Abstract 19423: Rodent Hind Limb Collaterals Develop as Intertwined Strands Accompanied by Veins

Abstract

Background: The process of angiogenesis and collateral remodeling in response to rodent hind limb ischemia has been intensely studied at micro- as well as at macroscopic level. Yet, a detailed investigation of the three-dimensional (3D) collateral vessel architecture in this model is lacking. Methods: To stimulate collateral growth in the rat hind limb the femoral artery was ligated. After sacrifice at day 3, 7 or 28, the systemic circulation was flushed via the cannulated abdominal aorta, the vasculature was filled with fluorescent cast material, and the frozen limbs were embedded in black gel. Registered stacks of episcopic fluorescence images, acquired with a custom-built imaging cryomicrotome (24 μm 3 voxel resolution). High-resolution 3D-renderings of the vascular cast were obtained after deblurring. Multiple collateral arteries and veins were identified between the muscular branch of the femoral and the saphenous artery and vein and diameters were assessed in four regions (near the stem and re-entry, at the mid-zone, and just proximal to the re-union of individual collaterals) using a semi-automatic approach based on vesselness filtering. Results: The gracilis collaterals form a complex network of intertwined vessels with highly individual architecture. Both pre-existing and new small collateral loops were commonly bundled as pairs of artery and vein. In early arteriogenesis, these bundles exhibited a multitude of small sprouting side branches. Arterial diameter increased with ischemia duration from 108 μm in healthy to 130 μm in 7-days post-ligation legs and 182 μm in 28-days post-FAL limbs (p<0.01) whereas collateral vein remained unchanged. Rather than an increasing number of long bridging collateral arteries over time, we noticed an increased arcade formation of paired arteries and veins. Conclusions: We examined 4 stages of neovascularization in 3D in high detail. Initial sprouting gave way to formation of intertwined collateral strands accompanied by veins. The proximity of collateral arteries to veins calls for investigation of the role of veins during arteriogenesis.