New developmental evidence clarifies the evolution of wrist bones in the dinosaur-bird transition.

From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal-anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal-anterior ossification does not support the dinosaur-bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal-posterior ossification develops from a cartilage referred to as "element x," but its position corresponds to distal carpal 3. The proximal-posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal-posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications.

Histochemical distribution of sialic acids and antimicrobial substances in porcine carpal glands.

The localization of sialic acids and antimicrobial products (lysozyme, IgA, lactoferrin, ß-defensin 2) as well as Rab3D in the carpal glands of pig was studied by sialoglycoconjugate histochemistry and immunohistochemistry. The secretory epithelium of the carpal glands consisted of dark and clear cells. The dark cells of these glands exhibited high levels of sialoglycoconjugates, including O-acetylated sialic acids, whereas the localization of sialic acids linked to α2-3Gal1-4GlcNAc was confined to a subpopulation of the dark cells. Furthermore, all antimicrobial substances and Rab3D were mainly detectable in a subpopulation of the dark cells. The results obtained are discussed with regard to the functional significance of these glands. Our findings suggested that Rab3D is involved in the secretory regulation of sialoglycoconjugates and antimicrobial substances. These secretory products may create a defensive barrier against microbial invasion and play an essential role in the preservation of skin integrity.

Influence of exercise on the distribution of technetium Tc 99m medronate following intra-articular injection in horses.

OBJECTIVE: To determine the effects of exercise on the distribution and pharmacokinetics of technetium Tc 99m medronate ((99m)Tc-MDP) following intra-articular (IA) injection in horses. ANIMALS: 5 horses. PROCEDURES: 1 antebrachiocarpal joint (ACJ)/horse was assigned to the exercised group (n = 5), and the contralateral ACJ was evaluated in the nonexercised group (5) after a minimum washout period of 7 days. Following IA injection of (99m)Tc-MDP (148 MBq), blood and scintigraphic images of the carpus were obtained at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 240, 360, 480, 600, 720, and 1,440 minutes. Plasma and scintigraphic radioactivity were determined over time, and pharmacokinetic parameters were generated via noncompartmental and compartmental analyses. Each horse was monitored via physical and lameness examination and ACJ synovial fluid analysis before injection and at days 1, 2, 3, and 7. RESULTS: Lameness was not observed. Mean ± SD synovial fluid WBC count increased at day 1 (exercised, 721 ± 234 cells/µL; nonexercised, 948 ± 223 cells/µL), but returned to baseline at days 3 and 7 Mean time to maximum plasma radioactivity was earlier in the exercised group (16.00 ± 2.35 minutes) than the nonexercised group (43.75 ± 3.64 minutes). Linear regression of the scintigraphic radioactivity-time curves revealed a greater negative slope in the exercised group within the first 25 minutes. There was no difference in absorption or elimination rate constants in a 2-compartment model. CONCLUSIONS AND CLINICAL RELEVANCE: IA injection of (99m)Tc-MDP was safe and effective for evaluating synovial solute distribution. Exercise significantly increased early transfer of (99m)Tc-MDP from the ACJ into plasma, although absorption and elimination rate constants were not affected. Exercise may affect synovial clearance and withdrawal times of medications administered IA.

Effects of exercise vs experimental osteoarthritis on imaging outcomes.

OBJECTIVE: To identify changes in imaging outcomes in a controlled model of osteoarthritis (OA) vs exercise. METHOD: Sixteen 2-year-old horses were randomly assigned to an exercise control (n=8) or an exercise OA (n=8) group. All horses had middle carpal joints arthroscopically explored and an osteochondral fragment was induced in one middle carpal joint of the OA group. All horses were treadmill exercised for the duration of the study (91 days). Clinical, radiographic, nuclear scintigraphic, computed tomographic and magnetic resonance imaging (MRI) examinations were performed and outcomes of these were compared between groups. Imaging results were correlated to clinical, biomarker and gross pathologic results. RESULTS: The OA group had significant increases in clinical outcomes and most imaging parameters. Specifically, the OA group showed significant increases in radiographic lysis and nuclear scintigraphic uptake. There was very little change in subchondral bone density, but a significant change in subchondral bone edema. Radiographic lysis, radial carpal bone edema and nuclear scintigraphy were strongly correlated with clinical changes and radial carpal bone edema was strongly correlated with changes in Type I and Type II collagen found in the synovial fluid. CONCLUSIONS: OA induced significant changes in imaging parameters beyond the adaptation seen with exercise. Bone edema detected with MRI was closely correlated with collagen biomarkers detected in the synovial fluid.

