We examined the distribution of parvalbumin in the pigeon thymus by light and electron microscopic immunohistochemistry. Tissues were also examined by conventional electron microscopy to determine the ultrastructure of immunoreactive cells. Parvalbumin immunoreaction was located in epithelial cells of the cortex, which formed dense mesh-like structures. Parvalbumin-positive epithelial cells were classified into 2 types. The first comprised elongated cells. In these, the nucleus was spindle-shaped, oval, or triangular, with a slightly irregular contour and contained rich heterochromatin peripherally. The cytoplasm was pale and processes extended laterally or ramified among the surrounding thymocytes. This type of cell formed the majority of immunoreactive cells. The other cell type consisted of polygonal epithelial cells. The nucleus was oval with deep indentations. Euchromatin occupied a large part of the nucleus. The cytoplasm contained numerous cell organelles compared with the elongated type, in particular, electron-dense vacuoles of various sizes and often bundles of tonofilaments. Both types of epithelial cell were interconnected by desmosomes. No secretory granules were found in the cytoplasm of elongated or polygonal cells. These results indicate the presence of heterogeneous group of parvalbumin-immunoreactive epithelial cells and suggest the likelihood of different functional roles for parvalbumin in the pigeon thymus.

Mammary glandular tissues and mammary secretions were obtained from sheep at 2–60 d after weaning to study the leucocyte phenotypes associated with mammary involution. From 2–4 d after weaning, neutrophils were the predominant leucocytes in the alveolar and ductal lumina. Lymphocytes were present in the alveolar and ductal epithelium, interalveolar and periductal areas. Most of the lymphocytes in the alveolar and ductal epithelium (IEL) were CD8+, some were CD45R+ and few were CD4+. In the periductal clusters and in the interalveolar areas most of the lymphocytes were CD4+. There was a significant increase (P < 0.05) in the percentages of CD45R+ granulated IEL from 2 to 7 d after weaning, and this paralleled the increase in the percentages of apoptotic cells in the glandular epithelium. By 7–60 d after weaning, most cells within the alveolar and ductal lumina were macrophages followed by predominantly CD8+ lymphocytes. CD8+ lymphocytes were still predominant in the alveolar and ductal epithelium while CD4+ cells were predominant in the interalveolar areas. Very few γδ+ T cells were observed at all the stages examined. The cells in the mammary secretions correlated with those observed in the alveolar and ductal lumina. At the early stages of involution, the neutrophils and macrophages were heavily laden with lipid droplets, casein and cellular debris. The most interesting feature was the presence of cells either with extensive cytoplasmic processes (LCA+ MHC class II+) or cytoplasmic veils (LCA+ MHC class II+CD1+), probably dendritic cells. It is concluded that the cellular constituents of the mammary gland at the latter part of involution may afford the mammary gland more resistance to infection than the lactating gland and the gland at early stages of involution. The CD45R+ IEL may trigger apoptotic cell death in the mammary glandular epithelium during mammary involution.

The ultrastructure and size distributions of collagen fibrils in Glisson's sheath were investigated in the rat liver to analyse the mechanical environment around the fibrils and their possible cells of origin. Glisson's sheath was found to contain 2 populations of collagen fibrils with different diameters and distinct localisations, namely fibroblast-associated and bile epithelium-associated. Fibroblast-associated collagen was composed of fibrils arranged in bundles and constituted the majority of the collagen in Glisson's sheath. Bile epithelium-associated collagen was represented by small dispersed groups of fibrils just beneath the basement membrane of the bile duct. The basement membrane of the bile duct was frequently reduplicated into a few or as many as 10 layers of laminae densae, with scattered collagen fibrils between these laminae. The diameters of the fibrils of both groups of collagen increased in relation to the calibre of the bile duct, whereas at any given place in Glisson's sheath bile epithelium-associated collagen fibrils had a smaller diameter compared with those of the fibroblast-associated fibrils. The increment in fibril diameter along the bile duct is considered to be correlated with the increase in mechanical stress acting on Glisson's sheath. The difference in diameter between the 2 populations as well as the incorporation of fibrils between the laminae densae of the basement membrane of the bile duct supports the view that the bile epithelium-associated collagen is produced by the epithelial cells of the bile duct, thus having a different origin from that of fibroblast-associated collagen. These findings provide the first evidence that the epithelial cells of the interlobular bile duct produce fibril-forming collagen. Furthermore, it is suggested that cholestasis stimulates the epithelial cells of interlobular bile duct to increased synthesis of fibril-forming collagen that is also produced by these cells under physiological conditions.

