Effects of in utero delta-9-tetrahydrocannabinol (THC) exposure on fetal and infant musculoskeletal development in a preclinical nonhuman primate model

Samantha A. Moellmer; Olivia L. Hagen; Parsa A. Farhang; Victoria R. Duke; Meghan E. Fallon; Monica T. Hinds; Owen J. T. McCarty; Jamie O. Lo; Karina H. Nakayama

doi:10.1371/journal.pone.0306868

Peer Review History

Original SubmissionMarch 15, 2024
2 May 2024 Decision Letter - Vinod K. Yaragudri, Editor PONE-D-24-10539Effects of in utero delta-9-tetrahydrocannabinol (THC) exposure on fetal and infant musculoskeletal developmentPLOS ONE Dear Dr. Nakayama, 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 Jun 16 2024 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. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In order to comply with PLOS ONE's guidelines for non-human primate experiments (http://journals.plos.org/plosone/s/submission-guidelines#loc-non-human-primates), please provide additional details regarding housing conditions, feeding regimens, environmental enrichment, and all relevant steps taken to alleviate suffering (anaesthesia, analgesia, details about humane endpoints, euthanasia, etc.). Also indicate how often animal care staff monitored the health and well-being of the animals and the criteria used to make such assessments. Lastly, specify the disposition of animals at the end of the study (e.g. euthanasia, returned to home colony, etc.), please provide this information for both mothers and infants involved in the study. If animals were euthanized following the study, please provide the method of sacrifice. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: This is a manuscript where the authors have the authors have assessed gene expression changes in skeletal muscle from fetal and infant rhesus monkeys that were exposed to delta-9-THC (THC) during gestation. The authors should clarify where in the central nervous system that CB2R is expressed. Most cannabinoid researchers do not believe that CB2R is expressed in neurons under normal, non-injury conditions. It is not accurate that THC is an antagonist of CB2R. The listed dosages are expressed in a non-standard manner. It would be better to express dosage of THC as mg/kg/day for comparison to other studies. It is not clear that animals consumed all of the cookies (containing THC). Was cookie (and THC) consumption confirmed and verified? The A260/A280, RIN, and DV200 values are very low. While I understand that these are comparable for work done using FFPE tissues, it does raise the possibility that some differentiated expressed genes (DEGs) were missed due to low RNA quality? This limitation should be mentioned in the discussion as well as the point that future work should/could address this question using fresh frozen tissue. In lines 196-198, the authors state that prenatal THC exposure leads to epigenetic gene expression changes. While this might be true, it seems to suggest that the DEGs observed in the current study were due to epigenetic changes, something for which there is no evidence for in the current work. Although there weren't glaring problems with the current manuscript, it also doesn't seem to represent a complete story. This pilot study would be much stronger and more compelling if there was more to the story. Reviewer #2: The endocannabinoid system (ECS) orchestrates crucial bodily functions and responses to external factors. While explored as a therapeutic target, understanding ECS modulation across tissues is essential. The team investigated the impacts of experimental exposure to natural plant THC (exogenous; a robust ligand of cannabinoid receptors) during pregnancy, on fetal and infant muscle development using a non-human primate model. The authors’ findings highlight subtle effects on muscle development, with notable changes in inflammation and cytokine signaling pathways, indicating potential tissue damage. Authors start with the premise that any therapeutic modulation of ECS by THC may heavily depend on patho/physiological state, based on studies in rodents. Specifically, considering that activation of CB1R through EC 2-AG can disrupt myoblast differentiation, the authors ask if enhanced activation via exposure to exogenous cannabinoids might affect early skeletal muscle development in primates. The team has used a chronic regimen using escalating oral dosing (constituted in chow/edibles) of 0-2.5 mg/7kg body weight per day over 11-12 weeks (plus maintenance with the highest THC dose) from conception until sample collection from, in time-mated, timed pregnant rhesus macaques. Authors sought to characterize (the impact of) neuroinflammatory response on fetal and infant stages of skeletal muscle development, evaluated by histomorphology and for differential gene expression using a Nanostring nCounter neuroinflammatory panel, to gain insights to shed light on key pathways impacted by THC exposure-induced stimulation of the ECS during early embryonic, fetal development. This important study, lays the groundwork for further exploration into the long-term, intergenerational consequences of prenatal THC exposure on neuroinflammatory profiles and functional outcomes in primates, and humans. Comments: Very important piece of work in the area. All aspects of the study design, data analysis, inference, presentation, literature citations are adequate. Methodology is rigorous and meet the requirements of PLOS One. ****** 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: No Reviewer #2: No ******** [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.0306868.r001
Revision 1
