The biology which underlies expert musical performance is coming increasingly under objective scrutiny. This is providing insights not only into the way in which a musician's skills are acquired and developed, but also more generally into how the areas of the brain involved in motor activity are modified by intensive, long term practice. The physical challenges faced by musicians are similar to those of professional sportsmen but as a group they provide some unique advantages for the study of motor control. Many start their training at a very early age when the central nervous circuitry is extremely plastic. To perform at the highest level, their motor skills must be honed by many hours of daily practice, but as the activity involved is not energetically demanding, physical fatigue is not generally a limiting factor. As a consequence, intensive training can be maintained over almost the entire lifespan. In keyboard and string players, the motor activity is focused primarily on the muscles which control the fingers and so consequent changes in the structure of the sensory and motor cortical maps are relatively easy to detect. In some instances these changes appear be maladaptive, and observations of the cortex of musicians suffering from focal dystonia has generated new theories about the origin of this condition and suggested new therapies for its treatment.
Encephalopathy is a common complication of sepsis and a frequent cause of death in intensive care units. Cerebral oedema is an important feature of septic encephalopathy, but it is not known whether it results from breakdown of the blood–brain barrier (BBB) or a failure of brain water clearance. Therefore the pathogenesis of septic encephalopathy-related cerebral oedema was investigated in an animal model.
Sepsis was induced by caecal ligation and puncture (CLP) in 10 male wistar rats and illness was allowed to develop over 24 h. Sham-operated (n = 10) and unoperated (n = 10) rats served as controls. 1 h before sacrifice, 4 rats from each group were injected with 1 mL of 2% Evans blue dye via a tail vein. 1 h later they were given a lethal dose of anaesthetic and perfused with fixative at a pressure of 105–110 mmHg and the brains removed. The remaining 6 rats from each group were perfused with fixative containing 2% lanthanum nitrate 24 h after surgery and the brains removed. Tissue from all brains was then processed into resin for microscopy. All procedures were authorised under the Home Office Animals (Scientific Procedures) Act 1986.
Rats subjected to CLP exhibited severe perimicrovessel oedema, swollen astrocyte endfeet, and occasional dark shrunken cells in the cerebral cortex, compared to controls. The perfused brains of all rats injected with Evans Blue appeared white, apart from the circumventricular organs which appeared blue. Dye particles did not penetrate the parenchyma of the cerebral cortex in any of the experimental groups. However, electron microscopy of tissue from lanthanum nitrate-perfused rats revealed particles of lanthanum in the neuropil surrounding cortical microvessels in CLP, but not control rats.
Evans blue binds to serum albumin and its absence from the cortical parenchyma following CLP indicates that the BBB is not permeable to serum proteins in these animals. The presence of lanthanum nitrate particles (MW 138.9) in the cortical parenchyma following CLP, suggests that the BBB is permeable to ions and solutes. However whether systemic sepsis causes cerebral oedema by opening paracellular or transcellular routes through the BBB remains to be investigated.
Damage to myelin sheaths and incomplete myelination, such as occur during demyelination and remyelination in Multiple Sclerosis (MS), leads to severe neurological deficits. Fibroblast growth factor (FGF-2) is upregulated in MS and is a key factor in the control of oligodendrocyte differentiation. It was hypothesised therefore that FGF-2 may be important in demyelination and the failure of remyelination in MS. Here we have examined the effects of FGF2 in vivo in the developing and adult anterior medullary velum (AMV), which roofs the IVth ventricle and is bathed by CSF. Rats aged postnatal day (P) 6–9 were deeply anaesthetised by isoflurane and FGF-2 was delivered by injection into the cerebrospinal fluid of the lateral ventricle, twice daily for 1–3 d. Sterile saline vehicle was injected for all control animals. After 1–3 d rats were humanely killed by perfusion of 4% paraformaldehyde via the left cardiac ventricle under terminal sodium pentobarbital, according to UK Home Office regulations. AMV were dissected free and immunohistochemistry was used to label oligodendrocytes, myelin, axons and oligodendrocyte progenitor cells (OPCs). FGF2 had a dual action on oligodendrocytes, promoting recruitment of immature oligodendrocytes after 2 d treatment, but causing the loss of these previously generated cells after 3 d treatment. In addition FGF-2 induced the loss of myelin-forming oligodendrocytes and myelin. Treatment with FGF2 for 2 d or 3 d induced a marked increase in the density of NG2 + OPCs within demyelinated lesions, and OPCs formed multiple contacts with demyelinated axons. Significantly, treatment with the GSK3β inhibitor indirubin effectively blocked the FGF2 mediated loss of myelin-forming oligodendrocytes and dramatically enhanced the FGF2-induced accumulation of premyelinating oligodendrocytes. This suggests that activation of GSK3β by FGF2 has an inhibitory action on the differentiation of myelin-forming oligodendrocytes, supporting a role for FGF-2 in the pathology of demyelination and the failure of remyelination in MS.
Supported by the Multiple Sclerosis Society.
The inwardly rectifying potassium channel (Kir) 4.1 is considered a specific glial potassium channel, and we have previously shown that Kir4.1 is crucial for the development of a highly negative resting membrane potential (RMP) in optic nerve glia. There is in vitro evidence that glial potassium currents are decreased following injury, and it is proposed that this is important for glial proliferation and reactive gliosis. Here we have tested whether Kir4.1 expression and glial RMP are altered in vivo following injury in the rat optic nerve. Rats aged postnatal day 15 were deeply anaesthetised by isoflurane and unilateral enucleation was performed. At 24 h, 3 and 50 d post-lesion animals were humanely killed by overdose of pentabarbitone, according to UK Home Office regulations. Optic nerves were removed and either treated for immunohistochemical analysis of Kir4.1 expression or for electrophysiological measurement of the glial RMP. Antibodies to Kir4.1 labelled both oligodendrocytes and astrocytes in control nerves, whereas Kir4.1 expression was markedly decreased at 24 h post-lesion and remained low up to 50 d post-lesion. For measurement of RMP, optic nerves were placed in oxygenated artificial cerebrospinal fluid and maintained in a brain slice chamber. Intracellular impalements were made along the length of the nerve and followed by ionophoretic injection of LRD for cellular identification. In control nerves, the RMP for astrocytes was −60.4 ± 2.4 mV (n = 32) and −63.0 ± 4.1 mV (n = 6) in oligodendrocytes. At 24 h post-lesion, the RMP was significantly depolarised in both astrocytes (−35.6 ± 3.1 mV, n = 21) and oligodendrocytes (−28.6 ± 4.8 mV, n = 5) in enucleated nerves. These results show that decreased expression of Kir4.1 is an early event in the glial injury response and correlates with glial depolarisation. This study supports our findings that Kir4.1 has a key role in glial differentiation and setting the glial RMP.
Supported by the BBSRC.
The inwardly rectifying potassium channel, Kir4.1, is almost exclusively a glial Kir within the CNS. The increase in expression of Kir4.1 during the first 3 week of postnatal development is believed to be concurrent with glial differentiation (Neusch, 2001 J Neurosci 21). Accordingly Kir4.1 is hypothesised to have a primary role in setting the highly negative resting membrane potential (RMP) of glia. We have previously shown that the glial RMP in the optic nerve of Kir4.1 knockout (KO) mice is significantly depolarised compared to wild-type (WT). Here we have examined whole cell inward currents of glia in cultures of optic nerve explants from Kir4.1 KO, heterozygote (HZ) and WT mice. Mice aged postnatal day (P) 8 to P12 were humanely killed by intraperitoneal injection of sodium pentabarbitone, according to UK Home Office regulations. Optic nerves were removed and explants cultured for 8–12 d in vitro when a mixed culture of astrocytes and oligodendrocytes in various stages of differentiation was evident. For perforated patch recordings, the pipette solution was supplemented with amphotericin B (20 µg/mL). Whole cell membrane currents were activated by applying a hyperpolarising voltage steps from −50 to −200 mV in artifical cerebrospinal fluid and following administration of BaCl2 (100 µm). Peak inward currents in KO cultures (n = 4) were significantly less than those recorded in WT cultures (n = 16), whilst cells from HZ cultures had intermediate inward currents (n = 12; P < 0.05). BaCl2 significantly decreased inward currents in cells from WT cultures (P < 0.05, n = 13) and HZ cultures (P < 0.05, n = 12). However, this inhibition was significantly less in HZ than in WT cultures (P < 0.05). In 3 of 4 cells in KO cultures, BaCl2 had no significant effect on inward current (P > 0.05) but 1 cell showed a reduction in inward current. These data provide evidence that Kir4.1 is crucial for the development of inward currents in optic nerve glia, and supports the hypothesis that an absence of Kir4.1 blocks glial differentiation.
Supported by the BBSRC.
The purpose of this project was to design and evaluate a clinical anatomy course for dental undergraduates delivered as part of a programme of vertical integration of basic sciences within the dental undergraduate curriculum in Newcastle. Final year undergraduates were exposed to a 6 week program of virtual anatomy tutorials mounted and run on Blackboard™. Six tutorials were designed by an anatomist and surgeon with special emphasis on providing clinical relevance to the anatomy. Students' learning was monitored by pre and post-tutorial tests. The pre-test was of a pure anatomical nature to focus the students' minds on the subject matter ahead. The post-tutorial test was a clinically relevant anatomy test where the links created in the tutorial were examined. Four virtual classrooms were provided as question and answer sessions once a fortnight. At the cessation of the course a purposive sample (n = 13) of the year-group was taken and in-depth qualitative interviews performed until data saturation occurred. All data were audio-recorded and transcribed verbatim. Interviews were conducted by one researcher and analysed by 2. The data were entered into a framework and both framework and constant comparative analysis were used to interpret it. The results of this qualitative analysis, which have been previously reported upon to the Society, were then used to generate a quantitative survey administered to the entire student group. The results of this quantitative study showed that the tutorials were rated highly overall by the students. However, they had a range of comments on the content of the tutorials and a lack of appreciation of the learning outcomes. The significance of the findings on content and on the communication of learning outcomes when designing virtual learning packages in anatomy will be discussed
This work was supported by a grant from LTSN-01.
The epithelial components of many organs such as the lung, kidney, pancreas, salivary, mammary and prostate glands begin as a simple structure. Through the process of branching morphogenesis, these epithelia form complex tree-like arrangements. It seems that these developing epithelia have functionally different cells at the ends of the buds compared to those elsewhere. In the branching ureteric bud of the kidney, for example, cells at the bud tips are capable of inducing nephrons while those of the stalk regions are not, and branching is largely restricted to the tip regions. Controlling cell identity within the developing epithelium may therefore be important if branching morphogenesis is to proceed normally.
