Intramuscular Administration of Alfaxalone Alone and in Combination for Sedation and Anesthesia of Rabbits (Oryctolagus cuniculus)

Abstract

This study compared alfaxalone, alone and in combination with other medications, for sedative and anesthetic properties after intramuscular administration in New Zealand white rabbits. In the main portion of the study, 6 female rabbits were assigned to 5 treatment regimens in a blinded crossover design. Alfaxalone (6 mg/kg IM) was administered alone and in combination with each of the following: 0.3 mg/kg butorphanol; 1 mg/kg midazolam; 0.2 mg/kg dexmedetomidine; and both 0.3 mg/kg butorphanol and 0.2 mg/kg dexmedetomidine. An additional 6 rabbits received 0.2 mg/kg dexmedetomidine for comparison. The median time to onset of recumbency ranged from 2.0 to 5.5 min, with times significantly shorter for animals that received alfaxalone with either midazolam or dexmedetomidine than for those given dexmedetomidine only. Duration of sedation (mean ± 1 SD) was: alfaxalone only, 40 ± 7.3 min; alfaxalone with butorphanol, 47.8 ± 9.9 min; alfaxalone with midazolam, 65.2 ± 6.5 min; alfaxalone with dexmedetomidine, 157.5 ± 22.4 min; alfaxalone with butorphanol and dexmedetomidine, 157.7 ± 22.3 min, and dexmedetomidine only, 93.7 ± 11.9 min. Response to noxious stimuli was absent in 2 of the rabbits given dexmedetomidine only, 4 of those given alfaxalone with dexmedetomidine, and all 6 of the animals dosed with alfaxalone, butorphanol, and dexmedetomidine; this last group displayed the longest absence of a toe-pinch response (57 ± 3 min).

Alfaxalone is a neuroactive steroid that acts as a positive allosteric modulator of GABA receptors, causing hyperpolarization of the neuron and producing a strong anesthetic effect.²⁴ This compound has been reintroduced into the veterinary market (Jurox, Kansas City, MO) after reformulation with hydroxypropyl β-cyclodextrin and is licensed in several countries for use as an anesthetic induction and maintenance agent in dogs and cats. In 2017, the alfaxalone license in the United Kingdom was extended to include the induction of anesthesia in rabbits.

Although its licensing around the world is predominantly for intravenous use, the formulation of the drug allows for it to be used intramuscularly.¹³ Alfaxalone can be administered through several parenteral routes and does not cause tissue irritation when extravasation occurs, thus offering a distinct advantage over other anesthetics, namely propofol.¹⁷ Alfaxalone has been safely used in a variety of species for intramuscular administration. In cats, a pharmacokinetic analysis comparing intramuscular with intravenous use showed that intramuscular administration offered high bioavailability (94.7%) and a half-life that was roughly 2.5 times greater than of intravenous use.¹⁸ Intramuscular administration resulted in deep sedation that lasted from 10 to 45 min.¹⁸ In dogs, intramuscular alfaxalone produced consistent and stable sedation, with dose-dependent cardiorespiratory depression.²² In pigs, intramuscular alfaxalone combined with dexmedetomidine produced smoother induction and deeper sedation than the combination of ketamine and dexmedetomidine.¹⁹ A study in cats comparing intramuscular dexmedetomidine and butorphanol combined with either alfaxalone or ketamine showed that alfaxalone provided effective sedation for castration procedures and yielded a smoother recovery than ketamine.¹⁴ In addition, alfaxalone has been investigated through several administration routes as an anesthetic option in small mammals including rats, ferrets, and guinea pigs.^2-4,9,15 Several studies have investigated the intravenous use of alfaxalone in rabbits.^8,11,23 However, only 2 studies to date have investigated the intramuscular use of alfaxalone in rabbits. One of these studies identified the potential for alfaxalone as an intramuscular sedative and induction agent in wild rabbits.¹⁶ The other study found that 6 mg/kg alfaxalone administered intramuscularly produced smooth and consistent sedation with minimal side effects; given these results, we chose this dose of alfaxalone for investigation in our current study.¹³

