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Background: Hypoadiponectinaemia is a risk factor for endocrinopathic laminitis, but the directionality and nature of its association with insulin dysregulation is unclear., Objectives: To investigate the effects of short-term induced hyperinsulinaemia and dexamethasone challenge on circulating [total adiponectin] and whole blood expression of adiponectin (AdipoR1 and AdipoR2), insulin, and insulin-like growth factor 1 (IGF-1) receptors in insulin-sensitive ponies., Study Design: In vivo experiment., Methods: Six never-laminitic, insulin-sensitive, native-breed UK ponies first underwent a dexamethasone challenge (0.08 mg/kg i.v.) with blood samples collected every 15 min over 3 h. After a 14-day washout period, hyperinsulinaemia was induced for 9 h via a euglycaemic-hyperinsulinaemic clamp (EHC), with blood samples collected every 30 min. Serum [insulin], plasma [total adiponectin], and plasma [IGF-1] were measured using validated assays and receptor gene expression was assessed via quantitative polymerase chain reaction (qPCR). Finally, whole blood was incubated with 10-1000 ng/mL dexamethasone for 3 h at 37°C to investigate its direct effects on gene expression., Results: There were no adverse effects observed during either protocol. Dexamethasone challenge did not alter circulating [insulin] or [total adiponectin] at any time-point, but significantly upregulated AdipoR1 and IGF-1R expression at 150 and 180 min. Ex vivo incubation of whole blood with dexamethasone did not alter expression of the genes examined. There was no change in [total adiponectin] or expression of the genes examined associated with EHC-induced hyperinsulinemia., Main Limitations: This was a small sample size that included only native-breed ponies; total adiponectin was measured rather than high-molecular-weight adiponectin., Conclusions: Short-term induced hyperinsulinaemia and dexamethasone challenge did not affect circulating [total adiponectin] in insulin-sensitive ponies. However, dexamethasone administration was associated with upregulation of two receptors linked to adiponectin signalling, suggesting that a physiological response occurred possibly to counteract dexamethasone-associated changes in tissue insulin sensitivity., (© 2023 MARS Horsecare and The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Microvesicles are small (up to 1 μm) vesicles found in plasma and other bodily fluids. They are recognised as part of the normal system of inter-cellular communication but altered numbers are also used as biomarkers of disease. Microvesicles have not been studied in detail in the horse but may be relevant to diseases such as laminitis. Identification of equine cell specific microvesicles was performed by developing a panel of cross reactive antibodies to use in flow cytometry to detect microvesicles of platelet, leucocyte and endothelial origin in plasma from healthy ponies and those predisposed to laminitis. The total number and proportion of microvesicles from the different cell types varied with season and there were more annexin V positive endothelial MV in non laminitic ponies compared to previously laminitic ponies. Development of this antibody panel and the technique for measuring microvesicles in the horse opens a new field for further investigation of these important structures in equine health and disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Certain individuals appear to be predisposed to recurrent pasture-associated laminitis. Previous studies have predominantly investigated risk factors only after disease occurrence., Objectives: To investigate risk factors for pasture-associated laminitis prior to disease occurrence., Study Design: Prospective cohort study., Methods: Non-laminitic ponies aged ≥7 years were recruited. Body condition score (BCS), height, weight, crest height and thickness were measured and an overnight dexamethasone suppression test performed. Plasma or serum adiponectin, leptin, triglyceride, basal insulin, insulin post-dexamethasone, insulin-like growth factor 1 (IGF-1), IGF binding protein 1 (IGFBP-1), IGFBP-3, C-reactive protein, von Willebrand's factor, soluble E-selectin and P-selectin concentrations were assayed. Follow-up data were obtained from owners annually for 3 years to ascertain occurrences of veterinarian-diagnosed pasture-associated laminitis. Data were analysed by multivariate logistic regression. Receiver operating characteristic (ROC) curves analysis was performed for significant risk factors and cut-off values determined., Results: A total of 446 animals with a median (interquartile range) age of 15 (10-20) years were recruited. Of these, 50.4% were mares and 49.6% were geldings. The most common breeds were Welsh (36.4%), Shetland (17.0%) and cob (9.4%). Overall, 72.2% of animals were overweight/obese (BCS 7-9/9), 27.3% were of ideal weight (BCS 4-6/9) and 0.5% were underweight (BCS 1-3/9). After 1, 2 and 3 years, respectively, 18 (4.0%), 30 (6.7%) and 44 (9.9%) animals were reported to have had laminitis. Plasma adiponectin, and serum basal (insulin) and (insulin) post-dexamethasone levels were significantly (P ≤ 0.05) associated with laminitis occurrence cumulatively after 1, 2 and 3 years. Use of the area under the ROC curves to distinguish animals that did and did not develop laminitis showed good (basal [insulin] after 1 year), fair (all others) or poor ([insulin] post-dexamethasone) levels of accuracy., Main Limitations: Animals were evaluated at a single time point and biomarkers were assayed using single assays., Conclusions: Risk factors for future laminitis prior to disease occurrence include low plasma adiponectin and high serum basal insulin or insulin post-dexamethasone concentrations., (© 2016 EVJ Ltd.)

