Equine osteoarthritis modifies fatty acid signatures in synovial fluid and its extracellular vesicles.

BACKGROUND: Individual fatty acids (FAs) and their derivatives (lipid mediators) with pro-inflammatory or dual anti-inflammatory and pro-resolving properties have potential to influence the health of joint tissues. Osteoarthritis (OA) is an age-associated chronic joint disease that can be featured with altered FA composition in the synovial fluid (SF) of human patients. The counts and cargo of extracellular vesicles (EVs), membrane-bound particles released by synovial joint cells and transporting bioactive lipids, can also be modified by OA. The detailed FA signatures of SF and its EVs have remained unexplored in the horse - a well-recognized veterinary model for OA research. METHODS: The aim of the present study was to compare the FA profiles in equine SF and its ultracentrifuged EV fraction between control, contralateral, and OA metacarpophalangeal joints (n = 8/group). The FA profiles of total lipids were determined by gas chromatography and the data compared with univariate and multivariate analyses. RESULTS: The data revealed distinct FA profiles in SF and its EV-enriched pellet that were modified by naturally occurring equine OA. Regarding SFs, linoleic acid (generalized linear model, p = 0.0006), myristic acid (p = 0.003), palmitoleic acid (p < 0.0005), and n-3/n-6 polyunsaturated FA ratio (p < 0.0005) were among the important variables that separated OA from control samples. In EV-enriched pellets, saturated FAs palmitic acid (p = 0.020), stearic acid (p = 0.002), and behenic acid (p = 0.003) indicated OA. The observed FA modifications are potentially detrimental and could contribute to inflammatory processes and cartilage degradation in OA. CONCLUSIONS: Equine OA joints can be distinguished from normal joints based on their FA signatures in SF and its EV-enriched pellet. Clarifying the roles of SF and EV FA compositions in the pathogenesis of OA and their potential as joint disease biomarkers and therapeutic targets warrants future studies.

Counts of hyaluronic acid-containing extracellular vesicles decrease in naturally occurring equine osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease with inadequately understood pathogenesis leading to pain and functional limitations. Extracellular vesicles (EVs) released by synovial joint cells can induce both pro- and anti-OA effects. Hyaluronic acid (HA) lubricates the surfaces of articular cartilage and is one of the bioactive molecules transported by EVs. In humans, altered EV counts and composition can be observed in OA synovial fluid (SF), while EV research is in early stages in the horse-a well-recognized OA model. The aim was to characterize SF EVs and their HA cargo in 19 horses. SF was collected after euthanasia from control, OA, and contralateral metacarpophalangeal joints. The SF HA concentrations and size distribution were determined with a sandwich-type enzyme-linked sorbent assay and size-exclusion chromatography. Ultracentrifugation followed by nanoparticle tracking analysis (NTA) were utilized to quantify small EVs, while confocal laser scanning microscopy (CLSM) and image analysis characterized larger EVs. The number and size distribution of small EVs measured by NTA were unaffected by OA, but these results may be limited by the lack of hyaluronidase pre-treatment of the samples. When visualized by CLSM, the number and proportion of larger HA-containing EVs (HA-EVs) decreased in OA SF (generalized linear model, count: p = 0.024, %: p = 0.028). There was an inverse association between the OA grade and total EV count, HA-EV count, and HA-EV % (rs = - 0.264 to - 0.327, p = 0.012-0.045). The total HA concentrations were also lower in OA (generalized linear model, p = 0.002). To conclude, the present study discovered a potential SF biomarker (HA-EVs) for naturally occurring equine OA. The roles of HA-EVs in the pathogenesis of OA and their potential as a joint disease biomarker and therapeutic target warrant future studies.

Evaluation of the effect of experimentally induced cartilage defect and intra-articular hyaluronan on synovial fluid biomarkers in intercarpal joints of horses.

