The aim of this study was to investigate the developmental characteristics of the mandibular condyle in sequential phases at the gene level using in situ hybridisation. At d 14.5 of gestation, although no expression of type II collagen mRNA was observed, aggrecan mRNA was detected with type I collagen mRNA in the posterior region of the mesenchymal cell aggregation continuous with the ossifying mandibular bone anlage prior to chondrogenesis. At d 15.0 of gestation, the first cartilaginous tissue appeared at the posterior edge of the ossifying mandibular bone anlage. The primarily formed chondrocytes in the cartilage matrix had already shown the appearance of hypertrophy and expressed types I, II and X collagens and aggrecan mRNAs simultaneously. At d 16.0 of gestation, the condylar cartilage increased in size due to accumulation of hypertrophic chondrocytes characterised by the expression of type X collagen mRNA, whereas the expression of type I collagen mRNA had been reduced in the hypertrophic chondrocytes and was confined to the periosteal osteogenic cells surrounding the cartilaginous tissue. At d 18.0 of gestation before birth, cartilage-characteristic gene expression had been reduced in the chondrocytes of the lower half of the hypertrophic cell layer. The present findings demonstrate that the initial chondrogenesis for the mandibular condyle starts continuous with the posterior edge of the mandibular periosteum and that chondroprogenitor cells for the condylar cartilage rapidly differentiate into hypertrophic chondrocytes. Further, it is indicated that sequential rapid changes and reductions of each mRNA might be closely related to the construction of the temporal mandibular ramus in the fetal stage.

Mutations in the gene for peripheral myelin protein 22 (PMP22) are associated with peripheral neuropathy in mice and humans. Although PMP22 is strongly expressed in peripheral nerves and is localised largely to the myelin sheath, a dual role has been suggested as 2 differentially expressed promoters have been found. In this study we compared the initial stages of postnatal development in transgenic mouse models which have, in addition to the murine pmp22 gene, 7 (C22) and 4 (C61) copies of the human PMP22 gene and in homozygous and heterozygous Trembler-J (TrJ) mice, which have a point mutation in the pmp22 gene. The number of axons that were singly ensheathed by Schwann cells was the same in all groups indicating that PMP22 does not function in the initial ensheathment and separation of axons. At both P4 and P12 all mutants had an increased proportion of fibres that were incompletely surrounded by Schwann cell cytoplasm indicating that this step is disrupted in PMP22 mutants. C22 and homozygous TrJ animals could be distinguished by differences in the Schwann cell morphology at the initiation of myelination. In homozygous TrJ animals the Schwann cell cytoplasm had failed to make a full turn around the axon whereas in the C22 strain most fibres had formed a mesaxon. It is concluded that PMP22 functions in the initiation of myelination and probably involves the ensheathment of the axon by the Schwann cell, and the extension of this cell along the axon. Abnormalities may result from a failure of differentiation but more probably from defective interactions between the axon and the Schwann cell.

This study was prompted by the prospect of transgenic pigs providing donor hearts for transplantation in human recipients. Autonomic innervation is important for the control of cardiac dynamics, especially in the conduction system. Our objective was to assess the relative distribution of autonomic nerves in the pig heart, focusing initially on the conduction system but addressing also the myocardium, endocardium and epicardium (see Crick et al. 1999). Quantitative immunohistochemical and histochemical techniques were adopted. All regions of the conduction system possessed a significantly higher relative density of the total neural population immunoreactive for the general neuronal marker protein gene product 9.5 (PGP 9.5) than did the adjacent myocardium. A similar density of PGP 9.5-immunoreactive innervation was observed between the sinus node, the transitional region of the atrioventricular node, and the penetrating atrioventricular bundle. A differential pattern of PGP 9.5-immunoreactive innervation was present within the atrioventricular node and between the components of the ventricular conduction tissues, the latter being formed by an intricate network of Purkinje fibres. Numerous ganglion cell bodies were present in the peripheral regions of the sinus node, in the tissues of the atrioventricular groove, and even in the interstices of the compact atrioventricular node. Acetylcholinesterase (AChE)-containing nerves were the dominant subpopulation observed, representing 60–70% of the total pattern of innervation in the nodal tissues and penetrating atrioventricular bundle. Tyrosine hydroxylase (TH)-immunoreactive nerves were the next most abundant neural subpopulation, representing 37% of the total pattern of innervation in the compact atrioventricular node compared with 25% in the transitional nodal region. A minor population of ganglion cell bodies within the atrioventricular nodal region displayed TH immunoreactivity. The dominant peptidergic nerve supply possessed immunoreactivity for neuropeptide Y (NPY), which displayed a similar pattern of distribution to that of TH-immunoreactive nerve fibres. Calcitonin gene-related peptide (CGRP)-immunoreactive nerves represented 8–9% of the total innervation of the nodal tissues and penetrating atrioventricular bundle, increasing to 14–19% in the bundle branches. Somatostatin-immunoreactive nerve fibres were relatively sparse (4–13% of total innervation) and were most abundant in the nodes, especially the compact atrioventricular node. The total pattern of innervation of the porcine conduction system was relatively homogeneous. A substantial proportion of nerve fibres innervating the nodal tissues could be traced to intracardiac ganglia indicative of an extensive intrinsic supply. The innervation of the atrioventricular node and ventricular conduction tissues was similar to that observed in the bovine heart, but markedly different to that of the human heart. It is important that we are aware of these findings in view of the future use of transgenic pig hearts in human xenotransplantation.