Reasons for Performing Study: Plasma fructosamine concentration ([fructosamine]) is believed to reflect medium term, average blood glucose concentration and in a previous study was higher in horses with active laminitis than in normal horses. Pituitary pars intermedia dysfunction (PPID) is associated with hyperglycaemia and laminitis., Objectives: To test the hypotheses that: [fructosamine] is higher in PPID cases than normal animals; furthermore, that within cases of PPID [fructosamine] is higher in those with active laminitis than nonlaminitic cases and in cases that have been affected by active laminitis in the preceding year than those that have not., Study Design: Observational, case-control/cross-sectional study., Methods: [Fructosamine] was measured in cases of PPID (n = 46) and normal animals (n = 139). A normal range was calculated; values were compared between the 2 groups and within the PPID group, between cases with and without active laminitis and between cases that had and had not been affected by active laminitis in the preceding year., Results: In normal animals mean [fructosamine] was 248.7 μmol/l; the normal range (mean ± 2 s.d.) was 195.5-301.9 μmol/l. Plasma [fructosamine] was not higher in PPID cases than in normal animals. In PPID cases, [fructosamine] was significantly (P = 0.006) higher in cases with active laminitis (mean ± s.d. 261.2 ± 39.2 μmol/l) compared with those without active laminitis (234.5 ± 32.9 μmol/), but [fructosamine] was not higher in cases that had been affected by active laminitis in the preceding year than those that had not., Conclusions: In horses affected by PPID, [fructosamine] is higher in cases with active laminitis than in cases without active laminitis., Potential Relevance: Clinical use of [fructosamine] is limited due to overlap with the normal range. Further studies into glucose and protein metabolism in cases of PPID are warranted., (© 2013 EVJ Ltd.)

Reasons for Performing Study: The mechanisms underlying individual animal predisposition to pasture-associated laminitis remain unclear; however, chronic inflammation is implicated., Objectives: To identify differences in the inflammatory profile of a group of previously laminitic ponies compared with control animals at pasture in late spring and winter., Methods: Previously laminitic (PL; n = 38 and 42) and nonlaminitic control ponies (NL; n = 41 and 39) were sampled in late spring and winter. Body condition score, height, weight and crest height and thickness were measured. Plasma concentrations of tumour necrosis factor-α, serum amyloid A, haptoglobin, insulin, adiponectin, triglyceride, fibrinogen, interleukin-17, interleukin-4 and interferon-γ were assayed by validated/standard methods. Factors independently associated with each cytokine were determined by multivariate analysis., Results: Plasma [adiponectin] was significantly influenced by laminitis status, being lower in PL (median [interquartile range] 2.1[1.4-3.2] μg/l) than in NL ponies (3.4 [2.6-4.1] μg/l; P<0.0001). No other cytokines or inflammatory markers were associated with laminitis status. Plasma fibrinogen and serum amyloid A concentrations were significantly (P = 0.04 and P = 0.01) higher in geldings (3.5 [3.0-4.0] g/l; 2.2 [0.5-3.6] mg/l) than in mares (3.0 [3.0-4.0] g/l; 1.5 [0.4-2.1] mg/l) and significantly (P = 0.04 and P<0.001) higher in winter (3.5 [3.0-4.0] g/l; 2.5 [0.9-3.6] mg/l) than in late spring (3.0 [3.0-3.5] g/l; 1.1 [0.3-1.9] mg/l). Serum haptoglobin concentration showed the same significant (P<0.001) seasonal difference (winter 2.1 [1.6-2.6 g/l; late spring 1.8 1.4-2.4 g/l) and was significantly (P = 0.01) inversely associated with weight. Serum interleukin-4 concentration was significantly (P<0.0001) higher in winter (2.0 [1.2-3.0] ng/l) than in late spring (0.0 [0.0-0.0] ng/l). Serum insulin concentration was significantly (P = 0.02) influenced by season (winter 31.7 [9.6-43.5] miu/l; late spring 84.0 [7.0-131.0] miu/l). Plasma triglyceride concentration was significantly (P = 0.02) higher in PL (0.5 [0.3-0.7] mmol/l) than in NL ponies (0.4 [0.2-0.5] mmol/l)., Conclusions: There were significant effects of season, gender and bodyweight on a number of proinflammatory mediators or markers of inflammation. The only marker influenced by laminitis status was adiponectin, and concentrations of this anti-inflammatory marker were lower in previously laminitic animals., Potential Relevance: Recurrent laminitis may be associated with reduced anti-inflammatory capacity rather than a proinflammatory state., (© 2012 EVJ Ltd.)

