The Effect of Repetitive Transcranial Magnetic Stimulation on Cognition in Diffuse Axonal Injury in a Rat Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The ms by Hyeong-Min and Colleagues describes the effects of rTMS on a rat model of axonal injury, focusing on a spatial memory test and some immunohistochemical and western blot analyses.

The general idea is interesting but the execution has some major weaknesses that need to be addressed.

The abstract is quite naive: no need to report all the average or the dead animal, but a core results paragraph is missing. The same with the discussion.

The introduction is missing some recent literature in the field of rTMS in rodents: there are some interesting data on both 1 Hz and high-frequency rTMS effects on the PFC in mice that would be of help.

Results and figure legends often report the same text or concept. Not necessary.

The Figure quality is very low and also need some more graphical appeal.

Immunohistochemical data are meaningless in their present form; though quantitative analysis have been done in WB, a quantification of the immuno might help to interpret the main finding.

 

Comments on the Quality of English Language

A language revision would help the reading of the text.

Author Response

Response to reviewers

Thank you for your helpful guidance and Reviewer’s informative comments for our manuscript.

And we tried to more completely solve the questions and comments from the reviewer during the given period.

 

Thank you for giving us the opportunity to revise and resubmit this manuscript. We appreciate the time and details provided by you and reviewers. We have incorporated the suggested changes into the revised manuscript to the best of our ability. The manuscript has certainly benefited from these insightful comments and suggestions. We look forward to working with you and the reviewer to move this manuscript closer to publication in Neurology International.

 

I have responded specifically to comments and suggestions as shown below. To make the changes easier to be identified, I have numbered them along each number from the reviewer’s questions and comments.

 

=========================================

Reviewer 1:

The ms by Hyeong-Min and Colleagues describes the effects of rTMS on a rat model of axonal injury, focusing on a spatial memory test and some immunohistochemical and western blot analyses.

The general idea is interesting but the execution has some major weaknesses that need to be addressed.

 

1. The abstract is quite naive: no need to report all the average or the dead animal, but a core results paragraph is missing. The same with the discussion.

[Author’s reply] Thank you for your precise comment. We condensed the abstract and discussion for brevity and key points were emphasized.

 

2. The introduction is missing some recent literature in the field of rTMS in rodents: there are some interesting data on both 1 Hz and high-frequency rTMS effects on the PFC in mice that would be of help.

[Author’s reply] Thank you for your valuable comment. We included recent literature about effect of various rTMS protocols in rodents. (Ref 18. Seewoo BJ, Feindel KW, Etherington SJ, Rodger J. Resting-state fMRI study of brain activation using low-intensity repetitive transcranial magnetic stimulation in rats. Scientific Reports. 2018;8(1).)

 

3. Results and figure legends often report the same text or concept. Not necessary.

[Author’s reply] Thank you for your precise comment. We removed redundant expressions in the results and figure legends for conciseness.

 

4. The Figure quality is very low and also need some more graphical appeal.

[Author’s reply] Thank you for your precise comment. We redraw the figures for higher quality.

 

5. Immunohistochemical data are meaningless in their present form; though quantitative analysis have been done in WB, a quantification of the immuno might help to interpret the main finding.

[Author’s reply] Thank you for your precise comment. We included the quantitative analysis with WB in the result part as table 1 which showed the ratio of immunoreactivity in hippocampal area compared to total area.

 

 

================================================

 

Thank you so much again.

Sincerely yours,

 

Min-Keun Song. MD, PhD

Department of Physical & Rehabilitation Medicine

Chonnam National University Medical School & Hospital

#160, Baekseo-Ro, Dong-Gu, Gwangju, 61469, Republic of Korea

Telephone: + 82 62 220 5186 (Gwangju office)

FAX: + 82 62 228 5975 (Gwangju office)

E-mail: drsongmk@jnu.ac.kr

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

 

The main purpose of the study was to examine the influence of rTMS applied to rats on diffuse axonal injury (DAI) after head trauma due to sudden acceleration and deceleration. The secondary purpose was to measure BDNF, VEGF, and MAP2 in the hippocampus after the DAI. The main dependent variable was a timed water maze test done after brain damage and then again in a post-test after the rTMS treatment. BDNF, VEGF, and MAP2 were measured post-euthanasia. MRI data were also collected, I am not sure of exactly why and on how many rats however.

