Showing papers in "Journal of Anatomy in 1999"

Peripheral nerve regeneration and neurotrophic factors

Abstract: 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.

Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload.

Abstract: 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.

Release of vasoactive substances from endothelial cells by shear stress and purinergic mechanosensory transduction

Abstract: 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

Morphometric and ultrastructural changes with ageing in mouse peripheral nerve

Abstract: Qualitative and quantitative information is reported on the morphological changes that occur in nerve fibres and nonneuronal cells of peripheral nerve during the lifetime of the mouse. Tibial nerves of mice aged 6–33 mo were studied. With ageing, collagen accumulates in the perineurium and lipid droplets in the perineurial cells. Macrophages and mast cells increase in number, and onion bulbs and collagen pockets are frequently present. Schwann cells associated with myelinated fibres (MF) slightly decrease in number in parallel with an increase of the internodal length from 6 to 12 mo, but increase in older nerves when demyelination and remyelination are common. The unmyelinated axon to myelinated fibre (UA/MF) ratio was about 2 until 12 mo, decreasing to 1.6 by 27 mo. In older mice, the loss of nerve fibres involves UA (50% loss of 27–33 mo cf. 6 mo) more markedly than MF (35%). In aged nerves wide incisures and infolded or outfolded myelin loops are frequent, resulting in an increased irregularity in the morphology of fibres along the internodes. In the mouse there is an adult time period, 12–20 mo, during which several features of degeneration progressively appear, and an ageing period from 20 mo upwards when the nerve suffers a general disorganisation and marked fibre loss.

Morphological variation in great ape and modern human mandibles

Abstract: Adult mandibles of 317 modern humans and 91 great apes were selected that showed no pathology. Adult mandibles of Pan troglodytes troglodytes, Pongo pygmaeus pygmaeus and Gorilla gorilla gorilla and from 2 modern human populations (Zulu and Europeans from Spitalfields) were reliably sexed. Thirteen measurements were defined and included mandibular height, length and breadth in representative positions. Univariate statistical techniques and multivariate (principal component analysis and discriminant analysis) statistical techniques were used to investigate interspecific variability and sexual dimorphism in human and great ape mandibles, and intraspecific variability among the modern human mandibles. Analysis of interspecific differences revealed some pairs of variables with a tight linear relationship and others where Homo and the great apes pulled apart from one another due to shape differences. Homo and Pan are least sexually dimorphic in the mandible, Pan less so than Homo sapiens, but both the magnitude of sexual dimorphism and the distribution of sexually dimorphic measurements varied both among and between modern humans and great apes. Intraspecific variation among the 10 populations of modern humans was less than that generally reported in studies of crania (74.3% of mandibles were correctly classified into 1 of 10 populations using discriminant functions based on 13 variables as compared with 93% of crania from 17 populations based on 70 variables in one extensive study of crania). A subrecent European population (Poundbury) emerged as more different from a recent European population (Spitalfields) than other more diverse modern populations were from each other, suggesting considerable morphological plasticity in the mandible through time. This study forms a sound basis on which to explore mandibular variation in Neanderthals, early Homo sapiens and other more ancient fossil hominids.

Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture.

Abstract: 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.

Stimulation of the middle meningeal artery leads to Fos expression in the trigeminocervical nucleus: a comparative study of monkey and cat

