Cell–matrix and matrix–matrix interactions are of critical importance in regulating the development, maintenance and repair of articular cartilage. In this study, we examined the structural colocalisation of type VI collagen and fibronectin in isolated chondrons and long-term agarose cultured chondrocytes extracted from normal adult canine articular cartilage. Using double labelling immunohistochemistry in conjunction with dual channel confocal microscopy and digital image processing we demonstrate that type VI collagen and fibronectin are distributed in a similar staining pattern and are colocalised at the surface of cultured chondrocytes and isolated chondrons. The results suggest that type VI collagen and fibronectin may play a role in both cell–matrix adhesion and matrix–matrix cohesion in the pericellular microenvironment surrounding articular cartilage chondrocytes.

Tartrate-resistant acid phosphatase (TRAP, Acp 5) is considered to be a marker of the osteoclast and studies using ‘knockout’ mice have demonstrated that TRAP is critical for normal development of the skeleton. To investigate the distribution of TRAP in the mammalian embryo, cryostat sections of 18 d murine fetuses were examined by in situ hybridisation, immunohistochemistry and histochemical reactions in situ. Abundant expression of TRAP mRNA was observed in the skin and epithelial surfaces of the tongue, oropharynx and gastrointestinal tract including the colon, as well as the thymus, ossifying skeleton and dental papillae. TRAP protein was identified at the same sites, but the level of expression in the different tissues did not always correlate with apparent enzyme activity. The findings indicate that abundant TRAP expression is not confined to osteoclasts in bone, but occurs in diverse tissues harbouring cells of bone marrow origin, including dendritic cells and other cells belonging to the osteoclast/macrophage lineage.

Development of desmin-positive hepatic stellate cells was studied in mice using double immunofluorescent techniques and in vitro cultures with special attention given to their cell lineages. Several studies recently reported on the presence of cells that are immunologically reactive with both antidesmin and anticytokeratin antibodies in young fetal rat livers, and suggested the possibility that these cells give rise to hepatocytes and hepatic stellate cells. At early stages of mouse liver development, stellate cells with desmin-positive filaments were scattered in the liver parenchyma. However, the stellate cells definitely differed from hepatoblasts and hepatocytes in terms of their morphology and expression of desmin and hepatoblast and hepatocyte-specific E-cadherin in the liver. Fetal hepatoblasts and hepatocytes did not react with antidesmin antibodies, nor did desmin-positive stellate cells express E-cadherin in vivo and in vitro. Thus it is likely that desmin-positive stellate cells and hepatoblasts belong to different cell lineages. In the fetal liver, the desmin-positive stellate cells surrounded blood vessels, and extended their processes to haematopoietic cells and megakaryocytes. Many, but not all, hepatoblasts and hepatocytes were observed to be associated with the stellate cells. At fetal stages, cellular processes positive for desmin in the stellate cells were also thick compared with those in the adult liver, in which desmin-positive stellate cells lay in Disse's space and were closely associated with all hepatocytes. These developmental changes in the geography of desmin-positive cells in the liver parenchyma and their morphology may be associated with their maturation and interactions with other cell types.

Bone is characterised by age-related morphological and histological changes. We have previously established an automated method of recording bone morphometry and histology from entire transverse sections of cortical bone. Our aim was to determine whether data acquired using this automated system were useful in the prediction of age. Ninety-six specimens of human femoral middiaphysis were studied from subjects aged 21–92 y. Equations predicting specimen age were constructed using macroscopic data (total subperiosteal area (TSPA), periosteal perimeter (PP), endosteal perimeter (EP), cortical bone area (CA) and moments of area) and microscopic data (the number, size and diversity of pores and intracortical porosity) together with sex, height and weight. Both TSPA and PP were independent predictors of age but the number of pores was not a significant predictor of age in any equation. The age predicted by these equations was inaccurate by more than 8 y in over half the subjects. We conclude that we could not predict age at a clinically acceptable level using data from our automated system. This most likely reflects an insensitivity to regional age-related changes in bone histology because we recorded data from each entire cortex. Automated bone measurement according to cortical region might be more useful in the prediction of age. The inclusion of TSPA together with PP as independent predictors of age raises the possibility that a future measure of periosteal shape at the femoral diaphysis could also be helpful in the prediction of age. The accuracy reached with the relatively simple methods described here is sufficient to encourage the development of image-analysis systems for the automatic detection of more complex features.

