In the early stages of embryonic development, many growth-promoting molecules must be provided by the maternal system. The molecules involved in growth processes may be either hormones or growth factors, or molecules that interact with such factors. The pregnancy related hormone, prolactin (PRL, MW 23 kDa) has been implicated in the control of embryonic growth. The growth-promoting potential of PRL and its species-specificity was investigated by culturing 9.5 d rat embryos in vitro for 48 h in depleted serum in the presence and absence of PRL from 3 different species. The growth-supporting capacity of the serum was reduced by removal of low molecular weight molecules by prolonged filtration of the serum using filters with a molecular weight exclusion of 30 kDa. This method provided a ‘semidefined’ medium (retenate) in which embryonic growth and development was significantly reduced, demonstrating that the low molecular weight fraction of serum may contain some growth-promoting factors. Addition of PRL (0.4–25.6 ng/ml) from different species (human, sheep and rat) to retenate significantly improved embryonic growth and development, suggesting that the developing embryo may utilise PRL. Amongst PRLs, rat PRL was found to be active at much lower concentrations than either of the other molecules, and human PRL had more effect in low concentrations than sheep PRL suggesting a species-specificity for this hormone. It may be that the PRL receptors of the rat embryos have greater affinity for the rat hormone as different responses for hormones from different species have been shown. These findings suggest that embryos may be able to utilise maternally derived PRL during organogenesis.

Sepsis stimulates an increase in the number and activity of mononuclear phagocytes in systemic host-defence organs. The present study was conducted to define the ultrastructural and cytochemical characteristics of the mononuclear phagocytes that sequester in the lung microvasculature of septic rats. Fourteen rats were challenged with a single intraperitoneal injection of saline (0.5 ml/100 g), E. coli (2×107/100 g) or glucan (4 mg/100 g), and euthanased 2, 4, or 7 d later. The lungs were inflation fixed and processed for transmission electron microscopy. Cellular morphology was used to identify the intravascular mononuclear phagocytes and acid phosphatase (AcPase) expression was monitored as an index of cellular differentiation and activation. Control rats contained a limited number of monocytes in the pulmonary vasculature. In contrast, large numbers of activated mononuclear phagocytes were seen in the microvasculature within 48 h of treatment with either microbial product. The recruited pulmonary intravascular mononuclear phagocytes (PIMP) exhibited AcPase-reactive Golgi complexes, accumulation of secretory vesicles and other features of cell activation consistent with enhanced biosynthetic activity. Subsequent electron microscopy, conducted 4 and 7 d posttreatment, suggested that a progressive decline in the number and activity of PIMPs then occurred. In order to quantify the sepsis-induced accumulation of AcPase-positive PIMP, the experimental challenges were repeated in 11 rats and, 48 h later, tissue samples were evaluated by light microscopy for tartrate-insensitive acid phosphatase. Control rats exhibited 0.148±0.107 AcPase-positive PIMP/alveoli. E. coli and glucan challenged animals exhibited significant (P<0.01) increases in AcPase-positive mononuclear phagocytes, with 0.782±0.073 and 0.636±0.170 PIMP/alveoli respectively. The results demonstrate that focal sepsis stimulates a significant, but transient, recruitment of activated mononuclear phagocytes into the rat pulmonary microvasculature.

Grafts of living or freeze-killed freshly dissected colonic smooth muscle from young inbred Fischer rats were implanted into the corpus striatum of adult Fischer rats. Sections of brain were examined electron microscopically 3 and 6 wk after implantation. At both times, living grafts were vascularised and contained healthy differentiated smooth muscle cells, fibroblasts, interstitial cells of Cajal and some macrophages. Large bundles of small nonmyelinated axons, identified as CNS axonal sprouts, could be observed in the brain at and near the interface between the living smooth muscle and the CNS tissue. Bundles of regenerating CNS axons, often associated with astrocyte processes, had grown into the grafts. Some axons within the grafts had matured, enlarged and become myelinated by oligodendrocyte processes or Schwann cells. In some cases, smooth muscle cells were observed in close and intricate association with axons. In contrast to the living grafts, grafts of freeze-killed smooth muscle, examined 3 and 6 wk after implantation, contained macrophages, fibroblasts, collagen and large amounts of cellular debris, but no living muscle cells, astrocytes or Schwann cells. The striatal neuropil around freeze-killed grafts did not contain large bundles of CNS axonal sprouts and bundles of axons were not observed within the freeze-killed graft. This study demonstrates that cells from the smooth muscle layers of the colon, in the absence of myenteric ganglia, can stimulate a vigorous regenerative response from CNS axons when implanted into the corpus striatum of adult rats.

