Neuroprotective Effects of Myricetin on PTZ-Induced Seizures in Mice: Evaluation of Oxidation, Neuroinflammation and Metabolism, and Apoptosis in the Hippocampus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

To the AAs

The neurobiological basis of epilepsy remains poorly understood. The Authors investigated on the role of caspase-8 (marker of neuronal apoptosis) and neuron-specific neuronal nuclear antigen (NeuN) (marker of neuronal survival) in an experimental murine model [pentyltetrazole (PTZ)-induced seizures)].  PTZ-induced seizures caused significant oxidative stress and inflammation, leading to neuronal damage. Biochemical analyses showed oxidative stress (MDA, SOD, GSH) and elevated proinflammatory cytokines levels (IL-1β, IL-6, TNF-α), along with a significant increase in caspase-8-positive and a decrease in NeuN-positive neurons in the hippocampus and other brain regions related with seizure severity. Myricetin (3,3′,4′,4,5,5′,7-hexahydroxyflavone), a flavonoid mainly extracted from Myrica rubra shows neuroprotective and anticonvulsant properties confirmed by its ability to regulate the BDNF-TrkB signalling pathway and modulate GABA receptor activity. The AAs tested the hypothesis that Myricetin administration would increase the synthesis of biological substances thereby enhancing GABAergic neurotransmission. The results were compared with the effects of a long-known ASM, i.e., VPA. Myricetin and VPA were reported to reduce oxidative stress markers and neuronal damage with fewer damaged neurons observed in the hippocampus, dentate gyrus, and other brain areas compared to the PTZ-only group. The topic is stimulating, the design (6 experimental groups including controls) appears to be appropriate. Laboratory methods and procedure appear to be sound. The cited literature is appropriate.

 

Minor points

1.      Title. In my personale opinion the title does not convey the full novelty of the results. I would encourage the AAs to rethink of it including the novelty of myricetin;

2.      I would suggest to take care that each of the tables and figures had a self-explanatory title with the proper abbreviations list.

3.      Although the Ms. is well written and well-organized, a few misspelling and style imperfections can be rarely spotted.  I would encourage the AAs to slightly re-edit and shorten the text (particularly Discussion and Conclusions) in order to improve the whole Ms readability and impact to the scientific community if published.

 

Comments on the Quality of English Language

1.      Although the Ms. is well written, a few misspelling and style imperfections can be rarely spotted.  I would encourage the AAs to slightly re-edit and shorten the text (particularly Discussion and Conclusions) in order to improve the whole Ms readability and impact to the scientific community if published.

Author Response

Dear Reviewer,

Thank you for your thorough and insightful review of our manuscript! We greatly appreciate your positive feedback and the valuable comments you provided. Your observations have been instrumental in helping us refine and enhance the quality of our work. We are grateful for the time and effort you invested in evaluating our study, and we believe that the revisions made in response to your comments have significantly strengthened our manuscript.

 

• Q1: In my personale opinion the title does not convey the full novelty of the results. I would encourage the AAs to rethink of it including the novelty of myricetin;

Answer: In response to your valuable comments and suggestions, we have made several revisions to our manuscript. We are very grateful for your feedback, which has significantly helped us improve our work. Specifically, we have revised the title of the manuscript to better highlight the action of Myricetin and its properties as discussed in the study.

• Q2: I would suggest taking care that each of the tables and figures had a self-explanatory title with the proper abbreviations list.

Answer: Thank You very much for your careful reviewing.

Corrected. We have refined the graphical materials, corrected the accuracy of the titles for all tables and figures, and included clear abbreviations with an accompanying list.

• Q3: Although the Ms. is well written and well-organized, a few misspelling and style imperfections can be rarely spotted. I would encourage the AAs to slightly re-edit and shorten the text (particularly Discussion and Conclusions) in order to improve the whole Ms readability and impact to the scientific community if published.

Answer: In this manuscript, we have tried to describe all our results as fully as possible and to discuss and debate our results to bring more attention to this interesting topic in the scientific community and to increase the citation rate for your journal. All, even the smallest details we have carefully checked and added, for the reader's full understanding of the present topic. However, after re-analysing the text of the manuscript according to your recommendations, particularly the Discussion and Conclusions sections, we have shortened some parts of the text to reduce its length. Thank you for your valuable comments!

 

Q4: Comments on the Quality of English Language

• Although the Ms. is well written, a few misspelling and style imperfections can be rarely spotted. I would encourage the AAs to slightly re-edit and shorten the text (particularly Discussion and Conclusions) in order to improve the whole Ms readability and impact to the scientific community if published.

Answer: Thank You very much for your careful reviewing.

Corrected. We have thoroughly reviewed the manuscript for English language quality    and, thanks to your recommendations, addressed any linguistic shortcomings.

