Rat Grimace Scale Scores Research Articles

An automated rat grimace scale for the assessment of pain.

Pain is a complex neuro-psychosocial experience that is internal and private, making it difficult to assess in both humans and animals. In pain research, animal models are prominently used, with rats among the most commonly studied. The rat grimace scale (RGS) measures four facial action units to quantify the pain behaviors of rats. However, manual recording of RGS scores is a time-consuming process that requires training. While computer vision models have been developed and utilized for various grimace scales, there are currently no models for RGS. To address this gap, this study worked to develop an automated RGS system which can detect facial action units in rat images and predict RGS scores. The automated system achieved an action unit detection precision and recall of 97%. Furthermore, the action unit RGS classifiers achieved a weighted accuracy of 81-93%. The system's performance was evaluated using a blast traumatic brain injury study, where it was compared to trained human graders. The results showed an intraclass correlation coefficient of 0.82 for the total RGS score, indicating that the system was comparable to human graders. The automated tool could enhance pain research by providing a standardized and efficient method for the assessment of RGS.

Rat Grimace Scale as a Method to Evaluate Animal Welfare, Nociception, and Quality of the Euthanasia Method of Wistar Rats.

Refinement of experimental procedures in animal research has the objective of preventing and minimizing pain/distress in animals, including the euthanasia period. This study aimed to evaluate pain associated with six methods of euthanasia in Wistar rats (injectable, inhalational, and physical), by applying the Rat Grimace Scale (RGS), comparing the scores, and determining the method with the highest score that might indicate pain for laboratory rodents. Sixty adult male and female Wistar rats were used and assigned to six treatments: pentobarbital, CO2, decapitation, isoflurane, ketamine + xylazine, and ketamine + CO2. Video recording to assess the RGS scores was performed in four events: basal: 24 h before the procedure; Ti1: three minutes before the procedure; Ti2: during the application of the euthanasia method; and Ti3: immediately after the application until LORR. The main findings of this study showed that, during Ti2, decapitation and ketamine + xylazine had the highest scores (0.6 ± 0.26 and 0.6 ± 0.16, respectively) (p < 0.0001), while at Ti3, CO2 (0.9 ± 0.18) and isoflurane (1.2 ± 0.20) recorded the highest scores (p < 0.0001). According to the present results, decapitation and ketamine + xylazine elicited short-term acute pain, possibly due to tissue damage caused by both methods (injection and guillotine). In contrast, isoflurane's RGS scores recorded during Ti3 might be associated with nociception/pain due to the pungency of the drug or to the pharmacological muscle relaxant effect of isoflurane. Further research is needed to establish a comprehensive study of pain during euthanasia, where RGS could be used minding the limitations that anesthetics might have on facial expression.

Hypothermia During General Anesthesia Interferes with Pain Assessment in Laboratory Rats (Rattus norvegicus)

Accurate pain assessment methods are necessary to ensure animal welfare and reliable data collection in animal research. The Rat Grimace Scale (RGS), a facial expression pain scale, allows effective identification of pain. However, the potential confounds of this method remain mostly unexplored. General anesthesia, which is used in many laboratory procedures, suppresses thermoregulation and results in hypothermia. We investigated the effects of isoflurane-induced hypothermia on RGS scores. Twenty (10 male and 10 female) Sprague-Dawley rats each received 30 min of anesthesia, followed by 30 min of observation after the return of sternal recumbency. Rats were randomized to receive warming with an electric heating pad or no warming during both periods. Unwarmed rats became hypothermic within 15 min after isoflurane exposure began and returned to normothermia within 15 min after returning to sternal recumbency. Warmed rats did not deviate from the normothermic range. The RGS scores of unwarmed rats were significantly higher than baseline levels for 3 h after anesthesia and were higher than those of warmed rats at 5 and 180 min after anesthesia. Hypothermia resulted in a larger proportion of rats crossing a predetermined analgesic intervention threshold. Our findings show that hypothermia induced by isoflurane anesthesia presents a confound to accurate RGS scoring. These results emphasize the importance of maintaining normothermia to avoid inflated pain scores and to obtain accurate pain assessment.