Interleukin-6 and tumour necrosis factor in synovial fluid from horses with carpal joint pathology.

The carpal joints are common sites of traumatic arthritis and osteoarthritis (OA) in athletic horses. The pro-inflammatory cytokines interleukin (IL)-6 and tumour necrosis factor (TNF) may be of great importance in the development of intra-articular lesions. The aim of the present study was to investigate possible associations between synovial fluid levels of bioactive IL-6 and TNF and different types of joint lesions seen in traumatic arthritis and OA. Synovial fluid was collected from horses with carpal lameness immediately before arthroscopic surgery. Articular cartilage, synovial membranes and intra-articular ligaments were assessed macroscopically at arthroscopy. Synovial fluid levels of IL-6 and TNF were determined by bioassays, and the cytokine levels between different grades of morphologic changes in each type of assessed tissue were compared. The highest levels of IL-6 were detected in joints with chip fractures. All joints with chip fractures also showed some degree of synovitis. Tumour necrosis factor bioactivity was low and not associated with any joint lesion. Hence, TNF is not useful as a biomarker indicating a specific joint lesion in equine traumatic arthritis or OA. We conclude that a dramatic increase of IL-6 in synovial fluid indicates the presence of osteochondral fragmentation, although low or undetectable levels of IL-6 do not exclude chip fractures. The role of IL-6 in the disease process of osteochondral fragmentation needs further investigation.

Ultrastructural immunolocalisation of bone sialoprotein in the osteocartilagenous interface of the equine third carpal bone.

REASONS FOR PERFORMING STUDY: One of the most common causes of lameness in racehorses is osteoarthritis (OA). Pathogenesis is not clear and pathological processes of the different joint tissues interact in often progressive events. The interface between cartilage and newly synthesised bone has been shown to be particularly enriched in bone sialoprotein (BSP), a cell-binding matrix protein. OBJECTIVES: To establish whether changes in the concentration of BSP may serve as a marker for early biochemical changes of the subchondral bone. METHODS: Articular cartilage, cartilage/bone interface and subchondral bone of the proximal third carpal bone from 3 Standardbred trotters were analysed ultrastructurally for the presence of BSP in normal and degenerative areas. RESULTS: A marked increase of BSP in the cartilage/bone interface with degenerative changes of the bone and cartilage compared to the morphologically intact cartilage/bone interface was noted, but levels of the protein were distinctly lower in the distal bone. CONCLUSIONS: The results indicate that BSP has the potential to be used as a marker for changes in bone metabolism in the subchondral bone. POTENTIAL RELEVANCE: Tools to monitor early biochemical changes within the connective tissues of the joint in vivo are essential in studies of the pathogenesis of OA. These could be used to monitor and understand such changes in relation to load, exercise, training programmes, inflammation and the development of OA.

Diffusion of mepivacaine between adjacent synovial structures in the horse. Part 1: forelimb foot and carpus.