Six muscles of the mallard duck (Anas platyrhynchos), the common coot (Fulica atra) and the yellow-legged gull (Larus cachinnans) were analysed morphometrically, with special emphasis on their functional implications and physiological needs. Oxidative fibres always had significantly smaller size than anaerobic fibres, although no differences in the number of capillaries per fibre were found. This resulted in greater capillary counts per unit of fibre area and perimeter in oxidative than anaerobic fibres, which indicates that the greater demand for oxygen supply may be achieved by decreasing the size of the muscle fibre rather than by increasing the number of associated capillaries. Fast oxidative fibres of the pectoralis and the triceps of the gull had greater sizes than the fast oxidative fibres of the mallard and the coot, which correlates with the difference in energetic demands between flapping and gliding flight. Greater fibre cross-sectional areas and perimeters seem suited to afford the long-lasting activity with low metabolic demands required during gliding. By contrast, mallards and coots attain a high oxidative metabolism, during sustained flapping flight, by reducing fibre size at the expense of a diminished ability for force generation. Between-species comparisons of the hindlimb muscles only yielded differences for the anaerobic fibres of the gastrocnemius, as an important adaptive response to force generation during burst locomotion. The need to manage sustained swimming abilities effectively may result in similar FOG fibre morphometry of the hindlimb muscles studied, indicating that a compromise between the oxygen flux to the muscle cell and the development of power is highly optimised in oxidative fibres of the bird species studied.

At the onset of their migration into the embryo, many neural crest cells are pluripotent in the sense that they have the capacity to generate progeny that consist of more than one cell type. More recently, we have found that there are pluripotent neural crest cell-derived cells even at sites of terminal differentiation. These findings support the notion that cues originating from the microenvironment, at least in part, direct neural crest cell type specification. Based on the rationale that growth factors that are known to support survival of neural crest cell derivatives may have additional functions in progenitor cell development, we have examined the action of pertinent growth factors. Trophic, mitogenic, antiproliferative and differentiation promoting activities were found. Stem cell factor (SCF) is trophic for pluripotent neural crest cells. Contrary to expectation, SCF plus a neurotrophin, rather than SCF alone, is trophic for committed melanogenic cells. Basic fibroblast growth factor (bFGF) is mitogenic both for pluripotent cells and committed melanogenic cells. However, the cells become dependent on another factor for survival. Whereas any neurotrophin tested can rescue bFGF-activated pluripotent neural crest cells, the factor that rescues melanogenic cells remains to be determined. Transforming growth factor β1 (TGF-β1) is a powerful antimitotic signal for all neural crest cells that overrides the bFGF/neurotrophin proliferative signal. Furthermore, SCF promotes differentiation of neural crest cells into cells of the sensory neuron lineage. Neurotrophin-3 (NT-3) specifically promotes high affinity uptake of norepinephrine by neural crest cells and is thus thought to play a critical role in the differentiation of sympathetic neuroblasts. In summary, our data indicate that neurotrophins and other pertinent growth factors affect survival, proliferation and differentiation of neural crest cells at multiple levels and in different lineages. Moreover, our findings emphasise the importance of the concerted action of combinations of growth factors, rather than of individual factors.

