动物造模,肾功能衰竭模型 z-bio 2021-03-26 716

　　1. Radical nephrectomy (1) Replication method Adult rats weighing about 250 g are anesthetized by intraperitoneal injection of 30 mg/kg body weight of sodium pentobarbital. After anesthesia, the rat was fixed on the back and the abdominal cavity was prepared. Disinfect, cut the skin along the left para-abdominal incision, expose the left kidney, cut the upper and lower poles of the capsule, remove one third of the kidney with the upper and lower poles, and use a gelatin sponge. The wound is compressed to stop bleeding, flushed with normal sodium and sutured by routine surgery. The muscle layer and skin close the abdominal cavity. One week later, the rat was anesthetized in the same manner, its back was fixed, and the skin was cut along the right abdominal incision to expose the right kidney. After ligating the renal pedicle, remove the right kidney. The incision was sutured and the abdominal cavity was closed by conventional surgery. Before and after modeling, the model rats were placed in a metabolic cage, and urine was collected for 24 hours to determine the 24-hour protein quantification. Venous blood samples were collected at the designated time of the experiment to measure blood creatinine (SCr) and blood urea nitrogen. (BUN); After anesthesia, the animals were sacrificed and collected by blood sampling. The animal’s residual kidney tissue specimen was fixed in a fixative, embedded in paraffin, and conventional tissue sections were prepared, stained with HE, and observed with an optical microscope.

　　(2) The characteristic of the model after the operation is that the rat wound has no bleeding or infection, but the weight is significantly reduced, the activity and food intake are reduced, and the hair is loose. One week after modeling, the animal may have proteinuria, serum urea nitrogen, creatinine and 24-hour urine protein gradually increased. Six weeks after the operation, the model animals entered the compensatory phase of renal function, electrolyte imbalance and anemia (decreased RBC, HGB and HGT, pale ears and tail), and increased blood pressure. Blood pressure reached the standard for renal hypertension within 8 weeks. The serum urea nitrogen, creatinine, 24-hour urine protein and blood pressure in the model increased significantly. One group of animals was 10 weeks after surgery. 140mmHg can reach 13.7kPa or more. Histopathological observation under the microscope showed that glomerular glomerulus telangiectasias, glomerular cell hypertrophy and foot process edema and fusion, moderate to severe glomerular mesangial hyperplasia and focal segments Related, manifested as glomerular degeneration and sclerosis, and showed glomerular mononuclear lymphocyte infiltration. Interstitium of renal tubules.

　　(3) Comparative medicine. After most nephrectomy, the nephrons have hemodynamic changes, leading to proteinuria filtration in the remaining nephrons, which ultimately damages the glomeruli and causes glomerular sclerosis. The main characteristics of the glomeruli are It is sclerosing CRF. The model is based on the glomerular filtration theory of renal failure, has the characteristics of glomerular hypertrophy and sclerosis, its performance is close to clinical reality, the preparation method is simple and easy, and the stability and reproducibility are good. , Wide application range. However, this model requires a two-step operation and is prone to side effects such as animal bleeding and death. This model takes a long time to prepare, and it is not easy to standardize the operation. Currently, in addition to 5/6 nephrectomy models, most CRF nephrectomy models can be created using 3/4, 4/5, and 7/8 nephrectomy. However, various nephrectomy models have unique characteristics in terms of timing of renal failure, changes in blood creatinine, urea nitrogen, urine protein content, and pathological damage to kidney tissue. This model has practical value in the timing of clinical CRF (reduction of renal tissue perfusion, ultrafiltration, inhibition of glomerular mesangial cell proliferation and antioxidants).

　　2. Methods of ligation and resection of renal artery branches

　　(1) Reproduction method Adult rats weighing 200 to 300 g were anesthetized intraperitoneally with 30 mg/kg body weight of sodium pentobarbital, and then lying on their backs after anesthesia. An incision was made along the left abdomen to fix the abdominal surgery area, regularly disinfected and depilated, opened the skin, exposed the left kidney after entering the abdomen, and separated the renal artery under the operating microscope. Keep the artery in the left kidney and ligate the rest. On all branches, the incision was sutured by conventional surgery and the abdominal cavity was closed. Two days later, the abdomen was anesthetized in the same way, the right renal hilum was ligated with 4-0 silk thread, the right kidney was taken out, and the abdomen was closed surgically. Before and after modeling, the model rats were placed in a metabolic cage, and urine was collected for 24 hours to determine the 24-hour protein quantification. Venous blood samples were collected at the designated time of the experiment to measure blood creatinine (SCr) and blood urea. Nitrogen (BIJN); After anesthesia, the animals are killed by blood sampling, the collected animal kidney tissue specimens are fixed with a fixative, embedded in paraffin, and conventional tissue sections are prepared, stained with HE, and then stained with an optical microscope. Observed.

　　(2) Postoperative model characteristics: the rats have no hemorrhage or infection in the wound, the weight is obviously reduced, the activity and food intake are reduced, and the hair is thin and uneven. The blood creatinine and blood pressure of model rats increased significantly after surgery, proteinuria might occur, and the kidneys were ligated to cause modern compensatory hypertrophy. Histopathological observation under the microscope showed that it was similar to the CRF model of gross nephrectomy. Pathological changes of glomerular vitreous degeneration and sclerosis. The process of creating this model is similar to most nephrectomy procedures. Most of the remaining kidneys after ligation are 1/6, 1/12, and 1/16, which is the same as removing 5/6, 11/12, and 15. /Kidney 16,

　　(3) Comparative medicine. The model replicated by this method has less bleeding than the CRF model of total nephrectomy, but the preparation method requires microsurgical techniques, the operation is complicated, the operation is easy to learn, and the model animal has a high mortality rate. . The postoperative hypertension is very serious, and the pathological damage of the kidney is also very serious. This model can be used to create CRF models with different degrees of disease and clinical symptoms, and to evaluate drug dosage and efficacy when using drugs to treat different degrees of CRF.

