Construction method of intraocular hypertension animal model

Abstract

The invention relates to the technical field of animal disease models, and discloses a construction method of an intraocular hypertension animal model, comprising the following steps: step A, selecting an experimental animal; step B, preparing experimental instruments and reagents; c, in an anesthesia mode, deep anesthesia is carried out on the animal, and subsequent deep anesthesia is carried out; d, intraocular pressure measurement, wherein the basic intraocular pressure value is measured through anterior chamber injection. Step E, performing anterior chamber injection, and using ofloxacin antibiotic oculentum to prevent infection after an operation; and F, intraocular pressure measurement: measuring the change of the intraocular pressure value after the anterior chamber injection. According to the method, the natural pathological process of open-angle glaucoma is simulated by injecting IFN-beta into the anterior chamber, the situation that acute intraocular pressure rise is caused mainly by blocking an aqueous humor flow outlet channel like other models is avoided, and the method can better reflect the real situation of glaucoma diseases; in addition, the model avoids intraocular pressure change caused by anatomical structure change, so that the accuracy and reliability of the model are improved.

Description

Technical Field

[0001] The invention relates to the technical field of animal disease models, and in particular to a method for constructing an animal model of intraocular hypertension. Background Art

[0002] Glaucoma is a group of heterogeneous diseases characterized by optic nerve damage and vision loss. It is also the leading cause of irreversible vision loss and blindness worldwide. By 2024, glaucoma has affected the quality of life of more than 118 million people. Primary open-angle glaucoma is the most common subtype of glaucoma, accounting for about 60-70%. Due to the insidious onset of primary open-angle glaucoma, early diagnosis is difficult, and drug treatment and surgery are less effective, the current research focus is on its pathogenesis and treatment methods.

[0003] After searching, the Chinese patent number CN109481071A discloses a method for establishing an animal model of chronic ocular hypertension, which includes: dilating the animal model eye, and then locally anesthetizing the ocular surface of the animal model eye after a predetermined time; after the pupil of the animal model eye is dilated, a magnet ring is placed above the eyeball of the animal model eye with the pupil as the center; silica magnetic microspheres are dispersed in physiological saline to obtain a silica magnetic microsphere suspension for standby use; the silica magnetic microspheres to be injected are evenly dispersed in the anterior chamber angle by the magnetic force of the magnet ring, and a chronic ocular hypertension animal model is obtained. The method for establishing an animal model of chronic ocular hypertension provided by the invention can significantly increase the time of high ocular pressure maintenance and obtain a stable and effective chronic ocular hypertension animal model.

[0004] After searching, the Chinese patent number CN118402497A discloses a method for preparing an animal model of intraocular hypertension, including: 1) mixing microbeads with natural polymer materials to prepare a colloidal solution, 2) injecting the colloidal solution into the anterior chamber of the animal's eye once. Compared with the traditional method of injecting magnetic beads into the anterior chamber to establish an animal model of intraocular hypertension, the method of this application can avoid the problem of leakage of magnetic bead suspension during injection, and reduce the risk of intraocular complications through a single injection. In addition, the operation is convenient, saving experimental time and cost.

[0005] The above-mentioned animal models of intraocular hypertension are mainly anterior chamber occlusion models, in addition to episcleral vein hypertonic saline injection models, aqueous humor outflow tract laser photocoagulation models, episcleral vein cauterization models, transgenic models, steroid drug-induced models, etc. However, most models are still mainly based on acute intraocular pressure increase caused by obstruction of aqueous humor outflow tract leading to retinal ganglion cell death, and cannot completely simulate the pathological process of natural development of glaucoma; in addition, these models also have the disadvantages of affecting the anatomical structure of animal aqueous humor circulation, high incidence of corneal endothelial decompensation, aggravated inflammatory response, unstable high intraocular pressure and short duration. Based on this, the present invention designs a method for constructing an animal model of intraocular hypertension to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing an animal model of intraocular hypertension, which solves the problem in the background art that the animal aqueous humor circulation cannot be simulated and affected.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] A method for constructing an animal model of intraocular hypertension comprises the following steps:

