Nicardipine co-administered with medetomidine-midazolam-butorphanol anesthesia attenuates post-anesthetic hypothermia in rats

Abstract

Medetomidine-midazolam-butorphanol (MMB) anesthesia is widely used in laboratory rodents but is frequently accompanied by peri- and post-anesthetic hypothermia. This study examined whether co-administration of nicardipine could attenuate hypothermia associated with MMB anesthesia in rats, thereby testing the concept that modulation of peripheral vascular tone can complement thermal management. Rats received MMB alone (0Ni) or MMB combined with nicardipine at 0.25 or 0.5 mg/kg. Body temperature was assessed under warmed and non-warmed conditions, and systolic blood pressure (SBP) and heart rate (HR) were monitored to evaluate cardiovascular effects. Under warmed conditions, body temperature profiles did not differ among groups. Under non-warmed conditions, however, nicardipine-treated rats maintained higher body temperatures during recovery, and both nicardipine groups showed significantly higher recovery-phase AUC values than the 0Ni group. Nicardipine did not significantly alter induction or recovery timing. In parallel, nicardipine produced a dose-dependent reduction in SBP and an increase in HR, particularly at 0.5 mg/kg. These findings suggest that nicardipine partially attenuates post-anesthetic hypothermia during recovery from MMB anesthesia under non-warmed conditions and may serve as a complementary approach to thermal management. However, the marked SBP reduction observed at 0.5 mg/kg warrants caution in practical application.

Introduction

Medetomidine–midazolam–butorphanol (MMB) anesthesia is widely used in laboratory rodents because of its ease of administration, reliable sedative and analgesic effects, and reversibility with atipamezole [1,2,3,4]. Consequently, MMB injectable anesthesia has become a standard alternative to inhalation anesthesia in many experimental settings. Despite these benefits, MMB is accompanied by several adverse effects, including metabolic disturbances (e.g., hyperglycemia) and peri-anesthetic hypothermia. Among these, peri-anesthetic hypothermia represents a major practical limitation of MMB anesthesia.

Hypothermia is one of the most common and physiologically significant adverse effects of anesthesia in small laboratory animals [5,6,7]. Rodents are particularly susceptible because of their high surface area-to-volume ratio, reduced metabolic heat production, and impaired thermoregulatory control. Even mild peri-anesthetic hypothermia can affect physiological stability, recovery, and experimental outcomes; therefore, its prevention is important from both welfare and refinement perspectives.

Injectable anesthetic regimens, including MMB, are reported to induce more pronounced and prolonged hypothermia than inhalational anesthesia [5]. During medetomidine-based anesthesia, α₂-adrenergic agonism suppresses central thermoregulatory control through hypothalamic pathways, lowers the thermoregulatory set point, and reduces metabolic heat production. In addition, medetomidine induces peripheral vasoconstriction [8], which may reduce perfusion and alter heat distribution. Together, these central and peripheral effects likely contribute to thermoregulatory instability during MMB anesthesia.

External warming is widely used to prevent peri-anesthetic hypothermia in laboratory rodents and represents a fundamental component of thermal management during anesthesia [6]. However, anesthesia-associated hypothermia involves not only heat loss from the body surface but also physiological alterations such as suppression of thermoregulatory control and changes in peripheral circulation. Therefore, in addition to conventional thermal support, pharmacological modulation of peripheral vascular tone may represent a complementary physiological strategy for stabilizing body temperature under anesthesia.

Nicardipine hydrochloride is a dihydropyridine L-type calcium channel blocker that induces peripheral vasodilation by inhibiting calcium influx into vascular smooth muscle cells, thereby reducing systemic vascular resistance and improving peripheral circulation [9]. Because nicardipine counteracts vasoconstriction, it may influence heat distribution and contribute to improved maintenance of body temperature during the peri-anesthetic period. In the context of MMB anesthesia, nicardipine-induced vasodilation may offset the peripheral vasoconstrictive effects of medetomidine and thereby attenuate hypothermia. However, the effects of pharmacological vasodilation on MMB-associated hypothermia have not been systematically evaluated in rats. We hypothesized that co-administration of nicardipine with MMB anesthesia would improve thermoregulatory stability by modifying peripheral circulation. If confirmed, such an effect could provide a complementary physiological approach to thermal management during injectable anesthesia, rather than replacing active warming.