This paper tests the hypothesis that the local analgesic agent mepivacaine diffuses between adjacent equine synovial structures in the forelimb and with greater frequency than latex, gelatine dye or contrast media. We report the incidence of diffusion of mepivacaine between the distal interphalangeal joint (DIPJ) and navicular bursa (NB) of the forelimbs and between the intercarpal (IC) and radiocarpal (RC) joints of 31 fresh equine cadavers. The DIPJ of one forelimb and the NB of the contra lateral forelimb and the RC joint of one forelimb and the IC joint of the contra lateral forelimb were injected with mepivacaine. After flexion and extension of the joints, synovial fluid was obtained from the synovial structures adjacent to the injected synovial structures. The concentration of mepivacaine in these samples was determined using an enzyme linked immunosorbent assay. For samples obtained by dilution of synovial fluid, the concentration of mepivacaine was determined by comparing the concentrations of urea in the diluted synovial fluid and the concentration of serum urea. Mepivacaine diffused from the DIPJ to the NB or from the NB to the DIPJ in 25/25 (100%) limbs. Mepivacaine diffused from the IC to RC joints in 24/25 (96%) limbs and from the RC to IC joints in 21/25 (84%) limbs. It was detected at concentrations >0.3 mg/l in 9/25 (36%) of IC joints after RC joint injection and in 25/25 (100%) of the NB after DIPJ injection; at concentrations >100 mg/l in 2/25 (8%) of IC and RC joints and 12/25 (48%) of NB following DIPJ injection; and at concentrations >300 mg/l in 1/25 (4%) in the IC joints following RC joint injection and in 11/25 (44%) of DIPJ following NB injection. The results show greater diffusion of mepivacaine between adjacent synovial structures than assumed from previous anatomical, latex injection and contrast arthrographic studies. This study showed that commonly performed intrasynovial analgesic techniques in the forelimb of the horse are not as specific as previously reported.

A joint myxoma in a dog.

Myxomas of the joints are extremely rare in domestic animals, only four cases, all in dogs, having been reported previously. This paper describes a myxoma originating from the synovium of the right radiocarpal joint of a mature female Dobermann pinscher with right front limb lameness. The tumour was excised surgically and no recurrence was detected during a 2-year follow-up period. Immunohistochemically, the tumour cells reacted with antibodies to vimentin and S100 protein but not with antibodies to cytokeratins (high and low molecular weight) or human callus keratin.

Release of lubricating synovial surfactant by intra-articular steroid.

This study was undertaken to determine whether glucocorticosteroids promote the secretion of lubricating surfactant, i.e. surface-active phospholipid (SAPL), into the joint. A standard clinical dose (100 mg) of methylprednisolone acetate (MPA) in 2.5 ml of saline was injected into the load-bearing right radiocarpal joint of five horses and 2.5 ml of saline injected into each of the contralateral joints used as controls. Synovial fluid (SF) was aspirated from all 10 joints before injection and at intervals of 16 and 32 h after injection, and then analysed by standard methods. All test joints showed an elevated level of SAPL, the increases averaging 112% after 16 h and 76% after 32 h, which were highly significant relative to the control joints. A large increase at 16 h was also found in proteolipid as a possible further marker of surfactant release. Significant quantities of proteolipid were also found in human SF. Since intra-articular steroids can dramatically improve joint mobility in both humans and horses, it is proposed that part of the benefit may be derived from improved lubrication arising from the remarkable ability of SAPL to lubricate under high load. Other possible benefits of elevating surfactant levels in the joints include control of cartilage hydration, promotion of macrophage activity and the ability to scavenge oxygen free radicals.

The effect of strenuous versus moderate exercise on the metabolism of proteoglycans in articular cartilage from different weight-bearing regions of the equine third carpal bone.