Studies of tetraploid[harr ]diploid (4n[harr ]2n) mouse chimaeras have demonstrated unequal contributions of 4n cells to different tissues of the midgestation conceptus. Such a pattern has also been reported in chimaeras as early as E3.5d, which show an enhanced contribution of 4n cells to the mural trophectoderm (Everett & West, 1996). In this study, sectioned 4n[harr ]2n and 2n[harr ]2n control chimaeric blastocysts were digitised and reconstructed in 3 dimensions (3-D). The 3-D images revealed only limited mixing of cells from the 2 contributing embryos of individual blastocysts in both chimaera groups. Consequently, the distribution pattern of the 2 cell types was dependent on the spatial relationship between the orientation of the blastocyst and the boundary between the 2 clusters of cells. The distribution patterns observed were not strikingly different for 4n[harr ]2n and 2n[harr ]2n chimaeras, each showing some transgenic positive cell contribution in all 3 identifiable developmental lineages. It was notable, however, that in all 4n[harr ]2n blastocysts at least some 4n cells were located adjacent to the blastocyst cavity. Such a consistent pattern was not evident in 2n[harr ]2n chimaeras. This study has demonstrated the value of 3-D reconstructions for the analysis of spatial relationships of 2 cell populations in chimaeric mouse blastocysts.

Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.

Conventional carbohydrate histochemistry and the binding patterns of 21 lectins were analysed to characterise the glycoconjugate content in the components of the vomeronasal organ of the armadillo Chaetophractus villosus. The mucomicrovillous complex of the sensory epithelium bound most of the lectins studied. No reaction was observed with Con A, PSA, S-Con A and SBA, and the sustentacular cells were stained with UEA-I, DSL, LEL, STL and Con A. The vomeronasal receptor neurons were labelled with S-WGA, WGA, PNA, UEA-I, STL, Con A, S-Con A, ECL and RCA120. The basal cell layer reacted with S-WGA, WGA, LCA, UEA-I, DSL, LEL, STL, Con A, JAC and VVA. The nonsensory epithelium exhibited a differential staining in relation to the different components. The mucociliary complex stained with ECL, DBA, JAC, RCA120, STL, LCA, PHA-E, PHA-L, LEL, BSL-I and VVA. However, SJA and UEA-I stained the mucus complex lining a subpopulation of columnar cells. The cytoplasm and cell membranes of columnar cells was labelled with DBA, DSL and LCA. The apical region of these cells exhibited moderate reactivity with LEL and SJA. None of the lectins bound specifically to secretory granules of the nonsecretory cells. Basal cells of the nonsensory epithelium were labelled with DSL, LEL, LCA, BSL- I and STL. The vomeronasal glands showed a positive reaction with WGA, DSL, LEL, LCA, DBA, PNA, RCA120 and SBA. Subpopulations of acinar cells were observed with ECL, S-WGA, Con A, S-Con A and DBA. PNA and RCA120 stained the cells lining the glandular ducts. In comparison with previous results obtained in the olfactory mucosa of the same group of armadillos, the carbohydrate composition of the vomeronasal organ sensory epithelium differed from the olfactory sensory epithelium. This is probably related to the different nature of molecules involved in the perireceptor processes.

The lectin binding characteristics of mouse placental cells were examined. Wax embedded tissue sections of placentae from d 14 pregnant mice were stained with 26 lectins, with a wide range of sugar specificities. Cell cultures prepared from d 14 mouse placentae and cultured for 24 h were stained with 7 of the lectins to determine if they could be used as markers for the different trophoblast cells in culture. In tissue sections all placental cell populations bound lectin but no lectin bound specifically to any single trophoblast population. All the lectins which bound to layer 1 cytotrophoblast lining the maternal blood spaces of the labyrinthine placenta also bound to the fetal endothelium of the labyrinthine placenta. Binding of lectin appeared strongest on the adluminal membrane of these cell populations suggesting a role for the carbohydrate moieties in nutrient transfer. Few lectins bound to junctional zone trophoblast. Overall, the binding of lectin to cultured cells did not correlate exactly with lectin binding to the cell populations in tissue sections. The value of lectins as markers for placental cells in culture was therefore found to be limited. Our findings indicate that carbohydrate expression by at least some placental cells may vary in culture from that expressed by the cells in vivo with obvious concerns for the validity of functional in vitro studies.

