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  • Author or Editor: Eric J. Abrahamsen x
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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 measure the effects of lowmolecular-weight inhibitors on the activity of bovine neutrophil matrix metalloproteinase 9 (MMP-9).

Sample Population—Bovine MMP-9 purified from bovine neutrophilconditioned medium.

Procedures—Neutrophils were degranulated by stimulation with phorbol ester. Enzyme purification was performed by use of gelatin affinity and gel-filtration chromatography. Activated enzyme was incubated with inhibitors prior to addition of substrate (gelatin fluorescein conjugate or fluorogenic peptide). Rates of enzymatic cleavage were determined by monitoring fluorescence as the reactions progressed. Values of IC50 (molar concentration of compound that inhibits specific activity by 50%) and KI (in vitro inhibition constant) were determined.

Results—Rates of enzymatic activity of monomeric and dimeric bovine MMP-9 measured by use of gelatin and peptide substrates were linear with respect to time and concentrations of enzyme and substrate. The MMP-9 was potently inhibited by hydroxamic acids (IC50 for gelatin, 29.2 to 55.7nM; IC50 for peptide, 4.8 to 24.6nM; KI, 0.2 to 0.5nM), whereas tetracyclines (IC50 for gelatin, 30.1 to 112.7MM; IC50 for peptide, 48.0 to 123.8MM; KI, 25.2 to 61.4µM) and chlorhexidine (IC50 for gelatin, 139.1MM; IC50 for peptide, 672.5MM to 1.7mM; KI, 495.0 to 663.0MM) had limited inhibition. Gelatinase-specific inhibitor SB-3CT had intermediate potency (IC50 for peptide, 185.0 to 290.0nM; KI, 66.5 to 86.0nM).

Conclusions and Clinical Relevance—Bovine MMP-9 was potently inhibited by hydroxamic acids and gelatinase inhibitor. These compounds may be useful as modulators of neutrophil-mediated protease activity in cattle.

Full access
in American Journal of Veterinary Research

Abstract

Objectives—To quantify direction and velocity of blood flow in hepatic veins in dogs under different hemodynamic conditions by use of pulsed-wave Doppler ultrasonography.

Animals—10 healthy dogs.

Procedure—Dogs were anesthetized, and venous flow velocities in the quadrate lobe were measured. Arterial blood pressure, right atrial pressure, pulmonary artery pressure, and cardiac output were measured simultaneously. The timing of each waveform during the cardiac cycle was used to identify velocity profiles. Peak waveform velocities were measured during conditions of light anesthesia with isoflurane (baseline; period 1), cardiovascular depression following administration of highdose isoflurane and esmolol IV (period 2), cardiovascular depression with crystalloid volume expansion (period 3), and high cardiac output induced with dobutamine (period 4). Hemodynamic measurements and maximum waveform velocities were compared among the 4 periods by use of an ANOVA and univariate and multivariate linear regression.

Results—During each study period, 4 distinct, lowvelocity waves were identified. Mean velocities recorded during period 1 were as follows: retrograde atrial contraction a-wave, 7.3 cm/s; antegrade systolic S-wave, 15.0 cm/s; retrograde venous return v-wave, 2.7 cm/s; and antegrade diastolic D-wave, 11.4 cm/s. Mean S:D ratio was 1.27. During periods 3 and 4, Swave velocity increased; D-wave velocity was highest during period 4.

Conclusions and Clinical Relevance—Consistent hepatic venous velocity profiles were observed in healthy dogs under different hemodynamic conditions. These findings provide baseline values that may be useful in evaluating clinical cases, but further study involving healthy, awake dogs and dogs with cardiac and hepatic diseases is required. (Am J Vet Res 2004;65:734–740)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize and purify covalent complexes of matrix metalloproteinase-9 (MMP-9) and haptoglobin released by bovine granulocytes in vitro.

Sample Population—Blood samples obtained from healthy cows and cows with acute and chronic inflammation to obtain WBCs and sera.

Procedures—WBCs were isolated by differential centrifugation, hypotonic lysis of RBCs, and degranulated by stimulation with phorbol ester (20 ng/mL). Cell-conditioned medium was subjected to affinity and gel chromatography and purified proteins subjected to SDS- PAGE gelatin zymography, western blot analysis, Coomassie blue staining, and peptide mass spectrometry for protein identification. Sera of cows hospitalized for acute and chronic septic conditions and of clinically normal cows were analyzed with similar methods.

Results—Matrix metalloproteinase-9 was released from neutrophils in vitro and migrated to a molecular mass of approximately 220 kd (prodimer), approximately 105 kd (promonomer), and > 220 kd (high–molecular mass complexes). These high–molecular mass complexes were composed of α- and β-haptoglobin and MMP-9 (ratio13:13:1). Complexes of MMP-9 and haptoglobin had biochemical properties of both its protein constituents (ie, enzymatic activity toward gelatin and hemoglobin binding). Complexes of MMP-9 and haptoglobin were also detected in sera of cows with acute inflammation, but not in clinically normal cows or cows with chronic disease.

Conclusions and Clinical Relevance—A fraction of neutrophil MMP-9 is released in complex with haptoglobin. The complex is present in granules and retains biological activity of its components. Detection of the complex in serum may provide an indicator of acute inflammation.

Full access
in American Journal of Veterinary Research