluorescence staining. Confirm whether normality and homogeneity of variance were assessed prior to one-way ANOVA.
4. Define all abbreviations (e.g., LNL, LVL) in the abstract as well as in the main text. Ensure consistent verb tense throughout the manuscript and correct minor grammatical errors.

 

Author Response

Dear Reviewer,
We sincerely thank you for your comments and suggestions. We have taken them into careful consideration and have revised the manuscript accordingly.

1. The following clarifications are provided: the mice were 4 weeks old at the start of the experiment; the full-thickness excisional wound was square-shaped, measuring 1 × 1 cm (area 1 cm²) with a depth of 3 mm; diabetes was defined as a blood glucose level exceeding 15 mmol/L, measured on days 10 and 12 post‑streptozotocin administration; the wound was created on day 10, i.e. after 10 days of established hyperglycemia (streptozotocin injections were performed on days 1–5); and the number of animals per group and time point was as follows: control – day 5 (n=5), day 10 (n=5), day 15 (n=6); diabetes – day 5 (n=6), day 10 (n=5), day 15 (n=5), with a total of 32 mice (16 per group). All these details have been incorporated into the respective subsections of "Materials and Methods".

2. We have expanded the discussion on how diabetes impairs wound regeneration, focusing on the chronicity of inflammation and the upregulation of matrix metalloproteinases, which degrade the extracellular matrix. This aspect has been further developed in the "Discussion" section.

3. All additional data requested have been incorporated into the "Materials and Methods" section under the subsection "Immunofluorescence Staining". Primary antibodies against CD31 (1:100) and CK14 (1:500) were incubated overnight at 4 °C, and secondary antibodies (1:1000) for 1 h at room temperature. The exposure time was 11.5 ms for the red channel, 17.257 ms for the green channel, and 11.5 ms for the blue channel. Regarding the assessment of normality, we tested all samples using the Shapiro–Wilk test and the Kolmogorov–Smirnov test. The analysis revealed that, due to the small group sizes (n = 5–6) and pronounced inter-individual variability in animal responses (particularly in the diabetes group), the data deviated significantly from a normal distribution (p < 0.05). Consequently, we entirely revised our statistical approach. We abandoned parametric analysis of variance (ANOVA) and Fisher's LSD post-hoc test. Instead, all comparisons were recalculated using the non-parametric Kruskal–Wallis test, followed by Dunn's post-hoc correction for multiple comparisons. The corresponding changes have been made in the "Materials and Methods" section, as well as in the figures and their respective legends.

The abstract contains no abbreviations; all major abbreviations used in the text have been defined at their first occurrence.

We hope that the revised version now meets your expectations and we remain at your disposal for any further clarification.

Yours sincerely, Elena I. Morgun

Reviewer 2 Report

Comments and Suggestions for Authors

I commend the authors for their manuscript entitled “Diabetic Non-Healing Wound Model in C57BL/6 Mice.” The study is interesting and may appeal to the journal's readership. However, I have several comments that should be addressed or clarified before the manuscript can be recommended for publication.

Given the scope of Methods and Protocols, the Materials and Methods section appears insufficiently detailed. Since the journal emphasizes methodological reproducibility, the authors are kindly asked to provide the following experimental details:

1. The introduction did not show the justification for this study.
2. Methods and Results
   1. The authors did not specify the total number of mice used in the study. This information is essential for animals and should be clearly stated.
   2. The authors should provide the age, sex, and average body weight of the mice in each experimental group, either in the main manuscript or as supplementary material.
   3. The authors did not specify how many animals were used per group, including the number of animals euthanized at each time point after wound induction.
   4. How did the authors confirm that diabetes was successfully induced following the low-dose streptozotocin treatment? The criteria used to define diabetic status should be clearly stated.
   5. On which day were the wounds created relative to the first day of streptozotocin treatment? In addition, do the 5-, 10-, and 15-day time points refer to days after the first streptozotocin injection or days post-wounding?
   6. The wound depth was not clearly described. The methodology reports wound area in square millimeters as length × breadth but does not indicate the wound's depth or anatomical extent.
   7. The authors monitored the wounds for only 15 days in a study described as a non-healing chronic wound model. This duration may not be sufficient to robustly model chronic wound healing. The authors should justify the use of this time frame, particularly given the contribution of the panniculus carnosus layer, which can support wound closure within this period even in STZ-treated mice. Maybe the authors could have used a different tissue. Moreover, by day 15, there was no significant difference between the treated and control groups across the reported experiments.
   8. Day 0 was not mentioned in the methodology, although it appears in the Results section on page 7, line 270. The authors should define Day 0 treatment clearly in the experimental design.
   9. The authors should provide evidence of ethical approval for the animal study, including the relevant approval number and institutional ethics committee details. If permitted by journal policy, a copy of the ethical clearance should be included as supplementary material.
3. Results and figures
   1. Figure 1 appears to be repeated twice. The figure presented on page 4, line 162, should likely be labeled as Figure 2.

