The craniosacral progression of muscle development influences the emergence of neuromuscular junction alterations in a severe murine model for spinal muscular atrophy.

AIMS: As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA. METHODS: Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice. RESULTS: During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes. CONCLUSION: SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis.

Electron-microscopic imaging of endothoracic fascia in the thoracic paravertebral space in rats.

BACKGROUND AND OBJECTIVES: Anesthesia and analgesia with paravertebral block are reportedly variable. Existence of an endothoracic fascia has been proposed as one of the possible mechanisms leading to variability. We undertook an electron-microscopy imaging study to investigate the endothoracic fascia in the thoracic paravertebral space (TPS) in rats. METHODS: Male Wistar rats were studied in accordance with the principles of laboratory animal care. After the rats were euthanized in a CO2 chamber, the thoracic paravertebral tissues were removed en bloc and cut into consecutive transverse sections of approximately 3 mm. Stereomicroscopy and electron-microscopy assessments were performed by 2 independent observers. RESULTS: The endothoracic fascia was consistently identified in all specimens. The fascia was located between the parietal pleura and the innermost intercostal muscles or ribs. Its thickness ranged from 15 to 27 µm (mean, 20 ± 3 µm). The endothoracic fascia divided the TPS in 2 compartments: one, extrapleural and anterolateral (EPC); another, subendothoracic and posteromedial (SETC). The spinal nerves with their ganglia were found within SETC, whereas the sympathetic ganglia were consistently located within the EPC. CONCLUSIONS: The endothoracic fascia in rats appears to divide the TPS into EPC and SETC. These anatomic characteristics may have implications in thoracic paravertebral blockade.

[Ultrastructural aspects of intercostal muscles in patients with COPD]. / Aspecte ultrastructurale ale muschilor intercostali la pacientii cu bronhopneumopatie obstructiva cronica (BPOC).

The studies to the electron microscope have shown that external and internal intercostal muscles present characteristic changes of ultra structural organization in COPD. The diameter of muscle fibers become unequal, sarcolemma shows frequently deep invaginations, having in the near sarcoplasma concentrations of mitochondria and tubes of the system T. Here and there, myocytes appear divided or with sarcomere frequently being in contraction state. Ultra structural changes are more emphasized to the external intercostal muscles, more requested than those internal. In this way, the results show that the intercostal breathing muscle are affected by the chronic obstructive pulmonary disease.

Effects of controlled mechanical ventilation on respiratory muscle contractile properties in rabbits.

OBJECTIVE: We examined in rabbits the effects of more than 48 h of mechanical ventilation on the contractile properties and fiber type adaptations of the respiratory muscles. DESIGN AND SETTING: Experimental prospective study in a university laboratory. ANIMALS AND INTERVENTIONS: Nineteen rabbits were randomly allocated to two groups: control (n=10) or mechanically ventilated (MV; n=9) for 51+/-3 h. MEASUREMENTS AND RESULTS: Respiratory muscles contractile properties were analyzed before and after a fatigue protocol using in vivo isometric 1-s tetanic contraction characteristics in both muscles: peak tetanic force, contraction time, relaxation time, and total contraction time. Both muscle fiber type proportions, diameter, and cross-sectional areas were measured using ATPase staining. The MV rabbits showed significant weight loss in both muscles, accompanied by a reduced peak tetanic force (9.96+/-3.2 vs. 7.44+/-2.2 N for diaphragm of control and MV animals respectively), fatigue resistance index, and increased relaxation time (57.5+/-8.7 vs. 85.8+/-9.4 ms for diaphragm of control and MV animals) and contraction time. These impairments in the MV group worsened after the fatigue runs. Both muscle showed a significant atrophy of type IIa and IIb fibers but a stability in type I fibers cross-sectional area. CONCLUSIONS: Mechanical ventilation in rabbits produces alterations in contractile properties of the diaphragm and 5th external intercostal muscle, increases both muscles fatigue, and promotes atrophy of type II fibers.

Effects of prolonged mechanical ventilation on respiratory muscle ultrastructure and mitochondrial respiration in rabbits.

