Assessment method of thyroid-related eye disease mouse model

Abstract

The invention provides a thyroid-related eye disease mouse model evaluation method. The thyroid-related eye disease mouse model evaluation method comprises the following steps: S1, appearance phenotype evaluation; s2, thyroid function index evaluation; s3, carrying out iconography evaluation; s4, performing immunological phenotype evaluation; and S5, carrying out histopathologic evaluation. Compared with a traditional research method only paying attention to single eye phenotype or single histological change, the thyroid-related eye disease mouse model evaluation method provided by the invention comprehensively reflects disease characteristics of a TAO mouse model from five dimensions of appearance phenotype, thyroid function, immune phenotype, histopathology and iconography; multi-dimensional comprehensive evaluation of the TAO mouse model is realized, and a scientific, comprehensive and standardized evaluation method is established.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an evaluation method of a mouse model of thyroid-associated ophthalmopathy. BACKGROUND

[0002] Thyroid-Associated Ophthalmopathy (TAO), also known as Graves' ophthalmopathy, is an organ-specific autoimmune disease characterized by inflammation, adipogenesis and muscle fibrosis of orbital tissues, and is the most important extra-thyroid manifestation of Graves' disease. The typical clinical manifestations of TAO include eyelid hyperemia and edema, exophthalmos, diplopia, and visual acuity decline. In severe cases, it can develop into corneal perforation or optic neuropathy, and even lead to blindness. The current clinical treatment of TAO mainly includes glucocorticoids, radiotherapy and surgical treatment. Although target drugs represented by Teprotumumab have been approved for use in some countries, their application population is still limited, the efficacy varies among individuals, and there are certain side effects. The development of new generation of targeted treatment strategies urgently needs to rely on reliable and repeatable animal models to carry out systematic pathogenesis research and intervention experiment verification.

[0003] Mouse models are important tools for TAO research, and have irreplaceable value in exploring pathogenesis, screening candidate drugs, and evaluating efficacy and toxicity before clinical translation. Due to the complexity of the pathogenesis of TAO and the chronic and prolonged course of the disease, various modeling strategies have been established, including TSHR protein immunization, TSHR gene immunization, TSHR cell immunization, and drug induction. These methods differ in modeling principles, operation procedures, phenotypic characteristics, and reproducibility. Therefore, if there is a lack of standardized evaluation system, it is difficult to accurately determine whether a model is reliable and whether it can truly reflect the clinical characteristics of the disease, which also limits the horizontal comparison between different studies and the optimization and improvement of the model. However, existing studies have not reached a consensus on the evaluation method of TAO mouse models, and there are significant differences in the selection of indicators, detection methods, and quantitative methods. In existing reports, common model evaluation indicators include changes in eye appearance (such as exophthalmos and retraction of the eyelid), histopathological features (such as adipose hyperplasia and muscle fibrosis), and serological indicators (such as total triiodothyronine TT3, total thyroxine TT4, thyroid-stimulating hormone TSH, and thyroid-stimulating hormone receptor antibody TRAb). However, there are significant differences in the selection of indicators, detection techniques, and quantitative methods between different studies, and most studies only focus on a single dimension or local phenotype, lacking a systematic evaluation method and stable detection means. In addition, the traditional evaluation of mouse models has the problem of directly copying the clinical diagnostic criteria of humans, but there are essential differences between mice and humans in terms of orbital anatomical structure and physiological function, which limits the reliability of the measurement results. For example, some studies have attempted to use magnetic resonance imaging (MRI) to measure the distance from the corneal apex to the outer canthus to evaluate the degree of exophthalmos in mice, but due to the anatomical characteristics of mice with the eye mainly protruding to the sides of the body, this measurement method has systematic errors and large measurement errors. Therefore, it is urgent to establish a standardized evaluation method for TAO mouse models that is scientific, reasonable, and suitable for the characteristics of mice. SUMMARY

[0004] To solve the problem of lack of systematic, standard, and quantifiable model evaluation method in the prior art, the present application provides an evaluation method for a mouse model of thyroid-associated ophthalmopathy.

[0005] The present application provides an evaluation method for a mouse model of thyroid-associated ophthalmopathy, comprising the following steps: S1, appearance phenotype evaluation: record the morphological changes of the mouse eye under fixed lighting conditions using a digital device, including overhead and side view photos; S2, thyroid function index evaluation: measure and record the body weight change of mice every week; detect the levels of total thyroxine, thyroid stimulating hormone and thyroid stimulating hormone receptor antibody in mouse serum; obtain mouse thyroid tissue, and perform hematoxylin-eosin staining to observe the morphological changes and integrity of thyroid follicle structure; S3, imaging evaluation: T2-weighted scanning of mouse orbit is performed by magnetic resonance imaging to measure the following parameters: exophthalmos, extraocular muscle transverse diameter, eyelid thickness; S4, immunological phenotype evaluation: obtain single cell suspension of mouse orbital tissue; use a combination of fluorescently labeled anti-CD45, CD3, CD4, IFNγ, IL-17a antibodies for staining; use flow cytometry to characterize the infiltration of T cells and their subsets in the orbital tissue; S5, histopathological evaluation: the mouse orbital tissue is stained as follows: H&E staining, calculate the ratio of adipose tissue area to optic nerve cross-sectional area; Masson staining, evaluate the degree of fibrosis of extraocular muscle tissue.

