INCREASED EXPRESSION OF DIRECT GENE-TRANSFER INTO SKELETAL-MUSCLES OBSERVED AFTER ACUTE ISCHEMIC-INJURY IN RATS

The direct injection of plasmid DNA into skeletal muscles represents a novel strategy that is potentially applicable to lower limb ischemic diseases. Most previous studies that involved skeletal muscle gene tra nsfer have used only normal animals, however, and the efficiency of ge ne transfer into the ischemic muscles has not yet been well characteri zed. Accordingly, we sought to determine the extent to which gene expr ession is altered by performing skeletal muscle transfection under isc hemic conditions. The femoral artery was ligated in one limb to induce limb ischemia in rats. The rectus femoris muscle of the ipsilateral l imb was transfected 0 to 14 days later with the plasmid pRSVLUC, which contains the firefly luciferase coding sequence. Muscles of the contr alateral nonischemic limb also were transfected in an identical fashio n to serve as controls. At the end of the study, the rectus femoris mu scle of the ischemic limb showed a significant reduction in weight com pared with the controls (0.99 +/- 0.02 mg vs 1.07 +/- 0.03 mg, p < 0.0 001), which demonstrates that the ligation of the femoral artery creat ed significant limb ischemia in this animal model. Luciferase expressi on was readily detected in all 98 transfected limb muscles from 49 rat s but not in nontransfected muscles or other organs. The relative luci ferase activity (ischemic limb to nonischemic limb) calculated for eac h rat was 1.64 +/- 0.49 at Day 0. It significantly increased after Day 4 (3.76 +/- 1.33), reached its peak at Day 7 (9.00 +/- 3.38, p < 0.05 ), and declined to the base-line levels by Day 14 (1.44 +/- 0.43). The se in vivo results indicate that gene expression after skeletal muscle transfection is significantly augmented by transfecting genes under i schemic conditions, which may have potential implications to increase the efficacy of gene therapy for lower limb vascular occlusive disease .