Introduction
Domestic rabbits have a 1.39% risk of anesthetic-related death, compared with 0.17% and 0.24% in dogs and cats, respectively.1 Drug-induced cardiorespiratory depression may be 1 factor contributing to this increased risk in domestic rabbits. Intubation is inherently difficult in rabbits, and the use of injectable sedatives and anesthetics (eg, opioids, benzodiazepines, α2-adrenergic receptor agonists, and dissociative anesthetics) is often necessary to facilitate intubation by providing sedation, immobilization, and muscle relaxation.2 Specific drug combinations such as medetomidine and ketamine are associated with variable and potentially severe adverse effects warranting the investigation of safer alternatives.3,4
Alfaxalone is a synthetic neuroactive steroid that binds to γ-aminobutyric acidA receptors and facilitates the inhibitory action of γ-aminobutyric acid, resulting in CNS depression.5 Alfaxalone is formulated with 2-hydroxypropyl-β-cyclodextrin and licensed for IV use in cats and dogs in the US and other countries. Alfaxalone is also approved for IV use in rabbits in the UK and Australia. Intramuscular administration is approved for cats in Australia; however, off-label IM use is common in other species.
Several studies investigated the sedative and anesthetic effects of IM administration of alfaxalone in rabbits. In 1 study,6 alfaxalone was administered IM to New Zealand White rabbits at doses of 4, 6, or 8 mg/kg. All doses resulted in smooth and rapid onset of sedation and dose-dependent respiratory depression; however, 1 rabbit experienced apnea followed by cardiac arrest after receiving a dose of 8 mg/kg. Another study7 of New Zealand White rabbits reported good quality of sedation with smooth recovery and minimal cardiorespiratory depression following IM administration of alfaxalone at 6 mg/kg as a sole agent.7 Furthermore, when the same dose was combined with butorphanol (0.3 mg/kg, IM) and dexmedetomidine (0.2 mg/kg, IM), no response to noxious stimuli occurred, indicating that a good anesthetic plane had been achieved. With this drug combination, the onset of effects was more rapid and duration was longer, compared with alfaxalone administration alone. The drug combination also resulted in a greater magnitude of cardiovascular and respiratory depression; however, values remained within reference range limits.7
Intravenous administration of alfaxalone in rabbits at 2 or 3 mg/kg following premedication with buprenorphine at 0.03 mg/kg IM resulted in a smooth and rapid induction of anesthesia with minimal cardiorespiratory effects and a high success rate of blind orotracheal intubation.8 However, to the authors' knowledge, no data exists on the suitability of IM administration of alfaxalone for orotracheal intubation of rabbits. Compared with IV administration, IM administration of alfaxalone is advantageous for induction of anesthesia in rabbits because it requires less animal handling and there is no need for placement of an IV catheter. Also, because alfaxalone is a non-controlled substance, potential drug abuse is limited.
The objective of the present study was to determine the dose of alfaxalone for IM administration combined with dexmedetomidine and hydro-morphone that would allow endoscopic-guided orotracheal intubation in rabbits without causing a decrease in respiratory rate or apnea. We hypothesized that, when administered IM with hydromor-phone (0.1 mg/kg) and dexmedetomidine (0.005 mg/ kg), increasing doses of alfaxalone (2, 5, or 7 mg/kg) would increase the success rate of endoscopic-guided orotracheal intubation in rabbits but would progressively depress respiratory rate and increase the occurrence of apnea.
Materials and Methods
Animals
This study was approved by the Louisiana State University Institutional Animal Care and Use Committee (Protocol No. 17-101). Fifteen sexually intact Miniature Lop rabbits were used for this study. Nine female and 6 male rabbits ranged from 4 to 9 months old and weighed (mean ± SD) 2.3 ± 0.3 kg. Physical examination, CBC, and serum biochemical analysis findings indicated that rabbits were healthy. At the time of arrival, rabbits were housed separately in pens grouped by sex and allowed to acclimatize for > 2 weeks prior to the experimental period. Rabbits had free access to fresh water. An Alicia Bermuda grass–alfalfa hay and a commercial pelleted diet (Rabbit Pellet 16%; Country Acres Feed Company) were fed twice daily.