OBJECTIVE: To investigate in rabbits whether prolonged mechanical ventilation (PMV) leads to ultrastructural changes in respiratory muscles and alters diaphragm mitochondrial respiration. DESIGN AND SETTING: Experimental prospective study in a university laboratory. ANIMALS AND INTERVENTIONS: We studied respiratory muscles of seven rabbits after 49+/-1 h of controlled mechanical ventilation. Ten nonventilated rabbits were used as a control group. MEASUREMENTS AND RESULTS: After mechanical ventilation electron-microscopic observations of the diaphragm and the external intercostal muscles revealed disrupted myofibrils, increased number of lipid vacuoles in the sarcoplasm, and smaller mitochondria with focal membrane disruptions. Volumetric and numerical densities of the mitochondria were significantly lower in the PMV group than the control group. Mitochondrial respiration was quantified in isolated diaphragm muscle-cell mitochondria using two respiratory substrates. There was no difference in oxygen consumption values in the three states of mitochondrial respiration between the two groups except for state 2 (basal state) with pyruvate/malate parameter (53.5+/-20 for the ventilated group vs. 33.8+/-10.2 nmol atom O/mg per minute for the control group). There was no significant difference between groups in ADP/O ratio or respiratory control ratio. CONCLUSIONS: PMV leads to respiratory muscle cell degeneration and minor changes in oxidative phosphorylation coupling in diaphragmatic mitochondria. These phenomena may mediate part of damage of respiratory muscles after inactivity related to PMV.

Postinspiratory activity of the parasternal and external intercostal muscles in awake canines.

Previous studies have shown in awake dogs that activity in the crural diaphragm, but not in the costal diaphragm, usually persists after the end of inspiratory airflow. It has been suggested that this difference in postinspiratory activity results from greater muscle spindle content in the crural diaphragm. To evaluate the relationship between muscle spindles and postinspiratory activity, we have studied the pattern of activation of the parasternal and external intercostal muscles in the second to fourth interspaces in eight chronically implanted animals. Recordings were made on 2 or 3 successive days with the animals breathing quietly in the lateral decubitus position. The two muscles discharged in phase with inspiration, but parasternal intercostal activity usually terminated with the cessation of inspiratory flow, whereas external intercostal activity persisted for 24.7 +/- 12.3% of inspiratory time (P < 0.05). Forelimb elevation in six animals did not affect postinspiratory activity in the parasternal but prolonged postinspiratory activity in the external intercostal to 45.4 +/- 16.3% of inspiratory time (P < 0.05); in two animals, activity was still present at the onset of the next inspiratory burst. These observations support the concept that muscle spindles are an important determinant of postinspiratory activity. The absence of such activity in the parasternal intercostals and costal diaphragm also suggests that the mechanical impact of postinspiratory activity on the respiratory system is smaller than conventionally thought.

Morphologic changes in intercostal muscle tissue associated with a viper (Agkistrodon halys blomhoffi) bite.

This study examined a 73-year-old woman who had been bitten in the right thumb by a viper (Agkistrodon halys blomhoffi). She suffered acute renal failure and respiratory failure after the bite. On the 20th day, respiratory failure persisted, although she had recovered from renal failure. Intercostal muscles were obtained by biopsy on the 40th day after the bite. Specimens were observed with enzyme-histochemical and electron microscopic techniques. The size of muscle fibers varied. Most of the smaller muscle fibers were enzyme-histochemically type 2C fibers. This was considered to be the regenerative stage of acute rhabdomyolysis. Ultrastructurally, two opposite types of changes, degeneration and regeneration, were mixed in the same areas. Muscle fibers might be continuously degenerated, and the regenerative fibers might also be degenerated. This study suggests that some slow allergic reactions to snake venom might bear some relation to the degenerative changes.

Composition of newly forming motor units in prenatal rat intercostal muscle.

