Anesthetic Activity of Alfaxalone Compared with Ketamine in Mice

Abstract

Alfaxalone encased in hydroxypropyl-β-cyclodextrin is a neuroactive steroid compound that has recently been approved in the United States for use as an anesthetic in dogs and cats. We evaluated the use of alfaxalone compared with ketamine, both alone and in combination with xylazine, for anesthesia of C57BL/6 mice. We assessed time to onset of anesthesia, duration of action, reflex responses, respiratory rate, and clinical signs. Alfaxalone (80 mg/kg IP) induced a light surgical plane of anesthesia in all mice, with a time to onset of 2.2 ± 0.2 min and duration of 57.1 ± 3.8 min, whereas ketamine (80 mg/kg IP) provided only sedative effects (time to onset, 5.4 ± 0.4 min; duration, 6.9 ± 0.8 min). Clinically, alfaxalone caused a spectrum of activities, including popcorn-like jumping movements after injection, intense scratching of the face, hyperresponsiveness to noise or touch, and marked limb jerking during recovery. Adding xylazine to the single-agent protocols achieved deep surgical anesthesia (duration: alfaxalone + xylazine, 80.3 ± 17.8 min; ketamine + xylazine, 37.4 ± 8.2 min) and ameliorated the adverse clinical signs. Our preliminary analysis suggests that, because of its side effects, alfaxalone alone is not a viable anesthetic option for mice. Although alfaxalone combined with xylazine appeared to be a more viable option, some mice still experienced mild adverse reactions, and the long duration of action might be problematic regarding the maintenance of body temperature and monitoring of recovery. Further studies evaluating different routes of administration and drug combinations are warranted.

Mice are the most common species used in experimental research, and many surgical procedures are performed in this small species. Inhaled and injectable anesthesia are 2 methods commonly available for general anesthesia. Chemical inhalants such as isoflurane and sevoflurane, administered by using anesthetic machines, are used for both brief and prolonged procedures. Drawbacks to this system include the cost of equipment, the requirement for a scavenging system to evacuate waste gases and protect personnel, and the increased training necessary to operate the systems correctly and maintain them appropriately. Injectable anesthesia has many advantages, such as requiring no extra equipment, dosing and administration are easily trained, and compounds and dosages can be tailored to the specific animal or research need. Many drugs are available for chemical anesthesia in rodents, and each drug has its unique characteristics regarding efficacy and safety. Because no current anesthetic drugs are specifically labeled for use in small laboratory species, such as mice, most anesthetics are used according to human or pet animal requirements, leading to difficulties with dosing, which are compounded by interstrain variability in responses, small body weights, and high metabolism³. Despite these difficulties, combinations of ketamine and xylazine are commonly used in mouse anesthesia.¹ This combination is only appropriate for mildly painful procedures, because a surgical plane of anesthesia is not achieved unless the dose is increased to 150 mg/kg ketamine, which is associated with a 40% fatality rate.^1,4,12,14,31

Alfaxalone (3a-hydroxy-5a-pregnane-11, 20-dione) is a steroidal anesthetic drug that acts through the allosteric binding site of the γ-amino-butyric acid subtype A (GABA_A) receptor.²⁹ First launched in 1971, the early version of alfaxalone was insoluble in water, and polyoxyl 35 castor oil was added to enhance solubility. However, this subsequent compound induced anaphylactoid reactions²¹ and was withdrawn from the market. The most recent commercial formulation complexes alfaxalone with 2-hydroxypropyl-β-cyclodextrin, thus enabling its solubilization in aqueous solution. This product is labeled for intravenous injection in dogs and cats. Additional data regarding alfaxalone use have been obtained in pigs,¹⁵ horses,⁷ and rats,¹⁸ but the data for mice are limited and debated. One recommendation regarding dosage for mice is 10 to 15 mg/kg IV,² but the Material Safety Data Sheets for alfaxalone indicate that the LD₅₀ in rats is 19 mg/kg IV. This limit leads to a very narrow margin of safety during intravenous injection, in addition to the fact that the intravenous route is not very practical for routine use in mice. The goal of the current project was to evaluate the efficacy and safety of intraperitoneal alfaxalone for general anesthesia in mice.