 

The main findings were that the rTMS group displayed lower (better) times in the water maze and had higher (better) BDNF, VEGF, and MAP2 levels post-euthanasia.

 

Overall, the study had a good design, was easy to understand, and appeared to be conducted carefully. It was also on an important topic and the result show promise for rTMS and concussion. It had few grammatical and topographical errors. I think the study adds to the literature on rTMS in animal models, will be of interest to readers of the journal, and has implications for rTMS in concussion in humans.

  I don’t think the study had any fatal flaws, but I do have some moderate and minor comments that the authors should address related to the writing style and organization along with some details of the methods and statistics. 

 

1. Overall, organization of the paper should be a little more systematic. In the abstract it seems that after the first purpose mentioned in lines 19 and 20 it should have mentioned a second purpose of measuring BDNF, VEGF, and MAP2.
2. Relatedly, MRI was not mentioned at all in the abstract, in the methods it was unclear why it was done, when it was done, on how many rats (all of them?) or if they just showed an example of an injured and non-injured rat in general. Then no mention of MRI was in the Discussion other than in the limitations. I assumed when first reading it that the MRI was to confirm brain injury and that it was equal between groups but this appears to not be the case. Please address this issue and give more details on MRI.
3. Was their any sort of a SHAM rTMS to the control group? This doesn’t seem as important as in human studies and it was between groups, but was anything done at all for a SHAM? Why were the parameters of TMS used chosen? Past study results?
4. How was resting motor threshold measured and determined to set the TMS intensity?? What muscle was used etc? More details are needed. Why was 50% of resting RMT chosen? Where was the coil placed on their head? What angle was it relative to the head?
5. Some paragraphs are only a single sentence or two in the Intro and Discussion. It makes them choppy. Consider combining them into larger paragraphs.
6. How was the sample size chosen? Was there a power analysis? Why only 9 rats per group?
7. The stats need to be written a little better. In section 2.7 spell out that it was a 2 group x 2 test Anova and what post hoc would be used (Fisher LSD?). For each statistical analysis spell out what dependent variables were analyzed by each separate type of statistical test.
8. There are many mistakes in the bibliography. Some journals abbreviated some are not. Capitalization is different for some titles compared with others. Titles of articles are sometimes captitalized in regard to the first letter of each word and sometimes not.

 

 

Comments on the Quality of English Language

minor proofreading needed

Author Response

Response to reviewer

Thank you for your helpful guidance and Reviewer’s informative comments for our manuscript.

And we tried to more completely solve the questions and comments from the reviewer during the given period.

 

Thank you for giving us the opportunity to revise and resubmit this manuscript. We appreciate the time and details provided by you and reviewers. We have incorporated the suggested changes into the revised manuscript to the best of our ability. The manuscript has certainly benefited from these insightful comments and suggestions. We look forward to working with you and the reviewer to move this manuscript closer to publication in Neurology International.

 

I have responded specifically to comments and suggestions as shown below. To make the changes easier to be identified, I have numbered them along each number from the reviewer’s questions and comments.

 

=========================================

 

 

Reviewer 2:

 The main purpose of the study was to examine the influence of rTMS applied to rats on diffuse axonal injury (DAI) after head trauma due to sudden acceleration and deceleration. The secondary purpose was to measure BDNF, VEGF, and MAP2 in the hippocampus after the DAI. The main dependent variable was a timed water maze test done after brain damage and then again in a post-test after the rTMS treatment. BDNF, VEGF, and MAP2 were measured post-euthanasia. MRI data were also collected, I am not sure of exactly why and on how many rats however.

 The main findings were that the rTMS group displayed lower (better) times in the water maze and had higher (better) BDNF, VEGF, and MAP2 levels post-euthanasia.

 Overall, the study had a good design, was easy to understand, and appeared to be conducted carefully. It was also on an important topic and the result show promise for rTMS and concussion. It had few grammatical and topographical errors. I think the study adds to the literature on rTMS in animal models, will be of interest to readers of the journal, and has implications for rTMS in concussion in humans.

