New phenotypic diversity associated with the mitochondrial tRNA(SerUCN) gene mutation.

We performed detailed clinical, histopathological, biochemical, in vitro translation and molecular genetic analysis in patients from two unrelated families harbouring the tRNA(SerUCN) 7472C-insertion mutation. Proband 1 developed a progressive neurodegenerative phenotype characterised by myoclonus, epilepsy, cerebellar ataxia and progressive hearing loss. Proband 2 had a comparatively benign phenotype characterised by isolated myopathy with exercise intolerance. Both patients had the 7472C-insertion mutation in identical proportions and they exhibited a similar muscle biochemical and histopathological phenotype. However, proband 2 also had a previously unreported homoplasmic A to C transition at nucleotide position 7472 in the tRNA(SerUCN) gene. This change lengthens further the homopolymeric C run already expanded by the 7472C-insertion. These data extend the phenotypic range associated with the 7472C-insertion to include isolated skeletal myopathy, as well as a MERRF-like phenotype.

Fifty year follow-up of a patient with central core disease shows slow but definite progression.

The follow-up of a patient with central core disease (CCD) over 50 years showed that although initially the condition was moderately non-progressive, progression of a significant degree did eventually occur. Histopathological and electron microscopic data were available from muscle biopsies carried out at the ages of 19 and 55 years, and show a marked predominance of type 1 fibres with central cores in most fibres at both ages. The four mutations within the RYR1 gene described in association with CCD and three of the more common malignant hyperthermia-associated mutations within RYR1 were not present.

The morphology of rat soleus neuromuscular junctions treated in vitro with purified beta-bungarotoxin.

Pairs of isolated rat soleus nerve-muscle preparations were maintained in vitro in oxygenated Ringer solution containing 3 microgram/ml beta-bungarotoxin prepared by the method of MacDermot et al. End-plate potentials and twitch tensions generated in response to continuous stimulation via the nerve at 0.5 Hz were recorded from one muscle, while miniature end-plate potential amplitude and frequencies were monitored in the corresponding unstimulated member of the pair. The results obtained were compared with the fine structure of end-plates from identical preparations sampled after varying periods of intoxication, and from unpoisoned muscles maintained for comparable times in vitro. The electron micrographs obtained were analyzed quantitatively using simple stereological techniques. Our results show that: (1) nerve terminal synaptic vesicle content declines for the first hour of intoxication but returns to near normal levels following conduction block; (2) there is a rapid increase in the 'coated' vesicle population of the nerve terminals, both attached to the plasmalemma and within the terminal cytoplasm, which persists to the end of the experiment; and that (3) there is no significant increase in the incidence of damaged mitochondria within the poisoned nerve terminals. These findings are discussed in relation to previous observations and the putative mechanisms of action of beta-bungarotoxin.

Emery-Dreifuss syndrome.

A young adult male is described with muscular dystrophy of probable X-linked recessive inheritance. An onset of muscle weakness in late adolescence was preceded by contractures of the neck and elbows dating back to childhood. The distribution of muscle weakness was proximal in the upper limbs and both proximal and distal in the lower. The mixed pattern of muscle involvement in the legs favours the view that cases of Emery-Dreifuss muscular dystrophy with proximal weakness in both the upper and lower limbs and X-linked scapuloperoneal muscular dystrophy represent the same disorder. A muscle biopsy in the present case showed unique appearances.

A fluorescence study of changes in noradrenergic sympathetic fibres in experimental peripheral nerve neuromas.

Experimental neuromas were produced in rats by sciatic nerve section and avulsion of the distal stumps. At intervals varying from 3 days to 8 weeks after nerve section, the developing neuromas were resected and processed for noradrenaline (NA) fluorescence microscopy by the sucrose-phosphate-glyoxylic acid (SPG) method. From serial longitudinal sections through the neuromas and the nerve proximally, counts of noradrenergic sympathetic axons were made, together with qualitative observations of axon sprouting and NA content. By 3 days after nerve section there was a massive sprouting of sympathetic axons, with increased NA content, particularly towards the distal tip of the neuroma. Axon counts remained high 1 week following section then fell to below normal levels at 2 weeks, returning towards normal 8 weeks after nerve section. These results are discussed in relation to the known pathophysiological interaction between sympathetic efferent and sensory afferent fibres, which develops in neuromas following nerve section.

