Determination of MMP-2 and -9 activities in synovial fluid of horses with osteoarthritic and arthritic joint diseases using gelatin zymography and immunocapture activity assays

REASON FOR PERFORMING STUDY: Matrix metalloproteinases (MMPs)-2 and -9 activities have been found elevated in synovial fluid from various joint diseases in man. However, in the horse few data are available. OBJECTIVES: To explore the clinical significance of MMP-2 and -9 activities in synovial fluid of horses with different forms of joint diseases. METHODS: Gelatin zymography and MMP-2 and -9 immunocapture activity assays were applied on synovial fluids from control joints and joints with aseptic joint disease (AJD) and septic arthritis (SA). Additionally, MMP-2 and -9 activities were measured in samples from SA to monitor the disease process. RESULTS: Zymographic analysis revealed that samples from AJD and SA contained significantly increased latent MMP-2 activity compared to controls. Samples from SA showed significantly increased monomeric latent MMP-9 activity compared with all other affected joints and controls. Trace amounts of MMP-9 activity, due to the active and dimer form, were detected in samples from SA; however, these bands were absent in samples from AJD and controls. Using immunocapture activity assays, MMP-2 and -9 activities were found to be significantly elevated in joints from SA compared to controls and AJD samples. MMP-2 activity in samples from AJD was significantly increased compared to controls. Both MMP activities decreased in the joints from SA in the course of successful therapy. CONCLUSIONS: Data from zymographic analysis confirmed that MMP-2 and -9 were elevated in equine joint diseases. Immunocapture activity assays have been shown to be suitable for the quantitative determination of MMP-2 and -9 activities in synovial fluid of horses. Both MMP-2 and -9 activities seem to be useful to indicate SA, and MMP-2 activity might be a suitable marker for AJD. POTENTIAL RELEVANCE: These findings encourage the potential use of MMP-2 and -9 as additional aids to clinical investigation. Further work is required to validate the clinical significance of MMP activities in the progress of different joint diseases in horses.     

Cartilage-derived retinoic acid-sensitive protein in equine synovial fluid from healthy and diseased joints

Reasons for performing study: More sensitive and specific diagnostic methods for early detection of changes in the joint cartilage are needed. Cartilage‐derived retinoic acid‐sensitive protein (CD‐RAP) is a potential marker of cartilage synthesis and regeneration. This is the first study on equine CD‐RAP. Objectives: To evaluate the ability of a commercially available human sandwich ELISA assay to detect equine CD‐RAP in synovial fluid from healthy and diseased joints. Methods: Synovial fluid was collected from 28 horses with no signs of joint disease and from 5 with induced inflammatory arthritis. CD‐RAP concentrations were measured using a human CD‐RAP ELISA. Intra‐ and interassay imprecision of the assay were evaluated by multiple measurements on pools of equine synovial fluid. Assay inaccuracy was determined by linearity under dilution. Results: The assay showed moderate to large intra‐ and interassay variation when applied to equine synovial fluid. Equine CD‐RAP was detected in synovial fluid from healthy horses ranged at 8.2–52 ng/ml. Repeated arthrocentesis (after injection of isotonic saline), age, joint or gender did not significantly affect CD‐RAP concentrations. Twelve hours after intra‐articular injection of lipopolysaccharide, concentrations of CD‐RAP were significantly lower than after injection of isotonic saline and remained significantly lower until the end of the study at 144 h. Conclusion and potential relevance: The assay is suitable for longitudinal monitoring of CD‐RAP concentration in individual horses. Disease significantly influenced CD‐RAP levels. Similar to previous results obtained in man, CD‐RAP seems to be a marker of cartilage synthesis and/or regeneration in horses.     