Previously published injectable rabbit sedation protocols use a range of sedatives, including ketamine, opioids, α2 agonists, and benzodiazepines.^6,7 One common injectable combination that is used in rabbits is ketamine–xylazine, but intramuscular injection of ketamine is reported to be painful,⁵ thus warranting investigation into alternative injectable anesthetics. The studies to date have described the safety of intramuscular alfaxalone alone in rabbits, demonstrating significant potential as both a sedative and anesthetic and merits further investigation into clinical applications and utility. The current study was designed to investigate and compare the sedative, anesthetic, and cardiovascular effects of intramuscular alfaxalone, both alone and in conjunction with other commonly used sedatives. Our hypothesis was that alfaxalone as part of a multimodal sedation protocol would produce more profound sedation and anesthetic effects than alfaxalone administered as a single agent.

Materials and Methods

Animals.

This project was approved by the IACUC at the University of Michigan, an AAALAC- accredited facility. Female New Zealand White Rabbits (n = 6; age, 3 mo; body weight, 2.3 to 2.9 kg) were used in the crossover portion of this study. An additional 6 age- and weight-matched rabbits were used in a noncrossover follow-up study. Rabbits were acquired from an inhouse breeding colony and were free of the following pathogens: Clostridium perfringens, Toxoplasmosa gondii, Francisella tularensis, Pasteurella multocida, myxoma virus, rabbit hemorrhagic disease virus, rotavirus, Treponema cuniculi, Encephalitozoon cuniculi, endoparasites, and ectoparasites. Rabbits were pair-housed in open caging (Allentown Caging, Allentown, PA) within a barrier facility, which was maintained on a 12:12-h light:dark cycle with constant temperature (20 ± 1 °C) and relative humidity maintained between 30% to 70%. Rabbits were fed 3/4 cup of pelleted feed (LabDiet 5326, PMI LabDiet, St Louis, MO) once daily and received hay and vegetables for enrichment daily.

Study design.

Rabbits were randomly assigned to treatment groups in a prospective, randomized, crossover study design, with a 1-wk washout period between treatments. The data recorder was blinded to the drug combination each rabbit received. All medications were administered intramuscularly into the caudal epaxial muscles, with no more than 1 mL administered per injection site, by using 3-mL syringes (Becton Dickinson, Franklin Lakes, NJ) and 25-gauge, 5/8-in. needles (Covidien, Dublin, Ireland). Rabbits received as many as 3 injections per anesthetic treatment, depending on the volume(s) administered. Treatment regimens included: alfaxalone (6 mg/kg; Alfaxan, Jurox, KS City, MO) only or with butorphanol (0.3 mg/kg; Torbugesic, Zoetis, Kalamazoo, MI); midazolam (1 mg/kg; Akorn, Lake Forest, IL); dexmedetomidine (0.2 mg/kg; Dexdomitor, Zoetis, Kalamazoo, MI); or both butorphanol (0.3 mg/kg) and dexmedetomidine (0.2 mg/kg). All rabbits were euthanized through overdose of intravenous sodium pentobarbital (Euthasol, Virbac, Ft Worth, TX) at the conclusion of the study, and epaxial muscles were examined grossly for abnormalities.

Animal preparation and data collection.

Body weights were obtained on the morning of testing, and rabbits were brought to the study room for a 30-min acclimation period. Respiratory rate (f_R) was recorded by visual observation; heart rate (HR), rectal temperature, and hemoglobin oxygen saturation (SpO₂) were monitored continuously by using noninvasive instrumentation (catalog no. V3395, TPR Monitor, Surgivet, Waukesha, WI). The SpO₂ probe was placed on the forepaw for all animals. The accuracy of the HR recorded from the monitor was confirmed periodically by manual auscultation every 15 min, or when issues with the probe were noted. Animals were placed on a circulating-water blanket (Gaymar T/Pump, Stryker, Kalamazoo, MI) set to 35 °C; the water blanket was turned off when patient temperature exceeded 40 °C. HR, f_R, and SpO₂ were recorded at baseline and at 5-min intervals until the rabbits had recovered from sedation. Rectal temperature was recorded at 5-min intervals after baseline. Mucous membrane color and capillary refill time were assessed periodically as part of routine anesthetic monitoring.