Abstract: The pain of a migraine attack is often described as unilateral, with a throbbing or pulsating quality. The middle meningeal artery (MMA) is the largest artery supplying the dura mater, is paired, and pain-producing in humans. This artery, or its branches, and other large intracranial extracerebral vessels have been implicated in the pathophysiology of migraine by theories suggesting neurogenic inflammation or cranial vasodilatation, or both, as explanations for the pain of migraine. Having previously studied in detail the distribution of the second order neurons that are involved in the transmission of nociceptive signals from intracranial venous sinuses, we sought to compare the distribution of second order neurons from a pain-producing intracranial artery in both monkey and cat. By electrically stimulating the middle meningeal artery in these species and using immunohistochemical detection of the proto-oncogene Fos as a marker of neuronal activation, we have mapped the sites of the central trigeminal neurons which may be involved in transmission of nociception from intracranial extracerebral arteries. Ten cats and 3 monkeys were anaesthetised with alpha-chloralose and the middle meningeal artery was isolated following a temporal craniotomy. The animals were maintained under stable anaesthesia for 24 h to allow Fos expression due to the initial surgery to dissipate. Following the rest period, the vessel was carefully lifted onto hook electrodes, and then left alone in control animals (cat n = 3), or stimulated (cat n = 6, monkey n = 3). Stimulation of the left middle meningeal artery evoked Fos expression in the trigeminocervical nucleus, consisting of the dorsal horn of the caudal medulla and upper 2 divisions of the cervical spinal cord, on both the ipsilateral and contralateral sides. Cats had larger amounts of Fos expressed on the ipsilateral than on the contralateral side. Fos expression in the caudal nucleus tractus solitarius and its caudal extension in lamina X of the spinal cord was seen bilaterally in response to middle meningeal artery stimulation. This study demonstrates a comparable anatomical distribution of Fos activation between cat and monkey and, when compared with previous studies, between this arterial structure and the superior sagittal sinus. These data add to the overall picture of the trigeminovascular innervation of the intracranial pain-producing vessels showing marked anatomical overlap which is consistent with the often poorly localised pain of migraine.

The transitional zone and CNS regeneration

Abstract: Most nerves are attached to the neuraxis by rootlets. The CNS–PNS transitional zone (TZ) is that length of rootlet containing both central and peripheral nervous tissue. The 2 tissues are separated by a very irregular but clearly defined interface, consisting of the surface of the astrocytic tissue comprising the central component of the TZ. Central to this, myelin sheaths are formed by oligodendrocytes and the supporting tissue is astrocytic. Peripheral to it, sheaths are formed by Schwann cells which are enveloped in endoneurium. The features of transitional nodes are a composite of those of central and peripheral type. The interface is penetrated only by axons. It is absent at first. It is formed by growth of processes into the axon bundle from glial cell bodies around its perimeter. These form a barrier across the bundle which fully segregates prospectively myelinated axons. Rat spinal dorsal root TZs have been used extensively to study CNS axon regeneration. The CNS part of the TZ responds to primary afferent axon degeneration and to regenerating axons in ways which constitute a satisfactory model of the gliotic tissue response which occurs in CNS lesions. It undergoes gliosis and the gliotic TZ tissue expands distally along the root. In mature animals axons can regenerate satisfactorily through the endoneurial tubes of the root but cease growth on reaching the gliotic tissue. The general objective of experimental studies is to achieve axon regeneration from the PNS through this outgrowth and into the dorsal spinal cord. Since immature tissue has a greater capacity for regeneration than that of the adult, one approach includes the transplantation of embryonic or fetal dorsal root ganglia into the locus of an extirpated adult ganglion. Axons grow centrally from the transplanted ganglion cells and some enter the cord. Other approaches include alteration of the TZ environment to facilitate axon regeneration, for example, by the application of tropic, trophic, or other molecular factors, and also by transplantation of cultured olfactory ensheathing cells (OECs) into the TZ region. OECs, by association with growing axons, facilitate their extensive regeneration into the cord. Unusually, ventral motoneuron axons may undergo some degree of unaided CNS regeneration. When interrupted in the spinal cord white matter, some grow out to the ventral rootlet TZ and thence distally in the PNS. The DRTZ is especially useful for quantitative studies on regeneration. Since the tissue is anisometric, individual parameters such as axon numbers, axon size and glial ensheathment can be readily measured and compared in the CNS and PNS environments, thereby yielding indices of regeneration across the interface for different sets of experimental conditions.

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage.

Abstract: 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.

Three dimensional analysis of microaneurysms in the human diabetic retina.