Intraperitoneal injection of sodium pentobarbital has the potential to elicit pain in adult rats (Rattus norvegicus).

An effective and pain-free killing method is required to achieve the goal of euthanasia, a “good death”. Overdose of sodium pentobarbital (PB) by intraperitoneal (IP) injection is a widely accepted technique in laboratory rats, but questions remain regarding pain associated with administration. As PB rapidly causes sedation and loss of consciousness, most studies have relied on indirect evidence of pain. The objective of this study was to assess pain associated with IP PB using an appropriate vehicle control. Adult male and female Sprague Dawley (SD) and female Wistar rats (N = 84) were block randomised by sex and strain to receive one of three treatments: 1) 800 mg/kg PB (pH 11), 2) saline or 3) vehicle controls (pH 11 or 12.5). Behavior (Rat Grimace Scale (RGS), writhing, back arching) was evaluated at baseline, before loss of righting reflex (LORR, PB group), and at 80s, 151s and 10 min post-injection (PI; saline and vehicle control groups). In the PB group, mean time to LORR was 78 ± 7.9 seconds. In the vehicle control groups, RGS scores were increased at 151s PI (SD: p = 0.0002, 95%CI 0.73 to 0.20) from baseline, as was relative frequency of writhing (SD: p < 0.0001; Wistar; p = 0.0004). RGS scores remained elevated 10 mins PI (SD: p = 0.0005, 95%CI 0.71 to 0.18; Wistar: p = 0.0234, 95%CI 0.91 to 0.07) but the relative frequency of writhing did not (p > 0.999). The RGS scores and the relative frequency of writhing remained low in the PB and saline groups (p > 0.05). These results show that, vehicle controls for IP PB result in signs associated with pain, pain may not be experienced following IP PB when LORR occurs quickly, and that the effects of PB limit behavioral pain assessments.

The method simulating spontaneous pain in patients with nociplastic pain using rats with fibromyalgia-like condition

The method shown in this article simulates spontaneous pain in patients with nociplastic pain using rats; the measurement with this method could be related to better translation of analgesic efficacies of therapeutic compounds between rats and humans. Nociplastic pain occurs in various disorders including fibromyalgia. Because the pain in patients occurs without an external stimulus, we assessed spontaneous pain in rats. The grimace scale, a methodology for rating facial expression, has been used for measuring spontaneous pain in animals. However, the responses in animals have been rather short-lived, and the scale has never been applied to animals exhibiting nociplastic pain. Here, we apply the rat grimace scale (RGS) to the reserpine-induced fibromyalgia-like rat, which induces nociplastic pain. The ratings of the orbital tightening, nose/cheek flattening, and changes in characteristics of ears and whiskers by three raters, who were blinded to the treatment allocated to rats, demonstrated substantial, long-lasting change in facial expression of rats. In this article, reference images for raters, and sample images used for rater training are provided. All raters independently indicated that the RGS score is significantly elevated with this methodology in reserpine-induced fibromyalgia-like rats.•The grimace scale, a method for rating facial expression, is applied to the reserpine-induced fibromyalgia-like rat, which manifests nociplastic pain.•Facial expression change in the reserpine-induced fibromyalgia-like rat is substantial and long-lasting.•Elevation of the RGS score in the reserpine-induced fibromyalgia-like rat may simulate spontaneous pain in patients with nociplastic pain.

