The following review articles are based on presentations given at a Symposium on Computer Modelling for Anatomists and Clinicians held in July 1997 at the Department of Anatomy, University of Edinburgh. The Symposium formed part of the Summer Meeting of the Anatomical Society of Great Britain and Ireland. There were 6 invited speakers, and 4 of these have produced review articles which are published in this issue of the Journal. In 3 of the presentations, recent advances in the use of 3D methodology to analyse mouse and human developmental anatomy were described. The reviews by Kaufman et al. and Baldock et al. (the latter to be published in a forthcoming issue of the Journal) complement each other in that they describe different aspects of a collaborative project (the Mouse Atlas Project, or MAP) involving the preparation of a text database of mouse developmental anatomy and a digital Atlas of normal mouse development. In the first of these presentations the principles underlying the methodology employed to reconstruct a range of early post-implantation mouse embryos spanning the stages between the primitive streak and the early limb-bud that had previously been serially sectioned was briefly described. Because of the efficiency of the computer technology involved (termed the warping program), the grey level images of the embryo histology could be arbitrarily resectioned to view the embryo as if sectioned in any orientation. Furthermore, all anatomically-defined tissues could be individually delineated (or painted) and viewed either in isolation or in combination with other appropriately labelled tissues and organs.

We have been involved with a group of computer scientists and anatomists in the development of computer-based methodologies that not only combine the advantages of scanning electron microscopy and conventional histology, but provide the additional dimension of tissue recognition. The latter is achieved by the appropriate labelling of tissues and structures by delineation or ‘painting’. Individually segmented anatomically defined tissues can be highlighted in a particular colour and viewed either in isolation or in combination with other appropriately labelled tissues and organs. Tissues can be shown in any orientation either as a transparent overlay on computer-generated histological sections or as 3-D images without the histological background. An additional feature of the system is that computer graphics technology combined with 3-D glasses now also allows the viewer to see the object under analysis in stereo. This facility has been found to be particularly helpful in drawing attention to topological relationships that had not previously been readily noted. As the mouse is now the mammalian model of choice in many areas of developmental research, it is of critical importance that a basic level of skill is available in the research community in the interpretation of serially sectioned material, for example, for the rapidly expanding field in which gene expression studies play a significant role. It is equally important that there is an understanding of the dynamic changes that occur in relation to the differentiation of the various organ systems seen in these early stages of development. What we emphasise here is the additional information that it is possible to gain from the use of this tool which, in our view, could not readily have been gained from the analysis of scanning electron micrographs or by studying conventional serial histological sections of similar stages of mouse embryonic development. The methodology has been developed as part of a large project to prepare a database of mouse developmental anatomy covering all stages from fertilisation to birth in order to allow the accurate spatial mapping of gene expression and cell lineage data onto the digital Atlas of normal mouse development. In this paper we show how this digital anatomical Atlas also represents a valuable teaching aid and research tool in anatomy.

Despite the fact that development of the human embryo heart is of considerable clinical importance, there is still disagreement over the process and the timing of events. It is likely that some of the conflicting accounts may have arisen from difficulties in describing and visualising 3-dimensional structures from 2-dimensional sections. To help overcome this problem and to improve our understanding of the development of the heart, we have devised techniques for the production of interactive 3D models reconstructed from serial histological sections of human embryos. Our method uses commercial software designed for the creation of 3D models and virtual reality environments. The ability to construct interactive visual images which both illustrate and communicate complex 3D information contributes to our understanding of the complex developmental changes occurring in embryogenesis.

This paper reviews recent work in radiological image registration and provides a classification of image registration by type of transformation and by methods employed to compute the transformation. The former includes transformation of 2D images to 2D images of the same individual, transformation of 3D images to 3D images of the same individual, transformation of images to an atlas or model, transformation of images acquired from a number of individuals, transformations for image guided interventions including 2D to 3D registration and finally tissue deformation in image guided interventions. Recent work on computing transformations for registration using corresponding landmark based registration, surface based registration and voxel similarity measures, including entropy based measures, are reviewed and compared. Recently fully automated algorithms based on voxel similarity measures and, in particular, mutual information have been shown to be accurate and robust at registering images of the head when the rigid body assumption is valid. Two approaches to modelling soft tissue deformation for applications in image guided interventions are described. Validation of complex processing tasks such as image registration is vital if these algorithms are to be used in clinical practice. Three alternative validation strategies are presented. These methods are finding application outside the original domain of radiological imaging.

