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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 (vascular endothelial growth factor, VEGF)¹⁴, chemokines and their receptors (CXCR4/CXCR7/CXCL12)⁴ have been the mainstay for studying vascular regeneration. However, a 3D model together with quantitative analysis of the vasculature would add critical anatomic information to gain a better understanding of the important relationship between hepatic parenchymal and vascular regeneration.

To visualize the hepatic vascular system, which requires contrasting the vascular trees, mice were injected with a radiopaque silicone rubber contrast agent directly into the portal or hepatic venous vascular tree. After polymerization of the silicone and explantation of the organ, the liver samples were subjected to µCT scanning using a CT scanner. The scans resulted in voxel image representations of the silicone-injection specimens⁹.

For quality control, the vascular system was first visualized in 3D using preclinical software. Segmentation was performed by setting a threshold between the soft tissue intensity and the vessel intensity. The resulting vessel mask was visualized using surface rendering. The software also allowed for manual determination of two parameters of vascular growth: maximal vessel length and radius.

A preclinical software was then used for 3D reconstruction of vascular trees and subsequent calculation of the supplying or draining vascular territories¹³. In addition, this software automatically determined certain parameters of vascular growth, such as the total length of all visible vascular structures also known as the total edge length or total vessel volume.

The silicone perfusion procedure was performed in naive mice and in mice that underwent 70% partial hepatectomy (PH). Livers were collected at different observation time points after resection for analyzing vascular and parenchymal liver regeneration using the aforementioned visualization and quantification technique.

The main goals of this film are to: (1) demonstrate the delicate injection-technique required to achieve optimal contrasting and (2) show the potential benefit resulting from a detailed analysis of the resulting specimen using µCT and histological serial sections. After watching this film, the reader should have a better understanding of how to inject silicone compound into a specific vascular system and of the usefulness and applicability of the technique.

Protokół

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. Portal Venous System Silicone Injection

1. Laparotomy
   1. Place the mouse in the anesthesia induction chamber and anaesthetize it with 2% isoflurane and 0.3 L/min oxygen.
   2. Fix the anaesthetized mouse on the operating table using tape with continuous inhalation of 2% isoflurane and 0.3 L/min oxygen. Check the toe-pinch withdrawal reflex of the mouse and start operation if the reflex is absent.
   3. Make a transverse incision on the abdomen using scissors for the skin layer and an electric coagulator for the muscle layer. Move out the intestines to the left side using cotton tips and cover the intestines with saline soaked gauze.
2. Catheter insertion
   1. Dissect portal vein under the microscope using micro-forceps. Place one 6-0 silk suture underneath the extrahepatic portal vein, in approximately 1 mm distance to its bifurcation and tie it loosely for later use.
   2. Inject prepared heparin-saline solution via penile vein (male) or inferior vena cava (female) for systemic heparinization for 5 min.
   3. Insert the 26-gauge (26 G) catheter with needle into portal vein and fix it with a clamp
3. Heparin-saline solution and silicone compound perfusion
   1. Load heparin-saline solution in 5 ml syringe and turn on perfusion device. Fill the catheter fully with heparin-saline solution to avoid air bubbles.
   2. Connect extension tube to the catheter and fix them tightly. Start heparin-saline perfusion with a perfusion rate of 0.4 ml/min.
   3. Ligate pre-placed 6-0 silk suture for double fixing the catheter and blocking blood flow from splenic vein and mesenteric vein. Euthanatize the mouse by exsanguination via perfusion under anesthesia.
   4. Rinse the liver using saline during the whole perfusion procedure in order to keep it moist.
   5. Add 0.1 ml curing agent into the MV-120 tube. Change heparin-saline syringe to silicone syringe.
   6. Start silicone perfusion with a perfusion rate of 0.2 ml/min for approximately 1 min to prefill the catheter system and to fill the portal vein system. Stop silicone perfusion when the vessels on the surface turn blue.
4. Sampling
   1. Keep the liver in-situ until the silicone is fully polymerized after approximately 15 to 30 min. Dissect ligaments connecting liver and adjacent organs with care to keep liver intact. Explant the liver and put it into formalin for fixation.

3. Hepatic Venous System Silicone Injection

1. Perform laparotomy as performed in step 2.1 and expose operative field fully.
2. Catheter insertion
   1. Dissect portal vein under the microscope using micro-forceps. Place one 6-0 silk suture underneath the extrahepatic portal vein, in approximately 1 mm distance to its bifurcation and tie it loosely for later use.
   2. Inject prepared heparin-saline solution via penile vein (male) or inferior vena cava (female) for systemic heparinization for 5 min.
   3. Insert one 26 G catheter (catheter 1) with needle into portal vein and fix it with a clamp. Insert another 26 G catheter (catheter 2) with needle into inferior vena cava and fix it with a clamp.
   4. Ligate the branches of inferior vena cava (including left and right renal veins) and its distal end using 6-0 synthetic, monofilament, nonabsorbable polypropylene suture.
3. Heparin-saline solution and silicone compound perfusion
   1. Load heparin-saline solution in 5 ml syringe and turn on perfusion device. Fill catheter 1 completely with heparin-saline solution to avoid air bubbles. Connect extension tube to catheter 1 and fix it tightly. Start heparin-saline perfusion at a rate of 0.4 ml/min.
   2. Ligate pre-placed 6-0 silk suture for double fixing the catheter and blocking blood flow from splenic vein and mesenteric vein. Euthanatize the mouse by exsanguination via perfusion under anesthesia.
   3. Rinse the liver using saline during the whole perfusion procedure in order to keep it moist. Add 0.1 ml curing agent into the MV-120 tube. Exchange heparin-saline syringe with silicone syringe.
   4. Place a clamp on the suprahepatic inferior vena cava to obstruct the outflow of the liver.
   5. Connect the extension tube to catheter 2 and start silicone perfusion with a perfusion rate of 0.2 ml/min for approximately 2 min to reach an objective hepatic vascular volume as reported. Stop silicone perfusion when the vessels on the surface turn blue.
4. Sampling
   1. Dissect hepatic ligaments avoiding any injury to the liver. Explant the liver and put it into formalin for fixation.

4. Micro-CT (µCT) Scanning

To scan the explanted liver sample using µCT, the following steps are needed.

1. Take the liver sample out of the fixation solution. Place the liver onto the µCT bed. Put the µCT bed with the liver sample into the µCT.
2. Acquire topogram before starting the scan. Use one subscan for the small liver sample and two subscans for large samples.
3. Select a µCT protocol with a high resolution (e.g., HQD-6565-390-90). This protocol acquires 720 projections with 1,032 x 1,012 pixels during one full rotation with the scanning time of 90 sec per sub-scan. Start the µCT scan.

5. Histological Serial Sections

1. Embed the liver specimen in paraffin as a whole after µCT scanning. Cut whole paraffin sample into 4µm sections, resulting in a series of 2,000 to 2,500 sections.
2. Stain sections with appropriate staining technique to visualize molecular events of interest such as Ki-67 as proliferation marker and HMGB1 as a marker of ischemic damage. Determine sequence of staining in respect to scientific question.
3. Use a whole slide scanner to digitalize the stained sections.
4. Perform 3D-reconstruction of vascular tree(s) (already feasible) and visualize 3D-distribution of molecular events in respect to vascular tree (research in progress).

Wyniki

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...

Dyskusje

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...

Materiały

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