A refinement approach in a mouse model of rehabilitation research. Analgesia strategy, reduction approach and infrared thermography in spinal cord injury.

The principles of Refinement, Replacement and Reduction (3R's) should be taken into account when animals must be used for scientific purpose. Here, a Reduction / Refinement approach was applied to the procedure of spinal cord injury (SCI), an animal model used in rehabilitation medicine research, in order to improve the quality of experiments, avoiding unnecessary suffering. The aims of this investigation were 1- to assess acute surgical pain in mice subjected to SCI, 2- to compare the efficacy of commonly used analgesia (three buprenorphine subcutaneous injection in 48 hours, 0,15 mg/kg each) with a combination of opioid and NSAID (one subcutaneous injection of 5 mg/kg carprofen before surgery followed by three buprenorphine subcutaneous injection in 48 hours, 0,15 mg/kg each) and 3- to test if Infrared Thermography (IRT) could be a potential new Refinement method to easily assess thermoregulation, an important metabolic parameter. Finally, we aimed to achieve these goals without recruiting animals on purpose, but using mice already scheduled for studies on SCI. By using behaviours analysis, we found that, despite being commonly used, buprenorphine does not completely relieve acute surgical pain, whereas the combination of buprenorphine and carprofen significantly decreases pain signs by 80%. IRT technology turned out to be a very useful Refinement tool being a non invasive methods to measure animal temperature, particularly useful when rectal probe cannot be used, as in the case of SCI. We could find that temperatures constantly and significantly increased until 7 days after surgery and then slowly decreased and, finally, we could observe that in the buprenorphine and carprofen treated group, temperatures were statistically lower than in the buprenorphine-alone treated mice. To our knowledge this is the first work providing an analgesic Refinement and a description of thermoregulatory response using the IRT technology, in mice subjected to SCI.

The conditioned place preference test for assessing welfare consequences and potential refinements in a mouse bladder cancer model.

Most pre-clinical analgesic efficacy assays still involve nociceptive testing in rodents. This is despite concerns as to the relevance of these tests for evaluating the pain-preventative properties of drugs. More appropriate methods would target pain rather than nociception, but these are currently not available, so it remains unknown whether animal pain equates to the negatively affective and subjective/emotional state it causes in humans. Mouse cancer models are common despite the likelihood of substantial pain. We used Conditioned Place Preference (CPP) testing, assessments of thermal hyperalgesia and behaviour to determine the likelihood that MBT-2 bladder cancer impacts negatively on mouse welfare, such as by causing pain. There was no CPP to saline, but morphine preference in tumour bearing mice exceeded that seen in tumour-free controls. This occurred up to 10 days before the study end-point alongside reduced body weight, development of hyperalgesia and behaviour changes. These effects indicated mice experienced a negative welfare state caused by malaise (if not pain) before euthanasia. Due to the complexity of the assessments needed to demonstrate this, it is unlikely that this approach could be used for routine welfare assessment on a study-by-study basis. However, our results show mice in sufficiently similar studies are likely to benefit from more intensive severity assessment and re-evaluation of end-points with a view to implementing appropriate refinements. In this particular case, a refinement would have been to have euthanased mice at least 7 days earlier or possibly by provision of end-stage pain relief. CPP testing was found to be a helpful method to investigate the responses of mice to analgesics, possibly on a subjective level. These findings and those of other recent studies show it could be a valuable method of screening candidate analgesics for efficacy against cancer pain and possibly other pain or disease models.

Behavioural effects of ovariohysterectomy and oral administration of meloxicam in laboratory housed rabbits.

This study aimed to develop a behaviour-based pain assessment system for rabbits following ovariohysterectomy. Behaviour was analysed to assess the severity and duration of pain induced and determine the effects of administration of meloxicam. The results suggest that pain associated with ovariohysterectomy induced changes in the frequency and duration of a number of behaviours. The most indicative was inactive pain behaviour, which was observed very infrequently prior to surgery compared to very frequently immediately following surgery. This strongly suggests that this increase is a direct response to the surgical pain and/or stress. The frequency of inactive pain behaviour also decreased over the four days post-surgery suggesting that pain is decreasing during this time. High dose meloxicam (initial 1mg/kg followed 0.5mg/kg/day) induced some degree of analgesia. However, higher doses of meloxicam or in combination with an opioid may be required to provide consistent analgesia in rabbits following soft-tissue surgery.

Effects of vasectomy surgery and meloxicam treatment on faecal corticosterone levels and behaviour in two strains of laboratory mouse.