At the completion of the study, male rabbits were castrated by the Louisiana State University Division of Laboratory Medicine personnel and all rabbits were made available for adoption.
Experimental treatment
For each rabbit, age, sex, body weight, and respiratory rate were recorded at the beginning of the experimental period (T = 0). Rabbits were randomly assigned9 to 3 treatment groups (5 rabbits/group). Rabbits received hydromorphone (0.1 mg/kg) and dexmedetomidine (Dexdormitor; 0.005 mg/kg) combined with 1 of 3 doses of alfaxalone (Alfaxan Multi-dose) as follows: low-dose alfaxalone (2 mg/kg; A2), mid-dose alfaxalone (5 mg/kg; A5), and high-dose al- faxalone (7 mg/kg; A7). Drug and doses were selected on the basis of clinical use at the veterinary teaching hospital and results of a pilot study with 2 rabbits. Drugs were mixed together immediately prior to injection and administered IM into the semimembranosus or semitendinosus muscle of the right or left hind limb of each rabbit by the same investigator (PQ-W).
Rabbits were placed in a small animal carrier and observed for 10 minutes to allow for maximal sedative effects. Subsequently, respiratory rate was recorded (T = 10) and quality of anesthesia was subjectively evaluated by use of a semiquantitative rating scale (Appendix 1). The rating scale consisted of 5 categories, and their scores were summed for a total anesthesia score ranging from 0 to 12, as follows: spontaneous posture (0 to 4), response to toe pinch (0 to 2), palpebral reflex (0 to 2), eye rotation (0 to 1), and jaw tone (0 to 3). The investigator assessing the anesthesia quality score (SNR) was blinded to the treatment group. Rabbits were then positioned in sternal recumbency with the head and neck extended. Lidocaine (2 mg) was blindly splashed over the arytenoid cartilages; 30 seconds later, the same investigator (PQ-W) attempted orotracheal intubation of rabbits with an endotracheal tube (Oral/ nasal endotracheal tube, cuffless, Murphy eye, 2.0 mm; Mallinckrodt) placed over a semirigid endo-scope (FM-1.9X6 Micro-Endoscope; MDS-VET). Once the endoscope was inserted into the trachea, the endotracheal tube was advanced off the endoscope and into the trachea. Only 1 intubation/rabbit was attempted. An intubation attempt was defined as the advancement of the endotracheal tube from the mouth, at the level of the incisors, to the opening of the larynx. End-tidal partial pressure of carbon dioxide was measured by use of a multiparameter monitor (CARESCAPE monitor B850; GE Healthcare). Intubation was considered successful when 1 to 6 consecutive end-tidal carbon dioxide readings were present on the capnograph and considered unsuccessful if no readings were present. For rabbits that were intubated, quality of intubation was subjectively evaluated by 1 investigator (PQ-W) with a semiquantitative rating scale ranging from 0 to 3 (Appendix 2).
Immediately following the end of the experiment, male rabbits were placed under general anesthesia with isoflurane in 100% oxygen for orchiectomy. Female rabbits were immediately extubated. All rabbits recovered under supervision and were placed back in their pen once they regained the ability to ambulate.
Statistical analysis
Statistical analysis was performed with commercially available software (JMP Pro 14.2.0; SAS Institute Inc). One-way ANOVA was used to analyze the continuous response variable (body weight) with the treatment group as the fixed effect. Respiratory rates at time 0 and 10 minutes were compared with a paired t test. Assumptions of these models (linearity, normality of residuals, and homoscedasticity of residuals) and influential data points were assessed by examining standardized residual and quantile plots, and the normality of residual was confirmed with the Shapiro-Wilk test. Ordinal scores (intubation and sedation scores) were analyzed by the Kruskal-Wallis test followed by post hoc pairwise comparisons. Sex against treatment groups was tested with the Fisher exact test. Values of P < 0.05 were considered significant. Data are reported as median (range) values.
Results
Rabbits completed the study without any adverse events. No significant differences were found in sex or body weight between rabbits of the 3 treatment groups. Respiratory rates were significantly (P < 0.001) lower at T = 10, compared with T = 0, for all treatment groups (Table 1). No apnea occurred in rabbits in any treatment group.
