Veterinary Sciences | Free Full-Text

Vet. Sci. 2023, 10(5), 310; https://doi.org/10.3390/vetsci10050310 (registering DOI)

Vet. Sci. 2023, 10(5), 310; https://doi.org/10.3390/vetsci10050310 (registering DOI)

Received: 22 February 2023
/
Revised: 5 April 2023
/
Accepted: 20 April 2023
/
Published: 24 April 2023

Round 1


Reviewer 1 Report

The manuscript can be accepted after minor revisions.

The number of rats used in this study should be increased

An English language review is required.

The conclusions should be expanded

Author Response

The number of rats used in this study should be increased.

Thank you for your comment.

The data obtained in this study with the normality test and the observed standard deviation suggest to the authors not having to increase the number of animals in this study.

In 2020, Wistorf et al., strengthened the importance of caring for the 3Rs within the framework of and the German Center for the Protection of Laboratory Animals (Bf3R). Hence the importance of a reduction in the size of N when the data behaves with a normal distribution and adequate standard deviation.

An English language review is required.

Thank you for your comment.

The article has undergone English language editing by MDPI. The text has been checked for correct use of grammar and common technical terms.

We send certificate of correction and edition of the English language issued by Basel, Switzerland.

The conclusions should be expanded

Thank you for your comment.

The conclusion has been expanded based on your suggestion.

The administration of a cannabinoid for 21 days alters the minimum alveolar concentration of isoflurane. We observed that the concentration of isoflurane required to prevent movement in response to a painful stimulus is higher in individuals who were repeatedly administered cannabinoid. The sparing effect of morphine on isoflurane is lower in rats constantly medicated with a cannabinoid. Therefore, it will be prudent to consider that individuals who have been under the effect of a synthetic cannabinoid may present different effects than expected when administering morphine, since cannabinoids decrease the sparing effect of morphine compared to individuals who do not consume cannabinoids. The sparing effect of dexmedetomidine on the minimum alveolar concentration of isoflurane is greater in rats repeatedly medicated with a cannabinoid. Because of this, it will be necessary to consider reducing the dose of isoflurane in patients who are constantly exposed to cannabinoids and are administered dexmedetomidine.

 

 

Author Response File: Author Response.docx


Reviewer 2 Report

Dear authors, the topic is very interesting and original. The results obtained can be the basis for subsequent applications in veterinary and human medicine. However, in my opinion, mat and met are described in a confusing and non linear way. Moreover, the statistical analysis described is poor.

Furthermore, not enough information was given in relation to the type of cannabinoid used (since the different types of cannabinoids have several pharmacokinetic/pharmacodynamic characteristics). In conclusion, I consider this manuscript fit for publication after improvements in writing mat and met and statistical analysis and an assessment of the English language by a native speaker. Other considerations are in the attached file.

Comments for author File: Comments.pdf

Author Response

Abstract:
– Change “on untreated and rats treated for 21 days” with “on untreated and treated rats for 21 days”

Thank you for your comment.

We have modified the paragraph based on your suggestion.

– Please edit the last paragraph “The sparing effect of morphine was lower and that of dexmedetomidine was greater on isoflurane in rats chronically medicated with a cannabinoid.” because it is unclear.

Thank you for your comment. We have modified the paragraph based on your suggestion.

The sparing effect of morphine on isoflurane was less, compared to dexmedetomidine were the sparing effect on isoflurane increase in rats chronically treated with a cannabinoid.

Keyword:

– Please change “Minimum Alveolar Concentration MAC of isoflurane” with “Minimum Alveolar Concentration if isoflurane”

Reviewer 2. Thanks for your comment. It has been modified based on your suggestion.

Introduction:

– “However, the reasons for these effects are still not entirely clear, and many studies have focused on elucidating these effects” Please edit this sentence to not repeat the word “effects”.

Thanks for your comment. It has been modified based on your suggestion.

However, the reasons are still not entirely clear, and many studies have focused on elucidating these effects.

– “Given the high possibility that a chronic consumer of Cannabis sativa, or its derivatives, requires general anesthesia and dexmedetomidine or an opioid and the lack of studies on this topic, we consider it important to study whether the isoflurane-sparing effects of morphine and dexmedetomidine, previously reported in the literature, are the same in cannabis users.” Please reword this paragraph because it is a little messy and unclear.

 

 

Reviewer 2. Thanks for your comment. It has been modified based on your suggestion.

