【Animal Modeling】-Establishment and application of rat model of partial dorsal root transection
动物造模,背根节切断模型
z-bo
2022-02-03
543
The first part of the source model source and principle introduction
Partial reverse introduction model (also called partial dorsal root removal model) is divided into two types. One is to cut the dorsal root and keep the dorsal root part, and the other is to remove the dorsal root part. In 1958, Liu and Chambers used a cat's partial dorsal root incision model (T10? L6, S1? Ca7 dorsal root was excised on one side, and L7 dorsal root was retained as a backup root) and observed the excision (T10→L6, S1→Ca7) ). After the animals survived for a few months, the remaining dorsal roots and the corresponding dorsal roots on the opposite side were cut, the ulcer areas of the two dorsal roots were compared, and the nerve fibers (derived from the spare dorsal root) were distributed in the spinal cord. I am allowed. The range and density of the dorsal root is much larger than that of the opposite side. This is because after cutting the adjacent dorsal roots, due to retention, there is a lateral bud at the center edge of the retained dorsal root (L7). During the process of protruding to the head, the tail end of the spinal cord is cut and crushed. . It is shown to make up close. The change of CNS end is the first evidence of lateral plasticity after spinal cord injury, or "plasticity" of damaged spinal cord.
Lateral buds are different from nerve regeneration, but the phenomenon of lateral buds may be an important factor in promoting nerve regeneration. Therefore, exploring the mechanism of spinal plasticity is of great value to understanding the mechanism of central nerve regeneration. In the following decades, most scholars further used ulcerative silver staining, ultrastructural quantification and horseradish peroxidase (HRP) tracking technology to confirm that the spinal cords of rats and cats are plastic. Wu Liangfang observed under an electron microscope the plasticity of the second layer of synaptic remodeling of the cat spinal cord after partial dorsal root transsection in the 1990s. Due to their high viability and fertility, mice are often used to create animal models. This experiment takes the creation of a rat dorsal root ganglion model as an example. Remove the left L1-L4 dorsal root ganglia and L6 dorsal root ganglia, while retaining the left L5 dorsal root ganglia.
Section 2 Model Creation One. Purpose
observe the plasticity of the rat spinal cord.
2. Experimental principle
After cutting part of the dorsal root, the center of the spare dorsal root is located on the head and tail of the spinal cord to supplement the center of the dorsal root that is broken due to the incision of the adjacent dorsal root. Form lateral buds. The nerve endings, the lateral buds after injury, are called spinal plasticity.
3. Experimental materials and methods
(1) Experimental materials
1. Preparation of reagents and sterile saline, iodophor, and penicillin. Use sterile saline to prepare 3.6% chloral hydrate (prepared as soon as possible to make chloral hydrate easier to dissolve in water).
2. Laboratory supplies: 5 ml disposable sterile syringe, 1 ml disposable sterile syringe, sterile cotton ball, disposable sterile drape cloth, 3-0 sterile surgical suture.
3. Equipment and equipment: animal body temperature maintenance equipment, ultraviolet disinfection lamp, shaver, electric heating constant temperature drying box.
4. Surgical instruments include bone forceps, toothed forceps, toothless forceps, nerve hooks, elbow eye scissors, micro scissors, microscope forceps, surgical scissors, homemade surgical hooks (1ml syringe head bent) folded at right angles.
5. Experimental animals: adult SD rats, female, weighing 280-300g.
(2) The specific method and operation steps of the experiment
1. Before surgery, prepare surgical instruments and cotton balls in an autoclave, and then put them in an oven to dry. One hour before the operation, the operating room was irradiated with ultraviolet light.
2. Operation process
1) Anesthesia: Grab the rat and inject 3 ml 3.6% chloral hydrate intraperitoneally, then return it to the mouse cage. (1) Choose a syringe with a fine needle to reduce trauma. Usually, a sterile disposable syringe with a specification of 5 ml is usually used.
(2) The injection site is usually located in the left lower abdomen or right lower abdomen to avoid damage to the bladder, pancreas, kidneys and other important organs. (3) The length of the needle inserted into the abdominal cavity is 1/3 to 1/2,
(4) After inserting the needle into the abdominal cavity, make sure that no blood is drawn to prevent the drug from directly entering the blood vessel.
(5) Do not inject liquid into the thigh muscles. If the liquid is not injected, the absorption of the drug will be very slow. If injected by mistake, another rat can be selected for anesthesia. The wrongly injected rats can be used on the second day of the experiment. It is not suitable to be anesthetized again today. Repeated anesthesia will increase animal mortality.
2) Preoperative hydration: After anesthetizing the animal, inject 5 ml of sterile saline into the abdominal cavity.
3) Fixation, skin preparation, and disinfection: Use rubber bands to fix the rat limbs on the abdomen on the dissection board. Gently pull the rat's tongue to one side to keep breathing as smooth as possible. Turn on the thermostat and set the temperature to 37°C and the operating room air conditioner to 28°C. Use a razor to remove the hair on the buttocks and tailbone.
Iodophor disinfects the shaving area, as shown in Figure 19-1.
4) Surgical procedures (Note: The following procedures need to compress the bleeding with a cotton ball immediately when encountering bleeding points).
(1) Cut 10 cm at the lumbar lumps of the spine.
(2) Anatomical location: The rat hip joint corresponds to the L6 vertebral body. Using ophthalmic scissors, close the left side of the spinous processes L1 to L6 and cut the fascia connected to it.
