History, Rest and Exercise Score (HRE-S) for assessment of disease severity in horses with trigeminal-mediated headshaking.

BACKGROUND: In horses with trigeminal-mediated headshaking (TMHS), clinical signs are likely to be expression of neuropathic facial pain. Currently, subjective assessment of disease severity is used as measure of compromise of animal's welfare. OBJECTIVES: To develop and validate a precise scoring system for TMHS: History, Rest and Exercise Score (HRE-S). The HRE-S consists of three subscores: history score (H-S), resting score (R-S) and exercise score (E-S). STUDY DESIGN: Retrospective observational study. METHODS: Seven masked observers with different experience used HRE-S to score 40 video recordings taken during rest and lungeing including five duplicates. Video recordings were taken from nine horses with TMHS and three controls. Inter- and intraobserver reliability and practicability of HRE-S were assessed. For every video recording severity of clinical signs was graded by every observer using an intuitive global-type-scale and interobserver reliability was calculated. Convergent validity was evaluated comparing HRE-S to groups created by an existing score (grade 0-3). Discriminant validity was analysed comparing HRE-S to groups created by intuitive global-type-scale. RESULTS: Reliability for HRE-S was excellent, irrespective of observers experience: Spearman's Rho = 0.946, p < 0.001 (intraobserver reliability) and intraclass correlation coefficient = 0.98, p < 0.001 (interobserver reliability). Interobserver reliability for intuitive global-type-scale was fair to substantial: Fleiss' κappa = 0.48 (R-S) -0.63 (E-S). Groups created by intuitive global-type-scale had significantly different R-S and E-S (p < 0.05), demonstrating discriminant validity. Convergent validity was proven as horses with grade 3/3 had significantly higher average E-S and total scores compared with an existing score than those with grade 0/3 or 1/3 (p < 0.001). MAIN LIMITATIONS: Retrospective nature, video recordings, sample size. CONCLUSIONS: HRE-S is a valid and reliable score evaluating disease severity in TMHS, independent of observers' experience.

Expression of Cannabinoid Receptors in the Trigeminal Ganglion of the Horse.

Cannabinoid receptors are expressed in human and animal trigeminal sensory neurons; however, the expression in the equine trigeminal ganglion is unknown. Ten trigeminal ganglia from five horses were collected post-mortem from an abattoir. The expression of cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and the cannabinoid-related receptors like transient receptor potential vanilloid type 1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARÉ£), and G protein-related receptor 55 (GPR55) in the trigeminal ganglia (TG) of the horse were studied, using immunofluorescence on cryosections and formalin-fixed paraffin-embedded (FFPE) sections. Neurons and glial cells were identified using fluorescent Nissl staining NeuroTrace® and an antibody directed against the glial marker glial fibrillary acidic protein (GFAP), respectively. Macrophages were identified by means of an antibody directed against the macrophages/microglia marker ionized calcium-binding adapter molecule 1 (IBA1). The protein expression of CB1R, CB2R, TRPV1, and PPARÉ£ was found in the majority of TG neurons in both cryosections and FFPE sections. The expression of GPR55 immunoreactivity was mainly detectable in FFPE sections, with expression in the majority of sensory neurons. Some receptors were also observed in glial cells (CB2R, TRPV1, PPARγ, and GPR55) and inflammatory cells (PPARγ and GPR55). These results support further investigation of such receptors in disorders of equine trigeminal neuronal excitability.

Expression of annexin-1 in equine leucocytes and the effects of the N-terminal annexin-1 peptide, Ac2-26, on equine neutrophil superoxide production.

N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil superoxide production was measured by the reduction of cytochrome (cyt) C following stimulation with Ac2-26 and the formyl peptide receptor (FPR) agonists, FMLP, WKYMVm and WKYMVM. Responses were examined in the presence of the pan-FPR antagonist, BOC-2, and the role of p42/44 MAPK in agonist-induced effects was determined using PD98059. The effect of Ac2-26 on superoxide production in response to serum-treated zymosan (STZ) was also investigated, and the roles of FPR and p42/44 MAPK ascertained. Annexin-1 was detected in both equine neutrophils and mononuclear cells using a polyclonal rabbit anti-human annexin-1 antibody. Ac2-26 (5x10(-5)M) induced superoxide production in cytochalasin B-primed (48+/-8 versus 21+/-9 (unstimulated cells) nmol cyt C/10(6) neutrophils) and un-primed cells (37+/-10 versus 11+/-5 nmol cyt C/10(6) neutrophils). FMLP and WKYMVm, but not WKYMVM, also caused superoxide production in primed neutrophils, suggesting the response was mediated by FPR receptor binding. This was supported by the marked inhibitory effect of BOC-2 on the responses to Ac2-26 and FMLP although, interestingly, the effects of WKYMVm were not significantly reduced (50+/-5 (WKYMVm) versus 45+/-5 (WKYMVm+BOC-2) nmol reduced cyt C/10(6) neutrophils). Inhibition of p42/44 MAPK activation with PD98059 significantly attenuated superoxide production in response to Ac2-26, FMLP and WKYMVm and Western blotting showed that Ac2-26 induced p42/44 MAPK activation. At a concentration which did not cause superoxide production, Ac2-26 (10(-5)M) significantly reduced the response to STZ (84+/-17% inhibition). This inhibitory effect was attenuated by both BOC-2 and PD98059. These results suggest that if activation of equine leucocytes in vivo leads to the release and subsequent cleavage of annexin-1, the N-terminal peptides formed could bind to neutrophil FPR and decrease free radical production in response to particulate stimuli. This could help to reduce local tissue damage but, as Ac2-26 can also stimulate superoxide production at higher concentrations in an FPR-dependent manner, the amount of free radical production may depend on the concentration of peptide present.