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  • Author or Editor: Mark B. Hurtig x
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Abstract

Objective—To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays.

Sample Population—25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses.

Procedure—A solid-phase DMMB assay was developed to measure sGAG concentrations in SF. Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Farndale method) and microplate DMMB assay.

Results—The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solidphase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays.

Conclusions and Clinical Relevance—The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solidphase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses. ( Am J Vet Res 2003; 64:894–899)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To study osteoarthritis in the equine medial femorotibial (MFT) joint after a single traumatic injury.

Animals—10 mature horses.

Procedure—In vitro explant cultures were used to determine injury threshold for stifle joint cartilage. Contusive impacts were applied to the medial femoral condyle (MFC), and horses were followed for 84 (n = 5) and 180 days (5). Synovial fluid samples were collected every 14 days for determination of sulphated glycosaminoglycan (sGAG) concentrations. Radiographic and lameness evaluations were performed. Gross and histologic descriptions, and immunohistochemistry, cartilage sGAG content determination, and cartilage aggregate modulus determination were performed at the MFC impact site (MFCi), MFC nonimpact site (MFCn), and medial tibial plateau (MTP).

Results—Synovial fluid sGAG concentration decreased significantly on days 14, 28, 42, and 56 in all horses. Macroscopic and microscopic articular lesions developed within all MFT joints. No radiographic abnormalities were observed. Mild lameness was evident in several horses. No significant differences were found between short-term and longterm cohorts of horses with respect to histologic scores and TUNEL results. On immunohistochemistry, MFCi was positive for COL2–¾Cshort. International Cartilage Repair Society scores differed significantly between short-term and long-term cohorts of horses. In all horses, sGAG concentrations were significantly decreased at the MFCi, compared with the MFCn.

Conclusions and Clinical Relevance—Use of contusive impacts on the MFC of horses results in cartilage lesions that are similar to those described clinically, supporting trauma as a contributing factor in the natural pathogenesis of osteoarthritis.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the anisotropic characteristics of the microarchitecture of the subchondral bone (SCB) plate and trabecular bone (TBB) of the distopalmar aspect of the metacarpal condyles in horses with different stages of SCB disease.

Sample Population—12 third metacarpal bone pairs from racing Thoroughbreds euthanized for diverse reasons.

Procedures—Both metacarpi were collected from horses with SCB changes that were mild (sclerosis and focal radiolucencies; n = 6) or severe (multifocal radiolucencies and articular surface defects; 6). Sample blocks of SCB plate and TBB were collected from the distopalmar aspect of both condyles and the sagittal ridge and examined via 3-D micro-computed tomography at 45-?m isotropic voxel resolution. For each sample, the angle between the principal orientation of trabeculae and the sagittal plane and the degree of anisotropy (DA) were calculated from mean intercept length measurements.

Results—Condylar samples had significantly lower angle (mean, 8.9°; range, 73° to 10.9°) than sagittal ridge samples (mean, 40.7°; range, 33.6° to 49.2°), TBB had significantly higher DA (mean ± SE, 1.75 ± 0.04) than SCB plate (1.29 ± 0.04), and mildly diseased TBB had higher DA (1.85 ± 0.06) than severely diseased TBB (1.65 ± 0.06).

Conclusions and Clinical Relevance—The highly ordered appearance of trabeculae within the condyles supports the concept that joint loading is primarily transmitted through the condyles and not the sagittal ridge. The sharp changes in the trajectories of the SCB trabeculae at the condylar grooves may be indicative of hypothetical tensile forces at this location contributing to the pathogenesis of condylar fractures. (Am J Vet Res 2010;71:1148—1153)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether a single contusive impact injury to the palmar aspect of the metacarpus would progress to post-traumatic osteoarthritis or palmar osteochondral disease in horses.

Animals—12 horses.

Procedures—In each horse, an impact injury was created on the palmar aspect of the medial metacarpal condyle of 1 randomly chosen limb with an impactor device under arthroscopic and fluoroscopic guidance. The opposite limb was sham operated as a control. A low to moderate amount of forced exercise was instituted, and horses were evaluated clinically via lameness examinations weekly for 5 months, then biweekly until endpoint, with synovial fluid analysis performed at 0, 1, 2, 3, 4, 6, 8, and 10 months and radiography at baseline and endpoint. Macroscopic examination, micro-CT, and sample collection for cartilage viability and sulfated glycosaminoglycan content, histologic evaluation, immunohistochemical analysis, and fluorochrome analysis were performed following euthanasia at 1 (3 horses), 4 (4), and 8 to 10 (5) months after surgery.

