Amy L. Miller

The Assessment of Post-Vasectomy Pain in Mice Using Behaviour and the Mouse Grimace Scale

Background Current behaviour-based pain assessments for laboratory rodents have significant limitations. Assessment of facial expression changes, as a novel means of pain scoring, may overcome some of these limitations. The Mouse Grimace Scale appears to offer a means of assessing post-operative pain in mice that is as effective as manual behavioural-based scoring, without the limitations of such schemes. Effective assessment of post-operative pain is not only critical for animal welfare, but also the validity of science using animal models. Methodology/Principal Findings This study compared changes in behaviour assessed using both an automated system (“HomeCageScan”) and using manual analysis with changes in facial expressions assessed using the Mouse Grimace Scale (MGS). Mice (n = 6/group) were assessed before and after surgery (scrotal approach vasectomy) and either received saline, meloxicam or bupivacaine. Both the MGS and manual scoring of pain behaviours identified clear differences between the pre and post surgery periods and between those animals receiving analgesia (20 mg/kg meloxicam or 5 mg/kg bupivacaine) or saline post-operatively. Both of these assessments were highly correlated with those showing high MGS scores also exhibiting high frequencies of pain behaviours. Automated behavioural analysis in contrast was only able to detect differences between the pre and post surgery periods. Conclusions In conclusion, both the Mouse Grimace Scale and manual scoring of pain behaviours are assessing the presence of post-surgical pain, whereas automated behavioural analysis could be detecting surgical stress and/or post-surgical pain. This study suggests that the Mouse Grimace Scale could prove to be a quick and easy means of assessing post-surgical pain, and the efficacy of analgesic treatment in mice that overcomes some of the limitations of behaviour-based assessment schemes.

Early detection of health and welfare compromises through automated detection of behavioural changes in pigs

Highlights • Recent developments in automatically detecting compromised pig health and welfare.• Five categories of behaviour are reviewed.• Behaviours mapped to sensors that are feasible for automated detection.• Progress towards levels of automation through detection, monitoring and fully automatic detection of behavioural change.• Challenges for automated detection of behavioural changes are multifaceted and require trade-offs to develop such systems.

The Mouse Grimace Scale: A Clinically Useful Tool?

Medical research has a heavy and continuing demand for rodent models across a range of disciplines. Behavioural assessment of pain in such models is highly time consuming, thus limiting the number of models and analgesics that can be studied. Facial expressions are widely used to assess pain in human infants. Recently the mouse grimace scale (MGS) has been developed and shown to be accurate and reliable, requiring only a short amount of training for the observer. This system therefore has the potential to become a highly useful tool both in pain research and clinical assessment of mouse pain. To date, the MGS has only been used as a research tool, however there is increasing interest in its use in cage-side clinical assessment. It is often wrongly assumed that MGS scores of animals not in pain (i.e. at baseline) are zero. Here, we aimed to assess the variability in baseline MGS scores between cohorts, sexes and strains of mice. Establishing the presence of a consistent baseline MGS score could lead to a valuable clinical pain assessment tool for mice when baseline information from the individual mouse may not be available as a comparator. Results demonstrated a significant difference in baseline MGS scores between both sexes (males > females) and strains of mice. The method used to score the facial action units (Live vs. retrospectively from still images) demonstrated significant differences in scores with live scores being significantly lower than retrospective scoring from images. The level of variation shown demonstrates the need for further research to be undertaken with regard to establishing baseline MGS scores for specific strains and sexes of mice, taking into account the method of scoring, prior to considering clinical implementation of this method in pain assessment.

A comparison of abdominal and scrotal approach methods of vasectomy and the influence of analgesic treatment in laboratory mice