Therefore, the present study evaluated the effects of nicardipine on body temperature, systolic blood pressure, and heart rate during and after MMB anesthesia in rats. In the following sections, the MMB regimen co-administered with nicardipine is referred to as MMB-Ni. By examining both thermoregulatory and cardiovascular responses over time, we aimed to clarify whether modulation of peripheral vascular tone contributes to attenuation of peri- and post-anesthetic hypothermia and to assess the potential adjunctive utility of nicardipine as a refinement approach in experimental animal anesthesia. Because external warming is routinely used to prevent peri-anesthetic hypothermia in laboratory rodents, the effects of nicardipine were evaluated under both warmed and non-warmed conditions. The warmed condition represents standard peri-anesthetic thermal support in which active heating was applied throughout the anesthetic period, whereas the non-warmed condition represents a hypothermia-prone setting in which any additional thermoregulatory contribution of nicardipine can be more readily detected. This design enabled us to assess whether nicardipine provides thermoregulatory effects beyond those achieved by warming alone, while recognizing that active warming remains the standard supportive measure.

Materials and Methods

Animals

Male Sprague-Dawley rats (Rattus norvegicus) (CLEA Japan, Tokyo, Japan) aged 8–15 weeks and weighing 253–430 g were used. Animals were housed under standard laboratory conditions at 25°C under a 12:12-h light/dark cycle, with free access to food and water. All procedures were conducted in accordance with institutional guidelines for animal experimentation and were approved by the Institutional Animal Care and Use Committee of Kitasato University (Approval No. 25-101).

Anesthesia and drug administration

Rats were anesthetized with a mixture of medetomidine (0.15 mg/kg; Dorbene Vet, Kyoritsu Seiyaku Co., Tokyo, Japan), midazolam (2 mg/kg; Midazolam Sandoz, Sandoz Japan Co., Ltd., Tokyo, Japan), and butorphanol (2.5 mg/kg; Vetorphale, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) administered intraperitoneally at a total volume of 5 ml/kg as previously described [1]. Nicardipine hydrochloride (Sawai Pharmaceutical Co., Ltd., Osaka, Japan) was mixed in the same syringe and co-administered at doses of 0 mg/kg (0Ni), 0.25 mg/kg (0.25Ni), or 0.5 mg/kg (0.5Ni). The nicardipine doses were selected on the basis of previous rat studies [10] and were intended to provide an exploratory range for evaluating physiological effects while avoiding excessive circulatory depression. The intraperitoneal route was adopted to allow single-injection co-administration with the MMB mixture. Atipamezole (0.3 mg/kg; Antisedan, Zoetis Japan, Tokyo, Japan) was administered intraperitoneally 60 min after MMB administration to antagonize medetomidine. Recovery time was defined as the latency from atipamezole administration to the return of the righting reflex (ATI-RORR). RORR was defined as the animal’s ability to spontaneously regain and maintain a prone posture after being gently placed in a supine position. The occurrence of RORR was confirmed independently by two veterinarians. After completion of the measurements, animals were monitored until full recovery and returned to their home cages.

Experimental groups

Animals were allocated to three nicardipine dose groups: MMB alone (0Ni), MMB plus nicardipine at 0.25 mg/kg (0.25Ni), and MMB plus nicardipine at 0.5 mg/kg (0.5Ni). Each rat remained in the same nicardipine group throughout the study. Warmed and non-warmed rectal-temperature sessions were performed within the same rat in a counterbalanced design, whereas tail-cuff cardiovascular measurements were obtained in a separate session under standardized mild warming.

Sample sizes were determined with reference to previous studies using similar MMB anesthesia protocols in rats [1, 5]. For body temperature analysis, 6–8 animals per group were included under warmed conditions and 7–8 animals per group under non-warmed conditions, depending on the analysis phase. For tail-cuff SBP and HR measurements, 5–8 animals per group were included. Warmed and non-warmed temperature sessions were counterbalanced across animals, and a washout period of at least 4 days was used. Because this interval exceeded at least five reported half-lives of the administered drugs [11, 12], carryover effects were considered minimal. Induction and recovery parameters were analyzed from the non-warmed temperature session.

