Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of β-aminopropionitrile-induced Aortic Aneurysm and Dissection

Summary

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Abstract

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in β-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

Introduction

The incidence of aortic dissection is 3 cases per 100,000 per year¹. Aortic dissection and aneurysmal diseases account for over 10,000 deaths in the United States each year, accounting for 1 - 2% of all deaths in Western countries². Aortic dissection is initiated by a tear in the intimal layer of the vessel with the propagation of blood through the layers of the aortic wall under physiologic pressures. Elevated patient pulse pressures are associated with an increased incidence of dissection and complications. Increased wall shear stress is associated with the aortic wall expansion leading to an aneurysm formation

Protocol

The protocols for animal handling were approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore (animal protocol number 0116024) and conducted according to AAALAC International standards.

1. Preparation of Reagents

1. Heparin
   1. Dilute 250 µL of 1000 U/mL heparin sulfate in 50 mL of phosphate buffered saline to make a final concentration of 5 U/mL.
   2. Warm the heparinized (5 U/mL) phosphate buffered saline, which will replace the blood in the vasculature in a water bath set to 37 °C.
   3. Prepare the pressure-controlled pump by connecting ....

Results

In order to evaluate this protocol, 20 male adult mice, of mixed background as previously described¹⁹ and of 20 - 30 weeks of age, with or without BAPN treatment, were perfused with a lead-based radiopaque silicone rubber (see the Table of Materials) using the protocol detailed above. They underwent micro-CT scanning on the following day (Figure 1 and Figure 2). There were.......

Discussion

Micro-CT imaging can be used to provide highly detailed and three-dimensional reconstructions of vascular pathology in animal models. Through the use of intravascular contrast-enhanced media, non-enhanced soft tissues, such as the lumen of a blood vessel, can be differentiated from those that are enhancing. While laser Doppler, microangiography, magnetic resonance angiography, histology with confocal, or two-photon microscopy may be used to assess vascular beds, they typically focus on a limited area of study and/or are .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microfil	Flow Tech, Inc	MV-122	We use yellow, a different color can be ordered as desired. Kit includes MV-Compound, MV-Diluent, and MV-Curing Agent.
Heparin (1000 U/mL)	Sagent Pharmaceuticals	25021-400-10
Phosphate buffered saline	Corning	21-031-CV
Isoflurane	Vet One, MWI	502017
3-Aminopropionitrile fumarate salt	Sigma-Aldrich	A3134
Single syringe pump	Fisher Scientific	14-831-200
27-gauge scalp vein set needle	Exel Int	26709	27G x 3/4", 12" tube
Inveon Micro-CT scanner	Siemens Medical Solutions
Osirix MD	Pimxmeo SARL		Version 8.0.2
Inveon Research Workplace	Siemens Medical Solutions		Version 4.2
Rodent Chow	Harlan Teklad	2018sx