New approaches to the study of human dystrophic muscle cells in culture.

Tissue culture provides a system for studying the growth and differentiation of muscle cells in a controlled environment. Several studies have been carried out on diseased muscle cells in culture in attempts to elucidate the aetiology of Duchenne muscular dystrophy (DMD) but the results were equivocal. Work in our laboratory in recent years has yielded an improved method for preparing primary muscle cell cultures from dissociated biopsies which permits the morphological and biochemical evaluation of these cultures at all stages of growth and development. Our results have shown abnormalities in cell behaviour, ultrastructure and creatine kinase synthesis. The background to these studies is reviewed. Recently we have developed a cell cloning procedure that allows the accumulation of a large number of cells from a single selected cell. We can with this technique monitor quantitative and qualitative cellular and cytochemical differences between individual cell types without the ambiguities inherent in the use of mixed cell populations. The results obtained with 4 different clonal preparations derived from dystrophic muscle have shown that a number of specific features were expressed by each of the 4 clones with respect to their growth pattern, ultrastructure, synthesis of muscle specific protein and cell surface antigen. These findings clearly illustrate the potential of these cloning procedures for studying the genetic expression of homogeneous cell populations derived from normal adult human muscle and patients with X-linked muscle disease.

Remyelination during optic nerve compression.

The structural changes produced in cat optic nerve by incremental chronic compression have been investigated by light and electron microscopy. Within the first week extensive demyelination occurred; some fibres underwent Wallerian degeneration and there was marked proliferation of astrocytic processes. After 5 weeks of compression remyelinated fibres were seen and occurred in all longer-term animals despite the maintenance of compression, but some partially and completely demyelinated fibres persisted. Myelin thickness had not returned to normal in the remyelinated fibres after 19 weeks and was often strikingly variable. The significance of these observations is discussed in the light of the recent demonstration of the restoration of central conduction by remyelination.

A mitochondrial myopathy with a deficiency of respiratory chain NADH-CoQ reductase activity.

This paper presents data on two sisters with a mitochondrial myopathy characterised by weakness, marked exercise intolerance and a fluctuating lactic acidaemia. Both patients also experienced episodes of increased weakness which could be brought on by unaccustomed activity, going without food or by taking small quantities of alcohol. Metabolic studies during exercise showed a marked and sudden rise in blood lactate and pyruvate levels. Biochemical studies in one case showed that mitochondrial respiratory rates were markedly decreased with all NAD-linked substrates tested but were normal with succinate and with TMPD + ascorbate. The mitochondrial cytochrome components were normal as determined by low temperature spectroscopy and the addition of uncoupler did not enhance state 3 respiratory rates utilising NAD-linked substrates. It was concluded, therefore, that the mitochondrial lesion was located at the level of the NADH-CoQ reductase complex.

A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells.

A quantitative and reproducible technique for establishing primary surface cultures from normal and diseased human muscle is described. Successful cultures were prepared from both fresh muscle and that stored up to 96 hr at 4 degrees C. The CPK activity of the muscle cells ranged between 0.5-3.0 micronmoles creatine per min per mg protein at 30 degrees C, thus indicating a high degree of differentiation. Spontaneous contractions were observed in 4 out of the 22 cultures established. Nerve cells were not required to achieve this level of differentiated function. No gross differences in plating efficiency, rate of myotube formation or CPK specific activity were found for the diseased muscle cells cultured so far. However, a 5--10-fold higher cell yield was obtained from muscles of patients with an inflammatory myopathy. The advantages of this technique for carrying out comparative studies on normal and dystrophic muscle cells are discussed.

Structural assessment of the urethral sphincter in women with urinary retention.

