PONE-D-24-12582Hypothermia improves neuronal network recovery in a human-derived in vitro model of the ischemic penumbraPLOS ONE

Dear Dr. Voogd,

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 all the points raised during the review process.

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Four reviewers evaluated the manuscript. They found it interesting, but all of them asked for further improvements. I do not think that extensive expriments are needed, but additional data were asked that can strengthen the paper, like viability test(s) or additional time-point in the experiments. Please carefully revise the text and make it more organized and clear.==============================

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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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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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

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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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 of Voogd et al. presents intriguing data, further confirming the previous finding that hypothermia applied during hypoxia may have neuroprotective effects. The topic is timely and of significant importance, as the prevalence of ischemic stroke is continuously increasing worldwide, however no neuroprotective agents have been demonstrated to impact clinical outcomes.

The authors monitored the electrophysiological activity throughout the hypoxic treatment until the end of the recovery period, thereby providing a complete, comprehensive picture of how temperature affects neuronal activity. However a similar comprehensive tracking of the number of synaptic puncta is missing from the study. In my opinion, it would be highly informative to investigate the number of synaptic puncta at the end of the recovery period as well. Furthermore, there is no information available regarding the viability of cells. Authors mentioned in the discussion, that under hypoxia, hyperthermia may induce apoptosis. Is it possible, that the loss of neuronal network functioning observed in this this study may be due to cell death?

Reviewer #2: The authors established a neuronal network model from hiPSC for MEA neuronal activity test in their previous publication. In this study, they used this model to investigate the effects of hypothermia and hyperthermia on neuronal activity after hypoxia.

1. In the title, “in vitro model of the ischemic penumbra” is incorrect. In the penumbra, blood flow is typically reduced to 30%-70%. The authors used 2% hypoxia which provides too little oxygen but too much glucose to accurately mimic penumbra situation in vitro. A modified oxygen-glucose deprivation (OGD) model with 5-8% hypoxia and 30% glucose content is recommended.

2. MEA technology has been utilized for over two decades, allowing numerous researchers to study neuronal activity under various physiological and pathological conditions, such as hypothermia, hyperthermia, and hypoxic stress, using either rodent, sheep or human neurons (e.g., PMID: 17081617; PMID: 17093117; PMID: 34975426; PMID: 32320794). It would be informative if the authors used patient-derived neurons, despite the difficulty in obtaining them from ischemic patients. Or, alternatively they may at least use primary human neurons from other diseases. iPSC-derived neurons remain quite artificial, and their results do not provide significant new insights in this field. It is already known that hypothermia is neuroprotective and promotes the recovery of neuronal activity after hypoxia, but it has the opposite effects under normal conditions. Conversely, hyperthermia is known to exacerbate neuronal damage after hypoxia.

3. To enhance the novelty of the present study, I recommend that the authors test the effects of different temperatures and oxygen levels in their model. The author noted that in some reports, even 36°C is neuroprotective after hypoxia. Providing a systematic view of temperature effects could be valuable for therapeutic applications.

Reviewer #3: About the manuscript PONE-D-24-12582, entitled ”Hypothermia improves neuronal network recovery in a human-derived in vitro model of the ischemic penumbra”, by Franca EJH et al., submitted to PLOS ONE.

While I consider it interesting, the manuscript is rather difficult to read. I would suggest to improve the whole presentation, clarifying the presentation of the results and very much the discussion.

General

The manuscript reports experiments with neuronal networks derived from human induced pluripotent stem cells, monitored with micro-electrode arrays, to investigate how treatment with normothermia, hypothermia and hyperthermia during hypoxia influences neuronal functionality, improving post-hypoxic recovery. The neuronal networks are also evaluated with immunocytochemistry, studying the effect of the treatment on synaptic puncta.

Methods

The description of the methods is detailed, but not well organised, including experimental steps that are not considered when describing the results. The authors describe the protocol for identifying excitatory and inhibitory neurons, but the phenotype is not confirmed nor used for evaluating the effects of hypoxia and/or temperature conditions.

Results

While the experimental timing is schematically described in Fig. 1, 2 and 4, it is not clear enough when the recording is performed. Is it continuously performed, as indicated in Fig.1e-g? But, when are the samples taken for building the graphics at Fig. 1i-k? Please, clarify that for all figures, completing and making it uniform along the figure legends.

Would one understand that the sampling in Fig. 1d, Fig.2b and Fig.4b was taken at the beginning or at the end of a period of 6h, 24h or 48h of asphyxia? Under continuous either normothermia, hypothermia or hyperthermia?, but a final 6h normoxia/normothermia period was also performed, at least for Fig. 4? Please, uniform the layout of the figures. In Fig. 1i-k; Fig. 2g-I and Fig. 4i-l, why not using the same scales? The dotted line represents 100% or 1? Also, in Fig. 4k?

The timing for Fig. 3 has to be clarified, making possible for the reader to rapidly understand when the tissue was fixed. After 6h, 24h and 48h of hypoxia? The labelling of synapsin ½ is not evident, at least in the figure I received. The scale bar in Fig. 3a is hardly seeing.

