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  • Author or Editor: Gayle W. Trotter x
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in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine relative amounts of mRNA expression of aggrecan, type-II collagen, matrix metalloproteinase (MMP) 1, and MMP3 in articular cartilage and synovial membrane samples from healthy equine joints and joints with osteoarthritis (OA) and to compare results of Northern blot hybridization with results of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay.

Sample Population—Articular cartilage samples from 8 pairs of joints (1 with OA and 1 healthy) from 6 horses and synovial membrane samples from 6 pairs of joints from 5 horses.

Procedure—RNA was extracted from samples by use of a modified Trizol procedure. Northern blot hybridization and the RT-PCR assay were performed; results were quantitated by use of glyceraldehyde 3- phosphate dehydrogenase as an internal standard.

Results—Articular cartilage samples from joints with mild or moderate OA yielded less total RNA than samples from joints with severe OA. Northern blot hybridization indicated that type-II collagen mRNA expression in articular cartilage samples from joints with OA was significantly greater than expression in samples from healthy joints. The RT-PCR assay identified low levels of MMP3 mRNA expression in 4 of 8 sets of articular cartilage samples and 4 of 6 sets of synovial membrane samples, whereas Northern blot hybridization identified MMP3 mRNA expression in only 1 of 6 sets of articular cartilage samples and 1 of 6 sets of synovial membrane samples.

Conclusions—A RT-PCR assay is more sensitive than Northern blot hybridization for detection of MMP3 mRNA expression in articular cartilage and synovial membrane and requires smaller samples. (Am J Vet Res 2000;61:900–905)

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in American Journal of Veterinary Research

Abstract

Objective—To determine response of interleukin-1α (IL-1α)-conditioned equine articular cartilage explants to insulin-like growth factor-1 (IGF-1).

Sample Population—Cartilage from the trochlea and condyles of the femur of a clinically normal 4-year-old horse.

Procedure—Effects of IGF-1 (0 to 500 ng/ml) after addition of IL-1α were evaluated by assessing matrix responses, using a sulfated glycosaminoglycan (GAG) assay, matrix 35SO4 GAG incorporation, and release of GAG. Mitogenic response was assessed by 3H-thymidine incorporation into DNA and fluorometric assay of total DNA concentration.

Results—Human recombinant IL-1α (40 ng/ml) increased the amount of labeled GAG released and decreased labeled and total GAG remaining in explants, and IL-1α decreased mitogenic response. Addition of IGF-1 counteracted effects seen with IL-1α alone. In general, IGF-1 decreased total and labeled GAG released into the medium, compared with IL-1α- treated explants (positive-control sample). Values for these variables did not differ significantly from those for negative-control explants. A significant increase in total and newly synthesized GAG in the explants at termination of the experiment was observed with 500 ng of IGF-1/ml. Labeled GAG remaining in explants was greater with treatment at 50 ng of IGF-1/ml, compared with treatment with IL-1α alone. Concentrations of 200 ng of IGF-1/ml abolished actions of IL-1α and restored DNA synthesis to values similar to those of negative-control explants.

Conclusions and Clinical Relevance—IGF-1 at 500 ng/ml was best at overcoming detrimental effects associated with IL-1α in in vitro explants. These beneficial effects may be useful in horses with osteoarthritis. (Am J Vet Res 2000;61:436–441)

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in American Journal of Veterinary Research

Objective—

To determine the outcome of horses after arthroscopic removal of abaxial fracture fragments of the proximal sesamoid bone and association of fracture grade with outcome.

Design—

Retrospective study.

Animals—

47 horses.

Procedure—

Information obtained from dorsopalmar and dorsoplantar radiographic views of metacarpophalangeal and metatarsophalangeal joints was used to classify fractures as grade 1 (< 15 mm long), grade 2 (15 to 25 mm long), and grade 3 (> 25 mm long) and type 1 (abaxial) and type 2 (apical-abaxial). Outcome was determined by whether the horse returned to its intended use, raced in the same class or higher (racehorses), or performed satisfactorily (nonracehorses). Number of starts, performance index, and money earned were also used to evaluate performance of racehorses.

Results—

Follow-up information was obtained for 41 horses (35 racehorses, 6 nonracehorses). Twenty-five racehorses were able to return to racing (16 in the same class, 9 in a lower class). All 6 nonracehorses were able to return to performance at the same level. Horses with small fracture fragments or fractures involving the abaxial aspect of the proximal sesamoid bone only had a more favorable outcome, compared with horses with large or apical-abaxial fractures.

