Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

Abstract

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10 6 cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodaminelabeled α-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213), the diameter of ectopic fibers (59.1 µm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

The lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes

“…After fixing in corks, muscles were immediately frozen in liquid nitrogen and stored at Ϫ80°C. To determine muscle fiber diameter, cryostat cross sections (5 m) of the lumbrical and EDL muscles were stained with hema-toxylin and eosin (H & E), as described by Fukushima et al (16). Images of stained myofibers (7 fields/section) were acquired under light microscope (Nikon Eclipse E800, Melville, NY; ϫ40 objective), analyzed with HL Image 97 software (Western Vision Software, Layton, UT), and processed with Adobe Photoshop CS 4.0 (Adobe Systems, Mountain View, CA).…”

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

“…Quantitative analysis of nuclear domains. Skeletal muscles were fixed overnight with 4% formaldehyde at 4°C, washed with PBS, pH 7.4, perfused, and then incubated for 1 h with a 5 g/ml Hoechst dye 33258 (Invitrogen, Carlsbad, CA) solution on DMEM containing 10% horse serum for nuclear staining (19). Then muscles were washed with DMEM and PBS, pH 7.4, and dissected under a stereoscope by separation of progressively thinner bundles of fibers.…”

Increased expression of acetylcholine receptors in the diaphragm muscle of mdx mice

“…For nAChR labeling the dissected muscles were rinsed for 10 min with Dulbecco's Minimum Essential Medium (DMEM, Gibco‐BRL, Life Technologies, Grand Island, New York) containing 10% horse serum (DMEM/HS) and incubated for 1 h in α‐bungarotoxin conjugated to tetramethylrhodamine (TRICT‐α‐BGT, Molecular Probes, Eugene, Oregon; 1 μg/ml) at room temperature 17. Muscles were then washed with DMEM/HS followed by PBS, and fixed in 4% paraformaldehyde in PBS at 4°C.…”