The maturity of current 3D rendering software in combination with recent developments in computer vision techniques enable an exciting range of applications for the visualisation, measurement and interactive manipulation of volumetric data, relevant both for diagnostic imaging and for anatomy. This paper reviews recent work in this area from the Image Sciences Institute at Utrecht University. The processes that yield a useful visual presentation are sequential. After acquisition and before any visualisation, an essential step is to prepare the data properly: this field is known as ‘image processing’ or ‘computer vision’ in analogy with the processing in human vision. Examples will be discussed of modern image enhancement and denoising techniques, and the complex process of automatically finding the objects or regions of interest, i.e. segmentation. One of the newer and promising methodologies for image analysis is based on a mathematical analysis of the human (cortical) visual processing: multiscale image analysis. After preprocessing the 3D rendering can be acquired by simulating the ‘ray casting’ in the computer. New possibilities are presented, such as the integrated visualisation in one image of (accurately registered) datasets of the same patient acquired in different modality scanners. Other examples include colour coding of functional data such as SPECT brain perfusion or functional magnetic resonance (MR) data and even metric data such as skull thickness on the rendered 3D anatomy from MR or computed tomography (CT). Optimal use and perception of 3D visualisation in radiology requires fast display and truly interactive manipulation facilities. Modern and increasingly cheaper workstations (<$10000) allow this to be a reality. It is now possible to manipulate 3D images of 2563 at 15 frames per second interactively, placing virtual reality within reach. The possibilities of modern workstations become increasingly more sophisticated and versatile. Examples presented include the automatic detection of the optimal viewing angle of the neck of aneurysms and the simulation of the design and placement procedure of intra-abdominal aortic stents. Such developments, together with the availability of high-resolution datasets of modern scanners and data such as from the NIH Visible Human project, have a dramatic impact on interactive 3D anatomical atlases.

In muscle spindles of the cat, independent control of dynamic and static components of the response of the primary sensory ending to stretch is provided by separate motor inputs to the various kinds of intrafusal muscle fibre: dynamic axons (γ or β) to the bag1 fibres and static axons to the bag2 (typically γ only) and chain (γ or β) fibres. Nonlinear summation of separately evoked effects during combined stimulation of dynamic and static motor axons appears to be due to mutual resetting by antidromic invasion of separate encoding sites, leading to partial occlusion of the momentarily lesser response by the greater. The encoding sites are thought to be located within the primary ending's preterminal branches which from first-order level are normally segregated to the bag1 fibre and to the bag2 and chain fibres. Here we describe the analysis of a special case that arose in a histophysiological study which had shown that the degree of occlusion was related to the minimum number of nodes between the putative encoding sites. Three-dimensional reconstruction of the primary ending revealed that the terminals of one chain fibre were derived entirely from the first-order branch that supplied the bag1 fibre, including one terminal that was shared directly with the bag1 (sensory cross-terminal). The other first-order branch supplied the bag2 and remaining chain fibres as normal. The degree of occlusion seen during simultaneous stimulation of a dynamic β axon and a static γ axon indicated that the encoding sites were separated by both first-order branches. Schematic reconstruction of the motor innervation revealed that the static γ axon was most unlikely to have supplied the chain fibre which shared sensory terminals with the bag1, but that these fibres also shared a motor input with histological characteristics of β type. Ramp-frequency stimulation of the dynamic β axon at constant length evoked a driving effect which persisted after fatiguing the extrafusal component and was therefore explicable on the basis of the observed pattern of motor innervation, though the identity of the axon could not be conclusively proved. Individually, instances of shared sensory terminals and motor input of bag1 and chain fibres are rare in the cat; their combination in a single spindle with correlated physiology is described here for the first time. The observation is considered in relation to the importance of dynamic and static segregation in motor control, since it may imply that there is a lower limit to the degree of segregation that the developmental programme can provide.

The effect(s) of finite exposure(s) to 22.0 °C on embryo weight gain, 3rd tarsus length, transfer of lipid from the yolk and uptake of specific lipid components by the liver, were measured in the chick embryo. Embryo growth was completely suspended over the period of temperature reduction, but resumed unimpaired when the optimum temperature of 37.5 °C was restored. Two successive periods of embryonic exposure to 22.0 °C (36 h at d 3 followed by 24 at d 10) were additive in their effect on growth retardation. Embryos subjected to temperature reductions on d 3 of incubation displayed significant changes in lipid concentrations and polyunsaturated fatty acid profiles within the liver when measured at d 18 of incubation. Yolk lipid uptake was significantly reduced but not suspended by embryonic exposure to 22.0 °C. It is suggested that the observed suspension of embryonic development was the result of a temperature dependent reduction in metabolism according to a Q10∼2, which is typical of an ectothermic organism.

