René van Weeren

Arthrogenic lameness of the fetlock: synovial fluid markers of inflammation and cartilage turnover in relation to clinical joint pain

REASONS FOR PERFORMING THE STUDY Joint pain is one of the most common causes of lameness in the horse but its pathogenesis is poorly understood. OBJECTIVES To investigate which synovial fluid markers may be related to the presence of clinically detectable joint pain in the horse. METHODS Concentrations of structural (CPII, C2C, GAG) and inflammatory markers (PGE2, LTB4, CysLTs, bradykinin and substance P) were measured in fetlock joint fluid from 22 horses in which lameness was localised to the fetlock region by perineural anaesthesia. Levels of these markers were then compared in horses that responded (n = 15) to those that did not (n = 7) to subsequent intra-articular anaesthesia (IAA). RESULTS Of all markers analysed, only substance P levels were significantly higher (P = 0.0358) in synovial fluid of horses that showed a positive response to IAA compared to those with a negative response to IAA. Notably, while PGE2 levels were found to be elevated in all 22 lame horses compared to sound controls (P = 0.0025), they were not related to the response to IAA. CONCLUSIONS While levels of PGE2 are elevated in synovial fluid of lame horses that respond to perineural anaesthesia, only substance P is related to joint pain as detected by the response to intra-articular anaesthesia. POTENTIAL RELEVANCE Substance P is associated with clinically detectable joint pain in the horse. Elevated levels of PGE2 in fetlock-lame horses, regardless of their response to IAA, indicate that either this mediator does not reflect intra-articular pain or that IAA might have limitations in differentiating between intra- and peri-articular sources of pain. Either way, a negative response to IAA may not exclude intra-articular pathology.

Dose- and time-dependent effects of genipin crosslinking on cell viability and tissue mechanics - toward clinical application for tendon repair

The crosslinking agent genipin is increasingly invoked for the mechanical augmentation of collagen tissues and implants, and has previously been demonstrated to arrest mechanical damage accumulation in various tissues. This study established an in vitro dose-response baseline for the effects of genipin treatment on tendon cells and their matrix, with a view to in vivo application to the repair of partial tendon tears. Regression models based on a broad range of experimental data were used to delineate the range of concentrations that are likely to achieve functionally effective crosslinking, and predict the corresponding degree of cell loss and diminished metabolic activity that can be expected. On these data, it was concluded that rapid mechanical augmentation of tissue properties can only be achieved by accepting some degree of cytotoxicity, yet that post-treatment cell survival may be adequate to eventually repopulate and stabilize the tissue. On this basis, development of delivery strategies and subsequent in vivo study seems warranted.

Kinetics and kinematics of the passage

REASONS FOR PERFORMING STUDY The load acting on the limbs and the load distribution between fore- and hindlimbs while performing specific dressage exercises lack objective assessment. HYPOTHESIS The greater a horses level of collection, the more load is shifted to the rear and that during the passage the vertical load on the limbs increases in relation to the accentuated vertical movement of the centre of mass. METHODS Back and limb kinematics, vertical ground reaction force and time parameters of each limb were measured in 6 Grand Prix dressage horses performing on an instrumented treadmill at the trot and the passage. Horses were ridden by their own professional rider. RESULTS At the passage, horses moved at a slower speed (-43.2%), with a lower stride frequency (-23.6%) and, therefore, higher stride impulses (+31.0%). Relative stance duration of fore- and hindlimbs and suspension duration remained unchanged. While at the trot the diagonal limbs impacted almost simultaneously, the hindlimbs always impacted first at the passage; the time dissociation between landing and lift-off remained unchanged. Because of the prolonged stride duration, stride impulse and consequently limb impulses were higher at the passage in the fore- as well as in the hindlimbs (+24.8% and +39.9%, respectively). Within the diagonal limb pair, load was shifted from the forehand to the hindquarters (percentage stride impulse carried by the forehand -4.8%). Despite the higher impulses, peak vertical forces in the fore- and hindlimbs remained unchanged because of the prolonged absolute stance durations in fore- and hindlimbs (+28.1% and +32.2%, respectively). CONCLUSIONS Based on the intralimb timing, the passage closely resembles the trot. Compared to other head-neck positions, the higher degree of collection resulted in a pronounced shift in impulse towards the hindquarters. Despite the higher limb impulses, peak forces acting on the limbs were similar to those observed at the trot. POTENTIAL CLINICAL RELEVANCE An understanding of load distribution between fore- and hindlimbs in relation to different riding techniques is crucial to prevent wear-and-tear on the locomotor apparatus.

