Rat pancreas transplantation modeling
Rat pancreas transplantation modeling
Rat pancreas transplantation modeling is mainly used for basic research on pancreas transplantation. Pancreas transplantation is currently an effective clinical treatment for type I diabetes mellitus and a surgical means to save type II diabetes mellitus in advanced stage with renal insufficiency, therefore, pancreas transplantation has been receiving widespread attention, and so far there are still many problems surrounding pancreas transplantation that need to be solved, and the basic research of these problems need to be carried out on suitable animal models, among which rats are one of the popular experimental animals in the field of organ transplantation because of their low cost, easy source, and similar immune system to humans. The rat is one of the popular experimental animals for organ transplantation because of its low cost, easy source, and similar immune system to that of humans. However, the establishment of rat pancreas transplantation model is a difficult experimental surgical technique with complicated operation and low success rate. Therefore, it is necessary to establish a standardized, easy-to-operate, stable and successful pancreas transplantation model to facilitate the basic research of pancreas transplantation.
Operation method
Rat pancreas transplantation modeling
Principle
Successful pancreas transplantation can provide diabetic patients or test subjects with pancreatic tissue with normal function, which can physiologically regulate insulin secretion, maintain normal blood glucose, and prevent or even reverse the occurrence and progression of diabetic complications. Currently, most of the models established are that the venous return of the donor pancreas adopts the return of body circulation by end-to-end anastomosis between the donor portal vein and the recipient inferior vena cava.
Materials and Instruments
Donor and recipient rats Move I. Selection of donor and recipient rats Different strains of animals were selected according to different research purposes. In the homologous transplantation model, two pure strains of healthy rats were generally used, such as Lewis rats, Brown Norway rats (BN), DA rats and so on, and Wistar and SD rats were also more frequently used in China. The recipient rats were prepared as diabetic models before the transplantation surgery. Establishment of diabetes model in rats 1. Method 1: 10% tetroxine was injected subcutaneously into the inguinal area of rats for two consecutive days at a dose of 100mg/kg, and blood glucose was measured every other day, and those whose blood glucose continued to be >16.7mmol/L for one week were treated as qualified diabetic recipient rats. 2. Method 2: Streptozotocin was injected intramuscularly at 70mg/kg, and those with qualitatively strong positive urine glucose and non-fasting blood glucose >20mmol/L lasting for 10 days were regarded as stable diabetic models. 3. Method 3: Recipient rats were injected with streptozotocin 55mg/kg intravenously at one time, and the non-fasting blood glucose was measured from the 7th d. If the blood glucose concentration exceeded 22mmol/L for 3 consecutive days, it was judged as the success of diabetes induction in rats. Preoperative preparation and anesthesia Before the operation, the donor fasted for 24h and drank 50g/L dextrose saline, the recipient fasted for 24h and drank 25g/L dextrose saline, and 1% sodium pentobarbital 40mg/kg was injected intraperitoneally to anesthetize both donor and recipient, and ether was added to assist the anesthesia when the anesthesia was too shallow. IV. Pancreatectomy 1. After successful anesthesia, enter the abdomen through the mid-abdominal incision. 2. 2. Turn the stomach upward, ligate and cut off the vascular connection between pancreas and greater omentum and the arteries and veins of gastric omentum, ligate the proximal end of duodenum, cut off the pylorus, ligate and cut off the left gastric blood vessel and esophagus at the junction of esophagus and stomach, ligate and cut off the short gastric artery and resect the whole stomach. 3. 3. The lower edge of the pancreas was freed from the spleen toward the head of the pancreas, the pancreatic duct was preserved into the duodenal duct for about 1.5cm, and the superior mesenteric vessels were ligated and cut off. 4. Ligate and cut off the common bile duct and the innominate hepatic artery, and free the portal vein to the bifurcation of the hepatic hilum. 5. Open the posterior peritoneum in front of the abdominal aorta, ligate and cut off the right and left renal and adrenal arteries, completely free the abdominal aorta where the celiac artery and the superior mesenteric artery are located, and inject heparin (25 U/L) into the dorsal penile vein. 6. Ligate the abdominal aorta below the opening of the superior mesenteric artery and above the opening of the celiac artery, cannulate the abdominal aorta below the opening of the superior mesenteric artery, and irrigate the grafts with 4℃ Euro Collins solution (or 4℃ equilibrium solution of 25U/L heparin solution), and at the same time, transverse the portal vein, irrigate the grafts until the effluent from the severed end of the portal vein turns clear and the grafts change from reddish to pale in color. 