动物造模,药效评价,自体血栓大脑中动脉闭塞 z-bio 2024-07-25 395

　　1. Modeling material animals: Wistar rats, half male and half female, weighing 250-350g; Medications: 6% chloral hydrate, thrombin, sodium citrate (0.109mol/L); Equipment: Physiological multi-channel instrument, spinal epidural catheter, 22G intravenous indwelling needle.

　　2. Modeling method

　　(1) Extraction of arterial blood: Animals were anesthetized by intraperitoneal injection of 6% chloral hydrate solution at a dose of 35mg/100g body weight. A midline incision was made in the neck, and the right common carotid artery (CCA) and internal carotid artery (ICA) were separated using conventional methods. The pterygopalatine artery was also separated and ligated. Free the distal end of the external carotid artery (ECA) and ligate it, block its branches, clamp CCA and ICA with a microvascular clamp, then cut open the ECA at the proximal end close to the ligation line, insert a 22G indwelling needle, open the microvascular clamp at the CCA, draw 0.5ml of arterial blood, ligate the proximal end of the ECA at 1mm from the bifurcation of the carotid artery, inject a small amount of heparin saline at the distal end, and open the vascular clamp.

　　(2) Preparation of improved thrombus: Immediately inject 0.5ml of arterial blood into an EP tube containing 0.75 μ l of sodium citrate (0.109mol/L), mix well, and centrifuge at 5000r/min for 1 minute. Take 160 μ l of platelet poor plasma, 10 μ l of RBC, and mix with 1 unit of thrombin (250000 units/L) and 30 μ l of calcium chloride (0.5mol/L) solution. Immediately inhale them into a plastic tube with an inner diameter of 0.58 or 0.65mm. After 2 minutes, transfer the thrombus to phosphate buffer solution to obtain a light red thrombus with a diameter of 0.35mm or 0.45mm. Cut it into small sections about 2mm long under a surgical microscope, and sequentially inhale 6 thrombi into PE50 tubes, with a distance of 2-3cm between the thrombi, for later use.

　　(3) Injecting thrombus: After the thrombus is prepared, the model group anesthetizes the animals again, exposes the neck artery, clamps the CCA and ICA with a vascular clip, and then unties or cuts the thread near the heart of the ECA. The indwelling needle is inserted and fixed again at the ECA incision, and the vascular clip at the ICA is opened after removing the needle core. The blood flowing back from the ICA can be seen. After the gas inside the indwelling needle is completely discharged, a PE50 tube is connected to the other end of the tube, which is connected to a syringe filled with 0.25ml phosphate buffer solution. The thrombus is injected sequentially (about 30 seconds), the ECA is ligated, the vascular clip at the CCA is opened, and the incision is sutured. The operation process of the sham surgery group is the same as that of the model group, but only 0.2ml of buffer solution is injected.

　　3. Modeling principle: Establish a middle cerebral artery occlusion model using rat autologous thrombus.

　　4. Changes after modeling MRI results: The modeled animals showed abnormal DWI signals 15 minutes after MCAO, gradually expanding from the tail, outer shell, and parietal cortex, and corresponding lactate signals were observed on H-MRS, with lactate peaks gradually deepening.

　　Neurofunctional deficit score: The model group scored significantly lower than the sham surgery group.

　　Thrombosis can be found in the MCA and ICA of the model group animals. 30 minutes after MCAO, thrombus can be seen located at the entrance of MCA and inside ICA, and MCA is completely occluded.

　　Pathological observation: The thrombus content is above 90%. After 6 hours of MCAO, HE staining revealed a significant amount of neuronal necrosis in the right hemisphere of the brain, with the lateral aspect of the striatum being the most pronounced.