Evaluation of storage conditions and periods for a three-drug anesthetic mixture in mice

Abstract

The three-drug anesthetic mixture (medetomidine, midazolam and butorphanol), developed as an injectable anesthetic for laboratory animals, has been verified from various perspectives and applied to mice and other laboratory animals. However, the effects of its storage conditions and periods on its efficacy have not yet been studied. This study investigated the mixture’s efficacy after storage under various conditions (room temperature, 4°C and −20°C) for 1 and 2 years. Mice in all groups were induced into a stable anesthetic state for at least 15 min after administration. The mice recovered from the anesthetized state 35 min after administration of the antagonist. These findings demonstrate the mixture’s stability under different storage conditions and durations, potentially improving laboratory mouse welfare.

Since the use of ketamine is strictly regulated in Japan, a three-drug anesthetic mixture containing medetomidine, midazolam, and butorphanol (MMB) was developed for use in mice [4]. The use of MMB in mice has been refined through verification of administration routes, strain differences, and temperature control [5, 6, 12]. Furthermore, its application is not limited to mice, and has been expanded to number of other laboratory animals, including cotton rats, rabbits, and chickens [3, 7, 9]. Previous studies show prospects of safe and sophisticated anesthesia-associated mouse experiments.

However, few studies have examined the effect of storage conditions and periods on MMB anesthetic. Previous reports investigating storage conditions for the drugs comprising MMB anesthetic in different formulations confirmed the stability of butorphanol for 15 days at room temperature and midazolam for 1 year at 4°C after filling and sealing [1, 11] Although the partial stability of these drugs could be inferred from previous reports, the optimal storage conditions and periods and use of MMB as a mixture are unknown. In this study, we investigated the effects of storage conditions (room temperature, refrigeration, freezing) and storage periods (1 year and 2 years) after preparation of MMB on its anesthetic effect.

MMB anesthetics and the antagonist included medetomidine hydrochloride (final concentration: 0.075 mg/mL; Domitor; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), midazolam (final concentration: 0.4 mg/mL; Dormicum; Maruishi Pharmaceutical Co., Ltd., Osaka, Japan), and butorphanol tartrate (final concentration: 0.5 mg/mL; Vetorphale; Meiji Animal Health Co., Ltd., Kumamoto, Japan), or atipamezole (final concentration: 0.075 mg/mL; Atipame Injection; Kyoritsu Seiyaku Co., Ltd., Tokyo, Japan) mixed with saline (Otsuka Normal Saline, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). The prepared MMB anesthetics were used immediately or stored sealed with Parafilm, protected from light, at room temperature (approximately 0–35°C), in a refrigerator (4°C), or a freezer (−20°C) in polypropylene tubes (Nippon Genetics, Tokyo, Japan). The storage periods were 1 and 2 years. All animal experiments in the present study were conducted under the guidance of the Animal Care and Use Committee of Yamaguchi University (Approval Number: 528). Five to six female ICR mice were purchased (Japan SLC Inc., Hamamatsu, Japan) and used for each group (8–15 weeks old). Mice were kept in Advanced Research Center for Laboratory Animal Science (Yamaguchi University, room temperature: 20–29°C, 12 hr light/dark cycle, 10 ventilation cycles/hr). The experimental procedure was performed as described by previous report [5]. Briefly, after the intraperitoneal administration of MMB anesthetics (dose: 100 µL/10 g Body Weight), scoring was performed based on the presence of 5 different reflexes (body-righting reflex, front paw reflex, hind paw reflex, tail reflex, and corneal reflex) every 5 min. After the administration of MMB anesthetics, the anesthetized state was observed for at least approximately 1 hr on a heating mat (35°C; KN-475-3-35, Natsume Seisakusho Co., Ltd., Tokyo, Japan) before the administration of the antagonist (dose: 100 µL/10 g Body Weight) and observation was continued until arousal (total score=0). After scoring, euthanasia was performed by cervical dislocation under isoflurane anesthesia. Statistical analysis was performed using Student’s t-test, with P<0.0083 (=0.05/6, Bonferroni-adjusted for multiple comparisons) considered statistically significant.