Immunohistochemistry of normal eccrine sweat glands was performed on paraffin sections of human skin. Immunoreactivity (ir) for neuron specific enolase, S100 protein (S100), regulatory peptides, nitric oxide synthase type I (NOS-I) and choline-acetyltransferase (ChAT) was found in small nerve bundles close to sweat glands. In the glands, secretory cells were labelled with anticytokeratin antibody. Using antibodies to S100, calcitonin gene-related peptide (CGRP) and substance P (SP) a specific distribution pattern was found in secretory cells. Granulated (dark) and parietal (clear) cells were immunopositive for CGRP, and S100 and SP, respectively. Immunoreactivity was diffuse in the cytoplasm for CGRP and S100, and peripheral for SP. Myoepithelial cells were not labelled. Electron microscopy revealed electron dense granules, probably containing peptide, in granulated cells. Using antibodies to NOS-I and ChAT, ir was exclusively found in myoepithelial cells. Immunoreactivity for the atrial natriuretic peptide was absent in sweat glands. These results provide evidence for the presence of both regulatory peptides involved in vasodilation and key enzymes for the synthesis of nitric oxide and acetylcholine in the secretory coil of human sweat glands. It is suggested that human sweat glands are capable of some intrinsic regulation in addition to that carried out by their nerve supply.

Since many human heart diseases involve both the intrinsic cardiac neurons and nerves, their detailed normal ultrastructure was examined in material from autopsy cases without cardiac complications obtained no more than 8 h after death. Many intracardiac nerves were covered by epineurium, the thickness of which was related to nerve diameter. The perineurial sheath varied from nerve to nerve and, depending on nerve diameter, contained up to 12 layers of perineurial cells. The sheaths of the intracardiac nerves therefore become progressively attenuated during their course in the heart. The intraneural capillaries of the human heart differ from those in animals in possessing an increased number of endothelial cells. A proportion of the intraneural capillaries were fenestrated. The number of unmyelinated axons within unmyelinated nerve fibres was related to nerve diameter, thin cardiac nerves possessing fewer axons. The most distinctive feature was the presence of stacks of laminated Schwann cell processes unassociated with axons that were more frequent in older subjects. Most unmyelinated and myelinated nerve fibres showed normal ultrastructure, although a number of profiles displayed a variety of different axoplasmic contents. Collectively, the data provide baseline information on the normal structure of intracardiac nerves in healthy humans which may be useful for assessing the degree of nerve damage both in autonomic and sensory neuropathies in the human heart.

The Trembler-J (TrJ) mouse has a point mutation in the gene coding for peripheral myelin protein 22 (PMP22). Disturbances in PMP22 are associated with abnormal myelination in a range of inherited peripheral neuropathies both in mice and humans. PMP22 is produced mainly by Schwann cells in the peripheral nervous system where it is localised to compact myelin. The function of PMP22 is unclear but its low abundance (∼5% of total myelin protein) means that it is unlikely to play a structural role. Its inclusion in a recently discovered family of proteins suggests a function in cell proliferation/differentiation and possibly in adhesion. Nerves from TrJ and the allelic Trembler (Tr) mouse are characterised by abnormally thin myelin for the size of the axon and an increased number of Schwann cells. We report ultrastructural evidence of abnormal Schwann cell-axon interactions. Schwann cell nuclei have been found adjacent to the nodes of Ranvier whereas in normal animals they are located near the centre of the internodes. In some fibres the terminal myelin loops faced outwards into the extracellular space instead of turning inwards and terminating on the axon. In severely affected nerves many axons were only partially surrounded by Schwann cell cytoplasm. All these features suggest a failure of Schwann cell–axon recognition or interaction. In addition to abnormalities related to abnormal myelination there was significant axonal loss in the dorsal roots.