Spontaneous pain-associated facial expression and efficacy of clinically used drugs in the reserpine-induced rat model of fibromyalgia

Fibromyalgia-associated chronic pain occurring without organic causes exerts negative effects on patients' quality of life, thereby necessitating the development of superior drugs. Since non-organic pain in patients with fibromyalgia occurs without external stimuli, an endpoint measure that reflects patients’ spontaneous pain should be implemented in preclinical research. The present study is the first to apply the rat grimace scale (RGS), a facial expression-dependent measure developed for quantifying spontaneous pain, to the rat with reserpine-induced myalgia, an animal model of fibromyalgia exhibiting non-organic pain. Animals were videotaped and still images of facial expressions were captured and scored in a blind fashion. The reserpine-induced myalgia rats exhibited a significant increase in the RGS score, which was sustained for 2 weeks or more after the induction of fibromyalgia-like state by reserpine injection. The period of RGS score elevation was similar to that of reduced paw withdrawal threshold (PWT) measured using the von Frey filament test, a conventional measure of evoked pain. The elevated RGS score and the decreased PWT were relieved by gabapentin (an α2δ subunit ligand) and duloxetine (a serotonin and noradrenaline reuptake inhibitor), but not by diclofenac (a nonsteroidal anti-inflammatory drug), buprenorphine (a mu-opioid receptor agonist), or diazepam (a benzodiazepine). The present study suggests that facial expressions in reserpine-induced myalgia rats simulate non-organic pain occurring spontaneously in patients with fibromyalgia. This finding achieves a coordination of pain measures between the animal model and patients with fibromyalgia and would improve the translation of analgesic efficacies between them.

Use of the Rat Grimace Scale to Evaluate Visceral Pain in a Model of Chemotherapy-Induced Mucositis.

Simple SummaryMucositis is a painful and often debilitating condition associated with cancer treatment. Management of associated symptoms is an important clinical consideration. Animal models are used in mucositis research to model the condition in humans in order to develop novel therapeutic agents to relieve symptoms. Previous animal studies have focused on disease severity and outcomes, but often failed to measure pain. The rat grimace scale (RGS) is a validated observational measure used to gauge pain levels experienced by rats. The aim of this study was to assess the rat grimace scale in a rat model of mucositis, and to examine whether changes in clinical signs and anxiety reflected the grimace responses recorded. We also aimed to determine whether the responses were pain-specific by administering potent opioid painkilling agents. In the present study rat grimace scores did not change significantly between treatments. Development of reliable pain assessment methods in animal models is urgently required to improve model relevance to human clinical practice, in addition to safeguarding animal welfare. The rat grimace scale (RGS) is a measure of spontaneous pain that evaluates pain response. The ability to characterize pain through a non-invasive method has considerable utility for numerous animal models of disease, including mucositis, a painful, self-limiting side-effect of chemotherapy treatment. Preclinical studies investigating novel therapeutics for mucositis often focus on pathological outcomes and disease severity. These investigations fail to measure pain, in spite of reduction of pain being a key clinical therapeutic goal. This study assessed the utility of the RGS for pain assessment in a rat model of mucositis, and whether changes in disease activity index (DAI) and open field test (OFT) reflected the grimace responses recorded. Sixty tumor-bearing female Dark Agouti rats were injected with either saline or 5-Fluourouracil alone, or with co-administration of opioid analgesics. Whilst differences in DAI were observed between treatment groups, no difference in RGS scores or OFT were demonstrated. Significant increases in grimace scores were observed across time. However, whilst a statistically significant change may have been noted, the biological relevance is questionable in terms of practical usage, since an observer is only able to score whole numbers. Development of effective pain assessment methods in animal models is required to improve welfare, satisfy regulatory requirements, and increase translational validity of the model to human patients.

Antinociceptive effects of the adenylyl cyclase inhibitor ST034307 on tooth-movement-induced nociception in rats