PURPOSE: The pathophysiology of urinary retention in women is generally unknown but a subgroup of women with urinary retention have been diagnosed as having so-called primary disorder of sphincter relaxation on the basis of an abnormal urethral sphincter electromyogram. It was suggested this sphincter overactivity could lead to work hypertrophy of the urethral rhabdosphincter and in this study we looked for any evidence of such muscle fiber hypertrophy. MATERIALS AND METHODS: In 9 women 18 to 45 years old (mean age 31.6) with urinary retention and overactive urethral sphincter electromyogram, light and electron microscopy were used to examine core needle biopsies of the urethral rhabdosphincter taken under transvaginal ultrasound control. Of the 9 patients only 5 biopsies processed for light microscopy and 4 processed for electron microscopy contained striated urethral muscle fibers. The results of these biopsies were compared to the morphology of a control specimen from a postmenopausal woman without a history of urinary retention. RESULTS: On light microscopy the urethral rhabdosphincter fiber diameter did not differ among patients (mean average 7.6 mum), was less than that reported in the literature (15 to 20), but did not differ from that of the control (mean 9.9). In all patients electron microscopy showed excessive peripheral sarcoplasm with lipid and glycogen deposition, and sarcoplasmic accumulation of normal mitochondria. These ultrastructural abnormalities were not seen in the control. CONCLUSIONS: To our knowledge this is the first morphological description of the urethral rhabdosphincter in a subgroup of women with urinary retention. Mean rhabdosphincter fiber diameter was approximately the same in patients and controls. This study does not support the previous theory that urethral sphincter overactivity in a subgroup of women with urinary retention leads to work hyperplasia of urethral rhabdosphincter fibers. An alternative hypothesis is suggested.

A Pacinian corpuscle in the human bladder lamina propria.

The fortuitous finding of a complex Pacinian corpuscle within the lamina propria of the human urinary bladder is described. It consisted of a complex of encapsulated nerve endings within the areolar connective tissue of the lamina propria immediately adjacent to the inner aspect of the detrusor muscle. It showed no structural evidence of directional sensitivity and was associated on its outer aspect with small unmyelinated axons containing small clear and dense-cored vesicles. This appears to be the first report of an encapsulated nerve ending within the lining of the adult human urinary bladder.

The role of the human bladder lamina propria myofibroblast.

OBJECTIVES: To describe the ultrastructure and relationship to nerves of the myofibroblast in the human bladder lamina propria, and discuss its possible role in bladder function, including sensing stretch, as the response of the bladder to stretch has been thoroughly investigated by afferent nerve recordings, but specialized stretch sensing organs have yet to be identified. MATERIALS AND METHODS: Flexible cystoscopic bladder biopsies were obtained from patients with detrusor hyper-reflexia and from controls. Systematic electron micrographs were obtained throughout the lamina propria, and the presence and location of cells with ultrastructural characteristics of myofibroblasts noted, together with their relation to surrounding nerves. RESULTS: Within the lamina propria there was a layer of cells with the cytological characteristics of both fibroblasts and smooth muscle cells, that included bundles of fine cytoplasmic filaments, dense bodies, linear arrays of subsurface vacuoles, and the presence of an interrupted basal lamina. This combination of features is characteristic of the myofibroblast. These cells had close contacts with unmyelinated axonal varicosities containing a mixture of clear and large dense-cored vesicles, or clear vesicles alone. CONCLUSIONS: There is a layer of cells with the ultrastructural characteristics of myofibroblasts within the human bladder lamina propria. Their close contacts with nerves containing both small clear, and small clear with dense-cored, vesicles implies they have both an efferent and an afferent nerve supply, possibly functioning as a bladder stretch receptor. Furthermore, because of their similarities with the interstitial cells of Cajal in the gut, which are claimed to modulate small intestinal function, we discuss other possible roles for bladder lamina propria myofibroblasts.

Ultrastructural localization of sympathetic axons in experimental rat sciatic nerve neuromas.