For an accurate assessment of jaw movement, it is crucial to understand the comprehensive formation of the masticatory muscles with special reference to the relationship to the disc of the temporomandibular joint. Detailed dissection was performed on 26 head halves of 14 Japanese cadavers in order to obtain precise anatomical information of the positional relationships between the masticatory muscles and the branches of the mandibular nerve. After complete removal of the bony elements, the midmedial muscle bundle in all specimens and the discotemporal muscle bundle in 6 specimens, derivatives of the temporalis, which insert into the disc were observed. On the anterior area of the articular capsule and the disc of the temporomandibular joint, the upper head of the lateral pterygoid, the midmedial muscle bundle of temporalis and the discotemporal bundle of temporalis were attached mediolaterally, and in 3 specimens the posterosuperior margin of the zygomaticomandibularis was attached to the anterolateral area of the disc. It is suggested that these muscles and muscle bundles contribute to various mandibular movements. Although various patterns of the positional relationships between the muscles and muscle bundles and the their innervating nerves are observed in the present study, relative positional relationships of the muscles and muscle bundles and of nerves of the mandibular nerve are consistent. A possible scheme of the developmental formation of the masticatory muscles based on the findings of the positional relationships between the muscles and the nerves is presented.

The accessory olfactory bulb (AOB) is the primary target of the sensory epithelium of the vomeronasal organ (VNO), and thus constitutes a fundamental component of the accessory olfactory system, which is involved in responses to behaviour-related olfactory stimuli. In this study we investigated the characteristics of the AOB, VNO, vomeronasal nerves (VNNs) and caudal nasal nerve (CdNN) in pigs and sheep, species in which olfaction plays a key behavioural role both in the neonatal period and in adulthood. The patterns of staining of the AOB by the Bandeiraea simplicifolia and Lycopersicon esculentum lectins were the same in the 2 species, whereas the Ulex europeus and Dolichos biflorus lectins gave different patterns. In both species, lectin staining of the AOB was consistent with that of the VNNs, while the CdNN did not label any of the structures studied. The entire sensory epithelium of the pig was labelled by Ulex europeus and Lycopersicum esculentum lectins, and all 4 lectins used labelled the mucomicrovillar surface of the sensory epithelium in sheep.

The aim of this study was to characterise pulmonary reimplantation injury in isolated, perfused rat lungs following 2 h of cold ischaemia, and 50 min. of in vitro reperfusion. The effects of 2 differently composed lung preservation solutions (low potassium Euro-Collins and Celsior; each n=5) were examined in comparison with untreated, nonischaemic control lungs (n=3). After fixation by vascular perfusion and tissue collection by systematic random sampling, the volume weighted mean volume (Vv) of alveoli and acinar pathways was estimated by light microscopic stereology using the method of point sampled intercepts in plastic embedded, Azan-stained material. Significantly higher Vv of alveoli and acinar paths was found in the Celsior group than in Euro-Collins preserved lungs. However, in the controls the size of acinar pathways was similar to Celsior preserved lungs whereas alveolar size was comparable to preservation with Euro-Collins. The between-animal coefficient of variation of alveoli was very low in controls and Celsior preserved but higher in the Euro-Collins group. Size distribution of alveoli and acinar paths in 15 size classes was largely homogeneous in all groups tested. In the Euro-Collins group the fractions of both class 1-alveoli and class 1-acinar paths significantly exceeded those of the other groups. Widely expanded alveoli (size classes 13–15) only occurred after preservation with Celsior whereas wider acinar paths (size class 15) were found in the Celsior group and in the controls. It is concluded that lung preservation with low-potassium Euro-Collins and Celsior solutions may act differently on distinct spaces in the distal gas-exchange regions of lungs. This may be due to selective effects on pulmonary surfactant activity and on elastic tissue elements in the alveolar ducts, respectively. Additionally, the method of point sampled intercepts is considered to be an efficient tool to evaluate the effects of different preservation solutions on lung parenchyma.

