Margarete Arras

General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit.

General anesthetics are widely used in clinical practice. On the molecular level, these compounds have been shown to modulate the activity of various neuronal ion channels. However, the functional relevance of identified sites in mediating essential components of the general anesthetic state, such as immobility and hypnosis, is still unknown. Using gene‐targeting technology, we generated mice harboring a subtle point mutation (N265M) in the second transmembrane region of the β3 subunit of the GABAA receptor. In these mice, the suppression of noxious‐evoked movements in response to the intravenous anesthetics etomidate and propofol is completely abolished, while only slightly decreased with the volatile anesthetics enflurane and halothane. β3(N265M) mice also display a profound reduction in the loss of righting reflex duration in response to intravenous but not volatile anesthetics. In addition, electrophysiological recordings revealed that anesthetic agents were significantly less effective in enhancing GABAA receptor‐mediated currents, and in decreasing spontaneous action potential firing in cortical brain slices derived from mutant mice. Taken together, our results demonstrate that a single molecular target, and indeed a specific residue (N265) located within the GABAA receptor β3 subunit, is a major determinant of behavioral responses evoked by the intravenous anesthetics etomidate and propofol, whereas volatile anesthetics appear to act via a broader spectrum of molecular targets.

Neuroserpin, a Neuroprotective Factor in Focal Ischemic Stroke

Because recent studies have indicated that tissue plasminogen activator (tPA) aggravates neurodegenerative processes in many neural pathologies, we studied whether the endogenous tPA antagonist neuroserpin has a neuroprotective effect in an animal model of focal ischemic stroke. After induction of a focal ischemic stroke in the mouse by occlusion of the middle cerebral artery, we found that microglial cells accumulated in the marginal zone of the infarct are the most important source for both plasminogen activators, tPA and uPA. To investigate the effect of neuroserpin on the size and the histology of the infarct we produced transgenic mice overexpressing neuroserpin approximately sixfold in the nervous system. In the brain of these mice the total tPA activity in the uninjured tissue was strongly reduced. After induction of a focal ischemic stroke in the transgenic mice by a permanent occlusion of the middle cerebral artery (MCA), the infarcts were 30% smaller than in the wild-type mice. Immunohistochemical analyses and in situ hybridization revealed an attenuation of the microglial activation in the reactive zone. Concomitantly, the microglial production of tPA and uPA, as well as the PA-activity in the infarct region was markedly reduced. Thus, our results indicate that neuroserpin reduces microglial activation and, therefore, the PA activity and has a neuroprotective role after focal ischemic stroke.

Higher heart rate of laboratory mice housed individually vs in pairs

Many studies have shown that housing mice individually over a long period significantly alters their physiology, but in most cases measurement has required human interference and restraint for sampling. Using a radio-telemetry system with implantable transmitters, we recorded heart rate (HR), motor activity (ACT) and body temperature (BT) of freely moving male mice (NMRI) housed either individually or in pairs with an ovarectomized female. Data for each parameter were collected at 5 min intervals for two consecutive 24 h periods. Even after several weeks of habituation to the social conditions, HR was increased in mice housed individually compared with mice housed in pairs, although their measured ACT did not differ. Additionally, BT tended to be reduced in individually-housed mice. When the data were analysed according to different ACT levels, HR was increased in individually-housed mice during phases of low and high, but not intermediate, motor activity. Furthermore, individually-housed mice had more, but shorter, resting bouts, indicating disruption of the normal circadian sleep pattern. Enhanced HR in individually-housed mice does not necessarily indicate stress, but might be an important physiological indicator of discomfort. The fact that individual housing alters basic physiological parameters in laboratory mice highlights the need to control for housing-dependent variation, especially in experiments that are sensitive to changes in these parameters.

Distinct molecular targets for the central respiratory and cardiac actions of the general anesthetics etomidate and propofol

General anesthetics are among the most widely used and important therapeutic agents. The molecular targets mediating different endpoints of the anesthetic state in vivo are currently largely unknown. The analysis of mice carrying point mutations in neurotransmitter receptor subunits is a powerful tool to assess the contribution of the respective receptor subtype to the pharmacological actions of clinically used general anesthetics. We examined the involvement of β3‐containing GABAA receptors in the respiratory, cardiovascular, hypothermic, and sedative actions of etomidate and propofol using β3(N265M) knock‐in mice carrying etomidate‐ and propofol‐insensitive β3‐containing GABAA receptors. Although the respiratory depressant action of etomidate and propofol, as determined by blood gas analysis, was almost absent in β3(N265M) mice, the cardiac depressant and hypothermic effects, as determined by radiotelemetry, and the sedative effect, as determined by decrease of motor activity, were still present. Taken together with previous findings, our results show that both immobilization and respiratory depression are mediated by β3‐containing GABAA receptors, hypnosis by both β3‐and β2‐containing GABAA receptors, while the hypothermic, cardiac depressant, and sedative actions are largely independent of β3‐containing GABAA receptors.

