Characterization of cultured smooth muscle cells obtained from the palmar digital arteries of horses

Dwayne H. Rodgerson Department of Large Animal Surgery and Medicine, Auburn University, Auburn, AL 36849.
Present address is the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136.

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 DVM, MS
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James K. Belknap Department of Large Animal Surgery and Medicine, Auburn University, Auburn, AL 36849.

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 DVM, PhD
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Genevieve L. Fontaine Department of Large Animal Surgery and Medicine, Auburn University, Auburn, AL 36849.
Present address is the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136.

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Dan L. Kroll Department of Large Animal Surgery and Medicine, Auburn University, Auburn, AL 36849.

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Abstract

Objective—To develop methods to isolate, culture, and characterize smooth muscle cells (SMC) from equine palmar digital arteries.

Sample Population—Segments of the medial or lateral palmar digital arteries from the forelimbs of 6 horses.

Procedure—To obtain smooth muscle explants, arterial segments were incised longitudinally. The tunica intima was gently scraped from the underlying tunica media, and explants were obtained from the tunica media. Approximately 18 to 24 explants were obtained from each palmar digital arterial segment. A substrate-attached technique was used to initiate primary culture of SMCCultured cells were identified as SMC, using light microscopy, electron microscopy, reverse transcriptase-polymerase chain reaction (RTPCR), and northern blot analysis. The replication index and serum dependence of equine SMC in culture was characterized by use of bromodeoxyuridine.

Results—The SMC of equine palmar digital arteries were successfully cultured, as confirmed by RT-PCR and northern blot analysis techniques for smooth muscle α-actin and detection of SMC-specific organelles during electron microscopy. When characterized by light and electron microscopy, SMC were found to have undergone phenotypic modulation to a more synthetic phenotype in culture while retaining characteristics of SMC.

Conclusions and Clinical Relevance—Culture of SMC from equine palmar digital arteries via an explant protocol is a viable technique for studying vascular biological mechanisms in horses. In vitro studies of SMC may aid investigators in determining cellular mechanisms involved in disease processes such as laminitis. (Am J Vet Res 2000;61:1602–1608)

Abstract

Objective—To develop methods to isolate, culture, and characterize smooth muscle cells (SMC) from equine palmar digital arteries.

Sample Population—Segments of the medial or lateral palmar digital arteries from the forelimbs of 6 horses.

Procedure—To obtain smooth muscle explants, arterial segments were incised longitudinally. The tunica intima was gently scraped from the underlying tunica media, and explants were obtained from the tunica media. Approximately 18 to 24 explants were obtained from each palmar digital arterial segment. A substrate-attached technique was used to initiate primary culture of SMCCultured cells were identified as SMC, using light microscopy, electron microscopy, reverse transcriptase-polymerase chain reaction (RTPCR), and northern blot analysis. The replication index and serum dependence of equine SMC in culture was characterized by use of bromodeoxyuridine.

Results—The SMC of equine palmar digital arteries were successfully cultured, as confirmed by RT-PCR and northern blot analysis techniques for smooth muscle α-actin and detection of SMC-specific organelles during electron microscopy. When characterized by light and electron microscopy, SMC were found to have undergone phenotypic modulation to a more synthetic phenotype in culture while retaining characteristics of SMC.

Conclusions and Clinical Relevance—Culture of SMC from equine palmar digital arteries via an explant protocol is a viable technique for studying vascular biological mechanisms in horses. In vitro studies of SMC may aid investigators in determining cellular mechanisms involved in disease processes such as laminitis. (Am J Vet Res 2000;61:1602–1608)

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