Vasectomized mice are needed in the production of genetically-modified animals. The BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement recommended that vasectomy should be performed via an incision in the scrotal sac, rather than via laparotomy, arguing that the former could be less painful due to minimal tissue trauma. This study was undertaken to assess the validity of this recommendation. Mice underwent vasectomy via either abdominal or scrotal approach surgery. Mice were filmed for 15 min presurgery and at one, 24 and 48 h postsurgery. Data were obtained using automated behaviour recognition software (HomeCageScan). Meloxicam was administered either alone or combined with acetaminophen prior to surgery. A third group received only saline subcutaneously. Postsurgery behaviour changes were compared between groups at each time point. Exploratory behaviours such as rearing, walking and sniffing were most greatly reduced at one hour following surgery whereas the duration of grooming increased. By 48 h these changes had largely subsided. Results indicated mice undergoing scrotal approach surgery fared better at one hour postsurgery, but the magnitude of this was relatively insignificant compared with the overall effects of surgery. If the observed behaviour changes resulted from pain, results suggested there was no significant advantage of scrotal versus abdominal approach vasectomy. These and other recently obtained data on the effects of non-steroidal anti-inflammatory drugs (NSAIDs) in mice suggest considerably larger doses of these or more potent analgesics, more precise monitoring of surgical outcomes, or a combination of these factors are needed to determine the extent of pain experienced by mice undergoing vasectomy.

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.

The Influence of Isoflurane Anaesthesia on the Rat Grimace Scale

Over 234,000 rats were used in regulated procedures in the UK in 2014, many of which may have resulted in some degree of pain. When using animals in research, there is an ethical and legal responsibility to alleviate or at least reduce pain to an absolute minimum. To do this, we must be able to effectively assess pain in an accurate and timely manner. The Rat Grimace Scale (RGS) is a pain assessment tool, which is suggested to be both accurate and rapid in pain assessment. Many procedures involve the use of general anaesthesia. To date, the effects of anaesthesia on the RGS have not been assessed, limiting its potential utility for assessing pain following anaesthesia. Forty-eight Lister hooded rats were used in this study (24 in part A and 24 in a separate part B). Rats were randomly assigned to one of two treatment groups in part A; short duration isoflurane exposure, short duration control exposure (air) and one of two treatment groups in part B; surgical duration isoflurane exposure or surgical duration control exposure (oxygen). Rats were placed into an anaesthetic induction chamber and isoflurane, or control gas piped into the chamber for either 4 (short duration exposure) or 12 minutes (surgical duration exposure). Following recovery, photographs of the rats’ faces were taken and then scored blindly using the RGS. Short duration isoflurane anaesthesia had no effect on RGS scores. However, when rats are anaesthetised for a longer duration, akin to a simple routine surgical procedure, the RGS score increases significantly and this increase remains on repeated exposure to this duration of anaesthesia over a 4-day period. This should be accounted for when using the RGS to assess pain in rats in the immediate time period following procedures involving the use of isoflurane anaesthesia.

Efficacy of intrathecal morphine in a model of surgical pain in rats

Concerns over interactions between analgesics and experimental outcomes are a major reason for withholding opioids from rats undergoing surgical procedures. Only a fraction of morphine injected intravenously reaches receptors responsible for analgesia in the central nervous system. Intrathecal administration of morphine may represent a way to provide rats with analgesia while minimizing the amount of morphine injected. This study aimed to assess whether morphine injected intrathecally via direct lumbar puncture provides sufficient analgesia to rats exposed to acute surgical pain (caudal laparotomy).In an initial blinded, randomised study, pain-free rats received morphine subcutaneously (MSC, 3mg.kg-1, N = 6), intrathecally (MIT, 0.2mg.kg-1, N = 6); NaCl subcutaneously (NSC, N = 6) or intrathecally (NIT, N = 6). Previously validated pain behaviours, activity and Rat Grimace Scale (RGS) scores were recorded at baseline, 1, 2, 4 and 8h post-injection. Morphine-treated rats had similar behaviours to NaCl rats, but their RGS scores were significantly different over time and between treatments. In a second blinded study, rats (N = 28) were randomly allocated to one of the following four treatments (N = 7): MSC, 3mg.kg-1, surgery; MIT, 0.2mg.kg-1, surgery; NIT, surgery; NSC, sham surgery. Composite Pain Behaviours (CPB) and RGS were recorded as previously. CPB in MIT and MSC groups were not significantly different to NSC group. MSC and MIT rats displayed significantly lower RGS scores than NIT rats at 1 and 8h postoperatively. RGS scores for MIT and MSC rats were not significantly different at 1, 2, and 8h postoperatively. Intraclass correlation value amongst operators involved in RGS scoring (N = 9) was 0.913 for total RGS score. Intrathecal morphine was mostly indistinguishable from its subcutaneous counterpart, providing pain relief lasting up to 8 hours in a rat model of surgical pain. Further studies are warranted to clarify the relevance of the rat grimace scale for assessing pain in rats that have received opioid analgesics.

