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The use of stereological tools to quantitatively estimate the injection size in neuroanatomical tract-tracing studies

B. Y. Martin, P. Negredo, J. Castro and C. Avendaño Department of Anatomy, Histology and Neuroscience, Autonomous University of Madrid, Spain

The accurate determination of the territory from which an injected tracer has been taken up by neurons (‘effective injection site’, or EIS) is a complex problem. The EIS varies for different tracers and survival times and it may not correspond to the tracer deposit which appears on microscopic examination (‘virtual injection site’, or VIS). A common criterion is to define a ‘core’ of the injection by the presence of uniform deposits of reaction product in the neuropil and cell bodies, and an injection ‘halo’ as the surrounding territory displaying an overall heavy staining of cell bodies within a more weakly and irregularly labeled neuropil. While the estimation of injection volume is manageable, a more difficult, and so far hardly considered, problem is to determine the number of neurons involved by the injection.

In this study, stereological methods were applied to iontophoretically delivered dextran injections in the brain. The cross-sectional area of the ‘core’ and ‘halo’ of the VIS was determined by point counting applying the Cavalieri estimator on a systematic sample of serial sections. Since a small tracer deposit usually allows distinguishing cell bodies within it, their number may be estimated by applying the optical fractionator on the same sections used to determine the injection size. However, neuron visualization can be hindered by various reasons, which will be discussed. An alternative strategy may then be applied that consists in estimating the number of neurons in adjacent, Nissl stained sections, or in a mirror VIS of the contralateral hemisphere. The latter can be generated by overlaying mirror images of the cross-sectional profiles of the injection site on roughly symmetrical spots of the contralateral brain region.

Supported by Grant BFU2004-05233-BFI from Spain's Ministry of Education.

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Morphometric evaluation for the use of magnetic resonance imaging to characterize neurodegeneration in mouse models

M. Carretero, E. J. Blanco, M. Rubio, J. J. Herrero, J. M. Riesco, D. Burks and J. Carretero Section of Physics and Mathematics, Department of Educational Sciences, University Pontificia of Salamanca; Department of Human Anatomy and Histology, Laboratory of Neuroendocrinology of the INCyL, Faculty of Medicine, University of Salamanca; Department of Surgery, Faculty of Medicine, University of Salamanca; and Laboratory of Neuroendocrinology, Institute Principe Felipe, Valencia, Spain

Common anatomical manifestations of neurodegenerative diseases such as Alzheimer include loss of neurons, formation of plaques and tangles, and inflammation. However detection and analysis of these pathological changes by histological methods can only be performed postmortem, thus complicating an accurate diagnosis in humans or limiting aging studies and therapeutic trials in the case of animal models. Therefore approaches allowing the direct, in vivo visualization of brain physiopathology would greatly facilitate assessment of disease status in humans and would enable researchers to fully exploit mouse models of neurodegenerative disorders. Here we present the use of magnetic resonance imaging (MRI) as an analytical tool in the characterization of neurodegeneration in the IRS-2 knockout model.

Insulin and IGF-I exert their various physiological effects by phosphorylation and activation of insulin receptor substrate (IRS) proteins. Deletion of IRS-2 in mice dramatically reduces the brain size due to impaired neuronal proliferation during development. The absence of IRS-2 in the adult brain produces an additional pathology: hyperphosphorylation and accumulation of the microtubule-associated protein tau. Young IRS-2 knockouts display defects in learning and memory as assessed by tests such as Morris water maze. We reasoned that IRS-2 deficiency might represent a reasonable mouse model of neurodegeneration. Therefore, we allowed these animals to age and subjected them to MRI at 18 months of age (Bruker Pharmascan system, Instituto Biomedicas, Madrid). This analysis revealed a very striking pathological difference in cerebral ventricles of aged KOs as compared to WT controls. We used these images to measure area, diameters and perimeter of lateral, medial and fourth ventricles and thus, have detected significant increases of these structures in the brains of IRS-2 KOs. Interestingly, enlarged ventricles have been reported for several neuronal disorders including schizophrenia and Alzheimer disease. Subsequent histological analysis of the same brains revealed other abnormalities including neuronal loss and amyloid deposits together with decreases in the thickness of cortical areas and basal ganglions. Thus, our initial observations suggest that MRI provides a valuable tool for in vivo detection of pathological changes in brains of mouse models suffering neurodegeneration processes.

Supported by I+D+I program: Project (CICYT)BMC2002-00872.

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The role of the actin-binding protein drebrin in growth cone pathfinding: a functional domain analysis

S. Geraldo and P. Gordon-Weeks MRC Centre for Developmental Neurobiology, King's College London, UK

Microtubule–actin filament interactions mediate a wide range of cell biological events from cell division to neuronal growth cone navigation. A diverse group of proteins that form a complex at the growing ends of microtubules (+ TIPs) enable and regulate this interaction. Although there is ample experimental evidence that microtubule–actin filament interactions are important for growth cone navigation, all the proteins involved have yet to be identified.

Drebrin (developmentally regulated brain protein) binds to the side of F-actin bundles and can displace other side-binding proteins such as fascin and tropomyosin. There is evidence that it interacts with myosin IIB but there is no evidence to show any involvement with microtubules. Overexpression of drebrin in fibroblasts and neuroblastoma cells causes remodelling of F-actin and induces the formation of filopodia.

We examined the expression of drebrin in primary neuronal cultures using immunofluorescence. We found that drebrin is highly concentrated in the transition zone of growth cones, a region where a dynamic interplay between microtubules and actin filaments takes place. Drebrin occasionally extended into the proximal region of some filopodia, particularly the subset of filopodia that are moving retrogradely over the dorsal surface of the growth cone. To identify functional domains in drebrin, we constructed a contiguous and nested set of deletion mutants. Transient transfection of these into COS-7 cells identified two domains that are independently sufficient to produce the same filopodial phenotype as the full-length protein, suggesting the existence of two different actin binding/remodelling domains. In live cell imaging of primary neuronal cultures, drebrin-YFP overexpression exhibited two different dynamic behaviours in growth cones: a retrograde flow from the periphery towards the central domain, and a proximal-distal extension along the base of some filopodia. This behaviour is consistent with the binding of drebrin to the proximal region of the core bundle of F-actin in filopodia. Future studies will focus on the interaction of drebrin with microtubules in growth cone pathfinding.

This research was supported by Fundação para a Ciência e a Tecnologia, Portugal and The Wellcome Trust.

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The role of clip-170 and dynein in the assembly of apico-basal microtubule arrays in polarized epithelial cells

D. A. Goldspink, G. Bellett, C. James and M. M. Mogensen School of Biological Sciences, University of East Anglia, Norwich, UK

Epithelial cell differentiation involves a dramatic rearrangement of the microtubule cytoskeleton, which is vital for the normal function of specialised cell types. In most nondifferentiated cells microtubules radiate from a centrally located centrosome, but in polarised epithelial cells the majority of the microtubules are noncentrosomal and organised in an apico-basal array. A release and capture model has been proposed to account for the generation of apico-basal microtubule arrays in polarised epithelial cells. Here microtubules are nucleated at the centrosome, released and relocated to apical noncentrosomal sites. Recent exciting evidence from our lab suggests that microtubule plus–end cortical interaction may be a vital intermediate step in the assembly process. This most likely involves microtubule + TIPs (plus-end tracking proteins) such as CLIP-170, cortical receptors and the motor protein dynein. The aims are to determine whether CLIP-170 is required for microtubule plus-end cortical capture and whether dynein acts by pulling released microtubules towards the cell base.

Microtubule regrowth experiments following nocodazole induced depolymerisation in cultured polarised MDCK cells showed an initial radial array, which extended towards the cell periphery and was subsequently replaced by an apico-basal array. Furthermore immunofluorescence and electron microscopy indicate that microtubule plus-end cortical contact occured at adherens junctions. Interestingly CLIP-170 localised not only to the plus-ends but also to cortical sites in a microtubule independent manner. Similarly distinct dynein clusters were evident along the cell cortex. Initial CLIP-170 depletion studies in ARPE-19 cells using siRNA showed no apparent affect on microtubule survival or organisation. However nocodazole regrowth experiments will be used to further analyse the affect of CLIP-170 depletion, especially with regard to cortical interaction. The localisation of dynein and CLIP-170 not only to the plus-ends of the microtubules but also to the cortex, suggests that these proteins may play key roles in facilitating the assembly of apico-basal arrays in polarized epithelial cells.

Funded by The Anatomical Society of Great Britain and Ireland.

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Development of an immunotoxin system for the selective ablation of NG2-glia to study their functions

G. Leoni 1,2 , A. M. Butt 1 and M. Rattray 2 1 School of Pharmacy and Biomedical Sciences, University of Portsmouth; and 2 Wolfson Centre for Age-Related Diseases, King's College London, UK

NG2-glia represent an abundant population of cells in the CNS with an undefined function. NG2-glia contact nodes of Ranvier in the white matter and synapses in the grey matter. Furthermore NG2-glia express ionotropic receptors such as AMPA and GABAA and functional glutamatergic and GABAergic synapses on NG2-positive cells have been demonstrated in hippocampal slices. These findings suggest that NG2-glia could have a structural role and be involved in the neuronal-glial signalling by modulating synaptic transmission. To test this hypothesis we wish to develop an immunotoxin system against the surface antigen NG2 for the selective ablation of NG2-glia in vivo and ex vivo. Here we have developed an in-vitro model to evaluate the efficacy of an immunotoxin system for the selective ablation of NG2-positive cells. Different cell lines were characterized for the expression of NG2 using Western blot and RT-PCR. The NG2-positive C6 cell line was exposed to a mouse primary anti-NG2 antibody with a secondary saporin-conjugated antimouse antibody (Mab-ZAP) in order to evaluate the efficacy of the immunotoxin system in determining cell death. An NG2-negative cell line was used as a control for the selectivity for the immunotoxin. LDH release assay and annexin binding were used to monitor cell death. Our results indicated that the Mab-ZAP in combination with an anti-NG2 antibody was effective in the induction of cell death in the NG2-positive C6 cell line. The development of a specific system for the selective ablation of NG2-positive cells is an approach to investigate the function of this newly identified population of glial cells. Future studies on organotypical cultures of brain slices and in-vivo by intracerebroventricular injections will give us the possibility to further investigate and clarify the NG2-glia functions.

Giampaolo Leoni is supported by a PhD studentship from the Anatomical Society of GB and Ireland.

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Different mechanisms of cell death during early development of vertebrate visual system

Y. Chavarria and R. Mayordomo Department of Morphology and Cell and Animal Biology, Plasencia Campus, University of Extremadura, Spain

The importance of programmed cell death on projecting neurons during vertebrate development of the nervous system has been well studied. This process is critical for the establishment of a definitive pattern of neuronal connection and is known as neurotrophic cell death. We know that it is regulated through competition of limited amount of trophy factors. Cell death occurring at earlier stages of neural development affecting proliferating neural precursor cells and recently born neuroblasts has also been recognized.

In this work we study programmed cell death, neurogenesis and proliferation throughout early development using chick embrionic retina as part of the central nervous system. We show characteristics of cell death and survival pathways at 2 different stages: early (day 5) and late (day 9) chick development, showing evidence of differential regulation mechanisms of cell survival between these proliferative and differentiative stages. Overall the results suggest that members of the family of insulin growth factors play an important role in the survival pathway, although the mechanisms of this antiapoptotic action are different for each stage.

