A method for the chemical isolation of individual muscle fibers and its application to a study of the effect of denervation on the number of nuclei per muscle fiber

Abstract

A method is described for isolating intact, individual skeletal muscle fibers from glutaraldehyde fixed muscle. This method was conceived to climinate the many limitations of determining muscle nuclear numbers in histological cross section. Glutaraldehyde fixed fibers are isolated by dissection while in a solution of low concentration guanidine in a borate buffer at high pH. Electron miscroscopy demonstrates that single fibers, isolated in this manner, are free of their microvasculature and connective tissue. Their basal laminas and the structures within them, including their satellite cells, are preserved. This method is employed to determine whether changes in nuclear numbers occur within the basal lamina of individual muscle cells from 1 to 28 days following denervation of mouse gastrocnemius muscle. The total number of nuclei located within the basal lamina of individual muscle fibers (i.e. muscle and satellite cell nuclei) does not change after denervation. This rules out the possibility that additional nuclei are arising from an influx of cells outside the basal lamina or by mitotic division of nuclei within the basal lamina. However, the possibility of a change in the ratio of satellite cell nuclei, to muscle cell nuclei, is not eliminated. Other possible applications of this isolation method are discussed.