Results—There was variability in impact lesion location, depth, and area on macroscopic inspection, but on histologic evaluation, cartilage defects were less variable. Mean sulfated glycosaminoglycan concentration from cartilage at the impact site was significantly lower than that at a similar site in control limbs. Higher concentrations of cartilage oligomeric matrix protein were observed in synovial fluid from impact-injured joints.

Conclusions and Clinical Relevance—The impact injury method caused mild focal osteoarthritic lesions in the metacarpophalangeal joint, but did not progress to palmar osteochondral disease at this site. Repeated injury is probably required for the development of palmar osteochondral disease.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether 3 variations of the 1,9-dimethylmethylene blue (DMMB) assay yield comparable results when measuring sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF).

Sample Population—25 samples of SF collected from affected joints of 13 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses.

Procedure—Sulfated glycosaminoglycan concentrations were measured by the direct spectrophotometric (ie, Farndale), microplate, and indirect DMMB assays in samples of SF collected from normal and affected joints and in samples digested with nucleases, papain, and hyaluronidase.

Results—All 3 assays reacted similarly to standard solutions of sGAGs and digestion of SF samples with nucleases, papain, and hyaluronidase. Nucleic acids were not important interfering substances, and papain and hyaluronidase could not be used interchangeably to digest SF. All 3 assays proved to have satisfactory precision (SD < 10%), but each DMMB assay resulted in significantly different measures of sGAG in equine SF.

Conclusions and Clinical Relevance—Samples of SF should be digested with papain or hyaluronidase prior to measurement via DMMB assay. Researchers currently are unable to compare clinical information when variations of the DMMB assay are used, because each DMMB assay yields substantially different sGAG concentrations in SF. Of the 3 assays examined here, we recommend use of the direct spectrophotometric DMMB assay. (Am J Vet Res 2003;64:900–906)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To use quantitative ultrasonography to evaluate the association between the speed of sound (SOS) at 9 sites in the third metacarpal bone (MCIII) of racing Thoroughbreds with workload accumulation and the effect that MCIII failure has on this association.

Animals—Sixty-two 2- and 3-year-old Thoroughbreds in racing condition.

Procedures—Cumulative work index (CWI) was used to calculate total workload (CWItotal) and also 3 independent CWIs for the various gaits (ie, trot [CWItrot], gallop [CWIgallop], and race [CWIrace]) used during training and racing. Speed of sound was monitored in horses during the 2007 racing season and compared with the CWIs via regression analysis. Sex, age, limb, and MCIII failure were included as covariates in the model.

Results—SOS was significantly associated with CWItotal at 8 sites and with independent CWIs of the various gaits at all 9 sites. Progression of SOS in MCIIIs with workload differed significantly in horses with clinical signs of metacarpal bone failure, compared with results for horses with clinically normal MCIIIs, in 1 site by use of CWItotal and in 5 sites by use of the independent CWIs for the various gaits.

Conclusions and Clinical Relevance—These results indicated that SOS in the MCIII of racing Thoroughbreds followed a constant pattern of progression as workload accumulated. With the development of more precise quantitative ultrasonography devices, SOS corrected for amount of activity may be used to identify horses at risk of bone failure.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize the mechanical properties of subchondral bone (SCB) of the distopalmar aspect of the condyles of the third metacarpal bone (MC3) and their correlations with structural aspects of MC3s in Thoroughbred racehorses.

Sample Population—12 pairs of MC3s from Thoroughbred racehorses euthanized for various reasons.

Procedures—MC3s were collected from horses with mild (n = 6) and with severe (6) SCB changes, as determined by micro–computed tomography (CT). Specimens of SCB plate and trabecular bone were cut from the distopalmar aspect of condyles and sagittal ridge and examined with 3-dimensional micro-CT. Specimens were tested in compression, and elastic modulus, yield stress, yield strain, and toughness were calculated. Apparent and true bone mineral density, bone volume fraction, trabecular thickness, trabecular separation, and connectivity were also calculated. Differences in mechanical properties among various classifications of bone were evaluated. Correlations between structural and mechanical variables were also assessed.

Results—No differences were detected between left and right forelimbs. Specimens from condyles had higher values for elastic modulus, yield stress, and toughness than did specimens of sagittal ridge. In SCB with severe changes attributable to SCB disease, SCB plate was weaker and trabecular bone was stronger than in SCB with mild changes. Microstructural and mechanical properties were significantly correlated.