The effect of handling method on the mouse grimace scale in two strains of laboratory mice

Pain assessment in laboratory animals is an ethical and legal requirement. The mouse grimace scale (MGS) is a new method of pain assessment deemed to be both accurate and reliable, and observers can be rapidly trained to use it. In order for a new pain assessment technique to be effective, we must ensure that the score awarded by the technique is only influenced by pain and not by other husbandry or non-painful but integral aspects of research protocols. Here, we studied 16 male mice, housed under standard laboratory conditions. Eight mice were randomly assigned to tail handling and eight to tube handling on arrival at the unit. On each occasion the mice were removed from their cage for routine husbandry, they were picked up using their assigned handling method. Photographs of the mouse faces were then scored by treatment-blind observers as per the MGS manual (see Nature Methods 2010, Vol. 7, pp 447–449), and scores from the two groups were compared. There was no significant difference in MGS scores between the mice that had been handled using a tube compared with the tail. Consequently, these methods of handling did not influence the baseline grimace score given, suggesting that these handling techniques are not confounding factors when establishing baseline MGS scores, further validating this technique.

Automated tracking to measure behavioural changes in pigs for health and welfare monitoring

Since animals express their internal state through behaviour, changes in said behaviour may be used to detect early signs of problems, such as in animal health. Continuous observation of livestock by farm staff is impractical in a commercial setting to the degree required to detect behavioural changes relevant for early intervention. An automated monitoring system is developed; it automatically tracks pig movement with depth video cameras, and automatically measures standing, feeding, drinking, and locomotor activities from 3D trajectories. Predictions of standing, feeding, and drinking were validated, but not locomotor activities. An artificial, disruptive challenge; i.e., introduction of a novel object, is used to cause reproducible behavioural changes to enable development of a system to detect the changes automatically. Validation of the automated monitoring system with the controlled challenge study provides a reproducible framework for further development of robust early warning systems for pigs. The automated system is practical in commercial settings because it provides continuous monitoring of multiple behaviours, with metrics of behaviours that may be considered more intuitive and have diagnostic validity. The method has the potential to transform how livestock are monitored, directly impact their health and welfare, and address issues in livestock farming, such as antimicrobial use.

Welfare Assessment following Heterotopic or Orthotopic Inoculation of Bladder Cancer in C57BL/6 Mice.

Few studies have assessed whether mice used as cancer models experience pain. Despite this possibility, the usual practice is to withhold analgesics as these are generally viewed as confounding. However, pain also alters cancer progression, so preventing it might not only be beneficial to welfare but also to study validity. Establishing the extent to which different cancer models result in pain is an important first step towards their refinement. We used conditioned place preference (CPP) testing and body-weight and behaviour analyses to evaluate the assumption that heterotopically implanted tumours result in less pain and fewer welfare concerns than those implanted orthotopically. C57Bl/6 mice received MB49Luc luciferase expressing bladder cancer cells or saline implanted subcutaneously or into the bladder. These tumour-bearing or control groups underwent 2 daily 45 minute conditioning trials to saline or morphine (2mg/kg) and then a 15 minute drug-free preference test on day 3 of a 3 day cycle, continuing until the study ended. Tumours were imaged and behaviour data obtained following preference tests. Development of preference for the morphine-paired chamber (morphine-seeking) was determined over time. Heterotopic tumour development had no effect on morphine-seeking, and although the restraint used for heterotopic inoculation caused greater initial weight losses than anaesthesia, these mice steadily gained weight and behaved comparatively normally throughout the study. Orthotopic tumour inoculation caused no initial weight losses, but over the final 7 days these mice became less active and lost more body weight than cancer-free controls. This indicated orthotopic implantation probably caused a more negative impact on welfare or conceivably pain; but only according to the current test methods. Pain could not be confirmed because morphine-seeking in the tumour-bearing groups was similar to that seen in controls. Imaging was not found to be an effective method of monitoring tumour development surpassing manual tumour inspection.