  I don’t think the study had any fatal flaws, but I do have some moderate and minor comments that the authors should address related to the writing style and organization along with some details of the methods and statistics. 

 

1. Overall, organization of the paper should be a little more systematic. In the abstract it seems that after the first purpose mentioned in lines 19 and 20 it should have mentioned a second purpose of measuring BDNF, VEGF, and MAP2.

[Author’s reply] Thank you for your precise comment. We revised the abstract to encompass secondary purpose of this study.

 

2. Relatedly, MRI was not mentioned at all in the abstract, in the methods it was unclear why it was done, when it was done, on how many rats (all of them?) or if they just showed an example of an injured and non-injured rat in general. Then no mention of MRI was in the Discussion other than in the limitations. I assumed when first reading it that the MRI was to confirm brain injury and that it was equal between groups but this appears to not be the case. Please address this issue and give more details on MRI.

[Author’s reply] Thank you for your valuable comment. I explained the details of MRI evaluation in the Method part and included the DAI evaluation using MRI in the discussion.

“Three rats were used in each group to confirm the DAI lesion.”

“DAI often lacks clear radiological changes, even in cases with prominent neurologi-cal impairment [24]. MRI can evaluate DAI in patients with non-hemorrhagic lesions who show normal findings on CT scans. In the MRI images of patients with DAI, gliosis is ob-served as increased T2 signals may appear in areas susceptible to shearing injury, such as the grey-white matter junction, corpus callosum, and brainstem [25]. Several rodent mod-els of TBI share similar imaging properties with human DAI [26,27], and there have been reports that MRI is effective in evaluating the appropriateness of DAI rat models [28]. In our study, gliotic changes were observed in the grey-white matter junction and deep brain tissue on brain MRI taken after DAI modeling, confirming the appropriateness of the modeling..”

 

23 Was their any sort of a SHAM rTMS to the control group? This doesn’t seem as important as in human studies and it was between groups, but was anything done at all for a SHAM? Why were the parameters of TMS used chosen? Past study results?

[Author’s reply] Thank you for your precise comment. In this study, no sham stimulation was administered to the control group. And the parameters of low frequency (1Hz) and low intensity (50% of resting motor threshold) used in this study were referenced from previous research.

(Ref 18. Seewoo BJ, Feindel KW, Etherington SJ, Rodger J. Resting-state fMRI study of brain activation using low-intensity repetitive transcranial magnetic stimulation in rats. Scientific Reports. 2018;8(1).

Ref 20. Yoon KJ, Lee YT, Han TR. Mechanism of functional recovery after repetitive transcranial magnetic stimulation (rTMS) in the subacute cerebral ischemic rat model: neural plasticity or anti-apoptosis? Experimental Brain Research. 2011;214(4):549-556.)

These details are mentioned in the method section.

4. How was resting motor threshold measured and determined to set the TMS intensity?? What muscle was used etc? More details are needed. Why was 50% of resting RMT chosen? Where was the coil placed on their head? What angle was it relative to the head?

[Author’s reply] Thank you for your valuable comment. We included additional details regarding TMS intensity, coil placement and related aspects in the method section.

5. Some paragraphs are only a single sentence or two in the Intro and Discussion. It makes them choppy. Consider combining them into larger paragraphs.

[Author’s reply] Thank you for your precise comment. We rewrote the sentences into larger paragraphs in Introduction and Discussion part.

 

6. How was the sample size chosen? Was there a power analysis? Why only 9 rats per group?

[Author’s reply] Thank you for your valuable comment. We included the method section with sample sized calculation and put the limitation about the small sample size.

 

7. The stats need to be written a little better. In section 2.7 spell out that it was a 2 group x 2 test Anova and what post hoc would be used (Fisher LSD?). For each statistical analysis spell out what dependent variables were analyzed by each separate type of statistical test.

[Author’s reply] Thank you for your precise comment. We clarified the statistical method.

“The repeated measure ANOVA, followed by post hoc tests, was utilized to analyze the behavioral data, and the Chi-square test was performed on the Western blot data.”

 

8. There are many mistakes in the bibliography. Some journals abbreviated some are not. Capitalization is different for some titles compared with others. Titles of articles are sometimes captitalized in regard to the first letter of each word and sometimes not.

