Exploring the Anxiolytic, Antidepressant, and Immunomodulatory Effects of Cannabidiol in Acute Stress Rat Models

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript that entitled by “Exploring the Anxiolytic, Antidepressant, and Immunomodulatory Effects of Cannabidiol in Acute Stress Animal Models”, conducted a research study to evaluate the effects of cannabidiol (CBD) on anxiety and depression-like behaviors in Wistar rats subjected to acute cold stress, as well as its impact on pro- and anti-inflammatory cytokines. Their findings suggest that CBD exerts anxiolytic and antidepressant effects, partially mediated by modulation of inflammatory processes, particularly IL-6. The manuscript is generally well-addressed and well-cited; However, I have some comments and suggestions.

Line 3: The research study conducted on rats. Therefore, I suggest changing the title to be "rat model" not “animal model” to be more specific.

Line : Please note that the author’s affiliation should show the address not names like "Prof. Dr. Elissay Yanev". Please revise.

Line 28: I suggest adding more keywords within permissible limit and easy reachable for citations by others such as depression; rat; IL-6

Line 48: upon the mentioned literature used at this manuscript, only FOUR references used for the literature. Please rewrite the introduction to be more informative, focused and specific.

Line 72: Please provide full details for each chemical or reagent separately mentioned on line 71 & 72 including the catalog number as well.

Line 75: Please verify the age of the rats and how many are in each group.

Line 103: Please add the catalog number and  the name of production company for ELISA kits.

Line 121: There is a significant difference between treated groups, specially the 5 &10 mg/Kg treated groups. Therefore, it’s important to mention (n), the number of animals in each group.

Line 122: I suggest changing the subtitles  of results to be 3 main assessment activity points: 1) social interaction/anti-anxiety activity, 2) Anti-depression activity test and 3) Pro anti-inflammatory activity.

Line 199: I suggest coloring the columns throughout the results to be more clear, characteristic and understandable.

Line 203: The discussion is too long. Please rewrite to be focus and specific.

Line 210: Please mention the references for these "majority of preclinical studies".

Line 231: Please verify the abbreviation of "GABA" as first time mentioned.

Line 240: Please note that abbreviation "FST" mentioned before. Please revise to mention either the abbreviation or the full definition and do it throughout the manuscript.

Line 261: By the end of this paragraph (line 248-261), please note that you didn't conduct any research study concerning the ROS and PPAR-α. Therefore, there is no significance to add them for discussion. Please revise to be more focused and specific.

Line 336: The conclusion usually restates the aim of research question, highlights the most important findings, and gives a concise and specific conclusion with future research directions. Please rewrite to get a full informative conclusion paragraph.

References: References number 36 and 37 that are incomplete. Please revise and check the DOI as well.

Comments on the Quality of English Language

Major editing of English Language required.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below.

Line 3: The research study conducted on rats. Therefore, I suggest changing the title to be "rat model" not "animal model" to be more specific.

Thank you for this suggestion. The title has been updated accordingly.

Line X: Please note that the author’s affiliation should show the address, not names like "Prof. Dr. Elissay Yanev." Please revise.

We appreciate your input. However, the current format reflects the official name of the department and their required format for affiliations. As such, we are unable to make this change.

Line 28: I suggest adding more keywords within permissible limits and easily reachable for citations by others, such as depression; rat; IL-6.

Thank you for the suggestion. Additional keywords have been included as recommended.

Line 48: Upon the mentioned literature used in this manuscript, only FOUR references were cited for the literature. Please rewrite the introduction to be more informative, focused, and specific.

We respectfully clarify that SEVEN references have been cited, focusing on various aspects, including CBD effects, anti-inflammatory properties, and antidepressive mechanisms. However, we have revised the introduction to ensure it is more focused and concise.

Line 72: Please provide full details for each chemical or reagent separately mentioned on lines 71 and 72, including the catalog number.

Full details, including catalog numbers, have been added as requested.

Line 75: Please verify the age of the rats and how many are in each group.

The age of the rats and group sizes have been verified and included in the manuscript.

Line 103: Please add the catalog number and the name of the production company for the ELISA kits.

The catalog numbers and production company details for the ELISA kits have been added.

Line 121: There is a significant difference between treated groups, especially the 5 & 10 mg/kg treated groups. Therefore, it’s important to mention (n), the number of animals in each group.

