Effects of early immobilization on the functional capacity of dystrophic (ReJ 129 dy/dy) mouse leg muscles.

Hindleg muscles of dystrophic (ReJ 129 dy/dy) mice were immobilized during the second post-natal week. Two months after remobilization histopathological features and isometric force characteristics of the m. extensor digitorum longus (EDL) and the m. tibialis anterior (TA) were studied. As a result of early transient immobilization significant differences were observed in muscle morphology and isometric force compared with untreated dystrophic muscles. Restriction of dynamic use of the muscles during this second post-natal week largely prevents the muscles of dystrophic mice from becoming affected by the disease process. Even after two months of remobilization pathology appears to be reduced.

Myosin isoforms in hindlimb muscles of normal and dystrophic (ReJ129 dy/dy) mice.

Myosin isoform expression was studied in hindlimb muscles of control (Dy/Dy) and dystrophic (dy/dy) mice of the ReJ129 strain during postnatal development. Three myosin heavy chain isoforms (fast II-B MHC, neonatal MHC, and slow or I MHC) were identified using monoclonal antibodies. Only original fibers, i.e., fibers formed during fetal life, were studied. Necrotic and regenerating fibers were excluded. The disappearance of neonatal MHC was found to be delayed in all muscles of dystrophic mice, except the soleus. The fraction of fibers containing I MHC was similar in control and dystrophic animals at all ages, except during the third postnatal week. The developmental increase in the fraction of fibers expressing II-B MHC was interrupted in dystrophic mice by two marked declines. The first occurred during the second postnatal week at the beginning of the main wave of fiber necrosis, and the second occurred at between 30 and 90 postnatal days.

Early immobilization of hindleg muscles of dystrophic mice: short-term and long-term effects.

Hindleg muscles of normal and dystrophic mice were immobilized unilaterally early during the postnatal period. After 1 week the casts were removed. Of one group of mice hindleg muscles were processed for histopathological and morphometrical evaluation at the end of the immobilization period. Hindlegs of other groups of mice were remobilized for various periods of time before the muscles were examined. In normal mice immobilization of calf muscles that were fixed in a shortened position resulted in atrophy of about 35% compared with untreated muscles. This was accompanied by a reduction in fibre number of about 15%. The antagonists that had been fixed extended, did not show those effects. Immobilization of dystrophic muscles minimized pathology in both agonists and antagonists, although atrophy developed. Upon remobilization the normal muscles resumed postnatal development. They did not deviate from normal, untreated muscles at the age of 3.5 months. Upon remobilization of dystrophic muscles pathology developed, but less severely than during the second and third postnatal week in untreated dystrophic muscles. Significant differences in morphometrical parameters compared with untreated dystrophic muscles were observed during the 3 months remobilization period studied.

Alterations in relative phosphocreatine concentrations in preclinical mouse muscular dystrophy revealed by in vivo NMR.

Using in vivo 31P NMR spectroscopy, the early postnatal development of lower hindleg muscles of normal and dystrophic mice was investigated. Ratios of phosphocreatine and inorganic phosphate, and of phosphocreatine and ATP increased exponentially during normal postnatal growth and differentiation. In dystrophic skeletal muscles, however, the ratios were already considerably lower during the early postnatal period, before histopathological features were observed. The ratios remained lower, relative to normal muscles, at least into the young adult stage. A deficiency in the sequestering of creatine or a defect in the phosphocreatine shuttle is proposed to explain the pathological features observed in this disorder.

Long term functional improvement of dystrophic mouse leg muscles upon early immobilization.

The long term effects of immobilization of one hindleg, during the second postnatal week, were investigated in dystrophic ReJ 129 dy/dy strain of mice. The muscles of the immobilized limb were compared with those of the contralateral, non-treated side and with those of naturally dystrophic age-mates, after 1, 2 and 3 months of remobilization. It appeared that the experimental animals made better use of their remobilized leg than of the contralateral leg for locomotion. The remobilized muscles were significantly less atrophic than the contralateral muscles and they also contained more muscle fibres. It is concluded that during postnatal growth and differentiation the dystrophic muscle fibres pass through a sensitive period. Immobilization during this period prevents the majority of the muscle fibres from becoming affected. Remobilization induces pathological features in the muscles, but they remain less damaged than the non-immobilized muscles.

Postnatal growth and differentiation in three hindlimb muscles of the rat. Characterization with biochemical and enzyme-histochemical methods.