[Author’s reply] Thank you for your valuable comment. We rewrote the bibliography.

 

================================================

 

Thank you so much again.

Sincerely yours,

 

Min-Keun Song. MD, PhD

Department of Physical & Rehabilitation Medicine

Chonnam National University Medical School & Hospital

#160, Baekseo-Ro, Dong-Gu, Gwangju, 61469, Republic of Korea

Telephone: + 82 62 220 5186 (Gwangju office)

FAX: + 82 62 228 5975 (Gwangju office)

E-mail: drsongmk@jnu.ac.kr

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled "The Effect of Repetitive Transcranial Magnetic Stimulation on Cognition in Diffuse Axonal Injury Rat Model" investigates the impact of rTMS on cognitive function in a rat model of diffuse axonal injury (DAI). The study appears well-conducted, with appropriate methodology and statistical analyses. However, there are a few areas that could be improved:

1. Sample size: The study used a relatively small sample size of 18 rats divided into two groups of 9 each. A larger sample size would increase the statistical power and robustness of the findings. The authors do acknowledge this limitation in the discussion section.
2. Intensity of rTMS: The study used a fixed intensity of 50% resting motor threshold for the rTMS. It would have been informative to explore the effects of varying rTMS intensities to determine the optimal intensity for cognitive improvements in DAI.
3. Assessment of DAI severity: The evaluation of DAI was done only through brain MRI, without quantifying the severity of the injury. Assessing the severity of DAI would provide more context for interpreting the cognitive outcomes and rTMS effects.
4. Long-term effects: The study looked at short-term effects of rTMS on cognitive function. It would be valuable to assess the long-term sustainability of the cognitive improvements and any potential long-term effects of rTMS treatment.
5. Mechanistic insights: While the study showed increased expression of BDNF, VEGF, and MAP2 after rTMS, further exploration into the detailed molecular mechanisms underlying the cognitive improvements would strengthen the findings.
6. Clinical translation: The authors could discuss the potential clinical translation of these findings and any challenges or considerations in applying rTMS for cognitive deficits in human DAI patients.

 

Author Response

Response to reviewer

 

Thank you for your helpful guidance and Reviewer’s informative comments for our manuscript.

And we tried to more completely solve the questions and comments from the reviewer during the given period.

 

Thank you for giving us the opportunity to revise and resubmit this manuscript. We appreciate the time and details provided by you and reviewers. We have incorporated the suggested changes into the revised manuscript to the best of our ability. The manuscript has certainly benefited from these insightful comments and suggestions. We look forward to working with you and the reviewer to move this manuscript closer to publication in Neurology International.

 

I have responded specifically to comments and suggestions as shown below. To make the changes easier to be identified, I have numbered them along each number from the reviewer’s questions and comments.

 

=========================================

Reviewer 3:

 The manuscript titled "The Effect of Repetitive Transcranial Magnetic Stimulation on Cognition in Diffuse Axonal Injury Rat Model" investigates the impact of rTMS on cognitive function in a rat model of diffuse axonal injury (DAI). The study appears well-conducted, with appropriate methodology and statistical analyses. However, there are a few areas that could be improved:

 

1. Sample size: The study used a relatively small sample size of 18 rats divided into two groups of 9 each. A larger sample size would increase the statistical power and robustness of the findings. The authors do acknowledge this limitation in the discussion section.

[Author’s reply] Thank you for your precise comment. We included the method section with sample sized calculation and put the limitation about the small sample size.

 

2. Intensity of rTMS: The study used a fixed intensity of 50% resting motor threshold for the rTMS. It would have been informative to explore the effects of varying rTMS intensities to determine the optimal intensity for cognitive improvements in DAI.

[Author’s reply] Thank you for your precise comment. We included the method section with several references.

“The resting motor threshold used to set the TMS intensity was measured from the gas-trocnemius muscle. Previous studies have suggested that an intensity of 80% of the resting motor thresh-old affects motor recovery in a stroke rat model [20]. However, we considered inflammation in acute TBI. An-other study has shown that low-intensity rTMS reduces inflammation in acute TBI [21]. Therefore, we fixed the intensity at 50% of the resting motor threshold..”  

