Disseminated intravascular coagulation, also known as DIC, is currently used to teach disseminated intravascular coagulation mainly by means of the rabbit animal model of acute DIC.
Principle
The basic principle of the methodology of the rabbit animal model of acute DIC is the injection of rabbit brain powder infusion into the marginal ear vein, which leads to the development of DIC by activating the exogenous coagulation system.
Appliance
The rabbit acute DIC animal model method can be used to investigate the pathogenesis of acute DIC.
Operation method
Animal model of acute DIC in rabbits
Principle
The basic principle of the methodology of the rabbit animal model of acute DIC is the injection of rabbit brain powder infusion into the marginal ear vein, which leads to the development of DIC by activating the exogenous coagulation system.
Materials and Instruments
Equipment: Move The basic process of the rabbit acute DIC animal model method can be divided into the following steps: (1) Rabbits were weighed and fixed on a rabbit table in the supine position. (2) 3% sodium pentobarbital solution (1 mL/kg) was injected intravenously through the ear margin for general anesthesia. (3) The hair on the surgical field of the neck was cut off, the trachea was routinely exposed, and a tracheal tube was inserted to maintain breathing; the common carotid artery on one side was isolated, and a silicone tube was inserted for blood sampling; the other common carotid artery on the other side was isolated, the common carotid artery was intubated, and a pressure transducer was connected to it, and blood pressure was traced on the BL-420 Biofunctional Laboratory System. (4) A longitudinal mid-abdominal incision of 5-8 cm was made on the right side next to the rectus abdominis muscle, the muscle was bluntly separated, the abdominal cavity was opened, the greater omentum was pushed open, and a section of free small intestinal collaterals was identified and pulled out of the abdominal cavity, which was then placed in the microcirculation constant-temperature perfusion tank connected to the BI-2000 Microcirculation Image Analyzer System, so that the mesentery was evenly spread out on a Plexiglas observation ring, and a fixation plate was pressed on to adjust the perfusion. Press on the fixation plate and adjust the level of the perfusion fluid so that it just covers the mesentery. (5) Observe the normal microcirculation of the mesentery under the microscope with the BI-2000 Microcirculation Image Analysis System. Record the blood pressure under normal condition. Blood was collected from the carotid artery, and the first few drops of blood were discarded. 4.5 mL of rabbit blood was then placed in a centrifuge tube containing 3.8% sodium citrate solution (0.5 mL) and mixed upside down without shaking. First take 20 μL for platelet counting, and then centrifuge the rest of the blood for 15 min (3,000 r/min), and take the plasma to test coagulation-related indexes. (6) Take 4% rabbit brain powder saline infusion, calculated at 2.0 mL/kg, dilute the total amount to 30 mL with saline, inject it into a vein at the edge of the ear (a scalp vein needle can be used), and finish the injection within 15 min. The injection rate was as follows: 1.0 mL/min for the first 5 min; 2.0 mL/min for the second 5 min; and 3.0 mL/min for the last 5 min. (7) Observe the microcirculation of the mesentery dynamically under the microscope 10x objective lens with BI-2000 Microcirculation Image Analysis System; observe the morphology of blood cells and platelets, the speed of blood flow, the formation of blood, and the phenomenon of plasma extravasation in the microcirculation under the objective lens 40x. (8) Blood pressure was recorded 15 min and 45 min after the injection of rabbit brain powder infusion, and blood samples were collected from the common carotid artery for platelet count and coagulation-related indexes. (9) At the end of the experiment, the animals were executed, and the thoracic and abdominal cavities were opened to observe the changes of each organ.
① animal experiment surgical instruments
② Test tube racks, test tubes
③ Graduated centrifuge tube, centrifuge
④ BL-420 Biological Function Experiment System
⑤ BI-2000 Microcirculation Image Analysis System
Reagents:
① 3% sodium pentobarbital solution
① 3% sodium pentobarbital solution ② 4% rabbit brain powder saline solution
③ 3.8% sodium citrate solution
Instead of injecting rabbit brain powder infusion into the vein at the ear margin, an equal amount of saline was injected, and the injection route, total amount, rate, and observation indexes were the same as those of the experimental group.
Caveat
(1) In this experiment, the preparation of rabbit brain powder infusion and injection speed have a great influence on the success or failure of the experiment.(1) Preparation of rabbit brain powder extract: weigh 400 mg of rabbit brain powder, the vigor (PT) should not be greater than 12 s, add 10 mL of saline, stir well and then put it into 37 ℃ constant temperature bath for 60 min, stir well every 15 min, then centrifuged (1000 r/min) for 5 min, and take the supernatant and filtered, for intravenous injection. The supernatant should be filtered and used for intravenous injection. Alternatively, the lyophilized rabbit brain thrombin should be diluted and injected intravenously.② The principle of injecting rabbit brain powder infusion is first slow and then fast, do not be too fast, otherwise it is very easy to cause sudden death of the animal. Closely observe the animal's respiration during injection, and adjust the injection speed as necessary.(2) Rinse the arterial cannula (5 mL syringe) with saline after each blood sample collection to prevent coagulation in the cannula. Be careful not to use anticoagulants, so as not to affect the results of the experiment.(3) There are many reagents, plasma samples and pipettes used in this experiment, so the same pipette should only be used to draw up a certain reagent or plasma sample to avoid cross use.(4) If the room temperature is lower than 20 ℃, the plasma should be kept warm for 1 min at 37 ℃ in a constant temperature water bath.
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