Laser microdissection-based expression analysis of key genes involved in muscle regeneration in mdx mice

We have used the mdx mice strain (C57BL/10ScSn-mdx) as an experimental subject for the study of reiterative skeletal muscle necrosis–regeneration with basement membrane preservation. In young mdx muscle, by means of Hematoxylin–Eosin staining, different types of degenerative–regenerative groups (DRG) can be recognized and assigned to a defined muscle regeneration phase. To evaluate the expression of known key-regulatory genes in muscle regeneration, we have applied Laser Capture Microdissection technique to obtain tissue from different DRGs encompassing the complete skeletal muscle regenerative process. The expression of MyoD, Myf-5 and Myogenin showed a rapid increase in the first two days post-necrosis, which were followed by MRF4 expression, when newly regenerating fibers started to appear (3–5 days post-necrosis). MHCd mRNA levels, undetectable in mature non-injured fibers, increased progressively from the first day post-necrosis and reached its maximum level of expression in DRGs showing basophilic regenerating fibers. TGFβ-1 mRNA expression showed a prompt and strong increase following fiber necrosis that persisted during the inflammatory phase, and progressively decreased when new regenerating fibers began to appear. In contrast, IGF-2 mRNA expression decreased during the first days post-necrosis but was followed by a progressive rise in its expression coinciding with the appearance of the newly formed myofibers, reaching the maximum expression levels in DRGs composed of medium caliber basophilic regenerating myofibers (5–7 days post-necrosis). mdx degenerative–regenerative group typing, in conjunction with laser microdissection-based gene expression analysis, opens up a new approach to the molecular study of skeletal muscle regeneration.