In order to understand cell identity within the branching epithelium, it would be helpful to identify some cell specific markers. We have identified a novel marker of the stalk regions of the ureteric bud, the lectin Dolichos biflorus agglutinin (DBA). Using confocal microscopy we have established that DBA binds to the stalk of the ureteric bud throughout kidney development in ex-vivo organ culture. We have also shown that DBA does not bind to ectopic tips that are induced when kidneys are grown in the presence of exogenous glial derived neurotrophic factor (GDNF), a molecule critical for branching morphogenesis in the kidney. Finally we have shown that, when signalling by GDNF is inhibited by application of either function-blocking antibodies or sodium chlorate, DBA staining extends throughout the ureteric bud. As DBA, the stalk marker, extends into the tips during sodium chlorate treatment the tip marker Wnt11 disappears, suggesting that the cells have changed differentiation state. We conclude that DBA is a useful marker of stalk regions of the ureteric bud during development in culture and that tip regions express stalk markers when branching morphogenesis is inactivated and that this change in cell behaviour is reversible.
Embryonic neurons can survive transplantation into the adult brain, where they grow, connect and influence host function, including recovery of symptoms induced by experimental lesions. On the basis of experimental animal studies, clinical trials have commenced in a number of human neurodegenerative diseases, with clear proof of concept provided by clinically beneficial effects in at least some patients with Parkinson's disease. Grafts were originally considered to work by neuronal replacement, but alternative less specific mechanisms have been identified including diffuse replacement of deficient neurochemicals, trophic stimulation of endogenous repair processes, and provision of alternative glial support for regeneration and remyelination. Evidence will be presented that true reconstruction of host brain circuitries can be achieved in at least one model system – lesions and grafts in the cortico-striatal system – such that the grafted cells become fully integrated functionally in the host neuronal circuits. Moreover the grafted cells can and do provide the neural substrate for new learning of motor skills and habits, demonstrated and both behavioural and physiological levels. On the basis of such results clinical trials are now under way seeking to achieve striatal repair in Huntingdon's disease. Safety has been demonstrated at several centres in France, UK and USA, with initial reports of efficacy from the French team. Experimental studies provide an essential complement to the clinical programmes not just to define and refine optimal transplant methodology but also to understand the mechanisms and limits of functional recovery so that optimal therapies can be designed to address the individual profile of deficits on a rational basis.
Our own studies in this area are supported by the MRC.
It has long been appreciated that studying the embryonic chick in ovo provides a multitude of advantages. These include the potential to control the embryo's environment and its kinetics independently of maternal influences. This allowed early workers to identify that movement exerted a pivotal influence on the development of the locomotor apparatus. With an increasing focus on the earliest detectable movements, we have exploited this system by developing novel models and schemes to examine the influence of defined periods of movement on musculoskeletal development. These models utilise drugs with well known neuromuscular actions to provoke hyperactivity (4-aminopyridine, AP) and either rigid (decamethonium bromide, DMB) or flaccid (pancuronium bromide, PB) paralysis, and have been used to examine the role of movement on joint, osteochondral and muscle development.
Our initial studies focused on the role of movement in joint formation, which showed that AP-induced hyperactivity had little, if any, effect on the timing or scope of joint cavity elaboration. This suggests that sufficient stimulus is derived from endogenous activity levels and that further mobilisation is without affect. In contrast the imposition of either rigid or flaccid paralysis prior to cavity formation completely blocked this process and produced, with prolonged paralysis, a fusion of cartilaginous elements and formation of continuous single cartilage rods across locations where joints would ordinarily form. The effect of these distinct forms of paralysis differed, however, when treatment was initiated after overt cavity formation; rigid, but not flaccid, paralysis partly conserved pre-cavitated joints, suggesting that ‘static’ stimulation derived from ‘spastic’ rigidity acts to preserve joint cavities. Another facet of these studies was the observation that DMB-induced rigid paralysis produces a uniform and specific pattern of limb deformity whilst PB, in contrast, generated a diverse range of fixed positional deformities.
Both forms of paralysis also diminish limb growth; our findings suggest that specific periods of development preferentially modify elaboration of specific osteochondral components. Changes in cartilage and bone accretion induced by 3 d periods of immobilisation, imposed at distinct developmental phases, provides support for an immobilisation-induced diminution in cartilage elaboration at an early phase, and for a relatively delayed influence of movement on osteogenesis. Immobilisation also exerts differential impact along the proximo-distal axis of the limb. Finally, our preliminary results support the possibility that embryonic hyperactivity influences the potential for postnatal muscle growth.
The extracellular matrix (ECM), and especially the connective tissue with its collagen, links tissues of the body together, and plays an important role in the force transmission and tissue structure maintenance especially in tendons, ligaments, bone and muscle. The ECM turnover is influenced by physical activity, and both collagen synthesis and degrading metalloprotease enzymes increase with mechanical loading. This can be shown by the use of microdialysis determination of propeptides and proteinases, as well as by biopsy verified incorporation of infused stable isotope amino acids. Local tissue expression and release of growth factors for ECM like IGF-1, TGF-beta and IL-6 is enhanced following exercise. For tendons, metabolic activity (detected by PET scanning), circulatory responses (measured by near infrared spectroscopy and dye dilution) and collagen turnover are demonstrated to be pronounced in humans after exercise. Tendon related blood flow is regulated by COX-2 mediated pathways, and glucose uptake is regulated by separate pathways in tendon than in skeletal muscle tissue. Chronic loading in the form of physical training leads both to increased collagen turnover, as well as – dependent on the type of collagen in question – some degree of net collagen synthesis. These changes will modify the mechanical properties and the viscoelastic characteristics of the tissue, decrease its stress, and likely make it more load-resistant. Mechanical properties of tendon fascicles vary within a given human tendon, and even show gender differences. The latter is supported by findings of gender related differences in activation of protein synthesis of collagen with exercise. Full understanding of these physiological processes will provide the physiological basis for understanding of tissue overloading and injury seen in both tendons and muscle with repetitive work and leisure time physical activity.
Human evolution and ingenuity both collaborated in providing biological/technological tools to move faster, despite the same actuator, and to better adapt our locomotion to very different environments. While evolution resulted in a versatile musculo-skeletal system featuring optimisation and energy-saving mechanisms to produce force and work more effectively, the passive tools we invented in the last 4–5 millennia have allowed an efficient and economic human powered locomotion both on/under water and in the air (Minetti, J Exp Biol 207, 2004). Other examples involve how to jump longer distances by using hand-held masses (Minetti & Ardigó, Nature 420, 2002) and how to throw objects further (a hand-thrown arrow barely reaches 25 m, while with the right bow the distance exceeds 1.3 km). In the past few years we reviewed the historic evolution of passive tools devoted to enhance human locomotion. Sometimes we built replicas of ancient tools (as in cycling and cross-country skiing) in order to study, through biomechanical and metabolic experiments, the progressive adaptation of (the same) muscle to the different machines and environments. The focus of my presentation will be on how limitations of the musculo-skeletal system have been attenuated both inside (gear ratio, tendons) and outside (crank, gears, poles, skis, fins) the body (Minetti et al. Proc R Soc B 268, 2001; Zamparo et al. J Exp Biol 205, 2002; Formenti et al. Proc Roy Soc B, in press, 2005). The relevance into the necessary mechanical work, the economy of transport and the related efficiency of motion/locomotion will be discussed.
The most important environmental influences on the musculoskeletal mass are food and exercise. In the last 25 years it has been possible to use stable tracer techniques to study the effects in human beings of interventions initially on muscle protein turnover and more recently on collagen in connective tissue. A parallel development was the characterisation of signal transduction mechanisms that regulate protein synthesis, breakdown, gene expression in animals or in vitro. Both lines of research are now increasingly integrated, which allows comprehensive studies into both the mechanisms and outcomes of interventions and diseases that affect the human musculoskeletal protein turnover and mass. We will present some of our recent findings concerning the effects of nutrition, mode of exercise and aging on different tissue components and also discuss the possible signalling mechanisms underlying them.
Horses are capable of locomotion within hours of birth, continue to grow until the fourth year of life, and may begin training for athletic competition in their second year. Therefore this presentation cites studies on the effects of exercise on bone, tendon, and articular cartilage. The response of these tissues to exercise has most usually been examined by clinical imaging and microscopy of various kinds.
In bone alterations in the mineral content, mineral density and the morphology of the mineralised tissue are the most common end points. Cortical bone density increases until around one year of age, and cortical content and density were increased 13 week after Thoroughbreds aged 2 years began training. Increase in apparent bone mineral density is massive in the major load paths of the epiphyses and cuboidal bones, despite the lower microscopic material density of the new bone, which is deposited on the pre-existing internal surfaces without prior osteoclastic resorption. With training of greater intensity, adaptive change is supervened by microdamage.
Collagen fibril diameter distribution in tendon and ligament changes during growth, but not after early training. The exact amount and type of protracted training that does cause reduction in mass average diameter (an early sign of progressive microdamage) has not been defined. Training is associated with increased tendon cross-sectional area, possibly due to slightly greater water content of noncollagenous or newly synthesised matrix.
Early training is associated with greater thickness of hyaline (but not calcified) articular cartilage, at least in some sites. The window for adaptation of cartilage to biomechanical influences may thus extend beyond very early life. However, cartilage appears to be the most susceptible to pathological alteration, since foals exposed to sprinting exercise sustained deleterious alterations to cartilage which were not temporary, and macroscopic scores of cartilage abnormality and collagen cross-linking of trained horses aged 2 years were inferior to those of control horses.
The effect of training exercise on the anatomical or patho-anatomical features of connective tissue structures is highly likely to be affected by the timing, type and amount of ‘natural’ exercise during growth and development which precedes the training.
Healthy articular cartilage has the ability to transfer enormous loads across synovial articulations without suffering damage. It is tempting to hypothesise that, as in other tissues with mechanical function, the emergence and maintenance of articular cartilage morphology is guided by a process of functional adaptation to mechanical stimuli and is not fully pre-programmed in the genome. We therefore tested the hypothesis that differences in cartilage morphology (primarily thickness) between species, subjects and anatomic sites can be explained on the basis of differences in the mechanical loading environment. Cartilage morphology was studied in quadrupeds and chimpanzees with different body weights (2.5 kg rabbit to 1200 kg rhinoceros), in 125 normal healthy subjects, in 14 top athletes (weight lifters and bobsleigh sprinters) versus 14 non-athletic volunteers, in 13 monozygotic twin pairs, and in 3 patients in whom ankle cartilage had been transplanted to the site of the knee more than 10 years earlier, because of a tibial osteosarcoma (van Nees rotation plasty). We show that humans possess the greatest known knee joint cartilage thickness among the species investigated, even when compared with quadrupeds 15 times heavier. We observed a relatively constant ratio between body weight and joint surface area across species, but not between body weight and cartilage thickness. There was no relevant difference in cartilage morphology between top athletes and non-athletic volunteers, despite the 30% higher extensor forces and muscle cross sectional areas in weight lifters and bobsleigh sprinters. There was, however, a remarkable similarity amongst monozygotic twins. After amputation, transplantation of ankle cartilage to the knee (in which the cartilage is commonly thicker), produced no increase in cartilage thickness in comparison with the contralateral ankle. These findings suggest that differences in cartilage form between species, subjects, and anatomical sites cannot be explained by differences in mechanical stimulation. Contrary to other tissues with a mechanical function, articular cartilage morphology and competence appears not to have the ability to adapt to changes in the mechanical environment in adolescence (where endochondral ossification occurs) and adulthood.