BACKGROUND: Inflammatory and degenerative activity inside the joint can be studied in vivo by analysis of synovial fluid biomarkers. In addition to pro-inflammatory mediators, several anabolic and anti-inflammatory substances are produced during the disease process. They counteract the catabolic effects of the pro-inflammatory cytokines and thus diminish the cartilage damage. The response of synovial fluid biomarkers after intra-articular hyaluronan injection, alone or in combination with other substances, has been examined only in a few equine studies. The effects of hyaluronan on some pro-inflammatory mediators, such as prostaglandin E2, have been documented but especially the effects on synovial fluid anti-inflammatory mediators are less studied. In animal models hyaluronan has been demonstrated to reduce pain via protecting nociceptive nerve endings and by blocking pain receptor channels. However, the results obtained for pain-relief of human osteoarthritis are contradictory. The aim of the study was to measure the synovial fluid IL-1ra, PDGF-BB, TGF-ß1 and TNF-α concentrations before and after surgically induced cartilage defect, and following intra-articular hyaluronan injection in horses. Eight Standardbred horses underwent bilateral arthroscopic surgeries of their intercarpal joints under general anaesthesia, and cartilage defect was created on the dorsal edge of the third carpal bone of one randomly selected intercarpal joint of each horse. Five days post-surgery, one randomly selected intercarpal joint was injected intra-articular with 3 mL HA (20 mg/mL). RESULTS: Operation type had no significant effect on the synovial fluid IL-1ra, PDGF-BB, TGF-ß1 and TNF-α concentrations but compared with baseline, synovial fluid IL-1ra and TNF-α concentrations increased. Intra-articular hyaluronan had no significant effect on the biomarker concentrations but a trend of mild improvement in the clinical signs of intra-articular inflammation was seen. CONCLUSIONS: Creation of the cartilage defect and sham-operation lead to an increase of synovial fluid IL-1ra and TNF-α concentrations but changes in concentrations of anabolic growth factors TGF-ß1 and PDGF-BB could not be documented 5 days after the arthroscopy. Intra-articular hyaluronan was well tolerated. Further research is needed to document possible treatment effects of intra-articular hyaluronan on the synovial fluid biomarkers of inflammation and cartilage metabolism.

Changes in biomarkers in equine synovial fluid two weeks after intra-articular hyaluronan treatment: a randomised double-blind clinical trial.

BACKGROUND: Inflammatory and degenerative activity inside the joint can be studied in vivo via analysis of synovial fluid (SF) biomarkers, which are molecular markers of inflammatory processes and tissue turnover. The aim of this study was to investigate the response of selected biomarkers in the SF after an intra-articular (IA) high-molecular-weight non-animal stabilized hyaluronic acid (NASHA) treatment. Our hypothesis was that prostaglandin E2 (PGE2), substance P, aggrecan chondroitin sulfate 846 epitope (CS846), and carboxypeptide of type II collagen (CPII) concentrations in SF would decrease more in the NASHA than in the placebo group. Twenty-eight clinically lame horses with positive responses to diagnostic IA anaesthesia of the metacarpophalangeal or metatarsophalangeal joints were randomized into treatment (n = 15) and control (n = 13) groups. After collection of baseline SF samples followed by IA diagnostic anaesthesia, horses in the treatment group received 3 ml of a NASHA product IA. Those in the placebo group received an equivalent volume of sterile 0.9% saline solution. The horses were re-evaluated and a second SF sample was obtained after a 2-week period. RESULTS: CS846 concentration decreased in the NASHA group only (P = 0.010). Both PGE2 and CPII concentrations decreased within the groups (PGE2, P = 0.010 for the NASHA group; P = 0.027 for the placebo group; CPII, P < 0.001 for NASHA group; P = 0.009 for placebo group). No significant treatment effect for any biomarker was found between groups. NASHA induced an increase in white blood cell count; this was significant compared with baseline (P = 0.021) and the placebo group (P = 0.045). CONCLUSIONS: Although the SF concentration of the cartilage-derived biomarker CS846 decreased in the NASHA group, no statistically significant treatment effect of any of the biomarkers were observed between treatment groups. The significant increase in SF white blood cell count after IA NASHA may indicate a mild inflammatory response. However, as no clinical adverse effects were observed, we conclude that IA NASHA was well tolerated.

Long-term follow-up on recovery, return to use and sporting activity: a retrospective study of 236 operated colic horses in Finland (2006-2012).

BACKGROUND: Surgical treatment of colic is expensive and complications may occur. Information on the prognosis and the use of the horse after surgery for colic is important for surgeons and owners. Current literature on return to athletic function after celiotomy is limited. The present study reviewed surgical cases of the Veterinary Teaching Hospital, Helsinki, Finland for 2006-2012. The aim was to follow the population of horses of different breeds for surgical findings, postsurgical complications, long-term recovery and prognosis. The findings and their influence on survival, return to previous or intended use and performance were assessed. RESULTS: Most of the operated horses (82.6%; 195/236) recovered from anesthesia and 74.9% (146/195) were discharged. The total follow-up time was 8 years and 10 months and the median survival time 79.2 months. Age of the horse, location of the abdominal lesion (small vs. large intestine), incidence of postoperative colic, surgical site infection, incisional hernia or convalescence time after surgery, did not significantly affect the probability of performing in the previous or intended discipline after the surgery. A majority of the discharged horses (83.7%) was able to perform in the previous or intended discipline and 78.5% regained their former or higher level of performance. Operated horses had 0.18 colic episodes per horse-year during the long-term follow-up. The incidence of colic was 20.0% within the first year after surgery. Horses operated for large intestinal colic were 3.3-fold more prone to suffer postoperative colic than horses operated for small intestinal colic. The majority of the owners (96.3%) were satisfied with the veterinary care and nearly all (98.5%) evaluated the recovery after the colic surgery to be satisfactory or above. CONCLUSIONS: If the horse survives to discharge, prognosis for long-term survival and return to previous level of sporting activity and performance was good after colic surgery in a population of horses of different breeds. None of the factors studied were found to decrease the probability of performing in the same or intended discipline after surgery. The majority of horses were able to return to their previous activity and perform satisfactorily for several years after surgery.

