Organ ischemia-reperfusion injury by simulating hemodynamic changes in rat liver transplant model. Ischemia-reperfusion injury is a common application after liver transplantation. Patients with extrahepatic organ involvement tend to stay longer in hospital, spend more and haves a worse prognosis.
The development of the complication is closely related to the length of the anhepatic phase of liver transplantation. However, basic research into the ischemia-reperfusion injury after liver transplantation has shown that the survival rate after liver transplantation is inversely related to the degree of injury to extrahaptic organs. Based on the definition of the anhaptic phase, we simulated the hemodynamic change in liver transplantation resulting in ischemia-reperfusion injury of extrahaptic organs.
Of this paper is to provide a detailed description of how to build an animal model of the anhaptic phase in rats, basic research into the ischemia-reperfusion injury after liver transplantation. Dr.Yuan Yuan is responsible for all video animal operations. Two ophthalmic forceps, straight and bending, and interocular lens, IOL forceps, ophthalmic scissors, one to two retractors, cotton swabs, 3-0 surgical sutures, iodoform gauze, suture needles, Biosystems when necessary, and interocular lens, IOL forceps is used to separate blood vessels because of roundheads.
Animal anesthesia machine, de-watering machine, embedding machine, pathological slicer, frozen machine, tissue slicer, air drying oven, neural optical microscope, the scanner, enflurane for general anesthesia, 4%paraformaldehyde solution, anyhydrous ethanol, xylene, high efficiency paraffin wax, hematoxylin-eosin dye solution, hydrochloric acid, ammonia, slide, cover glass, neutral gum and dying reagents, and consumable materials. Pipettes, centrifuge, automatic biochemical analyzer, alanine amino transferase, ALT kit, aspartate, amino transferase, AST kit, creatinine kit. After weighing, the rats are anesthetized with isoflurane by an animal anesthesia machine.
Following abdominal disinfection, make a median incision of three centimeters below the xiphoid process using forceps and scissors. Open the abdominal cavity, expose the liver using a retractor and mobilize the hepatogastric ligament. Use cotton swabs to flip the middle lobe of the liver gently and turn it upward to expose the porta habitis.
Identify the bile duct, portal vein, and the hepatic artery. Push the small intestine towards the left lower abdominal cavity using cotton swaps and move the inferior vena cava to the right renal vein, isolate the portal vein, hepatic artery and the inferior vena cava above the right renal vein with an intraocular lens, IOL, forceps marked with 3-0 silk thread each with a slip knot. Cut open the skin of the left and right lower extremity and expose the femoral vein using ophthalmic forceps.
Slowly inject low molecular weight heparin 625 IU/kg through the femoral vein to heparinize the whole body. Ligate the portal vein, hepatic artery, and inferior vena cava above the right renal vein with number 3-0 sutures, lasting for 45 minutes. Replace the small intestine in the abdominal cavity and cover it with gauze.
Inhalation anesthesia is reduced during these periods. After 45 minutes, release the portal vein, hepatic artery, and the inferior vena cava above the right renal vein. Suture the muscle and skin layer by layer and terminate the inhalational anesthesia.
Provide post-operative analgesia using subcutaneous morphine of 5 mg/kg every four hours Observe the rat until awake and keep fed under a temperature of 25 plus or minus two degrees Celsius and humidity of 50 plus or minus 10%Animal heating lamps is necessary. In this animal model, the rats were randomly divided into five groups according to ischemia for 15 minutes, I 15 group, 30 minutes, I 30 group, 45 minutes, I 45 group, 60 minutes, I 60, and a sham group with 10 rats in each group. Their survival rate of each group was observed 14 days after the operation.
All rats survived in the I 15, I 30 and sham groups. Eight survived for 14 days in the I 45 group and only two survived in the I 60 group. The results suggested that the rats could tolerate the anhepatic phase for 45 minutes at most.
During the experiment, Biosystems was used to record the changes of heart rate and blood pressure, right internal carotid artery intubation before and after the anhepatic phase. We found that the heart rate and mean arterial pressure, MAP of rats, changed dramatically after vascular ligation. After ligation, hepatic ischemia, congestion, and edema in the intestines, gastric varices and splenomegaly were evident.
80 rats were randomly divided into eight groups including ischemia for 45 minutes, T0, reperfusion for six hours, T6, 12 hours, T12, 24 hours, T24, 48 hours, T48, 72 hours, T72, seven days, T7, in 14 days, T14, respectively. The kidney, pancreas, small intestine, heart and lung tissues were taken and stained with HE after rats were sacrificed. Except for the heart, pathological scores were given according to references, respectively.
The results showed that the time of maximum injury of the extrahepatic organs varied. It was six to 24 hours after operation for the pancreas and 24 to 48 hours for the lungs. The intestinal tract and kidney were most severely injured after 45 minutes of ischemia.
There was no obvious abnormality of the intestinal mucosa 24 hours after the operation and the kidneys recovered after 48 hours. As for the heart, with the passage of time after reperfusion, local myocardial cell narcosis, cell fragmentation and dissolution, inflammatory cell infiltration, and local vasodilation and congestion were found in the tissue by 24 to 48 hours after the operation. Serum was collected and the levels of ALT, AST, creatinine, and amylase were detected by an automatic biochemical analyzer.
All indicators peaked at 24 to 48 hours, unlike the pathological changes. Although the levels were basically normal 48 hours after the operation, the pathological damage was still continuing. For summarize, the model in rats is simple and easier to use without microsurgery and provides basis to fundamental research of the ischemia-reperfusion injury of extrahepatic organs after hepatic ischemia.