Apoptosis is a form of naturally occurring cell death during development and it is characterised by extensive DNA fragmentation. Apoptosis is easily detected in the gill epithelium of brown trout embryos in ultrathin sections (Rojo et al. 1997). Here we provide the first biochemical evidence for apoptosis in the gill epithelium of brown trout embryos, using in situ end-labelling of DNA breaks (Gavrieli et al. 1992). Embryos at d 57 of development as well as those at hatching, were processed to analyse the distribution of apoptotic cells in the gills. The extent of apoptosis revealed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling method technique is considerably greater than apoptosis detected by nuclear morphology. This method revealed that apoptosis was frequent at hatching, although it was also present during embryonic development. The presence and distribution of stained nuclei were different depending on the developmental stage. In embryos of 57 d, apoptotic flattened nuclei were dispersed in the gill epithelium, whereas at hatching, they were mainly grouped in the tips of the filaments and in the gill arches. TUNEL also revealed a distinct pattern of nuclear staining: at hatching, the intense staining covered the entire cell, but in embryos it was restricted to the nucleus. These results show the functional relevance of apoptosis at hatching, when apoptosis seems to be the unique process by which cell numbers in the gill epithelium are adjusted, in order to prepare for the new extrinsic conditions affecting the free-living life of alevins.

Expression of surfactant proteins SP-A, SP-B and the transcription factors TTF-1 and HNF-3β was identified by immunohistochemistry in the developing chicken. SP-B, a small hydrophobic peptide critical for lung function and surfactant homeostasis in mammals, was detected in the epithelial cells of parabronchi in embryonic chicken lung from the 15th day of incubation, prior to the onset of the breathing movements and was expressed at high levels in the posthatching chicken lung. SP-A, an abundant surfactant protein involved in innate defence of the mammalian lung, was detected in the chick embryo in subsets of epithelial cells in the mesobronchus, starting from d 15 and was detected in the posthatching chicken lung. The transcription factors hepatocyte nuclear factor 3β (HNF-3β) and thyroid transcription factor-1 (TTF-1), both regulators epithelial cell differentiation and gene expression in mammalian species, were detected at the onset of lung bud formation (d 4 of incubation) and throughout lung development. Abundant nuclear expression was detected in nuclei of respiratory epithelial cells of developing bronchial tubules for both transcription factors. In contrast to the surfactant proteins, expression of both TTF-1 and HNF-3β decreased markedly in posthatching chicken lung. The expression of SP-A and SP-B in chick lung demonstrates the conservation of surfactant proteins in vertebrates. The temporospatial pattern of TTF-1 and HNF-3β overlaps with that of SP-A and SP-B, supporting their potential roles in chick lung development and demonstrating the conservation of regulatory mechanisms contributing to gene expression in respiratory epithelial cells in vertebrates.

The angioarchitecture of the coeliac sympathetic ganglion complex (CGC) of the common tree shrew (Tupaia glis) was studied by the vascular corrosion cast technique in conjunction with scanning electron microscopy. The CGC of the tree shrew was found to be a highly vascularised organ. It normally received arterial blood supply from branches of the inferior phrenic, superior suprarenal and inferior suprarenal arteries and of the abdominal aorta. In some animals, its blood supply was also derived from branches of the middle suprarenal arteries, coeliac artery, superior mesenteric artery and lumbar arteries. These arteries penetrated the ganglion at variable points and in slightly different patterns. They gave off peripheral branches to form a subcapsular capillary plexus while their main trunks traversed deeply into the inner part before branching into the densely packed intraganglionic capillary networks. The capillaries merged to form venules before draining into collecting veins at the peripheral region of the ganglion complex. Finally, the veins coursed to the dorsal aspect of the ganglion to drain into the renal and inferior phrenic veins and the inferior vena cava. The capillaries on the coeliac ganglion complex do not possess fenestrations.