Differential gene expression of the intermediate and outer interzone layers of developing articular cartilage in murine embryos.

Nascent embryonic joints, interzones, contain a distinct cohort of progenitor cells responsible for the formation of the majority of articular tissues. However, to date the interzone has largely been studied using in situ analysis for candidate genes in the context of the embryo rather than using an unbiased genome-wide expression analysis on isolated interzone cells, leaving significant controversy regarding the exact role of the intermediate and outer interzone layers in joint formation. Therefore, in this study, using laser capture microdissection (three biological replicates), we selectively harvested the intermediate and outer interzones of mouse embryos at gestational age 15.5 days, just prior to cavitation, when the differences between the layers should be most profound. Microarray analysis (Agilent Whole Mouse Genome Oligo Microarrays) was performed and the differential gene expression between the intermediate interzone cells and outer interzone cells was examined by performing a two-sided paired Students t-test and pathway analysis. One hundred ninety-seven genes were differentially expressed (≥ 2-fold) between the intermediate interzone and the outer interzone with a P-value ≤ 0.01. Of these, 91 genes showed higher expression levels in the intermediate interzone and 106 were expressed higher in the outer interzone. Pathway analysis of differentially expressed genes suggests an important role for inflammatory processes in the interzone layers, especially in the intermediate interzone, and hence in joint and articular cartilage development. The high representation of genes relevant to chondrocyte hypertrophy and endochondral ossification in the outer interzone suggests that it undergoes endochondral ossification.

Osteochondral Repair: Evaluation with Sweep Imaging with Fourier Transform in an Equine Model

PURPOSE To evaluate the status of articular cartilage and bone in an equine model of spontaneous repair by using the sweep imaging with Fourier transform (SWIFT) magnetic resonance (MR) imaging technique. MATERIALS AND METHODS Experiments were approved by the Utrecht University Animal Ethics Committee. Six-millimeter-diameter chondral (n = 5) and osteochondral (n = 5, 3-4 mm deep into subchondral bone) defects were created in the intercarpal joints of seven 2-year-old horses and examined with SWIFT at 9.4 T after spontaneous healing for 12 months. Conventional T2 maps and gradient-echo images were obtained for comparison, and histologic assessment of cartilage and micro-computed tomography (CT) of bone were performed for reference. Signal-to-noise ratio (SNR) analysis was performed, and a radiologist evaluated the MR images. Structural bone parameters were derived from SWIFT and micro-CT datasets. Significance of differences was investigated with the Wilcoxon signed rank test and Pearson correlation analysis. RESULTS SWIFT was able to depict the different outcomes of spontaneous healing of focal chondral versus osteochondral defects. SWIFT produced constant signal intensity throughout cartilage, whereas T2 mapping showed elevated T2 values (P = .06) in repair tissue (mean T2 in superficial region of interest in an osteochondral lesion = 50.0 msec ± 10.2) in comparison to adjacent intact cartilage (mean T2 = 32.7 msec ± 4.2). The relative SNR in the subchondral plate with SWIFT (0.91) was more than four times higher than that with conventional fast spin-echo (0.12) and gradient-echo (0.19) MR imaging. The correlation between bone volume-to-tissue volume fractions determined with SWIFT and micro-CT was significant (r = 0.83, P < .01). CONCLUSION SWIFT enabled assessment of spontaneous osteochondral repair in an equine model.