Autologous vein grafts are employed extensively to bypass stenoses in the arterial circulation. More recently arterial segments have been used for such bypass surgery. In this study the adaptation of regenerating vascular tissues in experimental autologous artery grafts (4 mm long and 1 mm in diameter) in 20 adult male Wistar rats was analysed. At 1, 2, 4, 8 and 16 wk after insertion, 4 grafts per time interval were removed, processed for high resolution light microscopy and the thicknesses of the media and neointima, as well as the area fractions of smooth muscle cells, were analysed morphometrically. All grafts were reendothelialised by 2 wk. Neointimal hyperplasia (a subendothelial layer of smooth muscle cells) developed in all grafts and reached its maximal thickness (40.4±4.7 μm) at 2 wk. The area fraction of smooth muscle cells in the neointima of the artery grafts did not change significantly at any time from 2 to 16 wk. The media underlying the neointima of the artery grafts remained relatively constant throughout the 16 wk duration of the experiment. Whilst the total wall thickness of the grafts reduced significantly between 2 and 4 wk after insertion, at all times the grafts were thicker than the host artery.

Vascular sympathetic neuroeffector associations have been examined in rat iris arterioles using serial section electron microscopy and reconstruction techniques. Examination of random sections showed that, of all profiles of varicosities (199) seen to lie closer than 4 μm to vascular smooth muscle cells, only a small proportion (29/199) were found in close association with vascular smooth muscle cells, where adjacent membranes were separated by less than 100 nm. However, serial section examination, from intervaricose region to intervaricose region, of 79 varicosities similarly observed lying within 4 μm of vascular smooth muscle cells showed that 54 formed close associations with vascular smooth muscle cells. In serial sections, all these varicosities were also closely associated with melanocytes and of the 25 remaining varicosities, 22 formed close associations with melanocytes alone, whilst 3 did not come into close association with any effector cell. The increased observation of close associations with vascular smooth muscle cells in serial sections, compared with random sections, is consistent with the demonstration that the area of contact only occupies, on average, a small percentage (5%) of the total surface area of the varicosity as seen in the 3-dimensional reconstructions. In both random and serial sections, close associations were observed between varicosities and vascular smooth muscle cells or melanocytes irrespective of whether fibres were present singly or in small nerve bundles. Three-dimensional reconstruction of associations of varicosities and vascular smooth muscle cells demonstrated several common features, such as accumulations of synaptic vesicles and loss of Schwann cell covering at the region of membrane facing the effector cell. The similarity in the appearance of the neuroeffector association seen in this study and those described in previous studies provides evidence for the existence of a common sympathetic neuroeffector association, irrespective of the receptor subtype involved in neurotransmission.

A spectrum of limb abnormalities ranging from adactyly, syndactyly, acrosyndactyly to nail hypoplasia was encountered in mouse embryos subjected to amniotic sac puncture at the corresponding gestational stage when human chorionic villus sampling (cvs) would normally be performed clinically. Previous skeletal studies revealed that, apart from the occasional incidence of fusion of 2 distal phalanges, syndactyly usually only affected the soft tissues within the interdigital spaces. A similar situation was also observed in cases of adactyly; while the skeletal elements of the digits were present, the soft tissues in the interdigital spaces failed to separate. A transient period of bradycardia is induced, possibly secondary to compression of the embryo by the extraembryonic membranes and uterine muscles following amniotic sac puncture. These factors, we believe, produce temporary hypoxia/ischaemia of the distal extremities, and may lead to the modification of the interdigital mesenchymal tissues within the autopods. In order to investigate the mechanism(s) underlying soft tissue syndactyly, limbs recovered at 0.5, 4, 8, 12, 24, or 36 h following amniotic sac puncture (ASP) were examined histologically. Vascular disruption in the form of localised areas of haemorrhage, vascular dilatation and congestion and the presence of fluid-filled cavities occurred in relation to the marginal vein and vascular plexus in the interdigital spaces. It is hypothesised that this interfered with the normal equilibrium of the preset programs of mitosis/cell death and apoptosis within the mesenchymal cells of the interdigital spaces. Apoptosis in these areas was inhibited in the majority of the experimental limbs analysed 4 h after ASP. Instead of undergoing necrosis/apoptosis, increased mitotic activity was usually observed from 8 h following ASP at the sites where apoptosis would normally be expected to be seen. The aberrant fate of the interdigital mesenchyme following ASP and the underlying mechanism(s) involved are discussed, as is the critical importance of an adequate vascular supply to the interdigital spaces during the morphogenesis of the autopod. We believe that this report contributes to understanding the mechanism(s) which lead to syndactyly following ASP, and the limb defects occasionally seen following cvs when this is undertaken during early gestation.