Median (range) respiratory rate (breaths/min) in rabbits at baseline and 10 minutes after administration of dexmedetomidine (0.005 mg/kg), hydromorphone (0.1 mg/kg), and either alfaxa-lone 2 mg/kg (A2 group), 5 mg/kg (A5 group), or 7 mg/kg (A7 group).
| Times | A2 group (n = 5) | A5 group (n = 5) | A7 group (n = 5) |
|---|---|---|---|
| Baseline (T = 0) | 160 (100–240) | 180 (160–210) | 170 (120–200) |
| 10 min after injection (T = 10) | 72 (28–168) | 36 (28–68) | 40 (32–48) |
Median (range) total anesthesia quality scores for rabbits in the A2 group, A5 group, and A7 group were 3 (2 to 5), 6 (5 to 6), and 6 (4 to 9), respectively (Table 2). Rabbits in the A7 group (P = 0.04) and A5 group (P = 0.02) had significantly higher total anesthesia quality scores, compared with rabbits in the A2 group.
Median (range) anesthesia quality scores in rabbits following administration of dexmedetomidine (0.005 mg/kg), hydromorphone (0.1 mg/kg) and either alfaxalone 2 mg/kg (A2 group), 5 mg/kg (A5 group), or 7 mg/kg (A7 group). The maximum possible anesthesia quality score is 12.
| Variable | A2 group (n = 5) | A5 group (n = 5) | A7 group (n = 5) |
|---|---|---|---|
| Spontaneous posture | 1 (1–2) | 2 (2–2) | 3 (2–4)* |
| Response to toe pinch | 0 | 0 | 0 |
| Palpebral reflex | 1 (0–1) | 1 (1–1) | 1 (1–2) |
| Eye rotation | 0 | 0 | 0 |
| Jaw tone | 1 (1–2) | 3 (2–3)* | 2 (2–3)* |
| Total anesthesia score | 3 (2–5) | 6 (5–6)* | 6 (4–9)* |
Significant (P < 0.05) difference from A2 group rabbits.
Median (range) values for each category of the anesthesia quality score on the basis of treatment group are presented (Table 2). Rabbits in the A7 group had a significantly (P = 0.03) higher spontaneous posture score, compared with rabbits in the A2 group. Jaw tone score was significantly higher in A7 group (P = 0.02) and A5 group (P = 0.02) rabbits, compared with A2 group rabbits. Scores for response to toe pinch, palpebral response, and eye rotation were not significantly different among groups.
Successful intubation occurred in 0 rabbits in the A2 group, 3 in the A5 group, and 4 in the A7 group, with median (range) intubation quality scores of 0 (0 to 0), 2 (0 to 3), and 2 (0 to 3), respectively. Rabbits in the A7 group had significantly (P = 0.02) higher intubation quality scores, compared with rabbits in the A2 group.
Discussion
In the present study, IM administration of alfaxa-lone in combination with hydromorphone and dexmedetomidine caused dose-dependent anesthetic induction of rabbits, facilitating endoscopic-guided orotracheal intubation with minimal changes in respiratory rate.
The total anesthesia quality scores for rabbits in the A5 group or A7 group were similar and higher than for rabbits in the A2 group. This indicated a dose-dependent effect progressing from mild sedation to anesthesia. For each dose, the onset of effects following IM administration of alfaxalone was smooth and rapid, with maximal sedative effects occurring within 10 minutes of injection. These findings were similar to those reported in other studies.6,10
Lateral recumbency (as indicated by spontaneous posture score) was achieved only in A7 group rabbits, whereas rabbits in the A2 group or A5 group remained in sternal recumbency. This finding is in contrast to data reported by Huynh et al,6 who reported rapid loss of consciousness and loss of righting reflex in rabbits administered a single dose of alfaxalone IM at either 4, 6, or 8 mg/kg. Ishikawa et al10 also reported lateral recumbency and loss of righting reflex at lower doses (1, 2.5, or 5 mg/kg, IM) of alfaxalone than the doses used in the present study. Furthermore, it was expected that addition of hydromorphone and dexmedetomidine in the present study would have an additive or synergistic effect on level of sedation, compared with alfaxalone administration alone. One reason for the observed differences is the methods used to evaluate spontaneous posture. Both aforementioned studies evaluated loss of righting reflex by positioning the rabbits in lateral or dorsal recumbency and documenting the resistance of the animals trying to obtain an upright position. In the present study, spontaneous posture was assessed without interference by human handling or positioning of the rabbits. It is possible that if rabbits in the A2 group or A5 group were placed in lateral recumbency, loss of righting reflex may have been observed.