Optimal hematocrit for maximal exercise performance in acute and chronic erythropoietin-treated mice

Erythropoietin (Epo) treatment increases hematocrit (Htc) and, consequently, arterial O2 content. This in turn improves exercise performance. However, because elevated blood viscosity associated with increasing Htc levels may limit cardiac performance, it was suggested that the highest attainable Htc may not necessarily be associated with the highest attainable exercise capacity. To test the proposed hypothesis that an optimal Htc in acute and chronic Epo-treated mice exists—i.e., the Htc that facilitates the greatest O2 flux during maximal exercise—Htc levels of wild-type mice were acutely elevated by administering novel erythropoiesis-stimulating protein (NESP; wtNESP). Furthermore, in the transgenic mouse line tg6 that reaches Htc levels of up to 0.9 because of constitutive overexpression of human Epo, the Htc was gradually reduced by application of the hemolysis-inducing compound phenylhydrazine (PHZ; tg6PHZ). Maximal cardiovascular performance was measured by using telemetry in all exercising mice. Highest maximal O2 uptake and maximal time to exhaustion at submaximal exercise intensities were reached at Htc values of 0.58 and 0.57 for wtNESP, and 0.68 and 0.66 for tg6PHZ, respectively. Rate pressure product, and thus also maximal working capacity of the heart, increased with elevated Htc values. Blood viscosity correlated with Apart from the confirmation of the Htc hypothesis, we conclude that tg6PHZ adapted better to varying Htc values than wtNESP because of the higher optimal Htc of tg6PHZ compared to wtNESP. Of note, blood viscosity plays a critical role in limiting exercise capacity.

Comparative analysis and physiological impact of different tissue biopsy methodologies used for the genotyping of laboratory mice

Genotyping of genetically modified mice and control of authenticity of the genetic background of congenic or coisogenic strains by polymerase chain reaction (PCR) is a routine procedure that can be performed with different tissue biopsies causing variable grades of trauma. In this study, some invasive and non-invasive sampling methods were compared, with the main focus on the impact on animal physiology. We compared ear punch, tail biopsy, hair plugging, mouth and rectum swabs and the simple restraint of the animals, scoring for the impact on heart rate (HR), core body temperature (BT) and motor activity by telemetry, during biopsy and for the following 6 h. Furthermore, in order to correlate the physiological impact with the practicability and reliability of the genotyping results, we performed a PCR analysis of the biopsy samples obtained by using the same collection procedures analysed by telemetry. All sampling methods and restraint induced significant increase in HR and BT and a slight increase in motor activity for 1 h, independent of the invasiveness of the method used. Genotyping of all biopsies allowed the proper identification of transgenic animals, tail biopsies, ear punches and hair follicles giving clear signals, the last method being fast, but also susceptible to cross contaminations during sampling by large numbers of animals. Restraint and all biopsy methods provoked similar physiological changes, indicating that the handling of the animals is of major importance and that the sampling procedure does not strongly influence the physiological parameters.

Mapping the contribution of β3-containing GABAA receptors to volatile and intravenous general anesthetic actions