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Morphometry of the recurrent laryngeal nerves of the rat

A. Pascual-Font, A. Merchán, E. Maranillo, J. Brillas, J. R. Sañudo and F. J. Valderrama-Canales Department of Human Anatomy and Embryology I, Faculty of Medicine, Complutense University of Madrid, Spain

In mammals the recurrent laryngeal nerves (RLN) are dissimilar in length between both sides. This asymmetry involves different time of arrival of the stimulus to the laryngeal musculature controlled by each nerve. Thus several explanations have been addressed to explain the closure of the glottis at the same time despite the different length of the nerves. However previous work on this topic lacked important data.

The present study compared the length (n = 10 rats) and the composition of myelinated fibres (n = 6) in the RLN of both sides, by means of light microscopy and computerized morphometric analysis.

The mean length for the right RLN was 2.79 cm and for the left RLN 3.63 cm, a mean difference of 0.84 cm longer for the left RLN than the right.

The whole population of morphometrically measured fibres was 1895. The mean number of fibres was higher in the left side than in the right (204 vs. 175) but that difference was not statistically significant. However the myelinated fibres of the right side were statistically bigger in diameter than the fibres of the left side, whereas the g ratio (ratio between diameter of the axon and diameter of the whole fibre) was almost equal in both sides with no statistical difference. These data suggest, at least in the rat, there are differences in the calibre of the myelinated fibres in the recurrent laryngeal nerves by side, but not in the number of them. All the data are combined and discussed in the context of the possible mechanisms for the compensation of the dissimilar length of both RLN.

Acknowledgements: This work has been supported by the Spanish FIS PI030035.

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History of the thalamic syndrome

J. B. Thiebaut 2 , J. F. Uhl 1 and O. Plaisant 1 1 Department of Anatomy, René Descartes University, Faculty of Medicine Paris V; and 2 Unité de traitement de la douleur Fondation Rothschild, Paris, France

We have reviewed the history of discovery of the thalamic syndrome by Déjérine. The description of the thalamic syndrome by Déjérine and Roussy in 1906 (in Roussy's thesis) established the anatomoclinical method a century ago.

Charcot (1825–1893) thought that the thalamus was not a ‘sensorial relay’as described by Luys’ theory. Dejerine (1849–1917), a student of Vulpian, frequently clashed with him on the subject. This was the beginning of a series of altercations stemming from controversy about the role of the thalamus, animated by the strong personalities of masters and students.

The thalamic syndrome drew the attention of neurologists around 2 major themes, the specificity of thalamic hemianesthesia, and the mechanisms of central pain. Head took up the Déjérine-Roussy concept but proposed a ‘deafferentation theory’ (as Foerster) in contrast with the ‘irritative theory’of Roussy. For L’Hermitte, the thalamus was more than a relay, it was an ‘analyser’. In 1996, Craig suggested the ‘thermosensory disinhibition hypothesis’. His confrontations with Wall were also very famous.

Today, central pain and abnormal movements are still discussed.

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Effects of the agu mutation on ageing and senescence in the brain and kidney in vivo

D. Russell, P. Shiels and A. P. Payne Departments of Biological and Life Sciences, and Surgery, University of Glasgow and Glasgow Royal Infirmary, UK

The AS/AGU rat, the result of a spontaneous mutation in the gene encoding PKC-γ, shows disordered locomotion which worsens with age and has been characterised neurochemically as having progressive deficits in dopaminergic and serotonergic systems. Since mutants also have a shorter life expectancy, one possibility is that they exhibit advanced senescence.

In this study, 3, 12 and 18-month-old-mutants were compared with control rats from the parent Albino Swiss (AS) strain for markers of ageing in kidneys and cerebellum. Renal pathology was examined using histological stains. Expression of the cyclin-dependant kinase inhibitor p16INK4a was quantified using immunocytochemistry with cell counting. Senescence-associated β-galactosidase (SA β-GAL), a marker for cell senescence, was applied to frozen sections of kidney and brain. Quantitative RT-PCR was also used to determine expression of senescence-associated genes p16 and p21. At 12 months pathological changes (tubular atrophy, thickened basement membrane, macrophage invasions, proteinaceous casts) were pronounced in AS/AGU kidneys compared to AS controls, but by 18 months both strains were affected. Immunocytochemistry for p16INK4a positive cells revealed a significantly higher number of cells in mutant kidneys compared to controls at 3 and 12 months, but not at 18 months. Similarly, there were age-related increases in SA β-GAL staining in kidneys and cerebella for both strains, but superimposed on a strain difference of higher levels in mutants. Increased p16INK4a gene expression was observed with age in both kidney and cerebellum, but with no strain differences. By contrast, p21 expression increased with age in mutant animals, but did not change in controls. The AS/AGU rat shows an ageing phenotype, with increased expression of senescence-associated markers and genes at an earlier age than the parent Albino Swiss strain. This suggests that the deficits in the nigro- and raphe-striatal systems reported in the past could be a product of accelerated ageing. Moreover the effects reported here in another brain region, and in the kidney, suggest that the AS/AGU rat (together with the control AS strain, from which it differs by a single point mutation) may prove an excellent model for investigating senescence-associated changes.

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Construction of a new recombinant adenovirus: possible uses in gene therapy

M. Perán 1 , C. Vélez 2 , H. Boulaiz 3 , J. A. Marchal 2 , F. Rodríguez-Serrano 2 , C. Melguizo 1 , J. C. Prados 3 , A. Martinez-Amat 2 , F. Hita 2 , O. Caba 3 and A. Aranega 3 1 Department of Neuroscience and Health Sciences, University of Almería; 2 Department of Health Sciences, University of Jaén; and 3 Department of Human Anatomy and Embryology, University of Granada, Spain

Gene therapy is an exciting new discipline with direct impact on the treatment of neurological disorders. Due to the difficulty in transfecting postmitotic cells by conventional methods the development of viral transfer systems which can also transfect neurons has become a key new methodology in neuroscience research. The recombinant adenovirus vectors have evolved a variety of complex and efficient means of delivering their genome to the nucleus of host cells and this gives them obvious potential as gene therapy agents.

Here we present the construction of a recombinant adenovirus vector with truncated subunit of the major inhibitory neurotransmitter receptor in the vertebrate brain, the γ-aminobutyric acid (GABAAR). The GABAAR is important not only because of its fundamental role in the regulation of brain excitability but also because of the fact that its function is allosterically regulated by pharmaceutically significant drugs.

The recombinant adenovirus allowed us to transfect neurons with very high efficiency and to carry out studies of neurotransmitter receptor dynamics on the cell surface in order to understand the complexity of the synapses.

In conclusion, the methodology used here is very relevant for the study of GABAAR processes and can also be employ to construct other recombinant adenovirus with specific DNA inserts to be used in research or even gene therapy.

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NG2-expressing glia in the mouse cerebellum

R. Wigley, A. Nishiyama and A. M. Butt Institute of Biomedical and Biomolecular Science (IBBS), School of Pharmacy and Biomedical Sciences, University of Portsmouth, UK

A novel class of glial cells has been identified in the adult CNS by their expression of the NG2 chondroitin sulphate proteoglycan. A number of lines of evidence indicate that NG2-expressing cells can differentiate into the 3 major cell types of the CNS, namely neurons, their supporting cells astrocytes, and oligodendrocytes, the myelinating cells of the CNS. This raises the possibility that NG2-glia, or a subpopulation of NG2-expressing cells, may act a reservoir of multipotent stem cells in the adult CNS. However adult NG2-glia are morphologically highly complex, postmitotic, and electrophysiologically active cells. This suggests that generation of neurons or glia from adult NG2-glia may involve de-differentiation or trans-differentiation in response to unresolved extracellular signals. The physiological and morphological properties of adult NG2-glia have not been fully resolved, and the mechanisms underpinning the differentiation programmes are unknown. It is known that adult NG2-glia form intimate contacts with neurons and astrocytes and respond to the neurotransmitter glutamate. It is our hypothesis therefore that glutamate activated intracellular signalling programmes may regulate the physiological and morphological properties of adult NG2-glia. In the first part of this study, we have begun to characterise the relationships between NG2-glia and neurons and glia in the cerebellar brain slice of mice in which NG2-glia express DsRed. Mice were humanely killed by lethal injection of pentobarbitone, according to UK Home Office guidelines. For immunohistochemistry, mice were perfusion fixed via the heart using 4% paraformaldehyde and brains were removed for sectioning. Confocal microscopy was used to analyse the relationships between NG2-glia, neurons and astrocytes. In the next stage of the study we will use live cell imaging of NG2-glia in brain slices to examine their response to glutamate. The information gained will contribute to our understanding of the factors controlling the function and fate of NG2-glia in the adult CNS.

Rebekah Wigley is in receipt of a BBSRC PhD Studentship.

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Measuring changes in synaptic connectivity

R. E. J. Dyball and G. S. Bhumbra Department of Physiology, Development and Neuroscience, University of Cambridge, UK

The classical studies of Cajal and others allowed scientists to visualise neuronal cell bodies, dendritic trees, and the pathways of axonal targets in the central nervous system To establish however, that neuron A makes functional contact with neuron B, additional methods are necessary. Once such method is based on the construction of the peri stimulus histogram (PSTH) by averaging the number of spikes occuring in specified time bins before and after an applied stimulus. The method made it possible to determine whether a connection was excitatory (an increased number of spikes after a stimulus) or inhibitory (a decreased number of spikes after a stimulus).

However not all connections are hard wired and that the behaviour of some synapses alter in response to physiological demand. For example some inputs to the suprachiasmatic nucleus, the mammalian circadian pacemaker, appear to alter at different times of day. It is thus necessary to measure such changes to establish whether a real change had occurred or not. We have developed a method that characterises stimulus-evoked spike activity by relating the timing of a spike with respect to the stimulus with the duration of the interspike interval that was terminated by that spike. This relationship is represented by the Phase Interval Stimulus Histogram (PhISH). Changes in the PSTHs evoked by stimulating caudal inputs to the suprachiasmatic nucleus that were very obvious but hard to quantify were characterised by applying information theory to the PhISH. The strength of the association between the presynaptic and postsynaptic neuron was quantified by the mutual information shared between stimulus and spike. For an individual cell, the mutual information increased from 0.06 bits at zeitgeber time (ZT) 0800–0.23 bits at ZT 1500. We have also applied the method to the synaptic inputs to the supraoptic nucleus during osmotic stimulation.

The approach allows precise quantification of synaptic connectivity and can be applied to recordings from any post synaptic cell. It will thus further our understanding of brain function.