We have re-evaluated, using the anterograde tracer biocytin, supraspinal efferent projections from the parabrachial complex (PBN) to gain new information about the nature of its connections and nerve terminal patterns. We selectively injected biocytin into the 3 main regions of the nucleus (lateral PBN, medial PBN and Kölliker-Fuse nucleus). We observed distinct groups of ascending and descending fibres of different calibre from the PBN running throughout the brain and reaching many brain areas involved in the regulation of autonomic function. Here we detected labelled bouton-like terminals and fibres with en-passage varicosities. The ascending efferents from the lateral PBN mainly reached the reticular, raphe and thalamic nuclei, the zona incerta (ZI), central nucleus of the amygdala (CeA) and lateral area of the periaqueductal grey (PAG). Thin descending efferents reached the ventral region of the solitary tract nucleus (STN). The ascending efferents from the medial PBN were seen in the raphe nuclei, reticular nuclei, ventral and lateral areas of the PAG, thalamic nuclei, and in the medial and lateral nuclei of the amygdala. Descending efferents were seen in the STN and in some reticular nuclei. The ascending projections from the Kölliker-Fuse targeted the ventral area of PAG, CeA, ZI, lateral hypothalamic area, ventromedial thalamic nucleus and, with only a few terminals, the ipsi and contralateral reticular area. A large number of descending efferents reached STN, caudal and paragigantocellular reticular nuclei. The higher sensitivity of biocytin compared with other types of markers allowed us to determine more effectively the distribution, nature and extent of the supraspinal PBN connections. This suggested that in several nerve circuits the PBN probably plays a more important role than previously thought.

A macroscopic, microscopic and scanning electron microscope study was performed on the pathological bone changes of the mandibles of wild red deer (n=61) exhibiting severe dental fluorosis. The animals originated from a highly fluoride polluted area in Central Europe (Ore mountains and their southern foreland, Czech-German border region) and constituted 11.2% of the studied red deer sample (n=545) from this area. Pathologically increased wear and fracture of fluorosed teeth caused a variety of mandibular bone alterations, including periodontal breakdown, periostitis, osteitis and chronic osteomyelitis. As a further consequence of severe dental attrition, opening of the pulp chamber and formation of periapical abscesses were occasionally observed. In case of severe periodontal breakdown, loss of teeth from the mandibles was found. In addition to the inflammatory bone changes, the occurrence of osteofluorotic alterations was also diagnosed in the specimens with the highest bone fluoride concentrations (>4000 mg F−/kg dry wt). These changes comprised extended apposition of periosteal bone onto the mandibular cortex as well as deformation of the mandibular body, which was attributed to a fluoride-induced osteomalacia. The present study provided circumstantial evidence that, in addition to fluoride induced dental lesions, the occurrence of marked periodontal disease and tooth loss is an important factor responsible for a reduction of life expectancy in severely fluorotic wild red deer.

A class of primitive progenitor cells with high proliferative potential in vitro (HPP-CFC), has been identified in fetal liver and adult bone marrow both in murine and human systems. The kinetic properties of HPP-CFC2 and the more mature granulocyte-macrophage colony forming units (CFU-GM) derived from murine fetal liver on d13, d15 and d19 of gestation, newborn liver and neonatal liver on d3 and d8 postpartum have been evaluated and compared with the kinetic properties of these progenitor cell populations derived from adult bone marrow. The frequency of HPP-CFC2 in fetal liver was found to be greatest on d15 of gestation then subsequently declined in newborn and neonatal liver. Similarly, the highest proportion of HPP-CFC2 engaged in DNA synthesis (53±3%) was detected in d15 fetal liver. This proportion decreased to 13±2% in the liver 1 wk after birth, which is comparable to the number of HPP-CFC2 derived from adult BM which were in S-phase (10±1%). Production of CFU-GM was found to be greater in adult bone marrow than in either fetal or newborn liver. While the proportion of CFU-GM in S-phase was high in all 3 tissue samples, the greatest proportion of cycling CFU-GM (50±2%) was detected in d15 fetal liver. These results suggest that HPP-CFC2 derived from fetal liver are actively cycling while HPP-CFC2 derived from adult bone marrow are relatively quiescent. In contrast, a high proportion of CFU-GM derived from fetal, newborn liver and adult bone marrow are engaged in DNA synthesis.

