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[Autotransplantation of previously denervated muscles in the rabbit].

Whole gastrocnemius muscles of rabbits, preliminarily denervated, were grafted. At the moment of grafting (60 days after the operation) the muscles were in the state of deep atrophy attended by distrophic changes. The autotransplantated muscles took at the site of grafting, their further reorganization provided progressive development of the muscle tissue within the transplant, its growth, and formation of definitive muscle fibers with nerve terminals. After a definite time some degenerative changes were observed in the transplant muscle tissue; as a result the muscle tissue was substituted by connective tissue. These data support the statement founded before on feasible free grafting of preliminary denervated whole muscles. However, deep denervation atrophy seems to influence the remote results of the transplantation.

Animals

Experimental myotonia induced in denervated muscles by 2,4-D.

Rats were denervated in one hind limb and injected with 2,4-dichlorophenoxyacetic acid (2,4-D). Isotonic tetanic contractions of the muscles treated with 2,4-D after more than 10 days of denervation revealed prolonged relaxation times similar to those of the intact side and characteristic of clinical myotonia. No myotonic discharges were observed in the muscles denervated for more than 10 days and treated with 2,4-D. The increase in threshold for action potential generation secondary to denervation is suggested as the factor limiting the initiation of the repetitive discharges.

2,4-Dichlorophenoxyacetic Acid

Alteration in the expression of GLUT-1 and GLUT-4 protein and messenger RNA levels in denervated rat muscles.

Denervation induces insulin resistance of the glucose transport process in skeletal muscle. To determine whether this is due to alterations in the expression of muscle glucose transporters (GLUT) in different fiber types, we evaluated the amount of GLUT-1 and GLUT-4 protein and messenger RNA (mRNA) in extensor digitorum longus (EDL) and soleus at 1, 2, and 3 days after sciatotomy. Denervation elevated the basal rate of 2-[1,2-3H]deoxy-D-glucose (2-DOG) uptake in the EDL and decreased the insulin-stimulated DOG uptake in both muscles. Denervation after 1 day did not modify the GLUT-1 or the GLUT-4 protein level in either muscle. However, it increased GLUT-1 mRNA by 66% and decreased GLUT-4 mRNA by 70% in the EDL, but not in the soleus (P < 0.05). After 2 days of denervation, by which time GLUT-1 mRNA was increased 2-fold and GLUT-4 mRNA was reduced by 70%, we observed a 2-fold increase in GLUT-1 protein (P < 0.01) in the EDL and a 40-45% decrease in GLUT-4 protein in both muscles (P < 0.01). These results indicate that modifications in the expression of GLUT-1 and GLUT-4 protein cannot explain the insulin resistance of the glucose transport process in the EDL or soleus 1 day after denervation. After 2 days of denervation, however, alterations in GLUT-1 and GLUT-4 protein levels may contribute to the change in basal and insulin-stimulated DOG uptake in both the EDL and the soleus muscles.

Animals

Is malignant hyperpyrexia muscle denervated?

To test the hypothesis that human muscular dystrophies may be secondary to denervation, the responses in vitro of muscle in human malignant hyperpyrexia to electrical and pharmacological stimuli have been compared with those of the denervated mouse soleus muscle. The results suggest that the muscle abnormality in malignant hyperpyrexia is different from that produced by denervation. This must cast doubt on the concept that other human muscular dystrophies are secondary to denervation.

Animals

Increased cyclic GMP in the end-plate region of denervated frog muscle.

Denervated frog sartorius muscles showed an approximately 2--3 fold increase of cyclic GMP in their end-plate rich regions which did not appear up to 5 weeks after denervation in the normally end-plate-free pelvic region. No increase in cyclic AMP was seen in these preparations. The results suggest that the increase of cyclic GMP is related to processes specific to the region in which end plates are normally present.

Acetylcholine

Regneration in free grafts of normal and denervated muscles in the rat: morphology and histochemistry.

