Cartilage canals in the medial coronoid process of young Golden Retrievers.

The aim of the study was to establish the location of cartilage canals in the medial coronoid process (MCP) of the ulna of young Golden Retrievers, a breed that is predisposed to fractures of the medial coronoid process (FMCP). To determine whether the presence of cartilage canals could be associated with the predilection site of FMCP, the right elbows of nine young Golden Retrievers (aged 4, 6, 8, 10, 13, 16, 18, 22 and 24 weeks) were dissected and, with no prior decalcification, the formaldehyde-fixed MCPs were embedded in methylmethacrylate. The entire MCPs were serially sectioned in the frontal plane from cranial to caudal and the sections (5 microm) were routinely stained. Between the ages of 5 and 10 weeks, three main cartilage canals were visible--medial, central and lateral. All originated from the periosteum of the distal parts of the MCP and ended proximally under the articular cartilage. Branches of the main canals were seen more cranially and caudally. At the age of 13 weeks, the central canal was absent, and the remaining canals showed a smaller diameter. From 16 weeks onwards, no cartilage canals were seen. No direct relationship could be established between the predilection site of FMCP (lateral part of the MCP) and the presence/absence of cartilage canals, since both medial and lateral canals disappeared at the same age. Further research is needed to elucidate the pathogenesis of FMCP.

Presence of SERCA and calcineurin during fetal development of porcine skeletal muscle.

Mechanisms involved in skeletal myofiber differentiation during fetal development of large animals are poorly understood. Studies in small animals suggest that the calcineurin (Cn) pathway is involved in myofiber differentiation. Neural activity is a prerequisite for Cn activity, implying maintenance of sustained low intracellular Ca(2+) concentrations. To study the role of Cn in fetal myofiber differentiation, we monitored the temporal and spatial distribution of Cn subunits, sarcoplasmic reticulum Ca(2+) ATPase (SERCA), phospholamban (PLB), and myosin heavy chain (MyHC) isoforms in relation to ingrowing nerves in porcine semitendinosus muscle (m. semitendinosus) at 55 and 75 days of gestation (dg) and at term. Immunofluorescence analysis revealed the presence of Cn subunits and SERCA isoforms at all analyzed stages. Cn distribution was not fiber-type specific, but expression became more prominent at term. At 75 dg, differential SERCA2 expression was accompanied by perinuclear PLB in primary fibers. SERCA1 was expressed in all fiber types at all stages. No specific MyHC isoform distribution was seen in relation to neuromuscular contacts, although neuromuscular contacts were present. From these results we speculate that in porcine m. semitendinosus differential SERCA2 expression precedes differential Cn expression. The question whether the Cn pathway is involved in prenatal myofiber differentiation needs further studies.

Myosin heavy chain isoforms in equine gluteus medius muscle: comparison of mRNA and protein expression profiles.

The major structural protein in skeletal muscle, myosin heavy chain (MyHC), is primarily transcriptionally controlled. We compared the expression of MyHC isoforms on the mRNA and protein level in biopsies from the m. gluteus medius from adult untrained horses. In transverse sections, the majority of fibers showed qualitatively identical mRNA and protein expression patterns. However, coexpression of 2a and 2d/x MyHCs was substantially more common at the protein than at the mRNA level, suggesting a fine-tuning of these two genes in normal muscle not subjected to any training protocol. Because transverse sections give a limited sampling of mRNA expression in the case of uneven distribution of transcripts in a muscle fiber, we also analyzed longitudinal sections. We present, for the first time, evidence that expression of MyHC mRNA and protein was equal along the length of the fiber. Hence, mRNA expression is not regulated by differential expression of isoforms by separate myonuclei. It is concluded that the number of protein hybrid fibers in equine gluteus medius muscle is controlled by alteration of the transcription pattern uniformly along the fiber, rather than by simultaneous transcription of genes. The differences with the results in muscle of small animals and humans are discussed.

Embedding of large specimens in glycol methacrylate: prerequisites for multi-signal detection and high-resolution imaging.

Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm(3)) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle.

Development of the subchondral bone layer of the medial coronoid process of the canine ulna.