The cortical representations of the vibrissae of the rat form a matrix in which each whisker has its own area of cortex, called a ‘barrel’. The afferent pathways from the periphery travel first to the trigeminal nuclei and thence via the ventroposteromedial thalamus (VPM) to the cortical barrels have been described in detail. We have studied the output from barrels by filling adjacent areas of the primary somatosensory cortex (SI) with either Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) and demonstrating the course and terminations of the axons that arise within the barrel fields. The method not only dramatically illustrates the previously described corticothalamic pathway to VPM but also demonstrates a strict topography in the cortical afferents to the thalamic reticular nucleus (RT). Cells supplying the RT projection are found below the barrels in layer IV. Connections to the posterior thalamus, on the other hand, have no discernible topography and are derived from cortical areas surrounding the barrels. Thus the outputs of these ‘septal’ areas return to the region from which they receive thalamic input. The corticocortical connections are also visible in the same material. Contralateral cortical connections arise from the cells of the septa between barrels. The projections to secondary somatosensory area (SII) are mirror images of the barrel pattern in SI with rather more overlap but nonetheless a recognisable topography.

The pulmonary arterial smooth muscle cell (SMC) cytoskeleton was studied in tissue from 36 piglets aged from within 5 min of birth to 21 d of age, and in 8 adults. An additional 16 piglets were made pulmonary hypertensive by exposure to hypobaric hypoxia (50.8 kPa) for 3 d. In conduit intrapulmonary elastic arteries α, β and γ actin, the 204, 200 and 196 kDa myosin heavy chain (MHC) isoforms and vinculin were localised by immunohistochemistry. The total actin content, the proportion of monomeric to filamentous α and γ actin and changes in the proportions of the MHC isoforms were determined biochemically. Dividing SMCs were localised and quantified using Ki-67. We found a transient reduction in immunohistochemical expression of γ actin, 204 kDa MHC isoform and vinculin at 3 and 6 d in the inner media, associated with a transient increase in Ki-67 labelling. The actin content also decreased at 3 and 6 d (P < 0.05), but there was a postnatal, permanent increase in monomeric actin, first the α then the γ isoform. The relative proportions of the MHC isoforms did not change between birth and adulthood in elastic pulmonary arteries but in muscular arteries the 200 kDa isoform increased between 14 d and adulthood. Pulmonary hypertension prevented both the immunohistochemical changes and the postnatal burst of SMC replication and prevented the transient postnatal reduction in actin content. These findings suggest that rapid remodelling of the actin cytoskeleton is an essential prerequisite of a normal postnatal fall in pulmonary vascular resistance.

X-ray microtomography was used to study the mineral concentrations in sequential slices of enamel of 5 mandibular incisors which showed an increase from ∼ 1.0 to ∼ 2.7 g cm−3 from the apex towards the incisal end. For points at the same distance from the apex, there were differences up to 0.6 g cm−3 between the teeth. The change of mean concentrations in the slices with distance could be modelled as (different) saturating exponentials. Under the assumption of a uniform growth rate of a mandibular incisor of 0.6 mm per day and a common time origin for the start of maturation (taken as a mineral concentration of 1 g cm−3), the distances were transformed to a common time frame to give a pooled data set. A single saturating exponential could be fitted to this pooled transformed data; this was: Cm = 2.84−1.94exp (−0.18d) where Cm is the mean mineral concentration (g cm−3) and d the time (days) from the start of maturation. This gives an asymptotic concentration of 2.84 g cm−3 towards the incisal end, with a time constant of 7.7 days. The mineral concentration distribution functions were found to be more positively skewed closer to the apex, but more negatively skewed towards the incisal end. The difference between the higher mineral concentration in the outer enamel and the enamel near the amelodentinal junction (ADJ) was ∼ 3%. The direction of maximum increase in concentration from the outer enamel surface to the ADJ meets the boundary of the ADJ at ∼ 80°. Three dimensional surface rendering of isodensity contours showed that the previously described C-shaped pattern of mineralisation is not solely a surface phenomenon, but extends through the depth of the enamel.