The number of animals in each group has been included in the "Materials and Methods" section.

Line 122: I suggest changing the subtitles of results to be three main assessment activity points: 1) social interaction/anti-anxiety activity, 2) anti-depression activity test, and 3) pro anti-inflammatory activity.

The subheadings in the results section reflect the employed tests and their outcomes. The discussion contextualizes these findings. We believe the current structure appropriately conveys the study's focus.

Line 199: I suggest coloring the columns throughout the results to make them clearer and more understandable.

Colors have been added to the columns to enhance clarity.

Line 203: The discussion is too long. Please rewrite to be focused and specific.

The discussion has been revised for better focus and conciseness.

Line 210: Please mention the references for these "majority of preclinical studies."

References have been added to support this statement.

Line 231: Please verify the abbreviation of "GABA" as it is first mentioned.

The full form of "GABA" has been included where it is first mentioned.

Line 240: Please note that the abbreviation "FST" was mentioned before. Revise to consistently use either the abbreviation or the full definition throughout the manuscript.

Consistent use of the abbreviation "FST" has been implemented throughout the manuscript.

Line 261: By the end of this paragraph (lines 248-261), please note that you didn’t conduct any research concerning ROS and PPAR-α. Therefore, there is no significance in adding them to the discussion. Please revise to be more focused and specific.

The discussion of ROS and PPAR-α has been removed to maintain focus and relevance.

Line 336: The conclusion usually restates the aim of the research question, highlights the most important findings, and provides a concise and specific conclusion with future research directions. Please rewrite for a more informative conclusion paragraph.

The conclusion has been rewritten to align with your recommendations, highlighting key findings and future directions.

References: References 36 and 37 are incomplete. Please revise and check the DOI as well.

References have been revised, and DOIs have been verified and updated.

Reviewer 2 Report

Comments and Suggestions for Authors

Exploring the Anxiolytic, Antidepressant, and Immunomodulatory Effects of Cannabidiol in Acute Stress Animal Models" by Hristina Zlatanova-Tenisheva et al. It appears to be a well-structured study investigating the effects of cannabidiol (CBD) on anxiety, depression, and inflammatory cytokine levels in rats subjected to acute cold stress. Here, the authors explore the potential of CBD as a therapeutic agent for stress-related disorders and its role in modulating inflammation. Male Wistar rats were administered CBD oil (2.5, 5, or 10 mg/kg) or olive oil (control) for 14 days. On day 15, rats in the stress groups were exposed to acute cold stress for 60 minutes. Behavioral assessments using the social interaction test and the forced swim test were conducted to evaluate anxiolytic and antidepressant effects, respectively. To assess inflammatory response, serum cytokine levels (IL-6, TNF-α, IL-1β, and IL-10) were measured using ELISA.The findings suggest that CBD administration:

• Significantly increased social interaction time, indicative of anxiolytic effects, at doses of 5 mg/kg and 10 mg/kg.
• Dose-dependently reduced immobility time in the forced swim test, suggesting antidepressant activity.
• Selectively lowered serum IL-6 levels, a key cytokine associated with stress and depression.

Overall, the study provides evidence supporting the potential of CBD in mitigating stress-induced anxiety and depression, possibly through the modulation of inflammatory processes. Here are some potential shortcomings of the study, along with suggestions for additional experiments, if possible.

Minor:

1.The study exclusively used male rats. Can authors explain the reason? It's crucial to investigate whether CBD's effects are comparable in female animals, as sex differences in stress responses and endocannabinoid system function are well-documented.

2.While the study included the social interaction test and the forced swim test, incorporating additional behavioral tests, such as the elevated plus maze, open field test, and sucrose preference test, could provide a more comprehensive assessment of anxiety- and depression-like behaviors

3.The study focused on cytokine levels but did not assess neurotransmitter levels (e.g., serotonin, dopamine, norepinephrine, GABA) in brain regions relevant to stress and mood regulation. This would provide valuable insights into the neurobiological mechanisms underlying CBD's effects, or the author can explain that without doing any experiment.

4.While three doses of CBD were tested, a more extensive dose-response curve analysis would be beneficial to identify the optimal dose for maximal therapeutic effects with minimal side effects. If possible, conduct a more extensive dose-response analysis to determine the optimal therapeutic dose. If the author can explain the reason for this, that would be helpful.