[0009] Step A. Select experimental animals, SPF-grade C57BL / 6J mice, male, aged 6 to 8 weeks; SD rats, male, aged 4 to 8 weeks; and Japanese large-eared white rabbits, male, aged 12 to 14 months, as modeling subjects; all animals were raised under standard environmental conditions, with free access to water and food, and the experiment began after 1 week of adaptive feeding, and the breeding and operation strictly followed the requirements of scientific research animal ethics;

[0010] Step B. Prepare experimental instruments and reagents, including but not limited to a small animal inhalation gas anesthesia machine, a stereo microscope, a 10 μl or 100 μl microsyringe, micro-instruments, a TonoLab rebound tonometer and a TonoVet rebound tonometer, and experimental reagents including proparacaine hydrochloride eye drops, levofloxacin eye drops, ofloxacin eye ointment, RecombinantMouse IFN-β, PBS buffer (without calcium and magnesium), and isoflurane;

[0011] Step C. Anesthesia method: deeply anesthetize the animal and then perform ocular surface anesthesia;

[0012] Step D. Measure the baseline intraocular pressure before the first anterior chamber injection. Use a TonoLab rebound tonometer to measure the baseline intraocular pressure, and the same operator should measure it within the same time period.

[0013] Step E. Anterior chamber injection, the experimental subject is mouse / rats, the animal is placed on the operating table of a dissecting microscope, a sterile cotton swab is dipped in iodine to disinfect the eyelid margin and expose the eyeball, and the conjunctival sac is flushed with ofloxacin antibiotic eye drops; a 34G needle is used to make a puncture about 2 mm outside the pupil edge to release some aqueous humor; IFN-β (mice: 1μl, rats: 3μl) is slowly injected through the original corneal puncture at an angle of 25° through a 10μl microsyringe connected to a 33G needle, and then 2μl of air is injected to form a bubble to seal the puncture; IFN-β is injected into the right eye as the experimental eye, and an equal volume of PBS is injected into the left eye as the control eye; after the operation, ofloxacin antibiotic eye ointment is used to prevent infection;

[0014] Procedure F. IOP measurement after anterior chamber injection. Experimental subjects were mice / rats. IOP was measured using a TonoLab rebound tonometer after inhalation anesthesia. The measurements were performed by the same operator during the same time period. IOP was monitored every other day after injection.

[0015] Preferably, step C comprises the following steps:

[0016] a. C57BL / 6J mice and SD rats were anesthetized by intraperitoneal injection of 40-50 mg / kg of 1% sodium pentobarbital;

[0017] b. Japanese white rabbits were anesthetized by intramuscular injection of 22-44 mg / kg ketamine hydrochloride;

[0018] c. After confirming that the animal is under deep anesthesia, use proparacaine hydrochloride eye drops for ocular surface anesthesia.

[0019] Preferably, in step E, when the experimental subject is a rabbit, the animal is placed on a surgical operating table and disinfected and prepared as for mice / rats; a 30G needle is used to make a puncture at the limbus of the cornea and sclera to release some aqueous humor; IFN-β (rabbit: 55μl) is injected through the original corneal puncture at an angle of 25° using a 100μl microsyringe connected to a 30G needle, and the puncture is closed by pressing with a sterile cotton swab; the rest of the operations are the same as for mice / rats.

[0020] Preferably, in step F, when the experimental subject is a rabbit, intraocular pressure measurement is performed after fixation in a rabbit box under topical anesthesia, and the rest of the measurement process is the same as that of mice / rat.

[0021] Preferably, in step E, when the experimental subject is a mouse / rat, after the injection of IFN-β, the needle stays in a suitable position in the anterior chamber for 30 seconds and then is carefully withdrawn to ensure that the air bubble seals the corneal puncture and prevents the injection reagent from flowing out.

[0022] Preferably, in step E, when the experimental subject is a rabbit, after the injection of IFN-β, a sterile cotton swab is also used to press and seal the puncture site to prevent the injection reagent from flowing back.

[0023] Preferably, in steps D and F, all intraocular pressure measurements are performed under the same conditions to ensure the accuracy and comparability of the measurement results.

[0024] Preferably, the experimental animals are placed on animal recovery pads after surgery and returned to the animal breeding room after recovery to ensure the health and safety of the animals; the method is suitable for scientific research, especially in ophthalmic disease research, drug development and treatment method evaluation.

