RESUMEN
OBJECTIVE: The aim of this study was to establish three-dimensional cultures originating from muscle biopsies and evaluate the viability and morphology. METHOD: Muscle biopsies from patients with suspected neuromuscular disorders were obtained and established as primary muscle tissue cultures. Tissue pieces, 1-2 mm of diameters, were placed in culture medium and subjected to sporadic stirring to prevent attachment and outgrowth as monolayer cells. Morphology and ability to attach to the surface were investigated by light microscopy. Viability was evaluated by (99m)Tc-tetrofosmin uptake. After 1 month, histology was evaluated by light microscopy and immunocytochemistry. The findings of a healthy muscle and a dystrophic muscle were compared. RESULTS: Initially, the tissue pieces were unshaped but formed spheroid-like structures during the culture period. For dystrophic muscle, attachment capacity to the surface was initially potent and decreased during the culture period, whereas control muscle showed weak attachment from the start that increased during the culture period. The uptake of (99m)Tc-tetrofosmin increased in control muscle, while it decreased in dystrophic muscle, during the culture period. The histological investigation demonstrated larger destruction of myofiber, weaker satellite cell activation and reduced myofiber regeneration in the dystrophic muscle as compared to the control muscle. CONCLUSION: The cellular components of the muscle tissue can survive and proliferate as spheroid-like primary cultures. The cellular composition resembles the in vivo condition, which allows studies of degeneration of the original fibers, and activation and proliferation of the satellite cells. The culture system may provide better understanding of the degeneration and regeneration processes in different muscle disorders and allow investigations of pharmacological interventions.
Asunto(s)
Técnicas de Cultivo de Célula , Músculo Esquelético/patología , Enfermedades Neuromusculares/patología , Adulto , Biopsia/métodos , Células Cultivadas , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Enfermedades Neuromusculares/diagnóstico , Compuestos Organofosforados/farmacocinética , Compuestos de Organotecnecio/farmacocinética , Radiofármacos/farmacocinética , Factores de TiempoRESUMEN
The aim of this study was to elucidate the mechanisms of action for potential targets of therapeutic intervention related to the arachidonic acid cascade in muscular dystrophy. Primary cultures from a Duchenne patient were used to study the expression of dystrophin-1, utrophin, desmin, neonatal myosin heavy chain (MHCn) and Bcl-2 during inhibition of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX). Hypo-osmotic treatment was applied in order to trigger Ca2+ influx and PLA2 activity. Inhibition of PLA2 and LOX with prednisolone and nordihydroguaiaretic acid (NDGA) caused a semi-quantitative increase of utrophin and Bcl-2-, and a dose-dependent, quantitative increase of desmin expression, an effect that was augmented by hypo-osmotic treatment. Our results indicate that LOX inhibitors, similarly to corticosteroids, can be beneficial in the treatment of muscular dystrophies.