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Electron microscopy has a strategic position in the diagnosis of neuromuscular disorders. In muscular fibers, the main abnormalities include vacuoles, inclusion bodies, and myofibrillar disorganization with or without abnormal inclusion material. Vacuolar changes include lipidic and glycogenic storage vacuoles, rimmed vacuoles, and lysosomal and autophagic vacuoles. Accumulation of abnormal inclusion material is found in nemaline myopathy, actinopathies, and hyaline body myopathy. Myofibrillar disorganization involves cores, multiminicores, and myosin chain depletion. Myofibrillar myopathies associate a pathologic pattern of myofibrillar dissolution and ectopic protein expression. They can be divided into two groups: myofibrillar myopathies with multiple expression proteins and myofibrillar myopathies with desmin and alphaB-crystallin expression only. In these two conditions, electron microscopy shows accumulation of a granulofilamentous material immunoreactive for desmin. At least three genes are implicated: desmin, alphaB-crystallin, and myotilin. Lastly, electron microscopy serves to identify changes, pathogenic or not, which are not shown up by light microscopy. Moreover, electron microscopy gives insight on pathophysiological mechanisms and can guide molecular genetics analysis.

Muscle biopsy is a simple procedure of great diagnostic interest when correct processing is obtained. Specimens for molecular genetics are very important. Paraffin embedded material is not sufficient in most of the cases except in some inflammatory processes. Histochemical techniques on frozen material are of major interest. Immunohistochemistry and electron microscopy can also be used. The aim of this paper is to describe the procedure of the muscle biopsy, the various techniques and the possible artefacts.

The pathological findings in 165 patients explored for malignant hyperthermia (MH) susceptibility are reported. The first group of 120 subjects were patients investigated for MH. These patients had suffered an attack of MH under anaesthetic or were members of families in which a subject had died of MH. In vitro contracture tests revealed 25 malignant hyperthermia susceptible (MHS) subjects, with positive contracture tests for halothane and caffeine, 5 malignant hyperthermia subjects with reaction to caffeine only (MHC), 3 malignant hyperthermia subjects with reaction to halothane only (MHH) and 87 malignant hyperthermia negative (MHN) subjects with normal contracture tests. The second group of 45 subjects had exertional heat stroke. In vitro contracture tests performed at least 3 months after the exertional heat stroke revealed 11 MHS, 6 MHC, 2 MHH subjects and 26 MHN. In both groups, whatever the in vitro contracture test results, pathological findings were heterogeneous and revealed various changes: rhabdomyolysis, mitochondrial myopathy, denervation, type II atrophy, AMPase deficiency, non-specific findings or normal features. Central core myopathy was only observed in the first subgroup and was the only disease significantly associated with MH. In contrast to previous reports, this study demonstrates the absence of a specific malignant hyperthermia or exertional heat stroke myopathy. Furthermore, the discovery of MHS subjects among the EHS group of patients highlights the need for systematic exploration of all these patients.

The expression of the CD24 molecule, a glycoprotein expressed at the surface of most B lymphocytes and differentiating neuroblasts, was studied in developing nerve and muscle (after 16 weeks of gestation), normal adult and various diseased human muscles using immunohistochemistry and Western blot analysis. Immunohistochemical studies demonstrated that: (1) in developing muscles, fibers did not express CD24, whereas only some mesenchymal areas, also expressing neural cell adhesion molecule (N.CAM) and vimentin, and developing nerves were positive; (2) in normal adult muscles, CD24 immunoreactivity was observed only in some unmyelinated nerve fibers--intra and extra fusal muscle fibers, satellite cells and neuromuscular junctions were negative; and (3) in all diseased muscles studied here, CD24 expression was always associated with a subpopulation of regenerative fibers. These fibers also expressed vimentin, desmin, developmental myosin heavy chain, N.CAM and its polysialylated isoforms (PSA-N.CAM). The number of CD24-positive fibers was always lower than that of PSA-N.CAM-positive fibers. Denervated fibers and vacuolated muscle fibers never expressed CD24. Western blot analysis indicated that the apparent molecular mass of CD24 antigen was different between muscle and developing nervous tissues, suggesting that CD24 glycosylation is tissue specific. Since the molecule was not expressed in developing human muscle fibers, it strongly suggests that regenerative and fetal myotubes are different with respect to the CD24 molecule expression.

