J. Anat (1990), 170, pp. 225-260 Printed in Great Britain
Proceedings of the Anatomical Society of Great Britain and Ireland DECEMBER 1989 The Winter Meeting for the session 1989/90 was held on Tuesday, Wednesday, and Thursday, 19, 20 and 21 December, 1989 at the Imperial College of Science, Technology and Medicine in London. The following are the abstracts of communications and posters presented at the meeting. COMMUNICATIONS 1. Effects of undernutrition on neuronal connectivity in the cerebelHar cortex of rats. By M. A. WARREN and K. S. BEDI. Department of Biomedical Science, University of Sheffield, and Department of Anatomy, University of Queensland, Australia Previously we (Bedi et al. J. Anat. 131, 1980) have reported that rats undernourished during early postnatal life have deficits in synapse-to-neuron ratios within certain layers of the cerebellar cortex. These deficits disappeared in animals given a period of nutritional rehabilitation. Whether or not the period of rehabilitation was crucial for this 'catch-up' remained uncertain. We have investigated this question by studying rats undernourished for a lengthy period of time. Male black and white hooded Lister rats were undernourished from birth to 150 days of age by restricting their diet to about half that eaten by well-fed controls. Some rats were allowed a period of nutritional rehabilitation between 75 and 150 days of age. Groups of rats were anaesthetised and killed by perfusion of 2-5% buffered glutaraldehyde at 21, 75 and 150 days of age. Stereological procedures at the light and electron microscopical levels were used to estimate synapse-to-neuron ratios in the granular layer of the cerebellar cortex. Twenty-one day old rats undernourished from birth had an average of about 258 synapses per neuron compared with 338 (P < 0-05) for well-fed controls. This deficit disappeared in rats undernourished continually until either 75 or 150 days of age. The 150 days old rats previously undernourished until 75 days had significantly (P < 0 05) more synapses per neuron than age matched controls. Synapse-to-neuron ratios approximately doubled between 21 and 150 days of age, with a peak at 75 days. Control, but not undernourished, rats showed a significant drop in the ratio between 75 and 150 days of age. These results suggest that although the deficit in synapse-to-neuron ratio is capable of 'catchup' to normal values despite continuing undernutrition, the early deprivation can nevertheless have long-lasting effects on the development of neuronal connectivity in the cerebellum.
2. The effects of age on cerebeliar neuron number. By R. R. STURROCK. Department of Anatomy and Physiology, University of Dundee The numbers per unit volume of Purkinje cells, granule cells, Golgi II cells and pale cells (Altman & Bayer, Expl Brain Res. 29, 1977) were estimated in the spinocerebellum, pontocerebellum and nodulus in mice aged 6, 15, 22, 25, 28 and 31 months. The number of stellate and basket cells per unit volume in the spinocerebellum of the same mice and the number of neurons in the deep cerebellar nuclei were also estimated. The number of Purkinje cells fell about 24% in all parts of the cerebellum between 6 and 31 months, with the decline in number beginning between 15 and 22 months of age. From 25 months of age Purkinje cells with distorted, hour-glass nuclei were common. There was no change in the volume density of granule cells, Golgi II cells or pale cells in any part of the cerebellum with age but stellate and basket cell number decreased by about 30 % between 6 and 31 months of age, with the decrease in number beginning between 22 and 25 months. The number of deep cerebellar neurons decreased by 35 % between 6 and 31 months of age with the decline in number occurring after 22 months.
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3. Changes in gene expression in the substantia nigra following damage of the ipsilateral striatum in the rat. By A. NAJLERAHIM, D. G. L. SHOWELL, P. J. HARRISON, A. J. L. BARTON and R. C. A. PEARSON*. Department of Anatomy, St Mary's Hospital Medical School, London and * Department of Biomedical Science, University of Sheffield Previous studies (Pearson et al. Brain Res. 400, 1987 and Brain Res. 412, 1987) showed that damage of the striatum of one side in rats leads to hypertrophy of the neurons of the ipsilateral pars reticulata of the substantia nigra (SNpr) and increased immunohistochemical staining of GABAergic axons in the superior colliculus and ventromedial nucleus of the thalamus, to which the enlarging cells project. We have now examined the level of specific mRNAs in these neurons in the SNpr by in situ hybridisation histochemistry. Twenty-one adult male Wistar rats were used for this study. Lesions of the left striatum were made as previously described. After survival periods of 21, 28 and 35 days, three experimental animals, together with three animals with lesions of the frontal cortex and one unoperated rat, were perfused through the heart with phosphate buffered saline (PBS), followed by 30 % sucrose in 20 mm phosphate buffer. In situ hybridisation histochemistry was performed as previously described (Largent et al. Proc. Natn. Acad. Sci. U.S.A. 85, 1988) using synthetic oligonucleotide probes directed against the mRNAs encoding alphal tubulin and glutamic acid decarboxylase (GAD). The specificity of the probes was confirmed by Northern analysis. The degree of labelling was quantified using an image analysis system (Image Manager PC, Sight Systems). Damage of the ipsilateral striatum led to significant increases in both mRNAs in the SNpr. 4. Changes in gene expression in the neocortex of the rat following local damage. By D. G. L. SHOWELL (supervised by R. C. A. PEARSON). Department of Anatomy and Cell Biology, St Mary's Hospital Medical School, London The technique of in situ hybridisation histochemistry (ISHH) has been used to study the effects of small areas of damage to the cerebral cortex on gene expression in the adjacent undamaged tissue. Twenty-four adult male Wistar rats were used in this study. In nineteen animals, under pentobarbitone anaesthesia, a small burr hole was made in the skull over the frontal pole, and the underlying cortex damaged by abrasion with a surgical needle. Five animals served as normal unoperated controls. Following survival times of one to six days, animals were perfused through the heart with phosphate buffered saline, followed by 30% sucrose in 20 mm phosphate buffer. ISHH was carried out as previously described (Largent et al. Proc. Natn. Acad. Sci. U.S.A. 85, 1988) on 1Opm sagittal cryostat sections. Synthetic oligonucleotide probes directed against the mRNAs encoding glutamic acid decarboxylase (GAD), the stimulatory alpha subunit of the GTPbinding protein (Gas), alpha1 tubulin (TUB), glutaminase (GluT) and aspartate aminotransferase (AspT) were used for ISHH. The specificity of the probes was confirmed by Northern analysis. In all cases the labelling was quantified on autoradiographs, using an image analysis apparatus (Image Manager PC, Sight Systems). The operated hemisphere was compared to the right, unoperated side, and to the normal unoperated control animals. Significant increases in GAD, TUB and Gas mRNAs were seen in the adjacent undamaged cortex on the operated side. The increases were maximal four days postoperatively, but declined by six days. 5. Muscarinic supersensitivity in the globus pallidus of MPTP-treated monkeys, revealed using (3H)-QNB autoradiography. By S. J. MELLOR and A. R. CROSSMAN. Experimental Neurology Group, Department of Cell and Structural Biology, University of Manchester (Figs. I and 2) Muscarinic cholinergic receptors can be visualised autoradiographically using the radiolabelled ligand [3H]quinuclidinyl benzilate (QNB) (Kuhar & Yamamura, Brain Res. 110, 1976). This study examines the status of central muscarinic receptors in parkinsonian monkeys. Nine monkeys (Macaca fascicularis) received systemic infusions of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) (1 8-27 0 mg/kg) (Burns et al. Proc. Natn. Acad. Sci. U.S.A. 80, 1983) rendering the animals parkinsonian. Six subsequently received dopamine (DA) replacement therapy, in the form of L-DOPA. Four monkeys received infusions of MPTP (0 5-2 5 mg/kg) into the right common carotid artery under general anaesthesia (Bankiewicz et al. Life Sci. 39, 1986) producing contralateral hemiparkinsonism. Four naive monkeys were used as controls. The animals were killed by barbiturate overdose, after which the brains were rapidly frozen. Cryostatcut sections (20 ,um) from eight rostrocaudal brain levels were preincubated in 50 mm phosphate buffer (pH 7 4, 2 x 30 minutes), and then transferred to the incubation medium of the same buffer
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Fig. 1 with 1 flm [3H]QNB, for 120 minutes. Sections were then washed in buffer, dried and exposed to tritium-sensitive film (Hyperfilm, Amersham) for 3-6 weeks at -20 'C. Histological staining using cresyl violet revealed a massive reduction in the number of cell bodies in the substantia nigra (SN) pars compacta of monkeys treated with MPTP. Analysis of [3HJQNB autoradiographs from animals treated unilaterally with MPTP revealed significant increases in ligand binding to the lesioned side. The greatest difference (47%) was in the medial pallidal segment (GPM). This is shown in Figure 1, a photograph of speCifiC [3H]QNB binding to a section from a monkey lesioned on the right side. Analysis of autoradiographs from animals treated systemically with MPTP revealed a significant increase in [3H]QNB binding to the GPM (89-96%) which was reduced by 16-L36 % after subsequent DA-replacement therapy. This is shown in Figure 2, a graphical representation of [3H]QNB binding to the GPM at four rostrocaudal brain levels (1-4). These results suggest an increased sensitivity of cholinergic receptors in the GPM of parkinsonian brains. Of the major afferent pathways to the GPM, only that from the pedunculopontine tegmental nucleus is associated with cholinergic function. The present study would suggest that this pathway is underactive in parkinsonism. Supported by the Medical Research Council. 6. Fetal rat adrenal cells maintained in culture continue to express tyrosine hydroxylase. By C. EARL and MARGARET BIRD. Department of Anatomy, The London Hospital Medical College For maximal effectiveness in the treatment of Parkinson's disease catecholamine (CA) releasing neurons grafted into, or close to, the striatum, should be capable of sustained synthesis and release of CA. We are exploring the optimal conditions for such grafting. In this study we have investigated the question of whether or not fetal adrenal medullary cells contain and continue to express in culture the enzyme tyrosine hydroxylase (TH) which is the rate limiting enzyme in the synthesis of CAs. Adrenal glands were removed from 18 day rat fetuses and explanted in culture in Dulbecco's modification of Eagles Medium (90 %) and horse serum (10 %) containing glutamine and nerve growth factor (NGF) 2 #sg/ml (Sigma). Cultures were examined at regular intervals and re-fed every 72 hours. Fourteen days after culturing, the cells were processed for immunocytochemistry
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"\ sMPTP s MPTP
Brain level Fig. 2
(Notter et al. Expi Brain Res. 76, 1989) at LM levels using a polyclonal antibody to TH and diaminobenzidine as the chromogen. Control cultures were identically treated but the primary antibody was replaced by phosphate buffered saline. TH immunoreactivity was intense and could be localised in the cell body of small cells and fine long processes which were in continuity with the cell bodies. Negative controls showed only a small amount of non-specific background staining. Thus, although we have not yet explored TH expression in adrenal cells maintained for periods longer than two weeks in culture it is clear that the removal of these cells to the abnormal environment of the culture dish does not prevent them from expressing the enzyme necessary for CA synthesis. 7. Anatomical evidence that the paramedian pontine reticular formation participates in the oculomotor system of the rat. By J. D. COOPER and 0. T. PHILLIPSON. Department of Anatomy, University of Bristol The paramedian pontine reticular formation (PPRF) has been demonstrated to play a major role in the brainstem control of horizontal eye movements in feline and primate species. The efferent connections of this region were investigated in the rodent, in the context of recent studies that have shown afferents to PPRF from prefrontal cortex lateral habenula, and interstitial nucleus of Cajal (INC), known to control vertical eye movements. Single stereotaxic micro-electrophoretic injections of the anterograde tracer Phaseolus vulgarisLeucoagglutinin (PHA-L) were made into the PPRF, of Wistar rats (n = 6), under chloral hydrate anaesthesia. Following 7-14 day survival times animals were re-anaesthetised and fixed by transcardial perfusion. Frozen sections were prepared to visualise transported PHA-L by immunocytochemistry using the avidin-biotin technique. Injections specifically confined to rostral PPRF at the level of the inferior colliculus, where habenular efferents terminate, resulted in bilateral terminal-like labelling (TLL) most marked ipsilateral to the injection site. Dense TLL was found more caudally in PPRF, reticulotegmental nucleus, supragenual nucleus dorsal to nucleus abducens, and dorsomedial inferior olive.
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Less prominent TLL was present in the nucleus of the posterior commissure, medial substantia nigra pars compacta, ventral tegmental area, interpeduncular nucleus, the mesencephalic reticular formation localised lateral to red nucleus, median and dorsal raphe pedunculopontine and laterodorsal tegmental nuclei, paraabducens nucleus, nucleus prepositus hypoglossi, and medial vestibular nucleus. Sparse TLL was present in the INC (including its rostral extension), the medial accessory, and somatic oculomotor nuclei, mesencephalic central grey, central and dorsal tegmental nuclei, and ventral horn of cervical spinal cord. This pattern of connections suggests that this region of rodent PPRF may play a role in the brainstem control of saccadic eye movements. This system may be influenced by efferents from frontal cortex, and from basal ganglia via the habenula.
