BACKGROUND: In vivo gene electrotransfer is frequently used in preclinical gene therapy. Many studies have attempted to optimize protocols efficiency at the same time as reducing muscle damage. Most of them have reported histological evidence of muscle degeneration and completion of regeneration within 15 days. The functional consequences have rarely been addressed, which may reflect the lack of appropriate techniques. Yet, it is important to characterize the changes induced by the procedure itself because it may interfere with therapy. We used multiparametric functional (mpf)-nuclear magnetic resonance (NMR) imaging to evaluate mice hindlimb muscle after electrotransfer of an empty plasmid. METHODS: NMR experiments were performed in a 4T Bruker magnet. Arterial spin labeling imaging of perfusion and blood oxygenation level dependent contrast and (31) P spectroscopy of phosphocreatine kinetics and pH were simultaneously acquired from the mice hindlimb during 2 min of electrically stimulated exercise and recovery. RESULTS: After 15 days, hindlimb cross-sectional area decreased by 10% compared to control mice. Specific force-time integral and end-exercise pH were identical in both groups, whereas oxidative capacities increased. Perfusion values doubled, and oxygenation significantly decreased. Histology revealed: (i) degeneration/regeneration; (ii) a decrease in type IIb fibers and an increase in type I and IIa fibers; and (iii) increased capillary density. CONCLUSIONS: In this model, loss in muscle mass was accompanied by important alterations of perfusion and bioenergetics. Fifteen days after electrotransfer, this was correlated with fiber type shift, capillary bed remodeling and degeneration/regeneration. mpf-NMR provides new insights into the functional consequences of standard electrotransfer and represents a powerful tool for optimization and longitudinal assessment of preclinical gene therapy protocols.
BACKGROUND: In vivo gene electrotransfer is frequently used in preclinical gene therapy. Many studies have attempted to optimize protocols efficiency at the same time as reducing muscle damage. Most of them have reported histological evidence of muscle degeneration and completion of regeneration within 15 days. The functional consequences have rarely been addressed, which may reflect the lack of appropriate techniques. Yet, it is important to characterize the changes induced by the procedure itself because it may interfere with therapy. We used multiparametric functional (mpf)-nuclear magnetic resonance (NMR) imaging to evaluate mice hindlimb muscle after electrotransfer of an empty plasmid. METHODS: NMR experiments were performed in a 4T Bruker magnet. Arterial spin labeling imaging of perfusion and blood oxygenation level dependent contrast and (31) P spectroscopy of phosphocreatine kinetics and pH were simultaneously acquired from the mice hindlimb during 2 min of electrically stimulated exercise and recovery. RESULTS: After 15 days, hindlimb cross-sectional area decreased by 10% compared to control mice. Specific force-time integral and end-exercise pH were identical in both groups, whereas oxidative capacities increased. Perfusion values doubled, and oxygenation significantly decreased. Histology revealed: (i) degeneration/regeneration; (ii) a decrease in type IIb fibers and an increase in type I and IIa fibers; and (iii) increased capillary density. CONCLUSIONS: In this model, loss in muscle mass was accompanied by important alterations of perfusion and bioenergetics. Fifteen days after electrotransfer, this was correlated with fiber type shift, capillary bed remodeling and degeneration/regeneration. mpf-NMR provides new insights into the functional consequences of standard electrotransfer and represents a powerful tool for optimization and longitudinal assessment of preclinical gene therapy protocols.
Authors: Aurea B Martins-Bach; Damien Bachasson; Ericky C A Araujo; Lucas Soustelle; Paulo Loureiro de Sousa; Yves Fromes; Pierre G Carlier Journal: Sci Rep Date: 2021-01-11 Impact factor: 4.379