We have examined the composition of rat intercostal motor units during the period of late gestation, when most muscle fibres are formed, in order to see the pattern of the contacts initially made between single motoneurons and myotubes. At this early stage, the muscle contains two types of myotubes, primary and secondary myotubes, and a major aim was to see whether individual motoneurons preferentially made contact with a particular myotube type. The technique used to define myotubes contacted by a single motoneuron was anterograde labelling of the neuron, followed by electron microscopic detection of labelled terminals and their postsynaptic targets. We find that prenatal motor units are inhomogeneous with respect to their primary/secondary myotube composition. Most individual motoneurons show many permutations of contact with primary myotubes, secondary myotubes, and undifferentiated cells, including single nerve terminals which contact both primary and secondary myotubes. Our results are interpreted in terms of changes to the composition of both the muscle and of the motor units during the final 5 days of gestation. We demonstrate that motoneurons necessarily make their initial contacts on primary myotubes, but that these are surprisingly sparse. As secondary myotubes appear and become innervated, motor units are at first all similar and all heterogeneous. However, primary myotubes are represented more often in motor units than in the muscle as a whole. This probably reflects the relative densities of polyinnervation of primary vs. secondary myotubes. By embryonic day 20, motor units have become divergent in composition, with some dominated by primary myotubes and others by secondaries. We propose that motoneurons initially establish contacts at random on either myotube type, but then begin to express preference for one type or the other and reorganise their periphery. Refining of motor unit composition towards homogeneity in the postnatal period probably involves other elements, such as mutability of muscle fibre and/or motoneuron characteristics as a function of usage and muscle position, perhaps influenced by sensory feedback mechanisms.

Extracellular Ca(2+)-dependent and independent calcium transient in fetal myotubes.

Spatio-temporal changes in the intracellular calcium concentration [Ca2+]i of dissociated mice myotubes from 14-day and 18-day-old fetuses were studied using digital imaging analysis of the Ca2+ indicator fura-2. Myotubes from 18-day-old fetuses displayed a transient [Ca2+]i increase upon electrical stimulation either in nominally calcium-free external solution or in Krebs solution containing 100 microM lanthanum. Thus, at this developmental stage, membrane depolarization appears to increase [Ca2+]i by stimulating Ca2+ release from the sarcoplasmic reticulum independently of extracellular Ca2+ influx. Similarly, myotubes from 14-day-old fetuses also showed a calcium transient upon electrical stimulation in Krebs solution. However, in 46% of these myotubes the calcium transient was abolished when Ca2+ entry through calcium channels was suppressed.

Structural and metabolic integrity of sheep external intercostal muscle during extended culture.

The objectives of this study were to examine the structural and metabolic integrity of isolated sheep external intercostal muscle bundles following variable lengths of preincubation (0 to 192 h). Samples of intact external intercostal muscle (10 to 15 g), with tendons attached, were prepared from growing wethers and maintained at their resting lengths during preincubation for 0 to 192 h. Protein synthesis (PS), protein degradation (PD), acetate oxidation and ultrastructural integrity of muscle samples were examined at 0 to 192 h, 0 to 96 h, 0 to 48 h and 0 to 96 h following isolation, respectively. Additionally, the effects of variable fetal calf serum (FCS) concentrations (0 to 20%; w/v) on PS and PD and acetate oxidation were examined. Rate of PS increased as preincubation time increased to 192 h; however, most of this increase was due to the proliferation of fibroblasts on the surface of the muscle sample. Addition of cytosine arabinoside to the incubation media prevented the fibroblast-dependent increase in PS; however, it did not entirely prevent the preincubation time-dependent increase in PS. Rate of PD increased greatly upon preincubation. The nitrogen balance of incubated muscles was negative at all times examined. Acetate oxidation was maintained through 12 h of preincubation and thereafter declined. Relatively normal myofibrillar structure was maintained through 48 h of preincubation; however, loss of mitochondrial integrity and dissolution of Z-disks at 48 h and at 96 h of preincubation were evident. Isolated tissues were able to respond to FCS concentration in medium following 48 h of preincubation.

The slow channel syndrome. Two new cases.

Two patients are described with a myasthenic syndrome that presented in early adult life. One patient had 2 asymptomatic first degree relatives with similar electrophysiological findings. Both patients had abnormal fatiguability, arm weakness being prominent; neither of them responded to anticholinesterase medication. An abnormal decrement at 3 Hz stimulation was present, and a single stimulus evoked a repetitive response. Electrophysiological studies on biopsied intercostal muscle showed miniature endplate potentials of normal amplitudes but with prolonged rise and decay times. Anticholinesterase staining (Case 1) was not reduced, and showed elongation of some endplates. Ultrastructural studies (Case 2) showed degeneration of junctional folds and diffusely thickened endplate basal lamina. Calcium deposits were not observed and myopathic changes were slight. The findings are consistent with a prolonged open time of the ACh-induced ion channel.

Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle.

Previous studies of denervated and cultured muscle have shown that the expression of the neural cell adhesion molecule (N-CAM) in muscle is regulated by the muscle's state of innervation and that N-CAM might mediate some developmentally important nerve-muscle interactions. As a first step in learning whether N-CAM might regulate or be regulated by nerve-muscle interactions during normal development, we have used light and electron microscopic immunohistochemical methods to study its distribution in embryonic, perinatal, and adult rat muscle. In embryonic muscle, N-CAM is uniformly present on the surface of myotubes and in intramuscular nerves; N-CAM is also present on myoblasts, both in vivo and in cultures of embryonic muscle. N-CAM is lost from the nerves as myelination proceeds, and from myotubes as they mature. The loss of N-CAM from extrasynaptic portions of the myotube is a complex process, comprising a rapid rearrangement as secondary myotubes form, a phase of decline late in embryogenesis, a transient reappearance perinatally, and a more gradual disappearance during the first two postnatal weeks. Throughout embryonic and perinatal life, N-CAM is present at similar levels in synaptic and extrasynaptic regions of the myotube surface. However, N-CAM becomes concentrated in synaptic regions postnatally: it is present in postsynaptic and perisynaptic areas of the muscle fiber, both on the surface and intracellularly (in T-tubules), but undetectable in portions of muscle fibers distant from synapses. In addition, N-CAM is present on the surfaces of motor nerve terminals and of Schwann cells that cap nerve terminals, but absent from myelinated portions of motor axons and from myelinating Schwann cells. Thus, in the adult, N-CAM is present in synaptic but not extrasynaptic portions of all three cell types that comprise the neuromuscular junction. The times and places at which N-CAM appears are consistent with its playing several distinct roles in myogenesis, synaptogenesis, and synaptic maintenance, including alignment of secondary along primary myotubes, early interactions of axons with myotubes, and adhesion of Schwann cells to nerve terminals.

Selective reinnervation of intercostal muscles transplanted from different segmental levels to a common site.

We transplanted external intercostal muscles from one of several thoracic (T) levels to the neck of adult rats. The cervical sympathetic trunk, which innervates the superior cervical ganglion, was cut, and its proximal end was apposed to the muscle. Preganglionic axons in the trunk reinnervated muscle fibers in the transplants. We determined the segmental origin of synaptic inputs to transplanted muscles by recording intracellularly from muscle fibers while stimulating individual ventral roots which supply axons to the trunk. In one series of experiments, T2 or T8 muscles were transplanted from the thorax to the neck of the same rat. While T2 and T8 muscles were reinnervated to a similar extent, they differed in the segmental origin of the innervation they received: T2 muscles received more inputs from rostral segments (T1 and T2) than did T8 muscles, and T8 muscles received more inputs from caudal segments (T4 to T6) than did T2 muscles. This difference between reinnervation of T2 and T8 muscles was detected both 2 to 4 weeks and 10 to 14 weeks after surgery. In a separate series, using rats of an inbred strain, T3, T4, or T5 muscles were transplanted from one rat to a separate host. Again, the average segmental origin of inputs to transplants from different levels differed systematically: it was most rostral to T3 muscles, intermediate to T4 muscles, and most caudal to T5 muscles. Finally, T3 and T5 muscles were soaked in a myotoxin, Marcaine, before reimplantation. This treatment kills muscle fibers but not myoblastic satellite cells; therefore, muscle fibers were replaced by regeneration. Marcaine-treated T3 and T5 muscles were successfully reinnervated but did not differ significantly in the segmental origin of their inputs. Our results show that adult mammalian muscles can be selectively reinnervated, and they raise the possibility that the selectivity is based on some positional quality that matches axons and muscles from corresponding segments. However, while differences among muscles survive denervation and transplantation, their expression or accessibility may change during regeneration.

