Healthy Equine Joints Research Articles

Acute joint inflammation induces a sharp increase in the number of synovial fluid EVs and modifies their phospholipid profile

Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis.LPS injection triggered a sharp increase of SF-EVs at 5-8 h post-injection, which started to decline at 24 h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0 h), SF-EVs collected at 5, 24, and 48 h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5 h and 24 h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation.

Highlights of recent clinically relevant papers

Equine Veterinary EducationVolume 35, Issue 6 p. 282-283 RESEARCH HIGHLIGHTS Highlights of recent clinically relevant papers Sue Wright, Corresponding Author Sue Wright [email protected] orcid.org/0000-0002-5513-8571 EVE Editorial Office Email: [email protected]Search for more papers by this author Sue Wright, Corresponding Author Sue Wright [email protected] orcid.org/0000-0002-5513-8571 EVE Editorial Office Email: [email protected]Search for more papers by this author First published: 03 May 2023 https://doi.org/10.1111/eve.13797Read the full textAboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References Fouquet, G., Abbas, G., Johnson, J.P., Pompermayer, E., Harel, C., Aldous, E. et al. (2022) Ultrasound-guided injection technique of the equine cervical nerve roots. Frontiers of Veterinary Science. Available from: https://doi.org/10.3389/fvets.2022.992208 eCollection 2022. Kolding, S.A., Sørensen, J.N., Kramer, J., McCracken, M.J., Reed, S.K. & Keegan, K.G. (2023) Prevalence and clinical significance of increasing head height asymmetry as a measure of forelimb lameness in horses when trotting in a straight line after palmar digital nerve block. Equine Veterinary Journal. Available from: https://doi.org/10.1111/evj.13921 Melly, V., Ortved, K.F., Manzi, T.J., Richardson, D.W., Stefanovski, D. & Wulster, K.B. (2023) Identification of a previously unreported site of subchondral bone injury in the dorsodistolateral calcaneus in thoroughbred racehorses. Equine Veterinary Journal. Available from: https://doi.org/10.1111/evj.13928 Miglinci, L., Reicher, P., Nell, B., Koch, M., Jindra, C. & Brandt, S. (2023) Detection of equine papillomaviruses and gamma-herpesviruses in equine squamous cell carcinoma. Pathogens, 12(2), 179. Available from: https://doi.org/10.3390/pathogens12020179 Wanstrath, M.A., Bauck, A.G., Smith, A.D. & Freeman, D.E. (2022) Surgical enlargement of the epiploic foramen in horses. Veterinary Radiology & Ultrasound, 52, 308– 314. Available from: https://doi.org/10.1111/vsu.13927 Wolkowski, D.D., McCarthy, R.D., Schoonover, M.J., Taylor, J.D. & Eastman, T.G. (2022) Effects of intra-articular injection of an acellular equine liquid amniotic allograft in healthy equine joints. Veterinary Surgery, 52, 62– 68. Available from: https://doi.org/10.1111/vsu.13918 Volume35, Issue6June 2023Pages 282-283 ReferencesRelatedInformation

Effects of intra-articular injection of an acellular equine liquid amniotic allograft in healthy equine joints.

Evaluate effects of acellular equine liquid amnion allograft (ELAA) injected into healthy equine joints. Randomized, blinded, controlled experiment. Eight healthy adult horses. One intercarpal joint (ICJ) of each horse was randomly assigned to be injected with 1.5ml of ELAA (treatment) while the contralateral ICJ was injected with 1.5ml of 0.9% NaCl (control). Subjective lameness evaluation, force plate analysis, and synovial fluid analysis, including interleukin-1 receptor antagonist (IL-1ra) analysis, were performed before (day 0) and at days 1, 3, 5, and 10. Synovial fluid analysis was also performed on days 20 and 30. No difference in subjective lameness (P=.75) and no decrease in peak vertical force or vertical impulse were seen in any limb on any day. Total nucleated cell count (TNCC) was increased in treatment joints on days 1 (P=.0007; T: 6039 cells/μl, C: 240 cells/μl) and 3 (P < .0001; T: 1119 cells/μl, C: 240 cells/μl). Log-10 transformed values for IL-1ra were higher in treated joints on days 1 (P=.0005; T: 3553.7pg/ml, C: 1890.1pg/ml) and 3 (P=.01; T: 2283.2pg/ml, C: 1250.7pg/ml). Injection of ELAA into the ICJ caused an increase in synovial fluid TNCC in comparison with saline control but no lameness was observed. There was increased IL-1ra on days 1 and 3 after ELAA injection. Intra-articular injection of ELAA into healthy equine joints results in no significant safety concerns. The observed increase in IL-1ra may provide beneficial effects in inflamed joints.

