Experimental animals: Six intact, adult beagle dogs (3 males and 3 females) that were 1 to 5
years of age [2.9 ± 1.5 (mean ± standard deviation) years old] and that weighed 8.6 to 16.8 kg (11.5 ± 3.2 kg)
were used for three drug treatments with at least 10 days between each treatment. All dogs used in the present
study exhibited the ideal body condition (body condition score 3/5) [2 (link)].
Each dog was received in the order of the IM treatment of a combination of alfaxalone-HPCD, medetomidine and
butorphanol (MBA), a combination of medetomidine and butorphanol (MB), and alfaxalone-HPCD alone (ALFX). All
dogs were judged to be in good physical condition based upon a physical examination. Food was withheld for 12 hr
before the each experiment, and water was continuously available. The dogs were cared for according to the
principles of the “Guide for the Care and Use of Laboratory Animals” prepared by Rakuno Gakuen University. The
Animal Care and Use Committee of Rakuno Gakuen University approved this study (approved No. VH22B17).
Instrumentation: All dogs were instrumented with arterial and central venous catheters under
general anesthesia prior to the administration of each IM drug solution. Anesthesia was induced by mask
induction using a vaporizer dial setting at 5% of sevoflurane (Sevoflo, DS Pharma Animal Health Co., Ltd.,
Osaka, Japan) with oxygen. Once anesthetized, the dogs were orotracheally intubated, and anesthesia was
maintained with vaporizer dial setting at 3.0–3.5% of sevoflurane with oxygen. The dogs were placed in left
lateral recumbency for catheter instrumentation. In each dog, 22-gauge catheters (Supercath, Medikit Co., Ltd.,
Tokyo, Japan) were placed into the left cephalic vein and the left dorsal pedal artery. In addition, an 18-gauge
catheter 30 cm in length (Intravascular Catheter Kit, Medikit Co., Ltd.) was placed into the cranial vena cava
through the right jugular vein after the cervical catheter site was aseptically prepared and infiltrated with 1
mg/kg of 2% lidocaine (2% Xylocaine Astrazeneca, Osaka, Japan). The position of the tip and insertion length of
the central venous catheter were approximated and confirmed by pressure waveform. The dogs were infused with
lactated Ringer’s solution (Solulact, Terumo Co., Tokyo, Japan) at a rate of 10 ml/kg/hr
through the catheter placed into the cephalic vein during the sevoflurane anesthesia. After the completion of
catheter placements, the sevoflurane was discontinued, and each dog was extubated when their laryngeal reflex
was functional. Each dog was then allowed to recover for 1 hr in a quiet room until the administration of its
allocated IM treatment.
Drug administration and data collection: Following the 1 hr recovery period, the dogs received
IM alfaxalone-HPCD (Alfaxan, Jurox Pty. Ltd., Rutherford, NSW, Australia) at 2.5 mg/kg (ALFX), medetomidine
(Domitor, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) at 2.5 µg/kg and butorphanol
(Vetorphal, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) at 0.25 mg/kg (MB), or alfaxalone-HPCD at 2.5 mg/kg,
medetomidine at 2.5 µg/kg and butorphanol at 0.25 mg/kg (MBA) in separate experiments. Maddern
et al. [16 (link)] showed that the anesthetic induction dose
of IV alfaxalone-HPCD can be decreased to around one third by premedication with low dose of medetomidine and
butorphanol in dogs. Therefore, the dose of IM alfaxalone-HPCD administration was set at 2.5 mg/kg (one third of
anesthetic IM alfaxalone-HPCD dose) in the present study. All IM drug solutions were administered to each dog at
total 0.3 ml/kg in volume under manual physical restraint. In the ALFX and MB treatments, the
IM drug solution was prepared by mixing appropriate amounts of each product and diluting with normal saline
(Otsuka normal saline, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) up to 0.3 ml/kg volume in
a single syringe. In the MBA treatment, the IM drug solution was prepared as a mixture of 0.1
ml of medetomidine (1 mg/ml), 2 ml of butorphanol (5
mg/ml) and 10 ml of alfaxalone (10 mg/ml). Thus, the final
dose of each drug given to the dogs was medetomidine 2.48 µg/kg, butorphanol 0.248 mg/kg and
alfaxalone 2.48 mg/kg in the MBA treatment. In each treatment, the IM drug solution was injected into the dorsal
lumbar muscle of the dog by using a syringe with a 23-gauge, 1-inch needle (TOP injection needle, TOP Co., Ltd.,
Tokyo, Japan). The dogs breathed room air and were endotracheally intubated with an endotracheal tube
[Endotracheal tube with cuff (I.D. 7.5 to 8.5 mm), Fuji Systems Corp., Tokyo, Japan] when possible. The
endotracheal tube was removed when the dogs regained their laryngeal reflex. Anesthetic and cardiorespiratory
effects were evaluated in the dogs before each IM treatment (baseline) and at 2, 5, 10, 15, 20, 30, 45, 60, 90
and 120 min after the administration of each IM drug solution.
