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  • Author or Editor: James K. Belknap x
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Abstract

Objective—To compare the levels of mRNA expression of cycooxygenase (COX)-1 and COX-2 in the digital laminae of normal horses and horses in the developmental stages of laminitis experimentally induced by administration of black walnut extract (BWE).

Sample Population—Samples of mRNA extracted from the digital laminae of 5 control horses and 5 horses at the onset of leukopenia after administration of BWE.

Procedure—Specimens of laminae were collected from anesthetized horses prior to euthanasia. Expression of COX-1 and COX-2 mRNA in laminae of control and affected horses was evaluated via realtime quantitative polymerase chain reaction techniques.

Results—Expression of COX-2 mRNA was significantly increased in the BWE-treated group, compared with that in control horses. In contrast to COX-2 regulation, COX-1 mRNA expression was not significantly different between groups. Interestingly, despite consistent clinical signs such as leukopenia in all BWE-treated horses, distinct differences in COX-2 mRNA expression were detected among those 5 horses (compared with values for control horses, the increase in COX-2 mRNA expression ranged from no increase to a 30-fold increase).

Conclusions and Clinical Relevance—Results indicated that there was a significant upregulation of COX-2 mRNA expression during the developmental stages of laminitis, with no significant change in expression of the COX-1 isoform. These data appear to provide support for aggressive use of nonsteroidal anti-inflammatory drugs in horses at risk for laminitis; further investigation into the clinical value of selective COX-2 inhibitors for treatment of laminitis in horses appears to be warranted. (Am J Vet Res 2004;65:1724–1729)

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 determine proinflammatory gene expression, endothelial adhesion molecule gene expression, and matrix metalloproteinase (MMP) concentrations in laminar specimens at 1.5 hours after administration of black walnut extract (BWE) and to compare these values with later time points.

Animals—25 horses.

Procedures—After nasogastric administration of BWE, anesthesia was induced at 1.5 hours in early time point (ETP) horses (n = 5), between 3 and 4 hours in developmental time point horses (5), and between 9 and 10 hours in acute onset of lameness time point horses (5). Anesthesia was induced at 3 and 10 hours after nasogastric administration of water in 2 groups of control horses (3-hour control group, n = 5; 10-hour control group, 5). Real-time quantitative PCR assay was performed on laminar specimens from control and ETP horses for cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1β, tumor necrosis factor-α, IL-6, IL-8, IL-10, MMP-2, and MMP-9 gene expression; and on laminar specimens from all groups for endothelial adhesion molecules, intercellular adhesion molecule (ICAM)-1, and E-selectin gene expression. Leukocyte emigration was assessed via CD13 immunohistochemistry, and gelatinase accumulation was determined by gelatin zymography.

Results—Laminar concentrations of IL-1β, IL-6, IL-8, COX-2, ICAM-1, and E-selectin mRNA were significantly increased in ETP horses, compared with control horses. Concentrations of IL-1β, IL-8, ICAM-1, and E-selectin mRNA peaked at 1.5 hours. In ETP horses, leukocyte emigration was present in 3 of 5 horses and pro–MMP-9 was detected in 2 of 5 horses.

Conclusions and Clinical Relevance—Results indicated that endothelial activation and laminar inflammation are early events in laminitis; MMP accumulation likely is a downstream event.

Full access
in American Journal of Veterinary Research

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 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

Abstract

Objective—To determine the effect of experimental infection with bovine viral diarrhea virus (BVDV) on llamas and their fetuses, evaluate seroprevalence of BVDV in llamas and alpacas, and genetically characterize BVDV isolates from llamas.

Design—Prospective study.

Animals—4 pregnant llamas for the experimental infection study and 223 llamas and alpacas for the seroprevalence study.

Procedure—Llamas (seronegative to BVDV) were experimentally infected with a llama isolate of BVDV via nasal aerosolization. After inoculation, blood samples were collected every other day for 2 weeks; blood samples were obtained from crias at birth and monthly thereafter. For the seroprevalence study, blood was collected from a convenience sample of 223 camelids. Isolates of BVDV were characterized by reverse transcription- polymerase chain reaction assay.

Results—Viremia and BVDV-specific antibody response were detected in the experimentally infected llamas, but no signs of disease were observed. No virus was detected in the crias or aborted fetus, although antibodies were evident in crias after colostrum consumption. Seroprevalence to BVDV was 0.9% in llamas and alpacas. Sequences of the llama BVDV isolates were comparable to known bovine isolates.

