The Effect of Insulin-like Growth Factor-1 on Protein Composition and DNA Content in Damaged Somatic Nerves

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study investigates the effects of insulin-like growth factor-1 (IGF-1) on protein composition, DNA content, and functional recovery in damaged somatic nerves. The research is well-designed and addresses an important gap in nerve regeneration therapeutics. However, several areas require clarification or improvement to strengthen the manuscript's impact and reproducibility.

Major Comments:

• The study lacks a sham-operated control group, which is critical for distinguishing the effects of surgical trauma from the nerve transection itself. Including this group would strengthen the validity of the findings.
• Clarify why only two concentrations of IGF-1 (50 and 100 ng/kg) were tested. A dose-response curve with additional concentrations would provide more robust evidence for the therapeutic window of IGF-1.
• The manuscript mentions ANOVA but does not specify post-hoc tests used for pairwise comparisons. Provide details (e.g., Tukey’s, Bonferroni) to ensure transparency.
• Include exact p-values in figures/tables rather than stating "p < 0.05." This aligns with current reporting standards.
• The proposed role of MAPK/ERK signaling is speculative, as no direct evidence (e.g., Western blotting for phosphorylated ERK) is provided. Include molecular data to support this claim.
• Discuss potential off-target effects of IGF-1, such as systemic metabolic changes, which could indirectly influence nerve regeneration.
• The Raman spectroscopy methodology needs elaboration. Specify laser wavelength, resolution, and number of scans per sample to ensure reproducibility.
• For electrophoretic protein separation, clarify how protein bands were quantified (e.g., densitometry) and normalized.
• Discuss whether IGF-1 improved motor/sensory function in vivo (e.g., gait analysis, reflex tests), as this is clinically relevant.

 

Minor Comments:

• Figure 1: Label the y-axis clearly (e.g., "Total protein content, μg/mg tissue").
• Figure 3: Use consistent color schemes for IGF-1 concentrations across all panels.
• Table 1: Add units for AP duration (ms) and clarify if values are mean ± SEM.
• The Introduction could better highlight the clinical relevance of somatic nerve injuries (e.g., prevalence, current treatment limitations).
• Define abbreviations at first use (e.g., PAG, AP) for readability.
• Update references to include recent studies (2023–2025) on IGF-1 and nerve regeneration.
• Check citation formatting for consistency (e.g., some journal names are italicized, others not)

Author Response

Dear reviewer, thank you for your valuable comments, which will certainly improve our manuscript.

Major Comments:

Comments 1: The study lacks a sham-operated control group, which is critical for distinguishing the effects of surgical trauma from the nerve transection itself. Including this group would strengthen the validity of the findings.

Response 1: Thank you for pointing this out. We agree with this comment. We conducted a series of experiments with a sham-operated control group. Nevertheless, in this experimental group, there was no significant change in the studied parameters in the sciatic nerve compared with the control. Based on this, these data were not presented in the article. Most likely, changes in blood parameters may occur in a sham-operated control group, but it was not part of the objectives of our study.

Comments 2: Clarify why only two concentrations of IGF-1 (50 and 100 ng/kg) were tested. A dose-response curve with additional concentrations would provide more robust evidence for the therapeutic window of IGF-1.

Response 2: Thank you for pointing this out. In our study, the effect of only two concentrations of IGF-1 (50 and 100 ng/kg) on nerve regeneration was tested, since the results of intramuscular administration of IGF-1 at a concentration of 75 ng/kg have practically no significant differences compared with 50 ng/kg. Based on this, it was decided to consider the effect of only the minimum and maximum concentrations of IGF-1.

Comments 3: The manuscript mentions ANOVA but does not specify post-hoc tests used for pairwise comparisons. Provide details (e.g., Tukey’s, Bonferroni) to ensure transparency.

Response 3: Agree. We have changed this point. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test for pairwise group comparisons. We have updated the text of the manuscript and marked our revisions in yellow.

Comments 4: Include exact p-values in figures/tables rather than stating "p < 0.05." This aligns with current reporting standards.

Response 4: Agree. We have changed this point. The changed data are presented in the article.

Comments 5: The proposed role of MAPK/ERK signaling is speculative, as no direct evidence (e.g., Western blotting for phosphorylated ERK) is provided. Include molecular data to support this claim.

Response 5: Thank you for pointing this out. Unfortunately, at this stage we have not performed Western blotting for phosphorylated ERK and plan to conduct a more detailed study of the effect of insulin-like growth factor-1 on various signaling pathways in the future. Nevertheless, our data on changes in the protein composition, quantitative DNA content, functional activity, and physicochemical condition of protein-lipid fraction of somatic nerves are consistent with literature data indicating activation of the mitogen-activated protein kinase signaling pathway (MAPK/ERK), which regulates cytoskeletal protein synthesis and axonal growth (Bogucka-Janczi, K.; 2023; Nishida, K.; 2023).

