Mouse Grimace Scale Scores Research Articles

Optogenetic cortical spreading depolarization induces headache-related behaviour and neuroinflammatory responses some prolonged in familial hemiplegic migraine type 1 mice

BackgroundCortical spreading depolarization (CSD), the neurophysiological correlate of the migraine aura, can activate trigeminal pain pathways, but the neurobiological mechanisms and behavioural consequences remain unclear. Here we investigated effects of optogenetically-induced CSDs on headache-related behaviour and neuroinflammatory responses in transgenic mice carrying a familial hemiplegic migraine type 1 (FHM1) mutation.MethodsCSD events (3 in total) were evoked in a minimally invasive manner by optogenetic stimulation through the intact skull in freely behaving wildtype (WT) and FHM1 mutant mice. Related behaviours were analysed using mouse grimace scale (MGS) scoring, head grooming, and nest building behaviour. Neuroinflammatory changes were investigated by assessing HMGB1 release with immunohistochemistry and by pre-treating mice with a selective Pannexin-1 channel inhibitor.ResultsIn both WT and FHM1 mutant mice, CSDs induced headache-related behaviour, as evidenced by increased MGS scores and the occurrence of oculotemporal strokes, at 30 min. Mice of both genotypes also showed decreased nest building behaviour after CSD. Whereas in WT mice MGS scores had normalized at 24 h after CSD, in FHM1 mutant mice scores were normalized only at 48 h. Of note, oculotemporal stroke behaviour already normalized 5 h after CSD, whereas nest building behaviour remained impaired at 72 h; no genotype differences were observed for either readout. Nuclear HMGB1 release in the cortex of FHM1 mutant mice, at 30 min after CSD, was increased bilaterally in both WT and FHM1 mutant mice, albeit that contralateral release was more pronounced in the mutant mice. Only in FHM1 mutant mice, contralateral release remained higher at 24 h after CSD, but at 48 h had returned to abnormal, elevated, baseline values, when compared to WT mice. Blocking Panx1 channels by TAT-Panx308 inhibited CSD-induced headache related behaviour and HMGB1 release.ConclusionsCSDs, induced in a minimally invasive manner by optogenetics, investigated in freely behaving mice, cause various migraine relevant behavioural and neuroinflammatory phenotypes that are more pronounced and longer-lasting in FHM1 mutant compared to WT mice. Prevention of CSD-related neuroinflammatory changes may have therapeutic potential in the treatment of migraine.

Deep Learning-Based Grimace Scoring Is Comparable to Human Scoring in a Mouse Migraine Model.

Pain assessment is essential for preclinical and clinical studies on pain. The mouse grimace scale (MGS), consisting of five grimace action units, is a reliable measurement of spontaneous pain in mice. However, MGS scoring is labor-intensive and time-consuming. Deep learning can be applied for the automatic assessment of spontaneous pain. We developed a deep learning model, the DeepMGS, that automatically crops mouse face images, predicts action unit scores and total scores on the MGS, and finally infers whether pain exists. We then compared the performance of DeepMGS with that of experienced and apprentice human scorers. The DeepMGS achieved an accuracy of 70–90% in identifying the five action units of the MGS, and its performance (correlation coefficient = 0.83) highly correlated with that of an experienced human scorer in total MGS scores. In classifying pain and no pain conditions, the DeepMGS is comparable to the experienced human scorer and superior to the apprentice human scorers. Heatmaps generated by gradient-weighted class activation mapping indicate that the DeepMGS accurately focuses on MGS-relevant areas in mouse face images. These findings support that the DeepMGS can be applied for quantifying spontaneous pain in mice, implying its potential application for predicting other painful conditions from facial images.

Welfare Assessment, End-Point Refinement and the Effects of Non-Aversive Handling in C57BL/6 Mice with Lewis Lung Cancer.

