The identification of myogenic precursor cells (mpc) is a key factor in determining the early events in the myogenesis and regeneration of skeletal muscle. Although satellite cells have long been established as the providers of myoblastic cells, very little is really known (apart from their anatomical location in relation to muscle fibres and their ability to migrate) about the precise role of satellite cells in myogenesis. Numerous techniques for labelling mpc have been devised, but none of these has proven to be completely reliable in firmly establishing the origin of myogenic cells. The use of tritiated thymidine to label DNA in proliferating mpc (which are not specifically distinguishable at the time) and the subsequent location of their labelled progeny in myotube nuclei has revealed a great deal of data on the timing of myogenesis, but not about the nature of mpc themselves. DNA synthesis can also be detected by antibodies to the thymidine analogue, bromodeoxyuridine, and also by antibody staining for proliferating nuclear cell antigen. Like tritiated thymidine, these other markers are not specific for muscle but are general markers for DNA synthesis. In situ hybridisation of various muscle-specific genetic markers and their products has been informative, as has immunolabelling of myogenin, MyoD1 and desmin. Desmin labelling has been particularly instructive in identifying mpc because it is one of the first muscle-specific proteins to be produced in mpc. This review covers some of the techniques mentioned above and their usefulness in determining the early events in myogenesis.

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.

Previous studies on ageing animal and human subjects have demonstrated a significant overall decline in neuronal numbers in the myenteric plexus of the enteric nervous system (ENS). Our study aimed to confirm this observation by counting myenteric neurons stained with the panneuronal markers PGP 9.5 and NADH-diaphorase. We also wished to examine the possibility that particular subpopulations of neurons are vulnerable. Therefore, we have immunostained and counted a number of nerve cell groups within the myenteric plexus of old and young Sprague Dawley rats using markers which reflect some of the neuronal phenotypes present, including ChAT and VIP. The number of neurons demonstrating NADH-diaphorase activity was significantly reduced (P<0.05) by approximately 15% in old rats. However, the number of neurons stained for PGP 9.5 immunohistochemistry was not reduced and demonstrated larger numbers of neurons than the NADH-diaphorase method. None of the other neuronal markers studied showed any significant reductions with age. In contrast to previous work, this study has gathered little evidence for extensive cell loss in the myenteric plexus of the aged rat, either in overall populations, or in any of the principal functional groups of neurons.

Collagen fibre organisation and fibril size were studied in the buccal gingival and hard palate mucosa of Macacus rhesus monkey. Light and electron microscopy analysis showed connective papillae exhibiting a similar inner structure in the different areas examined, but varying in distribution, shape and size. Moving from the deep to surface layers of the buccal gingival mucosa (free and attached portions), large collagen fibril bundles became smaller and progressively more wavy with decreasing collagen fibril diameter. This gradual diameter decrease did not occur in the hard palate mucosa (free portion, rugae and interrugal regions) where the fibril diameter remained constant. A link between collagen fibril diameter and mechanical function is discussed.

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.

A fluorescence confocal microscopy technique was employed to obtain subsurface images of nerve and microvascular structure in the vas deferens and colon of the living rat. The use of dual labelling with vital dyes and 2-channel confocal acquisition allowed differentiation of microscopic structure at both low and higher magnification. Characteristic staining patterns of nerves and blood vessels were repeatedly obtained in each tissue, suggesting the potential of this technique for studying morphological changes associated with surgical procedures and/or models of neuronal or vascular pathology.

The fibrillar architecture in the general matrix of softened cartilage has been compared with that of the normal matrix using both Nomarski light microscopy and transmission electron microscopy with combined stereoscopic reconstruction. A pseudorandom network developed from an overall radial arrangement of collagen fibrils is the most fundamental ultrastructural characteristic of the normal general matrix. This, in turn, provides an efficient entrapment system for the swelling proteoglycans. Conversely, the most distinctive feature of the softened matrix is the presence of parallel and relatively unentwined fibrils, strongly aligned in the radial direction. The presence of an optically resolvable fibrous texture in the softened cartilage matrix indicates the presence of discrete bundles of closely packed and aligned fibrils at the ultrastructural level of organisation. The general absence of such texture in the normal cartilage general matrix is consistent with the much greater degree of interconnectedness and related short-range obliquity in the fibrillar architecture, hence the importance of the term pseudorandom network. A mechanism of structural transformation is proposed based on the important property of lateral interconnectivity in the fibrils which involves both entwinement and nonentwinement based interactions. The previously reported difference in intrinsic mechanical strength between the normal and softened matrices is consistent with the transformation model proposed in this study.

