Résumé :
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In vivo gene electrotransfer (ET) is often used in preclinical gene therapy studies. Many studies attempted to optimize ET protocols to increase efficiency while reducing muscle damage. Most of them reported histological evidences of muscle degeneration and completion of subsequent regeneration within 15 days [1,2]. The functional consequences of this process have rarely been addressed [3]. In this work, we combined NMR imaging and spectroscopy [4] to quantitatively and non-invasively investigate long-term changes in mouse leg muscle function after ET of an empty plasmid, by measuring perfusion, oxygenation (BOLD), bioenergetics and force simultaneously. Both legs of 9 FVB mice were injected with an empty plasmid solution (Roche, pM1). ET was performed using plate electrodes (8 pulses 20 ms, 200V/cm, 2 Hz, [5]). Ten mice served as controls (Ctr). Mpf-NMR was performed 15 days after ET, in a 4T Bruker magnet. The exercise consisted of isometric 0.5 s contractions every 2.5 s. Arterial spin labeling perfusion imaging and 31P spectroscopy were interleaved during exercise (2 min) and recovery (10 min). The time constant for PCr recovery (tPCr) was used as an index of oxidative capacities. Gastrocnemius muscles were taken for fiber typing and capillary density assessment. Muscle size was decreased by 11% after ET. During exercise, neither specific force-time integral nor end-exercise pH differed, while tPCr was shortened in pM1 indicating increased oxidative capacities (Fig.1). In parallel, perfusion was almost doubled in pM1, and capillary oxygenation significantly decreased as shown by a more pronounced negative BOLD (Fig1B). Histology revealed numerous fibers with central nuclei in pM1, an increase in the proportion of type I and type IIa fibers, and in capillary density.In this standard model of ET, important alterations of both vascular and bioenergetics dynamics were shown. After 15 days, a time considered sufficient for recovery [1,2,3], these changes paralleled a shift toward oxidative fibers and a remodeling of the capillary network. Our mpf-NMR protocol provided a new insight into the functional consequences of ET and constitutes a powerful tool for the optimization of ET protocols and more globally the longitudinal assessment of preclinical gene therapy.[1]Mathiesen, Gene Therapy 1999 [2]Hartikka, et al. Mol Therapy 2001 [3] Schertzer et al. Molecular Therapy 2005 [4] Baligand et al. NMR in Biomed 2010 [5]Bloquel, et al. J Gene Med 2004.
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