Matrix metalloproteinase-9 in laminae of black walnut extract treated horses correlates with neutrophil abundance.

The research examines the correlation between the infiltration of white blood cells, specifically neutrophils, and a protein-digesting enzyme, matrix metalloproteinase-9 (MMP-9), in horse hoof tissue following the treatments with black walnut extract. It reveals that there is a strong correlation at the acute stage of laminitis, a hoof disease, with an increased presence of neutrophils and MMP-9 in the tissue, possibly giving rise to serious damage under certain conditions.

Understanding the Purpose and Approach

• The primary purpose of this study is to understand the relationship between the infiltration of specific white blood cells, particularly neutrophils, and the proteinase MMP-9 in the horse hoof tissue, called digital laminae, during both the early (prodromal) and intense (acute) stages of a hoof disease known as laminitis.
• The research involved treating horses with an aqueous solution derived from the heartwood of black walnut – a known inducer of laminitis – and collecting and examining samples of their hoof tissues.

Procedure and Techniques Used

• The hoof tissues were dissected at the onset of leukopenia (reduced white blood cells) and the start of observable lameness symptoms, mimic the course of laminitis.
• The tissue sections were scanned for neutrophils using immunohistochemistry with an anti-CD13 monoclonal antibody. In parallel, the same tissue extracts were assessed for MMP-9 activity through denaturing and non-denaturing gelatin zymography, a technique for identifying and measuring enzymes.
• Additionally, the researchers employed RT-qPCR, a common molecular biology technique, to check the gene expression level of both MMP-2 and MMP-9.

Findings and Outcomes

• The observations showed an absence of both MMP-9 and neutrophils in the control horse laminae. Conversely, evidence of higher levels of neutrophils and MMP-9 was found in the tissue samples from BWE-treated horses.
• This correlation was particularly noticeable at the acute stage of the disease when the MMP-9 gene expression was significantly increased, 15 times higher than normal.
• Increased MMP-2 gene or protein expression in the laminae was not observed following BWE treatment, stressing the specificity of MMP-9 in this case.
• The researchers noted that the majority of the MMP-9 detected in the laminae samples remained in its inactive form, suggesting that while this enzyme was accumulating, it did not directly contribute to the damage observed at these stages. Yet, the heightened presence of MMP-9 zymogen in the tissue increases the risk of possible catastrophic deterioration if conditions changed to activate these enzymes.