8. Axonal sprouting in the thalamus of adult rats following implantation of a peripheral nerve graft. By G. CAMPBELL, A. R. LIEBERMAN, P. N. ANDERSON and M. TURMAINE. Department of Anatomy and Developmental Biology, University College London (Fig. 3) Peripheral nerve grafts have been shown by retrograde HRP-labelling to provide a good environment for the regeneration of adult mammalian thalamic axons (Benfey et al. J. Neurocytol. 14, 1985; Campbell et al. unpublished). We have used electron microscopy to investigate the early stages of this regenerative process which must involve sprouting by injured CNS axons. Autografts of common peroneal or tibial nerve were implanted into caudo-lateral thalamus of Sagatal-anaesthetised adult rats. The distal, free end of the graft was laid beneath the scalp. Between five days and eight weeks later the animals were perfused and the brains were processed for EM. Large numbers of very small unmyelinated axons were present in the CNS parenchyma within a few hundred micrometres of the proximal (thalamic) tip of the graft at 5-14 days after grafting and in smaller numbers at four and eight weeks after grafting. These axons were smaller than unmyelinated axons of the control thalamus and were often tightly packed in bundles enclosed by astrocyte processes below the forming glia limitans. They were commonly impressed into the surface of astrocytes, myelin sheaths, oligodendrocytes and microglial cells (Fig. 3). At five days similar small unmyelinated axons were already present in the junctional zone between host brain and graft tissue and by eight days many small axons associated with Schwann cells and astrocytes were present in the junctional zone. At 14 days such axons were also present deep within the graft, associated predominantly with Schwann cells. By four weeks, regenerating axons had penetrated several millimetres towards the distal end of the graft along the columns of Schwann cells. On the basis of their ultrastructural features, location, size, number and fate we conclude that the small unmyelinated axons present in profusion around the graft in the early post-implantation period are newly formed regenerating axonal sprouts. Supported by Action Research for the Crippled Child. 9. Immunohistochemical localisation of galanin in the human brain. By S. M. GENTLEMAN, P. FALKAI, B. BOGERTS, M. T. HERRERO*, J. M. POLAK* and G. W. ROBERTSt (introduced by J. A. FIRTH). Division of Psychiatry, CRC, Harrow, * Department of Histochemistry, Royal Postgraduate Medical School, London and tDepartment of Anatomy, St. Mary's Hospital, London Galanin (GA) is an amidated 29 amino acid peptide originally isolated from porcine small intestine (Tatemoto et al. FEBS Lett. 164, 1983). No detailed mapping of this peptide in the human brain has yet been reported, although the distribution of GA-like immunoreactivity has been extensively studied in the central and peripheral nervous systems of the rat. In this study we describe the distribution of GA-like immunoreactivity in complete coronal of human brain. Brain tissue was obtained at autopsy from patients with no significant cerebrovascular or neurodegenerative disease (n = 9, 4 male, 5 female; age range 42-72 years). 20,um sections were incubated with a polyclonal antiserum raised in rabbits against synthetic galanin and developed using a modified peroxidase anti-peroxidase technique. GA-positive cell bodies were largely restricted to the hypothalamus with the densest concentrations in the supraoptic, ventromedial, and posterior areas. Smaller numbers of positive cells were seen in the paraventricular nucleus and the anterior and lateral areas of the hypothalamus. The only other major concentration of immunoreactive cells was found in the basal nucleus of Meynert. Fibre staining was more widespread with immunoreactive fibres seen
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throughout the hypothalamus and in the diagonal band of Broca, septum, amygdala, hippocampus, stria terminalis and the fornix. Positive fibres were occasionally seen scattered throughout the neocortex. The prevalence of galanin in the hypothalamus suggests that it may have a neuromodulatory role on hypothalamo-pituitary function in man, similar to that reported in rats. The presence of the peptide in the basal forebrain and septo-hippocampal pathway along with its recently reported colocalisation in cholinergic neurons of the basal nucleus of Meynert is also of interest with respect to Alzheimer's disease. 10. Localisation of the nuclei of origin of efferent nerve fibres to the sternocleidomastoid/trapezius muscle group in the rat. By R. C. RINTOUL (supervised by J. ROBERTSON-RINTOUL). Department of Biology and Preclinical Medicine, University of St. Andrews This study utilised a wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) conjugate to investigate the motor innervation of sternomastoid (SM), cleidomastoid (CM) and clavotrapezius (CT) in the albino rat. In one group of animals the fully innervated SM, CM or CT was injected with WGA-HRP; in another group SM, CM or CT was injected following section of the spinal accessory nerve (SAN). Prior to surgical exposure of the elected muscle, animals were anaesthetised by intraperitoneal injection of 2,2,2-tribromoethanol. Twenty-four hours later the animals were killed by administration of ether. Following appropriate neurohistochemistry the brainstem and spinal cord were examined for labelling of neural structures. Motoneuron labelling from the two sets of experiments showed that in the rat SM, CM and CT receive a motor innervation through the ventral roots of the upper cervical spinal nerves in addition to that through the SAN. The positions of nuclei formed by SAN motoneuron somata and cervical efferent motoneuron somata were plotted. The spinal accessory nucleus (nspA) and the nucleus of cervical efferents, both supplying SM and CM, extended caudally from the lower medulla; the former as far as C2, the latter until C3. In close apposition throughout their lengths, rostrally the nuclei lay dorsomedially
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within the ventral grey horn and caudally, ventrolaterally. The shift in their positions occurred at the Cl/C2 border. The nspA supplying CT lay solely in a ventrolateral position within the ventral horn between C2 and C4. The nucleus of cervical efferents to CT paralleled the nspA between C3 and C4. The likelihood of a dual motor innervation of sternocleidomastoid in man and its clinical significance is discussed. 11. The effect of separation from the target muscle on the survival of developing motoneurons. By M. B. LoWRIE (introduced by J. A. FIRTH). Department of Anatomy and Cell Biology, St Mary's Hospital Medical School, London Following sciatic nerve crush at birth in the rat, the reinnervated soleus muscle remains severely impaired because of motoneuron loss (Lowrie et al. Dev. Brain Res. 31, 1987). If the nerve is injured in the same way at five days or later the muscle recovers almost completely (Lowrie et al. J. Physiol. 331, 1982). In this study the period of separation following nerve crush at five days was varied and its effect upon the survival of motoneurons was investigated. Using ether anaesthetic and aseptic precautions, the sciatic nerve was crushed in one leg of 5 days old rats, the other leg serving as control. In half of the animals nerve crush was repeated at a second operation 5-7 days later. Two to four months later, the motoneurons innervating the soleus muscle of both legs were labelled retrogradely by intramuscular injection of HRP under chloral hydrate anaesthetic. Two days later the animals were killed by anaesthetic overdose and frozen sections of the spinal cord were stained for HRP. Counts of labelled motoneurons in the 'experimental' and 'control' anterior horns of the spinal cord showed that a single nerve crush at five days did not affect the number of motoneurons (exp. horn: 57 + 3-7; cont. horn: 59 + 5 1; mean + S.E., n = 6), but when the period of disconnection was prolonged by a second nerve crush the number of motoneurons was reduced to 59 % of the contralateral control side (exp. horn: 30 + 3.9; cont. horn: 52 + 2-9, n = 6). Tension recording experiments performed on a different group of animals showed that after prolonged denervation the strength of the reinnervated muscles was reduced to 48 % (± 4-8, n = 9) of control. Thus motoneuron loss after postnatal nerve injury is due to disrupted interaction with the muscle rather than axonal injury per se, and the longer the motoneuron is deprived of its target the more likely it is to die. 12. Reinnervation of developing rat molars. By C. D. JOHNSTON (supervised by P. D. A. OwENS). School of Basic Medical Sciences/Anatomy, The Queen's University of Belfast Recent studies have shown reinnervation of dentinal tubules in adult rats (Berger & Byers, Pain 15, 1983) and cats (Holland et al. J. Physiol. 386, 1987) following alveolar nerve transections but the extent of this reinnervation is debated. The present study investigated the ability of regenerating nerve fibres to reinnervate predentine of developing rat molar teeth as well as quantifying the extent of reinnervation. Using ether anaesthesia, intramandibular transection of the right inferior alveolar nerve was performed in eleven 20 days old rats. At intervals of 5, 15, 30 and 50 days afterwards, groups of rats were anaesthetised and perfusion-fixed and the mesial cusp of the right first molar tooth was processed for light and transmission electron microscopy. At each interval, the percentage innervation was calculated from a sample of at least 54 tubules in the predentine. These values were compared with those obtained from similar sites in control animals of the same ages. Degeneration of almost all pulpal myelinated axons and dentinal unmyelinated axons had occurred within five days of surgery. By 15 days after transection there was evidence of some pulpal reinnervation by myelinated axons but less than 2% of predentinal tubules showed reinnervation (control figures 31 6 %). At 30 days after surgery however the figure for predentinal reinnervation was approximately 17-7 % (control figures 44-7 %); and by 50 days after transection (70 days of age) innervation was slightly less than half the level observed in normal 70 day teeth (28-4 % versus 47-3 % respectively). These values are currently being reassessed with a larger sample but it is concluded that in immature rats innervation of predentine does occur after nerve transection. However, these findings indicate that contrary to the results of Berger & Byers (1983) such reinnervation does not attain control levels and that the process is slower in the younger animals. This work was supported by the MRC.
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13. Nerve damage in leprosy. By C. L. CRAWFORD and M. J. HOBBS. Department of Anatomy, Charing Cross and Westminster Medical School, London (See also p. 260) When a peripheral nerve is sectioned there is paralysis of muscles and loss of sensation in a localised area of the skin. In leprosy, according to standard textbooks, restricted segments of individual nerves are damaged. The disability should therefore be similar to that following nerve section except that more than one nerve is usually involved. Yet the characteristic feature of leprosy is the mutilation of the fingers and toes with trophic ulcers on the feet. Radiologically, there is extensive bony loss including in some cases tarsal disintegration (Charcot's joints). These features are never seen after nerve section even in proximal injury to the sciatic nerve. In leprosy, it is therefore necessary to postulate some additional form of nerve damage to account for this sensory denervation. In 1923, Monrad-Krohn described a form of sensory loss of 'glove and stocking' distribution and these findings have been confirmed in Nigerian patients. Only superficial modalities are affected. This sensory loss can coexist with the lesions to peripheral nerve segments or occur separately, and is important because (1) the onset may be acute involving both hands and feet simultaneously. Unless recognised and treated with steroids permanent loss of pain sensation can occur leading to the sequelae already described. (2) Currently, it has been suggested that loss of protective sensation can be restored if restricted segments of peripheral nerves are removed and grafts of muscles inserted. This is unlikely to be successful as the whole of the distal axon is destroyed. Moreover motor nerve fibres will inevitably be killed and there is no guarantee that they will reinnervate muscle. Nerve fibres subserving deep modalities of sensation will also be destroyed during these operations.
14. Junctional complexes in the perineurium of the sural nerve in human diabetic neuropathy: a study by freeze-fracture electron microscopy. By N. G. BEAMISH, C. STOLINSKI* and P. K. THOMAS. Department ofNeurological Science, Royal Free Hospital School of Medicine and * Department of Anatomy and Cell Biology, St Mary's Hospital Medical School, London The endoneurial environment of peripheral nerve is protected by blood-nerve and perineurial diffusion barriers. The water content of peripheral nerve is known to be increased in human diabetic neuropathy (Griffey et al. JAMA 260, 1988) and in streptozotocin-induced diabetes in rats (Anand et al. J. Neurol. Sci. 83, 1988). The perineurial and vascular diffusion barriers have been studied in experimental diabetes with protein tracers. An earlier study reporting that it was increased (Seneviratne, J. Neurol. Neurosurg. Psych. 35, 1972) was not confirmed by Jakobsen et al. (Exp. Neurol. 60, 1978) and Sima & Robertson (Acta Neuropathol. (Berl.) 44, 1978). Vascular permeability to small molecules is increased (Rechthand et al. J. Neuropath. Exp. Neurol. 46, 1987). Endoneurial vascular permeability has been found to be increased in human diabetic neuropathy (Poduslo et al. Proc. Natn. Acad. Sci. U.S.A. 85, 1988). In this study we have investigated the intercellular contacts in the perineurium in human diabetic neuropathy using freeze-fracture electron microscopy. Sural nerve biopsies from organ-donor patients were obtained with the approval of the Ethics Committee at the Royal Free Hospital and from patients with diabetic neuropathy with their informed consent and the approval of the Ethics Committee at King's College Hospital. The specimens were fixed in glutaraldehyde, cryoprotected in glycerol and frozen in melting propane at - 180 'C. The tissue was fractured at - 120 'C and platinum-carbon replicas produced. Gap junctions, which had previously not been found in adult human perineurium (Gabriel et al. J. Anat. 146, 1986), were observed infrequently, both in control and diabetic perineurium. Tight (occluding) junctions were observed in both, and there was no obvious alteration in their numbers in the diabetic nerves. However, the configuration of some of the tight junction networks in the diabetic nerves differed from the controls in that they displayed an excessive number of curvilinear strands which were non-branching. These changes could reflect an alteration in barrier properties. We are indebted to the Medical Research Council and Joint Standing Research Committee of St Mary's Hospital for financial support. 15. The neurotransmitters and neuropeptides in the anterior major pelvic ganglion of the male guinea-pig. By D. DHAMI (introduced by B. S. MITCHELL). Human Morphology, University of
The nerves which supply the pelvic viscera and external genitalia of the male guinea-pig either pass through or synapse within the anterior and posterior major pelvic ganglia. The current
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investigation was carried out to identify certain neurotransmitters and neuropeptides in the anterior major pelvic ganglia. An overdose of Euthatal was administered to pre-pubertal male Hartley guinea-pigs which were then perfused transcardially with 4 % paraformaldehyde. Anterior major pelvic ganglia (AMPG) and gut tissues were dissected out and quenched in 2-methyl butane cooled by liquid nitrogen prior to cryostat sectioning. Gut tissues were used as positive controls. The histochemical method of Karnovsky & Roots (J. Histochem. Cytochem. 12, 1964) was used to detect acetylcholinesterase activity, while indirect immunofluorescence techniques were used to localise tyrosine hydroxylase-, neuropeptide Y-, vasoactive intestinal peptide-, substance P-, atrial natriuretic factor-, or enkephalin leucine 5-like immunoreactivities. Neuronal perikarya within the AMPG displayed acetylcholinesterase activity. There was an abundance of tyrosine hydroxylase-like immunoreactivity in some neuronal perikarya, and in nerve terminals abutting unlabelled neuronal perikarya. Neuropeptide Y and vasoactive intestinal peptide were present in neuronal perikarya, nerve fibres, and in nerve terminals which were abutting labelled and unlabelled neuronal perikarya. Substance P and atrial natriuretic factor were detected in nerve fibres, and in nerve terminals which abutted unlabelled neuronal perikarya. Isolated neuronal perikarya were immunoreactive for atrial natriuretic factor. Enkephalin leucine5 immunoreactivity was absent from the ganglia. The differing intensities of fluorescent specks abutting unlabelled neuronal perikarya suggest that the immunoreactivity could be in nerve terminals or the processes of other cells eg. 'small intensely fluorescent cells'. The precise localisation of the fluorescence may be clarified by immuno-electron microscopy studies. The abundance of tyrosine hydroxylase immunoreactive and vasoactive intestinal peptide immunoreactive neuronal perikarya in different regions of certain serial sections suggests the possibility that the AMPG is polarised. 16. The morphology, neurotransmitter and neuropeptide content of the paracervical ganglion of the marmoset (Callithrix jaccus). By B. S. MITCHELL and V. V. STAUBER. Human Morphology, University of Southampton The autonomic innervation of the pelvic viscera in the female marmoset (Callithrix jaccus) has not been extensively investigated. In the present study paracervical ganglion tissues from three adult female marmosets were examined using microscopical and immunohistological techniques. Animals were given an overdose of Euthatal anaesthetic and perfused transcardially with a 2 % glutaraldehyde :4% paraformaldehyde mixture or 4% paraformaldehyde. Uterovaginal and associated paracervical tissues were processed to paraffin wax or Araldite for light or electron microscopy or were sectioned on a cryostat for immunohistology or histochemistry. An indirect immunofluorescence method was used to demonstrate tyrosine hydroxylase-, neuropeptide Y-, vasoactive intestinal peptide-, substance P-, and enkephalin-like immunoreactivities. Acetylcholinesterase activity was demonstrated using a histochemical method (Karnovsky & Roots, J. Histochem. Cytochem. 12, 1964). The ganglia appeared as clusters of neuronal perikarya in loose connective tissue lying between the uterovaginal junction, the rectum and the urinary bladder. The ganglion formations contained up to 30 or so neuronal perikarya which were surrounded by closely applied satellite cells, except where there were nerve terminals. Numerous nerve terminals and varicosities typical of autonomic ganglia were present which contained a predominance of small, round clear vesicles and a few vesicles possessing an electron-dense core. Axoaxonal, axodendritic and axosomatic synapses were observed. Low amounts of acetylcholinesterase activity were detected in many neuronal perikarya and nerve fibres. Tyrosine hydroxylase immunoreactivity was detected in some small intensely fluorescent cells (SIF cells) and in nerve fibres, though in few neuronal perikarya. Neuropeptide Y was detected in weakly fluorescent neuronal perikarya. Abutting unlabelled neuronal perikarya were weakly fluorescent specks indicating substance P immunoreactivity and stronger fluorescent specks indicating enkephalin immunoreactivity. Vasoactive intestinal peptide immunoreactivity was absent from the ganglionic tissues examined. Our previous studies on guinea-pig paracervical ganglion suggest morphological similarities with the marmoset, but whether the low neuropeptide immunoreactivity of the marmoset ganglion indicates functional dissimilarity requires clarification.