In order to delineate the effects of death on the immunofluorescence of autonomic nerves supplying the human ventricular myocardium, we studied percutaneous myocardial samples obtained postmortem from 5 individuals within 3 h of death. Subsequent samples were obtained daily from the same individuals up to a total of 5–11 d. The antibodies employed included those against protein gene product 9.5 to demonstrate nervous tissue, dopamine β-hydroxylase and tyrosine hydroxylase to reveal catecholaminergic neural tissue and neuropeptide Y. An indirect immunofluorescence technique using the avidin-biotin method was employed. The density of myocardial protein gene product 9.5 immunoreactive nerves declined on the 7th day, and became markedly diminished by the 11th day. Immunoreactive dopamine β-hydroxylase nerves decreased on the 5th day, and were difficult to idenitify by the 9th day. The density of tyrosine hydroxylase and neuropeptide Y containing nerves rapidly diminished on the 3rd and 4th days, and became undetectable by the 7th and 8th days, respectively. The present results indicate that, depending on the type of antibodies used, immunohistochemical techniques can be used on human hearts obtained up to within 6 d of death to study cardiac innervation.

The objective of this paper was to analyse sex differences of the thickness of the subchondral mineralised tissue zone (SMZ), and to find out whether systematic changes of SMZ thickness are associated with naturally occurring, non-full-thickness cartilage lesions of human patellae. In 32 methyl-methacrylate-embedded specimens (16 normal, 8 with focal medial, and 8 with lateral lesions) the SMZ thickness was determined, using a binocular macroscope and an image analysing system. In each case, the thickness distribution was reconstructed throughout the entire joint surface. The maximal and mean SMZ thicknesses were significantly higher in males than in females (P<0.01). In normal patellae and those with lateral lesions, the thickness was significantly thicker laterally than medially (P<0.05), but it was not in specimens with medial damage. Patellae with medial damage exhibited a significantly lower total mean and lateral mean (P<0.05). A lower SMZ thickness was found directly beneath medial lesions than beneath lateral ones, but the local thickness was always in the range of that observed in normal specimens. We conclude that differences of patellar SMZ thickness exist between males and females. Naturally occurring cartilage lesions appear, however, not to be associated with local changes of SMZ thickness, but they may be associated with an altered regional distribution pattern within the joint surface.

Many studies demonstrate that differentiation of certain sensory receptors during development is induced by their nerve supply. Thus the navigational accuracy of pioneering fibres to their targets is crucial to this process. The special gustatory elements of the facial and glossopharyngeal nerves are used extensively as model systems in this field. We examined the chorda tympani, the gustatory component of the facial nerve, to determine the precise time course of its development in mice. The transganglionic fluorescent tracer DiI was injected into the anterior aspect of the mandibular arch of fixed embryos aged between 30 and 50 somites (E10–E12). It was allowed to diffuse retrogradely via the geniculate ganglion to the brainstem for 4 wk, before the distribution of DiI was determined using confocal laser scanning microscopy. Geniculate ganglion cells were first labelled at the 34 somite stage (E10). Pioneering chorda tympani fibres that arise from these cells passed peripherally and followed an oblique course as they grew towards the mandibular arch. At the 36 somite stage (E10.5), the peripheral component followed an intricate postspiracular course and passed anteriorly to arch over the primitive tympanic cavity, en route to the lingual epithelium. From the 36 to 50 somite stages (E10.5–E12), it consistently traced in the fashion of a ‘U’ bend. The central fascicle also traced at the 36 somite stage (E10.5) and just made contact with the brainstem. At the 40 somite stage (E11), the central fibres clearly chose a route of descent into the spinal trigeminal tract and branched into the solitary tract. Pioneering chorda tympani fibres contact the lingual epithelium when the target is primordial. The lingual epithelium may be a source of a neurotropic factor that attracts peripheral chorda tympani fibres to the sites of putative papillae. However, the chorda tympani is probably not a vital influence on the subsequent differentiation of gustatory papillae, since the papillae are elaborated 5 d later at E15 in murine embryos. The early morphology of the nerve is true to the amniote vertebrate phenotype.

The purpose of this study was to quantify and map the gross fibre architecture of the cranial dura mater (CDM) using small angle light scattering (SALS). In SALS, HeNe laser light is passed through the tissue, and the resultant scattering pattern is analysed to determine the preferred fibre direction and degree of orientation. The dura mater was found to be a complex structure with fibre orientations ranging from highly aligned to nearly random. In the temporal region, 80% of the samples (n=20) were found to have regions composed of highly oriented fibres with a mean fibre direction of 6.3°±0.8° with respect to the sagittal plane (i.e. almost parallel to the superior sagittal sinus). These highly aligned regions were found in symmetric anatomical locations about the median sagittal sinus and had similar fibre orientations over both hemispheres. Although our sample size was small, we found that the size of the symmetric regions, which covered 14.4±1.6% of the total CDM area, was not influenced by subject's age or sex. The presence of these highly oriented fibre regions in CDM may be due to mechanical forces exerted on dura mater during its development. These forces may have induced realignment of the collagen fibres in the direction of tensile pull, although the exact basis for the unique gross fibre architecture of CDM remains unknown.