Evaluation of the inflammatory response to two intra-articular hyaluronic acid formulations in normal equine joints.

Intra-articular (IA) hyaluronic acid (HA) is commonly used to treat equine arthritis. Inflammatory response or "joint flare" is a recognized potential side effect. However, the incidence and severity of inflammation following IA HA injection in horses is not well documented. This study compared the effects of two IA HA formulations of different molecular weight (MW) and a saline control on clinical signs and synovial fluid markers of inflammation in normal equine joints. Eight adult horses each had three healthy fetlock joints randomly assigned to treatment with either 1.4 mega Dalton HA, 0.8 mega Dalton HA or saline control once weekly for three weeks. Clinical evaluation and synovial fluid analysis were performed by blinded assessors. Outcomes of interest were lameness score, joint effusion score and synovial fluid white cell count and differential, total protein, viscosity and serum amyloid A. Joints injected with HA developed significant mild-to-moderate inflammatory responses often associated with lameness and joint effusion compared with saline control joints. The higher MW HA formulation elicited a significantly greater inflammatory response than the lower MW HA after the first injection. In HA injected joints, viscosity remained poor for the entire study. Both IA HA formulations in this study induced an inflammatory response in healthy equine joints. This may have implications for the use of HA in equine joints. The findings in this study are limited to the two HA formulations used. Further investigation of different HA formulations and the use of HA in normal and arthritic equine joints iswarranted.

Gene Therapy for Osteoarthritis: Pharmacokinetics of Intra-Articular Self-Complementary Adeno-Associated Virus Interleukin-1 Receptor Antagonist Delivery in an Equine Model.

Toward the treatment of osteoarthritis (OA), the authors have been investigating self-complementary adeno-associated virus (scAAV) for intra-articular delivery of therapeutic gene products. As OA frequently affects weight-bearing joints, pharmacokinetic studies of scAAV gene delivery were performed in the joints of the equine forelimb to identify parameters relevant to clinical translation in humans. Using interleukin-1 receptor antagonist (IL-1Ra) as a secreted therapeutic reporter, scAAV vector plasmids containing codon-optimized cDNA for equine IL-1Ra (eqIL-1Ra) were generated, which produced eqIL-1Ra at levels 30- to 50-fold higher than the native sequence. The most efficient cDNA was packaged in AAV2.5 capsid, and following characterization in vitro, the virus was injected into the carpal and metacarpophalangeal joints of horses over a 100-fold dose range. A putative ceiling dose of 5 × 1012 viral genomes was identified that elevated the steady-state eqIL-1Ra in the synovial fluids of injected joints by >40-fold over endogenous levels and was sustained for at least 6 months. No adverse effects were seen, and eqIL-1Ra in serum and urine remained at background levels throughout. Using the 5 × 1012 viral genome dose of scAAV, and green fluorescent protein as a cytologic marker, the local and systemic distribution of vector and transduced cells following intra-articular injection scAAV.GFP were compared in healthy equine joints and in those with late-stage, naturally occurring OA. In both cases, 99.7% of the vector remained within the injected joint. Strikingly, the pathologies characteristic of OA (synovitis, osteophyte formation, and cartilage erosion) were associated with a substantial increase in transgenic expression relative to tissues in healthy joints. This was most notable in regions of articular cartilage with visible damage, where foci of brilliantly fluorescent chondrocytes were observed. Overall, these data suggest that AAV-mediated gene transfer can provide relatively safe, sustained protein drug delivery to joints of human proportions.

