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

Abstract

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.

Intussusceptive Pillar Formation in Developing Porcine Glomeruli

“…However, a very promising correlative and quantitative technique has been recently developed, which could be used in the setup of the porcine metanephros in order to study IA. This microangio-CT-based imaging approach for the 3D visualization of the entire vasculature down to the capillary level has already been used in the murine hind limb [38] and kidney [39]. This technique allows for the definition of sites of interest within the tissue which can then be further analyzed with optical or electron microscopy.…”

Ex vivo microangioCT: Advances in microvascular imaging

“…In summary, the entire field of vascular biology has a considerable demand for new and trustworthy high-resolution vascular 3D imaging, which would allow rapid visualizing and quantification of the microvasculature [8].…”

“…(see above). Until recently, visualizing capillaries in situ by microCT, without prior tissue maceration, seemed unachievable [8,[17][18][19][20].…”

“…Once the skeleton is obtained, a more detailed analysis of the vasculature can be performed. Not only the hierarchy of the vascular tree, but also the parameters like connectivity, tortuosity, changes in vascular pattern can be therefore analyzed [8]. The analysis of whole vascular networks and parts thereof, down to "microvascular units", the smallest functional units for blood flow regulation [21], can be performed and used for future simulations of the mechanisms of the blood flow regulation at different levels.…”

“…interest. In a recent study, it was used for revealing the sites of local injection of VEGF-transduced myofibroblasts [8]. Later on, the defined sites of interests within single muscles were rescanned at a higher resolution and subvolumes within the volumes of interest were used for the straightforward quantitative analysis (Fig.…”