Thank You so much!

 

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors main role was to investigate the potentially neuroprotective effect of Myricetin in PTZ-induced acute epileptic seizures. The manusctipt provides results of promising neuroprotective capabilities of Myrecitin pretreatment. The Authors performed complex behavioral, morphological and molecular research to test their hypothesis about Myricetin. The design of the study was well thought and the illustration of the experimental design (Fig.1) is really nice.

Mayor comments/questions:

1. The size of the manuscript is way too extensive, it has to be reduced a little bit. This could be done by simplifying some sentences and explanations because in some parts the manuscript it is a little bit over-explaned or a repetitive.

2. The description of the statistics say that: „All data are presented as the mean ± standard deviation, representative of six replicates.”  How was the control data made out of six replicates when the tissue homogenates were examined? If the 3 h, 24h, 3 day and 5 day groups all had six control replicate that means at least 24 animals supposed to be in the control group, but according to the manuscript the control group had 12 animals.

3. If it is possible a supplementary file should be made with the all the measurment results because the datas showed in the manuscript was the representative of six replicates but according to the results (and materials) the mortality of group III was 25% and of group IV was 20 % which means that out of 60 animals of each group 45 animals survived and (should’ve) presented results from group III and 48 animals from group IV.

4. The figure legends in Figure 3 and Figure 6 the text indicates that: „The bars represent mean ±SD” but there are no error bars in those graphs. Please correct those figure descriptions.

5. The figures (graphs) should be harmonised because now everything is all over the place. Some graphs have an upper and right side frame but other graphs don’t. The colour of the bars in each group should be consistent throughout the manuscript because for example in Fig. 3 and Fig 6 Group II has a light red colour but in Fig. 4 and Fig 5 Group II is purple. That could be confusing to the readers.

6. Figure 4 is confusing. According to the text, the graphs should show us that treatments of Myricetin and Valproic acid are able to delay the begining of seizures. But the graphs show that PTZ-group has the longest latency in every seizure type. Should not this be the opposit way? On the other hand, the numbers (mean±SD) in the text are not matching to the numbers that the bars indicate in the graphs. Also markings of the graphs (A,B,C) are missing.

7. Figure 12 A, B and C should be included in one figure/picture instead of being 3 individual big figure to decrease the length of the manuscript.

8. In the results (Line 636) the text said: „The comparison drug (Valproic acid) showed the best histological and histochemical picture at all terms of the study (Figures 13 A, B).” But Fig. 13 A. does not show any results about Valproic acid, only Fig. 13B has shows results of Valproic acid. Please correct the sentence.

9. In the results (Line 653) the text said: „The degree of optical density of NeuN staining varied depending on the region and time point. In the CA3 region exhibited the weakest labeling of hippocampal neurons, characterized by light nuclei, that indicate about neuronal loss (p<0.05)” According to this part there was some quantification of the NeuN staining but data of these quantitative results are not showed in the manuscript. Fig.14A only shows the histological staining and Fig. 14B the number of NeuN positive cells. If the density of the NeuN staining was measured and had significant difference and it was mentioned in the manuscript the results of that should be spresented in the article or as a supplementary data.

10. In the resume section (line 716) the text said: In the PTZ-induced model, all animals (n=40) showed a significant increase in the number of damaged neurons (“dark”, caspase-8-positive) compared to control values in almost all structures. How could be „n=40” if 60% of the PTZ-induced animals died during the seizure which means 24 animals survived to provide samples in this group? Please clarify this in the manuscript.

Minor comments:

1. There are some minor grammatic and text editing issues that should be corrected.

- For example (Materials and Methods; Line 265-267): „Primary mouse monoclonal antibody to NeuN/Neuronal Marker (1B7, 1:100) and polyclonal rabbit antibody to Caspase 8 (CAP4; 1:50); according to the protocol of manufacturer”.  The verb is missing from this sentence.

2. The catalog number/reference number of the purchased poducts (e.g. antibodies) must be included in the manuscipt.

3. Please indicate what type of antigen retrieval was used in the immunohisochemical experiment.

4. The morphological pictures are missing the scale bar. Although the text indicates that the magnification is 400x in every picture some pictures are visibly not the same.

-  For example in Figure 15A there is no way that the day 3 pictures of PTZ-only and PTZ+Myricetin have the same magnification. Please check and correct (if it is necessary) the indication of the magnifications in the figure legends.

Comments on the Quality of English Language

- Line 82: " For morphofunctional assessment of the state of neural tissue is used a neuron-specific neuronal nuclear antigen (NeuN, Neuronal Nuclei)."   This sentence is grammatically incorrenct. To be correct „for morphofunctional assessment of the state of neural tissue a neuron-specific neuronal nuclear antigen (NeuN, Neuronal Nuclei) is/was used” should have been written.