Eight normal human spinal cords were studied. Spinal segments were identified and embedded in paraffin wax. Serial cross sections were cut at 25 μm and stained by cresyl violet. Motor columns were reconstructed adapting Elliott's (1942) methods. Motor columns were classified into the medial and lateral divisions and were numbered sequentially from medial to lateral at the level of Cl. In the cervical cord, 8 motor columns were traced. Column 1, corresponding to the medial column, presented 3 subdivisions designated as 1a, 1b and 1c with ventral, dorsal and lateral positions respectively. Columns 1a and 1b extended throughout the cervical region while 1c was confined to 3rd, 4th and 5th cervical segments. At the level of C3, 1c was a discrete column situated lateral to 1a and 1b but at C4 and C5 it became displaced medially close to the medial margin of the ventral horn. In cross section, it presented smaller medial and large lateral part. With the help of clinical and developmental evidence an attempt was made to correlate column 1c with the phrenic nucleus.

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.

The manipulation mechanism of the giant panda (Ailuropida melanoleuca) was examined by means of CT (computed tomography) and 3-dimensional (3-D) Volume Rendering techniques. In the 3-D images of the giant panda hand, not only the bones but also the muscular system was visualised. Sections of the articulated skeleton were obtained. It was demonstrated that the hand of the panda is equipped with separately moulded manipulation units as follows: (1) the radial sesamoid (RS), the radial carpal, and the first metacarpal (R–R–M) complex; and (2) the accessory carpal (AC) and the ulnar (A–U) complex. When the giant panda grasps anything, the R–R–M complex strongly flexes at the wrist joint, the RS becomes parallel with the AC, and the phalanges bend and hold the object. It is shown that the well-developed opponens pollicis and abductor pollicis brevis muscles envelop and fix the objects between the R–R–M complex and the phalanges during grasping.

Fibre optic confocal imaging (FOCI) enabled subsurface fluorescence microscopy of the skin of hairless mice in vivo. Application of acridine orange enabled imaging of the layers of the epidermis. The corneocytes of the stratum corneum, the keratinocytes in the basal layers and redundant hair follicles were visualised at depths greater than 100 μm. Cellular and nuclear membranes of keratinocytes of the skin were visualised by the use of acridine orange and DIOC5(3). Imaging of the skin after injection of FITC-dextran revealed an extensive network of blood vessels with a size range up to 20 μm. Blood cells could be seen moving through dermal vessels and the blood circulation through the dermal vascular bed was video-taped. The fluorescent dye 4-di-2-ASP showed the presence of nerves fibres around the hair follicles and subsurface blood vessels. Comparison was made between images obtained in vivo using FOCI and in vitro scanning electron microscopy and conventional histology. FOCI offers the potential to study dynamic events in vivo, such as blood flow, skin growth, nerve regeneration and many pathological processes, in ways which have not previously been possible.

The present immunoperoxidase cytochemical study describes the distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP) and vimentin, in the brain and spinal cord of the adult lizard, Podarcis sicula. GFAP immunoreactivity is abundant and the positive structures are mainly represented by fibres of different lengths which are arranged in a rather regular radial pattern throughout the CNS. They emerge from generally immunopositive radial ependymoglia and are directed from the ventricular wall towards the meningeal surface. The glial fibres give origin to endfeet which are apposed to the blood vessel walls and subpial surface where they form the continous perivascular and subpial glia envelopes, respectively. In the optic tectum and spinal cord, star-shaped astrocytes coexist with radial glia. In the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependyma. While vimentin immunoreactive elements are almost completely absent in the brain except for a few diencephalic radial fibres, the spinal cord ependyma exhibits a clearly vimentin positivity and no GFAP staining. In the Podarcis CNS the immunocytochemical response of the astroglial intermediate filaments appears typical of mature astroglia cell lineage since it fundamentally expresses GFAP immunoreactivity. Moreover, this immunocytochemical study shows that the Podarcis fibre pattern with predominant radial glial cells is morphologically more immature than in avians and mammalians, a condition suggesting that reptiles represent a fundamental step in the phylogenetic evolution of vertebrate astroglial cells.