Abstract: The retinal vasculature of postmortem normal human and diabetic eyes was studied using an immunohistochemical technique in conjunction with confocal laser scanning microscopy. The technique, which stained for von Willebrand factor, allowed both large areas of the retinal vasculature to be visualised and abnormalities to be studied in detail without disturbing the tissue architecture. Only one microaneurysm, defined as any focal capillary dilation, was observed in 10 normal eyes but numerous microaneurysms were seen in 4 out of 5 diabetic retinas; counts varied between 0 and 26 per 0.41 mm2 sample area. Microaneurysms were classified into 3 categories according to morphology: saccular, fusiform and focal bulges. Most were saccular, these having no preferred orientation. The majority of microaneurysms were associated with just 2 vessels suggesting they were unlikely to develop at vascular junctions. The majority were observed to originate from the inner nuclear layer and were therefore in the deeper part of the inner retinal capillary plexus. Variation in the staining of microaneurysms may correlate with endothelial dysfunction seen clinically as dye leakage during fluorescein angiography.

Sequential changes in trace metal, metallothionein and calmodulin concentrations in healing skin wounds.

Abstract: Metalloenzymes have an important role in repair and regenerative processes in skin wounds. Demands for different enzymes vary according to the phase in the healing cascade and constituent events. Sequential changes in the concentrations of calcium, copper, magnesium and zinc were studied in the incisional wound model in the rat over a 10 d period. Copper levels remained low (< 10 microg/g dry weight) throughout, but calcium, magnesium and zinc increased from wounding and peaked at about 5 d at a time of high inflammation, granulation tissue formation and epidermal cell proliferation. Metal concentrations declined to normal by 7 d when inflammation had regressed, re-epithelialisation of the wound site was complete and the 'normalisation' phase had commenced. Although the wound was overtly healed by 10 d, the epidermis was still moderately hyperplastic. In view of competitive binding of trace metals at membrane receptors and carrier proteins, the ratios or balance between these trace metals was examined and the significance is discussed. Using immunocytochemistry, we demonstrated increases in metallothionein immunoreactivity as an indication of zinc and copper activity in the papillary dermis and in basal epidermal cells near the wound margin 1-5 d after wounding. This is consistent with metalloenzyme requirements in inflammation and fibrogenesis. Calmodulin, a major cytosolic calcium binding protein was highest in maturing keratinocytes and in sebaceous gland cells of normal skin; it was notably more abundant in the epidermis near the wound margin and in re-epithelialising areas at a time when local calcium levels were highest.

Diffusion tensor imaging in biomechanical studies of skeletal muscle function

Abstract: In numerical simulations of skeletal muscle contractions, geometric information is of major importance. The aim of the present study was to determine whether the diffusion tensor imaging (DTI) technique is suitable to obtain valid input with regard to skeletal muscle fibre direction. The accuracy of the DTI method was therefore studied by comparison of DTI fibre directions in the rat tibialis anterior muscle with fascicle striation patterns visible on high-resolution magnetic resonance imaging (MRI) and with fibre directions in an actual longitudinal section (ALS) through the same muscle. The results showed an excellent qualitative agreement between high-resolution MRI and DTI. Despite less accurate quantitative comparison with ALS, it was concluded that DTI does indeed measure skeletal muscle fibre direction. After the experiment, it was possible to determine an appropriate voxel size (0.9 mm3) that provided enough resolution and acceptable accuracy (5°) to use DTI fibre directions in biomechanical analyses. Muscle deformation during contraction, resulting from a finite element simulation with a mesh that was directly generated from the experimental data, has been presented.

Median artery revisited.

