Silencing of dentate gyrus inhibits mossy fiber sprouting and prevents epileptogenesis through NDR2 kinase in pentylenetetrazole kindling rat model of TLE

Chen Zhang; Zixian He; Zheren Tan; Fafa Tian

doi:10.1371/journal.pone.0284359

Peer Review History

Original SubmissionNovember 11, 2022
29 Dec 2022 Decision Letter - Andreia Cristina Karklin Mortensen, Editor PONE-D-22-30742Silencing of dentate gyrus inhibits mossy fiber sprouting and prevents epileptogenesis through NDR2 kinase in pentylenetetrazole kindling rat model of TLEPLOS ONE Dear Dr. Fafa Tian, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by February 20, 2023. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: The manuscript “Silencing of dentate gyrus inhibits mossy fiber sprouting and prevents epileptogenesis through NDR2 kinase in pentylenetetrazole kindling rat model of TLE”, by Zhang et al establishes mechanistic studies the role of mossy fiber sprouting on epileptogenesis using chemogenetic approaches. The authors conclude that silencing of the dentate gyrus inhibits sprouting and prevents epileptogenesis through NDR2, suggesting a potential therapeutic strategy. To be amenable for publication, please address the criticisms and suggestions by reviewer 1. Additionally, the English language must be thoroughly revised by a native speaker, as several sentences and paragraphs are not comprehensible. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript titled “Silencing of dentate gyrus inhibits mossy fiber sprouting and prevents epileptogenesis through NDR2 kinase in pentylenetetrazole kindling rat model of TLE”, by Fafa Tian and colleagues demonstrates an experimental epileptogenesis study. The authors point out that the role of DG’s excitability in epileptogenesis have not yet been well investigated, and underlying mechanism have not been elucidated. After careful reading, I suggest the following to improve the current manuscript: - In the following text: “…The commonest type of refractory epilepsy that can be found in adults is the temporal lobe epilepsy (TLE)[3]”… Are these adults’ humans or experimental animals? Please detail this type of information. - What country is the “National Institutes of Health for the care and use of laboratory animals” from? The authors already cited the animal experimental rules and protocol number. - Where is Central South University? Please add in the text. - There are many mentions of animal ethics committees, such as: “Ethics Committee of Xiangya Hospital, Central South University (Protocol Number: 20153213”); “guidelines of the World Medical Association Declaration of Helsinki”; “Department of experimental animals, Central South University”; “ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines”. Please explain all these committees and guidelines. - How many animals per group (I, II and III) were used? - In the following text: ”…and the others were euthanized after the last observation of PTZ-induced seizure...”. After all, how many animals were euthanized? - What was the stereotaxic device used (company, brand, model, etc.)? - There are many errors in English grammar, concordance, typing, etc. (examples: "sterotaxic" device), please correct. Check and standardize in the manuscript. I strongly recommend that you ask the assistance of a native English speaker to revise the whole manuscript. - Where is "OBiO Technology" from? Where is MultiScience from? Please check and standardize the origin of the equipment’s and drugs in the manuscript. - How much rats died during the course of PTZ kindling? - What equipment is used for coronal sections frozen cryosections? - Photomicrographs were captured for each animal with a microscope. What was this microscope? - What are Beyotime, Servicebio, etc? - In Figure 1, it would be better to indicate some structure, cell, etc. - In figures 3 and 5 indicate with symbols what we are seeing. In figure 3 the images are small. It would be good if the authors put an excerpt for each image with higher resolution. - The findings indicated that chemogenetic inhibition in DG alleviates epileptogenesis in PTZ kindling rat model. How much were the values in, for example, percentage? - The MFS and NDR2 expression in hippocampus were investigated. Is it more coherent to express with numbers, percentage, not only was it significant, how much better or worse, if it increased or decreased significantly how much was it? - One more example: The expression of NDR2 was confirmed in DREADD+CNO+NDR2 group. Data showed that the hippocampal level of NDR2 was significantly increased in DREADD+CNO+NDR2. How much? - In the last item (3.4) of the "results" the authors put the values in percentage! Therefore, it is also possible to place the results in this way for the other results, in addition to indicating the statistic as they did. - Please discuss in the “Discussion”: Some authors consider that “Mossy fiber sprouting (MFS”) is a cell death marker phenomenon. Others, however, consider that the neuron is recovering, including a form of neuronal plasticity, regenerating. How do the authors see this aspect? - Put in the “Discussion”: The “...regulators of axonal growth may be a potential target to prevent and cure TLE.” How specifically can this occur? - A counterpoint to the previous consideration can be the study by Rodrigues et al. 2002 (doi: 10.1016/j.eplepsyres.2004.02.001): "...present results are the first behavioral description—and comparison—of seizures induced by ICV and subregional hippocampal formation (dorsal × ventral) bicuculline microinjection in rats. Also, this is the first attempt to block the seizures originated by focal bicuculline in the dorsal hippocampus with GABAergic drugs microinjected in the “substantia nigra pars reticulata “ (SNPR) of rats...". How do the authors see this manipulation (dorsal x ventral) and the one they propose to resolve, in part, temporal lobe epileptic (TLE) seizures? Is there any link between the two ideas (hippocampus-DG….SNPR)? Therefore, due to such a connection with another area close to the brain. - Still in this direction, regulators of axonal growth may be a potential target to prevent and cure TLE are they efficient due to connections (pathways- axons) linking the hippocampus to the SNPR. Please discuss. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Wagner Ferreira dos Santos ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0284359.r001
Revision 1
16 Jan 2023 Author Response Reviewer #1: The manuscript titled “Silencing of dentate gyrus inhibits mossy fiber sprouting and prevents epileptogenesis through NDR2 kinase in pentylenetetrazole kindling rat model of TLE”, by Fafa Tian and colleagues demonstrates an experimental epileptogenesis study. The authors point out that the role of DG’s excitability in epileptogenesis have not yet been well investigated, and underlying mechanism have not been elucidated. After careful reading, I suggest the following to improve the current manuscript: - In the following text: “…The commonest type of refractory epilepsy that can be found in adults is the temporal lobe epilepsy (TLE)[3]”… Are these adults’ humans or experimental animals? Please detail this type of information. Thanks for your suggestion. Those adults is human.The sentence has been rewritten. - What country is the “National Institutes of Health for the care and use of laboratory animals” from? The authors already cited the animal experimental rules and protocol number. Thanks for your question. “National Institutes of Health is NIH of USA. Although this study was conducted in China, but the animal research ethics standards of United States is worthy of our reference. - Where is Central South University? Please add in the text. CSU is located in Changsha, China. Corresponding content has been added in manuscript. - There are many mentions of animal ethics committees, such as: “Ethics Committee of Xiangya Hospital, Central South University (Protocol Number: 20153213”); “guidelines of the World Medical Association Declaration of Helsinki”; “Department of experimental animals, Central South University”; “ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines”. Please explain all these committees and guidelines. Thanks for your question. This study was conducted in China.As we all known, Western countries have more excellent practical experience and theory in animal protection, so the animal research and care standards in this study refer to the famous guidelines you mentioned, and finally the project was approved by Ethics Committee of Xiangya Hospital.The experiments were conducted in Department of experimental animals, Central South University. - How many animals per group (I, II and III) were used? Thanks for your question,12 rats were used in group I , 11 rats were used in group II, 56rats were used in group III,30 rats were used in group IV. - In the following text: ”…and the others were euthanized after the last observation of PTZ-induced seizure...”. After all, how many animals were euthanized? Thanks for your question.96 rats were euthanized. - What was the stereotaxic device used (company, brand, model, etc.)? Thanks for your question. The stereotaxic device was purchased from Yuyan Instruments, China.The brand information was added. - There are many errors in English grammar, concordance, typing, etc. (examples: "sterotaxic" device), please correct. Check and standardize in the manuscript. I strongly recommend that you ask the assistance of a native English speaker to revise the whole manuscript. I am so sorry for my poor English.The manuscript has been polished. - Where is "OBiO Technology" from? Where is MultiScience from? Please check and standardize the origin of the equipment’s and drugs in the manuscript. Thanks for your question and suggestion.Those companies is from China.The origin of drugs and equipments has been added in methods section. - How much rats died during the course of PTZ kindling? Thanks for your question.13 rats died. Most of them is from group IV. - What equipment is used for coronal sections frozen cryosections? Leica CM1950. - Photomicrographs were captured for each animal with a microscope. What was this microscope? Thanks for your question. The Nikon eclipse C1 was used. - What are Beyotime, Servicebio, etc? Those are Chinese technology companies. - In Figure 1, it would be better to indicate some structure, cell, etc. Thanks for your suggestion, the layers were indicated by smybols in revised Figure 1. - In figures 3 and 5 indicate with symbols what we are seeing. In figure 3 the images are small. It would be good if the authors put an excerpt for each image with higher resolution. Thanks for your suggestion, symbols were added to figure 3and 5,the high resolution figure 3 was uploaded. - The findings indicated that chemogenetic inhibition in DG alleviates epileptogenesis in PTZ kindling rat model. How much were the values in, for example, percentage? The kindling rate was decreased from 91.7%(group