The postnatal development, between 0 and 90 days, of three hindlimb muscles and diaphragm of the rat was investigated with respect to fiber types and diameter (histochemistry) and substrate oxidation rates and enzyme activities (biochemistry). The process of muscle fiber differentiation into mature patterns was evaluated by visual classification into 3 or 4 groups having different staining intensities for 3 enzyme-histochemical reactions, enabling 26 fiber types to be distinguished. These exhibited specific sizes and growth rates that varied among the muscles. One of the hindleg muscles (flexor digitorum brevis) remained much more immature than soleus and extensor digitorum longus. The histochemical and biochemical findings correlated well. The capacity for pyruvate and palmitate oxidation, and the activities of cytochrome c oxidase and citrate synthase, increased markedly between 9 and 37 days in soleus and extensor digitorum longus (except citrate synthase in the latter) but not in flexor digitorum brevis. Creatine kinase activity increased in all hindlimb muscles. Both the capacity and the activity of pyruvate oxidation (determined in homogenates and intact isolated muscles, respectively), were in accordance with the fiber type composition. In contrast to oxidation capacity, the activity of pyruvate oxidation decreased after birth until the mature stage, when a value of 18-42% of that of early postnatal muscles was recorded.

Postnatal growth and differentiation of muscle fibres in the mouse. II. A histochemical and morphometrical investigation of dystrophic muscle.

Postnatal development of three hind legs muscles, the soleus, plantaris, and gastrocnemius, of dystrophic mice (ReJ 129) was investigated with histochemical and morphometric methods. The results were compared with normal postnatal development. Especially during the second week postnatally, there was severe fibre necrosis with no apparent preference for any particular fibre type. This period of necrosis was shortly followed by a wave or regeneration during the third week that could not, however, compensate for the loss of fibres. In dystrophic animals of 4-5 months of age, the number of fibres was reduced by 40-70%. Cross sectional areas of dystrophic muscles rarely, if ever, exceeded values for normal animals 14 days of age, while body weights were also drastically reduced. Growth and differentiation of the nonaffected fibres proceeded almost normally during the first month. During the second month, the "slow' fibres in the soleus muscle, and the "fast-oxidative-glycolytic' fibres in the plantaris muscle were hypertrophied, while, incidentally, some "fast-glycolytic' fibres showed hypertrophy; but in this case the average size of the fibre type was not increased. After two months, a general fibre atrophy was observed. The fate of the regenerated fibres was difficult to trace, especially in muscles older than one month. It is assumed that a number of them were capable of developing into "adult' fibre types histochemically. During the course of the disease the percentage of "intermediate' fibres increased markedly, whereas nearly all "fast-glycolytic' fibres disappeared. Because of these shifts in fibre profiles, the plantaris and the gastrocnemius muscles obtained a rather "juvenile' and "oxidative' aspect. Changes in the histochemical character of the soleus muscle were less spectacular. In dystrophic muscles, no new fibre types were found, compared with normal muscles. Rather, fibre types were present at the wrong moment, or occurred in quantities unusual for the age concerned.

Postnatal growth and differentiation of muscle fibres in the mouse. I. A histochemical and morphometrical investigation of normal muscle.

A histochemical classification of muscle fibres based on three enzymes (ATPase, pre-inc. pH 4:35; succinic dehydrogenase and alpha-glycerol-phosphate-dehydrogenase) was used to describe postnatal growth and differentiation of muscle fibres. The m. soleus, m. plantaris and m. gastrocnemius were examined in normal mice from birth to the young adult stage. At birth, differentiation of the gastrocnemius muscle was in a more advanced stage than that of the plantaris and the soleus muscles, while the last of these was the least developed. During growth, as well as in the (young) adult, there was a distinct relation between fibre type and size, which, however, differed per muscle (region). The development of muscle fibres was a gradual process, rather than a succession of distinct stages, although a change in fibre type was often accompanied by a change in size. Differentiation of fibres already occurred perinatally, and the "adult fast' fibre types appeared during the second week postnatally, varying with the muscle region. During development, a percentage of fibres remained as a population that was histochemically and morphometrically intermediate between the fast-oxidative-glycolytic and fast-glycolytic adult fibres. A model is presented in which the most probable pathways of development are depicted.

Inhibition of the expression of pathology in dystrophic mouse leg muscles by immobilization.

Normal and dystrophic calf muscles of young mice of the Bar Harbor strain Re 129 were immobilized with the foot in an extended position. Two weeks after treatment the muscles were examined morphologically. Immobilization resulted in the inhibition of fibre necrosis and, consequently, of regeneration. In the contralateral leg these processes progressed unhampered and gave rise to typical dystrophic features. As a result of treatment there was some muscle fibre atrophy. The results clearly indicate that injury of muscle fibres is of a true "myogenic" nature, and that mechanical, i.e. contractive, activity is an important factor inducing damage to the sarcolemma of dystrophic muscle fibres.