The formation of the first epidermal generations and in particular of the shedding complex in the developing scales of the lizards Anolis lineatopus and Podarcis muralis was studied by electron microscopy. The initially linear and bilayered epidermis turned into symmetric papillae, which became asymmetric scales. The outer periderm was more electrondense than the following layer, provisionally named ‘inner periderm’ but probably derived from the basal layer. Coarse thick filaments progressively filled the cytoplasm of the inner periderm or formed reticulate bodies resembling avian peridermal granules. Peridermal cells cornified slightly and could be shed in ovo. From the basal layer various suprabasal epithelial layers were produced. The first layer contained keratohyalin-like granules and was identified as a clear layer. Beneath it a spinulated (Anolis) or serrated (Podarcis) oberhäutchen differentiated. The clear and ‘oberhäutchen’ layers constitute the first shedding complex. The first epidermal layers that were shed consisted of flaking periderm or periderm together with the clear layer. The differentiation of a mesos layer was under way before hatching, when the epidermal morphology resembled a stage 4–5 of the adult shedding cycle. Fibroblasts under the inner side of the scale made few contacts with the basement membrane and their cytoplasmic elongations were mostly oriented parallel to the dense lamina. Instead, fibroblasts under the basal layer of the outer scale surface (BLOS) made numerous contacts with the basement membrane, suggesting that more dermal–epidermal interactions take place on this side of the scale.

Wound healing has been studied in the surface ectoderm overlying the midbrain region of stages 16–20 chick embryos by light microscopy, scanning and transmission electron microscopy, and immunofluorescent techniques. The embryos were divided into 6 groups, i.e. stages 16–17 for groups I, V and VI, and stages 19–20 for groups II, III and IV. For groups I and II embryos, a longitudinal incision about 0.6 mm was made close to the dorsal midline and the embryos incubated for varying periods of time up to 24 h. To determine the role of actin in the process of healing, selected groups I and II embryos were stained with FITC phalloidin and the wound margins examined using a confocal microscope. Wounds of all embryos in group I and about 20% in group II healed completely within 24 h of reincubation. The process of healing involved a change in the shapes of the ectodermal cells at the wound ends. This appeared as a zipping-up of the wound from both ends. In about 80% of group II embryos where healing did not occur, wound gaping was marked. Intense actin staining (actin cable) was observed at the wound margins of groups I and II embryos suggesting that the actin purse-string mechanism may play a role during wound healing in this epithelial model. The role of tension in wound healing was also determined by placing 2 secondary wounds about 0.5–0.7 mm long close to, and at right angles to the ends of the primary wound in groups III and V embryos. The procedure decreased the tension within the ectodermal cells at the wound ends. Groups IV and VI embryos served as controls for groups III and V embryos, respectively. Healing of both primary and secondary wounds after reduction of tension was rapid. Most primary wounds in group V embryos healed completely within 3 h of reincubation and the rate of reepithelialisation after the reduction of tension was about 160% more than that in group VI (control) embryos. Similarly, most primary wounds in group III embryos were almost closed within 6 h of reincubation. Here, the rate of reepithelialisation was 80% more than that in group IV (controls). Thus tension is an important factor in wound healing in this model.

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.

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.

We sought to study the effect of synovectomy on the surface morphology of articular cartilages of the rabbit knee using scanning electron microscopy (SEM). Fifteen rabbits were surgically synovectomised and allowed to regenerate their synovia during time intervals ranging from 3 to 44 wk. Cartilage specimens were shaved from 5 distinct articular sectors of synovectomised, contralateral and sham-operated knees and prepared for SEM using tannic acid. Applying structural reinforcement by tannic acid was found to secure the in vivo surface morphology of the cartilages and thus production of surface irregularities during preparation was excluded. The surface morphology of cartilages both from the synovectomised and contralateral joints was found to differ from that of intact healthy rabbits. A ‘chaotic’ nature of the altered cartilaginous morphology persisted as late as 44 wk postsynovectomy. Cartilages from sham-operated joints did not differ detectably from normal cartilages.

The endocrine pancreas of the toad consists of rounded islets of various sizes embedded in the exocrine tissue. Isolated cells are also present. At least 4 types of endocrine cell are distinguishable by shape, size and electrondensity of the secretory granules as well as by their immunoreactivity with different antisera: insulin, somatostatin, pancreatic polypeptide, and glucagon cells. Insulin cells can be divided into 2 types according to their cytoplasmic electrondensity. Colocalisation of different hormones in the same cell is rarely observed. The close contact between endocrine and exocrine cells and the scarcity of nerve supply is indicative of a paracrine control of hormone secretion.

In the acinus of the sheep and mouse liver, lymphatic vessels are restricted to the portal tracts. Vessels less than about 25 μm across form a network around portal venules, and are closely associated with the limiting plate of hepatocytes. The perisinusoidal space of Disse is continuous with the interstitial space of the portal tracts at the origin of the sinusoids. It seems likely that excess interstitial fluid derived from the sinusoids flows along the perisinusoidal space of Disse to enter the portal tracts near the portal venules, and then enters the small lymphatics which lie adjacent to those venules. It then enters the larger vessels, which are adjacent to hepatic arterioles and bile ductules.

