The distribution of mRNAs and antigens of tissue type (t) and urokinase type (u) plasminogen activators (PA) plus their corresponding inhibitors, type-1 (PAI-1) and type-2 (PAI-2) were studied in human and rhesus monkey placentae by in situ hybridisation and immunocytochemistry. Specific monkey cRNA and antibodies against human tPA, uPA, PAI-1 and PAI-2 were used as probes. The following results were obtained. (1) All the molecules tPA, uPA, PAI-1 and PAI-2 and their mRNAs were identified in the majority of the extravillous cytotrophoblast cells of the decidual layer between Rohr's and Nitabuch's striae and in cytotrophoblast cells of the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (2) Expression of uPA and PAI-2 was noted in villous trophoblast whereas tPA and PAI-1 were mainly concentrated where detachment from maternal tissue occurs. (3) No expression of tPA, uPA, PAI-1 and PAI-2 was observed in the basal plate endometrial stromal cells, chorionic plate connective tissue cells, septal endometrial stromal cells or villous core mesenchyme. (4) The distribution of probes observed following in situ hybridisation is generally consistent with the immunofluorescence pattern of the corresponding antigens and no significant interspecies differences were noted. It is possible that both decidual and extravillous trophoblast cells of placentae of human and rhesus monkey are capable of producing tPA, uPA, PAI-1 and PAI-2 to differing extents. Coordinated expression of these genes in the tissue may play an essential role in the maintenance of normal placentation and parturition. The differences in distribution we observed are consistent with the suggestion that coordinated expression of tPA and its inhibitor PAI-1 may play a key role in fibrinolytic activity in the early stages of placentation and separation of placenta from maternal tissue at term. On the other hand, uPA with its inhibitor PAI-2 appears mainly to play a role in degradation of trophoblast cell-associated extracellular matrix, and thus may be of greatest importance during early stages of placentation.

The role of neurotrophic factors in the maintenance and survival of peripheral neuronal cells has been the subject of numerous studies. Administration of exogenous neurotrophic factors after nerve injury has been shown to mimic the effect of target organ-derived trophic factors on neuronal cells. After axotomy and during peripheral nerve regeneration, the neurotrophins NGF, NT-3 and BDNF show a well defined and selective beneficial effect on the survival and phenotypic expression of primary sensory neurons in dorsal root ganglia and of motoneurons in spinal cord. Other neurotrophic factors such as CNTF, GDNF and LIF also exert a variety of actions on neuronal cells, which appear to overlap and complement those of the neurotrophins. In addition, there is an indirect contribution of GGF to nerve regeneration. GGF is produced by neurons and stimulates proliferation of Schwann cells, underlining the close interaction between neuronal and glial cells during peripheral nerve regeneration. Different possibilities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. The studies reviewed in this article show the therapeutic potential of neurotrophic factors for the treatment of peripheral nerve injury and for neuropathies.

Nitric oxide (NO) is a unique biological messenger molecule. It serves, in part, as a neurotransmitter in the central and peripheral nervous systems. Neurons containing NO have been identified histochemically by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reactivity or immunohistochemically by the antibody for neuronal NO synthase (n-NOS). Previous histochemical or pharmacological studies have raised the possibility that NO may play an important role in the neural pathways of the lower urinary tract. There is also considerable evidence to suggest that n-NOS is plastic and could be upregulated following certain lesions in the lower urinary tract. The present review summarises the distribution of n-NOS containing neurons innervating the urinary bladder and the changes of the enzyme expression in some experimentally induced pathological conditions.

In 1895, well over 100 years ago, Willem Roux published his collected works on the developmental mechanics of organisms in 2 volumes. Volume 1 is largely dedicated to functional adaptation and is a condensation of his investigations into causes of the size and shape of organs and tissues, postulating the influence of functional demand, mediated by mechanical stimuli, on the shaping of organs and tissues. In this classic work he contributed to the understanding of the control of the structural development and organisation of blood vessels, muscles and bone. This work has been a source of inspiration for many investigators over the years. Well known examples are Wolff's law and Pauwel's theory on trajectories. Over the past 2 decades these hypotheses and concepts have been reappraised using 2 main approaches. Firstly, in muscles and tendons a qualitative approach with classical Newtonian mechanics combined with the anatomical configuration of these structures has been used to study the direction and the nature of the mechanical stresses in the tissues, be they compressive, tensile or shear. In bone and blood vessels these stresses are less accessible and often require computer modelling to calculate the mechanics at a cellular level. Secondly, molecular biology has demonstrated, both in tissue culture and in animal experiments, that mechanical stimuli can bring about cascades of messages in and between cells, but the experimental control of mechanical stresses in biological experiments is far from simple and limits the conclusions that can be derived. In order to approach a complete picture, the gap between these 2 approaches must be bridged. In this respect modern imaging techniques are helpful because they offer the possibility of studying the shape and change of shape over time in living organisms in greater detail.