Comments 6: Discuss potential off-target effects of IGF-1, such as systemic metabolic changes, which could indirectly influence nerve regeneration.

Response 6: Thank you for pointing this out. We agree with this comment. According to literature data, multiple injections of IGF-1 led to lower weight gain and adipose tissue deposition in mice in the Alzheimer's disease model, compared with control animals injected with saline solution (Kim Y.K., Jo D., Arjunan A. et al., 2024). In addition, IGF-1 affects the immune status by shifting the cytokine balance towards an anti-inflammatory response (Bæk O., Rasmussen M.B., Gerts T. et al., 2023).

Comments 7: The Raman spectroscopy methodology needs elaboration. Specify laser wavelength, resolution, and number of scans per sample to ensure reproducibility.

Response 7: Agree. We have changed this point. We have updated the text of the manuscript and marked our revisions in yellow.

The Raman spectrometer is equipped with a short-focus high-aperture monochromator with a focal length of less than 250 mm. A laser with a wavelength of 532 nm and a radiation power of 10 mW was used to excite the spectrum. The signal was detected using a CCD detector (1024 × 256 pixels). The spectra were recorded in the range of 100-3200 cm^-1 with a spectral resolution of at least 2 cm ^-1. At least 5 scans were performed for each sample to ensure statistical reliability and reproducibility of the results.

Comments 8: For electrophoretic protein separation, clarify how protein bands were quantified (e.g., densitometry) and normalized.

Response 8: Thank you for your valuable comment. The color intensity of the stripes corresponds to the amount of protein in each stripe. The gels were scanned using the gel documentation system software (GelDoc XR+; Bio-Rad, Hercules, California, USA). The program (Image lab) allows you to perform densitometry – measuring the intensity of the stripes. Image lab allows to automatically normalizing the total protein on the same gel.

Comments 9: Discuss whether IGF-1 improved motor/sensory function in vivo (e.g., gait analysis, reflex tests), as this is clinically relevant.

Response 9: By the 21st day of observation, motor activity does not recover, since it takes much longer to improve sensory function. Nevertheless, measuring the action potential, which is the most important indicator of restoring the functional activity of a nerve, makes it possible to register regeneration processes long before improving motor function, predicting the course and degree of restoration of the functions of the damaged nerve conductor. The objective of our study was to record the nerve impulse and the parameters of the action potential in the early stages after cutting, since it is during this period that the further course of the pathological process and the completeness of the restoration of normal nerve functioning will depend on the degree of regeneration.

Minor Comments:

Comments 1: Figure 1: Label the y-axis clearly (e.g., "Total protein content, μg/mg tissue").

Response 1: Agree. We have changed this point.

Comments 2: Figure 3: Use consistent color schemes for IGF-1 concentrations across all panels.

Response 2: Agree. We have changed this point.

Comments 3: Table 1: Add units for AP duration (ms) and clarify if values are mean ± SEM.

Response 3: We have changed this point. The values are mean ± SEM.

Comments 4: The Introduction could better highlight the clinical relevance of somatic nerve injuries (e.g., prevalence, current treatment limitations).

Response 4: Agree. We have changed this point and marked our revisions in yellow.

Comments 5: Define abbreviations at first use (e.g., PAG, AP) for readability.

Response 5: Agree. We have carefully reviewed the entire article.

Comments 6: Update references to include recent studies (2023–2025) on IGF-1 and nerve regeneration.

Response 6: Agree. We have changed this point and marked our revisions in yellow.

Comments 7: Check citation formatting for consistency (e.g., some journal names are italicized, others not).

Response 7: Agree. We have changed this point and marked our revisions in yellow.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Marina Parchaykina et al. are reporting their study on assessing the effect the IGF-1 on protein composition and DNA content in damaged somatic nerves. Overall, the study is well designed and presented. The following are specific comments the reviewer would like to share. 

1. In Figure 2 and Figure 4, Electropherogram data is presented to show the protein fractions. Would it be possible to have the Electropherogram data quantified for quantitative comparisons?
2. Figure 6 used the RS spectrum to indicate the orderliness of fatty acid chains forming a hydrophobic layer in the membrane. Would it be beneficial to have both the physicochemical conditions assessed before and after the damage that causes the complete loss of the nerve conduction in the distal nerve conductor?
3. Authors’ interpretations of the mechanism behind the damage of somatic nerves caused by the exposure to IDF-1 and preliminary validations are highly desired. In the current manuscript, only literature data is mentioned that the activation of MAPK/ERK signaling pathway can result in the increased expression of the genes related to the remyelination and recovery of the functional activity of damaged nerve fibers. An in depth discussion of the mechanism behind will further leverage the significance of the current story. 