The axon determines whether or not it is myelinated by the Schwann cell. At maturity there is a positive correlation between sheath thickness and axon calibre. This correlation is initially very low or absent, but gradually strengthens during development. This increase could come about because the axon continuously controls Schwann cell myelinating activity, so that a given axon calibre is associated with a particular myelin sheath thickness, an interaction which would entail the Schwann cell continuously monitoring and responding to axon size. This seems unnecessarily complex. This theoretical study shows that the strong correlation between the 2 parameters within a given myelinated fibre population may come about in a much simpler way than outlined above. This is demonstrated by modelling the growth and myelination of a hypothetical population, utilising data from earlier studies on cervical ventral motoneuron axon development. The hypothesis tested shows that the only instructive interactions by the axon on the Schwann cell necessary for the strong correlation between the 2 parameters to emerge are for the initiation of myelination, its continuation and its termination. These could result from a single stimulus being switched on, persisting for a time and being switched off. Under this influence, the Schwann cell is assumed to proceed to form the myelin sheath at a constant rate which it itself inherently determines, in the absence of any quantitative influence exerted by the axon. This continues until the stimulus for myelination ceases to emanate from the axon. The validity of the hypothesis is demonstrated, because the resulting myelin-axon relationships correspond closely to those observed during development.

In human placenta, 3D spatial relationships between villi and the maternal vascular bed determine intervillous porosity and this, in turn, influences haemodynamics and transport. Recently-developed stereological methods were applied in order to examine and quantify these relationships. Placentas were collected after 37 wk from control pregnancies and those associated with maternal diabetes mellitus classified according to duration and severity (White classification scheme). Two principal questions were addressed: (1) are normal spatial arrangements maintained in well-controlled diabetes mellitus? and (2) do arrangements vary between diabetic groups? To answer these questions, tissue sections cut at random positions and orientations were generated by systematic sampling procedures. Volume densities of villi (terminal+intermediate), intervillous spaces and perivillous fibrin-type fibrinoid deposits were estimated by test point counting and converted to global volumes after multiplying by placental volumes. Design-based estimates of the sizes (volume- and surface-weighted volumes) of intervillous ‘pores’ were obtained by measuring the lengths of point- and intersection-sampled intercepts. From these, theoretical numbers of pores were calculated. Model-based estimates (cylinder model) of the hydraulic diameters and lengths of pores were also made. Second-order stereology was used to examine spatial relationships within and between villi and pores and to test whether pair correlation functions deviated from the value expected for ‘random’ arrangements. Estimated quantities did not differ significantly between diabetic groups but did display some departures from control values in non-insulin-dependent (type 2) diabetic placentas. These findings support earlier studies which indicate that essentially normal microscopical morphology is preserved in placentas from diabetic subjects with good glycaemic control. Therefore, it is likely that fetal hypoxia associated with maternal diabetes mellitus is due to metabolic disturbances rather than abnormalities in the quantities or arrangements of maternal vascular spaces.

The orientation of the superior articular processes in thoracic and lumbar vertebrae differs. The present study was undertaken to investigate the possible mechanism for the change from a posterolaterally facing superior articular surface in the thoracic region to a posteromedially facing curved articular surface in the lumbar region. The material of the study consisted of dry macerated bones of 44 adult human vertebral columns. The orientation of the superior articular process and its relation to the mamillary tubercle (process) was examined between T9 and L5 vertebrae in each column. An abrupt change from the thoracic to lumbar type of articular process was observed in 3 columns (7%). Forty-one (93%) columns showed a gradual change extending over either 2 or 3 successive vertebrae. The present study suggests that the change in the orientation of the superior articular process, from the coronal to the sagittal plane (sagittalisation), occurs due to the change in the direction of weight transmission through zygapophyseal joints at the thoracolumbar junction. It was observed that the gradual sagittalisation of the superior articular process in the transitional zone brought it close to the mamillary tubercle which eventually fused with it. Thus the study suggests that the characteristic posteromedially facing concave superior articular process of lumbar vertebrae may have formed because of the fusion of the articular process and the mamillary tubercle.