CN-121971600-A - Standardized NMOSD animal model construction method and drug screening application

Abstract

The invention discloses a standardized NMOSD animal model construction method and drug screening application, which selects female Lewis rats of 6-8 weeks old, injects Asian race highly pathogenic AQP4-IgG through a spinal sheath internal tube, continuously doses for five days, and verifies model pathogenicity through behavioural scoring, immunohistochemistry and Western Blot. The rat model is simple and convenient to operate, can stably simulate the glucocorticoid treatment resistance phenotype, has core pathological indexes which are highly consistent with those of clinically refractory NMOSD patients, and has the drug screening application covering grouping intervention, multidimensional evaluation and quantitative judgment. The invention solves the defects of poor repeatability, insufficient race specificity and the like of the existing model, provides a standardized tool for NMOSD mechanism research and targeted drug research and development, and has remarkable application value.

Inventors

• SONG HONGLU
• CHUAI YUCAI

Assignees

• 中国人民解放军联勤保障部队第九八〇医院

Dates

Publication Date

20260505

Application Date

20260209

Claims (5)

1. 1. The construction method of the standardized NMOSD animal model is characterized by comprising the following steps: Step 1, selecting and preprocessing experimental animals, namely selecting 6-8 week old female Lewis rats, purchasing 120-180g of the rats from experimental animal suppliers with legal qualification, adaptively feeding the rats for 1 week, and carrying out modeling after confirming no nerve dysfunction through three triple standards of tail tension test, gait coordination observation and limb activity evaluation every day; Step 2, spinal sheath catheterization, namely, after a rat is anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5 ml/kg), the rat is prone to be fixed on a constant-temperature operating table (maintaining the body temperature to be 37+/-0.5 ℃), the occipital is dehaired and disinfected, skin (length of 1.5-2.0 cm) is incised along the median line behind the neck, subcutaneous tissue and muscle are passively separated, the area between the atlas (C1) and occipital is exposed, fascia and fat on the surface of the rat are gently scraped by a cotton swab, the atlas occipital is exposed, a PE10 catheter (with the inner diameter of 0.28mm and the outer diameter of 0.61 mm) with the length of 10.0cm is slowly inserted into the subarachnoid space, the insertion depth of 7.0cm is ensured to be accurately positioned at the lesion target position corresponding to the first lumbar vertebra, the medical suture is used for fixing the catheter, muscle and skin are sutured in layers, penicillin sodium (10 ten thousand U/min) is injected into the muscle after operation to prevent infection, the rat is adaptively fed for 2 days, abnormal behaviors such as fever, catheter fall off, limb paralysis and the like are observed, and the following steps are entered; Step 3, model construction and drug administration, namely injecting Asian high pathogenic AQP4-IgG into subarachnoid space through a catheter, wherein the injection dosage is 5-10 mu L/dose (antibody concentration is 1 mu g/mu L), the injection is carried out 1 time per day for 5 days, the administration time is fixed to 9:00-10:00 am, and the rats are gently fixed for 10min after administration, so that the reflux of the drugs is avoided; setting a control group, namely setting a rAb-53 monoclonal antibody control group (rAb-53 monoclonal antibody is injected at the same dose) and a PBS blank control group (PBS with the same amount is injected), wherein the administration mode and the frequency are consistent with those of a model group; and 5, model verification: performing behavioral scoring on a daily basis (0-5 min for no abnormality, 1min for tail weakness, 2 min for hind limb weakness but standing, 3 min for hind limb paralysis, 4 min for limb weakness, 5min for dying or dying) by adopting a model of 0-5 min for 7-10 days after modeling, wherein the model group score is more than or equal to 3 min; On the 10 th day after modeling, randomly selecting 6 rats from each group, killing the tissues (0.5 cm) of the spinal cord lesion, detecting the positive expression rate of the C3 protein and the demyelinating area ratio of MBP (myelin markers) through immunohistochemistry, wherein the positive expression rate of the C3 protein in the model group is more than or equal to 70%, and the demyelinating area ratio of the MBP is more than or equal to 40%; The Western Blot detects the expression level of GFAP (astrocyte activation marker) and NeuN (neuron damage marker) in spinal cord tissues, the relative expression quantity of the GFAP protein of the model group is more than 2.0 times of that of the rAb-53 monoclonal antibody control group, the relative expression quantity of the NeuN protein is less than 0.6 times of that of the control group, and the model is confirmed to accord with the pathological characteristics of NMOSD.
