Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

Summary

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using µCT and histological serial sections.

Abstract

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (µCT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.

The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after µCT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Introduction

Liver regeneration is often determined by measuring the increase of liver weight and volume and by assessing the hepatocyte proliferation rate¹⁶. However, liver regeneration is not only inducing parenchymal regeneration but also vascular regeneration⁶. Therefore, vascular growth should be further investigated with respect to its role in the progression of liver regeneration. Visualization of the hepatic vascular system is critical to advancing our understanding of vascular regeneration. Numerous indirect methods have been developed to study the underlying molecular mechanisms of hepatic vascular regeneration. Traditionally, detection of cytokines....

Protocol

Procedures involving animal subjects have been approved by Thüringer Landesamt für Verbraucherschutz Abteilung Tiergesundheit und Tierschutz, Germany. Because the portal venous system was visualized separately from the hepatic venous system, separate animals were needed for the different vascular trees.

1. Reagents Preparation

1. Heparin-saline solution
   1. Add 0.1 ml heparin into 10 ml saline (5 IU/ml).
2. Silicone compound mixture
   1. Add 2 ml MV-120 in one 5 ml tube. Dilute MV-120 by adding 3 ml MV diluent resulting in a 40% solution.

2....

Results

Quality Criteria

The quality of silicone injection can be judged with the naked eye during the procedure. The small vessels on liver surface fill gradually with the blue compound. If the normal vascular structure was observed on the liver surface, the silicone rubber injection quality was good. If the perfusion volume was insufficient, the small vessels on the liver surface were not fully filled. In contrast, ov.......

Discussion

Contrasting the vascular tree by silicone injection and µCT scanning has been introduced in tumor models and neurological disease models frequently to study the angiogenic progression^5,7,8,10. Improvements in methodology of silicone injection were made in the present study for visualizing and quantifying vascular growth after partial hepatectomy in mice.

There are a number of critical steps needing attention to achieve good perfusion quality. First of all, systemic heparinizati.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PERFUSOR® VI	B.BRAUN	87 222/0
Pipetus®-akku	Hirschmann	9907200
Pipets	Greiner	606180
micro scissors	Fine Science Tools (F·S·L)	No. 14058-09
micro serrefine	Fine Science Tools (F·S·L)	No.18055-05
Micro clamps applicator	Fine Science Tools (F·S·L)	No. 18057-14
Straight micro forceps	Fine Science Tools (F·S·L)	No. 00632-11
Curved micro forceps	Fine Science Tools (F·S·L)	No. 00649-11
needle-holder	Fine Science Tools (F·S·L)	No. 12061-01
1 ml syringe	B.Braun	9161406V
5 ml syringe	B.Braun	4606051V
extension and connection lines	B.Braun	4256000	30 cm, inner ø 1.2 mm
6-0 silk (Perma-Hand Seide)	Ethicon	639H
6-0 prolene	Ethicon	8711H
Microfil® MV diluent	FLOW TECH, INC
Microfil® MV - 120	FLOW TECH, INC	MV - 120 (blue)
MV curing agent	FLOW TECH, INC
Heparin 2500 I.E./5 ml	Rotexmedica	ETI3L318-15
Saline	Fresenius Kabi Deutschland GmbH	E15117/D DE
Imalytics Preclinical software	Experimental Molecular Imaging, RWTH Aachen University, Germany
HepaVision	Fraunhofer MEVIS, Bremen, Germany
NanoZoomer 2.0-HT Digital slide scanner	Hamamatsu Electronic Press, Japan	C9600
Tomoscope Duo CT	CT Imaging GmbH, Erlangen, Germany	TomoScope® Synergy