During the course of study, rabbits were placed in lateral recumbency, rotating laterality every 60 min. The righting reflex response was assessed every 5 min by rotating the animal into dorsal recumbency; any attempt the rabbit made to right itself into sternal recumbency was noted as a positive righting reflex. The response to toe and ear pinches was tested at 5-min intervals. The toe pinch was first assessed by applying a mosquito hemostat to the interdigital webbing of a hindfoot for 2 s, applying enough pressure to engage the first click of the locking mechanism on the hemostat. Any purposeful movement or vocalization was considered a positive response. Purposeful movement was limited to gross movement of the head, body, or extremities. Movements such as twitching, shivering, or stiffening or reflex withdrawal of the limb were regarded as nonpurposeful and not considered as a positive response. When no positive response to the hindfoot pinch occurred, the pinch procedure was repeated on the ear pinna. When a lack of response was confirmed, the animal continued to undergo testing until a positive response was recorded. Each animal underwent at least 30 min of pinch–response testing, and from that time forward until the first positive response occurred; thereafter, that variable was no longer tested, to avoid causing pain to the animal. Any response that was deemed inconclusive was retested at the next 5-min interval; a positive response at this time resulted in a positive score being recorded for the previous timepoint, whereas a negative response was recorded as negative for both time points. The overall depth of sedation or anesthesia was evaluated according to American Society of Anesthesiologists guidelines by using a combination of stimuli responses, gross movement, and physiologic variables.¹

Times to loss and return of righting reflex were recorded to the nearest minute throughout the study. The total sedation time was calculated as the time from righting reflex loss to righting reflex return. The total sedation time was ended after 180 min of sedation. The total duration of absence of ear or toe pinch response was calculated by subtracting the time when righting reflex was lost from the time point when first pinch response was noted. The quality of anesthetic induction and recovery was scored for each anesthetic trial; scoring system criteria are defined in Figures 1 and 2.

Figure 1.

Sedation and anesthetic induction scoring system.

Figure 2.

Sedation and anesthetic recovery scoring system.

Follow-up study.

Six experimentally naïve age- and weight-matched female rabbits received dexmedetomidine at 0.2 mg/kg IM; however, the data recorder could not be blinded to this treatment group because it was assessed separately from the main study. The methods and data collection were performed as described for the main study.

Statistical analyses.

All statistical analyses were performed by using Prism 7.0 (GraphPad Software, San Diego, CA). Statistical methods were reviewed by University of Michigan Consulting for Statistics, Computing, and Analytics Research Center. Normality was assessed through Wilk–Shapiro testing. Rabbits that were included in the crossover study were assessed through repeated-measures one-way ANOVA with Tukey posthoc testing, corrected for multiple comparisons. Induction and recovery scores were analyzed by using Friedman testing with Dunn multiple comparisons for animals included in the crossover portion of the study. Data from rabbits dosed with dexmedetomidine only were analyzed separately from the groups used in the crossover study, given that the experimental designs differed. The data from dexmedetomidine-only rabbits was compared with each individual treatment from the crossover study by using unpaired t tests. Induction and recovery scores were compared with those from each remaining treatment through Mann–Whitney tests. A P value of less than 0.05 was regarded as statistically significant.

Results

Induction and recovery score.

Mean induction scores for each group ranged from 1 to 2, and there was no statistical difference between any of the groups. Recovery scores (mean ± 1 SD) for rabbits given dexmedetomidine only (2.7 ± 0.5) were significantly higher than all other groups (alfaxalone only, 1 ± 0 [P = 0.002]; alfaxalone with butorphanol, 1.7 ± 0.5 [P = 0.033]; alfaxalone with midazolam, 1.3 ± 0.5 [P = 0.013]; alfaxalone with dexmedetomidine, 1.5 ± 0.5 [P = 0.022]; and alfaxalone with butorphanol and dexmedetomidine, 1 ± 0 [P = 0.002]). One individual rabbit reacted adversely to injection twice; no other injection reactions were noted throughout the remainder of the study.