Conclusions and Clinical Relevance—A marked gradient in mechanical properties of SCB from horses, which could be involved in the pathogenesis of condylar fractures, was detected. Mechanical properties of SCB from the distal aspect of MC3s can be predicted to some extent via micro-CT.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize the microstructure of subchondral bone (SCB) plate and trabecular bone (TBB) of the distopalmar aspect of the condyles of third metacarpal bones (MC3s) from Thoroughbred racehorses at 2 different stages of SCB disease via micro–computed tomography (CT).

Sample Population—12 pairs of MC3s from Thoroughbred racehorses euthanized for various reasons.

Procedures—MC3s were collected from horses with mild (n = 6) or severe (6) SCB disease, as determined via micro-CT. Cubic (6 × 6 × 6-cm) specimens of SCB plate and TBB were cut from the palmar aspect of condyles and sagittal ridges and examined with 3-dimensional micro-CT. For each specimen, apparent bone mineral density (aBMD), true BMD (tBMD), bone volume fraction (BVF), trabecular thickness (TBT), trabecular separation (TBS), and connectivity (CN) were calculated.

Results—Condyles had higher aBMD, tBMD, BVF, and TBT and lower TBS than did the sagittal ridge. In bone specimens with mild SCB changes, SCB plate had higher aBMD, TBT, and CN and lower TBS than did TBB. In bone specimens with severe SCB disease, TBB had higher aBMD and TMD and lower TBS than it did in bone specimens with mild disease, and values were similar to those for SCB plate in bone specimens with severe disease.

Conclusions and Clinical Relevance—The microstructure of SCB of the distopalmar aspect of metacarpal condyles of horses varied according to the severity of changes identified via micro-CT. With mild SCB disease, sclerosis existed in the SCB plate of the condyles; with severe disease, sclerosis also invaded condylar TBB.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the prevalence and severity of osteoarthritis in the metacarpophalangeal joints of Thoroughbred racehorses via development and validation of a quantitative macroscopic evaluation system.

Sample Population—Metacarpophalangeal joints from 50 Thoroughbred racehorses.

Procedures—Joints were collected from horses that died or were euthanized within 60 days of racing. Metacarpophalangeal joints were assessed for osteoarthritic degeneration by use of macroscopic and histologic scoring systems, polarized light microscopy, and cartilage biochemical analysis. The global macroscopic score for the entire metacarpophalangeal joint was based on factors that reflected the size and severity of lesions as well as the involvement of weight-bearing surfaces.

Results—One-third of all 2- and 3-year-old horses had partial-or full-thickness cartilage lesions and osteoarthritis. Osteoarthritis severity increased until age 6 in this population. Significant correlations were found between macroscopic grade and age, cause of death, glycosaminoglycan depletion, and loss of superficial cartilage zone polarized light intensity.

Conclusions and Clinical Relevance—The macroscopic system devised for this study had good correlations with quantitative methods. Two-and 3-year-old horses had full-thickness cartilage lesions that may have been career limiting. Year-to-year attrition and a small population of older horses may have led to underestimation of the prevalence of osteoarthritis in older horses. The macroscopic scoring system was reliable when used by nonexpert and expert users. (Am J Vet Res 2010;71:1284–1293)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine characteristics, incidence rate, and possible associations with selected demographic characteristics of catastrophic musculoskeletal injuries (CMIs) in Thoroughbred racehorses.

Animals—76 Thoroughbreds with CMIs.

Procedures—Incidence rates of CMIs during racing or training were calculated with number of CMIs as the numerator and overall numbers of races or training events during 2004 and 2005 as the denominators. Exact 95% confidence intervals were calculated. Associations between incidence and dichotomous exposure factors, nominal factors, and ordinal factors were determined. Only univariable associations were examined.

Results—76 horses were euthanized because of CMI and represented 2.36 and 1.69 deaths/1,000 racing starts in 2004 and 2005, respectively. Of these, 57 were euthanized within 60 days before or after a race, which yielded a point incidence of 1.05/1,000 racing starts and 0.39/1,000 training starts.

Conclusions and Clinical Relevance—Incidence rate of CMIs at 2 Ontario racetracks was similar to that at other North American racetracks. A cumulative death rate of 1 to 2 deaths/wkshould be considered typical when designing prevention strategies and offers a baseline value for measuring improvement.

Full access
in American Journal of Veterinary Research