[0025] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0026] 1. In the present invention, the method simulates the natural pathological process of open-angle glaucoma by injecting IFN-β into the anterior chamber, unlike other models that mainly cause acute intraocular pressure increase by blocking the aqueous humor outflow tract. This method can better reflect the true situation of glaucoma; in addition, the model does not destroy the normal anatomical structure of the aqueous humor outflow tract, avoids changes in intraocular pressure caused by changes in the anatomical structure, and thus improves the accuracy and reliability of the model.

[0027] 2. In the present invention, the method is simple to operate, has a high success rate, and a relatively short experimental period, and a stable animal model of high intraocular pressure can be obtained in a relatively short time; and because the method is standardized in experimental conditions, animal selection, and operating procedures, the experimental results have good repeatability, which is conducive to scientific researchers to conduct in-depth research in the field of glaucoma.

[0028] 3. In the present invention, the method inhibits the activity of trabecular meshwork cells by injecting IFN-β into the anterior chamber of the experimental animal, thereby affecting its normal aqueous humor regulation function, and ultimately causing a sustained and stable increase in intraocular pressure. This method induces the occurrence of open-angle glaucoma from the pathogenesis of glaucoma, providing researchers with a more accurate, reliable and easy-to-operate animal model, which is helpful for in-depth research on the pathogenesis of glaucoma, drug development and treatment evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a front perspective structural schematic diagram of the present invention;

[0030] Figure 2 For the present invention Figure 1 The enlarged structural diagram at A in the middle;

[0031] Figure 3 The intraocular pressure changes within 29 days after the intracameral injection of Mouse IFN-β 5000U / mL or PBS in Example 1. (A) The trabecular meshwork (TM) cells were treated with gradient concentrations of IFN-α for 24 h , 36h , 48 h After 1, 3 and 5 days (n=3, mean±SD), CCK8 was used to detect changes in cell activity; (B) TM cells were treated with gradient concentrations of IFN-β for 24 h , 36 h , 48 h , 3d, and 5d later (n=3, mean±SD), CCK8 was used to detect changes in cell activity;

[0032] Figure 4 Type I IFN, mainly IFN-β, affects the extracellular matrix and cytoskeletal proteins of TM cells. (A) After TM cells were treated with IFN-α (1000, 2500 and 5000 U / mL) or IFN-β (1000, 2500 and 5000 U / mL) for 5 days (n=4, mean ± SD), the expression levels of Collagen I, CTGF and α-SMA in TM cells were detected by WB; (B) After TM cells were treated with 2500 U / mL IFN-β for 5 days, the immunofluorescence staining of Vimentin, F-actin, Fibronectin and Collagen I in the cells; (B) Scale bar, 50 μm; Nuclear staining: DAPI;

[0033] Figure 5 To induce autophagy in TM cells by IFN-β, (A) TM cells were pretreated with the autophagy inhibitor CQ (0 μM, 10 μM, 20 μM, 30 μM, 40 μM) for 1 h, and then IFN-β (5 U / mL, 2500 U / mL) was added for 36 h and 5 d (n=3, mean ± SD), and the changes in cell activity were detected. (B) Changes in the expression of autophagy-related proteins in TM cells treated with IFN-β (5u / mL, 2500u / mL, 36h) (n=3, mean±SD); Changes in the expression of autophagy-related proteins in TM cells treated with IFN-β (5U / mL, 2500U / mL) for 5d (n=5, mean±SD); (C) Transmission electron microscopy observation of typical autophagy morphological changes in TM cells (N: nucleus, N: nucleus, Nu: nucleolus, M: mitochondria, RER: rough endoplasmic reticulum, ASS: autolysosome, AP: autophagosome) after treatment with 5 or 2500U / mL IFN-β; (C) Scale bar, 5μm;

[0034] Figure 6IFN-β increases intraocular pressure by activating autophagy in TM cells. (A) Flow chart of animal experiment of injection of 5000U / mL IFN-β into the anterior chamber of the eye of mice. IFN-β (5000u / ml, injected once every 7 days) or PBS was injected into the anterior chamber of the eye of mice, and the changes in intraocular pressure levels were monitored before and 28 days after injection (n=10); (B) Representative images of increased LC3 expression in TM cells were observed in frozen sections of the anterior segment of the eye of mice 28 days after injection of 5000U / mL IFN-β; (D) Mice were divided into CQ group and saline group. After intraperitoneal injection of autophagy inhibitor CQ or saline for 2 weeks, IFN-β (5000U / mL, injected once every 7 days) was injected into the anterior chamber of the eye of the experimental eye, and PBS (control, injected once every 7 days) was injected into the anterior chamber of the eye of the control eye, with 5 eyes in each group. Changes in intraocular pressure levels were monitored before and 4 weeks after injection (n=5, mean ± SD). DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Embodiment 1;