A 3-dimensional computerised system with landmark representation of the soft-tissue facial surface allows noninvasive and fast quantitative study of facial growth. The aims of the present investigation were (1) to provide reference data for selected dimensions of lips (linear distances and ratios, vermilion area, volume); (2) to quantify the relevant growth changes; and (3) to evaluate sex differences in growth patterns. The 3- dimensional coordinates of 6 soft-tissue landmarks on the lips were obtained by an optoelectronic instrument in a mixed longitudinal and cross-sectional study (2023 examinations in 1348 healthy subjects between 6 y of age and young adulthood). From the landmarks, several linear distances (mouth width, total vermilion height, total lip height, upper lip height), the vermilion height-to-mouth width ratio, some areas (vermilion of the upper lip, vermilion of the lower lip, total vermilion) and volumes (upper lip volume, lower lip volume, total lip volume) were calculated and averaged for age and sex. Male values were compared with female values by means of Student's t test. Within each age group all lip dimensions (distances, areas, volumes) were significantly larger in boys than in girls (P < 0.05), with some exceptions in the first age groups and coinciding with the earlier female growth spurt, whereas the vermilion height-to-mouth width ratio did not show a corresponding sexual dimorphism. Linear distances in girls had almost reached adult dimensions in the 13–14 y age group, while in boys a large increase was still to occur. The attainment of adult dimensions was faster in the upper than in the lower lip, especially in girls. The method used in the present investigation allowed the noninvasive evaluation of a large sample of nonpatient subjects, leading to the definition of 3-dimensional normative data. Data collected in the present study could represent a data base for the quantitative description of human lip morphology from childhood to young adulthood.

The histology and structure of 38 spleens of the dromedary (aged 0.5–15 y) were studied in relation to age. The spleen was found to have a thick capsule (292±106 mm) divided into an outer layer (113±39 mm) composed mainly of connective tissue and an inner layer (180±81 mm) consisting mainly of smooth muscle cells. Vascular and avascular trabeculae extend from the capsule, the former containing arteries and nerves but no trabecular veins, the latter being divided structurally into primary and secondary trabeculae. Subcapsular and peritrabecular blood sinuses around primary and vascular trabeculae are unique to the camel spleen. The central artery emerges from the periarterial lymphatic sheath and branches into up to 4 penicilli which extend as sheathed arterioles (42±8 μm). These are found near or surrounded by blood sinusoids of the red pulp. A wide marginal zone surrounds the white pulp and contains sheathed arteries but no marginal sinuses. The red pulp is characteristically divided into cords by secondary trabeculae and contains venous sinusoids of different sizes. The camel spleen is of a sinusal type that can store blood. The thick muscular capsule and trabeculae pump the stored blood according to the body's need. Both closed and open circulations are found. The venous return is unique as the blood flow is from the venous sinusoids of the red pulp to the peritrabecular sinuses to the subcapsular sinuses to the splenic vein. No significant structural differences related to age were found.

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.

Membranous (M) cells in follicle-associated epithelium (FAE) play an important role in the mucosal immunity through transport of a variety of foreign antigens to the underlying mucosa-associated lymphoid tissue (MALT). We aimed to investigate the ultrastructure of M cells in the FAE covering nasal-associated lymphoid tissue (NALT) both in specific pathogen-free (SPF) rats and in conventional environment-adapted (SPF-CV) rats aged 8–38 wk. In NALT of both SPF and SPF-CV rats, FAE included the nonciliated microvillous cell, which appears to be an analogue of M cell previously described in other MALT. In SPF rats, M cells increased in number only slightly with age, and they maintained morphological uniformity irrespective of age. In SPF-CV rats, M cells selectively increased in number resulting in prominent expansion of FAE surface area in parallel with the duration of maintenance in a conventional environment. In addition, M cells in SPF-CV rats showed heterogeneity in their surface morphology such as the length and number of microvilli and cell surface area and outline. In addition, the FAE was stratified by various subtypes of M cells, which were characterised by several subcellular alterations including the presence of many keratin filaments, homogeneous dark bodies and extensive cytoplasmic interfoliation with wide intercellular spaces filled with amorphous proteinaceous material. These characteristics of M cells in SPF-CV rat were intimately related with a preferential influx of immunocompetent cells into the FAE, which was not seen or was very rare in SPF rats irrespective of age. The results suggest the possibility that NALT may effectively carry out the mucosal immune response against antigenic stimuli of different magnitude through the unique dynamics of M cells which seem to be influenced by the infiltration of immunocompetent cells.