Effects of medical ozone upon healthy equine joints: Clinical and laboratorial aspects.

ObjectiveThe aim of this study was to verify whether transient inflammatory reactions induced by intra-articular medicinal ozone administration affect joint components, by in vivo evaluation of inflammatory (prostaglandin E2, Substance P, Interleukin-6, Interleukine-1, Tumor Necrosis Factor), anti-inflammatory (Interleukin-10) and oxidative (superoxide dismutase activity and oxidative burst) biomarkers and extracellular matrix degradation products (chondroitin sulphate and hyaluronic acid) in synovial fluid.MethodsThe effects of medicinal ozone were analyzed at two ozone concentrations (groups A and B, 20 and 40 μg/ml, respectively), using oxygen-injected joints as controls (group C); each group received ten treatments (15 ml gas per treatment). Physical evaluation, evaluation of lameness, ultrasonography, and synovial fluid analysis were performed.ResultsAll joints presented mild and transient effusion throughout the study. Group B exhibited the highest lameness score on day 14 (P<0.05), detected by the lameness measurement system, probably because of the higher ozone concentration. All groups exhibited increased ultrasonography scores on day 14 (P < 0.05). Groups A and B exhibited increased proteins concentrations on day 21 (P<0.05). There was no change in hyaluronic acid concentration or the percentage of high-molecular weight hyaluronic acid throughout the experiment. Chondroitin sulfate concentrations decreased in group B, and did not change in group A and C, indicating that neither treatment provoked extracellular matrix catabolism. Cytokine and eicosanoid concentrations were not significantly changed.ConclusionsThe ozonetherapy did not cause significant inflammation process or cartilage degradation, therefore, ozonetherapy is safe at both evaluated doses.

Autologous processed plasma: cytokine profile and effects upon injection into healthy equine joints.

This experimental controlled study was performed to evaluate the composition of autologous processed plasma (APP), and the effects of APP intra-articular injection into healthy equine metacarpophalangeal joints. The effects on joints were analysed with a short-phase protocol and a prolonged-phase protocol using saline-injected joints as controls. For the short protocol, horses received one intra-articular APP injection. Synovial fluid samples were collected prior to the injection and 3, 6, 24, 48, and 16 h after treatment. For the prolonged protocol, the joints received three weekly injections of APP, and samples were collected at 0, 7, 14, 21, and 28 days before APP administration. IL1-ra level was found to be increased in APP compared to plasma. Upon intra-articular administration of APP, transient (up to 24 h) increases in white blood cell (WBC) counts along with elevated protein and prostaglandin E2 (PGE2) concentrations were observed in the treated joints. Over the 28-day observation period, APP did not elicit changes relative to baseline levels, but WBC counts, PGE2 and chondroitin sulphate concentrations were lower than those found in the control. In conclusion, APP intra-articular injection induced a mild and transitory inflammatory response but no inflammation reaction was observed over a longer period of treatment and observation.

Effects of repeated intra‐articular administration of amikacin on serum amyloid A, total protein and nucleated cell count in synovial fluid from healthy horses

