Review Reports - Minimum Alveolar Concentration of Isoflurane in Rats Chronically Treated with the Synthetic Cannabinoid WIN 55,212-2

Round 1

Reviewer 1 Report

Can you also include a comment on the dose of cannabinoid used in your study or a comment that a limitation of the study is that a single dose was used.

Minimum alveolar concentration of isoflurane in rats chronically treated with the synthetic cannabinoid WIN 55,212-2

Suggestions:

Add a full stop at the end of the title

Change from ‘isoflurane MAC’ to ‘MAC of isoflurane throughout’

Change from ‘…the end expiratory samples at time of tail clamping under…’ to ‘…end expiratory samples at the time of tail clamping under…’

Change ‘…and other group 8 days after stopping treatment for 21 days with WIN 5…’ to ‘…and in rats 8 days after stopping treatment with WIN 5…’

Change ‘Results: The MACISO was 1.32 ± 0.06. In the MACISO+WIN55 group the MAC increase to 1.69 ± 0.09 (28%). After 8 days stopping treatment, MAC did not decrease significantly 1.67 ± 0.07 (26%). Conclusions: The administration for 21 days of WIN 55,212-2 increases the MAC of isoflurane in rats; this effect does not disappear after 8 days of discontinuing treatment with the synthetic cannabinoid.’ To Results: The MACISO was 1.32 ± 0.06. In the MACISO+WIN55 group the MAC increased to 1.69 ± 0.09 (28%, p value = xxx). Eight days after stopping treatment with WIN55, the MAC did not decrease significantly 1.67 ± 0.07 (26%, p value = xxx). Conclusions: The administration of WIN 55,212-2 for 21 days increases the MAC of isoflurane in rats. This effect does not disappear 8 days after discontinuation of treatment with the synthetic cannabinoid.

Change ‘was discovered in 1993 (5); subsequently, the first (6) and second endogenous cannabinoids were discovered (7).’ To ‘was discovered in 1993 (5). Subsequently, the first (6) and second endogenous cannabinoids were discovered (7).’

Change ‘The objective of this study was to evaluate the interaction of chronically administered cannabinoid agonist on the minimum alveolar concentration…’ to ‘The objective of this study was to evaluate the interaction of a chronically administered cannabinoid agonist on the minimum alveolar concentration …’

Change ‘In this study, with protocol number 3492/2013CHT, 24 male Wistar rats weighing 310 ± 20 g was used. This experiment was approved by the Animal Research Ethics Committee for Animal Experimentation of the Faculty of Veterinary Medicine of the University of the State of Mexico.’ To ‘This experiment was approved by the Animal Research Ethics Committee for Animal Experimentation of the Faculty of Veterinary Medicine of the University of the State of Mexico (protocol number 3492/2013CHT). Twenty four male Wistar rats weighing 310 ± 20 g were used.’

Provide reference for the Guide for the care and Use of laboratory Animals.

Change’ Anesthetic induction was performed by placing each rat in the induction chamber providing 5% isoflurane (Forane; Baxter Laboratories, USA) with a continuous oxygen flow of 5 L/min. Once the animal was anesthetized and lost jaw tone, it was removed from the induction chamber and placed in dorsal recumbence for endotracheal intubation. The oral cavity was opened and with the aid of an otoscope, the larynx was visualized and a flexible blunt-tip wire guide was inserted inside and used to direct the endotracheal catheter (16G Teflon catheter: Introcan; B-Braun, Brazil), which was secured to the maxilla by means of adhesive tape.’ To ‘Anesthesia was induced by placing each rat in the induction chamber and delivering 5% isoflurane (Forane; Baxter Laboratories, USA) with a continuous oxygen flow of 5 L/min. Once the animal was adequately anesthetized, it was removed from the induction chamber and placed in dorsal recumbency for endotracheal intubation. The oral cavity was opened and with the aid of an otoscope, the larynx was visualized and a flexible blunt-tip wire guide was inserted and used to direct the endotracheal catheter (16G Teflon catheter: Introcan; B-Braun, Brazil), which was secured to the maxilla by means of adhesive tape.’

