Enhancing cardiovascular research with whole-organ imaging

Abstract

Purpose of review There have been tremendous advances in the tools available for surveying blood vessels within whole organs and tissues. Here, we summarize some of the recent developments in methods for immunolabeling and imaging whole organs and provide a protocol optimized for the heart. Recent findings Multiple protocols have been established for chemically clearing large organs and variations are compatible with cell type-specific labeling. Heart tissue can be successfully cleared to reveal the three-dimensional structure of the entire coronary vasculature in neonatal and adult mice. Obtaining vascular reconstructions requires exceptionally large imaging files and new computational methods to process the data for accurate vascular quantifications. This is a continually advancing field that has revolutionized our ability to acquire data on larger samples as a faster rate. Summary Historically, cardiovascular research has relied heavily on histological analyses that use tissue sections, which usually sample cellular phenotypes in small regions and lack information on whole tissue-level organization. This approach can be modified to survey whole organs but image acquisition and analysis time can become unreasonable. In recent years, whole-organ immunolabeling and clearing methods have emerged as a workable solution, and new microscopy modalities, such as light-sheet microscopy, significantly improve image acquisition times. These innovations make studying the vasculature in the context of the whole organ widely available and promise to reveal fascinating new cellular behaviors in adult tissues and during repair.

Blood flow modeling reveals improved collateral artery performance during the regenerative period in mammalian hearts

“…To utilize 3D CFD for modeling coronary blood flow at high resolution and with controlled parameter perturbations, we required a method to image the entire intact artery tree in three dimensions. A whole-organ immunostaining and clearing method based on iDISCO was optimized for cardiac tissue using postnatal day 6 (P6) mice 37,39 , which allowed us to image smooth muscle actin (α-SMA)-positive arterial smooth muscle cells throughout the heart using Light sheet microscopy (Fig. 1a).…”

“…Whole heart vasculature staining was performed following the modified iDISCO+ protocol previously described 37,39 . For all following steps, tissue was always agitated unless noted otherwise.…”

“…To date, the vascular labeling methods utilized have not had the resolution required to generate detailed anatomic models suitable for CFD modeling. However, recent innovations in tissue clearing and whole-organ microscopy now provide the possibility of generating sufficiently high resolution images suitable for CFD model building [37][38][39][40] . Thus, CFD is perfectly poised to push forward our understanding of collateral function.…”

CXCL12drives natural variation in coronary artery anatomy across diverse populations

“…When dominance is determined in unknown; we hypothesized it would be established during heart development with the formation of the coronary arteries. Since angiograms and non-invasive imaging procedures are not performed during healthy pregnancies, we investigated whether dominance is presence during fetal stages by performing whole-organ immunofluorescence that allowed visualization of the entire artery tree in 3D ( Figure 4A and B ) 23,24 . Tracing primary and secondary branches of the right and left coronary arteries of each heart revealed a pattern reminiscent of the adult anatomy.…”

Editorial: New technological developments and their impact in vascular research

“…Recent methodological advances in tissue clearing have effectively addressed this challenge in a manner to enable visualization of entire organs. Coronado and Red-Horse [7] in this series of reviews, provide a detailed compilation of techniques and approaches, including iDISCO to visualize vessels in organs like the heart. Included in the review are detailed stepwise procedures.…”