Materials and Methods

Animals and housing.

This study was approved by the IACUC of Colorado State University. Male and female C57BL/6NCrl mice (age, 8 to 10 wk) were purchased from Charles River Laboratories (Wilmington, MA) and housed in an AAALAC-accredited animal facility. Mice were allowed to acclimate for 1 wk after their arrival at the facility and were housed under standard conditions in IVC. Irradiated pelleted feed (no. 2918, Harlan Teklad) and sterile water were provided without restriction, and all cages contained sterilized aspen bedding (Harlan Teklad Sani-chip 7090) to a depth of 1/4 in., and sterilized paper napkins were provided for nesting. Room environments are maintained at 21.7 ± 1.7 °C, 20% to 40% humidity (IACUC-approved exemption based on local environmental conditions), and 12:12-h light:dark cycle.

Dose determination and initial safety assessment.

Initially, a pilot dosing study was performed where each of 4 mice (2 male and 2 female, randomly assigned and blinded) received 4, 8, 16, and 40 mg/kg IP of alfaxalone to judge the safety and efficacy of this drug route and dose. Based on the results of these initial doses, a limited ED50 study using higher doses was undertaken. Mice (n = 18) were pooled and randomly allocated into 3 mixed-sex groups (n = 6 each group) to evaluate the effectiveness of 3 higher doses of alfaxalone (20, 40, and 80 mg/kg IP). The effect criterion for this phase was defined as loss of the righting reflex (LORR); anesthetic depth was not evaluated.

Comparison of alfaxalone and ketamine alone and in combination with xylazine.

The main study was conducted in a crossover design with 2 phases, each having 2 groups of 4 male mice and 3 female mice per group. In the first phase, each group of mice received either alfaxalone (80 mg/kg IP) or ketamine (80 mg/kg IP); after a 48-h washout period, the groups were switched. The second phase occurred 1 wk later, used the same groups, and compared 80 mg/kg alfaxalone + 10 mg/kg xylazine with 80 mg/kg ketamine + 10 mg/kg xylazine, with a washout period of 96 h between sessions. Sterile normal saline was used to dilute each drug to an appropriate concentration at the time of use. For the combination regimens, the 2 drugs were mixed together in the same syringe and introduced into the mice at the same time.

Time after drug injection until LORR was used as the onset of action time; LORR was confirmed by turning over the recumbent mouse and noting no righting attempts in 30 s. The immobilized mice were placed on a circulating warm-water pad to aid in maintenance of body temperature then were evaluated every 5 min for the tail-pinch reflex, muscle tone of rear leg, the toe-pinch reflex, and respiratory rate. All evaluations were performed by the same investigator. Reflexes were tested by firmly pinching a distal foot or tail between 2 fingers. Muscle tone was determined by gently palpating with the tip of a finger the caudal thigh, lumbar, and temporal muscles for the presence or absence of tension. Respiratory rate was determined by observation of chest wall movement. The duration of action was determined as the total time of LORR or absent toe pinch reflex, depending on the depth of anesthesia achieved. All unexpected observations were recorded. To protect against heat loss, all mice were kept on the warming pad until voluntary movement occurred.

Statistical analysis.

GNU PSPP open source software (Free Software Foundation; https://www.gnu.org/software/pspp/) was used for statistical analysis. General linear models were used to analyze the onset and the duration of action; data were normally distributed, and Pvalue was set at < 0.05. The paired t test was used to test significant differences between drug groups, and an independent t test was used to evaluate the effect of mouse sex on responses. To describe the character of anesthetic effect or adverse effect based on reflexes or clinical signs, the number of affected animal were summarized as descriptive statistics.

Results

Dose determination and safety study.