The Symposium was organised in such a way that for different tissues recent advances using different approaches could be presented, helping to identify future directions for this field of morphological research. Review articles based on 2 of the 6 contributions to the Symposium are published here. One has already appeared (Benjamin & Ralphs, J. Anat. 193, pp. 481–494).

The study of the underlying mechanisms by which cells respond to mechanical stimuli, i.e. the link between the mechanical stimulus and gene expression, represents a new and important area in the morphological sciences. Several cell types (‘mechanocytes’), e.g. osteoblasts and fibroblasts as well as smooth, cardiac and skeletal muscle cells are activated by mechanical strain and there is now mounting evidence that this involves the cytoskeleton. Muscle offers one of the best opportunities for studying this type of mechanotransduction as the mechanical activity generated by and imposed upon muscle tissue can be accurately controlled and measured in both in vitro and in vivo systems. Muscle is highly responsive to changes in functional demands. Overload leads to hypertrophy, whilst decreased load force generation and immobilisation with the muscle in the shortened position leads to atrophy. For instance it has been shown that stretch is an important mechanical signal for the production of more actin and myosin filaments and the addition of new sarcomeres in series and in parallel. This is preceded by upregulation of transcription of the appropriate genes some of which such as the myosin isoforms markedly change the muscle phenotype. Indeed, the switch in the expression induced by mechanical activity of myosin heavy chain genes which encode different molecular motors is a means via which the tissue adapts to a given type of physical activity. As far as increase in mass is concerned, our group have cloned the cDNA of a splice variant of IGF that is produced by active muscle that appears to be the factor that controls local tissue repair, maintenance and remodelling. From its sequence it can be seen that it is derived from the IGF gene by alternative splicing but it has different exons to the liver isoforms. It has a 52 base insert in the E domain which alters the reading frame of the 3′ end. Therefore, this splice variant of IGF-1 is likely to bind to a different binding protein which exists in the interstitial tissue spaces of muscle, neuronal tissue and bone. This would be expected to localise its action as it would be unstable in the unbound form which is important as its production would not disturb the glucose homeostasis unduly. This new growth factor has been called mechano growth factor (MGF) to distinguish it from the liver IGFs which have a systemic mode of action. Although the liver is usually thought of as the source of circulating IGF, it has recently been shown that during exercise skeletal muscle not only produces much of the circulating IGF but active musculature also utilises most of the IGF produced. We have cloned both an autocrine and endocrine IGF-1, both of which are upregulated in cardiac as well as skeletal muscle when subjected to overload. It has been shown that, in contrast to normal muscle, MGF is not detectable in dystrophic mdx muscles even when subjected to stretch and stretch combined with electrical stimulation. This is true for muscular dystrophies that are due to the lack of dystrophin (X-linked) and due to a laminin deficiency (autosomal), thus indicating that the dystrophin cytoskeletal complex may be involved in the mechanotransduction mechanism. When this complex is defective the necessary systemic as well as autocrine IGF-1 growth factors required for local repair are not produced and the ensuing cell death results in progressive loss of muscle mass. The discovery of the locally produced IGF-1 appears to provide the link between the mechanical stimulus and the activation of gene expression.

The distribution of glycoconjugates was examined in the nonsensory regions of the rat cochlea during postnatal development using biotin-conjugated lectins. Temporal bones of rats at postnatal d 1 and at wk 2, 4 and 6 were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for paraffin wax embedding. The dewaxed sections were incubated with 7 biotinylated lectins, followed by avidin-biotin- peroxidase complex. A different staining pattern was observed in the stria vascularis, spiral ligament and spiral limbus in the age groups examined. The staining intensity varied between lectins and the reaction product exhibited limited disparity. The staining intensity for WGA increased with age in all the 3 nonsensory regions. The staining patterns for the other lectins differed in the various nonsensory regions examined indicating tissue specificity. The limited variations in the lectin binding patterns after 2nd wk of postnatal life also indicate that the changes in the carbohydrate moieties are established during the fetal period of cochlear development and limited changes take place during postnatal maturation of the nonsensory regions.