Abstract: This study confirms that the median artery may persist in adult life in 2 different patterns, palmar and antebrachial, based on their vascular territory. The palmar type, which represents the embryonic pattern, is large, long and reaches the palm. The antebrachial type,which represents a partial regression of the embryonic artery is slender, short, and terminates before reaching the wrist. These 2 arterial patterns appear with a different incidence. The palmar pattern was studied in the whole sample (120 cadavers) and had an incidence of 20%, being more frequent in females than in males (1.3:1), occurring unilaterally more often than bilaterally (4:1) and slightly more frequently on the right than on the left (1.1:1). The antebrachial pattern was studied in only 79 cadavers and had an incidence of 76%, being more frequent in females than in males (1.6:1); it was commoner unilaterally than bilaterally (1.5:1) and was again slightly more prevalent on the right than on the left (1.2:1). The origin of the median artery was variable in both patterns. The palmar type most frequently arose from the caudal angle between the ulnar artery and its common interosseous trunk (59%). The antebrachial pattern most frequently originated from the anterior interosseous artery (55%). Other origins, for both patterns, were from the ulnar artery or from the common interosseous trunk. The median artery in the antebrachial pattern terminated in the upper third (74%) or in the distal third of the forearm (26%). However, the palmar pattern ended as the 1st, 2nd or 1st and 2nd common digital arteries (65%) or joined the superficial palmar arch (35%). The median artery passed either anterior (67%) or posterior (25%) to the anterior interosseous nerve. It pierced the median nerve in the upper third of the forearm in 41% of cases with the palmar pattern and in none of the antebrachial cases. In 1 case the artery pierced both the anterior interosseous and median nerves.

The electron microscope appearance of the subchondral bone plate in the human femoral head in osteoarthritis and osteoporosis.

Abstract: The subchondral bone plate supports the articular cartilage in diarthrodial joints. It has a significant mechanical function in transmitting loads from the cartilage into the underlying cancellous bone and has been implicated in the destruction of cartilage in osteoarthritis (OA) and its sparing in osteoporosis (OP), but little is known of its composition, structure or material properties. This study investigated the microscopic appearance and mineral composition of the subchondral bone plate in femoral heads from patients with OA or OP to determine how these correspond to changes in composition and stiffness found in other studies. Freeze-fractured full-depth samples of the subchondral bone plate from the femoral heads of patients with osteoarthritis, osteoporosis or a matched control group were examined using back scattered and secondary emission scanning electron microscopy. Other samples were embedded and polished and examined using back-scattered electron microscopy and electron probe microanalysis. The appearances of the samples from the normal and osteoporotic patients were very similar, with the subchondral bone plate overlayed by a layer of calcified cartilage. Osteoporotic samples presented a more uniform fracture surface and the relative thicknesses of the layers appeared to be different. In contrast, the OA bone plate appeared to be porous and have a much more textured surface. There were occasional sites of microtrabecular bone formation between the trabeculae of the underlying cancellous bone, which were not seen in the other groups, and more numerous osteoclast resorption pits. The calcified cartilage layer was almost absent and the bone plate was apparently thickened. The appearance of the osteoarthritic subchondral bone plate was, therefore, considerably different from both the normal and the osteoporotic, strongly indicative of abnormal cellular activity.

Localisation and quantitation of autonomic innervation in the porcine heart I: conduction system.

