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  • Author or Editor: Genevieve L. Fontaine x
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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)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin- (IL)-1β from cultured equine smooth muscle cells (SMC).

Sample Population—Segments of palmar digital artery harvested from 6 clinically normal adult horses.

Procedure—Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 µg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-α, and IL-1β was performed, using isolated total cellular RNA.

Results—Although no message was detected for IL-1β or TNF-α in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1β and TNF-α mRNA.

Conclusions and Clinical Relevance—Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis. (Am J Vet Res 2001;62:1957–1963)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To study expression of interleukin-1beta (IL-1β) in the digital laminae of horses in the prodromal stage of experimentally induced laminitis.

Animals—8 healthy adult horses with no signs of laminitis.

Procedure—Black walnut extract was administered via nasogastric tube to 4 horses, and water was administered to the remaining 4 (controls). Complete blood counts and physical examinations were performed every 30 minutes after administration of black walnut extract or water. General anesthesia was induced when total WBC count decreased by 30% in horses given the black walnut extract and 3 hours after water administration in control horses. The left forefoot was perfusion fixed with neutral-buffered 10% formalin, and paraffin-embedded sections of the digit were used for in situ hybridization with an equine-specific IL-1β probe.

Results—IL-1β mRNA expression was observed in perivascular cells of the small laminar venules and capillaries in all 4 horses given black walnut extract and in interstitial cells remote from the microvasculature in 1 of the 4. Other cellular components of the laminar tissue and cellular components of the digital arterioles and veins did not exhibit IL-1β mRNA expression. Expression of IL-1β mRNA was not detected in laminae from control horses.

Conclusions and Clinical Relevance—Results suggest that IL-1β mRNA is expressed by perivascular cells in the laminar tissues of horses in the prodromal stage of experimentally induced laminitis. This provides evidence of an inflammatory process during the prodromal stage of laminitis, indicating that local digital proinflammatory cytokine expression may be an initiating factor in laminitis.(Am J Vet Res 2001;62: 714–720)

Full access
in American Journal of Veterinary Research