The immunological problems of pig hearts supporting life in human recipients have potentially been solved by transgenic technology. Nevertheless, other problems still remain. Autonomic innervation is important for the control of cardiac dynamics and there is evidence suggesting that some neurons remain intact after transplantation. Previous studies in the human heart have established regional differences in both general autonomic innervation and in its component neural subpopulations. Such studies are lacking in the pig heart. Quantitative immunohistochemical and histochemical techniques were used to demonstrate the pattern of innervation in pig hearts (Sus scrofa). Gradients of immunoreactivity for the general neural marker protein gene product 9.5 were observed both within and between the endocardial, myocardial and epicardial plexuses throughout the 4 cardiac chambers. An extensive ganglionated plexus was observed in the epicardial tissues and, to a lesser extent, in the myocardial tissues. The predominant neural subpopulation displayed acetylcholinesterase activity, throughout the endocardium, myocardium and epicardium. These nerves showed a right to left gradient in density in the endocardial plexus, which was not observed in either the myocardial or epicardial plexuses. A large proportion of nerves in the ganglionated plexus of the atrial epicardial tissues displayed AChE activity, together with their cell bodies. Tyrosine hydroxylase (TH)-immunoreactive nerves were the next most prominent subpopulation throughout the heart. TH-immunoreactive cell bodies were observed in the atrial ganglionated plexuses. Endocardial TH- and NPY-immunoreactive nerves also displayed a right to left gradient in density, whereas in the epicardial tissues they showed a ventricular to atrial gradient. Calcitonin gene-related peptide (CGRP)-immunoreactive nerves were the most abundant peptide-containing subpopulation after those possessing NPY immunoreactivity. They were most abundant in the epicardial tissues of the ventricles. Several important differences were observed between the innervation of the pig heart compared with the human heart. These differences may have implications for the function of donor transgenic pig hearts within human recipients.

Metalloenzymes have an important role in repair and regenerative processes in skin wounds. Demands for different enzymes vary according to the phase in the healing cascade and constituent events. Sequential changes in the concentrations of calcium, copper, magnesium and zinc were studied in the incisional wound model in the rat over a 10 d period. Copper levels remained low (<10 μg/g dry weight) throughout, but calcium, magnesium and zinc increased from wounding and peaked at about 5 d at a time of high inflammation, granulation tissue formation and epidermal cell proliferation. Metal concentrations declined to normal by 7 d when inflammation had regressed, re-epithelialisation of the wound site was complete and the ‘normalisation’ phase had commenced. Although the wound was overtly healed by 10 d, the epidermis was still moderately hyperplastic. In view of competitive binding of trace metals at membrane receptors and carrier proteins, the ratios or balance between these trace metals was examined and the significance is discussed. Using immunocytochemistry, we demonstrated increases in metallothionein immunoreactivity as an indication of zinc and copper activity in the papillary dermis and in basal epidermal cells near the wound margin 1–5 d after wounding. This is consistent with metalloenzyme requirements in inflammation and fibrogenesis. Calmodulin, a major cytosolic calcium binding protein was highest in maturing keratinocytes and in sebaceous gland cells of normal skin; it was notably more abundant in the epidermis near the wound margin and in re-epithelialising areas at a time when local calcium levels were highest.