BackgroundAgents belonging to diverse chemical classes are used clinically as general anesthetics. The molecular targets mediating their actions are however still only poorly defined. Both chemical diversity and substantial differences in the clinical actions of general anesthetics suggest that general anesthetic agents may have distinct pharmacological targets. It was demonstrated previously that the immobilizing action of etomidate and propofol is completely, and the immobilizing action of isoflurane partly mediated, by β3-containing GABAA receptors. This was determined by using the β3(N265M) mice, which carry a point mutation known to decrease the actions of general anesthetics at recombinant GABAA receptors. In this communication, we analyzed the contribution of β3-containing GABAA receptors to the pharmacological actions of isoflurane, etomidate and propofol by means of β3(N265M) mice.ResultsIsoflurane decreased core body temperature and heart rate to a smaller degree in β3(N265M) mice than in wild type mice, indicating a minor but significant role of β3-containing GABAA receptors in these actions. Prolonged time intervals in the ECG and increased heart rate variability were indistinguishable between genotypes, suggesting no involvement of β3-containing GABAA receptors. The anterograde amnesic action of propofol was indistinguishable in β3(N265M) and wild type mice, suggesting that it is independent of β3-containing GABAA receptors. The increase of heart rate variability and prolongation of ECG intervals by etomidate and propofol were also less pronounced in β3(N265M) mice than in wild type mice, pointing to a limited involvement of β3-containing GABAA receptors in these actions. The lack of etomidate- and propofol-induced immobilization in β3(N265M) mice was also observed in congenic 129X1/SvJ and C57BL/6J backgrounds, indicating that this phenotype is stable across different backgrounds.ConclusionOur results provide evidence for a defined role of β3-containing GABAA receptors in mediating some, but not all, of the actions of general anesthetics, and confirm the multisite model of general anesthetic action. This pharmacological separation of anesthetic endpoints also suggests that subtype-selective substances with an improved side-effect profile may be developed.

Assessment of postsurgical distress and pain in laboratory mice by nest complexity scoring

Preliminary studies have suggested a correlation between postsurgical pain and nest building behaviour in laboratory mice. However, there is no standardized measure for estimating pain by means of nest building performance. Here, we investigated nest building under various conditions, and scored nest complexity to assess postsurgical pain. Mice of both sexes, different strains [C57BL/6J, DBA/2J, and B6D2-Tg(Pr-mSMalphaActin)V5rCLR-25], and kept under different housing conditions, showed no differences in their latency to use the offered nest material. Healthy female C57BL/6J mice were engaged 4.3% of the day with nest building and showed three peaks of this behaviour: in the beginning and middle of the light phase, and in the second half of the dark phase. For assessment of postsurgical pain, female C57BL/6J mice underwent a sham embryo transfer +/− different doses of the analgesic carprofen or control treatment. Nest complexity scoring at 9 h after the experimental treatments (i.e. at the end of the light phase) resulted in less than 10% of animals with noticeably manipulated nest material (nestlet) after surgery and more than 75% of healthy mice having built identifiable-to-complex nests or had noticeably manipulated nestlets, while animals after anaesthesia-only showed intermediate nest complexity. Carprofen analgesia resulted in no (5 mg/kg) or only slight (50 mg/kg) improvement of nest complexity after surgery. Thus, nest complexity scoring can be incorporated into daily laboratory routine and can be used in mice as a sensitive tool for detecting reduced wellbeing and general condition, but probably not for determining the efficacy of pain treatment.

Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination.

Summary Airway access is needed for a number of experimental animal models, and the majority of animal research is based on mouse models. Anatomical conditions in mice are small, and the narrow glottic opening allows intubation only with a subtle technique. We therefore developed a microscopic endotracheal intubation method with a wire guide technique in mice anaesthetized with halothane in oxygen. The mouse is hung perpendicularly with its incisors on a thread fixed on a vertical plate. The tongue is placed with a pair of forceps between the left hands thumb and forefinger and slightly pulled, while the neck and thorax are positioned using the third and fourth fingers. By doing so, the neck can be slightly stretched, which allows optimal visualization of the larynx and the vocal cords. To ensure a safe intubation, a fine wire guide is placed under vision between the vocal cords and advanced about 5 mm into the trachea. An intravenous 22G × 1 in. plastic or Teflon catheter is guided over this wire. In a series of 41 mice, between 21 and 38 g, the success rate for the first intubation attempt was >95%. Certainty of the judgement procedure was 100% and success rate was higher using the described method when compared with a transillumination method in a further series. The technique is safe, less invasive than tracheostomy and suitable for controlled ventilation and pulmonary substance application.

Inhibitory Ligand-Gated Ion Channels as Substrates for General Anesthetic Actions

General anesthetics have been in clinical use for more than 160 years. Nevertheless, their mechanism of action is still only poorly understood. In this review, we describe studies suggesting that inhibitory ligand-gated ion channels are potential targets for general anesthetics in vitro and describe how the involvement of y-aminobutyric acid (GABA)(A) receptor subtypes in anesthetic actions could be demonstrated by genetic studies in vivo.