Articular cartilage degeneration in the middle carpal joint is a common problem in racing horses. This study evaluated the effect of exercise on the in-vitro synthesis of the large aggregating proteoglycans (aggrecan) and two small proteoglycans, biglycan and decorin, in articular cartilage taken from three weight bearing regions of the third carpal bone of horses which were subjected to moderate or strenuous exercise. Twelve Standardbred horses free from clinical and radiographic disease of the middle carpal joint were subjected to an 8 week moderate exercise program. The horses were then randomly assigned to two groups: group A--continued moderate exercise and group B--strenuous exercise for 17 weeks. Horses were then rested for 16 weeks. Full-depth articular cartilage explants from the dorsal radial facet (DRF), dorsal intermediate facet (DIF) and palmar condyle (PC) of the third carpal bone were collected and cultured. Cartilage proteoglycan content and release into culture media were measured. Newly synthesized proteoglycans were labeled with 35SO4(2-) for 48 h and analyzed by size exclusion and hydrophobic chromatography, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and autoradiography. Histologic sections of adjacent osteochondral regions were evaluated for evidence of arthritic change. No histologic abnormalities or differences in proteoglycan content were detected in any of the articular cartilage regions examined. There was however, a significant reduction (P < 0.05) in aggrecan synthesis and a concomitant increase in decorin synthesis (P < 0.05) in articular cartilage from the DRF of group B animals. There was no change in biglycan synthesis, aggrecan hydrodynamic size or ability to aggregate in any articular cartilage region. This study has demonstrated that strenuous exercise in horses can lead to a disturbance in the biosynthesis of proteoglycans in articular cartilage regions subjected to high contact stresses (DRF). These metabolic abnormalities, which persisted for 16 weeks after cessation of exercise, could have deleterious effects on the biomechanical properties of the tissue. We suggest that the observed alteration in articular cartilage metabolism in CRF cartilage of strenuously exercised horses could represent a predisposing factor for cartilage degeneration and osteoarthritis at a later stage.

Effect of tumor necrosis factor antibody on synovial fluid cytokine activities in equine antebrachiocarpal joints injected with endotoxin.

Six horses received intra-articular injections of a mixture of 1 micrograms of endotoxin/5 mg of equine tumor necrosis factor (eqTNF) monoclonal antibody in 1 antebrachiocarpal joint and an equal volume (2 ml) of 1 micrograms of endotoxin/5 mg of control antibody in the opposite joint. Synovial fluid sample collection (1 ml) was accomplished by use of an indwelling, intra-articular catheter at postinjection hours (PIH) 0, 1, 1.5, 2, 5, and 8, and by arthrocentesis at PIH 24. Joint fluid samples were analyzed for nucleated cell count, protein concentration, and TNF, interleukin 6 (IL-6), IL-1, and IL-1-inhibitory activities. To monitor local inflammation, each carpus was graded semiquantitatively for swelling prior to each sample collection. Tumor necrosis factor, IL-1, or IL-1-inhibitory activity was not detected in any synovial fluid sample collected before endotoxin/antibody was administered. However, low IL-6 activity (< 100 U/ml) was found in 2 of 12 preinjection samples. In joints injected with endotoxin/control antibody mixture, maximal mean +/- SEM activities for TNF (1,019 +/- 310 U/ml), IL-1 (173 +/- 102 U/ml), and IL-6 (10.8 +/- 3.1 x 10(4) U/ml) were observed at PIH 2, 5, and 8, respectively. Tumor necrosis factor and IL-1 activities returned to baseline values by PIH 8 and 24, respectively; however, IL-6 activity remained high. Interleukin 1 inhibitory activity (27.4 +/- 2.25 IU/ml) was detected in all PIH-24 samples from control joints, but was not detected at any other time in control joints (limit of detection, 20 IU/ml). Tumor necrosis factor activity was not detected in any synovial fluid sample from joints treated with endotoxin/eqTNF antibody. In contrast, endotoxin IL-1 inhibitory activity (PIH 24) was higher in eqTNF antibody-treated joints (41.0 +/- 7.7 IU/ml) than in control joints, but the difference was not significant. Mean WBC count and protein concentration in control and treated joints were maximal at PIH 8. The curves for mean values of WBC count and total protein concentration were not significantly different in treated versus control joints. Swelling in each treated joint was either less than or the same as that in the opposite control joint at even, time in the initial 8 PIH. There was significant (P = 0.043) difference between treated and control joints at PIH 5 and 8. These results describe a profile of synovial fluid TNF, IL-1, IL-6 bioactivities, and IL-1-inhibitory activity during the initial 24 hours of synovitis induced by intra-articular administration of endotoxin in horses. Our eqTNF monoclonal antibody was effective in neutralizing TNF activity in synovial fluid when administered intra-articularly with endotoxin in horses. The induction of IL-1, IL-1 inhibitory activity IL-6, WBC, and total protein concentration responses are largely independent of TNF activity in synovial fluid of horses receiving endotoxin intra-articularly.