Abstract: This study was prompted by the prospect of transgenic pigs providing donor hearts for transplantation in human recipients. Autonomic innervation is important for the control of cardiac dynamics, especially in the conduction system. Our objective was to assess the relative distribution of autonomic nerves in the pig heart, focusing initially on the conduction system but addressing also the myocardium, endocardium and epicardium (see Crick et al. 1999). Quantitative immunohistochemical and histochemical techniques were adopted. All regions of the conduction system possessed a significantly higher relative density of the total neural population immunoreactive for the general neuronal marker protein gene product 9.5 (PGP 9.5) than did the adjacent myocardium. A similar density of PGP 9.5-immunoreactive innervation was observed between the sinus node, the transitional region of the atrioventricular node, and the penetrating atrioventricular bundle. A differential pattern of PGP 9.5-immunoreactive innervation was present within the atrioventricular node and between the components of the ventricular conduction tissues, the latter being formed by an intricate network of Purkinje fibres. Numerous ganglion cell bodies were present in the peripheral regions of the sinus node, in the tissues of the atrioventricular groove, and even in the interstices of the compact atrioventricular node. Acetylcholinesterase (AChE)-containing nerves were the dominant subpopulation observed, representing 60–70% of the total pattern of innervation in the nodal tissues and penetrating atrioventricular bundle. Tyrosine hydroxylase (TH)-immunoreactive nerves were the next most abundant neural subpopulation, representing 37% of the total pattern of innervation in the compact atrioventricular node compared with 25% in the transitional nodal region. A minor population of ganglion cell bodies within the atrioventricular nodal region displayed TH immunoreactivity. The dominant peptidergic nerve supply possessed immunoreactivity for neuropeptide Y (NPY), which displayed a similar pattern of distribution to that of TH-immunoreactive nerve fibres. Calcitonin gene-related peptide (CGRP)-immunoreactive nerves represented 8–9% of the total innervation of the nodal tissues and penetrating atrioventricular bundle, increasing to 14–19% in the bundle branches. Somatostatin-immunoreactive nerve fibres were relatively sparse (4–13% of total innervation) and were most abundant in the nodes, especially the compact atrioventricular node. The total pattern of innervation of the porcine conduction system was relatively homogeneous. A substantial proportion of nerve fibres innervating the nodal tissues could be traced to intracardiac ganglia indicative of an extensive intrinsic supply. The innervation of the atrioventricular node and ventricular conduction tissues was similar to that observed in the bovine heart, but markedly different to that of the human heart. It is important that we are aware of these findings in view of the future use of transgenic pig hearts in human xenotransplantation.

Supra and infralevator neurovascular pathways to the penile corpora cavernosa.

Abstract: The aim of this study was to provide a comprehensive description of both penile innervation and vascularisation. Eighty-five male cadavers were examined through gross and microscopic anatomical analysis. The pelvic nerve plexus had both parasympathetic and sympathetic roots. It was distributed to the external urethral sphincter giving rise to cavernous nerves which anastomosed in 70% of the cases with the pudendal nerve in the penile root. Accessory pudendal arteries were present in the pelvis in 70% of the cases, anastomosing in 70% of the cases with the cavernous arteries that originated from the pudendal arteries. Transalbugineal anastomoses were always seen between the cavernous artery and the spongiosal arterial network. There were 2 venous pathways, 1 in the pelvis and 1 in the perineum with a common origin from the deep dorsal penile vein. It is concluded that there are 2 neurovascular pathways destined for the penis that are topographically distinct. One is located in the pelvis and the other in the perineum. We were unable to determine the functional balance between these 2 anastomosing pathways but experimental data have shown that they are both involved in penile erection. These 2 neurovascular pathways, above and below the levator ani, together with their anastomoses, form a neurovascular loop around the levator ani.

Localisation and quantitation of autonomic innervation in the porcine heart II: endocardium, myocardium and epicardium.

Abstract: The immunological problems of pig hearts supporting life in human recipients have potentially been solved by transgenic technology. Nevertheless, other problems still remain. Autonomic innervation is important for the control of cardiac dynamics and there is evidence suggesting that some neurons remain intact after transplantation. Previous studies in the human heart have established regional differences in both general autonomic innervation and in its component neural subpopulations. Such studies are lacking in the pig heart. Quantitative immunohistochemical and histochemical techniques were used to demonstrate the pattern of innervation in pig hearts (Sus scrofa). Gradients of immunoreactivity for the general neural marker protein gene product 9.5 were observed both within and between the endocardial, myocardial and epicardial plexuses throughout the 4 cardiac chambers. An extensive ganglionated plexus was observed in the epicardial tissues and, to a lesser extent, in the myocardial tissues. The predominant neural subpopulation displayed acetylcholinesterase activity, throughout the endocardium, myocardium and epicardium. These nerves showed a right to left gradient in density in the endocardial plexus, which was not observed in either the myocardial or epicardial plexuses. A large proportion of nerves in the ganglionated plexus of the atrial epicardial tissues displayed AChE activity, together with their cell bodies. Tyrosine hydroxylase (TH)-immunoreactive nerves were the next most prominent subpopulation throughout the heart. TH-immunoreactive cell bodies were observed in the atrial ganglionated plexuses. Endocardial TH- and NPY-immunoreactive nerves also displayed a right to left gradient in density, whereas in the epicardial tissues they showed a ventricular to atrial gradient. Calcitonin gene-related peptide (CGRP)-immunoreactive nerves were the most abundant peptide-containing subpopulation after those possessing NPY immunoreactivity. They were most abundant in the epicardial tissues of the ventricles. Several important differences were observed between the innervation of the pig heart compared with the human heart. These differences may have implications for the function of donor transgenic pig hearts within human recipients.