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17. Histological and histochemical studies of the rat intercostal muscles following selective nerve root excisions. By B. C. MSAMATI. Department of Anatomy, University of Zimbabwe Medical School, Harare, Zimbabwe Denervation of skeletal muscle has been the subject of many careful studies and extensive work has also been reported on light and electron-microscopic structure in the normal muscle, following denervation and tenotomy. However, in spite of this enormous literature, reports on histochemical changes following denervation of intercostal muscles of the rat have not been reported and histochemical studies following selective nerve root lesions are lacking. In the present study the structural and histochemical reactions of these muscles were studied using twenty-six adult rats matched for sex, age and strain. After limited laminectomies (three segments) and removal of zygapophyseal joints which were carried out under methoxyflurane anaesthesia using a Boyles' machine, the animals were subjected to dorsal radisectomy alone: dorsal radisectomy combined with ganglionectomy; and dorsal radisectomy combined with ventral radisectomy. The animals were then killed at planned intervals by stunning them on the head. Results of denervation of intercostal muscles indicate that after de-afferentation over a 12 week period extrafusal muscle fibres do not show any histological or histochemical change in the enzyme profiles of muscle fibres, as indeed is the case in other muscles of the limbs and trunk. Histological and histochemical observations in intrafusal muscle fibres showed selective denervation reactions to motor and sensory complements. Intrafusal nuclear bag fibres showed 'targeting' associated with histological changes mainly confined to the equatorial regions of these fibres. When both dorsal and ventral roots were excised, histological and histochemical changes in extrafusal fibres confirmed the progressive reduction of contractile elements indicated by a fall in fibre size, and an increased connective tissue component. Intrafusal fibres showing Bag 1 and Bag 2 fibres were indistinguishable and histological changes involving these fibres were generalised along the length of the fibres. 18. The use of a replication deficient retrovirus to mark cells during muscle development. By P. WIGMORE and P. MARTIN*. Department of Anatomy, University of Aberdeen, and * ICRF Developmental Biology Unit, University of Oxford Striated muscle fibres form by the fusion of mononuclear cells (myoblasts). Evidence from studying avian muscle in vitro (Miller et al. J. Cell Biol. 103, 1986) suggests that several different populations of cells are present which appear at different times and differentiate to produce different fibre types. To identify and analyse the interactions of cell populations in vivo it is necessary to mark cells and to be able to follow their subsequent lineage. Most available markers are diluted by cell division but recently techniques have been developed to insert marker genes into cells using viruses as vectors. The advantage of this technique is that the gene being used as a marker is copied at cell division and therefore suffers no dilution. Replication deficient viruses are unable to replicate within a cell and so, after the initial infection no new virus particles are released. The viral genome containing the marker gene is therefore restricted to the subsequent lineage of the infected cell. In the present study a replication deficient retrovirus (made available by Dr R. Beddington, ICRF, Oxford) was used to mark cells of developing muscle. This virus contains the gene for bacterial , galactosidase and cells producing this enzyme stain blue with a simple histochemical test. Newborn mice were anaesthetised by cooling and concentrated virus was injected into the distal hind limb muscles (lumbricals and flex. dig. brevis). These muscles are relatively immature at birth and form the majority of their fibres postnatally. Four days after the injection clones of marked cells and marked muscle fibres were observed. This technique will enable the analysis of different populations of cells within muscle and the distribution of clones
of cells as they fuse into new fibres. 19. The effects of genetic sex and androgens on skeletal muscle tissue. By N. C. STICKLAND and P. J. O'SHAUGHNESSY. Department of Veterinary Basic Sciences, The Royal Veterinary College, London Previous studies have shown that certain muscles may exhibit different muscle fibre type proportions between the sexes. Experiments on the effects of either male castration or injection of androgens into female animals have led investigators to conclude that these differences are due to
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levels of androgens. However these conclusions are based on invasive techniques which may involve secondary factors, and results have usually only been apparent on a few highly 'androgensensitive' muscles. The sex-reversed (Sxr) mouse is genetically (X/X) female but phenotypically male due to the presence of the testis determining gene (Tdy). Serum testosterone in the Sxr male is in the low normal range and this animal provides the opportunity to examine whether factors other than androgens affect sexual dimorphism in muscle. The biceps brachii (predominantly a fast muscle) and soleus (a slow muscle), neither of known high androgen-sensitivity, were removed immediately post mortem from ten normal (X/Y) males, ten normal (X/X) females, and ten (X/X) Sxr males. The muscles were sectioned and serial sections reacted for myosin adenosine triphosphatase, glycogen phosphorylase and succinic dehydrogenase activities. These tests enabled the fibre type proportions of each muscle to be assessed as well as the mean size of each fibre type. In m. soleus the proportion of slow fibres in the Sxr male was similar to that in the normal male, being less than that in the normal female. Furthermore the mean sizes of each fibre type in the Sxr male were similar to the normal male, the fast fibres being larger and the slow fibres being smaller than the normal female. In m. biceps brachii only normal females contained a few slow fibres. However, in contrast to m. soleus, the fast fibres (of which there were two types in the biceps) were larger in the normal male than in both the Sxr male and normal female. In conclusion, it would seem from this study that both muscles were sensitive to androgens with respect to fibre type differentiation but that one muscle (m. soleus) was more sensitive to androgens as far as muscle fibre growth was concerned. An alternative explanation is that genetic sex may be more important than androgen levels in determining muscle fibre growth in some muscles. 20. The morphology of cultured metrial gland explants and their emigrant granulated metrial gland cells. By D. D. Y. MUKHTAR and I. J. STEWART. Human Morphology, University of Southampton Granulated metrial gland (GMG) cells migrate from explants of mouse metrial gland tissue which are maintained in tissue culture (Mukhtar & Stewart, Cell Tiss. Res. 253, 1988). We report here on our studies of the morphology of cultured metrial gland explants and of the GMG cells which migrate from these explants onto plastic culture dishes. Metrial glands were dissected from mice killed by scheduled methods at days 10 to 16 of pregnancy. Quarter metrial glands (explants) were cultured in Minimal Essential Medium supplemented with 10% fetal calf serum and antibiotics. The cultures were maintained at 37 °C in a humid atmosphere of 5 % CO2 in air. After 24-96 hours the cultures were fixed and processed for light microscopy or for scanning electron microscopy. Within 24 hours of culture extensive changes had occurred in the explants. These changes resulted in the explants having a central degenerative core with a few layers of healthy cells, including some GMG cells, at the periphery. Scanning electron microscopy of the explants revealed GMG cells which appeared to be emerging from the explant and others which appeared to be crawling over the surface of the explant. The GMG cells which were on the surface of the explant and on the culture dish had a variable but distinct morphology. In particular, the cells had a very rounded form. Many cells had pseudopodia and filopodia, structures which are often associated with motile cells. The surface morphology of a few GMG cells was fairly smooth but most had numerous projections on their upper surface. These projections had a variety of forms, with some cells having small finger-like projections, some having more leaf-like projections and others having a combination of both. The possible role of these surface projections in cell-cell interactions was considered. Supported in part by the Wessex Medical School Trust. 21. Patterning cell adhesion and movement on artificial substrata; a simple method. By S. T. BRITLAND, G. MOORES*, P. CLARK* AND P. CONNOLLY (introduced by R. J. SCOTHORNE). Department of Electronics and Electrical Engineering and * Department of Cell Biology, Glasgow University It is known that the topography and adhesiveness of artificial substrata can each modulate the behaviour of cells in vitro (Clark et al. Development 99, 1987). However, recent studies have shown 9-2
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that it is possible to promote the attachment of cells and direct their subsequent outgrowth by patterning surface chemistry alone (Kleinfeld et al. J. Neurosci 8, 1988). The complex and lengthy procedures involved in preparing substrata in this way have been a disadvantage to what is potentially a powerful technique in cell biology. We have developed a method whereby patterning of surface chemistry on artificial substrates, which creates areas of differential adhesion for cell culture, can be completed in approximately two hours. Photolithographic techniques, normally employed by the microelectronics industry in fabricating integrated circuits, coupled with silane chemistry were used to produce the high resolution patterning on planar quartz. The attachment of cultured BHK cells and their subsequent movement was monitored in order to evaluate the quality and precision of surface preparation. Observations showed that both aspects of cell behaviour could be strongly influenced. Artificial, non-topographic boundaries were produced that cells seldom crossed. This made it possible to align cells and confine their movement within tracks of width approximately equal to the dimensions of the cells themselves (- 10 ,m). Successful control of cell adhesion, outgrowth and movement on artificial substrata has important implications in many research areas. In addition to furthering the basic understanding of cell behaviour, specific applications for this technique are currently being realised in studies on the electrical properties of cardiac cells in culture and neural cell communication. 22. Identification of plasminogen activator activity in human osteoblasts using a single cell assay: effects of retinoic acid and parathyroid hormone-related peptide. By NARANI SIVAYOHAM (supervised by J. A. GALLAGHER). Department ofHuman Anatomy and Cell Biology, University of Liverpool Human osteoblasts were isolated from fragments of cancellous bone dissected from femoral heads obtained at surgery. Plasminogen activator (PA) was detected by a recently developed single cell assay in which cells were overlain with an agar gel containing finely dispersed fibrin clots. The cells with the fibrin overlay were incubated for 1-2 days, then fixed and stained with Coomassie brilliant blue R250. On destaining the area of fibrinolysis appeared as a clear zone surrounding the active cell. The area of each lytic zone was measured by point counting and the percentage of positive cells was estimated by random sampling. The results show that unstimulated human osteoblasts produce PA. The proportion of PA positive cells in unstimulated cultures was low. This varied from 0-9 % depending on donor and length of time in primary culture. When the cells were stimulated with 10-7 M or 10-6 M retinoic acid there was generally a large increase in PA activity. Three experiments were carried out in which retinoic acid was added. In all there was an increase in percentage lytic area; however an increase in the percentage of active cells was observed in only two of the experiments. Parathyroid hormone-related peptide (PTHrP) is a hormone implicated in hypercalcaemia of malignancy. PTHrP has recently been shown to increase PA activity in rodent cells. However, in this study, when PTHrP was added to human bone cells at 3 ng/ml and 33 ng/ml no consistent effect was observed. 23. Early patterns of humeral and femoral remodelling in the rat induced by the synthetic retinoid N-ethylretinamide (NER). By J. TURTON, R. MARIAN HICKS, E. KATCHBURIAN* and L. REAMt. School of Pathology, Middlesex Hospital Medical School, * Department of Anatomy and Histology, The London Hospital Medical College and t Department of Anatomy, Wright State University, School of Medicine, Dayton, Ohio, U.S.A. (Fig. 4) Synthetic retinoids are used in the treatment of a variety of skin disorders including psoriasis, ichthyoses, nodulocystic and conglobate acne. Adverse effects, 'hypervitaminosis A', are associated with retinoid therapy and include mucocutaneous, ocular, hepatic and skeletal changes. Reported skeletal findings are long bone remodelling, premature epiphyseal closure, hyperostosis and calcification of tendons and ligaments. A previous study (J. Anat. 167, 1989) showed that the retinoid 1 3-cis-retinoic acid induced femoral remodelling in rats killed at 78 weeks. We have now investigated early remodelling changes in the humerus and femur of rats fed dietary NER for 15 weeks.