Simple SummaryMice are widely used to study various types of cancer to develop effective treatments, but this can cause them to experience pain or poor welfare, which may lead to inconsistent or negative findings. This study used a variety of welfare monitoring methods to determine if, or more importantly when mice developing lung cancer experienced poor welfare so that similar future studies can be ended before this occurs. It also aimed to determine whether non-aversive handling, also known as tunnel handling (lifting mice in a plastic tube rather than by the tail) is an effective method of refinement, leading to improved welfare and improved consistency of results. Compared to normal mice, those developing lung cancer showed behaviour changes consistent with poor welfare including a general lack of movement and facial expression changes (grimacing). Most noticeably, cancer caused a reduction in food consumption and the ability of the mice to build a suitable nest. These combined factors suggested the mice began to suffer around 4 days before the study ended. Tunnel handling did not improve our results consistency or enhance welfare, but importantly, it had no negative effects. The findings suggest similar studies should be ended when mice appear to make reduced quality nests.Cancer-bearing mice are at risk of developing anxiety, pain, or malaise. These conditions may not only harm welfare but could also undermine data quality and translational validity in studies to develop therapeutic interventions. We aimed to establish whether, or at what point mice developing lung cancer show these symptoms, what measures can best detect their onset, and if data quality and animal welfare can be enhanced by using non-aversive handling (NAH). Welfare was monitored using various daily methods. At the beginning and end of the study, we also scored behaviour for general welfare evaluation, recorded nociceptive thresholds, and applied the mouse grimace scale (MGS). Cancer caused a decline in daily welfare parameters (body weight, and food and water consumption) beginning at around 4 days prior to euthanasia. As cancer progressed, rearing and walking declined to a greater extent in cancer-bearing versus control mice, while grooming, inactive periods, and MGS scores increased. A decline in nest building capability and food consumption provided a particularly effective means of detecting deteriorating welfare. These changes suggested a welfare problem arose as cancer developed, so similar studies would benefit from refinement, with mice being removed from the study at least 4 days earlier. However, the problem of highly varied tumour growth made it difficult to determine this time-point accurately. There were no detectable beneficial effects of NAH on either data quality or in terms of enhanced welfare.

Effects of Three Consecutive Days of Morphine or Methadone Administration on Analgesia and Open-Field Activity in Mice with Ehrlich Carcinoma.

This study assessed the exploratory behavioral responses in BALB/c mice inoculated with Ehrlich ascitic carcinoma after 3 consecutive days of treatment with morphine or methadone. Fifty-three female mice, 60 ± 10 d old, were used. Seven days after intraperitoneal tumor inoculation (2 × 106 cells), the animals were randomized into 7 groups: morphine 5 mg/kg (MO5), morphine 7.5 mg/kg (MO7.5), morphine 10 mg/kg (MO10), methadone 2.85 mg/kg (ME2.85), methadone 4.3 mg/kg (ME4.3), methadone 5.7 mg/kg (ME5.7), and 0.9% NaCl (Saline) (n = 7). Drug treatments were administered subcutaneously every 6 h for 3 d. The animals were evaluated for analgesia using the mouse grimace scale (MGS) and for general activity using the open field test. The MGS was performed before tumor inoculation (day 0), on day 7 at 40, 90, 150, 240, and 360 min after drug injection, and on days 8 and 9 at 40, 150, 240, and 360 min after drug injection. The open field test was performed before tumor inoculation (day 0), on day 7 after inoculation at 40, 90, 150, 240, and 360 min after drug injection, and on days 8 and 9 after inoculation at 40, 150, and 360 min after drug injection. MGS results indicated that administration of morphine promoted analgesia for up to 240 min. Conversely, methadone reduced MGS scores only at 40 min. All tested doses promoted a significant dose-dependent increase in the total distance traveled and the average speed, and increase that was markedly pronounced on days 8 and 9 as compared with day 7. The frequencies of rearing and self-grooming decreased significantly after morphine or methadone administration. Despite the difference in analgesia, both drugs increased locomotion and reduced the frequency of rearing and self-grooming as compared with the untreated control animals.

Reliability of the Mouse Grimace Scale in C57BL/6JRj Mice.

