正常マウスで70％肝切除後の血管と実質再生の可視化

Visualization of vascular and parenchymal regeneration after 70%partial hepatectomy in normal mice. Traditionally, liver regeneration is assessed by measuring the increase of liver weight and volume as well as by determination of the hepatocyte proliferation rate. Nowadays, we are interested in vascular liver regeneration in mice and want to visualize and quantify vascular growth.

Thus, we establish the workflow for imaging vascular regeneration. The workflow consists of four steps:contrasting the vascular tree, acquisition of CT scans, 3D-reconstruction, and quantitative and qualitative analysis of resulting images. The workflow starts with contrasting the vascular tree.

First we expose the liver, either a normal or a resected one, then we inject the polymerizing silicone contrast compound into either portal vein or hepatic vein, depending on the vascular system of interest. Next, the explanted livers are subjected to scanning in a MicroCT. After scanning, we perform a 3D vascular reconstruction using Imalytics preclinical software.

If the technical quality of the resulting images is good enough, 3D reconstruction can be performed using HepaVision. This program is used in clinics for planning hepatobiliary surgery. In addition to the 3D visualization of the vascular tree, this program allows visualization and calculation of the liver volume, of total liver and each lobe, based on the supplying and draining vessels.

After reconstruction, we can determine additional parameters indicative of vascular growth such as the length and diameter of a specific vessel with the help of computational techniques. This will allow a quantitative description of vascular growth. On the other hand, formalin fixed Microfil samples can be cut into serial sections.

The serial sections can be stained and digitalized using a whole slide scanner. After rigid and elastic registration of serial sections, 3D vascular trees of both portal venous and hepatic venous systems can be reconstructed. In a future step, we want to explore the spatial distribution of molecular events.

One example of a molecular event of interest in hepatic regeneration is the appearance of the proliferation marker Ki-67 in hepatocyte nuclei and its spatial distribution in the 3D reconstructed vascular trees. Next, we are going to give a detailed introduction of how to perform the Microfil injection. Preparing the reagents.

For the Microfil compound mixture, we need MV diluent, MV compound, in our case, the blue MV-120, and curing agent. We also need pipets, tube, and a pipettor. Prepare two milliliter MV-120 in one five-milliliter tube.

Then dilute it by adding three milliliter MV diluent. Mix them by shaking gently. For heparin saline solution, add 1 milliliter heparin into 10 milliliter saline.

Preparing the operating table. First, we need a microscope, second, a set of consumables is needed. 6-0 silk and 6-0 polypropylene sutures, five milliliter and one milliliter syringes, 30 gauge and 21 gauge needles, extension tube for connecting syringe and catheter, 26 gauge catheter with needle insertion, cotton tips and gauze.

Third, surgical instruments are needed. Micro instruments, clamps, and electric coagulator. Fourth, a perfusion pump is needed.

Preparing the animal. Place the mouse in the anesthesia induction chamber and anesthetize the mouse with 2%isoflurane and 0.3 milliliter per minute oxygen. Fix the anesthetized mouse on the operating table using tapes, with continuous inhalation of 2%isoflurane and 0.3 milliliter per minute oxygen.

Microfil injection, portal venous system. 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.

Dissect the portal vein under the microscope. Place 6-0 silk suture underneath the extra hepatic portal vein in approximately one millimeter distance to its bifurcation. Tie it loosely for later use.

Inject the heparin saline solution by a penile vein for systemic heparinization for five minutes. This animation will demonstrate the procedure of catheter insertion. First, place the 6-0 silk suture underneath the extra hepatic portal vein, then insert the 26 gauge catheter into the portal vein and fix it using a clamp.

Ligate the pre-placed suture for fixing the catheter and blocking the blood flow from the splenic vein and the mesenteric vein. Here we will demonstrate the surgical procedure. Insert the 26 gauge catheter with needle.

Insert the catheter further and move out the needle slowly at the same time. Fix the catheter using a clamp. Fill the catheter completely with heparin saline solution to avoid air bubbles.

Connect the extension tube to the catheter and fix them tightly. Turn on the perfusion pump. Start heparin saline perfusion with a flow rate of 0.4 milliliter per minute.

Observe the right lobes becoming pale immediately after perfusion. Rinse the liver using saline to keep it moist during the whole perfusion procedure. Euthanize the mouse by exsanguination via perfusion under anesthesia.

Continue heparin perfusion for several minutes until the whole liver becomes pale. Before Microfil perfusion, add 0.1 milliliter curing agent into prepared Microfil compound to accelerate the polymerizing procedure. Load Microfil compound in five-milliliter syringe and extension tube.

Get rid of the air bubbles by injecting the heparin saline solution into the catheter, the same as before. Connect the tube with the catheter and fix it tightly. Start Microfil injection with a flow rate of 0.2 milliliter per minute for approximately one to two minutes.

Observe the blue agent slowly spreading into the portal vein branches. Stop the perfusion when the blue vessel structure appears on the surface of each liver lobe as indicated a red circle. Leave the liver for at least 15 minutes for Microfil compound polymerization.

Microfil injection, hepatic venous system. The procedure basically follows the same steps as the injection to the portal venous system. Place a 6-0 silk suture underneath the extra hepatic portal vein in approximately one millimeter distance to its bifurcation.

Then insert a catheter to the portal vein and fix it via clamp. Insert another catheter into the inferior vena cava and fix it with a clamp as well. Ligate the pre-placed suture for fixing the catheter and blocking the blood flow from the splenic vein and the mesenteric vein.

Place a clamp on the suprahepatic inferior vena cava to obstruct the outflow of the liver. Ligate branches of the inferior vena cava, including both renal veins and the distal end of the inferior vena cava. Flush the liver using heparin saline solution via catheter one.

Perfuse the hepatic venous system with Microfil compound via catheter two. Results, Micro CT reconstruction. We obtained 3D vascular reconstructions using Imalytics preclinical software after Micro CT scanning.

Vascular regeneration in both portal and hepatic venous systems appeared as elongation and widening of the main vascular structures in combination with an outbranching of smaller vessels. On post-operative day two, the vascular trees of the remnant lobes elongated. By post-operative day seven, additional small branches from the same main portal vein and the hepatic vein became visible.

Results, 3D color coded reconstruction of the hepatic vascular tree and the dependent hepatic territories. Each liver lobe was easily visualized in all three dimensions after color coded reconstruction by HepaVision. As indicated in the table, hepatic volumes of the total liver and each lobe were determined, as shown here, for hepatic venous system of a normal liver.

Results, serial sections and reconstruction. On the other hand, the formalin fixed Microfil sample was subjected to serial sectioning. The stained serial sections were digitalized using a whole slide scanner and used for vascular tree reconstruction of both systems.

These are cross-sections of a stack of mouse liver serial sections before and after rigid and elastic registration. After computer registration, portal venous and hepatic venous systems can be reconstructed in a single 3D model. This manually and computationally time consuming step needs further optimization.

The next step, namely the visualization of the spatial distribution of molecular events in respect to the vascular trees is currently under development. This step can be envisioned as a fusion of Micro CT information derived from the contrasted vascular tree with histological information or as a 3D visualization of purely histologic information. In this case, the Micro CT information would serve as quality control for the demanding, histology based, 3D reconstruction.

Once achieved, we have a tool for better investigating liver regeneration. Especially the spatial distribution of molecular events and the hepatic parenchyma in respect to vascular regeneration.