The presence of a cholinergic innervation of arterioles within the gut wall is suggested by pharmacological studies of nerve mediated vasodilatation, but attempts to identify nerve cells that give rise to cholinergic vasodilator fibres have yielded discrepant results. In the present work, antibodies to the vesicular acetylcholine transporter protein (VAChT) were used to investigate the relationships of immunoreactive nerve fibres to submucosal arterioles. Comparison was made with cerebral arteries, which are known to be cholinergically innervated. Double labelling immunohistochemical techniques revealed separate VAChT and tyrosine hydroxylase (TH) immunoreactive (IR) fibres innervating all sizes of arteries of the submucosa of the stomach, ileum, proximal colon, distal colon and rectum as well as the cerebral arteries. Arterioles of all digestive tract regions had greater densities of TH-IR innervation than VAChT-IR innervation. In the ileum, double labelling for VAChT-IR and VIP-IR or calretinin-IR showed more VAChT-IR than either VIP-IR or calretinin-IR fibres. Calretinin-IR and VAChT-IR were colocalised in a majority of calretinin-IR axons, but VIP-IR and VAChT-IR were not colocalised. All calretinin-IR nerve cells in submucous ganglia were immunoreactive for choline acetyltransferase, but only 1–2% of VIP-IR nerve cells were immunoreactive. Extrinsic denervation of the ileum did not alter the distribution of VAChT-IR fibres, but it eliminated TH-IR fibres. Removal of myenteric ganglia (myectomy) did not alter the distribution of fibres with VAChT or TH-IR. This work thus provides evidence for cholinergic innervation of intrinsic arterioles throughout the digestive tract and indicates that the fibres in the small intestine originate from submucosal nerve cells.

This report deals with the as yet undetermined issue of whether cell-surface associated microtubules in certain cochlear epithelial cells are centrosomally nucleated and subsequently migrate to microtubule-capturing sites located at the surface regions in question. Alternatively, the cells may possess additional nucleating sites which are noncentrosomal and surface-associated. These alternative possibilities have been investigated for highly polarised epithelial cells called supporting cells in the mouse and guinea pig organ of Corti using antibodies to pericentrin and γ-tubulin. There is substantial evidence that both proteins are essential components of microtubule-nucleating sites in cells generally. Each mature supporting cell possesses a large microtubule array that is remotely located with respect to its centrosome (more than 10 μm away). The antibodies bind to a cell's centrosome. No binding has been detected at 2 other microtubule-organising centres that are associated with the ends of the centrosomally-remote microtubule array while it is being constructed. Such arrays include thousands of microtubules in some of the cell types that have been examined. If all a cell's microtubules are nucleated by its centrosome then the findings reported above imply that microtubules escape from the centrosomal nucleating site and migrate to a new location. Furthermore, capture of the plus and minus ends of the errant microtubules is taking place because both ends of a centrosomally-remote microtubule array are attached to sites that are precisely positioned at certain cell surface locations. Minus ends are locating targets with an exactitude comparable to that which has been demonstrated for plus ends in certain cell types. These cells apparently operate a single control centre strategy for microtubule nucleation that is complemented by precise positioning of plus and minus end-capturing sites at the cell surface.

In tonsils of the soft palate of pigs high endothelial venules (HEV) occur throughout the parafollicular region. In some regions of the venules the endothelial lining is uniformly high; in other areas, mainly the nuclear region of the endothelial cell protrudes into the lumen and the cytoplasmic region is attenuated. In this portion of the venule endothelial cells may traverse the lumen of the vessel to form intravascular bridging processes. The endothelial cells are characterised by pinocytotic vesicles, many surface microvilli, cytoplasmic processes and local cell-cell contacts with adjacent endothelial cells. These specialised HEV may play a role in the unusual pattern of lymphocytes from tonsillar tissue into blood. In the tonsils of the soft palate of pigs, HEV may mediate both lymphocyte migration to and from the lymphoid tissue. Further investigation is required to determine the importance of these 2 migration pathways.

The Summer meeting of the Anatomical Society of Great Britain and Ireland was held at the Anatomy Department, Edinburgh University from 15 to 17 July 1997. It included a symposium on ‘Computer Modelling for Anatomists and Clinicians’ on Wednesday 16 July. The following are abstracts of communications and demonstrations presented at the meeting.

Anatomical Society of Great Britain

15–18 April, 1998. Joint meeting with the Nederlandse Anatomen Vereniging in Rolduc, Netherlands, in cooperation with the South African Anatomical Society.

24–26 July, 2000. University of Cambridge.

International Society for the Study of the Lumbar Spine

MacNab/Larocca Research Fellowship Award. Deadline: March 1, 1998

1998 Smith & Nephew Travelling Fellowship Award. Deadline April 1, 1998.