[0006] In an embodiment of the present application, in step S1, the overhead view photo is used to evaluate the exophthalmos, and the side view photo is used to evaluate the eyelid lesion.

[0007] In an embodiment of the present application, the appearance phenotype evaluation is determined by the following criteria: Under the overhead view, if the outlines of both eyes are clearly prominent, it is judged as exophthalmos positive, and each eye is scored 1 point; Under the side view, if eyelid edema is visible and the thickness of a single eyelid reaches or exceeds 1.5 mm, each eye is scored 1 point; If the mouse has eyelid skin ulceration and bleeding, each eye is scored 1 point; The total score of both eyes of each mouse is calculated, and if the total score of the appearance of the model mice is ≥2 points, it is considered that the appearance is consistent with the typical eye manifestations of TAO.

[0008] In an embodiment of the present application, in step S2, enzyme-linked immunosorbent assay is used to detect the levels of total thyroxine, thyroid stimulating hormone and thyroid stimulating hormone receptor antibody in mouse serum.

[0009] In an embodiment of the present application, the criteria for thyroid function index evaluation include: (1) If the ratio of the body weight of the model mice to the average body weight of the control mice at the same period is less than 1, and the difference in body weight between the two groups is statistically analyzed with p value <0.05, it is determined that the body weight change is consistent with the hypermetabolic state caused by hyperthyroidism; (2) if the serum TRAb level is more than 5 times of the control group, the TT4 level is more than 1 times of the control group, and the difference in serum TRAb level between the two groups is statistically analyzed with p value <0.05, it is determined that the peripheral blood thyroid function status of the mouse meets the characteristics of hyperthyroidism caused by Graves disease; (3) if at least two of the following pathological changes are observed by hematoxylin-eosin staining, it is determined that the thyroid histology has changed to meet the characteristics of the TAO model: thyroid follicle lumen atrophy; follicular epithelial cells proliferate from flat or cubic to high columnar; capillary dilation, hyperemia, and lymphocyte infiltration are observed in the thyroid parenchyma.

[0010] In an embodiment of the present application, in step S3, the exophthalmos degree is the vertical distance from the most protruding point of the mouse eyeball to the line connecting the inner and outer canthi on the axial view; the extraocular muscle transverse diameter is the diameter of the thickest part of the mouse extraocular muscle measured on the coronal view; and the eyelid thickness is the thickness of the mouse eyelid measured on the axial view.

[0011] In an embodiment of the present application, the criteria for the imaging evaluation include: if the ratio of any one of the exophthalmos degree, the extraocular muscle diameter, and the eyelid thickness between the modeling group and the control group is greater than 1, and the statistical analysis p value is <0.05, it is determined that the mouse presents the imaging performance of TAO.

[0012] In an embodiment of the present application, the criteria for the immunological phenotype evaluation include: if the following three indicators are met at the same time, it is determined that the mouse eye socket is in a highly activated inflammatory state, which meets the immunological phenotype changes of TAO: the proportion of CD3⁺CD45⁺T cells in CD45⁺immune cells is increased; the proportion of IFN-γ⁺CD4⁺Th1 cells in CD4⁺T cells is increased; the proportion of IL-17A⁺CD4⁺Th17 cells in CD4⁺T cells is increased; and the ratio of the above three indicators between the modeling group and the control group is greater than 1, and the statistical analysis p value is <0.05.

[0013] In an embodiment of the present application, in step S5, the ratio of adipose tissue area to optic nerve cross-sectional area is calculated by H&E staining, and the degree of fibrosis of the extraocular muscle tissue is evaluated by Masson staining.

[0014] In an embodiment of the present application, the criteria for the histopathological evaluation include: One of the following two conditions is met, that is, the mouse is determined to have orbital tissue lesions characteristic of TAO: (1) The ratio of retrobulbar adipose tissue area / nerve area is more than 1.5 times of the control group, and the statistical analysis p value is less than 0.05; (2) The ratio of fibrous collagen in extraocular muscle tissue is more than 1.5 times of the control group, and the statistical analysis p value is less than 0.05.

[0015] Compared with the prior art, the embodiments of the present application have the following beneficial effects: The present application provides an evaluation method for a mouse model of thyroid-associated ophthalmopathy, compared with the traditional research method which only focuses on a single eye phenotype or a single histological change, the present application comprehensively reflects the disease characteristics of the TAO mouse model from five dimensions of appearance phenotype, thyroid function, immune phenotype, histopathology and imaging, realizes the multidimensional comprehensive evaluation of the TAO mouse model, and establishes a scientific, comprehensive and standardized evaluation method.

