动物造模,药效评价,抗原诱导EAE模型 z-bio 2024-07-29 510

　　(1) Induction of EAE model in Wistar rats by immunization with guinea pig spinal cord homogenate

　　① Experimental animals and reagents: Experimental 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 use, complete fresh adjuvant (CFA) was added to the CFA by adding freeze-dried powder of Mycobacterium tuberculosis, resulting in a final concentration of 4mg/L.

　　② Preparation of Guinea Pig Whole Spinal Cord Homogenate - Complete Freund's Adjuvant: After excessive anesthesia with ketamine, guinea pigs were perfused with pre cooled 0.9% sodium chloride solution until no blood flowed out. The guinea pig spinal cord was removed, and the spinal membrane was removed. The pre cooled physiological saline was used to prepare a 50% (m/V) guinea pig spinal cord physiological saline homogenate, which was then mixed with an equal amount of CFA to make a water in oil emulsion, 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 rats, for a total of 0.4ml. At the same time, 0.1ml of pertussis vaccine was injected intradermally into the dorsal surface of the feet. The experimental control group rats were injected with equal amounts of CFA and physiological saline into the skin of each of the four foot pads.

　　(2) Induction of EAE animal model in DA rats sensitized with guinea pig spinal cord homogenate as antigen: ① Experimental animals. Dark Agouti (DA) rats, female 8-10 weeks old, weighing 250-300g Preparation of antigen adjuvant emulsion. Guinea pig spinal cord homogenate (GPSCH) was uniformly mixed with Freund's complete adjuvant as an antigen adjuvant emulsion. After anesthesia with pentobarbital sodium (50mg/kg, intraperitoneal injection), guinea pigs were perfused with pre cooled ice physiological saline until the outflow fluid from the right atrium was clear. The guinea pig spinal cord was carefully removed, the spinal membrane and blood vessels were removed, weighed, and an equal amount of complete Freund's adjuvant (CFA) was added. The mixture was repeatedly injected with a syringe to create an oil in water emulsion, which became the antigen adjuvant emulsion Establishment of EAE animal model in DA rats. After routine disinfection of the skin at the tail root of DA rats, 0.2ml of antigen adjuvant emulsion was injected subcutaneously at a single point. On the day of immunization and after immunization, observe the diet and activity of all animals daily, and weigh their bodies.

　　(3) Preparation of crab eating monkey EAE model using homologous monkey brain white matter homogenate: ① Experimental animals. Crab eating macaques, female from June to October, with a body weight of 2.5-3.0kg Preparation of antigen adjuvant emulsion. Healthy crab eating monkey brain, fresh and sterile stored at 70 ℃, gray matter at 18 ℃, monkey brain white matter mixed with PBS according to body mass/volume=1/1.5 to make monkey brain white matter homogenate, CFA added according to volume/volume=1/1, repeatedly whipped with a syringe to make it in oil in water state, which is the antigen adjuvant emulsion Establishment of EAE animal model in crab eating macaques. The ways in which sensitized experimental animals develop EAE include subcutaneous, intradermal, intramuscular, intraperitoneal, and intravenous injection. Numerous animal experiments have shown that subcutaneous injection of allergens at multiple sites has the highest incidence of EAE induction. We use subcutaneous injection of 0.2ml into the armpit and groin of immunized animals, with 3-4 injections per site. Repeat injection once every 5-7 days Clinical grading criteria for monkey EAE. Divided into 6 levels, 0 levels, normal; Level 1, drowsiness, anorexia, and decreased body mass; Level 2, ataxia and tremor; Level 3, visual impairment, hemiplegia, and paraplegia; Level 4, quadriplegia; Level 5, in a dying state. a. Pathological examination of EAE. Autopsy was performed on EAE monkeys 10 days after onset. The brains, spinal cords, and optic nerves were collected and fixed with 40ml/L formaldehyde solution for 6 hours. After rinsing with running water, the brains were cut in half from the sagittal sinus. Then, the frontal, temporal, parietal, occipital, cerebellar, brainstem, and spinal cord were cut into 0.5cm thick tissue blocks according to the coronal plane. The blocks were dehydrated, transparent, infiltrated, embedded, and made into wax blocks using conventional methods. b. Dyeing method. Observe inflammatory changes using HE routine staining and observe myelin sheath changes using Luxol fast blue staining method. c. Electron microscopy sectioning and observation. Quickly trim the freshly cut optic nerve into tissue blocks of 1mm × 1mm × 1mm size and fix them in a pre fixative solution of 25g/L glutaraldehyde and 20g/L paraformaldehyde for at least 3 hours. After removal, fix with osmium tetroxide for 1.5 hours, dehydrate with gradient ethanol, embed with epoxy resin Epon 812, slice with an AO ultra-thin slicer, stain with uranium acetate and lead citrate, and finally observe with a Hitachi H-600 transmission electron microscope.