Culture and characterization of equine terminal arch endothelial cells and hoof keratinocytes.
Abstract: To develop methods to isolate, culture, and characterize equine hoof endothelial cells (EC) and keratinocytes. Methods: Cells harvested from the forelimbs of 8 horses. Methods: EC were obtained via catheters placed in the palmar digital arteries of the disarticulated lower portion of the forelimbs from 4 horses that had been heparinized prior to euthanasia. Phosphate-buffered saline solution was used to remove and discard RBC from blood vessels, and collagenase was used to loosen and flush EC from the vasculature. Hoof keratinocytes were obtained from 4 recently euthanatized horses by use of dispase/trypsin dissociation of the coronary band epidermis. Use of an extracellular matrix gel as a culture flask attachment factor was important to the success of hoof keratinocyte cultures. Results: EC from the palmar digital arteries were successfully cultured and characterized by in vitro morphology, uptake of a fluorescence-labeled acetylated-low density lipoprotein, and lack of expression of von Willebrand factor and smooth muscle actin. Hoof keratinocytes were characterized by morphology in culture and expression of keratin proteins, as determined by immunochemical reaction. Keratinocyte cultures were also positive for vimentin expression. Conclusions: Culture techniques to isolate and characterize hoof cells should aid investigators in their study of equine hoof pathobiologic features, especially as it relates to laminitis.
Publication Date: 1999-01-26 PubMed ID: 9918161
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research aims to develop methods for the isolation, culture, and characterization of equine hoof cells, specifically endothelial cells and keratinocytes. These methods could support further studies on diseases affecting horses’ hooves, such as laminitis.
Methodology
- The researchers harvested cells from the forelimbs of eight horses. They divided the horses into two equal groups, one for endothelial cells (EC) extraction, and one for hoof keratinocytes.
- For the EC extraction, catheters were placed in the palmar digital arteries of the disarticulated lower part of the forelimbs. The horses were heparinized before euthanasia. Then, they used a phosphate-buffered saline solution to remove red blood cells from blood vessels and collagenase to flush EC from the vasculature.
- Hoof keratinocytes were sourced from the coronary band epidermis of four euthanatized horses. The researchers used dispase/trypsin dissociation for this process.
- The hoof keratinocyte cultures relied on an extracellular matrix gel as an attachment factor in the culture flask.
Results
- The researchers successfully cultured endothelial cells from the palmar digital arteries. They characterized these cells based on their in vitro morphology, their ability to uptake a fluorescence-labeled acetylated-low density lipoprotein, and their lack of expression of von Willebrand factor and smooth muscle actin.
- Hoof keratinocytes were characterized by their morphology in culture and their expression of keratin proteins, as determined by an immunochemical reaction. They also showed positive vimentin expression.
Conclusion
- The techniques developed in this study to isolate and characterize equine hoof cells could be a significant aid in further investigations into the pathology of horse hoof diseases, specifically laminitis. As a common and serious ailment among horses, a deeper understanding of laminitis could lead to more effective preventative measures or treatments.
Cite This Article
APA
Wunn D, Wardrop KJ, Meyers K, Kramer J, Ragle C.
(1999).
Culture and characterization of equine terminal arch endothelial cells and hoof keratinocytes.
Am J Vet Res, 60(1), 128-132.
Publication
Researcher Affiliations
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman 99164-7060, USA.
MeSH Terms
- Animals
- Arteries / cytology
- Cell Culture Techniques / methods
- Endothelium, Vascular / cytology
- Extracellular Matrix
- Forelimb
- Hoof and Claw / blood supply
- Hoof and Claw / cytology
- Horse Diseases / pathology
- Horses
- Immunohistochemistry
- Keratinocytes / cytology
- Microscopy, Phase-Contrast / veterinary
Citations
This article has been cited 3 times.- Reisinger N, Schaumberger S, Nagl V, Hessenberger S, Schatzmayr G. Concentration Dependent Influence of Lipopolysaccharides on Separation of Hoof Explants and Supernatant Lactic Acid Concentration in an Ex Vivo/In Vitro Laminitis Model. PLoS One 2015;10(11):e0143754.
- Leise BS, Watts MR, Roy S, Yilmaz AS, Alder H, Belknap JK. Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis. Equine Vet J 2015 Jul;47(4):478-88.
- Visser MB, Pollitt CC. Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes. BMC Vet Res 2010 Mar 15;6:16.
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