Acute laminitis occurs secondary to a variety of insults, including gastrointestinal diseases, pleuritis, retained placenta, carbohydrate overload, and exposure to shavings from black walnut trees.1 Results of recent studies2,3 indicate that peripheral WBCs become activated during the prodromal stage of acute laminitis induced by administration of an aqueous BWHE and leave the circulation. Concomitant with this leukopenic response are increases in leukocyte production of radical oxygen species, numbers of WBCs in laminar soft tissues, and expression of inflammatory mediators in the same tissues.3–5 These early inflammatory events precede the development of morphologic changes in the digit, including interstitial edema, epidermal cell necrosis, and separation and loss of the basement membrane, that characterize laminitis.6–10 Although the laminar tissue is a highly specialized tissue able to withstand large biomechanical forces, it remains part of the common integument, which includes the skin, ergots, and the chestnuts. Consequently, a broader examination of integument in horses given laminitisinducing substances may provide additional insight into the pathogenesis of the disease.
Normal equine integument contains few neutrophils,5,11 and these cells may be difficult to detect under pathologic conditions because they rapidly undergo apoptotic degeneration.10 However, the presence of these cells in tissues may be documented by monitoring the presence of MPO, a leukocyte-specific lysosomal enzyme.12 Stimulation of neutrophils leads to their activation and release of MPO, which catalyzes the production of reactive oxygen species that can cause local tissue damage, including disruption of the laminar basement membrane.13 Although MPO concentrations have been used as a marker of neutrophil presence in other equine tissues and plasma,12,14–17 concentrations of MPO in equine laminar tissue and skin have not, to our knowledge, been previously reported.
The purpose of the study reported here was to compare results obtained with 2 new laboratory techniques designed to monitor changes in MPO concentration or activity. These techniques were used to characterize temporal changes in MPO in plasma, laminar tissues, and skin obtained from control horses and horses given BWHE. Hypotheses of this study were that administration of BWHE results in increased concentration and activity of MPO in plasma, laminae, and skin, compared with values from control horses; that tissue concentrations of MPO in horses given BWHE increase after the onset of leukopenia; and that concentrations of MPO in the skin correlate with values in the laminar tissues in individual horses. We elected to study the effects of BWHE to be able to directly compare our results with the results of recently published studies3,4,18 that have implicated activation of peripheral WBCs in pathophysiologic causes of acute laminitis.
Black walnut heartwood extract
Specific immunologic extraction followed by enzymatic detection
Becton-Dickinson, Franklin Lakes, NJ
Glen Mills Inc, Clifton, NJ
Microplate wells, Thermo Labsystems, Waltham, Mass
Sigma Chemical Co, St Louis, Mo
Multiscan Ascent plate reader, Thermo Labsystems, Waltham, Mass
Molecular probes, Eugene, Ore
Fluoroscan Ascent instrument, Thermo Labsystems, Waltham, Mass
GraphPad Prism, version 3.03, GraphPad Software, San Diego, Calif.
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