Body temperature measurement

Body temperature was measured as rectal temperature during anesthesia and throughout the recovery period using a MouseOx Plus monitoring system equipped with a rectal temperature probe (Mouse Rectal Probe, part no. 015002; Starr Life Sciences, Oakmont, PA, USA).

Measurements were performed under two thermal conditions:

(1) with external warming using a heating pad maintained at approximately 37°C

(2) without external warming.

The warmed condition was used to simulate standard peri-anesthetic thermal support [13], in which animals were placed on a heating pad (M110, Shiraimatsu Co., Ltd., Osaka, Japan) set to approximately 37°C from anesthetic administration until 60 min after injection. Immediately after atipamezole administration, animals were removed from the heating pad. In contrast, the non-warmed condition represented limited thermal support and allowed assessment of hypothermia-prone responses. Comparison of these two conditions allowed evaluation of nicardipine under routine thermal management and under limited thermal support.

Statistical analyses of body temperature data are described in the Statistical analysis section.

Cardiovascular measurements

Systolic blood pressure and heart rate were measured using a tail-cuff system (BP-98A-L, Natsume Seisakusho, Tokyo, Japan) at 10-min intervals for 60 min after anesthetic administration. Measurements were performed under standardized mild warming (37°C) using the system’s warming chamber/tube to promote tail perfusion and stable signal acquisition. Tail-cuff measurements were conducted in a separate session from the rectal-temperature experiments to avoid confounding from the warming required for cardiovascular recording. In the present study, cardiovascular analysis focused on SBP and HR, whereas induction and recovery parameters were analyzed from the non-warmed temperature session.

Statistical analyses of SBP and HR are described in the Statistical analysis section.

Statistical analysis

Data are presented as mean ± SEM, except for induction and recovery parameters (Fig. 1), which are shown as mean ± SD to illustrate inter-individual variability. Because repeated measurements were obtained from the same animals over time, time-course data for systolic blood pressure, heart rate, and body temperature were analyzed using LMMs. In these models, group, time, and their interaction (group × time) were included as fixed effects, and animal identity was included as a random intercept to account for within-subject correlation.

Fig. 1.

Effects of nicardipine on induction and recovery parameters during MMB anesthesia. (A) Latency to immobilization. (B) Latency to loss of righting reflex (LORR). (C) Time from atipamezole administration to recovery of the righting reflex (ATI-RORR latency). Rats received MMB anesthesia alone (0Ni), MMB with 0.25 mg/kg nicardipine (0.25Ni), or MMB with 0.5 mg/kg nicardipine (0.5Ni). n=7 rats per group. Bars represent mean ± SD and dots indicate individual animals. Group differences were analyzed using Welch’s ANOVA followed by Holm-corrected pairwise comparisons. Significant differences would be indicated by symbols (*: 0Ni vs 0.25Ni; †: 0Ni vs 0.5Ni; ‡: 0.25Ni vs 0.5Ni; Holm-adjusted P<0.05); however, no significant differences were detected and therefore no symbols are shown.

When a significant group × time interaction was detected, post hoc comparisons between groups at individual time points were performed with multiplicity correction using the Holm method.

In addition, area under the curve (AUC) values were calculated for each animal using the trapezoidal rule to provide a summary measure of overall changes across the observation period. AUC values were compared between groups using pairwise Welch’s t-tests with Holm correction. Induction and recovery parameters were analyzed using Welch’s ANOVA followed by pairwise Welch’s t-tests with Holm correction. Statistical analyses were performed using Python (SciPy, pandas, and statsmodels libraries). Holm-adjusted P<0.05 was considered statistically significant.