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Distribution of p73 expression in rat circumventricular organs

A. Castañeyra-Perdomo 1,2 , E. M. Carmona-Calero 1,2 , D. M. García 1 , I. González-Marrero 2 , P. Fernández-Rodríguez 2 , L. Castañeyra-Ruiz 2 , H. Pérez-González 1 , A. Castañeyra-Ruiz 2 , T. A. Ruiz-Mayor 2 , J. M. González-Toledo 1 and R. Ferres-Torres 1 1 Department of Anatomy, Faculty of Medicine, University of La Laguna, Tenerife; 2 Department of Biotechnology, Institute of Investigative Sciences of Puerto del Rosario, Fuerteventura; and 3 Neurology Service, HUC La Laguna, Tenerife, Canary Isles, Spain

The circumventricular organs are brain structures located in the recess of the 3rd and 4th ventricles and are as follows: the organum vasculosum of the lamina terminalis (OVLT) located in anteroventral region of the 3rd ventricle, the subfornical organ (SFO) located under the fornix commissure, the subcommissural organ (SCO) lying under the posterior commissure, the median eminence (ME) in the floor of the 3rd ventricle, and the area postrema (AP) lying over the central canal at end of the 4th ventricle. The choroid plexus (CP), the pineal gland and the pituitary posterior lobe are also considered to be circumventricular organs. They have common characteristics such as: differentiation of the ependyma, rich in neuropeptides and catecholamines, lack of a blood brain barrier (except the SCO), secretory organs connecting the blood, brain and CSF. The p73 protein is a member of a family of transcription factors which also includes p53 and p63. p73 has full-length transactivating (TA) and an N-terminally truncated (DeltaN) isoforms, with pro- and antiapoptotic activities, respectively. It has been reported that TAp73 isoform is necessary for the development and maintenance of the ventricular wall. The purpose of present work is to study p73 expression in the circumventricular organs that are differentiations of brain ventricle epithelium.

Brains from Wistar-Kyoto rats (WKY) were used. The paraffin sections containing the circumventricular organs were immunohistochemically processed with antip73. p73 bands were identified in the circumventricular organ extracts by Western blot.

We found that p73 is expressed in the SFO, SCO, PC, and OVLT. p73 expression was more intensive in the SCO than the other circumventricular organs, whereas in the AP and the OVLT p73 expression was scarce. It is also known that TAp73 in the CSF may play an important role in the maintenance of the adult ventricular wall as well as in the development of the proliferating neuroepithelium, The present results that more TAp73 was found in the SCO and PC than the OVLT and AP, may have the explanation that those are circumventricular organs in which structural components are mainly formed by ependyma and neuroepithelium.

Acknowledgements: this work was supported by the Fundación Canaria de Investigación y Salud (FUNCIS) project no. 74/03.

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Does hypoxia prime mouse motor nerve terminal loss?

R. Baxter and S. H. Parson Institute of Membrane and Systems Biology, University of Leeds, UK

Changes to or loss of synaptic connectivity as a result of neuronal death causes either aberrant or complete loss of function of postsynaptic targets. It is this loss of synaptic connectivity that causes the punitive symptoms seen in neuropathological disorders. Recently changes to and loss of nerve terminals have been shown to occur before cell body death and prior to the manifestation of clinical symptoms in a number of neuropathies, e.g. Alzheimer's disease. Understanding the mechanisms and triggers of nerve terminal loss are therefore of key importance. Using the neuromuscular junction as a model synapse, mouse skeletal muscle explants with long distal nerve stumps were maintained for > 3 h. We have shown that nerve terminals appear to be lost quickly in response to adenosine triphosphate (ATP) and hypoxia. In wild type mice, this appears as a significant reduction in neurofilament and synaptic vesicle immunoreactivity, indicative of gross morphological change in > 3 h. Real time monitoring of nerve terminals using transgenic Thy-1/16(YFP)/C57Bl mice that express yellow fluorescent protein in all their neurons reveal that degenerative changes can occur over minute timescales. This work suggests that nerve terminals may be very quickly lost during ischemia where increases in extracellular ATP are coupled with reduced oxygen availability. Using YFP transgenic mice we will investigate previous unpublished findings that 3′-O-(4-benzoyl)benzoyl adenosine triphosphate (BzATP) causes adult motor nerve terminal withdrawal. Future work will include investigations into the morphological and cellular events induced by ischaemic insult, and in particular, the involvement of the cytoskeleton as a possible mediator of nerve terminal stability, degeneration and withdrawal.

This project is supported by the Anatomical Society of Great Britain and Ireland.

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Morphometric analysis of the nasal cavity in a collection of human skulls

M. G. Fernández García, M. D. Cabañas Armesilla and M. I. Maestre López Department of Anatomy and Human Embryology 2, Faculty of Medicine, Complutense University of Madrid, Spain

The morphological characteristics of the skull are of interest to the scientific community in anthropometric studies for population analysis of ethnic groups.

We previously studied metric variability at the individual and population levels, discriminating for sex and age (Fernandez 2001).

We have now studied the nasal cavities in skulls from the Department of Anatomy and Human Embryology II of the UCM to reconsider the variables used.

Our sample consists of 34 adult skulls, of unknown sex and age. This study followed the protocol described by Martin-Saller (1957) and a new protocol designed by the investigating group. We estimated age based on the closing of the cranial sutures (Meindl and Lovejoy 1985). For the diagnosis of sex the qualitative criteria by Boyd and Trevor (in Brigg, 1989) were used, which according to these authors achieve 98% accuracy in differentiating the morphology of the cranium. SAS 9.1.3 was used for statistics.

The variables that contribute best to the study are the average length and width of the nasal cavities, but other qualitative features have been used.

We concluded: (1) The length and width of the nasal cavity contribute information on population variability, in relation to sex, age, and ethnic group; (2) The length of the nose is a significant variable for population studies.

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Analysis of variability of the human bony palate

M. I. Maestre López, M. D. Cabañas Armesilla and M. G. Fernández García Department of Anatomy and Human Embryology 2, Faculty of Medicine, Complutense University of Madrid, Spain

We analysed the morphology of the human hard palate by means of statistical programs, and recorded malformations and other pathology in a sample of skulls in the Department of Anatomy and Human Embryology II, and the Museum of Villa of our Faculty. We aimed to study the palate morphometrically, analyse variability statistically and determine any relation between the anatomy of the orbital and nasal cavities with that of the hard palate. 34 adult skulls were chosen at random from a collection of 160 skulls amassed over almost a century. This study followed the protocol of Martin-Saller (1957) and a new protocol designed by the investigating group. For the diagnosis of sex, we used the qualitative criteria of Boyd and Trevor (in Brigg, 1989), and SAS 9.1.3 for the statistics.

Our results were as follows. A low correlation (r < 0.4) indicated no direct relationship between the measured quantitative variables of palate morphology. There was linear independence between all quantitative variables. A low correlation also indicated no direct relationship between malformations and the variables measured, with the exception of vertical length, in that long verticality gave rise to a more pointed palate. More of the collection of skulls needs to be studied to establish any definite relationships in the variables studied here.

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Sex determination by means of the proximal phalanx of the hallux

D. G. Monreal Redondo 1 , F. J. Fernández Camacho 2 , J. A. Sánchez Sánchez 3 , S. Ramirez Varela 4 and R. Fernández-Baíllo 5 1 Department of Morphological Sciences and Physiology, European University of Madrid; 2 Department of Human Anatomy and Embryology, University of Alcala; 3 Department of Legal Medicine and Toxicology, Complutense University of Madrid; 4 Medical Surgery, Spanish Army; and 5 Biostatistic Specialist, Logos Medical Centre, Madrid, Spain

Our objective was to propose a method for sex determination using the proximal phalanx of the big toe in cases where other useful bones may be unavailable. We recorded 18 linear measurements, 5 indices and 2 angles of the proximal phalanx of the big toe in 47 amputated lower limbs. Demographic and pathological criteria of the sample were known from the clinical history.

By means of univariate analysis we could detect statistically significant differences of means for 16 linear measurements and the torsion angle, with higher values in males. Using Fisher's stepwise discriminant analysis we obtained a discriminant function, which with only 3 variables (distal tibial facet height, proximal transverse curved length and valgus diaphyseal deviation angle) identified sex correctly for 87% in matrix and 83% in jack-knife classifications.

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Total soft tissue components as in vivo determinants of body bone mass by means of DXA

S. Aguado, C. Alonso, R. Rodríguez and L. Gómez-Pellico Department of Human Anatomy and Embryology. University of Alcalá. Alcalá de Henares, Spain

Body weight is one of the best predictors of bone mass in both sexes. It is fundamental to know what component of the soft tissue (total body fat mass and total lean body mass) explains better the bone mass for each sex according to age. This will help us predict bone growth in young people, and prevent bone disease in adults. Our goal was to determine the contribution of each soft tissue component to the total body bone mass in both sexes and according to age.

For this we took a population sample of 459 healthy persons (140 men and 319 women) separated into 3 age groups: young people (16–20 years old), young adults (41–55 years) and senior adults (61–65 years). Full body densitometry using dual X-ray absorptiometry (DXA) was performed in each individual, over a mean time of 20 min, to obtain the total values of each body component. The body components we analysed were: total body fat mass (TBFM), total lean body mass (TLBM), total bone mineral content (TBMC), and TBMC adjusted to body size (HTBMC).

We performed a multiple linear regression analysis to determine to what degree TBFM and TLBM are determinants of HTBMC. The results indicate that TLBM is the main determinant of HTBMC in both sexes, with the exception of young adult men and women. The development and maintenance of muscular mass is important in all ages and in both sexes, and it is important to avoid sudden body weight losses, especially in premenopausal and menopausal women in whom fat mass is the main determinant of bone mass.

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An anatomical variation of the clavicle

E. López-Viriero, M. Manzano, L. Arraez and J. Lara COT Service of Valme Hospital, Seville; and Department of Anatomy, Complutense University, Madrid, Spain

We present the case of a boy aged 16 years who presented for a clinical opinion on a mass in the upper right part of the thorax, originally found by an orthopaedic surgeon when he was 7 years old. The mass had increased in size in the meantime. On examination we could palpate a hard, immobile mass, well delimitated and not painful. It projected anterosuperiorly in the region of the right clavicle. In a plain radiograph we observed a mass with a smooth curve, convex superiorly, that we interpreted as an abnormal development of the right clavicle, compared with the left. Computerised tomography revealed a clavicular enlargement at its acromial extremity. MRI confirmed this anterosuperior enlargement. We interpret this finding as an anatomical variation of the right clavicle. We present this case for its rarity and difficult diagnosis. In our case there were no known familial antecedents of what we may term ‘short clavicle syndrome’.

P20

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Markers of locomotor activity in a Roman population from Caesaraugusta (Zarazoga)

J. L. Nieto, S. Baena, A. I. Cisneros and P. Galve Department of Human Anatomy and Histology, School of Medicine, University of Zaragoza, Spain

Activity markers include structural modifications in the periosteum and cortical surface of the bone in places where muscles, tendons or ligaments are inserted, especially exostosis or depression which may be very localised and easy to detect. We present a study of osseous remains of the Roman period from 10 graves dating from the 3rd century AD from Caesaraugusta (Zaragoza). The average age is 25 years (± 4.2). We highlight as musculoskeletal stress markers: Robustness of the long bones; Presence of numerous holes in the epiphysis, and craterformation in the humerus, femur and tibia; Humeral and femoral periostitis, and calcaneal and patellar tendinitis with osseous marks of tendinous fibres; Appearance of spicules by ossification of insertion tendons; Microtraumatism and stress injury in metatarsals.

P21

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The structure and innervation of the muscular origin of tibialis anterior in the rat

H. M. Shaw, R. M. Santer, A. H. D. Watson and M. Benjamin School of Biosciences, Cardiff University, UK

The term enthesis is usually associated with the bony attachment of a tendon or ligament; however, some muscles attach to bone via a small amount of loose connective tissue. Such ‘fleshy’ entheses have not been widely studied. This region is of particular interest as this may be the site where pain arises in overuse injuries, such as shin splints. The purpose of this study is to investigate the structure and innervation of the fleshy attachment of tibialis anterior in the rat.

