Analysis of uncoupling protein 2-deficient mice upon anaesthesia and sedation revealed a role for UCP2 in locomotion

Abstract

General anaesthesia is associated with hypothermia, oxidative stress, and immune depression. Uncoupling Protein (UCP2) is a member of the mitochondrial carrier family present in many organs including the spleen, the lung and the brain. A role of UCP2 in the activation of the inflammatory/immune cells, in the secretion of hormones, and in the excitability of neurons by regulating the production of reactive oxygen species has been discussed. Because of the side effects of anaesthesia listed above, we aimed to question the expression and the function of UCP2 during anaesthesia. Induction of anaesthesia with ketamine (20 mg/kg) or isoflurane (3.6%) and induction of sedation with the α2 adrenergic receptor agonist medetomidine (0.2 mg/kg) stimulated infiltration of immune cells in the lung and increased UCP2 protein content in the lung, in both immune and non-immune cells. UCP2 content in the lung inversely correlated with body temperature decrease induced by medetomidine treatment. Challenge of the Ucp2(-/-) mice with isoflurane and medetomidine revealed an earlier behavioral recovery phenotype. Transponder analysis of body temperature and activity showed no difference between Ucp2(-/-) and control mice in basal conditions. However, upon an acute decrease of body temperature induced by medetomidine, Ucp2(-/-) mice exhibited increased locomotion activity. Together, these results show that UCP2 is rapidly mobilized during anaesthesia and sedation in immune cells, and suggest a role of UCP2 in locomotion.

Figure 1. Induction of UCP2 expression in the lung and in the spleen upon analgesia and sedation.

A, Western blot analysis of UCP2 (upper panel) and COX I (lower panel) expression in the lung of C57B6/J mice. Mice (5 per group) were injected with ketamine (20 mg/kg) in combination with the α2 adrenergic receptor agonist medetomidine (0.2 mg/kg). Fifteen minutes after induction of anaesthesia, the mice were awoken by injection of 0.4 mg/kg of the α2 adrenergic receptor antagonist atipamezole and euthanatized 2, 3 and 4 hours after arousal from anaesthesia. The lungs were collected and mitochondrial preparation and Western blot analysis were performed. B, Graphic representation of the UCP2/COX I ratio from the experiment in panel A. C, UCP2/COX I ratio in the lung of mice 3 hours after injection of 0.2 mg/kg, medetomidine (Met.), 30 min after intravenous injection of 20 mg/kg of ketamine (Ket. i.v.), 3 hours after intramuscular injection of 20 mg/kg ketamine (Ket. i.m.), or 3 hours after 5 min inhalation of 3.6% isoflurane (Isof.). COX I subunit was used to normalize the amount of mitochondrial proteins. D, UCP2 expression in the spleen of mice 3 hours after injection of 0.2 mg/kg medetomidine (Met.), 200 mg/kg of ketamine (Ket. 200), 20 mg/kg of ketamine (Ket. 20) or 5 min inhalation of 3.6% isoflurane (Isof.). Porin was used to normalize the amount of mitochondrial proteins.

Figure 2. Immune cells contribute to UCP2 expression in lung during analgesia or sedation.

A, Irradiated C57B6/J mice were transplanted with the bone marrow from Ucp2^+/+ (C57B6t(Ucp2^+/+)) or Ucp2^−/− (C57B6t(Ucp2^−/−)) mice as described in (6). Two months after transplantation, the mice were intramuscularly injected with medetomidine (0.2 mg/kg) and ketamine (20 mg/kg). Fifteen minutes after anaesthesia, the mice were awoken by injection of atipamezole and euthanized 3 hours later. UCP2/COX I protein ratio was established by Western blot using the anti UCP2-605 and the anti COX I antibodies. B, Three hours after induction of anaesthesia or sedation, the awoken mice were intraperitoneally injected with pentobarbital (50 mg/kg) and bronchoalveolar lavage was immediately performed. Monocytes were identified and counted under a microscope.

Figure 3. UCP2 induction correlates with sedation-induced hypothermia.

Mice were intramuscularly injected with medetomidine (0.2 mg/kg). A, Rectal body temperature was recorded at indicated time after injection. B, Mice (5 on each time point) were euthanized and lung mitochondria were analyzed for their UCP2/porin protein content by Western blot using the anti UCP2-605 and the anti porin antibodies.

Figure 4. Locomotion and body temperature of Ucp2^−/− mice during medetomidine induced sedation.

One week before the start of the experiment, transponders were implanted in mice (five per group). A, Activity and B, body temperature of Ucp2^+/+ and Ucp2^−/− 24 hours before medetomidine injection. Data were plotted every 2 hours. Mice were subsequently injected with medetomidine (180 µg/kg) and C, the physical activity and D, the body temperature were recorded every 5 min for 7 hours. Time 0 in panels B and D corresponds to the time of medetomidine injection. Note, using this measurement system, the detection limit of temperature was 30°C. * P<0.05 between genotypes for a given time.

Figure 5. Ucp2 ^−/− mice recover faster than wild type mice upon arousal from isoflurane-induced anaesthesia.

Graphic representation of arousal and locomotion times of Ucp2 ^+/+ and Ucp2 ^−/− mice after isoflurane anaesthesia. Mice inhaled isoflurane 3.6% for five minutes in an anesthetic chamber. Anaesthesia was interrupted by taking the mice out of the chamber. The mice were laid down on their back and from this time-point, arousal (rotation of the mice onto their feet) and locomotion times were recorded.