Currently, animals with chronic pain can be medicated with cannabis sativa or its derivatives. If one of these patients requires general anesthesia, The interaction of cannabis derivatives with anesthetic agents is unknown. We consider it important to study whether the sparing effects of morphine and dexmedetomidine, previously reported in the literature, are the same in medicated cannabis patients. We hypothesize that the isoflurane-sparing effects of morphine and dexmedetomidine are different in rats that are repeatedly administered a synthetic cannabinoid.

Mat and Met

– Please correct paragraph alignment.

– “This was maintained for 15 min to allow equilibrium to be established between the alveolar gas, arterial blood, and the spinal cord” What do you mean by “spinal cord”? You mean the passage through the blood- brain barrier? it’s not clear.

Thank you for your comment.

The word spinal cord has been changed to central nervous system.

For better reader understanding

– “A positive response was considered if the rat shook or moved its head, trunk or pelvic limbs or thoracic limbs. A negative response was considered if the rat moved its tail…” Why were no objective scores established?

Thank you for your comment.

In fact, objective scores were not established, since the methodology of the evaluation of the positive and negative response was validated in a study carried out by Edmon I. Eger et al. in 1965, in the study “Minimum Alveolar Anesthetic Concentration: a Standard of Anesthetic Potency”, which literally says As a result, the stimulus was applied for at least 30 to 40 seconds, usually for a minute. A positive response was a gross purposeful muscular movement, usually of the head (jerking or twisting) or extremities (running or clawing). Coughing, swallowing, or chewing were not included as positive responses.

– “If we observed a negative response, we decreased the isoflurane concentration by 10%” What do you mean by 10%? 10% of what? Please, explain better.

Thank you for your comment.

His statement is correct, and it is explained based on the article published by Eger et al. in 1965, with study “Minimum Alveolar Anesthetic Concentration: a Standard of Anesthetic Potency”. Indicating that the MaC was taken to be the concentration midway between the highest concentration allowing and the lowest concentration preventing a positive response.

For example, if no response were obtained at 1.00 per cent halothane the concentration was lowered to 0.80 per cent and the stimulus re-applied. If movement then occurred MAC was said to be 0.90 per cent (a maximum possible error in the true. MAC of about 10 per cent).

In addition to the previous study, recently. Rioja et al. in 2006 with the study “Cardiorespiratory and minimum alveolar concentration sparing effects of a continuous intravenous infusion of dexmedetomidine in halothane or isoflurane-anesthetizes rats”, determined the MAC ISO and MAC HAL using a method described by Quasha et al., 1980 In which, a supramaximal noxious stimulus was applied to the animal at a specific end-tidal concentration of each volatile agent. Performed by tail clamping with 20 cm full-length hemostat. The tail was always clamped close to the base and to a full ratchet lock, but at different sites, for 60 s. If the response was positive, the delivery ISO or HAL concentration was increased by 0.1-0.2%, and, if the response was negative, the concentration of the volatile anesthetic was decreased by 0.1-0.2%.

– Why didn’t you measure the baseline MACIso in dex group?

Thank you for your comment.

The paragraph has been modified for better understanding.

The isoflurane control group (MAC (ISO) was measured before morphine administration, was considered the control group for all treatment groups.

It has been modified in the manuscript based on your suggestion for better understanding.

The description of the administration of cannabinoids in the two study groups is repetitive. Please reword the paragraph.

Thank you for your comment.

The paragraph has been modified for better understanding.

For groups the MAC (ISO + CANN + MOR) and the MAC (ISO + CANN + DEX) was intraperitoneally (i.p.) administered 1 mg/kg of WIN (mesylate salt, Sigma–Aldrich, St. Louis, MO, USA) every 24 hours for 21 days, in accordance with Lawston et al. [18]. For the MAC (ISO + CANN + MOR) the measurement was performed 24 hours after the last treatment (day 21). Then, 45 min prior to MAC measurement, 3 mg/kg of morphine was administered i.v. The MAC of isoflurane (MAC (ISO + CANN)) was measured before morphine administration. While for the MAC (ISO + CANN + DEX) the measurement was performed 24 hours after the last treatment (day 22), 30 min after the continuous intravenous infusion of 0.25 μg/kg/min of dexmedetomidine i.v.

Statistical analysis

– Why didn’t you calculate the sample size suitable for the study? This aspect is important, especially in experimental studies.

Thank you for your comment.

Its observation has been addressed in the section on materials and methods.

The sample size was determined by the method described by Charanand Biswas in relation to clinical studies.

Charan J, Biswas T. How to calculate sample size for different study designs in medical research?.Indian J Psychol Med.