The attachment site was removed from 5 male Wistar rats at 12 week of age, fixed in 4% paraformaldehyde, stored overnight in 10% sucrose buffer and cryosectioned. Sections were immunolabelled with antibodies to protein gene product 9.5 (PGP 9.5), substance P (SP), CGRP and neurofilament 200 (NF200). Antibody binding was detected with; Vectastain avidin/biotin kit or FITC-conjugated goat antirabbit Fab fragments. Reference sections were stained with toluidine blue and Masson's Trichrome.

The muscle itself was attached indirectly to the fibrous layer of the periosteum via a small amount of loose connective tissue; this in turn attached to the osteogenic layer of the periosteum which subsequently attached to the underlying bone. In contrast to the aneural nature of fibrocartilaginous entheses, ‘fleshy’ entheses were innervated with a large number of both nociceptive (SP and CGRP) and mechanoreceptive (NF200) nerve fibres. Nociceptive fibres may play a significant role in pain caused by microtrauma and pathology associated with this region. In addition muscle spindle-like structures immunoreactive to NF200 close to the enthesis were common. These structures may play a role similar to Golgi tendon organs in monitoring stretch at myotendinous junctions.

P22

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Isokinetic behaviour of the triceps surae muscle after surgery of the calcaneal tendon

J. Carrascosa, A. Slocker, E. Gómez Martín and L. Gómez Pellico Department of Anatomy and Embryology. Alcala University, Madrid, Spain

An important role is played by the calcaneal tendon-plantar system in the longitudinal stabilisation of the ankle, bearing compression forces (trabecular-bone), and stretching (ligaments and muscles). We studied its performance in 25 patients with calcaneal tendon rupture, one year following reparative surgery. All patients underwent the same surgical technique, subsequent immobilisation with a plaster boot, and rehabilitation (an average of 5 weeks).

Muscle strength was assessed, together with the range of articular movement and the agonist/antagonist relation during plantar and dorsal flexion of the ankle joint on the healthy and lesioned sides, by isokinetic analysis. A densitometric study of bone mineral density (BMD) was made in the posteroinferior portion of the calcaneum. The isokinetic results showed a reduction in muscular force and in the range of plantar flexion, and an alteration of the agonist/antagonist relation on the operated side. As was to be expected, given the known negative effect of immobilisation on skeletal BMD (Leblanc, 1990), the densitometric study revealed reduced BMD in the assessed region of the calcaneum on the lesioned side compared with the healthy side, even a year following surgery.

We conclude that we need: (1) to take account of the agonist muscular group in a protocol for calcaneal tendon rehabilitation; and (2) to strengthen the triceps surae to reach higher values for moment of force and range of articular movement, in order to achieve less bone mass reduction.

P23

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Verification of a computer model of the formation of foot pressure using a novel foot marker system

P. Caravaggi and R. Crompton Department of Human Anatomy and Cell Biology, Liverpool University, UK

The bases for the creation of a new 3D computer model of the foot have been laid down: the aim of the project is to predict the relationship between human foot pressure and human footprint form.

The task of simulating contact between the soft tissues of the sole of the foot and rigid bodies (ground and bones) has been undertaken via hybrid finite element analysis (FEA) and multibody dynamic analysis. A material model for the heel pad soft tissues has been identified and its mechanical behaviour successfully tested via FEA. Although the interaction between FEA and the software for multibody dynamic analysis has proved to be quite challenging and deformations at the heel pad difficult to reproduce, preliminary dynamic pressure values predicted by the model are close to experimental data from pressure plates.

In order to replicate its complex in vivo kinematics a foot marker protocol has been identified in terms of three 6-DOF and one 5-DOF segments. The movement of hindfoot (talus and calcaneus), midfoot (navicular, cuboid and 3 cuneiforms), metatarsals and hallux has been tracked by the motion capture system. In order to work out the range of movement of the ankle joint the lower limb has also been tracked: during forward dynamic analysis ankle joint motion is important as it affects the position of the insertion of the main muscles bringing about the foot movement with respect to the joint centre.

The 3D data from the motion capture system have been used to drive the model in an inverse dynamic analysis. A plantar aponeurosis, the elastic material properties of which have been taken from the literature, has been added to the model as it is one of the main structures affecting the rigidity of the longitudinal arch; its length changes over time and hence energy stored and released during the stride can be computed by the software. Although skin movement markers are a source of errors and the model's morphology doesn't match the real one, it has been possible to have an insight of the complex foot biomechanics without any invasive methods.

This research is supported by the Anatomical Society of Great Britain and Ireland.

P24

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Nervous relationships of the short saphenous vein: implications for management of chronic venous disease

J. F. Uhl, C. Gillot, O. Plaisant and V. Delmas Department of Anatomy, University René Descartes, Faculty of Medicine Paris V, France

The aim of this study was to describe the anatomical relationships of the short saphenous vein in the lower limb as an aid in locating important anatomical landmarks in surgical treatment and therapy involving sclerosing agents. Embryology was illustrated by the ‘angio-guiding nerves’ theory. Descriptive anatomy was based on the dissection of 120 unembalmed cadavers after latex injection.

We found the area around the apex of the calf presented the highest risk and was the most hazardous. The veins of the popliteal fossa were very close to both the medial head of gastrocnemius and the tibial nerves. At the ankle level, the origin of the short saphenous vein was plexiform, located deep below the fascia and close to the sural nerve.

In conclusion clinicians need to be aware of potentially hazardous areas in the treatment of venous disease involving a surgical approach to the short saphenous vein, and should determine the possible presence of a ‘short saphenous artery’ with a high risk for the injection of a sclerosing agent. These findings emphasise the usefulness of ultrasonographic mapping prior to an invasive procedure for treatment of chronic venous disease.

P25

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An anatomical study of the posterior septal space of the heart

S. H. El-Maasarany and E. Azab Department of Anatomy and Embryology, Faculty of Medicine, Alexandria University, Egypt

Radiofrequency catheter ablation has become the treatment of choice in patients with drug-resistant atrio-ventricular (AV) nodal re-entrant tachycardia. This procedure risks not only the myocardial fibres but also the components of the AV conduction system and/or its vascular elements. The aim of the present work was to study the anatomy of the posterior septal space (PSS) to delineate its shape, walls and contents with special consideration to the origin and course of the atrio-ventricular nodal artery (AVNA).

Twenty embalmed human hearts (16 males and 4 females) with ages from 25 to 60 years were dissected at the crux of the heart to demonstrate its shape, boundaries and the contents of the PSS. The arteries in the space were dissected to their destination.

The PSS proved to be an inclined 4 sided pyramidal space, having its base posteroinferiorly and its apex anterosuperiorly. In addition to the base the space had 4 walls (right upper, right lower, left upper and left lower) formed by the walls of the 4 chambers of the heart: the right atrium, right ventricle, left atrium and left ventricle, respectively. The base of the space which was lozenge-shaped was formed by the visceral layer of the serous pericardium. The right coronary artery (RCA) crossed the base from its right to its left angle. While crossing the base the RCA looped upwards to lie inferior and adherent to the terminal part of the coronary sinus and connected to it by a fibrous band. The posterior interventricular artery, the ventricular branches of the RCA, the middle cardiac vein and the ventricular veins, all crossed tortuously the base of the space to their final destination. AVNA was found to branch from either the RCA or from its posterior interventricular branch. A double AVNA was encountered in 14 specimens of the 20. The larger AVNA branched from the RCA as it looped upwards, and passed forward in the PSS along its upper angle, i.e. the angle between the right atrium and the left atrium. The smaller AVNA branched from the posterior interventricular artery and passed forward in the space along the right lower wall, i.e. related to the wall of the right ventricle. The course of the AVNAs should be taken into consideration during surgical procedures involving the PSS and also in radiofrequency ablations.

P26

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Computerized atlas of the superficial venous network of the lower limb

M. Hamid, J. F. Uhl, C. Gillot and O. Plaisant Department of Anatomy, University René Descartes, Faculty of Medicine Paris V, France

The aim of this work was to build a computerized anatomical atlas of the superficial venous network of the lower limb as described in Gillot's atlas. Descriptive anatomy was based on 120 unembalmed cadavers after latex injection, anatomical dissection and drawing. Anatomical plates and drawings are interactive, and an electronic index is available in 3 languages (English, French and Spanish).

The CD-ROM is divided into 2 parts: (1) 14 theoretical drawings providing anatomical basics and animations for beginners. It includes embryology, and modal anatomy of the great and small saphenous vein territories; (2) 19 interactive dissection plates with dissections of normal limbs (Plates 15–23), dissections with varicose veins (Plates 25–30), and dissections including the deep venous system (Plates 32–44). A search index is available for direct access to these plates according to anatomical variations.

P27

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Endothelial function in small veins

S. Hammond, T. M. Mayhew and W. R. Dunn School of Biomedical Sciences, University of Nottingham, UK

In small arteries, many hormones induce the release from endothelium of a variety of substances that cause vasodilatation. These include nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factor (EDHF). Although the latter factor has yet to be identified, roles for potassium ions, hydrogen peroxide, epoxyeicosatrienoic acids and gap junctions have been proposed. The present study has investigated the vasodilator substances released in rat mesenteric veins. Veins were isolated, cannulated and pressurized to 10 mmHg in a pressure myograph. After a period of equilibration, they were preconstricted with noradrenaline until their internal diameter was 50% of the original. Thereafter the responses to bradykinin (10−10 M to 10−6 M), in the presence or absence of the NO synthase inhibitor, l-NAME (10−4 M) and/or KCl (45 mm) were examined. Indomethacin, the cyclooxygenase inhibitor (10−6 M) was present in all experiments. The presence of either l-NAME or KCl alone significantly reduced the maximum vasodilator response of the vein to bradykinin. In the presence of both l-NAME and KCl the response to bradykinin was abolished.

Maximum Relaxation

TreatmentControlTreatmentnP
  1. * l-NAME (10−4 M) present during control experiments.

L-NAME (10−4 M)80.8 ± 4.254.1 ± 9.16< 0.05
KCl (45 mm)81.0 ± 3.131.5 ± 3.34< 0.05
L-NAME + KCl40.0 ± 8.1* 9.2 ± 4.37< 0.05

Bradykinin produced a vasodilator response in rat mesenteric vein which was partially blocked by l-NAME and by raised extracellular potassium, and abolished by the combination. This indicates that endothelium-dependent vasorelaxation is mediated by nitric oxide and EDHF. Future studies will be directed at determining the identity of EDHF in veins. In particular the role of gap junctions will be assessed by examining connexin expression using immunofluorescence with confocal microscopy and immunogold labeling with transmission electron microscopy. Studies will be extended to human vessels and used to compare small veins isolated from women with normal pregnancies with those from pregnancies complicated by pre-eclampsia. This is important since pre-eclampsia may be associated with increased venous tone which results in increased cardiac output and, subsequently, elevated blood pressure.

SH is the grateful recipient of an Anatomical Society of Great Britain and Ireland PhD Studentship.