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Rats fed control diet
Cortical Time Cortical bone (weeks) bone I I I -n O r I
II I~~~~~~~~~~ * I~~~~~~~~~~~ -
aI I I
*. I ---N-
I.I':11i '~~~~~~~~~~~~~I 1
Fig. 4 Weanling, F344 female rats were fed a ground diet. Ten rats were killed by CO2 overdose at the beginning of the experiment (week 0); 60 animals were fed a 2 5 mmol NER/kg diet and 60 a placebo (control) diet. Ten rats per group were killed at week 2, 4, 6, 9, 12, and 15. A further 20 rats were fed NER diet for 9 weeks and placebo diet from 9 to 15 weeks; 10 rats were killed at week 12 and 15. At PM, body measurements were recorded and humeri and femora removed. The bones were sectioned transversely and cortical bone thickness, medullary cavity diameter and diaphysis diameter measured using a binocular microscope. Neither body weight nor the length of the body, tail, hind limb, humerus or femur was reduced in NER-fed rats. The weight, shaft diameter and medullary cavity diameter of both the humerus and femur were reduced in NER-fed animals over the 15 week period but cortical bone thickness was increased. In rats fed retinoid diet for 9 weeks and then placebo diet, the beginning of a reversal of the retinoid effects on both bones was evident between 9 and 15 weeks. Diagramatic representations of transverse sections of the humeral diaphyses, illustrating the patterns of remodelling changes are shown (Fig. 4). In placebo-fed rats there was normal remodelling with slight periosteal deposition, significant endosteal deposition and an increase in cortical bone thickness over the 15 week period. In NER-fed rats there was early and progressive periosteal resorption with accompanying gross endosteal deposition resulting in an additional increase in cortical thickness which almost occluded the medullary cavity. When NER diet was replaced at 9 weeks with placebo diet the retinoid-induced remodelling effects began to reverse. In conclusion, NER induced early and progressive humeral and femoral remodelling during the 15 week period. In the humerus remodelling consisted of periosteal resorption and significant endosteal deposition which reduced the diameter of the medullary cavity. 24. The relationship between sex, body size and the auricular surface of the ilium. By R. S. ALI (supervised by S. M. MACLAUGHLIN). Department of Anatomy, U.M.D.S. (Guy's Campus), London Given an intact adult human skeleton, one might expect to achieve close to 100 % accuracy of correct sex prediction. This accuracy will decrease substantially if the skeleton is either incomplete or fragmentary. The innominate is generally considered to be the most reliable skeletal element for sex determination. The most sexually dimorphic area of the innominate is widely accepted to be the os pubis. However, it is covered by a relatively thin layer of cortical bone, and thus does not tend to survive inhumation readily. However, the area surrounding the acetabulo-cristal buttress does persist. This buttress is a dense bar of bone which transmits axial body weight, via the auricular surface of the ilium to the lower appendicular skeleton. As a direct result of its close proximity to the buttress, the auricular surface of the ilium tends to survive inhumation well. Beal (J. Am. Osteop. Ass. 81, 1982) reported that 'The dimensions and relative proportions of
Proceedings of the Anatomical Society of
the sacro-iliac articular surfaces do not vary significantly for males and females'. Other studies have reported that the articular surfaces of the sacro-iliac joint display low levels of sexual dimorphism, and as a result, this joint has generally been considered of little value for sex identification. Most if not all diarthrodial articulations tend to show some degree of sexual dimorphism by virtue of the larger overall body size of the male. In this investigation, the relationship between the dimensions and relative proportions of the articular limbs and sex and body size were examined in a skeletal sample of documented sex. The results showed not only a significant sex difference in limb dimensions but also a complicated relationship between relative limb proportions and body size. These results are discussed. 25. The blood supply to the patella. By M. BROOKES, G. BRIDGEMAN and F. W. HEATLEY* Department of Anatomy U.M.D.S. Guy's Campus, London, and * Rayne Institute, St Thomas' Hospital, London Six cadaveric lower limbs donated in life from individuals 65-85 years old, were perfused intra-arterially with a 40 % suspension of barium sulphate in water. The knee joints in life were held to be normal. Angiography of whole patellae with skin and muscle attachments in situ, shows the presence of a peri-patellar extrinsic vascular plexus. An anastomotic peripatellar arterial ring as commonly represented is not present. Microangiography shows that marginal arteries pass to the bone, but vascular penetration into the three borders of the patella is negligible. The dominantarterial supply is from about ten central arteries which pierce the cortex anteriorly. A lesser blood supply comes from a small group of apical arteries. The two groups form an intrinsic vascular anastomosis in the patellar marrow. The central arteries are branches of the inferior lateral genicular artery. The apical arteries lie behind the ligamentum patellae, and are branches of the anterior tibial recurrent artery. The circumflex fibular artery can anastomose with both. It follows that the major blood supply to the whole patella comes from an infero-lateral source. The evidence suggests that in surgical procedures on the knee, vigorous haemostasis of prepatellar central arteries is to be avoided: a medial parapatellar incision is a safe approach; in lateral release procedures, care should be taken to conserve the infero-lateral blood supply to the
patella. 26. The medial and lateral muscle fans as stabilisers of the hip joint. By J. W. MORRENHOF and A. HUSON*. Orthopaedic Department, Erasmus University, Rotterdam and * Department of Anatomy and Embryology, University of Leiden, The Netherlands The gluteus medius muscle is often described as the main stabiliser of the hip joint of the supporting leg during the stance phase. Clinical evidence for this can be seen in the Trendelenburg gait of patients suffering from an insufficiency of the gluteus medius. However, action of the gluteus medius solely will be inadequate and additional activity of other muscles is needed for a mechanically exact prescribed motion. For this reason we developed a kinematical model comprising the geometrical data of the hip joint and of the insertions of 12 muscles, some of which were subdivided and represented by two or more different lines of action. These muscle lines were grouped into three muscle fans, one lateral and two medial fans. Moving the joint while keeping each of these fans alternately in isometric conditions the three degrees of freedom of the joint were reduced to only one. This resulted in a strictly defined path of motion of the femur. For each run this femur track could be calculated together with the concomitant length changes of the muscles constituting the other (antagonistic) fans. Analysis of the resulting curves showed clearly that a medial muscle fan, in combination with the gluteus medius muscle, is able to stabilise the human hip joint in an area around its neutral position. As a motion can be regarded as a series of consecutive positions, and as the leg can be brought from one stable position into a new stable position by a combined action of a medial and a lateral muscle fan, these fans must be able to produce a mechanically stable motion of the hip joint. 27. Testis-conditioned media influence the differentiation of mouse ovaries in vitro. By SANDRA J. TAVENDALE, SARAH MACKAY, and R. A. SMITH. Department of Anatomy, University of
Glasgow Mammalian gonads develop along female pathways unless subjected to masculinising influences (Jost et al. Rec. Prog. Horm. Res. 29, 1973). The first sign of male gonadal differentiation is the
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appearance of the presumptive Sertoli cells and their aggregation to form testicular cords in which germ cells become enclosed. This is followed by the formation of a tunica albuginea and Leydig cell differentiation. Ovarian differentiation occurs slightly later when groups of germ cells are partitioned within indistinct ovigerous cords (Mackay & Smith, Cell Diff. & Devel. 27, 1989). Female mouse germ cells enter meiosis at approximately Day 14 p.c. (post coitum); male germ cells undergo mitotic arrest at this stage as T-spermatogonia. In the present study, fetal gonads were removed from inbred CBA mice killed by cervical dislocation. Day 11 5 p.c. and Day 13 p.c. ovaries were maintained for 4-7 days in conditioned media (CM), generated by culturing Day 13 p.c. or Day 17 p.c. testes for 48 hours in Williams' E medium. Cultured ovaries were assessed by light and electron microscopy and by planimetric analysis. Electrophoretic analysis of CM gave several bands unique to male samples in the molecular weight range 65-80 kDa. Ovarian differentiation was modified by male CM: in Day 11 5 p.c. ovaries, cultured in Day 17-19 p.c. CM, tunica albuginea developed, but germ cell numbers and gonadal volume were not affected as compared with controls. Whereas, in Day 13 p.c. ovaries cultured in 13-15 p.c. CM., reductions in oocyte numbers and gonadal volume were noted compared to controls, although no tunica albuginea developed nor was meiosis inhibited. These results are consistent with those of Charpentier & Magre (C.r. Seanc. Acad. Sci., Paris 309, 1989) in the rat.
28. The repair and developmental effects of fracture on Meckel's cartilage in the chick embryo. By R. R. J. COUSELY (supervised by D. J. WILSON). School of Basic Medical Sciences/Anatomy, The Queen's University of Belfast Meckel's cartilage forms the initial skeletal rudiment of the mandible in both avian and mammalian embryos. It has been suggested that as this cartilaginous rod elongates, it dictates the growth and morphogenesis of the mandibular process. In addition, the morphogenesis of the cartilage indirectly influences that of the mandibular membrane bones, which form adjacent to, but not in contact with the cartilage (Thorogood, Cartilage, vol. 2, ed. B. K. Hall, Academic Press, 1983). The growth and morphogenesis of the cartilage itself are largely under intrinsic control (Hall, Can. J. Zool. 62, 1984; Noden, Devl. Biol. 96, 1983). Its origin from neural crest cells and its failure to undergo hypertrophy makes Meckel's cartilage distinct from the appendicular cartilages. In view of this, Meckel's cartilage on the right side of the mandible was fractured in vitro on Day 7 of incubation (Stages 29-30, Hamburger & Hamilton, J. Morph. 88, 1951). Observations were made of subsequent repair in the mandible over a seven day period, using Alcian green wholemount preparations, wax histology and scanning electron microscopy. The reconstitution of the cartilaginous rod and surrounding tissues was complete within 16 hours in 88% of cases. This repair had apparently occurred primarily by fusion of the cartilaginous matrix and secondarily by perichondrial activity. In the remaining cases, however, the mandible was deviated significantly to the operated side and there was a variable reductive effect on its outgrowth. This suggests that mandibular growth and morphogenesis closely parallel those of Meckel's cartilage. The total length and basic shape of the fractured cartilage, however, were not significantly different from the contralateral cartilage, suggesting that the growth and morphogenesis of the cartilage, itself, are largely under intrinsic control. Histological examination of these non-united and mal-united fractures indicated that mesenchymal intervention and subsequent fibroblastic proliferation may have been causal in the resultant dysmorphogenesis. Bone was also observed in the fracture gap after the initiation of mandibular osteogenesis, but seemed to be the result of tissue relocation. Irrespective of whether fracture healing had occurred, re-epithelialisation of the wound site was complete within 48 hours after surgery. Overall, surgery had resulted in minimal haematoma formation, an absence of large-scale cell death, no scar tissue, and no cellular blastema or fracture callus. Hence the speed of repair and sequelae of such embryonic wounds differ markedly from adult wounds and are consequences of the concurrent developmental processes occurring during the period of investigation. 29. Behaviour of grafts of rat ectoplacental cone explanted to the chick intraembryonic coelom. By M. D. BARBER (supervised by R. J. SCOTHORNE). Department of Anatomy, University of Glasgow Rodent trophoblast is commonly regarded as an actively invasive, cytolytic and phagocytic tissue, on the basis of its behaviour when explanted to ectopic sites (e.g. Kirby, J. Anat. 97, 1963). However, some of its apparent destructiveness may be due to the operative procedure. This
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possibility has been tested by explanting rat (Albino Swiss x Dark Agouti) ectoplacental cone (EPC) at 8 5 and 9 5 days p.c. to the intraembryonic coelom of 3-5 days old chick embryos (n = 31), using Dossel's method (Science, 120, 1954), which causes no operative damage. Embryos carrying grafts of EPC, or comparably sized grafts of chick chorion, or of various tissues from 10 days old chick embryos (as controls) were studied at intervals up to 4 days in serial wax sections, with 3-D computer reconstruction and by TEM. Control grafts were usually located in the coelom, adherent to the coelomic mesothelium, which was disrupted, but there was no invasion. Rat EPC transformed into giant cells, forming generally compact masses either lying within the pericardial cavity or embedded in, e.g. liver, lung bud or heart. From the main graft, flattened tongues of cells extended for up to 700 ,um (after three days), usually along basement membranes or tissue interfaces, in a manner different from that seen in utero. Giant cells frequently lay in close contact with host cells, without evidence of phagocytic or cytolytic damage. Despite frequent intimate contact with blood vessels, these were only occasionally breached and haemorrhage was unusual, so that many chicks with 8 5 day EPC grafts survived for up to four days; 9 5 day EPC grafts caused higher mortality. The findings raise some doubts about traditional views of the aggressive 'invasiveness' of rat trophoblast. 30. The development of mesonephric tubules and their possible contribution to gonadal and adrenal cortical anlagen in the CBA mouse. By C. SMITH (supervised by S. MACKAY). Department of Anatomy, University of Glasgow This study investigated the histogenesis of the mesonephric tubules from the intermediate mesoderm, and the possible 'transdifferentiation' of tubule epithelial cells to gonadal somatic cells (Wartenberg., Archs. Anat. Micros. Morph. exp. 74, 1985). and/or adrenal cortical cells (Upadhyay et al. Anat. Rec. 202, 1982). Embryos at Stages 13-21 (Theiler) were obtained from inbred CBA mice killed by cervical dislocation. Material was processed for light and electron microscopy and the development of basal laminae was studied by immunocytochemical localisation of laminin. Mesonephric tubules first appeared, at Stage 15, as an unorganised mass of condensed mesenchyme. The cells became aligned, elongated and formed junctions. A basal lamina appeared around the cranial tubules at Stage 16, being seen first round the dorsal margins of tubules and only by late Stage 20 at the ventral margins, by which time population of the gonadal blastema was complete. The cells of the ventral margins of tubules were morphologically identical to other mesenchymal cells of the mesonephros. Gonadal somatic cells and adrenal cortical cells appeared to be derived from mesonephric mesenchyme, from the ventral margins of tubules and from the coelomic
epithelium. Caudal tubules degenerated by apoptosis and there was no cranio-caudal sequence of
degeneration. It is concluded that cells of the ventral margins of mesonephric tubules did not show an epithelial phenotype during the period of population of the gonadal blastema by mesenchyme. Thus it is not necessary to postulate 'transdifferentiation' of these cells. Further, the mesonephros did not appear to be the only source of gonadal somatic cells and adrenal cortical cells in the mouse.
31. The developing pronephros in the Chelonian Caretta caretta. By P. COLLINS, D. A. GOULDING and F. S. BILLETT*. Human Morphology and *Department of Biology, University of
Southampton (Fig. 5) The developing kidney is classically described as having three parts which succeed each other both spatially and temporally: the pronephros, mesonephros and metanephros. Whilst the pronephros is active in larval teleosts and amphibians it has always been described as vestigal in amniote embryos. This study describes the differentiation, maturation and subsequent degeneration of the pronephros in the Chelonian Caretta caretta. Caretta caretta eggs were collected for a study on the relationship between sex ratio and incubation temperature. Embryos were examined at intervals throughout development and staged according to Miller (J. D. Miller, Biology of Reptilia, vol. 14, J. Wiley & Sons, 1985). The embryos were fatally anaesthetised with MS222 or Sagatal according to age and prepared for scanning electron microscopy. Subsequently the specimens were re-embedded and sectioned for light and transmission electron microscopy.
