-->PONE-D-25-61424-->-->Chronic oral cannabidiol delays or prevents seizures in a mouse model of CLN2 disease-->-->PLOS One

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[JTD and JDC received generous gifts from the Lehrman Family Fund to conduct this research. Special thanks to Stephen and Sandra Lehrman for their support.

JDC also received funding from the Batten Disease Support and Research Association to support this research.

MW received funding from the Washington University in St. Louis IDDRC - NIH P50 HD103525].

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[The authors give a special thanks to Stephen and Sandra Lehrman and the Lehrman Family Fund for providing financial support to complete this work. Thanks also to the Batten Disease Support and Research Association for research support, and to Dr. Russell B. Williams at the Proteomics and Mass Spectrometry Facility of the Donald Danforth Plant Science Center who performed LC-MS/MS analysis on instruments purchased with DDPSC funds.]

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[JTD and JDC received generous gifts from the Lehrman Family Fund to conduct this research. Special thanks to Stephen and Sandra Lehrman for their support.

JDC also received funding from the Batten Disease Support and Research Association to support this research.

MW received funding from the Washington University in St. Louis IDDRC - NIH P50 HD103525]

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Reviewers' comments:

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: N/A

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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 manuscript addresses an important and clinically relevant question, namely whether chronic oral cannabidiol (CBD) can delay seizure onset and reduce seizure burden in a mouse model of CLN2 disease (Cln2R207X). The study has notable strengths, including the use of continuous video-EEG monitoring and objective outcome measures such as time to first seizure and survival. The observation that CBD delays seizure onset and that a subset of treated animals remains seizure-free is potentially meaningful.

However, in its current form, the manuscript presents several methodological and analytical issues that limit the robustness of the conclusions. In particular, concerns related to statistical treatment (especially pseudoreplication), multiple-comparison control, incomplete reporting of randomization and blinding procedures, inconsistencies in ethical descriptions, and incomplete characterization of dosing and intake must be addressed before the work can be considered for publication. For these reasons, I recommend major revision.

Section-by-section comments

Title and Abstract

The title is generally appropriate, but the wording “delays or prevents seizures” may overstate the findings. Since seizure prevention was observed only in a subset of animals, a more cautious formulation (e.g., “delays seizure onset and reduces seizure burden”) would be more accurate.

In the abstract, the use of expressions such as “nearly significant” (e.g., regarding survival) should be avoided. Results with p values above the significance threshold should be described as not statistically significant, ideally accompanied by effect size estimates or confidence intervals. In addition, it should be clearly stated that the reported dose of 300 mg/kg represents a target or estimated dose, based on voluntary consumption, rather than a precisely administered amount.

Introduction

The introduction provides a solid background on CLN2 disease and the rationale for evaluating CBD. However, the main hypothesis should be stated more explicitly. It is currently unclear whether the study primarily aims to test an anticonvulsant effect, an anti-inflammatory mechanism, or both.

The translational justification for the selected dose also requires clarification. The equivalence between the mouse dose and an approximate human dose should be supported with appropriate references and accompanied by a discussion of the limitations inherent to interspecies scaling and oral bioavailability differences.

Methods

This section requires substantial clarification to meet standards of reproducibility and rigor.

Randomization and blinding.

The manuscript should explicitly state whether animals were randomly assigned to treatment groups and describe the randomization procedure used. It should also clarify whether investigators performing EEG analysis, histological quantification, and cytokine measurements were blinded to group allocation.

CBD administration and intake monitoring.

Because CBD was administered via a gelatin-based edible with reported consumption of approximately 60–75%, it is essential to provide the actual intake data. These data should ideally be included as supplementary material, showing variability by animal and by sex, and explaining how the effective dose was calculated and adjusted over time. Without this information, it is difficult to interpret exposure consistency.

Ethical procedures and euthanasia.

There are inconsistencies in the manuscript regarding euthanasia methods (references to pentobarbital, Fatal-Plus, and CO₂). These should be reconciled, clearly indicating which methods were used for which experimental groups and confirming that all procedures were approved under the same institutional protocol.

Statistical analysis.

The statistical section requires substantial revision. The primary endpoint should be clearly defined (e.g., time to first seizure). Secondary and exploratory outcomes should be explicitly labeled as such.

Effect sizes and confidence intervals should be reported where applicable (e.g., hazard ratios for survival analyses). The rationale for choosing parametric versus nonparametric tests should be clarified.

Most importantly, the manuscript must address the issue of pseudoreplication. Analyses of seizure duration appear to treat individual seizures as independent observations, even though multiple seizures originate from the same animal. This violates the assumption of independence. These data should be reanalyzed using either:

animal-level summaries (e.g., median seizure duration per animal), or

mixed-effects or clustered models with animal as a random effect.

Without such correction, conclusions regarding seizure duration are not statistically valid.

Results

Brain CBD concentrations.

Measurement of brain CBD levels by LC–MS/MS is a strength of the study. However, additional information is needed regarding the timing of tissue collection relative to last ingestion, inter-animal variability, and whether sampling was standardized. Displaying individual data points would improve transparency.

Neuroinflammatory markers (GFAP and CD68).

Negative findings are acceptable, but methodological details require clarification, including how thresholding was defined, how many sections per animal were analyzed, and whether quantification was performed blinded. Reporting effect sizes and confidence intervals, even in the absence of significance, would strengthen interpretation.