The aim of this study was to examine the kinetics of neovascularisation of splenic autoimplants into the abdominal cavity after splenectomy in mice. Sixty-eight female Swiss mice were submitted to splenectomy. The spleen from each animal was sliced and the slices were implanted into the abdominal cavity. Groups of animals were killed after 1, 2, 3, 5, 7, 15, 21, 28, 42, 56, 70 and 84 d. Fluorescent polystyrene microspheres were injected via the orbital venous plexus before killing and the splenules were removed 5 min later for light and electron microscopy. Mesenteric blood vessels were injected with coloured latex to study the origin of the nutrient vessels. Three days after the implant the microspheres were observed at the periphery and then migrating to the internal parts of the implant in the subsequent days. The blood supply to the implants originated from branches of the splenic, short gastric, mesenteric and gastroepiploic arteries. It is concluded that revascularisation of splenic autografts proceeds centripetally, starting as early as 3 d after implantation.

The results obtained using morphometric variables which describe fin ray regeneration patterns are reported for individual fin ray amputations in the goldfish (Carassius auratus) and zebrafish (Brachydanio rerio). Classical and updated experiments are compared to verify previous morphogenetic models of cell tractions (Oster et al. 1983) or epidermis-mesenchyme induction (Saunders et al. 1959) applied to the limb of other vertebrates. Position-dependent patterns within the fin of Carassius auratus are analysed under a comparative protocol using morphometric methods. Conditions in which the apical epidermis is separated from blastema may differentiate small fin rays, thus suggesting this epidermis is involved in blastemal formation. Blastemal cells differentiating as lepidotrichia forming cells (LFCs) may also be related to morphological changes in covering epidermis. Long-range interactions from neighbouring fin ray blastemas or short-range interactions within the blastema, may be postulated through the analysis of segmentation.

Vein-to-artery graft surgery is used widely to by-pass arterial stenoses, but such grafts can fail over a prolonged period as a result of excessive neointimal hyperplasia causing thrombosis and graft occlusion. It has been suggested that neointimal hyperplasia, in vein grafts, is a result of the vessel wall adapting to the higher intraluminal pressure of the arterial circulation, compared with the venous circulation. Autologous artery grafts have been used to bypass arterial stenoses. Initially it was assumed that donor artery segments would not develop neointimal hyperplasia as they are already adapted to the arterial circulation but this is not so. In this study we postulated that surgical or postsurgical trauma was the cause of neointimal hyperplasia in autologous artery-to-artery grafts. In addition, as artery grafts are pre-adapted to the arterial circulation, autologous artery-to-artery grafts in hypertensive rats should develop similar levels of neointimal hyperplasia as seen in normotensive rats. Artery-to-artery grafts were placed in a series of 20 spontaneously hypertensive rats (SHR). In a separate series of sham grafting experiments the effects of anoxia and clamp trauma were assessed in SHR and WKy normotensive control rats. Finally, clamping, anoxia and anastomosis trauma were assessed in a similar series of rats. In the artery-to-artery graft series there was no difference in neointimal thickness between the SHR and that previously reported for normotensive rats. Minimal neointimal hyperplasia was demonstrated in the sham grafted series of rats and only slightly more in the single anastomosis series. It was only in the full grafting procedure that considerable neointimal hyperplasia developed. These data demonstrate that neointimal hyperplasia in artery-to-artery grafts is not exacerbated by the hypertension. In addition, trauma appears to be the initiator of neointimal hyperplasia and the extent of trauma correlates with the degree of neointimal hyperplasia.

The human vomeronasal organ is of interest because of its potential role in sex pheromone detection. Due to the scarcity of early human material, studies of its development have concentrated on fetal rather than embryonic stages. The availability of embryonic specimens in the Walmsley Collection has enabled us to study the development of the vomeronasal organ (VNO) in human embryos between Carnegie Stages 17 and 23. Embryos at Carnegie Stage 17 or below showed no evidence of a VNO. One embryo with characteristics intermediate between Carnegie stages 17 and 18 was the earliest to show evidence of a VNO, in the form of a shallow indentation. All embryos at Carnegie Stages 18 or later had VNOs. Three-dimensional computer reconstructions were made of the VNO in each specimen where this was possible. This in part depended on the plane of section. The total volume and lumen volume were measured from these reconstructions and the volume of the vomeronasal epithelium was calculated by subtraction. A generally consistent increase in total volume and epithelial volume was observed with increasing developmental stage. The lumen contributed rather little to the total volume at these stages.