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In 8-somite embryos, Miller Stage 12, podocyte precursor cells can be seen in the somatopleuric layer of the intraembryonic coelom wall just lateral to the pericardial cavity. By Miller Stage 14 the podocytes cover blood vessels which bulge into the coelom and they have primary branches. Also at this stage the first pronephric tubules invaginate from the coelom wall. Between Miller Stages 19 and late 21 the glomus is developed to its fullest extent (Fig. 5a) and is completely covered with podocytes, these have both primary and secondary branches and pedicles which interdigitate with those of neighbouring cells (Fig. 5b). Lateral to each glomus is ciliated tract which conceals the ostia of six ciliated pronephric tubules (Fig. 5a, c). Degeneration of the pronephros begins at Miller Stage 22, the cilia become sparse and the podocytes lose their pedicles. By late Stage 23, cilia are seen infrequently, the podoctyes have no branches and are coated with extracellular matrix. Ultrastructural features of the pronephric podocytes and pronephric tubules are similar to those of corresponding cells in the metanephric kidney. This study provides the first account of pronephric podocytes in a reptilian embryo and the results suggest that the pronephros reaches a functional state in the developing amniote Carreta caretta.
32. Development of the septal leaflet of the tricuspid valve in the human heart. By JOANNA M. R. JoNEs (supervised by G. Moscoso and R. H. ANDERSON). Department of Anatomy, University College London Knowledge concerning the formation of the leaflets of the atrioventricular valves in the human heart is remarkably vague. Indeed, much of what is written is unequivocally wrong. In the light of the uncertainty, I have studied the stages of differentiation of the tricuspid valve, concentrating on its septal leaflet, using scanning electron microscopy supplemented by examination of serial histologic sections. The formation of the valve was examined in the context of the differentiating right ventricle and atrioventricular junction. In all, 29 hearts from human fetuses were prepared by a standardised protocol avoiding fixation artefacts and studied by scanning electron microscopy. They spanned the time period from 41 days to 12 weeks after the last menstrual
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period. A further six hearts, covering the period of 10 to 12 weeks of development, were prepared for light microscopy. The most significant finding is that, at the time of closure of the embryonic interventricular communication (7- weeks), there is no morphological evidence of any structure destined to become the septal leaflet of the tricuspid valve. The first sign of formation of the leaflet, seen at eight weeks of development, is appearance of a bilobed prominence occupying the septal surface. Subsequently, delamination occurs at the inferior margin of the prominence and extends superiorly, forming the primordium of the leaflet. Thereafter, delamination extends towards the apex, forming a structure which is the precursor of the chordae tendineae and papillary muscles. Longitudinal splits occur within the precursor to produce the immature tension apparatus. This is matched later by a supero-inferior delamination which lifts the tension apparatus away from the septal myocardium. Formation of the leaflet proper is completed in the twelfth week of
development. 33. Epidermal growth factor and its B-loop fragment are capable of promoting embryonic angiogenesis. By ROSEMARY STEWART (supervised by D. J. WILSON). Schools of Basic Medical Sciences/Anatomy and Clinical Dentistry/Dental Surgery, The Queen's University of Belfast The angiogenic effects of epidermal growth factor (EGF) were studied, using the chick extraembryonic vasculature as a model. Millipore filter discs containing 10 ng-l ,ag of EGF were placed onto the advancing edge (sinus terminalis) of the area vasculosa of 3-day chick embryos, and the effect examined macroscopically and histologically 24 hours after disc application. The capillary density at the site of application increased significantly, and the effect was seen to be dose-dependent: a similar but more marked response was observed in the vessel cross-sectional area per unit length. This change in vascularity was accompanied by tortuous folding of the mesoderm and endoderm (which normally lie parallel to the ectoderm in a trilaminar arrangement) into the yolk substance. This may indicate precocious development of all three layers (ectoderm, mesoderm and endoderm) of the membrane, and that the proliferative effects of EGF may not be confined to the vascular endothelium. A synthetic peptide corresponding to amino acid residues 20-31 (the 'B-loop') of the parent EGF molecule was also capable of inducing a considerable increase in capillary density in the chick vitelline membrane, when examined 24 hours after administration. Again the effect was dose-dependent, but EGF 20-31 was less potent in its promotion of angiogenesis (being effective in the range 1 ,ug-l mg). An inactive 11-amino acid synthetic peptide (corresponding to the Nterminal region of pancreatic polypeptide) assayed alongside the 20-31 fragment had no effect on the membrane capillary density and so EGF 20-31 appears to be specific in its angiogenic action. In addition, the effects of somatostatin and its analogue Sandostatin, which are known to inhibit EGF-induced proliferation of some cell lines in vitro, were investigated. Both these compounds inhibited the outgrowth of the capillary network (Stewart, Nelson & Wilson, J. Anat. 164, 1989) and caused the formation of avascular areas behind the advancing sinus terminalis. The effects of these peptides suggest that endogenous EGF-like factors may be involved in the normal stimulation of growth of the area vasculosa capillary network. Supported by grants from the Wolfson Foundation and the Humane Research Trust. 34. Cytoskeletal changes associated with secretion in rat basophilic leukaemia cells. By J. P. BENNETT and J. CLINTON (introduced by J. A. FIRTH). Department of Anatomy and Cell Biology, St Mary's Hospital Medical School, Imperial College, University of London Rat basophilic leukaemia cells (RBL cells) grown in tissue culture have an IgE-mediated secretory response analogous to that of normal basophils and mast cells. RBL cells contain IgE receptors on their plasma membrane, and if provided with specific IgE they acquire secretory responsiveness to the polyvalent antigen. In this study we used a monoclonal IgE directed against dinitrophenol (DNP) to prime cells, and a DNP-albumin complex to elicit secretion. We found that the time-course of secretion consisted of three phases. There was a lag of approximately 1 minute following addition of DNP-albumin during which no secretion was observed. The majority of secretion then occurred over the following 5 minutes, with half the final level being reached at about 3-5 minutes. Finally there was a termination phase when small but measurable levels of secretion continued for around an hour. Unstimulated RBL cells have large numbers of microvillus-like projections on their surface,
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approximately 0 08 ,um in diameter as determined from scanning electron micrographs. During secretion these disappear and instead there are large cell surface ruffles, 1-10 lum in length. This change in cell surface morphology precedes actual secretion, with the first ruffles being observed during the lag phase. At the end of secretion, in the termination phase, the small projections reappear. Using immunofluorescence we found that the ruffles associated with secretion contain significant amounts of actin and the actin-binding protein, gelsolin. There is some evidence that this ruffling, which may be an essential part of the secretory process in RBL cells, involves recruitment of phosphorylated myosin to the regions of ruffle formation. 35. Collagen fibril populations in repaired extensor tendons of rabbit hind limb. By KAREN WILSON and M. J. MOORE. Department of Anatomy, Marischal College, Aberdeen. A method has been devised in which the process of tendon repair can be observed at various stages. This method has been previously introduced to the Society (Wilson et al. J. Anat. 161, 1988) and involved removing the middle third from a proximal stump of a transected tendon, folding this 'tongue' over and stitching it to the distal stump to re-establish continuity. This report concerns the changes which occur in the collagen fibril populations subsequent to the repair. The operation was carried out under halothane anaesthesia to the hind limb, third toe extensor digitorum longus tendon of seventeen adult female New Zealand White rabbits. The animals were killed using 1 ml Hypnorm intravenously under halothane anaesthesia and tissue removed at 14 days (n = 5), 30 days (n = 6), and 60 days (n = 6) postoperatively. Contralateral tendons were used as controls. The sites of specific interest in this study were (a) the remaining tendon strips, (b) the window between them, (c) the tongue, and (d) the regenerated girth which appeared on either side of the tongue. The tissue was prepared for electron microscopy by conventional techniques. A systematic random sample of collagen fibril diameters was measured to give frequency distributions. Compared with controls two types of fibril diameter distribution were observed. Firstly, in regions (b) and (d), a narrow range of diameters, the mean of which changed only little throughout the experimental period. Secondly, in regions (a) and (c), a broad range of diameters similar to controls, but with marked increase in the percentage of small diameter fibrils. The presence of the small fibril population in the experimental areas is the result of differing stimuli. Initially, in regions (b) and (d) the tendon responds to injury and replaces 'lost' tissue with a translucent material composed of small diameter fibrils (< 40 nm). The tissue in regions (a) and (c), by an increase in stress per unit cross-sectional area, is stimulated to produce small diameter fibrils. The distribution of collagen fibril diameters does not regain control values in any region. The model has provided valuable information about the way in which tendons respond to changed mechanical requirements following this method of repair. Karen Wilson was supported by the Scottish Home and Health Department.
36. A comparative biochemical and ultrastructural study of proteoglycan;collagen interactions in corneal stroma. By J. E. ScoTT and T. R. BOSWORTH. Chemical Morphology, Cell and Structural Biology, University of Manchester The interfibrillar proteoglycans (PGs) of corneal stroma exert a swelling pressure that keeps the collagen fibrils a constant distance apart, this being essential for transparency. We have examined the PG: collagen relationships in mouse, rat, guinea-pig, rabbit, cat, dog, sheep, pig and cow using the PG stains Alcian blue (AB) and Cupromeronic blue (CB) for light and electron microscopy, respectively, in critical electrolyte concentration methods. Biochemical analyses quantified glycosaminoglycans (GAGs), proteoglycans (PGs) and collagen. In all species PGs were associated at the a, c, d, and e bands along the collagen fibril, keratan sulphate (KS) PG at the a and c bands and chondroitin (deramatan) sulphate (CS-DS) PGs at the d and e bands. The occupancy of the a, c bands increased with increasing corneal thickness. The d band was heavily populated in all species. These findings quantitatively accord with the biochemical analyses. The ratio KS/total GAGs increased with stromal thickness, supporting the hypothesis that 02. which diffuses from outside into the stroma, determines the KS: CS-DS balance. KS biosynthesis consumes no 02, and it can be made in conditions of 02 lack or low NAD: NADH ratio (Scott & Haigh, J. Anat. 158, 1988). The ratio of DS: CS diminishes with stromal thickness, also suggesting an 02 dependant step, perhaps involving NAD NADH ratios at the epimerisation step from CS.
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NW-' R.: 1,
Fig. 6 37. The incidence and histology of the brochs of human enamel. By J. A. P. JAYASINGHE (introduced by A. UE)4 Department of Anatomy and Developmental Biology, University College, London
Brochs (microelevations at the cervical margin of the enamel; Boyde, J. Anat. 109, 1971) were investigated using different types of microscopy to reveal their incidence and distribution, morphology and histology. Examination of 751 permanent teeth under the stereo binocular microscope revealed that 40% had brochs, while the distribution among premolars, molars, incisors and canines was 67 %, 35 %, 23 % and 12 % respectively. Their incidence was less on the buccal surface. In 48 % of the premolar teeth the broch patches were scattered on the surface, while the rest were arranged in bands around the cervix. The maximum width of the band observed was 1 mm. The density in these bands was usually about 133 mm2. Their average height was 10 ,um, and the average diameter was 32 ,sm. The distribution of brochs is similar in contralateral teeth from one individual. The forms of the brochs were extremely varied, and were well illustrated by SEM (Fig. 6a-d. These features have not previously been reported in deciduous teeth, but during this study they were discovered in incisors (Fig. 6d), canines (Fig. 6c) and molars (Figs. 6a,b). Unlike the case in permanent teeth, brochs were usually confined to one surface only and most deciduous brochs had an intact surface enamel layer.
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245 "IA ~....,
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38. Ultrastructure of melanocytes in normal individuals and patients with alopecia areata. By D. J. TOBIN, D. A. FENTON* and MARION KENDALL. Cell Biology Unit, Department of Biochemistry, UMDS and * Dowling Skin Unit, St Thomas's Hospital, London (Fig. 7) Hair follicle melanocytes, normally found around the connective tissue invagination of the actively growing hair follicle, differ from epidermal melanocytes by exhibiting periodic activity in melanin synthesis although both have a neural crest origin. An ordered sequence of melanin deposition is characteristic of normal melanosomes and all stages are routinely observable in normal melanocytes. Alopecia areata is thought to be an autoimmune disorder, precipitating premature involution of the hair follicles with consequent shedding of hairs. Pigmentary abnormalities may be associated including vitiligo, ocular depigmentation, sparing of white hair and regrowth of depigmented hair. It has been hypothesized that melanocytes may be involved in the pathomechanism of alopecia areata (Messenger et al. Br. J. Dermatology 110, 1984) although no ultrastructural evidence has been documented. Scalp biopsies (4 mm punch) were taken, with consent, under local anaesthesia (Lignocaine hydrochloride 2 % w/v) from three normal adults, and ten patients with untreated acute alopecia areata. Light and electron microscopy revealed morphological changes in affected cells including increased numbers of bizarre melanosomes, compared with normal melanocytes, and marked nuclear and cytoplasmic hyperchromatism (Fig. 7 a). Bizarre melanosomes had incomplete or 'aborted' melanisation resulting in poor pigment deposition and a disrupted, enlarged and rounded shape (Fig. 7b). Other atypical melanosome effects included marked pigment displacement into peribulbar and dermal papilla melanophages. In the dermal papilla clumped melanin granules formed giant spherical complexes without discernible membranes, which were sometimes associated with lymphocytes. An unusual outer root sheath distribution, below the critical level, was seen in some affected hair follicles. These morphological changes indicate an active involvement of melanocytes in the etiology of alopecia areata. 39. A novel epidermal growth factor-secreting cell lineage induced from human gastrointestinal mucosa by chronic ulceration. By N. A. WRIGHT, CHRISTINE PILE and G. ELIA. ICRF Histopathology Unit, Lincoln's Inn Fields, London Epidermal growth factor, and its human homologue, urogastrone (EGF/URO) is secreted by the gut-associated salivary and Brunner's glands. Recombinant EGF/URO is a powerful stimulator of cell proliferation and differentiation in the rodent and neonatal human intestine. However, EGF/URO is not absorbed from the adult gut and has no action when given via the gut lumen; thus the physiological role of secreted EGF/URO is unknown. We now report that ulceration of the epithelium anywhere in the human gastrointestinal tract induces the development of a novel cell lineage from gastrointestinal stem cells. This lineage initially appears as a bud from the base of intestinal crypts, adjacent to the ulcer, and grows locally as a tubule, ramifying to form a new, small gland, and ultimately emerges onto the mucosal surface. The lineage produces neutral mucin, shows a unique lectin-binding profile and immunophenotype, is non-proliferative, and contains and secretes abundant immunoreactive EGF/URO. We propose that all gastrointestinal stem cells can produce this cell lineage following mucosal ulceration, secreting EGF/URO to stimulate cell proliferation, regeneration and ulcer healing. This cell lineage is very commonly associated with gastrointestinal mucosal ulceration, and we conclude that a major in vivo role for EGF/URO is to stimulate ulcer healing throughout the gut via induction of this cell lineage in the adjacent mucosa. DEMONSTRATIONS D. 1. A sampling scheme for studying the functional morphology of the coprodaeum in hens on highand low-NaCI diets. By T. M. MAYHEW, V. DANTZER*, V. S0DRING_ELBROND* and E. SKADHAUGEt. Department of Anatomy, University of Aberdeen and Departments of * Anatomy and t Animal Physiology and Biochemistry, The Royal Veterinary and Agricultural University, Copenhagen (Fig. 8) The avian coprodaeum (cop) forms part of the cloaca. In the coprodaeum, absorption of Na from the intestinal lumen is variable. On a high-salt (HS) diet, the epithelium (epi) absorbs little
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Remove coprodaeum and open lengthwise
Cut in half
ro.. Fig. 8
Na but on a low-salt (LS) diet, absorption increases dramatically (100-fold). Physiological studies have shown that Na permeability increases at the apical plasma membrane of epithelial cells. Morphological studies suggest that the microvillous surface area (Smic) expands on the LS diet. This study presents a sampling scheme for combining physiological estimates of Na absorption with stereological estimates of Smic obtained from the same coprodaeum. The scheme is summarised in Fig. 8. The coprodaeum was removed from hens killed by decapitation. It was divided into two roughly equal halves whose fresh weights and volumes were measured. Halves were assigned randomly for physiology or morphometry. Following fixation in buffered glutaraldehyde, fixed weight and volume (VC0,) were determined and shrinkage factors were calculated. Systematic random samples of tissue were obtained for light (LM) and electron microscopy (EM) and embedded in resin at known orientations so that they could be sectioned vertically (Baddeley et al. J. Microsc. 142, 1986). For this purpose, the muscularis layers provided the 'horizontal' references. Stained LM sections were projected (linear magnification: M = 75) on to a wall and tracings made of vertical strips. Tracings were analysed to estimate epithelial volume density, V,Pi/ VJ',. Vertical EM sections were used to estimate epithelial surface density, Sepj/ V,pj (M = 4536) and the microvillous amplification factor, Smi(/Sep, (M = 31150). From macroscopic, LM and EM estimates, values of Sm.c were calculated.