Digital vasoconstriction, ischaemia and hypoxia may predispose to acute laminitis. Laminitis incidence varies seasonally, peaking in spring and summer. Direct seasonal influences on equine digital artery (EDA) contractility have not been investigated. This study assessed seasonal variation in maintenance of phenylephrine (PHE)-induced tone in isolated EDAs under hypoxic (95% nitrogen) and hyperoxic (95% oxygen) conditions. The objective was to measure change in arterial tone over time after constriction to a plateau with PHE. Tone was measured at plateau and over time and percentage change calculated. Hyperoxic EDAs maintained PHE-induced tone over 1 h with no seasonal variation. Hypoxic EDAs relaxed in fall (median [inter-quartile range] 59% [44-77%] decrease from plateau; P=0.008), contracted in spring (65% [20-192%] increase from plateau; P=0.03) and did not significantly change tone in winter (18% [0-28%] decrease; P=0.13). Continued contraction under hypoxic conditions in spring may contribute to digital vasoconstriction., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Identification of ponies (Equus caballus) at increased risk of pasture-associated laminitis would aid in the prevention of the disease. Insulin resistance has been associated with laminitis and could be used to identify susceptible individuals. Insulin resistance may be diagnosed by feeding supplementary water-soluble carbohydrate (WSC) and measuring blood glucose and insulin concentrations. The aim of this study was to assess the glycemic and insulinemic responses of 7 normal (NP) and 5 previously laminitic (PLP), mixed breed, native UK ponies fed glucose, fructose, and inulin [1 g/(kg·d) for 3 d] or no supplementary WSC (control) in spring and fall after a 7-d adaptation to a pasture or hay diet. Blood samples were taken for 12 h after feeding on each day, and baseline and peak concentrations and area under the curve (AUC) for glucose and insulin were recorded. Linear mixed models were used for statistical analysis. Differences between PLP and NP groups were most marked after glucose feeding with differences in peak glucose (P = 0.02) and peak insulin (P = 0.016) concentrations. Season and diet adaptation also affected results. Peak concentrations of glucose and insulin occurred 2 to 4 h after WSC feeding. Peak insulin concentration was greater and more variable in fall, particularly in PLP adapted to fall pasture. Baseline glucose and insulin concentrations varied between individuals and with season and diet adaptation but were not greater in PLP than NP. Insulin AUC was greater in PLP than NP after feeding both glucose and fructose (P = 0.017), but there were no differences between PLP and NP in glucose AUC. Glycemic and insulinemic changes were less (P ≤ 0.05) after feeding fructose than glucose, although differences between PLP and NP were still evident. Minimal changes in glucose and insulin concentrations occurred after inulin feeding. Measurement of peak insulin 2 h after feeding of a single dose of glucose (1 g/kg) may be a simple and practical way to aid identification of laminitis-prone ponies before the onset of clinical disease, particularly when ponies are adapted to eating fall pasture.

Certain individual animals appear predisposed to recurrent pasture-associated laminitis, but the exact mechanisms underlying their predisposition remain a fundamental question in laminitis research. The risk of endocrinopathic laminitis can be reduced if steps are taken to improve insulin sensitivity and reduce inflammation using strategies based on exercise and diet. Exercise has been shown to reduce insulin resistance and suppress inflammation, and dietary manipulation can significantly affect insulin sensitivity., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The endothelium plays a major role in the pathogenesis of endotoxemia. Binding of endotoxin (lipopolysaccharide; LPS) to endothelium initiates a range of signalling events, including activation of p38 mitogen activated protein kinases (MAPKs) that are involved in the initiation of inflammatory responses. In the present study we have examined whether clinically relevant concentrations of LPS can activate p38 MAPK in equine endothelial cells and have investigated the role of the kinase in neutrophil adhesion and mediator release. Cultured equine digital vein endothelial cells (EDVEC) were exposed to LPS and phosphorylation of p38 MAPK was assessed by Western blotting using phospho-specific antibodies. Neutrophil adhesion was quantified by assaying myeloperoxidase and the release of prostacyclin (PGI(2)) was measured by radioimmunoassay of its stable breakdown product 6-keto-PGF(1alpha). The effects of the p38 MAPK inhibitors, SB203580 