Results

Effects of nicardipine on induction and recovery times

To determine whether nicardipine affects anesthetic induction and recovery profiles, we first compared the latency to immobilization, loss of righting reflex (LORR), and recovery time following atipamezole administration among three groups: 0Ni (MMB alone) and MMB-Ni at 0.25 and 0.5 mg/kg (Figs. 1A–C). The mean induction times were comparable across groups. The latency to immobilization was 217.7 ± 69.6 s in 0Ni, 180.3 ± 74.1 s in 0.25Ni, and 148.6 ± 32.4 s in 0.5Ni. Similarly, the latency to LORR was 243.7 ± 72.5 s, 214.1 ± 90.5 s, and 162.6 ± 33.0 s, respectively. Recovery time after atipamezole administration was also similar among groups (216.4 ± 128.0 s in 0Ni, 159.4 ± 75.7 s in 0.25Ni, and 192.1 ± 60.0 s in 0.5Ni). Welch’s ANOVA with Holm-corrected post hoc comparisons revealed no significant differences among groups for any of these parameters, indicating that nicardipine did not measurably alter induction or early recovery characteristics (Figs. 1A–C). These analyses were conducted using data obtained from the non-warmed session only. Having confirmed that nicardipine did not affect the temporal profile of anesthesia, we next examined its effects on post-anesthetic hypothermia.

Body temperature

Body temperature was measured during anesthesia and recovery under warmed and non-warmed conditions (Figs. 2 and 3). Under warmed conditions, body temperature remained largely stable within a narrow range during anesthesia in all groups (Fig. 2A). Linear mixed-effects model (LMM) analysis revealed significant effects of time, whereas differences among nicardipine doses were limited. During recovery after RORR, body temperature showed a transient early decline (notably in the 0Ni and 0.25Ni groups) followed by gradual rewarming toward baseline across groups (Fig. 2B). Although minor time-point differences were detected by post hoc analysis, the overall temperature profiles were similar among groups. AUC analysis revealed no significant differences among groups during either the early recovery period (0–30 min) or the late recovery period (30–120 min) (Figs. 2C and D). No significant differences in AUC during anesthesia were detected (data not shown).

Fig. 2.

Body temperature during and after MMB anesthesia under warmed conditions. (A) Body temperature during anesthesia. (B) Body temperature during recovery after return of the righting reflex (RORR). (C) Area under the curve (AUC) of body temperature during early recovery (0–30 min). (D) AUC of body temperature during late recovery (30–120 min). Body temperature was measured in rats receiving MMB alone (0Ni), MMB with 0.25 mg/kg nicardipine (0.25Ni), or MMB with 0.5 mg/kg nicardipine (0.5Ni). During anesthesia: 0Ni (n=8), 0.25Ni (n=6), 0.5Ni (n=6). During recovery: 0Ni (n=8), 0.25Ni (n=6), 0.5Ni (n=6). Data are presented as mean ± SEM. The same animals were analyzed for both the anesthesia and recovery phases within each experiment. Significant differences are indicated by symbols as defined in Fig. 1.

Fig. 3.

Body temperature during and after MMB anesthesia under non-warmed conditions. (A) Body temperature during anesthesia. (B) Body temperature during recovery after return of the righting reflex (RORR). (C) Area under the curve (AUC) of body temperature during early recovery (0–30 min). (D) AUC of body temperature during late recovery (30–120 min). Body temperature was measured in rats receiving 0Ni, 0.25Ni, or 0.5Ni. During anesthesia: 0Ni (n=8), 0.25Ni (n=8), 0.5Ni (n=7). During recovery: 0Ni (n=8), 0.25Ni (n=8), 0.5Ni (n=7). Data are presented as mean ± SEM. The same animals were analyzed for both the anesthesia and recovery phases within each experiment. Significant differences are indicated by symbols as defined in Fig. 1.

Under non-warmed conditions, body temperature decreased over time during anesthesia in all groups (Fig. 3A), and overall differences among groups were modest, although post hoc analysis indicated higher temperatures in the 0.25Ni group than in the 0Ni group at several late time points. During recovery after RORR, body temperature gradually increased over time (Fig. 3B). Rats receiving nicardipine tended to maintain higher body temperatures compared with the 0Ni group. Post hoc comparisons demonstrated significantly higher body temperatures in the 0.25Ni group compared with the 0Ni group at multiple time points during recovery (Holm-adjusted P<0.05), whereas the 0.5Ni group showed a similar upward shift but did not reach significance at individual time points. AUC analysis showed significantly higher cumulative body temperature in both nicardipine groups compared with the 0Ni group during both the early recovery period (0–30 min) and the late recovery period (30–120 min) (Figs. 3C and D). No significant differences were observed between the 0.25Ni and 0.5Ni groups in the AUC analysis. To explore a possible physiological mechanism underlying the temperature differences observed under non-warmed conditions, cardiovascular parameters were next examined.