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Preliminary findings are presented for hens (3 7-64 kg body weight) fed on HS and LS diets. These suggest that the LS diet induces larger and/or more numerous microvilli because the total microvillous surface is roughly twice that seen in hens on the HS diet. Supported by E.E.C. grant ST2J-0392-C (EDB) and by grant 13-7224 from the Danish Agricultural and Veterinary Research Council. D. 2. A morphometric study on placental diffusing capacity for oxygen in maternal diabetes. By MOIRA R. JACKSON, T. M. MAYHEW*, F. B. SORENSENt and J. G. KLEBE4. Department of Human Anatomy, University of Oxford, * Department of Anatomy, University of Aberdeen, t Stereological Research Laboratory, University of Aarhus and t Department of Obstetrics and Gynaecology, Aarhus University Hospital, Denmark In this study, placental dimensions relevant to gaseous exchange have been quantified in order to assess whether or not the duration and severity of maternal diabetes influence oxygen diffusing capacity (Dp). Thirty-four organs delivered after normal, uncomplicated pregnancies served as controls. Using the White classification, five diabetic groups were identified: classes A (least severe, n = 22), B (n = 11), C (n = 7), D (n = 12) and F/R (most severe, n 7). Mothers were closely followed to obtain comprehensive clinical, biochemical, anthropometric, haematological, physiological and gestational age information. Diet and insulin dosages were recommended individually. At delivery, infant sex and weight were noted. Systematic random samples of placentae were immersion-fixed in formalin, embedded in paraffin, cut at 4,m and stained by the Masson trichrome technique. Random samples of micrographs ( x 250) and transparencies ( x 2000) were analysed by stereological methods. Estimates of Dp (in ml/min/torr) were obtained using a slight modification of an earlier method (Mayhew et al. J. Anat. 139, 1984). The materno-fetal oxygen pathway was divided into six serially-arranged compartments (maternal erythrocytes, maternal plasma, trophoblast, stroma, fetal plasma, fetal erythrocytes) whose partial resistances were summed to obtain total resistance to diffusion, Rp. The reciprocal of Rp is Dp. Comparisons between groups were drawn after analyses of variance and Student's t test. No differences between control and diabetic groups were found for birth weights but placental weights in classes A, B and D were greater than in controls. Values of Dp in classes A, B, C and D ranged from 4-4 to 4-9 ml min-' torr'1 and were also greater than in controls (value 3 9 ml min-' torr-'). In the most severe class, total Dp was only 3-4 ml min-' torr-'. Coefficients of variation amounted to about 30 % of their group mean Dp values. When normalised for birth weights (in kg), specific placental diffusing capacities did not vary significantly across all groups. However, values appeared to be smallest in class F/R organs (1l0 ml min-' torr-' kg-', c.f. 1l1 ml min-' torr-I kg-' in controls and 1 3 ml min-' torr-' kg-' in other White classes). Study supported by a research assistantship awarded to T. M. M. by The Cunningham Trust. =
D. 3. Endocytosis of HRP-labelled immunoglobulin-G by the syncytiotrophoblast of the perfused human placenta. By LOPA LEACH, B. M. EATON, J. A. FIRTH* and SOLI F. CONTRACTOR. Department of Obstetrics and Gynaecology, Charing Cross and Westminster Medical School and * Department ofAnatomy and Cell Biology, St Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London Selected lobules of term human placenta were extracorporeally perfused and immunoglobulinG complexed to horseradish peroxidase was added to the maternal side. The IgG-HRP was visualised using diaminobenzidine cytochemistry after different perfusion durations. IgG-HRP was found bound to microvilli and coated pits of the syncytiotrophoblast. Endocytosis into coated vesicles and tubulovesicular bodies occurred within the first ten minutes. Subsequently, IgG-HRP was found in multivesicular bodies and by thirty minutes began to appear in basal vesicles, the frequency of the latter event increasing with time. Routing of IgG-HRP into Golgi regions or lysosomes was not observed. IgG-HRP was found in microdomains of the basal plasma membrane after thirty minutes. The pattern of uptake and routing observed suggests receptormediated endocytosis followed by transcytosis of IgG-HRP across the syncytiotrophoblast. Unconjugated HRP was located in early endocytotic compartments similar to those involved in uptake of IgG-HRP but did not bind to microvillar regions and was not transcytosed across the syncytiotrophoblast.
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D. 4. The effect of change in pH of the medium on the uptake, transport and digestion of immunoglobulin-G (IgG) by the rat visceral yolk cultured as a closed vesicle. By A. TOOMEY, MARGARET K. PRATTEN and A. P. GULAMHUSEIN. Department of Anatomy, University of Leicester The transfer of passive immunity in the rat occurs in a pH dependent fashion by the neonatal gut (Rodewald, J. Cell Biol. 71, 1976) and also via a less well-defined system, the visceral yolk sac, which is the principal site for antibody selection and uptake. In order to investigate the latter, rat visceral yolk sacs were explanted at 9 5 days from animals killed by terminal anaesthesia in an ether chamber and cultured as closed vesicles (Dunton et al. J. Anat. 145, 1986) up to 17 5 days at 37 °C in a medium consisting of a 50:50 mixture of rat serum and Medium 199. The cultured yolk sacs were then placed in media of differing pH (Medium 199 buffered to pH 56-8 4) to equilibrate for four hours before the addition of 1251I-labelled IgG to the culture medium. After a 15 hour incubation period, the yolk sacs were harvested. Samples of the exocoelomic fluid, the incubation medium and the visceral yolk sac tissue were taken to determine total radioactivity and TCA soluble counts (digested material). The total protein content of the yolk sac tissue was also determined. Uptake of IgG peaked at pH range between 60-7 0 and thereafter the rate decreased with increasing pH. At lower pH (6-7) a large proportion of the IgG taken up by the yolk sac tissue passed intact into the exocoelomic fluid, while at higher pH (8-0 and above) most of the molecule was digested. Ultrastructural examination of 17 5 day visceral yolk sac cultured at 37 °C in a medium (pH 7 40) containing colloidal gold-labelled IgG revealed the presence of gold particles in apically located coated pits, vesicles and large vacuoles, the latter being part of the lysosomal compartment of the visceral yolk sac endodermal cell. The results indicate that the passage of intact IgG across the visceral yolk sac is a pH-dependent process, where slightly acid conditions favour binding to the receptor and subsequent transport to the embryonic face of the yolk sac tissue.
D. 5. Astrocytes remove waste material from within neurons. By C. C. NOLAN, J. B. CAVANAGH and A. W. BROWN (introduced by M. E. CAVANAGH). Toxicology Unit, MRC Laboratories, Carshalton, Surrey Ultrastructural studies of the effects of triethyllead (TEL), trimethyltin (TMT) and acrylamide on rodents have revealed a new aspect of neuronal-astrocytic interaction, whereby perineuronal astrocytes insert processes into neuronal perikarya to remove abnormal material. Animals were killed by perfusion fixation under deep ether anaesthesia. Within 48 hours of a single dose of TEL (19 mg/kg) to rats large numbers of dense bodies formed in the perikaryon and dendrites of hippocampal pyramidal neurons, only a few of which became necrotic. The dense bodies were removed by selective transfer to astrocyte processes inserted into the neuronal cytoplasm. These intrusions became most numerous at four days. The majority of neurons survived these changes, returning to apparent normality by seven days (Nolan & Brown, Neuropath. Appi. Neurobiol. 15, 1989). Removal of similar dense bodies from hippocampal neurons by astrocyte intrusions also occurs after TMT poisoning in rats (10 mg/kg) and gerbils (4 mg/kg). Astrocyte intrusions have also been seen removing clusters of abnormal SER from perikarya of cerebellar Purkinje neurons within 24-48 hours of a single (90 mg/kg) dose of acrylamide (Cavanagh & Gysbers, J. Neurocytol. 12, 1983). Glial intrusions may be a general mechanism for the removal of accumulations of abnormal material from neurons, requiring as yet unknown neuron-astrocyte signals. Preliminary studies on the effects of the protease inhibitor Leupeptin, which causes dense body accumulation in both neurons and glia, have so far failed to reveal any direct involvement of astrocytes in recovery, suggesting that the precise nature of the abnormal material and the astrocytes' capacity to respond are both critical to the initiation of this response. D. 6. Quantitative analysis of the microvasculature of pelvic sympathetic ganglia in young and aged rats. By JANE A. GRIFFITHS and R. M. SANTER. Department ofAnatomy, University of Wales College of Cardiff Hypogastric and paracervical ganglia were removed under ether anaesthesia from rats of 4 and 24 months of age. Frozen sections of the ganglia were cut and incubated in a Gomori-type medium for the demonstration of alkaline phosphatase (AP) activity. The reaction product was visualised
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in this multiple sequence enzyme histochemical reaction by successive treatment with cobalt chloride and ammonium sulphide followed by counterstaining with 0 4% cresyl violet. AP was confined to the endothelial cells of the microvasculature and consequently revealed the ganglionic microvascular bed clearly even though the intensity of the reaction product was reduced in the 24 months rats. A quantitative analysis of the microvasculature was made by measuring the length of the stained microvessels and their diameter and then calculating the volume of tissue sampled. From these measurements, the density of the microvasculature in mm/mm3 was calculated and the total luminal volume, luminal surface area and the surface area of endothelium available for gas exchange to a given volume of blood subsequently derived from the data. In the hypogastric ganglion but not in the paracervical ganglion there was a. significant increase in microvessel diameter (P < 0 002) in the 24 months animals and in both ganglia the density of the microvascular bed decreased significantly (P < 0-001) in old age. These results are compared with those from an earlier study of the microvasculature in other sympathetic ganglia in young and aged rats (Santer & Kabeer, Expl Brain Res. 16, 1987). D. 7. The effect of age on succinate dehydrogenase activity in neurons of the rat nodose ganglion determined by a quantitative enzyme histochemical method. By D. M. BAKER and R. M. SANTER*. Department of Surgery, City Hospital, Nottingham and * Department of Anatomy, University of Wales College of Cardif Nodose ganglia (NG) were removed under ether anaesthesia from Wistar rats, frozen and cryostat sectioned at 10,um. The sections were incubated in a phosphate buffered medium containing sodium succinate and nitroblue tetrazolium to demonstrate succinate dehydrogenase (SDH) (EC 18.104.22.168). Readings of the final reaction product intensity in neuronal perikarya were taken using a Vickers M85 microdensitometer at the isobestic wavelength of 540 nm. Optimum conditions were first validated to ensure accurate quantitation of SDH activity with respect to the stain intensity in the cryostat sections of NG. This included verification of the Beer-Lambert laws and obtaining typical enzyme reaction curves with variating temperature, pH and substrate concentrations. Sections of NG from six 6 months and six 24 months rats were then identically processed to demonstrate SDH. Enzyme kinetic values were derived from the data using the Hans plot. For 6 months rats Vmax = 5*44 + 0 52 (absolute units of integrated optical density per minute) and Km = 0-097 + 0-022 mm. For 24 month rats Vmax = 2-87+0-63 (absolute units) and Km = 0-067 + 0031 mm. Km values do not vary with age but Vmax show a significant decrease (P < 0-01) with age. SDH activity can be used as an indicator of overall metabolic activity and this result therefore suggests that, with age, there is a decrease in metabolic activity of rat visceral sensory neurons as well as in postganglionic sympathetic neurons (Baker & Santer, J. Anat. 146, 1986; J. Anat. 164, 1989). D. 8. Bicuspid value innervation patterns. By J. C. FOLAN,* Y-F. WANG, J. Y. JEW and T. H. WILLIAMS. * Department of Anatomy, University College Dublin, Ireland and Department of Anatomy, University of Iowa, Iowa City, USA Whole-mount preparations facilitate investigation of terminal innervation patterns. The atrioventricular valves serve as a model for autonomic networks and provide the potential for studying a series of contributions made to the networks by transmitter specific nerves. The objective of the present study was to compare the patterns of innervation in the bicuspid (mitral) valves of four species, mouse, rat, guinea-pig and opossum. The hearts were removed under Nembutal anaesthesia and wholemount preparations of the bicuspid valves stained for acetylcholinesterase (AChE). The best and most uniformly stained segments of valve nerve networks were drawn using a Camera Lucida/Nikon Optiphot system. Video images of the drawings were then converted to a digital image format using a Gould IP8500 image processor. From these images the densities of nerve fibres per segment were calculated. The acetylcholinesterase-stained nerve plexus consists of thick bundles of nerve fibres forming a large mesh which we term a 'primary plexus'. The smaller meshwork formed by finer nerve strands that apparently come off the primary plexus is the 'secondary plexus' with the final free nerve endings and specialisations that branch from this secondary plexus forming a tertiary component. The basal zone of the valve adjacent to the fibromuscular atrioventricular ring displayed the most dense plexus of nerves with AChE-positive fibres being seen across the width of the valves. In the intermediate zone of the valve only moderate numbers of nerve fibres were present, more numerous in the cuspal areas and less numerous in the intervening commissural