Effects of exercise and polysulfated glycosaminoglycan on repair of articular cartilage defects in the equine carpus.

Our aim was to determine if mild to moderate postoperative exercise and intra-articular polysulfated glycosaminoglycan result in improved repair of large, experimentally induced osteochondral defects in a weight-bearing surface of equine joints. Arthroscopic debridement was used to produce full-thickness defects in a weight-bearing area of the radial carpal bones in 18 ponies. The ponies were randomly assigned to two groups balanced for age: nine animals in the exercise and nine in the no exercise group. Six ponies in each group were medicated weekly for 5 weeks with an intra-articular injection of polysulfated glycosaminoglycan in one middle carpal joint beginning at the time of operation. Walking (twice daily) was begun 6 days postoperatively, and by the twelfth week postoperatively the ponies were trotting for 25 min and walking for 15 min twice daily. At the time of the ponies' death, 17 weeks postoperatively, each defect had an average of 50-75% coverage with repair tissue. Exercised, medicated joints had a significantly smaller area of coverage with repair tissue than exercised, nonmedicated joints. Cartilaginous repair tissue from exercised ponies contained significantly more glycosaminoglycan and type-II collagen (r = 0.53, p < 0.05). The ratio of hydroxylysine to hydroxyproline was significantly lower and the ratio of collagen content to total protein was significantly higher in the repair tissue of medicated joints than in the repair tissue of nonmedicated joints; this is consistent with the presence of less type-II collagen in the repair tissue in medicated joints. We concluded that postoperative exercise was beneficial and that the immediate postoperative use of intra-articular polysulfated glycosaminoglycan was detrimental to the development of cartilaginous repair tissue in large osteochondral defects of equine joints.

Regional perfusion of the equine carpus for antibiotic delivery.

Regional perfusion of carpal tissues by forced intramedullary administration of fluids was evaluated in 10 horses. Results of subtraction radiography after perfusion with a contrast medium demonstrated that perfusate was delivered to the carpal tissues by the venous system. Perfused India ink was distributed uniformly in the antebrachiocarpal and middle carpal synovial membranes. Histologically, the ink was within the venules of the synovial villi. Immediately after perfusion with gentamicin sulfate (1 g), the gentamicin concentrations in the synovial fluid and synovial membrane of the antebrachiocarpal joint were 349 +/- 240 micrograms/mL and 358 +/- 264 micrograms/g, respectively. When gentamicin concentrations in the synovial fluid of the antebrachiocarpal joint and serum were measured 0, 0.5, 1, 4, 8, 12, and 24 hours after carpal perfusion, the mean peak gentamicin concentration in the synovial fluid was 589 +/- 429 micrograms/mL. At hour 24, the mean gentamicin concentration in the synovial fluid was 4.8 +/- 2.0 micrograms/mL. The resulting peak gentamicin concentration in the serum was 23.7 +/- 14.5 micrograms/mL immediately after the perfusion; it decreased below the desired trough level of 1 micrograms/mL between hours 4 and 8.

Iodide and albumin kinetics in normal canine wrists and knees.

The clearance rates of free iodide and of radioiodinated serum albumin were measured in the knee and wrist joints of 9 normal adult dogs. Iodide clearance from the knee was 3 times greater than that from the wrist. In contrast, radioiodinated serum albumin clearance from the knee was only slightly greater than that from the wrist. Interpreted as respective indices of effective synovial plasma flow and lymphatic drainage, these values indicate that the filtration fraction is normally greater in microvessels of the wrist than in those of the knee. These findings complement the results of companion studies of Starling forces that indicate a higher pressure microvascular bed in the wrist than in the knee.