In situ hybridisation study of type, I, II, X collagens and aggrecan mRNas in the developing condylar cartilage of fetal mouse mandible

Abstract: The aim of this study was to investigate the developmental characteristics of the mandibular condyle in sequential phases at the gene level using in situ hybridisation. At d 14.5 of gestation, although no expression of type II collagen mRNA was observed, aggrecan mRNA was detected with type I collagen mRNA in the posterior region of the mesenchymal cell aggregation continuous with the ossifying mandibular bone anlage prior to chondrogenesis. At d 15.0 of gestation, the first cartilaginous tissue appeared at the posterior edge of the ossifying mandibular bone anlage. The primarily formed chondrocytes in the cartilage matrix had already shown the appearance of hypertrophy and expressed types I, II and X collagens and aggrecan mRNAs simultaneously. At d 16.0 of gestation, the condylar cartilage increased in size due to accumulation of hypertrophic chondrocytes characterised by the expression of type X collagen mRNA, whereas the expression of type I collagen mRNA had been reduced in the hypertrophic chondrocytes and was confined to the periosteal osteogenic cells surrounding the cartilaginous tissue. At d 18.0 of gestation before birth, cartilage-characteristic gene expression had been reduced in the chondrocytes of the lower half of the hypertrophic cell layer. The present findings demonstrate that the initial chondrogenesis for the mandibular condyle starts continuous with the posterior edge of the mandibular periosteum and that chondroprogenitor cells for the condylar cartilage rapidly differentiate into hypertrophic chondrocytes. Further, it is indicated that sequential rapid changes and reductions of each mRNA might be closely related to the construction of the temporal mandibular ramus in the fetal stage.

Regional differences in fibre type composition in the human temporalis muscle.

Abstract: 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 microm2, 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 microm2 and 1206 microm2, 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 alpha + 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.

Increase in liver pigmentation during natural hibernation in some amphibians

Abstract: The amount/distribution of liver melanin in 3 amphibian species (Rana esculenta, Triturus a. apuanus, Triturus carnifex) was studied during 2 periods of the annual cycle (summer activity–winter hibernation) by light and electron microscopy, image analysis and microspectrofluorometry. The increase in liver pigmentation (melanin content) during winter appeared to be correlated with morphological and functional modifications in the hepatocytes, which at this period were characterised by a decrease in metabolic activity. These findings were interpreted according to the functional role (e.g. phagocytosis, cytotoxic substance inactivation) played by the pigment cell component in the general physiology of the heterothermic vertebrate liver and, in particular, in relation to a compensatory engagement of these cells against hepatocellular hypoactivity during the winter period.

Epidermal differentiation during carapace and plastron formation in the embryonic turtle Emydura macquarii.

