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^3,4. Consequences of aortic dissection include the occlusion of blood flow to distant organs including the brain, kidneys, bowels, and limbs, the formation of chronic aneurysms, rupture, or death^5,6,7.

At present, the biochemical and cellular processes involved in the initiation and progression of aortic aneurysms and dissections are still poorly understood. Reproducible animal models of aortic aneurysm and dissection are key to understanding their pathophysiology. β-aminopropionitrile (BAPN) is a lysyl oxidase inhibitor, which prevents the cross-linking of elastin and collagen and has been shown to significantly alter the structure of the vessel wall extracellular matrix and its biomechanical integrity^6,8. Rodents treated with BAPN have been utilized as a common animal model of aortic aneurysm and dissection^9,10.

Vascular imaging modalities are instrumental in identifying vascular pathology, confirming vessel patency, and evaluating organ perfusion. Recently, micro-computed tomography (micro-CT) has been utilized to study the vasculature of mice and similarly sized animals. Unlike bone, the axial imaging of blood vessels by computed tomography is limited, as intraluminal blood is inherently relatively radiolucent. When combined with intravascular contrast agents, however, micro-CT enables detailed three-dimensional reconstructions of animal vasculatures for the study of macro-anatomic vascular pathology¹¹.

The selected contrast agent (see the Table of Materials) is a radiopaque silicone rubber that contains lead chromate and lead sulfate. Upon perfusion in the presence of a catalyst, it quickly hardens to form a cast of the vasculature with minimal alterations in the macro-anatomic architecture of the vessels, making the vasculature highly radiopaque in contrast to the background tissues when examined radiographically. This contrast agent is advantageous because it is easy to handle and avoids the tissue degradation and vessel loss due to breakage often associated with vascular cast corrosion. As it cures with minimal shrinkage¹², vessels cleared of blood remain patent and allow for an accurate assessment of the animal macro-vasculature in non-survival experiments. Previous work has successfully used radiopaque silicone rubber-contrast in a variety of animal studies. Specifically, applicability in visualizing the coronary, glomerular, placental, and cerebral circulations^{11,12,13,14,15} has been shown. In this paper, we detail the methodology of open left-ventricular puncture for the intravascular perfusion of lead-based radiopaque silicone rubber to quantitatively characterize BAPN-induced aortic pathology in a mouse model by micro-CT.

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 the required tubing and 2 empty 10-mL syringes, 1 for the heparinized-saline buffer and 1 for the contrast agent.
   4. Fill the tubing with the warm heparinized phosphate buffered saline and remove the air bubbles from the tubing of the pressure pump.
2. Contrast agent
   NOTE: Please refer to the Table of Materials for the contrast agent kit constituents.
   1. Mix a pigmented compound with a diluent to achieve a 1:6 dye to diluent ratio.
   2. Immediately prior to use (step 2.3.12), add 200 µL of a curing agent to each 5-mL aliquot of the diluted pigmented compound and mix them well (4% by volume).
      NOTE: The manufacturer reported the working time is 40 min. As the silicone-rubber contrast agent begins to polymerize 20 min after the addition of the curing agent, it is important to prepare the solution immediately prior to its infusion.
3. BAPN drinking water
   1. Dissolve β-aminopropionitrile (BAPN) in drinking water to create a final concentration of 3 g/L (adapted from protocols previously described in the literature)^9,16,17.
   2. Administer the BAPN-containing drinking water to a group of mice once they are 4 weeks of age until the time of perfusion for micro-CT.