Koelle's copper thiocholine method performed with a low-pH phosphate buffer, followed by osmification of the precipitate: revival of two abandoned procedures.

Using a glutaraldehyde-fixed mouse neuromuscular junction, a fine precipitate of copper thiocholine was obtained with Koelle's medium prepared by a mixture of phosphate buffer (pH 5.6-5.9) and copper glycine solution (strongly acidic). The final pHs of these incubation media were very low, being situated between 3.8 and 4.2, respectively. It is well known that phosphate buffer, at such a low pH value, has no buffering effect on the acetic acid of enzymatic hydrolysis. This probably caused a sharp drop of the pH value in the vicinity of the enzymatic site and allowed a fine localization of copper thiocholine, the precipitation of which is pH dependent. Furthermore, the osmification of copper thiocholine in the same phosphate buffer provided a finely localized electron dense product. The chemical nature of the osmified copper thiocholine is discussed.

Types of human intrafusal muscle fibers.

The histochemical and fine structural profiles of human intrafusal muscle fibers were studied. Spindles were located in freshly frozen specimens taken from biopsied normal external intercostal muscles, and periodic 10- and 50-mum-thick cross sections were processed alternately for enzyme histochemical and electron microscopic examination. Nuclear bag fibers were of two types, bag1 and bag2, histochemically, and they displayed two distinct types of ultrastructure. Nuclear chain fibers were histochemically and ultrastructurally homogeneous. Regional differences in enzymatic staining and ultrastructure occurred along individual intrafusal fibers. Human bag1 and bag2 fibers appear to be analogous to the two types of nuclear bag fiber identified in animal spindles and are considered to have different roles in spindle function. The presence of three types of intrafusal fibers should be taken into account when studying spindle abnormalities in human neuromuscular disorders.

Intercostal muscle biopsy in human neuromuscular disease. Histochemical and electron microscopic studies.

External intercostal muscle biopsies were examined histochemically and by electron microscopy. The use of this muscle allowed correlation with physiological and pharmacological studies on the same specimens. Changes observed in musclar dystrophy and motor neurone disease resembled those previously described in biopsied limb muscle and underline the particular usefulness of this preparation in the study of human neuromuscular disease.

Relationship of the sarcoplasmic reticulum to fibril and triadic junction development in skeletal muscle fibers of fetal monkeys and humans.

Examinations of stages of fibril development in muscle fibers of seven Rhesus monkey and six human fetuses reveal SR tubules encircling the Z lines at all stages of fibril development. The encircling SR tubules are continuous with the SR network of tubules which is found surrounding fibrils at all stages of development observed. The SR tubules encircling the Z lines show connections (electron-opaque strands) with the Z lines. The developing triadic junction shows a progressive increase in complexity of structures within the junction. First, membranes of T and SR become apposed with no visible structure between them- Second, tenuous connections are found traversing the space between apposed membranes. Third, well developed bridges are seen traversing the space. And finally, an intermediate density midway between the apposed membranes and parallel to them is found in favorable sections. Junctions between T tubule membranes were also observed and the structures in these junctions are somewhat similar to those found in junctions between T and SR membranes. The change in orientation of triads from predominantly longitudinal to predominantly transverse is complete in the 18-week monkey fetus and incomplete in the latest stage (28-week) of fetal development observed in humans.

Hyperthyroid myopathy. Intracellular electrophysiological measurements in biopsied human intercostal muscle.

Morphological and electrophysiological studies were performed on intercostal muscle biopsies from 2 thyrotoxic patients. The diseased fibers had numerous areas of subsarcolemmal glyogen accumulations and abnormal membranous projections. Both Type I and Type II muscle fibers were atrophied. Diseased fibers were substantially depolarized and when artifically hyperpolarized showed earlier inactivation of the sodium conductance as a function of membrane potential, and a critical depolarization potential more depolarized than in normal fibers. When stimulated at 20 pulses/sec, or faster, the diseased fibers could not generate normal action potentials due to membrane depolarization and the appearance of a marked after-hyperpolarization. Muscle weakness associated with hyperthyroidism is attributed to the reduced membrane excitability.