ObjectiveThis study aimed to investigate the antinociceptive effects of the selective adenylyl cyclase type 1 (AC1) inhibitor ST034307 on tooth movement nociception through orofacial nociceptive behavior tests and molecular examination. MethodsWe placed fixed nickel-titanium alloy closed-coil springs around the incisors of male Sprague-Dawley rats to induce tooth movement. We subsequently administered ST034307 (3 mg/kg), for 2 days, intraperitoneally, and then subjected the rats to a battery of behavioral tests (n = 10/group) to assess orofacial nociception. The changes in the expression of key molecules in the anterior cingulate cortex were measured by ELISA (n = 8/group) and Western blotting (n = 8/group). ResultsTooth movement increased face-grooming activities and rat grimace scale scores. Tooth movement was also associated with enhanced cyclic adenosine monophosphate (cAMP) generation as well as protein kinase A (PKA) activation. Moreover, the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and expression of N-methyl-d-aspartate (NMDA) receptors in the anterior cingulate cortex increased during tooth movement. ST034307 significantly decreased mouth wiping and rat grimace scale scores, accompanied by reductions in cAMP generation, PKA activation, AMPA receptor phosphorylation, and NMDA receptor expression in the anterior cingulate cortex. ConclusionsThese results suggest that adenylyl cyclase type 1 plays an important role in the development of orthodontic tooth movement nociception. Furthermore, ST034307 can be used as an effective pharmacotherapy for orthodontic nociception.

Grading facial expression is a sensitive means to detect grimace differences in orofacial pain in a rat model

Although pre-clinical models of pain are useful for defining relationships between biological mechanisms and pain, common methods testing peripheral sensitivity do not translate to the human pain experience. Facial grimace scales evaluate affective pain levels in rodent models by capturing and scoring spontaneous facial expression. But, the Rat Grimace Scale (RGS) has not assessed the common disorder of temporomandibular joint (TMJ) pain. A rat model of TMJ pain induced by jaw loading (1 hr/day for 7 days) was used to investigate the time course of RGS scores and compare them between different loading magnitudes with distinct peripheral sensitivity profiles (0N–no sensitivity, 2N–acute sensitivity, 3.5N–persistent sensitivity). In the 3.5N group, RGS is elevated over baseline during the loading period and one day after loading and is correlated with peripheral sensitivity (ρ = −0.48, p = 0.002). However, RGS is not elevated later when that group exhibits peripheral sensitivity and moderate TMJ condylar cartilage degeneration. Acutely, RGS is elevated in the 3.5N loading group over the other loading groups (p < 0.001). These findings suggest that RGS is an effective tool for detecting spontaneous TMJ pain and that spontaneous pain is detectable in rats that develop persistent TMJ sensitivity, but not in rats with acute resolving sensitivity.

Application of the Rat Grimace Scale as a Marker of Supraspinal Pain Sensation after Cervical Spinal Cord Injury.

Experimental models of neuropathic pain (NP) typically rely on withdrawal responses to assess the presence of pain. Reflexive withdrawal responses to a stimulus are used to evaluate evoked pain and, as such, do not include the assessment of spontaneous NP nor evaluation of the affective and emotional consequences of pain in animal models. Additionally, withdrawal responses can be mediated by spinal cord reflexes and may not accurately indicate supraspinal pain sensation. This is especially true in models of traumatic spinal cord injury (SCI), wherein spastic syndrome, a motor disorder characterized by exaggeration of the stretch reflex that is secondary to hyperexcitability of the spinal reflex, can cause paroxysmal withdrawals not associated with NP sensation. Consequently, the aim of this study was to utilize an assessment of supraspinal pain sensation, the Rat Grimace Scale (RGS), to measure both spontaneous and evoked NP after a contusion SCI at cervical level 5 in adult male rats. Spontaneous and evoked pain were assessed using the RGS to score facial action units before and after the application of a stimulus, respectively. Rodents exhibited significantly higher RGS scores at week 5 post-injury as compared to baseline and laminectomy controls before the application of the stimulus, suggesting the presence of spontaneous NP. Additionally, there was a significant increase in RGS scores after the application of the acetone. These data suggest that the RGS can be used to assess spontaneous NP and determine the presence of evoked supraspinal pain sensation after experimental cervical SCI.