Conclusions and Clinical Relevance—Findings suggest that llamas may be infected with BVDV but have few or no clinical signs. Inoculation of llamas during gestation did not result in fetal infection or persistent BVDV infection of crias. Seroprevalence to BVDV in llamas and alpacas is apparently low. The most likely source for BVDV infection in camelids may be cattle. (J Am Vet Med Assoc 2003;223:223–228)

Restricted access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine whether increased gene expression of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) in laminae of horses with starch gruel–induced laminitis was accompanied by increased enzyme activity and substrate degradation.

Sample—Laminae from the forelimb hooves of 8 healthy horses and 17 horses with starch gruel–induced laminitis (6 at onset of fever, 6 at onset of Obel grade 1 lameness, and 5 at onset of Obel grade 3 lameness).

Procedures—Gene expression was determined by use of cDNA and real-time quantitative PCR assay. Protein expression and processing were determined via SDS-PAGE and quantitative western blotting. Protein distribution and abundance were determined via quantitative immunofluorescent staining.

Results—ADAMTS-4 gene expression was increased and that of versican decreased in laminitic laminae, compared with expression in healthy laminae. Catalytically active ADAMTS-4 also was increased in the tissue, as were ADAMTS-4–cleavage fragments of versican. Immunofluorescent analyses indicated that versican was depleted from the basal epithelia of laminae of horses at onset of Obel grade 3 lameness, compared with results for healthy laminae, and this was accompanied by regional separation of basal epithelial cells from the basement membrane. Aggrecan gene and protein expression were not significantly affected.

Conclusions and Clinical Relevance—Changes in gene and protein expression of ADAMTS-4 and versican in the basal epithelium of laminitic laminae indicated a fundamental change in the physiology of basal epithelial cells. This was accompanied by and may have caused detachment of these cells from the basement membrane.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate the pharmacokinetic-pharmacodynamic parameters of enrofloxacin and a low dose of amikacin administered via regional IV limb perfusion (RILP) in standing horses.

Animals—14 adult horses.

Procedures—Standing horses (7 horses/group) received either enrofloxacin (1.5 mg/kg) or amikacin (250 mg) via RILP (involving tourniquet application) in 1 forelimb. Samples of interstitial fluid (collected via implanted capillary ultrafiltration devices) from the bone marrow (BMIF) of the third metacarpal bone and overlying subcutaneous tissues (STIF), blood, and synovial fluid of the radiocarpal joint were collected prior to (time 0) and at intervals after tourniquet release for determination of drug concentrations. For pharmacokinetic-pharmacodynamic analyses, minimum inhibitory concentrations (MICs) of 16 μg/mL (amikacin) and 0.5 μg/mL (enrofloxacin) were applied.

Results—After RILP with enrofloxacin, 3 horses developed vasculitis. The highest synovial fluid concentrations of enrofloxacin and amikacin were detected at time 0; median values (range) were 13.22 μg/mL (0.254 to 167.9 μg/mL) and 26.2 μg/mL (5.78 to 50.0 μg/mL), respectively. Enrofloxacin concentrations exceeded MIC for approximately 24 hours in STIF and synovial fluid and for 36 hours in BMIF. After perfusion of amikacin, concentrations greater than the MIC were not detected in any samples. Effective therapeutic concentrations of enrofloxacin were attained in all samples.

Conclusions and Clinical Relevance—In horses with orthopedic infections, RILP of enrofloxacin (1.5 mg/kg) should be considered as a treatment option. However, care must be taken during administration. A dose of amikacin > 250 mg is recommended to attain effective tissue concentrations via RILP in standing horses.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the expression and distribution of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), its substrates aggrecan and versican, and their binding partner hyaluronan in laminae of healthy horses.

Sample—Laminae from the forelimb hooves of 8 healthy horses.

Procedures—Real-time quantitative PCR assay was used for gene expression analysis. Hyaluronidase, chondroitinase, and keratanase digestion of lamina extracts combined with SDS-PAGE and western blotting were used for protein and proteoglycan analysis. Immunofluorescent and immunohistochemical staining of tissue sections were used for protein and hyaluronan localization.

Results—Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in laminae. The ADAMTS-4 was predominantly evident as a 51-kDa protein bearing a catalytic site neoepitope indicative of active enzyme and in situ activity, which was confirmed by the presence of aggrecan and versican fragments bearing ADAMTS-4 cleavage neoepitopes in laminar protein extracts. Aggrecan, versican, and hyaluronan were localized to basal epithelial cells within the secondary epidermal laminae. The ADAMTS-4 localized to these cells but was also present in some cells in the dermal laminae.

Conclusions and Clinical Relevance—Within digital laminae, versican exclusively and aggrecan primarily localized within basal epithelial cells and both were constitutively cleaved by ADAMTS-4, which therefore contributed to their turnover. On the basis of known properties of these proteoglycans, it is possible that they can protect the basal epithelial cells of horses from biomechanical and concussive stress.

Full access
in American Journal of Veterinary Research