(3) As shown in Figure 19-3, use micro-scissors to expose the lateral process of L1, L2, L3, L4, and L6 on the left side, and wipe the rest of the pedicle muscles with a neural hook.
(4) Use the longer pointed bone to bite the transverse processes of L1, L2, L3, L4 and L6 to the dorsal root ganglion and central branch and a small segment of spinal nerve (about 1 mm) to fully expose (the spinal nerve of the dorsal root) and the previous Path mix). Do not open the bone window too wide. Increasing it will increase trauma.
Working point: The horizontal process of the upper and lower vertebral bodies on the left can be represented by two "C" patterns.
The four parts a, b, c, d of the living body are adjacent to each other. After completely cutting off the two parts b and d with a sharp nose, you can see the posterior root part along the cranial side of the posterior root part. The central and surrounding branches of the dorsal root segment (about 1 mm long) are exposed at the tail. use
(5) The hook gently pulls the branch around the dorsal root ganglia. At this time, there is a gap between the dorsal root ganglia and the spinal cord. The gap is covered with translucent dura mater. Use the tip of a 1 ml syringe to gently scrape the dura mater covering the gap to show that the cerebrospinal fluid is obviously flowing out.
Note: Do not pull the hook excessively. It is recommended to check the gap between the dorsal root ganglia and the spinal cord. Otherwise, the anterior and posterior roots connected to the spinal cord will be torn off. The stump is usually located in the spinal canal and can only be seen when the rat is dissected.
(6) Use micro scissors to gently cut the central branch of the dorsal root while keeping the hook fixed. Be especially careful when disconnecting. The front root cannot be cut. The gap between the central branch of the dorsal root and the front root is very narrow. Microscope scissors must be inserted in this gap to cut off the dorsal root. Central branch. Lift the dorsal root ganglion gently with micro tweezers, and cut the other end of the dorsal root ganglion with micro scissors. At this time, the spinal nerve is actually the anterior root, so please be careful not to cut the anterior root directly. When fused with the dorsal root, it forms a mixed nerve that mixes motor and sensory. Once it leaves the eye, the spinal nerve splits into ventral and dorsal branches. Remove the dorsal root and see the front root clearly.
(7) Cut the left L1, L2, L3, L4, and L6 dorsal root nodes in sequence according to the above steps, leaving the left L5 dorsal root node. (8) Generally, when the L6 transverse process is exposed for the first time, the depth of anesthesia of the rat is not enough to maintain the operation, and 1 ml of 3.6% chloral hydrate can be injected intraperitoneally.
(9) Suture the muscle and skin layer by layer. (10) Disinfect the wound with iodine again, and inject 200,000 U of penicillin intraperitoneally twice a day for 1 week after the operation.
(11) Put it back in the squirrel’s basket and put the new bedding. After feeding and drinking the rats, observe their condition every day.
Operation points: Make sure to place it next to the heater to maintain body temperature. However, a certain distance must be maintained so that the body temperature does not become too high. You can put your hand on the mouse to see if the temperature is correct.
(12) Clean surgical equipment and operating room.
IV. Postoperative state of the animal The animal wakes up after anesthesia, and it can be observed that the right hind limb is completely normal and the left hind limb is soft. If the hind limbs are paralyzed, it may be due to spinal shock (usually recoverable after 1 week) or paraplegia caused by excessive trauma to the spinal cord during the operation. The mouse in this case needs to be removed).
5. Evaluation of nerve function
The content includes measurement of sciatic nerve conduction velocity, thermal stimulation of shoe sole, and mechanical stimulation of shoe sole.
6. Experience and experience
This model has the characteristics of trauma, bleeding and long operation time. It takes about 3 hours to create the model. Sturdy mice should be used, weighing more than 280g. During this process, you will need to carefully stop the bleeding and make a small cotton ball to stop the bleeding. When separating the nerve roots, please pay attention to maintain the animal's body temperature and keep it gentle. It should always be stored in a greenhouse after surgery.
Try to choose a unilateral afferent model that will reduce trauma, improve survival and help restore hind limb function after surgery. One of the things that is often overlooked is spine integrity. To remove the dorsal root segment, the transverse process must be removed during the operation. The lateral process is one of the links connecting the upper and lower vertebral bodies, thereby compromising the integrity of the spine. This is destruction. Hind limb movement requires not only the support of the nervous system, but also the support of the skeletal system. When the bilateral dorsal root ablation model is selected, the skeletal system is completely destroyed, and the unilateral dorsal root ablation model can also play a non-surgical compensation role.
is better than female rats because there may be a period of retention of urine after surgery. The female urethra is shorter than the male urethra, so it is easier to squeeze urine. If antibiotics are used to prevent infection before surgery, penicillin is the first choice, while third-generation cephalosporins are banned because they can affect blood clotting and cause bleeding during surgery. I will. The author used cefotaxime sodium for preoperative prophylaxis, and the animal died due to almost bleeding during the operation. Finding relevant information further confirms that the third-generation cephalosporins affect the coagulation system. Penicillin powder should begin to disintegrate immediately after dissolving in the liquid, so the penicillin solution should be prepared for immediate use.
It is very important to hydrate before surgery. This will reduce blood loss and provide some blood volume replacement. Replenishing blood volume, maintaining body temperature, ensuring smooth breathing and maintaining sufficient depth of anesthesia are the cornerstones of any successful animal surgery.
