-->PONE-D-26-12414-->-->Inhibition of SUR1–TRPM4 attenuates astrocyte swelling and reactivity under oxygen-glucose deprivation/reoxygenation-->-->PLOS One-->

Dear Dr. Tapechum,

-->

Thank you for submitting your manuscript to PLOS ONE. During preliminary evaluation of your manuscript, I have found 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 editorial screening process.

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We look forward to receiving your revised manuscript.

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Alexander A. Mongin, Ph.D.

Academic Editor

PLOS One

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-->PONE-D-26-12414R1-->-->Inhibition of SUR1–TRPM4 attenuates astrocyte swelling and reactivity under oxygen-glucose deprivation/reoxygenation-->-->Dear Dr. Tapechum,

Thank you for submitting your manuscript to PLOS ONE. Your work has been evaluated by two experts in the field. 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.-->

I would like to draw your particular attention to the following points:

[1] Both reviewers expressed concerns about novelty. These concerns do not need to be addressed because PLOS ONE does not require novelty or major conceptual breakthroughs for publication, but instead focuses on methodological rigor and the soundness of the conclusions. However, all other issues raised by the reviewers should be specifically addressed in detailed point-by-point responses.

[2] Reviewer 2 raised a valid concern regarding the limitations of using an immortalized cell line in place of primary astrocytes. This limitation should be explicitly acknowledged in the manuscript text. I would also advise against referring to CTX-TNA2 cells as “astrocytes”; terms such as “CTX-TNA2 cells” or “astroglial cell line” would be more appropriate.

[3] Reviewer 2 also cautioned against excessive generalization of the in vitro OGD findings to astrocyte pathology in situ. You may speculate about the potential significance and applicability of your findings, but statements such as (lines 459–461) “Our findings further indicate that SUR1–TRPM4 is involved in both astrocyte swelling and astrocyte reactivity following ischemic injury” should be avoided or substantially softened.

[4] Please address the Editor’s concerns regarding statistical analyses. Most journals have now transitioned to reporting SD rather than SEM. In all cases where control data were normalized to 100% or unity (e.g., Fig. 1A, Fig. 3C, Fig. 4A,B, Fig. 6B–D), one-way ANOVA is not an appropriate statistical test. Instead, you may use one-sample t-tests with Bonferroni correction for multiple comparisons or consult a statistician regarding alternative approaches.

[5] Please clarify what is meant by the term “intracellular concentration” as presented in Fig. 2D.

[6] It appears that in the Western blot analyses, β-actin loading controls were probed on separate membranes. If this is the case, it must be clearly stated in the Methods and/or figure legends.

[7] Both reviewers expressed concerns regarding the resolution and/or composition of the figures. Personally, I found the high-resolution versions generally acceptable (perhaps the reviewers did not access them). Nevertheless, I agree that several figures, particularly Figure 7, will be difficult to read when printed and may even be problematic in the online format. Please consider reorganizing these figures to improve overall legibility.

Please submit your revised manuscript by Jun 24 2026 11:59PM. 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.

Please include the following items when submitting your revised manuscript:

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Alexander A. Mongin, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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-->2. 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

Reviewer #2: Partly

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: (No Response)

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-->4. 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

Reviewer #2: Yes

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-->5. 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

Reviewer #2: Yes

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-->6. 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 authors employ an astrocytic cell line and an in vitro model of ischemia–reperfusion, combined with pharmacological interventions, to demonstrate that glibenclamide-sensitive SUR1-TRPM4 channels are not only involved in astrocytic swelling but also contribute to ischemia–reperfusion-induced ROS production, ER stress, and inflammatory responses.

Overall, the manuscript is clearly written and well organized. The experimental design is described in sufficient detail, and the results are presented and discussed in a comprehensive manner. The data appear robust and convincing, and the experiments were conducted rigorously, with appropriate replication across treatment conditions.

Major Comments

Figure quality

The quality of the figures is currently insufficient for publication. Several images are of low resolution, and some labels are difficult to read. All figures should be replaced with high-resolution versions to ensure clarity and readability.

Redundancy of astrocyte swelling experiments

The role of glibenclamide in reducing astrocyte swelling via SUR1-TRPM4 channels has already been well established in the literature. Repeating these findings may not add substantial novelty. The authors may consider reducing this section and instead placing greater emphasis on the less well-characterized downstream effects.

Lack of mechanistic insight into downstream signaling

While the study convincingly demonstrates downstream effects such as ROS production, ER stress, and inflammation, the underlying signaling mechanisms remain unclear and are not addressed in the manuscript. For example, it would be important to consider whether these effects are mediated by changes in intracellular calcium levels. Additionally, the factors responsible for the effects of the conditioned media are not identified. Including experiments to explore these mechanisms would significantly strengthen the manuscript and provide valuable mechanistic insight.