Change ‘The carotid artery was exposed via surgical cut-down and catheterized using a 24- gauge catheter (Introcan; B-Braun, Brazil); this was connected to a pressure transducer system for direct blood pressure monitoring and the collection of …’ to ‘The carotid artery was exposed surgically and catheterized using a 24- gauge catheter (Introcan; B-Braun, Brazil). This catheter was connected to a pressure transducer system for direct blood pressure monitoring and the collection of …’

Change ‘The mean value of the MACISO+WIN55+8D group was 1.67% ± 0.07; there was no statistically difference when compared to the MACISO+WIN55 group (p=0.6995), but a significant difference was found compared with the MACISO (p<0.0001).’ to ‘The MAC value of the MACISO+WIN55+8D group was 1.67% ± 0.07 and there was no difference when compared to the MACISO+WIN55 group (p=0.6995), but a significant difference was found when compared with the MACISO (p<0.0001).’

Change ‘MAC is defined as the alveolar concentration of an inhalatory anesthetic necessary to prevent movement in 50% of subjects exposed to a supramaximal noxious stimulus and represents an index of potency of anesthetic agents’ to ‘MAC is defined as the minimum alveolar concentration of an inhaled anesthetic required to prevent movement in 50% of subjects exposed to a supramaximal noxious stimulus and represents an index of potency of anesthetic agents’

Change ‘The use of cannabinoids has increased in a recreational and therapeutic way (2), to the knowledge of the authors, there are no reports of the effect of chronic administration of cannabinoids on the requirements of inhalation anesthetics.’ To ‘ While the use of cannabinoids has increased in a recreational and therapeutic way (2), to the knowledge of the authors, there are no reports of the effect of chronic administration of cannabinoids on the requirements of inhalation anesthetics.’

Change ‘this investigation, we observed that 21 days of administering the synthetic cannabinoid WIN 55,212-2 increased the isoflurane MAC in rats’ to ‘this investigation, we observed that after 21 days of administering the synthetic cannabinoid WIN 55,212-2 MAC of isoflurane in rats increased.’

Change ‘WIN 55,212-2 cause an increase in the central levels of norepinephrine, Page and collaborators (20) showed that rats treated with WIN 55,212-2 for 8 days presented an increase in noradrenergic activity, furthermore they demonstrated that repeated administration of WIN 55,212-2 stimulates CB1 cannabinoid receptors in cell bodies of the locus coeruleus and in nerve terminals containing norepinephrine, generating an increase in norepinephrine efflux.’ To ‘WIN 55,212-2 causes an increase in the central levels of norepinephrine: Page and collaborators (20) showed that rats treated with WIN 55,212-2 for 8 days had an increase in noradrenergic activity. Furthermore they demonstrated that repeated administration of WIN 55,212-2 stimulates CB1 cannabinoid receptors in cell bodies of the locus coeruleus and in nerve terminals containing norepinephrine, generating an increase in norepinephrine efflux.’

Change ‘Norepinephrine levels in nervous terminals modulates MAC response of isoflurane as Miller et al’ to ‘Norepinephrine levels in nervous terminals modulate the MAC response of isoflurane as Miller et al’

Change ‘demonstrated in a previous work, in experiments following the administration of alpha-methyldopa, reserpine and iproniazid, they demonstrated that the requirements of inhalational anesthetics are related to norepinephrine levels.’ To ‘demonstrated in a previous work, in experiments following the administration of alpha-methyldopa, reserpine and iproniazid. These authors demonstrated that the requirements of inhalational anesthetics are related to norepinephrine levels.’

Change ‘It´s also known, diverse neurotransmitters such as norepinephrine, might manifest some of their actions by strongly inhibiting TWIK-related acid-sensitive K+ channels (TASK) and thus influence neuronal excitability.’ To ‘In addition, diverse neurotransmitters such as norepinephrine, may manifest some of their actions by strongly inhibiting TWIK-related acid-sensitive K+ channels (TASK) and thus influence neuronal excitability.’

Change ‘Similarly, Inhalation anesthetics activate and cannabinoid agonists inhibit TASK channels’ to ‘Similarly, inhalation anesthetics activate and cannabinoid agonists inhibit, TASK channels’

Change halothane MAC and cyclopropane MAC to MAC of halothane and MAC of cyclopropane

Change ‘for a lees period of time’ to ‘for a shorter period of time.’

Change ‘observed in de isoflurane MAC’ to ‘observed in MAC of isoflurane’

Author Response

Can you also include a comment on the dose of cannabinoid used in your study or a comment that a limitation of the study is that a single dose was used.

Thank you for your comment.

We have placed the following comment in the last paragraph of the discussion.