【Animal Modeling】-Establishment of rat brain contusion model and behavioral evaluation
动物模型,脑挫伤模型
z-bo
2022-02-19
1369
1. The purpose of the experiment is to use SD rats as experimental animals, and use a self-made free fall injury device to further develop a standardized and reproducible traumatic brain injury (TBI) animal model and behavior evaluation system. Build detailed. Study the mechanism of brain contusion and laceration to lay the foundation.
2. Experimental principle
Direct external force hits the cerebral cortex of rats and damages the brain.
3. Experimental materials and methods
(1) Experimental materials
1. Required equipment, automatic sterilization pot (MLS-3020), electric heating constant temperature blast drying box.
2. Necessary reagents hydrate chloral hydrate powder, 0.9% sodium chloride solution, medical iodine disinfectant.
3. Reagents for preparing 3.6% chloral hydrate solution: Weigh 3.6 g of chloral hydrate powder, dissolve it in 100 ml of sterile 0.9% sodium chloride solution and mix well. Store at room temperature.
4. Experimental animals SD male rats, weighing (160±10) g.
(II) Experiment specific methods and operating procedures
1. Preparation of TBI model
(1) Anesthesia: 3.6% chloral hydrate (1 ml/100 g) was injected intraperitoneally for anesthesia.
(2) Skin preparation: After anesthesia, shave with an electric razor.
(3) Towel disinfection: regularly disinfect the scalp with a medical iodine disinfectant, and spread a sterile towel.
(4) Expose the wound: Use a scalpel to incise the skin along the sagittal suture to expose the left parietal bone, scrape the periosteum, and suture the front Font bone and herringbone cross to the left of the sagittal suture. Clearly expose the rear Font door and the sagittal suture, 1mm transverse to the midpoint of the front and rear door, and use a dental drill to cut a 5mm diameter bone window (to avoid damage to the dura), use a cotton ball to stop bleeding.
(5) Injury: A rat brain trauma model was created using the free fall impact injury method. The weight of the hammer was 50 g and the impact height was 30 cm. The contact surface between the lower edge of the hammer and the rat brain is a spherical surface with a diameter of 4 mm. Place the rat on the bottom plate of the free fall injury device. The head of the rat faces the free fall injury device and the tail faces the operator. Grasp the head of the mouse with your index finger and thumb and fix it directly under the striker. Before each impact, disinfect the impact rod with a medical iodine disinfectant. The lower edge is located on the left side of the skull, above the rat skull window. A hammer weighing 50 grams will fall freely on the impact rod from 30 cm, damaging the brain. Remove the piston rod immediately after impact.
(6) Close the incision: After removing the firing pin, disinfect the incision with medical iodine disinfectant and suture the scalp again.
(7) Postoperative treatment: After the operation, place the rat next to the incubator, keep the body temperature at 37°C, return to the cage after fully awakening, and then raise it. Ampicillin (20 IU dose) was injected intraperitoneally into each rat every day to prevent infection for 3 days.
2. The behavioral assessment is performed on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th day after the operation using the NSS (Neural Dysfunction Severity Score) standard (18 points system) to perform the TBI model in 15 days Rat behavior study. The higher the score, the more severe the damage.
(3) Precautions in the experiment
(1) During anesthesia, the needle should be inserted into the abdominal cavity at a 45-degree angle from the inner side of the hind limb to the upper side (head), and a clear feeling of shedding should be felt. I can. Leave the needle for 1 minute and then immediately remove it to avoid injecting the drug into the hind leg muscles that affect the anesthesia effect.
(2) When opening the bone window to damage the sagittal sinus, causing hemorrhagic shock in rats or increasing the mortality of experimental rats, please operate the dental drill carefully. Please do not accidentally injure the sagittal sinus and bleeding, please stop the operation immediately, press the cotton ball on the skull wound for 5-10 minutes, and continue the operation after hemostasis.
(3) The pipette tip can be placed on the dental drill, so the drill only exposes the tip of the pipette tip by 2mm. Therefore, when the bone drill opens the window, please deepen the drill and brain. You can effectively avoid excessive force damage to the cortex. Fourth, the experimental results obtained (1) No animal died after injury, and the postoperative survival rate was high.
(2) Most NSS scores on the first day after injury are 7-9 points, and most of them recover to 2-3 points 13-15 days after surgery.
5. Result analysis
(1) The rat TBI model created in this way is highly reliable and has extremely low animal mortality.
(2) Using a weight of 50g and an amazing height of 30cm can cause moderate brain damage and obvious symptoms of nerve damage.
6. Experience and experience
(1) Preparations before starting the experiment, such as preparing laboratory animals, sterilizing surgical instruments, preparing anesthetics, preparing antibiotics, etc., are very important. You can only use it when you are ready. The experiment went smoothly.
(2) The most important step in preparing an animal model is to drill a bone window. This is directly related to the success of the model preparation. During the opening of the bone window, accidental damage to the sagittal sinus can cause excessive bleeding from the head wound. Animals die easily within 24-48 hours after surgery. If the bone window is too small, the impact bar will get stuck on the skull during impact, and the purpose of damaging the cortex will not be achieved.
