Striving for humane deaths for laboratory mice: hypobaric hypoxia provides a potential alternative to carbon dioxide exposure.

Killing is often an unavoidable and necessary procedure for laboratory mice involved in scientific research, and providing a humane death is vital for public acceptance. Exposure to carbon dioxide (CO2) gas is the most widely used methodology despite well proven welfare concerns. Consequently, the continued use of CO2 and its globally permitted status in legislation and guidelines presents an ethical dilemma for users. We investigated whether killing with hypobaric hypoxia via gradual decompression was associated with better welfare outcomes for killing laboratory mice. We compared the spontaneous behaviour of mice exposed to CO2, decompression or sham conditions, and used analgesic or anxiolytic interventions to determine their relative welfare impact. Gradual decompression resulted in longer times to unconsciousness and death and the pharmacological interventions support the notion of a minimally negative animal experience, while providing further evidence for pain and anxiety associated with exposure to CO2. Decompression resulted in moderate ear haemorrhage, but our welfare assessment suggests this may happen when mice are unconscious. Hence, gradual decompression could be the basis of significant refinement for killing laboratory mice. Future work should corroborate behaviour with neurobiological markers of loss of consciousness to verify the conscious phase of concern for animal welfare.

Effects of acute chemotherapy-induced mucositis on spontaneous behaviour and the grimace scale in laboratory rats.

Intestinal mucositis is a frequent side-effect of chemotherapy treatment. Many oncological research programs aim to identify novel treatments for this distressing condition, and these programs frequently use rat models. Little is known about the presence and progression of pain in these models and how this can best be treated by analgesic therapy. We used a number of behaviour-based methods of pain assessment to determine which tools were best suited for pain identification. Baseline measures for behavioural assessment, rat grimace score and sociability were determined through analysis of continuously recorded video data and an applied social interaction test (n = 16). Mucositis was then induced by intraperitoneal injection of 5-fluorouracil (150 mg/kg) and further behavioural analyses undertaken. An assessment of enrichment interaction was also made by determining the mass of a plastic chew toy gnawed both pre- and post-chemotherapy injection. Behavioural scoring was performed 1, 6, 12, 24 and 48 h after injection, with facial expression being scored at the 12, 24 and 48 h time-points. Sociability testing was performed once during the post-injection period. No significant differences were found in grimace scores between baseline and later daily measures. Behaviours similar to those previously reported post-laparotomy were observed. Writhing, twitching and back-arching behaviours were most evident in rats affected by mucositis and were increased in frequency (respective P values: 0.002, 0.004 and 0.008) 48 h after chemotherapy injection compared with baseline, implying that pain onset occurred around this time-point. Social investigatory behaviour was also increased (P = 0.002) following disease onset. Each day, rats post-5FU injection gnawed a greater percentage of their Nylabone enrichment by weight than the saline-injected control rats (P = 0.046). These data suggest that, of the tools tested, behavioural assessment scoring may find greatest utility in rodent models of intestinal mucositis and should be investigated further.

Using the mouse grimace scale to assess pain associated with routine ear notching and the effect of analgesia in laboratory mice.

Social housing is recommended where possible for laboratory mice. In order to achieve this, mice must be individually identifiable. Although, various methods are available, permanent identification is often required, such as ear notching. This method is likely to be painful and to date there is limited literature on pain assessment and alleviation for this routine husbandry practice. Here we aimed to determine if the mouse grimace scale (MGS) could be used to assess pain in C57BL/6 mice following routine ear notching. Langford et al. found that very acute noxious stimuli (i.e. < 10 min in duration) did not produce a change in MGS score in comparison to baseline. Here, no significant difference was found between MGS scores at baseline and immediately post ear notching, potentially indicating that the pain associated with ear notching is either too acute to assess using the MGS tool or the practice is not painful. Studies in other species indicate that ear notching is painful, therefore, unless we can confidently conclude that the process of ear notching is not painful, we should err on the side of caution and assume it is painful due to the large number of mice ear-notched and potential welfare consequences. Alternative methods of assessing pain following this routine practice should be used in order to assess both the potential pain in mice, and the effectiveness of analgesics or local anaesthetics to relieve any associated pain.