- Primary mouse monoclonal antibody to NeuN/Neuronal Marker (1B7, 1:100) and polyclonal rabbit antibody to Caspase 8 (CAP4; 1:50); according to the protocol of manufacturer”.  The verb (was used) is missing from this sentence.

Author Response

Dear Reviewer,

Thank you for your thorough and insightful review of our manuscript! We greatly appreciate your positive feedback and the valuable comments you provided. Your observations have been instrumental in helping us refine and enhance the quality of our work. We are grateful for the time and effort you invested in evaluating our study, and we believe that the revisions made in response to your comments have significantly strengthened our manuscript.

Mayor comments/questions:

Q1: The size of the manuscript is way too extensive, it has to be reduced a little bit. This could be done by simplifying some sentences and explanations because in some parts the manuscript it is a little bit over-explaned or a repetitive.

Answer: In this manuscript, we have tried to describe all our results as fully as possible and to discuss and debate our results to bring more attention to this interesting topic in the scientific community and to increase the citation rate of your journal. All, even the smallest details we have carefully checked and added, for the reader's full understanding of the present topic. However, after re-analysing the text of the manuscript according to your recommendations, particularly the Discussion and Conclusions sections, we have shortened some parts of the text to reduce its length. Thank you for your valuable comments!

 

 

Q2: - The description of the statistics say that: „All data are presented as the mean ± standard deviation, representative of six replicates.”  How was the control data made out of six replicates when the tissue homogenates were examined? If the 3 h, 24h, 3 day and 5 day groups all had six control replicate that means at least 24 animals supposed to be in the control group, but according to the manuscript the control group had 12 animals.

Answer: In this paragraph, thanks to your valuable comments, we discovered an error by our translation team (it was made by the translators who translated the manuscript text from our native language - unfortunately, having no medical background, they made an inaccuracy that was confusing). We re-analyzed the manuscript and made corrections, in particular, it was about 6 groups - we fixed this problem! Thank you for your attention to detail and accuracy!

 

 

Q3. If it is possible a supplementary file should be made with the all the measurment results because the datas showed in the manuscript was the representative of six replicates but according to the results (and materials) the mortality of group III was 25% and of group IV was 20 % which means that out of 60 animals of each group 45 animals survived and (should’ve) presented results from group III and 48 animals from group IV.

Answer: We apologize for any confusion this may have caused. We revised the manuscript to clarify the methodology and presentation of the data. We have ensured that the data presented in the manuscript accurately reflect the measurements and analyses performed on the surviving animals in each group. This includes a detailed explanation of how representative data were selected to ensure clarity and accuracy. We have also corrected any inconsistencies related to mortality rates and sample size. We believe these changes address the reviewer's concerns and increase the transparency of our study.

The Materials and Methods section presents data on the number of animals directly involved in the experiment. We originally had more animals (n=340) because, according to other authors, we expected high mortality after PTZ injections. We have corrected the number of animals and added explanations of these points in the text. For example, we took 110 animals in PTZ group II (we considered the work of other authors where they noted a high percentage of mortality.) We had a mortality rate of 60%, i.e. 44 animals were left. But later, according to the protocols of preclinical studies, as well as to simplify statistical tests, the exact number of animals was left in each group (edits on these points are made in the text), for example, in the PTZ group we included 40 animals for further study (i.e. 4 animals were withdrawn from the experiment).

 

Q4. The figure legends in Figure 3 and Figure 6 the text indicates that: „The bars represent mean ±SD” but there are no error bars in those graphs. Please correct those figure descriptions.

Answer: Corrected.

 

Q5. The figures (graphs) should be harmonised because now everything is all over the place. Some graphs have an upper and right side frame but other graphs don’t. The colour of the bars in each group should be consistent throughout the manuscript because for example in Fig. 3 and Fig 6 Group II has a light red colour but in Fig. 4 and Fig 5 Group II is purple. That could be confusing to the readers.

Answer: Corrected.

 

Q6. Figure 4 is confusing. According to the text, the graphs should show us that treatments of Myricetin and Valproic acid are able to delay the begining of seizures. But the graphs show that PTZ-group has the longest latency in every seizure type. Should not this be the opposit way? On the other hand, the numbers (mean±SD) in the text are not matching to the numbers that the bars indicate in the graphs. Also markings of the graphs (A,B,C) are missing.

Answer: Thank you for your attention to detail. We identified and corrected the errors in Figure 4, ensuring the graphs now accurately reflect the delay in seizure onset for the Myricetin + PTZ and Valproic Acid + PTZ groups. We have also corrected the mean±SD values in the text and added the appropriate graph labels. We appreciate your feedback, which helped us improve the manuscript.