P28

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Exogenous maternal insulin induces vascular leakage in the perfused human placenta

M. Uppal, D. B. J. Hughes and L. Leach Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, University of Nottingham, UK

Increased vascular leakage and altered junctional molecular profiles are features of feto-placental vessels in pregnancies complicated by Type1 insulin-dependent diabetes. It remains unclear if this is solely attributable to the complications of the diabetic milieu, or if exogenous insulin therapy, may paradoxically contribute to vascular disturbances. The aim of this study was to investigate whether presence of exogenous insulin in the maternal perfusate would result in changes in vascular leakage, alteration in localisation of the key adherens junctional molecule, vascular endothelial (VE-) cadherin and trans-placental transfer of insulin. Microvascular beds of normal term placentae were perfused in a dual independent closed circuit system, in the presence (n = 3) and absence (n = 3) of human recombinant insulin (1 U/mL) introduced to the maternal reservoir. After 20 min perfusion, a 76 Mr dextran tracer (0.5 mg/mL) was introduced to the fetal circuit for a further 10 min The beds were perfusion fixed and duly processed for imaging. Perfusate (maternal and fetal) were sampled at 1 and 20 min by radioimmunoassay. Insulin-treated placenta demonstrated a 3-fold increase in tracer leakage from the fetal vascular compartment, with > 60% of vessels showing leaks. These vessels also exhibited loss of junctional VE-cadherin. Within the 20 min duration, 0.08% of insulin was transferred from the maternal to the fetal venous outflow. Thus high concentrations of insulin in the maternal blood bathing the placenta, may disrupt adherens junctions and increase tracer leakage in feto-placental vessels; moreover trans-placental transfer of insulin may complicate the fetal homeostasis in diabetes.

P29

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Visualization of external lumbar vertebral venous plexus using magnetic resonance imaging and computed tomography

P. J. Toussaint, O. Ami, M. Hamid, C. Gillot, J. F. Uhl and O. Plaisant Department of Anatomy, University René Descartes, Faculty of Medicine – Paris V, France

Lumbar vertebral venous plexus are traditionally visualized with phlebography for anatomical and morphological study. Current imaging techniques allow better visualization of these vessels in a noninvasive, rapid and practical manner. In this study we have tried to assess the anatomy of the lumbar venous plexus using multislice computed tomography (CT) without contrast injection, and angiography by magnetic resonance imaging (MRI) in order to determine the sensitivity of the latter technique.

MR and CT images of the lumbar area in 2 pregnant women were assessed for quality of visualisation of the vertebral venous plexus. One pregnant woman underwent a multislice CT scan, without the use of contrast agent. The other expectant patient underwent MRI (1.5T) examination using a two-dimensional (2D) time-of-flight protocol. The lumbar area was also imaged using 3D phase contrast, as well as 2D time-of-flight, MRI in a healthy male volunteer to evaluate the sensitivity of this technique for detecting lumbar veins in an uncompressed situation.

Lumbar vessel morphology and venous anatomy were analysed, and the results compared for the 2 imaging methods. Multislice CT and MR angiography both showed clear delineation of the main lumbar arteries (on both sides) in the pregnant women. MR angiography also showed similar sensitivity for detection of main lumbar vessels in the male subject. Lumbar veins in L2, L3 and L4 are clearly visible on the localizer slice, 2D TOF, and 3D phase contrast sections. MR angiography successfully delineated all parent and adjacent veins of the lumbar area (L1 to L4). However breath-hold contrast-enhanced dynamic two-dimensional TOF MR angiography would probably improve lumbar vein visualization and help in the anatomical assessment of the lower lumbar venous plexus.

P30

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Morphogenesis of the human tensor veli palatini muscle (specimens measuring 15–76 mm crown rump length)

C. De la Cuadra-Blanco, J. J. Pozo-Kreilinger, M. D. Peces-Peña, J. R. Mérida-Velasco, I. Sánchez-Montesinos, J. R. Ruiz-Mezcua and J. F. Rodríguez-Vázquez Departments of Anatomy and Embryology I and II, Faculty of Medicine, Complutense University of Madrid; and Departments of Anatomy and Embryology I and II, Faculty of Medicine, University of Granada, Spain

Development of the palate has been studied by many authors, although few studies have studied the musculature of the palate velum in any depth. The aim of our work is to study in humans development of the tensor veli palatini muscle. The study was carried out by light microscopy on serial sections of 21 human specimens: 7 embryos and 14 fetuses ranging from 15 to 76 mm CRL (7–13 week development).

At the start of our study, with embryos of 15 mm CRL (O’Rahilly's stage 18), we observed a well defined muscular blastema of the tensor veli palatini muscle innervated by the mandibular nerve. In embryos of 20 mm CRL (O’Rahilly's stage 20) the hamulus of the pterygoid process appeared as a mesenchymal condensation and the tensor veli palatini muscle was reflected and turns back on itself.

At the end of the embryonic period, 30 mm CRL (O’Rahilly's stage 23), the hamulus, in the process of chondrification, served to reflect the tendon of the tensor veli palatini muscle. At the start of the fetal period, 9th week of development, the palatine aponeurosis, where the tensor veli palatine muscle is inserted, was perfectly defined. Thus development of the tensor veli palatini muscle is parallel to development of the palate velum.

P31

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Morphogenesis of the human lacrimal apparatus

M. D. Peces Peña 1 , C. de la Cuadra Blanco 2 , J. R. Ruiz Mezcua 2 , J. J. Pozo 2 and J. R. Mérida Velasco 2 Departments of Anatomy and Embryology I1and II2, Complutense University of Madrid, Spain

The tear film is essential for maintaining normal function of the cornea as well as the refractive optical integrity of the eye. It is composed of 3 layers. The middle aqueous layer is synthesized fundamentally by the lacrimal gland and forms the secretory system of the lacrimal apparatus. The tears that have not evaporated are drained into the lacrimal puncta, lacrimal canaliculi, lacrimal sac and lacrimonasal duct which form the excretory system of the lacrimal apparatus.

The purpose of this study was to analyse and systematise the development of the 2 components of the human lacrimal apparatus: the secretory and excretory system. The study was performed by light microscopy on serial sections of 51 human specimens: 33 embryos and 18 fetuses ranging from 8 to 137 mm CR (5–16 week of development) from the collection of the Embryology Institute of the Complutense University of Madrid were used.

Three stages were identified in the morphogenesis of the lacrimal apparatus.

Secretory system: (1) The presumptive glandular stage (O’Rahilly stage 19–20); (2) The bud stage (O’Rahilly stage 21–23); and (3) The glandular maturity stage (weeks 9–16). We confirmed that the development of the lacrimal gland originates in the surface ectoderm and not from other structures and it is an example of epithelial–mesenchymal interaction.

Excretory system: (1) The formative stage of the lacrimal lamina (O’Rahilly stage 16–18); (2) The formative stage of the lacrimal cord (O’Rahilly stage 19–23); and (3) The maturative stage of the excretory lacrimal system.

We confirm that the lacrimal duct originates from a single cord of epithelial cells and also observed mesenchymal condensation around the primordium of the lacrimal canaliculi.

P32

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The role of the notochord in vertebral column patterning

B. Senthinathan, S. Harris and R. Keynes Department of Physiology, Development and Neuroscience, University of Cambridge, UK

The influence of the anterior–posterior polarity of the somites on the patterning of the dorsal vertebral column and spinal peripheral nerves is well known. In the ventral vertebral column however, the mechanisms underlying segmentation of the vertebral bodies and intervertebral discs are still unclear despite the widespread acceptance of Remak's classical theory of ‘resegmentation’. In teleost fish there is recent evidence that the notochord, and not somite polarity, is responsible for patterning the ventral vertebral column, and there are indications in the older literature for a similar role of the notochord in amniote vertebrates. We have also found that unilateral reversal of the anterior–posterior polarity of the sclerotome in chick embryos produces discs with sclerotome-specific periodicity laterally, but that align nonetheless at the notochord/midline, consistent with an instructive role for the notochord in vertebral patterning.

Our main goal therefore is to determine whether the notochord is required for segmentation of the ventral sclerotome in amniote vertebrates. Excision of the notochord in the chick embryo is known to result in morphologically unsegmented cartilage ventrally. However the notochord contributes cells to the intervertebral disc (nucleus pulposus) and it remains possible that segmental patterning still takes place in notochordectomized embryos at stages preceding the failure of discs to form.

A marker for segmentation of the ventral sclerotome is the Pax1 gene which is expressed in the sclerotome-derived intervertebral disc anlagen. We have therefore assayed for early sclerotome segmentation using in situ hybridisation to the Pax1 mRNA transcript in notochordectomized chick embryos. We found that although restriction of Pax1 expression to the disc anlagen was disrupted, residual segmental expression of Pax1 remained, suggesting that segmentation is intrinsic to the sclerotome and independent of the notochord. We are now carrying out further, more stringent, tests to determine if axial structures are necessary to impart segmental patterning of the ventral midline in amniote vertebrates.

Acknowledgements: The Anatomical Society of Great Britain and Ireland for funding; Rudi Balling for cDNA; Baker, Keynes and Lewis Laboratory for assistance.

P33

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Determining the molecular code behind digit anatomy

L. A. McDonald, M. F. Fisher and C. Tickle Department of Cell and Developmental Biology, University of Dundee, UK

A fundamental problem in biology is how structures develop in the correct place during embryonic development. How does a little finger form at one edge of the hand and a thumb at the other? There is experimental evidence in mouse and chick that the number and pattern of digits that develop across the A-P axis of the limb depends on Shh signalling. However, relatively little is known about the genes that are expressed in response to Shh and how expression of these genes is translated into digit anatomy.

We are working on 2 strategies to find genes that are downstream of Shh and control digit anatomy. First, using the chick as a model, we are investigating the hypothesis that digit patterning in vertebrates is analogous to a signalling cascade in Drosophila wing development. I am focussing on the role of Iroquois-1 (Irx1), a vertebrate homologue of a gene that is expressed in response to Hh signalling in the fly. Irx1 is expressed in the distal part of the chick limb at the time when digit primordia are developing, and I have obtained evidence that Irx1 expression depends on Shh signalling in the early limb bud. In insects, Hh signalling is mediated by Dpp (a homologue of Bmp2), and I have shown that the effects of Shh on Irx1 expression may also be mediated by Bmps in the chick wing. I am currently testing whether Irx1 plays a role in determining digit identity by carrying out genetic manipulations in the chick limb.

The second approach is to find genes that encode A-P pattern involves microarrays. In collaboration with D. Burt and Ark Genomics, Roslin, tissue was collected from regions containing the primordia of digits 2 + 4 in the chick, and microarray analysis was performed to compare gene expression profiles of cells in these different primordia. Initial screens via in situ hybridisation have uncovered a number of genes with expression patterns restricted to specific primordia, and these provide new candidates for genes encoding digit anatomy.