[0016] In view of the problem that the existing evaluation method directly applies human clinical evaluation standards to mice and ignores species differences, the present application optimizes and standardizes the MRI measurement method of key parameters such as exophthalmos degree, extraocular muscle transverse diameter and eyelid thickness based on the characteristics of mouse orbit, fully considers the anatomical structure and physiological function characteristics of mouse orbit, and cooperates with high-field small animal MRI to realize non-invasive, accurate and repeatable image evaluation, thereby reducing the system error of measurement.

[0017] The evaluation method for a mouse model of thyroid-associated ophthalmopathy provided by the present application evaluates various pathophysiological characteristics of the TAO mouse model, including eye shape change, thyroid function state, orbital tissue structure change, immune cell infiltration and tissue fibrosis degree, which can more accurately and comprehensively reflect the disease progression process of TAO.

[0018] The evaluation method for a mouse model of thyroid-associated ophthalmopathy provided by the present application unifies and standardizes the collection process, analysis method and interpretation standard of each index, especially formulates clear parameter definition and software processing path in the aspects of image parameter analysis and pathological image quantification, which is helpful to improve the data consistency and experimental repeatability between different studies, and is convenient for popularization and application between different laboratories.

[0019] The evaluation method for a mouse model of thyroid-associated ophthalmopathy provided by the present application is simple and easy to implement, suitable for different experimenters and different experimental platforms, and has good popularization and application prospect.

[0020] The evaluation method of the thyroid-associated ophthalmopathy mouse model provided by the application can verify the reliability of various TAO mouse models, and is helpful to screen animal models more in line with the characteristics of clinical diseases, and provides a more reliable experimental basis for the pathogenesis research and drug research of TAO.

[0021] The evaluation method of the thyroid-associated ophthalmopathy mouse model provided by the application fully considers the species characteristics of mice and the complexity of TAO diseases, and selects pathological evaluation indexes closely related to each other for evaluation, thereby filling the blank of the current standardized evaluation system of TAO models.

[0022] The evaluation method of the thyroid-associated ophthalmopathy mouse model provided by the application is not only suitable for in-depth research on the pathogenesis of TAO, but also can be widely applied to the early screening and efficacy verification of new target drugs, and has good scientific research application prospect and clinical transformation potential. At the same time, the evaluation method provided by the application can verify the reliability of various TAO mouse models, and is helpful to screen animal models more in line with the characteristics of clinical diseases, and provides a more reliable experimental basis for the pathogenesis research and drug research of TAO. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a front view and a side view of mice in different groups; Figure 2 It is a result graph of hyperthyroidism of mice; wherein, Figure 2 A graph in the middle is a result graph of thyroid tissue slices of mice in different groups and hematoxylin and eosin (H&E) staining, and evaluation of thyroid follicle lesion; Figure 2 B graph in the middle is a result graph of evaluation of body weight of mice in different groups; Figure 2 C graph in the middle is a result graph of evaluation of thyroid stimulating hormone receptor antibody (TRAb), serum total thyroxine (TT4) and thyroid stimulating hormone (TSH) in serum of mice in different groups by serum ELISA method; Figure 3 It is a result graph of evaluation of orbital lesion of mice by orbital magnetic resonance; wherein, Figure 3 A graph in the middle is that the perpendicular distance of the mouse eyeball most protruding point to the inner and outer canthus connecting line (shown by the yellow reference line) is taken as the mouse eyeball protrusion (shown by the red arrow) on the axial view; Figure 3 B graph in the middle is that the diameter (shown by the red clip head) of the thickest part of the mouse extraocular muscle (the yellow line shows the outline of the extraocular muscle) is measured on the coronal view to evaluate the thickening of the extraocular muscle; Figure 3 C graph in the middle is that the mouse eyelid thickness (shown by the red arrow) is measured on the axial view to evaluate the eyelid edema; Figure 4 It is a result graph of evaluation of immunological changes of the mouse orbit; wherein, Figure 4Figure A is based on CD3 + Cells account for CD45 + Image showing the results of orbital T-cell infiltration assessment based on cell proportion; Figure 4 Figure B is based on IFNγ + Cells account for CD4 + Image showing the results of orbital Th1 cell infiltration assessment based on cell proportion; Figure 4 The middle C figure is based on IL-17a + Image showing the results of assessing orbital Th17 cell infiltration based on the proportion of CD4+ cells; Figure 5 This is a diagram showing the results of the histopathological assessment; in which... Figure 5 Figure A shows orbital tissue sections from different groups of mice, stained with hematoxylin and eosin (H&E) to assess the extent of retrobulbar fat proliferation. The ratio of adipose tissue area to optic nerve area was used to evaluate the relative extent of retrobulbar fat proliferation. Figure 5 Figure B shows tissue sections of extraocular muscle from different groups of mice stained with Masson stain. Muscle fibers appear red, while collagen fibers formed by fibrosis appear blue. The results of extraocular muscle fibrosis were assessed based on the proportion of collagen fibers.