and PD169316, on neutrophil adhesion and 6-keto-PGF(1alpha) release were examined, as was the effect of an anti-E-selectin antibody on neutrophil adhesion to LPS-activated EDVEC. LPS treatment significantly increased p38 MAPK phosphorylation, which was maximal after a 1h LPS incubation using a concentration of 10(-5)g/ml (EC(50) = 2 x 10(-7)g/ml). Neutrophil adhesion to LPS-stimulated endothelial cells (maximal at 10(-6)g/ml; EC(50) = 6 x 10(-10)g/ml) was significantly inhibited in the presence of p38 MAPK inhibitors (reduced from a maximum of 33+/-6% to 13+/-4% adhesion at 10(-6)M SB203580 and to 21+/-4% adhesion at 10(-6) M PD169316), or an anti-E-selectin antibody (reduced from 17+/-1% adhesion to 6+/-1% adhesion). 6-keto-PGF(1alpha) release was increased in a concentration-dependent manner following LPS exposure (maximal at 10(-6)g/ml; EC(50) = 1 x 10(-9)g/ml), and was significantly inhibited by p38 MAPK blockade (reduced from 1.6+/-0.3 x 10(-9)g/ml to 4+/-1 x 10(-10)g/ml and 4+/-2 x 10(-10)g/ml with 10(-6) M SB203580 or 10(-6) M PD169316, respectively). This study has demonstrated that clinically relevant concentrations of LPS activate p38 MAPK in equine endothelial cells and that both neutrophil adhesion to LPS-activated EDVEC and PGI(2) release are dependent upon p38 MAPK phosphorylation. These results reveal a key role for p38 MAPK in the response of the endothelium to LPS and suggest that inhibition of this kinase may reduce inflammatory events in the endotoxemic horse.

Reasons for Performing Study: Lipopolysaccharide (LPS) infusion reduces digital perfusion, but the mediators responsible remain undetermined., Objectives: To identify vasoconstrictor mediators released following LPS infusion and relate their appearance in plasma to digital blood flow alterations., Methods: Blood flow in the lateral digital vessels of 6 Thoroughbred horses, following a 30 min infusion of LPS (E. coli 055:B5; 30 ng/kg), was measured using Doppler ultrasonography. Concomitant measurements of hoof wall and coronary band surface temperatures (HWST and CBST) were made. Serial blood samples were collected and plasma LPS, tumour necrosis factor alpha (TNFalpha), 5-HT, thromboxane B2 (TxB2) and endothelin measured., Results: Plasma LPS concentrations reached a maximum of 13.2 pg/ml during the infusion, followed by an increase in plasma TNFalpha concentration. Digital arterial and venous blood flow decreased by 43 and 63%, respectively; HWST and CBST similarly decreased. Systemic blood pressure remained unaltered. Plasma concentrations of TxB2 and 5-HT increased, coinciding with the onset of digital hypoperfusion. Plasma endothelin concentrations remained unchanged., Conclusions: The temporal relationship between the onset of digital hypoperfusion and increases in plasma 5-HT and TxB2 concentrations is consistent with these platelet-derived mediators being associated with LPS-induced laminitis., Potential Relevance: These experimental data support the use of anti-platelet therapy in the prevention of laminitis associated with endotoxaemic conditions.

Reasons for Performing Study: Disturbances of digital blood flow are thought to be fundamental to the pathophysiology of acute laminitis. However, factors linking the initiating events in the equine hindgut with these disturbances in the foot remain to be determined., Hypothesis: Amine compounds, formed by bacteria in the equine hindgut, have digital vasoconstrictor effects in vivo., Methods: Tryptamine (1.6 microg/kg/min) and phenylethylamine (2.13 microg/kg/min) were infused i.v. into standing nonsedated horses. Digital blood flow was measured by Doppler ultrasound and foot surface temperature was monitored. Plasma 5-hydroxytryptamine (5-HT) concentrations were measured by HPLC., Results: Tryptamine and phenylethylamine infusions had no effect on systemic arterial blood pressure or heart rate, but caused significant decreases in digital arterial blood flow (mean +/- s.e. 29.2 +/- 8.5 and 18.4 +/- 6.8%, respectively). Both amines also caused decreases in dorsal hoof wall temperature (0.6 +/- 0.1 and 0.5 +/- 0.1 degrees C for tryptamine and phenylethylamine, respectively) and concomitant increases in plasma 5-HT concentration., Conclusions: Tryptamine and phenylethylamine caused reduction of digital blood flow, effects which may have been mediated, in part, via displacement of 5-HT from platelets., Potential Relevance: Amine compounds occurring in the equine hindgut, if released into the circulation following carbohydrate overload, could contribute to selective digital vasoconstriction. Further work in ponies and horses, with naturally occurring laminitis, is necessary to determine whether amines represent a therapeutic target in this disease.