In Thoroughbred racehorses the third metacarpal bone (MC3) midshaft dorsal cortex (DC) thickens in response to fast exercise. This change can be measured radiographically (Davies et al. Equine Vet J Suppl 30 1999). The linear relationship between exercise speed and peak DC surface strains (Davies et al. Acta Anatomica, 146 1993), suggests that DC thickening occurs as an adaptation to the increased strain associated with faster exercise. Hence the change in DC shape should plateau at the bone volume required to withstand the maximum exercise speed for each horse. This experiment was designed to measure the speed of change in DC thickness in racehorses, and to test whether the DC reached a plateau that was sustained through subsequent exposures toracing speed exercise. Weekly lateral MC3 radiographs were taken from racehorses in a commercial racing stable between September 1996 and May 2005. DC thickness was measured at 2.5 cm distal to the nutrient foramen, and corrected for magnification. The time of change in DC was from the first measurement to the point where there was no further significant increase.
40 horses were chosen that had not been exposed to racing before measurements commenced and did not show clinical shin soreness during training. All horses with a continuing increase in DC thickness or less than 10 week of data from the start of the plateau were excluded. Mean time to plateau in DC thickness was 501 d (SE 29, range 220–900). Mean increase in DC was 3.9 mm (SE 0.3, range 1–7.5) on the left and 3.7 mm (SE 0.2, range 1.2–7.2) on the right. Rate of change was 8.8 µm (SE 0.8, range 2.9–24.1) per day on the left, and 8.4 µm (SE 0.8, range 1.9–32.1) per day on the right. In most horses the medulla did not change significantly in width between the first and last measurements so these bone growth rates reflect periosteal bone growth on the DC surface. Some horses have been followed for several years through their racing and subsequent careers with no further change in DC thickness.
This experiment was approved by the Animal Experimentation Ethics Committee of the University of Melbourne.
Tarsal slab fractures are a significant problem in Thoroughbred racehorses, but their aetiology is unknown. This project investigated if adaptive remodelling occurred in this site, looked for predisposing morphological variation and assessed novel imaging methods.
Hind limbs were obtained from 36 Thoroughbred horses, with known signalment and exercise history, destroyed for reasons other than hock fractures. All specimens were radiographed. Nine pairs of hocks were imaged using computed tomography (CT) and magnetic resonance imaging (MRI). Dorsolateral and dorsomedial slabs were cut from the dorsal aspect the central (Tc) and third (T3) tarsal bones. A quantitative index of apparent bone mineral density (ABD) was obtained from digital radiographs of 2 or 3.5 mm plane parallel sections. Backscattered electron microscopy (BSE) was performed on sections embedded in polymethylmethacrylate. anova was used to test for significant relationships between the ABD and other parameters.
Digital radiography revealed a wide variation in ABD of the compact and cancellous bone of the distal tarsal bones. Training caused an increased density of both compact and cancellous bone, more apparent on the dorsolateral aspect. There were also morphological variations between specimens. The ABD was significantly influenced by training status (P < 0.001) and age (P < 0.001); and there was an increase in ABD with training within each age group. BSE revealed creases and indentations in the calcified cartilage layer, associated with a high concentration of healing microfractures. Many microfractures were evident within the calcified cartilage of T3, in comparison to Tc. In comparison to radiography, both CT and MRI imaged in planes that allowed independent determination of regional changes within the bones.
Thus marked adaptive remodelling occurs within the distal tarsus, and is most marked in the prediliction site for slab fractures. BSE demonstrated potential stress-risers. We propose that tarsal fractures in racing Thoroughbreds are another form of fatigue fracture.
Stretch injury to the myotendinous junction is a common problem in athletics. The major risk factor for recurrent injury appears to be the primary injury itself. Physicians, physical therapists, athletic trainers, and athletes alike continue to search for optimal treatment and prevention strategies. Acute inflammation is regarded as the body's generalised protective response to tissue injury. An especially important and unexplored aspect of inflammation following injury is the role of inflammatory cells in extending injury and possibly directing muscle repair. It has been suggested that the inflammatory reaction, although it typically represents a reaction to damage and necrosis, may even bring about some local damage of its own and therefore increase the possibility for scarring and fibrosis. Limiting certain aspects of inflammation may theoretically reduce muscle degeneration as well as signals for muscle scarring. In this presentation we will focus on the role of neutrophils in injury and repair of stretch-injured skeletal muscle. A minimally invasive model that generates a reproducible injury to rabbit skeletal muscle is presented. We present a plausible theory that neutrophil-derived oxidants resulting from the initial stretch injury are responsible for extending the damage. An anti-CD11b antibody that blocks the neutrophil's respiratory burst is employed to reduce myofibre damage. An intriguing area that is currently being explored in our laboratory and others is the potential role for neutrophils to contribute to muscle growth and repair. It may be possible that neutrophils facilitate muscle repair through removal of tissue debris as well as by activation of satellite cells. Recent and ongoing investigations point to IL-6 as a possible key cytokine in muscle inflammation and repair. Studies to elucidate a clearer understanding of this possibility will be reviewed.
Entheses (insertion sites, osteotendinous junctions, osteoligamentous junctions) are sites of stress concentration at the region where tendons and ligaments attach to bone. Consequently they are commonly subject to overuse injuries (enthesopathies) that are well documented in a number of sports. In this review we focus on the structure-function correlations of entheses on both the hard and soft tissue sides of the junction. Particular attention is paid to mechanical factors that influence form and function and thus to exploring the relationship between entheses and exercise. We deal with the molecular parameters which are indicative of adaptation to mechanical stress, explain the basis on which entheses are classified and the concept of an ‘enthesis organ’. The latter is a collection of tissues adjacent to the enthesis itself, which jointly serve the common function of stress dissipation. The application of the enthesis organ concept to understanding enthesopathies is an important recent advance. Emphasis is placed on similarities which exist at many entheses between signs of fibrocartilage degeneration and the early changes that are characteristic of osteoarthritic articular cartilage. Novel roles of adipose tissue at entheses are reviewed (including space-filling and possible proprioceptive functions) and a distinction made between endotenon, epitenon, meniscoid and insertional angle fat. The basic anchorage function of entheses is discussed in detail and comparisons are explored between entheses and other ‘anchorage’ sites, both in living organisms and in non-biological materials. The ability of entheses for self-repair is addressed and evidence presented which suggests that tissue turnover can continue into old age. Finally a range of enthesopathies common in sport are reviewed (e.g. tennis elbow, golfer's elbow, jumper's knee, plantar fasciitis and Achilles insertional tendinopathies) and potential links between muscle and enthesis injury are suggested. This highlights the importance of considering the muscle-tendon-bone unit as a whole when evaluating pathology in any one area and not considering each region in isolation.
Sarcopenia, the loss of muscle mass associated with ageing, is one of the main causes of muscle weakness and reduced locomotor ability in old age. Although this condition is mainly driven by neuropathic processes, nutritional, hormonal and immunological factors as well as a reduction in physical activity, contribute to this phenomenon. Sarcopenia alone, however, does not fully account for the observed muscle weakness since the loss of force is greater than that accounted by the decrease in muscle size. As a consequence a reduction in the force per unit area, both at single fibre and at whole muscle level, is observed. We recently suggested that at whole muscle level this reduction in intrinsic force is the result of the combined effect of changes in (1) muscle architecture (2) tendon mechanical properties (3) neural drive (reduced agonist and increased antagonist muscles’ activity), and (4) single fibre specific tension. Whereas several studies support the role of the last 2 factors in the loss of intrinsic muscle force with ageing, only recently have alterations in muscle architecture and in tendon mechanical properties been shown to contribute to the above phenomenon (Reeves et al. Exp. Physiol. 89, 2004; Narici et al. Scand. J. Medical Sci. Sport in press, 2005). As a matter of fact sarcopenia of the human plantarflexors, represented by a 25.3% reduction in muscle volume, was found to be associated with a decrease in fibre fascicle length (10.2%) and pennation angle (13.2%). These architectural alterations were accompanied by a 10% decrease in tendon stiffness, attributable to alterations in tendon material properties, as suggested by a 14% decrease in Young's modulus (Narici et al. Scand. J. Medical Sci. Sport in press, 2005). However, when the combined effect of the decrease in fascicle length and in tendon stiffness on the length-tension relation was estimated, no shift in the optimum length for maximum force generation was found, suggesting a compensation between the decrease in fascicle length and the increase tendon compliance. Most of these changes may be reversed by 14 weeks of resistive training, for both fibre fascicle length and tendon stiffness were found to be increased by 8–10% and 64%, respectively. Similarly with ageing-induced disuse, training had no effect on the estimated relative length-tension properties of the muscle, indicating that the effects of increased tendon stiffness and increased fascicle length cancelled out each other (Reeves et al. Exp. Physiol. 89, 2004).
The major purpose of this study was to determine the relationships between the static electromyographic (EMG) activities of trunk muscles and the angles of trunk flexion in a range of positions. Previous authors have reported trunk muscle activation patterns during the stoop lift movement when trunk position changes relative to an external reference. When bending to prepare for a stoop lift, trunk flexor torque due to upper body weight increases as the trunk is flexed. In this study trunk flexor torque was constant because the upper body was maintained in a horizontal position while the lower body was strapped to a board whose inclination was varied between the horizontal and vertical positions in 10° increments. The length of the trunk flexor muscles increased as the trunk was flexed. Twenty young adults (10 male and 10 female) performed a total of 20 isometric contractions; 2 trials in each of 10 different postures while surface EMG of their vertebral extensor (erectores spinae and multifidi) and hip extensor (gluteus maximus and the hamstrings) muscles were recorded bilaterally. There were no significant differences between the normalised EMG values from the right and left sides, indicating that the tasks were performed symmetrically. As the trunk was flexed from 0o (lower body horizontal) to 90° (lower body vertical), the EMG of the vertebral extensor muscles reduced to 50% of their initial values, while the hip extensor muscles reduced to 20% of their initial values. Only 4 of the subjects (all male) exhibited flexion-relaxation of the erectores spinae muscles within the range of 0–90°, however all 20 subjects exhibited myoelectric silence of the vertebral extensors at full trunk flexion. The results for all muscles indicate a gradual change from active trunk stabilisation to an increasing contribution from the connective tissue structures. The contributing structures to the passive resistance to vertebral flexion include the thoracolumbar fascia, erector spinae aponeurosis, postvertebral ligaments, and the posterior annulus. It is mainly the connective tissue components of the hip extensor muscles that contribute to passive pelvic stabilisation because all the hip ligaments become slack during hip flexion.