[0024] Figure 6 This is a diagram showing the results of exploring the regulatory axis of autoantibodies, immune environment, and pathological changes in the TAO mouse model; among them, Figure 6 Image A shows the H&E staining results of orbital tissue, indicating that the orbital fat in the model group showed obvious inflammatory edema, accompanied by a large number of lymphocyte infiltrations. Figure 6 Figure B shows the immunohistochemical staining results of CD3 staining of extraocular muscles extracted from different groups of mice, indicating that the extraocular muscles of the model group contained a relatively high amount of CD3. + T cell infiltration (red arrow, yellow staining indicates CD3) + T cells); Figure 6 Figure C shows the results of further treatment of the model group mice with the blocking monoclonal antibody TRAb to inhibit the effect of TRAb in vivo, and further evaluation of the infiltration of immune cell subsets Th1 and Th17 cells. Figure 7 Imagej software was used to calculate the tissue area. Figure 8 Image showing the results of calculating the optic nerve area using Imagej software; Figure 9 Imagej software was used to calculate the total tissue area. Figure 10 Imagej software is used to calculate the area of ​​the fibrous indigo-stained region. Detailed Implementation

[0025] The application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the application, but do not limit the application in any form. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the application. These are within the scope of protection of the application.

[0026] Example 1 Construction of TAO mouse model The experimental animals were 6-week-old female BALB / c mice (purchased from Shanghai South Model Organisms Technology Co., Ltd.) and were raised in a standard experimental animal center, maintained under 12-hour alternating light and dark, with an ambient temperature of 21 °C and a humidity of 55%.

[0027] The construction of the TAO mouse model in this application and the prior art are mainly improved as follows: ① The concentration of the recombinant adenovirus (sequence name Tshr(289aa), transcript ID NM_000369.5) mother liquor is increased (from the original ≥10 10  pfu / mL to ≥10 11  pfu / mL), and the dilution ratio is adjusted from 1:50 to 1:5 to increase the final administration concentration; ② The administration interval is shortened, and the total modeling period is compressed from the original scheme of 34 weeks to 21 weeks, thereby improving the modeling efficiency.

[0028] In this application, the virus titer detection method is as follows: The 293 cells were inoculated in a 96-well plate at 1×10 4 cells per well; the virus was diluted to 8 consecutive gradient concentrations to infect the cells and cultured in a 37°C CO2 incubator; on day 10, microscopic observation was performed, and the cytopathic effect (CPE) results were analyzed and recorded; the virus titer calculation formula is as follows: For a 100 μl sample, the titer T = 10 1+d (s-0.5) d = log 10 dilution = 1 (for a 10-fold dilution) s = sum of positive rates (from the first 10-fold dilution) Convert TCID50 / ml to PFU / ml: T = a × 10 b TCID50 / ml = a × 10 b-0.7 PFU / ml The specific modeling method is as follows: the experimental group mice were injected intramuscularly with an adenovirus expressing human thyroid stimulating hormone receptor (Ad-hTSHRα) (10 11 pfu / mL) was used to establish the model, and the control group was established using the same dose of adenovirus expressing green fluorescent protein (Ad-EGFP).

[0029] The specific modeling process is as follows: ① The adenovirus mother liquor is diluted with PBS at a volume ratio of 1:5 to obtain an adenovirus working solution; ② On the first day of the 0th week, 25 uL of the Ad-TSHR diluent was injected into the left and right large muscles of the experimental animals; ③ Repeat step ② at weeks 3, 6, 9, 12, 15, 18, and 21, a total of 8 times to complete model construction. After the construction is completed, the mice exhibit signs such as exophthalmos and eyelid edema.

[0030] Example 2 The present embodiment provides an evaluation method for a thyroid-associated ophthalmopathy mouse model, which is the thyroid-associated ophthalmopathy mouse model constructed in Example 1.

[0031] The evaluation method for the thyroid-associated ophthalmopathy mouse model includes the following steps: S1, appearance phenotype evaluation: record the changes in the eye morphology of the mice under fixed lighting conditions using a digital device, including overhead and side view photos; S2, thyroid function index evaluation: measure and record the body weight changes of the mice every week; detect the levels of total thyroid hormone, thyroid stimulating hormone, and thyroid stimulating hormone receptor antibodies in the serum of the mice; obtain the thyroid tissue of the mice and perform hematoxylin-eosin staining to observe the changes in the structure and integrity of the thyroid follicles; S3, imaging evaluation: perform T2-weighted scanning of the mouse orbit using magnetic resonance imaging to measure the following parameters: exophthalmos degree, extraocular muscle cross-sectional diameter, and eyelid thickness; S4, immunological phenotype evaluation: obtain a single-cell suspension of the mouse orbital tissue; use a combination of fluorescently labeled anti-CD45, CD3, CD4, IFNγ, and IL-17a antibodies for staining; use flow cytometry to characterize the infiltration of T cells and their subsets in the orbital tissue; S5, histopathological evaluation: perform the following staining on the mouse orbital tissue: H&E staining to calculate the ratio of adipose tissue area to optic nerve cross-sectional area; Masson staining to evaluate the degree of fibrosis in the extraocular muscle tissue.