P34

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Correlation of oestrogen receptor coactivator AIB1, oestrogen receptor α, and aromatase with cellular proliferation and apoptosis in human prolactinomas

E. J. Blanco 1 , C. M. Hernández 1 , M. Rubio 1 , J. M. Riesco 1 , M. Carretero 2 , E. Pérez 3 , J. J. Herrero 3 , J. Font de Mora 4 , D. J. Burks 4 and J. Carretero 1 1 Department of Human Anatomy and Histology, Laboratory of Neuroendocrinology of Institute of Neurosciences of Castilla y León. Faculty of Medicine, University of Salamanca; 2 Section of Physics and Mathematics, Department of Educational Sciences, University Pontificia of Salamanca; 3 Department of Surgery, Faculty of Medicine, University of Salamanca; and 4 Institute Príncipe Felipe, Valencia Spain

Although differential responses of hormone-dependent tumours to sex steroids could be strongly influenced by the relative amounts of coactivator protein AIB1, AIB1 expression and its correlation with aromatase (the enzyme responsible for the local aromatization of testosterone to oestradiol) and oestrogen receptor α in these tumours are not yet established. The aim of the present study was to investigate the protein expression of the coactivator AIB1 in human prolactinomas and compare this expression with the expression of aromatase and oestrogen receptor α and with the cellular proliferation in these tumours. Of a series of 87 adenomas studied 56% showed a high population of prolactin-positive cells and were classified as prolactinomas because they show hyperprolactinemia. Moreover 80% of the adenomas were aromatase-positive tumours. Interestingly 100% of the prolactinomas were aromatase-positive tumours. Western blotting with antiaromatase antibodies revealed a doubling in expression of aromatase in pituitary tumours compared to normal human pituitary gland. All prolactin- and aromatase-positive tumours were positive for oestrogen receptor α and AIB1. 81% of prolactinomas were PCNA-positive tumours. Nuclear expression of AIB1 was the predominant expression in PCNA-positive prolactinomas. 7% of prolactinomas were non PCNA-positive, showed cytoplasmic reaction for AIB1, and were caspase 3-positive. Our results demonstrate for the first time the pituitary expression of AIB1 in prolactinomas and their correlation with the cellular proliferation of the tumour, suggesting the possibility that AIB1 could be involved in the growth of the tumour and that an abnormally high conversion of testosterone into oestradiol mediated by aromatase in pituitary cells occur.

Supported by the FIS Spanish program No. PI0-21803.

P35

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Coexpression of aromatase, oestrogen receptor α and dopaminergic receptor D2L in human prolactinomas

J. M. Riesco 1 , L. González 1 , D. Prieto 1 , S. Gutierrez 1 , R. González 1 , C. M. Hernández 1 , M. Rubio 1 , M. Carretero 2 and J. Carretero 1 1 Department of Human Anatomy and Histology, Laboratory of Neuroendocrinology of Institute of Neurosciences of Castilla y León, Faculty of Medicine, University of Salamanca; and 2 Section of Physics and Mathematics, Department of Educational Sciences, University Pontificia of Salamanca, Spain

In previous studies we demonstrated the immunocytochemical expression of aromatase in pituitary cells and prolactinomas in the rat, suggesting that aromatase could be involved in the genesis of prolactinomas in rodents. The aim of this study is demonstrate the expression of the enzyme in cells of human prolactinomas by immunocytochemistry and in situ hybridization, and the relationship between immunocytochemical expression of aromatase and dopamine receptor D2L and/or oestrogen receptor α in human prolactinomas. Immunocytochemistry and in situ hybridization demonstrated that aromatase was expressed in 100% of human prolactinomas, which moreover express oestrogen receptor α Dopamine receptor D2L was expressed in 98% of human prolactinomas. These findings demonstrate the ability of human prolactinomas to bind dopamine through dopamine receptor D2L and the synthesis of aromatase in human prolactinomas and its functional ability to develop the estrogenic effects mediated by oestrogen receptor α suggesting that these effects could be involved in the growth of human prolactinomas.

Supported by the FIS Spanish program No. PI0-21803.

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Regulation by gonadal steroids of aromatase p450 expression in the rat pituitary

Rubio 1 , E. Pérez 2 , E. J. Blanco 1 , M. Carretero 3 , J. M. Riesco 1 , J. J. Herrero 2 , D. J. Burks 4 and J. Carretero 1 1 Department of Human Anatomy and Histology and Laboratory of Neuroendocrinology, Institute of Neurosciences of Castilla y León and 2 Department of Surgery, Faculty of Medicine, University of Salamanca; 3 Section of Physics and Mathematics, Department of Educational Sciences, University Pontificia of Salamanca; and 4 Institute Príncipe Felipe, Valencia, Spain

Rat aromatase immunohistochemical expression is different in male and female adult rats. In order to analyze if these differences are related to the presence of gonadal steroids, a study was carried out on pituitaries of adult castrated and gonadal steroid-treated castrated rats using immunohistochemistry, Western blotting and in situ hybridization for rat aromatase P450. Rat aromatase P450 mRNA was detected in the pituitary of male and female rats. Evidences for sex-related variations of mRNA were observed, the mRNA signal was more abundant in males than in females, moreover the male pituitaries showed more immunohistochemical positive cells than females and, by Western blotting, the enzyme was more abundant in male than in female rats. With the 3 methods assayed, ovariectomy elicited a considerable increase in the reaction to aromatase in females; in male rats castration reduced the number of reactive cells, although the reaction persisted. Treatment with gonadal steroids after castration modified aromatase expression in the sense that in testosterone-treated castrated males the expression of aromatase increased while in castrated females treated with estradiol it decreased. Our results demonstrate the synthesis of aromatase in the pituitary and its immunohistochemical expression in the gland of adult rats, suggesting that the expression of this enzyme is sex-dependent and can be modified by castration and gonadal steroid administration. This in turn suggests that aromatase may be involved in the regulation of adenohypophysial cytology by gonadal steroids.

Supported by the FIS Spanish program No. PI0-21803.

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Annexin 1: a local paracrine regulator of adrenal size and corticosterone secretion: evidence from annexin 1-null mice

J. F. Morris 1 , E. Davies 1 , S. Omer 1 , J. Buckingham 2 and H. Christian 1 1 Department of Physiology, Anatomy and Genetics, University of Oxford; and 2 Department of Neuroendocrinology, Imperial College of Science Technology and Medicine, London, UK

Annexin 1 (ANXA1) is a member of the annexin family of phospholipid- and calcium-binding proteins with a clear role in early delayed (30 min-3 h) inhibitory feedback of glucocorticoids in the hypothalamus and pituitary gland. Adrenal gland tissue from ANXA1-null transgenic mice, in which a beta-galactosidase (beta-gal) reporter gene is controlled by the ANXA1 promoter, and from wild-type control mice was used to explore the potential role of ANXA1 in adrenal function. Strong expression of ANXA1 mRNA and protein in the adrenal gland was revealed by RT-PCR and Western blotting. Immunofluorescence labelling of ANXA1 in wild-type, and beta-Gal expression in ANXA1-null adrenals localized intense staining in the outer cortical cell layers. Immunogold electron microscopy identified cytoplasmic and nuclear ANXA1 labelling in outer zona glomerulosa and in subcapsular cells. Exposure of adrenal segments in vitro to dexamethasone (0.1 µM, 3 h) caused an increase in the amount of ANXA1 in the intracellular compartment and attached to the surface of the cells. Corticosterone release in response to ACTH (10 and 100 pm) was significantly greater from ANXA1-null adrenal cells tan from wild-type, but basal and angiotensin II (10 nm) -stimulated aldosterone release from ANXA1-null adrenal cells did not differ from wild-type controls. Morphometric studies demonstrated that the cortex of ANXA1-null adrenal glands was smaller than those in wild-types and all three zones (glomerulosa, fasciculata and reticularis) were significantly reduced in size. These results suggest ANXA1 is a locally produced paracrine peptide which regulates adrenocortical size and corticosterone secretion.

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Localization of renal COX-1 expression in control and NSAID treated CD-1 mice

M. Meskell 1 , R. Ettarh 2 and A. McGarvey 3 1 School of Nursing, Midwifery and Health Systems, and 2 School of Medicine and Medical Sciences, University College Dublin; and 3 Department of Anatomy, Royal College of Surgeons in Ireland, Dublin, Ireland

The enzyme cyclooxygenase mediates the biosynthesis of prostaglandins in the kidney and supports the role of these lipid mediators in the maintenance of physiological renal functions. The use of cyclooxygenase-inhibiting NSAIDs is limited by unwanted renal side-effects. In order to further understand the mechanisms that underlie these renal side-effects intrarenal localisation of murine COX-1 was examined by immunohistochemistry following treatment with indomethacin and nimesulide. Animals were killed by cervical dislocation 24 h after administration of the drug and processed for immunohistochemistry.

In control samples, COX-1 immunoreactivity was present in the glomerular parietal epithelial cells. There was prominent staining in the glomerulus in indomethacin treated group, and in the glomerular parietal epithelial cells in both treated groups. Reactivity to COX-1 in the proximal and distal convoluted tubule was similar in all groups, although there was greater staining apparent in the thin limb of the loop of Henle in control samples. No COX-1 expression was seen in the thick limb of the loop of Henle or collecting ducts in any group. These findings indicate that NSAIDs do not alter the distribution of COX-1 in the kidney but may affect the level of expression of this enzyme in some parts of the nephron.

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High-resolution chromosome structure after premature condensation induced by caliculin: a protocol

G. Mazzotti, G. Teti, M. Zago and M. Falconi Department of Anatomical Sciences, University of Bologna, Italy

In recent years many attempts to elucidate the higher order structure of chromosomes have been made by scanning electron microscopy (SEM), but one limit in understanding the real organization is due to the procedures used in preparing chromosome samples. Although the function and composition of chromosomes has been well known for many years, the structure and organization is still a matter of discussion. The purpose of the present work was to study the structure of in situ human chromosome by transmission electron microscopy (TEM) and field emission in lens scanning electron microscopy (FEISEM) prepared by a caliculin A (CLA) protocol. CLA is an inhibitor of protein phosphatases which lead to a premature chromosome condensation (PCC) in all phases of the cell cycle when they are inactivated. Thus it is possible to analyze both metaphase and G2-PCC chromosomes.

HL60 cells were arrested in metaphase, fixed with glutaraldehyde and then processed for FEISEM or TEM analysis. FEISEM allows higher resolution images of biological samples without any metal coating, in comparison to traditional scanning electron microscopy.

TEM images of CLA treated samples showed condensed areas inside the cells corresponding to different chromosomes. The areas were connected each other. Some cells were blocked in the G2 phase of cell cycle and showed smaller areas corresponding to different chromosomes separated from each other. In no images was it possible to observe a fibre organization inside the chromosome. For FEISEM observation cells were fixed with glutaraldehyde, cryoprotected, frozen and cryosectioned. In comparison to TEM, FEISEM allowed us to distinguish fibres of different diameter overlapping each other.

We suggest that CLA in combination with FEISEM is a powerful tool for the investigation of chromosome structure with the aim of elucidating its organization and condensation. To understand fundamental events in cell kinetics or in regulation of gene expression it appears crucial to clarify whether the transition from nucleosome chromatin to metaphase chromosome occurs through a hierarchical order of condensation or through a single compaction of low order chromatin fibres.

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Counting your gold: comparing the relative labelling indices of cellular compartments in a mixture of organelles and membranes

T. M. Mayhew School of Biomedical Sciences, University of Nottingham, UK

Immunogold-labelling TEM offers high-resolution localisation of defined antigens in cellular compartments and the particulate nature of colloid gold probes allows quantification. By relating numbers of gold particles to cell ultrastructure a key question can be addressed: ‘Do gold particles within cells label compartments randomly or preferentially?’ Using quantitative immunoelectron microscopy to examine the distribution of gold particles, preferentially labelled compartments can be identified by estimating labelling densities (LDs) and relative labelling indices (RLIs). Hitherto this approach has been confined to studies in which all compartments were of the same category (organelles or membranes) rather than a mixture of categories (organelles and membranes). In reality some antigens translocate between membranes and organelles and the problem then arises of relating gold particles to a common reference (e.g. compartment profile area). Here a possible solution to this problem is proposed. It relies on multistage systematic uniform random sampling to randomise the positions and orientations of encounters between sections and specimens. Observed distributions of gold particles are determined simply by counting particles associated with each compartment. An expected distribution of gold particles is created by randomly superimposing test points and counting those hitting each compartment. To this end membranes are treated as ‘organelles’, i.e. as volumes delimited by boundaries at a fixed distance either side of the true membrane. To fix this distance, a subsample of ‘clear’ membrane images (local vertical windows) is selected and an estimate of their LD (gold particles per profile area) is obtained. Using factors estimated by intersection counting or by goniometry membrane counts are corrected for the effects of image loss due to membrane tilt from the direction of the electron axis. The LDs of chosen compartments (organelles and membranes) are used to calculate RLI values and, once corrections have been made, observed and expected gold counts are used to calculate compartmental chi-squared values. Those making substantial contributions to total chi-squared help to identify the compartments which are preferentially labelled. Since RLI = 1 for random labelling, these compartments must also have an RLI > 1.

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Postgraduate continued learning of anatomy: preferences in Spanish primary care

J. Carretero, J. M. Riesco, E. Blanco, M. Rubio and R. Vázquez Department of Human Anatomy and Histology, Faculty of Medicine, University of Salamanca, Spain

The need for convergence in the European Area of Higher Education promoted our team to apply for a project financed by the Junta de Castilla y León aimed at analysing the requirements needed for the teaching of human anatomy and the preferences in teaching methodologies during undergraduate and postgraduate stages.

After assessing and analysing the results obtained from questionnaires completed by specialists in primary care, the most relevant results are as follows. Those interviewed showed a marked trend to incorporating computer-assisted teaching; they rejected, providing scores lower than 2.4/5, teaching based exclusively on Problems Based Learning (PBL); they favourably evaluated the a posteriori incorporation of the learning of anatomical knowledge from teaching based on PBL; they were favourable to integration of the teaching of Anatomy into that of other disciplines, with no marked differences between the opinions of those favourable to a vertical integration and those who would prefer a horizontal integration.

Regarding continued training courses the interviewees preferred ‘nonface-to-face’ on-line courses and chose the following 3 topics over the rest, ordered by preference: (1) Courses on the anatomical basis for exploration of the locomotor apparatus; (2) Courses on surface anatomy as a basis for palpatory physical exploration; (3) Courses on the anatomical basis of the somatic constitution and body postures and their repercussions in spinal pathology, and in the use of ergonomics at work and at home. The interviewees rejected, with scores lower than 2.5/5, courses on the dissection of some parts of the human body.

The contrast of continued training in primary care with graduate teaching is seen on comparing these results with those obtained among the graduate students of clinical courses.

Supported by Junta de Castilla y León.

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Contributions of the Romanian medical school to the development of basic and applied neuroanatomy

A. R. M. Chirculescu 1,2 and M. Chirculescu 1 1 Department of Anatomy, Faculty of Medicine, C. Davila University, Bucharest, Romania; and 2 Department of Physiology, Anatomy and Genetics, University of Oxford,UK

Gr.T. Popa (1892–1948) was first to describe the hypothalamo-pituitary portal system (with Una Fielding, 1930). Ion Th. Niculescu (1895–1957) described the periventriculo-hypophyseal and infundibulo-hypophyseal tracts (with I. Raileanu, 1925) and the tuberal commissure (with Maria Niculescu, 1929), as complementary details to the supraoptico-hypophyseal and paraventriculo-hypophyseal tracts, described by Ramon y Cajal (1905). He also described the peri-retro-paleorubrum of the red nucleus (with Charles Foix, 1925), studied the tubero-mammillary area angioarchitectonic and the development of the myelin tracts in the human fetus (1926).

V. Babes described independently of Negri the intracytoplasmic inclusions in hippocampal pyramidal and cerebellar Purkinje cells, which are pathognomonic for rabies infections (1912).

Gheorghe Marinescu (1864–1938) published ‘Studies on Evolution and Involution of the Nerve Cell’ (1900) and the first monograph on pathology of the neuron ‘The Nerve Cell’ (1909), with introductory remarks by Ramon y Cajal. He also described acromegaly as related to pituitary eosinophilic adenoma, at the time when Pierre Marie considered it a consequence of pituitary necrosis. He also described a somatotopy in the facial motor nucleus. An association of congenital malformations of the CNS and limbs is known as Marinescu's syndrome.

Toma Ionescu (Thomas Jonnesco or Thoma Ionnesco, 1860–1926), one of the contributors to Poirier's and Charpy's Handbook of Anatomy (vol. IV, 1, 1894), was both Professor of Anatomy and Surgery in Bucharest. As Professor of Anatomy, he described the variant of the cervical sympathetic chain, which divides to let the vertebral artery pass from the origin toward the transverse canal (the slit of Drobnick and Ionescu) and the space where the stellate ganglion usually lies (supra-retro-pleural fossa of Sebileau and Thoma Ionnesco). As a clinician and surgeon, he initiated the ‘general rachi-anaesthesia’ (GRA − 1908) or general spinal analgesia (Br. Medical J. 1909), and developed an original technique for cervical sympathectomy (1908). This anaesthesia allowed him to practice surgery on the head, neck, thorax and upper limbs.

A.R.M.C.'s visit to Oxford was supported by a Senior Visiting Fellowship from the Anatomical Society of Great Britain and Ireland.

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Communicating clinical anatomy to a lay audience

D. J. R. Evans and D. J. Lawrence-Watt Brighton and Sussex Medical School, Brighton, UK

In recent years there has been a dramatic increase in the time devoted to teaching oral communication skills within medical curricula worldwide, with the ability to convey medical knowledge to a lay audience being one of the main features of communication training. Communicating with patients through written means however, does not appear to be a learning focus in many programmes, despite its fundamental importance in creating understanding of medicine within the general population. The result of such an omission is that newly qualified doctors often lack some of the special skills required when, writing letters to or on behalf of patients, writing articles in the general press and producing patient focussed information. At Brighton and Sussex Medical School we have explored one way in which patient-centred written communication can be integrated into part of the early training years of medical students. Within the Reproduction and Locomotion module, a core module undertaken by students in their second year, the coursework element is focussed on writing for a lay audience. Students are ‘commissioned’ by the editor of a fictitious newspaper to write a short article of no more than 500 words in response to a letter from a member of the public regarding a clinical condition associated with either reproductive or locomotor anatomy. Students are required to respond in lay terms describing what the particular condition is, how it arises, the consequence of the condition to the well being of the patient and how the condition may be alleviated or rectified. The resulting articles have been very effective, of an extremely high standard, and at a level making them very readable to a lay audience without being patronising. Qualitative feedback has demonstrated that students find it a challenging yet very useful and enjoyable experience in developing their written communication skills. It is hoped that developments such as this within the medical curriculum will enhance the capacity of all potential doctors to communicate more effectively with patients in both the written and spoken form.

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A strategy towards professionalism in the dissecting room

M. Heyns Division of Basic Medical Sciences, Queen's University Belfast, UK

The School of Medicine and Dentistry uses an approach of directed self learning for teaching anatomy, and students are given the opportunity to learn anatomy from human dissection. The students have to prepare for the dissection sessions by prereading the relevant sections in the prescribed textbook as well as using other resources made available on the university intranet. This is followed by 4 h of dissection per week. These practical sessions take place in the Dissecting Room (DR), where the approximately 140 students are grouped around 20 dissection tables.

In an attempt to address the issue of large numbers around the table and the inactivity of some of the students, as well as the lack of preparation by some students, a strategy was developed to engage all students around the table. Each group is seen as a team, and the staff member coordinating the session will appoint a leader for such a session. The leader will allocate roles to each member as they see fit. These roles typically include dissectors, a reader to display relevant text or diagrams, a summariser to recapitulate the events taking place at regular intervals, a note taker to document any anatomical variations observed as well as any issue of clinical relevance and an operational manager to oversee the maintenance of the working area, washing of instruments, stacking of chairs and general cleanliness.

This team approach to the dissection session proved to be well received by the students and staff. For the staff it also reduced the administrative burden of maintaining the attendance register. The students reflected positively on the skills and competencies gained such as cooperative learning, development of leadership skills, team building, communication skills, responsibility, mutual respect, increased motivation and preparedness.

The value of such a strategy can be further enhanced by including structured tasks around the dissection taking place and the roles can be expanded to simulate other professions in the health system in order to foster interprofessional understanding and respect. Such self-directed teams can also be utilised to nurture the development of the attributes of professionalism, such as altruism, confidentiality and trustworthiness.

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Anatomy instruction in physiotherapy students: a preliminary study in the context of the European higher education area

C. Melguizo 1 , J. Prados 2 , F. Rodríguez 3 , F. Hita 3 , M. Peran 1 , H. Boulaiz 2 , C. Velez 3 , J. A. Marchal 3 , O. Caba 2 , R. Ortiz 2 , A. Martinez 3 , E. Carrillo 2 , L. Alvarez 2 and A. Aranega 2 1 Department of Neuroscience and Health Sciences, University of Almería; 2 Department of Anatomy and Embryology, School of Medicine, University of Granada; and 3 Department of Health Sciences, University of Jaén, Spain

The future integration of the Spanish University System into the European Higher Education Area means a change in the current educational model including a flexible system which allows compatibility of degrees and higher competition among them. In this system pupils will have a better and higher contribution in real learning useful for professional activity. These changes imply new relationships between pupils and teachers, new methodologies, new teaching strategies and different evaluation systems. The success of this project will depend on the knowledge of our present situation. Our study is the first approaching the status of human anatomy and embryology in the physiotherapy undergraduate degree. This analysis has been carried out on students from the first course of the degree and specifically in the Structure and function of the human body, Skeletal and muscle system anatomy and General human anatomy subjects at the Universities of Almeria and Jaen. By means of the evaluation of a test, we have determined the students’ opinion of the appropriateness of the subjects to the degree, to the methods used in practice and theory, to the assessment and tutorial system. From this analysis we can obtain the following conclusions which are very similar in the different Universities included in this study. This conclusion can summarized in the following way: (1) the pupils have a high opinion of the usefulness of the subject contents in human anatomy and embryology; (2) pupils accept the new technologies far from the traditional educational systems; (3) the pupils have a positive appreciation of the written examination instead of oral tests or continuous assessment. These results will allow the Professors of Anatomy and Embryology to establish the pattern for improvement of the quality in the learning process in the context of the European Higher Education Area.

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Nursing student's perceptions on the use of anatomical prosections as an educational tool

M. Meskell 1 , J. E. OConnor 2 and L. McKelvey 1,2 1 School of Nursing, Midwifery and Health Systems, and 2 School of Medicine and Medical Sciences, University College Dublin, Ireland

Human anatomy is taught in Stage 1 of undergraduate nursing programs at University College Dublin. Dissection room based practical sessions were recently introduced as part of this module. Nursing candidates were surveyed to assess the value of these sessions as an educational tool. Student's reactions and concerns regarding the dissection room were also evaluated.