The effects of lithium on vascular development were examined using the chick embryo area vasculosa in shell-less culture as an experimental model. Embryos were explanted after 48 h in ovo and LiCl (50, 100, 150 and 200 μg in 10 μl water) was applied to the centre of the blastodisc. Controls were untreated or given equimolar amounts of NaCl. At 24 h and 48 h after treatment, untreated and NaCl controls were identical, having well developed extraembryonic vessels. At doses of 100 μg and greater, LiCl significantly inhibited normal vascular development and expansion of the area vasculosa in the majority of explants. In many specimens blood islands continued to form but their assembly into primitive vessels was prevented, indicating that lithium affects the mechanism regulating the assembly of vascular endothelium. At the same time the embryos were alive but retarded in development compared with controls. When LiCl (150 μg) was applied to cultures explanted after 72 h in ovo (when the primary vascular network had already formed through vasculogenesis) no adverse effects were seen. This suggests that lithium affects vasculogenesis but not angiogenesis. Treatment with myo-inositol completely reversed the effects of lithium in a time dependent manner indicating that the phosphatidylinositol second messenger cycle may be involved in the cellular events of vasculogenesis. Finally the results of this study show that the yolk sac vasculature is particularly vulnerable to lithium and the consequent effects of this interference on embryonic development are discussed.

This review is concerned with the development of the rat corticospinal tract (CST). The CST is a long descending central pathway, restricted to mammals, which is involved both in motor and sensory control. The rat CST is a very useful model in experimental research on the development of fibre systems in mammals because of its postnatal outgrowth throughout the spinal cord as well as its experimental accessibility. Hence mechanisms underlying axon outgrowth and subsequent target cell finding can be studied relatively easily. In this respect the corticospinal tract forms an important example and model system for the better understanding of central nervous system development in general.

The anatomy of the lymphoid organs was studied during the course of detailed dissections of 50 beach-stranded bottlenose dolphins, Tursiops truncatus. Constant lymph nodes occur in 4 groups, based on their location and structure. These groups are somatic, including nodes of the cervical region and pelvic recess; lung-associated, included marginal, diaphragmatic and hilar nodes; visceral, including the mesenteric, pancreatic, pericolic and porta hepatis nodes; and aortic arch nodes. Lymphatic drainage of the lung is primarily to the marginal and diaphragmatic nodes. The mesenteric node mass is well-endowed with capsular and trabecular smooth muscle, and a network of muscle fascicles within the organ implies an important contractile function in the circulation of lymph. In addition to constant nodes, occasionally nodes are found in relation to the thoracic aorta, the kidney, and under the scapula. Gut-associated structures include dorsal and ventral oropharyngeal tonsils, mucosal aggregates in the straight segment of the intestine (colon) and anal tonsils; this gut-associated lymphoid tissue tends to involute with age, being greatly reduced by puberty. Formed lymphoid organs include the thymus and the spleen, the latter being relatively small in relation to body size. None of these structures is unique among cetaceans, but the anal tonsils are particularly well developed in T. truncatus. The lymphoid aggregates in the colon resemble the arrangement in the vermiform appendix, which is lacking in most cetaceans, and may have functions analogous to that organ.

The pattern of variation of certain vertebral measurements along the vertebral column is known to differ in man and mouse. This paper investigates changes in this pattern in 7 species of small mammals and attempts to correlate them with locomotor adaptations and limb dimensions.

The evidence for release of vasoactive substances from endothelial cells in response to shear stress caused by the viscous drag of passing fluids is reviewed and, in particular, its physiological significance both in short-term regulation of blood vessel tone and in long-term regulation of cell growth, differentiation, proliferation, and cell death in pathophysiological conditions is discussed. A new concept of purinergic mechanosensory transduction, particularly in relation to nociception, is introduced. It is proposed that distension of tubes (including ureter, vagina, salivary and bile ducts, gut) and sacs (including urinary and gall bladders, and lung) leads to release of ATP from the lining epithelium, which then acts on P2X2/3 receptors on subepithelial sensory nerves to convey information to the CNS.

We report a change in the vascularisation of the adipose depots surrounding the popliteal lymph node that has, and the contralateral node that has not, been exposed to a simulated immune challenge. The percentage of the depot that consists of vessels, as measured by image analysis, decreases over a period of 2 d after immune stimulus, then increases in a biphasic manner over the next 2–3 wk. By 1 mo after the stimulus, the vascularisation has returned to baseline values. The adipose tissue surrounding both the stimulated and the unstimulated lymph nodes shows a similar pattern, but the unstimulated depot lags by 3–6 d in reaching its maximum vascularisation. These data support the hypothesis that perinodal adipose tissue is involved in peripheral immune responses.

