and the KA, make it potentially useful as a marker of differentiation in the epidermal keratinocytes of the hoof lamellae. Moreover, altered UEA reactiv- ity might be consistent with tissue metaplasia and the resultant widening of the keratinized axis of primary epidermal lamellae during lamellar wedge formation.5 On the other hand, a marker that iden- tifies the less differentiated epidermal cell popula- tion would be equally useful. Antibodies against the intermediate filament protein K14 specifically label this cell type. The abnormal localization of K14 to suprabasal cells in the lamellar wedge is consistent with the presence of dysplastic epidermal cells in this tissue.5 Altered epidermal differentia- tion might contribute to laminitis disease pathogenesis. Since the cytolinker desmoplakin interconnects intermediate filaments with desmosomal adhesion complexes, antibodies immunoreactive with this protein are useful in the localization of the plasma membranes of epidermal cells interlinking with other epidermal cells. The restriction of desmo- plakin staining to the non-basal SEL cell mem- branes is consistent with its absence from hemidesmosomes linking these cells to the base- ment membrane.22 We are currently using combi- nations of these lectins and immunoreagents to investigate K14 and desmoplakin localization dur- ing laminitis pathogenesis.
5. Conclusion
Fluorescently-conjugated lectins and antibodies are useful for the identification of tissue, cellular, and subcellular microanatomy in equine hoof lamellar tissue. Structures that can be specifically labelled include connective tissue extracellular matrix, epi- dermal basal cells, and subcellular components of epidermal cells such as cell membranes, cytoskeletal intermediate filaments, and cytolinkers that join the cytoskeleton to adhesion complexes. Ongoing stud- ies in our laboratory are using these markers to investigate laminitis histopathology and molecular pathogenesis.
Acknowledgments
The authors acknowledge Christopher Pollitt (Queensland, Australia) for contributing some of the horse tissue used in this study. Technical assis- tance by Mark Modelski and Susan Megee is grate- fully acknowledged. We also thank Gordon Ruthel (Philadelphia, PA) for technical advice on confocal microscopy. This work was funded, in part, by grants from the Bernice Barbour Foundation, Inc. (0048.42008), Grayson-Jockey Club Research Foun- dation, and the University of Pennsylvania Fund for Laminitis Research.
Conflict of Interest
The Authors declare no conflicts of interest. The manuscript was not reviewed by any financial spon- sors prior to submission.
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6. Senoo M, Pinto F, Crum CP, et al. p63 is essential for the proliferative potential of stem cells in stratified epithe- lia. Cell 2007;129:523–536.
7. Wang L, Pawlak E, Johnson PJ, et al. Effects of cleavage by a disintegrin and metalloproteinase with thrombospon- din motifs-4 on gene expression and protein content of versican and aggrecan in the digital laminae of horses with starch gruel-induced laminitis. Am J Vet Res 2012;73: 1047–1056.
8. Pawlak E, Wang L, Johnson PJ, et al. Distribution and processing of a disintegrin and metalloproteinase with throm- bospondin motifs-4, aggrecan, versican, and hyaluronan in equine digital laminae. Am J Vet Res 2012;73:1035–1046.
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http://www.aaep.org/images/files/ Research%20Study%20-%20Final%20-%2010%2007%2009. pdf. Accessed December 1, 2009.
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18. Hashimoto Y, Reese S, Bragulla H, et al. Eine lectinhisto- chemische untersuchung der epidermis von haut und huf pferdes. Anat Histol Embryol 1992;21:238–245. 19. Virtanen I, Kariniemi AL, Holtho
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