The anesthetic, sedative, and analgesic agents used in the present study were as follows:
ketamine hydrochloride (Ketalar, Sankyo Lifetech Co., Ltd., Tokyo, Japan), xylazine
(Celactar, Bayer Yakuhin Ltd., Tokyo, Japan), pentobarbital sodium (Somnopentyl, Kyoritsu
Seiyaku Co., Ltd.), medetomidine hydrochloride (Domitol, Meiji Seika Pharma Co., Ltd.,
Tokyo, Japan), midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan), butorphanol
(Vetorphale, Meiji Seika Pharma Co., Ltd.), and isoflurane (Isoflu, DS Pharma Animal
Health Co., Ltd., Osaka, Japan). All agents were kept at room temperature before use.
Animals were divided into four groups corresponding to each anesthetic protocol as
follows: ketamine hydrochloride and xylazine combined (K/X; ketamine hydrochloride 100
mg/kg and xylazine 10 mg/kg); pentobarbital monoanesthesia (50 mg/kg); medetomidine,
midazolam, and butorphanol combined (M/M/B; medetomidine 0.3 mg/kg, midazolam 4 mg/kg, and
butorphanol 5 mg/kg); and inhalant anesthesia using isoflurane (5% for induction and 2%
for maintenance). In the M/M/B group, mice were administered atipamezole (Antisedan,
Zoetis Japan Inc., Tokyo, Japan) at a dose of 0.3 mg/kg 30 min after the administration of
M/M/B. All injectable anesthetics were administered intraperitoneally. The dose and
concentration of each agent were as reported previously in mice [4 (link), 5 (link), 17 (link)]. Before administration, the concentration of M/M/B, K/X, and
pentobarbital sodium was adjusted to 6 ml/kg by diluting with saline. In the M/M/B
anesthetic group, a mixture of medetomidine, midazolam, and butorphanol with saline was
prepared and then concurrently administered. Similarly, the mixture of ketamine
hydrochloride and xylazine was adjusted with saline before concurrent administration.
Isoflurane anesthesia was administered using a commercially available rodent inhalant
anesthesia apparatus (SomnoSuite Small Animal Anesthesia System, Kent Scientific
Corporation), which has a digital vaporizer and internal air-flow pump. The vaporized
anesthetic gas was introduced into the induction chamber and nose mask (Kent Scientific
Corporation) at a flow rate of 32 ml/min. The nose mask was covered with a latex membrane
that had a hole in the center to fit closely around the nose. Initially, mice were induced
with isoflurane at a 5% concentration. Once loss of the postural reaction and righting
reflex was confirmed, the mice were rapidly transferred to the nose mask, and anesthesia
was maintained with 2% isoflurane (Fig. 1 ![]()
ketamine hydrochloride (Ketalar, Sankyo Lifetech Co., Ltd., Tokyo, Japan), xylazine
(Celactar, Bayer Yakuhin Ltd., Tokyo, Japan), pentobarbital sodium (Somnopentyl, Kyoritsu
Seiyaku Co., Ltd.), medetomidine hydrochloride (Domitol, Meiji Seika Pharma Co., Ltd.,
Tokyo, Japan), midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan), butorphanol
(Vetorphale, Meiji Seika Pharma Co., Ltd.), and isoflurane (Isoflu, DS Pharma Animal
Health Co., Ltd., Osaka, Japan). All agents were kept at room temperature before use.
Animals were divided into four groups corresponding to each anesthetic protocol as
follows: ketamine hydrochloride and xylazine combined (K/X; ketamine hydrochloride 100
mg/kg and xylazine 10 mg/kg); pentobarbital monoanesthesia (50 mg/kg); medetomidine,
midazolam, and butorphanol combined (M/M/B; medetomidine 0.3 mg/kg, midazolam 4 mg/kg, and
butorphanol 5 mg/kg); and inhalant anesthesia using isoflurane (5% for induction and 2%
for maintenance). In the M/M/B group, mice were administered atipamezole (Antisedan,
Zoetis Japan Inc., Tokyo, Japan) at a dose of 0.3 mg/kg 30 min after the administration of
M/M/B. All injectable anesthetics were administered intraperitoneally. The dose and
concentration of each agent were as reported previously in mice [4 (link), 5 (link), 17 (link)]. Before administration, the concentration of M/M/B, K/X, and
pentobarbital sodium was adjusted to 6 ml/kg by diluting with saline. In the M/M/B
anesthetic group, a mixture of medetomidine, midazolam, and butorphanol with saline was
prepared and then concurrently administered. Similarly, the mixture of ketamine
hydrochloride and xylazine was adjusted with saline before concurrent administration.
Isoflurane anesthesia was administered using a commercially available rodent inhalant
anesthesia apparatus (SomnoSuite Small Animal Anesthesia System, Kent Scientific
Corporation), which has a digital vaporizer and internal air-flow pump. The vaporized
anesthetic gas was introduced into the induction chamber and nose mask (Kent Scientific
Corporation) at a flow rate of 32 ml/min. The nose mask was covered with a latex membrane
that had a hole in the center to fit closely around the nose. Initially, mice were induced
with isoflurane at a 5% concentration. Once loss of the postural reaction and righting
reflex was confirmed, the mice were rapidly transferred to the nose mask, and anesthesia
was maintained with 2% isoflurane (
Vital signs monitoring during isoflurane anesthesia in mice. The rectal probe and
pulse oximeter are located at the colorectum and tail base, respectively.
