Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Summary

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (μCT) imaging.

Abstract

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels^1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample^3-5. One method which circumvents these issues is micro-Computed Tomography (μCT). μCT imaging can scan at resolutions <10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)^6-11. However, imaging vessels by ex vivo μCT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by μCT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for μCT imaging.

Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) ^12-14, or ex vivo via a Langendorff perfusion system ^15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network – arteries, capillaries, and veins alike.

Protocol

1. Preparations before starting

1. Fill each side of the pressure perfusion apparatus with Vasodilator buffer (4mg/L Papaverine + 1g/L Adenosine in PBS) or 4% Paraformaldehyde (PFA) in PBS, respectively.
2. Prepare a 1/2cc Insulin Syringe (with a permanently attached 29G ½" needle) by filling it with 0.1 ml of 1:100 Heparin (5000U/ml stock) and bending the needle to ~120 degree angle with the bevel up. Do the same with a 1 ml syringe (with a 26G ½" needle) filled with 0.3 ml saturated KCl solution.

2. Exposing the heart and cannulating the aorta

1. Anesthetize the mouse using your anesthetic of ch....

Discussion

Cardiac tissue has a very high metabolic demand, and therefore requires a constant supply of nutrients and oxygen from the blood delivered by the coronary vasculature. Diseases of the coronary vessels, which decrease coronary function due to vessel stenosis and blockage, can lead to tissue hypoxia and ischemia, and put affected patients at risk for myocardial infarction and irreparable damage to the heart muscle. A better understanding of the diseased state of these vessels is necessary, and critical to our ability to st.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 ml syringes	BD Biosciences	BD-309602
1/2cc insulin syringes with permanently attached 29G ½’ needles	BD Biosciences	BD-309306
2" x 2" Gauze pads	Med101store.com	SKU 2208
24G ¾" Angiocath IV catheter	BD Biosciences	BD-381112
26G ½"gauge needles	BD Biosciences	BD-305111
Adenosine	Sigma-Aldrich	A9251	1g/L in PBS for Vasodilation Buffer (with Papaverine)
Angled Graefe Forceps	Fine Science Tools	11052-10
Cotton-tipped applicators: 6" non-sterile	Cardinal Health	C15055-006
Curved Surgical Scissors	Fine Science Tools	14085-09
Dissecting stereoscope and light source	Nikon Instruments	NA	NA
Dissecting Tray, 11.5 x 7.5 inches	Cole-Parmer	YO-10915-12	Filled with tar for pinning down the mouse
Fine Curved Forceps	Aesculap	FD281R	Need two
Heparin, 5000 U/ml stock	APP Pharmaceuticals	NDC 63323-047-10	1:100 dilution in water
KCl	Fisher Scientific	P217	Saturated solution in H2O
Ketamin (Ketaset), 100 mg/ml stock	Fort Dodge Animal Health	NDC 0856-2013-01	Mixed as 130 mg/kg body weight, with Xylazine in 0.9% saline
Microfil	FlowTech	MV-122 (yellow). Other color options are also available.	Mix 1:1 by weight, with 10% by volume of curing agent. Prepare just before injection, and vortex to ensure it is well mixed
Non-sterile Suture: 6-0, braided silk	Harvard Apparatus	723287
Papaverine	American Regent Inc.	NDC 0517-4010-01	4mg/L in PBS for Vasodilation Buffer (with Adenosine)
Paraformaldehyde	Sigma-Aldrich	P6148	Prepared as 4% solution
Perfusion Apparatus			See figure 2
Spring Scissors	Fine Science Tools	15018-10
Xylazine (Anased), 20 mg/gl stock	Lloyd, Inc.	NADA #139-236	Mixed as 8.8 mg/kg body weight, with Ketamin in 0.9% saline