Arthroscopic treatment of temporomandibular joint sepsis in a horse

OBJECTIVE: To report arthroscopic debridement and lavage of a septic temporomandibular joint (TMJ) in a horse. DESIGN: Clinical case report. ANIMALS: A 12-year-old Thoroughbred mare. METHODS: Arthroscopic investigation of the dorsal joint pouch of the right TMJ was made through a stab incision into the dorsocaudal compartment. Mechanized resection of synovium and fibrinous debris combined with copious lavage, and intra-articular and systemic antibiotic administration was used to treat the septic TMJ. RESULTS: Arthroscopic debridement and lavage of the TMJ, in combination with intra-articular and systemic antimicrobials resulted in resolution of sepsis. Eight months post-operatively, there was no clinical evidence of degenerative joint disease or ankylosis of the TMJ. CONCLUSION: TMJ sepsis is rare in horses, however, standard arthroscopic equipment can be used in the management of this condition with minimal complications. CLINICAL RELEVANCE: Arthroscopic debridement and lavage should be considered for evaluation and initial treatment of TMJ sepsis in horses.     

Idiopathic infectious arthritis of the coxofemoral joint in a mature horse

The authors are aware of only one previously described case of idiopathic infectious arthritis of the coxofemoral joint in a mature horse. The case described herein presented with clinical signs that localised pain to the proximal limb, subtle increased radiopharmaceutical uptake at the coxofemoral joint and an increase of the major acute phase protein, serum amyloid A in the horse's blood biochemistry. The latter finding led the authors to search initially for an infective process. Ultrasound guided aspiration of the coxofemoral joint yielded infected synovial fluid. Despite the duration of the condition, nonsurgical treatment allowed the horse to return to its previous, relatively modest, level of activity. This case is presented to supplement the single previously published case report of idiopathic adult coxofemoral infectious arthritis in the horse.     

Use of stem cells in equine musculoskeletal disorders

Stem cell therapies for musculoskeletal disorders are becoming commonplace in the horse. In order to decipher the many options available for stem cell therapy and interpret results of accumulating experimental and clinical data, practitioners should have a basic understanding of stem cells.     

Deformation of the equine pelvis in response to in vitro 3D sacroiliac joint loading

Reasons for performing study: Sacroiliac joint injuries can cause poor performance; however, the interaction between pelvic mechanics and the sacroiliac joint is poorly understood. Objective: To measure pelvic displacement during 3D sacroiliac joint loading. Methods: Nine reflective triads were attached rigidly to bony prominences in sacropelvic specimens harvested from 14 horses for stereophotogrammetric analysis of triad displacements and joint kinematics. The sacrum was coupled to a load cell and mounted vertically within a material testing system (MTS). A pneumatic actuator was used to apply 90 Nm moments to the ischial arch to simulate nutation‐counternutation and left and right lateral bending of the sacroiliac joints. Axial rotation of the sacrum was induced by torsion of the upper MTS fixture. Vectors of marker displacement within orthogonal planes of motion were measured during loading of the sacropelvic specimens. Comparisons in the magnitude and direction of triad displacements were made between paired left‐right markers and paired loading conditions. Results: Nutation‐counternutation of the sacroiliac joint caused vertical displacement of the ischial tuberosities and cranial‐caudal displacement of the wings of the ilium. Lateral bending induced rotational displacement within the horizontal plane of all pelvic landmarks, relative to the sacrum. Axial rotation of the sacrum caused elevation of the wing of the ilium ipsilateral to the direction of sacral rotation and depression of the contralateral ilial wing. Significant paired left‐right differences occurred during most sacroiliac joint loading conditions. Comparable magnitudes of pelvic displacement were measured during nutation‐counternutation, left and right lateral bending, and left and right axial rotation. Conclusions: The equine pelvis is not a rigid structure and asymmetric pelvic deformation occurs during most sacroiliac joint movements. Clinical relevance: Bony pelvic deformation should be considered a normal response to any sacroiliac joint movement.     

Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery

OBJECTIVE: To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow-derived mesenchymal stem cells (BMD-MSCs). SAMPLE POPULATION: Articular cartilage, synovium, and bone marrow from 15 adult horses. PROCEDURES: Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1-deficient adenovirus vector encoding the beta-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1-deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection. RESULTS: -Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors. CONCLUSION AND CLINICAL RELEVANCE: Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.     

