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. 2022 Jun 6;7(3):289–293. doi: 10.1089/can.2021.0014

The Impact of Cannabidiol on the Induction of Isoflurane Anesthesia and Recovery in Wistar Rats

Matjaž Uršič 1, Alenka Babič 1, Tilen Vake 1, Tomaž Snoj 1,*
PMCID: PMC9225406  PMID: 33998903

Abstract

Background:

Beside others, neuroinhibitory and sedative effects of CBD were documented.

Aim and Methods:

The aim of the study was to assess the dose-related effects of CBD premedication on the course of isoflurane anesthesia. Wistar rats were pretreated with different doses of CBD 1 h before isoflurane anesthesia. In the pretreatment, animals were given CBD at doses of 100, 20, 10, or 2 mg kg−1. Before the fifth (control) anesthesia, the animals were given only mid-chain triglyceride oil, which served as a solvent in the CBD formulation. The induction time was determined, and on awakening, the time to appearance of the flexion reflex and the recovery from anesthesia were determined.

Results:

Statistical analysis showed a significantly shorter induction time if animals were pretreated with 20 mg kg−1 CBD. In addition, pretreatment with 100 mg kg−1 CBD resulted in a prolonged induction time, while on awakening, delayed appearance of reflexes and prolonged recovery from anesthesia compared to pretreatment with 20 mg kg−1 CBD were observed.

Conclusions:

The results indicate that the influence of CBD on the course of isoflurane anesthesia depends on the dose and can reduce the induction time. Although this study was performed in laboratory rats, in clinical practice, these data should be considered when CBD-treated patients undergo isoflurane anesthesia.

Keywords: anesthesia, cannabidiol, induction, isoflurane, recovery

Introduction

CBD is one of several phytocannabinoids that were found in Cannabis sativa L.1 Although the pharmacologic mechanism of CBD is still a topic of numerous studies, its multipotent action is currently confirmed.2–4 The activity is associated with interaction with cannabinoid receptor 1 (CB1) and 2 (CB2), vanilloid and serotonin receptors, G protein-coupled receptors 55 (GPR55), mitochondrial sodium-calcium exchanger (NCX), and modulation of glutamate-gamma-aminobutyric acid (GABA) systems and serotonergic signaling. It affects intracellular calcium concentration through CB1 and mitochondrial NCX. Also, interactions with cannabinoid receptors and GABA and serotonergic systems influence the activity of the central nervous system. Moreover, intracellular calcium mobilization enhances endocannabinoid anandamide production.4–6 Thus, CBD interacts with several neuroactive systems and directly and indirectly modulates the endocannabinoid system.

The literature describes the analgesic, antipyretic, anti-inflammatory, anticarcinogenic, antiallergic, antioxidant, neuroprotective, anxiolytic, antidepressant, and sedative effects of CBD.2,7,8 In several countries, CBD is approved for the cotreatment of multiple sclerosis and the prevention of epileptic seizures in humans.2,7–10 In addition, it is widely believed that cannabinoids are associated with many therapeutic effects; however, not all of these effects have been scientifically confirmed.9

CBD is also used in veterinary medicine. Thus, it is used for the management of acute and chronic pain, anxiety, and seizure treatments, as an antiemetic and appetite stimulant, and as an anticancer drug, predominantly in dogs.11,12

Although the pharmacological effects of CBD are currently intensively studied, little is known about its effects on the course of general anesthesia. This issue is important when CBD-treated patients are anesthetized. It was reported that in humans, the medicinal and recreational use of cannabis and cannabinoids have significantly increased the need for propofol and fentanyl use during anesthesia and for postoperative analgesia.13 Malor et al. reported prolonged ether anesthesia in mice after premedication with delta-9-tetrahydrocannabinol (THC), but not with CBD.14 In addition, CBD was found to have no effect on anesthesia induced by ketamine, thiopentone, propanidid, and alfathesin, but prolonged anesthesia induced by pentobarbital in mice.15

Since neuroinhibitory and sedative effects of CBD were reported,2,7,8,11 we hypothesized that its use might affect induction time and recovery from anesthesia. Thus, the aim of our study was to determine the dose-related effects of CBD premedication on the course of isoflurane anesthesia. The work was performed on Wistar rats, which served as a mammal model that provides basic information about the effects of CBD use before anesthesia in pets and humans.

Materials and Methods

The study was approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector, and Plant Protection under approval number U34401-15/2019/8. During the experiment, all ethics standards were considered in accordance with Directive 2010/63/EU and “Animal Research: Reporting In Vivo Experiments” recommendations.

