动物造模,药效评价,静脉血栓 z-bio 2024-08-21 654

　　Venous thrombosis is closely related to slow blood flow, increased coagulation factor activity, and vascular wall damage. Therefore, experimental models of venous thrombosis can be formed by methods such as ligation of veins, interruption or obstruction of blood flow, damage to vascular walls, activation of coagulation factors, etc. The commonly used experimental animals include rats, rabbits, and dogs, and pigs can also be used.

　　1 Rabbit Neck Venous Thrombosis Model

　　(1) Method of replication: Rabbits weighing 2.5-3kg. Anesthetize by injecting pentobarbital sodium (at a dose of 25-30mg/kg body weight) or 20% Uratan (at a dose of 5ml/kg body weight) through the ear vein. Take a supine position, cut off the hair on the neck, and disinfect the skin in the surgical area. Make a longitudinal incision on the ventral side of the neck, separate the external jugular vein, which is 1.5-2cm long, and separate the cranial branches (maxillary and extramaxillary veins). Insert non-invasive blood vessels into the proximal end of the external jugular vein and the internal and external maxillary veins, temporarily blocking blood flow while the venous lumen is filled. Use toothless forceps to clamp the venous wall 10 times to cause mechanical damage. Subsequently, 0.2ml (40U) of thrombin was injected into the maxillary vein, and the puncture needle was removed. The maxillary vein was ligated and observed for 15 minutes. After seeing the formation of free blood clots in the venous lumen, open the blood flow in the external jaw and external neck veins; At the previously blocked proximal end of the external jugular vein, incomplete ligation with silk thread was performed to reduce the vascular lumen by about one-third, in order to slow down blood flow. After the surgery, the surrounding tissues were checked for no bleeding, and the incision was sutured and disinfected gauze was applied externally. Postoperative animals are fed with normal feed and do not require anticoagulants or antibiotics.

　　Observation and testing can be conducted at different times after modeling treatment (such as 1d, 3d, 7d, and 15d): ① Observe the swelling of the conjunctiva in experimental rabbits. ② Cut the simulated venous segment specimen under anesthesia, observe the overall appearance of the blood vessels, and longitudinally cut open to observe the changes in the luminal surface and thrombus with the naked eye Fix the sample in a 10% formaldehyde solution for pathological examination.

　　(2) Model features: After surgery, the rabbit's bilateral conjunctiva is highly swollen, the pterygium is swollen, and the eyeballs protrude and shed tears. The appearance of the modeled blood vessels is dark purple, with obvious dilation of the diameter and gradual thickening of the vessel wall. Thrombosis begins to fill the entire lumen of the vein, appearing purple red and without adhesion to the vessel wall. Over time, the thrombus adheres to the vessel wall and retracts. The surface of the vascular endothelium is rough and pale. Under the light microscope, endothelial cells can be seen peeling off, exposing the subendothelial layer. After 7 days of injury, endothelial cells gradually cover the surface around the thrombus. Inflammatory cell infiltration is evident in the vascular wall and interstitial spaces.

　　(3) The comparative medicine model is based on the three elements of thrombosis formation, which cause mechanical damage to the blood vessel wall, increased coagulation factor activity, and slowed blood flow velocity, thereby forming venous thrombosis. Due to the significant differences in venous volume between small animals such as rats and humans, especially during modeling surgery, blood vessel walls are prone to contraction, resulting in minimal or difficult thrombosis formation. Therefore, it is impossible to systematically observe the functional and morphological evolution at different postoperative stages. Rabbits have higher external jugular vein pressure and sufficient blood flow compared to other limb veins. Thrombosis forms rapidly and fills the entire vascular lumen, which is beneficial for studying and comparing the functional and morphological changes of venous thrombosis in different stages using thrombolytic therapy. From the pathological evolution process at different time points of 24h, 72h, 7d, and 15d after surgery, this model conforms to the pathological evolution of human venous thrombosis. At 24 hours, 72 hours, and 7 days after surgery, the conjunctiva of both eyes of the model animal was highly swollen, with protruding eyeballs. This was often caused by thrombosis blocking the venous lumen, blood reflux disorders, and compensatory function not yet established, leading to edema. This is consistent with the clinical symptoms of deep vein thrombosis in the lower limbs, which causes swelling in the patient's limbs. If the swelling of the conjunctiva is graded or counted, it may be more conducive to experimental observation and comparative research.

