Differential Effects of α²-adrenoceptors in the Modulation of the Thermoregulatory Response in Mice Induced by Meperidine

Permission for this study was received from the local Animal Care Committee at the Ministry for Agriculture and Environment, Kiel, Germany, before the initiation of work. The generation of mouse lines lacking a single α²-adrenoceptor (α^2A-, α^2B-, or α^2C-adrenoceptor knock out) has been described previously.11,12The knock-out mice were obtained from L. Hein (Freiburg, Germany), and maintained in a specified pathogen-free facility at our institution where all experiments were performed. Also, wild-type mice (129S2/SvHsd) were analyzed as a control group.

Ten mice of each group weighing 26–35 g were investigated. Mice were housed 3–4 animals per cage and maintained on a 12-h light-dark cycle with free access to water and food. All experiments were performed between 09:00 and 16:00 h. The animals were weighed before the experiments. In a first set of experiments, animals received saline or meperidine (20 mg/kg) (Dolantin; Sanofi-Aventis, Frankfurt, Germany), respectively. To rule out that a possible effect of meperidine is mediated via  opioid receptors, in a second set of experiments, the opioid receptor antagonist naloxone hydrochloride (125 μg/kg) (Naloxon Curamed; Curamed Pharma, Karlsruhe, Germany) and the α²-adrenoceptor antagonist atipamezole (2 mg/kg) were injected 20 min after the injection of saline/meperidine.

To investigate possible effects of different opioids on the thermoregulatory threshold temperature that can be reversed by naloxone, we also analyzed fentanyl (50 μg/kg) (Fentanyl-Janssen; Janssen-Cilag, Neuss, Germany) plus naloxone (125 μg/kg) as an additional control.

To rule out that 125 μg/kg naloxone was not sufficient to completely reverse opioid receptor–mediated effects, a control group of 10 α^2C-adrenoceptor knock-out mice received meperidine plus different naloxone doses (125 vs.  1250 μg/kg). All drugs were administered intraperitoneally (0.1 ml/10 g body weight).

Each animal was analyzed with all six different drug regimens (saline, meperidine, saline plus naloxone, meperidine plus naloxone, fentanyl plus naloxone, and meperidine plus atipamezole) and therefore served as its own control. Before a new experiment was started, animals had at least 2 weeks to recover from injections of pharmaceuticals. After injection of saline or meperidine, the animals were returned to their cages until starting the measurements 40 min later. In a second set of experiments, 20 min after injection of saline (for saline plus naloxone experiment), meperidine (for meperidine plus naloxone or meperidine plus atipamezole), or fentanyl (for fentanyl plus naloxone) as the first drug, mice were injected with naloxone or atipamezole as the second drug and immediately returned to their cages until start of measurements 20 min later (fig. 1). To prevent an inappropriate decline of body temperature, cages were warmed by a heating lamp.

Forty minutes after injection of the first drug, mice were positioned in a gas-tight plexiglas chamber (internal diameter, 4 cm; length, 7 cm). A constant oxygen flow of 100 ml/min was delivered to the chamber. Mixed expired carbon dioxide was recorded continuously by capnography (Phasein Multigas sensor; Armeda Medizintechnik, Hannover, Germany). Body temperature was measured and recorded continuously by a rectal temperature probe (Sirecust 402; Siemens, Danvers, MA). Ice was positioned around the plexiglas chamber to provoke a thermoregulatory response by whole body cooling. No animal was allowed to get colder than 32°C. According to previous studies in rats,13the thermoregulatory threshold was defined as the temperature (°C) at which a sustained increase in expiratory carbon dioxide can be measured (fig. 1). The maximum response intensity of nonshivering thermogenesis was calculated as the ratio of the maximum and minimum carbon dioxide plateau during cooling.

To determine the thermoregulatory threshold, plots of mixed expired carbon dioxide (mmHg) as a function of time were analyzed by three blinded observers and the median value of the three observers was taken for further analysis. Within-group data were analyzed using repeated-measures ANOVA, with Dunnett test for comparison to control (precooling baseline values). Differences between the treatment groups were evaluated using one-way ANOVA followed by Bonferroni correction for multiple comparisons. To analyze differential effects between different genotypes, a 4 × 6 ANOVA was performed. Results are presented as mean ± SD; P < 0.05 was considered significant. GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) was used to perform statistical analyses.

Baseline body temperatures before and after intraperitoneal injection were neither significantly different between mice of all groups, nor between wild-type and α²-adrenoceptor knock-out mice.