 

3. Assessment of DAI severity: The evaluation of DAI was done only through brain MRI, without quantifying the severity of the injury. Assessing the severity of DAI would provide more context for interpreting the cognitive outcomes and rTMS effects.

[Author’s reply] Thank you for your precise comment. We didn’t perform the assessment of severity of DAI in this study. We performed only Morris water maze test for measurement of cognitive impairment. So, we included the limitation about the lack of assessment of DAI.

 

4. Long-term effects: The study looked at short-term effects of rTMS on cognitive function. It would be valuable to assess the long-term sustainability of the cognitive improvements and any potential long-term effects of rTMS treatment.

[Author’s reply] Thank you for your precise comment. We included the limitation section with lack of the long-term effects of rTMS treatment and explained that further study should be needed.

 

5. Mechanistic insights: While the study showed increased expression of BDNF, VEGF, and MAP2 after rTMS, further exploration into the detailed molecular mechanisms underlying the cognitive improvements would strengthen the findings.

[Author’s reply] Thank you for your precise comment. We included the discussion section with more references.

“Despite these limitations, the current research identified significant alterations in escape latency fol-lowing low-frequency rTMS, thus highlighting the efficacy of the molecular-biological approach. In other words, our study demonstrated that BDNF, MAP2, and VEGF increased following low-frequency rTMS. BDNF supports learning and memory through synergistic interactions between neuronal activity and syn-aptic plasticity [47], MAP2 influences neuronal plasticity through its involvement in the initial differentiation of neurons [48], and VEGF is recognized for its neuroprotective effects against excitotoxicity associated with neurodegeneration [49]. BDNF, MAP2, and VEGF each play a unique role, but they also interact to protect neurons and maintain cognitive function. Elevated expression levels of BDNF, VEGF and MAP2 after rTMS may align with the neuroprotective impact of low-frequency rTMS and suggest a molecular biomecha-nism for the protective effect against the inflammatory process following acute brain inju-ry. Therefore, there is a need for further research into strategies for enhancing cognitive function in patients with DAI through the appropriate regulation and interaction of these molecules.”

 

6. Clinical translation: The authors could discuss the potential clinical translation of these findings and any challenges or considerations in applying rTMS for cognitive deficits in human DAI patients.

[Author’s reply] Thank you for your precise comment. We included the discussion section just as below;

“Additionally, it is essential to understand potential challenges or safety issues that may arise when applying these techniques to humans.”

 

================================================

 

Thank you so much again.

Sincerely yours,

 

Min-Keun Song. MD, PhD

Department of Physical & Rehabilitation Medicine

Chonnam National University Medical School & Hospital

#160, Baekseo-Ro, Dong-Gu, Gwangju, 61469, Republic of Korea

Telephone: + 82 62 220 5186 (Gwangju office)

FAX: + 82 62 228 5975 (Gwangju office)

E-mail: drsongmk@jnu.ac.kr

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made a lot of changes and have done an adequate job of addressing all of my prior comments. The manuscript is much improved.

Comments on the Quality of English Language

Minor english proofreading and minor formatting proofing are needed

Author Response

Thank you for reviewing my manuscript.

English editing was done and I included the certification of English editing service.

 

Thank you so much again.

Sincerely yours,

 

Min-Keun Song. MD, PhD

Department of Physical & Rehabilitation Medicine

Chonnam National University Medical School & Hospital

#160, Baekseo-Ro, Dong-Gu, Gwangju, 61469, Republic of Korea

Telephone: + 82 62 220 5186 (Gwangju office)

FAX: + 82 62 228 5975 (Gwangju office)

E-mail: drsongmk@jnu.ac.kr

Reviewer 3 Report

Comments and Suggestions for Authors

The authors responded to my comments well. Thank you.

Author Response

Thank you for your reviewing.

 

Thank you so much again.

Sincerely yours,

 

Min-Keun Song. MD, PhD

Department of Physical & Rehabilitation Medicine

Chonnam National University Medical School & Hospital

#160, Baekseo-Ro, Dong-Gu, Gwangju, 61469, Republic of Korea

Telephone: + 82 62 220 5186 (Gwangju office)

FAX: + 82 62 228 5975 (Gwangju office)

E-mail: drsongmk@jnu.ac.kr