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- Author or Editor: Alicia L. Bertone x
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Abstract
Objective—To create high-quality sequence data for the generation of an equine gene expression microarray and evaluate array performance by use of lipopolysaccharide (LPS) exposure of synoviocytes.
Sample Population—Public nucleotide sequence database from Equus caballus and synoviocytes from clinically normal adult horses.
Procedure—Computer procurement of equine gene sequences, probe design, and manufacture of an oligomicroarray were performed. Array performance was evaluated by use of patterns for equine synoviocytes in response to LPS.
Results—Starting with 18,924 equine gene sequences, 3,098 equine 3' sequences were annotated and met the inclusion criteria for an expression microarray. An equine oligonucleotide expression microarray was created by use of 68,266 of the 25-oligomer probes to uniquely identify each gene. Most genes in the array (68%) were expressed in equine synoviocytes. Repeatability of the array was high (r, > 0.99), and LPS upregulated (> 5-fold change) 84 genes, many of which were inflammatory mediators, and downregulated (> 5-fold change) 14 genes. An initial pattern of gene expression for effects of LPS on synoviocytes consisted of 102 genes.
Conclusions and Clinical Relevance—Use of a computer algorithm to curate an equine sequence database generated high-quality annotated species-specific gene sequences and probe sets for a gene expression oligomicroarray, which was used to document changes in gene expression associated with LPS exposure of equine synoviocytes. The equine public database was expanded from 290 annotated genes to > 3,000 provisionally annotated genes. Similar curation and annotation of public databases could be used to create other species-specific microarrays. (Am J Vet Res 2004;65:1664–1673)
Abstract
OBJECTIVE To investigate effects of hyaluronic acid (HA) or HA combined with chondroitin sulfate (CS) and N-acetyl-d-glucosamine (NAG) by use of a lipopolysaccharide (LPS) in vitro method.
SAMPLE Monolayer cultures of synovial cells from 4 adult horses.
PROCEDURES Synovial cell cultures were untreated or treated with HA alone or HA-CS-NAG for 24 hours, subsequently unchallenged or challenge-exposed with 2 LPS concentrations (20 and 50 ng/mL) for 2 hours, and retreated with HA or HA-CS-NAG for another 24 hours. Cellular morphology of cultures was evaluated at 0, 24 (before LPS), 26 (after LPS), and 50 (24 hours after end of LPS) hours. At 50 hours, cell number and viability and prostaglandin (PG) E2, interleukin (IL)-6, matrix metalloproteinase (MMP)-3, and cyclooxygenase (COX)-2 production were measured.
RESULTS LPS challenge exposure induced a significant loss of characteristic synovial cell morphology, decrease in cell viability, and increases in concentrations of PGE2, IL-6, MMP-3, and COX-2. Cells treated with HA or HA-CS-NAG had significantly better viability and morphology scores and lower concentrations of PGE2, MMP-3, IL-6, and COX-2 than untreated LPS challenge-exposed cells. Cells treated with HA had significantly better morphology scores at 50 hours than cells treated with HA-CS-NAG. Cells treated with HA-CS-NAG had significantly superior suppression of LPS-induced production of PGE2, IL-6, and MMP-3 than cells treated with HA alone.
CONCLUSIONS AND CLINICAL RELEVANCE HA and HA-CS-NAG protected synovial cells from the effects of LPS. Treatment with HA-CS-NAG had the greatest anti-inflammatory effect. These results supported the protective potential of HA and HA-CS-NAG treatments.
Abstract
Objective—To evaluate the association between subjective lameness grades and kinetic gait parameters and assess the variability in kinetic parameters in horses with experimentally induced forelimb lameness.
Animals—32 horses.
Procedures—Forelimb lameness was induced in each horse via injection of lipopolysaccharide into 1 metacarpophalangeal joint (40 experimental trials). Subjective lameness grading and 13 kinetic gait parameters (force plate analysis) were assessed before (baseline) and at 12, 18, and 24 hours after lipopolysaccharide injection. While horses were trotting, kinetic gait analysis was performed for 8 valid repetitions at each time point. Repeated-measures analyses were performed with 8 repetitions for each kinetic parameter as the outcome, and lameness grades, time points after lipopolysaccharide injection, and repetition order as explanatory variables. Sensitivity and specificity of kinetic parameters for classification of horses as sound or lame (in relation to subjective lameness scores) were calculated. Between- and within-horse variabilities of the 13 kinetic parameters were assessed by calculation of coefficients of variation.
Results—Subjective lameness grades were significantly associated with most of the kinetic parameters. Vertical force peak and impulse had the lowest between- and within-horse coefficients of variation and the highest correlations with subjective lameness grade. Vertical force peak had the highest sensitivity and specificity for lameness classification. Vertical force peak and impulse were significantly decreased even in horses with mild or unobservable lameness.