[0037] See also Figure 1-Figure 2 In an embodiment of the present invention, a method for constructing an animal model of intraocular hypertension comprises the following steps:

[0038] Step A. Select experimental animals, SPF-grade C57BL / 6J mice, male, aged 6 to 8 weeks; SD rats, male, aged 4 to 8 weeks; and Japanese large-eared white rabbits, male, aged 12 to 14 months, as modeling subjects; all animals were raised under standard environmental conditions, with free access to water and food, and the experiment began after 1 week of adaptive feeding, and the breeding and operation strictly followed the requirements of scientific research animal ethics;

[0039] Step B. Prepare experimental instruments and reagents, including but not limited to a small animal inhalation gas anesthesia machine, a stereo microscope, a 10 μl or 100 μl microsyringe, micro-instruments, a TonoLab rebound tonometer and a TonoVet rebound tonometer, and experimental reagents including proparacaine hydrochloride eye drops, levofloxacin eye drops, ofloxacin eye ointment, RecombinantMouse IFN-β, PBS buffer (without calcium and magnesium), and isoflurane;

[0040] Step C. Anesthesia method: deeply anesthetize the animal and then perform ocular surface anesthesia;

[0041] Step D. Measure the baseline intraocular pressure before the first anterior chamber injection. Use a TonoLab rebound tonometer to measure the baseline intraocular pressure, and the same operator should measure it within the same time period.

[0042] Step E. Anterior chamber injection, the experimental subject is mouse / rats, the animal is placed on the operating table of a dissecting microscope, a sterile cotton swab is dipped in iodine to disinfect the eyelid margin and expose the eyeball, and the conjunctival sac is flushed with ofloxacin antibiotic eye drops; a 34G needle is used to make a puncture about 2 mm outside the pupil edge to release some aqueous humor; IFN-β (mice: 1μl, rats: 3μl) is slowly injected through the original corneal puncture at an angle of 25° through a 10μl microsyringe connected to a 33G needle, and then 2μl of air is injected to form a bubble to seal the puncture; IFN-β is injected into the right eye as the experimental eye, and an equal volume of PBS is injected into the left eye as the control eye; after the operation, ofloxacin antibiotic eye ointment is used to prevent infection;

[0043] Step F. Intraocular pressure measurement. The experimental subjects are mice / rats. The intraocular pressure is measured using a TonoLab rebound tonometer after inhalation anesthesia. The measurements are made by the same operator during the same time period; the baseline intraocular pressure value is measured before the first anterior chamber injection, and the intraocular pressure is monitored every other day after the injection.

[0044] Step C includes the following steps:

[0045] a. C57BL / 6J mice and SD rats were anesthetized by intraperitoneal injection of 40-50 mg / kg of 1% sodium pentobarbital;

[0046] b. Japanese white rabbits were anesthetized by intramuscular injection of 22-44 mg / kg ketamine hydrochloride;

[0047] c. After confirming that the animal is under deep anesthesia, use proparacaine hydrochloride eye drops for ocular surface anesthesia.

[0048] In step E, when the experimental subject is a rabbit, the animal is placed on the operating table and disinfected and prepared as for mice / rats; a 30G needle is used to make a puncture at the limbus of the cornea and sclera to release some aqueous humor; IFN-β (rabbit: 55μl) is injected through the original corneal puncture at an angle of 25° using a 100μl microsyringe connected to a 30G needle, and the puncture is closed by pressing with a sterile cotton swab; the rest of the operations are the same as for mice / rats.

[0049] In step F, when the experimental subject is a rabbit, intraocular pressure measurement is performed after fixation in a rabbit box under topical anesthesia, and the rest of the measurement process is the same as that of mice / rat.

