Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Summary

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Abstract

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.

To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (µCT) scanner.

When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

Introduction

The focus of this technique is the visualization of pulmonary arterial architecture using a polymer-based compound in mice. While extensive work has been performed on systemic vascular beds such as brain, heart, and kidney^1,2,3,4,5, less information is available regarding the preparation and filling of the pulmonary arterial network. The aim of this study, therefore, is to expand upon previous work^6,7,8 and provide....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (ACUC) of the National Heart Lung and Blood Institute.

1. Preparation

1. Inject the mouse intraperitoneally with heparin (1 unit/g mouse body weight) and allow it to ambulate for 2 min.
2. Euthanize the animal in a CO₂ chamber.
3. Arrange the mouse in a supine position on a surgical board and secure all four limbs to the board with tape. Use magnification for fine dissection.

2. Exposing lungs and trachea

1. Spray the ventral side of the mouse with 70% ethanol to minim....

Results

A successful cast will exhibit uniform filling of the entire pulmonary arterial network. We demonstrate this in C57Bl/6J mice ranging in age: Postnatal day P90 (Figure 4A), P30 (Figure 4B), P7 (Figure 4C), and P1 (Figure 4D). By controlling the rate of flow and visually monitoring the fill in real-time, reliable endpoints of the most distal vasculature were achieved (

Discussion

Executed properly, this method yields striking images of pulmonary arterial networks, allowing for comparison and experimentation in rodent models. Several critical steps along the way ensure success. First, investigators must heparinize the animal in the preparatory stage to prevent blood clots from forming in the pulmonary vasculature and chambers of the heart. This allows for the complete arterial transit of polymer compound. Second, when puncturing the diaphragm and removing the ribcage, take care to protect the lung.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1cc syringe	Becton Dickinson	309659
20ml Glass Scintillation Vials	Fisher	03-340-25P
30G Needle	Becton Dickinson	305106
50mL conical tubes	Cornin	352098	For sample Storage and scanning
60cc syringe	Becton Dickinson	309653
7-0 silk suture	Teleflex	103-S
Analyze 12.0 Software	AnalyzeDirect Inc.	N/A	Primary Software
Amira 6.7 Software	Thermo Scientific	N/A	Alternative Sofware
CeramaCut Scissors 9cm	Fine Science tools	14958-09
Ceramic Coated Curved Forceps	Fine Science tools	11272-50
CO2 Tank	Robert's Oxygen Co.	n/a
Dual syringe pump	Cole Parmer	EW-74900-10
Dumont Mini-Forceps	Fine Science tools	11200-14
Ethanol	Pharmco	111000200
Formalin	Sigma - Life Sciences	HT501128
Gauze	Covidien	441215
Hemostat	Fine Science tools	13013-14
Heparin (1000USP Units/ml)	Hospira	NDC 0409-2720-01
Horos Software	Horos Project	N/A	Alternative Sofware
induction chamber	n/a	n/a
Kimwipe	Fisher	06-666	fiber optic cleaning wipe
Labelling Tape	Fisher	15966
Magnetic Base	Kanetec	N/A
Micro-CT system	PerkinElmer	Quantum GX
Microfil (Polymer Compound)	Flowech Inc.	Kit B - MV-122	8 oz. of MV compound; 8 oz. of diluent; MV-Curing Agent
Micromanipulator	Stoelting	56131
Monoject 1/2 ml Insulin Syringe	Covidien	1188528012
Octagon Forceps Straight Teeth	Fine Science tools	11042-08
Parafilm	Bemis company, Inc.	#PM999
PE-10 tubing	Instech	BTPE-10
Phospahte buffered Saline	BioRad	#161-0780
Ring Stand	Fisher	S13747	Height 24in.
Sodium Nitroprusside	sigma	71778-25G
Steel Plate	N/A	N/A	16 x 16 in. area, 1/16 in thick
Straight Spring Scissors	Fine Science tools	15000-08
SURFLO 24G Teflon I.V. Catheter	Santa Cruz Biotechnology	360103
Surgical Board	Fisher	12-587-20	This is a converted slide holder
Universal 3-prong clamp	Fisher	S24280
Winged Inf. Set 25X3/4, 12" Tubing	Nipro	PR25G19
Zeiss Stemi-508 Dissection Scope	Zeiss	n/a