2. Surgical Procedure

1. Animal preparation
   1. Wean the mice at 3 weeks of age, maintain them on a 12 h light/12 h dark cycle, and feed them standard rodent chow. For the BAPN-treated group, administer freshly prepared BAPN drinking water for 16 - 26 weeks ad libitum. Provide the control animals with standard drinking water ad libitum.
2. Anesthetic technique
   NOTE: 24 h prior to the CT analysis, the following procedure is performed. Surgical procedures are enacted to prepare the specimen for a postmortem intracardiac perfusion.
   1. Induce an anesthesia via an induction tank with 100% O₂ and 3% isoflurane delivered via a precision vaporizer. After the anesthesia induction, discontinue the isoflurane and flush the chamber with O₂. Maintain the anesthesia with 2 - 2.5% isoflurane and 1 L/min of O₂ via a nose cone.
   2. Attach both the induction chamber and the facemask to a charcoal scavenger for waste gas adsorption to protect personnel. Ensure an adequate anesthetic plane by demonstrating that there is no response to noxious stimuli (toe pinch).
   3. Prepare an operative field consisting of a surgical tray and the necessary surgical instruments.
   4. Transfer the animal to the surgical field and position it in dorsal recumbency.
3. Operative technique
   1. Using scissors, make a midline incision through the skin and soft tissue from midway between the pubic symphysis to the sternal notch, extending through the skin and soft tissue overlying the sternum.
   2. Using scissors, create a hole in the diaphragm at the xiphoid process to enter the thoracic cavity.
   3. Use scissors to dissect the diaphragm of the ventral chest wall, bilaterally.
   4. Cut through the costal cartilages to separate the ribs from the sternum at the right sternal border.
   5. Apply a fine hemostatic clamp to the tip of the sternum (near the xiphoid process) and move the hemostat cranially so that it is positioned over the head of the mouse. This will retract the thymus and sternum away from the heart, exposing the heart and great vessels for further manipulation.
   6. Sharply dissect any attachments between the heart and the chest wall.
   7. Connect the 27-gauge IV catheter needle to a syringe pre-loaded with 10 mL of heparinized phosphate buffered saline (5 U/mL) and fill all tubing with the buffer in order to remove the air bubbles from the tubes of the pressure pump.
   8. Use care while preparing the fluid as bubbles in the fluid line may impede the filling of smaller vessels. Limit the number of vessels that are damaged during the animal preparation, as this will cause the contrast agent to leak out of the severed vessels, changing the volume required for a complete filling and introducing artifacts to the final imaging.
   9. Puncture the left ventricle with a 27-gauge needle that is stabilized with a right-angle clamp. Immediately incise the right ventricle or the inferior vena cava to drain the heparin solution and blood.
      NOTE: Heparin is used as an anticoagulant to prevent the blood from clotting in the vessels after the animal's death.
   10. Perfuse the animal at a constant rate of 2 mL/minute using a single syringe pump. Note the visible blanching of the organs. Continue the perfusion until the perfusate draining from the venous circulation is free of blood (about 5 - 6 mL). Stop the pump.
   11. Disconnect the IV catheter tubing from the 10-mL syringe, taking care not to disrupt the needle's position in the left ventricle.
   12. Immediately after the complete exsanguination, separate the contrast agent solution into 5-mL aliquots and add the curing agent at this time (see step 1.2). Mix them well. Draw up 5 mL of the contrast agent mix into a 10-mL syringe and perfuse the animal with it.
   13. For a complete filling of the vessels (arteries and veins), continue the infusion past the point when it can be seen exiting the venous solution. Look for signs of a successful perfusion including the visualization of a casting agent in the coronary arteries, pulmonary arteries, intestine, and liver vasculature.
   14. The contrast agent will cure after approximately 20 min at room temperature. Upon curing, harvest the individual organs, as needed, and fix them in 10% neutral buffered formalin. Fix whole carcasses if the samples are not used for micro-CT scanning the following day. If the carcasses will be used the subsequent day, position them on a metal tray and place them in a refrigerator at 4 °C to cure overnight.

3. Micro-CT Scanning and Parameters

NOTE: The specific image acquisition parameters will be dependent upon the machine in use.

1. Acquire X-ray computed tomography images of each mouse the day following the perfusion using a micro-CT scanner using an X-ray tube voltage of 55 kVp, a current of 150 μA, a system magnification factor of 2.19, and a CCD camera pixel binning factor of 2. This yields an effective pixel size of 29 µm.
   1. Lay the mouse carcass supine on the micro-CT scanner table and obtain a scout X-ray scan.
   2. Focus the detector field of view of 57.4 mm (axial) x 37.1 mm (transaxial) on the torso to image the full length of the aorta.
   3. Acquire 180 image projections with a rotation increment of 2 degrees and a time per projection of 2800 ms.
2. Reconstruct the images using a modified Feldkamp algorithm; the reconstructed voxel size is 29 x 29 x 29 µm³ (slice thickness = 29 µm) using the Multimodal 3D Visualization plug-in for the software used here.

4. Post-processing and Rendering

1. Convert the CT data to a DICOM format using the appropriate software.
2. Analyze the images to identify whether an aneurysm was present. Measure the minor axis diameter at the widest point of the aortic arch, descending thoracic aorta, and abdominal aorta as previously described¹⁸ (Figure 1).
   NOTE: In our study, images were analyzed by two independent observers (one blinded) utilizing a DICOM viewer to identify whether an aneurysm was present. The minor axis diameter was measured at the widest point of the aortic arch, descending thoracic aorta, and abdominal aorta as previously described¹⁹ (Figure 1). Mean non-aneurysmal arterial segments of BAPN-untreated mice established normal vessel diameters serving as the age-matched control values.
3. Aneurysms are defined as a localized or diffuse dilation of the aortic segments to diameters greater than 50% of the reference diameter. Locate these based on the above measurements.

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