Behaviour was assessed in 32 C57BL/6JCrl and 32 C3H/HeN male mice 1 h following vasectomy; saline or meloxicam was administered 30 min prior to surgery at 5, 10, or 20 mg kg(-1). Faeces were collected 24 h prior to, and 3, 6, 9, 12, 24 h following, vasectomy for measurement of faecal corticosterone. Peak corticosterone levels were significantly higher in mice that underwent vasectomy and received saline (p<0.001) or meloxicam at 5 or 10 mg kg(-1) (p=0.021, and p<0.001, respectively) compared with normal un-operated controls. Mice that underwent vasectomy and received 20 mg kg(-1) meloxicam had peak corticosterone levels that were not different from normal un-operated mice (p=0.254). Discriminant analysis was used to identify behaviours responsible for group separation; these were summed to create two behaviour scores. Score 2 (the frequency of flinching, writhing, rear leg lift and press 2) was thought to be pain related; mice that underwent vasectomy and received saline exhibited significantly more of these behaviours than the normal controls (p=0.032), and the mice that received meloxicam (at any dose). Strain differences were observed in both the stress response to vasectomy and the behavioural changes; the C3H/HeN mice had higher pain scores (behaviour Score 2) and peak corticosterone responses than the C57BL/6JCrl mice. We have demonstrated that significant changes occur in the behaviour of mice following vasectomy, and these changes are reduced by use of meloxicam. Vasectomy elicits a rise in corticosterone levels that was only reduced by the highest dose of meloxicam.

Behavioural assessment of the effects of tumour growth in rats and the influence of the analgesics carprofen and meloxicam.

Very little is known concerning the occurrence of pain in cancer research models. We wished to establish whether a behaviour-based approach, originally developed to assess postoperative pain, could be used to determine positive effects of the analgesics carprofen and meloxicam in rats that might be experiencing pain during tumour development in an orthotopic model of bladder cancer. An invasive but non-metastatic rat bladder cancer cell line was surgically implanted into the bladder wall of 57 inbred Fisher344 rats. The rats underwent daily clinical assessments. When clinical signs consistent with chronic pain were apparent, behavioural data were collected from 44 animals during 2 x 10 min periods, immediately before and one hour after a subcutaneous injection of either physiological saline (0.9%; 0.2 ml/100 g), carprofen (5 mg/kg) or meloxicam (2 mg/kg). Treatment-associated behaviour changes were then compared between groups. The lack of active behaviour, both before and after each treatment, was consistent with established clinical signs of pain. The rats were so inactive following the treatment that the behavioural technique we had previously developed was of comparatively little use in determining either pain severity or analgesic efficacy. One very prominent effect, however, was an increase in ventral abdominal licking in the control (saline) group. As this was absent in rats given meloxicam or carprofen, and has previously been considered to indicate pain emanating from damaged tissue, it was concluded that the analgesic-treated rats gained at least some benefit from the drug treatments, but it was not possible to gauge the extent of this. Handling for examination or treatment may have intensified pain in rats in the control group, and so this should be avoided whenever possible. It is likely that post-surgical pain differs markedly from cancer pain, so a different set of behavioural markers may be needed to assess it effectively. More intensive behaviour monitoring may help to develop a suitable technique for detecting the onset of, and assess the severity of pain that may occur during tumour development.

Evaluation of a short duration behaviour-based post-operative pain scoring system in rats.

We have recently demonstrated dose-related analgesic-induced reductions in the occurrence of 7 behavioural activities following midline laparotomy in rats. For these behaviours to be useful in evaluating pain in laboratory rats they must be shown to occur after different types of surgery, and frequently enough to allow rapid scoring of animals. Here, the relevant behaviours were used to test the analgesic efficacy of meloxicam with a variation of our previous laparotomy model. As part of an unrelated project, 57 male Fischer rats were divided into groups to receive either saline (0.2 ml/100g s.c.), meloxicam (0.5, 1 or 2 mg/kg s.c.) or carprofen (2.5, 5, or 10 mg/kg s.c.) 1h before surgery. Behaviour data were collected for 10 min following 25 min of recovery from isoflurane anaesthesia. The cumulative frequencies of back arching, fall/stagger, writhe and poor gait were used to compute a composite behaviour score. Irrespective of whether analyses included only 5 or all 10 min of the observation period, the relevant behaviours occurred significantly more often in rats given saline or low dose meloxicam than in those given 1 or 2 mg/kg of meloxicam, or any dose of carprofen. We conclude that this technique of quantifying post-surgery behaviour is an effective pain scoring method following abdominal surgery in rats, and that 1 mg/kg meloxicam significantly attenuates laparotomy induced pain. Since only a short observation period is required, this approach represents an important practical advance in assessing abdominal pain severity and clinical drug potency.