Clinical Implications—

Overall, horses with abaxial fractures of the proximal sesamoid bone have a favorable prognosis for return to racing, but only a fair prognosis for return to racing in the same class, after arthroscopic removal of fracture fragments. Successful results can be expected for nonracehorses. (J Am Vet Med Assoc 1998:213:1016-1021)

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in Journal of the American Veterinary Medical Association

Abstract

Objectives

To clone equine interleukin 1α (IL-1α) and equine interleukin 1β (IL-1β) and determine their full-length cDNA sequences.

Procedure

The mRNA isolated from lipopolysaccharide-stimulated cultured equine monocytes was reverse transcribed, and a cDNA library was constructed in a λ phage. The cDNA library was screened by means of plaque hybridization with radiolabeled human IL-1α and IL-1ß cDNA probes. The cDNA nucleotide sequences for equine IL-1α and equine IL-1β were determined by use of the dideoxy chain termination technique. The cDNA sequences were analyzed, using computer software, for sequence characteristics and compared with sequences reported for other species.

Results

The cDNA for equine IL-1α was 1,728 base pairs in length with an ORF encoding a peptide of 270 amino acids with a predicted molecular mass of 30.823 kd. The cDNA for equine IL-1β was 1,473 base pairs in length with an ORF encoding a peptide of 268 amino acids with a predicted molecular mass of 30.342 kd. Similarity between amino acid sequence of equine IL- 1α and sequences for IL-1 α of other species ranged from 62.5 to 82.2%; similarity between amino acid sequence of equine IL-1ß and sequences for IL-1β of other species ranged from 62.5 to 66.4%. Similarity between amino acid sequences of equine IL-1α and equine IL-1β was 26%.

Conclusions and Clinical Relevance

Results establish a basis for studying the roles of interleukin 1 in healthy and diseased joints in horses. (Am J Vet Res 1998;59:704-711)

Free access
in American Journal of Veterinary Research

Abstract

Objectives

To clone equine interleukin 1 receptor antagonist (IL-1 ra) and determine its full-length cDNA sequence.

Procedure

A cDNA library derived from lipopolysaccharide-stimulated equine monocytes was screened by means of plaque hybridization to radiolabeled equine IL-1ra DNA probes generated by means of the polymerase chain reaction. The cDNA nucleotide sequence for equine IL-1ra was determined by use of the dideoxy chain termination technique, analyzed by use of computer software for sequence characteristics, and compared with sequences reported for IL-1ra of other species.

Results

The cDNA for equine IL-1ra was 1,614 base pairs in length with an ORF encoding a peptide of 177 amino acids with a predicted molecular mass of 20.427 kd. Similarity between the amino acid sequence of equine IL-1ra and sequences for human, murine, rat, and lapine IL-1ra was 76%. Similarity between sequence for equine IL-1ra and sequences for equine interleukin-1α and equine interleukin-1ß were 22.6 and 24.6%, respectively.

Conclusion

Comparison of the sequence for equine IL-1ra with sequences for IL-1ra of other species indicated a high degree of conservation.

Clinical Relevance

Results establish a basis for studying the roles of interleukin-1 in healthy and diseased joints in horses. (Am J Vet Res 1998;59:712-716)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate the effects of a commercially defined, serum-free medium additive on equine articular cartilage explants, compared with effects of serum-free and serum-supplemented media.

Animals

Articular cartilage from a 3-year-old, mixed breed horse euthanatized for reasons other than musculoskeletal disease or sepsis.

Procedure

Media were changed every 48 hours, and the glycosaminoglycan (GAG) content was determined in media collected at each time point. Glycosaminoglycan synthesis by explant chondrocytes, and residual GAG content of articular cartilage (as a measure of explant GAG loss) were determined at the end of the study (day 8).

Results

Articular cartilage explants in serum-free medium and the commercial supplemented medium had significantly lower GAG synthesis and GAG content than did those incubated in serum-supplemented medium. There were no significant differences in GAG synthesis and content between serum-free and commercial supplemented medium groups. When comparing medium GAG content for all treatment groups, the GAG content in serum-free medium on day 8 was significantly greater than that in commercial supplemented medium, but significant differences were not evident in percentage of release of GAG (as an indicator of GAG degradation) among all 3 treatment groups.