　　3, freezing plus excision method

　　(1) Preoperative measurement of anesthetized adult male rats without proteinuria with a weight of 200-300 g by intraperitoneal injection of sodium pentobarbital 30 mg/kg by replication method. After anesthesia, the rat was placed on the operating table in a prone position, and the operating area on the back was disinfected and depilated regularly. An incision is made to separate the muscle layer on the left side of the back, exposing the left kidney. Use the left side. Lift up gently from the corresponding part of the abdomen with your index finger, and the kidney will slide out. On the outside of the skin incision, peel off the renal fascia, use a cold knife to immerse the upper and lower poles of the animal's left kidney and the outer front and back four parts of the animal's left kidney into a liquid nitrogen bottle. After freezing each area for 40 seconds, the kidneys were reset and stratified. Suture the surgical incision. Two weeks later, anesthesia was performed in the same manner, the renal pedicle was ligated, and the right kidney was taken out. A stable CRF model can be established 6-12 weeks after surgery. For blood biochemical measurement, blood samples were collected at a given time in the experiment, left kidney tissue samples were collected, fixed in fixative, and routine tissue sections were performed and examined under an optical microscope.

　　(2) There is no wound infection or bleeding after the operation, but the weight loss is obvious, the food intake and activity are reduced, and the hair becomes loose and uneven. After two weeks, the blood creatinine and blood urea nitrogen of the model animals began to increase, indicating a gradual development. The blood creatinine and blood urea nitrogen peaked in 4 to 6 weeks and kept the peak fluctuations. Histopathological observation under the microscope showed glomerular hypertrophy, vitreous degeneration and sclerosis, and mesangial matrix hyperplasia and hypertrophy. (3) Comparative medicine. The model design has higher surgical requirements, and there is the disadvantage of bleeding when establishing a CRF model through subtotal nephrectomy. The manufacturing method is simpler than the CRF model of total nephrectomy, but the degree of cold damage to the kidney, such as the time of the cold knife in liquid nitrogen, the range of the cold knife placed on the kidney surface, and the size of the contact surface. Will be as follows. Everything will affect the degree of damage in the model. If there is a difference, a unified standard is needed when creating the model. This model can create CRFs of different severity by controlling the freezing time, without the risk of bleeding during surgery, but requires special equipment to manufacture and induce immunity from necrotic tissue in situ, so this mechanism cannot be ruled out. This model is suitable for clinical research of CRF drug treatment and screening.

　　4. Electrocoagulation and resection (1) The replication method is to inject pentobarbital sodium intraperitoneally at a dose of 30 mg/kg body weight to anesthetize adult mice weighing about 25 g. After anesthesia, the mouse is angry and immobilized. Regarding regular disinfection and depilation of the surgical site on the back and abdomen of the operating table. An incision was made on the right side of the dorsal side to expose the right kidney. Peel off the surrounding fat tissue and kidney capsule to form a needle. Electrocautery is used to coagulate tissues other than 2 mm around the hilar. The depth of the entire surface of the right kidney is about 1 mm. The abdominal kidney returns to the abdominal cavity, and the muscle layer and skin are sutured regularly. surgery. The operation process is controlled within about 10 minutes. Then, after 12 to 15 days, a second operation was performed in the same manner, anesthetized, and after entering the abdomen, the left kidney door was ligated with silk thread, the left kidney was removed, and the abdominal cavity was closed. Surgery. The modeling process takes approximately 18 weeks. Blood samples were collected at the scheduled time of the experiment for blood biochemical measurement. Kidney tissue samples were collected, fixed in fixative, sliced regularly and examined under an optical microscope.

　　(2) After the model features are modeled, if the wound of the model animal has no bleeding, dehiscence or infection, it will usually heal spontaneously within a week. Within one week after the electrocautery, the model animals lost weight during the observation period, their food intake and activity were reduced, their hair was loose, and their weight gain slowed down. Anemia may occur 2 weeks after modeling, and blood urea nitrogen and blood creatinine may rise, and the measured value of blood urea nitrogen may be 4 times higher than before modeling. With the increase of urine output and urine protein content, model animals may have parathyroid function. Hyperactivity, such as high blood pressure, hypocalcemia, elevated alkaline phosphatase and other clinical symptoms. The surface of the electrocoagulated kidney is pale, swollen and deformed by visual observation. The pathological observation under the microscope showed that at the 18th week, most of the renal cortex of the kidney tissue of the model animal was necrosis and atrophy of the glomerulus. The renal tubules disappeared, and the renal interstitial fibrosis. (3) The use of electrocautery in comparative medicine can cause necrosis of the renal cortex of the kidney of model animals, destroy most of the glomerulus and renal tubular tissue structure, and compensate for the function of the residual nephron, which is gender-specific. The main feature of CRF is glomerular sclerosis and fibrosis of the renal matrix tissue, which eventually forms a small kidney. Models are easy to create and can be created in large quantities at once. The function of the model is stable and reliable, reaching the level of moderate to severe CRF, the success rate of the model is high, and the possibility of surgical bleeding is small. Or infectious diseases, which have objective indicators similar to human chronic renal failure. This method allows the creation of the same rat CRF model and draws more blood than the mice used for index testing. This model is suitable for clinical research on CRF drug treatment, screening and evaluation.