Evaluation of anesthetic effect: Anesthetic effect was evaluated by the degree of
neuro-depression, the quality of anesthetic induction including the ease of endotracheal intubation and the
quality of recovery from anesthesia. The neuro-depression produced with each IM treatment was subjectively
evaluated by using an existing composite measurement scoring system in dogs [30 (link)]. The scoring system consisted of 6 categories: spontaneous posture, placement on side, response to
noise, jaw relaxation, general attitude and nociceptive response to toe-pinch. These categories were rated with
a score of 0 to 2 for jaw relaxation, 0 to 3 for placement of side, general attitude and toe-pinch response and
0 to 4 for spontaneous posture and response to noise based on the responsiveness expressed by the dogs [30 (link)]. Total neuro-depressive score was calculated as the sum of the scores for
the 6 categories (a maximum of 19). The qualities of anesthetic induction and recovery were assessed using
numerical scoring systems previously used in dogs [28 (link)]. A well-trained
observer (N. H.) was responsible for evaluation of the anesthetic effect of the treatments using these scoring
systems.
In addition, we recorded the periods of time before the dogs lay down in lateral recumbency (Time until onset
of lateral recumbency), were intubated (Time until intubation), appeared the first spontaneous movement (Time
until spontaneous movement), and appeared their head lift (Time until head lift) and unaided standing (Time
until unaided standing), after the start of each IM treatment. The durations of acceptance of endotracheal
intubation and maintenance of lateral recumbency were also recorded as the periods of time from the intubation
to extubation (Duration of intubation) and from the onset of lateral recumbency to head lift (Duration of
lateral recumbency).
Measurements of cardiorespiratory valuables: Lead II electrocardiography (ECG), heart rate
(HR; beats/min), respiratory rate (RR; breathes/min), rectal temperature (RT;°C), systolic arterial blood
pressure (SABP; mmHg), diastolic arterial blood pressure (DABP; mmHg), mean arterial blood pressure (MABP; mmHg)
and central venous blood pressure (CVP; mmHg) were recorded before and after the IM treatment. RR was counted by
observing thoracic movements. ECG, HR, RT, SABP, DABP, MABP and CVP were recorded by a patient monitoring system
(DS-7210, Fukuda Denshi Co., Ltd., Tokyo, Japan). SABP, DABP and MABP were directly measured by connecting the
catheter placed in the left pedal artery to a pressure transducer (BD DTXTM Plus DT-4812, Japan
Becton, Dickinson and Co., Fukushima, Japan). In addition, CVP was also measured by connecting the catheter
placed in the cranial vena cava to a pressure transducer. These pressure transducers were placed at the level of
the right atrium.
Arterial blood samples (0.5 ml each) were anaerobically withdrawn from the arterial catheters
into a heparinized syringe before and after the IM treatment. The blood samples were analyzed immediately after
collection to measure arterial pH (pHa), partial pressure of arterial oxygen (PaO2; mmHg) and carbon
dioxide (PaCO2; mmHg), and arterial plasma lactate concentration (Lac; mmol/l) using
a blood gas analyzer (GEM Premier 3000, Instrumentation Laboratory, Tokyo, Japan). In addition, arterial
bicarbonate concentration (HCO3; mmol/l), base excess (B.E.;
mmol/l) and arterial oxygen saturation (SaO2;%) were analyzed. The pHa,
PaO2 and PaCO2 were corrected for the rectal temperature determined immediately after
the blood collection. In the same way, central venous blood samples (0.5 ml each) were
anaerobically withdrawn from the central venous catheters and immediately analyzed to obtain central venous
oxygen saturation (ScvO2;%) using the blood gas analyzer.
Statistical analysis: The total neuro-depressive score was reported as the median ± quartile
deviation and was analyzed by the Friedman test to assess changes from baseline values with time for each
treatment. Difference in the total neuro-depressive score and the qualities of anesthetic induction and recovery
amongst the treatments were compared by the Friedman test with the Scheff test for post hoc comparisons.
Anesthetic effect times and cardiorespiratory variables were reported as the mean ± standard deviation. The
times were compared by paired t test and one-way (treatment) factorial ANOVA with the
Bonferroni test for post hoc comparisons among treatments. The cardiorespiratory variables were analyzed using
two-way (treatment and time) repeated measure ANOVA followed by the Bonferroni test. Observations and/or
perceived adverse effects related to drug administration were compared between treatments by using the chi
square test. The level of significant was set at P<0.05.
years of age [2.9 ± 1.5 (mean ± standard deviation) years old] and that weighed 8.6 to 16.8 kg (11.5 ± 3.2 kg)
were used for three drug treatments with at least 10 days between each treatment. All dogs used in the present
study exhibited the ideal body condition (body condition score 3/5) [2 (link)].
