3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

Summary

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

Abstract

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.

A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure^1,2, alterations in brain vasculature of a Alzheimer's disease mouse model³, portal vein abnormalities in portal hypertensive and cirrhotic mice⁴, developmental steps in rat lymphatic maturation between immature and adult lungs⁵, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury⁶.

Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

Protocol

1. Prepare Cannula

1. Warm a 1-inch long section of PE10 tubing with your fingertips and stretch it so that the tubing becomes thin.
   Note: the size of the vessel or duct to be cannulated will determine the degree of stretching required. The cannula must be well-stretched for bile duct casts but may only be required to be moderately stretched to fit within the larger portal vein.
2. Cut the stretched tubing at a diagonal to generate a beveled tip.
3. Cut the other edge of the tubing to create a 5-6 inch segment.
4. Insert a 32 gauge, ½ inch hypodermic needle into the non-beveled edge of the tubing.

Discussion

We have described specific examples for how the portal vein and intrahepatic bile duct systems of the liver can be cast, but this technique can be applied to virtually any other ductal or vascular system with slight adaptations. Previous work has demonstrated the feasibility of this technique in multiple species, including mouse^1,2,7-9, duckling^10,11, rabbit^12,13, dog¹⁴, and pig¹⁵, and in many organs, including nasal gland¹⁴, heart¹⁶, bladder

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PE10 polyethylene tubing	Fisher Scientific	1417012P
5-0 surgical black braided silk	Roboz Surgical	SUT-15-1
Steriject 32 G x 13 mm needle	Air-tite	TSK3213
Spring scissors	Fine Science Tools	15000-08
Number 5 forceps	Fine Science Tools	91150-20
Mercox II kit	Ladd Research	21247
Benzyl alcohol	Fisher Scientific	1816-04	Only required for BABB-clearing
Benzyl benzoate	MP Biomedicals, LLC	154839	Only required for BABB-clearing
Phosphate-buffered saline (PBS)
Modeling clay
Scale
Laboratory scissors
15 ml cap polypropylene tubes
4% paraformaldahyde
15% potassium hydroxide (KOH)			Only required for maceration
Razor blade
100% methanol
3 ml luer lock syringe