Correlative Imaging of the Murine Hind Limb Vasculature and Muscle Tissue by MicroCT and Light Microscopy

A detailed vascular visualization and adequate quantification is essential for the proper assessment of novel angiomodulating strategies. Here, we introduce an ex vivo micro-computed tomography (microCT)-based imaging approach for the 3D visualization of the entire vasculature down to the capillary level and rapid estimation of the vascular volume and vessel size distribution. After perfusion with μAngiofil®, a novel polymerizing contrast agent, low- and high-resolution scans (voxel side length: 2.58-0.66 μm) of the entire vasculature were acquired. Based on the microCT data, sites of interest were defined and samples further processed for correlative morphology. The solidified, autofluorescent μAngiofil® remained in the vasculature and allowed co-registering of the histological sections with the corresponding microCT-stack. The perfusion efficiency of μAngiofil® was validated based on lectin-stained histological sections: 98 ± 0.5% of the blood vessels were μAngiofil®-positive, whereas 93 ± 2.6% were lectin-positive. By applying this approach we analyzed the angiogenesis induced by the cell-based delivery of a controlled VEGF dose. Vascular density increased by 426% mainly through the augmentation of medium-sized vessels (20-40 μm). The introduced correlative and quantitative imaging approach is highly reproducible and allows a detailed 3D characterization of the vasculature and muscle tissue. Combined with histology, a broad range of complementary structural information can be obtained.