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 kidney1,2,3,4,5, 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 work6,7,8 and provide a detailed written and visual reference that investigators can easily follow to produce high-resolution images of the pulmonary arterial tree.
While numerous methods exist for labeling and imaging lung vasculature, such as magnetic resonance imaging, echocardiography, or CT angiography9,10, many of these modalities fail to adequately fill and/or capture the small vessels, limiting the scope of what can be studied. Methods such as serial sectioning and reconstruction provide high resolution but are time/labor-intensive11,12,13. Surrounding soft tissue integrity is compromised in traditional corrosion casting10,13,14,15,16. Even animal age and size become factors when attempting to introduce a catheter or, the resolution is lacking. The polymer injection technique, on the other hand, fills arteries to the capillary level and when combined with µCT, allows for unparalleled resolution5. Samples from mouse lungs as young as postnatal day 14 have been successfully casted8 and processed in a matter of hours. These can be rescanned indefinitely, or even sent for histological preparation/electron microscopy (EM) without compromising the existing soft tissue17. The main limitations to this method are the upfront cost of CT equipment/software, challenges with accurately monitoring intravascular pressure, and the inability to acquire data longitudinally in the same animal.
This paper builds on existing work to further optimize the pulmonary artery injection technique and push age/size related boundaries down to postnatal day 1 (P1) to yield striking results. It is most useful for teams that want to study arterial vascular networks. Accordingly, we provide new guidance for catheter placement/stabilization, increased control over fill rate/volume, and highlight notable pitfalls for increased casting success. Resulting casts can then be used for future characterization and morphologic analysis. Perhaps more importantly, this is the first visual demonstration, to our knowledge, that walks the user through this intricate procedure.
Protocol
Representative Results
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This research was supported in part by the NHLBI Intramural Research Program (DIR HL-006247). We would like to thank the NIH Mouse Imaging Facility for guidance in image acquisition and analysis.
Materials
| 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 | SkyScan | 1172 | |
| 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 |
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