Jaw tone scores for rabbits in the A5 group and A7 group were similar and higher than that of rabbits in the A2 group. Decreased jaw tone at these higher doses provided sufficient muscle relaxation to facilitate opening the mouth, and this was likely a contributing factor to successful intubation in these rabbits. Ishikawa et al10 reported poor or slight jaw relaxation in rabbits receiving alfaxalone IM at 1 and 2.5 mg/ kg (poor) or 5 mg/kg (slight), whereas in the present study jaw tone was minimal or none in rabbits administered alfaxalone IM at 5 or 7 mg/kg in combination with hydromorphone and dexmedetomidine. These findings indicated that alfaxalone administration alone may not provide sufficient jaw relaxation to allow intubation, and higher doses and the addition of muscle relaxants such as α2-adrenergic receptor agonists or opioids may be necessary to increase the success rate of intubation. Even though jaw tone scores were similar for rabbits in the A5 group and A7 group, more A7 group rabbits were successfully intubated. As mentioned, A7 group rabbits exhibited a higher spontaneous posture score, indicating a deeper plane of anesthesia, which likely contributed to the greater success rate of intubation in A7 group rabbits.
All rabbits in the present study responded to the toe pinch with a complete withdrawal of the fore-limb, indicating that response to noxious stimulation was still present at all alfaxalone doses tested. These findings are in accordance with other studies assessing a withdrawal response following a single IM injection of alfaxalone at similar doses in rabbits.6,7 However, in a study by Bradley et al,7 when alfaxalone (6 mg/kg) was combined with butorphanol (0.3 mg/kg) and dexmedetomidine (0.2 mg/kg) and administered IM, the withdrawal response was consistently absent. This suggests that sufficient muscle relaxation and analgesia was present in the drug protocol to suppress noxious stimulation associated with the toe pinch. This lack of response could possibly be a result of the relatively high dose of dexmedetomidine used, which provides dose-dependent analgesia in addition to muscle relaxation. In the present study, the dose of dexmedetomidine was comparatively lower than that used in the previous study7 and may have not been sufficient to abolish the toe pinch withdraw.
Variable IV doses of alfaxalone allowing for orotracheal intubation have been reported, ranging from 2 to 10 mg/kg.8,11 These differences may reflect age-related or breed-specific characteristics and type or dose of premedication used. Rabbits are frequently difficult to intubate, largely a result of their unique oropharyngeal anatomy.12 They have a long, narrow oral cavity with a large, fleshy tongue and large incisors, making visualization of the larynx difficult. As such, an adequate plane of anesthesia to allow proper extension of the head and opening of the mouth is required to visualize the larynx with a scope and to facilitate intubation. In the present study, none of the rabbits in the A2 group could be intubated. However, 3 of 5 rabbits in the A5 group and 4 of 5 rabbits in the A7 group were successfully intubated. On the basis of this dose-related progression, we speculated that IM doses of alfaxalone > 7 mg/kg may further increase the success rate of intubation; however, the risk of respiratory complications such as hypoventilation or apnea may also increase in a dose-dependent fashion, and further studies are needed to ascertain these risks. In addition, increasing doses of alfaxalone will increase the total injectate volume. With the only currently available concentration (10 mg/mL) of alfaxa-lone, larger doses may become impractical to inject or result in pain or discomfort, as is the case in dogs and cats.13,14
Even though rabbits in the A5 group and A7 group exhibited similar total anesthesia quality scores, a higher number of rabbits were successfully intubated in the A7 group versus the A5 group. Rabbits in the A5 group had lower individual total anesthesia score values (maximum score = 6/12) and overall a narrow range of values (5 to 6), whereas rabbits in the A7 group had higher individual total anesthesia score values (maximum score = 9/12) and a wider range of values (4 to 9), resulting in similar median total anesthesia scores.