[0050] In step E, when the experimental subject is a mouse / rats, after the injection of IFN-β, the needle stays in the appropriate position in the anterior chamber for 30 seconds and then is carefully withdrawn to ensure that the bubble seals the corneal puncture to prevent the injection reagent from flowing out; when the experimental subject is a rabbit, after the injection of IFN-β, a sterile cotton swab is also used to press and seal the puncture to prevent the injection reagent from flowing back.

[0051] In step F, all intraocular pressure measurements were performed under the same conditions to ensure the accuracy and comparability of the measurement results.

[0052] The experimental animals were placed on animal recovery mats after surgery and returned to the animal breeding room after recovery to ensure the health and safety of the animals. This method is suitable for scientific research, especially in ophthalmic disease research, drug development and treatment evaluation.

[0053] The basal intraocular pressure of mice was 11.1±1.12 mmHg. On the second day after intracameral injection of IFN-β (5000 U / mL) into C57BL / 6J mice, the intraocular pressure of the experimental eyes increased significantly compared with that of the control eyes (P<0.05). The intraocular pressure of the experimental eyes then increased steadily, reaching a peak level of 16.08±3.16 mmHg about 14 days after modeling, and remained higher than that of the control eyes until the 28th day of observation. Figure 2 As shown; during the observation period, the cornea of ​​the mice was in good condition, and no endophthalmitis, iris adhesion, cataract, etc. occurred.

[0054] Working principle of this embodiment: In terms of animal selection, this method carefully selected SPF-grade C57BL / 6J mice, SD rats and Japanese white rabbits as experimental subjects, and these animals were within a specific age range to ensure the consistency and repeatability of the experiment. All animals were raised under standard environmental conditions and were adaptively fed to minimize stress reactions before the experiment.

[0055] During the experimental preparation stage, this method used a series of sophisticated experimental instruments and reagents. These instruments include small animal inhalation gas anesthesia machine, stereo microscope, micro-injection, micro-instrument and rebound tonometer, which together ensure the accuracy and reliability of the experiment. At the same time, the reagents required for the experiment, such as proparacaine hydrochloride eye drops, levofloxacin eye drops, ofloxacin eye ointment, Recombinant Mouse IFN-β, PBS buffer and isoflurane, have been strictly screened and prepared to meet the experimental needs.

[0056] This method uses a deep anesthesia strategy to ensure that the animals maintain a stable physiological state during the experiment. For different types of animals, the anesthesia method is also different. For example, C57BL / 6J mice and SD rats are anesthetized by intraperitoneal injection of sodium pentobarbital, while Japanese white rabbits are anesthetized by intramuscular injection of ketamine hydrochloride; after confirming that the animals are in deep anesthesia, ocular surface anesthesia is performed to reduce pain during surgery.

[0057] Next is the anterior chamber injection stage, which is a method of injecting IFN-β into the anterior chamber of the animal by precisely controlling the injection location and injection volume. For mice and rats, a 34G needle is used to make a puncture about 2mm outside the pupil edge, and some aqueous humor is released to reduce intraocular pressure. Then, IFN-β is slowly injected through a microsyringe connected to a 33G needle, and then air is injected to form a bubble to seal the puncture. For rabbits, a 30G needle is used to make a puncture at the limbus of the cornea and sclera, and a larger volume of IFN-β is injected. These operations must be performed under a microscope to ensure the accuracy and safety of the operation.

[0058] In terms of intraocular pressure measurement, this method uses a rebound tonometer for non-contact intraocular pressure measurement to avoid causing additional harm to the animals; the measurement process is performed by the same operator within the same time period to ensure the accuracy and comparability of the measurement results. By regularly monitoring changes in intraocular pressure, the effects of IFN-β on intraocular pressure and the stability of the animal model can be evaluated.

[0059] After surgery, the animals were placed on a recovery mat and waited for recovery, and were returned to the animal breeding room for close observation. At the same time, this method is also applicable to scientific research, especially in the study of ophthalmic diseases, drug development and treatment evaluation, providing powerful tools and support for research in related fields.