2. 2. The construction method according to claim 1, wherein the AQP4-IgG of the African race is derived from Asian race NMOSD patients which recur within 12 months after glucocorticoid impact treatment in the step 3, the purity is more than or equal to 95% by Protein A column chromatography and molecular sieve chromatography purification, and the capacity of inducing primary astrocytes to produce the C3 Protein is more than 1.5 times of that of rAb-53 monoclonal antibody (verified by Western Blot quantification).
3. 3. The construction method according to claim 1, wherein the success rate of the spinal sheath catheterization in the step 2 is more than or equal to 90%, the survival rate of rats after the operation is more than or equal to 95%, and the catheter fixing adopts a dual mode of suture fixing and medical glue reinforcement, so that the catheter is prevented from falling off during the intervention.
4. 4. The use of the standardized NMOSD animal model constructed according to the method of claim 1 for screening glucocorticoid therapy resistant NMOSD drugs, characterized by comprising the steps of: Step 1, model grouping and drug intervention: Selecting an NMOSD animal model constructed in claim 1 (behavioural score is 3.0-3.5), randomly dividing into a drug group to be tested, a glucocorticoid control group, a normal saline blank group and a model control group, wherein each group is not less than 6, the drug group to be tested is given with drugs to be tested according to different concentration gradients (low concentration, medium concentration and high concentration), the administration route is selected from intraperitoneal injection, gastric lavage or intravenous injection according to the drug characteristics, the glucocorticoid control group is lavage with dexamethasone at a dose of 30mg/kg, 1 time per day, the blank group is given with normal saline in an equivalent amount, the model control group is not given, only conventional feeding is performed, each group is continuously interfered for 14 days, and the feeding environment is kept consistent during the intervention period (temperature 22+/-2 ℃, humidity 50+/-5% 12h illumination/12 h dark period); Step2, behavioural assessment: 2 blind observers independently perform behavioural scoring at fixed time in the morning during the intervention period, and take an average value, calculate the score lifting rate ((post-intervention score-pre-intervention score)/pre-intervention score multiplied by 100 percent), and compare and analyze the improvement effect of the drugs to be tested with different concentrations on the nerve function of the model rat; And 3, detecting pathological indexes: After the intervention, all rats were sacrificed and spinal cord lesions and adjacent normal tissues were removed for the following tests: Immunohistochemistry, after dewaxing to water, Blocking endogenous peroxidase, repairing citrate buffer solution antigen, adding primary antibody (C3 protein antibody dilution ratio 1:500, MBP antibody dilution ratio 1:800), incubating at 4deg.C overnight, incubating for 1h with fluorescent labeled secondary antibody, dying nuclei with DAPI, observing and counting C3 protein positive expression rate and MBP demyelination area ratio under a fluorescence microscope; Western Blot, RIPA lysate (containing 1% protease inhibitor) is used for extracting total protein, 20 mug is taken for SDS-PAGE electrophoresis after BCA method, membrane transfer is carried out to PVDF membrane, 5% skimmed milk is blocked for 1h, primary antibody (GFAP antibody dilution ratio 1:1000, neuN antibody dilution ratio 1:1000 and GAPDH antibody dilution ratio 1:2000) is added for 4 ℃ incubation overnight, HRP-labeled secondary antibody is used for incubation for 1h, ECL color development is carried out, and image J software is used for quantitatively analyzing the band gray value, so as to calculate the relative expression quantity of target protein; histopathological staining by HE staining to observe the inflammatory infiltration degree of spinal cord tissue, and Luxol Fast Blue (LFB) staining to further verify the repair of myelin lesions; And 4, judging the effective medicaments: If the to-be-detected drug can improve the behavioral score of a model rat by more than or equal to 30%, the C3 protein expression is reduced by more than or equal to 40%, the MBP demyelination area ratio is reduced by more than or equal to 30%, HE (high-level) staining shows that inflammatory infiltration is remarkably reduced, LFB (low-level fiber) staining shows that myelin sheath repair is good, the drug is judged to be an effective drug for glucocorticoid therapy resistant NMOSD (N-type nuclear magnetic resonance imaging), and the drug which can reach the index under low concentration is preferentially selected as a candidate targeting drug.
5. 5. The method of claim 4, wherein the agent comprises one or more of a C3aR inhibitor, a microglial cell inhibitor, a complement pathway antagonist, an anti-inflammatory agent, and a myelin repair promoter, and wherein the agents with different mechanisms of action are used in combination to evaluate the synergistic therapeutic effect.