Time to loss of righting reflex and total sedation time.

The time to loss of righting reflex was rounded to the nearest minute and reported as mean values with standard deviation. Total sedation time was assessed as the time to the loss of righting reflex until the time of the return of the righting reflex. Data were rounded to the nearest minute and are presented in Table 1.

Table 1.

Time to sedation (loss of righting reflex) and duration of sedation (min; mean ± 1 SD [95% CI])

	Group A	Group AB	Group AM	Group AD	Group ABD	Group D
Time to sedation	4.0 ± 1.2 (2. 7–5.3)	4.2 ± 1.2 (2.8–5. 6)	2.5 ± 1.0 (1.4–3.6)	2.8 ± 1.6 (1.0–4.6)	2.0 ± 0.6 (1.3–2.7)	5.5 ± 1.6b (3.7–7.4)
Total sedation time	40.0 ± 7.3 (31.6–48.4)	47.8 ± 9.9 (36.4–59. 2)	65.2 ± 6.5d (57.7–72.7)	158 ± 22.4c (131–183)	158 ± 22.3c (132–183)	93.7 ± 11.8a (80.0–107)

A, alfaxalone; B, butorphanol; D, dexmedetomidine; M, midazolam

Data represent time from baseline (time 0) until animals regained the righting reflex.

ANOVA performed for each variable. Time to sedation: F = 0.204, DF_n = 1.85, DF_d = 9.25; total sedation time: F = 0.43, DF_n = 1.51, DF_d = 7.56.

Superscripted lowercase letters indicate significant (P < 0.05) differences from designated groups.

^aDifferent from all groups.

^bDifferent from groups AM, AD, and ABD.

^cDifferent from groups A, AB, AM, and D.

^dDifferent from group A.

Response to toe and ear pinches.

For both toe- and ear-pinch responses, all rabbits given alfaxalone only or with butorphanol or midazolam responded at all assessed timepoints. For the toe-pinch response, 4 of the 6 animals dosed with alfaxalone and dexmedetomidine did not respond, with a mean return time of 47.5 ± 13.1 min (95% CI, 3.1 to 60.2 min) for those that did not react. All animals treated with alfaxalone, butorphanol, and dexmedetomidine lost the toe-pinch response, with a mean return time of 56.7 ± 6.7 min (95% CI, 49.0 to 64.3 min). For the dexmedetomidine-only group, 2 of the 6 animals lacked a toe-pinch response, with a mean response return time of 25.0 ± 10.0 (95% CI, –6.8 to 23.4 min) min for the 2 animals that did not react. Statistically significant differences were present between the alfaxalone–butorphanol–dexmedetomidine animals and the alfaxalone-only, alfaxalone–butorphanol, alfaxalone–midazolam, and dexmedetomidine-only groups (ANOVA: F = 0.7197, DF_n = 1.088, DF_d = 5.44; P < 0.0001); there was no significant difference between rabbits given alfaxalone and dexmedetomidine and the other groups.

For the ear-pinch response, 4 of the 6 animals dosed with alfaxalone and dexmedetomidine did not respond, with a mean return of response in those 4 animals of 34.8 ± 13.4 min (95% CI, 0.5 to 45.8 min). In the alfaxalone–butorphanol–dexmedetomidine group, all rabbits developed a lack of response, with a mean of 36.7 ±17.7 min (95% CI, 16.3 to 57.0 min). In the dexmedetomidine-only group, 2 of the 6 animals demonstrated a lack of response, with a mean return of response of 25.0 ± 10.0 (95% CI, –6.8 to 23.4 min). Statistically significant differences occurred between the alfaxalone–butorphanol–dexmedetomidine group and those given alfaxalone only or with butorphanol or midazolam (ANOVA: F = 0.9478, DF_n = 1.828, DF_d = 9.14; P = 0.0285) or dexmedetomidine only (P = 0.0165); there was no significant difference between rabbits dosed with alfaxalone and dexmedetomidine and the other groups.

Physiologic data.