For cases of parametric statistics, n=30 when the values ​​of a data set are normally distributed based on the Shapiro-Wilk test.

The number of animals included in the study was determined according to the methodology described by Charan and Biswas.

Results

– In the graphs it is not clear what is compared to the # symbol. It is recommended to enter a symbol for each group or specify whether the difference is with respect to the control group.

Thanks for your comment. It has been modified based on your suggestion.

 

 Figure 1. Effect of the cannabinoid WIN on the MAC of isoflurane. MAC (ISO) was 1.32 ± 0.06 and 1.69 ± 0.09 in the MAC (ISO + CANN) group. An increase in the minimum alveolar concentration was observed. [*] Statistically significant compared to the control group p <0.0001.

Figure 2. Sparing effect of morphine in un medicated rats and rats medicated for 21 days with the cannabinoid WIN. MAC (ISO + MOR) was 0.97 ± 0.02 (26% less than MAC (ISO)). MAC (ISO + CANN + MOR) was 1.55 ± 0.08 (8% less than MAC (ISO + CANN)). The sparing effect of morphine on isoflurane was lower in rats constantly medicated with a cannabinoid compared to the effect of morphine in rats not treated with the cannabinoid. [*] Statistically significant between MAC ISO + MOR and the MAC ISO group p <0.0001. (#) Statistically significant. between MAC ISO + CANN + MOR and MAC ISO + CANN group p 0.0094  

 

Figure 3. Sparing effect of dexmedetomidine in un medicated rats and rats medicated for 21 days with the cannabinoid WIN. MAC (ISO + DEX) was 0.68 ± 0.10 (48% less than MAC (ISO)).  MAC (ISO + CANN + DEX) was 0.67 ± 0.08 (60% less than MAC (ISO + CANN)). The sparing effect of dexmedetomidine on the minimum alveolar concentration of isoflurane was greater in rats repeatedly medicated with a cannabinoid compared to the effect of dexmedetomidine in rats not treated with the cannabinoid. [*] Statistically significant between the MAC Iso + Dex and the MAC ISO group p <0.0001. (#) Statistically significant  between MAC ISO + CANN + DEX and the MAC ISO + CANN group p <0.0001.

 

 

Reviwer 2. Thanks for your comment. It has been modified based on your suggestion.

 (*) Statistically significant compared to the control group.

 

Reviwer 2. Thanks for your comment. It has been modified based on your suggestion.

 (*) Statistically significant compared to the control group.

 

 

Reviwer 2. Thanks for your comment. It has been modified based on your suggestion.

 (*) Statistically significant compared to the control group.

 

 

Reviwer 2. Thanks for your comment. It has been modified based on your suggestion.

 (*) Statistically significant compared to the control group.

 

Author Response File: Author Response.docx


Reviewer 3 Report

Dear authors

I do consider your research quite interesting, but the article is poorly written and structured. Also, you should clarify the specific MAC that you would like to assess. Did you calculate the sample size? It is not specify in your design. Both the results and discussion are not well structured, thus the reader could find difficult to interpret your results.

I apologize for my final decision about your article, but I encourage to re-check the study and make all the improvements that are needed.

Kind regards

Author Response

Dear authors

I do consider your research quite interesting, but the article is poorly written and structured. Also, you should clarify the specific MAC that you would like to assess. Did you calculate the sample size? It is not specify in your design. Both the results and discussion are not well structured, thus the reader could find difficult to interpret your results.

I apologize for my final decision about your article, but I encourage to re-check the study and make all the improvements that are needed.

Kind regards

 

Appreciable reviewer

 

Thanks for your comments.

 

We have made changes to the manuscript based on three reviewers, hoping that these may have improved our work.

Author Response File: Author Response.docx


Reviewer 4 Report

 

Simple summary: Put parentheses around MAC (iso+cann+Mor) to DEX),

Introduction

Please use plasma concentration not ‘plasmatic’.

Methods

Anesthetic Procedure – ‘Each rat was placed in an induction chamber …’

‘Animals always breathed spontaneously’

You used a pressure transducer – please state how you calibrated the transducer

‘We placed another catheter in the trachea’ – what size and was this percutaneously or through the endotracheal tube?

‘A painful noxious stimulus’ – strictly when the animal is anesthetized it cannot be painful. The usual definition of the noxious stimulus is ‘supramaximal’ – i.e the stimulus should be greater than the nociceptive threshold.

‘The person who evaluated the MAC’ – please define this more clearly. If you had a negative response followed by a positive response did you then go back up to find the next negative response. This then counted as two points?