The ultrastructural localization of sympathetic axons was investigated in normal rat sciatic nerves and experimental sciatic nerve neuromas. The best ultrastructural localization of noradrenaline in the dense-cored vesicles of sympathetic axons was accomplished following pretreatment of rats with nialamide and 5-hydroxy dopamine, followed by fixation according to the modified chromaffin technique of Tranzer and Richards (1976). After such preparation, sympathetic axons containing 5-hydroxy dopamine-labelled dense-cored vesicles could be identified in normal sciatic nerve. Large accumulations of labelled dense-cored vesicles were also found in acute neuromas, up to 1 week after nerve section. Much smaller numbers of dense-cored vesicles could be identified in chronic neuromas from 2 to 3 weeks following nerve section. Sympathetic axons could also be identified following electron probe X-ray microanalysis of the tissue sections, using chromium detection as the marker for the noradrenaline-containing dense-cored vesicles. Unusual configurations of Schwann cell subunits, which enclosed myelinated fibres and sympathetic axon sprouts within the same basal lamina, were identified in the acute neuromas, 3-7 days after nerve section. Such configurations may be of relevance to the pathophysiological interaction which develops between sympathetic efferent and sensory fibres in peripheral nerve neuromas.

Remyelination during optic nerve compression.

An inflatable silicone rubber balloon implanted in the orbit has been used to simulate an expanding orbital tumour. The changes produced in the compressed cat optic nerve have been investigated by light and electron microscopy. Within the first week extensive demyelination occurred; some fibres degenerated completely and there was marked proliferation of astrocytic processes. Some partially and completely demyelinated axons were seen throughout the period studied but after 5 weeks many axons had become remyelinated. Remyelinated fibres were seen in all longer-term animals despite the continued presence of the orbital mass. The significance of these observations is discussed in the light of the physiology of conduction in demyelinated and remyelinated central axons.

The characterization of experimental gliosis by quantitative nuclear magnetic resonance imaging.

Quantitative nuclear magnetic resonance imaging techniques were used to study the development of astrocytic gliosis following a cortical freezing injury in 4 cats, and the findings compared with the histological and ultrastructural appearances of the affected white matter. In the first week after lesioning, the acutely oedematous white matter was apparent as a region of signal abnormality with very prolonged T1' and T2' relaxation times. Following resolution of this oedema, the images remained normal for a period of approximately three months. After this latent period they became abnormal once again, and the abnormal signal was most evident in images emphasizing differences between tissues in T1' and proton density, but not T2'. The light and electron microscopic appearances of the corresponding tissue were of astrocytic gliosis in each animal. Measurement of the relaxation times, T1' and T2', of the abnormal regions revealed an increase in T1' without a corresponding change in T2'. The T2' magnetization decay characteristics were consistently monoexponential, in contrast to the clearly biexponential T2 decay which has previously been demonstrated in the presence of acute vasogenic oedema. These findings suggest that the pattern of change of the relaxation times and T2 magnetization decay might provide a means of distinguishing between lesions in multiple sclerosis which are predominantly gliotic, and those containing significant amounts of oedema.

Chronic optic nerve compression. An experimental study.

Chronic progressive compression of the optic nerve in the cat was produced by an inflatable silicon rubber balloon implanted in the orbit. Within the first week the predominant pathological change was demyelination. Both partially and completely demyelinated fibres were seen at all stages of the experiments but by the fifth week some axons had been remyelinated by oligodendrocytes despite the continued presence of the balloon. The myelin sheaths of the new internodes were abnormally thin and short, and many showed an atypical paranodal organization. There was evidence of breakdown of some of the new internodes. The pathophysiology of optic nerve compression in man is reviewed.

Magnetic resonance imaging of experimental cerebral oedema.

Triethyl tin(TET)-induced cerebral oedema has been studied in cats by magnetic resonance imaging (MRI), and the findings correlated with the histology and fine structure of the cerebrum following perfusion-fixation. MRI is a sensitive technique for detecting cerebral oedema, and the distribution and severity of the changes correlate closely with the morphological abnormalities. The relaxation times, T1 and T2 increase progressively as the oedema develops, and the proportional increase in T2 is approximately twice that in T1. Analysis of the magnetisation decay curves reveals slowly-relaxing and rapidly-relaxing components which probably correspond to oedema fluid and intracellular water respectively. The image appearances taken in conjunction with relaxation data provide a basis for determining the nature of the oedema in vivo.