Cytokine and chemokine panel.

This section raises a major concern due to multiple testing. With 12 analytes analyzed, the risk of false-positive findings is substantial. Appropriate correction for multiple comparisons (e.g., false discovery rate) should be applied. Alternatively, the authors should clearly define these analyses as exploratory. Claims regarding IL-1β, IL-6, or GROα should be interpreted cautiously unless corrected analyses support them.

Seizure outcomes.

The delay in time to first seizure is a key strength of the study and should be presented with full survival analysis, including numbers at risk and, if possible, hazard ratios with confidence intervals.

The analysis of seizure duration requires reanalysis, as noted above, to avoid pseudoreplication. The current degrees of freedom strongly suggest that individual seizures were treated as independent observations, which is inappropriate.

Survival analysis.

The manuscript inconsistently reports survival using both a comparison of mean days and a log-rank test. The survival analysis should be standardized, prioritizing Kaplan–Meier curves and Cox regression where applicable. Statements implying “near significance” should be avoided.

Discussion

The discussion is generally balanced but should further temper mechanistic interpretations. While the absence of changes in GFAP, CD68, or cytokines is acknowledged, conclusions should avoid implying mechanistic anti-inflammatory effects. These should instead be framed as hypotheses for future investigation.

Limitations should be more explicitly acknowledged, including:

variability in actual CBD intake,

lack of dose–response evaluation,

small sample sizes for pharmacokinetic measurements,

absence of sex-stratified analyses,

limited power for survival outcomes.

Conclusions

The conclusions should be revised to reflect the data more conservatively. A suggested framing would be that CBD administration was associated with delayed seizure onset and that a subset of animals remained seizure-free during the observation period, while no clear modulation of glial or inflammatory markers was detected under the conditions tested.

Figures, consistency, and references

All figures should clearly report sample sizes. Kaplan–Meier plots should include numbers at risk. Figure numbering should be checked for consistency, as discrepancies appear between the text and figure labels.

The reference list should be reviewed for duplication and formatting inconsistencies (notably repeated citations related to gene therapy studies).

Reviewer #2: This manuscript by Dearborn et al. examines the effects of chronic oral CBD (300 mg/kg) in mouse model of CLN2 disease. The authors report that long-term CBD treatment significantly delays or prevents seizure onset and leads to a near-significant extension of lifespan, despite no improvement in classical neuroinflammatory or neuroimmune markers. The study addresses a clinically relevant question, and provides evidence for an anti-seizure effect of CBD. However, some aspects of the experimental design, data analysis, and interpretation require clarification to strengthen the conclusions.

• The study employs a single relatively high dose of CBD (300 mg/kg) without a dose–response analysis, and this approach limits interpretation of both efficacy and therapeutic window. Notably, recent preclinical studies have reported antiseizure effects of CBD at substantially lower doses (120 mg/kg), including dose-dependent effects (e.g., 12 vs 120 mg/kg) and subthreshold dosing strategies (once vs twice a day). The authors are encouraged to discuss these studies in more detail, comparing their dosing strategy with existing literature and clarifying whether the chosen dose reflects disease severity, pharmacokinetic considerations, or other experimental constraints. Please see:

Costa, AM. et al, Antiseizure Effects of Fully Characterized Non-Psychoactive Cannabis sativa L. Extracts in the Repeated 6-Hz Corneal Stimulation Test. Pharmaceuticals 2021, 14, 1259. https://doi.org/10.3390/ph14121259

Costa, AM. et al, Antiseizure Effects of Cannabidiol Leading to Increased Peroxisome Proliferator-Activated Receptor Gamma Levels in the Hippocampal CA3 Subfield of Epileptic Rats. Pharmaceuticals 2022,15, 495. https://doi.org/10.3390/ph15050495

Lucchi, C. et al, Subthreshold Cannabidiol Potentiates Levetiracetam in the Kainic Acid Model of Temporal Lobe Epilepsy: A Pilot Study. Pharmaceuticals 2024, 17, 1187. https://doi.org/10.3390/ph17091187

• Voluntary oral delivery via gelatin cubes likely introduces variability in individual drug intake. The authors should clarify whether intake was monitored and discuss how this variability may affect seizure-related outcomes.

• The primary outcome (time to first seizure) shows a significant treatment effect, whereas seizure frequency and seizure duration display limited or only marginal effects. The authors should discuss possible reasons for this outcome. In particular, it should be consider whether the dosing regimen may have influenced seizure frequency outcomes. Notably, preclinical studies reporting reductions in seizure number have employed multiple daily administrations of CBD (e.g., twice-daily dosing at ~12 h intervals) to achieve more stable drug exposure and better mimic clinical treatment paradigms. Finally, the authors should clarify whether EEG analysis and seizure scoring were performed blinded to treatment condition. Please see Costa et al., 2022.

• Indicate sex distribution across groups and whether sex-dependent effects were assessed.

• Improve clarity of Figure 3 (especially panel b) regarding symbols and endpoints.

• Standardize terminology for the CLN2 mouse model throughout the manuscript.

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Reviewer #1: No

Reviewer #2: No

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Chronic oral cannabidiol delays seizure onset and reduces seizure burden in a mouse model of CLN2 disease

PONE-D-25-61424R1

Dear Dr. Dearborn,

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,

Giuseppe Biagini, MD

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

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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

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

Reviewer #1: 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

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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

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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)

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Reviewer #1: No

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