74 students (67 female and 7 male) responded to the questionnaire. 97% of those surveyed were of Irish nationality; other nationalities included Ivorian, Nigerian and Philipino. 54% of students reported that they had no previous exposure to dead bodies before entering the dissection room. The majority of students (69%) were apprehensive at the thought of visiting the dissection room for the practical class.

Loss of appetite was reported by 11% of students prior to entering the dissection room. 58% of students surveyed reported that the smell in the dissection room disturbed them in some way. The sight of the anatomical specimens also caused some disturbance (15%) as did touching the specimens used (16%). 8% of nursing candidates reported dizziness as a physical symptom during the practical session. Other physical symptoms such as sweating, trembling and recurring or disturbing visual images of cadavers did cause some distress among nursing candidates surveyed, although this was minimal.

Two open-ended questions were asked in order to determine the value of anatomy and the use of anatomical specimens by the students. An overwhelming majority (95%) of students reported that Anatomy as a subject is relevant to the nursing profession and is important to comprehend in detail prior to commencing clinical placement. 92% of students surveyed thought that the use of anatomical specimens aids better visualisation of internal structures.

Research findings suggest that although these nursing candidates were apprehensive about practical sessions an overwhelming majority found them to be very beneficial. These findings suggest that anatomy has a pivotal role to play in nursing education in Ireland.

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Selection for medical studies and success in the subject of human gross anatomy

B. Mompeó Department of Morphology, University of Las Palmas de Gran Canaria, Spain

The admission system for medical studies in our country is based on high school grades and an Access to University Essay, not including other factors such as motivation or personality of the students. We developed this study aiming to know if, the success of students in their earlier stages of education have a direct relationship with the success in the subject of human gross anatomy.

Our objectives were (1) To determine the predictive value of this admission method in relation to the academic performance in the first year subject human gross anatomy; (2) To determine if there is any relationship between the scores obtained in the human gross anatomy and other factors like gender, educational background, leisure activities and motivation for medical studies.

A questionnaire was given to the students the first day of classes.

The main topics were marks for admission to the Medicine Faculty, educational background, hobbies and motivation for choosing medical studies. The answers were scored and studied in relationship with the final marks obtained for the students in Human Gross Anatomy.

The results showed that the admission scores were not related to the results obtained in human gross anatomy. Factors like gender, motivation for choosing medical studies and educational background showed differences in their relationships with the admission marks and with the results obtained in the subject studied. We conclude that factors others than the previous success in the earlier stages of education can affect the academic success in the subject of human gross anatomy.

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The impact of the use of virtual practicals in anatomical learning

R. Sola and M. C. Mias Department of Human Anatomy, Faculty of Medicine, University of Lleida, Spain

The authors analyse their experience following the introduction of virtual practicals in the practical learning of one anatomical subject, in order to adapt the programme to the European programme during the 2005–2006 academic year.

The introduction of virtual practicals has needed work by the anatomical teachers with a team of virtual learning experts of our University in order to design the multimedia practicals, programming the work according to the dates of classes and the time to make them. At the beginning of the course an introductory class has been offered to the students in order to explain the use of the virtual platform necessary.

In this study we analysed the students’ participation, the delivery of practicals in the designated time; the academic or informatic problems of the platform, and the possibility of development of the system in the future. We have found that 85% of students matriculating in this subject delivered their exercise within the time limit. The remaining 15% have used the virtual platform to solve their problems. 10% of students submitted the practicals in paper format, and 9% used an E-mail: system different from the virtual platform. Students that didn't submit the practicals didn't make the final exam.

We believe that the use of virtual practicals in anatomical learning is well accepted by the students, because the participation is high, but it is necessary to remediate the knowledge of some students about the virtual platform. We also think that the virtual practicals assist improvement in theoretic and practical anatomical knowledge, because they permit to the student to control their own progress and organise their time to obtain results. The teachers could follow the efforts of students through the delivery of the practicals and by the virtual consultations during the course.

In conclusion we think virtual practicals must be considered in anatomical learning as a tool in the learning process, because they help in learning knowledge and competences and act as an evaluation tool to both teacher and student.

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Learning the language of anatomy

A. T. Wilkinson Division of Basic Medical Sciences/Anatomy, Queen's University Belfast, Northern Ireland, UK

Each subject discipline has its own language, which must be learnt in the process of becoming a member of that professional community. In anatomy many terms are based on Latin or Greek, subjects no longer generally taught in the schools of English-speaking countries. Thus students learning anatomy for the first time often struggle with the terminology. A study was therefore conducted to investigate whether knowing the meaning of anatomical terms helped students in their learning of the subject.

23 second-year biomedical science students doing a module on topographical anatomy were given a list of largely unfamiliar anatomical terms and asked to write down (or guess) both the meaning of the term and the anatomical structure it describes. Their approach to the task was then discussed. On a second occasion, 19 students were given a new list of terms. They attempted to relate each of these to a known English word and tried again to work out the anatomical structure. They were also asked whether they thought that knowing the meaning of the anatomical terms assisted in their learning of anatomy.

Correct identification of anatomical structures that had already been introduced in the topographical module was good (65–91% of students), whereas for structures that had not yet been covered, identification was poor (0–39%). Correct meanings for the terms ranged from 0% (e.g. sartorius) to 83% (hepatis). For the second list, correctly providing an English word related to the anatomical term ranged from 0% (genu) to 95% (dura), while giving the correct meaning of the term ranged from 0% (e.g. capitulum, genu) to 68% (brevis). All students thought that knowing the meaning of anatomical terminology assisted their learning.

The study demonstrated that these students were not adept at guessing the meanings of anatomical terms either directly or through association with known English words unless they had met the terms previously. Many fell into the traps found commonly in learning a second language such as deceptive transparency (mistaken identification), or confusing words with morphologically similar lexical forms. Although student perception is that understanding is rewarded by better retention when learning the language of anatomy, more evidence needs to be provided before this can be confirmed.

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Anatomical teaching for medical students from the perspective of European Union enlargement

A. R. M. Chirculescu 1,2 , M. Chirculescu 1 and J. F. Morris 2 1 Department of Anatomy, Faculty of Medicine, C. Davila University, Bucharest, Romania; and 2 Department of Physiology, Anatomy and Genetics, University of Oxford, UK

When the decision to extend the European Union was taken, a series of cooperative programmes focused on education were organized and financed (Tempus, Erasmus – Socrates, etc.) to elicit and develop bilateral mobility of teaching staff and students (undergraduate and PhD). Their aim was to facilitate contacts and develop partnerships, at both levels. They were designed to evaluate competency and professionalism, as a concrete starting point for ensuring a minimal compatibility of education standards between EU members and candidates, and a common basis for a valid and equitable European Transfer Credit System generally accepted in all European countries. This raises questions of core curriculum or benchmarks, organization of courses in units, and methods of teaching and assessment. There is also a tendency for additional criteria to be evaluated (manual, presentation, writing, oral communication, IT competence skills, data analysis and interpretation, ability in team work, individual initiative and originality).

The Romanian anatomy teaching ensures a good training using lectures, dissection and demonstration on prosections, radiological and living anatomy, in a classical traditional manner. But horizontal and vertical integration of courses is almost missing. Many possibilities are not used, including problem-based learning around common clinical conditions, virtual anatomy tutorials, virtual dissection, seminars, small groups, personal and academic tutorials, project-based tasks (‘learning by doing’), learning from experience, Personal Development Planning Portfolios, and Honours degree classification. All these and others omitted here, do not need special investments and expenses so they can be introduced, one by one, in the future.

One particularly important aspect is to define the teaching aims of anatomy courses, in order to provide background for other basic sciences and for clinical disciplines (not only surgery) and make it compatible in terms of uniform grading and equal opportunities for all groups of students. This should be based on ‘core’ information and ‘additional’ information, used in logical ways, to arrive at diagnoses, treatments, and management plans.

A.R.M.C.'s visit to Oxford was supported by a Senior Visiting Fellowship from the Anatomical Society of Great Britain and Ireland.

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Workshop in clinical anatomy of the brain

V. Macchi, A. Porzionato, A. R. Parenti and R. De Caro Department of Human Anatomy and Physiology, University of Padova, Italy

A workshop in Clinical anatomy of the brain has been developed as part of the training program for the first year residents in neurology 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 10 residents who work in 5 teams on 10 plastinated brains, 10 embalmed brains and 1 unembalmed, unfixed brain (harvested from the cadavers 24 h after death). The workshop is organized into 2 sessions of 2 h each in length, one on the whole brains and the second on the sectioned brains. Each session has 5 parts: in the first part (30 min), a team of the 2 residents analyzed the plastinated specimen following a clinically oriented worksheet, in the second part (20 min), the teacher showed to the residents each step of the worksheet. In the third part (20 min), each resident recognized, on an embalmed specimen, a selection of the clinical points listed in the worksheet, correlated to selected brain pathologies. In the fourth part (25 min), the workshop included presentations on the specimens that illustrate anatomical variability and illustration of the clinical points on the unembalmed brain. In the last section (25 min), the residents are requested to indicate on radiological images (CT and MR of the brain, CT and MR angiography) the clinical points. The guidelines in planning the workshop have been: (1) the selection of brain pathologies (hydrocephalus, acustic neuroma, primary cerebral haemorrhage) and of the involved anatomical structures; and (2) the correlation between the axial radiological images and the sectioned anatomical specimens.

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Structure and assessment of a short intense clinical anatomy course immediately before clinical studies

J. F. Morris and A. R. M. Chirculescu Department of Physiology, Anatomy and Genetics, University of Oxford, UK

In many traditional courses topographical anatomy is one of the first subjects studied, and students have often forgotten much of what they have learned when confronted by the need to recall the information during clinical studies. Oxford University wishes to maintain a strong science base to medical studies; students take an honours medical science course before starting clinical studies and so this problem was particularly acute even though the science is always related to function and clinical problems. To overcome the problem, Oxford has divided students’ topographical anatomy learning into a first year course, in which the underlying principles of body structure are studied, with appropriate illustrations from various but not all regions, and a newly designed 3- week clinical anatomy course which students take in June after their final science exams and before starting clinical attachment in September.

The course aims to ensure that students start clinical training with a high level of knowledge of those features of topographical anatomy that are particularly relevant to examination of patients, diagnosis of disease processes with an anatomical element, and simple clinical procedures. To achieve this, the course is intensive, 8 h per day for 3 week. Each day follows a similar pattern: orientation lecture, morning and afternoon practical session each interspersed with a short (30 min) lecture by a practising clinician illustrating the clinical use to which the anatomy being studied will be put. During the 3-h practical sessions, half the students follow an electronic learning programme which includes clinical scenarios, test questions and model answers; the other half undertake guided study of prosections; the groups then exchange. Each element of the body is studied in relation to common clinical conditions and procedures to which it is subject.

Assessment of student progress occurs at the end of each week of study with an on-line set of 20, 5-part, extended match or MCQ questions many of which are based on an illustration of either normal anatomy or consequences of an anatomical lesion. All questions are extensively assessed by clinically qualified staff. One great advantage of electronic assessment has been the detailed analysis that is rapidly generated and which informs the generation and modification of questions.

Dr Chirculescu was a Senior Visiting Fellow of the Anatomical Society of Great Britain and Ireland.