The medial coronoid process (MCP) of the ulna takes part in the weight-bearing function of the elbow in quadrupedal animals. In this study, the timing of development of a solid subchondral bone layer (SBL) of the MCP in the dog is investigated, as this might be important in the pathogenesis of the fractured medial coronoid process, a common disease in young dogs of larger breeds. The SBL is considered to make an important contribution to the strength of the MCP. In this study, the SBL is visualized at the humeral articular side (H-side) and in the radial notch (RN) via three-dimensional reconstructions of micro-CT scans (34 mum voxel size) in nine young golden retrievers. After micro-CT scanning, the area was investigated histologically. Gradually, the appearance of the SBL on the H-side changes from a trabecular aspect to an even surface with gaps and finally to a completely even surface. The surface in the RN is still rough at 24 weeks after birth, although some consolidation has occurred. Initially, the enchondral ossification activity, as observed in the histological sections, is high, but later, when the intertrabecular spaces are filled in with calcified cartilage and bone, activity is less evident. Some vessels penetrated the SBL, but it is unclear if they account for all the gaps in the surface seen in micro-CT. In addition, the formation of a cortical structure of the proximal ulnar shaft could be visualized. The bony cortex is already even at the mediocaudal side of the proximal ulna 4 weeks after birth, but remains trabecular at the dorsal side until 6 weeks later. We hypothesize that the observed differences in the formation of an even SBL or cortex can be explained by mechanical factors. A smooth cortical layer has an even thickness and will be stronger than a cortex with varying thickness. In the MCP, compressive forces exerted by the humerus may be responsible for the early smoothening on the H-side. In the proximal ulna, the resistance to bending in the sagittal plane will depend mainly on the caudal surface of the ulna (and dorsal surface of the radius), surfaces furthest away from the neutral axis. At least the caudal ulna smoothened first, thus providing stiffness against bending at an early age.

Architecture and mineralization of developing trabecular bone in the pig mandibular condyle.

Architecture and mineralization are important determinants of trabecular bone quality. To date, no quantitative information is available on changes in trabecular bone architecture and mineralization of newly formed bone during development. Three-dimensional architecture and mineralization of the trabecular bone in the mandibular condyle from six pigs of different developmental ages were investigated with micro-CT. Anteriorly in the condyle, a more advanced state of remodeling was observed than posteriorly, where more active growth takes place. Posteriorly, the bone volume fraction increased with age (r=0.87; P<0.05) by an increase of trabecular thickness (r=0.88; P<0.05), while the number of trabeculae declined (r=-0.86; P<0.05). Anteriorly, despite an increase in trabecular thickness (r=0.97; P<0.001), there was no change in bone volume fraction due to a simultaneous decline in trabecular number (r=-0.84; P<0.05) and increase in trabecular separation (r=0.95; P<0.01). Posteriorly, rods were remodeled into plates as expressed by the structure model index (r=-0.97; P<0.001), whereas anteriorly, a plate-like structure was already present in early stages. The trabecular structure had a clear orientation throughout the developmental process. The global degree of mineralization increased both anteriorly (r=0.86; P<0.05) and posteriorly (r=0.89; P<0.05). We suggest that the degree of mineralization does not depend on the bone volume, but on the thickness of the trabeculae as the mineralized centers of trabeculae were getting larger and more highly mineralized with age compared to their appositional layers. This indicates that besides apposition of new bone material on the surface of trabeculae, the mineralized tissue in their centers still changes and matures.

Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

Cartilage-free areas in the elbow joint of young golden retrievers.

The present study describes cartilage-free areas on the ulnar trochlear notch and the humeral condyle of eight very young golden retrievers with otherwise healthy elbow joints. Remarkably, the youngest dog with full-thickness cartilage-free areas was only 8 weeks old. The younger dogs showed no macroscopic abnormalities on the locations that were affected in the older dogs. Two kinds of cartilage modifications were found. Cartilage-free areas at the edges of the articular cartilage layer were present on the humeral capitulum and on two locations of the ulna, (the medial and lateral at the base of the anconeal process, and the trochlear notch near the lateral coronoid process, which was fractured in two cases). Histological examination showed that these cartilage-free areas were filled with dense supportive tissue. Synovial cells covered this tissue as well as the surrounding hyaline cartilage. The synovial membrane covering the areas was macroscopically enlarged, but histological examination revealed no signs of inflammation. The second type of modification consisted of discoloration of the articular surface at the humeral trochlea. Histological examination revealed that in this area the articular surface was composed of fibrocartilage instead of hyaline cartilage. Apparently, there are locations within the elbow joint in which articular cartilage is not necessary for normal joint functioning. The presence of fibrocartilage on the articular surface of the humeral condyle is a surprising finding, for which no explanation has yet been found.

The equine hind limb is actively stabilized during standing.

Horses spend much of their life standing, and they are believed to be able to keep their limbs straight without muscular effort. We tested the hypothesis that the stifle (knee) and hock (tarsal) joints could be stabilized merely with the help of a passive lock mechanism whereby the patella is secured behind a hook, formed by the medial femoral trochlea. In anaesthetized animals and isolated limbs the stifle and hock flex readily under compression. In isolated limbs this collapse was prevented by a small force applied to the patella, mimicking the action of the vastus medialis muscle. In vivo, when the limb was planted loosely on the ground none of the muscles with a connection to the patella was active. However, during weight-bearing the vastus medialis (but no other muscle) was active, providing the necessary traction to stabilize the stifle. The required tension was estimated to be less than 2% of the force that would be needed in absence of a lock mechanism. Diagnosis and treatment of patellar fixation should include the possibility of overactive vastus medialis muscle as a possible cause of the disorder.