[0060] Embodiment 2;

[0061] In the embodiment of the present invention, the experimental subjects were male C57BL / 6J mice aged between 6 and 8 weeks. These animals were kept under standard laboratory conditions. First, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (10 mL / kg), and then given oxybrocaine hydrochloride eye drops for local anesthesia of the ocular surface. A tunnel incision was formed by gently puncturing the cornea with a 34G needle at a distance of 2 mm from the pupil. Subsequently, 1uL reagent + 2μL air was slowly injected from the original puncture port with a 10μL microsyringe connected to a 33G needle. After injection, the needle remained in place for about 1 minute and then slowly withdrew. IFN-β treatment experiment: the right eye was the experimental eye, injected with IFN-β, and the left eye was the control eye, injected with an equal volume of PBS. After injection, antibiotic eye ointment was applied to the eyes. The changes in intraocular pressure of mice were detected with a tonometer every 1 day before and after the anterior chamber injection, and a total of 28 days were detected.

[0062] Embodiment 3;

[0063] See also Figure 1-Figure 6 , by gradient concentrations of IFN-α ( Figure 3 A) and IFN-β ( Figure 3 B) After treating TM cells for different time periods, the changes in cell activity were detected by CCK-8. The CCK-8 results showed that the activity of TM cells gradually decreased with the increase in the concentration and treatment time of IFN-α and IFN-β. IFN-β had a stronger effect on cell activity. In addition, IFN-β could significantly inhibit cell growth at both low and high concentrations.

[0064] Western blot results showed that after TM cells were treated with gradient concentrations of IFN-α and IFN-β, the expression levels of extracellular matrix proteins CTGF, α-SMA and Myocilin in the IFN-β group were significantly decreased compared with the control group ( Figure 4 A), the expression levels of Collagen I, CTGF and α-SMA in the IFN-β group were significantly lower than those in the IFN-α group.

[0065] This suggests that IFN may cause elevated intraocular pressure by affecting the activity and function of TM cells. After TM cells were treated with 2500 U / mL IFN-β for 5 days, the morphological changes of TM cells were observed using a fluorescence microscope ( Figure 4 B). Normal TM cells are slender and spindle-shaped, but after 5 days of IFN-β treatment, the cell morphology changed from spindle to polygonal and the cell body became larger, the filamentous structure of Vimentin protein increased, and the structure of Fibronection protein was disordered and sparse. These results show that compared with IFN-α, IFN-β has a more significant effect on TM cells.

[0066] Next, 5U / mL and 2500U / mL of IFN-β were selected for subsequent in vitro experiments. After TM cells were co-treated with 5 or 2500U / mL of IFN-β and cell death pathway inhibitors, the changes in cell activity were measured ( Figure 5 A). After 36 h of co-treatment, it was observed that the autophagy inhibitor CQ was able to reverse the decrease in cell activity caused by IFN-β. However, the autophagy inhibitor BafalomycinA1, the pyroptosis inhibitor MCC950, the ferroptosis inhibitor Ferrostatin-1, and the apoptosis inhibitor Z-VAD did not reverse the decrease in TM cell activity. Western bolt results showed that the LC3-II / LC3-I ratio in the IFN-β-treated group increased compared with the control group ( Figure 5B). This suggests that autophagy in TM cells is activated. Electron microscopy results showed that TM cells treated with 5 or 2500U / mL IFN-β showed typical autophagy activation characteristics, including increased autophagic lysosomes, increased intracellular vacuoles, and deterioration of cell status. The above results indicate that IFN-β causes trabecular meshwork dysfunction by activating autophagy.

[0067] High intraocular pressure is a key risk factor for glaucoma, and the trabecular meshwork is responsible for the circulation of aqueous humor. To study the effects of IFN-β on intraocular pressure levels and the trabecular meshwork, an animal model was established by injecting 5000 U / mL of IFN-β into the anterior chamber of the eyes of mice. IOP changes were measured every 1 day before and after injection for 28 days ( Figure 6 A). The intraocular pressure of the IFN-β-injected eyes began to increase from day 6 and maintained at this level until day 28. Increased LC3 expression was found in TM cells of the IFN-β-injected eyes ( Figure 6 B), which confirmed that increased intraocular pressure was significantly correlated with autophagy. In order to further clarify the mechanism by which IFN-β causes increased intraocular pressure, the autophagy inhibitor CQ or an equal amount of normal saline was intraperitoneally injected at the same time as the anterior chamber injection of IFN-β. The changes in intraocular pressure were detected every 1 day before and after the intracameral injection for a total of 4 weeks. The results of intraocular pressure monitoring showed that from the 8th day to the 28th day after the injection of IFN-β, the intraocular pressure of the experimental eyes of the mice in the normal saline group was significantly higher than that of the control group, while the intraocular pressure of the experimental eyes of the mice in the CQ group was not significantly different from that of the control group ( Figure 6 D).