[0032] Specifically, step S1 includes the following steps: after the mouse modeling is completed, record the changes in the eye morphology of the mice under fixed lighting conditions (illumination of about 30 lux) using a high-definition digital device (SONY ILCE-6700 APS-C), and the photographing angles include: ① perpendicular to the mouse head (overhead), 20 cm away from the mouse head, for recording and evaluating the exophthalmos degree; ② the mouse head side, 20 cm away from the mouse head, for recording and evaluating the eyelid lesion.

[0033] Specifically, step S2 includes the following steps: measuring and recording the weight change of the mice every week to evaluate the systemic metabolic state of the mice. After euthanasia of the mice, peripheral blood is collected and serum is separated, and the levels of thyroid function-related indicators such as total thyroxine (TT4), thyroid stimulating hormone (TSH) and thyroid stimulating hormone receptor antibody (TRAb) in the serum are detected by enzyme-linked immunosorbent assay (ELISA) for quantitative evaluation of the hyperthyroid state and autoimmune activation of the mice. The above detection indicators use the following commercial kits and strictly follow the instructions: Mouse TRAb ELISA Kit (Cat No: ELK0450, ELK Biotechnology), Mouse TT4 ELISA Kit (Cat No: ELK0451, ELK Biotechnology) and Mouse TSH ELISA Kit (Cat No: ELK2284, ELK Biotechnology). At the same time, a transverse incision is made above the larynx and below the seventh cartilage ring to expose and remove the thyroid tissue. The obtained thyroid tissue is fixed in 4% paraformaldehyde and then paraffin-embedded, and after sectioning, the morphological changes and integrity of the thyroid follicle structure are observed under Hematoxylin and Eosin staining (H&E staining).

[0034] Specifically, step S3 includes the following steps: the mice are induced anesthesia using 5% isoflurane and maintained using a concentration of 1.5%-2%. Under anesthesia, the mouse orbit is scanned using a 9.4T Bruker BioSpec small animal imaging system, equipped with a 660mT / m gradient system and a ramp time of 110μs. A four-pole volume excitation coil with an inner diameter of 86mm is used for radio frequency excitation, and a four-pole mouse brain surface coil is used for signal acquisition. The scanning parameters are as follows: T2-weighted imaging (T2WI), echo time (TE) 33ms, repetition time (TR) 2.5s, field of view (FOV) 20mm×20mm, matrix 256×256, average number 4 times. The animal is continuously monitored for respiration and body temperature during scanning, and the body temperature is maintained at 37 °C by a hot air system. Data acquisition and management use Paravision360 software. Subsequently, based on the MRI image, the exophthalmos degree, extraocular muscle cross-sectional diameter, eyelid thickness and other parameters of the mouse are quantitatively measured: ① Exophthalmos degree (A): the vertical distance (red arrow) from the most protruding point of the mouse eyeball to the line connecting the inner and outer canthi (shown by the yellow reference line) is measured on the axial slice and statistically analyzed; ② Extraocular muscle cross-sectional diameter (B): the diameter (red clip head) of the thickest part of the mouse extraocular muscle (yellow line showing the outline of the extraocular muscle) is measured on the coronal slice and statistically analyzed; ③ Eyelid thickness (C): the thickness of the eyelid (red clip head) is measured on the coronal slice and statistically analyzed. Figure 3 Figure 3 Figure 3 ​​C) Measure the eyelid thickness of mice (red arrow shown) in axial section and perform statistical analysis.

[0035] Specifically, step S4 includes the following steps: after euthanasia of the mouse, the orbital complex (including eyeball, intraorbital segment of optic nerve, extraocular muscles and adipose tissue) is completely separated in the orbital region. After the orbital tissue is cut into small pieces, it is placed in a working concentration of 1 mg / ml collagenase D (11088858001, Roche) and shaken at 37°C for 30 min. Then 5 times the volume of PBS balancing solution is added to terminate the digestion, and a cell suspension is obtained. Red blood cells are lysed by using red blood cell lysis solution (Bi Yun Tian C3702) at room temperature for 5 minutes. Then 2 times the volume of PBS balancing solution is added to terminate, count and adjust to 1 x 10 7 Specifically, step S5 includes the following steps: the mouse orbital tissue is completely obtained using the same method as described above, fixed in 4% paraformaldehyde and paraffin-embedded, and sectioned perpendicular to the optic nerve direction: ① H&E staining: the ratio of adipose tissue area to optic nerve cross-sectional area is calculated using Imagej software to evaluate the degree of adipose hyperplasia and perform statistical analysis; ② Masson staining: the area ratio of fibrosis area (collagen fibers formed by fibrosis lesions are dyed blue) in retrobulbar adipose tissue and retrobulbar muscle tissue is calculated using Imagej software to quantitatively evaluate the degree of fibrosis of extraocular muscle tissue and perform statistical analysis.