Simple SummaryThe protection and welfare of laboratory animals is ethically and legally required. Any possible impairment of well-being caused by husbandry or experiment must be reduced to a minimum. A prerequisite for ameliorating animal welfare is that we recognize the current well-being state of the animals by using measuring tools, for instance, the grimace scale. Facial expressions of mice, the most frequently used laboratory animals, can be analyzed using the Mouse Grimace Scale, which consists of five action units, i.e., orbital tightening, nose and cheek bulge, ear position, and whisker change. Since it is important that the animal welfare measuring tools are reliable and objective, we investigated the reliability of the Mouse Grimace Scale. Our results indicated good agreement between all observers applying the Mouse Grimace Scale. However, when investigating the action units individually, the best agreement was achieved for orbital tightening and the poorest agreement for nose as well as cheek bulge, depending on the observers’ experience levels. Against this background, we critically discuss factors that potentially influence the reliability of the Mouse Grimace Scale to improve its application.To maintain and foster the welfare of laboratory mice, tools that reliably measure the current state of the animals are applied in clinical assessment. One of these is the Mouse Grimace Scale (MGS), a coding system for facial expression analysis. Since there are concerns about the objectivity of the MGS, we further investigated its reliability. Four observers (two experienced and two inexperienced in use of the MGS) scored 188 images of 33 female and 31 male C57BL/6JRj mice. Images were generated prior to, 150 min, and two days after ketamine/xylazine anesthesia. The intraclass correlations coefficient (ICC = 0.851) indicated good agreement on total MGS scores between all observers when all three time points were included in the analysis. However, interrater reliability was higher in the early post-anesthetic period (ICC = 0.799) than at baseline (ICC = 0.556) and on day 2 after anesthesia (ICC = 0.329). The best agreement was achieved for orbital tightening, and the poorest agreement for nose and cheek bulge, depending on the observers’ experience levels. In general, experienced observers produced scores of higher consistency when compared to inexperienced. Against this background, we critically discuss factors that potentially influence the reliability of MGS scoring.

Improvement of the Mouse Grimace Scale set-up for implementing a semi-automated Mouse Grimace Scale scoring (Part 1).

The Mouse Grimace Scale (MGS) has been widely used for the noninvasive examination of distress/pain in mice. The aim of this study was to further improve its performance to generate repeatable, faster, blinded and reliable results for developing automated and standardized pictures for MGS scoring and simultaneous evaluation of up to four animals. Videos of seven C57BL/6N mice were generated in an experiment to assess pain and stress induced by repeated intraperitoneal injection of carbon tetrachloride (CCl4). MGS scores were taken 1 h before and after the injection. Videotaping was performed for 10 min in special observation boxes. For manual selection, pictures of each mouse were randomly chosen for quality analysis and scored according six quality selection criteria (0 = no, 1 = moderate, 2 = full accordance); the maximum possible score was 12. Overall, 609 pictures from six videos were evaluated for MGS scoring quality; evaluation was performed by using the picture selection tool or by manual scoring. With manual scoring, 288 pictures (48.3% of all randomly generated pictures) were deemed scorable using MGS (mean score = 22.15 ± SD 6.3). To evaluate the algorithm, ratings from different rater groups (beginner, medium-level trained, professional) were compared with the automated image generated. These differences were not significant (p = 0.1091). This study demonstrates an improved set-up and a picture selection tool that can generate repeatable, not-observer biased and standardized pictures for MGS scoring.

Evaluating analgesic efficacy and administration route following craniotomy in mice using the grimace scale

Most research laboratories abide by guidelines and mandates set by their research institution regarding the administration of analgesics to control pain during the postoperative period. Unfortunately, measuring pain originating from the head is difficult, making adequate decisions regarding pain control following stereotaxic surgery problematic. In addition, most postsurgical analgesia protocols require multiple injections over several days, which may cause stress and distress during a critical recovery period. Here we sought to (1) assess the degree of postoperative pain following craniotomy in mice, (2) compare the efficacy of three common rodent analgesics (carprofen, meloxicam and buprenorphine) for reducing this pain and (3) determine whether the route of administration (injected or self-administered through the drinking supply) influenced pain relief post-craniotomy. Using the mouse grimace scale (MGS), we found that injectable analgesics were significantly more effective at relieving post-craniotomy pain, however, both routes of administration decreased pain scores in the first 24 h postsurgery. Specifically, buprenorphine administered independently of administration route was the most effective at reducing MGS scores, however, female mice showed greater sensitivity to carprofen when administered through the water supply. Although it is necessary to provide laboratory animals with analgesics after an invasive procedure, there remains a gap in the literature regarding the degree of craniotomy-related pain in rodents and the efficacy of alternative routes of administration. Our study highlights the limitations of administering drugs through the drinking supply, even at doses that are considered to be higher than those currently recommended by most research institutions for treating pain of mild to moderate severity.