[0068] The working principle of Example 3 of the present invention is: IFN-β was injected into the anterior chamber of the eye of the mouse and it was found that the intraocular pressure began to increase on the 12th day and remained at this level for 3 weeks. 28 days after the injection of IFN-β, the expression of LC3 in the TM cells of the injected eye increased. In summary, the research results show that IFN-β affects the normal function and structure of TM cells by activating autophagy, ultimately leading to increased intraocular pressure levels.

[0069] Working principle: The overall working principle of this program is based on injecting IFN-β into the anterior chamber of experimental animals, which affects the activity and function of TM cells by activating cell autophagy, thereby interfering with their normal aqueous humor circulation, and ultimately leading to a continuous and stable increase in intraocular pressure, thereby simulating the occurrence process of open-angle glaucoma.

[0070] In the selection of experimental animals, the protocol carefully selected different types of animals such as SPF-grade C57BL / 6J mice, SD rats, and Japanese large-eared white rabbits, and strictly controlled them within a specific age range to ensure the consistency and repeatability of the experiment. All animals were raised under standard environmental conditions and fed adaptively to minimize stress reactions before the experiment. This step provides stable and reliable experimental subjects for subsequent experimental operations.

[0071] During the experimental preparation stage, the program used a series of sophisticated experimental instruments and reagents, including small animal inhalation gas anesthesia machine, stereo microscope, micro-injection, micro-instrument and rebound tonometer, etc., to ensure the accuracy and reliability of the experiment. At the same time, the reagents required for the experiment, such as proparacaine hydrochloride eye drops, levofloxacin eye drops, ofloxacin eye ointment, Recombinant Mouse IFN-β, PBS buffer and isoflurane, etc., were strictly screened and prepared to meet the experimental needs.

[0072] During the experimental operation, the protocol adopted a deep anesthesia strategy to ensure that the animals maintained a stable physiological state during the experiment. For different types of animals, the anesthesia method is also different to ensure the anesthesia effect while minimizing the harm to the animals. Then, by precisely controlling the injection position and injection volume, IFN-β is injected into the anterior chamber of the animal. This step is the key to the experiment because it directly affects the subsequent experimental results.

[0073] By regularly monitoring changes in intraocular pressure, the program evaluated the effects of IFN-β on intraocular pressure and the stability of the animal model. The experimental results showed that after the injection of IFN-β, the intraocular pressure of the experimental eyes increased significantly and remained stable for a certain period of time. At the same time, through histological analysis and biochemical testing, the effects of IFN-β on the activity and function of trabecular meshwork cells, as well as the role of biological mechanisms such as autophagy, were further confirmed.

[0074] Finally, according to the experimental conclusions and data, the program successfully achieved its purpose: that is, by injecting IFN-β into the anterior chamber, the occurrence process of open-angle glaucoma was simulated. Compared with other existing open-angle glaucoma animal models, this model has the advantages of simple operation, high success rate, short cycle and high repeatability. More importantly, it induces the occurrence of open-angle glaucoma from the pathogenesis of glaucoma without destroying the normal anatomical structure of the aqueous humor outflow tract, providing a more realistic and reliable animal model for the study of glaucoma.