For each group, HR and f_R data were collected every 5 min. All rabbits experienced a marked decrease in respiratory rate (that is, decrease greater than 100 breaths per minute) after administration of any of the drug combinations examined. Due to the variation in time of total sedation, we selected 4 time points to more precisely compare the data: just prior to intramuscular injection (baseline), 10 min after administration of drug(s), halfway through the duration of sedation for each animal (rounded up to the nearest time point), and at the last recorded monitoring interval prior to the return of the righting reflex. The data for HR and f_R can be found in Tables 2 and 3, respectively. The mean midpoint time for each group analyzed was: alfaxalone only, 23 min; alfaxalone with butorphanol, 27 min; alfaxalone with midazolam, 34 min; alfaxalone with dexmedetomidine, 79 min; alfaxalone with butorphanol and dexmedetomidine, 81 min; and dexmedetomidine alone, 50 min. The mean last time point for each group was: alfaxalone only, 43 min; alfaxalone with butorphanol, 51 min; alfaxalone with midazolam, 67 min; alfaxalone with dexmedetomidine, 158 min; alfaxalone with butorphanol and dexmedetomidine, 159 min; and dexmedetomidine only, 96 min. There was no difference observed between all groups in rectal temperature, which ranged from 38° to 40 °C. Maintaining a consistent SpO₂ reading was problematic, and due to uneven group numbers and missing data points, we were unable to perform statistical analysis for this variable. SpO₂ data are presented in Figure 3; animal numbers vary from 4 to 6 at any given time point presented. During the course of study, all rabbits were observed to have pink to light pink mucous membranes and capillary refill times of less than 2 s. All animals recovered well after each anesthetic trial and did not have any adverse health conditions during the course of the crossover study. Postmortem examination of the epaxial muscles of the rabbits used in the crossover study did not reveal any gross abnormalities.

Table 2.

Heart rate data (bpm; mean ± 1 SD [95% CI]) at baseline and selected time points after sedation

	Group A	Group AB	Group AM	Group AD	Group ADB	Group D
Baseline	308 ± 20 (256–360)	309 ± 20 (258–359)	312 ± 18 (267–357)	292 ± 23 (232–352)	286 ± 21 (230–339)	231 ± 10a (206–256)
10 min after injection	285 ± 10 (258–311)	290 ± 22 (235–345)	277 ± 11 (249–305)	146 ± 8b (124–167)	153 ± 7b (134–171)	133 ± 3d (126–140)
Midpoint	273 ± 13 (238–307)	281 ± 25 (216–346)	263 ± 13 (230.1–296.3)	168 ± 10c (142–192)	193 ± 6b (178–208)	148 ± 5d (135–162)
Last time point	255 ± 12 (225–286)	240 ± 21 (185–294)	235 ± 13 (202–268)	205 ± 4e (194–216)	190 ± 4e (178–201)	162 ± 4a (152–172)

A, alfaxalone; B, butorphanol; D, dexmedetomidine; M, midazolam

ANOVA performed for each time point. Baseline: F = 0.4828, DF_n = 2.32, DF_d = 11.58; 10 min, F = 2.531, DF_n = 1.53, DF_d = 7.67; midpoint: F = 3.508, DF_n = 1.48, DF_d =7.42; last time point, F = 4.97, DF_n = 1.87, DF_d = 9.33.

Superscripted lowercase letters indicate significant (P < 0.05) differences from designated groups.

^aDifferent from all groups

^bDifferent from groups A, AB, and AM

^cDifferent from groups A and AM

^dDifferent from groups A, AB, AM, and ABD

^eDifferent from group A.

Table 3.