Experimental animals were purchased from an approved supplier of laboratory rats (Envigo, Italy). Sample size calculation was performed using the Sample size calculator ClinCalc (ClinCalc LLC, IL). Based on a previously performed pilot study, the mean and standard deviation of 60±7 sec regarding the induction time of rats anaesthetized with isoflurane were taken to calculate the sample size. With a power of 80% and alpha error of 0.05, power analysis revealed that a sample of 11 rats was necessary to detect a 10% decrease in induction time. Due to possible animal loss during general anesthesia, one additional animal was included in the study. Thus, a group of 12 Wistar rats was used. All animals were females, and at the beginning of the study, their age was 9–10 weeks. The animals arrived at our laboratory 10 days before the beginning of the experiment. They were kept under standard laboratory conditions with controlled temperature (22°C±2°C) and relative humidity (30–70%). Lighting was artificial with a sequence of 12-h light and 12-h dark. Water and conventional diet for laboratory rodents (Altromin, Germany) were available ad libitum. The animals were housed in polypropylene cages (66×40×20 cm) with six rats in each cage.

The CBD oil solution was purchased at the pharmacy (University Medical Centre, Ljubljana, Slovenia). The CBD was dissolved in mid-chain triglyceride (MCT) oil at a concentration of 100 mg mL−1. CBD used to make the CBD solution has been certified to contain less than 0.5% impurities. In our laboratory, CBD was further diluted in MCT oil to concentrations of 20.0, 4.0, 2.0, and 0.4 mg mL−1. The volume of the applied dose was 0.5 mL 100 g body weight−1.

Isoflurane (Isoflurin 1000 mg g−1; Vetpharma Animal Health, Spain) was purchased from an approved supplier of veterinary drugs.

Animals were fasted for 4 h before the experiment. The experiment was performed in the following order:

  • The tested substance (CBD) was administered using oral gavage.

  • The animal was left in the home cage for 60 min.

  • The induction chamber of an isoflurane inhalation anesthesia system (IsoFlo, Eickemeyer, United Kingdom) was filled with 4% isoflurane in oxygen.

  • Sixty minutes after CBD administration, the experimental animal was placed in the induction chamber, and the gas flow was set to 4 L per minute.

  • When the animal stopped showing any sign of vigilance, the level of anesthesia was evaluated by the presence/absence of reflexes. Thus, every 5 sec, the flexion reflex test was performed by pinching the fourth toe on the left hind limb with forceps. To avoid subjective testing influence, the procedure was always performed by the same person (the assessor). The time between the first contact with isoflurane and the point of absence of the flexion reflex was considered the induction time.

  • After the absence of flexion reflex was observed, the animal was still exposed to 4% isoflurane for 15 sec.

  • After 15 sec, the anesthetized rat was taken from the chamber and placed on the warming plate.

  • During recovery, the flexion reflex test was performed every 5 sec.

  • The period from leaving the chamber to the first flexion response was noted and considered the time to appearance of the reflexes.

  • The flexion reflex test was continued every 5 sec.

  • The time when the animal started to move was considered the recovery from anesthesia.

The described procedure was repeated five times on each animal. The period between each procedure was 5 to 7 days. In particular, 100, 20, 10, and 2 mg kg−1 CBD were administered orally by gavage. Anesthesia without CBD pretreatment was performed as a control procedure. In the last procedure, the animals received solely MCT oil (0.5 mL 100 g−1), which was used as a solvent in the CBD formulation. Similar CBD doses have been used in previously published studies.15,16 The CBD doses were applied in the same order as stated above. After the experiment, rats were euthanized with carbon dioxide and exsanguination.

Statistical analysis was performed using SPSS Statistics package version 24 (IBM, IL, USA). Data distribution was estimated by the Shapiro–Wilk test. Repeated measures analysis of variance (ANOVA) with Fisher's least significant difference (LSD) post hoc test was used to evaluate the impact of the CBD dose on the induction time, the appearance of the flexion reflex, and the recovery period. The results are expressed as the mean±standard error (SE). p<0.05 was considered significant.

Results

The results of observed induction time, the time when reflexes appeared on awakening, and the recovery period (in seconds) in CBD-pretreated rats are shown in Figures 1 and 2.

FIG. 1.

FIG. 1.

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The induction time (in seconds) in rats subjected to isoflurane anesthesia and pretreated with different doses of CBD. Results are expressed as the mean±SE. Value indicated with * differs significantly from the control. SE, standard error.

FIG. 2.

FIG. 2.

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The time when reflexes appeared on awakening and the recovery (in seconds) in CBD-pretreated rats.

The induction time in rats that were pretreated with CBD in a dose 20 mg kg−1 differs significantly (p<0.05) from control measurements. In addition, the induction time in rats pretreated with CBD at a dose of 100 mg kg−1 was significantly longer than the induction times during anesthesia at 2, 10 (p<0.05), and 20 (p<0.01) mg kg−1, but not significantly longer compared with the control group.

On awakening, the appearance of the reflexes was significantly delayed, and the recovery period was significantly longer (p<0.05) in rats pretreated with 100 mg/kg CBD than in animals pretreated with 20 mg/kg CBD. No significant differences were observed compared to the control.