7. After the completion of irrigation, the graft and spleen were cut intact, and the graft was stored in Euro Collins at 4℃. V. Donor pancreas implantation 1. Vena cava - bladder drainage type pancreas transplantation (1) The lower abdomen was entered through a median incision, and the bowel was pushed upward with saline gauze, the bladder and seminal vesicle glands were pushed downward and secured with a pull-hook, and the abdominal aorta and inferior vena cava about 1.5 cm above the bifurcation of the iliac blood vessels were freed, and heparin (25 IU/L) was injected through the dorsal vein of the penis until the whole body was heparinized. (2) The upper and lower ends of the free vessels were blocked by vascular clips, the donor portal vein was anastomosed to the recipient's inferior vena cava with a 9-0 noninvasive microsuture, and the donor abdominal aorta was anastomosed to the recipient's abdominal aorta with an 8-0 noninvasive microsuture. When the pancreas was moved during the anastomosis, the spleen connected to the transplanted pancreas was pulled to avoid direct clamping of the transplanted pancreas. A small amount of ZT glue can be applied to the surface of the anastomosis and the donor abdominal aorta to close the anastomotic gap and the nutrient vessels of the abdominal aorta, resect the spleen attached to the graft, and loosen the vascular clamps distally and then proximally to restore the blood supply of the grafted pancreas. (3) The donor duodenal segment was anastomosed side-to-side with the top of the recipient's bladder (3-0 silk single-layer continuous suture), the anastomotic opening was about 0.5 cm, and the graft was covered with the greater omentum, and the abdomen was closed to end the operation. 2. Portal vein-intestinal drainage pancreas transplantation (1) Mid-abdominal incision into the abdomen, open the abdominal cavity, below the left renal vein, isolate the abdominal aorta, longitudinally clamp the segment of the artery with a modified Lee's forceps, longitudinally incise the artery to close it, flush the lumen with a balanced salt solution containing heparin, isolate the superior mesenteric vein at a low level, proximally clamp it with microvascular clamps, distally ligate it with a 3-0 silk ligation, and at the same time at the same level and height, ligate the superior mesenteric artery. (2) The superior mesenteric vein was cut off immediately after the ligation of the silk thread, the lumen was also flushed with a balanced salt solution containing heparin, the ischemic small intestine and the ileum were cut off (30--40%), and the two severed ends were anastomosed end to end with a 7-0 nylon thread, and the grafts were removed from the iced water, and the grafts were covered with wet gauze with ice shavings. (3) Venous suture was performed first, and it should be ensured that there was no torsion after venous anastomosis. The portal vein of the donor and the superior mesenteric vein of the recipient were sutured end-to-end interruptedly with 10-0 nondestructive microscopic suture, and in the process of suture, the venous lumen was irrigated with saline and the edges of the blood vessels were gently separated with microscopic forceps to avoid narrowing of the anastomosis. The abdominal aorta of the pancreas donor and the abdominal aorta of the recipient were sutured end-to-end with 9-0 non-invasive microsuture. (4) After the anastomosis was completed, the venous clamp was released first, then the arterial clamp, and the arterial anastomosis was lightly compressed with a dry gelatin sponge for 1--2 min. (5) Finally, the duodenal opening of the donor pancreas was anastomosed end to end with the proximal jejunum of the recipient, and the other end was ligated. The spleen attached to the donor pancreas was resected, and the abdomen was closed to end the operation. Postoperative monitoring and management The function of the transplanted pancreas was monitored according to the needs of the experimental study design, and if the transplanted pancreas functioned normally, the fasting blood glucose value was normal and the C-peptide value was normal before rejection occurred. Caveat 1. After the operation, pay attention to the heat preservation of the recipient animal, which can be placed on the electric blanket and radiated by the heat of the electric lamp for 24 h. Generally, the animal wakes up from anesthesia within 1 h after the operation. 2. 30--40ml of sugar saline containing 25g/L glucose and 4.5g/L sodium chloride (containing 150mg of oxpiperazine penicillin) should be injected subcutaneously every day after the operation, and normal diet should be started in 36-48h depending on the condition of the animals. Common Problems 1. Rats that have undergone successful surgery are generally able to move normally after they have awakened from anesthesia and are responsive to external stimuli. After blood glucose returns to normal, the rats' fur becomes shiny and their weight gradually increases. Rats with unsuccessful surgeries showed messy and dull-colored fur, arched backs, little activity, and poor responsiveness to external stimuli. 2. Before the formal modeling experiments, basic microsurgical skills were practiced and pre-tests were conducted for more than 4 times, aiming to further improve the microsurgical techniques and find out the best surgical methods and conditions. Formal experiments were conducted after the surgical plan was matured. For more product details, please visit Aladdin Scientific website.
Streptomycin 10% tetracycline Glucose saline 1% pentobarbital sodium Ether 25 U L heparin solution Sterile water
Microsurgery kit Surgical microscope Ophthalmic scissors Surgical instruments
3. Source paper "Establishment of rat pancreas transplantation model and experimental study on the role of chemokines in transplantation rejection".