Rat liver ultrastructure was investigated after partial hepatectomy (PH), by scanning and transmission electron microscopy. Portal pressure was monitored before and after PH and, after killing performed at 6, 12, 24, 48 h and 10 d, regenerating livers were fixed by portal vein perfusion under haemodynamic conditions identical to those existing in vivo. An early and persistent increase in portal pressure after PH was found (P<0.01 for normal vs sham-operated controls). Ultrastructural study showed sinusoid dilatation and disappearance of the sieve-plate arrangement of small endothelial pores, thus leaving the parenchymal liver cell surface directly exposed to portal blood. Widening of sinusoids, endothelial fenestrations, intercellular spaces and spaces of Disse, was accompanied by dilatation of bile canaliculi. At 10 d, liver ultrastructure had returned to normal. Our observations suggest that a rise in portal pressure, as a consequence of PH, may be related to the observed ultrastructural changes in the liver.

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.

Muscle fibre type composition and distribution in the biceps brachii (long head) and triceps brachii (long head) of the rat and rabbit were investigated using the following histochemical techniques: myosin ATPase, with preincubation at pH 10.4 and 4.35; succinate dehydrogenase (SDH) and glycogen phosphorylase. The muscle fibres were classified into slow-twitch (SO), fast-twitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG and FOg) and fast-twitch oxidative fibres (FO). Significant differences in the regional distribution of muscle fibre types have been observed between the rat and the rabbit. In the rat, SO fibres were restricted to the deep regions of both biceps and triceps brachii, whereas FG fibres were located in the intermediate and superficial regions (the superficial regions contained the highest percentages of FG fibres). In the rabbit, SO and FG fibres were spread over the entire muscle, although SO and FG fibres were most abundant in the deep and superficial regions respectively. These findings indicate that the biceps and triceps brachii are more regionalised in the rat than in the rabbit.

Atrioventricular (AV) nodal conduction time is known to be modulated by the autonomic nervous system. The presence of numerous parasympathetic and sympathetic nerve fibres in association with conduction tissue in the heart is well authenticated. In this study, confocal microscopy was used to image the distribution of antibodies directed against the general neuronal marker PGP 9.5, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and β1 and β2-adrenoreceptors. Serial 12 μm sections of fresh frozen tissue taken from the frontal plane of the rat atrioventricular node, His bundle and bundle branches were processed for histology, acetylcholinesterase (AChE) activity and immunohistochemistry. It was found that the AV and ventricular conduction systems were more densely innervated than the atrial and ventricular myocardium as revealed by PGP 9.5 immunoreactivity. Furthermore, the transitional cell region was more densely innervated than the midnodal cell region, while spatial distribution of total innervation was uniform throughout all AV nodal regions. AChE-reactive nerve processes were found throughout the AV and ventricular conduction systems, the spatial distribution of which was nonuniform exhibiting a paucity of AChE-reactive nerve processes in the central midnodal cell region and a preponderance in the circumferential transitional cell region. TH-immunoreactivity was uniformly distributed throughout the AV and ventricular conduction systems including the central midnodal and circumferential transitional cell regions. β1-adrenoreceptors were found throughout the AV and ventricular conduction systems with a preponderance in the circumferential transitional cell region. β2-adrenoreceptors were localised predominantly in AV and ventricular conduction systems with a paucity of expression in the circumferential transitional cell region. These results demonstrate that the overall uniform distribution of total nerve processes is comprised of nonuniformly distributed subpopulations of parasympathetic and sympathetic nerve processes. The observation that the midnodal cell region exhibits a differential spatial pattern of parasympathetic and sympathetic innervation suggests multiple sites for modulation of impulse conduction within this region. Moreover, the localisation of β2-ARs in the AV conduction system, with an absence of expression in the circumferential transitional cell layer, suggests that subtype-specific pharmacological agents may have distinct effects upon AV nodal conduction.

This study was undertaken to develop a functional model of otitis media with effusion (OME) in the pig (Sus scrofa), with the purpose of investigating the origin of lymphocytes populating the middle ear during the course of an inflammatory process. The relevance of the model to the human condition of OME is to a large extent dependent on the anatomical and physiological similarities between the middle ear cavity and the pharyngeal lymphoid tissue of the pig and man. Anatomical specimens were collected from 7 young Large White pigs to determine the gross anatomy of the middle ear cavity and the histological characteristics of the middle ear mucosa. It was found that the anatomy of the 3 parts of the middle ear cavity in man and in the pig is broadly similar, although some minor differences were observed. The porcine eustachian tube was seen to be cartilaginous throughout its length in contrast to the part osseous, part cartilaginous structure found in man; the porcine ossicles were slightly different in shape to those of man and the air cell system was situated inferior to the tympanic cavity in the pig as opposed to posteriorly in man. This paper describes the structure and morphology of the pig middle ear cavity and compares and contrasts it with that of man. The minor differences observed are of anatomical importance but do not diminish the usefulness of the pig middle ear cleft as a potential model for human middle ear disorders.

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.