动物造模,药效评价,肝衰竭 z-bio 2024-08-26 417

　　(1) Model method: Adult rats were anesthetized by intraperitoneal injection of ether or ketamine at a dose of 100mg/kg body weight. They were fixed in a supine position and opened along a transverse incision in the upper abdomen. The abdominal wall blood vessels were cauterized to stop bleeding using an electrocoagulation device. Expose the liver, cut off the falciform ligament, coronal ligament, and interlobar ligament, expose the hepatic hilum, first ligate and cut off the ventral bile duct of the portal vein head branch, expose the portal vein head branch and accompanying hepatic artery, and ligate the portal vein head branch and accompanying hepatic artery with 5-0 silk thread. Then carefully separate the right branch of the portal vein that enters the right upper and lower lobes from the accompanying hepatic artery, and ligate the right branch of the portal vein or its right upper lobe branch with 5-0 silk thread. At this point, it can be seen that the ligated liver lobe is in an ischemic state. Insert a 5.5 gauge needle into the portal vein above the ligation line, inject 10ml/kg body weight of 5% glucose sodium chloride solution or sodium lactate Ringer's solution until the color of the liver lobes turns white, then quickly ligate with 5-0 silk thread and remove the liver lobes. A total of 85% to 95% of liver tissue was excised. After checking for no bleeding, suture the abdominal wall incision. Postoperative observation of rats' mental state, activity, liver function changes, and 12 hour survival rate. The success of the surgery in rats is marked by the ability to wake up and turn over. Failure to wake up and stand up, as well as death within 12 hours, is considered a failure of the surgical procedure. Or healthy piglets were anesthetized by intraperitoneal injection of 30mg/kg body weight of barbiturates. An infusion channel was established in the right upper limb, oxygen was administered through a nasal cannula, and the pig was fixed in a supine position. The pig was then placed layer by layer through a midline incision in the upper abdomen, and the portal vein and right renal vein were freed. Inject 0.25mg heparin into the portal vein, take a silicone plastic tube (15cm long, 0.8cm diameter) filled with 2% heparin physiological saline, and insert it into the inferior vena cava 2-3cm from the distal end of the free right renal vein and fix it. The other end is inserted about 1.5cm from the proximal end to the distal end of the portal vein and fixed. Immediately open the bypass tube, check for obvious bleeding, and then close the abdomen. Two days later, a second stage surgery was performed. After entering the abdomen, all liver ligaments (including the left and right triangular ligaments, coronal ligaments, falciform ligaments, and hepatogastric ligaments) were cut off, and the hepatic artery branch towards the liver was sutured. Finally, the hepatic artery and gastroduodenal artery were ligated to completely block liver blood flow, and femoral artery catheterization was performed to monitor blood pressure. At 1, 4, and 7 hours after complete blockage of hepatic blood flow, small pieces of liver tissue were harvested and fixed with 10% formaldehyde and 0.25% glutaraldehyde, respectively. Then close your abdomen. During the operation, 500ml of balanced saline solution and 800000 U of penicillin were intravenously administered to the experimental pigs. After death, the bodies were dissected and liver specimens were collected for further examination. During the modeling process, the general condition and time of death of the model animals can be dynamically observed, and whole blood can be extracted to prepare serum for biochemical testing. After modeling, the animals were euthanized and their livers were harvested for histological examination.