Body temperature and mouse scoring systems as surrogate markers of death in cecal ligation and puncture sepsis

BackgroundDespite increasing ethical standards for conducting animal research, death is still often used as an endpoint in mouse sepsis studies. Recently, the Murine Sepsis Score (MSS), Mouse Clinical Assessment Score for Sepsis (M-CASS), and Mouse Grimace Scale (MGS) were developed as surrogate endpoint scoring systems for assessing pain and disease severity in mice. The objective of our study was to compare the effectiveness of these scoring systems and monitoring of body temperature for predicting disease progression and death in the cecal ligation and puncture (CLP) sepsis model, in order to better inform selection of surrogate endpoints for death in experimental sepsis.MethodsC57Bl/6J mice were subjected to control sham surgery, or moderate or severe CLP sepsis. All mice were monitored every 4 h for surrogate markers of death using modified versions of the MSS, M-CASS, and MGS scoring systems until 24 h post-operatively, or until endpoint (inability to ambulate) and consequent euthanasia.ResultsThirty percent of mice subjected to moderate severity CLP reached endpoint by 24 h post-CLP, whereas 100% undergoing severe CLP reached endpoint within 20 h. Modified MSS, M-CASS, and MGS scores all increased, while body temperature decreased, in a time-dependent and sepsis severity-dependent manner, although modified M-CASS scores showed substantial variability. Receiver operating characteristic curves demonstrate that the last recorded body temperature (AUC = 0.88; 95% CI 0.77–0.99), change in body temperature (AUC = 0.89; 95% CI 0.78–0.99), modified M-CASS (AUC = 0.93; 95% CI 0.85–1.00), and modified MSS (AUC = 0.95; 95% CI 0.88–1.01) scores are all robust for predicting death in CLP sepsis, whereas modified MGS (AUC = 0.78; 95% CI 0.63–0.92) is less robust.ConclusionsThe modified MSS and body temperature are effective markers for assessing disease severity and predicting death in the CLP model, and should thus be considered as valid surrogate markers to replace death as an endpoint in mouse CLP sepsis studies.

Roles of TRPV1 and TRPA1 in Spontaneous Pain from Inflamed Masseter Muscle

Craniofacial muscle pain, such as spontaneous pain and bite-evoked pain, are major symptoms in patients with temporomandibular disorders and infection. However, the underlying mechanisms of muscle pain, especially mechanisms of highly prevalent spontaneous pain, are poorly understood. Recently, we reported that transient receptor potential vanilloid 1 (TRPV1) contributes to spontaneous pain but only marginally contributes to bite-evoked pain during masseter inflammation. Here, we investigated the role of transient receptor potential ankyrin 1 (TRPA1) in spontaneous and bite-evoked pain during masseter inflammation, and dissected the relative contributions of TRPA1 and TRPV1. Masseter inflammation increased mouse grimace scale (MGS) scores and face wiping behaviors. Pharmacological or genetic inhibition of TRPA1 significantly attenuated MGS but not face wiping behaviors. MGS scores were also attenuated by scavenging putative endogenous ligands for TRPV1 or TRPA1. Simultaneous inhibition of TRPA1 by AP18 and TRPV1 by AMG9810 in masseter muscle resulted in robust inhibition of both MGS and face wiping behaviors. Administration of AP18 or AMG9810 to masseter muscle induced conditioned place preference (CPP). The extent of CPP following simultaneous administration of AP18 and AMG9810 was greater than that induced by the individual antagonists. In contrast, inflammation-induced reduction of bite force was not affected by the inhibition of TRPA1 alone or in combination with TRPV1. These results suggest that simultaneous inhibition of TRPV1 and TRPA1 produces additive relief of spontaneous pain, but does not ameliorate bite-evoked pain during masseter inflammation. Our results provide further evidence that distinct mechanisms underlie spontaneous and bite-evoked pain from inflamed masseter muscle.

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.

Using the mouse grimace scale and behaviour to assess pain in CBA mice following vasectomy