[0036] Specifically, step S5 includes the following steps: the mouse orbital tissue is completely obtained using the same method as described above, fixed in 4% paraformaldehyde and paraffin-embedded, and sectioned perpendicular to the optic nerve direction: ① H&E staining: the ratio of adipose tissue area to optic nerve cross-sectional area is calculated using Imagej software to evaluate the degree of adipose hyperplasia and perform statistical analysis; ② Masson staining: the area ratio of fibrosis area (collagen fibers formed by fibrosis lesions are dyed blue) in retrobulbar adipose tissue and retrobulbar muscle tissue is calculated using Imagej software to quantitatively evaluate the degree of fibrosis of extraocular muscle tissue and perform statistical analysis.

[0037] wherein the ImageJ software calculates the adipose tissue area and the optic nerve cross-sectional area, and distinguishes the fibrosis area as follows: adipose: the range of whole adipose tissue in the orbital tissue is circled using Imagej software, and the tissue area (yellow line marked range) is calculated, then the optic nerve area (yellow line marked range) is calculated using the same method, and then the relative retrobulbar fat area is calculated as (adipose tissue area-optic nerve area) / optic nerve area for comparison between groups; Figure 7 Figure 8 adipose: the range of whole adipose tissue in the orbital tissue is circled using Imagej software, and the tissue area (yellow line marked range) is calculated, then the optic nerve area (yellow line marked range) is calculated using the same method, and then the relative retrobulbar fat area is calculated as (adipose tissue area-optic nerve area) / optic nerve area for comparison between groups;​ Fibrosis: Open the Masson staining image using Imagej software, adjust the threshold value, and calculate the total area of the tissue (red area) and the fibrosis blue staining area (red area) respectively. The quantitative analysis method is the area of blue staining area / total tissue area. Figure 9 Figure 10 The area of blue staining area / total tissue area.

[0038] The results of step S1 are as follows: The TAO mouse model can be seen on the appearance of the eyeball to the two sides of the obvious protrusion, in the overhead view of the double eye contour clear convex, as a positive performance, each eye protrusion eye each record 1 points; If the mouse can be seen eyelid edema and single eyelid thickness reaches or exceeds 1.5mm, each eye each record 1 points; If the mouse eyelid skin ulcer, bleeding, each eye each record 1 points. Figure 1 The total score of each mouse double eye is calculated. The double eye cumulative highest score of each mouse is 6 points, and the appearance score of the control group mouse should be 0 point. If the appearance score of the model group mouse is greater than or equal to 2 points, it is considered that the appearance is consistent with the typical eye performance of TAO.

[0039] The results of step S2 are as follows: H&E staining observation shows that the thyroid tissue of TAO modeling mouse appears typical pathological changes, including: (1) thyroid follicle cavity atrophy; (2) follicular epithelial cells from flat or cubic to high columnar hyperplasia; (3) capillary expansion, hyperemia in thyroid parenchyma, with lymphocyte infiltration Figure 2 A). The above three pathological changes each record 1 point, and the cumulative score of thyroid tissue of single mouse is 3 points. If the score is greater than or equal to 2 points, it is determined that the thyroid histology has appeared the changes consistent with the characteristics of TAO model.

[0040] The mouse appears weight loss after TAO modeling. If the ratio of the body weight of the modeling group mouse to the average body weight of the control group mouse in the same period is less than 1, and the difference in body weight between the two groups reaches a significant level (p<0.05) by statistical analysis, it is determined that the body weight change is consistent with the high metabolic state caused by hyperthyroidism Figure 2 B).

[0041] The TAO modeling group mouse can detect obvious hyperthyroidism and immune activation characteristics in the peripheral blood. When the serum TRAb level is more than 5 times that of the control group, the TT4 level is more than 1 times that of the control group, and the difference between the two is statistically significant (p<0.05), it is determined that the thyroid function state of the mouse peripheral blood is consistent with the characteristics of Graves' disease induced hyperthyroidism, and it is suggested that it is in the state of autoimmune activation. In addition, the decrease of TSH level in peripheral blood serum of modeling group mouse can be used as supporting evidence Figure 2 C).

