动物造模,急性肾盂肾炎动物模型 z-bio 2021-03-26 423

　　1 Simple infection model

　　(1) Reproduction method Rats weighing about 200g are prohibited from drinking water for 24 hours, and then the penis of the rat is tied with a thin thread in advance. Under ether inhalation anesthesia, the animal is placed on the back and fixed on the surgical board. Routinely disinfects the abdominal skin and dehairs, cuts it along the middle of the animal's lower abdomen, fully exposes the bladder, and uses a syringe to directly inject the large intestine with a concentration of 1000000/ml into the bladder. Escherichia (O111B4) bacterial solution, each injection volume is about 0.5ml, surgical incision is sutured, and abdominal cavity is closed. Loosen the penis thread after 1 hour. After the operation, the animals returned to normal drinking and eating. The animals were sacrificed at a predetermined time, the kidneys on both sides were taken, and the renal capsule was carefully removed; then the kidneys were cut longitudinally, half of the kidneys were homogenized and cultured for bacteria, and the other half was fixed in fixative for regular tissue sectioning and HE staining. Observe under a light microscope. At the same time, bladder puncture was used for urine bacterial culture.

　　(2) Model characteristics 3 days after surgery, bacteria can be cultured in urine, and red round colonies are grown on MacConkey's medium; 80% of animals are still positive for bacterial culture at 7 days, and only a few animals are cultured at 15 days. Positive. Bacterial culture of kidney tissues The bacterial culture of both kidneys of most animals was positive, and the culture of one kidney of some animals was positive. By the 15th day, there were still a few positive kidney bacteria cultures. Gross specimens showed that the kidneys of the model animals had increased in volume to varying degrees, and there was no obvious abscess on the kidney surface; the longitudinal section showed dilated renal pelvis, mucosal hyperemia, and unclear boundaries between renal cortex and medulla. Microscopic pathological observation showed that the submucosal and interstitial areas of the renal pelvis were dominated by neutrophils with a small amount of lymphocytes infiltration of inflammatory cells, telangiectasia, congestion, and interstitial edema on the 3rd day. On the 7th day, lymphocytes were infiltrated under the mucosa of the renal pelvis and in the interstitium, and a small amount of eosinophils and interstitial capillaries were seen. Interstitial lymphocyte infiltration, proliferation and expansion of capillaries were still seen at 15 days. A small amount of fibroblasts and granulation tissue are formed.

　　(3) Comparative medicine E. coli-induced retrograde pyelonephritis animal models are commonly used methods, but these acute pyelonephritis (APN) models are all based on unilateral ureteral ligation, the preparation process is more complicated, and There are obvious differences in kidney disease on both sides. This model uses simple intravesical injection without ligation of the unilateral ureter. A small dose of bacteria is directly injected into the bladder and maintained for a certain residence time. The bacteria can retrogradely invade the renal pelvis, and typical inflammatory processes can be seen in the renal tissue. Cell infiltration—lymphocyte infiltration—mainly granulation tissue formation), but the acute phase of inflammation lasts for a short period of time (3 to 4 days), the degree of disease is relatively mild, no obvious suppurative foci are formed, and there is a certain self-repair trend, similar In the clinically common light APN.

　　2 Surgery plus infection model

　　(1) Replication method Preparation of Escherichia coli (O111B4) bacterial solution: inoculate the bacterial species in a broth medium, incubate in a 37°C, 5% CO2 incubator for 12 hours, and prepare a bacterial solution with a concentration of 1000000/ml by the inclined plate method stand-by. Male rats weighing about 200g should be anesthetized by injecting pentobarbital sodium at a dose of 30 mg/kg body weight into the abdominal cavity after 18 hours of drinking water. Animals lie on their backs and fix the surgical board, routinely disinfect and dehair the abdominal skin, and spread sterile surgical towels. Make a 2cm median incision along the lower abdomen, and cut the abdominal wall layer by layer to enter the abdominal cavity, fully exposing the left ureter and bladder; thread needles from both sides of the middle ureter to the lateral abdomen and draw out the sutures. Keep the two needles as far as possible. Similar, without ligation temporarily, clamp the penis with an arterial clip, and slowly inject 0.75ml of bacterial solution into the bladder with a TB needle. Then tighten both ends of the suture outside the abdominal wall to "ligate" the ureter with an appropriate degree of tightness. The abdominal wall incision was sutured layer by layer, and the penile artery clamp was loosened. After the operation, the animals resumed regular feeding, and they were free to drink and eat. Twenty hours after the operation, the ureteral ligature outside the abdominal wall was removed to reopen the ureter. Clamp the penis of the rat with arterial clamps at the specified time, anesthetize with ether, lie supine on the operating table, open the chest and abdomen under aseptic conditions, draw blood and urine, cut the left and right kidneys and spleen for visual inspection , Bacterial culture and histopathological examination.