Conclusions

Commercial supplemented medium had effects on articular cartilage matrix GAG loss into medium equal to those of serum-supplemented medium (eg, both lost articular cartilage explant GAG to a similar degree). However, residual articular cartilage GAG content was higher in serum-supplemented medium, as was GAG synthesis. Commercial supplemented medium appears to either lack the proper ingredients to maintain steadystate GAG synthesis, or lacks proper concentrations of these ingredients. (Am J Vet Res 1996;57:1261–1265)

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in American Journal of Veterinary Research

Summary

Three doses of sodium monoiodoacetate (mia) were used to induce degenerative changes in articular cartilage in middle carpal joints of horses. Twelve young (2- to 5-year-old) horses, free of lameness, were randomly allotted to 3 groups. One middle carpal joint of each horse was injected with 0.9% NaCl solution (control joint). The contralateral middle carpal joint was injected with 0.09 mg of MlA/kg of body weight (group 1); 0.12 mg/kg (group 2); or 0.16 mg/kg (group 3). After mia administration, horses were allowed ad libitum exercise in a 2-acre paddock for 12 weeks. At the end of the study, gross and microscopic tissue changes were evaluated and biochemical analyses of articular cartilage were done. Grossly, diffuse partial thickness articular cartilage lesions were observed in group-2 (n = 2) and group-3 (n = 4) horses, but not in group-1 horses. Articular cartilage uronic acid content was significantly (P < 0.03) decreased in all mia-injected joints, compared with controls. Articular cartilage matrix staining with safranin-O was decreased in 3 of 4 mia- injected joints of group-1 horses and in all mia-injected joints of group-2 and group-3 horses, compared with controls (P < 0.06). Microscopic degenerative changes in articular cartilage were not significantly different between mia-injected and control joints in group-1 horses, but were increased (P<0.06) in all MlA-injected joints of group-2 and group-3 horses, compared with controls. Qualitatively, decreased matrix staining and degenerative changes were more severe in group-3 horses. On the basis of articular cartilage gross and microscopic changes, as well as biochemical changes, 0.12 mg of mia/kg injected intra-articularly was determined to induce moderate degrees of articular cartilage degeneration. This model of chemically induced articular cartilage injury could be useful for evaluating treatment effects of anti-arthritic drugs in horses.

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in American Journal of Veterinary Research

SUMMARY

The use of periosteal autografts to resurface osteochondral defects was investigated in 10 horses (2 to 3 years old), and the repair tissue was characterized morphologically. Middle carpal joint arthrotomies were made, and osteochondral defects were induced bilaterally on the distal articular surface of each radial carpal bone. Each defect measured approximatively 1 cm2 and extended 3 mm into the subchondral bone plate. Residual subchondral bone plate of control and principal defects was perforated by drilling. A sterile fibrin adhesive was made by mixing a fibrinogen component and a thrombin component. A periosteal autograft was harvested from the proximal portion of the tibia and was glued onto the recipient osseous surface, with its cambium facing the joint cavity. Control defects were glued, but not grafted. Horses were walked 1 hour daily on a walker, starting at postoperative week 7 and continuing for 9 weeks. Sixteen weeks after the grafting procedure was done, carpal radiography was performed, after which horses were euthanatized. Quality of repair tissue of control and grafted defects was evaluated and compared grossly, histologically, and histochemically. Using a reticule, the proportions of various repair tissue types filling each defect were quantitated.

Seven weeks after the grafting procedure was done, bilateral arthroscopy revealed synovial adhesions and marginal pannus formation in control and grafted defects. None of the autografts was found floating unattached within the respective middle carpal joints. At 16 weeks, the gross appearance of most grafted and nongrafted defects was similar, and repair was dominated by a fibrous pannus. In 4 grafted defects, bone had formed either concentrically within the defect or eccentrically in the fibrous adhesions between the defect and the joint margin. Histologically, all grafted and nongrafted defects were repaired similarly by infiltration of a mixture of fibrous tissue, fibrocartilage, and bone. Fibrous tissue was the predominant tissue in most defects and its mean proportion was 56 and 59% in the grafted and nongrafted defects, respectively. Fibrocartilaginous tissue in the deeper layers approximated 20%, and woven bone at the base of the defect was 20% in all defects. Histochemically, difference in staining for proteoglycans was not observed between grafted and nongrafted defects. Little remaining original periosteal graft tissue was evident at the defect sites. The only distinguishing feature of grafted defects was the presence of islands of bone formation either at the defect site (n = 2 horses), or in somewhat dorsally displaced tissue that was incorporated in fibrous adhesions (n = 2 horses). It was concluded that use of periosteal autografts did not improve the healing of osteochondral defects of the distal portion of the radial carpal bone. The repair tissue produced in grafted and nongrafted defects was similar and was principally fibrous in nature.

Free access
in American Journal of Veterinary Research