Effect of autologous adipose tissue-derived mesenchymal stem cells on neovascularization of artificial equine tendon lesions

AIMS To investigate whether autologous adipose tissue-derived mesenchymal stem cells (AT-MSCs) treatment of tendon lesions increases neovascularization during tendon healing. MATERIALS & METHODS A standardized surgical model was used to create lesions in both front limb superficial digital flexor tendons (SDFTs) of nine horses. Either AT-MSCs or control substance was injected intralesionally 2 weeks post-surgery. Color Doppler ultrasonography of SDFTs was performed at regular intervals. Horses were euthanized 22 weeks post-treatment and SDFTs were harvested for histology. RESULTS The color Doppler ultrasonography signal was significantly more extensive at 2 weeks post-treatment and the number of vessels counted on histologic slides was significantly higher at 22 weeks post-treatment in AT-MSC-treated SDFTs. CONCLUSION Our findings indicate that AT-MSC treatment has a beneficial effect on neovascularization of healing tendons.

Variation in training regimens in professional showjumping yards

REASONS FOR PERFORMING STUDY Training regimens of showjumping horses under field conditions are largely undocumented. OBJECTIVES The aims of this study were to quantify and compare training regimens used in professional-level showjumping yards, with respect to time exercised and type of activity. STUDY DESIGN Prospective cohort study. METHODS A prospective 6-month cohort study of showjumping horses in 4 European countries (The Netherlands, Sweden, Switzerland, Great Britain) was designed to analyse training and health data, in yards with several horses in training and riders competing at professional level. Riders documented the daily frequency and duration of all physical activities of the horses. Variation in training routines were compared between riders, location and time. Mixed-models analysis was used to examine factors associated with total time exercised and time spent in flatwork. RESULTS In 4 countries, the 31 participating riders trained 263 European Warmbloods. The total days at risk (e.g. days in which the horses were considered fit for exercise) was 39,262. Mean time spent in daily exercise, including ridden work, lungeing and treadmill exercise, varied between riders from 19-52 min/day at risk. There was considerable variation in activities and level of heavy work and light exercise, i.e. turnout. Total time exercised and time spent in flatwork differed with month, country and proportion of days lost to training. Low variation of activities was associated with decreased total time trained and increased time spent in flatwork. CONCLUSIONS Riders at this elite professional level of showjumping used training regimens that vary substantially in time spent training and other physical activities and showjumping horses are challenged differently during training despite competing at the same level. Whether all training regimens prepare the horses equally for the demands of competition remains to be determined.

A prospective study on fitness, workload and reasons for premature training ends and temporary training breaks in two groups of riding horses.

Little is known about wastage in riding horses and the factors like fitness and workload that may reduce injuries and maximise welfare. To evaluate fitness, workload and reasons for premature training ends (PTEs) and temporary training breaks (TTBs) during a nine week training period, two groups of riding horses were used: Group A consisting of 58 horses used for student equitation courses (32 with training prior to admission and 26 without) and Group B consisting of 26 horses owned by two riding schools (school-I and school-II). To assess fitness, all horses performed a standardised exercise test (SET) at the start (SET-I) and end of the training period (SET-II) measuring heart rate (HR bpm) and speed (m/s). In addition, all horses were monitored daily during the training period for their health and workload. In Group A, trained horses had significantly lower HRs in SET-I (P=0.05) compared to untrained horses and in SET-II, trained horses tended to have lower HRs than untrained horses, though this was not statistically significant (P=0.057). During the training period all horses received an identical workload. A total of 19.0% of Group A horses ended the training period prematurely for veterinary reasons (PTEV); of those untrained horses had earlier a PTEV in the training period (after 2.8 ± 1.3 weeks) than trained horses (after 4.1 ± 1.5 weeks, P=0.030). In Group B, school-I and school-II horses did not differ significantly in fitness level nor in workload. More school-II horses ended the training period prematurely for veterinary reasons (n=7; 70%) compared to school-I horses (n=4; 25%, P=0.032), although seven (63.6%) of these horses were still continuously used in riding lessons. In both groups (A and B), small injuries (without a temporary training break) were significantly associated with premature training ends for veterinary reasons later on: in Group A small injuries preceded 27.3% of the PTEVs (P=0.005) and in Group B small injuries preceded 54.5% of the PTEVs (P=0.030). In conclusion, as all horses in each subgroup had the same workload, the occurrence of PTEV seemed not associated with the workload. In Group A horses, level of fitness seems to be an important factor for the point in time injuries will occur during the training period. In all horses, injuries were more likely when a temporary training break was not taken following seemingly minor injuries. Since a lot of injured Group B horses were used in riding lessons against veterinary advice, this may indicate that riding school owners have different perception on welfare and if true this may cause serious welfare problems.

