Effect of the association of cell therapy and low-intensity training on the dystrophic muscle of mdx mice

Author(s)

Priscilla Avelino Ferreira Pinto

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2022-05-24

Examining board members

Carlos Eduardo Ambrosio; Fabiana Fernandes Bressan; Adriano Polican Ciena; Thaís Peixoto Gaiad Machado; Atanásio Serafim Vidane

• Advisor: Carlos Eduardo Ambrosio

Abstract

Duchenne Muscular Dystrophy (DMD) is a genetic disorder, still incurable, that leads to progressive muscle degeneration and chronic inflammatory process. Cell therapy (CT) has been performed; however, cell viability has been limited by the presence of muscle fibrosis and inflammatory process. Low-intensity training (LIT) has been shown to reduce inflammatory areas and intramuscular fibrosis in the muscle of mdx mice. Thus, the objective was to evaluate the effect of LIT associated with CT with mesenchymal stem cells (MSC) in the dystrophic muscle of mdx mice. MSCs were obtained from the canine yolk sac, cultured, and characterized by growth curve, cell viability, colony-forming unit test, and immunophenotypic characterization by flow cytometry. Thirty-two male mdx mice were randomly divided into 4 groups: mdxT+CT group trained mdx mice that received MSC; mdxT group trained mdx mice; mdxS+CT group sedentary mdx mice that received MSC; and mdxS group sedentary mdx mice. A control group with wild-type mice (WT group) was also performed, without any intervention. The training protocol was performed on a horizontal treadmill at 9m/min, 2x/week, 30min/day, for 8 weeks. CT was performed after 4 weeks of training, intramuscularly in the quadriceps femoris muscle (QD). In vivo functional tests were performed. Morphometric analysis of the percentage of fibers with central core and percentage of the area of intramuscular fibrosis of the QD, cranial tibial, gastrocnemius, and diaphragm muscles were performed. In addition, we also analyzed the expression of inflammatory cytokines IL1, IL4, IL6 and TNFα and markers related to muscle regeneration IGFβ, TGFβ and MyoD by real-time polymerase chain reaction (RT-qPCR). No statistical difference was found between the dystrophic groups for the functional tests, being statistically different from the WT group. There was no statistical difference between the dystrophic groups regarding the percentage of fibers with central core, being statistically different from the WT group. As for the percentage of area of intramuscular fibrosis, the mdxT+CT group had a smaller area compared to the mdxS group in the QD muscle, and a smaller area than the mdxT group in the gastrocnemius muscle. However, the trained groups had a larger area compared to the sedentary groups in the cranial tibialis muscle. There was no difference in the percentage of fibrosis area of the dystrophic groups in the diaphragm. Based on the results found, the proposed therapeutic association was able to reduce the percentage of intramuscular fibrosis area in the QD muscle compared to the sedentary group, which did not receive MSCs, in addition to having a smaller area of fibrosis than the trained group. without CT in the gastrocnemius muscle, showing a possible paracrine effect of MSCs in the muscle close to the application. In addition, the trained animals that received cell therapy showed no statistical difference from the group that received cell therapy and remained sedentary, supporting that the association of low-intensity physical training was not able to negatively alter the effect of cell therapy. (AU)

• FAPESP's process: 18/11752-7 - Effect of association of cellular therapy and low-intensity training on MDX mouse dystrophic muscle
• Grantee: Priscilla Avelino Ferreira Pinto
• Support Opportunities: Scholarships in Brazil - Doctorate