Systolic blood pressure

Because nicardipine is a peripheral vasodilator, systolic blood pressure (SBP) was measured over time to evaluate its cardiovascular effects during MMB anesthesia (Fig. 4A). LMM analysis revealed significant effects of time and group, as well as a significant group × time interaction (P<0.05), indicating that the temporal profile of SBP differed among groups. Overall, SBP values were lower in MMB-Ni animals than in the 0Ni group, with the most pronounced reduction observed in the 0.5Ni group. Although post hoc comparisons suggested differences between groups at certain time points, these differences were not consistently significant after correction for multiple comparisons and should therefore be interpreted with caution. AUC analysis demonstrated significant differences among all groups (Holm-adjusted P<0.05; Fig. 4C), indicating a dose-dependent reduction in SBP associated with increasing nicardipine dose. These findings support cautious interpretation of the higher nicardipine dose from a circulatory-safety perspective.

Fig. 4.

Cardiovascular responses during MMB anesthesia. (A) Systolic blood pressure (SBP). (B) Heart rate (HR). (C) Area under the curve (AUC) of SBP during 10–50 min after MMB administration. (D) AUC of HR during 10–50 min after MMB administration. SBP and HR were measured in rats receiving 0Ni, 0.25Ni, or 0.5Ni. 0Ni (n=5), 0.25Ni (n=7), 0.5Ni (n=8). Data are presented as mean ± SEM. Statistical analyses are described in the Statistical analysis section. Tail-cuff measurements were obtained under standardized mild warming (37°C) to ensure reliable signal acquisition. Significant differences are indicated by symbols as defined in Fig. 1.

Heart rate

To assess the effect of nicardipine on cardiac dynamics after MMB administration, heart rate (HR) was analyzed over time (Fig. 4B). LMM analysis revealed significant effects of group, time, and their interaction, indicating that HR trajectories differed among groups. AUC analysis showed limited between-group differences, with a significant increase observed only between the 0.5Ni and 0.25Ni groups. Pairwise comparisons at individual time points were not consistently significant after Holm correction, suggesting that the effect of nicardipine on HR was modest and better characterized as an overall temporal pattern rather than discrete time-point differences. Differences between the 0Ni and 0.25Ni groups were limited and sporadic. In the AUC analysis, overall HR was significantly higher in the 0.5Ni group than in the 0.25Ni group (Holm-adjusted P<0.05; Fig. 4D), whereas no significant differences were detected between 0Ni and either nicardipine group after Holm correction. Thus, although time-point comparisons suggested transient differences, these were not consistently supported by the AUC analysis after Holm correction.

Discussion

In this study, co-administration of nicardipine partially attenuated post-anesthetic hypothermia during recovery from MMB anesthesia under non-warmed conditions, supporting the study’s central concept. Specifically, nicardipine improved recovery-phase thermal profiles without measurably altering anesthetic induction or recovery timing within the present observation window. Together, these findings support the view that pharmacological modulation of peripheral vascular tone may complement conventional thermal support in managing hypothermia associated with injectable anesthesia. Nicardipine also produced dose-dependent reductions in systolic blood pressure and compensatory increases in heart rate; however, these cardiovascular findings should be interpreted primarily as mechanistic and safety-related context for the thermal effect.

The most salient finding is that nicardipine-treated animals exhibited higher body temperatures during recovery when external warming was withheld. Under non-warmed conditions, both the 0.25Ni and 0.5Ni groups showed significantly higher cumulative temperatures (AUC) during early and late recovery compared with the 0Ni group. This effect was not observed under warmed conditions, suggesting that nicardipine’s contribution to thermal stability is most apparent when conventional thermal support is limited.