Mice used in biomedical research should have pain reduced to an absolute minimum through refinement of procedures or by the provision of appropriate analgesia. Vasectomy is a common and potentially painful surgical procedure carried out on male mice to facilitate the production of genetically modified mice. The aim of our study was to determine if 0.05mg/kg buprenorphine would ameliorate pain associated changes following abdominal vasectomy and to determine if the mouse grimace scale is an appropriate tool for the assessment of pain in this model. Eight male CBA mice underwent abdominal vasectomy as part of a genetically modified mouse-breeding programme. Here we assessed pain using a previously validated behaviour-based method and the mouse grimace scale. All mice received buprenorphine (0.05mg/kgs.c.) pre-surgery. Behaviour and grimace scores were compared between baseline (pre-surgery), 30min, 5h, 24h and 25h post surgery. Following 24h post-op, all mice were administered 5mg/kg meloxicam (s.c.) as additional analgesia. Significant increases in specific pain behaviours and mouse grimace scale score were found 30min post surgery. At 5h post surgery, scores were returning to baseline levels. Frequency of rearing was significantly decreased at both 30min and 5h post surgery compared to baseline, demonstrating a longer lasting change in normal exploratory behaviour. Buprenorphine (0.05mg/kg) was ineffective at ameliorating these pain-associated changes in CBA mice and should be considered inadequate at this dose. By 24h post surgery, pain associated behaviours, grimace scale and rearing had all returned to baseline levels. There was no change in pain behaviours or MGS following administration of meloxicam indicating that an additional dose of meloxicam does not appear to offer benefit at this point. Using the mouse grimace scale to assess pain in mice, appeared to be effective in the immediate post vasectomy period in CBA mice demonstrating the same duration of increased score as the pain associated behaviours.

Quantification of pain in sickle mice using facial expressions and body measurements

Pain is a hallmark feature of sickle cell disease (SCD). Subjects typically quantify pain by themselves, which can be biased by other factors leading to overtreatment or under-treatment. Reliable and accurate quantification of pain, in real time, might enable to provide appropriate levels of analgesic treatment. The mouse grimace scale (MGS), a standardized behavioral coding system with high accuracy and reliability has been used to quantify varied types of pain. We hypothesized that addition of the objective parameters of body length and back curvature will strengthen the reproducibility of MGS. We examined MGS scores and body length and back curvature of transgenic BERK sickle and control mice following cold treatment or following treatment with analgesic cannabinoid CP55,940. We observed that sickle mice demonstrated decreased length and increased back curvature in response to cold. These observations correlate with changes in facial expression for the MGS score. CP55,940 treatment of sickle mice showed an increase in body length and a decrease in back curvature concordant with MGS scores indicative of an analgesic effect. Thus, body parameters combined with facial expressions may provide a quantifiable unbiased method for objective measure of pain in SCD.

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.

The effect of isoflurane anaesthesia and buprenorphine on the mouse grimace scale and behaviour in CBA and DBA/2 mice

Prevention or alleviation of pain in laboratory mice is a fundamental requirement of in vivo research. The mouse grimace scale (MGS) has the potential to be an effective and rapid means of assessing pain and analgesic efficacy in laboratory mice. Preliminary studies have demonstrated its potential utility for assessing pain in mouse models that involve potentially painful procedures. The next step in validation is to determine if the other procedures that are integral to these models, i.e. anaesthesia or analgesia, result in any changes in MGS score which would need to be taken into account when using this tool to assess post-procedural pain. Here, spontaneous behaviour and MGS data for CBA and DBA/2 mice were recorded at baseline and following either isoflurane anaesthesia (suitable to perform abdominal surgery) or 0.05mg/kgs.c. buprenorphine. In line with previous studies, isoflurane anaesthesia alone had limited effects on the spontaneous behaviour in either strain of mice. Administration of buprenorphine resulted in increased periods of activity e.g. walking and chewing bedding in CBA mice. These effects were not demonstrated in DBA/2 mice. In comparison, buprenorphine alone had no impact on MGS score in either strain of mice, however DBA/2 mice showed a significant increase in MGS score following isoflurane anaesthesia. The presence of this increased MGS score must be taken into account when attempting to use the MGS to assess pain in DBA/2 mice. Further work should be carried out to establish the presence of this isoflurane effect in other strains and the potential influence of gender on the MGS. This further validation is necessary prior to implementation of this technique in clinical scenarios.

Meloxicam prevents COX-2-mediated post-surgical inflammation but not pain following laparotomy in mice.