In wild-type mice receiving saline, the thermoregulatory threshold temperature was 36.3 ± 0.4°C. Additional injection of naloxone did not change threshold temperature significantly (36.2 ± 0.5°C). After administration of meperidine, the thermoregulatory threshold decreased to 35.0 ± 0.6°C (P < 0.01). This effect could not be abolished by additional administration of naloxone (35.2 ± 0.7°C) (fig. 2).

In α^2B- and α^2C-adrenoceptor knock-out mice, the thermoregulatory threshold temperature after injection of saline was comparable (36.3 ± 0.7°C vs.  36.3 ± 0.8°C). No significant changes could be detected in both groups after additional injection of naloxone. Administration of meperidine decreased the threshold temperature to 35.3 ± 0.6°C (α^2B-adrenoceptor knock out) (P < 0.05) and 35.4 ± 0.7°C (α^2C-adrenoceptor knock out) (P < 0.05), respectively. In α^2B-adrenoceptor knock-out mice, the additional administration of naloxone after meperidine was followed by a minimal and not significant increase of threshold temperature (35.5 ± 0.6°C). In α^2C-adrenoceptor knock-out mice meperidine plus naloxone administration resulted in an alleviated decline of threshold temperature, which was not significant compared with saline administration anymore (fig. 2).

In contrast, in α^2A-adrenoceptor knock-out mice, meperidine application alone did not result in a significant decline of the thermoregulatory threshold temperature (36.2 ± 0.6°C vs.  36.5 ± 0.4°C in the saline control group). No significant differences could be detected between threshold temperatures of α^2A-adrenoceptor knock-out mice in all analyzed groups (fig. 2).

In wild-type, α^2B-, and α^2C-adrenoceptor knock-out mice, the thermoregulatory threshold after meperidine plus atipamezole injection was significantly higher than after meperidine alone, whereas atipamezole had no effect in α^2A-adrenoceptor knock-out mice. There also was no significant difference between saline control and meperidine plus atipamezole in wild-type, α^2B-, and α^2C-adrenoceptor knock-out mice. This indicates that atipamezole abolished the effects of meperidine in wild-type and α^2B- and α^2C-adrenoceptor knock-out mice (fig. 2).

The application of fentanyl plus naloxone had no significant effect on the thermoregulatory threshold temperature in wild-type and α²-adrenoceptor knock-out mice compared with saline control.

An extraordinarily high dose of 1250 μg/kg naloxone compared with 125 μg/kg following saline or meperidine injection had no significant effect on the thermoregulatory threshold temperature in α^2C-adrenoceptor knock-out mice (fig. 3).

The maximum response intensity of nonshivering thermogenesis did not show any significant differences between the analyzed groups. Maximum response intensity also was comparable between wild-type and α²-adrenoceptor knock-out mice (table 1).

Finally, the analysis of a differential effect by a 4 × 6 ANOVA revealed no significant interaction between both drugs applied and mice genotype (interaction term, P = 0.2986).

The main findings of our study are as follows:

1. Meperidine decreased the thermoregulatory threshold temperature in wild-type and α^2B- and α^2C-adrenoceptor knock-out mice. This effect was not abolished by additional administration of the opioid receptor antagonist naloxone but by additional administration of the α²-adrenoceptor antagonist atipamezole.

2. Naloxone had no effect on meperidine-induced decrease of thermoregulatory threshold in wild-type and α^2B-adrenoceptor knock-out mice, but did in α^2C-adrenoceptor knock-out mice.

3. In contrast, in α^2A-adrenoceptor knock-out mice, meperidine had no significant effect on thermoregulatory threshold temperature. Furthermore, naloxone administration did not show any effect.

4. The maximum intensity of nonshivering thermogenesis was comparable in wild-type and α²-adrenoceptor knock-out mice.

The underlying mechanisms of meperidine’s impact on thermoregulation, including the thermoregulatory threshold temperature, remain to be elucidated. In fact, meperidine is more effective in treatment of postanesthetic shivering than equianalgesic doses of other opioids, especially specific μ-opioid receptor agonists.3,14,15It has been suggested, that additional κ-opioid receptor activation by meperidine was responsible for this effect.4,16However, even high-dose naloxone expected to block κ-opioid receptors did not completely reverse the antishivering action of meperidine in volunteers.4 

In addition to its agonistic effects at opioid receptors, meperidine shows local anesthetic activity and anticholinergic properties,17,18but this does not fully explain meperidine’s special antishivering action.19 