Two patients with mitochondrial encephalomyopathy due to complexes I and IV deficiencies received 150 mg/d of coenzyme Q10 (CoQ). We studied them with a bicycle ergometer exercise test and 31P NMR spectroscopy before and after 10 months of treatment. Before treatment, we observed a low phosphocreatine/inorganic phosphate (PCr/P(i)) resting value along with abnormally high resting lactate concentration. During exercise, there was a pronounced acidosis with delayed kinetics of postexercise recovery for blood lactate, pH, PCr, and PCr/P(i) ratio. Oxygen uptake during exercise was reduced while the lowering of the ventilatory threshold indicated an early activation of glycolysis. After treatment, the bicycle ergometer exercise test indicated a significant improvement with a decrease in resting blood lactate level, an increase in oxygen consumption during exercise, and an increase in the kinetics of lactate disappearance during the recovery period. A shift of the ventilatory threshold to higher workload was present. 31P NMR spectroscopy confirmed the improvement, showing a significant increase in the PCr/P(i) ratio at rest and in the kinetics of recovery for pH, PCr, and PCr/P(i) ratio following exercise in patient 1. For patient 2, we observed a less pronounced acidosis correlated with a lesser amount of Pi produced during exercise. These observations indicate an improvement of mitochondrial function and a shift from high to low glycolytic activity in both patients consequent to CoQ treatment.

Muscles from Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) patients were analysed using monoclonal and polyclonal antibodies raised against different regions of the dystrophin molecule. On blot, two of the antibodies detected a protein of Mr 400K in muscle extracts from all patients, including a BMD patient with a deletion which spanned more than 40% of the central rod domain of the Xp21 encoded dystrophin. Immunocytochemical labelling of tissue sections from the same patients showed that the same two antibodies labelled a protein at the surface membrane of smooth muscle fibers in blood vessels of both BMD and DMD muscles. Thus we have demonstrated a 400K blood vessel-associated protein, which is immunologically homologous with dystrophin, for at least two epitopes from the carboxy terminal and the central rod domains must be encoded by another gene than the dystrophin gene.

Neural cell adhesion molecules (NCAM) are known to play a pivotal role in regulating cell-cell interactions in various tissues. The diversity of NCAM is made by alternative splicing of a single gene and by post-translational modifications. The spatio-temporal expression of the various isoforms is developmentally regulated and may modulate cell interactions. We investigated the expression of NCAM isoforms, in particular polysialylated and phosphatidylinositol-anchored isoforms, in developing psoas and quadriceps human muscle from 15 weeks of gestation to term. In parallel, we examined the expression of the myosin heavy chain phenotype (another developmentally regulated system) to determine whether polysialylated-NCAM molecules (the so-called embryonic NCAM) and developmental myosin heavy chains are coexpressed. Our results showed an expression of polysialylated-NCAM and phosphatidylinositol-anchored isoforms during the early stages of myotube maturation. The expression of polysialylated-NCAM on developing myotube was always associated with the expression of developmental myosin heavy chains. However, the loss of polysialylated-NCAM from maturing myotubes was not correlated with the disappearance of the developmental myosin heavy chains, but rather with the appearance of an adult myosin heavy chain phenotype. The relationship between polysialylated-NCAM and myosin heavy chain phenotype was similar in psoas and in quadriceps muscles. We observed that maturation of quadriceps muscle takes place earlier than psoas. Biochemical analysis showed that phosphatidylinositol-anchored molecules were never polysialylated; this indicates different roles of these isoforms in muscle development.

Slowly progressive myopathy with tubular aggregates is rare and dominantly or recessively inherited. Three sporadic cases are reported in the present study occurring in 2 men and 1 young woman. All patients had proximal limb weakness without severe atrophy. They also complained of exercise-induced stiffening and cramps of their leg muscles. In 1 case severe cardiomyopathy caused unfavorable clinical course and death. Serum creatine kinase activity was normal and electromyogram showed only slight myopathic changes. Tubular aggregates were found to be the sale morphological abnormality. They were present in type II fibres in 1 case and in type I and type II fibres in the others. An immunocytological study with a polyclonal antibody against Ca2+ SR-ATPase showed positivity of the tubular aggregates with this antibody. A quantitative analysis (SAMBA 2000 alcatel TITN) was carried out on frozen sections stained for calcium. It showed a lower calcium content in tubular aggregates than in other part of the fibre. Slowly progressive myopathy with tubular aggregates may be distinguished from other diseases where tubular aggregates are the sale structural change, such as myopathies with myasthenic features and some neuromuscular diseases with exercise intolerance without progressive course. Usually, tubular aggregates are not a specific finding; they have been described in various disorders in association with other structural changes.