Ultrasound of the calcaneus may be used as a cheap, ionising radiation-free and easy to use indicator of skeletal status, and hence of osteoporotic fracture risk. At present ultrasound is not widely used as it suffers from high precision errors. As ultrasound parameters are determined in part by bone mineral density (BMD), an increase in the accuracy and precision of BMD measurements should reduce the precision error associated with ultrasound measurements. The aim of this study was to define an anatomical site on the calcaneus at which accurate and precise measurements of BMD can be made. Ten dry calcanei and 10 cadaveric feet were scanned using a DXA scanner; 9 anatomically defined regions (1 cm2) were selected in the posterior part of the calcaneus for analysis. The centre of region 1 was positioned halfway along the line joining the anterior border of the calcaneal tubercle and the peak of the posterior superior tubercle, and the remaining 8 regions were placed around this central area. The BMD in these 9 regions was compared with the whole bone BMD and the variability of BMD within each of the 9 regions was measured. The reproducibility of the technique was assessed by taking 10 repeated measurements of 2 bone and 2 cadaveric specimens, each specimen being removed and repositioned between measurements. Region 1 was found to be the most representative of total BMD in cadaveric feet. This region also showed the least variability of BMD and consistently gave the lowest coefficients of variation in the reproducibility study both in the bone and the cadaveric specimens. This region is hence the most suitable site on the calcaneus for measuring absolute values of and changes in BMD. The surface position of region 1 was found to be consistently 5/9 along the line at 45° to the vertical, from the lateral malleolus to the heel. The identification of the surface location of region 1 relative to anatomical landmarks of the foot has enabled the same anatomical site to be measured in all subjects. This allows meaningful intersubject comparisons to be made. Preliminary data suggest that precision errors using ultrasound are also reduced when measurements are taken at this region of the calcaneus. The reduction in the precision error of ultrasound assessment of skeletal status may provide a cheap and safe way to identify individuals at risk from osteoporotic fracture.

The papillary layer of the oral part of the human soft palate was studied in 31 subjects of different age by means of histological, immunohistochemical and scanning electron microscopical methods. For scanning electron microscopy a new maceration method was introduced. Results determine epithelial thickness, height and density of connective tissue papillae and their 3-dimensional architecture inside the lining epithelium as well as the collagenous arrangement of the openings of the glandular ducts. The individual connective tissue papillae of the soft palate are compared with the connective tissue boundary on the other side of the oral cavity. The connective tissue plateaux carrying a variable number of connective tissue papillae were found to be the basic structural units of the papillary body. The function of the epithelial-connective tissue interface and the extracellular matrix arrangement in the lamina propria are discussed in order to promote the comparability of normal with pathologically altered human soft palates.

Changes in vaginal epithelium are known to occur during the normal oestrous or menstrual cycle and in ovariectomised animals in response to various hormones. However, vaginal changes due to exogenous gonadotrophins superimposed on the normal hormonal milieu as occurs in in vitro fertilisation programmes have not previously been demonstrated. Female rats were hyperstimulated with follicle stimulating hormone and human chorionic gonadotrophin prior to mating. Control mated animals were not injected. Vaginal tissue was collected at 4.5, 5.5 and 6.5 d after mating. Tissue was processed for light microscopy. Hyperstimulation by the exogenous gonadotrophins caused mucification, a decrease in the number of epithelial layers and an increase in the thickness of the epithelium on d 4.5 of pregnancy. The mitotic index of the epithelial cells was depressed in the hyperstimulated rats with respect to control animals. Exposure to high levels of oestradiol and an altered progesterone[ratio ]oestradiol ratio appear to have caused the changes in the vaginal epithelium.

During the course of a systematic study on the human vertebrobasilar arterial system (De Caro et al. 1995), our attention was drawn in one case to a strange morphology of the ventrolateral aspect of one side of the medulla. While initially our impression was that we were facing an actual malformation, review of the structure of the brainstem as described in most standard anatomical texts showed that it could correspond to the circumolivary fasciculus (Williams et al. 1995).

The subject was a 63-y-old man who had died from rupture of an atherosclerotic aneurysm of the abdominal aorta. The medical history was negative for neuropsychiatric pathology. Brain examination showed severe atherosclerosis of the cerebral arteries and hypoplasia of the initial segment of the right posterior cerebral artery. A recent infarct was found in the white matter of the right occipital lobe. After removal of the vertebrobasilar arterial system, the right pyramid showed a superficial groove extending from about 2 mm lateral to the foramen caecum obliquely downwards to the right anterolateral sulcus of the medulla, at the level of the superior margin of the inferior root of the hypoglossal nerve (Fig. 1a). Due to this groove, the lateral part of the pyramid formed a ridge that crossed the anterolateral sulcus of the medulla passing between the 2 main roots of the hypoglossal nerve and then continued, below the inferior pole of the olive, over the lateral region of the medulla ending at the level of the posterolateral sulcus (Fig. 1b). The general appearance was that of a small cord forming a loop around the anterior margin and the inferior pole of the olive, with a descending part (8 mm long), a horizontal part (6 mm long) and with a transverse diameter of 3 mm. After formalin fixation, the brainstem was cut in 8 transverse sections. At the level of the medulla the right pyramid appeared larger than the contralateral and the small lateral cord was separated from the main part of the pyramid by a thin whitish transverse stria (Fig. 1c). Macroscopic examination of the cerebellum showed nothing of relevance.