Intact soleus and extensor digitorum longus muscles in the rat were freely grafted to the contralateral leg after either no preliminary treatment or 14 days prior denervation. Normal muscle grafts during the first week were characterized by a central zone of degenerating original muscle fibers (disappearing by 7-9 days) and a peripheral zone, containing regenerating muscle as well as small numbers of surviving original muscle fibers. A radial gradient of regeneration was establihed, with more mature muscle at the periphery and less mature muscle toward the center. Denervated grafts were characterized by rapid degeneration (within 2-3 days) of original muscle fibers in the central area, rapid appearance of regenerating muscle fibers (e.g. cross striations by 5 days) with uniform levels of differentiation throughout the graft and larger numbers of surviving original muscle fibers at the periphery. During the first week, stages of muscle differentiation in denervated grafts were attained 1-2 days earlier than comparable stages in normal grafts. Later stages of muscle differentiation were similar in both types of grafts. Histochemical studies revealed a loss of enzyme activity (phosphorylase, ATPase and SDH) in the center of early (2-4-day) normal and denervated grafts. Denervated grafts, however, possessed a thicker peripheral rim of enzymatically active surviving muscle fibers than normal grafts. In both types of grafts the old muscle fibers in the center were replaced by enzymatically active regenerating muscle fibers which stained uniformaly (ATPase) until 30 days. By 60 days a mixed fiber pattern had developed. Muscle spindles were found within the grafts.

Adenosine Triphosphatases

Experimental study of denervated muscle atrophy following severance of posterior rami of the lumbar spinal nerves.

The morphologic changes in denervation atrophy of paravertebral muscles after severance of the posterior rami in cats were investigated, using histochemical methods and electromyography. Using a paraspinal approach, three branches of the posterior rami on the left side were cut under microscopy at one, two, or three levels (L2 approximately L4). Muscle atrophy was evaluated, using the percent wet weight and the percent diameter of muscle fibers as parameters. Myosine ATPase stain was used to observe reinnervation. Four weeks after surgery, the range and severity of muscle atrophy increased proportionally to the number of posterior rami severed. Muscle atrophy was revealed at one or two levels caudal to the injured nerve level. At 12 and 24 weeks, muscle atrophy recovered gradually. In more than two-level injury groups, however, recovery of percent wet weight reached up to 80% even after 24 weeks, despite the fact of reinnervation demonstrated in some parts of the denervated muscles.

Animals

Firing rates of human motor units in partially denervated muscle.

Single motor unit firing rates were measured from the first dorsal interosseous muscle (FDI) of normal subjects and patients with partial denervation of that muscle. Motor unit discharges were recorded at various levels of voluntary, stationary, isometric contraction of the FDI. The mean increase in firing rate associated with an increased muscle tension of 100 gm was significantly greater in severely weak muscle. However, when an "adjustment" was made for the degree of weakness, reduced firing rate responses were observed which correlated with the degree of muscle weakness.

Electromyography

Experimental myasthenia in Balb/c mice immunized with rat acetylcholine receptor from rat denervated muscle.

A new model of an autoimmune disease of the neuromuscular junction was obtained by injection of acetylcholine receptor purified from rat denervated muscles into Balb/c mice. Anti-rat, then anti-mouse acetylcholine receptor antibodies, appear in mouse serum during the immunization procedure. Electrophysiological investigations performed on immunized mice reveal a neuromuscular block similar to that found in myasthenia gravis. Not a single mouse with objective signs of muscular weakness was lacking anti-mouse acetylcholine receptor antibodies but no correlation was found between their level and the severity of the disease.

Action Potentials

Studies on the mechanism of fibrillation potentials in denervated muscle.