On the other hand, α²-adrenoceptor agonists such as clonidine or dexmedetomidine have demonstrated their potency in treatment of shivering and their ability in decreasing both the shivering and the vasoconstriction threshold.20A previous study from our group demonstrated that meperidine decreased the thermoregulatory threshold temperature in wild-type mice. According to our present results, this effect was abolished by administration of the α²-adrenoceptor antagonist atipamezole, indicating an involvement of α²-adrenoceptors in the mediation of meperidine actions.10This conclusion is supported by the fact that meperidine acts as an agonist at α²-adrenoceptors in vitro  in clinically relevant concentrations, showing highest affinity to the α^2B-adrenoceptor subtype (α^2B, 8.6 ± 0.3 μm; α^2C, 13.6 ± 1.5 μm; α^2A, 38.6 ± 0.7 μm).5 

As a result of these in vitro  binding studies, mice with modified α²-adrenoceptor genes have become important tools in elucidating α²-adrenoceptor subtype-specific functions. In agreement with our results with meperidine, Hunter et al.  8did not detect hypothermic effects in mice with a functional α^2A-adrenoceptor knock out in response to varying doses of dexmedetomidine. In contrast, in wild-type and α^2B- and α^2C-adrenoceptor knock-out mice, thermoregulation was influenced, whereas Sallinen et al.  21reported a slight attenuation of the hypothermic response in α^2C-adrenoceptor knock-out mice. These results point to the α^2A-adrenoceptor as the primary mediator of the thermoregulatory effects of α²-adrenoceptor agonists, although the α^2C-adrenoceptor subtype also may be involved.9 

In our study, in α^2A-adrenoceptor knock-out mice no significant decline of the thermoregulatory threshold temperature after meperidine administration could be detected, whereas the threshold temperature decreased in wild-type- and α^2B- and α^2C-adrenoceptor knock-out mice. The fact that this decrease in wild-type and α^2B-adrenoceptor knock-out mice was not abolished by naloxone but atipamezole administration indicates that nonopioid receptor pathways are involved.

To rule out that the high naloxone dose4that we applied was too small and not capable of blocking all μ- and κ-opioid receptors, we compared the effects of 125 μg/kg versus  1,250 μg/kg naloxone without any significant difference. Average duration of our experiments from naloxone administration was 45 min, so a repetitive injection seemed unnecessary. On the other hand, meperidine plus naloxone injection led to a small increase of threshold temperature compared with meperidine alone in α^2C-adrenoceptor knock-out mice. In α^2B-adrenoceptor knock-out mice, this tendency also was detectable but not significant. This indicates that opioid receptors—possibly the κ-subtype—in addition are likely to be involved in the mediation of meperidine’s effects.

From the current data, no change of the maximum response intensity of shivering as the ratio of the maximum and minimum carbon dioxide plateau during cooling could be observed. In agreement with our results, Ikeda et al.  22did not detect any changes in the maximum intensity of shivering after meperidine and also alfentanil application. However, both drugs proved to decrease the thermoregulatory threshold. This indicates that antishivering activity does not necessarily include both an effect on the thermoregulatory threshold and on the maximum intensity or gain (defined as incremental intensity increase) of shivering.

Our study may be criticized because of the relatively small number of animals investigated, resulting in a certain variability of the results. Furthermore, naloxone was initially administered in only one defined high dosage to block both μ- and κ-opioid receptors, according to previous studies in humans and mice.4Small differences between meperidine and meperidine plus naloxone may not have been detected as result of the sample size. As a result, we cannot conclude that the effects of meperidine on thermoregulation in mice are mediated exclusively by α²-adrenoceptors. Furthermore, data from animal studies should be projected to humans with some limitations. Whereas adult humans use shivering as a defense against hypothermia and nonshivering thermogenesis plays only a small role, rodents use nonshivering thermogenesis as their primary mechanism to resist hypothermia. Therefore, it should be clearly stated that our results in mice have only very limited impact on the peri- or postoperative thermoregulation in humans.

In conclusion, the present data indicate that meperidine’s ability to decrease the thermoregulatory threshold temperature in wild-type mice is not abolished by additional administration of the opioid receptor antagonist naloxone. In α^2A-adrenoceptor knock-out mice, meperidine administration had no significant effect on the thermoregulatory threshold temperature. This indicates an important role of the α^2A-adrenoceptor in the modulation of the thermoregulatory response induced by meperidine in mice.

The authors thank Lutz Hein (Professor, Institute of Pharmacology and Toxicology, University of Freiburg, Freiburg, Germany) for providing the α²-adrenoceptor knock-out mice for this study, and Maria Kiosz (Technician, Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Germany) for her help.