Three-Dimensional Perfusion Imaging of Microcirculatory Networks Within Axially Vascularized Artificial Tissue

Evaluating the complex, three-dimensional (3D) architecture of <i>de novo</i> angiogenesis in artificially engineered tissue remains a significant challenge, as conventional methods like 2D histology and microimaging techniques are limited. For axial vascularization techniques, a reproducible method for complete visualization of the microcirculatory system is needed. We present an integrated workflow for high-resolution 3D visualization of neovascularization within arteriovenous (AV) loop-based tissue constructs in a rat model. An intravascular perfusion with a cationic near-infrared fluorescent dye, MHI148-polyethylenimine, was used to 3D label the patent vasculature. Following perfusion-fixation and explantation, the construct was rendered optically transparent using an ethyl cinnamate-based clearing protocol. The fluorescent signal was then imaged using confocal and light-sheet fluorescence microscopy at 7 and 28 days postimplantation. Our workflow successfully achieved high-contrast, 3D visualization of the microvascular network, allowing for whole-mount and segmental analysis of the vascular tree. At day 7, imaging delineated solely the AV loop axis while by day 28, a dense and complex, interconnected capillary plexus from the central axis demonstrated a progressive neovascularization. Downstream processing compatibility was confirmed through successful rehydration and 3D nuclear counterstaining. This workflow offers a powerful and reproducible method for detailed structural assessment of microvascular networks in large engineered constructs, overcoming key limitations of existing techniques.