[0042] ​The results of step S3 are as follows: The quantitative analysis of MRI of the orbit shows that the exophthalmos degree, the diameter of the extraocular muscle and the thickness of the eyelid of the TAO model mice are significantly increased compared with the control group Figure 3 A-C), indicating that the orbital connective tissue, extraocular muscle and eyelid are involved, which is consistent with the clinical features of TAO. If the ratio of any one of the above three indicators between the model group and the control group is greater than 1 and has a statistically significant difference (p<0.05), it can be determined that the mouse presents the imaging performance of TAO.

[0043] The results of step S4 are as follows: Flow cytometry analysis shows that the local orbital of the TAO model mice presents a highly activated inflammatory state, and the T cell infiltration is significantly increased compared with the control group Figure 4 A), and presents a dominant immune activation pattern of Th1 Figure 4 B) and Th17 Figure 4 C). The local orbital is in a highly activated inflammatory state only when the following three indicators satisfy the conditions that the ratio between the model group and the control group is greater than 1 and has a statistically significant difference (p<0.05), which is consistent with the immunological phenotype changes of TAO: (1) the proportion of CD3⁺CD45⁺T cells in CD45⁺immune cells is increased; (2) the proportion of IFN-γ⁺CD4⁺Th1 cells in CD4⁺T cells is increased; (3) the proportion of IL-17A⁺CD4⁺Th17 cells in CD4⁺T cells is increased.

[0044] The results of step S5 are as follows: H&E staining of the orbital tissue shows that the ratio of retrobulbar adipose tissue area / nerve area of the TAO model mice is increased compared with the control group, indicating that the orbital adipose tissue of the TAO mice is actively proliferating Figure 5 A). Masson staining shows that the proportion of fibrous collagen in the extraocular muscle tissue is significantly increased, indicating that the extraocular muscle presents obvious fibrosis Figure 5 B). If one of the following two conditions is met, it can be determined that the mouse presents the orbital tissue lesions consistent with the characteristics of TAO: (1) the ratio of retrobulbar adipose tissue area / nerve area is 1.5 times or more than that of the control group, and has a statistically significant difference (p<0.05); (2) the proportion of fibrous collagen in the extraocular muscle tissue is 1.5 times or more than that of the control group, and has a statistically significant difference (p<0.05).

[0045] Further, the verification experiment of the "autoantibody-immune environment-pathological change" regulation axis is carried out in this embodiment: H&E staining of the orbital tissue shows that the orbital tissue of the model group presents obvious tissue inflammatory edema changes compared with the control group, and there are a large number of immune cell infiltrations Figure 6 A); further, immunohistochemistry is used to detect CD3 +Analysis of T cell infiltration revealed a large number of T cell infiltrations in the diseased extraocular muscles of the model group. Figure 6 (B) This suggests a correlation between changes in immune cells and pathological changes in extraocular muscles. After treating model mice with a competitive blocking antibody against the autoantibody TRAb (K1-70, HY-P990534, MedChemExpress) (20 mg / kg body weight, intraperitoneal injection once a week), the proportion of pathogenic T cell subsets (Th1 and Th17 cells) in the orbit significantly decreased, suggesting that the TAO autoantibody TRAb is closely related to immunopathogenesis and orbital pathological changes.

[0046] The validation experiment of the "autoantibody-immune environment-pathological change" regulatory axis provides biological mechanism support for the evaluation method provided in this embodiment.

[0047] The evaluation method provided in this embodiment includes a multi-dimensional comprehensive evaluation standard, including: Basic criteria for determining success: ① Changes in physical appearance (see step S1), ② Changes in imaging features (see step S3), ③ Changes in histopathological characteristics (see step S5). These three indicators are the core evaluation indicators for establishing the TAO mouse model, and all three must be met simultaneously for successful modeling.

[0048] Supporting criteria: ① Changes in thyroid function (see step S2), ② Changes in immunophenotypic pattern (see step S4). These two indicators are enhanced validation criteria for the TAO mouse model, used to improve the model's integrity and consistency with the pathogenesis of human TAO.

[0049] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A method for evaluating a mouse model of thyroid-associated ophthalmopathy, characterized by, Comprising the following steps: S1, appearance phenotype evaluation: record the morphological changes of mouse eyes under fixed lighting conditions using a digital device, including overhead view and side view photos; S2, thyroid function index evaluation: measure and record the body weight changes of mice every week; detect the levels of total thyroid hormone, thyroid stimulating hormone and thyroid stimulating hormone receptor antibody in mouse serum; obtain mouse thyroid tissue, and perform hematoxylin-eosin staining to observe the morphological changes and integrity of thyroid follicle structure; S3, imaging evaluation: perform T2 weighted scanning of mouse orbital using magnetic resonance imaging, and measure the following parameters: exophthalmos degree, extraocular muscle transverse diameter, eyelid thickness; S4, immunological phenotype evaluation: obtain single cell suspension of mouse orbital tissue; use a combination of fluorescently labeled anti-CD45, CD3, CD4, IFNγ, IL-17a antibodies for staining; use flow cytometry to characterize the infiltration of T cells and their subsets in the orbital tissue; S5, histopathological evaluation: perform the following staining on mouse orbital tissue: H&E staining to calculate the ratio of adipose tissue area to optic nerve cross-sectional area; Masson staining to evaluate the degree of fibrosis of extraocular muscle tissue.

