To stage and quantify regenerative myogenesis in FES-Induced Functional Recovery of Human Long-Term Permanent Dener- vated Muscle

Following denervation, skeletal muscle undergoes rapid loss in both mass and contractile force, with an accompanying series of changes in structure, biochemistry and physiology. Morphologic features of the long-term denervated muscle suggest that the original fibers are lost and those seen are the results of repeated cycles of cell death and regeneration. Markers of myogenic events in adult muscles are activated satellite cells, presence of embryonic myosin and myogenic transcription factors. Electrical stimulation of permanent denervated muscle increases the mean size of the myofibers, maintains the sarcomeres and possibly prevents apoptosis/necrosis and secondary degeneration. We here describe histological, immunohistochemical and molecular methods, which identify markers of damage, regeneration, and repair on a muscle sample of just a few milligrams. A suitable source is a small biopsy, whose cryostat sections allow fiber typing and histopathology by immunochemical analyses. A few additional serial cryostat sections are used for molecular analyses. By light morphometry percent fat and/or interstitial tissue area, and fiber size distribution are determined. Myosin ATPases and immunohistochemistry are used to quantify fiber types, including early or late regenerated myofibers. By electron microscopy we identify abnormalities of sarcomeric organization, nuclear distribution, mitochondrial content, and activated satellite cells. Myosin heavy chain profiles are indicators of regenerative events and of muscle adaptation to changing demands. Total protein content, myosin: total protein and myosin: actin ratios are determined by SDS-PAGE to reliably complement or substitute morphometry in quantifying muscle trophism in skeletal muscle biopsies. Taken together the results of morphometry and molecular analyses solidly demonstrate that two-year-long FES substantially reverses the severe atrophy expected in four-year denervated human muscles, and that generative/regenerative myogenic events significantly contribute to the functional recovery of long-term paralyzed human muscles.