Before evaluating the temperature variable, please, describe clearly on the effect of hypoxia, I guess Control versus Normothermia, describing the time course. Clarify, please, the issue of the effect of 48h of hypoxia, inducing only 50% of decrease of synaptic puncta?

Is that a severe or mild effect? How should be that when comparing with in vivo models? Any specific effect on excitatory or inhibitory neurons, or neurons versus glial cells?

Discussion

I would recommend to start by discussing the relevance and advantages of the experimental mode, also summarising the obtained results, making clear what are we learning with the results. Also, please, explain about the relevance of the effects observed after hypothermia, producing outcomes surpassing the control condition?

How long does it take for seeing a deleterious effect of hyperthermia? 48h (as seeing in Fig.3b-d??

Discuss please about the physiological relevance of the obtained results. What is the relevance of an increase in synaptic puncta following 48h hypoxia + hypothermia?

In general, the discussion is floppy, referring to issues that are not considered by the selected experimental protocol. I would suggest to concentrate on the observed results, expanding then on their relevance, physiologically and/or clinically.

The discussion in lines 382-396 should be improved, expanding the idea that hypothermia would reinforce inhibitory synapses? How is that? considering what is shown in Fig. 3 and 4? What’s about effects on excitatory and/or inhibitory neurons?

How to deal with the discussion expressed in line 406-412 and the effects shown in Fig. 2?

I would suggest that the authors focus on what was observed in the study.

The issue of a therapeutic strategy before a clinical outcome could perhaps deserve some commentaries.

The conclusion should be more convincing. Hypothermia should prevent synaptic loss or rather increase functional efficacy?

The final sentence “freezing the penumbra” is justifying the title? Indeed, the issue of penumbra is just taken at the end of the article. How should the chosen experimental approach be a model for describing what is occurring in the penumbra? Is the issue of “penumbra” justified for being inserted in the title?

Reviewer #4: Review of “Hypothermia improves neural network recovery…”

This manuscript evaluates the effects of hypothermic (34C) and hyperthermic (39C) conditions on human-derived neuronal network plated on microelectrode arrays during the application of hypoxia. Functional readouts are performed prior, during and after application of 48hrs of hypoxic conditions at normothermic, hypothermic and hyperthermic temperatures. The results show that the application of hypothermia had some beneficial effects on the network’s electrophysiology, specifically the firing and burst rate whereas the application of hyperthermia had a generally significantly worse outcome than normothermia.

The manuscript is generally well-written and organized, and the data presented is by en large clear. The study is somewhat limited in its focus (primarily focusing on electrical activity and 2 antibody stains), and I have a few comments I’d like the authors to address before I recommend this manuscript to be published:

1. While the data in Fig. 1 shows that there is a clear benefit of applying hypothermic (34C) conditions to the neuronal networks during under hypoxic conditions - it is unclear how viable the culture is 24hrs or even 48hrs after the application of hypothermia. It would significantly strengthen the paper if the authors could provide data further out then just 6hrs (see for example Scimone et al. PlosOne, 2020 - application of hypothermia to TBI).

2. The statement on Line 427 that hypothermia enhances functional recovery during hypoxia might be misleading if the long-term outcome of the culture is unaltered, i.e., if the long-term (24-48hr) outcome does not show any statistical significant effect from normothermia under hypoxic conditions.

3. It would be very helpful for all figures showing time on the x-axis (e.g., Fig.1 e-g, etc) to show the full time data (10min initially, 48 hrs + 6 hrs post) and delineate the different regimes (with shaded regions for example) clearly on the plots. Please also add raster plots for the 6hrs post hypoxia for all groups.

4. It would be great to have some general viability data alongside the electrophysiological data to assess the general health of the neural cell populations in particular at the final time point.

5. Line 442 in the conclusion “freezing the penumbra” is somewhat misleading as we are talking about a small change (37C to 34C) in temperature (not 0C), which would have significant adverse effects.

6. The fact that hypothermia had such a strong effect on the control samples is somewhat concerning. Some loss in cell function (and most likely cell viability) is certainly expected (this is also shown in the Scimone et al as an in vitro example) but coming back to my point 1., without showing the long term, stable benefit of the application of hypothermia the overall study impact might be significantly diminished. Also, I was somewhat surprised to see, on the flip side, the almost positive effect of hyperthermia on the control cases - some more elaboration on what the authors think might be happening here within the context of theirs and other, similar in vitro culture systems will be useful.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Hypothermia improves neuronal network recovery in a human-derived in vitro model of oxygen-deprivation

PONE-D-24-12582R1

Dear Dr. Voogd,

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.

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Kind regards,

Mária A. Deli, M.D., 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 #4: 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 #4: Yes

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

Reviewer #1: Yes

Reviewer #4: 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 #4: 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 #4: 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: (No Response)

Reviewer #4: (No Response)

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Reviewer #1: No

Reviewer #4: No

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