Infrared spectroscopy reveals both qualitative and quantitative differences in equine subchondral bone during maturation

The collagen phase in bone is known to undergo major changes during growth and maturation. The objective of this study is to clarify whether Fourier transform infrared (FTIR) microspectroscopy, coupled with cluster analysis, can detect quantitative and qualitative changes in the collagen matrix of subchondral bone in horses during maturation and growth. Equine subchondral bone samples (n = 29) from the proximal joint surface of the first phalanx are prepared from two sites subjected to different loading conditions. Three age groups are studied: newborn (0 days old), immature (5 to 11 months old), and adult (6 to 10 years old) horses. Spatial collagen content and collagen cross-link ratio are quantified from the spectra. Additionally, normalized second derivative spectra of samples are clustered using the k-means clustering algorithm. In quantitative analysis, collagen content in the subchondral bone increases rapidly between the newborn and immature horses. The collagen cross-link ratio increases significantly with age. In qualitative analysis, clustering is able to separate newborn and adult samples into two different groups. The immature samples display some nonhomogeneity. In conclusion, this is the first study showing that FTIR spectral imaging combined with clustering techniques can detect quantitative and qualitative changes in the collagen matrix of subchondral bone during growth and maturation.

Young Friesian horses show familial aggregation in fitness response to a 7-week performance test

The aim of this study was to monitor the fitness level of young Friesian horses and to assess whether fitness data are predictive for final performance score and whether familial aggregation of response to training could be detected. Sixty-six young Friesian horses, the offspring of six different stallions (A, B, C, D, E and F), underwent a 7-week performance test. The horses were given a performance score for their ability for dressage (in weeks 5 and 7; 0-110 points) and were evaluated for fitness using standardised exercise tests (SETs) at the beginning (week 2, SET-I) and the end (week 6, SET-II) of the period. Heart rate (HR, beats/min) was measured in both SETs, and plasma lactate concentration (LA, mmol/L) was measured only in SET-II. Fitness of the horses improved moderately but significantly between SET-I and SET-II (P=0.015). There was a large heterogeneity in responsiveness to training; some horses were high responders, whereas others were non- or low responders. There was a familial aggregation of HR canter-1 response to training (P=0.039), while the HR of stallion Cs offspring decreased significantly more than those of stallions A (P=0.09), D (P=0.013) and F (P=0.009). Horses that were reluctant to exercise did not differ in HR or LA concentrations compared to those that completed the SET, which may have been a sign of overreaching. HR had no predictive value for the performance score, but horses that did not reach the anaerobic threshold in SET-II scored significantly better (73.8 ± 5.6 points) than horses that did not (69.9 ± 5.9 points, P=0.025). The findings demonstrate for the first time in the horse a familial aggregation of HR response to training, as has been reported previously in humans. Familial aggregation suggests a genetic influence on the effect of training on fitness in horses. HR could not predict final performance score, but LA concentrations during SET-II were predictive.