Cannabigerol Reduces Acute and Chronic Hypernociception in Animals Exposed to Prenatal Hypoxia-Ischemia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Firstly, I would like to thank the authors for their comprehensive and insightful study. That said, I have a few points for further consideration and improvement:

1. Please provide university email addresses for future correspondence instead of personal ones

2. Line 20: Change "open field tests" to "open-field tests" (hyphen needed for consistency).

3. Lines 42-44: Improve the flow and avoid repetition etc. starting several times with the word Pain

4. Remove line spacing from your manuscript

5. Even thought the content of the introduction is very to the point, the authors have to improve the flow in general by better linking the content and ensuring that sentences are more smoothly connected.

6. Use subscript formatting for the numbers in Δ⁸-THC and Δ⁹-THC. Make sure every instance of these compounds is formatted this way.

7. Overall, the study design, interpretation  and the methods used are appropriate for the research purpose. However, while the authors show that CBG modulates specific pain-related genes, more in-depth molecular analyses or receptor studies could have strengthened their findings. In other words, the study does not fully elucidate the precise signaling pathways involved. This can be further discussed in the results section with one extra paragraph. 

Comments on the Quality of English Language

The English language are very good. 

Author Response

# Reviewer 1

Firstly, I would like to thank the authors for their comprehensive and insightful study. That said, I have a few points for further consideration and improvement:

1.Please provide university email addresses for future correspondence instead of personal ones

Response: Thank you for emphasizing the importance of using institutional email addresses for future correspondence. We have carefully considered your suggestion and updated the title page to include the institutional email addresses as requested.

2. Line 20: Change "open field tests" to "open-field tests" (hyphen needed for consistency).

Response: Sorry for this mistake.The hyphen has been added, and the correction is highlighted in line 22.

3. Lines 42-44: Improve the flow and avoid repetition etc. starting several times with the word Pain.

Response: The sentences have been restructured to ensure a more cohesive narrative without repeatedly starting with the word "Pain." We believe this adjustment enhances the readability of the text.

4. Remove line spacing from your manuscript.

Response: The correction has been made.

5. Even thought the content of the introduction is very to the point, the authors have to improve the flow in general by better linking the content and ensuring that sentences are more smoothly connected.

Thank you for your important feedback. We have revised the introduction to improve the overall flow by better linking the content and ensuring smoother transitions between sentences. These changes aim to create a more cohesive narrative that clearly connects the limitations of previous research with the objectives of our study. Please refer to lines 40 until 97, for the revised text. We hope this revision meets your expectations and enhances the clarity and coherence of the introduction.

6. Use subscript formatting for the numbers in Δ⁸-THC and Δ⁹-THC. Make sure every instance of these compounds is formatted this way.

Response: Sorry for the mistake. We have corrected the formatting in all instances as requested.

 

7. Overall, the study design, interpretation and the methods used are appropriate for the research purpose. However, while the authors show that CBG modulates specific pain-related genes, more in-depth molecular analyses or receptor studies could have strengthened their findings. In other words, the study does not fully elucidate the precise signaling pathways involved. This can be further discussed in the results section with one extra paragraph. 

Response: Thank you for your constructive feedback. While our study focused on the initial screening of targets involved in pain and inflammatory response modulated by CBG, we agree that a further discussion of potential signaling pathways would enhance the depth of our findings. We will add content between lines 479 until 509 in the discussion section to explore these mechanisms in more detail, referencing relevant literature to provide clearer context for our study and to suggest directions for future research

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review this manuscript. All my comments can be seen in the attachment. 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

All my comments regarding English language can be seen in the attachment. 

Author Response

# Reviewer 2

We sincerely appreciate your valuable feedback and are pleased to inform you that we have made the following revisions to the manuscript. All formatting errors have been corrected, including the addition of missing periods and the adjustment of spacing. We have also provided further details on the apparatus used in the animal behavior experiments (line 172) and the substances used (line 128 until 134). The experimental timeline has been added as requested (line 137 until 150). Furthermore, all abbreviations have now been written out in full the first time they appear. In this revised version, all significant results are consistently reported as *p<0.05. The number of animals per group for each experiment is specified in the figure legends.

Additionally, we carefully reviewed the figures and implemented the necessary corrections based on your suggestions.

With regard to some specific questions:

Why the authors used two types of rodents? What was the age of the animals?