Conclusions and Clinical Relevance—Among the kinetic gait parameters, vertical force peak and impulse had the best potential to reflect lameness severity and identify subclinical forelimb gait abnormalities. (Am J Vet Res 2005;66:1805–1815)
Abstract
Objective
To determine whether irradiation with a low-intensity diode laser, which produces radiation at a wavelength of 810 nm, will induce nonthermal enhancement of chondrocyte metabolism.
Sample Population
144 grossly normal articular cartilage expiants aseptically harvested from the femoral condyles of 6 adult horses.
Procedure
Treated cartilage expiants were irradiated with a diode laser at 1 of 7 fluence levels that ranged from 8 to 1,600 J/cm2. Expiants were incubated for 24 or 72 hours, labeled for 24 hours with [35S]Na2SO4, and assayed for newly synthesized sulfated glycosaminoglycan (GAG; measured incorporation of 35SO4) and endogenous GAG, chondroitin 6-sulfate (CS), and keratan sulfate (KS) content, using a dimethylmethylene blue assay. Laser-induced temperature changes were measured during irradiation with a diode laser and a neodymiumyttrium aluminum garnet (Nd:YAG) laser, which produces radiation at a wavelength of 1,064 nm, using conditions that were reported in previous studies to increase explant metabolism.
Results
After incubation for 24 or 72 hours, rate of 35SO4 uptake or endogenous GAG, CS, or KS content in irradiated expiants was not significantly different than in nonirradiated expiants. Cartilage temperature increased < 4.75 C during diode laser application. Cartilage temperature increased 5 to 12 C during Nd:YAG laser application.
Conclusions
Minimal thermal increases in cartilage expiants with use of a low-intensity diode laser resulted in no change in proteoglycan metabolism of chondrocytes. An increase in tissue temperature over a narrow range with use of a Nd:YAG laser may have contributed to the metabolic alteration of chondrocytes reported in previous studies. (Am J Vet Res 1998;59:1613-1618)
Abstract
Objectives
To determine oxygen metabolism, permeability, and blood flow in isolated joints in response to interleukin 1β (IL-1β) and contribution of innervation.
Sample Population
One metacarpophalangeal (MCP) joint of 24 adult horses.
Procedure
The MCP joint was isolated for 6 hours in a pump-perfused, auto-oxygenated, innervated or denervated preparation. Isolated joints were assigned to the following 4 groups: control, control-denervated, inflamed, and inflamed-denervated, and inflammation was induced by intra-articular injection of IL-1β. Circuit arterial and venous pressures, flows, and blood gas tensions, synovial fluid production, and intra-articular pressure were measured. Total vascular resistance; oxygen delivery, consumption, and extraction ratio (ER); and permeability surface area product were calculated. Synovial membrane blood flow was determined at 0, 60, and 330 minutes. Synovial membrane wet-to-dry ratio was obtained, and permeability to macromolecules was determined by intra-articular injection of Evans blue albumin and fluorescein isothiocyanate-conjugated dextran.
Results
Oxygen delivery and synovial membrane blood flow progressively increased but were not different among groups. Oxygen consumption and ER significantly increased in inflamed joints, as did intraarticular pressure and synovial fluid production. Inflamed joints had greater wet-to-dry ratio. Albumin permeability significantly increased in the villous synovial membrane of the inflamed groups, and dextran permeability was increased in the innervated groups, with a trend toward increased permeability in inflamed groups.
Conclusion
Inflammation significantly increased oxygen demand, which was initially met by increased ER. Permeability to small molecules was increased with inflammation; innervation increased permeability to large molecules. Use of an isolated joint model enabled documentation of the physiologic responses of the joint to acute inflammation. (Am J Vet Res 1998;59:1307–1316)
SUMMARY
Lactase, maltase, sucrase, and alkaline phosphatase activities were determined in the intestinal mucosa from 3 locations in the small intestine and 4 locations in the large intestine 1 year after extensive large-colon resection (group 1; n = 5) and 1 year after sham operation (group 2; n = 3) in horses.
Lactase, maltase, and sucrase activities were similar (P > 0.05) between group-1 and group-2 horses in all locations measured in the intestinal tract. Alkaline phosphatase activity in the remaining large colon of group-1 horses was significantly (P < 0.05) greater than the activity in the large colon of group-2 horses. Decreased apparent digestion of phosphorus and a negative phosphorus balance are persistent features of large-colon resection in horses. Increases in alkaline phosphatase activity in the remaining colon of horses with extensive large-colon resection may be a specific functional adaptive mechanism that attempts to counteract the derangements in phosphorus metabolism.