Abstract: As part of a large comparative study on the development of reptilian skin, we provide the first ultrastructural description of differentiation of the epidermis of the carapace and plastron in the Chelonia, using the Australian pleurodiran turtle Emydura macquarii as a model. The epidermis is initially composed of an external flat peridermis and a basal layer of cuboidal cells. During differentiation, the peridermis darkens, flakes off and is partially lost before hatching. Four to 6 layers of flat cells containing lipids and mucus form from the basal layer beneath the external peridermis. Because such cells are found only during embryogenesis, we have referred to these layers as embryonic epidermis. They contain reticulate bodies made of a meshwork of coarse filaments similar to those described in the inner peridermis of lizard and bird embryos. In advanced embryos, cells of the embryonic epidermis condense into a thin dark stratum which is subsequently lost after hatching. The lowermost 2 layers of the embryonic epidermis keratinise, as for a typical lepidosaurian α-layer. A splitting zone is progressively formed beneath the α-layer to separate the embryonic epidermis from the underlying β-layer. Patterns of cytodifferentiation of the β-synthesising cells over the carapace and plastron essentially resemble those of the lepidosaurian epidermis. The β-keratin matrix initially accumulates among ribosomes as round bodies not clearly surrounded by a membrane. These bodies appear not to be derived from the Golgi apparatus. Melanosomes and other dark granules of uncertain nature are present among early differentiating β-cells. The round β-keratin bodies merge with the dense bodies to produce the definitive variegated pattern of the mature β-keratin layer. The histochemistry suggests that calcium combines with organic molecules within β-keratinising cells to harden the tissue. In contrast to the β-keratin cells of lizards and snakes, cells of the mature β-keratin layer of E. macquarii maintain their cell boundaries in part or completely, a characteristics shared with the β-keratin layer of Sphenodon and crocodilians.

Expression of tissue type and urokinase type plasminogen activators as well as plasminogen activator inhibitor type-1 and type-2 in human and rhesus monkey placenta.

Abstract: 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

Immuno-inflammatory cell dynamics during cutaneous wound healing.

Abstract: The process of wound healing begins with an inflammatory reaction that is principally dependent on cellular immune elements. Although the involvement in wound healing of leucocytes that mediate nonspecific immunity (e.g. neutrophils and macrophages) is well known, the participation of cells which prime the immune reaction, i.e. the lymphocytes, requires further investigation. This study was performed to examine the temporal sequence and kinetics of these cells during cutaneous wound repair. The model selected was a full-thickness skin excisional wound made on the flanks of female Wistar rats. At time points ranging from 3 h to 2 wk wound samples were processed for polyester wax-embedding. Target antigens were identified and monitored quantitatively in sections stained immunohistochemically. Monoclonal antibodies against neutrophils, macrophages, pan T cells and cytotoxic populations of lymphocytes were used. The results showed that these cells are involved in the process of wound healing in a distinctive dynamic pattern. The accumulation of CD3+ T lymphocytes in the wound bed was mainly associated with the phase of granulation tissue formation. Intraepithelial CD3+ T lymphocytes were detected in considerable numbers within the regenerating epidermis. The cytotoxic cell populations (OX8+) were classified morphologically into the cytotoxic/suppressor subset of T cells and NK cells. The OX8+ T cells were shown to have a kinetic pattern similar to CD3+ T lymphocytes but of a lower magnitude. The accumulation of OX8+ NK cells was confined to the early inflammatory phase of repair. It is concluded that CD3+ T lymphocytes as well as OX8+ cytotoxic populations of the immune system are involved in the process of cutaneous wound healing in temporal sequences which suggest that they may be involved in its modulation.

Immunolocalisation of NHE3-like immunoreactivity in the gills of the rainbow trout (Oncorhynchus mykiss) and the blue-throated wrasse (Pseudolabrus tetrious).

Abstract: Na+/H+ exchange has been implicated in models of ion transport across the branchial epithelium of marine and freshwater fishes. In this preliminary study, we present immunohistochemical data using a polyclonal antibody raised against NHE3 which show NHE3-like immunoreactivity (IR) in the gills from a freshwater and a marine teleost species. In both species, branchial epithelial cells demonstrating NHE3-like IR were localised predominantly to the junction between the filament and the secondary lamellae. However, there was a marked difference in the morphology of the NHE3-like immunoreactive epithelial cells between the species. This morphological difference between the species suggests functional differences in the exchanger, which may be related to marine versus freshwater environments.