1. 1. For this type of wound-healing study, representative macroscopic images of the wounds at each time point for both experimental groups are essential. These images would improve the clarity of the study and allow readers to better relate the macroscopic wound progression to the microscopic and histological findings.
   2. The authors should provide the equations used to calculate the reported wound-healing metrics, including wound bed closure, relief index, re-epithelialization, and any other derived parameters.
   3. Across the reported metrics, there is no significant difference between the treated and untreated control groups. Therefore, the novelty and methodological advantage of this model are not sufficiently clear. The authors should clarify what distinguishes this study from previously reported diabetic wound models and explain the added value of the proposed protocol.
   4. The discussion is insufficient as the authors did not show how their findings relate to the published works. Also, the discussion and conclusion lack a discussion of the study's limitations and a future perspective. 

Overall, the manuscript addresses a relevant topic, but the issues outlined above should be addressed.

Author Response

Dear Reviewer,
We sincerely thank you for your valuable comments and suggestions. We have taken them into careful consideration and have revised the manuscript accordingly.
1. The absence of a single model that simultaneously accounts for the diabetic background, square wound geometry with angular inhibition of contraction, and an optimized size of 1 × 1 cm that excludes epimorphic regeneration served as the rationale for the present study; this rationale is discussed in more depth in the Introduction.
2. We have clarified all methodological details and incorporated them into the relevant subsections of "Materials and Methods". Specifically, we now explicitly state that a total of 32 male C57BL/6 mice (4 weeks old, average body weight 20–22 g) were used, with 16 animals in the control group and 16 in the diabetic group. The number of animals per group and per time point (days 5, 10, and 15 post‑wounding) is provided in Table 1 (control: n=5, 5, 6; diabetes: n=6, 5, 5 for days 5, 10, and 15, respectively). Diabetes was defined as blood glucose >15 mmol/L, measured on days 10 and 12 after the first streptozotocin injection; only animals meeting this criterion were included in the diabetic group. The wound (1 × 1 cm, 3 mm deep) was created on day 10 after the first STZ injection, and the 5‑, 10‑, and 15‑day time points refer to days after wounding, with day 0 representing the day of wound creation. This has been clearly defined in the text.

Regarding the duration of observation, we chose the 15‑day time frame because preliminary experiments and literature data indicate that in this model the most pronounced differences in inflammatory and regenerative parameters occur within the first two weeks, while beyond this period secondary complications (e.g., infection or excessive contraction) may confound interpretation. Although no significant differences were observed between groups at day 15 in some assays, this time point remains critical for assessing late-stage healing dynamics, and we have now provided a rationale for this choice in the Discussion. Finally, we confirm that all animal procedures were approved by the IDB RAS Institutional Animal Care and Use Committee (Protocol No. 74 of September 2023 and Protocol No. 88 of December 2024); a copy of the approval will be provided as supplementary material if required by the journal. All the above clarifications have been integrated into the revised manuscript.

3. The novelty and methodological advantages of our model (standardized wound shape, reproduction of delayed inflammation during the proliferative phase in contrast to classical models, and suitability for preclinical testing), a detailed comparison of our results with published data on the role of neutrophils, reactive oxygen species, and metalloproteinases in chronic wounds, including direct comparison with other models, as well as the study limitation (observation up to day 15) and future perspectives (use of the model for evaluating new therapeutics and extending the observation period) — all of this is thoroughly described in the Discussion section.

