Inhibition of lamellar separation caused by endotoxins by Polymyxin B in an ex vivo/ in vitro model of equine laminitis

Abstract

Pasture containing high non-structural carbohydrates (starch, sugar and fructan) is a common trigger factor of the development of laminitis. The horse cannot digest fructans directly in the small intestine; therefore they are rapidly fermented in the hindgut. This leads to a change of the conditions, which results in release of endotoxins. When endotoxins are absorbed into the bloodstream, this release can result in impaired circulation, particularly in the feet. Polymyxin B (PMB) is an antibiotic which can inhibit the effect of endotoxins. Therefore, the aim of our study was to test the effects of endotoxins on lamellar separation and to investigate if PMB can inhibit this effect. Hoof explants were cultured in 24 well plates at 37 °C and 5% CO₂ Different concentrations of LPS (lipopolysaccharides) of Escherichia coli O55:B5 (2.5–20 μg/ml), PMB (25–750 μg/ml) and LPS (10 and 20 μg/ml) combined with PMB were added to the explants. After 24 hours, explants were mechanically tested for its integrity. All explants cultured only in medium remained intact. In explants incubated with 10–40 μg/ml LPS, the number of separated explants was significantly higher than in explants incubated with medium (P< 0.01). Incubation of explants with 5, 2.5 and 1.25 μg/ml LPS had no influence on lamellar separation (P > 0.01). The effect of separation in explants cultured with 20 μg/ml LPS could be inhibited with 500 μg/ml PMB (P< 0.01). PMB concentrations from 100–750 μg/ml were able to inhibit lamellar separation induced by 10 μg/ml LPS (P< 0.001). In lower concentrations PMB was not able to inhibit the effect of 20 or 10 μg/ml LPS. Endotoxins seem to have an influence on the lamellar separation, but this effect can be inhibited by PMB.