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areas. The distal valve nerve networks arborised extensively, with some nerve fibres extending towards the chordae tendineae and free edge of the valve cusp. Only in the guinea-pig and oppossum did these fibres reach the free margin of the valve cusp where they either ended directly as free nerve endings or lay parallel to the free edge of the cusp often running between adjacent chordae tendineae. Free nerve endings with no light microscopic evidence of specialisation were present throughout the mouse, rat, guinea-pig and oppossum bicuspid valves. Some nerve endings in the oppossum showed evidence of specialisation, with brush-like arborisations leading to presumed free terminals seen chiefly in the distal zone of the valve cusps. Although some general tendencies were apparent we have demonstrated considerable interspecies heterogeneity in the autonomic terminal networks of the bicuspid valve. Noradrenergic, Substance P-like and VIP-like peptidergic nerve contributions are currently being studied as an extension of this project. Supported by NIH grant DK38123. D. 9. Interaction of rat and mouse granulated metrial gland cells with immunoglobulin G and albumin in vitro. By E. ADAM and S. PEEL. Human Morphology, University of Southampton Mouse granulated metrial gland (GMG) cells possess receptors which bind immunoglobulin (IgG) and this raises the question of the function of these receptors, particularly in view of the cytotoxic activity of mouse GMG cells. This work examines rat and mouse GMG cell interaction with IgG in, respectively, rat and mouse serum in vitro. By varying the length of time and the temperature at which the cells were incubated in serum, and by detecting IgG and albumin by immunofluorescence techniques, the involvement of receptors in the interaction of GMG cells and these proteins has been assessed. Cytoplasmic IgG, and to a lesser extent albumin, was demonstrated in rat GMG cells after 0 5 and 5 hours incubation at 37 °C in medium containing rat serum but no endocytosis was detected when incubations were done at 4 °C. Incubation at 4 °C, however, resulted in surface binding of IgG. When rat GMG cells which had been incubated for 0 5 hours at 4 IC in rat serum were washed and incubated further for 4-5 hours at 37 °C in fetal calf serum, cytoplasmic IgG was detected suggesting that the IgG was initially bound by receptors and subsequently internalised. Similar binding and subsequent internalisation of albumin was not detected. When mouse GMG cells were incubated (0-5 or 5 hours) at 37 °C in medium containing mouse serum no evidence for the endocytosis of IgG or albumin was found but IgG was detected as a 'cap' at one edge of most GMG cells. If incubations were carried out at 4 °C IgG was detected around the entire periphery of most GMG cells: albumin was rarely detected. IgG and albumin were not detected after incubation at 4 °C in mouse serum for 05 hours followed by 4-5 hours in fetal calf serum at 37 'C. Receptor mediated endocytosis of IgG is shown by rat, but not mouse GMG cells. D. 10. Variation in exudate associated with dietary fat in the rat small intestine. By S. NUNN, F. A. SAGHER*, J. A. DODGE* and K. E. CARR. School of Basic Medical Sciences/Anatomy and * Department of Child Health, the Queen's University of Belfast Many workers clean small intestine specimens by a variety of methods prior to examination using scanning electron microscopy (SEM), in order to maximise mucosal detail. In this study specimens were not cleaned as the true interface between the lumen and the underlying tissue would be through such material. This layer has been termed mucosal exudate and consists of mucus, micro-organisms and cells/debris. Female Sprague-Dawley rats aged three weeks were fed iso-caloric diets in which 40% of the total calories were provided as either butter, corn oil or olive oil, while one group was kept on a low-fat standard diet as controls. There were five animals in each group. After 8-5 weeks the animals were anaesthetised using intraperitoneal sodium pentabarbitone and then killed by cardiac puncture. Portions of jejunum and ileum were fixed in 3 % glutaraldehyde in 0-2 M cacodylate buffer, pH 7-4, dehydrated through a graded ethanol series, critical point-dried, sputter-coated with gold and examined in a JEOL JSM-840A SEM. Specimens were also prepared for resin histology and semithin sections were stained with toluidine blue and basic fuchsin. Measurements of crypt length and villous height were prepared and goblet cell counts made. Marked variation was found in the exudate layer between animals on the different dietary regimes. In those fed on butter the jejunal villi were predominantly surrounded by mucous sheets;
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mucus was more patchily distributed in the ileum. In the corn oil-fed animals jejunal mucus appeared similar to that of butter-fed rats, although it contained more particulate debris with fewer bacteria. The ileum had more mucus than the jejunum and this ileal mucus was stringier and less tenacious, with more debris present. Rats fed on the olive oil diet showed a similar pattern to those on corn oil. Changes in crypt length, villous height and goblet cell numbers were most marked after the butter diet. It can be concluded that dietary fat plays a role in the nature and extent of exudate in the rat small intestine. This work was supported by the Medical Research Council.
D. 11. A microscopical study of the persorption of iron particles in rat jejunum. By J. S. MCCULLOUGH and K. E. CARR. School of Basic Medical Sciences/Anatomy, The Queen's University of Belfast Persorption is a phenomenon whereby large particles (> 5 ,/m) are transported paracellularly across the intestinal epithelium into the portal circulation (Volkheimer & Schulz, Digestion 1, 1968). There have been few microscopical investigations into this process and consequently the present study seeks to present preliminary data on such uptake of large iron particles (6-9 ,um) by rat intestinal epithelium. Seven weeks old, Sprague-Dawley rats were fed a suspension of particulate iron in milk (5 g/ 100 ml) and sampled 6-24 hours later. The jejunum was removed, fixed, and processed for wax histology. The presence of iron was detected by staining with Perls' Prussian Blue. In addition, single sections were cut on to coverslips, dewaxed and mounted on aluminium stubs. After carbon coating, the sections were viewed in a JEOL JSM-840A SEM, using the backscattered electron detector. Densely staining blue particles of iron were detected predominantly at the villous tips. They occurred especially in villous creases and were often associated with goblet cells. Some particles were seen in the villous stroma. The mesenteric lipid accumulations and blood vessels were frequently found to contain many particles. The SEM backscattered detector confirmed the presence of iron particles in the aforementioned sites. It is clear that the study does provide some evidence that particles of iron, too large to be absorbed, are nevertheless transported from the gut lumen to the mesenteric blood vessels. The fact that the main entry point occurs at the villous tips may be significant in that this is also the region of epithelial cell sloughing. Such an area of weakness may more easily allow large particles to break past into the stroma. This work was supported by the Department of Health and Social Services. D. 12.The effects of irradiation on the bacterial colonisation of the rabbit oesophagus: a scanning electron microscope study. By S. NUNN, RuTH ST.C. GILMORE and K. E. CARR. School of Basic Medical Sciences/Anatomy, The Queen's University of Belfast Previous scanning electron microscopy (SEM) studies of the normal rabbit oesophagus have showed that the number and distribution of micro-organisms associated with the surface of the lining epithelium vary along the length of the tube. A study using SEM and transmission electron microscopy (TEM) was undertaken to examine the effects of whole-body irradiation on the bacterial flora of the oesophagus. Adult female New Zealand rabbits were subjected to y-radiation from a 60Co source, being restrained during exposure in a ventilated box. The doses used were 3 Gy, 6 Gy and 10 Gy. Sham control animals were handled and restrained in the same way as the irradiated group, but were not subjected to irradiation. Untreated control animals also were used. There were three animals in each group. Thirty-six hours after irradiation the animals were killed by a blow to the head. The oesophagus was removed and fixed in 0 2 M glutaraldehyde/sodium cacodylate buffer at pH 7-4. Specimens for SEM were dehydrated through an ethanol series, critical-point dried, sputter-coated with gold and examined in a JEOL JSM-840A SEM. Sections for TEM were routinely prepared and viewed in a JEOL CXIOOII TEM. The degree of bacterial colonisation was found to be dependent on both the level of dose and the site examined. Bacterial numbers increased from the lowest in controls, becoming progressively higher in the sham control, 3 Gy and 6 Gy groups. After 10 Gy, the number was reduced compared to the lower doses. The distribution of bacteria also was found to be affected by the amount of local damage to the epithelial surface. Areas where the microridge patterns on the
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surface of the cells were relatively normal in appearance were associated with more bacteria than areas where the patterns showed substantial disruption. These results suggest that the numbers and distribution of bacteria in the oesophagus are affected by irradiation, and further work is in progress to try to determine the mechanisms behind ihese observations. This work was supported by the Medical Research Council. D. 13. The pattern and differentiation of the developing vasculature in the chick hind limb. By FIONA McNEILL, J. J. MCCULLAGH and D. J. WILSON*. Schools of Basic Medical Sciencesl Anatomy and *Clinical Dentistry/Dental Surgery, The Queen's University of Belfast The role of the developing vasculature in the patterning of the limb skeleton is controversial. It has been argued that the vasculature dictates skeletal pattern (Caplan, Cell Differ. 16, 1985) and clinical reports have implicated abnormal vascular development in the formation of skeletal abnormalities in the limb (Hootnick et al. Teratology 27, 1984). There have been few accounts of early vascular development in the hindlimb and these have concentrated on the temporal appearance of vessels, with little regard for the state of vascular differentiation (e.g. Levinsohn et al. Am. J. Anat. 169, 1984). The present study examines early embryonic limb development between 4-8 days incubation (Stages 22-35, Hamburger & Hamilton, J. Morph. 88, 1951). The vascular pattern was revealed by India ink injection whilst the state of vascular differentiation was examined by resin histology. The results showed that at four days (Stage 22) the hind limb bud was supplied by a capillary plexus, which at five days (Stage 25/26) has been replaced by a single central vessel derived from the dorsal aorta and drained by anterior and posterior marginal vessels. At Day 6 the central artery has bifurcated close to the distal end of the limb; these vessels then unite to form a metatarsal arch which supplies branches to the adjacent sides of all four digits. Histological examination of the vessels revealed that although by Stage 28/29 the future arterial pattern is well established, i.e. similar to final adult pattern, vessel differentiation even by Stage 31 is still relatively immature, showing only early differentiative features such as endothelial cell shape changes and pericyte apposition. These results show that vascular pattern in the developing limb bud resembles the adult pattern from an early stage; but differentiation of recognisable arteries does not occur until well after skeletal differentiation. In view of this it seems unlikely that the vasculature has an influential role in determining skeletal pattern. D. 14. Calcitonin-induced area reduction and the associated cytoskeletal rearrangements in rat osteoclasts in vitro. By J. CLINTON, N. N. ALI* and J. P. BENNETT (introduced by J. A. FIRTH). Department of Anatomy and Cell Biology, St Mary's Hospital Medical School, Imperial College, University of London and * Bone Unit, Royal Veterinary College, London An osteoclast (OC) enriched cell preparation was obtained from long bones of 1-4 days old rats, killed by decapitation. Individual OCs were recognised under the microscope by their large multinucleate appearance. Using indirect immunofluorescence with antibodies to actin, gelsolin and tubulin, cytoskeletal distributions were studied in OCs, either untreated or treated with 0 3 4ug/ml calcitonin (CT). Untreated cells were well spread, displaying intense lamellipodial ruffling and motility in timelapse video microscopy. With anti-actin and anti-gelsolin labelling, fluorescence was bright and uniform around the nuclei and in the ruffled borders. Microtubules visualised by anti-tubulin labelling, generally radiated out from the centre of the cell and did not extend directly to the plasma membrane. CT treatment ceased motile activity, and the lamellipodia retracted causing a reduction in cell area, typically around 30%. The response to CT had a half-time of 15-30 minutes, and was complete by 15-2 hours. The change in cell appearance was accompanied by a cytoskeletal rearrangement. Actin appeared to diminish in the lamellipodia after CT addition, but remained present in the processes which persisted after lamellipod retraction. There was a lighter staining region inside the cell periphery and uniform labelling of the central cytoplasm. Gelsolin was also reduced in the lamellipodia, but was relatively concentrated in a ring at the base of the retraction fibres, and again appeared uniformly in the cell centre. The distribution of microtubules altered little, except for appearing to be more tightly compacted as the cytoplasm retracted. These data indicate that CT initiates a response of specific cytoskeletal components and that they may play an active role in the molecular mechanisms by which the cell responds to CT.