Experimental design:

‘This is the optimal dose’ The reference you quote used doses of 1, 3 and 10 mg/kg and they obtained the greatest reduction with the 10 mg/kg. Please state why you chose the 3 mg/kg as the ‘optimal’ dose.

How is the dose of 0.25 µg/kg/min ‘optimal’ – this was the only dose tested in the reference quoted and it is a massive dose compared with what is used in humans. Your dose would be 15 µg/kg/hr and typically in humans the doses are less than 1 µg/kg/hr? These doses in people have minimal effect on the vasculature whereas you clearly had significant vasoconstriction causing the bradycardia. Doses of up to 100 µg/kg have been used in rats with a 90% reduction in MAC (e.g. The alpha 2-adrenoceptor agonist dexmedetomidine increases the apparent potency of the volatile anesthetic isoflurane in rats in vivo and in hippocampal slice in vitro. Savola MK, MacIver MB, Doze VA, Kendig JJ, Maze M. Brain Res. 1991 May 10;548(1-2):23-8.) 

It appears that WIN 55,2212-2 is a CB1 and CB2 agonist and its effects are dose dependent. It may therefore not be a good substitute for THC or CBD. The authors should spend some time in the discussion evaluating the dose and duration of their administration on its relevance to marijuana use in the human population (WIN55,2122, a Dual Modulator of Cannabinoid Receptors and G Protein-Coupled Inward Rectifier Potassium Channels. An D, Peigneur S, Tytgat J. Biomedicines. 2021 Apr 28;9(5):484. doi: 10.3390/biomedicines9050484.)

Statistical analysis. Please state how you are reporting your results (mean ±SD).

Discussion

You state that ‘These results indicate that the repeated use of a cannabinoid modifies the isoflurane-sparing effect of dexmedetomidine’ but you could argue that the effect of dexmedetomidine is independent of the cannabinoid because there was no difference in the MAC with or without the WIN. You use the reduction of MAC after the WIN as different from the reduction without the WIN but if the effect of dexmedetomidine is independent of the WIN then this comparison is irrelevant. You also state that the dexmedetomdine changes MAC in a way that is independent of its alpha-2 agonist effects yet studies have shown that using an alpha-2 antagonist reverses the MAC reduction (e.g. The effect of imidazoline receptors and alpha2-adrenoceptors on the anesthetic requirement (MAC) for halothane in rats. Kagawa K, Mammoto T, Hayashi Y, Kamibayashi T, Mashimo T, Yoshiya I. Anesthesiology. 1997 Oct;87(4):963-7. doi: 10.1097/00000542-199710000-00032.)

‘The authors we believe it…’ replace with ‘The authors believe that it…’

‘We feel that’ –  there is no place for ‘feelings’ in a scientific paper – We think that…

 

 

 

Author Response

Simple summary: Put parentheses around MAC (iso+cann+Mor) to DEX),

 

Thank you for your comment.

 

Has been corrected based on your suggestion.

 

Introduction

Please use plasma concentration not ‘plasmatic’.

 

Thank you for your comment.

 

Has been corrected based on your suggestion

 

Methods

Anesthetic Procedure – ‘Each rat was placed in an induction chamber …’

 

Thank you for your comment.

 

Has been corrected based on your suggestion

 

‘Animals always breathed spontaneously’

 

Thank you for your comment.

 

Has been corrected based on your suggestion

 

 

You used a pressure transducer – please state how you calibrated the transducer

 

Thank you for your comment.

 

Has been corrected based on your suggestion:

 

Before beginning each experiment, the transducer was calibrated zeroed exposing the transducer to atmospheric pressure and placing it to the heart level.

 

‘We placed another catheter in the trachea’ – what size and was this percutaneously or through the endotracheal tube?

 

Thank you for your comment.

 

Has been corrected based on your suggestion:

 

Intubation is performed through the orotracheal route

 

“Through the catheter previously placed in the trachea, we collected an endotracheal gas sample and used a gas analyzer …”

 

 

‘A painful noxious stimulus’ – strictly when the animal is anesthetized it cannot be painful. The usual definition of the noxious stimulus is ‘supramaximal’ – i.e the stimulus should be greater than the nociceptive threshold.

 

Thank you for your comment.

 

Has been corrected based on your suggestion.

 

The person who evaluated the MAC’ – please define this more clearly.

 

 

Thank you for your comment.

 

Has been corrected based on your suggestion.

 

“(that applies supramaximal stimulus and observed if the response was positive or negative)”…

 

 

If you had a negative response followed by a positive response did you then go back up to find the next negative response. This then counted as two points?

