To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30- microm-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased sampling design. To determine if there are age-related differences in muscle fiber types, morphometric characteristics of muscle fibers were examined on thin sections stained for myofibrillar ATPase. Autopsy samples of vastus lateralis muscle from six young (28.7 +/- 2.3 years) and six old (70.8 +/- 1.3 years) persons who had suffered sudden death were analyzed. Numbers of satellite cells per fiber length (Nsc/Lfib) and number of satellite cells per total number of nuclei (satellite cell nuclei + myonuclei) (Nsc/Nnucl) were significantly lower in the old group (p < 0.05). We demonstrate the importance of proper sampling and counting in estimation of sparsely distributed structures such as satellite cells. Our results support the hypothesis that the satellite cell fraction declines during aging.

Human skeletal muscle stem cells from healthy donors aged 2-82 years (n = 13) and from three children suffering from Duchenne Muscular Dystrophy (DMD) were implanted into soleus muscles of immunoincompetent mice and were also expanded in vitro until senescence. Growth of implanted cells was quantified by structural features and by the amount of human DNA present in a muscle. Proliferative capacity in vitro and in vivo was inversely related to age of the donor. In vitro, a decline of about two mean population doublings (MPDs) per 10 years of donor's age was observed. Muscle stem cells from DMD children were prematurely aged. In general, cell preparations with low or decreasing content in desmin-positive cells produced more MPDs than age-matched high-desmin preparations and upon implantation more human DNA and more nonmyogenic than myogenic tissue. Thus, a "Desmin Factor" was derived which predicts "quality" of the human muscle tissue growing in vivo. This factor may serve as a prognostic tool.

The aim of this study was to determine whether capillarity in the denervated and reinnervated rat extensor digitorum longus muscle (EDL) is scaled by muscle fiber oxidative potential. We visualized capillaries adjacent to a metabolically defined fiber type and estimated capillarity of fibers with very high oxidative potential (O) vs fibers with very low oxidative potential (G). Capillaries and muscle fiber types were shown by a combined triple immunofluorescent technique and the histochemical method for NADH-tetrazolium reductase. Stacks of images were captured by a confocal microscope. Applying the Ellipse program, fibers were outlined, and the diameter, perimeter, cross-sectional area, length, surface area, and volume within the stack were calculated for both fiber types. Using the Tracer plug-in module, capillaries were traced within the three-dimensional (3D) volume, the length of capillaries adjacent to individual muscle fibers was measured, and the capillary length per fiber length (Lcap/Lfib), surface area (Lcap/Sfib), and volume (Lcap/Vfib) were calculated. Furthermore, capillaries and fibers of both types were visualized in 3D. In all experimental groups, O and G fibers significantly differed in girth, Lcap/Sfib, and Lcap/Vfib, but not in Lcap/Lfib. We conclude that capillarity in the EDL is scaled by muscle fiber size and not by muscle fiber oxidative potential.

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

A total of 106 fascicles at 6 predetermined areas of the vastus lateralis muscle from 9 healthy men, aged 18 to 40 years, were analyzed. Fibers in a fascicle were divided into layers according to their relation to the perimysium. In each layer the proportions of type 1 and 2, subdivided into 2a, 2b, and 2c fibers were determined and normalized by the fiber type proportion in the whole fascicle. A consistent arrangement of fiber types within the fascicles was obtained, regardless of subject, sampling site, fiber type proportion, and fascicle size. A high proportion of 2b fibers on the border, a prevalence of type 1 fibers in the layer beneath, and a rather uniform distribution of 2a fibers in all layers are the main characteristics of the distribution of fibers in a fascicle. Developmental processes in fiber type differentiation most probably constitute the basis for fiber type arrangement, which can further be influenced by local factors.