Proceedings of the Anatomical Society of
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D. 15. Cytoskeletal proteins in bone cells in vivo. By SHEILA J. JONES, M. LOUISE TAYLOR and A. BOYDE. Department of Anatomy and Developmental Biology, University College London (Fig. 9) Conventional fluorescence microscopy of bone cells in situ does not permit three-dimensional localisation of immunolabelled cytoskeletal proteins owing to superimposition of the stain and the complexity of the bone matrix :cell interface. We have, therefore, applied confocal fluorescence microscopy to the study of whole bones because of its usefulness in serial optical sectioning. Hemicalvaria were removed from neonate rats and rabbits, and periosteal tissues dissected away from part of the bone. The bones were fixed (3 % formaldehyde in PBS with 2 % sucrose), permeabilised (Hepes Triton X- 100 buffer), immunolabelled for actin (incubated with TRITC-labelled phalloidin 10,ug/ml) or vinculin (incubated with mouse monoclonal anti-vinculin antibodies and then TRITC-labelled goat anti-mouse IgG), rinsed in PBS and mounted in glycerol for examination in a confocal laser optical microscope using a 60/1.4 oil immersion lens. Actin distribution: periosteal cells had prominently labelled stress fibres (Fig. 9a); underlying osteoblasts had fine microfibrils mainly parallel to the long axis of the cells and in processes, densest in the cytoplasm adjacent to the bone matrix and ending at intercellular contacts with adjacent osteoblasts or osteocytes (Fig. 9b); osteocytes had most specific staining in the processes and sub-plasmalemmal zones (Fig. 9 c); resorbing osteoclasts showed brightly stained ruffled borders (Fig. 9d). Vinculin distribution (Fig. 9e, f: osteoblasts, osteocytes, and osteoclasts on bone surface) was more diffuse in the cytoplasm of osteoblasts (and osteocytes) in vivo than in vitro; in osteoclasts, diffuse cytoplasmic staining was similar to that found in early cultures of isolated bone cells, and similar, if less pronounced, cell-substratum contacts were present; periosteal cells had 'arrowheads' of labelled vinculin. Confocal fluorescence microscopy proved invaluable for the localisation of cytoskeletal proteins and the appraisal of intercellular and cellbone matrix interactions and relationships without the need for any disturbance of the complex three-dimensional organisation of the different but interacting layers of bone cells. D. 16. Cell-substratum contacts of osteoclasts and osteoblasts: a correlative study of reflection and fluorescence images using confocal light microscopy. By SHEILA J. JONES, M. LOUISE TAYLOR, T. R. ARNETT and A. BOYDE. Department of Anatomy and Developmental Biology, University College London (Fig. 10) Isolated osteoblasts and osteoclasts form specialised attachment foci when seeded on to glass or plastic and maintained in vitro. The sites of these may be visualised after fixation of the cells by fluorescence immunolocalisation of, for example, vinculin. The attachment foci are strikingly different in the two cell types, the osteoblasts typically having an 'arrowhead' and the osteoclasts a 'podosomal' configuration. The aim of this study was to examine cultured bone cells when alive or after fixation and staining for vinculin by reflection and fluorescence imaging to see whether a correlation could be made between the images. Bone cells were isolated mechanically from either the bony regions of growing deer antler or from chick long bones, seeded on to glass coverslips and cultured for periods up to 24 hours. The cells were examined live by confocal reflection imaging, or were fixed in 3% formaldehyde in PBS with 2% sucrose, permeabilised in Hepes Triton X- 100 buffer and incubated first with mouse monoclonal anti-vinculin antibodies and then with TRITC-labelled goat anti-mouse IgG. Control cultures for non-specific staining were also prepared. Immunolabelled specimens were imaged by both reflection and fluorescence in a confocal laser microscope. Interference reflection images were obtained on live (Fig. 10a, b) and fixed cells (Fig. 10c, e), confocal microscopy enabling the selection of different optical planes and the discard of out-of-focus information if desired. Specialised contact zones showed to great advantage as black patches at the cell-glass interface (Fig. 10a: live deer osteoclast), and interference patterns related to cell thickness variation could also be displayed (Fig. lOb: live deer osteoclast). The correlation between the interference reflection images for cell-substratum contacts (Fig. 10c: lamellar edge of chick osteoclast; Fig. lOe: chick osteoblast) and the regions of vinculin staining (Fig. lOd, J) are illustrated. At these relatively short culture periods, vinculin is also dispersed throughout the cytoplasm of the osteoclasts, showing the nuclei in negative contrast. Interference reflection contrast mode confocal imaging proved a rapid and sensitive method for the detection of close contacts of cultured osteoclasts and osteoblasts.
Proceedings of the Anatomical Society of
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D. 17. Histological studies on the radial and ulnar attachments of the triangular fibrocartilage complex of the wrist. By M. BENJAMIN, E. J. EVANS and D. J. PEMBERTON*. Department of Anatomy, University of Wales College of Cardif and * Department of Traumatic and Orthopaedic Surgery, University of Wales College of Medicine The triangular fibrocartilage complex (TFCC) of the wrist includes the articular disc, dorsal and volar radio-ulnar ligaments, meniscus homologue, ulnar collateral ligament, and the sheath of extensor carpi ulnaris (Palmer & Werner, J. Hand Surg. 6, 1981). Its gross structure has often been described, but we know little of its histology. Its attachments to the radius and ulna are of particular interest in view of its role in stabilising the inferior radio-ulnar joint and accommodating pronation and supination. The present demonstration is based on a study of serial sections of eight dissecting room specimens (ages 54-85 years) with well-preserved TFCC. The articular disc arises from the radius as a fibrocartilaginous extension of the superficial zone of hyaline articular cartilage. This hyaline cartilage is continuous from the ulnar notch to the carpal surface of the radius. The disc arises from the sharp border between these two articular surfaces. It is thus not directly attached to bone and forms a labrum that extends the carpal articular surface. The dorsal and volar parts of the disc are thicker, and have clearly longitudinally-orientated fibres. These parts of the disc are attached to the radius via calcified and uncalcified fibrocartilage and probably correspond to the radio-ulnar ligaments. Such regions of the TFCC are presumably important in strengthening the disc and creating a more secure attachment to the radius. Strands of collagen fibres from the supero-medial part of the disc turn proximally to attach directly to bone in the roughened area at the base of the ulnar styloid process. Medial to this, is vascular, loose connective tissue through which run other collagen fibres that attach to the base and sides of the styloid process via fibrocartilage. In most of our specimens, the tip of the styloid process was covered by at least some articular cartilage and separated from the meniscus homologue by a prestyloid recess. This contrasts with the findings of Mohiuddin & Janjua (The Hand 14, 1982).
D. 18. Histological studies on the attachment zone of the quadriceps tendon and patellar ligament in man. By E. J. EVANS, M. BENJAMIN, D. J. PEMBERTON* and R. DONTHINENI RAO. Department of Anatomy, University of Wales College of Cardiff, and * Department of Traumatic and Orthopaedic Surgery, University of Wales College of Medicine In order to investigate the relationship between mechanical factors and the presence of fibrocartilage at the attachment sites of ligaments and tendons, the quadriceps tendon/patellar ligament complex was examined by routine histology. The results were then considered in relation to the amount of movement that occurs at each attachment site. Strips of tissue, approximately 5 mm wide were taken from the central portion of each tendon/ligament from dissecting room cadavers of both sexes (aged 71-89) and sectioned in a sagittal plane. The largest quantity of fibrocartilage was in the quadriceps tendon. Here, the tissue was most characteristic of the central region of the attachment zone, but was also found in its deep portion. There was less fibrocartilage at the patellar attachment of the patellar ligament. At its tibial attachment, the tissue was almost entirely restricted to a prominent, deep wedge at the proximal end. A substantial portion of the ligament was attached directly to bone without any fibrocartilage. At the patellar attachment, the small quantity of fibrocartilage was diffusely distributed, though some was present in the deep regions of all specimens. Both simple observation and radiological measurements show that there is only a small change in the angle between the patellar ligament and the patella during flexion and extension of the knee. However, there is a larger change in the angle between the quadriceps tendon and the patella and between the patellar ligament and the tibia. Thus, the amount of fibrocartilage was greatest at the most mobile attachments. The tissue may prevent collagen fibres from splaying out near the bony interface during movement and also minimise local concentrations of stress. The wedge of fibrocartilage at the tibial attachment of the patellar ligament could resist pressure on deep fibres by superficial ones.
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D. 19. The ultrastructural architecture of interendothelial junctions of rat cardiac microvasculature as demonstrated by ruthenium red and lanthanum nitrate. By CATHERINE E. SARRAF*, BARBARA J. WARDt and J. A. FIRTH. Departments of * Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, t Anatomy, St Bartholomew's Medical College, Charterhouse Square, London and Anatomy and Cell Biology, St Mary's Hospital Medical School, Norfolk Place, London Ruthenium red and lanthanum nitrate are electron-dense cationic dyes that cause precipitation of cell surface mucopolysaccharides, and thus label them in situ at electron microscope level. When tissue is treated before dehydration and embedding, the cell surfaces exhibit an amorphous covering 6-10 nm deep. Visualisation of the glycocalyx of rat cardiac vascular endothelium was achieved during perfusion of isolated rat hearts under both oxygenated and hypoxic conditions; animals were killed by cervical dislocation. Luminal, abluminal and junctional surfaces were stained, as were endothelial vesicles that opened on to cell surfaces. The results obtained by these inorganic, nonimmune agents have been contrasted and the role of mucopolysaccharides in the ultrastructural architecture of transport pathways through endothelial cell junctions has been examined. Lanthanum nitrate was found to be a superior stain for demonstrating these features in this tissue. D. 20. Glutamate-like immunoreactivity in dorsal root ganglia neurons of the rat. By M. A. KAIKAI and R. HowE. Department of Preclinical Veterinary Sciences, University of Edinburgh Localisation of sensory glutamatergic neurons is difficult to demonstrate by autoradiography because the satellite glia accumulate the radiolabelled aspartate or glutamate with little penetration into neurons (Duce & Keen, Neuroscience 8, 1983). We have studied the distribution of glutamate-positive structures in the DRG and spinal cord by autoradiography and immunohistochemistry. Ten weeks old male Wistar rats were anaesthetised and killed by transcardiac perfusion with Krebs bicarbonate pH 7 4. The fourth lumbar DRG were dissected and immersed in a medium containing 7 2 x 10- M-D[2,3-3H]aspartic acid (Amersham) and incubated at 37 °C under 02/CO2 for 30 minutes. The tissue was processed for light microscopic autoradiography (LM-ARG). Two sets of experimental animals were used for immunohistochemistry; one group was injected with 14 7 x 10-4 M of D-glutamate solutions one hour before perfusion and one hour before the second group. The rats were perfused with paraformaldehyde (4 %)-glutaraldehyde (0-5 %) fixative buffered in 0 14 M phosphate buffer, pH 74. 8 ,tm frozen sections were reacted with polyclonal anti-glutamate serum (1:100) and the antigen-antibody complex visualised by immunofluorescence or the peroxidase-anti-peroxidase (PAP method). LM autoradiographs revealed dense silver grains in the satellite glia with uptake in about 1 % of neurons. Glutamate-immunofluorescence was localised in all satellite glia surrounding glutamate-like immunoreactive (glu-LIR) and non-immunoreactive neurons. The majority of glu-LIR DRG neurons measured 10-35 ,um in diameter. The positive cells in animals pretreated with D-glutamate formed approximately 35 %, 48 %, 50 % and 55 % of the total cells in cervical29 thoracic6, lumbar2 and sacral, respectively. Untreated rats showed lower percentages and there was an increase of 10-37 % after injection of cold glutamate. Sections from the different segments of the spinal cord showed immunoreactive structures in the dorsal horn. In the thoracic region intensely stained fibres and interneurons occurred in the substantia gelatinosa (SG). Pretreatment with glutamate showed concentrated immunoreaction in the meninges and in the walls of the spinal arteries. The control experiments provided good evidence that a significant proportion of small diameter, class B neurons and spinal interneurons are glutamatergic although the sensory modality these cells transmit is unknown.
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D. 21. Microvascular morphology during tumour induced angiogenesis. By R. WILLIAMS, D. ROBERTSON* and A. J. S. DAVIES* (introduced by J. A. FIRTH). Department of Surgery, St Mary's Hospital and * The Institute of Cancer Research, London Angiogenesis is a feature of many physiological and pathological processes. It is of fundamental importance in the growth of solid cancers. Histological sections reveal little about the events that occur during vascular growth. An experimental model was developed in mouse to study changes in omental microvasculature in response to an ascitic tumour. Part of murine omentum normally comprises an avascular membrane, approximately 30 ,um in thickness. Proliferative changes in the capillary plexus bordering this membrane were induced by intraperitoneal injection of 107 irradiated Landschutz cells. The lectin, Dolichos biflorus agglutinin (DBA), specifically recognises N-acetyl galactosamine residues on endothelial cells in 'SWR' mice. A peroxidase conjugate of DBA was used to delineate blood vessels in whole mounts and ultrathin sections. Capillary endothelial cells and their processes were intensely stained. After tumour cell injection, new blood vessels were seen to originate as outgrowths from pre-existing capillary loops. Early 'sprouts' comprised cytoplasmic extensions from the abluminal surface. Fine filaments extended from the apex of each sprout. Ostensibly these filaments made contact with those of other sprouts, possibly directing growth so that adjacent sprouts converge and anastomose to form new capillary loops. On EM, DBA was concentrated in relation to the extraluminal plasma membrane. Endothelial cells in areas of angiogenic activity appeared much thicker than normal, surrounding an irregular narrow lumen partly filled by membranous processes. The described in vivo model provides a new insight of early angiogenesis. Endothelial cells can be distinguished with a precision not previously available. In thin whole-mounts, relationships can be observed that are not easily appreciated in transverse section. EM of selected areas of interest permits a second more detailed perspective.
D. 22. Intestinal morphology - assessing differences in normal mice using a mathematical model. By K. E. CARR, J. S. MCCULLOUGH, W. A. MCCALLION, J. A. STEELE, G. R. DICKSON, W. R. HANSON*, S. P. HuMEt and A. C. NELSONI. School of Basic Medical Sciences/Anatomy, The Queen's University of Belfast, *Department of Therapeutic Radiology, Rush Presbyterian St Luke's Chicago, USA., tMRC Cyclotron Unit, Hammersmith Hospital, London and IDepartment of Bioengineering, University of Washington, Seattle, USA In a previous communication to the Society, a mathematical model was used to calculate a Morphological Index (MI), which acted as a measure of structural integrity in the intestine. Tissues may also be described individually to provide partial MIs for epithelium, connective tissue, muscle and nerve, which show where major differences occur. Previously the model was used to look at the effects of radiation on the intestine. However, this paper uses it to compare intersite and interstrain differences in normal mice. Using resin sections, comparisons were made between the duodenum and jejunum of female HC/CFLP mice and the jejunum of female HC/CFLP and male B6D2F1 mice. One way ANOVA demonstrated that the number of Paneth cells and Auerbach nerve clusters were significantly greater in jejunum than in duodenum of HC/CFLP mice. Interstrain jejunum comparisons showed that statistical differences existed in the number of villi, Paneth, goblet and endocrine cells, as well as enterocytes, Auerbach clusters and submucosal arterioles. These results clearly showed that there were fewer intersite than interstrain differences. Calculation of the MI also reflected this. For example, in HC/CFLP mice, the MI for jejunum was 43 when compared to 100 for duodenum, whereas the MI for B6D2F, mouse jejunum was 16 when compared to 100 for HC/CFLP mice. An MI of less than 100% produced by comparison of two control groups does not imply that one is damaged, but only that it has a different balance of tissues and cells. Partial MIs showed that the greatest interstrain differences were located in the epithelium. The versatility of the model is clear in providing an overall figure for differences between samples and also a partial figure which can show those tissues that exhibit the greatest changes.
Proceedings of the Anatomical Society of Note concerning Communications 13: Nerve damage in leprosy. By C. L. Crawford and M. J. Hobbs
To avoid misunderstanding, Professor R. E. M. Bowden stresses that repair of a lepromatous nerve by muscle grafting will only be undertaken in the following circumstances: (i) Complete loss of all modalities of sensation in the distribution of the nerve. (ii) After rigorous, recorded and objective pre-operative examination and with equally rigorous follow-up examination according to a detailed protocol. (iii) With the informed consent of the patient.