Serum amyloid A (SAA) in synovial fluid has recently been used as a marker for septic arthritis in horses but the effects of repeated intra-articular (IA) administration of amikacin on synovial SAA concentrations are unknown. To report the effect of repeated IA administration of amikacin on SAA, total protein (TP), nucleated cell count (NCC) and differential NCC in synovial fluid of healthy equine joints. A controlled, 2 period crossover study was performed on 5 clinically healthy horses. Each intercarpal joint received one of 2 treatments every 48 h for 5 consecutive times: arthrocentesis alone (control group) or arthrocentesis combined with IA administration of 500 mg of amikacin (treatment group). Clinical and lameness examinations were performed daily. Serum SAA and synovial SAA, TP, NCC and differential NCC were measured and statistically compared. Significance level was set at P < 0.05. Horses remained healthy and nonlame throughout the study. Baseline values for all variables were not significantly different between groups. Values for TP in the treatment group were significantly higher than in the control group after the first sample (P < 0.05). In both groups NCC increased significantly (P < 0.05) after the first sample. No significant changes were identified in differential NCC. In both groups, all synovial and most serum SAA concentrations remained below the lower limit of quantification. Repeated IA administration of amikacin caused increased values of TP and NCC in synovial fluid, with some TP concentrations falling within the range reported for septic arthritis. In contrast, synovial SAA concentrations did not increase in either group. Synovial SAA could serve as a more reliable marker than TP and NCC when evaluating a joint previously sampled or treated with amikacin.

Synovial Prostaglandin E2 Concentrations in Horses Fed Long Chain Polyunsaturated Fatty Acids: An Inquiry Study

Synovial Prostaglandin E2 Concentrations in Horses Fed Long Chain Polyunsaturated Fatty Acids: An Inquiry Study

Influence of repeated arthrocentesis and exercise on synovial fluid concentrations of nitric oxide, prostaglandin E2 and glycosaminoglycans in healthy equine joints

The importance of osteoarthritis (OA) in the horse and the difficulty in its early diagnosis have led to a search for potential biomarkers of joint disease. If the levels of such markers are to be interpreted accurately, clinicians and researchers need to know whether they are influenced by environmental factors and/or interventions such as exercise and repeated arthrocentesis. To investigate the influence of repeated arthrocentesis and exercise on nitric oxide (NO), prostaglandin E2 (PGE2) and glycosaminoglycan (GAG) concentrations in synovial fluid (SF) from normal equine joints. SF was collected from the left metacarpophalangeal (MCP), radiocarpal and tarsocrural joints of 16 horses. Half of the horses were exercised and arthrocentesis was repeated 14, 14.5, 17 and 24 days after the start of the exercise programme, in both exercised and control horses. Nitric oxide was determined in SF from the MCP joint only and PGE2 and GAG concentrations were determined in SF from all joints. Repeated arthrocentesis caused an increase in NO concentration in the MCP joint on Day 145, in PGE2 concentrations in the radiocarpal and tarsocrural joints on Day 145 and the release of GAGs into SF of the MCP and radiocarpal joints on Day 17. Exercise resulted in an increase in PGE2 levels in all joints but did not influence the other parameters measured. Repeated arthrocentesis is a potential confounding factor for the use of synovial NO, PGE2 and GAG concentrations as markers of joint disease. Based on this study, such a confounding effect can be avoided if one week or more separates arthrocentesis procedures. Moderate exercise causes a transient rise in PGE2 in SF.

Effects of continuous oral administration of phenylbutazone on biomarkers of cartilage and bone metabolism in horses

To evaluate the effects of continuous oral administration of phenylbutazone on serum and synovial fluid biomarkers of skeletal matrix metabolism in horses. 11 adult female horses without clinical or radiographic evidence of joint disease. Horses were randomly assigned to control or treatment groups. Phenylbutazone was administered orally twice daily at a dose of 4.4 mg/kg for 3 days to the treatment group and subsequently at a dose of 2.2 mg/kg for 7 days. Serum and radiocarpal synovial fluid samples were obtained at baseline and thereafter at regular intervals for 4 weeks. Biomarkers of cartilage aggrecan synthesis (chondroitin sulfate 846) and type II collagen synthesis (procollagen type II C-propeptide) and degradation (collagen type II cleavage) were assayed. Biomarkers of bone synthesis (osteocalcin) and resorption (C-terminal telopeptide of type I collagen) were also measured. No significant differences were found between control and treatment groups or temporally for the biomarkers chondroitin sulfate 846, procollagen type II C-propeptide, collagen type II cleavage, and C-terminal telopeptide of type I collagen in serum or synovial fluid. A significant increase in osteocalcin concentration occurred in synovial fluid during treatment in the treated group. No treatment effect was detected for serum osteocalcin concentration. Results suggested that continuous phenylbutazone administration at recommended doses altered some biomarkers in healthy equine joints after short periods of administration. Increased osteocalcin concentration may indicate an undetermined anabolic effect of phenylbutazone administration on periarticular bone or transient induction of osteogenesis in articular chondrocytes or a mesenchymal subpopulation of synoviocytes.