The streptozotocin (STZ)-diabetic rat, the most commonly employed model of experimental diabetic neuropathy, is characterised by a reduction in nerve conduction velocity, pain threshold and blood flow. Whether or not structural abnormalities underlie these functional abnormalities is unclear. 10 adult male Sprague–Dawley STZ-diabetic rats (diabetes duration 27 d) and 10 age-matched (23 wk) control animals were studied. Motor nerve conduction velocity (m s−1) was significantly reduced in diabetic (41.31±0.8) compared with control (46.15±1.5) animals (P<0.001). The concentration of sciatic nerve glucose (P<0.001), fructose (P<0.001) and sorbitol (P<0.001) was elevated, and myoinositol (P<0.001) was reduced in diabetic compared with control animals. Detailed morphometric studies demonstrated no significant difference in fascicular area, myelinated fibre density, fibre and axon areas as well as unmyelinated fibre density and diameter. Endoneurial capillary density, basement membrane area and endothelial cell profile number did not differ between diabetic and control animals. However, luminal area (P<0.03) was increased and endothelial cell area (P<0.08) was decreased in the diabetic rats. We conclude there is no detectable structural basis for the reduction in nerve conduction velocity, pain threshold or blood flow, observed in the streptozotocin diabetic rat.

A new stereological method is proposed which combines vertical slice projections with the fractionator to estimate the total capillary length in a skeletal muscle. The method was demonstrated on the soleus muscle of a Wistar rat. The implementation required capillary highlighting, tissue sampling, and data acquisition in the form of intersection counts between capillary projections and cycloid test lines. The capillaries were demonstrated using vascular perfusion (with gelatine) of the hind leg of the rat. The sampling procedure followed the fractionator design, namely a multistage systematic sampling design with a known sampling fraction at each stage. To make the design unbiased, vertical slices were used; for efficiency, the vertical axis was chosen parallel to the main axis of the muscle. As prescribed to avoid bias, the cycloid test lines were superimposed on the slice projections, viewed under the light microscope, with their minor axes normal to the vertical axis. The estimation precision was compared for different sampling and subsampling fractions. The proposed method was globally highly efficient, unbiased, and easy to implement.

The frequency and distribution of 11 endocrine cell populations were studied in the intestine of differently aged buffalo, grouped on the basis of diet: 2-d-olds (suckling), 5-mo-olds (weaning) and 5-y-olds (ruminant adult diet). The endocrine cell populations were identified immunocytochemically using antisera against 5-hydroxytryptamine (5-HT), somatostatin, gastrin, cholecystokinin (CCK), COOH-terminal octapeptide of gastrin/CCK, neurotensin, motilin, gastric inhibitory polypeptide (GIP), secretin, glucagon/glicentin (GLU/GLI) and polypeptide YY (PYY). In adult buffalos the regional distribution of endocrine cells is similar to that of other adult ruminants. During postnatal development, these cell types showed the following changes in their frequency and distribution: (1) 5-HT, neurotensin and gastrin/CCK immunoreactive cells (i.c.) showed a decrease in frequency with age; (2) somatostatin i.c. frequency remained stable with age; (3) motilin, GIP, secretin and CCK i.c. showed a slight increase in frequency with age; (4) GLU/GLI and PYY i.c. decreased in frequency with age in the small intestine, caecum and proximal colon and an increase in frequency in the rectum. It was hypothesised that the endocrine cell types, whose presence and localisation is substantially stable in all examined ages, probably contain substances that are strictly necessary for intestinal function. In contrast the hormones contained in the cell populations that decreased with age, are probably involved in physiological needs during the milk and weaning diet or play a role in intestinal growth.

Elastic fibres, which are intimately associated with collagen, a major component of the urethra, have been assumed to contribute to the resting urethral closure pressure. The Miller stain for elastin was used to demonstrate elastic fibres in cryostat sections of guinea pig bladder base, vesicourethral junction (VUJ) and urethra. Computerised image analysis was employed to objectively quantify these fibres. Both male and female guinea pigs showed significantly greater amounts of circularly disposed elastic fibres in the VUJ than in the other 2 regions examined. This particular disposition of fibres may be responsible for imparting resiliency and plasticity to the VUJ, allowing it to distend and recoil repeatedly in response to urine outflow. Furthermore, the elastic fibres may be partly responsible for the passive occlusive force in this region. Elastic fibres in the distal urethra were not quantified because of their relative paucity. Sagittal sections of the urethra revealed a mass of longitudinally arranged elastic fibres localised almost exclusively within the mucosa, submucosa and longitudinal smooth muscle layer. Functionally, this arrangement may exist to facilitate urethral length changes that occur in micturition.

