PONE-D-24-20382Morphine self-administration is inhibited by the antioxidant N‐acetylcysteine and the anti-inflammatory Ibudilast; an effect enhanced by their co-administration.PLOS ONE

Dear Dr. Ezquer,

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PLOS ONE

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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

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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

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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 research done by the authors is well planned and justified, and the results are clearly described and showed in the figures. The paper itself is very interesting and provides the readers with new information regarding the dependence and new possibilities in lowering the adverse effects of opioid withdrawal and decreasing the level of dependence in using easy, safe and affordable measures. My suggestion to the authors is to reorganize or add extra information in the discussion section regarding the limitation of the study (example - M1/M2, ibudilast clearance) and a conclusions part in the end of the discussion to summarize the study, and give the reader an answer to the question stated by the authors while planning the study - yes, NAC might be helpful, but also, shortly, by means of what mechanisms/processes? In my opinion this will make the discussion less results-like and more pleasant to read and understandable.

Reviewer #2: Quintanilla and colleagues tested the effect of NAC, ibudilast and their combination on rats subjected to prolonged morphine self-administration in a TBC paradigm. Both drugs reduced morphine intake and the combination showed further efficacy. Then they they demonstrated that NAC, ibudilast and their combination reverted the effect of morphine on

biomarkers of oxidative stress and glial activation in the hippocampus. They also reported that morphine decreased the level of GLT1 in the accumbens, and demonstrated that the combination of NAC and ibudilast, but not the single drugs, could revert this effect of morphine.

The study is overall interesting and soundly built. There are a number of points that however, require further attention.

Authors should better rationalize the choice for working in female only. The fact that male rats consume less morphine than females (lines 162-163) does not mean they don’t consume morphine or that males are not subject to opioid addiction risks. If there is not a stronger rational, this should be recognized as a study limitation.

Morphine has a low oral bioavailability. Although oral morphine is used in pain therapy, the oral way is not the route normally chosen by people affected by OUD. To which extent the oral way is used to abuse morphine? Also, when a patient assuming morphine for treatment purposes develops OUD, they often switch to other opioids cheaply available on the black market, like heroin.

Lines 208-209, please move consumption data to the result section.

Supplementary Figure 1 show two factors, the morphine concentrations and the day each concentration was maintained, therefore a two-way anova would be a better choice here. However, it looks like not all concentration were maintained the same number of days, if that’s the case authors should consider using the average intake of each concentration and keep the one-way ANOVA.

Experiment in figure 1: Baseline training; please provide statistic. NAC loading phase lasted two days, why using a one-way anova? NAC + Ibudilast phase, please clarify to which day the significant difference between groups refer to.

Same for figure 2.

In TBC paradigm food intake is used as natural reinforcer control, did you check food intake during treatments?

Please provide the rational for the euthanasia time point. Were rats still under morphine when sacrificed?

Result section usually report exclusively results. In this regard, what is written in

Line 348-352 and 366-367 look better suited for sections like introduction or discussion.

Line 957 the df do not correspond to what a one way ANOVA with 5 groups of 6 rats each would yield.

Line 932 please check df.

The classical view of dichotomous microglia M1/M2 states has been challenged as too simplistic to describe the multidimensional activation profiles of microglia. Authors may want to have a look at PMID: 36327895 and if they found the arguments in there reasonable, they could reconsider the terminology used in this work.

Also, opioid induced switch of microglia morphology toward a reactive state has been recently reported by others (PMID: 38452987). In this case authors reported that opioids affected microglia morphology but not density.

There are typos scattered over the manuscript please correct.

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Reviewer #1: No

Reviewer #2: No

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Morphine self-administration is inhibited by the antioxidant N‐acetylcysteine and the anti-inflammatory ibudilast; an effect enhanced by their co-administration.

PONE-D-24-20382R1

Dear Dr. Ezquer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication after fixing the minor comments from reviewer 2. 

Please address the comments and return the revised manuscript by Nov 16 2024 11:59PM.

Kind regards,

Shao-Jun Tang

Academic Editor

PLOS ONE

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: No

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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: Thank you for providing the revised manuscript. The authors have made all of the revisions suggested by the reviewers. I recommend the paper for publication.

Reviewer #2: Authors appropriately addressed my major points. I have only few remaining minor comments to make.

Line 230: Authors specified that animals were euthanized one hour after the last recorded morphine intake, it remains unclear however whether morphine was withdrawn during that hour (i.e. rats had one hour of abstinence) or not. This is a crucial information for the reproducibility of the study and I warmly recommend to clarify it.

Line 314 and Fig 1: when ANOVA finds no significant effects, it does not require to be followed by post-hoc tests. This apply also to other session of the manuscript were a non-significant effect is reported.

Both factors and their interaction of two-way ANOVA should be reported, whether they are significant or not.

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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: No

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Morphine self-administration is inhibited by the antioxidant N‐acetylcysteine and the anti-inflammatory ibudilast; an effect enhanced by their co-administration.

PONE-D-24-20382R2

Dear Dr. Ezquer,

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.

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Kind regards,

Shao-Jun Tang

Academic Editor

PLOS ONE

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Reviewers' comments: