Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Transplantation is often the only effective therapy for end stage organ disease. To understand the complex mechanisms inherent to injury of in-liver transplantation, robust small animal transplant models have been developed to recapitulate the physiology and pathophysiology of allograft injury. Innovative strategies will need to be developed to deeply interrogate the mechanistic pathways involved in tissue injury observed in transplantation and to expand the donor pool.

The use of mice to study transplantation is vital as it allows for the use of gene edited and transgenic mouse lines to study the role of specific molecular pathways on the pathophysiology of allograft injury. There are a number of technical challenges with mouse liver transplantation, including the small diameter of the vascular anastomosis, and the subsequent risk of thrombosis leading to allograft failure. While many technologies such as ultrasound angiography have been employed, microcomputer tomography has advantages including the ability to attain high resolution images of the vascular anastomosis in the entire abdomen.

These studies can be performed longitudinally to evaluate vascular patency and profusion of the liver over time. There are many challenges to performing accurate and clear micro CT angiography studies in rodents. We have found the use of cardiac gating and the adjustment of iso fluorine concentrations to decrease the respiratory rate produce the clearest images.

We have also found that utilizing rodent specific contrast timing for specific phases has also improved visualization. To begin, place the anesthetized donor mouse under the high powered microscope. Using a pair of scissors, perform a midline laparotomy from the xiphoid process to the pubic synthesis.

Then make a transverse incision below the ribs to create a cross like pattern. After retracting the xiphoid process, eviscerate the intestines and place them on the wet gauze sponge on the left side of the abdominal cavity. To mobilize the liver, separate it from all its ligamentous attachments.

Once the proper hepatic artery or PHA is exposed, using curved forceps, skeletonize it. After ligating the PHA, dissect the entire length of the common bile duct or CBD. Insert the bile duct stent in the CBD and secure it with a 10-0 nylon suture.

Then ligate the distal aspect of CBD. Using a wet gauze sponge, retract the right hepatic lobe toward the xiphoid and expose the inferior vena cava or IVC. Mobilize the intrahepatic inferior vena cava or IHIVC away from the retroperitoneum and cauterize the right adrenal vein.

Once the right renal artery and vein are dissected and ligated, cut both and the remaining ligamentous attachments. After removing the right kidney, inject 0.5 milliliters of cold histidine tryptophan ketoglutarate solution with 100 units of heparin through the portal vein or PV.After one minute of injection, cut the portal vein superior to the splenic and superior mesenteric veins. To perfuse the liver, slowly inject a cold histidine tryptophan ketoglutarate preservation solution into the IHIVC until the fluid coming from the PV is clear.

Place a micro clamp or fine suture on the IHIVC just superior to the right renal vein, and cut just inferiorly to the clamp. Once the CBD is cut distally to the stent, identify and ligate the cystic duct with a 10-O nylon suture. With forceps, grasp the dome of the gallbladder, and using spring scissors, cut the cystic duct above the suture to complete the cholecystectomy.

Retract the liver inferiorly to expose the suprahepatic inferior vena cava or SHIVC and cut it. Preserving an adequate length for the anastomosis in the recipient mice. After removing the additional ligamentous attachments, place the liver in cold saline.

After shaving and disinfecting the surgical site of an anesthetized recipient mouse, use a midline laparotomy to expose the proper hepatic artery or PHA. Once the liver is mobilized, ligate the left phrenic and paraesophageal vasculature with a 10-0 nylon suture. Next, retract the liver inferiorly and dissect the suprahepatic inferior vena cava or SHIVC from the retroperitoneum.

Retract the liver superiorly and dissect the infrahepatic inferior vena cava or IHIVC from the retroperitoneum. Then ligate the right adrenal vein and expose the liver hilum. Ligate the PHA with 10-0 nylon suture.

Dissect the common bile duct or CBD from the portal vein or PV and ligate it with a 7-0 suture near the bifurcation, creating adequate space for the CBD anastomosis. Once the IHIVC is clamped, temporarily ligate the PV with a 7-0 suture. Using a 30 gauge needle, flush 0.5 milliliters of heparin saline into the native liver's portal vein.

Once the SHIVC and IHIVC are clamped, cut the native SHIVC, IHIVC, PV and any remaining ligamentous attachments to the recipient's native liver. Place the donor liver allograft in the abdominal cavity and retract the hilum of the donor allograft to expose the PV.Once the donor and native PV are flushed, insert the previously made cuff of the donor PV into lumen of the recipient liver PV.After securing the anastomosis, perform a hand sewn anastomosis using a 10-0 nylon suture with the donor and native SHIVC. Finally, complete anastomosis of the superior wall.

Then remove the ligation suture of the PV, followed by the vascular clamps from SHIVC to begin reperfusion. Perform hand sewn anastomosis for IHIVC as demonstrated for SHIVC. After completing the reconstruction, remove the micro clamp or suture from the IHIVC to reperfuse.

Once a ductotomy is created, insert the donor CBD stent into the recipient CBD lumen and secure the anastomosis with a 10-0 suture. To begin, use tape to attach an ECG pad to the left, right for and right hind limbs of the anesthetized mouse with the transplanted liver. To ensure proper QRS complexes in the source setup tab, select ECG as the source check to logic lead and choose the lead that offers the clearest ECG curve.

Under the trigger setup tab, confirm that Channel A is checked, then choose the desired output and select Channel A trigger parameters. Then under the display setup tab, adjust the display setting for a clear signal view. Take the box next to ECG set to 500.

Set the presented imaging parameters. Ensure that the threshold for ECG is below the hysteresis value and above the peak of the ST segment. After placing the mouse into the scanner, press Update Image.

Then acquire an x-ray scout image of the mouse to select the appropriate field of view and anatomical scan coverage. To perform micro CT angiography image acquisition, set the presented parameters. To reconstruct the micro CT images, upload the image and set blue bars to straddle the anatomical area of interest.

Open the reconstruction utility screen and select the data file to reconstruct. Turn on the volume of interest outline to optimize the image boundary. Then choose a 40 micron voxel size, hand projector filter and Gaussian volume filter, 80 micron.

Select Slice Preview to reduce file size and determine the field of view based on the volume of interest outline. Next, coelomate the x-ray image to remove unwanted space or areas. Go to Advanced and click Image-Based Gating.

Choose 10 phases for cardiac gating. Update the file name and then push the volume reconstruction button. Finally, rename the file to include parameter choices and select volume reconstruction.

A successful micro CT angiography scan provides detailed information on vascular anastomosis. The contrast seen through the IVC and PV demonstrates the patency of the suprahepatic and intrahepatic anastomosis. Additionally, 3D reconstruction of successful micro CT scans provides detailed information about vascular anatomy.