　　(2) Characteristics of the model On the 3rd day after the operation, the bladder urine of the model animals can cultivate bacteria and grow red round colonies on MacConkey's medium. On the 7th day, the positive rate of bacterial culture can be as high as 80% or more, and this high positive rate can be maintained for more than 15 days. The bacterial culture of the kidney tissue on the ligated side also showed bacterial growth, and the positive bacterial culture of the kidney on the unligated side was rare and the duration was short. Bacterial cultures of heart blood and spleen were negative. The volume of the left kidney of the model animal was enlarged to varying degrees compared with the right kidney. The surface of the left kidney showed different numbers of tiny abscesses, and some kidneys had larger abscesses protruding from the surface of the kidney; the renal pelvis of the cut surface was enlarged, the mucosa was congested and swollen, and there were inside the renal pelvis. Purulent secretions, small abscesses or cone-shaped inflammatory foci can be seen in some kidney sections. There was no obvious abnormality in the appearance and cut surface of the right kidney. Microscopic pathological observation showed that the left kidney renal pelvis and calyx mucosa of the model animals were hyperemia and swelling, some of which showed focal purulent secretions on the mucosal surface, and a large number of neutrophil infiltration under the mucosa. Renal interstitial vascular congestion, abscesses in most kidneys, cone-shaped inflammatory foci of different sizes are still seen in some renal papillae; renal tubules are dilated to varying degrees, with the distal convoluted tubule as the weight; more neutrophil infiltration around the tubules , Some renal tubular epithelial cells are necrosis and shedding, pus cells and a few casts can be seen in the lumen. There was no obvious damage to the glomerulus. On the 7th day after the operation, the left renal interstitial inflammatory cells of the animal decreased, and neutrophils were mainly seen under the renal pelvic mucosa and around the renal tubules, accompanied by more lymphocytes and monocytes. Fibrocytes, in addition to neutrophils, there are also a large number of lymphocytes and monocytes in the focus. The congestion and swelling of the renal pelvis mucosa was reduced, the submucosal neutrophil cell infiltration was significantly reduced, and more monocytes and lymphocytes appeared. There was no obvious histomorphological change in the right kidney tissue.

　　(3) Comparative Medicine Pyelonephritis is a clinically complicated, common and harmful urinary tract infection disease. If APN is not treated properly, it can often relapse or become chronic, and cause renal failure in patients. Although many treatment measures including various antibiotics have been taken clinically, the incidence, recurrence rate and slowing down rate of pyelonephritis have not been significantly reduced. To replicate animal models similar to human pyelonephritis, it is important to investigate and study this disease. The pathogenesis and treatment methods are of great significance. This model uses rats as experimental animals because of its moderate size and easy identification of the ureter, especially the anatomical characteristics of the urinary tract of rats. It is prone to spontaneous urine reflux, which is conducive to causing retrograde infection and infection with human pyelonephritis. The way is close. When making this model, the amount of bacteria injected into the bladder needs to be adjusted appropriately according to the virulence of the bacteria, otherwise it is easy to cause sepsis or hematogenous pyelonephritis in the animal; at the same time, to ensure the successful operation of the model, it is necessary to avoid bladder spasm and the tightness of ureteral ligation. Should be appropriate, it is very necessary to control the temperature and humidity during the operation. The animal model made by this method is similar to human APN in terms of pathogenic bacteria, infection route and pathological changes, and the model making method is simple and reproducible. It is a suitable animal model for studying human pyelonephritis.