Local infiltration of the surgical wounds with levobupivacaine, dexibuprofen, and norepinephrine to reduce postoperative pain: A randomized, vehicle–controlled, and preclinical study

Postoperative pain may lead to poor wound healing, insomnia, and increased postoperative hospitalization. The objective of the study was to explore the effect of local infiltration of the surgical wound with levobupivacaine, dexibuprofen, and norepinephrine in postoperative pain. A randomized, parallel experimental design was applied in 40, 9–11–week–old Wister albino rats. A laparotomy was performed in all groups of 10 rats each. During surgery, the sutured muscle was infiltrated with 40μL of a normal saline (vehicle group), a normal saline containing 0.25% v/v levobupivacaine, 0.2mg/mL dexibuprofen, and 0.1mg/mL norepinephrine (treatment group) before skin closure. The same combination (negative control group) and a 10–fold higher dose (positive control group) were administered systematically. Rat Grimace Scale scores, based on facial expression, 24h after suturing of the tissues, histopathology and tensile strength of the surgical wound, two weeks after suturing of the tissues were measured. The one–way ANOVA and the two–tailed paired t–test/the Dunnett multiple comparisons test were used to compare the Rat Grimace Scale scores for pain within the group and between groups. The Kruskal–Wallis test and the one–way ANOVA/the Dunnett multiple comparisons test were used to compare the histopathology study and the tensile strength. The difference for statistical analysis was considered significant at the 95% of confidence level. The mean Rat Grimace Scale score at 24h for the treatment, negative control, and positive control groups was 1.945 (p=0.0025; q=3.257), 1.1 (p=0.1), and 1.95 (p=0.0021 q=3.547) respectively. The reduction in postoperative pain started within 1h after the suturing of the tissues in the treatment group. There were significant difference for epidermal and dermal regeneration (p=0.043), granulation tissue thickness (p=0.025), and angiogenesis (p=0.002) between treatment group and vehicle group. Tensile strength for the treatment group was 0.82±0.013N/cm2(p=0.003; q=5.231). The rapid infiltration of surgical wounds with a low–dose levobupivacaine, dexibuprofen, and norepinephrine may reduce postoperative pain and increase the wound regeneration process.

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 role of periodontal ASIC3 in orofacial pain induced by experimental tooth movement in rats.

This study aimed to clarify the roles of Acid-sensing ion channel 3 (ASIC3) in orofacial pain following experimental tooth movement. Sixty male Sprague-Dawley rats were divided into the experimental group (40g, n = 30) and the sham group (0g, n = 30). Closed coil springs were ligated between maxillary incisor and molars to achieve experimental tooth movement. Rat grimace scale (RGS) scores were assessed at 0, 1, 3, 5, 7, and 14 days after the placement of the springs. ASIC3 immunostaining was performed and the expression levels of ASIC3 were measured through integrated optical density/area in Image-Pro Plus 6.0. Moreover, 18 rats were divided into APETx2 group (n = 6), amiloride group (n = 6), and vehicle group (n = 6), and RGS scores were obtained compared among them to verify the roles of ASIC3 in orofacial pain following tooth movement. ASIC3 expression levels became significantly higher in the experimental group than in sham group on 1, 3, and 5 days and became similar on 7 and 14 days. Pain levels (RGS scores) increased in both groups and were significantly higher in the experimental group on 1, 3, 5, and 7 days and were similar on 14 days. Periodontal ASIC3 expression levels were correlated with orofacial pain levels following experimental tooth movement. Periodontal administrations of ASIC3 antagonists (APETx2 and amiloride) could alleviate pain. This study needs to be better evidenced by RNA interference of ASIC3 in periodontal tissues in rats following experimental tooth movement. Moreover, we hope further studies would concentrate on the pain perception of ASIC3 knockout (ASIC3-/-) mice. Our results suggest that periodontal ASIC3 plays an important role in orofacial pain induced by experimental tooth movement.

The relationship between the Rat Grimace Scale and mechanical hypersensitivity testing in three experimental pain models.