Morphology of the lymphoid organs of the bottlenose dolphin, Tursiops truncatus

Abstract: 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.

Development of early postnatal peripheral nerve abnormalities in Trembler-J and PMP22 transgenic mice.

Abstract: Mutations in the gene for peripheral myelin protein 22 (PMP22) are associated with peripheral neuropathy in mice and humans. Although PMP22 is strongly expressed in peripheral nerves and is localised largely to the myelin sheath, a dual role has been suggested as 2 differentially expressed promoters have been found. In this study we compared the initial stages of postnatal development in transgenic mouse models which have, in addition to the murine pmp22 gene, 7 (C22) and 4 (C61) copies of the human PMP22 gene and in homozygous and heterozygous Trembler-J (TrJ) mice, which have a point mutation in the pmp22 gene. The number of axons that were singly ensheathed by Schwann cells was the same in all groups indicating that PMP22 does not function in the initial ensheathment and separation of axons. At both P4 and P12 all mutants had an increased proportion of fibres that were incompletely surrounded by Schwann cell cytoplasm indicating that this step is disrupted in PMP22 mutants. C22 and homozygous TrJ animals could be distinguished by differences in the Schwann cell morphology at the initiation of myelination. In homozygous TrJ animals the Schwann cell cytoplasm had failed to make a full turn around the axon whereas in the C22 strain most fibres had formed a mesaxon. It is concluded that PMP22 functions in the initiation of myelination and probably involves the ensheathment of the axon by the Schwann cell, and the extension of this cell along the axon. Abnormalities may result from a failure of differentiation but more probably from defective interactions between the axon and the Schwann cell.

Skeletal maturity in Pakistani children.

Abstract: Skeletal maturity in 750 normal Pakistani children (400 males, 350 females) aged 1–18 y was determined by the Greulich–Pyle atlas system. Male children during first year and female children during first 2 y of life matured in conformity with Greulich–Pyle standards. After that period mean bone ages were lower than the American standards up to 15 y in males and 13 y in females (at or around puberty), which may be due to malnutrition, ill health or other environmental factors. After puberty bone ages were higher than the American standards indicating earlier maturity in Pakistani than Western children. Hence for the proper evaluation of skeletal age in a given region, a longitudinal study on individuals in that region to establish normal standards is necessary.

Histochemical and immunohistochemical analysis of the mechanism of calcification of Meckel's cartilage during mandible development in rodents

Abstract: It is widely accepted that Meckel's cartilage in mammals is uncalcified hyaline cartilage that is resorbed and is not involved in bone formation of the mandible. We examined the spatial and temporal characteristics of matrix calcification in Meckel's cartilage, using histochemical and immunocytochemical methods, electron microscopy and an electron probe microanalyser. The intramandibular portion of Meckel's cartilage could be divided schematically into anterior and posterior portions with respect to the site of initiation of ossification beneath the mental foramen. Calcification of the matrix occurred in areas in which alkaline phosphatase activity could be detected by light and electron microscopy and by immunohistochemical staining. The expression of type X collagen was restricted to the hypertrophic cells of intramandibular Meckel's cartilage, and staining with alizarin red and von Kossa stain revealed that calcification progressed in both posterior and anterior directions from the primary centre of ossification. After the active cellular resorption of calcified cartilage matrix, new osseous islands were formed by trabecular bone that intruded from the perichondrial bone collar. Evidence of such formation of bone was supported by results of double immunofluorescence staining specific for type I and type II collagens, in addition to results of immunostaining for osteopontin. Calcification of the posterior portion resembled that in the anterior portion of intramandibular Meckel's cartilage, and our findings indicate that the posterior portion also contributes to the bone formation of the mandible by an endochondral-type mechanism of calcification.