The cellular ensheathment of capillaries in the 3 outer capillary layers of the central retina of the adult tree shrew Tupaia belangeri was studied quantitatively by transmission electron microscopy. Using a stereological approach, the relative surface of capillary basal lamina ensheathed by Müller cells and by nonmacroglial cells (collectively termed non-Müller cells) was estimated in 5 animals. The participation of Müller cells was distinctly different in the 3 capillary layers studied. In the outermost capillary layer 1, the mean (standard deviation) percentage surface coverage by non-Müller cell processes was 46.8 (15.3)%. Much less of the capillary basal lamina was ensheathed by non-Müller cells in capillary layers 2 and 3 (3.0 (2.1)% and 0.3 (0.3)% respectively). The observed total variation of the stereological estimates for the surface fraction of Müller cells (expressed as the between-subject coefficient of variation) was significantly higher in capillary layer 1 (28.8%) compared with capillary layers 2 (2.2%) and 3 (0.3%). In capillary layer 1, the high observed total variation was due to a high biological variation among animals for the fractions of both Müller cell and non-Müller cell ensheathment. The rare occurrence of direct contacts between the capillary basal lamina and the perikarya of either microglial cells (capillary layer 3) or amacrine cells (capillary layer 2) corresponded well to the low stereological values obtained for the relative capillary surface ensheathed by non-Müller cells in these capillary layers. Previously, extensive and frequent contacts between the basal lamina of capillaries belonging to capillary layer 1 and horizontal cells had been observed in single sections. The present study quantitatively demonstrates a marked paucity of macroglial investment of capillaries located in capillary layer 1 of Tupaia. It can be concluded that horizontal cells ensheath most of the capillary surface not invested by Müller cells.

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.

The degrees of mediolateral asymmetry of the patellar articular facet, as well as the median and lateral articular angles of the facet, were compared across samples of recent humans and of Pleistocene archaic and modern fossil humans. All samples exhibit considerable variability in these patellar proportions. The articular angles are similar across the different samples, but there is a trend towards decreasing lateral angles with decreasing robusticity. The archaic humans exhibit significantly more symmetry of the medial and lateral facets than do any of the recent human samples. However, given the variability in medial versus lateral patellofemoral contact forces documented for extant humans and the roles of the distal oblique portions of vastus medialis and vastus lateralis in patellar stabilisation, it is unclear to what extent this variation in patellar articular proportions may affect knee kinesiology. The contrasts may be related to different levels of patellar stability and/or musculoskeletal hypertrophy, but they appear unlikely to have affected primary knee function.

In most mammalian species the cardiac skeleton is composed of coarse collagen fibres, fibrocartilage, and pieces of hyaline cartilage. Bone, the os cordis, is a regular constituent of the ruminant heart. The cardiac skeleton of the otter (Lutra lutra) has not previously been described. The skeleton in 30 otter hearts was studied by x-ray analysis and light microscopy. Serial sections were cut parallel to the atrioventricular plane and histochemical staining methods were performed to identify connective tissue fibres, glycosaminoglycans, mineral deposits, and bone. Age and sex of the animals under investigation were considered. The otter heart skeleton was composed of coarse collagen fibres with intercalated pieces of fibrous and/or hyaline cartilage, calcified cartilage, and lamellar bone with red or white marrow. Pieces of hyaline cartilage were not clearly defined: a perichondrial layer was missing and coarse connective tissue continuously transformed into fibrous and hyaline cartilage. In both sexes the amount of cartilage and bone were found to increase with age.

Our results establish the presence of bony material in the heart skeleton of the otter, a small mammalian species. This finding indicates that differentiation of bone is not exclusively related to the size of the organ. Increasing amounts of calcified cartilage and bone correlated with increasing age.