1. Intracellular electrodes were used to study the origin of fibrillation potentials in chronically denervated rat muscle. 2. Fibrillation potentials were observed to start from spontaneous biphasic membrane potential oscillations. Each action potential was followed by an after-hyperpolarization which in turn served as a pre-potential for the next spike. The critical level (threshold) for the initiation of the first spike in a train was lower than that of the next and subsequent spikes. 3. A correlation was found between the level of membrane potential and the critical level for action potential generation. This relation was most marked around the resting membrane potential (minus 60 to minus 80 mV) where 10 mV hyperpolarization caused a 9 m V increase in the critical potential level. At higher membrane potentials the correlation was less pronounced. In innervated muscles a similar correlation existed but it was less marked and was present only at membrane polarizations below the resting potential. 4. Increasing the external calcium concentration from 2 to 8 mM reduced the membrane potential-critical level relationship in denervated fibres towards that of innervated ones. 5. As critical level changes with membrane hyperpolarization, the rate of rise of the action potential increased, suggesting a progressive removal of sodium inactivation. 6. It is suggested that a mechanism similar to anode break excitation is important for the induction and maintenance of fibrillation potentials.

Action Potentials

The relation between ATPASE activity and light chains of myosin in developing, adult and denervated muscles of several animals species.

Ca2+ATPase activity and light chains of myosin, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in developing, adult and denervated fast, slow and cardiac muscles of the rat, guinea-pig, cat, rabbit and chick were studied. It has been shown that in normal adult muscles the electrophoretic pattern of light chains of myosin reflects the myosin ATPase activity only when muscles from the same animal species are compared. In homologous muscles from adult animals differing in size, the size-dependent difference in myosin ATPase activity is not revealed in the electrophoretic pattern. Both in developing and in denervated muscle, changes in myosin ATPase activity are either connected with changes in the pattern of light chains of myosin or this pattern does not change. This relation is different in fast and slow muscles and also differs in chick and rabbit muscles. There are several possibilities of explaining the relation between ATPase activity of myosin and the pattern of light chains of myosin. The observation that myosin from the soleus muscle of 1-month-old rabbit contains light chains corresponding to both fast and slow type of myosin, indicates that the change in myosin ATPase activity during development is due to changes in the ratio between the fast and slow type of myosin.

Adenosine Triphosphatases

Denervated muscle: rate of propagation and effects of manganese ions and glycerol on the fibrillatory activity of frog semitendinosus.

Denervated frog's semitendinosus muscles were studied. Fibrillation potentials generate propagated spikes which are conducted at a rate similar to that calculated for innervated muscles. The twitch tension per unit area was similar in denervated and innervated single fibres; the average value corresponded with values reported in the literature for fibres stimulated at 125/sec. Manganese ions (1-3 mM), selectively inhibit fibrillation potentials and have no appreciable effect upon evoked spikes. Mn2+ also inhibits (40-50%) the twitch responses and abolishes the resting tension of muscle fibres. The latter effects were observed in denervated as well as in innervated preparations when the fibres studied belonged to fasicles located at the site of entrance of the nerve. Disruption of the transverse tubular system by glycerol treatment does not interfere with the generation of fibrillation potentials, although the effects on twitching are similar to those reported in the literature. A possible mechanism is proposed to explain the generation of fibrillation potentials in frog denervated muscles.

Animals

Ultrastructure of an iguana fast twitch denervated muscle.

This ultrastructural study was undertaken to investigate the morphological changes which occur in the fast twitch gastrocnemius muscle of the reptile Iguana iguana after nerve section. It was found that initial degenerative alterations appeared in muscle fibers two weeks after denervation and progressed along the two months of the investigative period. They consisted of disorganization of contractile and sarcotubular elements and the appearance of autophagic vacuoles with mitochondrial debris. However, even two months after nerve section some myofibrils and mitochondria looked normal. Our results suggest that although the general course of denervation atrophy in iguana gastrocnemius is similar to that in other twitch muscles of vertebrates, the chronology of the process shows that iguana fast twitch skeletal muscles exhibit an intermediary position among the vertebrates in relation to their velocity of response to denervation.

Animals