The study of laminitis in horses has been hampered by the inability to serially characterize the progression of histologic changes and molecular events in the laminae of an individual affected horse. Histologically, stretching of the lamellae, dermal edema, hemorrhage, changes to the basal cells, presence of WBCs in the dermis, and signs of basement membrane detachment have been observed in specimens collected from horses euthanized during different stages of acute laminitis.1–4 Molecular events are similarly altered during the course of disease in experimentally induced laminitis.5–7 However, substantial variation from horse to horse evaluated within the same time frame has confounded attempts to characterize the sequence of early events without euthanasia of large numbers of experimental horses at various time points. Serial sampling of an individual horse would allow study of the patterns of events in early laminitis that may proceed at different rates among horses.
Laminar biopsy specimens have been collected through the hoof wall in standing animals.8–10 In cattle, tissues have been harvested from the sole-heel junction.9,10 Single laminar biopsy specimens have been collected in standing horses by use of a technique whereby the hoof wall was thinned with a drill and the biopsy specimen was obtained by cutting a square of tissue with a scalpel blade.8 The effects of collection of serial laminar biopsy specimens in horses have not been reported.
In laminitis, skin distant from laminae has molecular, biochemical, and leukocyte migration changes similar to laminae.11–13 Events in the skin appear to parallel events occurring in the relatively inaccessible lamellar tissue in experimental models.12 Results of a recent study14 indicate a lack of superoxide dismutase in equine laminar tissues, suggesting a tissue-specific susceptibility to damage by inflammatory leukocytes during laminitis. These recent findings support continued investigation into the similarities and differences between these closely related tissues.
We hypothesized that multiple biopsy specimens could be harvested from a single hoof during a period of 24 hours for morphologic examination and RNA recovery without inducing undue pain or causing morphologic changes in adjacent tissues that would be biopsied at later times. The purpose of the study reported here was to determine the feasibility of performing serial laminar and skin biopsies on an individual horse and whether sampling affected adjacent tissues.
AnaSed, 100 mg/mL, Lloyd Laboratories, Shenandoah, Iowa.
PromAce, 10 mg/mL, Fort Dodge Animal Health, Fort Dodge, Iowa.
Dormosedan, 10 mg/mL, Pfizer Animal Health, Exton, Pa.
Carbocaine, 2% (20 mg/mL), Pfizer Animal Health, Exton, Pa.
Esmark Bandage, McKesson Medical-Surgical Corp, Richmond, Va.
Dremel Lithium-Ion cordless drill, Dremel, Racine, Mich.
Miltex Dermal Biopsy Punch, 6 mm diameter, Miltex Inc, Bethpage, NY.
BD Beaver Mini-Blade Blades, 8D Ophthalmic Systems, Waltham, Mass.
Phenylbutazone tablets, The Butler Co, Dublin, Ohio.
Equi-Thane “Super-Fast” Instant Shoe, Vettec Hoof Care Products, Oxnard, Calif.
Tri-Reagent, Schering-Plough Animal Health, Kenilworth, NJ.
Mo Bio Laboratories Inc, Carlsbad, Calif.
Invitrogen, Carlsbad, Calif.
TaqMan, Applied Biosystems, Foster City, Calif.
BHQ-1, Biosearch Technologies, Novato, Calif.
Gluck Equine Research Center, University of Kentucky, Lexington, Ky.
Applied Biosystems, Foster City, Calif.
SAS, version 9.1, SAS Institute Inc, Raleigh, NC.
Galey FD, Whiteley HE, Goetz TE, et al. Black walnut (Juglans nigra) toxicosis: a model for equine laminitis. J Comp Pathol 1991;104:313–326.
French KR, Pollitt CC. Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study. Equine Vet J 2004;36:230–235.
Garner HE, Hahn AW, Salem C, et al. Cardiac output, left ventricular ejection rate, plasma volume, and heart rate changes in equine laminitis-hypertension. Am J Vet Res 1977;38:725–729.
Blikslager AT, Yin C, Cochran AM, et al. Cyclooxygenase expression in the early stages of equine laminitis: a cytologic study. J Vet Intern Med 2006;20:1191–1196.
Fontaine GL, Belknap JK, Allen D, et al. Expression of interleukin-1B in the digital laminae of horses in the prodromal stage of experimentally induced laminitis. Am J Vet Res 2001;62:714–720.
Kyaw-Tanner M, Pollitt CC. Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase. Equine Vet J 2004;36:221–225.
Morgan SJ, Hood DM, Wagner IP, et al. Submural histopathologic changes attributable to peracute laminitis in horses. Am J Vet Res 2003;64:829–834.
Singh SS, Murray RD, Ward WR. Gross and histopathological study of endotoxin-induced hoof lesions in cattle. J Comp Pathol 1994;110:103–115.
Black SJ, Lunn DP, Yin C, et al. Leukocyte emigration in the early stages of laminitis. Vet Immunol Immunopathol 2005;109:161–166.
Johnson PJ, Ganjam VK, Slight SH, et al. Tissue-specific dysregulation of cortisol metabolism in equine laminitis. Equine Vet J 2004;36:41–45.
Wattle OS. Cytokeratins of the matrices of the chestnut (torus carpeus) and periople in horses with acute laminitis. Am J Vet Res 2001;62:425–432.
Loftus JP, Belknap JK, Stankiewicz KM, et al. Laminar xanthine oxidase, superoxide dismutase and catalase activities in the prodromal stage of black-walnut induced equine laminitis. Equine Vet J 2007;39:48–53.
Chirgwin SR, Nowling JM, Coleman SU, et al. Effect of immunostimulatory oligodeoxynucleotides on host responses and the establishment of Brugia pahangi in Mongolian gerbils (Meriones unguiculatus). J Parasitol 2003;89:483–489.
Leutenegger CM, Mislin CN, Sigrist B, et al. Quantitative real-time PCR for the measurement of feline cytokine mRNA. Vet Immunol Immunopathol 1999;71:291–305.
Douglas JE, Thomason JJ. Shape, orientation and spacing of the primary epidermal laminae in the hooves of neonatal and adult horses (Equus caballus). Cells Tissues Organs 2000;166:304–318.
Riggs LM, Franck T, Moore JN, et al. Neutrophil myeloperoxidase measurements in plasma, laminar tissue, and skin of horses given black walnut extract. Am J Vet Res 2007;68:81–86.
Quinlivan M, Nelly M, Prendergast M, et al. Pro-inflammatory and antiviral cytokine expression in vaccinated and unvaccinated horses exposed to equine influenza virus. Vaccine 2007;25:7056–7064.
Bogaert L, Van Poucke M, De Baere C, et al. Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids. BMC Biotechnol 2006;6:24–30.
Waguespack RW, Cochran A, Belknap JK. Expression of the cyclooxygenase isoforms in the prodromal stage of black walnut-induced laminitis in horses. Am J Vet Res 2004;65:1724–1729.
Waguespack RW, Kemppainen RJ, Cochran A, et al. Increased expression of MAIL, a cytokine-associated nuclear protein, in the prodromal stage of black walnut-induced laminitis. Equine Vet J 2004;36:285–291.
Loftus JP, Belknap JK, Black SJ. Matrix metalloproteinase-9 in laminae of black walnut extract treated horses correlates with neutrophil abundance. Vet Immunol Immunopathol 2006;113:267–276.
Vick MM, Murphy BA, Sessions DR, et al. Effects of systemic inflammation on insulin sensitivity in horses and inflammatory cytokine expression in adipose tissue. Am J Vet Res 2008;69:130–139.
Smith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 1996;271:33157–33160.