The integument, as an organ system, is composed of the avascular epidermis and the vascular dermis, which are intimately connected at the basement membrane. The hoof of horses is a specialized modification of the integumentary system and includes interdigitations of the dermis and epidermis that are termed laminae. These interdigitations dramatically increase surface area, dissipate the load, and increase the weight-bearing strength of the hoof.1,2 The findings of previous studies3,4 of acute laminitis in horses indicate that enzymatic degradation of the basement membrane of the hoof results in dermal-epidermal separation and the subsequent loss of the integrity of laminar interdigitations, which consequently compromises weight bearing.
Proteases such as MMP-2 and MMP-9 have been implicated in the disruption of the basement membrane that occurs during episodes of laminitis in horses.5–8 The MMPs are secreted in a latent (pro-MMP) form and require an activation step before they gain full enzymatic activity. Matrix metalloproteinase-2 is normally present in laminar tissue (constitutively produced by cells such as keratinocytes and fibroblasts) and is involved in growth and remodeling of the hoof. Matrix metalloproteinase-9 is produced by neutrophils, stored in tertiary granules, and released upon cellular activation.9 In recent studies10,11 of acute laminitis in horses, the presence of MMP-2 and MMP-9 in laminar tissue varies depending on whether the disease is naturally occurring or experimentally induced by means of carbohydrate overload or BWHE administration. Compared with findings in horses without laminitis, increases in MMP-2 and MMP-9 activities were detected in laminar tissue obtained from several horses with naturally occurring acute or chronic laminitis10 and in 2 horses with carbohydrate overload–induced laminitis.11 Tissues obtained from the latter 2 horses also had increased expression of MMP-2, although the authors did not indicate the Obel grade of lameness in the horses at the time that the samples were collected.12 In contrast, laminar tissue obtained from horses after administration of BWHE had increased expression and activity of MMP-9 but not MMP-2, compared with controls.6
Recent studies6,13,14 have revealed that activation of peripheral blood leukocytes is involved in the development of acute laminitis. Experimental induction of laminitis in horses via administration of BWHE has been associated with peripheral leukopenia13 followed by increases in myeloperoxidase and MMP-9 activities in both skin and laminar tissue.6,14 Both enzymes are produced in neutrophils, stored in cytoplasmic granules within those cells, and released upon cellular activation. Although changes in activities of those enzymes have been detected in horses following experimental induction of laminitis, changes associated with endotoxemia in horses have not been assessed, to our knowledge. Endotoxemia, a condition that is commonly associated with the development of laminitis under clinical conditions,15 is characterized by activation and emigration of leukocytes from the peripheral circulation.16
Because horses with diseases that are characterized by clinical evidence of endotoxemia (eg, colitis, enteritis of the proximal portion of the small intestine, and intestinal strangulation obstruction) appear to be at increased risk for development of laminitis17 and endotoxin has been detected in horses that develop laminitis subsequent to carbohydrate overload,18 there has been considerable interest in defining the relationship, if any, between endotoxemia and the development of laminitis. The purpose of the study reported here was to compare the characteristics and enzymatic products of leukocytes detected in the skin and laminar tissues of horses administered BWHE and horses administered purified LPS (endotoxin). Because experimentally induced endotoxemia and administration of BWHE result in leukopenia of similar severity and duration but only the latter induces clinical signs of laminitis, we hypothesized that there would be significant differences in the distribution and enzyme products of activated leukocytes in horses treated with BWHE or LPS. Consequently, we compared leukocyte emigration and MMP-2 and MMP-9 gene expressions and activities during the period in which horses developed Obel grade 1 lameness following administration of BWHE or LPS.
Black walnut heartwood extract
Pellet pestle, Kimble/Kontes, Vineland, NJ.
Versagene total RNA purification kit, Gentra Systems, Minneapolis, Minn.
High-capacity cDNA archive kit, Applied Biosystems, Foster City, Calif.
Taqman Custom Probe, Applied Biosystems, Foster City, Calif.
Primer Express software, Perkin-Elmer Biosystems, Waltham, Mass.
NCBI. Equine MMP-2/9 nucleotide sequence. Available at: www.ncbi.nlm.nih.gov/sites/gquery. Accessed March 8, 2008.
ABI Prism 7700 sequence detection system, Applied Biosystems, Foster City, Calif.
TaqMan Ribosomal RNA Control Reagent, Applied Biosystems, Foster City, Calif.
Polytron tissue homogenizer, Glen Mills Inc, Clifton, NJ.
Coomassie brilliant blue R-250 staining solution, Bio-Rad Laboratories, Richmond, Calif.
Bio-Rad Fluor-S MAX 2 MultiImager, Bio-Rad Laboratories, Richmond, Calif.
Mouse anti-horse CD13 monoclonal antibody, AbD Serotec, Raleigh, NC.
HRP-conjugated goat anti-mouse IgG, Pierce Biotechnology, Rockford, Ill.
SuperSignal West Dura extended duration chemiluminescence substrate solution, Pierce Biotechnology, Rockford, Ill.
Goat anti-mouse IgG, biotin conjugated, AbD Serotec, Raleigh, NC.
Quantity One quantitation software, Bio-Rad Laboratories, Richmond, Calif.
GraphPad Prism, version 3.03, GraphPad Software, San Diego, Calif.
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