Overall, a major revision would be recommended. 

Author Response

Dear reviewer, thank you for your valuable comments, which will certainly improve our manuscript.

Comments 1: In Figure 2 and Figure 4, Electropherogram data is presented to show the protein fractions. Would it be possible to have the Electropherogram data quantified for quantitative comparisons?

Response 1: The color intensity of the stripes corresponds to the amount of protein in each stripe. The gels were scanned using the gel documentation system software (GelDoc XR+; Bio-Rad, Hercules, California, USA). The program (Image lab) allows you to perform densitometry – measuring the intensity of the stripes. Image lab allows to automatically normalizing the total protein on the same gel.

Comments 2: Figure 6 used the RS spectrum to indicate the orderliness of fatty acid chains forming a hydrophobic layer in the membrane. Would it be beneficial to have both the physicochemical conditions assessed before and after the damage that causes the complete loss of the nerve conduction in the distal nerve conductor?

Response 2: The objective of our study was to register the conduction of a nerve impulse, the parameters of the action potential, and to study the physicochemical state of nerve fibers in the early stages after cutting, since it is during this period that the further course of the pathological process and the completeness of the restoration of normal nerve functioning will depend on the degree of regeneration. According to literature data, nerve fibers undergo degeneration over a short length of the proximal and throughout the distal segment. After cutting, a part of the axons located distal to the injury site loses its shell, consisting mainly of Schwann cells and extracellular matrix. This is due to a deficiency of retrograde neurotrophic factors coming from Schwann cells along nerve fibers. Without trophic axonal support, Schwann cells lose myelin and undergo structural and functional destruction within 48 hours [Ding Z., Jiang M., Qian J. et al., 2024; Zhai X., Wang Y.; 2024]. Such processes are associated with a violation of the central innervation, due to which a clear regulation and control over the functional state of the nerve conductors is carried out. Therefore, it seems advisable to study degenerative processes and the effect of IGF-1 in the proximal nerve segment, since it retains its connection with the central nervous system and, judging by its ability to conduct an action potential as a result of changes in the physico-chemical state of the nerve fiber membrane, it is possible to assess the intensity of regenerative processes.

Comments 3: Authors’ interpretations of the mechanism behind the damage of somatic nerves caused by the exposure to IDF-1 and preliminary validations are highly desired. In the current manuscript, only literature data is mentioned that the activation of MAPK/ERK signaling pathway can result in the increased expression of the genes related to the remyelination and recovery of the functional activity of damaged nerve fibers. An in depth discussion of the mechanism behind will further leverage the significance of the current story.

Response 3: Agree. We have changed this point and marked our revisions in yellow (4. Conclusions)

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have thoughtfully addressed several of the previous comments, improving the clarity and rigor of the manuscript. However, minor improvements are needed: clarify technical details (e.g., IGF-1 administration volume, RS-spectroscopy peak selection), expand the discussion on IGF-1's cell-type-specific mechanisms, and address limitations (e.g., lack of in vivo functional assays). Figures and tables should include scale bars and p-values, respectively, and the conclusion could better highlight translational potential. Grammatical edits (e.g., "nerve transaction" → "transection") are also recommended. With these revisions, the manuscript will be suitable for publication

Author Response

Comments 1: Minor improvements are needed: clarify technical details (e.g., IGF-1 administration volume, RS-spectroscopy peak selection)

Response 1: Dear reviewer, thank you for your valuable comments, which will certainly improve our manuscript. We have changed this point. We have updated the text of the manuscript and marked our revisions in green.

Comments 2: Expand the discussion on IGF-1's cell-type-specific mechanisms, and address limitations (e.g., lack of in vivo functional assays)

Response 2: Thank you for your valuable comment. We have updated the text of the manuscript and marked our revisions in blue.

Comments 3: Figures and tables should include scale bars and p-values, respectively

Response 3: Thank you for pointing this out. We have modified Table 1 to include p-values to emphasize this point. In Figures 2 and 4, we felt that the scale bars and p-values were unnecessary because they express a qualitative assessment, whereas the quantitative assessment is reflected in Figures 1, 3, 5, 7, and 8, where we already provide p-values. Figure 9 already includes p-values and scale bars.

Comments 4: The conclusion could better highlight translational potential.

Response 4: Thank you for your valuable comment. We have updated the text of the manuscript (4. Conclusions) and marked our revisions in green.

Comments 5: Grammatical edits (e.g., "nerve transaction" → "transection") are also recommended.

Response 5: Thank you for your valuable comment. We have updated the text of the manuscript and marked our revisions in green.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have addressed reviewer's comments and suggestions posted. No additional questions to report. 

Author Response

Dear reviewer, thank you for your time, valuable comments, and the attention you paid to our research.