In the initial experiment, alfaxalone doses of 4, 8, 16, and 40 mg/kg IP were administered (n = 1 per dose); only the 40-mg/kg dose induced LORR. Because a fifth mouse given the 40-mg/kg dose did not achieve LORR, higher doses were explored. The mice recovered normally, with no obvious side effects from intraperitoneal administration of this drug. Groups of 6 mice were then given alfaxalone at 20, 40 or 80 mg/kg IP; LORR occurred in all 6 mice in the 80-mg/kg group, 4 of those in the 40-mg/kg group, and none of those given 20 mg/kg. All mice recovered normally, with no obvious side effects. In light of these results, we chose the 80-mg/kg dose of alfaxalone for comparison with ketamine in the main study.

Comparison of alfaxalone and ketamine.

Single-drug regimens.

At 80 mg/kg alfaxalone, all mice showed LORR and lost the tail-pinch reflex but did not lose either the toe-pinch reflex or muscle tone. LORR was rapid and within a few minutes of intraperitoneal injection (Table 1). The onset of action was similar between female mice (mean ± 1 SD, 2.17 ± 0.17 min) and male mice (2.38 ± 0.52 min). Alfaxalone's duration of action was significantly (P = 0.02) longer in female mice (67.0 ± 2.3 min) than in male mice (46.0 ± 1.2 min). The 80-mg/kg IP dose of ketamine failed to induce loss of consciousness in 5 of the 14 mice, and muscle tone was present in all mice. Neither the onset nor duration of action of ketamine differed between sexes. The onset of action was significantly (P < 0.001) shorter for alfaxalone (2.22 ± 0.15 min) than ketamine (5.44 ± 0.41 min), whereas duration of action was significantly (P < 0.001) longer for alfaxalone (57.1 ± 3.8 min) than ketamine (6.9 ± 0.8 min; Table 2).

Table 1.

Time to onset and duration of action (mean ± 1 SD) of alfaxalone and ketamine alone and in combination with xylazine in male (n= 8) and female (n= 6) mice

	Time to onset (min)		Duration (min)
	Male	Female	Male	Female
Alfaxalone	2.4 ± 0.5	2.2 ± 0.4	46.0 ± 3.3a	67.0 ± 5.5a,b
Ketamine	5.3 ± 1.7c	5.6 ± 0.9c	8.8 ± 2.2	5.4 ± 1.5
Alfaxalone + xylazine	2.3 ± 0.2	2.3 ± 0.2	73.1 ± 5.6d	89.8 ± 6.6d
Ketamine + xylazine	4.1 ± 0.4e	4.0 ± 0.4e	39.9 ± 3.1	34.2 ± 2.2

^aSignificantly (P < 0.001) greater than corresponding value in ketamine-treated mice of same sex.

^bSignificantly (P = 0.02) greater than corresponding value in alfaxalone-treated male mice.

^cSignificantly (P < 0.001) greater than corresponding value in alfaxalone-treated mice of same sex.

^dSignificantly (P < 0.0001) greater than corresponding value in mice of same sex treated with ketamine and xylazine.

^eSignificantly (P < 0.001) greater than corresponding value in mice of same sex treated with ketamine and xylazine.

Table 2.

Anesthetic effects in male (n = 8) and female (n = 6) mice

	No. of male/no. of female mice (overall %) that lost the reflex
	Righting reflex	Tail pinch reflex	Muscle tone	Toe pinch reflex	Respiratory rate (breaths/min)
Alfaxalone	8/6 (100%)	8/6 (100%)	8/6 (100%)	0/0 (0%)	134 ± 4.94
Ketamine	4/5 (64%)	3/2 (36%)	0/0 (0%)	0/0 (0%)	215 ± 6.26a
Alfaxalone + xylazine	8/6 (100%)	8/6 (100%)	8/6 (100%)	8/6 (100%)	134 ± 2.77
Ketamine + xylazine	8/6 (100%)	8/6 (100%)	8/6 (100%)	8/6 (100%)	138 ± 4.35

The respiratory rate (mean ± 1 SD) shown is that at the deepest anesthetic plane.

^aSignificantly (P< 0.001) different from corresponding values from other treatment groups.

Combination regimens.