Each dog was received in the order of the IM treatment of a combination of alfaxalone-HPCD, medetomidine and
butorphanol (MBA), a combination of medetomidine and butorphanol (MB), and alfaxalone-HPCD alone (ALFX). All
dogs were judged to be in good physical condition based upon a physical examination. Food was withheld for 12 hr
before the each experiment, and water was continuously available. The dogs were cared for according to the
principles of the “Guide for the Care and Use of Laboratory Animals” prepared by Rakuno Gakuen University. The
Animal Care and Use Committee of Rakuno Gakuen University approved this study (approved No. VH22B17).
Instrumentation: All dogs were instrumented with arterial and central venous catheters under
general anesthesia prior to the administration of each IM drug solution. Anesthesia was induced by mask
induction using a vaporizer dial setting at 5% of sevoflurane (Sevoflo, DS Pharma Animal Health Co., Ltd.,
Osaka, Japan) with oxygen. Once anesthetized, the dogs were orotracheally intubated, and anesthesia was
maintained with vaporizer dial setting at 3.0–3.5% of sevoflurane with oxygen. The dogs were placed in left
lateral recumbency for catheter instrumentation. In each dog, 22-gauge catheters (Supercath, Medikit Co., Ltd.,
Tokyo, Japan) were placed into the left cephalic vein and the left dorsal pedal artery. In addition, an 18-gauge
catheter 30 cm in length (Intravascular Catheter Kit, Medikit Co., Ltd.) was placed into the cranial vena cava
through the right jugular vein after the cervical catheter site was aseptically prepared and infiltrated with 1
mg/kg of 2% lidocaine (2% Xylocaine Astrazeneca, Osaka, Japan). The position of the tip and insertion length of
the central venous catheter were approximated and confirmed by pressure waveform. The dogs were infused with
lactated Ringer’s solution (Solulact, Terumo Co., Tokyo, Japan) at a rate of 10 ml/kg/hr
through the catheter placed into the cephalic vein during the sevoflurane anesthesia. After the completion of
catheter placements, the sevoflurane was discontinued, and each dog was extubated when their laryngeal reflex
was functional. Each dog was then allowed to recover for 1 hr in a quiet room until the administration of its
allocated IM treatment.
Drug administration and data collection: Following the 1 hr recovery period, the dogs received
IM alfaxalone-HPCD (Alfaxan, Jurox Pty. Ltd., Rutherford, NSW, Australia) at 2.5 mg/kg (ALFX), medetomidine
(Domitor, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) at 2.5 µg/kg and butorphanol
(Vetorphal, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) at 0.25 mg/kg (MB), or alfaxalone-HPCD at 2.5 mg/kg,
medetomidine at 2.5 µg/kg and butorphanol at 0.25 mg/kg (MBA) in separate experiments. Maddern
et al. [16 (link)] showed that the anesthetic induction dose
of IV alfaxalone-HPCD can be decreased to around one third by premedication with low dose of medetomidine and
butorphanol in dogs. Therefore, the dose of IM alfaxalone-HPCD administration was set at 2.5 mg/kg (one third of
anesthetic IM alfaxalone-HPCD dose) in the present study. All IM drug solutions were administered to each dog at
total 0.3 ml/kg in volume under manual physical restraint. In the ALFX and MB treatments, the
IM drug solution was prepared by mixing appropriate amounts of each product and diluting with normal saline
(Otsuka normal saline, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) up to 0.3 ml/kg volume in
a single syringe. In the MBA treatment, the IM drug solution was prepared as a mixture of 0.1
ml of medetomidine (1 mg/ml), 2 ml of butorphanol (5
mg/ml) and 10 ml of alfaxalone (10 mg/ml). Thus, the final
dose of each drug given to the dogs was medetomidine 2.48 µg/kg, butorphanol 0.248 mg/kg and
alfaxalone 2.48 mg/kg in the MBA treatment. In each treatment, the IM drug solution was injected into the dorsal
lumbar muscle of the dog by using a syringe with a 23-gauge, 1-inch needle (TOP injection needle, TOP Co., Ltd.,
Tokyo, Japan). The dogs breathed room air and were endotracheally intubated with an endotracheal tube
[Endotracheal tube with cuff (I.D. 7.5 to 8.5 mm), Fuji Systems Corp., Tokyo, Japan] when possible. The
endotracheal tube was removed when the dogs regained their laryngeal reflex. Anesthetic and cardiorespiratory
effects were evaluated in the dogs before each IM treatment (baseline) and at 2, 5, 10, 15, 20, 30, 45, 60, 90
and 120 min after the administration of each IM drug solution.