Respiratory rate data (breaths per minute; mean ± 1 SD [95% CI]) at baseline and selected time points after sedation

	Group A	Group AB	Group AM	Group AD	Group ABD	Group D
Baseline	196 ± 7 (178–214)	183 ± 14 (146–219)	210 ± 3 (203–217)	216 ± 9 (193–239)	212 ± 4 (202–222)	191 ± 7b (174–208)
10 min after injection	35 ± 2 (30.4–40.2)	26 ± 5 (13.1–39.0)	27 ± 4 (17.6–37.1)	44 ± 3 (37.0–51.0)	22 ± 3d (15.1–28.9)	75 ± 8a (53.3–96.0)
Midpoint	38 ± 4 (27.3–46.7)	30 ± 4 (19.1–40.9)	36 ± 1 (32.3–40.0)	79 ± 22 (22.9–136)	35 ± 3 (26.9–42.5)	76 ± 12 (45.4–107)
Last time point	101 ± 23 (41.0–161)	52 ± 11 (23.5–80.5)	81 ± 13 (49.0–113.7)	95 ± 12 (63.3–127)	48 ± 17 (4.4–91.6)	129 ± 16c (88.4–169)

A, alfaxalone; B, butorphanol; D, dexmedetomidine; M, midazolam

ANOVA performed for each time point. Baseline: F = 0.645, DF_n = 1.562, DF_d = 7.81; 10 min: F = 0.766, DF_n = 2.29, DF_d = 11.45; midpoint: F = 0.996, DF_n = 1.16, DF_d = 5.82; last time point: F = 1.82, DF_n = 2.58, DF_d = 12.9.

Superscripted lowercase letters indicate significant (P < 0.05) differences from designated groups.

^aDifferent from all other groups.

^bDifferent from groups AM, AD, and ABD

Different from groups AB, AM, and ABD

^dDifferent from group A.

Figure 3.

Peripheral oxygen saturation after intramuscular administration of alfaxalone alone and with other drugs.

Discussion

This study builds on initial investigations of alfaxalone administered intramuscularly in rabbits.^13,16 The current study demonstrated that alfaxalone in combination with other sedatives produces prolonged and reliable sedation and, in some combinations, can provide anesthesia. Compared with the use of alfaxalone alone, the addition of midazolam increased the duration of sedation by an average of 25 min, and the addition of dexmedetomidine increased the duration of sedation by an average of 117 min. Both midazolam and alfaxalone are GABA receptor agonists, and there is a presumed additive effect when acting at a similar receptor during coadministration.¹⁰ However, we did not examine the use of midazolam alone to confirm to this presumed additive effect. In contrast, because we investigated both alfaxalone and dexmedetomidine individually, we were able to observe a potential synergistic effect of these drugs when co-administered, given that the duration of sedation in rabbits dose with both alfaxalone and dexmedetomidine was far longer than for either single-agent group. The combination of butorphanol and alfaxalone, with or without dexmedetomidine, had no effect on sedation duration but did influence the response to an adverse stimulus, as evidenced by the consistent lack of response to toe pinch in the alfaxalone–butorphanol–dexmedetomidine group. The absence of toe pinch response is likely due to opioid receptor activation with suppression of pain transmission and enhanced central induced sedation.⁶ In our study, alfaxalone–butorphanol–dexmedetomidine was the only combination that consistently produced a lack of toe- and ear-pinch responses, which lasted as long as 1 h in some rabbits. This finding is important, and this combination regimen has promising potential for use in short procedures that are deemed painful, such as tissue biopsy and superficial wound repair. The addition of a local anesthetic in conjunction with this protocol could also be beneficial for these types of procedures. However, further characterization of this drug combination would be prudent before we would recommend it for use in more invasive surgical procedures. Although only alfaxalone–butorphanol–dexmedetomidine reliably produced an absence of toe and ear-pinch responses, all of the alfaxalone–sedative combinations used in this study demonstrated great potential for use in common diagnostic or research-related imaging procedures, such as ultrasonography, radiography, CT, and other noninvasive modalities that require patient immobility. According to the American Society of Anesthetists guidelines for defining sedation and anesthesia, the following regimens were considered to provide moderate sedation: alfaxalone only, alfaxalone–butorphanol, alfaxalone–midazolam, and dexmedetomidine only. Conversely, alfaxalone–dexmedetomidine and alfaxalone–butorphanol–dexmedetomidine provided deep sedation for a prolonged period, with alfaxalone–butorphanol–dexmedetomidine providing consistent anesthesia for as long as 1 h.