 

Q7. Figure 12 A, B and C should be included in one figure/picture instead of being 3 individual big figure to decrease the length of the manuscript.

Answer: Corrected.

 

Q8. In the results (Line 636) the text said: „The comparison drug (Valproic acid) showed the best histological and histochemical picture at all terms of the study (Figures 13 A, B).” But Fig. 13 A. does not show any results about Valproic acid, only Fig. 13B has shows results of Valproic acid. Please correct the sentence.

Answer: Thank you for your attention to this line of text and detail, indeed we have corrected the inaccurate text and made edits to the text! Thank you

 

Q9. In the results (Line 653) the text said: „The degree of optical density of NeuN staining varied depending on the region and time point. In the CA3 region exhibited the weakest labeling of hippocampal neurons, characterized by light nuclei, that indicate about neuronal loss (p<0.05)” According to this part there was some quantification of the NeuN staining but data of these quantitative results are not showed in the manuscript. Fig.14A only shows the histological staining and Fig. 14B the number of NeuN positive cells. If the density of the NeuN staining was measured and had significant difference and it was mentioned in the manuscript the results of that should be spresented in the article or as a supplementary data.

Answer: We thank you for your detailed feedback. After revising our manuscript, we realised that the wording regarding the optical density of NeuN staining may have inadvertently led to misleading thoughts about additional studies. We have revised the text, double-checking everything beforehand to clarify that our observations were based on qualitative assessment of NeuN-positive cells and not on quantitative measurements of staining intensity. We appreciate your understanding and have made the necessary changes to ensure the accuracy and clarity of our results.

 

Q10. In the resume section (line 716) the text said: In the PTZ-induced model, all animals (n=40) showed a significant increase in the number of damaged neurons (“dark”, caspase-8-positive) compared to control values in almost all structures. How could be „n=40” if 60% of the PTZ-induced animals died during the seizure which means 24 animals survived to provide samples in this group? Please clarify this in the manuscript.

Answer: We have corrected the “confusion” with the number of animals (see above). Thank you!

 

 

Minor comments:

 

Q11. There are some minor grammatic and text editing issues that should be corrected.

 

- For example (Materials and Methods; Line 265-267): „Primary mouse monoclonal antibody to NeuN/Neuronal Marker (1B7, 1:100) and polyclonal rabbit antibody to Caspase 8 (CAP4; 1:50); according to the protocol of manufacturer”.  The verb is missing from this sentence.

Answer: Corrected.

 

Q12. The catalog number/reference number of the purchased poducts (e.g. antibodies) must be included in the manuscipt.

Answer: Corrected.

 

Q13. Please indicate what type of antigen retrieval was used in the immunohisochemical experiment.

Answer: Corrected.

 

 

Q14. The morphological pictures are missing the scale bar. Although the text indicates that the magnification is 400x in every picture some pictures are visibly not the same.

-  For example in Figure 15A there is no way that the day 3 pictures of PTZ-only and PTZ+Myricetin have the same magnification. Please check and correct (if it is necessary) the indication of the magnifications in the figure legends.

Answer: Thank you for your valuable comments, we have tweaked the graphics to improve readability and audience understanding.

 

Q15. Comments on the Quality of English Language

- Line 82: " For morphofunctional assessment of the state of neural tissue is used a neuron-specific neuronal nuclear antigen (NeuN, Neuronal Nuclei)."   This sentence is grammatically incorrenct. To be correct „for morphofunctional assessment of the state of neural tissue a neuron-specific neuronal nuclear antigen (NeuN, Neuronal Nuclei) is/was used” should have been written.

Answer: Corrected.

 

Q16- Primary mouse monoclonal antibody to NeuN/Neuronal Marker (1B7, 1:100) and polyclonal rabbit antibody to Caspase 8 (CAP4; 1:50); according to the protocol of manufacturer”.  The verb (was used) is missing from this sentence.

Answer: Corrected.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

To the AAs

The Authors have taken very good care of the raised constructive criticism by this reviewer. I would believe that the Ms key messages are strengthened with the article presentation being clearly improved.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate that the Authors considered the suggested corrections. With these changes the Authors greatly improved the quality of the manuscipt. The results became more coherent and more understandable for the readers.

I nocited that the title of the manuscript is also changed (I assume due to the request of another reviewer) and it is much better than the original one.

Comments on the Quality of English Language

Some minor grammatic issues are still presented in the manuscript.

For example: Methods sections (Line 267): "For antigen retrieval was used Citrate buffer, pH 6.0, 10×" This sentence is incorrect in this form. "For antigen retrieval Citrate buffer, (pH 6.0; 10×) was used" would be the grammatilcally correct sentence.