Antibodies directed against neural cell adhesive molecules (NCAM) and in particular a monoclonal antibody recognizing polysialylated isoforms, were used to characterize the expression of these molecules in normal and diseased human muscles. Normal subjects as well as patients with inflammatory, dystrophic and denervating diseases were examined. By immunohistochemistry the main observations were (1) satellite cells expressed the non-sialylated form of NCAMs; (2) regenerative fibers strikingly expressed NCAMs and their sialylated isoforms both on membranes and in the cytoplasm; (3) in denervated muscles, fibers in atrophic groups and some fibers in acute denervation expressed NCAMs on their membrane but not the highly sialylated form; (4) finally, some fibers in myotonic dystrophy and fibers with rimmed vacuoles also expressed NCAMs. Biochemical approaches, using enzymes such as endoglycosidase N and phosphatidylinositol phospholipase C combined with immunoblot analysis allowed visualization of the nature of the expressed isoforms. We have shown that non activated cells, i.e. satellite cells and denervated fibers do not express polysialylated NCAMs. This post-translational modification may be only observed in activated or regenerating fibers. This would parallel the sequence of NCAM expression occurring in normal myogenic pathways.

The authors report 5 cases of stress myalgias associated with tubular aggregates seen in muscle biopsy specimens. A few similar cases are reported in the literature and a close relationship is established by some authors between the morphologic anomaly present in type II fibers and the lack of tolerance to stress. However, tubular aggregates are not specific and found in other disorders, especially the hyperkaliemic periodic paralysis where all types of fibers are affected. The origin of the aggregates is unknown. They could come from the endoplasmic reticulum containing an excess of calcium and form aggregates in order to prevent a muscle contraction.

The muscle biopsy performed in a 58-year-old woman with a myopathy involving pelvic girdle and lower limbs displayed unusual intermediate filament aggregates by light and electron microscopy. No cardiac involvement was detected. The filamentous aggregates selective for type 1 fibers were found in subsarcolemmal and intermyofibrillar areas closely related to Z bands. Immunohistochemical study by light and electron microscopy using polyclonal and monoclonal antibodies and avidin-biotin peroxidase method revealed that aggregates consisted of desmin filaments. Desmin positive material was unstained with vimentine antibodies.

A predominance and/or an atrophy of type I fibers and a loss of deep tendon reflexes are often observed in different types of congenital myopathy. Various data indicate that both findings can be linked: dysfunction of the myotatic reflex can induce predominant involvement of type I fibers. In order to specify the mechanism of the loss of tendon reflex, an investigation of the Hoffmann reflex (H reflex) was performed in one case of centronuclear myopathy and in one case of congenital type fiber disproportion with type I hypertrophy. The Achilles tendon reflex was absent but the H reflex showed normal recruitment amplitude and latency. The Jendrassik maneuver reinforced the H reflex. These results indicate the involvement of muscle spindles or impairment of the fusimotor system. Nuclear bag intrafusal fibers have common characteristics with type I extrafusal fibers. Both types of fibers could be involved simultaneously in congenital myopathies, thus explaining the loss of tendon reflex.

Inclusion body myositis is now a well-known disease but its incidence is underestimated. We report 12 cases with clinical heterogeneity. Three groups of patients could be described. The first one corresponded to asymmetrical muscle involvement and distribution with a slow clinical course (4 cases). The second was characterized by a polymyositis-like syndrome (3 cas), but steroid therapy was ineffective. The last group mimicked a chronic spinal muscular atrophy (4 cases). One patient showed a scapuloperoneal syndrome. Both myopathic and neurogenic EMG patterns were present in 6 patients; a neurogenic pattern was found in 4 cases and a myopathic pattern in 2 cases. In all patients, muscle biopsies showed rimmed vacuoles with eosinophilic inclusions. In 9 cases ultrastructural studies displayed abnormal filaments of 15-18 nm in diameter in the vacuoles. Intranuclear filaments were rarely observed. The significance of the filaments is unknown and their specificity is doubtful because they are present in other myopathies with rimmed vacuoles (some distal myopathies and oculopharyngeal muscular dystrophies). Finally a rich inflammatory exudate was present in 8 patients only.