Discussion

Information about the impact of CBD pretreatment on the course of general anesthesia is important for clinical praxis since some people daily use CBD or cannabis. The results of this study showed a significantly reduced induction time if CBD was applied at a dose of 20 mg kg−1. This effect might be the result of its sedative effect.7,11,15 Lower doses (2 and 10 mg kg−1) seem to be too low to affect the course of isoflurane anesthesia, although a dose-dependent, but not significant effect can be observed (Fig. 1). In contrast, at the dose of 100 mg kg−1, prolonged induction was observed. However, it did not differ significantly from that of the control, but was significantly longer than that obtained with doses of 2, 10, and 20 mg kg−1 CBD (Fig. 1). Dose-dependent opposite effects of CBD have been already described. Zuardi et al. studied the anxiolytic effects of CBD in humans and described an inverted U-shaped dose–response curve.16 The pharmacologic pathways and the dose–response effects of CBD are not yet completely clear. As mentioned above, CBD interacts with cannabinoid, some serotonin, orphan GPR55, and vanilloid receptors and modulates anandamide production and the activity of GABAergic and serotonergic systems. It was reported that at high, but not low doses, CBD interacts with vanilloid receptor 1, exhibits allosteric interactions with serotonin 5-HT1A receptor, and facilitates glutamate release.2,4–6,16,17 Dose-dependent activation of various pharmacological pathways might lead to dose-dependent opposite effects. However, further studies are needed for a complete explanation.

Although expected, on awakening from isoflurane anesthesia, the period to the flexion reflex, and the recovery period in animals treated with different doses of CBD did not differ significantly from those of the control (Fig. 2). However, the period to appearance of the flexion reflex and the recovery period were longer after pretreatment with 100 mg kg−1 CBD than after 20 mg−1 kg CBD. Ibera et al. described a higher bispectral index in humans who were previously treated with high doses of cannabinoids.18 Although the studies cannot be directly compared, both show increased use of anesthetics when high doses of cannabinoids were previously used.

We believe that the observed prolonged recovery from anesthesia at a dose 100 mg kg−1 is not due to the sedative effect of a high dose of CBD, but a result of significantly longer induction time compared to that observed with a dose 20 mg kg−1 (Fig. 1). Since isoflurane is a highly lipophilic compound, the prolonged recovery period might result from longer isoflurane exposure (longer induction) in the case of pretreatment with 100 mg kg−1 CBD. Although the induction time was longer with 2 mg kg−1 CBD than with 10 mg kg−1, the recovery time was, contrary to expectations, longer after the application of 10 mg kg−1 (Fig. 2). Considering the presumption that longer isoflurane exposure causes longer recovery time, longer recovery time should be expected after treatment with 2 mg kg−1. The difference in recovery times between treatments is small and not significant. Thus, the longer recovery time after treatment with 10 mg kg−1 may result from chance, but further studies are needed to fully resolve this issue.

In this study, female Wistar rats were a mammalian model used to evaluate the CBD pretreatment effects on the course of isoflurane anesthesia. Since cannabinoids express greater motor effects in females,19 similar effects should also be expected in males. In addition, because a species-specific response to CBD pretreatment is possible, the results of this study cannot be directly applied to humans or target animals (dogs). Further studies are needed to clarify these issues. Moreover, to avoid the influence of previous treatments and other external factors on the results, other randomization procedures in CBD dosing should also be performed in further studies. Similarly, as in the experiment dealing with CBD anxiolytic activity in humans,16 the results of our study also show that CBD causes an inverted U-shaped dose–response curve. However, this study in general indicates that CBD influences isoflurane anesthesia. Furthermore, since the appropriate CBD dose reduces the induction time, new premedication protocols for general anesthesia that include CBD could be established. However, further targeted studies to provide detailed information on the possible use of CBD for this purpose are needed.

Conclusion

In conclusion, the results of our study show significant dose-dependent effects of CBD pretreatment on the course of general anesthesia with shorter induction after 20 mg kg−1 CBD and no effect after 2, 10, and 100 mg kg−1 CBD. In addition, CBD causes an inverted U-shaped dose–response curve, as was already described previously.

Acknowledgments

The authors would like to thank Katarina Babnik and Boštjan Drolc for technical assistance.

Abbreviations Used

ANOVA

analysis of variance

CB1

cannabinoid receptor 1

CB2

cannabinoid receptor 2

CBD

cannabidiol

GABA

gamma-aminobutyric acid

GPR55

G protein-coupled receptors 55

LSD

least significant difference

MCT

mid-chain triglyceride

NCX

mitochondrial sodium-calcium exchanger

SE

standard error

THC

delta-9-tetrahydrocannabinol

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This research was funded by Slovenian Research Agency, grant no. P4-0053.

Cite this article as: Uršič M, Babič A, Vake T, Snoj T (2022) The impact of cannabidiol on the induction of isoflurane anesthesia and recovery in Wistar rats, Cannabis and Cannabinoid Research 7:3, 289–293, DOI: 10.1089/can.2021.0014.

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