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.

Individual housing of female mice: influence on postsurgical behaviour and recovery.

Individual housing of laboratory mice may increase vulnerability to surgical stress, and interfere with postsurgical recovery. To analyse the effect of housing conditions on recovery, pair- and single-housed female C57BL/6J mice underwent a minor laparotomy +/- analgesia, anaesthesia only or no treatment. Animals were monitored using non-invasive methods during the immediate postsurgical period to assess pain and general impairment. While no appearance or posture abnormalities were observed postexperiment, home cage behaviours were affected distinctly. Discriminant analysis identified self-grooming, locomotion, climbing and resting as mainly responsible for experimental group separation. Behavioural rhythmicity was disrupted, and behaviours related to wellbeing, such as nest building, climbing and burrowing, decreased. Behavioural pain signs (e.g. press) increased. Most behavioural alterations showed a gradation between treatments, e.g. burrowing latency ranged from an intermediate level following anaesthesia only and surgery with analgesia, to pronounced prolongation after surgery without analgesia. Significantly lower burrowing performance after surgery without analgesia in individually-housed animals indicates better recovery in pairs. Social interaction in pairs--an important component of normal behaviour (64%) and a potential indicator for direct social support--was nearly absent (0.3-0.5%). While anaesthesia and surgery resulted in clear changes in behaviour, differences between housing conditions were minor. Hence, despite a tendency towards better recovery in pairs, we found no distinct negative effect of individual housing. In conclusion, both housing conditions are acceptable during the period immediately following minor surgery, though social housing is always preferable in female mice.

Multiparameter telemetry as a sensitive screening method to detect vaccine reactogenicity in mice.

Refined vaccines and adjuvants are urgently needed to advance immunization against global infectious challenges such as HIV, hepatitis C, tuberculosis and malaria. Large-scale screening efforts are ongoing to identify adjuvants with improved efficacy profiles. Reactogenicity often represents a major hurdle to the clinical use of new substances. Yet, irrespective of its importance, this parameter has remained difficult to screen for, owing to a lack of sensitive small animal models with a capacity for high throughput testing. Here we report that continuous telemetric measurements of heart rate, heart rate variability, body core temperature and locomotor activity in laboratory mice readily unmasked systemic side-effects of vaccination, which went undetected by conventional observational assessment and clinical scoring. Even minor aberrations in homeostasis were readily detected, ranging from sympathetic activation over transient pyrogenic effects to reduced physical activity and apathy. Results in real-time combined with the potential of scalability and partial automation in the industrial context suggest multiparameter telemetry in laboratory mice as a first-line screen for vaccine reactogenicity. This may accelerate vaccine discovery in general and may further the success of vaccines in combating infectious disease and cancer.

Burrowing behavior as an indicator of post-laparotomy pain in mice.

Detection of persistent pain of a mild-to-moderate degree in laboratory mice is difficult because mice do not show unambiguous symptoms of pain or suffering using standard methods of short-term observational or clinical monitoring. This study investigated the potential use of burrowing performance - a spontaneous and highly motivated behavior - as a measure of post-operative pain in laboratory mice. The influence of minor surgery on burrowing was investigated in adult C57BL/6J mice of both genders in a modified rodent burrowing test (displacement of food pellets from a pellet-filled tube) within the animal's home cage. Almost all (98%) healthy mice burrowed (mean latency 1.3 h, SEM 0.5 h). After surgery without pain treatment, latency of burrowing was significantly prolonged (mean Δ latency 10 h). Analgesic treatment using the anti-inflammatory drug carprofen (5 mg/kg bodyweight) decreased latency of burrowing after surgery (mean Δ latency 5.5 h) to the level found in mice that had been anesthetized (mean Δ latency 5.4 h) or had received anesthesia and analgesia (mean Δ latency 4.6 h). Analgesia during surgery was associated with a significantly earlier onset of burrowing compared to surgery without pain treatment. A distinct gradation in burrowing performance was found ranging from the undisturbed pre-operative status to the intermediate level following anesthesia/analgesia and surgery with analgesia, to the pronounced prolongation of latency to burrow after surgery without pain relief. In conclusion, post-surgical impairment of general condition, probably mainly attributable to pain, can be conveniently assessed in laboratory mice on the basis of the burrowing test.

Optimized surgical techniques and postoperative care improve survival rates and permit accurate telemetric recording in exercising mice.

BACKGROUND: The laboratory mouse is commonly used as a sophisticated model in biomedical research. However, experiments requiring major surgery frequently lead to serious postoperative complications and death, particularly if genetically modified mice with anatomical and physiological abnormalities undergo extensive interventions such as transmitter implantation. Telemetric transmitters are used to study cardiovascular physiology and diseases. Telemetry yields reliable and accurate measurement of blood pressure in the free-roaming, unanaesthetized and unstressed mouse, but data recording is hampered substantially if measurements are made in an exercising mouse. Thus, we aimed to optimize transmitter implantation to improve telemetric signal recording in exercising mice as well as to establish a postoperative care regimen that promotes convalescence and survival of mice after major surgery in general. RESULTS: We report an optimized telemetric transmitter implantation technique (fixation of the transmitter body on the back of the mouse with stainless steel wires) for subsequent measurement of arterial blood pressure during maximal exercise on a treadmill. This technique was used on normal (wildtype) mice and on transgenic mice with anatomical and physiological abnormalities due to constitutive overexpression of recombinant human erythropoietin. To promote convalescence of the animals after surgery, we established a regimen for postoperative intensive care: pain treatment (flunixine 5 mg/kg bodyweight, subcutaneously, twice per day) and fluid therapy (600 microl, subcutaneously, twice per day) were administrated for 7 days. In addition, warmth and free access to high energy liquid in a drinking bottle were provided for 14 days following transmitter implantation. This regimen led to a substantial decrease in overall morbidity and mortality. The refined postoperative care and surgical technique were particularly successful in genetically modified mice with severely compromised physiological capacities. CONCLUSION: Recovery and survival rates of mice after major surgery were significantly improved by careful management of postoperative intensive care regimens including key supportive measures such as pain relief, administration of fluids, and warmth. Furthermore, fixation of the blood pressure transmitter provided constant reliable telemetric recordings in exercising mice.

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.

Should laboratory mice be anaesthetized for tail biopsy?

Tail biopsies are routinely taken to genotype genetically modified mice. However, the effect of this procedure on the wellbeing of the animals has rarely been investigated. Thus, it has not yet been clearly demonstrated to what extent the mice suffer from tail biopsy (TB) and for how long. The aim of our study was to assess the impact of a single TB on the physiological and behavioural parameters of adult mice and to investigate whether or not anaesthesia can be beneficial. Body weight (BW) curves, daily food/water consumption and telemetric measurements of heart rate, body core temperature, and locomotor activity were recorded for three days following TB, both with and without anaesthesia with methoxyflurane (MOF) or diethylether (ether). Additionally, the impact of anaesthesia alone was characterized. TB without anaesthesia induced an increase in heart rate and locomotor activity for 1 h. Body core temperature was elevated for 2 h. In contrast, heart rate was increased for up to 4 h after anaesthesia. Body core temperature remained altered for up to 20 h after exposure to ether and for 44 h after exposure to MOF. BW was slightly reduced after MOF. Cases of death occurred exclusively under ether at a rate of 7%. Our results indicate a short-lived impact of a TB, whereas anaesthesia with either MOF or ether induced remarkable alterations in the parameters analysed. In conclusion, these types of anaesthesia did not improve mouse wellbeing following tail biopsy.