14 May 2024 Author Response We thank the reviewers for their insightful comments and suggestions to improve the quality of our manuscript (PONE-D-24-10539) “Effects of in utero delta-9-tetrahydrocannabinol (THC) exposure on fetal and infant musculoskeletal development”. Based on reviewer feedback, we have improved the manuscript by clarifying background information and expanded on future work that can be done to progress the field. We are happy to submit the current revised version and hope to meet the high-quality standards of your journal. Please find below our point-by-point responses to reviewer comments below: Journal requirements 1. In order to comply with PLOS ONE's guidelines for non-human primate experiments (http://journals.plos.org/plosone/s/submission-guidelines#loc-non-human-primates), please provide additional details regarding housing conditions, feeding regimens, environmental enrichment, and all relevant steps taken to alleviate suffering (anaesthesia, analgesia, details about humane endpoints, euthanasia, etc.). Also indicate how often animal care staff monitored the health and well-being of the animals and the criteria used to make such assessments. Lastly, specify the disposition of animals at the end of the study (e.g. euthanasia, returned to home colony, etc.), please provide this information for both mothers and infants involved in the study. If animals were euthanized following the study, please provide the method of sacrifice. Thank you for highlighting this, we have provided additional details in the Methods section regarding housing conditions, feeding regimens, steps to alleviate suffering, monitoring of animal welfare, and disposition of animals. Here is the excerpt: “Animals were observed at least twice a day by trained animal care technicians and every effort is made by both veterinary and research staff to minimize pain and distress in the rhesus macaques as clinically needed. During the course of the study, clinical signs of distress in the pregnant dams such as weight loss, anorexia, cachexia, self-injury, or failure to thrive were assessed by ONPRC Division of Comparative staff and the ONPRC Behavioral Management Program to ensure psychological health and well-being. When possible, positive reinforcement training was used to train rhesus macaques to cooperate with various procedures, thus reducing the stress associated with those procedures. Rhesus macaques were housed in full contact pairs with a compatible social partner for the duration of the study in accordance with the Guide for the Care and Use of Laboratory Animals” “This study used indoor-housed adult, female rhesus macaques (n=18) of similar size (~6-7 kg) maintained on a standard chow diet (TestDiet, St. Louis, Missouri) given twice a day in addition to fresh produce or other food enrichment daily, with and tap water available ad libitum” “All methods of euthanasia utilized at the ONPRC are consistent with the American Veterinary Medical Association Guidelines for the Euthanasia of Animals , sodium pentobarbital was delivered intravenously followed by exsanguination under a deep surgical plane of anesthesia.” 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Thank you for noticing that discrepancy. We will be sure to provide the correct grant numbers for the awards upon resubmission. 3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. The Nanostring nCounter raw and normalized data has been made available on Kaggle, doi: 10.34740/kaggle/dsv/7853952. We will add this information into our data availability statement. Reviewer #1 This is a manuscript where the authors have the authors have assessed gene expression changes in skeletal muscle from fetal and infant rhesus monkeys that were exposed to delta-9-THC (THC) during gestation. Specific comments follow: 1. The authors should clarify where in the central nervous system that CB2R is expressed. Most cannabinoid researchers do not believe that CB2R is expressed in neurons under normal, non-injury conditions. Thank you for this comment, we have made clarifications regarding where in the central nervous system CB2R is expressed in the introduction. Here is the excerpt: “There is conflicting evidence surrounding CB2R expression in the central nervous system, with some studies suggesting CB2R is weakly expressed in hippocampal and brain stem neurons under normal physiological conditions [2, 3]. However, under inflammatory conditions or injury, CB2R is highly expressed on reactive microglia [4, 5]. Outside of the nervous system, CB2R is highly expressed in peripheral immunological tissues [4].” 2. It is not accurate that THC is an antagonist of CB2R. Thank you to the reviewer for pointing out this discrepancy. We have made this clarification in the introduction. Here is the excerpt: “THC is the main psychoactive component in cannabis and acts as a partial agonist on CB1R and CB2R [12, 13]. In addition to its role as a partial agonist on the CB2R receptor, THC can inhibit the activity of cannabinoid ligands on CB2R, thereby also acting as a weak antagonist in select settings [14].” 3. The listed dosages are expressed in a non-standard manner. It would be better to express dosage of THC as mg/kg/day for comparison to other studies. Thank you for this suggestion, we have made this correction throughout the manuscript. 4. It is not clear that animals consumed all of the cookies (containing THC). Was cookie (and THC) consumption confirmed and verified? Yes, cookies were administered in the morning prior to daily chow and complete ingestion was confirmed. These details were added into the methods. Here is the excerpt: “Cookies were administered in the morning prior to daily chow to ensure complete ingestion.” 