Equine neutrophil elastase in plasma, laminar tissue, and skin of horses administered black walnut heartwood extract

Laminitis is a local manifestation of a systemic inflammatory response that is characterized by neutrophil activation and movement of neutrophils into the laminar tissues. Given the evidence for the involvement of neutrophils in the development of laminitis, we measured concentrations of neutrophil elastase, a serine protease released from the azurophilic granules of neutrophils, in plasma, skin and laminar tissues obtained from control horses and horses given black walnut heartwood extract (BWHE) to induce laminitis. Healthy horses (5–15 years old) were randomly assigned to 4 groups: 3 experimental groups given BWHE via nasogastric tube, and a control group given an equal volume of water. The experimental groups consisted of horses euthanized 1.5 h (n = 5), 3 h (n = 6) or 12 h (n = 10) after BWHE administration. Control horses (n = 7) were euthanized 12 h after intragastric administration of water. Plasma samples were collected in all horses of the control and 12 h BWHE groups at 0, 1, 2, 3, 4, 6, 8, 10, and 12 h after treatment, and laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia in all 1.5 and 3 h BWHE horses, in 6 of the 12 h BWHE horses and in 5 of the control horses. Plasma and tissue concentrations of neutrophil elastase were determined using an equine specific ELISA, and statistical significance was set at p < 0.05. Plasma concentrations of neutrophil elastase in the BWHE group were significantly higher at 6 and 8 h compared to the control group and at 8 and 10 h compared to time 0. Concentrations of neutrophil elastase in skin and laminar tissue were significantly higher in the 3 and 12 h BWHE groups compared to the control group. Concentrations of neutrophil elastase were significantly higher in the skin than in the lamina in the 12 h BWHE horses. The administration of BWHE thus results in significant increases in the concentration of neutrophil elastase in the circulation, skin and laminar tissue. These results confirm a role for neutrophils in the developmental phase of laminitis, and the systemic nature of the inflammatory process. Furthermore, neutrophil elastase may play a key role in the disintegration of the hoof basal membrane and be a target for the development of new treatments for laminitis.