The enthesis (bony insertion site) is a common site of overuse injuries in sport and a region that is also commonly affected in patients with ankylosing spondylitis. Because insertional tendinopathies can be very painful, it is often assumed that entheses are richly innervated. However there is little evidence to confirm this assumption, and thus we have investigated the nerve supply to the Achilles tendon enthesis in the rat. This insertion site is one of the most complex of tendon attachments. Together with adjacent structures, it forms part of an ‘enthesis organ’. The other structures include a sesamoid fibrocartilage in the tendon, a periosteal fibrocartilage on the calcaneus, and Kager's fat pad which protrudes into the retrocalcaneal bursa. Enthesis organs were removed from 3 male Wistar rats at each of the following ages – neonates, 4 week, 12 week and 24 month. The tissue was prepared for indirect immunofluorescence and cryosections cut in the sagittal plane. The presence of nerve fibres in the different parts of the enthesis organ was evaluated in serial sections immunolabelled with polyclonal antibodies against protein gene product 9.5, substance P, calcitonin gene related peptide, and neurofilament 200. At all ages, the enthesis itself and the sesamoid and periosteal fibrocartilages were not innervated, but the fat pad was supplied by nerve fibres immunoreactive to substance P, calcitonin gene related peptide, and neurofilament 200. We suggest that healthy entheses are not innervated because insertion sites experience high levels of mechanical load. However, the striking innervation of the fat pad suggests that it may have an unheralded proprioceptive role monitoring changes in insertional angle between tendon and bone that occur as a result of foot movements. Prominent mast cells seen in toluidine blue sections within the fat pad may play a role in neurogenic inflammatory responses.
Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle. The mechanical response of tendons to inactivity differs depending whether the measures are made in vivo or in vitro. We investigated the hypothesis that this difference was the result of an artefact of the testing process and did not represent what occurred in vivo. To test this hypothesis a novel approach was used to determine the mechanical properties of the TA tendon by optically measuring the end-to-end mechanical strains as well as the local strains at specific regions of excised TA tendon units. When the end-to-end strain of normal TA tendon is determined across the entire tendon length the stress-strain response curve shows considerably more extensibility than when strain is measured across only the mid-section of the tendon (mid-tendon). The strains in the region close to the muscle (muscle-tendon) are 5 times greater than the strains in either the mid-tendon or near the bone (bone-tendon). 5 weeks of denervation resulted in a 3.9-fold increase in end-to-end stiffness and a 70% reduction in the toe region as a result of a drastic reduction of the strain level in the muscle-tendon region. When only the mid-tendon regions are compared the control tendon is initially stiffer. The stress-strain response measured in the mid-tendon region of a normal TA tendon is therefore not indicative of its overall ability to deform in vivo as it transmits forces from muscle to bone.
Continuous measurement of muscle length has traditionally been achieved using animal preparations and invasive methods (e.g. sonomicroscopy) in which the muscle and tendon are exposed. There has been no technique for measuring continuous changes in muscle contractile length in vivo in a non-invasive manner. Ultrasonography has been used for several years to measure discrete, and more recently, continuous changes in tendon length and aponeurosis length. We have recently developed ultrasound tracking to measure tiny, continuous, naturally occurring changes in muscle contractile length during quiet standing. Here we use externally applied perturbations and frequency analysis to investigate the spatial and temporal resolution of the technique. Subjects were strapped to a vertical backboard while they maintained a low, constant level of left ankle torque. A pneumatic actuator applied rapid, square pulse, ankle rotations of defined magnitude and 0.2 s duration to the left ankle joint. Tracked changes in gastrocnemius and soleus muscle length follow the temporal profile of the perturbations with some temporal jitter (±23 ms) and scale progressively (5–400 ìm) with the size of the ankle rotation (0.03–0.7 degs). In a second experiment we tracked a wire oscillating in water with known peak to peak amplitudes of 1.5 ìm to 8 mm. The ultrasound tracking procedure had near 100% accuracy at all amplitudes for frequencies up to 3 Hz and showed roll off in accuracy at higher frequencies consistent with an effective sampling frequency of 12 Hz. This technique is non invasive and is suitable for measuring continuous changes in contractile element length in the spatial and frequency range of voluntarily controlled movements. As well as automating the tracking process, the technique is particularly useful for measuring small movements that cannot be measured accurately by eye.
Osteoarthritis (OA) is a crippling disease ultimately affecting the articular cartilage in both young and old. The disease is multifactorial, the end point characterised by the degradation of the articular cartilage covering the ends of bones.
We have been studying the role of the Notch family of cell surface signalling molecules during cartilage development, growth and disease. Our studies have shown that Notch signalling is vital for chondrogenesis and that Notch signalling controls the proliferation and differentiation of chondroprogenitor cells. In the present study we have documented the distribution of Notch family members in human osteoarthritic tissue and demonstrated that Notch signal modulation modulated the matrix kinetics of both human OA tissue and cytokine-induced GAG loss in an in vitro model.
Human OA cartilage was obtained after knee arthroplasty with the relevant ethical approval, fixed in 10% NBFS and processed into wax. Sections were stained with toluidine blue and immunolabelled labelled with antibody to Notch 1. Label for Notch 1 was present in the surface zone and the deep zone of the articular cartilage and was strongly expressed in the clusters of chondrocytes typical of OA cartilage suggesting a recapitulation of the embryonic phenotype in these clones.
OA tissue was also used to establish micromass cultures (2 × 105 cells 10 µL−1) which were treated with the notch signal modulator DAPT for up to 7 d. Analysis of GAG release from DAPT treated samples revealed decreased GAG loss to the media and increased GAG retention in the micromass. Additionally, IL1-induced GAG loss from bovine explants treated with DAPT was reduced to controls levels.
These results suggest that the Notch family of cell signalling molecules will provide a novel target for therapeutic treatment of OA after initial diagnosis by reducing the degree of cartilage degeneration.
Proximal patellar tendinopathy is a both an overuse injury common in sport and an inflammatory enthesopathy that occurs in patients with ankylosing spondylitis. It particularly affects a region in the posteromedial part of the patellar attachment of the patellar tendon, though the reason for this is unclear. We investigated whether it relates to an unequal pattern of force transmission between the patella and the patellar tendon that is reflected by the trabecular architecture of the patella, or to regional differences in the histology of the enthesis. Trabecular structure was determined from radiographs taken with a Faxitron MX-20 machine from the patellae of dissecting room cadavers and in magnetic resonance images of living volunteers. Structural and fractal analyses were performed on the Faxitron images using MatLab software. Regional differences at the enthesis in the thickness of the zone of uncalcified fibrocartilage and the subchondral plate were determined histologically in cadaveric material. The radiological investigations showed that the amount of bone and the trabecular thickness were greatest medially, and that, there were fewer antero-posteriorly or supero-inferiorly aligned trabeculae in the lateral part of the patella. The histological analysis of the proximal patellar tendon enthesis showed that uncalcified enthesis fibrocartilage was most prominent medially and that the subchondral plate was thinner laterally. Overall, the results indicate that mechanical stress at the enthesis is asymmetrically distributed and greater on the medial than the lateral side. This is in line with the site of pathology in proximal patellar tendinopathy.
This work was supported by Action Medical Research and Search.
Recent developments in medical curricula have led to marked changes in the teaching of gross anatomy. This has resulted from the belief that anatomy is largely ‘content-driven’ and not ‘skills-based’. Medical educationalists especially have seen anatomy as being ‘stuffed with facts’ and therefore in need of being diminished in importance within medical curricula that have reduced factual overload. However the changes in anatomy have not only involved decreasing the time available to teach and learn the subject but have often required the employment of new teaching methods without the use of human cadavers. But how do students, laypersons and anatomists view the downgrading of anatomy and the loss of dissecting room teaching?
Thurstone and Chave attitude analyses for medical students at Cardiff and Paris (Moxham & Plaisant, Anat. Sci Intern. 79, 200) indicate that, both before any tuition in anatomy and after course completion/examination, the students consider anatomy to be fundamentally important for clinical medicine. For the layperson, the findings of a recent survey (Hennon & Moxham, J. Anat. 206, 2005) suggest that laypersons have a reasonable understanding and knowledge of anatomy and believe strongly that anatomy is crucial for medical education. Furthermore, they believe that medical students should experience anatomy by dissection of cadavers and are concerned that this may not occur. For professional anatomists, whether they regard themselves as being ‘traditionalists’ or ‘modernists’, they favour the use of human cadaveric dissection above other teaching methods (Patel & Moxham, Clin. Anat. in press, 2005), the order of preference for teaching methods being: practical lessons using cadaveric dissection by students; practical lessons using prosection; tuition based upon living and radiological anatomy; electronic tuition using computer aided learning (CAL); didactic teaching alone (e.g. lectures/class room based tuition); use of models. Most importantly, anatomists recognise that, while no particular teaching method is perceived as being most efficacious with respect to imparting anatomical information, for higher academic and skills-based aims the teaching of anatomy by student dissection of the human cadaver is perceived as being very advantageous.
Therefore, to return to the question of why should the study of anatomy require the examination/dissection of cadaveric material by students? The answer is because students want it, the layperson/prospective patient requires it, and because anatomists know that it remains the best way of delivering both anatomical knowledge and important educationally and medically related skills (including, for example, providing medical humanities projects and dealing with issues related to death).
Over several years a concept of teaching gross anatomy has been developed and repeatedly evaluated.
In the first year of the medical curriculum a topic is dealt with in lectures, followed by dissection (26 afternoon sessions) of the same region in the next week coordinated with clinicians talking about the clinical relevance partly including the presentation of patients (e.g. cystic fibrosis patients when the lung is being dissected). Thus a total of 48 h by clinicians in the presence of an anatomist are given. In parallel the students examine each other in the living anatomy seminars. They not only deal with the projection of organs on the body surface, testing joints and the peripheral nervous system but also use an ultrasound machine to visualise the liver, spleen, kidney etc. and also get their first training in using the otoscope and ophthalmoscope.
A special seminar offered to female students on the living anatomy of women includes self examination of the breast and discussions on contraception. This type of seminar can only be performed in small groups.
In several evaluations (at the end of the course, at the end of the preclinical curriculum and before leaving the medical school, as well as finally after 7 years working in a hospital as a medical doctor) it has been documented that this concept is not only highly appreciated but also graded as one of the most relevant courses in medical training with respect to practical clinical relevance.