The induction of sedation or anesthesia in all alfaxalone-treated groups was both smooth and fairly rapid, with all animals achieving sedation quicker than for dexmedetomidine only. Similarly recovery scores were low for all alfaxalone-containing regimens, indicating a smooth recovery for patients. This outcome is in contrast to the dexmedetomidine-treated group, which included several animals whose recoveries were characterized by paddling, strong startle responses, and multiple attempts to right themselves. This finding is important, because recovery from sedation can be a stressful event for animals, and providing a smooth recovery improves overall animal welfare related to the anesthetic event. Overall, the intramuscular injections were well tolerated, and other than 2 episodes in the same rabbit, there were no observed adverse injection reactions. Regarding the crossover study, all rabbits continued to gain weight throughout the study, and no injection site reactions or other health concerns occurred, demonstrating that repeated intramuscular administration of alfaxalone in rabbits appears to be well tolerated. Furthermore, gross examination of the injection sites postmortem did not reveal any abnormalities; however histologic examination of the tissues was not performed and thus any microscopic pathology could not be evaluated.

The HR and f_R data analyzed during our study revealed a distinct difference between the animals that received dexmedetomidine and those that did not. This difference in HR is unsurprising, given that dexmedetomidine causes both centrally mediated bradycardia and reflexive bradycardia due to its vasoconstrictive action on α₂ receptors.⁶ Comparing among the groups that did not receive dexmedetomidine showed no differences in HR or f_R throughout the study, with a gradual, nonsignificant decrease in HR and increase in f_R until the animals awoke from sedation. Comparing among the groups that received dexmedetomidine revealed than animals given alfaxalone–dexmedetomidine or alfaxalone–butorphanol–dexmedetomidine had higher HR than those given dexmedetomidine only, which may be a compensatory mechanism if the groups with higher HR had lower SpO₂. The rabbits treated with alfaxalone–butorphanol–dexmedetomidine had the lowest f_R among the groups that received dexmedetomidine and consequently this group had the highest HR. Another potential cause for increased HR in these groups is decreased blood pressure, which was not assessed in this study. No animals exhibited a period of apnea or cyanosis throughout the study, but without more defined advanced monitoring, whether they experienced hypoxemia is unclear. Although there were distinct differences in HR and f_R among the groups, overall the variables monitored were considered within acceptable limits for anesthetized rabbits, considering the profound effects that dexmedetomidine can have on HR.

We acknowledge several limitations to the current study. During the course of our study, we had difficulties in using the pulse oximetry probe to achieve accurate, consistent, and physiologically appropriate readings. This difficulty resulted in disparity in our data and missing data points and, consequently, statistical analysis of this parameter was not possible. We noted that, in general, rabbits that received dexmedetomidine had lower SpO₂ readings (Figure 3). Treatment with alfaxalone only or coadministered with butorphanol or midazolam appeared to have mild to minimal effects on peripheral oxygen saturation, in comparison to dexmedetomidine only, alfaxalone–dexmedetomidine, or alfaxalone–butorphanol–dexmedetomidine. The accurate measurement of SpO₂ in rabbits receiving dexmedetomidine may have been confounded by the vasoconstrictive properties of the drug, which have been shown to decrease SpO₂ in people.²¹ The exact amount by which dexmedetomidine may decrease SpO₂ readings in rabbits due to its vasoconstrictive properties currently is unknown. In addition, the lower SpO₂ in these groups may have been the result of hypoxemia, which has been reported as a side effect of dexmedetomidine in some species.⁶ Furthermore, the rabbits that received dexmedetomidine were more heavily sedated, with some experiencing periods of anesthesia, so it is reasonable to presume that these animals may have experienced true hypoxemia due to respiratory depression. All rabbits had pink to pale-pink mucous membranes and acceptable capillary refill times throughout the study, but these measures do not definitively confirm cardiorespiratory stability.⁶ Future studies should attempt to characterize the cardiorespiratory adverse effects of these drug combinations through direct blood pressure monitoring, blood gas evaluation, and analysis of end-tidal CO₂. When sedating rabbits with any of the alfaxalone drug combinations investigated in this study, we recommend having both airway support devices and oxygen delivery systems available in the event that hypoxemia occurs and supplemental oxygen delivery is necessary. Investigators might also provide flow-by oxygen or delivery by mask in these situations, given that there is little to no negative side effects of providing such therapy during a sedative or anesthetic event. We also recognize that the duration of sedation associated with the drug combinations that included dexmedetomidine may exceed what is necessary in most clinical settings. Although not part of this study, in the scenario of prolonged sedation, pharmacologic reversal of dexmedetomidine with atipamezole should expedite recovery.

