Optimization of Vascular Casting for Three-Dimensional Fluorescence Cryo-Imaging of Collateral Vessels in the Ischemic Rat Hindlimb

Abstract

Development of collateral vessels, arteriogenesis, may protect against tissue ischemia, however, quantitative data on this process remain scarce. We have developed a technique for replicating the entire arterial network of ischemic rat hindlimbs in three dimensions (3D) based on vascular casting and automated sequential cryo-imaging. Various dilutions of Batson's No. 17 with methyl methacrylate were evaluated in healthy rats, with further protocol optimization in ischemic rats. Penetration of the resin into the vascular network greatly depended on dilution; the total length of casted vessels below 75 µm was 13-fold higher at 50% dilution compared with the 10% dilution. Dilutions of 25-30%, with transient clamping of the healthy iliac artery, were optimal for imaging the arterial network in unilateral ischemia. This protocol completely filled the lumina of small arterioles and collateral vessels. These appeared as thin anastomoses in healthy legs and increasingly larger vessels during ligation (median diameter 1 week: 63 µm, 4 weeks: 127 µm). The presented combination of quality casts with high-resolution cryo-imaging enables automated, detailed 3D analysis of collateral adaptation, which furthermore can be combined with co-registered 3D distributions of fluorescent molecular imaging markers reflecting biological activity or perfusion.

Rapid stromal remodeling by short‐term VEGFR2 inhibition increases chemotherapy delivery in esophagogastric adenocarcinoma

“…Subsequently, an incision was made in the vena cava and the vasculature was gently flushed with wash buffer for 30 min. The corrosion casting medium Mercox (Ladd Research, Williston, Vermont, USA) was labeled with UV‐blue fluorescent dye (vasQtec, Zürich, Switzerland, 0.275 mg·mL −1 ), prepared (3 mL resin + 25 µL catalyst) in a syringe, and the vasculature was slowly filled with Mercox at 100 mmHg using a handmade pressure system (Schwarz et al , ). Forty minutes after infusion, Mercox had polymerized and the tumor‐containing right hind limb was harvested and fixed for 48 h in 4% PFA and equilibrated for 48 h in 20% sucrose in PBS at 4 ºC.…”

Topologic and Hemodynamic Characteristics of the Human Coronary Arterial Circulation

“…As a next step, the topological tree was extracted from the digital reproduction of the fluorescent vascular cast. For this, dark current artifacts were eliminated and optical blurring was corrected by deconvolving the images with a system-specific point-spread function (Matlab; The MathWorks Inc., Natick, MA, United States) (Schwarz et al, 2017). Subsequently, the vessels were segmented ( Figure 1B) in three steps.…”

“…More extensive work came from the group of Kassab, who in a series of studies described these characteristics in the arterial, capillary, and venous coronary bed of pigs, also based on corrosion casts, followed by extensive modeling and hemodynamic analyses (Kassab et al, 1993(Kassab et al, , 1999Kassab and Fung, 1994;Kalsho and Kassab, 2004;Kassab, 2005;Mittal et al, 2005a,b;Kaimovitz et al, 2008;Namani et al, 2018). Following initial work on manual segmentation of the coronary vasculature based on corrosion casts, our lab has developed an imaging cryomicrotome that allows for extensive 3D recording of branching structures (Spaan et al, 2005) and applied this technique for the study of network characteristics in several species and organs (van Horssen et al, 2010(van Horssen et al, , 2014van den Wijngaard et al, 2011;Hakimzadeh et al, 2014;Bedussi et al, 2015;Schwarz et al, 2017), culminating in the current work on the human heart.…”

Timely Visualization of the Collaterals Formed during Acute Ischemic Stroke with Fe3O4 Nanoparticle‐based MR Imaging Probe

“…Recent achievements in molecular imaging have offered new approaches for noninvasively studying the nononcological diseases, such as the hindlimb ischemic and myocardial infarction diseases . For example, magnetic iron oxide nanoparticles and upconversion nanoparticles have been used for imaging the atherosclerotic plaques and even for differentiating the vulnerable plaques from stable ones .…”