Regeneration and innervation in cultures of adult mammalian skeletal muscle coupled with fetal rodent spinal cord

Abstract

In a collagen-substrate culture, long strips of teased adult skeletal muscle fibers oriented toward ventral-root nerve fibers of fetal rodent spinal cord explant will regenerate rapidly as the neural outgrowth makes contact with the muscle. Trophic enhancement of the early regenerative capacity of muscle is relatively nonspecific and can also be produced by contacts with a variety of nonneural fetal cells. Myotubes of neurally deprived muscle can regenerate, show fibrillatory contractions, and develop transient cross striations at their peak of development in vitro, but soon afterwards, as in vivo, they atrophy. Further differentiation, maturation, and long-term maintenance require innervation, in vitro as well as in vivo. In culture this is dependent upon the organotypic development of both the central and peripheral nerve network of the spinal cord complex. Although electrophysiologic studies show that neuromuscular transmission can occur early in the second week after coupling, the junctional structures are primitive and definitive loci of cholinesterase activity are not discernable (at the light microscopic level) until about a week later. During the following weeks in culture gradual maturation of the motor endplate structure occurs, including increased complexity of nerve terminals and postsynaptic specializations, as illustrated by silver impregnation, cholinesterase staining, and other histologic techniques. The ordered progression of skeletal muscle regeneration and maturation through spinal cord innervation in vitro is quite comparable to basic aspects of regeneration in vivo. Organotypic cultures provide, therefore, a reliable model system for studies of some of the mechanisms underlying muscle regeneration and the restoration of functional neuromuscular relations which may be difficult to analyze in situ.