 

By addressing these shortcomings and conducting further research, we can gain a more comprehensive understanding of CBD's therapeutic potential for stress-related disorders and its mechanisms of action.

Comments on the Quality of English Language

well written

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below.

The study exclusively used male rats. Can authors explain the reason? It's crucial to investigate whether CBD's effects are comparable in female animals, as sex differences in stress responses and endocannabinoid system function are well-documented.

Thank you for your insightful comment. In the present study, male rats were selected to adhere to ethical guidelines and to address specific considerations regarding the inclusion of female animals. Female rats, due to their reproductive capacity, may raise ethical concerns related to the potential transgenerational transmission of study-related effects. The exclusive use of male rats aimed to mitigate these complexities while ensuring the scientific validity of the results. We acknowledge the importance of examining sex differences and plan to include female animals in future studies to comprehensively evaluate their unique biological responses and to explore potential generational effects in a manner that is both scientifically rigorous and ethically responsible.

While the study included the social interaction test and the forced swim test, incorporating additional behavioral tests, such as the elevated plus maze, open field test, and sucrose preference test, could provide a more comprehensive assessment of anxiety- and depression-like behaviors.

We appreciate your suggestion. The comprehensive evaluation of behavioral outcomes indeed requires a wide range of tests. However, addressing all such assessments within the scope of a single manuscript is not feasible. The behavioral tests used in this study were specifically chosen as screening tools to assess the potential impact of CBD on anxiety- and depression-like behaviors. These findings serve as a foundation for further research. We have systematically planned additional studies incorporating specialized behavioral tests, such as the elevated plus maze, open field test, and sucrose preference test, to expand and deepen our understanding of the effects of CBD in this context.

The study focused on cytokine levels but did not assess neurotransmitter levels (e.g., serotonin, dopamine, norepinephrine, GABA) in brain regions relevant to stress and mood regulation. This would provide valuable insights into the neurobiological mechanisms underlying CBD's effects, or the authors can explain this limitation without additional experiments.

Thank you for highlighting this important aspect. At present, our research team lacks the capability to perform the extensive analyses required to evaluate neurotransmitter levels, their precursors, or RNA expression. We are actively collaborating with laboratories equipped with the necessary expertise and facilities to conduct these advanced investigations. Future studies will incorporate these analyses alongside additional behavioral tests, such as the Vogel conflict test for assessing GABAergic neurotransmission, to elucidate the role of specific neurotransmitter pathways. This collaborative approach is expected to provide a more comprehensive understanding of the neurobiological mechanisms underlying CBD’s effects.

While three doses of CBD were tested, a more extensive dose-response curve analysis would be beneficial to identify the optimal dose for maximal therapeutic effects with minimal side effects. If possible, conduct a more extensive dose-response analysis to determine the optimal therapeutic dose. If the authors can explain the reason for this, that would be helpful.

We appreciate your thoughtful comment. As mentioned earlier, the tests employed in this study primarily served as screening methods. For this reason, only three doses of CBD were examined at this stage. Once the effects of CBD on anxiety- and depression-like behaviors are confirmed, further behavioral assessments will be conducted to investigate the underlying mechanisms of action. Following these analyses, a comprehensive dose-response study will be undertaken to explore the relationship between CBD dosage and its behavioral effects. This sequential approach allows us to build a robust foundation before conducting more extensive dose-response analyses.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is improved. Thanks for taking my comments into your consideration as well!. I have only one comment:

Line 68: At the end of introduction, please add short sentence for highlighting your main conclusions to follow the journal guidelines.

Comments on the Quality of English Language

Minor English language required.

Author Response

Thank you for taking the time to review our manuscript and for your valuable feedback.

Line 68: At the end of introduction, please add short sentence for highlighting your main conclusions to follow the journal guidelines.

A sentence has been added, and the preceding paragraph has been slightly revised to avoid repetitions. The changes have been highlighted in blue.

Minor English language required.

While we are happy to make the necessary revisions, we would appreciate further clarification on the specific aspects that need attention. Is it related to grammar, sentence structure, or perhaps the length or clarity of certain sentences? For context, the manuscript has been reviewed using generative AI tools for spelling and grammatical accuracy, and after the first round of review, a native English speaker was consulted to further improve the language. Nonetheless, we are keen to address any remaining concerns to ensure the text is clear and aligns with the journal’s standards. Your guidance on this matter would be greatly appreciated.