The presence of collagen in enameloid distinguishes it clearly from true enamel, but little is known about the phylogenetic relationship between these 2 tissues. It has previously been reported that amelogenins are the principal proteins of the enamel matrix, that type I collagen and chondroitin sulphates are the predominant matrices in dentine, and that amphibian and reptilian aprismatic enamels contain no sulphated glycoconjugates, although certain sulphated substances are secreted into mammalian prismatic enamel during matrix formation. The larval urodele (Triturus pyrrhogaster) teeth are known to be composed of enameloid, dentine, and enamel-like tissue. To characterise the tooth matrices, the localisation of amelogenin-like proteins, type I collagen, and sulphated glycoconjugates was investigated. Chondroitin sulphates and fine fibrils immunoreactive for type I collagen were elaborated as the enameloid matrix inside the dental basement membrane. After the matrix had been deposited in full thickness, coarse collagen fibrils also immunoreactive for type I collagen and chondroitin sulphates were deposited below as the first dentine matrix. Further, enamel-like matrix with no collagen fibrils or sulphated glycoconjugates but strongly immunoreactive for amelogenins was deposited on the dentine. Although no immunolabelling for amelogenins was found over the enameloid matrix, at least at the formation stage, the zone of coarse collagen fibrils of dentine was partially immunoreactive as observed in mammalian mantle dentine. From the ontogeny and matrix constituents of larval urodele teeth, it is suggested that enameloid is originally a dentine-like tissue.

Na+/H+ exchange has been implicated in models of ion transport across the branchial epithelium of marine and freshwater fishes. In this preliminary study, we present immunohistochemical data using a polyclonal antibody raised against NHE3 which show NHE3-like immunoreactivity (IR) in the gills from a freshwater and a marine teleost species. In both species, branchial epithelial cells demonstrating NHE3-like IR were localised predominantly to the junction between the filament and the secondary lamellae. However, there was a marked difference in the morphology of the NHE3-like immunoreactive epithelial cells between the species. This morphological difference between the species suggests functional differences in the exchanger, which may be related to marine versus freshwater environments.

Variations in the arterial pattern of the upper limb are common and have been reported by several investigators (Fuss et al. 1985; Poteat, 1986; Tountas & Bergman, 1993; Rodriguez-Baeza et al. 1995). These variations are often associated with anomalies in the arrangement of the nerves of the brachial plexus (Miller, 1939; Lengele & Dhem, 1989). The presence of a superficial brachial artery (Schwyzer & De Garis, 1935; Skopakoff, 1959; Fuss et al. 1985) and the usual pattern of its branching in the upper arm or forearm have also been reported (McCormack et al. 1953; Keen, 1961; Karlson & Niechalev, 1982; Lippert & Pabst, 1985; Rodriguez-Baeza et al. 1995). The great variability of this arterial pattern may be attributed to the failure of regression of some paths of the embryonic arterial trunks (Tountas & Bergman, 1993; Rodriguez-Baeza et al. 1995).

The aim of the present report is to describe the concomitant appearance of 3 unusual variations in the same upper limb of a male cadaver. In this arm: (1) a superficial brachial artery terminated its course by dividing into 3 branches at the cubital fossa; (2) the definitive brachial artery had an unusual origin; and (3) the ulnar nerve was abnormally formed from 2 roots. This novel variation is compared with other anatomical variations in the arterial supply of the upper limb. In a series of routine dissections of 100 embalmed human cadavers the following variations were observed in the right upper limb of a male subject.

Anatomical Society of Great Britain and Ireland

Future meetings

5–7 January 2000. Royal Holloway College, University of London. This will include a symposium on phenotypic changes in epithelial development.

24–26 July 2000. Department of Anatomy, University of Cambridge; this will be a tripartite meeting with the Anatomische Gesellschaft and the Nederlandse Anatomen Vereniging. This will include a symposium on the new anatomical basis of the emergence of behaviour, and one entitled ‘Anatomy: the challenges for the 21st century.’