An alternative method of endotracheal intubation of common marmosets (Callithrix jacchus).

Endotracheal intubation was carried out in 11 common marmosets (Callithrix jacchus). A commercially available tilting stand and a Miller laryngoscope blade were used to visualize the larynx. Anaesthesia was induced with alphaxalone (10.6 ± 1.6 mg/kg intramuscularly, followed by 3.2 ± 1.2 mg/kg intravenously). The diameter of the proximal trachea easily fitted an endotracheal tube made from readily available material (a 12 G 'over the needle' catheter). Once the tip of the endotracheal tube was at the level of the vocal folds, the tube had to be gently rotated through a 180° angle in order to pass through the larynx into the trachea. Assessment of the dimensions of the larynx and trachea, and comparison with external anatomical features of the animals (n = 10) showed that the length of the trachea could be predicted by multiplying the craniosacral length of the marmoset by a factor of 0.42.

A preliminary investigation into the practicality of use and duration of action of slow-release preparations of morphine and hydromorphone in laboratory rats.

Significant advances have been made in our ability to assess pain and administer appropriate pain relief in laboratory animals. However, providing long-lasting analgesia using a route that does not involve animal restraint remains difficult. The objective of this study was to investigate whether oral administration of slow-release morphine or hydromorphone results in increased thermal nociception in laboratory rats. The results showed that 64 mg/kg morphine and 16 mg/kg hydromorphone induced comparable increases in foot withdrawal latencies for up to three hours postadministration; however, slow-release morphine increased response latencies for up to 11 hours. Whether these dose rates provide clinically effective pain relief has yet to be determined; however, these data suggest that using slow-release preparations could be an effective and highly practical method of elevating pain thresholds for a relatively prolonged period.

Evaluation of the efficacy of a novel electronic pain assessment device, the Pain Gauge, for measuring postoperative pain in rats.

One of the major challenges for individuals working with laboratory animals is the recognition and alleviation of pain. The Pain Gauge is marketed as a pain assessment device that measures electrodermal activity. To establish whether the Pain Gauge is effective in assessing postoperative pain in laboratory rats, preoperative and postoperative pain gauge scores ('pain scores') were obtained from 67 rats. Rats were randomly assigned to one of three experimental groups (laparotomy, craniotomy or control) and to one of four analgesic groups (meloxicam [2 mg/kg s.c.] or parecoxib [1, 5 or 20 mg/kg i.v.]). Five consecutive 'pain scores' were obtained from each animal at each of five time points (preprocedure, and at 1, 2, 3 and 4 h postoperatively). Overall there was a significant difference between 'pain scores' at different time points; mainly a decrease at 1 h postoperatively compared with the preoperative scores. There was no overall increase in postoperative 'pain scores' in the rats that were most likely to suffer from postoperative pain (rats given a lower dose of analgesic that underwent a surgical procedure) compared with rats that did not undergo a potentially painful procedure (rats in anaesthesia-only/control group). Therefore it was concluded that the Pain Gauge is ineffective in assessing postoperative pain in rats in this study.

Degrees of aversion shown by rats and mice to different concentrations of inhalational anaesthetics.

The distress associated with the induction of anaesthesia with halothane, isoflurane, enflurane and carbon dioxide was investigated in rats and mice by measuring the level of aversion they displayed on exposure to low, medium and high concentrations of these agents. The animals were exposed to each agent in a test chamber containing air or gas mixtures, which they were able to enter and leave at will, and the level of aversion was assessed in terms of the initial withdrawal and total dwelling times in the chamber. Comparisons between the anaesthetic and air-control treatments indicated that concentrations of the agents recommended for the rapid and efficient induction of anaesthesia were associated with some degree of aversion. Carbon dioxide was by far the most aversive gas for both rats and mice, with the least aversive being halothane for rats, and halothane and enflurane for mice. With all the anaesthetics, the level of aversion increased as the concentration increased.