Haematogenous septic arthritis in foals: Short‐ and long‐term outcome and analysis of factors affecting prognosis

Synovial sepsis represents a major cause of morbidity and mortality in foals; however, there are no studies focusing on foals particularly at risk during the first 2 months of life. Our objective was to analyse outcome in foals aged <2 months with haematogenous septic arthritis and to identify prognostic factors and compare the effects of two different lavage procedures on outcome. Sixty foals with synovial sepsis were used for our retrospective study in which medical records, online data and telephone interviews were utilised to analyse prognostic factors, and determine short‐ and long‐term survival and athletic performance of foals treated at Evidensia Equine Specialist Hospital, Helsingborg, between 2008 and 2014. Overall survival to discharge was 80% (48/60). Time to long‐term follow‐up ranged from 6 months to 6.5 years. Forty/60 (67%) horses survived long‐term. Four/8 (50%) of the nonsurvivors at long‐term follow‐up had been subjected to euthanasia due to sequelae of joint sepsis. Of the long‐term survivors, 37/40 (92.5%) achieved athletic soundness. Overall long‐term outcome for athletic soundness was 37/60 (62%). Prognostic factors based on historical, clinical or laboratory data, or the use of through‐and‐through needle lavage compared to endoscopic lavage as the first procedure, could not be identified. The present study demonstrates a more favourable prognosis than previously reported, with 80% of foals with synovial sepsis surviving to discharge, and approximately 60% achieving athletic soundness. However, the study was limited by a small population size, which may account for the failure to verify statistically significant prognostic factors.     

Identity of the E-series prostaglandin produced by equine chondrocytes and synovial cells in response to a variety of stimuli

Although an E-series prostaglandin has previously been identified in equine inflammatory exudate, the identity of this eicosanoid as PGE2 has not been confirmed. The objective of this study was the specific characterisation of the prostaglandin produced by equine cells in the presence of an inflammatory stimulus. By using two radioimmunoassays, a relatively non-specific PGE2 assay and a more specific PGE1 assay, it has been possible to identify the E-series prostaglandin produced by equine chondrocytes and synovial cells, in response to a variety of stimuli, as PGE2.     

百里香酚对山羊子宫内膜上皮细胞的体外毒活性研究

为了探究百里香酚对山羊子宫内膜上皮细胞(goat endometrial epithelial cells,g EECs)的毒性作用,采用不同浓度的百里香酚作用于g EECs后,观测其对细胞生长曲线,细胞形态学变化,以及细胞膜结构完整性的影响。结果表明,百里香酚对EECs的毒性作用与其浓度呈剂量依赖效应(y=-0.008x+1.2202,R~2=0.9767),即随着药物浓度的增加细胞的增殖抑制率增大,药物对细胞的半数增殖抑制率(IC50)为100μg/m L。当50、100μg/m L的百里香酚作用于EECs时,g EECs的细胞核、细胞浆的形态均未产生明显变化,但当150μg/m L的百里香酚作用于g EECs时,细胞膜破裂,细胞死亡;而且药物浓度≥100μg/m L时细胞LDH的释放量显著高于正常细胞组,g EECs细胞膜的完整性受到破坏。百里香酚作用于山羊子宫内膜上皮细胞后呈现一定的毒性作用,且毒性作用和药物的浓度有关。百里香酚作用于g EECs的安全范围在0~100μg/m L,研究结果为百里香酚治疗奶牛等经济动物的炎症性疾病的临床用药浓度提供理论依据。     

Successful treatment of infectious (Salmonella type III: 44) polyarthritis and osteomyelitis in a 4‐week‐old foal

This article describes the acute onset of infectious polyarthritis and osteomyelitis in a 4‐week‐old foal. Analysis of synovial fluid obtained from the left femoropatellar and right tarsocrural joints combined with clinical signs consisting of joint effusion and lameness yielded a diagnosis of septic arthritis. Bacterial culture of synovial fluid from the left stifle revealed Salmonella type III: 44. Rapid, sustained clinical improvement was noted following discontinuation of empirical antimicrobial therapy (potassium penicillin and amikacin sulphate) and initiation of treatment with ceftiofur and ampicillin. The importance of combining knowledge of veterinary pharmacology and microbiology so that appropriate antimicrobials may be selected with regard to the local environment in which they are to eradicate infection is emphasised. Despite frequent reference to amikacin sulphate as an effective antimicrobial for treating infections in foals caused by Salmonella, factors are discussed that explain why amikacin may not be clinically effective for treating infectious arthritis caused by Salmonella.