Immunohistochemical determination of the expression of endothelin receptors in bronchial smooth muscle and epithelium of healthy horses and horses affected by summer pasture-associated obstructive pulmonary disease

To immunohistochemically determine the expression of endothelin (ET) receptors in bronchial smooth muscle and epithelium of healthy horses and horses affected by summer pasture-associated obstructive pulmonary disease (SPAOPD). Tissue specimens obtained from 8 healthy and 8 SPAOPD-affected horses. Horses were examined and assigned to healthy and SPAOPD groups. Horses were then euthanatized, and tissue specimens containing bronchi of approximately 4 to 8 mm in diameter were immediately collected from all lung lobes, fixed in zinc-formalin solution for 12 hours, and embedded in paraffin. Polyclonal primary antibodies against ET-A or ET-B receptors at a dilution of 1:200 and biotinylated IgG secondary antibodies were applied to tissue sections, followed by the addition of an avidin-biotin immunoperoxidase complex. Photographs of the stained slides were digitally recorded and analyzed by use of image analysis software to determine the intensity of staining. Two-way ANOVA was used for statistical analysis. The left diaphragmatic lung lobe of SPAOPD-affected horses had a significantly greater area of bronchial smooth muscle that immunostained for ET-A, compared with that for healthy horses. All lung lobes of SPAOPD-affected horses, except for the right diaphragmatic lobe, had significantly greater staining for ET-B receptors in bronchial smooth muscle, compared with results for healthy horses. This study revealed overexpression of ET-A and, in particular, ETB receptors in the bronchial smooth muscle of SPAOPD-affected horses, which suggested upregulation of these receptors. These findings improve our understanding of the role of ET-1 in the pathogenesis of SPAOPD.