The assessment of facial expressions associated with pain has been used to evaluate pain in humans and has recently found application in non-human mammals. These so called 'grimace scales' have the potential to be developed into a widely accepted non-invasive method of measuring pain in laboratory rodents. Currently, common methodologies to assess pain rely on nociceptive tests that assess stimulus evoked withdrawal responses. These tests, however, are limited to the assessment of a reflexive response without an affective component. This study aimed to use the recently developed Rat Grimace Scale (RGS) and assess its relationship with a conventional nociceptive test (the application of von Frey filaments). Fifty-two adult, male Wistar rats were randomized to one of five treatment groups: intraplantar carrageenan, intraplantar complete Freund's adjuvant (CFA), plantar incision, anaesthetic control and saline injection control. The RGS and response to mechanical hypersensitivity testing was evaluated at predetermined time points before and after treatment until withdrawal responses returned to baseline levels. The RGS score significantly increased in all pain models. The peak RGS score also coincided with the development of paw hypersensitivity. However, mechanical hypersensitivity persisted after RGS scores returned to baseline. This study confirms that the three pain models induce pain in rodents and showed that peak pain coincided with peak mechanical hypersensitivity. However, mechanical hypersensitivity remained once pain subsided, mimicking the human experience of CFA injection. These findings further our understanding of the roles of, and relationship between, these assays in the assessment of nociception and pain.

G0200305 セメントレス人工股関節における大腿部痛の原因究明

A cause of thigh pain after cementless THA is still uncertain. The final goal of this study is clear up the cause for thigh pain and the development of painless cementless stem. For that, the objective of this paper is to make the animal model of thigh pain and to establish the method of evaluating the pain. We made special pin that pushes from the inside and expands the rat bone. The rat that the pin was implanted was bred with the cage. The loadcells were arranged on four corners of the cage in order to calculate rat's dynamics parameters, namely, rat's position, stay rate, total movement distance, crosspoint and errorpoint. There is the resulting rat grimace scale score (RGS score) as an index that evaluates the pain that the rat feels. RGS score is a method of evaluating the pain from the rat's facial expression. On the other hand, the method of the proposal in this study is a method of evaluating the pain from the activity of the rat. This new index shall be designated as "the resulting rat activity scale score (RAS score)". RAS is more objective and more quantitative.

Psychometric assessment of the Rat Grimace Scale and development of an analgesic intervention score.

Our limited ability to assess spontaneous pain in rodent models of painful human conditions may be associated with a translational failure of promising analgesic compounds in to clinical use. If measurement of spontaneous pain behaviours can be used to generate an analgesic intervention score their use could expand to guide the use of analgesics, as mandated by regulatory bodies and ethical and welfare obligations. One such measure of spontaneous pain, the Rat Grimace Scale (RGS), has recently been described and shown to exhibit reliability. However, reliability of measurement scores is context and content specific, and further testing required to assess translation to a heterogenous setting (different model, raters, environment). The objectives of this study were to perform reliability testing with the Rat Grimace Scale in a heterogenous setting and generate an analgesic intervention score for its use. In a randomised, blinded study, sixteen adult female rats received one of three analgesia treatments (0.05 mg/kg buprenorphine subcutaneously, 1 mg/kg meloxicam subcutaneously, 0.2 mg/kg oral buprenorphine in jelly) peri-operatively (telemetry unit implantation surgery). Rats were video-recorded (before, 1–6 and 12 hours post-operatively) and images collected for independent scoring by three blinded raters using the RGS, and five experts based on “pain/no pain” assessment. Scores were used to calculate inter- and intra-rater reliability with an intraclass correlation coefficient and generate an analgesic intervention score with receiver operating characteristic curve analysis. The RGS scores showed very good inter- and intra-rater reliability (0.85 [0.78–0.90 95% CI] and 0.83 [0.76–0.89], respectively). An analgesic intervention threshold of greater than 0.67 was determined. These data demonstrate that the RGS is a useful tool which can be successfully employed in a heterogenous setting, and has the potential to guide analgesic intervention.