The successful establishment of rat OLT model makes some research projects difficult to perform in human be feasible[19]. But the experiment operation process is long and boring, especially by the single operator under direct vision. Beginners need to have enough patience and confidence to train, and achieve stability after a certain number of operations. The goal of many researchers is to achieve proficiency in methods of operation in the shortest possible time, while reducing the number of experimental animals to obtain the purpose of cost-effective[20]. This requires that beginners should not only fully understand the theory and experience of our predecessors, but also put into hard work and appropriate methods of training.
Most beginners have no micro-operation experience, therefore the theory learning and the use of micro-devices, vascular anastomosis techniques, should be carried out as early as possible[21-22]. With regard to the use of surgical microscope or magnifying glass, the author's experience has shown that suprahepatic inferior vena cava of 200-250 g rats was approximately 0.6 cm wide, it can completely sutured under direct vision, and is not limited by the changed view of surgical microscope by the beginners. Much more important is the use of micro-devices and vascular anastomosis methods.
The stability of anesthesia is essential for rat OLT. Donor anesthesia adopted chloral hydrate 3 mL/kg through intraperitoneal injection, quick onset and maintaining a long time, not increasing the secretion of rat airway secretions. The rat’s breath slowed down was taken as the control index, if the effect is not apparent the dose could increase by 0.1-0.2 mL. Receptor was anesthetized using ether inhalation, rapid onset, full fasting before transplantation is an important way to prevent an increase of digestive secretions, atropine is not necessarily required. Rats can be placed in transparent containers with a damp cotton ball, it can be conducive to observe the rat’s anesthetized conditions, and will not make rat suffocation. Once agitation occurred at the time of surgery, the semi-open ether anesthesia was also used in rats, with a quiet and slow breathing as the basis. Before the start of anhepatic phase, rats were allowing more inhaled ether for a few seconds, controlled breathing slowed, basically it is safe through the anhepatic phase. Good surgical exposure is critical to the success model. By use of lumbar pad on-demand, self-made abdominal retractor, we achieve good results.
Donor rat systemic heparinization is necessary. With regard to the timing and dosage of heparin in rats, there is not a unified conclusion. In the present study, intravenous injection of heparin saline was given; according to Kamada et al[23] described the dose of 30 U/mL, a total injection of 2 mL, can achieve satisfactory results. A uniform texture donor liver is a prerequisite to the success of liver transplantation. Any traditional methods of flipping the liver, even if more gentle, will be localized pressure causing mechanical injury in liver tissue and micro-thrombosis, so that affects liver perfusion and the success rate of transplantation[24-25]. In this study, firstly perfusion liver through abdominal aortic, without flipping the liver, warm ischemia time was 0 minute, liver perfusion was uniform and soft. When separation, to deal with portal and hepatic inferior vena cava firstly, although flipping the liver, but no significant effect on quality of donor liver, nor worry about graft unexpected bleeding. Abdominal aortic perfusion have a dual role of perfusion, markedly improved quality of donor liver[26]. Liver perfusion method used intravenous drip hanging to an altitude of about 1 m, taking ≤ 60 drops/minute constant infusion, accurately controlling the amount, speed and pressure of perfusion to ensure the quality of donor liver. Tokunaga et al[27] reported that the effective perfusion is not dependent on the increase of perfusion fluid, slow and evenly infusion is an important guarantee to obtain uniform texture of donor liver.
Suprahepatic inferior vena cava suture is the core technology of liver transplantation, it require skilled and fast[28-30]. Superior inferior vena cava of the donor liver was isolated by a "one-step in vivo" method, it was directly cut close to the diaphragm, without diaphragm ring, that can obviously save the trimming time of the liver ex vivo, reduce the cold ischemia time. And the superhepatic vena cava stump was regular, without diaphragm, vascular plasticity is good which is beneficial for anastomosis and does not easily cause anastomotic stenosis. The two suprahepatic vena cava should maintain a distance about 0.8 cm, it was reported that the superhepatic vena cava of donor liver could be retained approximately 0.2 cm, receptors’ may retain about 0.5 cm[8]. That prevented vein posterior wall adhered and revealed unclearly after corners fixed. When suture, the needle distance should be uniform to avoid wrinkling endoleak of vascular wall, ensure a smooth lining and to reduce post-transplant thrombosis.
Anhepatic phase is also the key to the success of liver transplantation. Liu et al[31] reported that the receptor celiac abdominal aorta was blocked during anhepatic phase , which can increase rat tolerance, thereby enhancing the success rate of the model. We believe that on the basis of technical proficiency, such approach is not necessary. Kamada et al[8] proposed anhepatic phase should not exceed 26 minutes, otherwise the rats were very difficult to survive. The author's experience also shows that a direct relationship between the duration of anhepatic phase and the survival. Moreover, the skilled techniques of portal vein casing is also very important, the key of applying rubber fixation to install sleeve is that donor-receptor vein debouch were in a linear line, first posterior sets, then turned on the anterior wall of homeopathy, so that the portal vein is easy to cover into the cuff. In this experiment, about 1 minute is enough to complete the installation of the portal vein casing.
Biliary tract infection and bile leakage is an important factor for long-term survival after OLT, the length of donor liver bile duct can not be too long, the surrounding tissue can not be over-stripping. Ensure tension-free, non-reversed, drainage unobstructed. Wrapped using the greater omentum with good blood circulation is feasible. Post-operative rehydration was performed mainly in the cases of the longer anhepatic phase. It was not requird if the hemodynamics of receptor was stable after operation. Rewarming after surgery is also very important for the recovery of rat heart and lung function.
In summary, abundant theory, good anesthesia, adequate surgical exposure, good quality of donor liver, skilled vascular suture technique is the key to establish stable rat OLT model.