Articular cartilage undergoes cycles of compressive loading during joint movement, leading to its cyclical deformation and recovery. This loading is essential for chondrocytes to perform their normal function of maintenance of the extracellular matrix. Various lines of evidence suggest the involvement of the cytoskeleton in load sensing and response. The purpose of the present study is to describe the 3-dimensional (3D) architecture of the cytoskeleton of chondrocytes within their extracellular matrix, and to examine cytoskeletal responses to experimentally varied mechanical conditions. Uniformly sized explants of articular cartilage were dissected from adult rat femoral heads. Some were immediately frozen, cryosectioned and labelled for filamentous actin using phalloidin, and for the focal contact component vinculin or for vimentin by indirect immunofluorescence. Sections were examined by confocal microscopy and 3D modelling. Actin occurred in all chondrocytes, appearing as bright foci at the cell surface linked to an irregular network beneath the surface. Cell surface foci colocalised with vinculin, suggesting the presence of focal contacts between the chondrocyte and its pericellular matrix. Vimentin label occurred mainly in cells of the deep zone. It had a complex intracellular distribution, with linked networks of fibres surrounding the nucleus and beneath the plasma membrane. When cartilage explants were placed into organ culture, where in the absence of further treatments cartilage imbibes fluid from the culture medium and swells, cytoskeletal changes were observed. After 1 h in culture the vimentin cytoskeleton was disassembled, leading to diffuse labelling of cells. After a further hour in culture filamentous vimentin label reappeared in deep zone chondrocytes, and then over the next 48 h became more widespread in cells of the explants. Actin distribution was unaffected by culture. Further experiments were performed to test the effects of load on the cytoskeleton. Explants were placed in culture and immediately subjected to static uniaxial radially unconfined compressive loads of 0.5, 1, 2 or 4 MPa for 1 h using a pneumatic loading device. Loads greater than 0.5 MPa maintained the vimentin organisation over the culture period. At 0.5 MPa, the chondrocytes within the explant behaved as in free-swelling culture. The rapid change in vimentin organisation probably relates to rapid swelling of the explants—under free-swelling conditions, these reached their maximum swollen size in just 15 min of culture. The chondrocytes' response to change in tissue dimensions, and thus to their relationship to their immediate environment, was to disassemble their vimentin networks. Loading probably counteracts the swelling pressure of the tissue. Overall, this work suggests that chondrocytes maintain their actin cytoskeleton and modify their vimentin cytoskeleton in response to changing mechanical conditions.

The structure of the developing thymus of the marine teleost, Diplodus puntazzo, was studied by light and transmission electron microscopy. The first anlage of the thymus developed by d 20 postfertilisation (p.f.) as a group of undifferentiated cells dorsal to the epithelium of the branchial chamber. The organ increased significantly in size around d 51–66 p.f. and differentiation of cortex and medulla occurred concomitantly. On the basis of their localisation, 4 main types of epithelial cell were distinguished: (1) limiting, adjacent to the connective capsule; (2) medullary and cortical reticular cells; (3) nurse cells, located in the corticomedullary boundary; (4) Hassall-like corpuscles. The majority of medium to large blast-like lymphoid cells were localised in the medulla, while small lymphocytes were housed in the cortical region. These morphological features were maintained at later stages. However, in juveniles in the medulla we observed reticular epithelial cells with cysts and rare Hassall-like corpuscles. The study was designed to obtain more information concerning the histology of the developing thymus of sharpsnout seabream and give a concise description of the differentiation of epithelial cells and lymphoid cells in the thymic parenchyma.

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.

After aldehyde prefixation, pretreatment with cryoprotectant and subsequent freeze-substitution with OsO4 in acetone (AC-FS), extensive gap junction-like close membrane appositions are frequently found in the basal infolding of the salivary gland epithelium, although the desmosomal intercellular space had the same width as with conventional electron microscopy. The intercellular space between podocyte pedicles and endothelial cells at the renal glomerular filtration site was narrower by the total width of 2 laminae lucidae following AC-FS than with conventional electron microscopy and was occupied by a homogeneous lamina densa without a lamina lucida, although no marked difference was discernable in the thickness of the lamina densa itself between the 2 preparative procedures. In addition, a decrease in the thickness of the glycocalyx was evident in the intestinal epithelial microvilli following AC-FS. It is thus likely that osmication in acetone at freezing temperatures remove the glycocalyx and related structures to a variable extent, and that this loss is responsible for reducing the intercellular spaces at some of the simple appositions narrower to the dimensions of the gap junction. It is also responsible for disappearance of the lamina lucida of the basement membrane.

