Effect of Pulsed Electromagnetic Field Stimulation on Splenomegaly and Immunoglobulin E Levels in 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis Mouse Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. What is the reason for choosing the model of  DNCB-induced atopic dermatitis (AD)?

2. What is the significance of studying the effects of pulsed electromagnetic fields (PEMF) on immune factors.

3. It may be necessary to increase the detection of relevant inflammatory factors to evaluate the therapeutic effect.

4. What is the therapeutic effect of PEMF on AD？

Author Response

reviewer 1

 

1. What is the reason for choosing the model of DNCB-induced atopic dermatitis (AD)?

Answer : This is a very important and insightful question. 2,4-Dinitrochlorobenzene (DNCB) is a well-known agent used to induce atopic dermatitis (AD) because it can reliably produce all six representative symptoms of AD: pruritus and dead skin cells, lichenification, excoriation, erythema, edema, and erosion. Numerous studies, including the ones we cited, have employed DNCB to induce AD. In our previous histological studies, all symptoms of AD were observed with the use of DNCB. Therefore, we believe DNCB is the optimal agent for inducing the AD model.

 

2. What is the significance of studying the effects of pulsed electromagnetic fields (PEMF) on immune factors.

Answer : As mentioned in our paper, there is currently no method to completely cure atopic dermatitis (AD). Due to the autoimmune nature of AD, patients often suffer from a significantly reduced quality of life. Therefore, we have devised alternative or complementary methods to drug therapy. One such method is pulsed electromagnetic field (PEMF) stimulation. If PEMF stimulation can influence the modulation of immune factors in the body, it could emerge as an effective new treatment for AD.

Moreover, modern society is increasingly aging. Various conditions such as skin necrosis from diabetes, rheumatoid arthritis, and systemic inflammation due to obesity are all closely related to inflammation. Thus, by researching the immune-modulatory and anti-inflammatory effects of PEMF stimulation, we aim to propose a novel treatment applicable to a variety of diseases. This is the ultimate goal and significance of our research.

 

3. It may be necessary to increase the detection of relevant inflammatory factors to evaluate the therapeutic effect.

Answer : Your observation is indeed very accurate. Our research team is currently devising various methods to detect related inflammatory markers in future studies. However, it is important to note that the anti-inflammatory mechanisms of PEMF have not yet been elucidated in previous studies. Additionally, except for our preliminary research, there are no cases where PEMF has been applied to AD. Therefore, we focused on histological results in our preliminary studies.

The paper we have submitted now represents the next step, focusing on the immunological outcomes as foundational research. If this sequential approach progresses as planned, we will be able to proceed with our next study. As you pointed out, we are planning future research, and if successful results are obtained, we will submit our findings to your esteemed journal with more significant outcomes.

 

4. What is the therapeutic effect of PEMF on AD？

Answer : Since our study is the first to apply PEMF stimulation to AD, there are still many outcomes that need to be elucidated. Therefore, our research aims to sequentially verify these therapeutic effects.

1. From our preliminary research, we have observed a reduction in abnormal lesions and inflammation depth in skin tissue.
2. Additionally, in the currently submitted paper, we have noted a decrease in serum IgE levels, spleen hypertrophy, and splenocytes, which play a central role in the immune response.
3. These two studies collectively demonstrate the anti-inflammatory effects of PEMF from both histological and immunological perspectives.

To provide a detailed explanation of PEMF stimulation, a clear understanding of its mechanisms is necessary. Unfortunately, the mechanisms of PEMF are not yet fully elucidated, which is why they were not included in our paper. We are conducting extensive research to uncover the mechanisms of PEMF.

However, the effects of PEMF on blood flow enhancement, vasodilation, and anti-inflammatory properties have been validated in some previous studies. Therefore, we investigated whether PEMF could produce anti-inflammatory effects in autoimmune diseases such as AD. While it is true that there is a lack of evidence regarding the mechanisms of PEMF, our study represents the first application of PEMF to AD and is in the early stages of elucidating its mechanisms. We appreciate your understanding of this context.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this paper, the authors examined the effects of PEMF on DNCB-induced IgE production. They found the treatment with PEMF inhibited the increase in spleen weight and IgE protuction.  These results were interesting but I have several concerns.

Major points:

1) The Methods were unclear.  The authors stated PEMF treatments were 14 days, but figure 1 showed "7weeks".

2) The concentration of DNCB was stated at 0.1-1%.  How did the authors sensitized?

3) Was IgE measured   total IgE or antigen-specific IgE?

4) The authors should determined the igE levels before PEMF treatment.

5) Did PEMF treatment induce the release of glucocorticoid

6) There were no Hz -dependency.  The authors should check more low Hz.

Minor comment:

1) Trypan blue assay was basal way.  Therefore, FIg. 3 was not necessary. If possible, the authors should show histochemical analysis of spleen specimen.

Author Response

reviewer 2

1) The Methods were unclear. The authors stated PEMF treatments were 14 days, but figure 1 showed "7weeks".

Answer : Your observation is very precise and accurate. It is my mistake with no excuse. As per your suggestion, I have corrected the notation from "7 weeks" to "8 weeks" in Figure 2. Your feedback has been incredibly valuable to me. Thank you very much.

