GaAs 904-nm laser irradiation improves myofiber mass recovery during regeneration of skeletal muscle previously damaged by crotoxin

This work investigated the effect of gallium arsenide (GaAs) irradiation (power: 5 mW; intensity: 77.14 mW/cm(2), spot: 0.07 cm(2)) on regenerating skeletal muscles damaged by crotoxin (CTX). Male C57Bl6 mice were divided into six groups (n = 5 each): control, treated only with laser at doses of 1.5 J or 3 J, CTX-injured and, CTX-injured and treated with laser at doses of 1.5 J or 3 J. The injured groups received a CTX injection into the tibialis anterior (TA) muscle. After 3 days, TA muscles were submitted to GaAs irradiation at doses of 1.5 or 3 J (once a day, during 5 days) and were killed on the eighth day. Muscle histological sections were stained with hematoxylin and eosin (H&E) in order to determine the myofiber cross-sectional area (CSA), the previously injured muscle area (PIMA) and the area density of connective tissue. The gene expression of MyoD and myogenin was detected by real-time PCR. GaAs laser at a dose of 3 J, but not 1.5 J, significantly increased the CSA of regenerating myofibers and reduced the PIMA and the area density of intramuscular connective tissue of CTX-injured muscles. MyoD gene expression increased in the injured group treated with GaAs laser at a dose of 1.5 J. The CTX-injured, 3-J GaAs laser-treated, and the CTX-injured and treated with 3-J laser groups showed an increase in myogenin gene expression when compared to the control group. Our results suggest that GaAs laser treatment at a dose of 3 J improves skeletal muscle regeneration by accelerating the recovery of myofiber mass.

Differential Expression of Genes Involved in the Degeneration and Regeneration Pathways in Mouse Models for Muscular Dystrophies

The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmd (mdx) , SJL/J, Large (myd) and Lama2 (dy2J) /J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-beta 1 and Pro-collagen 1 alpha 2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-beta 1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle.

Local Injections of Adipose-Derived Mesenchymal Stem Cells Modulate Inflammation and Increase Angiogenesis Ameliorating the Dystrophic Phenotype in Dystrophin-Deficient Skeletal Muscle

The effects of adipose-derived mesenchymal stem cells (ADMSC) transplantation on degeneration, regeneration and skeletal muscle function were investigated in dystrophin-deficient mice (24-week-old). ADMSC transplantation improved muscle strength and, resistance to fatigue. An increase in fiber cross-sectional area and in the number of fibers with centralized nuclei and augment of myogenin content were observed. In ADMSC-treated muscles a decrease in muscle content of TNF-alpha, IL-6 and oxidative stress measured by Amplex(A (R)) reagent were observed. The level of TGF-beta 1 was lowered whereas that of VEGF, IL-10 and IL-4 were increased by ADMSC treatment. An increase in markers of macrophage M1 (CD11 and F4-80) and a decrease in T lymphocyte marker (CD3) and arginase-1 were also observed in ADMSCs-treated dystrophic muscle. No change was observed in iNOS expression. Increased phosphorylation of Akt, p70S6k and 4E-BP1 was found in dystrophic muscles treated with ADMSC. These results suggest that ADMSC transplantation modulates inflammation and improves muscle tissue regeneration, ameliorating the dystrophic phenotype in dystrophin-deficient mice.