First, scoring with the MMB anesthetic immediately after preparation for control data acquisition showed that the anesthetic could be used for more than 3 hr (Fig. 1a). A transient decrease in the score was observed at 10 min after administration; however, stable anesthesia with a score of 4 or higher was obtained from 15 min. On average, scores of 1 or less were obtained 20 min after antagonist administration, and all mice recovered from anesthesia 35 min after antagonist administration. Similar to fresh preparation, the MMB anesthetic stored under each condition for 1 year also produced anesthetic effects for more than 3 hr (Fig. 1b). For anesthetic induction, a stable anesthetic state was obtained with a score of 4 or higher at an average of 15, 5, and 10 min after administration of MMB anesthetics stored at room temperature, 4°C and −20°C, respectively. These mice recovered 10–15 min after the antagonist administration. MMB anesthetics stored under each condition for 2 years exhibited an anesthetic state with a score of 4 or higher, similar to those observed in other storage periods (Fig. 1c). For induction of anesthesia, all groups under all storage conditions had a score of 4 or higher 10 min after administration (Table 1). All groups recovered 15–25 min after the antagonist administration.

Fig. 1.

Time course of anesthesia scores. (a) Time course of anesthesia scores in mice using freshly prepared medetomidine, midazolam and butorphanol (MMB) anesthetics. (b) Time course of anesthesia scores in mice using MMB anesthetics stored for 1 year under each storage condition. Storage conditions are shown at the top of the graph (from left to right: room temperature, 4°C, and −20°C). (c) Time course of anesthesia scores in mice using MMB anesthetics stored for 2 years under each storage condition. Storage conditions are shown at the top of the graph (from left to right: room temperature, 4°C, and −20°C). All values are shown as mean ± SE. The timing of the antagonist administration is indicated by dashed lines.

Table 1. Average time to induction of anesthesia.

Experimental Group	Induction Time (Score ≥4, min)	P value
Control	8.0 ± 1.8
1 year, Room temperature	9.0 ± 3.7	0.83
1 year, 4°C	8.0 ± 2.7	1
1 year, −20°C	8.0 ± 1.8	1
2 year, Room temperature	8.0 ± 1.2	1
2 year, 4°C	10.0 ± 2.7	0.57
2 yaer, −20°C	10.0 ± 3.9	0.66

All values are shown as mean ± SE.

In the present study, we examined the effects of storage conditions and periods on the anesthetic efficacy of MMB. The results showed that an anesthetic state sufficient for performing surgical procedures could be induced regardless of its storage conditions and period. Specifically, the prepared MMB anesthetic can be stored at room temperature, refrigerated, or frozen for at least 2 years. Since midazolam is stable at temperatures between −1 and 38°C for 1 year before opening [10], the chemical structure of midazolam may be stable with respect to storage temperature. However, storage in a closed container is required because stability is lost in unsealed syringes due to crystallization or other factors [8]. Our results may be applicable to alfaxalone and remimazolam, which are improved anesthetics for MMB [2, 13].

In summary, this study examined the effect of MMB anesthesia kept under various storage conditions and periods using mice and demonstrated that MMB anesthetics with the present formulation can be stored for 2 years at room temperature or can be refrigerated or frozen. Our results revise the report of Kirihara et al. who suggested that MMB anesthetics can be stored at 4°C for 8 weeks [5]. While our findings demonstrated that the prepared anesthetic mixture can be stored for up to 2 years under various condition, this does not imply repeated use over the entire 2-year period. For long-term storage, it is crucial to take precautions such as protecting from light, ensuring airtight sealing and avoiding contamination. By paying attention to those precautions, we could reduce the frequency of preparing the anesthetic mixture. Reducing the number of anesthetic preparations will contribute towards animal welfare by allowing us to focus on animal care.

CONFLICT OF INTEREST

The authors have no conflicts of interest to disclosure.