The ‘Centre for Body Donation at Paris’ was established in 1953 by Professor A. Delmas. On 15 January 1981 it became part of the University-Descartes in Paris. Before the Centre was established, unclaimed cadavers were used for both teaching and research. Now body donations require consent when the donor is alive. All cadavers are anonymous when they are admitted to the Donation Centre.
Since 1995, as a precautionary measure, a blood test from the donor body is required. As a result about 10% of bodies cannot be used for anatomy and are immediately cremated. Since 1st June 1998 it is required that all anatomical material is cremated. The method of disposal is always known and approved by the donors. The ashes of the cremated cadavers are buried at the Thiais cemetery, where the head-stone of a grave acknowledges the generosity of the donors.
Use of anatomical materials has two main aims, teaching and research. Research is sometimes part of the teaching procedures and consequently cannot be differentiated from teaching. Most of the research protocols are undertaken on unembalmed cadavers. Dissections for the second year of medical school (the first out of three cycles of medical school in France) are always undertaken on embalmed cadavers. The second, third and specialty courses use unembalmed cadavers. Paramedical students, especially physical therapists or chiropractic students, utilise both kinds of cadavers. For example, 836 bodies were admitted in 2003 at the Centre for Body Donation in Paris. 167 were used for teaching in the second year of medical school. Dissection is part of the programme in most, but not all, the 13 medical schools in Paris. 498 bodies were utilised for the other teaching programmes and 285 were part of a research programme.
The work of the Centre for Body Donation is essential for medical teaching programmes, for most of the teaching of surgical techniques and anaethesiology, and for research. It is rarely required, however, for other medical specialties. In addition, dental students attend regularly to dissect and paramedical professions, such as Chiropratic, Physiotherapy, Osteopathy and Etiopathy, do part of their teaching at the Centre.
With acknowledgements and thanks to E Bordereaux.
In our Division of clinical functional anatomy, cadavers are used in anatomical teaching as follows: (1) The Division provides an Anatomical Museum, which is open to the public. The main group of visitors are students of high schools, which visit the museum in the course of their biology classes, but also medical students and even the ‘plain’ public. The museum displays several prosected specimens, ancient and recent skeletons of both humans and animals. Old anatomical instruments and art works by an academic artist accompany the exhibition. (2) In anatomical lectures and other classes for undergraduate students, prosected anatomical specimens are used whenever appropriate. Additionally, the Division provides ‘Anatomical Demonstrations’, which accompany the ‘Main Lecture’. Therefore the Division maintains a large stock of different specimens. These specimens were and are prepared either by the faculty of the department or by medical students in an elective class (study group for extended instruction). (3) A large number of cadavers is used for the 135-h undergraduate dissection course, which is now attended by approximately 280 students a year. The students start with the dissection of isolated joint specimens, continue with a whole corpse in order to dissect the locomotor apparatus and finally dissect another whole corpse for topographical anatomy. Eight students work therefore on one corpse. (4) The Division offers several minor electives, mainly in the form of lectures, and a major elective, the study group for extended instruction. In this elective the students have to dissect several specific specimens either for scientific or educational purposes. Furthermore the students act as tutors in the dissection course and several other classes. (5) Another large number of cadavers is used in up to 20 different workshops and hands-on courses per year for national and international postgraduate medical education, for instance for the ESRA workshops on regional anaesthesia. In these workshops specific medical techniques can be trained directly on cadavers and accompanying prosected specimens can be studied.
Peninsula Medical School opened in September 2002, enrolling 130 students into a 5-year undergraduate course. Controversially, cadavers were not used in anatomy teaching: instead, living anatomy and medical imaging were the main methods employed. The course has now been running for 3 years, and this is therefore a good moment to review the lessons learned from this approach.
The decision was not taken in order to reduce costs, and as expected the course has developed in a resource intensive way. In particular extensive use of radiologists and other clinicians in teaching roles has proved expensive. Student engagement with peer examination has been high. Use of professional life models within the class has proved valuable even beyond expectations. A number of innovative approaches to teaching living anatomy have been devised. Use of live ultrasound in teaching has been explored and protocols developed to address safety, consent and observations requiring further investigation.
Students have not yet graduated from the course, and so no information is available on their level of performance as clinicians. In general, they are performing as expected in professional level exams. We intend to engage with the ASGBI Anatomy Outcomes Project, which may enable direct comparisons of cognitive anatomical knowledge to be made.
Inside and outside Maastricht University (UM) the questions of necessity and cost of teaching anatomy using human bodies, are repeatedly raised.
About necessity: Students ask for more undergraduate teaching (Prince et al. Medical Educ., 2003) and clinicians for more courses for their residents to upgrade insufficient anatomy knowledge. They feel that plastic models, textbooks and atlases do not suffice to really understand 3D anatomy. Beside living anatomy sessions and modern imaging techniques, human bodies are still used for practicals. Bodies show clinically relevant relationships that are not mentioned in textbooks (Mameren, Anat Rec., New Anatomist, 2004). Residents use unfixed bodies for practicing surgical procedures.
About expensiveness: The faculty allocates money to Research Institutes and to the Institute for Medical Education (IME). Teaching money is strictly separated from research money. The IME allocates it as ‘teaching hours’ to curriculum elements: lectures, practicals, construction of learning resources and the organisation of blocks, clusters and clerkships (teaching and supervising). Not included are costs of committees of education/exams, IME organization and the curriculum year 6. Costs of anatomy teaching were compared to costs of clinical (treating patients) and non-clinical (not treating patients) departments and to the skills laboratory. 74% is allocated to health care groups (53% to UM clinical departments and 21% to affiliated hospitals). 49% is used just for financing clerkships (74% minus 25% allocated to UM clinical departments mainly for use in year 1–3).The skills lab uses 9% of all teaching money, the anatomy department only 2%. If the IME needs to rearrange budgets, of course the big spenders should be examined not the anatomy teaching. Cutting of anatomy teaching is not cost effective. Decrease of the budget for anatomy teaching will cause increased demands of students and residents and (if not met) a decreased quality of their clinical anatomy knowledge.
In human standing the calf muscles soleus and gastrocnemius actively prevent forward toppling about the ankles. It has been generally assumed that these postural muscles behave like springs with dynamic stiffness reflecting their mechanical properties, reflex gain including higher derivatives, and central control. In this scheme, standing balance is achieved primarily by the mechanical stiffness of the activated calf muscles and this stiffness is enhanced by peripheral feedback resulting from elongation of the contractile element of the calf muscles as the body sways forwards.
We have used an ultrasound scanner and automated image analysis to record the tiny muscular movements occurring in normal standing and during large voluntary sways. This new, non-invasive technique resolves changes in muscle length as small as 10 µm without disturbing the standing process. This technical achievement has allowed us to test the long-established mechano-reflex, muscle-spring hypothesis that contractile element length changes in a spring-like way during sway of the body.
We show that the postural muscles do not behave in the expected spring-like manner. During forward bodily sway the contractile portion of the muscles on average shortens rather than lengthens (paradoxical muscle movements). These paradoxical movements are the consequences of a compliant Achilles tendon. The stiffness of the Achilles tendon is tantalisingly close to counteracting the unbalancing torque of gravity, but actually leaves the body unstable. Rather than modulating the stiffness of a mechano-reflex spring, the nervous system appears to control standing balance using an impulsive process that is associated with a mean central processing duration of 400 ms. The frequency of calf muscle adjustments is the same as that associated with voluntary control of hand movements under visual guidance and suggests that postural control may be more similar to voluntary control of movement than has previously been realised.
Our current understanding of the functional anatomy of entheses largely relates to the structure of their soft tissues and less attention has been paid to the bone, despite its key role in anchorage. Here we report variations in bone structure in limb entheses from dissecting room cadavers and evaluate their functional significance. Over 20 different entheses were assessed radiologically and structural and fractal analyses were performed on the images using MatLab software. Over 40 entheses were also studied histologically. The radiographs showed that at many entheses (a) bone quantity and trabecular thickness were greater distally than proximally (b) there was a greater proportion of longitudinally-orientated trabeculae in the superficial, compared with the deep, parts of entheses. Together, these results highlight important regional variations in mechanical stress in the bone at insertion sites that are critical in promoting secure anchorage. In particular the superficial network of longitudinal trabeculae that we have demonstrated beneath many attachment sites may be fundamental in linking hard and soft tissues. The histological survey showed that there was a substantial layer of cortical bone at fibrous entheses that was richly vascularised, with many vessels reaching the hard/soft tissue boundary. In contrast, compact bone was virtually absent at most fibrocartilaginous entheses and there was only a thin subchondral plate. Nevertheless this was often thicker distally than proximally. Particularly at the smaller fibrocartilaginous entheses, bone was often absent in local areas so that avascular enthesis fibrocartilage contacted vascular marrow directly. Complete bony absence was less obvious at larger fibrocartilaginous entheses. We conclude that the structure of bone at entheses is highly variable and this probably reflects variations in force transmission between different entheses. The local absence of subchondral bone at fibrocartilaginous entheses could have a bearing on the inflammatory responses that occur in enthesopathies.
This work was supported by Action Medical Research and Search.
The aim of this study was to assess the ability of global (stature) and local (arm length) body size measures, and actual measurements of muscle size to explain differences in elbow joint torque in pre-pubertal children. Based on the principles of geometric similarity, the square of a linear dimension is often deemed adequate to control for differences in muscle size. Conclusions from past studies which have not directly measured muscle size may have clouded our understanding of sex and age related differences in strength.
38 pre-pubertal children (18 boys and 20 girls), mean (SD) age, 9.6 (0.3) years; stature 1.38 (0.07) m; humerus length 28.5 (1.6) cm; body mass 32.89 (7.87) kg volunteered to participate in the study. All children completed maximal voluntary concentric isokinetic elbow flexion and extension tests at 30°/s. The peak torques were gravity corrected. Isometric flexion and extension torques were also measured at 90° and 10° of joint flexion, respectively. 33 of the children underwent magnetic resonance imaging for the determination of maximal elbow flexor and extensor muscle cross sectional areas (CSAs). Elbow flexor and extensor muscle volumes were also estimated due to the proportionality between volume and physiological CSA (and lack of muscle architectural data in children). Allometric scaling was used to assess whether global and local linear dimensions (stature and arm length) as opposed to muscle CSA or volume influenced the interpretation of differences in elbow flexion and extension torques.
Use of stature in the allometric models suggested that torques increased to a greater extent than predicted muscle size (stature2). In contrast, flexion and extension torques were proportional to arm length2. Significant sex differences in the relation of isometric elbow flexion torque to stature or arm length were observed. These sex differences were eliminated when muscle CSA or volume were used as covariates. Inclusion of either muscle CSA or volume in the allometric models indicated direct proportionality to isokinetic and isometric elbow flexion and extension torques. When examining differences in strength in children, use of linear dimensions as opposed to actual measurements of muscle size may yield different conclusions regarding factors affecting strength development.
