The overall aim of this procedure is to perform partial orthotopic liver transplantation in rats as a reproducible experimental model for transplantation research. This is accomplished by first ex explaning, the liver from the donor rat. The second step is the ex vivo preparation of the liver graft, including a 50%liver resection.
Next, the native liver of the recipient rat is excised. The final step is the implantation of the partial liver graft into the recipient rat. Ultimately, serum alanine, aminotransferase levels are measured to assess hepatocellular damage to the liver graft after transplantation.
Generally, individuals new to this procedure, we struggle because of the complexity throughout that requires advanced microsurgical skills. To begin this procedure, place the anesthetized donor rat on a heating pad after confirming by a toe pinch that the animal is fully anesthetized, fix the upper arms using the magnetic fixator retraction system. Shave the fur from the entire abdominal area of the rat and sterilize the corresponding skin with a povidone iodine solution.
Open the abdomen by midline incision with bilateral extensions. Place a five milliliter syringe under the back of the rat so that the supra hepatic vena caver is elevated Ventrally using a mosquito forceps clamp and pull the process towards the head and apply the subcostal retractors to open the surgical field. Due to the need to frequently manipulate the retractors and the five milliliter syringe during the procedure, surgical drapes are not used around the incision working under a surgical microscope at 16 x magnification, dissect the falciform ligament and left triangular ligament like eight and divide the left phrenic vein, retract the median and left lateral lobes upward with a wet gauze swap.
Using bipolar forceps coagulate and divide the paraesophageal vessels between the left lateral and anterior coordinate lobe. Move the intestines outside the abdomen to the left side of the rat and cover them with a wet gauze swab. Swab, retract the right lateral lobe upward with a wet gore swab.
Isolate the infra hepatic vena caver from the retroperitoneal tissue and ligate the right adrenal vein, which will be divided later just before the graft removal to insert a stent into the bile duct, first ligate the bile duct at the level of the branching of the gastroduodenal artery. Make a small incision in the anterior wall of the bile duct proximal to the ligated point then while holding the anterior wall of the incision with a straight micro forceps in the left hand, use a curved micro forceps in the right hand to insert a stent into the duct. Secure it with a six zero silk thread.
Keep one of the cut ends of the thread on the bile duct at a length of four millimeters so that the thread can be held during the later anastomosis. Liberate the poral vein from the pori and splenic veins by ligating and dividing them ligate and divide the gastroduodenal artery. Then isolate the common hepatic artery from the pancreatic head to its root.
Rotate the liver to the right with cotton swabs and dissect the ligament around the back of the liver and esophagus us at the completion of preparations. For liver excision. Remove the retractors, the mosquito forceps for the xiphoid process and the five milliliter syringe under the back of the rat.
Return the intestines to the abdominal cavity. Inject 500 IU of heparin natrium in two milliliters of normal saline solution through the penile vein. About three minutes later, reset the five milliliter syringe, the mosquito forceps and the retractor ligate the common hepatic artery proximal to its root.
Keep one of the cut ends of the thread ligated for the common hepatic artery. Long after clamping the infra hepatic vena caver. Close to the right renal vein with a mosquito forceps.
Clamp the portal vein with a disposable micro vessels clamp below the stump of the splenic vein. Incise the anterior wall of the portal vein and insert 18 gauge catheter into the portal. Vein perfuse the liver in situ with 60 milliliters of cold histidine trytophan ketoglutarate solution.
At a hydrostatic pressure of 20 centimeters, water immediately cut the diaphragm, the intrathoracic vena caver, and the anterior wall of the infra hepatic vena caver open to allow the perfusion solution to be rinsed out of the liver. Clamp the infra hepatic vena caver with a micro vessels clamp just below the liver. Excise the liver by dissecting the infra hepatic vena caver.
The poral vein, the diaphragm, the remaining ligaments, the right adrenal vein and the common hepatic artery. Place the excise liver in cold HTK solution in a metal cup mounted in a plastic box full of crushed ice org. Procedures for the liver graft are performed in the metal cup filled with ice cold HTK solution for the attachment of a cuff to the portal vein clamp the portal venous trunk with a DeBakey bulldog clamp and place the clamp in a bridging position over the cup.
Put the poral vein through the cuff and clamp the al vein again together with the extension of the cuff at the 12 o'clock position, avert the wall of the poral vein over the cuff to position the stump of the splenic vein outside of the cuff at the seven o'clock position and secure the portal vein with a six zero silk thread to insert a stent into the hepatic artery. Fix the liver by clamping both edges of the diaphragm by forceps and pull the common hepatic artery straight by holding the ligated thread with the DeBakey bulldog clamp with straight micro scissors. Make a small incision in the anterior wall of the common hepatic artery with the left hand.
Hold the anterior wall of the incision with a straight micro forceps and with the right hand, insert a stent into the common hepatic artery. Using a curved micro forceps, secure the stent with a six zero silk thread and keep one of the cut ends of the thread at a length of four millimeters. Flush the liver through the arterial catheter with five milliliters of cold HTK solution for the 50%liver resection.
First clamp the posterior coordinate lobe with a mosquito forceps to fix it in place. Then ligate. Its pedicle with a four zero silk thread and resect the lobe.
The anterior corate lobe is removed in the same manner. Next, rotate the plastic box 90 degrees. Clamp the right edge of the diaphragm and the left portion of the median lobe.
Make a small incision at the upper edge of the border of the bilateral portions of the medial lobe and then remove the left portion after ligation. After ligation of the pedicle with a four zero silk thread, remove the left lateral lobe, cauterize the resected liver surface carefully with bipolar forceps. The liver mass is now reduced by approximately 50%for plasty of the supra hepatic vena cava.
