动物造模,药效评价,脑脊髓炎 z-bio 2024-08-07 259

　　（1） Guinea pig model of allergic encephalomyelitis

　　Method 1

　　1. Modeling material animal: guinea pig, weighing about 400g, male or female not limited; Drug: Freund adjuvant, physiological saline; Equipment.

　　2. Preparation of spinal cord FA emulsion: Heparin guinea pigs under sterile conditions, then perfuse the thoracic aorta with physiological saline until there is no blood, remove the spinal cord, remove the spinal membrane, weigh, and homogenize the spinal cord with a glass homogenizer. Prepare a 50% suspension with physiological saline; Mix the suspension of spinal cord tissue with FA in a 1:1 ratio and repeatedly inject it with a syringe to form an emulsion.

　　Inject 0.1ml of spinal cord FA emulsion into each of the four paw pads of guinea pigs, with a total of 0.4ml injected into each guinea pig. After injection, feed them regularly.

　　3. The principle of modeling is that autoimmune causes allergic encephalomyelitis in animals.

　　4. Changes after modeling: After half a month of modeling, anti spinal cord antibodies gradually appeared in the serum, and skin tests showed a delayed response. In addition, guinea pigs gradually developed hind limb paralysis, and even urinary and urinary incontinence, making them prone to death.

　　Method 2

　　1. Modeling material animals: rats, 300-350g; Guinea pigs, half male and half female, weighing 250-350g; Drug: Freund's complete adjuvant.

　　2. Modeling method: Preparation of basic myelin protein (MBP) antigen: After euthanizing rats, the spinal cord is quickly removed and homogenized into a 50% homogenate using a homogenizer. The mixture is then mixed with an equal amount of Freund's complete adjuvant (BCG 10mg/ml) and injected into a water in oil emulsion using a syringe.

　　Inject MBP antigen subcutaneously into the hind paw of guinea pigs, with 0.2ml on each side, to create an experimental tal allergic encephalomyelitis (EAE) model.

　　3. The principle of modeling is that heterologous proteins can cause allergic encephalomyelitis in animals.

　　4. Biochemical changes after modeling: The level of cytokine IL-1 β in the brain tissue of the model group was significantly higher than that of the control group [control group (0.51 ± 0.02) ng/mg, model group (2.85 ± 0.70) ng/mg].

　　The level of TNF - α in the brain tissue of the model group was significantly higher than that of the control group [1.13 ± 0.28) ng/mg, and the model group (2.83 ± 0.90) ng/mg].

　　（2） Experimental allergic encephalomyelitis model in SD rats

　　1. Modeling material animals: SD rats, 10 weeks old, female, weighing 200-230g; Drug: Complete Freund Adjuvant (CFA).

　　2. Modeling method: SD rat spinal cord homogenate (SD-SCH) preparation: SD rats were euthanized by intraperitoneal injection of excess 10% chloral hydrate, and the spinal cord and brain were weighed. Mix 1g of rat spinal cord and brain white matter with 1ml of phosphate buffered solution (PBS) in an ice bath using an electric homogenizer (3000r/min), and homogenize for 3 minutes (1 minute each time, with an interval of 1 minute) to prepare rat spinal cord and brain homogenate. Mix the homogenate thoroughly with an equal volume of Freund's complete adjuvant to prepare an induction emulsion.

　　The model group rats were subcutaneously injected with induction emulsion 0.006ml/kg twice into the hind foot pads, and supplemented with 0.2ml pertussis bacillus (2 × 10 ^ 10 cells/ml) once into the dermis on the back of the hind foot pads. The normal control group was injected with a mixture of PBS and CFA in equal amounts.

　　3. The principle of modeling is that autoimmune causes allergic encephalomyelitis in animals.

　　4. Changes after modeling: The animals in the model group showed weight loss on the 1st and 2nd day after sensitization, gradually increasing and stabilizing before onset of the disease. The control group observed for 70 days without any clinical symptoms. The weight of the model group rats suddenly decreased before the onset of the disease, and the degree of weight loss was consistent with the severity of clinical symptoms; As the condition improves, weight gradually increases. The clinical symptoms mainly include varying degrees of appetite loss, reduced activity, weight loss, tail weakness, relaxation, and sagging; Stumbling, weakness in the hind limbs, paralysis, and in severe cases, a state of near death or even death may occur. The duration of two episodes of illness in the model group animals was 16-20 days and 10-14 days, respectively; The incidence rate was 85%, the recurrence rate was 46.67%, and the mortality rate was only 5.88%.

　　5. Pathological changes after modeling: The pathological changes in the model group animals mainly manifested as vasculitis with peripheral myelin sheath loss. The main lesions included: ① "sleeve like" changes in blood vessels, with a large number of inflammatory cells densely surrounding small blood vessels, especially small veins, forming a "sleeve like" appearance; ② Glial cell nodules, with multiple nodules formed due to glial cell proliferation visible; ③ Demyelination changes, with patchy demyelinating areas of varying sizes visible on myelin staining; ④ Neuronal degeneration, especially in the brainstem or spinal cord, with significantly increased cell bodies, rounded morphology, displaced nuclei, disappearance of nucleoli, disintegration of Nissl bodies into granular or completely absent cells, and pale cytoplasmic staining; ⑤ Satellite phenomenon, a phenomenon formed by glial cells and neutrophils surrounding degenerated neurons; ⑥ Lattice cells, which are abundant in inflammatory lesions, are formed by the proliferation of microglia and the phagocytosis of myelin sheath damaging products such as lipids and neutral fats.

　　The lesions in the model rats mainly invade the white matter and gray white matter junction of the brain and spinal cord, as well as the junction of the cortex and cortex medulla, and even the deep medulla. The meninges of the brain and the periphery of the lateral ventricles are also affected. The lesion also invades the cerebellum, brainstem, and optic chiasm, which is consistent with observed clinical manifestations such as ataxia and convulsions.

　　Electron microscopy shows scattered lesions, significant edema in tissue gaps, especially around small blood vessels, swelling of endothelial cell mitochondria, and blurred tight junctions. Multiple wandering lymphocytes and monocytes are distributed outside the vascular lumen. The cytoplasm of lymphocytes in the parenchyma increases and protrudes, becoming activated lymphocytes. Visible activated lymphocytes are close to neurons, and the surrounding tissue is loose and edematous. The myelin sheath has a loose layered structure, with loss and fusion, and the alternation of light and dark disappears. Swelling of mitochondria within the axon, unclear microtubule structure, or complete disappearance of organelles. The cytoplasmic structure of neurons is loose, the endoplasmic reticulum expands and degranulates, and neurons undergo degeneration. Release of the inner layer of the same myelin sheath and separation of axonal myelin sheaths.

　　Magnetic resonance imaging (MRI) scans have shown that lesions in animal models are mostly distributed around the ventricles, with varying sizes and slightly blurred edges. There are scattered long elliptical T1 and T2 abnormal signal shadows, which may have significant space occupying effects. This is similar to the imaging changes in human MS (mass spectrometer), indicating the reliability of the model.