During MMB anesthesia, medetomidine likely contributes to this instability through both central and peripheral mechanisms. Centrally, activation of α₂-adrenoceptors suppresses hypothalamic thermoregulatory pathways, lowers the thermoregulatory set point, and reduces metabolic heat production. Peripherally, medetomidine induces vasoconstriction via α₂-adrenergic actions [8, 14], which may reduce peripheral perfusion and alter redistribution of heat between the core and peripheral compartments.

In the present study, temperature differences became evident primarily during recovery rather than during anesthesia and were consistently observed under non-warmed conditions. These findings suggest that the effect of nicardipine was related less to increased heat production than to processes governing heat redistribution within the body [15]. This interpretation is consistent with the general understanding of anesthesia-associated hypothermia, in which a substantial portion of the temperature decrease after anesthetic induction is attributed to redistribution of heat from the core to peripheral tissues [15]. Nicardipine-induced vasodilation may partially counteract medetomidine-induced vasoconstriction, improve peripheral perfusion, and thereby influence recovery-phase temperature profiles [15].

This interpretation is supported by the concurrent cardiovascular changes observed in the present study. Consistent with the vasodilatory action of nicardipine [9, 10], SBP decreased in a dose-dependent manner, especially in the 0.5Ni group, whereas HR tended to increase, likely reflecting a compensatory baroreflex response [16,17,18]. Notably, although both nicardipine groups showed higher recovery-phase temperature AUC values than 0Ni under non-warmed conditions, no significant thermal advantage of 0.5Ni over 0.25Ni was detected. This pattern suggests that partial modulation of vascular tone may be sufficient to influence recovery-phase thermal trajectories, whereas the higher dose may predominantly increase circulatory impact without proportionally enhancing thermoregulatory benefit. Therefore, from a practical perspective, 0.25 mg/kg may represent the more balanced adjunctive dose, although this interpretation should be made cautiously because the present study was not specifically powered to detect modest differences between nicardipine doses for thermal endpoints.

Maintenance of physiological stability during and after anesthesia is an essential component of refinement in laboratory animal research [7]. Hypothermia is one of the most common complications associated with injectable anesthesia in rodents and can affect recovery, metabolism, and overall physiological condition [5, 6]. The present findings support the concept that nicardipine co-administration can serve as an adjunctive physiological measure to partially attenuate post-anesthetic hypothermia during MMB anesthesia under non-warmed conditions. This effect should be regarded as complementary to, rather than a substitute for, active warming. Because nicardipine did not reproduce the protection achieved with active warming and induced a dose-dependent reduction in SBP, adequate thermal support should remain the primary supportive measure. The generalizability of this approach to other anesthetic regimens [19, 20] and experimental contexts remains to be determined.

Several limitations should be acknowledged. First, the present study relied on non-invasive measurements and did not include direct assessment of peripheral perfusion, cardiac output, oxygen consumption, respiratory variables, or blood-gas parameters; therefore, the proposed mechanism cannot be demonstrated directly from the current dataset. Second, cardiovascular measurements were obtained by tail-cuff recording under standardized mild warming and did not include pre-anesthetic baseline values or MAP. Accordingly, the cardiovascular data should be interpreted as relative between-group comparisons under warmed measurement conditions. Third, body temperature and cardiovascular measurements were not obtained simultaneously in the same session, and transient temperature changes immediately after removal from the heating pad in the warmed condition were not strictly controlled, which limits direct linkage of thermal and hemodynamic responses. Finally, because atipamezole was administered 60 min after MMB, the recovery-phase temperature data reflect post-reversal conditions; whether similar effects are observed under other peri-anesthetic workflows requires further study.

In summary, nicardipine co-administration partially attenuated post-anesthetic hypothermia during recovery under non-warmed conditions, supporting its potential as an adjunctive approach to thermal management during MMB anesthesia. This thermoregulatory benefit was observed without measurable changes in anesthetic induction or recovery timing. Because nicardipine also reduced SBP, further evaluation of dose selection and safety is warranted before routine application.

Conflict of Interest

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.