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated ‘scoop’-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the ‘scoop’. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation.

Secretory granule formation in pancreatic acinar cells is known to involve massive membrane flow. In previous studies we have undertaken morphometry of the regranulation mechanism in these cells and in mast cells as a model for cellular membrane movement. In our current work, electron micrographs of pancreatic acinar cells from ICR mice were taken at several time points after extensive degranulation induced by pilocarpine injection in order to investigate the volume changes of rough endoplasmic reticulum (RER), nucleus, mitochondria and autophagosomes. At 2–4 h after stimulation, when the pancreatic cells demonstrated a complete loss of granules, this was accompanied by an increased proportion of autophagosomal activity. This change primarily reflected a greatly increased proportion of profiles retaining autophagic vacuoles containing recognisable cytoplasmic structures such as mitochondria, granule profiles and fragments of RER. The mitochondrial structures reached a significant maximal size 4 h following injection (before degranulation 0.178±0.028 μm3; at 4 h peak value, 0.535±0.109 μm3). Nucleus size showed an early volume increase approaching a maximum value 2 h following degranulation. The regranulation span was thus divided into 3 stages. The first was the membrane remodelling stage (0–2 h). During this period the volume of the RER and secretory granules was greatly decreased. At the intermediate stage (2–4 h) a significant increase of the synthesis zone was observed within the nucleus. The volume of the mitochondria was increasing. At the last step, the major finding was a significant granule accumulation in parallel with an active Golgi zone.

Anatomical and electromyographic studies point to regional differences in function in the human temporalis muscle. During chewing and biting the anterior portions of the muscle are in general more intensively activated and they are capable of producing larger forces than the posterior portions. It was hypothetised that this heterogeneity in function is reflected in the fibre type composition of the muscle. The composition and surface area of different fibre types in various anteroposterior portions of the temporalis muscle were investigated in 7 cadavers employing immunohistochemistry with a panel of monoclonal antibodies against different isoforms of myosin heavy chain. Pure slow muscle fibres, type I, differed strongly in number across the muscle. In the most posterior portion of the muscle there were 24% type I fibres, in the intermediate portion 57%, and in the most anterior portion 46%. The mean fibre cross-sectional area (m-fcsa) of type I fibres was 1849 μm2, which did not differ significantly across the muscle. The proportion of pure fast muscle fibres, type IIA and IIX, remained more or less constant throughout the muscle at 13% and 11% respectively ; their m-fcsa was 1309 μm2 and 1206 μm2, respectively, which did not differ significantly throughout the muscle. Pure type IIB fibres were not found. The relative proportion of hybrid fibres was 31% and did not differ significantly among the muscle portions. Fibre types I+IIA and cardiac α+I+IIA were the most abundant hybrid fibre types. In addition, 5% of the type I fibres had an additional myosin isoform which has only recently been described by means of electrophoresis and was named Ia. In the present study they were denoted as hybrid type I+Ia muscle fibres. It is concluded that intramuscular differences in type I fibre distribution are in accordance with regional differences in muscle function.

The interosseous nerve of birds innervates a string of Herbst corpuscles located near the interosseous membrane between the tibia and fibula. Fibre composition of this nerve was assessed including both myelinated and unmyelinated axons. The diameter of the whole nerve is ∼100 μm. Complete data were obtained for 3 nerves. The mean total number of myelinated fibres and unmyelinated axons was 2872±53. The mean number of myelinated fibres was 280±20 and that for unmyelinated axons was 2600±47. There was a broad distribution of diameters for myelinated fibres ranging from ∼2 μm to 10 μm with a distinct peak at ∼3–5 μm and a less prominent second peak at 6–8 μm. Similarly, myelin sheath thickness distribution showed 2 peaks, one at 0.6–0.8 μm and another at 1.4–1.6 μm. It is suggested that the group represented by the second peak innervates the Herbst corpuscles. The group of smaller myelinated fibres and the unmyelinated axons are assumed to innervate other types of receptors, some of which may be nociceptors.