2. The method of evaluating a mouse model of thyroid-associated ophthalmopathy according to claim 1, wherein In step S1, the overhead view photo is used to evaluate the exophthalmos degree, and the side view photo is used to evaluate the eyelid lesion condition.

3. The method of evaluating a mouse model of thyroid-associated ophthalmopathy according to claim 2, wherein The appearance phenotype evaluation is determined by the following criteria: Under overhead view, if the outlines of both eyes are clearly protruding, it is judged as exophthalmos positive, and each eye is scored 1 point; Under side view, if eyelid edema is visible and the thickness of a single eyelid reaches or exceeds 1.5 mm, each eye is scored 1 point; If the mouse has eyelid skin ulceration and bleeding, each eye is scored 1 point; Calculate the total score of both eyes of each mouse, and if the total score of the appearance of the model group mice is ≥2 points, it is considered that the appearance is consistent with the typical eye manifestations of TAO.

4. The method of claim 1, wherein the mouse model of thyroid-associated ophthalmopathy is evaluated by, In step S2, enzyme-linked immunosorbent assay is used to detect the levels of total thyroid hormone, thyroid stimulating hormone and thyroid stimulating hormone receptor antibody in mouse serum.

5. The method of evaluating a mouse model of thyroid-associated ophthalmopathy according to claim 4, wherein The criteria for thyroid function index evaluation include: (1) If the ratio of the body weight of the model group mice to the average body weight of the control group mice at the same period is less than 1, and the difference in body weight between the two groups is statistically analyzed with p value <0.05, it is determined that the body weight change is consistent with the hypermetabolic state caused by hyperthyroidism; (2) If the serum TRAb level is more than 5 times that of the control group, the TT4 level is increased by more than 1 times compared with the control group, and the difference in serum TRAb level between the two groups is statistically analyzed with p value <0.05, it is determined that the peripheral thyroid function state of the mouse is consistent with the characteristics of hyperthyroidism caused by Graves' disease; (3) If at least two of the following pathological changes are observed by hematoxylin-eosin staining of the thyroid, it is determined that the thyroid histology has changed in accordance with the characteristics of the TAO model: Atrophy of thyroid follicle lumen; Follicular epithelial cells proliferate from flat or cuboidal to high columnar; Thyroid parenchyma shows capillary dilation, hyperemia, and lymphocyte infiltration.

6. The method of claim 1, wherein the mouse model of thyroid-associated ophthalmopathy is evaluated by, In step S3, the exophthalmos is the vertical distance from the most protruding point of the mouse eyeball to the line connecting the inner and outer canthi on the axial section; the extraocular muscle diameter is the diameter of the thickest part of the extraocular muscle measured on the coronal section; and the eyelid thickness is the thickness of the eyelid measured on the axial section.

7. The method of evaluating a mouse model of thyroid-associated ophthalmopathy according to claim 6, wherein The criteria for the imaging evaluation include: If the ratio of any of the three indicators, exophthalmos, extraocular muscle diameter, and eyelid thickness, between the model group and the control group is greater than 1, and the statistical analysis p value is <0.05, it is determined that the mouse presents the imaging performance of TAO.

8. The method of claim 1, wherein the mouse model of thyroid-associated ophthalmopathy is evaluated by, The criteria for the immunological phenotype evaluation include: If the following three indicators are met at the same time, it is determined that the mouse eye orbit is in a highly activated inflammatory state, which meets the immunological phenotype changes of TAO: The proportion of CD3⁺CD45⁺T cells in CD45⁺immune cells increases; The proportion of IFN-γ⁺CD4⁺Th1 cells in CD4⁺T cells increases; The proportion of IL-17A⁺CD4⁺Th17 cells in CD4⁺T cells increases; And the ratio of the above three indicators between the model group and the control group is greater than 1, and the statistical analysis p value is <0.

05.

9. The method of claim 1, wherein the mouse model of thyroid-associated ophthalmopathy is evaluated by, In step S5, the ratio of adipose tissue area to optic nerve cross-sectional area is calculated by H&E staining, and the degree of fibrosis in the extraocular muscle tissue is evaluated by Masson staining.

10. The method of evaluating a mouse model of thyroid-associated ophthalmopathy according to claim 9, wherein The criteria for the histopathological evaluation include: One of the following two conditions is met, that is, it is determined that the mouse has orbital tissue lesions characteristic of TAO: (1) The ratio of retrobulbar adipose tissue area to optic nerve area is 1.5 times or more than that of the control group, and the statistical analysis p value is <0.05; (2) The proportion of fibrous collagen in the extraocular muscle tissue is 1.5 times or more than that of the control group, and the statistical analysis p value is <0.05.

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