Intravenous administration of alfaxalone can cause apnea in dogs and cats, especially following rapid IV administration.15,16,17 In rabbits, apnea was also shown to occur, even when IV doses of alfaxa-lone were administered slowly over a period of 60 seconds.8,10,18 In contrast, IM administration of alfaxa-lone is less likely to cause apnea, presumably as a result of slower drug uptake and absorption; however, clinically relevant, dose-dependent respiratory depression may occur, especially when other sedative agents are used in combination.6,7,10,11 In the present study, no apnea occurred. However, respiratory rates were lower following administration of all 3 drug protocols but remained within normal physiologic values for rabbits (ie, 30 to 60 breaths/min).19 Other studies10,20 evaluating similar IM doses of alfaxalone had similar findings. Reasons for decreased respiratory rates reported in some studies and the lack of respiratory adverse effects observed in others, including the present study, can only be speculated and may be a result of several factors, as follows: differences in breed-specific or strain-specific drug sensitivity, age- or sex-related factors, environmental factors (eg, stress and study design), and presence of underlying or subclinical disease.
Several limitations were identified in the present study. Bias could have been introduced into the study design because the investigator performing intubation (PQ-W) was aware of the treatment group. In addition, only young and healthy Miniature Lop rabbits were used in the present study, indicating that results can only be applied to this subset of animals. Potential differences in drug-related effects may arise in older rabbits, different breeds of rabbits, or rabbits with clinical illness or underlying disease. The suitability of drug protocols tested in the present study were only evaluated for endoscopic-guided orotracheal intubation, when in fact there are several intubation techniques used in rabbits. The intubation success rates with different techniques could vary with the drug protocols used in the present study. Finally, the evaluation of respiratory depression in the present study was only made on the basis of respiratory rate and presence of apnea, whereas other methods of respiratory function monitoring, such as capnography and blood gas analysis, would provide better measurements of the depressant effects on ventilation and oxygenation.
In conclusion, when administered with hydro-morphone (0.1 mg/kg) and dexmedetomidine (0.005 mg/kg), increasing IM doses (2, 5, and 7 mg/kg) of alfaxalone progressively increased the success rate of endoscopic-guided orotracheal intubation. Furthermore, increasing the dose of alfaxalone had no significant effect on respiratory rate.
Acknowledgments
Financial support was provided by the Louisiana State University Veterinary Clinical Sciences Competitive Organized Research Program (CORP) grant.
The authors declare no conflicts of interest.
The authors thank Dr. Sarah Keeton for her technical assistance, as well as Dr. Kriscelle Mendoza for performing orchiectomies of male rabbits and for assistance in the adoption of rabbits.
References
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Appendix 1
Semiquantitative scoring system to quantify the degree of anesthesia quality in rabbits. Scores for each category were summed to determine the total anesthesia quality score for each treatment group (maximum possible score of 12).
| Category | Score | Description |
|---|---|---|
| Spontaneous posture | 0 | Normal |
| 1 | Sitting with head up | |
| 2 | Sternal recumbency, head down | |
| 3 | Lateral recumbency, response to stimuli | |
| 4 | Lateral recumbency, no response to stimuli | |
| Response to toe pinch | 0 | Complete withdrawal of limb |
| 1 | Partial withdrawal of limb | |
| 2 | No withdrawal of limb | |
| Palpebral reflex | 0 | Strong |
| 1 | Weak | |
| 2 | Absent | |
| Eye rotation | 0 | Central |
| 1 | Ventromedial | |
| Jaw tone | 0 | Unable to open mouth |
| 1 | Able to open mouth with strong resistance, unable to extrude tongue | |
| 2 | Able to open mouth with minimal resistance, possible tongue extrusion, chewing or tongue retraction | |
| 3 | Able to open mouth and extrude tongue with no resistance |
Appendix 2
Semiquantitative rating scale to quantify the degree of intubation quality in rabbits.
| Score | Description |
|---|---|
| 0 | Swallowing or coughing not allowing intubation |
| 1 | Minimal swallowing or coughing not allowing intubation |
| 2 | Minimal swallowing or coughing allowing intubation |
| 3 | No swallowing or coughing allowing intubation |
