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Endotoxin-induced changes of type VII collagen– cleaving matrix metalloproteinases in lamellar tissue of extracorporeally perfused equine limbs

Bianca Patan-Zugaj Dr Med Vet1, Felicia C. Gauff DVM, PhD2, Monika Egerbacher Dr Med Vet3, and Theresia F. Licka Dr Med Vet4,5
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  • 1 Equine University Clinic, Department for Companion Animals and Horses, University of Veterinary Medicine, A-1210 Vienna, Austria.
  • | 2 Equine University Clinic, Department for Companion Animals and Horses, University of Veterinary Medicine, A-1210 Vienna, Austria.
  • | 3 Institute of Anatomy, Histology and Embryology, University of Veterinary Medicine, A-1210 Vienna, Austria.
  • | 4 Equine University Clinic, Department for Companion Animals and Horses, University of Veterinary Medicine, A-1210 Vienna, Austria.
  • | 5 Department of Veterinary Clinical Studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin Midlothian, EH25 9RG, Scotland.

Abstract

OBJECTIVE To investigate the effect of lipopolysaccharide (LPS) on type VII collagen– cleaving matrix metalloproteinases (MMPs) in the lamellar tissue of extracorporeally perfused equine limbs.

SAMPLE 10 right forelimbs and 3 left forelimbs collected from 10 adult horses after slaughter at a licensed abattoir.

PROCEDURES Extracorporeal perfusion of the isolated equine limbs was performed for 10 hours under physiologic conditions (control-perfused limbs; n = 5) and with the addition of 80 ng of LPS/L of perfusate (LPS-perfused limbs; 5). Lamellar tissue specimens were then collected from the dorsal aspect of the hooves. Additionally, corresponding control specimens were collected from the 3 nonperfused left forelimbs. Immunohistochemical analysis was performed on paraffin-embedded tissue blocks with antibodies against total (latent and active) MMP-1, MMP-2, MMP-8, and MMP-9 as well as antibody against active MMP-9. Intensity of immunohistochemical staining was scored, and stain distribution in the lamellar tissue was noted.

RESULTS Staining intensity of total and active MMP-9 was significantly increased in LPS-perfused versus control-perfused limbs. No such difference was identified for MMP-1, MMP-2, and MMP-8.

CONCLUSIONS AND CLINICAL RELEVANCE Of the 4 MMPs that are capable of degrading type VII collagen, MMP-9 was the only one for which production increased in the lamellar tissue of isolated equine limbs perfused with versus without a clinically relevant concentration of LPS. These results suggested that MMP-9 may be involved in initiation of pathological changes in lamellar tissue in endotoxin-induced laminitis, whereas MMP-1, MMP-2, and MMP-8 may be less relevant.

Abstract

OBJECTIVE To investigate the effect of lipopolysaccharide (LPS) on type VII collagen– cleaving matrix metalloproteinases (MMPs) in the lamellar tissue of extracorporeally perfused equine limbs.

SAMPLE 10 right forelimbs and 3 left forelimbs collected from 10 adult horses after slaughter at a licensed abattoir.

PROCEDURES Extracorporeal perfusion of the isolated equine limbs was performed for 10 hours under physiologic conditions (control-perfused limbs; n = 5) and with the addition of 80 ng of LPS/L of perfusate (LPS-perfused limbs; 5). Lamellar tissue specimens were then collected from the dorsal aspect of the hooves. Additionally, corresponding control specimens were collected from the 3 nonperfused left forelimbs. Immunohistochemical analysis was performed on paraffin-embedded tissue blocks with antibodies against total (latent and active) MMP-1, MMP-2, MMP-8, and MMP-9 as well as antibody against active MMP-9. Intensity of immunohistochemical staining was scored, and stain distribution in the lamellar tissue was noted.

RESULTS Staining intensity of total and active MMP-9 was significantly increased in LPS-perfused versus control-perfused limbs. No such difference was identified for MMP-1, MMP-2, and MMP-8.

CONCLUSIONS AND CLINICAL RELEVANCE Of the 4 MMPs that are capable of degrading type VII collagen, MMP-9 was the only one for which production increased in the lamellar tissue of isolated equine limbs perfused with versus without a clinically relevant concentration of LPS. These results suggested that MMP-9 may be involved in initiation of pathological changes in lamellar tissue in endotoxin-induced laminitis, whereas MMP-1, MMP-2, and MMP-8 may be less relevant.

Contributor Notes

Address correspondence to Dr. Patan-Zugaj (bianca.patan-zugaj@vetmeduni.ac.at).