The major purpose of this study was to compare the effects of oscillating and static isometric tasks on thigh muscle activation patterns. Many articles report knee torques in flexion and extension but very few report medial and lateral rotation torques, with even fewer reporting combinations of flexion/extension and medial/lateral rotation. A specially designed ‘Geniculandum’ with a fixed, instrumented ski boot was used to calculate static knee torques in 3 independent planes of motion. Eight female subjects performed 15 isometric experimental tasks in the seated position with a knee angle of 90°. There were 9 static tasks and 6 oscillating (0.5 Hz) torque productions at 50% of maximum voluntary contraction (MVC). The tasks included single and dual degrees of freedom. Myoelectric activities of 6 muscles acting across the knee (vastus medialis – VM, vastus lateralis – VL, rectus femoris – RF, semitendinosus/semimembranosus – ST/SM, biceps femoris – BF and tensor fasciae latae – TFL) were recorded using surface electromyography. MVCs were 157 ± 47 Nm for knee extension, 84 ± 35 Nm for flexion, 11 ± 3 Nm for lateral rotation, and 8 ± 1 Nm for medial rotation. Compared with slow isokinetic strengths of the nonsurgical knee of male tendon transfer patients (Osternig et al. Arch.Phys. Medical Rehab. 62, 1981), the female subjects had strengths that were 97% (flexion), 63% (extension), 24% (lateral rotation) and 21% (medial rotation). The relatively weak knee rotatory muscles could be a factor that contributes to the high incidence of noncontact knee ligament injuries in female sports. At 50% MVC, the muscles that were most active during single degree of freedom tasks were VM (ext. 40% Max EMG), VL (ext. 45% Max EMG) and BF (flex. 55% Max EMG), while others were most active during dual degree of freedom tasks; RF (ext. + lat. rot. 55% Max EMG), ST/SM (flex. + med. rot. 70% Max EMG) and TFL (ext. + lat. rot. 30% Max EMG). Generally, levels of muscle activation increased when isolated flexion-extension oscillating tasks were performed, in comparison to the static isometric tasks. However, activity levels tended to decrease during oscillating 2-degree of freedom tasks, such as oscillating lateral/medial rotation with a constant extension or flexion torque.
During osteoarthritis friction-free articulation of the joint is reduced leading to a loss of mechanical stimuli as the joint becomes painful to move. An important component of the synovial fluid within the joint cavity is hyaluronan which acts as a lubricating molecule and helps to maintain a constant synovial fluid volume. Previously, differential hyaluronan synthesis at the presumptive joint line has been shown to be mediated by mechanical stimuli and it is likely that continued mechanical stimuli and subsequent HA synthesis is required to maintain friction-free articulation of the joint. Indeed, a number of studies have already highlighted the benefits of combined walking, aerobic and muscle strengthening exercise in improving the flexibility of the joint and in reducing joint pain. The synovium and synovial fluid from OA knees show alterations in HA synthesis and decreases in HA concentration respectively which could be a result of the loss of mechanical stimuli. Thus this study aimed to examine the in vitro effects of mechanical stimuli on synovial cells from osteoarthritic human knees to try and understand their response to short periods of mechanical stimuli in terms of media HA concentration and differential HAS gene expression.
Cells were subjected to 10 min of strain using a 4-point bending mechanical loading jig to apply a defined and controlled mechanical stimulus to the cells. The physiological strain of 4000µɛ increased media HA concentrations 24 h post strain whereas a higher strain of 6000µɛ did not maintain increased media HA concentrations. QPCR data showed significant increases in all 3 HAS gene mRNA expression 24 h post strain.
The data from this study demonstrates an important role for mechanical stimuli in generating the necessary signals to synthesise HA in OA synovial cells. These data suggest that the development of exercise based therapies engendering defined physiological strains could prove beneficial in the non-invasive treatment of OA.
Iliotibial band (ITB) syndrome is a common cause of knee pain, particularly among runners and cyclists. It is viewed as an overuse injury caused by repetitive friction of the iliotibial tract (ITT) as it ‘rolls over’ the lateral femoral epicondyle during knee flexion and extension. Here, we reinvestigate ITT anatomy and propose an alternative theory. Gross anatomical observations, both on dissecting room cadavers and on the knees of younger individuals at surgery, together with magnetic resonance imaging studies have demonstrated the universal presence of strong fibrous connections that pass from the ITT, through a layer of fat to the lateral aspect of the femur – including the epicondyle. This conflicts with the view that any fibrous links are pathological adhesions between the ITT and the femur. Histological studies showed that the fibrous tissue both attached to and traversed the periosteum, and that the epicondyle was covered by fibrocartilage. As the ITT is not a discrete structure, but a region of increased tension in the fascia lata that is anchored to the femur as well as the tibia, we suggest that it cannot roll over the epicondyle. It creates the illusion of doing so, because stress levels shift between anterior and posterior fibres during knee flexion and extension. Our finding that a bursa was absent between the ITT and the epicondyle in all knees is in line with our theory and challenges the suggestion that oedema in individuals with ITB syndome necessarily indicates an inflamed bursa. We suggest that ITB syndrome has more in common with enthesopathies and may be associated with degenerative change at a fibrous connection between the ITT and the femur. Our findings also challenge the concept that the ITT can be stretched along its length from tensor fasciae latae to Gerdy's tubercle. Part of the stretching load must be dissipated through the fibrous links.
Kager's (pre-Achilles) fat pad is a prominent mass of adipose tissue lying anterior to the distal portion of the Achilles tendon. Proximally it relates to the region where the tendon is commonly ruptured and distally it protrudes into the retrocalcaneal bursa. This is a region implicated in insertional tendinopathies. We report the findings of an anatomical, magnetic resonance imaging and ultrasound study which lead us to suggest that the fat pad has a mechanical role in protecting the Achilles tendon and its blood supply from injury. Ultrasound studies show that the fat pad is moved passively during plantarflexion and dorsiflexion of the foot by the muscular contractions of flexor hallucis longus (FHL): the movement is most obvious during weight-bearing. By recording ultrasound movies, we have detected a J-shaped line of force transmission through the fat pad, which allows contractions of the muscle belly of FHL to drive fat distally into the retrocalcaneal bursa during plantarflexion. Lateral movements of Kager's pad are minimised by the deep fascia but distal movements are promoted by the wedge-shaped, fibrous tip of the pad with its deep synovial infolds. As the plantarflexed foot returns to the anatomical position, the fatty wedge is retracted from the bursa. The deep fascia plays an important role in limiting lateral movements of the fat pad (but maximising distal movements), and fibrous connections linking the fat to the Achilles tendon, anchor and stabilise the fat more proximally. We suggest that the mobility of the wedge-shaped tip of Kager's pad reduces kinking of the Achilles tendon during plantarflexion of the foot and that the relative stability of the more proximal fat, protects the blood vessels coursing through it to supply the Achilles tendon.
The direct transformation of one tissue type into another – metaplasia – is an uncommon mode of skeletogenesis under normal (non-pathological) circumstances. As part of a larger study on the evolutionary developmental biology of the amniote exoskeleton (dermoskeleton), we investigated the development of skeletal plates (osteoderms) within the dermis of Alligator mississippiensis. Osteoderm development is considerably delayed compared with the rest of the skeleton. Prior to osteoderm formation, the dermis is already well structured. Towards the hypodermis, large bundles of collagen form orthogonal or herring bone horizons that demonstrate strong positive reactions for various connective tissue stains, albeit not Alcian blue. Towards the epidermis, the collagen bundles are thinner, more poorly stratified, and stain less intensely. Incipient centres of ossification develop within the keels, dorsal to the thick collagen network, beginning around one year in age. Incipient osteoderms do not demonstrate a regular periosteum, nor is there any sign of osteoid or cartilage. Compared with other dermal elements there are relatively few skeletogenic cells, most of which resemble fibroblasts (fusiform morphology). Osteoderms also demonstrate Sharpey-like fibres, suggesting the incorporation of preexisting collagen matrix of dermis into the mineralization front. The unusual pattern of osteoderm development is best characterized as tissue metaplasia.
This work was supported by a Jurassic Foundation Grant to MKV and an NSERC Operating Grant to BKH.
This investigation evaluated the sub-clinical abnormality of increased numbers of immature neutrophils observed in patients with rheumatoid arthritis treated with enteric coated sulfasalazine. Neutrophil maturity of patients and controls was compared, by determining mean nuclear lobularity in peripheral blood smears. Immaturity was defined as a decreased nuclear lobation. White blood cell and neutrophil counts were also made. Subjects were patients medicated with sulfasalazine (n = 27). Controls (n = 20) were healthy, non-medicated clinical and academic staff. Determination of mean lobe number involved assessment of 300 neutrophils per individual. For subject and control groups, means of mean lobe numbers, mean white cell and neutrophil counts were determined. Means for each group were compared using the Mann–Whitney U test; variances using F ratios. Means of mean of lobe numbers of the patient population were significantly different (P < 0.0001) from controls. 89% of patients had mean lobe numbers below the control range, but total white cell and neutrophil counts in patients and controls did not differ significantly. Thus patients medicated with sulfasalazine typically have increased immature neutrophils, but have normal white cell and neutrophil counts compared with controls. For one patient, mean lobe numbers were obtained before and after sulfasalazine medication commenced, and the mean lobe number decreased with treatment. Consequently this effect is probably drug-induced. The mean lobe number is used here as a measure of a patients response to such drug-induced sulfasalazine treatment and possible toxicity.
Previous studies investigated cellular relationships of the presumptive motor exit points (MEP) and sensory entry zones (SEZ) of both spinal and cranial nerves of the developing rat neural tube (O'Donoghue et al. Journal of Anatomy 4, 2004; Int J Develop Neurosci 22, 2004). They showed that presumptive MEPs are not prefigured by the presence of any cells apposed to the presumptive glia limitans. In this they differ from presumptive SEZs, at which cell clusters (Boundary Caps) are found, apposed to the glia limitans. These cells have possible roles in the guidance and segregation of centripetal dorsal rootlet axons (Golding & Cohen, Mol Cell Neurosci 9, 1997; Niederländer & Lumsden, Development 122, 1976).
This study examines corresponding areas of the developing chick neural tube. Transverse semithin sections of E2 to E4 chick neural tubes were taken at both spinal and cranial levels. As in the rat, the presumptive MEPs are not prefigured by any cells apposed to the glia limitans at the loci of either cranial or spinal presumptive exit zones. By contrast Boundary Cap cell clusters were present at the SEZs at all levels. It was only after emergence of the motor rootlets from the CNS that peripheral cells became associated with them, initially at a level distal to the CNS surface. The length of bare rootlet between the surface and these clusters decreased over time, as the axons became segregated by peripheral cells, which became associated with them. The age at which this association took place was markedly earlier in the chick than in the rat, in accordance with the more rapid developmental rate of the former.
