Comparison of characteristics and enzymatic products of leukocytes in the skin and laminar tissues of horses administered black walnut heartwood extract or lipopolysaccharide.

This research is a comparative study on the leukocyte behavior and enzymatic byproducts in the skin and laminar tissues of horses, post administering black walnut heartwood extract (BWHE) and lipopolysaccharide (LPS). The findings suggest that BWHE administration leads to an increase in CD13-positive leukocyte numbers and MMP-9 expression and activity in horses’ laminae and skin, implying the development of laminitis involves mechanisms beyond endotoxemia-induced leukocyte activation.

Objective and Methodology

• The primary aim of the study was to compare the characteristics and enzymatic products of leukocytes in the skin and the laminae of horses after administering BWHE and LPS.
• The study included 25 healthy horses, aged between 5 to 15 years. These horses were divided into three groups, with each group receiving a different form of treatment: LPS, BWHE, or water (acts as control group).
• The horses were euthanized twelve hours post-treatment or when Obel grade 1 lameness was observed. Skin and laminar tissue samples were then collected for analysis.

Experimental Procedures and Techniques

• Leukocyte movement from blood vessels into tissues or ‘leukocyte emigration’ was determined through CD13 immunohistochemical analysis.
• Expression and activities of matrix metalloproteinases (MMP-2 and MMP-9), enzymes that break down extracellular matrix in normal physiological processes, such as tissue remodeling, as well as disease processes like arthritis, was assessed via reverse transcription PCR assays and gelatin zymography – a technique used to detect hydrolytic enzymes, based on substrate degradation.

Findings and Conclusion

• Significantly higher numbers of CD13-positive cells and increased MMP-9 gene expression and activity were observed in the tissues of horses receiving BWHE compared to the other two groups.
• The LPS-treated horses did not show any increase in comparison to the control group.
• The results suggest that BWHE administration leads to increases in CD13-positive leukocyte numbers and MMP-9 expression and activity in laminar tissue and skin in horses, while similar effects were not observed following LPS administration.
• This discovery suggests that the underlying mechanisms of laminitis development go beyond leukocyte activation caused by endotoxemia alone, highlighted by the differing leukocyte behaviors in horses post BWHE and LPS administration.