 

Thank you for your comment.

 

This never happened. To modify the concentration of isoflurane, two equal responses were always sought, either positive or negative, never different.

 

Experimental design:

 

‘This is the optimal dose’ The reference you quote used doses of 1, 3 and 10 mg/kg and they obtained the greatest reduction with the 10 mg/kg. Please state why you chose the 3 mg/kg as the ‘optimal’ dose.

 

Thank you for your comment.

 

In the work we refer to, they did not test the 10 mg dose of morphine with aspirin, for this reason we did not know how morphine behaved at those doses administered with other drugs, therefore we decided to use the 3 mg dose.

 

How is the dose of 0.25 µg/kg/min ‘optimal’ – this was the only dose tested in the reference quoted and it is a massive dose compared with what is used in humans. Your dose would be 15 µg/kg/hr and typically in humans the doses are less than 1 µg/kg/hr? These doses in people have minimal effect on the vasculature whereas you clearly had significant vasoconstriction causing the bradycardia. Doses of up to 100 µg/kg have been used in rats with a 90% reduction in MAC (e.g. The alpha 2-adrenoceptor agonist dexmedetomidine increases the apparent potency of the volatile anesthetic isoflurane in rats in vivo and in hippocampal slice in vitro. Savola MK, MacIver MB, Doze VA, Kendig JJ, Maze M. Brain Res. 1991 May 10;548(1-2):23-8.) 

 

Thank you for your comment.

 

That’s right, the dose indicated in the referenced article was used only as a starting point of a known effect and to be able to determine if the treatment with the cannabinoid modified it.

 

 

 

It appears that WIN 55,2212-2 is a CB1 and CB2 agonist and its effects are dose dependent. It may therefore not be a good substitute for THC or CBD. The authors should spend some time in the discussion evaluating the dose and duration of their administration on its relevance to marijuana use in the human population (WIN55,2122, a Dual Modulator of Cannabinoid Receptors and G Protein-Coupled Inward Rectifier Potassium Channels. An D, Peigneur S, Tytgat J. Biomedicines. 2021 Apr 28;9(5):484. doi: 10.3390/biomedicines9050484.)

 

 

 

Thank you for your comment.

 

Has been corrected based on your suggestion.

 

“The results of this study should be interpreted with caution since WIN is a synthetic cannabinoid and may not reflect the effect of consuming natural cannabinoids”.

 

 

Statistical analysis. Please state how you are reporting your results (mean ±SD).

 

Thank you for your comment.

 

Has been corrected based on your suggestion.

 

 

Discussion

You state that ‘These results indicate that the repeated use of a cannabinoid modifies the isoflurane-sparing effect of dexmedetomidine’ but you could argue that the effect of dexmedetomidine is independent of the cannabinoid because there was no difference in the MAC with or without the WIN. You use the reduction of MAC after the WIN as different from the reduction without the WIN but if the effect of dexmedetomidine is independent of the WIN then this comparison is irrelevant.

 

Thank you for your comment.

 

It’s right. the effect of dexmedetomidine on isoflurane is maintained despite treatment with the cannabinoid and not only that, it is greater in rats medicated for 21 days with WIN.

 

 

 

You also state that the dexmedetomdine changes MAC in a way that is independent of its alpha-2 agonist effects yet studies have shown that using an alpha-2 antagonist reverses the MAC reduction (e.g. The effect of imidazoline receptors and alpha2-adrenoceptors on the anesthetic requirement (MAC) for halothane in rats. Kagawa K, Mammoto T, Hayashi Y, Kamibayashi T, Mashimo T, Yoshiya I. Anesthesiology. 1997 Oct;87(4):963-7. doi: 10.1097/00000542-199710000-00032.)

 

Thank you for your comment.

 

What we really mean is that  dexmedetomidine lowers the requirements of inhalational anesthetics by processes other than inhibiting noradrenaline given that locus coeruleus is not the only place where α2-adrenoceptor agonists exercise their mechanisms of action

 

‘The authors we believe it…’ replace with ‘The authors believe that it…’

‘We feel that’ –  there is no place for ‘feelings’ in a scientific paper – We think that…

 

Thank you for your comment.

 

Has been corrected based on your suggestion.

 

“The authors think…”

Author Response File: Author Response.docx

Round 2


Reviewer 2 Report

As result of the changes made, it is my opinion that the manuscript could be published in this form.

Read more here: Source link

Leave a Reply

Your email address will not be published. Required fields are marked *