In the adult PNS, damaged nerves regenerate their axons and may re-establish synapses with their target tissues. From an early stage in the CNS, injury-induced changes in the surrounding tissue, predominantly in glial cells, are inhibitory to axon regrowth.
This study is a morphometric assessment, using unbiased stereological methods, of the effects of NT3, an endogenous neurotrophin, on PNS and CNS tissue responses to damage. Six rats received nerve crush of the C6–8 dorsal roots, proximal to the DRG. The test group (n = 3) received NT3 infusion, via osmotic mini-pump, into the subarachnoid space close to the dorsal spinal root transitional zone (DRTZ). The untreated group (n = 3) served as controls.
Following injury, NT3 treatment was associated with increases of the volume fractions of blood vessels (180%), of cellular reticulum (50%) and of phagocytes (90%), compared with untreated material.
In both groups, disrupted myelin volume was lower in the PNS than in the CNS compartment. In the PNS, mean disrupted compact myelin volume in treated tissue was only 7% of that in control tissue, indicating that NT3 enhances its removal/processing This finding is in accordance with the increased vascularisation and phagocyte incidence in treated tissue, shown above.
NT3 was associated with significantly increased axon size. Mean nonmyelinated axonal diameters were 1.3 µm and 1.4 µm in CNS and PNS tissue, respectively, compared with corresponding measurements of 0.8 µm and 1.1 µm, respectively, in untreated tissue. Myelinated axon diameters were significantly larger in CNS and PNS components of treated tissue (1.8 µm and 2.2 µm, respectively) compared with control values (1.0 µm and 1.3 µm, respectively).
Myelin sheath thickness was greater (50%) in CNS tissue of treated animals, suggesting an upregulation of oligodendrocyte activity by NT3. By contrast, in its presence, sheath thickness in PNS tissue was lower (50%) than in control tissue. This could suggest a downregulation of Schwann cell activity by NT3. Alternatively the greater proportion of early myelinating axons associated with the presence of NT3 could, at the stage studied, skew the distribution towards the smaller end of the spectrum, thereby lowering the mean value.
NT3 treatment thus enhances removal of breakdown products as well as the growth of regenerating axons. It may also stimulate the onset of their myelin sheath formation.
In the PNS, the Schwann cell response to nerve injury creates an environment, which is supportive of axon regrowth. In the CNS, glial cell responses contribute to the creation of a milieu inhibitory to regenerating axons; an outstanding feature being astroglial hypertrophy.
This study is a morphometric assessment, using unbiased stereological methods, of the effect of the endogenous neurotrophin 3 (NT3) on glial cell responses to crush injury of C6, 7 and 8 dorsal spinal nerve roots, proximal to the dorsal root ganglion (DRG). The dorsal root transitional zone (DRTZ) was studied, as it allows assessment of responses in the PNS and CNS and at the interface between the two compartments. Six rats were allowed to survive for 7 d following nerve crush surgery. The treated group (n = 3) received an infusion of NT3, via an osmotic mini-pump, into the subarachnoid space adjacent to the DRTZ. The untreated group (n = 3) were controls.
In the absence of NT3, responses were considerably more marked than in its presence: the mean volume of astrocytic cell processes was significantly larger (57%); and mean mitochondrial and RER volumes were also larger (111% and 50%, respectively). Oligodendrocyte mean cell, nuclear and mitochondrial volumes were also larger (119%, 26% and 247%, respectively). These findings suggest that NT3 has a stabilising effect and limits the increase in metabolic activity resulting from injury. In treated tissue, the oligodendrocyte proportion was increased by 7% and the astrocyte contribution was correspondingly reduced. This could reflect enhanced survival of oligodendrocytes; it is known that they have a susceptibility to injury-invoked cell death via the necrosis pathway.
NT3 also had a pronounced effect on responses in the PNS compartment: Mean Schwann cell volume was significantly smaller (65%), and mean mitochondrial and RER volumes were lower (48% and 58%, respectively) in treated than in untreated tissue, reflecting reduced metabolic activity.
The astrocytic hypertrophic response to nerve damage is a major component of the inhibitory effect of on regeneration in the CNS. This study documents its reduction by NT3, and shows the extent to which NT3 may contribute to the successful passage of regenerating axons between PNS and CNS compartments.
The main types of glia in the central nervous system (CNS) are astrocytes and oligodendrocytes. In addition there is a recently discovered population of novel NG2-glia that specifically express the NG2 chondroitin sulphate proteoglycan. NG2-glia, like astrocytes, are stellate cells with multi-branching processes that form multiple contacts with neurons and glia. Moreover both astrocytes and NG2-glia express ionotropic glutamate receptors (iGluR) and respond to neuronal activity. We have previously shown that ATP and glutamate mediate Ca2+ signalling in unidentified optic nerve glia. The aim of the present study was to investigate calcium signalling in identified astrocytes and NG2-glia in transgenic mice in which enhanced green fluorescent protein (EGFP) and Ds-Red are driven by the promoters for glial fibrillary acidic protein and NG2 respectively. Mice, aged postnatal day (P) 10–16, were humanely killed by pentobarbitone overdose according to Home Office guidelines. Optic nerves were isolated intact and the pia was carefully split to allow loading of the calcium sensitive dye Fura-2 AM. Nerves were maintained in a brain slice chamber and superfused with oxygenated artificial cerebrospinal fluid. ATP (107 cells from 4 nerves) and glutamate (100 cells from 4 nerves) evoked an increase in astroglial and NG2-glial cytosolic calcium ([Ca2+]i), in a dose-dependent manner. ATP (100 µm) evoked a rapid and transient increase in [Ca2+]i to 86 ± 4.2 nm (mean ± SEM), whereas glutamate (100 µm) evoked a slower and prolonged increase to 34 ± 4.9 nm. Mechanical stimulation of astrocytes mediated a calcium wave that was propagated to neighbouring astrocytes and NG2-glia, at distances up to 110 µm at a rate of 30 µm/s. These calcium waves were inhibited by suramin, a general P2X/Y receptor antagonist, and altered but not blocked by NBQX, an iGluR antagonist. These results indicate a role for both ATP and glutamate in calcium signalling between astrocytes and NG2-glia, which may be important in regulating their growth, proliferation and differentiation.
Supported by the Anatomical Society of Great Britain and Ireland.
We have previously shown that undernutrition during early life causes a permanent deficit in the total number of dentate granule cells. However it is unknown whether this deficit is due to neuronal cell death and/or to fewer cells being born during the period of neurogenesis. We have therefore used stereological methods combined with specific labelling techniques to examine the numbers of apoptotic cells in specific regions of the hippocampal formation. Rats were undernourished by restricting their daily food intake to about half that eaten by well-fed controls. Control and undernourished rats were killed on postnatal day 21 and their brains fixed in 4% paraformaldehyde. Serial sections through the hippocampal formation were labelled with the TUNEL technique to distinguish apoptotic cells. All care and animal handling procedures were approved by the institutional Animal Ethics Committee in line with Australian NHMRC procedures. There were about 21 500 and 57 000 TUNEL-positive cells in the dentate gyrus granule cell layer of control and undernourished rats respectively. The difference between these values was statistically significant by anova. In the CA3 + CA2 region, there were about 22 000 and 19 500 TUNEL-positive cells in control and undernourished rats respectively. The difference between these values was not statistically significant. Furthermore it was observed that the majority of the TUNEL-positive cells in the dentate gyrus were located close to the border between the dentate gyrus granule cells and hilus of the hippocampal formation. Our results show that undernutrition during gestation and lactation can result in an increase in the level of TUNEL-positive apoptotic cells in the rat dentate gyrus.
The aim of the present study was to evaluate distribution of apoptotic neurons in the nuclei of the brainstem in order to identify possible spatial or age-related patterns. Apoptosis was investigated, with a terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, in human brainstems taken at autopsy from 8 infants (range of age: 1 month-2 y), who died of sudden infant death syndrome (SIDS) (n = 4) and of definitely identified causes (n = 4), and from 18 adults (range of age: 32–79 y). The procedures were approved by the Ethics Committee of the University of Padova. The nuclei examined included the hypoglossal (XII), dorsal motor nucleus of the vagus (DMNV), principal inferior olivary nucleus (PION), medial inferior olivary nucleus (MION), dorsal inferior olivary nucleus (DION), nucleus of the spinal trigeminal tract (NSTT), cuneate (Cu) and vestibular (Ve). For each nucleus, a TUNEL index was expressed as the percentage of TUNEL positive neurons to the total number of neurons counted. Statistical analysis was performed with anova one-way test and Student-Newman-Keuls test for multiple comparisons.
In all the nuclei considered, the TUNEL index was significantly higher in adults with respect to infants (P < 0.01). At the level of the inferior olivary nuclei and NSTT, SIDS cases had a higher TUNEL index than the other infant cases (P < 0.05). Both in adults and infants, the nuclei which showed a high TUNEL index were the Cu, NSTT and Ve, while the DMNV was lower. The PION presented a higher TUNEL index than the other two olivary nuclei in both the series.
The high incidence of apoptosis in the inferior olivary nuclei and NSTT of SIDS victims is consistent with previous reports and has been ascribed to hypoxic-ischaemic insults at the level of watershed areas (Waters et al. Pediatr. Res. 45, 1999). Data from both infants and adults show the same spatial pattern of apoptosis in the brainstem, with a higher TUNEL index in Cu, NSTT and Ve. These findings support the hypothesis of different vulnerability or timing of cell death of the nuclei of the brainstem and may be explained on the basis of the characteristics of blood supply of the nuclei. The higher incidence of apoptosis in adults may be ascribed to the effect of ageing on neuronal survival or to hypoxic-ischaemic insults.