A case of an infantile type III glycogenosis (Forbes disease), confirmed by morphologic and biochemical studies, had light-microscopic, histochemical, and electron-microscopic evidence of multicore structures and type 1 fiber predominance with hypotrophy. This association is discussed with relation to the unusual clinical findings. The authors conclude that two distinct disease entities--Forbes disease and multicore myopathy--may coexist.

Two recent publications have shown the advantage of understanding the deficit in AMP desaminase in rheumatology. On this subject, the authors report 4 cases of deficit in AMP desaminase. The first one includes a semiology made of pain, and stiffening, the second case is discovered in the course of a primary muscular disease. The third case is present during the first stage of a spinal cord compression. The fourth case is a muscular deficit accompanied with a histological picture of inflammation, considered initially as a chronic polymyositis, but explained secondarily as a pseudo-polymyositis form of facio-scapulo-humeral dystrophy. In this respect, the cases from the literature are divided into three groups: asymptomatic, infraclinical forms, forms occurring in the course of specific diseases (muscular diseases, spinal cord diseases, inflammations of the connective tissues, metabolic diseases), apparently isolated forms. In the latter, emerges a semiology made of pain, cramps, stiffening of the lower extremities occurring on exertion. However, the specificity of the symptoms remains to be discussed.

Concentric laminated bodies are described in five cases (one case previously reported and four new ones) of various muscle diseases. A general review of previous reports on these structures is made. They appear to be unspecific bodies generally noticed in abnormal skeletal muscle in various disorders; nevertheless they were found in normal muscle fibres of the mothers of children affected by myopathy. They have a complex filamentous structure. According to the authors, their myofilamentous nature is uncertain but seems more probable than a mitochondrial or lipidic one.

We report the cases of a 46 year old woman and of a 18 year-old boy who met the criteria for Kearns-Sayre syndrome. Additional atypic features were present in one case: family history, psychosis and acute respiratory failure. In both cases histoenzymatic analysis of the muscle biopsy and biochemical studies of mitochondria isolated from the muscle sample demonstrated mitochondrial myopathy associated with combined partial deficiency of complexes I and IV of the electron transfer chain. Although there is no correlation between clinical and biological data in the mitochondrial myopathies our cases confirm that such defects are involved in Kearns-Sayre syndrome. Improvement with coenzyme Q10 therapy in these patients is reported.

Muscle changed found in five patients with periodic paralysis are described. Three patients had familial hypokaliemic paralysis, one had familial periodic paralysis with hyperkaliemia, and one had periodic attacks of muscular weakness with thyrotoxicosis. According to the stage in the clinical course at which biopsy was performed, several pathological patterns are described. Vacuolization was the main feature in the early stages of periodic paralysis. Later in the clinical course, tubular aggregates were the characteristic findings. In addition, degenerative changes of the muscle fibres were found in patient exhibiting a permanent myopathic condition. In thyrotoxic periodic paralysis, the longer the clinical course, the more severe the changes appeared to be. Possible mechanisms leading to such structural abnormalities remain in question.

CT Scan examination in 145 cases of neuromuscular diseases yielded the following results: Diagnosis between myogenic and neurogenic process is inconstant and cannot be considered as absolute. In myogenic diseases the X ray density of muscle is early decreased with a preservation of muscle outline. In neurogenic diseases muscle volume is early decreased. Coexistence of atrophic and hypertrophic muscles indicates primarily muscle disease. Some patterns of involvement appear to be frequent. In Duchenne's dystrophy a contrast exists between atrophic "empty" or hypertrophic muscles during the ambulatory period and "ghostly" muscles during the terminal period. In facio-scapulo-humeral muscular dystrophy, tibialis anterior and hamstring muscles have often a decreased density and psoas muscles are normal or hypertrophic. In myotonic dystrophy a hypodense perifemoral crescent is frequently observed. Diagnosis between limb-girdle myopathy ("empty" muscles with preserved limits, hypertrophic muscles, hypodense gastrocnemius medialis muscles) and chronic spinal amyotrophy (irregular and atrophic muscles without selective involvement and hypertrophic muscles) is tentatively proposed but is not considered to be clear-cut. Muscle involvement has an asymmetric distribution in amyotrophic lateral sclerosis and is rather symmetric in peripheral neuropathies.