A randomised, double-blinded, placebo-controlled clinical study on intra-articular hyaluronan treatment in equine lameness originating from the metacarpophalangeal joint.

BACKGROUND: Intra-articular inflammation resulting in lameness is a common health problem in horses. Exogenous intra-articular hyaluronic acid has been shown to provide an analgesic effect and reduce pain in equine and human osteoarthritis. High molecular weight non-animal stabilized hyaluronic acid (NASHA) has gained popularity in the treatment of human arthritic conditions due to its long-acting pain-relieving effects. The aim of this study was to compare the response to treatment of lameness localized in the equine metacarpophalangeal joint injected with non-animal stabilized hyaluronic acid (NASHA) and placebo (saline). Twenty-seven clinically lame horses with a positive response to diagnostic intra-articular anaesthesia of the metacarpophalangeal joint and with no, or at most mild, radiographic changes in this joint were included in the study. Horses in the treatment group (n = 14) received 3 mL of a NASHA product intra-articularly, and those in the placebo group (n = 13) received an equivalent volume of sterile 0.9% saline solution. RESULTS: The change in the lameness score did not significantly differ between NASHA and placebo groups (P = 0.94). Scores in the flexion test improved more in the NASHA group compared with placebo (P = 0.01). The changes in effusion and pain in flexion were similar (P = 0.94 and P = 0.27, respectively) when NASHA and placebo groups were compared. A telephone interview follow-up of the owners three months post-treatment revealed that 14 of the 21 horses (67%) were able to perform at their previous level of exercise. CONCLUSIONS: In the present study, a single IA NASHA injection was not better than a single saline injection for reducing lameness in horses with synovitis or mild osteoarthritis. However, the results of this study indicate that IA NASHA may have some beneficial effects in modifying mild clinical signs but more research is needed to evaluate whether the positive effect documented ie. reduced response in the flexion test is a true treatment effect.

Determination of the unsaturated disaccharides of hyaluronic acid in equine synovial fluid by high-performance liquid chromatography and fluorescence detection.

BACKGROUND: The purpose of this study was to develop and validate an analytical method to determine the presence of hyaluronic acid derived disaccharides in equine synovial fluid. FINDINGS: A high-performance liquid chromatography method for the determination of hyaluronic acid derived unsaturated disaccharides in equine synovial fluid was developed and validated. The method is based on the measurement of unsaturated disaccharides released by digestion of linear hyaluronic acid molecules. The method showed linearity (r(2) = 0.996) over the full working concentration range 0.89-30 mg/l. Relative standard deviation of intra- and inter-day precision ranged from of 4.3-6.7% and 7.1-7.8% respectively. The detection limit was 0.3 mg/l corresponding to 20 mg/l in synovial fluid. Accuracy of the assay was 97-103%. This method was evaluated by determining the concentration of unsaturated disaccharides from hyaluronic acid in synovial fluid of horses with lameness in the metacarpo-/metatarsophalangeal joint localized with positive response to intra-articular anesthesia. CONCLUSIONS: The described method is valid for determination of hyaluronic acid derived disaccharides in equine synovial fluid. This method was applied to a larger research project dealing with a new form of intra-articular therapy in horses with arthritic diseases.

Application of optical coherence tomography enhances reproducibility of arthroscopic evaluation of equine joints.

BACKGROUND: Arthroscopy is widely used in various equine joints for diagnostic and surgical purposes. However, accuracy of defining the extent of cartilage lesions and reproducibility in grading of lesions are not optimal. Therefore, there is a need for new, more quantitative arthroscopic methods. Arthroscopic optical coherence tomography (OCT) imaging is a promising tool introduced for quantitative detection of cartilage degeneration and scoring of the severity of chondral lesions. The aim of this study was to evaluate the inter-investigator agreement and inter-method agreement in grading cartilage lesions by means of conventional arthroscopy and with OCT technique. For this aim, 41 cartilage lesions based on findings in conventional and OCT arthroscopy in 14 equine joints were imaged, blind coded and independently ICRS (International Cartilage Repair Society) scored by three surgeons and one PhD-student. RESULTS: The intra- and inter-investigator percentages of agreement by means of OCT (68.9% and 43.9%, respectively) were higher than those based on conventional arthroscopic imaging (56.7% and 31.7%, respectively). The intra-investigator Kappa coefficients were 0.709 and 0.565 for OCT and arthroscopy, respectively. Inter-investigator Kappa coefficients were 0.538 and 0.408 for OCT and arthroscopy, respectively. CONCLUSIONS: OCT can enhance reproducibility of arthroscopic evaluation of equine joints.