Minor Comments

Therapeutic context of glibenclamide

In the final part of the Introduction, the authors suggest that glibenclamide may have therapeutic potential. However, this concept has already been proposed in earlier studies, and the compound has been under preclinical investigation for brain ischemia for some time. The text should be revised to better reflect the current state of the field.

Figure 8 – pSTAT3 interpretation

Figure 8 does not fully reflect the described results. Specifically, pSTAT3 levels appear to be increased not only in cells treated with conditioned media but also - more prominently - in ischemic astrocytes themselves. This aspect should be clearly represented in the figure and/or clarified in the figure legend.

Reviewer #2: 1. This m/s is not particularly novel, as earlier studies have shown similar results. As the authors know, Simard’s group has published work identical to the current findings (e.g., SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells, Volodymyr Gerzanich,….J. Marc Simard; Cell swelling and a nonselective cation channel regulated by internal Ca2+ and ATP in native reactive astrocytes from adult rat brain. JNeurosci 21: 6512–6521), and the authors cited some of them, except for the pharmacological manipulation.

2. The cell line used in this study is unacceptable. The rat astrocyte cell line CTX-TNA2 (ATCC® CRL-2006™) is an immortalized, type 1 astrocyte line derived from the cortex of 1-day-old Sprague-Dawley rats. These adherent cells, often used, exhibit a fibroblast-like morphology. More importantly, only 20% of these cells exhibit glial fibrillary acidic protein (GFAP) immunoreactivity. Thus, this cell line can't be used for any GFAP-related applications. As the authors know, these cells often lack specific functions of normal cells, such as metabolic pathways or tissue-specific markers, due to changes in gene expression and metabolic rearrangement. Since these cells are derived from tumors, the results should have been validated in primary cells to ensure physiological relevance.

3. The argument that inhibition of SUR1–TRPM4 attenuates astrocyte swelling in injury-like settings, and the strongest evidence is in activated astrocytes and post-ischemic brain tissue rather than in a pure OGD-only astrocyte culture model, is hard to accept. Because the in vitro system lacks blood components. This needs to be reversed.

4. The manuscript is poorly written and lacks methodological details. For example, the authors noted that they used 96-well plates. If that’s the case, how many plates were used per condition? TomoDish imaging dishes, what does n=4 mean?

5. The major issues when using Astrocyte-conditioned medium (ACM) with naive cells are reproducibility and a lack of standardization in its secretome composition. Because ACM is produced by cultured astrocytes, which often default to a reactive or immature phenotype in 2/3D systems, the precise cocktail of secreted proteins, lipids, and cytokines can vary significantly depending on astrocyte culture conditions, passage number, and basal media. It's unclear how the authors address these issues.

6. The authors used a microplate reader for ROS measurement. But flow cytometry is a reliable tool for measuring ROS using the Invitrogen Total Reactive Oxygen Species (ROS) Assay Kit (Cat. No. 88-5930-74). Because the fluorescent signal will now be retained after cell permeabilization.

7. Provide the original number for Fig. 1A instead of expressing it as a percentage.

8. It is difficult to compare legends with figures because the legends are far from the figures. This may be due to the journal's style (the journal should look into it).

9. The fluorescence images are of poor quality (Fig 7 specifically). The astrocytes may have been stained with May-Grunwald to better assess morphology or density. Because we don’t know what cells we are looking at.

10. It's unclear what concentrations of Gli were used (it should be explicitly mentioned in all figure legends).

11. Fig. 9 needs to be redrawn. Astrocyte death and reactivity can occur under the same conditions, particularly in severe neurodegenerative diseases, stroke, and traumatic brain injuries. While reactive astrogliosis is traditionally viewed as a protective, hypertrophy-driven (growth) response to damage, it often transitions into a pathological state in which reactive astrocytes lose their supportive functions, become neurotoxic, and eventually die. This raises the question: Did the authors measure the neurotoxic substances associated with this condition? If so, what are they? Since this is an in vitro condition, what may have occurred in vivo?

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-->7. 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:  Helena Pivonkova

Reviewer #2: Yes:  Arumugam R. Jayakumar

[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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Inhibition of SUR1-TRPM4 attenuates astrocyte swelling and reactivity under oxygen-glucose deprivation/reoxygenation

PONE-D-26-12414R2

Dear Dr. Tapechum,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support.

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,

Alexander A. Mongin, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. 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

Reviewer #2: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. 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

Reviewer #2: Yes

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-->5. 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

Reviewer #2: Yes

**********

-->6. 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: (No Response)

Reviewer #2: The authors made significant improvements to their initial submission. My concerns were addressed appropriately.

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-->7. 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: No

Reviewer #2: Yes:  Arumugam Radhakrishnan Jayakumar

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