Description

Standardized NMOSD animal model construction method and drug screening application Technical Field The invention belongs to the technical field of autoimmune disease animal models, and particularly relates to a construction method of a standardized neuromyelitis optica (NMOSD) animal model and application of the standardized neuromyelitis optica (NMOSD) animal model in drug screening, which are particularly suitable for research and development of glucocorticoid therapy resistant NMOSD targeted drugs, pathogenesis analysis and treatment scheme optimization, and can be widely applied to related researches of biological medicine enterprises, scientific research institutions and clinical medical institutions. Background The diseases of the optic neuromyelitis spectrum (NMOSD) are autoimmune diseases with central nervous system inflammatory demyelination and axonal injury as main pathological characteristics, the incidence rate of the diseases is obviously higher than that of caucasian species in Asian species, and about 30 percent of patients respond poorly to glucocorticoid treatment, the diseases are continuously relapsed, the nerve function is deteriorated, and the diseases belong to refractory NMOSD. The patients need targeted therapeutic drugs, but the development of related drugs is slow, and one of the core bottlenecks is the lack of standardized animal models with strong clinical relevance. In the prior art, the construction of an NMOSD animal model mainly depends on non-standardized patient serum antibodies or pathogenic antibodies (such as rAb-53 monoclonal antibodies) derived from caucasian species, and a plurality of defects which are difficult to overcome exist: 1. The model repeatability is poor, the antibody titer and pathogenicity heterogeneity of the non-standardized serum antibody are strong, the models constructed by antibodies from different batches and different patients have obvious differences in key indexes such as behavioural score, pathological injury degree and the like, so that experimental results of different laboratories can not be mutually verified, and the research efficiency is seriously influenced; 2. The existing model is mostly constructed based on Caucasian antibodies, and the gene background, immunophenotype and pathogenic antibody characteristics of Asian NMOSD patients are obviously different from the Caucasian antibodies, so that the model can not accurately reflect the disease progress rule and treatment response characteristics of Asian people, and the screened medicines have poor curative effect in clinical application; 3. The model is not related to clinical treatment response, the constructed model is generally sensitive to glucocorticoid treatment, and the refractory NMOSD phenotype of 'glucocorticoid treatment resistance' in clinic cannot be simulated, so that the drug screening direction deviates from clinical requirements; 4. the existing model is not perfect in evaluation system, the verification of the existing model only depends on behavioral scores, and the detection of molecular indexes (such as complement C3 protein, myelin markers MBP, astrocyte activation markers GFAP and the like) directly related to the pathogenic mechanism of NMOSD is lacking, so that the pathological characteristics of the model and the action targets of medicines cannot be accurately evaluated; 5. The operation difficulty is high, the existing model mostly adopts a mouse as an experimental object, the mouse is small in size, the operation space of the spinal sheath internal catheterization is narrow, the catheter positioning difficulty is high, the modeling success rate is low, the postoperative nursing difficulty is high, and the popularization and the large-scale application of the model are not facilitated; 6. The lack of quantification of pathological indexes in the prior art is that the description of the pathological characteristics of the model is mostly qualitative judgment (such as obvious inflammatory infiltration and myelin sheath damage existence), quantification standards are not established, and the stability of the model and the intervention effect of the medicine cannot be objectively evaluated. NMOSD-based pathogenic mechanism research proves that after the AQP4-IgG is combined with the AQP4 protein on the surface of astrocytes, the complement pathway is activated, the astrocytes are induced to produce a large amount of C3 protein, the C3 protein further activates microglial cells, an 'astrocyte-microglial cell' pathogenic loop is formed, and finally, neuronal damage and demyelination are caused. The capability of the Asian species of highly pathogenic AQP4-IgG to induce the production of C3 protein is obviously stronger than that of the Caucasian species of antibodies, which is also an important reason for high incidence rate and more serious disease of the Asian species of NMOSD. Therefore, an NMOSD animal model which is based on Asian race highly pathogenic AQP