First, fix the position of the liver by clamping both edges of the diaphragm with a mosquito forceps. Then trim the anterior wall of the supra hepatic vena cava by removing the corresponding diaphragm. Attach 2 7 0 polypropylene sutures from the outside to the inside of both corners as stay sutures for the later anastomosis.
After that, trim the posterior wall of the supra hepatic vena cava. Store the liver graft at four degrees Celsius in HTK solution in a cold water bath. Prepare the recipient for surgery in a similar way as for the donor operation and open the abdomen by midline incision.
Place a wet gore swab over the right side of the duodenum and whole intestines. To obtain a surgical field around the infra hepatic bena caver. Put the left lateral and median lobes into the left sub phrenic cavity and retract the right lateral lobe upward with a wet gaze swab.
Isolate the infra hepatic vena caver from the retroperitoneal tissue legate and divide the right adrenal vein with the wet gauze and cotton swabs. Rotate the liver to the left and dissect the ligament around the back of the liver. Transect the bile duct just below the branch from the chordate lobe.
Keep one of the cut ends of the thread ligated for the bile duct of four millimeters long ligate and divide the gastroduodenal artery and the proper hepatic artery at a three millimeter distance to the branching from the common hepatic artery to make a wide structure of the artery at the end of the common hepatic artery clamp the infra hepatic vena caver with a metal micro vessels clamp just above the right renal vein and the portal vein at the level of its bifurcation in the liver hilum by a mosquito forceps clamp the supra hepatic vena cava together with the diaphragm by a peripheral vascular clamp and fix the clamp in a lump of oil-based clay. Reduce the anesthesia with isof fluorine to 0.4 volume percent. Excise the recipient native liver by dissecting the sup hepatic vena cava at the border between the supra hepatic vena cava and the liver.
The portal vein just above the jaw of the mosquito forceps and the infra hepatic vena cava slightly below the middle point between the liver and the right renal vein. Place the liver graft ortho topically. The reconstruction of vessel is the most difficult and important part of this procedure.
It has to be done very carefully but quickly For the anastomosis of the supra hepatic vena cava by continuous suture. Place the stay suture on the recipient supra hepatic vena cava from the inside to the outside by using the attached seven zero polypropylene at the right corner of the graft, followed by tying a knot in the same way. Place the second stay suture at the left corner.
This will be the starting stitch of a running suture. To widen the anastomosis, grasp and maintain the sutures using DeBakey bulldog clamps at both corners with gentle traction superior laterally. Next, pierce the suture at the left corner through the wall on the graft side from the outside to the inside closely to the knot outside and suture the posterior row of the satic vena caver Intraluminal with seven to eight stitches to the right corner.
Make the first few stitches carefully so that the inside lumens face each other at the right corner. Pierce the seven zero polypropylene through the vessel on the graft side to the outside. Suture the anterior row from the outside from right to left with approximately 10 stitches.
Flush the inside with lactated ringer solution. To remove air bubbles, make the last stitch close to the stay suture and then tie them together for the reconstruction of the portal vein clamp the portal vein by a micro vessels clamp and fix the mosquito forceps in the clay. Next inci the anterior wall of the portal vein just below the jaw of the mosquito forceps.
Wash the inside of the recipient portal vein and the cuff with lactated ringer solution. Insert the cuff into the portal vein and secure it with a circumferential six zero silk thread. Lastly, reperfuse the liver.
Remove the five milliliter syringe from the back of the rat and increase the concentration of isof fluorine to 0.8 volume percent for the reconstruction of the hepatic by a stent technique. First, hold the thread of the proper hepatic artery by mosquito forceps and fix it in the clay. Then clamp the recipient common hepatic artery.
Make a small incision in the bifurcation of the Y structure at the end of the common hepatic artery to make a funnel shaped opening. Hold the stent placed in the graft common hepatic artery with a curved micro forceps. Slide the stent into the recipient common hepatic artery and secure it with a six zero silk thread Tie, one end of this thread and the four millimeter thread on the graft common hepatic artery together so that both common hepatic arteries get closer to each other with reduced tension of the anastomotic site.
After that, release the clamp amp for the anastomosis of the infra hepatic vena caver by a continuous suture anastos in the same fashion as for the supra hepatic vena caver, but use more stitches with finer bites. After dec clamping, increase the concentration of anesthesia to 1.0 volume percent for the reconstruction of the bile duct. Hold the thread of the recipient bile duct by a mosquito forceps and fix it in the clay.
Make a small incision in the bile duct at the appropriate level so that the reconstructed bile duct would not be too long. Insert the stent and secure it with a six zero silk thread. Tie this thread and the thread on the graft duct.
Together now all the reconstruction procedures are finished. Close the abdominal incision by continuous four zero Vicryl sutures into layers. Immediately after the operation, treat the recipient rat with a subcutaneous injection of sphero sodium and buprenorphine in a total of 1.5 milliliters of normal saline solution.
Allow the rat to recover for 60 minutes in a special intensive care unit cage with warm air and an oxygen supply before returning it to a normal cage. All recipient rats survived until planned euthanasia for blood sampling at 1 3 24 and 168 hours after portal reperfusion. Serum samples were analyzed for alanine aminotransferase levels which reflect the degree of hepatocellular damage to the liver grafts after transplantation.
As shown in this graft, a LT levels reached a peak at 24 hours and then declined to within normal limits of 168 hours. After this video, you should have a good understanding of how to perform partial topic liver transplantation in rats as a reproducible experimental model for transplantation research.