       (2) Model features: Rats wake up quickly after surgery and are able to turn over and move around. However, liver failure occurs 1-2 hours later, followed by liver coma 12 hours later, characterized by mental exhaustion, body curling up, erect fur, reduced activity, refusal to drink or eat, and yellow urine color. The survival rate of model animals undergoing 85% liver tissue resection at 12 hours was 93%, and all animals died at 48 hours; The survival rates of model animals undergoing 95% liver tissue resection at 2, 4, 6, and 12 hours were 92%, 75%, 58%, and 50%, respectively, and all animals died at 24 hours. Model piglets die within 12-16 hours after complete blockage of human liver blood flow. 12 hours after surgery, the levels of alanine aminotransferase (ALT), blood ammonia (NH3), and total bilirubin (TBIL) in rats increased, while the level of blood glucose (GLU) decreased. After hepatic blood flow obstruction in model piglets, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), Amm, urea nitrogen (BUN), creatinine (CRE), and prothrombin time (PT) showed a progressive increase, while albumin (ALB) and fibrinogen (FIB) showed a progressive decrease. At the time of death 12 hours after surgery in rats, the residual liver tissue had a dark red color, swollen capsule, and surface bleeding. Under the microscope, the structure of liver lobules can be seen to be damaged, with large areas of degeneration and necrosis of liver cells, loose cytoplasm, infiltration of inflammatory cells in the necrotic area, dilation of liver sinusoids in a mesh like pattern, and visible congestion and bleeding. After completely blocking the blood flow into the liver of the model pig for 1 hour, the structure of the liver lobules and the arrangement of liver cells were still normal, the Diess gap was slightly dilated, and the liver cells were cloudy and swollen, but the nuclear membrane and nucleolus were still clear; After 4 hours under light microscopy, the liver lobule structure still exists, the arrangement of liver cells is not yet disordered, the liver plate structure exists, the interstitial space between the liver cells is significantly enlarged, the central vein collapses, the cytoplasm of liver cells shows vacuolar degeneration, the nucleus shrinks, and the nucleolus disappears; Under electron microscopy, homogenization of glycogen granules in the cytoplasm, degranulation of rough endoplasmic reticulum, mitochondrial edema, blurred cristae, partial mitochondrial dissolution, significant reduction of microvilli in intercellular bile ducts, aggregation of nuclear chromatin, nuclear deformation, and formation of nuclear pseudoinclusions can be observed. After 7 hours, under the microscope, the structure of liver lobules was disordered, the arrangement of liver cells was disordered, the liver plates were dissociated, and the dilation of the Diesl gap was more obvious. The turbidity, swelling, and vacuolar degeneration in the cytoplasm of liver cells were more significant, and the nuclear chromatin was clustered. Some liver cell membranes were incomplete and showed signs of dissolution and necrosis. When death was caused by liver failure and liver tissue microscopy was performed immediately, it was found that the structure of the liver lobules in the model pig was unclear, the liver cells were reduced, disordered, the liver plates were dissociated, the Dieselt's gap was dilated, and focal and patchy extensive liver cell necrosis was visible.

　　(3) Acute liver failure in comparative medicine is a syndrome characterized by diffuse hepatocyte necrosis and/or sudden severe liver function damage caused by multiple factors, with a mortality rate of over 75% in patients. Its etiology is mainly caused by hepatitis virus, but can also be caused by one-time or repeated trauma, excessive medication or toxins. At present, there are two main types of animal models for acute liver failure: surgical and drug-induced liver injury. Among them, total or partial liver resection is the most classic and commonly used modeling method. Total liver resection can undoubtedly lead to liver failure, but the characteristics exhibited by this model are far from the pathological process of acute liver failure in clinical practice, such as irreversible course of disease, short duration of liver coma, absence of damaged or dead liver cells in circulation, and continuous release of toxic substances. Abnormal liver function only manifests 2-4 hours before the death of the model animal. Given these shortcomings, animal models of acute liver failure with partial hepatectomy have been studied and established to replace them. Under normal circumstances, when liver function is normal, animals can tolerate liver resection of 70% to 75% or less. If liver resection exceeds 75%, fatal liver failure will occur. When liver resection exceeds 85%, portal shunting is required, and almost 100% of animals die within 48 hours. This model underwent partial liver resection, with 85% to 95% of the liver tissue removed. After 6-12 hours, the animal's liver and kidney function showed abnormalities, progressively worsening, and subsequently leading to liver coma. Histomorphological examination showed that the liver lobule structure was disordered, liver cells decreased, liver plates dissociated, and the Dieselt's gap expanded significantly. Focal and patchy extensive liver cell necrosis was visible, and all animals died within 24-48 hours after surgery. Partial liver resection has physiological and pathological characteristics that are closer to clinical practice compared to total liver resection models, and there is a possibility of reversal. For 90% liver resection rats, continuous transplantation of liver cells into the spleen for 3 days before surgery can result in a survival period of over 28 days for the 40% model. So far, various animal models of partial liver resection have been successfully replicated and established, such as rats, rabbits, dogs, and pigs. Among them, small animal models are more suitable for studying the pathophysiological processes of acute liver failure and the effects of drug interference on this process, while large animal models are more commonly used to evaluate the efficacy and safety of artificial liver technology. The animal model of liver failure using partial hepatectomy has its unique advantages, including short replication period, quantifiable liver resection, no specific pathological changes in the remaining liver tissue, animal symptoms and related blood indicators that are consistent with the clinical manifestations of liver failure, good repeatability and stability of the model, and high success rate.