Evaluation of anesthetic effect: Anesthetic effect was evaluated by the degree of
neuro-depression, the quality of anesthetic induction including the ease of endotracheal intubation and the
quality of recovery from anesthesia. The neuro-depression produced with each IM treatment was subjectively
evaluated by using an existing composite measurement scoring system in dogs [30 (link)]. The scoring system consisted of 6 categories: spontaneous posture, placement on side, response to
noise, jaw relaxation, general attitude and nociceptive response to toe-pinch. These categories were rated with
a score of 0 to 2 for jaw relaxation, 0 to 3 for placement of side, general attitude and toe-pinch response and
0 to 4 for spontaneous posture and response to noise based on the responsiveness expressed by the dogs [30 (link)]. Total neuro-depressive score was calculated as the sum of the scores for
the 6 categories (a maximum of 19). The qualities of anesthetic induction and recovery were assessed using
numerical scoring systems previously used in dogs [28 (link)]. A well-trained
observer (N. H.) was responsible for evaluation of the anesthetic effect of the treatments using these scoring
systems.
In addition, we recorded the periods of time before the dogs lay down in lateral recumbency (Time until onset
of lateral recumbency), were intubated (Time until intubation), appeared the first spontaneous movement (Time
until spontaneous movement), and appeared their head lift (Time until head lift) and unaided standing (Time
until unaided standing), after the start of each IM treatment. The durations of acceptance of endotracheal
intubation and maintenance of lateral recumbency were also recorded as the periods of time from the intubation
to extubation (Duration of intubation) and from the onset of lateral recumbency to head lift (Duration of
lateral recumbency).
Measurements of cardiorespiratory valuables: Lead II electrocardiography (ECG), heart rate
(HR; beats/min), respiratory rate (RR; breathes/min), rectal temperature (RT;°C), systolic arterial blood
pressure (SABP; mmHg), diastolic arterial blood pressure (DABP; mmHg), mean arterial blood pressure (MABP; mmHg)
and central venous blood pressure (CVP; mmHg) were recorded before and after the IM treatment. RR was counted by
observing thoracic movements. ECG, HR, RT, SABP, DABP, MABP and CVP were recorded by a patient monitoring system
(DS-7210, Fukuda Denshi Co., Ltd., Tokyo, Japan). SABP, DABP and MABP were directly measured by connecting the
catheter placed in the left pedal artery to a pressure transducer (BD DTXTM Plus DT-4812, Japan
Becton, Dickinson and Co., Fukushima, Japan). In addition, CVP was also measured by connecting the catheter
placed in the cranial vena cava to a pressure transducer. These pressure transducers were placed at the level of
the right atrium.
Arterial blood samples (0.5 ml each) were anaerobically withdrawn from the arterial catheters
into a heparinized syringe before and after the IM treatment. The blood samples were analyzed immediately after
collection to measure arterial pH (pHa), partial pressure of arterial oxygen (PaO2; mmHg) and carbon
dioxide (PaCO2; mmHg), and arterial plasma lactate concentration (Lac; mmol/l) using
a blood gas analyzer (GEM Premier 3000, Instrumentation Laboratory, Tokyo, Japan). In addition, arterial
bicarbonate concentration (HCO3; mmol/l), base excess (B.E.;
mmol/l) and arterial oxygen saturation (SaO2;%) were analyzed. The pHa,
PaO2 and PaCO2 were corrected for the rectal temperature determined immediately after
the blood collection. In the same way, central venous blood samples (0.5 ml each) were
anaerobically withdrawn from the central venous catheters and immediately analyzed to obtain central venous
oxygen saturation (ScvO2;%) using the blood gas analyzer.
Statistical analysis: The total neuro-depressive score was reported as the median ± quartile
deviation and was analyzed by the Friedman test to assess changes from baseline values with time for each
treatment. Difference in the total neuro-depressive score and the qualities of anesthetic induction and recovery
amongst the treatments were compared by the Friedman test with the Scheff test for post hoc comparisons.
Anesthetic effect times and cardiorespiratory variables were reported as the mean ± standard deviation. The
times were compared by paired t test and one-way (treatment) factorial ANOVA with the
Bonferroni test for post hoc comparisons among treatments. The cardiorespiratory variables were analyzed using
two-way (treatment and time) repeated measure ANOVA followed by the Bonferroni test. Observations and/or
perceived adverse effects related to drug administration were compared between treatments by using the chi
square test. The level of significant was set at P<0.05.