A case of centronuclear myopathy is presented, showing a rapid course in a 47-year-old male. Clinical and muscular findings were consistent with the diagnosis of centronuclear myopathy. On nerve biopsy, there was evidence of a regenerative process. Electron microscopy showed proliferation of Schwann cells with numerous Büngner's bands and other anomalies. This rare case with peripheral nerve changes may provide evidence to support the hypothesis of peripheral nerve involvement in centronuclear myopathy.

In a case of Fabry's disease, microscopic, ultrastructural, and biochemical studies of a muscle biopsy were performed, as well as microscopic, ultrastructural, and morphometric studies of a nerve biopsy. Pleomorphic lipid inclusions were observed in muscle fibers, fibroblasts, and endomysial capillaries. Moreover, the thermolabile isoenzyme A of alpha-D-galactosidase was almost completely absent. In the nerve specimen, polymorphous lysosomes were noted in perineural cells, in fibroblasts, and in endothelial and perithelial cells in association with some nonspecific degenerative changes. The morphometric data revealed a loss of large myelinated fibers, an uncommon finding in Fabry's disease, and a decrease of the average diameter of the unmyelinated fibers, which was related to axonal sprouting. The relationship between the pain attacks and the increased number of the small unmyelinated fibers is discussed.

Hypertrophy of the calf associated with sciatica is rare. In the present case there was hypertrophy, predominantly of type I fibers, which showed a high percentage to the detriment of type II fibers. In the previously reported cases, the type of hypertrophied fibers was variable and pseudomyotonic discharges were inconstantly recorded. The mechanism underlying the hypertrophy is unknown. Inequality of size of muscle fibers could result from proximity of atrophic fibers and those with compensatory hypertrophy. The increased proportion of type I fibers could be the consequence of compensatory hypertrophy induced by lesions of synergic muscles and/or muscle compartments.

Unmyelinated afferent fibres are the most numerous of the whole afferent component. Their function in nociception is now well established. However some recent data allow to think that their function is much more complex. Cutaneous unmyelinated afferences are evaluated in controls and in 3 groups of patients (painful neuropathies, painless neuropathies with hypesthesia, diabetic neuropathies). Amyelinic myelinic ratio is not in accordance with the gate control theory. Muscular afferent fibres are studied, especially the lateral gastrocnemius soleus nerve of cat. Their predominance is obvious. Our study was conducted by recording single unit activity from a micro electrod inserted in dorsal root ganglia. But 45% of unmyelinated fibres are not activated by various algesic stimuli. It is suggested that they have a function in cardio-pulmonary adaptation to effort and possibly segmental vascular control as homonymous reflex effect on moto-neuron. Their several properties are a possible explanation of muscular cramps.

In order to investigate the pathological basis of muscle hypertrophy in Duchenne dystrophy, 9 biopsy specimens of the lateral gastrocnemius and 7 of the vastus lateralis were compared. All patients had calf hypertrophy and normal strength in gastrocnemius-soleus, whereas the quadriceps biopsied were all atrophied and weak. The patients' ages ranged from 4 to 11 years. The pathological and histochemical changes were assessed semi-quantitatively. Comparison of the gastrocnemius and quadriceps groups showed that the number of hypercontracted fibres, the degree of endomysial fibrosis and the degree of fat infiltration were significantly higher in the quadriceps. The fibre type differentiation was better in the gastrocnemius group. The mean fibre diameter was above normal in all gastrocnemius biopsies and showed no increase with age. In the quadriceps, fibre hypertrophy was found early in the disease but had changed into fibre atrophy in the three oldest patients. When present, fibre hypertrophy involved both fibre types. The amount of fat-fibrosis per unit area was increased in both groups, but more severely so in the quadriceps. These results indicate that there is no true muscle hypertrophy in the gastrocnemius, in which the fat-fibrosis component was increased in all patients and that the dystrophic process is more active in the quadriceps. The finding of persistent fibre hypertrophy in the gastrocnemius is discussed with respect to the postural abnormalities observed in the lower limbs in Duchenne dystrophy.