Inflammation is thought to be a major contributor to post-surgical pain, so non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics. However, compared to rats, considerably less is known as to how successfully these prevent pain in mice. A fluorescent COX-2 selective probe was used for the first time to evaluate the post-surgical anti-inflammatory effects of meloxicam, and automated behaviour analyses (HomeCageScan; HCS), the Mouse Grimace Scale (MGS) and body weight changes to assess its pain-preventative properties. Groups of 8-9 BALB/c mice were subcutaneously injected with saline (0.3 mL) or meloxicam at (1, 5 or 20 mg/kg) 1 h before a 1.5-cm midline laparotomy. The probe or a control dye (2 mg/kg) was injected intravenously 3 h later. Imaging was used to quantify inflammation at 7, 24 and 48 h following surgery. HCS data and MGS scores were respectively obtained from video recordings and photographs before surgery and 24 h later. Post-surgical inflammation was dose dependently reduced by meloxicam; with 5 or 20 mg/kg being most effective compared to saline. However, all mice lost weight, MGS scores increased and behavioural activity was reduced by surgery for at least 24 h with no perceivable beneficial effect of meloxicam on any of these potentially pain-associated changes. Although meloxicam prevented inflammation, even large doses did not prevent post-laparotomy pain possibly arising due to a range of factors, including, but not limited to inflammation. MGS scoring can be applied by very naïve assessors and so should be effective for cage-side use.

Refinement of analgesia following thoracotomy and experimental myocardial infarction using the Mouse Grimace Scale.

New Findings What is the central question of this study? There is an ethical imperative to optimize analgesia protocols for laboratory animals, but this is impeded by our inability to recognize pain reliably. We examined whether the Mouse Grimace Scale (MGS) provides benefits over a standard welfare scoring system for identifying a low level of pain in the frequently used murine surgical model of myocardial infarction. What is the main finding and its importance? Low‐level pain, responsive to analgesia, was detected by MGS but not standard methods. In this model, most of the pain is attributable to the thoracotomy, excepted in mice with very large infarcts. This approach represents a model for assessing postsurgical analgesia in rodents. The Mouse Grimace Scale (MGS) was developed for assessing pain severity, but the general applicability to complex postsurgical pain has not been established. We sought to determine whether the MGS provides benefits over and above a standard welfare scoring system for identifying pain in mice following experimental myocardial infarction. Female C57BL/6J mice (n = 60), anaesthetized with isoflurane, were subjected to thoracotomy with ligation of a coronary artery or sham procedure. A single s.c. dose of buprenorphine (1.1 mg kg−1) was given at the time of surgery and pain assessed at 24 h by MGS and a procedure‐specific welfare scoring system. In some animals, a second dose of 0.6 mg kg−1 buprenorphine was given and pain assessment repeated after 30 min. The MGS was scored from multiple photographs by two independent blinded observers with good correlation (r = 0.98). Using the average MGS score of both observers, we identified a subset of mice with low scores that were not considered to be in pain by the welfare scoring system or by single observer MGS. These mice showed a significant improvement with additional analgesia, suggesting that this low‐level pain is real. Pain attributable to the myocardial injury, as opposed to thoracotomy, persisted at 24 h only in mice with large infarcts >40%. In conclusion, the use of a multi‐observer, post hoc version of the MGS is a sensitive tool to assess the efficacy of postsurgical analgesic protocols. Following surgical induction of myocardial infarction, we identified a significant proportion of mice that were in low‐level pain at 24 h that were not identified by other assessment methods.

Effects of "Danzhi Decoction" on Chronic Pelvic Pain, Hemodynamics, and Proinflammatory Factors in the Murine Model of Sequelae of Pelvic Inflammatory Disease.

Objective. To evaluate the effect of Danzhi decoction (DZD) on chronic pelvic pain (CPP), hemodynamics, and proinflammatory factors of sequelae of pelvic inflammatory diseases (SPID) in murine model. Methods. SPID mice were randomly treated with high-dose DZD, mid-dose DZD, low-dose DZD, aspirin, and vehicle for 3 estrous circles. The Mouse Grimace Scale (MGS) was performed to evaluate CPP; blood flows of the upper genital tract, pelvic wall, and mesentery were used to assess hemodynamics in SPID mice; expressions of vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and osteopontin (OPN) were measured by Western blot and immunochemistry. Results. Treatment with dose-dependent DZD significantly decreased the MGS scores, accelerated blood flows of the pelvis, and reduced expressions of VEGF, Ang-2, and OPN in the upper genital tract. Conclusions and Discussions. DZD was effective in relieving CPP and improving hemodynamics of the pelvic blood-stasis microenvironment in SPID mice. There was a relationship between CPP and the pelvic blood-stasis microenvironment. Furthermore, DZD might play a positive role in the anti-inflammatory process.

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

BackgroundCurrent 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 FindingsThis 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.ConclusionsIn 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.