BackgroundThe assessment of pain is critical for the welfare of horses, in particular when pain is induced by common management procedures such as castration. Existing pain assessment methods have several limitations, which reduce the applicability in everyday life. Assessment of facial expression changes, as a novel means of pain scoring, may offer numerous advantages and overcome some of these limitations. The objective of this study was to develop and validate a standardised pain scale based on facial expressions in horses (Horse Grimace Scale [HGS]).Methodology/Principal FindingsForty stallions were assigned to one of two treatments and all animals underwent routine surgical castration under general anaesthesia. Group A (n = 19) received a single injection of Flunixin immediately before anaesthesia. Group B (n = 21) received Flunixin immediately before anaesthesia and then again, as an oral administration, six hours after the surgery. In addition, six horses were used as anaesthesia controls (C). These animals underwent non-invasive, indolent procedures, received the same treatment as group A, but did not undergo surgical procedures that could be accompanied with surgical pain. Changes in behaviour, composite pain scale (CPS) scores and horse grimace scale (HGS) scores were assessed before and 8-hours post-procedure. Only horses undergoing castration (Groups A and B) showed significantly greater HGS and CPS scores at 8-hours post compared to pre operatively. Further, maintenance behaviours such as explorative behaviour and alertness were also reduced. No difference was observed between the two analgesic treatment groups.ConclusionsThe Horse Grimace Scale potentially offers an effective and reliable method of assessing pain following routine castration in horses. However, auxiliary studies are required to evaluate different painful conditions and analgesic schedules.

Highlights 1. There are conceptual and methodological problems for animal pain assessment 2. Facial expression scales are valid, reliable pain recognition and evaluation tools 3. Consistent and careful development of facial expression scales is required 4. Facial expression scales need feasibility testing to aid use in clinical practice 5. Guidelines are provided for development and use of facial expression scales Conceptual and methodological issues relating to pain assessment in mammals: the development and utilisation of pain facial expression scales.

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Simple SummaryAcute laminitis is a common equine disease characterized by intense foot pain. This work aimed to investigate whether the Horse Grimace Scale (HGS), a facial-expression-based pain coding system, can be usefully applied to assess pain associated with acute laminitis in horses at rest. Ten horses, referred as acute laminitis cases with no prior treatment, were assessed at the admission and at seven days after the initial evaluation and treatment. The authors found that the Horse Grimace Scale is a potentially effective method to assess pain associated with acute laminitis in horses at rest, as horses showing high HGS scores also exhibited higher Obel scores, and veterinarians classified them in a more severe painful state. AbstractAcute laminitis is a common equine disease characterized by intense foot pain, both acutely and chronically. The Obel grading system is the most widely accepted method for describing the severity of laminitis by equine practitioners, however this method requires movement (walk and trot) of the horse, causing further intense pain. The recently developed Horse Grimace Scale (HGS), a facial-expression-based pain coding system, may offer a more effective means of assessing the pain associated with acute laminitis. The aims of this study were: to investigate whether HGS can be usefully applied to assess pain associated with acute laminitis in horses at rest, and to examine if scoring HGS using videos produced similar results as those obtained from still images. Ten horses, referred as acute laminitis cases with no prior treatment, were included in the study. Each horse was assessed using the Obel and HGS (from images and videos) scales: at the admission (before any treatment) and at seven days after the initial evaluation and treatment. The results of this study suggest that HGS is a potentially effective method to assess pain associated with acute laminitis in horses at rest, as horses showing high HGS scores also exhibited higher Obel scores and veterinarians classified them in a more severe painful state. Furthermore, the inter-observer reliability of the HGS total score was good for both still images and video evaluation. There was no significant difference in HGS total scores between the still images and videos, suggesting that there is a possibility of applying the HGS in clinical practice, by observing the horse for a short time. However, further validation studies are needed prior to applying the HGS in a clinical setting.

Circulating microRNAs (miRNAs) are emerging as promising biomarkers for several disorders and related pain. In equine practice, acute laminitis is a common disease characterised by intense pain that severely compromises horse welfare. Recently, the Horse Grimace Scale (HGS), a facial expression-based pain coding system, was shown to be a valid welfare indicator to identify pain linked to acute laminitis. The present study aimed to: determine whether miRNAs can be used as biomarkers for acute pain in horses (Equus caballus) affected by laminitis; integrate miRNAs to their target genes and to categorise target genes for biological processes; gather additional evidence on concurrent validity of HGS by investigating how it correlates to miRNAs. Nine horses presenting acute laminitis with no prior treatment were recruited. As control group, nine healthy horses were further included in the experimental design. Samples were collected from horses with laminitis at admission before any treatment ('pre-treatment') and 7 days after routine laminitis treatment ('post-treatment'). The expression levels of nine circulating miRNAs, namely hsa-miR-532-3p, hsa-miR-219-5p, mmu-miR-134-5p, mmu-miR-124a-3p, hsa-miR-200b-3p, hsa-miR-146a-5p, hsa-miR-23b-3p, hsa-miR-145-5p and hsa-miR-181a-5p, were detected and assessed as potential biomarkers of pain by quantitative PCR using TaqMan® probes. The area under the receiver operating curve (AUC) was then used to evaluate the diagnostic performance of miRNAs. Molecular data were integrated with HGS scores assessed by one trained treatment and time point blind veterinarian. The comparative analysis demonstrated that the levels of miR-23b-3p (P=0.029), miR-145-5p (P=0.015) and miR-200b-3p (P=0.023) were significantly higher in pre-treatment and the AUCs were 0.854, 0.859 and 0.841, respectively. MiR-200b-3p decreased after routine laminitis treatment (P=0.043). Combining two miRNAs in a panel, namely miR-145-5p and miR-200b-3p, increased efficiency in distinguishing animals with acute pain from controls. In addition, deregulated miRNAs were positively correlated to HGS scores. Computational target prediction and functional enrichment identified common biological pathways between different miRNAs. In particular, the glutamatergic pathway was affected by all three miRNAs, suggesting a crucial role in the pathogenesis of pain. In conclusion, the dynamic expression of circulating miR-23b-3p, miR-145-5p and miR-200b-3p was detected in horses with acute laminitis and miRNAs can be considered potentially promising pain biomarkers. Further studies are needed in order to assess their relevancy in other painful conditions severely compromising horse welfare. An important implication would be the possibility to use them for the concurrent validation of non-invasive indicators of pain in horses.