2) The concentration of DNCB was stated at 0.1-1%. How did the authors sensitized?

Answer : This is a very important question. We encountered several challenges with DNCB sensitization, and I would like to explain the reasons and solutions.

 

1. We applied 1% DNCB topically to all experimental animals uniformly.
2. However, the degree of natural recovery varied among individuals, resulting in inconsistent AD symptoms across subjects.
3. Therefore, after the initial application of 1% DNCB, we adjusted the concentration of DNCB for each individual based on their specific symptom severity, while maintaining a constant volume of 200μl.
4. From the fourth week onwards, to ensure uniform AD symptoms across all subjects, we made fine adjustments to the DNCB concentration. We adjusted the concentration between 0.1% and 1% to induce consistent AD symptoms in all individuals.

 

When applying 1% DNCB, the AD symptoms induced varied significantly between individuals. Thus, we aimed to induce AD uniformly and observe the effects of PEMF stimulation.

 

 

3) Was IgE measured total IgE or antigen-specific IgE?

Answer : We measured total IgE for the following reasons:

1. There is no specific antigen test for DNCB.
2. As you correctly pointed out, a specific antigen test for DNCB would be more accurate. However, specific antigen tests are typically used for diagnosing patients by considering their medical history, symptoms, age, constitution, dietary habits, and other test findings. In our study, the experimental animals had controlled variables such as age, gender, genetics, and environment. Therefore, we believed that measuring total IgE to assess the increase or decrease in IgE specific to the single antigen DNCB was accurate.

 

4) The authors should determined the igE levels before PEMF treatment.

Answer : That is an excellent point. Our research team intended to measure IgE levels before PEMF treatment as you suggested. However, the amount of blood required for IgE measurement was insufficient with either retro-orbital bleeding or tail bleeding. In other words, we could not collect enough blood for IgE measurement without sacrificing the experimental animals. We considered splitting the experiment into two parts to measure IgE levels before PEMF treatment, but this approach would compromise the reliability of the data due to differences between the individual subjects.

 

Your point is very precise and well-taken, but we hope you understand the practical difficulties in conducting the experiment under these circumstances.

 

 

5) Did PEMF treatment induce the release of glucocorticoid

Answer : Our experiment did not investigate glucocorticoids. It is true that glucocorticoids, as steroid hormones, contribute to anti-inflammatory effects. However, our study has not yet included endocrine analysis. In future experiments, we plan to incorporate your valuable feedback and conduct endocrine analysis along with investigations of various inflammatory markers. Thank you for your insightful feedback.

 

6) There were no Hz dependency. The authors should check more low Hz.

Answer : Our study is the first to apply PEMF stimulation to AD, and it is true that there are still many elements that need to be elucidated. Additionally, the mechanisms of PEMF are not yet fully understood. Therefore, we prioritized verifying the effects of PEMF within the 10-100Hz range, considering the biological frequencies. As you pointed out, lower frequencies might maximize the effects, but frequencies above 100Hz could also yield better results. We will continue to research the effects of various frequencies based on biological frequencies.

 

Minor comment:

 

1) Trypan blue assay was basal way. Therefore, FIg. 3 was not necessary. If possible, the authors should show histochemical analysis of spleen specimen.

Answer : As you correctly pointed out, this is a fundamental method. However, we aimed to visually verify the ratio of live cells to dead cells for the following reasons:

1. Splenocytes are extremely sensitive cells, and if not observed immediately after extraction, the number of dead cells increases rapidly. Therefore, we standardized the extraction time across all subjects and presented visual evidence of this process.
2. In the sham group, a significantly higher number of splenocytes was observed compared to other groups. Thus, we needed to determine the appropriate dilution ratio. Through numerous trials, we found the optimal dilution ratio and aimed to provide evidence of this.

While it might appear to be unnecessary data, we hope you can understand our intentions once more.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper is interesting and in my opinion it could be published in Applied Sciences, but before accepting for publication some minor revisions suggested below should be made.

It should be clarified how the number of mice in particular groups was estimated, as it seems that 5 mice in one group is not sufficient to obtain statistical significance of differences of values of analyzed parameters between particular groups.

In figure 1 it is not clear if the mice were inserted in the coil in a cage (one per cage). It should be also described what was the material used for making the cages.

It should be explained why the particular physical parameters of magnetic field stimulation were selected (frequency, magnetic induction, time of a single exposure and total number of exposures in therapeutic cycle).

In Figures 4-6 the presentation of statistical significance is not sufficiently clear - it should be clarified what was the statistical significance of differences between AOO, PEMF 15 Hz, PEMF 75 Hz and Control groups and Sham group, as well as AOO, PEMF 15Hz and PEMF 75 Hz groups and Control group.

The references are relatively old - only 8 of 32 references have been published in last 10 years. It is necessary to add some more actual references. Moreover most of references are not presented in uniform style according to Journal's guidelines - it must be corrected. Especially in many references the names of the Journals are lacking - instead of Journal's names the statements such as: Wiley Online Library, Taylor & Francis, journals.plos.org, aafp.org, nature.com etc. are used - it is not acceptable in scientific article.