All reflexes (Table 1) were abolished in all mice by both combination regimens. For this reason, the period of absent toe-pinch reflex was used to determine the duration of action. Sex had no effect on the onset or duration action for either regimen, however the duration of action for alfaxalone+xylazine was even higher in female mice (89.8 ± 6.9 min) than male mice (73.1 ± 5.6 min) than for alfaxalone alone. The onset of action of alfaxalone+xylazine (2.29 ± 0.13 min) was significantly (P < 0.0001) shorter than that for ketamine+xylazine (4.07 ± 0.27 min). The duration of action of alfaxalone+xylazine (80.3 ± 4.8 min) was significantly (P < 0.0001) longer than that for ketamine+xylazine (37.43 ± 2.19 min; Table 2).

Observation during anesthesia.

Unexpected events occurred during the induction and recovery phases of alfaxalone anesthesia. Specifically, 10 of the 14 mice that received alfaxalone alone showed mild to moderate signs during induction, including 4-legged vertical hopping immediately after injection, muscle shivering, scratching at the face, and repetitive jerking of one or more legs. The behavior occurred within 1 min of injection and ceased by 30 s before LORR. The group given alfaxalone+xylazine had fewer mice that showed adverse behavior ( 8 of 14), and the severity of the episodes was decreased from shivering and jerking to shivering only. The behavior did not appear to cause any health problems in the mice, and the signs resolved completely as the mice recovered. All mice dosed with alfaxalone or alfaxalone+xylazine recovered safely and, once ambulatory, were able to access food and water normally. Mice that received ketamine or ketamine+xylazine did not show any seizure-like activity during induction or recovery.

Discussion

This study found that, as a single agent, alfaxalone has improved anesthetic effects over ketamine. Although single-agent ketamine is typically ineffective and therefore not routinely used for rodent anesthesia, we used it here so that we could compare it with alfaxalone to demonstrate differences in administration effects. Ketamine has a wide recommended dosage range in rodents, and we chose 80 mg/kg for simplicity because we used a dose of 80 mg/kg for alfaxalone.¹⁰ At 80 mg/kg IP, alfaxalone resulted in a light surgical plane of anesthesia for 57 min, with a 2-min onset, whereas ketamine did not anesthetize any mice to a surgical level. These results suggest that alfaxalone alone can provide appropriate anesthesia depth and duration for general or minor surgical procedures in mice; however the side effects seen in this study may prevent its use as a single agent.

Interestingly, the intraperitoneal route of alfaxalone used here provided a longer duration of anesthesia than that in previous work using the intravenous route.¹⁸ Encasing alfaxalone with 2-hydroxypropyl-β-cyclodextrin changes its solubility from nonsoluble to soluble in water. Because of the arrangement of toroids in the structure of 2-hydroxypropyl-β-cyclodextrin, its interior is less hydrophilic than the exterior,¹⁹ such that alfaxalone is isolated inside the compound, whereas the outside remains miscible in water. The solubility modification could cause a variation in the bioavailability or pharmacokinetics of alfaxalone in different matrices such as the abdominal cavity. In plasma, 2-hydroxypropyl-β-cyclodextrin is immediately degraded by hydrolysis and catalytic enzymes,²⁸ which free the alfaxalone as a bolus in the blood stream.¹⁸ However, 2-hydroxypropyl-β-cyclodextrin may not readily permeate the membranes in the peritoneal cavity, due to its chemical structure, molecular weight, and low partition coefficient. 2-hydroxypropyl-β-cyclodextrin has to be degraded before the free form of alfaxalone can penetrate lipophilic membranes.²⁶ This phenomenon likely allows alfaxalone to act as a controlled-release drug when given intraperitoneally, thus achieving the long-lasting effect seen in our study. In addition, the significant difference in alfaxalone's duration of action that we noted supports previous work in rats, where male rats required a much higher dose to reach and maintain an anesthetic plane of anesthesia than did female rats.⁶ The previous authors postulated that the interactions of steroidal anesthetics with synaptic membranes, which might be modulated by sex steroids, may be more specific than currently thought.⁶ Those previous results and hypothesis explain why the female mice in our study had a longer duration of anesthesia even though both sexes were given the same dose.