In small rodents such as the rat the majority of the sympathetic and parasympathetic postganglionic neurones innervating the pelvic viscera are located in a single ganglion, the major pelvic ganglion (MPG). Neurones of the MPG are almost completely devoid of dendrites and consequently their preganglionic inputs are predominantly axosomatic, this feature assisting quantitative assessment. It is possible to distinguish the parasympathetic and sympathetic phenotype of neurones in the MPG with a high degree of certainty as less than 1% contain both the cholinergic and noradrenergic markers choline acetyltransferase and tyrosine hydroxylase (TH) respectively (Keast et al. Neurosci. Letts. 196, 1995). TH-negative neurones can therefore be classified as parasympathetic. To investigate the effects of age on the number and distribution of cholinergic preganglionic terminals on parasympathetic and sympathetic MPG neurones we have used a double immunostaining protocol on 12 µm cryosections of the MPG from 3 and 24 month Wistar rats fixed in 4% paraformaldehyde. Sympathetic neurones were identified by immunoreactivity to a monoclonal anti-TH antibody and preganglionic terminals by antibodies to the vesicular acetylcholine transporter (VAChT) or to met-enkephalin (ENK), a peptide co-localised in a significant proportion of the preganlionic input. Digital images were subjected to image analysis to determine neurone area, bouton area and for counting purposes. The total number of MPG neurones receiving VAChT inputs did not change with age, nor did the numbers of parasympathetic and sympathetic neurones receiving VAChT inputs. There was an overall decrease in the number of ENK inputs to both parasympathetic and sympathetic MPG neurones. The mean area of VAChT-immunoreactive boutons did not change with age and was significantly (P < 0.0001) greater for those contacting sympathetic neurones compared to those contacting parasympathetic neurones. The mean VAChT bouton area was significantly greater than the area of ENK-immunoreactive boutons in both age groups. These results suggest that the overall preganglionic input, in terms of terminal boutons, to the MPG remains stable with increasing age but that the ENK component may suffer from age-associated attrition.
Previous studies have suggested that maturation of peptidergic afferent pathways in the urinary bladder of the neonatal rat might be an important factor in the development of the adult micturition pattern. In addition, maturation of peptidergic afferent pathways may play a significant role in the maturation of micturition reflexes, of which the afferent part plays a very important role in the initiation of the reflex. Nerve growth factor (NGF) is an essential neurotrophic protein involved in differentiation, survival and functional maintenance of peripheral sensory and sympathetic neurons. As part of an extensive investigation into the effects of ageing on the sensory innervation of the bladder, we have examined the effects of age upon NGF protein levels in the bladder of male rats. Two groups of male Wistar rats, 3 and 24+ months of age, were used in this study. After perfusion under terminal anaesthesia with phosphate-buffered 4% paraformaldehyde, the urinary bladders were removed, weighed and processed for 2-site enzyme-linked immunoassay (ELISA). The average weight of the urinary bladder in young rats was 108.0 ± 5.0 mg and this was significantly (P < 0.05) less than the mean weight (258.0 ± 30.0 mg) of aged rat urinary bladder. The difference between these age groups was reflected by an increase of 85% in NGF protein level with age. The content of NGF per urinary bladder and per milligram bladder wet weight was significantly higher (P < 0.05) in aged rat urinary bladder than in young rats. This difference represents an increase in NGF protein level of about 8 times with age. This increase in NGF levels may be due to an increase in the uptake of NGF by sensory neurons and/or an increase in NGF expression in the hypertrophied smooth muscle of the detrusor muscle or in the urothelium of aged rats. This high level of trophic support may well contribute to the almost unchanged density of sensory innervation that we (Mohammed & Santer, Eur. J. Morphol. 39, 2001) have observed in aged rats.
The developing kidney changes position during development, moving from the pelvis to the abdomen. This study quantifies this rostral displacement to determine if it is relative, due to differential growth, or true, possibly necessitating a mechanism for driving the migration.
Fetal white mice were studied, 6–8 at each of 19 regular, 6 h intervals from E11.25 to E16.00. Using serial longitudinal sections, the positions of 3 renal points (the midpoint of the ureteric bifurcation and the upper and lower renal poles) were determined in terms of (1) segmental vertebral level, and (2) distance from the anterior margin of the foramen magnum, measured along the central axes of the vertebral bodies.
The mean segmental level of the ureteric bifurcation ascended progressively at a diminishing rate over time, from S1/S2 to L3/L4. Most of the displacement took place between E11.25 and E11.75. Mean absolute distances from the foramen magnum to each renal point increased progressively with age at gradually increasing rates, showing that true rostral displacement of the kidney does not occur.
In the cervical region, a rapid decrease in vertebral column flexion commenced at E11.75. Elsewhere and later, the decrease was more gradual. Since this change in vertebral orientation did not coincide with the rapid phase of rostral renal displacement, it is unlikely to be a significant cause of the latter.
When first identifiable, at E13.00, the adrenal primordium lay 3 vertebral segments rostral to the upper renal pole, being separated from it by loose mesenchymal tissue. Between then and E14.00, this interval decreased to a narrow gap. This close apposition is likely to contribute to ending renal displacement. The adrenal gland itself is prevented from rostral displacement by its apposition to the diaphragm.
Longitudinal growth of the vertebral column takes place throughout the period studied and is likely to be the major contributor to the relative change in position of the developing kidney. The presence of extensive, loose mesenchymal tissue between the two structures facilitates this.
The findings clearly show that the rostral displacement of the developing kidney can be explained by differential growth processes alone.
During development of the mammalian secondary palate, hyaluronan (HA) plays a critical role by increasing turgor pressure within the palatal shelves resulting in shelf elevation. HA is known to interact with several extracellular matrix (ECM) proteins which exist as different isoforms resulting from differential RNA splicing of the same gene. CD44 is a widely expressed cell surface glycoprotein that has potential roles in processes such as receptor-mediated uptake of hyaluronan, cell migration, proliferation and adhesion, presentation of certain growth factors, and cell–signalling via interaction with cytoskeletal components. The presence of CD44 splice variants has previously been reported during a range of embryonic developmental events, including the development of the limb bud, the tooth germ, the hair follicle and the nasal septum. For the first time we have demonstrated the presence of CD44 transcripts containing variant exons in the developing secondary palate. Previous immunohistochemical studies have detected CD44 in the developing secondary palate. In this study, using semi-nested RT-PCR, we have shown the presence, both before and after palate shelf elevation, of CD44s and numerous splice variants containing exons v1-v10. Expression of splice variants was found to be transient and dynamic during palatogenesis, particularly during palatal shelf elevation. More marked differential expression of CD44 v1, v2, v3, v8 and v9 containing splice variants was noted pre- and post- shelf elevation. We therefore propose that there are distinct roles for these variants during the complex process of palate development. As CD44 is the major HA receptor of the hyaladherin family, we suggest that various forms of CD44 have critical, and possibly distinct, functional roles in cellular events such as migration, proliferation and adhesion that are of utmost importance during various aspects of embryogenesis.
In male rats the levator ani (LA) muscle is one of the androgen-sensitive muscles of the pelvis whose likely function is to enhance penile erection. However the functional significance of its attachment to the dorsal surface of the rectum, after which it is named, is unclear. As a consequence of reduced circulating testosterone levels in old age or following orchidectomy the muscle fibres of the LA are known to decrease in size. We have sought to characterise the deterioration of the LA in aged male rats by a range of immunohistochemical, enzyme histochemical and histological methods and to measure the force of contraction generated by the LA in vitro. Wistar rats of 3, 12, 17, 21 and 24 month (n = 6 for each age group) were used in the study. The LA consists primarily of Type II fibres and 5–10% Type I fibres and this pattern of fibre composition, as revealed by succinate dehydrogenase histochemistry, does not changes with age. Overall the muscle fibre cross-sectional area decreased by 44% in old age and this deterioration is primarily attributable to decreases in Type II fibre areas. Enzyme histochemical staining intensity for myosin ATPase did not decrease with age but immunostaining for sarcomeric myosin was less intense.Immunostaining with antibodies against 4 connective tissue glycosaminoglycans (GAG) revealed that there is considerable variation in GAG staining within the muscle and between the central and peripheral parts in particular, with epimysial staining showing the most variability. Generally there were increased intensities of GAG staining in the aged group and more connective tissue overall. Electrical stimulation of isolated LA in vitro demonstrated a significant decline in maximum tetanic force of 40% and a fall in specific force of 42.0% respectively in 24 month old rats compared with 3 month old rats. Interestingly, the age-related decline in specific force was evident in rats at 21 month old, whereas the fall in maximum tetanic force only became evident in 24 month old rats. These results demonstrate that the LA exhibits a dramatic deterioration in both structural and functional properties in old age.
A workshop in the Clinical Anatomy of the Heart has been developed as part of the training program for the first year residents in Cardiology at the University of Padova. The purpose of the workshop is to offer a direct experience of practical anatomy despite a shortage of cadavers. It is planned for the 12 residents who work in 6 teams on 12 plastinated hearts, 12 embalmed hearts and 2 unembalmed, unfixed hearts (harvested from the cadavers 24 h after death, stored frozen at − 12 C and removed from the freezer 12 h before the workshop). The workshop is organised into 2 tutorials of 2 h each in length, one on the external and the second on the internal examination of the heart. Each tutorial has 5 parts: in the first part (20 min), the team of the 2 residents analyse the plastinated specimen following a clinically oriented worksheet, in the second part (30 min), the teacher shows to the residents each step of the worksheet. In the third part (20 min), each resident recognises, on an embalmed specimen, a selection of the topographical points of the worksheet. In the fourth part (25 min), the workshop includes presentations on the specimens that illustrate anatomical variability and illustration of the topographical points on the unembalmed heart. In the last section (25 min), the resident are requested to indicate on radiological images (chest x-ray, CT and MR of the heart, CT and MR angiography, ultrasonography) the topographical points, making a comparison between the radiological images and the morphology.
The main guidelines in planning the workshop have been: (1) the selection of the anatomical landmarks relevant from the topographical point of view for the external examination; and (2) the anatomo-radiological correlation to present the heart in anatomical orientation.
Teaching anatomy using the cadaver at the dissecting room has two main instruments: prosections and dissections.
How the dissection is done or who prepares the prosections are some of the two cornerstones in anatomical departments, leaving apart the trouble of getting enough number of cadavers or the amount of time spent in doing anatomical practices.
At the moment research is the main goal for academic staff in many anatomical departments and in most cases technicians have not enough qualifications to do prosections. Therefore, who will prepare prosections?
In order to solve this problem, we have created a method based on the students' own work. It tries to satisfy the lack of prosections at the same time that with a minimum number of cadavers and time (30 h) the students can acquire knowledge, skills and attitudes related to the future medical competences.
Each group of students dissected an anatomical region during their practice. This allowed us to obtain new prosections and pictures of the cadavers while they were being dissected. With this iconographic material and the help of 3 students, we created a free-access website (http://www.ucm.es/info/morfos/atlasdiseccion/index.htm) to be used as a guide for following year's students.
The web-site already includes the next chapters: how to perform a dissection, anatomical variations, gallery of images, and a link to each dissection student work group, as well as a dissection guide for many regions through a simple and intuitive index page.
Presentation of each region is organised by layers (from skin to deep structures) and every layer shows a photograph and a brief explaining text. The most interesting structures have been labelled and related anatomic variations cited in some cases.
We are currently increasing the number of regions described. We are also inserting clinical cases with anatomical interest, radiological images, review questions and related links.