动物造模,药效评价,脑血管痉挛 z-bio 2024-08-21 371

　　(1) Method of replication: Male mice weighing 25-30g.

　　1) The replication of SAH was anesthetized by intraperitoneal injection of pentobarbital sodium (60mg/kg body weight), and the skin in the surgical area was disinfected. Cut open and separate the right femoral artery, and insert a tube to extract 60 μ l of autologous blood for intracranial injection (with the least leakage and lowest mortality rate in mice when injecting 60 μ l into the skull). The mouse's head is tilted forward by 30 degrees, and a midline incision is made at the back of the head to expose the skull. Use a No.30 puncture needle to puncture from the midline of the lower part of the head to the left and downward at a 45 ° angle into the greater occipital cistern. Extract 60 μ l of autologous blood from the femoral artery (followed by intraperitoneal injection of 60 μ l of physiological saline to supplement normal fluid volume) and slowly inject it into the occipital cistern for about 1 minute. When injected with 20 μ l, the animal's breathing becomes abnormal, and when injected with 60 μ l, respiratory arrest may occur. After injection, immediately tilt the animal's head forward at 75 ° for 10 minutes to allow blood to spread in the occipital cistern. Slowly withdraw the puncture needle, administer 2-3 drops of gentamicin (at a dose of 2.50 × 10000U/kg body weight) to the wound, ligate the femoral artery and withdraw the catheter, carefully suture the incision to prevent leakage of cerebrospinal fluid and blood. Maintain animal body temperature at 37 ℃ during surgery. The animals regained consciousness and gradually resumed normal feeding 1 hour after surgery, with a normal postoperative mortality rate of less than 5%.

　　2) Cerebral angiography can be performed at 1, 6, 12 hours and 1, 1.5, 2, 3, 4, 5, and 7 days after SAH. Left ventricular catheterization is performed, and 10% formaldehyde solution is infused at a rate of 5.5 ml/min for 2 minutes. Stereoscopic microscopy is used to measure the anterior cerebral artery, middle cerebral artery, and basilar artery.

　　3) Histological examination of animals was first performed by lavage with 0.1mol/L phosphate buffer solution, followed by lavage with a mixture of 4% paraformaldehyde and 2.5% glutamate in 0.1mol/L phosphate buffer solution at a rate of 5.5ml/min. Quickly remove the mouse brain and continue to fix it in the above liquid (4 ℃) for 24 hours. Separate the anterior cerebral artery, middle cerebral artery, and basilar artery, make 0.5 μ m thick sections, and stain with toluidine blue.

　　(2) The model features blood clots above the anterior cerebral artery and basilar artery, as well as near the middle cerebral artery, within 2 days after SAH. Blood clots are most common and cleared slowly around the anterior cerebral artery, while blood clots at the top of the brain disappear after 3-4 days of SAH. The anterior cerebral artery begins to contract 1 hour after SAH, and its diameter can decrease by 30% to 40%, lasting for 1-3 days. By the 7th day, the vessel diameter gradually returns to normal. The contraction of the middle cerebral artery and basilar artery reaches 20% to 25% at 6 hours after SAH and lasts for 1-1.5 days. Pathological histology examination showed that after SAH 1 day, the inner elastic layer of the vascular wall significantly wrinkled, the lumen narrowed, and the wall thickened. There are no significant abnormalities in the smooth muscle cells of blood vessels. Accurate operation can avoid damage to the brainstem, spinal cord, and cerebellum.

　　(3) Up to now, there is no animal model in comparative medicine that can fully mimic human DCV. According to Schwartz's description, a good DCV model should have: ① persistent subarachnoid hemorrhage. ② The amount of bleeding is controllable. ③ The bleeding mechanism is similar to the rupture of a hemangioma The distribution of blood is consistent with subarachnoid hemorrhage Easy to operate and reasonably priced. The ideal experimental method should be to directly puncture the blood vessel wall to allow blood to flow out. However, this method has a high animal mortality rate. This model simulates the rupture of a vascular tumor by injecting blood directly into the occipital cistern, with blood distribution similar to SAH and causing sustained vascular spasm. The pathological changes are similar to those of human DCV. And it has the characteristics of good repeatability, easy operation, low cost, and low mortality rate.