Mice that received alfaxalone showed distinct behaviors after injection, including scratching at the face and twitching of the leg muscles. Another study also noted the leg twitching behavior and classified it as myoclonic jerk.⁵ Myoclonic jerk can be seen as large-amplitude rhythmic movement due to rapid contraction and relaxation of the muscle. The brainstem, cerebellum, and cortex are involved in the effect of the GABA neurotransmitter.¹⁷ Reduction of GABA by GABA_A receptor antagonists also results in myoclonus. Two GABA_A receptor antagonists, pentylenetetrazole³⁰ and flurothyl, are usually used for inducing myoclonic models.²⁵ The myoclonus induced by flurothyl can be inhibited by diazepam and other GABA_A receptor agonists at the benzodiazepine allosteric binding site.⁹ Conversely, many antimyoclonic drugs are GABA_A receptor agonists, such as diazepam, phenobarbital, gabapentin, valproate, and tiagabine. However, tiagabine at high doses caused myoclonus in rats,²³ and the same result occurred in mice with the administration of high-dose muscimol, a GABA_A receptor agonist.²² These results demonstrate that myoclonus could result from either increasing or decreasing GABA_A receptor transmission. Because there are 2 types of neurons, excitatory and inhibitory, the paradoxical myoclonus seen with alfaxalone administration may be due to inhibition of inhibitory neurons, which causes disinhibition and thus muscular contractions.²⁷ Alfaxalone has been reported to cause muscle tremors and limb rigidity in horses and twitching and paddling in dogs but only occasional involuntary muscle movement in cats.^16,20,24 Although the seemingly involuntary movements in the mice in this study might be comparable to those in horses, dogs, and cats, the significant hopping reaction seen shortly after intraperitoneal administration of alfaxalone appears to be a unique reaction, given that the mice were still conscious. Perhaps the formulation was slightly painful when given in this manner, although the reports of intramuscular administration of alfaxalone without significant signs of pain in cats and rabbits make this hypothesis unlikely.¹¹

Combining xylazine with alfaxalone resulted in prolonged surgical anesthesia and ablated the adverse myoclonic-like activity associated with single-agent alfaxalone. Myoclonus was greatly reduced when xylazine was combined with alfaxalone. Given their pharmacology, xylazine is an optimal choice for combination with alfaxalone, because other commonly used anesthetics, such as benzodiazepines and barbiturates, have an allosteric binding site on GABA_A receptor that might aggravate the myoclonus in mice. Xylazine is an α₂-adrenergic agonist, and its anticonvulsive effect is due to its action on the amygdala center.¹³ However, α₂-agonists have both convulsant and anticonvulsant effects. In rats, the convulsant effect was observed at a dose of 0.3 mg/kg xylazine, and the anticonvulsant effect occurred at doses of 3 to 20 mg/kg.¹³ A possible explanation for this pattern is that xylazine is not α₂-specific at high doses. The low-dose effect of xylazine is associated with α₂-receptor activation, whereas the high dose effect is associated with α₁-receptor activation.¹³

It is important to note that the duration of anesthesia was quite long with the combination regimen. We did not measure body temperature in this study, although thermal support was provided. We acknowledge that some degree of hypothermia might have occurred despite thermal support, thus perhaps prolonging the anesthetic times. This issue is an avenue for exploration in future studies.

From our preliminary analysis, alfaxalone appears to have a wide margin of safety, but it may not be a viable single-agent option for mouse anesthesia in light of the adverse myoclonic signs that we observed; however, further studies of different routes of administration or drug combinations may be warranted. The combination of alfaxalone with xylazine appeared to be a more viable option, although mild seizure-like activity still occurred in some mice, and because the combination's long duration of action might be problematic in terms of body temperature maintenance and recovery monitoring, these factors need to be considered carefully before choosing this regimen. Although the ED50 study performed here only assessed doses of 20, 40, and 80 mg/kg, evaluation of additional doses in the 40- to 80-mg/kg range would be helpful to determine whether a lower dose of alfaxalone combined with xylazine decreases the duration of anesthesia and further ameliorates the myoclonic signs. If so, users might then tailor the drug doses depending on the expected duration of the surgical procedure, thus achieving a beneficial refinement regarding rodent surgical management.