The second paragraph of the Conclusions is not directly supported by the obtained results and in my opinion it should be moved to Discussion section.

 

Author Response

reviewer 3

1.The paper is interesting and in my opinion it could be published in Applied Sciences, but before accepting for publication some minor revisions suggested below should be made.

It should be clarified how the number of mice in particular groups was estimated, as it seems that 5 mice in one group is not sufficient to obtain statistical significance of differences of values of analyzed parameters between particular groups.

 

Answer : Your observation is very sharp and important. Each group consisted of five subjects, but our results showed clear differences even within these small groups because we performed multiple repeated measurements. However, as you pointed out, the sample size is indeed a limitation. In future studies, we plan to increase the number of subjects per group based on our initial findings to conduct more detailed research. Since our study is the first to apply PEMF to AD, we hope you can consider it as an initial step for future research.

 

2.In figure 1 it is not clear if the mice were inserted in the coil in a cage (one per cage). It should be also described what was the material used for making the cages.

 

Answer : Figure 1 focuses on describing the PEMF system and coil, emphasizing the shape of the coil and the configuration of the system. We used the most commonly utilized plastic cages for housing the experimental animals. These cages are universally used by researchers, and to our knowledge, there are no papers that specifically describe the materials used to make the cages. Could you please explain the reason for the need to describe the materials used in the cages?

 

 

3.It should be explained why the particular physical parameters of magnetic field stimulation were selected (frequency, magnetic induction, time of a single exposure and total number of exposures in therapeutic cycle).

 

Answer : This is a good point. However, the optimal treatment parameters and mechanisms for PEMF are still unknown. Therefore, we aimed to detect the effects within the 10-100Hz frequency range based on biological frequencies. While other prior studies do propose specific frequencies and exposure times, these claims are specific to different diseases and mechanisms, making them not entirely applicable to our study. Since there is no clear evidence for the optimal stimulation time and treatment cycle yet, we referred to the parameters from previous studies and will continue to research in various ways to find the optimal treatment parameters.

 

4. In Figures 4-6 the presentation of statistical significance is not sufficiently clear - it should be clarified what was the statistical significance of differences between AOO, PEMF 15 Hz, PEMF 75 Hz and Control groups and Sham group, as well as AOO, PEMF 15Hz and PEMF 75 Hz groups and Control group.

 

Answer : We have described the statistical significance between groups as accurately as possible. AOO, as mentioned in the manuscript, refers to the application of Acetone and Olive Oil without DNCB, and it was included to clarify any potential issues related to AOO. As a result, it did not differ from the Control group. Additionally, no significant differences were observed in the PEMF stimulation groups based on different frequencies. Therefore, while the anti-inflammatory effects of PEMF on AD were observed as stated in the manuscript, the lack of significant differences between frequencies suggests that further studies are needed to determine if similar results occur with other inflammatory markers. However, significant differences were observed between the PEMF groups and the Sham group that did not receive PEMF stimulation. These results indicate that PEMF stimulation accelerated the recovery of AD symptoms compared to the Sham group, where AD was induced similarly.

Could you please clarify the specific reasons why you believe the presentation of statistical significance is insufficient? If I have misunderstood your intent, this response may not address your concerns. I would appreciate it if you could provide more detailed information about your expectations.

 

5. The references are relatively old - only 8 of 32 references have been published in last 10 years. It is necessary to add some more actual references. Moreover most of references are not presented in uniform style according to Journal's guidelines - it must be corrected. Especially in many references the names of the Journals are lacking - instead of Journal's names the statements such as: Wiley Online Library, Taylor & Francis, journals.plos.org, aafp.org, nature.com etc. are used - it is not acceptable in scientific article.

 

Answer : It is true that some of our references are outdated. We aimed to find the most fundamental sources in immunology, primarily selecting papers with high citation counts. This is because the factors we are observing are based on the foundational principles of immunology, which do not change significantly with recent research. If this response does not align with your intent, I apologize. Could you please explain what issues arise from citing older papers?

 

We appreciate your meticulous feedback. Although we used the Mendeley program to format our references according to the Applied Science journal's requirements, it appears the program did not meet the journal's specifications. Following your suggestion, we have revised all cited references to match the journal's format. Please review the updated references. Thank you again for your valuable feedback.

 

6. The second paragraph of the Conclusions is not directly supported by the obtained results and in my opinion it should be moved to Discussion section.

 

Answer : We agree with your opinion because we initially included this information in the discussion section. However, the second paragraph of the conclusion emphasizes that since our study is the first to apply PEMF stimulation to AD, further research is needed to determine the optimal parameters. Therefore, we aimed to conclude our paper by highlighting the limitations of our study and the need for improvements, as well as explaining the significance of our research.

 

We would appreciate it if you could consider our reasoning and review the conclusion section accordingly.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The figure 3 is no meaning. If the authors wanted to show, the real photo should be added with the data.  I think this data is better to be shown as a supplimental  figure.

Author Response

I will accept your opinion and remove Figure 3. Thank you for your review.

Author Response File: Author Response.pdf