动物造模,抗原诱导EAE模型 z-bio 2023-11-21 1052

　　(1) Immunization of Wistar rats with guinea pig spinal cord homogenate to induce EAE model

　　① Laboratory animals and reagents: Laboratory animals. Female Wistar rats, 6-8 weeks old, weighing 130-200g; Guinea pig, female, weighing 330-400g. Reagents. BCG freeze-dried powder, pertussis vaccine, complete Freund's adjuvant. Before the use of complete freund adjuvant (CFA), freeze-dried powder of Mycobacterium tuberculosis was added to the CFA to achieve a final concentration of 4mg/L.

　　② Preparation of guinea pig whole spinal cord homogenate - complete Freund's adjuvant: After excessive anesthesia with ketamine, the guinea pig was perfused with pre cooled 0.9% sodium chloride solution until there was no blood flow. The guinea pig spinal cord was removed from the spinal cord, and pre cooled physiological saline was used to prepare 50% (m/V) guinea pig spinal cord physiological saline homogenate. After mixing with an equal amount of CFA, an oil in water emulsion was prepared, which is the immune antigen.

　　③ Induction method: The experimental group injected 0.1ml of immune antigen into the skin of each of the four foot pads of the rats, with a total of 0.4ml for one mouse. At the same time, 0.1ml of pertussis vaccine was injected into the skin of the back of the foot pads. The experimental control group rats were injected with an equal amount of CFA and physiological saline into each of the four foot pads.

　　(2) Using guinea pig spinal cord homogenate as antigen to induce EAE in DA rats: ① Experimental animals. Dark Agouti (DA) rats, female 8-10 weeks old, weighing 250-300g Preparation of antigen adjuvant emulsions. Mix guinea pig spinal cord homogenate (GPSCH) and Freund's complete adjuvant evenly as an antigen adjuvant emulsion. After anesthesia with pentobarbital sodium (50mg/kg, intraperitoneal injection), the guinea pigs were infused with pre cooled ice physiological saline into the heart until the right atrial outflow was clear. The spinal cord of the guinea pigs was carefully removed, the spinal membrane and blood vessels were removed, the weight was weighed, and an equal amount of complete Freund's adjuvant (CFA) was added. Repeatedly beating with a syringe was used to make the antigen adjuvant emulsion in an oil-in-water state Establishment of a DA rat EAE animal model. After routine disinfection of the skin at the root of the tail of DA rats, a single subcutaneous injection of 0.2ml of antigen adjuvant emulsion was performed. On the day of immunization and after immunization, all animals were observed daily for their diet, activity, and weighed.

　　(3) Using homologous monkey brain white matter homogenate to create a crab eating monkey EAE model: ① Experimental animals. Crab eating macaques, female from June to October, with a body weight of 2.5-3.0kg Preparation of antigen adjuvant emulsions. Healthy crab eating monkey brain, stored fresh and sterile at 70 ℃, with gray matter at 18 ℃. PBS was added to the white matter of the monkey brain at a body mass/volume ratio of 1/1.5 to produce a homogenized white matter of the monkey brain. CFA was added at a volume/volume ratio of 1/1, and repeatedly beaten with a syringe to form an oil in water emulsion of the antigenic adjuvant Establishment of an EAE animal model for crab eating macaques. The pathways for producing EAE in sensitized experimental animals include subcutaneous, intradermal, intramuscular, intraperitoneal, and intravenous injection. Numerous animal experiments have confirmed that subcutaneous injection at multiple sites can cause EAE