New blood vessels can be induced to invade ischemic skeletal muscle

Despite intense investigation of angiogenesis, little effort has been made to exploit this phenomenon in ischemic tissue. The few studies on this topic have focused primarily on the development of collateral arteries in existing arterial beds. A previous study showed that a perfused muscle flap transposed to an ischemic limb formed vascular connections, which were demonstrated with angiography, between the arteries of the flap and the limb vasculature. Microsphere studies suggested that these vascular connections increased the resting perfusion of the ischemic limb. This study is designed to confirm histologically that such connections develop and to quantitate the number and dimensions of these new vessels.

Through a midline laparotomy, the right common iliac artery was ligated and divided in 18 male New Zealand white rabbits. An abdominal-wall muscle flap based on the left inferior epigastric artery was transposed to the right thigh. On the seventh day, contrast dye was injected into the flap artery of eight rabbits and an arteriogram was obtained. The tissue of the remaining rabbits was perfusion-fixed at 3 days (n = 2), 7 days (n = 4), and 14 days (n = 4). Thin sections of the flap-thigh muscle interface were stained with hematoxylin and eosin and for alpha-actin and proliferating cell nuclear antigen and were examined microscopically.

An arteriogram confirmed vascular connections between the flap and the native limb circulation in seven of the eight rabbits. Histologic evaluation of the flap-thigh muscle interface showed no new vessels on the third day. On the seventh day, 6.8 +/- 4.8 new vessels (positive alpha-actin staining, red blood cells in lumen) were seen per 40 x field; the vessels averaged 10.2 +/- 5.2 microns in diameter. On the fourteenth day, there were 7.3 +/- 3.8 vessels per 40 x field (p = 0.46), but the vessel diameter increased to 20.7 +/- 10.6 microns (p = 0.013). Proliferating cell nuclear antigen staining confirmed that these were proliferating vessels.

Within seven days, new vessels that were more mature than capillaries (stained for alpha-actin, a smooth muscle cell protein) formed between the flap and the thigh muscle. These new connecting vessels continue to enlarge in diameter between 7 and 14 days, but the stimulus to form new vessels appeared to decline or disappear before the fourteenth day. Attempts to sustain this phenomenon with angiogenic factors are underway.