Effect of endotoxin on leukocyte activation and migration into laminar tissue of isolated perfused equine limbs

Abstract

Objective-To investigate effects of endotoxin on leukocyte activation and infiltration of the laminar tissue in isolated perfused equine limbs. Sample-10 right forelimbs and 3 left forelimbs collected from 10 healthy adult horses after slaughter at a licensed abattoir. Procedures-Isolated right forelimbs were randomly assigned to 2 groups (5 forelimbs/group): perfusion of the distal portion for 10 hours with 80 ng of endotoxin/L and perfusion under the same conditions without endotoxin. After perfusion, samples for immunohistochemical detection of leukocytes (by use of antibodies against calprotectin and myeloperoxidase) and transmission electron microscopy were collected from the laminar tissue of the dorsal aspect of the hooves. Additionally, control samples were collected from the 3 nonperfused left forelimbs. Results-Samples of laminar tissue from the endotoxin perfusion group had significantly higher scores for calprotectin and myeloperoxidase staining than did control samples and samples perfused without endotoxin. Ultrastructural examination revealed endotoxininduced damage of the epidermal basal cells with loss of cell contacts including hemidesmosomes and anchoring filaments and a resulting separation of parts of the basement membrane. Additionally, local breakdown of the basement membrane was detected at the location of leukocyte adherence. Conclusions and Clinical Relevance-In isolated perfused equine limbs, endotoxin at a clinically relevant concentration induced a distinct inflammatory reaction with intravascular and extravascular accumulation of leukocytes in the laminar tissue, similar to that seen during the developmental phase of laminitis. Therefore, endotoxin should be considered as a causative factor for some types of laminitis.