Measurement of Articular Cartilage Stiffness of the Femoropatellar, Tarsocrural, and Metatarsophalangeal Joints in Horses and Comparison with Biochemical Data

To determine normal cartilage stiffness values in different weight-bearing and non-weight-bearing areas of 3 different equine joints, and to evaluate the relationship between cartilage stiffness and glycosaminoglycan (GAG) and collagen content. Compressive stiffness of the articular cartilage was measured in 8 horse cadaver femoropatellar (FP), tarsocrural (TC), and metatarsophalangeal (MT) joints. Gross evaluation, collagen content, GAG content, and histologic appearance were assessed for each measurement location. Eight equine cadavers (4 intact females, 4 castrated males; 7 Quarter Horse or Quarter Horse type, 1 Arabian; aged 4-12 years, weighing 400-550 kg). The articular surfaces of 8 equine cadaver FP, TC, and MT joints were grossly evaluated for signs of articular cartilage pathology. Stiffness at preselected sites (FP joint-6 sites; TC joint-3 sites; MT joint-4 sites) was determined using an arthroscopic indentation instrument. Biochemical composition (collagen, GAG content) and histologic evaluation (modified Mankin score) were assessed for each measurement site. All cartilage from all sites evaluated was determined to be normal based on macroscopic and histologic assessments. No significant correlation between Mankin scores and cartilage stiffness values was observed. Site differences in cartilage stiffness were measured in all 3 joints (P<.001). GAG or collagen content had a significant positive correlation with stiffness values in 6 of 13 sites (P<.05, r>0.622, r2>0.387). Relative cartilage stiffness values measured in healthy equine joints are site dependent and can be measured using an indentation device intended for arthroscopic application. An indentation instrument provided an objective means of determining relative compressive stiffness of articular cartilage. Further research needs to be performed to confirm the site and joint differences observed in this study in clinically normal horses and to determine if the tester can be used clinically to predict articular cartilage pathology.

Evaluation of subchondral bone mineral density associated with articular cartilage structure and integrity in healthy equine joints with different functional demands

To determine and correlate subchondral bone mineral density and overlying cartilage structure and tensile integrity in mature healthy equine stifle (low magnitude loading) and metacarpophalangeal (high magnitude loading) joints. 8 healthy horses, 2 to 3 years of age. Osteochondral samples were acquired from the medial femoral condyle (FC) and medial trochlear ridge (TR) of the stifle joint and from the dorsal (MC3D) and palmar (MC3P) aspects of the distal medial third metacarpal condyles of the metacarpophalangeal joint. Articular cartilage surface fibrillation (evaluated via India ink staining) and tensile biomechanical properties were determined. The volumetric bone mineral density (vBMD) of the underlying subchondral plate was assessed via dual-energy x-ray absorptiometry. Cartilage staining (fibrillation), tensile moduli, tensile strength, and vBMD were greater in the MC3D and MC3P locations, compared with the FC and TR locations, whereas tensile strain at failure was less in MC3D and MC3P locations than FC and TR locations. Cartilage tensile moduli correlated positively with vBMD, whereas cartilage staining and tensile strain at failure correlated negatively with vBMD. In areas of high joint loading, the subchondral bone had high vBMD and the articular cartilage surface layer had high tensile stiffness but signs of structural wear (fibrillation and low failure strain). The site-dependent variations and relationships in this study support the concept that articular cartilage and subchondral bone normally adapt to physiologic loading in a coordinated way.