The hot plate test was conducted using mice because the available equipment in our lab is designed for small rodents. For the following steps, we used rats. This approach is commonly adopted in the literature, including in previous publications from our group (https://doi.org/10.2147/JPR.S295265,  doi: 10.1016/j.ejphar.2017.09.018), as the pharmacological screening provides only an initial overview of the effects of substances tested. In our study, we used mice aged 4-6 weeks (line 108) and rats aged 30 to 45 days (line 143 and 144), which aligns with the established methodologies referenced.

Do they have a period for acclimatization prior the experiment? For example, 7 days prior to the begening of the experiment.

Response: Thank you for your observation. The animals were born and remained in the animal facility of our department until the day of the experiment. Acclimatization refers to the time the animals were placed in the experimental room to become familiar with the environment and room temperature. We have rewritten this section to improve understanding and flow on page 3, lines 116-117.

The authors should specify what were the substances used in this experiment, and how were the animals randomized.

Response: Sorry for this mistake. We have rewritten this section, adding the substances used to bring more clarity on lines 128-134. The animals were randomized according to what is described in the experimental model (line 137 until 150).

Why this experiment was not performed on the rats?

Response: Thank you for your observation. Our laboratory has a hot plate device for mice, which is why we used this protocol with mice for the initial pharmacological screening. This screening was intended to provide a preliminary assessment of the CBG extract's effects. For a more detailed analysis, we subsequently conducted further tests using rats, as described in the manuscript.

Locomotor activity

Why this experiment was not performed on mice?

Thank you for your question. The initial pharmacological screening was performed on mice due to the availability of a hot plate device specifically designed for small rodents in our laboratory. However, all subsequent experiments were carried out using rats. This decision was based on the availability of equipment and the anatomical suitability of rats for the hypoxic-ischemic surgeries and spinal nerve ligation procedures. Rats are better suited for these types of surgical interventions due to their larger size and anatomical features, which makes them a more appropriate model for these specific experimentais procedures.

What are the specification of the apparatus? Dimenions? What is the nature of the box? Plastic? Colour of the walls?

Response: Thanks for the suggestion. We have corrected and clarified the specifications for the open field equipment.

 

Acetylsalycylic acid was used as a solution? Suspension? In what vehicle? What was the vehicle used in control group?

Response: Thank you for bringing this detail to our attention. Acetylsalicylic acid (ASA) was dissolved in dimethyl sulfoxide (DMSO), which was the vehicle used for both the treatment and the control groups. The ASA was prepared as a solution in this vehicle. We have included this information in line 133-134.

How the authors got the spinal cord?

Response: We have rewritten this section and added a separate paragraph to the text specifically for this explanation.

 

What test was applied for the parametric distribution?

For parametric distributions in our statistical analysis, we used the Shapiro-Wilk test to assess whether the data followed a normal distribution, which is a key assumption for parametric tests. We have included this information in line 283-286.

 

 

SPSS version 16.0 software

Why the authors used two different softwares?

Response: Thank you for pointing that out. We made a mistake, but it has already been corrected. We only used one software. The corrected information has been updated from line 280 until 289 in the statistics section.

The authors have to specify what is KS.
Response: Dear Reviewer, apologies for the oversight. We have revised this section, and it is no longer necessary to specify what KS refers to.

In the second phrase of the paragraph the authors used “ANOVA” without specifying what this represents, and now, the authors specify what is ANOVA.

Response:Thank you for your feedback. We have revised this section to make it clearer.

Results should be expressed as mean±SD.

Thank you for the suggestion that results should be expressed as mean ± SD, however, we chose to express our results as mean ± standard error of the mean (SEM) for several reasons:

1- SEM provides a measure of how accurately the sample mean represents the population mean. Unlike standard deviation, which reflects the spread of individual data points, SEM considers the sample size, indicating how much the sample mean may vary from the true population mean. This makes it a more relevant metric when aiming to generalize findings.

2- SEM is particularly useful in parametric statistical tests where the goal is to compare group means. By accounting for sample size, SEM helps in interpreting the reliability of mean differences between groups, which is a crucial aspect in our study.

3- In graphical representations, error bars using SEM make it easier for readers to visually assess the overlap between groups. This is particularly important in determining the potential for statistical significance.