We hope that the revised version now meets your expectations and we remain at your disposal for any further clarification.

Yours sincerely, Elena I. Morgun

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled "Diabetic Non-Healing Wound Model in C57BL/6 Mice" aspires to present an animal model of non-healing wounds that recapitulates the pathogenesis of human chronic wounds. However, the use of diabetic mice for studying chronic wounds has been introduced more than forty years ago and is still being used; I am just quoting randomly some examples of the numerous studies, some of which use the same mice strain as the present manuscript and the same method for diabetes induction: Weringer et al. Acta Endocrinol (Copenh). 1982 Jan;99(1):101-8. doi: 10.1530/acta.0.0990101; Tsuboi et al. J Dermatol. 1992 Nov;19(11):673-5. doi: 10.1111/j.1346-8138.1992.tb03757.x; Ko et al. Diabetes Metab J. 2011 Jun;35(3):226-35. doi: 10.4093/dmj.2011.35.3.226. I could not find any originality in the present manuscript, and the authors did not make any attempt to show which is the novelty of their study. For example, a presentation of previous studies using similar models is absolutely necessary in the Introduction, following a thorough search of the literature. The authors cite only eight other references, which corresponds to a poor presentation of the literature; moreover two of the references are self-citations, i.e., self-citation rate is 25%. The authors refer to their previous attempts to develop models of ischemic non-healing wounds by using flaps with impaired circulation, however it is not clear what is the relation of these studies with the current manuscript. A discussion of the current findings in relation to other studies using diabetic mice would clearly be more useful for the reader.

The main conclusion of the authors as stated in the Abstract is that their "findings confirm the relevance of this model to human pathology and support its applicability for preclinical studies of drugs aimed at promoting wound regeneration" (lines 22-24). This statement, however, needs to be slightly understated. For example, since up to 90% of excisional wounds in mice close by contraction (due to the panniculus carnosus) while cutaneous wounds in humans heal mainly by formation of granulation tissue and re-epithelization (see Zomer and Trentin, J Dermatol Sci. 2018 Apr;90(1):3-12. doi: 10.1016/j.jdermsci.2017.12.009), a putative drug enhancing contraction would be very efficient in the mouse model but less efficient in humans.

Finally, the term "non-healing wound" used throughout the text (including the title) is somewhat misleading, since even in diabetic animals wound closure has been achieved at the final time-point of the study. The term "delayed healing" is obviously more accurate.

 

Author Response

Dear Reviewer,

We thank you for your valuable comments, which have helped us improve the manuscript. We have made the following revisions:

1. Novelty and literature review. We agree that diabetic models have been used for decades. In the revised Introduction, we have added the historical references you suggested (Weringer et al., 1982; Tsuboi et al., 1992; Ko et al., 2011), expanded the literature review (from 8 to 24 references), and clearly stated the novelty of our approach – this is the first model combining three parameters: STZ-induced diabetes in C57BL/6 mice, a square 1×1 cm wound to minimize contraction, and a size sufficient for testing constructs but below the threshold for WIHN induction. The self-citation rate has been reduced.

2. Relationship with previous work. We have clarified that our previous ischemic model served as a foundation but had limitations (suture artifacts, bleeding), which have been addressed in the new diabetic model.

3. Comparison with other studies. In the Discussion, we have provided a detailed comparison of our histological parameters (re-epithelialization index, relief index, angiogenesis, granulation tissue layers) with data from classical models, demonstrating that our model better reproduces the delay in inflammation.

4. Contraction in mice vs. healing in humans. We acknowledge this limitation, have added a discussion of the differences in healing mechanisms with reference to Zomer and Trentin (2018), toned down our conclusions, and now recommend the model for preclinical studies with appropriate caveats rather than claiming direct predictive value for humans.

5. Terminology. We agree that "non-healing" is imprecise and have replaced it with "delayed healing" throughout the text, including the title.

All changes have been thoroughly reflected in the revised manuscript.

Kind regards,
Elena I. Morgun

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing the comments. Good luck

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been fundamentally revised. The revised manuscript is significantly improved compared to the original version.