[0075] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations may be made to the embodiments without departing from the principles and spirit thereof, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an animal model of intraocular hypertension, characterized in that: The following steps are involved: Step A. Select experimental animals, SPF-grade C57BL / 6J mice, male, aged 6 to 8 weeks; SD rats, male, aged 4 to 8 weeks; and Japanese large-eared white rabbits, male, aged 12 to 14 months, as modeling subjects; all animals were raised under standard environmental conditions, with free access to water and food, and the experiment began after 1 week of adaptive feeding, and the breeding and operation strictly followed the requirements of scientific research animal ethics; Step B. preparing experimental instruments and reagents, including but not limited to a small animal inhalation gas anesthesia machine, a stereo microscope, a 10 μl or 100 μl microsyringe, micro-instruments, a TonoLab rebound tonometer and a TonoVet rebound tonometer, and experimental reagents including proparacaine hydrochloride eye drops, levofloxacin eye drops, ofloxacin eye ointment, Recombinant Mouse IFN-β, PBS buffer (without calcium and magnesium), and isoflurane; Step C. Anesthesia method: deeply anesthetize the animal and then perform ocular surface anesthesia; Step D. Measure the baseline intraocular pressure before the first anterior chamber injection. Use a TonoLab rebound tonometer to measure the baseline intraocular pressure, and the same operator should measure it within the same time period. Step E. Anterior chamber injection, the experimental subject is mouse / rats, the animal is placed on the operating table of a dissecting microscope, a sterile cotton swab is dipped in iodine to disinfect the eyelid margin and expose the eyeball, and the conjunctival sac is flushed with ofloxacin antibiotic eye drops; a 34G needle is used to make a puncture about 2 mm outside the pupil edge to release some aqueous humor; IFN-β (mice: 1μl, rats: 3μl) is slowly injected through the original corneal puncture at an angle of 25° through a 10μl microsyringe connected to a 33G needle, and then 2μl of air is injected to form a bubble to seal the puncture; IFN-β is injected into the right eye as the experimental eye, and an equal volume of PBS is injected into the left eye as the control eye; after the operation, ofloxacin antibiotic eye ointment is used to prevent infection; Step F. Intraocular pressure measurement. The experimental subjects are mice / rats. The intraocular pressure is measured using a TonoLab rebound tonometer after inhalation anesthesia. The measurements are made by the same operator during the same time period; the baseline intraocular pressure value is measured before the first anterior chamber injection, and the intraocular pressure is monitored every other day after the injection.

2. The method for constructing an animal model of intraocular hypertension according to claim 1, characterized in that: The step C comprises the following steps: a. C57BL / 6J mice and SD rats were anesthetized by intraperitoneal injection of 40-50 mg / kg of 1% sodium pentobarbital; b. Japanese white rabbits were anesthetized by intramuscular injection of 22-44 mg / kg ketamine hydrochloride; c. After confirming that the animal is under deep anesthesia, use proparacaine hydrochloride eye drops for ocular surface anesthesia.

3. The method for constructing an animal model of intraocular hypertension according to claim 1, characterized in that: In step E, when the experimental subject is a rabbit, the animal is placed on the operating table and disinfected and prepared as for mice / rats; a 30G needle is used to make a puncture at the limbus of the cornea and sclera to release some aqueous humor; IFN-β (rabbit: 55μl) is injected through the original corneal puncture at an angle of 25° using a 100μl microsyringe connected to a 30G needle, and the puncture is closed by pressing with a sterile cotton swab; the rest of the operations are the same as for mice / rats.

4. The method for constructing an animal model of intraocular hypertension according to claim 1, characterized in that: In step F, when the experimental subject is a rabbit, intraocular pressure is measured after fixation in a rabbit box under topical anesthesia, and the rest of the measurement process is the same as that of mice / rat.

5. The method for constructing an animal model of intraocular hypertension according to claim 1, characterized in that: In step E, when the experimental subject is a mouse / rat, after the injection of IFN-β, the needle stays in the appropriate position in the anterior chamber for 30 seconds and then is carefully withdrawn to ensure that the air bubble seals the corneal puncture and prevents the injection reagent from flowing out.

6. The method for constructing an animal model of intraocular hypertension according to claim 3, characterized in that: In step E, when the experimental subject is a rabbit, after the injection of IFN-β, a sterile cotton swab is also used to press and seal the puncture site to prevent the injection reagent from flowing back.

7. The method for constructing an animal model of intraocular hypertension according to claim 1, characterized in that: In step F, all intraocular pressure measurements are performed under the same conditions to ensure the accuracy and comparability of the measurement results.

8. The method for constructing an animal model of intraocular hypertension according to claim 1, characterized in that: The experimental animals were placed on animal recovery mats after surgery and returned to the animal breeding room after recovery to ensure the health and safety of the animals.

9. The method for constructing an animal model of intraocular hypertension according to claim 1, characterized in that: The method is suitable for use in scientific research, particularly in ophthalmic disease research, drug development, and treatment evaluation.

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