4- The use of SEM in presenting experimental data is a standard approach in pharmacological research, as seen in numerous journals, ensuring that our presentation aligns with established practices in the field.

By adopting SEM, we ensure that our data presentation is consistent with the standards of pharmacological research and provides an accurate and precise representation of our findings.

 

In Material&Methods the authors did not describe that they performed the experiment at diferent

Response:Thank you for your feedback. We have corrected and rewritten the text, adding the missing information in line 161.

Lines 257-258 - In open field testing performed with single oral administration 50 mg/kg of CBG or Vehicle

Response: We have made adjustments to the text and believe that this improves it.

What about the distance, the time spent in a freezing state?

Thank you for your question regarding the distance traveled and time spent in a freezing state. The open field test is widely used to assess locomotion, anxiety, and stereotypical behaviors such as grooming and rearing in rodents. Time spent in a freezing state is a well-established parameter for evaluating anxiety-like behavior, where prolonged freezing suggests heightened anxiety or fear response.

Our focus in this study was on locomotor activity, specifically assessing the number of quadrants crossed (ambulation) within a defined area, such as the open field, during a set period of time. This measure provides a general indication of the animal’s overall movement and exploratory behavior.

We based our experimental approach on well-established references in the field, including:

• Kraeuter AK, Guest PC, Sarnyai Z. The Open Field Test for Measuring Locomotor Activity and Anxiety-Like Behavior. Methods Mol Biol. 2019;1916:99-103. doi:10.1007/978-1-4939-8994-2_9
• Khatian, N., Aslam, M. Effect of Ganoderma lucidum on memory and learning in mice. Clin Phytosci 5, 4 (2019). https://doi.org/10.1186/s40816-019-0101-7

 

The authors cannot state this since they only assayed the number of quadrants crossed.

Thank you for your note, we decided to remove this part of the manuscript.

Why the authors used only 5 individuals? It is not statistically significant.

Thank you for your observation. The statistical tests we used can be reliably performed with a minimum of 3 samples per group for meaningful analysis. The decision to use 5 animals per group was guided by our commitment to the Principles of Russell and Burch (1959), known as the 3Rs—Reduction, Replacement, and Refinement—in the use of animals. Additionally, we followed guidelines from previous studies within our group, which support using this sample size for preliminary research.

For example, the study by Fontes-Dantas et al. (2023), titled "SARS-CoV-2 Spike protein induces TLR4-mediated long-term cognitive dysfunction recapitulating post-COVID-19 syndrome in mice" (Cell Rep. 42(3):112189, https://doi.org/10.1016/j.celrep.2023.112189), effectively applied similar protocols with 4-5 animals per group, demonstrating the feasibility and scientific rigor of this approach.

Line: 449-450 “demonstrated that CBG had a superior antinociceptive effect in the carrageenan-induced inflammatory pain model, surpassing CBC. What is CBC?

Response: We apologize for the error; we have rewritten this section and made the necessary adjustment, clarifying that CBC stands for cannabichromene.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I suggest the authors add a section for the limitations of the study. 

Author Response

Dear Reviewer,

Thank you for your suggestion, we included a study limitation topic

Here is the suggestion for the text 

While this study provides valuable insights into the antinociceptive effects of CBG in acute and chronic pain models, it is not without limitations. First, the study primarily used rodent models, which, although widely accepted in preclinical research, may not fully replicate human pain conditions. Thus, the translational applicability of the results to clinical settings requires further validation. Second, while the study explored both male and female subjects, sex-related differences in cannabinoid receptor expression and hormonal variations, particularly in females, could have introduced variability in responses. A more comprehensive analysis of the role of sex hormones in CBG's effects is needed to better understand this variability. Additionally, the dosing regimen focused on a single dose of 50 mg/kg CBG. Exploring a wider range of doses could provide more nuanced information regarding its efficacy and safety. Furthermore, the pharmacokinetic profile of CBG was not assessed, leaving potential gaps in understanding its bioavailability and metabolism in vivo. Finally, while molecular analyses were performed, the study did not explore the potential long-term effects of CBG on neural tissues beyond the 14-day treatment period. Further studies are warranted to investigate the sustained efficacy and safety of CBG in chronic pain models over an extended period. These limitations should be addressed in future research to strengthen the understanding of CBG's therapeutic potential in pain management.