5. The A260/A280, RIN, and DV200 values are very low. While I understand that these are comparable for work done using FFPE tissues, it does raise the possibility that some differentiated expressed genes (DEGs) were missed due to low RNA quality? This limitation should be mentioned in the discussion as well as the point that future work should/could address this question using fresh frozen tissue. This is an excellent point, and we have now included additional information outlining this in the discussion. Here is the excerpt: “This study utilized FFPE tissues, despite known challenges with RNA quality. We strategically employed Nanostring nCounter technology, a method specifically optimized to extract high-quality gene expression data from FFPE tissues. While this approach was effective, it’s possible that some differentially expressed genes (DEGs) may not have been detected. Future studies utilizing fresh frozen tissues and RNA sequencing could further enhance our understanding of THC-induced differential gene expression in skeletal muscle.” 6. In lines 196-198, the authors state that prenatal THC exposure leads to epigenetic gene expression changes. While this might be true, it seems to suggest that the DEGs observed in the current study were due to epigenetic changes, something for which there is no evidence for in the current work. Thank you for this comment. We agree that discussing epigenetic changes may be misleading to readers in suggesting the DEGs are caused by epigenetic changes. We have decided to remove details of THC and epigenetic changes. 7. Although there weren't glaring problems with the current manuscript, it also doesn't seem to represent a complete story. This pilot study would be much stronger and more compelling if there was more to the story. We appreciate this feedback. This pilot study lays the groundwork for future exploration into the effects of prenatal THC-exposure on musculoskeletal development. As per your recommendation, we have added a limitations and future directions section into the discussion. Here is the excerpt: “This study utilized FFPE tissues, despite known challenges with RNA quality. We strategically employed Nanostring nCounter technology, a method specifically optimized to extract high-quality gene expression data from FFPE tissues. While this approach was effective, it’s possible that some differentially expressed genes (DEGs) may not have been detected. Future studies utilizing fresh frozen tissues and RNA sequencing could further enhance our understanding of THC-induced differential gene expression in skeletal muscle.” Reviewer #2 The endocannabinoid system (ECS) orchestrates crucial bodily functions and responses to external factors. While explored as a therapeutic target, understanding ECS modulation across tissues is essential. The team investigated the impacts of experimental exposure to natural plant THC (exogenous; a robust ligand of cannabinoid receptors) during pregnancy, on fetal and infant muscle development using a non-human primate model. The authors’ findings highlight subtle effects on muscle development, with notable changes in inflammation and cytokine signaling pathways, indicating potential tissue damage. Authors start with the premise that any therapeutic modulation of ECS by THC may heavily depend on patho/physiological state, based on studies in rodents. Specifically, considering that activation of CB1R through EC 2-AG can disrupt myoblast differentiation, the authors ask if enhanced activation via exposure to exogenous cannabinoids might affect early skeletal muscle development in primates. The team has used a chronic regimen using escalating oral dosing (constituted in chow/edibles) of 0-2.5 mg/7kg body weight per day over 11-12 weeks (plus maintenance with the highest THC dose) from conception until sample collection from, in time-mated, timed pregnant rhesus macaques. Authors sought to characterize (the impact of) neuroinflammatory response on fetal and infant stages of skeletal muscle development, evaluated by histomorphology and for differential gene expression using a Nanostring nCounter neuroinflammatory panel, to gain insights to shed light on key pathways impacted by THC exposure-induced stimulation of the ECS during early embryonic, fetal development. This important study, lays the groundwork for further exploration into the long-term, intergenerational consequences of prenatal THC exposure on neuroinflammatory profiles and functional outcomes in primates, and humans. Comments: Very important piece of work in the area. All aspects of the study design, data analysis, inference, presentation, literature citations are adequate. Methodology is rigorous and meet the requirements of PLOS One. Thank you for these comments and summary. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0306868.r002
25 Jun 2024 Decision Letter - Vinod K. Yaragudri, Editor Effects of in utero delta-9-tetrahydrocannabinol (THC) exposure on fetal and infant musculoskeletal development PONE-D-24-10539R1 Dear Dr. Nakayama, 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. 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For example please add "preclinical nonhuman primate model" or "in rhesus macaques" to the title. 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 ******** 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: Yes ****** 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: (No Response) ****** 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 ******** https://doi.org/10.1371/journal.pone.0306868.r003
Formally Accepted
9 Jul 2024 Acceptance Letter - Vinod K. Yaragudri, Editor PONE-D-24-10539R1 PLOS ONE Dear Dr. Nakayama, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Vinod K. Yaragudri Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306868.r004

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