I) to 63.6(group III with CNO 10mg/kg)，the mean latency of kindling was prolonged from 15.4days（group I）to 21.0（group III with CNO 10mg/kg）. - The MFS and NDR2 expression in hippocampus were investigated. Is it more coherent to express with numbers, percentage, not only was it significant, how much better or worse, if it increased or decreased significantly how much was it? Thanks for your suggestion,The median timm score and relative level of protein had been added in result section to show the difference between groups. - One more example: The expression of NDR2 was confirmed in DREADD+CNO+NDR2 group. Data showed that the hippocampal level of NDR2 was significantly increased in DREADD+CNO+NDR2. How much? Thanks for your suggestion,The median timm score and relative level of protein had been added in result section to show the difference between groups. - In the last item (3.4) of the "results" the authors put the values in percentage! Therefore, it is also possible to place the results in this way for the other results, in addition to indicating the statistic as they did. Thanks for your suggestion. Findings were presented with such way in other part of result section. - Please discuss in the “Discussion”: Some authors consider that “Mossy fiber sprouting (MFS”) is a cell death marker phenomenon. Others, however, consider that the neuron is recovering, including a form of neuronal plasticity, regenerating. How do the authors see this aspect? Thanks for your suggestion. Previous studies found that because of hippocampal cell death granule cells in DG lose their target cells in CA4 and CA3 regions, which resulted in the formation of MFS ,and extent of MFS correlates with neuronal cell loss in patients with epilepsy.Although presence of MFS implies the neuronal loss in hippocampus, MFS is the best-studied form of axonal plasticity and reorganization in epilepsy, which contributed to development of epilepsy. - Put in the “Discussion”: The “...regulators of axonal growth may be a potential target to prevent and cure TLE.” How specifically can this occur? Thanks for your suggestion, relative contents has been added in discussion section. - A counterpoint to the previous consideration can be the study by Rodrigues et al. 2002 (doi: 10.1016/j.eplepsyres.2004.02.001): "...present results are the first behavioral description—and comparison—of seizures induced by ICV and subregional hippocampal formation (dorsal × ventral) bicuculline microinjection in rats. Also, this is the first attempt to block the seizures originated by focal bicuculline in the dorsal hippocampus with GABAergic drugs microinjected in the “substantia nigra pars reticulata “ (SNPR) of rats...". How do the authors see this manipulation (dorsal x ventral) and the one they propose to resolve, in part, temporal lobe epileptic (TLE) seizures? Is there any link between the two ideas (hippocampus-DG….SNPR)? Therefore, due to such a connection with another area close to the brain. From the perspective of seizure model creation, IVC injection can induce seizures faster and have more diverse seizure manifestations. However, due to the wide range affected by IVC injections, the relationship between specific brain regions and epileptic manifestations cannot be established. So I think it is brilliant to conduct the local injection in DHF and AHiPM, and make a comparison in seizure behaviors between IVC injection and local injection,which can better help us understand the origins of seizure behaviors heterogeneity. - Still in this direction, regulators of axonal growth may be a potential target to prevent and cure TLE are they efficient due to connections (pathways- axons) linking the hippocampus to the SNPR. Please discuss. Thanks for your ssuggestion. Substantia nigra is a mesencephalic structure inserted along several circuits which appear to play a key role in epilepsy. The function of substantia nigra pars reticulata (SNpr) has been associated with the control of seizures. Behavioral seizures induced in the hippocampus are blocked by a GABAergic receptor agonist muscimol microinjected in the SNpr. Moreover, injection of exogenous DA into SNpr induced seizures and a significant reduction of gene expression for GluR1, GluR2 and NMDAR1 subunits in rat hippocampal subfields including CA1 through CA4 and DG,which indicated hippocampus-SNpr pathway may be involved in mechanism of epilepsy. Whether the connection between hippocampus and SNpr is involved in therapeutic effect of modulator of axonal growth deserves further study. https://doi.org/10.1371/journal.pone.0284359.r002
29 Mar 2023 Decision Letter - Andreia Cristina Karklin Mortensen, Editor Silencing of dentate gyrus inhibits mossy fiber sprouting and prevents epileptogenesis through NDR2 kinase in pentylenetetrazole kindling rat model of TLE PONE-D-22-30742R1 Dear Dr. Fafa Tian We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Andreia Cristina Karklin Mortensen, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0284359.r003
Formally Accepted
3 Apr 2023 Acceptance Letter - Andreia Cristina Karklin Mortensen, Editor PONE-D-22-30742R1 Silencing of dentate gyrus inhibits mossy fiber sprouting and prevents epileptogenesis through NDR2 kinase in pentylenetetrazole kindling rat model of TLE Dear Dr. Tian: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Andreia Cristina Karklin Mortensen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0284359.r004

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