Several limitations to this study are in regard the overall study design. The dexmedetomidine-only group was not included in the initial experiment but was added after the main crossover study was completed. We added the dexmedetomidine-only group due to the marked differences between the animals that did not receive dexmedetomidine and those given alfaxalone in combination with dexmedetomidine or both dexmedetomidine and butorphanol; these differences warranted further investigation to address whether these effects could be explained by the inclusion of dexmedetomidine alone. Because published data are unavailable currently regarding the duration of sedation and other physiologic effects of dexmedetomidine at the dose examined, we added the dexmedetomidine-only group to the study. Consequently, the investigators were not blinded to this group's treatment, and all of the statistics had to be run separately to correctly compare the dexmedetomidine-only data with the original study. Another limitation of this study is that a single observer scored the anesthetic induction and recovery quality, and thus we did not validate our scoring system with any statistical metrics for assuring accuracy and reliability. Despite this limitation, there is an abundance of literature in veterinary species that anesthetic induction with alfaxalone produces rapid and smooth sedation after intramuscular injection, and it appears that this effect also occurs in rabbits. A further limitation of this study is that we studied only a single dose of each drug. Studying multiple doses of these drugs is necessary to further characterize any additive or synergistic effects that were potentially observed in this study. By definition, an additive effect occurs when 2 drugs acting at the same receptor site produce an effect greater than the sum of their individual effects; synergy occurs when 2 drugs act at different sites.^12,20 However, to further characterize these interactions, the investigation of various dosages of these medications is necessary.¹² In addition, other metrics, such as an anesthetic depth scoring system and electrophysiologic monitoring, could have been performed to further characterize the level of sedation or anesthesia.

Another limitation of this study is that, because only young, healthy, female rabbits were used, we do not know whether potential differences in the effects of these drug combinations occur in males or older animals. The average duration of sedation in our study was 40 min for the 6-mg/kg alfaxalone group, which is lower than the 51.8 min reported elsewhere for the same dosage.¹³ That study used male rabbits that were similar in age to our animals, so the difference may be due to sex-associated differences in sedation duration with intramuscular alfaxalone in rabbits. In addition, the relatively low numbers of animals in each study group may help to explain this observed difference. Furthermore, we did not assess the utility of the drugs in our study in animals with comorbidities, so the safety of these drug combinations in clinically ill rabbits is unknown at this time. One study that examined alfaxalone administered intramuscularly to 81 wild rabbits noted the lack of any adverse outcomes with this regimen.¹⁶ Although the health status of the wild rabbits is unknown, it is reasonable to assume that some animals may have had comorbidities that were clinically undetected. It may be necessary to augment the doses we described here when administering them to animals with clinical illness, similar to strategies used with other sedatives and anesthetics.

In conclusion, alfaxalone, administered alone and in combination with other sedatives, produces effective and consistent sedation and short-term anesthesia in rabbits when administered intramuscularly. Alfaxalone yielded smooth induction and recovery from sedation or anesthesia, and each protocol we presented provided fairly consistent depth and duration of sedation. There are many potential applications for the different drug combinations that we investigated in this study. Further assessment in conjunction with nonpainful procedures, such as diagnostic imaging, may demonstrate a wide range of clinical utility. The combination of alfaxalone, dexmedetomidine, and butorphanol demonstrated the potential for use in short, minor surgical procedures. Further investigation into this combination, and other combinations (for example, with local anesthetics), is warranted to help fully characterize the surgical procedure potential for intramuscular administration of alfaxalone in rabbits.