　　2 inferior vena cava occlusion thrombus model

　　(1) Method of replication: Rats weighing 250-300g. After intraperitoneal injection of pentobarbital sodium (at a dose of 50-60mg/kg body weight) or chloral hydrate (at a dose of 350-400 mg/kg body weight), the patient is anesthetized and fixed in a supine position. The abdominal hair is cut off, and the skin in the surgical area is disinfected. Cut along the white line of the abdomen into the abdomen, separate the inferior vena cava, ligate the inferior vena cava with thick silk thread below the left renal vein, suture the abdominal wall, and reopen the abdominal cavity 2-6 hours after ligation. Pinch the blood vessel 2cm below the ligation line, cut open the lumen, remove the thrombus, and weigh the dry weight of the thrombus.

　　(2) The characteristics of the model are that the thrombus formation rate is generally 60% to 80% around 3 hours after ligation, and 100% at 6 hours after ligation. This model focuses on the percentage of thrombus formation, thrombus mass, coagulation and fibrinolysis systems, and is commonly used to study the effects and mechanisms of drugs in inhibiting thrombus formation in vivo.

　　Three vein occlusion thrombus model

　　1. Rabbit model

　　Method of replication: Rabbits weighing 2.5-3kg. Anesthetize by injecting pentobarbital sodium (at a dose of 25-30mg/kg body weight) or 20% Uratan (at a dose of 5ml/kg body weight) through the ear vein. Fix in supine position, remove hair from both sides of the thigh, and disinfect the skin in the surgical area. Longitudinal incision on the inner side of the thigh, blunt separation of subcutaneous tissue and muscle, 2cm free bilateral femoral veins, blocked with vascular clips at the distal and proximal ends for 12 hours, and then removed. After confirming thrombus formation through imaging, thrombolytic therapy can be administered. Obtain femoral vein segments with thrombosis at different stages after modeling, and evaluate the effects of thrombolytic therapy on vascular wall inflammatory response and morphological changes in the acute phase of venous thrombosis by detecting venous patency, presence of wall attached thrombosis, smooth muscle actin, collagen deposition, endothelial cell damage, inflammatory cell infiltration, and scanning electron microscopy observation.

　　2. Dog model

　　The replication method involves experimental dogs weighing 15-30kg. After intravenous injection of pentobarbital sodium anesthesia at a dose of 30mg/kg body weight, fix in a supine position, remove hair from one side of the thigh, and disinfect the skin in the surgical area. Longitudinal incision on the inner side of the thigh, separating the femoral vein by about 2-3cm, ligating its proximal and distal ends, and releasing the ligature after 48 hours. The success rate of thrombus formation model production is 97% to 100%. By observing the degree of venous patency, presence or absence of wall thrombus, deposition of collagen fibers, expression of smooth muscle actin, and degree of endothelial cell damage, this study aims to investigate and compare the effects of catheter direct thrombolysis and systemic thrombolysis on the morphology of the venous wall after acute deep vein thrombosis, as well as their recent therapeutic effects.

　　3. Model of foreign body thrombosis in femoral vein

　　The replication method can use dogs and rabbits as experimental animals. After animal anesthesia, prepare and disinfect the skin. Cut open the skin along the direction of the femoral vein, bluntly separate subcutaneous tissue and muscle, fully expose the femoral vein, clamp the blood vessels with vascular clamps at the proximal and distal ends respectively, insert spiral copper wire (self-made) into the proximal end, suture the blood vessels, and after confirming the recanalization of the blood vessels, suture them layer by layer. The principle of modeling is based on local blood stasis, hypoxia, damage to endothelial cells, and the insertion of foreign objects (copper wires), which activate internal and external coagulation mechanisms and promote thrombus formation.

　　4. Thrombosis model induced by femoral vein electrical stimulation

　　Method of replication: Healthy rats weighing 250-300g. First, administer dexamethasone sodium phosphate injection (at a dose of 1mg/kg body weight) intramuscularly to rats once a day for 7 consecutive days. On the 8th day, femoral vein thrombosis was performed: After anesthesia, the rats were fixed in a supine position, and the hair on one side of the thigh was cut off. The skin in the surgical area was disinfected. A longitudinal incision was made on the inner side of the thigh, and the main trunk of the free femoral vein was about 1.5cm long. The positive and negative electrodes of the experimental in vivo thrombus formation analyzer were placed at the distal and proximal ends of the femoral vein, respectively. Direct current (1.5mA) was applied and stimulated continuously for 7 minutes. The principle is to apply glucocorticoids to cause hypercoagulability in the blood, coupled with electrical stimulation to form blood clots. Explore the pharmacological effects of drugs against venous thrombosis based on changes in thrombus wet weight and blood fibrinolysis indicators.