Name: visualization of vascular and parenchymal regeneration after 70% partial hepatectomy in normal mice
Uploaded: 2016-09-13T13:00:00
Description: Watch this Scientific Journal Video about Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice at JoVE.com

The authors acknowledge funding by the German Ministry of Education and Research (BMBF) via the systems biology network "Virtual Liver", grant numbers 0315743 (ExMI), 0315765 (UK Jena), 0315769 (MEVIS).The authors also thank Frank Schubert for technical support.

Procedures involving animal subjects have been approved by Th&#252;ringer Landesamt f&#252;r 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
<ol>
	<li>Heparin-saline solution
	<ol>
		<li>Add 0.1 ml heparin into 10 ml saline (5 IU/ml).</li>
	</ol>
	</li>
	<li>Silicone compound mixture
	<ol>
		<li>A...

<ol>
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Contrasting the vascular tree by silicone injection and &#181;CT scanning has been introduced in tumor models and neurological disease models frequently to study the angiogenic progression5,7,8,10. 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...

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 &#181;CT 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 &#181;CT 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.

<table border="0" cellpadding="0" cellspacing="0" width="692">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PERFUSOR&#174; VI</td>
			<td style="width:191px;">B.BRAUN</td>
			<td style="width:119px;">87 222/0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipetus&#174;-akku</td>
			<td style="width:191px;">Hirschmann</td>
			<td>9907200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipets</td>
			<td style="width:191px;">Greiner</td>
			<td style="width:119px;">606180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro scissors</td>
			<td>Fine Science Tools (F&#183;S&#183;L)</td>
			<td>No. 14058-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro serrefine</td>
			<td>Fine Science Tools (F&#183;S&#183;L)</td>
			<td>No.18055-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro clamps applicator</td>
			<td>Fine Science Tools (F&#183;S&#183;L)</td>
			<td>No. 18057-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight micro forceps</td>
			<td>Fine Science Tools (F&#183;S&#183;L)</td>
			<td>No. 00632-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved micro forceps</td>
			<td>Fine Science Tools (F&#183;S&#183;L)</td>
			<td>No. 00649-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">needle-holder</td>
			<td>Fine Science Tools (F&#183;S&#183;L)</td>
			<td style="width:119px;">No. 12061-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1ml syringe</td>
			<td style="width:191px;">B.Braun</td>
			<td style="width:119px;">9161406V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5ml syringe</td>
			<td style="width:191px;">B.Braun</td>
			<td style="width:119px;">4606051V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">extension and connection lines</td>
			<td style="width:191px;">B.Braun</td>
			<td style="width:119px;">4256000</td>
			<td>30cm, inner &#248;1.2mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6-0 silk (Perma-Hand Seide)</td>
			<td style="width:191px;">Ethicon</td>
			<td style="width:119px;">639H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6-0 prolene</td>
			<td style="width:191px;">Ethicon</td>
			<td>8711H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microfil&#174; MV diluent</td>
			<td style="width:191px;">FLOW TECH, INC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microfil&#174; MV - 120</td>
			<td style="width:191px;">FLOW TECH, INC</td>
			<td style="width:119px;">MV - 120 (blue)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MV curing agent</td>
			<td style="width:191px;">FLOW TECH, INC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heparin 2500 I.E./5ml</td>
			<td style="width:191px;">Rotexmedica</td>
			<td>ETI3L318-15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Saline</td>
			<td style="width:191px;">Fresenius Kabi Deutschland GmbH</td>
			<td style="width:119px;">E15117/D DE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imalytics Preclinical software</td>
			<td colspan="3">Experimental Molecular Imaging, RWTH Aachen University, Germany</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HepaVision</td>
			<td style="width:191px;">Fraunhofer MEVIS, Bremen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoZoomer 2.0-HT&#160;Digital slide scanner</td>
			<td style="width:191px;">Hamamatsu Electronic Press, Japan&#160;</td>
			<td style="width:119px;">C9600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tomoscope Duo CT&#160;</td>
			<td>CT Imaging GmbH, Erlangen, Germany</td>
			<td style="width:119px;">TomoScope&#174;&#160;Synergy</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of vascular and parenchymal regeneration after 70% partial hepatectomy in normal mice

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 (&#181;CT) 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 &#181;CT 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.

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...

Watch this Scientific Journal Video about Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice at JoVE.com

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

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 &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

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