Rapid Whole-Mount High-Resolution Imaging of Small Animal Vasculature for Quantitative Studies

Summary

This protocol introduces a rapid method for quantitative whole-mount three-dimensional vascular imaging using light-sheet fluorescence microscopy. The efficacy of the method is demonstrated using the pharyngeal arch artery system of the chick embryo model, with hemodynamic forces quantified via computational fluid dynamics.

Abstract

In small animal models of cardiovascular development and diseases, subject-specific computational simulations of blood flow enable quantitative assessments of hemodynamic metrics that are difficult to measure experimentally. Computational fluid dynamic simulations shed light on the critical roles of mechanics in cardiovascular function and disease progression. Acquiring high-quality volumetric images of the vessels of interest is central to the accuracy and reproducibility of morphological measurement and flow quantitation results. This study proposes a rapid, cost-effective, and accessible method for whole-mount high-resolution imaging of small animal vasculature using light-sheet fluorescence microscopy. The modified iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) light-sheet sample preparation protocol involves (1) labeling vasculature with a fluorescent agent, (2) preserving the sample, and (3) rendering the sample transparent. Unlike classical iDISCO+, which uses immunohistochemical staining, the authors label vascular endothelium with FITC-tagged poly-L-lysine, an affordable non-specific fluorescent dye that is highly resistant to photo-bleaching, in a process termed "endo-painting." The rapid labeling reduces sample preparation time from approximately four weeks to less than 3 days. Furthermore, the use of minimally hazardous solvent ethyl cinnamate (ECi) as the clearing agent and imaging solution makes the samples safer to handle and compliant with a wider range of imaging facilities. The proposed protocol is applied to obtain highly resolved light-sheet fluorescence microscopy image stacks of the cardiovascular system in chick embryos ranging from day 3 (HH18) to day 8 (HH34). This study further demonstrates the suitability of this method for vascular quantitation through 3D reconstruction and computational hemodynamic modeling of a day 5 (HH 26) chick embryo.

Introduction

Volumetric imaging is necessary for accurate studies of cardiovascular physiology and disease. Quantitative imaging produces high-resolution image stacks with intact volumetric dimensions. Samples must both be preserved to maintain their in-vivo morphology and lumen volume as well as imaged in a uniform high resolution capacity. From high-resolution imaging stacks, the user can generate high-fidelity three-dimensional vascular renderings that allow for a complete display of vessel shapes, structure, and connectivity¹.

Cardiovascular structures possess complex three-dimensional anatomical features that cannot....

Protocol

The Office of Laboratory Animal Welfare interprets Public Health Service policy as applying to the chick model as a "vertebrate animal" only after hatching. These embryos are similarly exempt from Institutional Animal Care and Use Committee (IACUC) jurisdiction. The relevant National Institutes of Health frequently asked questions can be accessed at: http://grants.nih.gov/grants/olaw/faqs.htm#ApplicabilityofthePHSPolicy.

1. Embryo collection, labeling and fixation

1. Fashion pulled 0.75 inner diameter glass capillary rods into cut microneedles using a microforge^1,

Results

The rapid whole-mount high-resolution imaging protocol presented here (Figure 1, Table 1) produces clearly outlined vessel lumens as shown in Figure 2, Figure 3, and Figure 4, where the chick embryo vasculature endothelium is GFP fluorescent and therefore outlined in green across embryo stages from early to mature heart development (Figure 4). It is important to find the ri.......

Discussion

The ability to study biology in 3D is critical to an accurate understanding of morphological complexity, inner organ structure, and vascular connections. Accurate and reliable 3D vascular images are also central to subject-specific computational hemodynamic simulations, which are often the only reliable means of quantifying key hemodynamic parameters such as wall shear stress and pressure distribution. Here, the authors introduce a rapid and accessible sample preparation method for high-resolution 3D vascular imaging in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#5 forceps	Fine Science Tools	11252-30
#55-forceps	Fine Science Tools	11295-51
0.03 inch inner diameter silicone tubing	VWR	32829-182
20 μL pipette tips	VWR	76322-134
35 mm Petri dish	VWR	10799-192
5 mL plastic syringe	VWR	BD 309646
Dichloromethane (DCM)	Sigma-Aldrich	270997	Refer to MSDS. Stored in side cabinet under fume hood
Ethyl cinnamate (ECi)	Sigma-Aldrich	112372	Stored at 4 °C
Fine Curved scissors	Fine Science Tools	14061-09
FITC-poly-L-lysine	Sigma-Aldrich	P3069	Store at -20 °C (powder, stock solution), 4ºC (working solution)
Fluoresent microscope	EVIDENT SCIENTIFIC	MVX10
Glass capillary tubes (0.75 mm ID)	Sutter Instrument	FG-GB100-75-10
Lightsheet microscope	Zeiss	Z.1 system
Methanol	Sigma-Aldrich	M1775	Refer to MSDS. Stored in flammable cabinet under fume hood
Microforge	Narishige International USA, Inc.	MF2
Micromanipulator	World Percision Instrrument	M3301R
Paraformaldehyde (PFA) 4%	Thermo Scientific	J19943.K2	Refer to MSDS. Stored at -20 °C (powder), 4 °C (4% working solution)
Phosphate buffered saline (PBS)	Cytiva	SH30256.01	Stored on benchtop
SimVascular			open source software www.simvascular.org
Tyrode’s Solution			Made in-house