Construction method of glaucoma animal model and animal model and application thereof

Abstract

The invention relates to the technical field of animal models, in particular to a construction method of a glaucoma animal model, the glaucoma animal model and application of the glaucoma animal model. The invention discloses a construction method of a glaucoma animal model. The construction method comprises the following steps: (1) injecting a suspension containing magnetic nano microspheres into an anterior chamber of an eye of an experimental animal in an anterior chamber injection manner; and (2) attracting the magnetic nano-microsphere solids by using a magnet to enable the magnetic nano-microspheres to be distributed at room corners, then feeding the experimental animal for 35-40 days, and establishing an animal model. According to the animal model, the magnetic nano-microspheres can be guided to the anterior chamber corner through the magnet, aggregation of the magnetic nano-microspheres in other positions of the anterior chamber is effectively reduced, and the problem that latex microspheres are difficult to position is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and in particular to a method for constructing a glaucoma animal model, and the animal model and application thereof. Background Art

[0002] Glaucoma is a progressive optic neuropathy caused by pathologically high intraocular pressure, characterized by apoptosis of retinal ganglion cells and gradual loss of visual function. The pathological mechanism of glaucoma is not very clear at present, and it is difficult to study the pathogenesis directly in humans. Therefore, animal models can be used to simulate the occurrence of glaucoma, understand its mechanism, and develop new treatment technologies.

[0003] Common animal models of glaucoma include:

[0004] 1. Animal Model of Trabecular Meshwork Occlusion

[0005] Methods: By increasing the expression of fibronectin in trabecular meshwork cells, fibronectin accumulates inside and outside the cells, blocking the anterior chamber angle.

[0006] Advantages: The clinical phenotype is similar to that of open-angle glaucoma (OAG): open angle, increased intraocular pressure, damaged trabecular meshwork, accumulation of extracellular matrix, etc.; the method is simple to operate.

[0007] Disadvantages: High intraocular pressure only lasts for a short time and is only mild to moderate; moreover, hormones need to be applied multiple times for a long time, which is rather cumbersome; it is also prone to complications caused by multiple hormones.

[0008] 2. Autologous blood cell intracameral injection model

[0009] Method: Autologous blood is extracted from experimental animals and injected into the anterior chamber. The denatured blood cells cannot pass through the aqueous humor outflow channel as easily as normal red blood cells, and are blocked in the trabecular meshwork, causing increased intraocular pressure.

[0010] Advantages: Rapid increase in intraocular pressure; no intraocular inflammatory response.

[0011] Disadvantages: The rapid drop in intraocular pressure is not conducive to the study of the slow degeneration of the optic nerve in the fundus; blood cells suspended in the anterior chamber can easily block the pupil, which is not conducive to the observation of the fundus.

[0012] 3. Latex Microsphere Intracameral Injection Model

[0013] Method: By injecting latex microspheres into the anterior chamber, the outflow of aqueous humor is hindered by blocking the chamber angle, thereby increasing intraocular pressure.

[0014] Advantages: The method is simple and easily tolerated by animals; no special equipment is required and it can be used on a variety of experimental animals; different degrees of high intraocular pressure models can also be obtained by injecting different numbers of latex microspheres.

[0015] Disadvantages: Since latex microspheres are difficult to accumulate in the anterior chamber angle continuously, the intraocular pressure in the injected eye fluctuates significantly and the intraocular pressure level is unstable; there is a phenomenon that latex microspheres accumulate in the anterior chamber and hinder observation of the fundus; when the intraocular pressure is high, corneal ulcers or even necrosis are prone to occur.

[0016] 4. Magnetic Microsphere Injection Model

[0017] In order to overcome the problem of microspheres gathering in the anterior chamber and obstructing fundus observation, simple polypropylene latex microspheres are replaced with magnetic microspheres, which are injected into the anterior chamber and guided to the anterior chamber angle with a handheld magnet. This can increase the effect of blocking the anterior chamber angle while reducing the accumulation of microspheres in other locations in the anterior chamber.

[0018] Advantages: Avoids the inconvenience and inflammatory response caused by repeated injections of simple polypropylene microspheres; the method is simple and easily tolerated by animals; high intraocular pressure is maintained for a longer time; the degree of increased intraocular pressure can be controlled.

[0019] Disadvantages: The magnetic ball in the anterior chamber blocks the pupil; the extent of the increase in intraocular pressure and the degree of ganglion cell loss will vary with the age and genetic background of the animal.

[0020] To this end, the present application provides a method for constructing a glaucoma animal model, and its animal model and application. Summary of the invention

[0021] In order to overcome the shortcomings of the prior art, the present invention provides a method for constructing an animal model of glaucoma, and an animal model and application thereof. The animal model described in the present application can guide magnetic nano-microspheres to the anterior chamber angle by a magnet, effectively reducing the aggregation of magnetic nano-microspheres in other locations of the anterior chamber, and solving the problem that latex microspheres are difficult to position.

[0022] The technical solution adopted by the present invention to solve the technical problem is:

[0023] In a first aspect, the present application provides a method for constructing a glaucoma animal model, comprising the following steps:

[0024] (1) Injecting a suspension containing magnetic nanoparticles into the anterior chamber of the eye of the experimental animal by intracameral injection;

[0025] (2) The magnetic nano-microsphere solid is attracted by a magnet so that the magnetic nano-microsphere is distributed in the anterior chamber angle, and then the experimental animal is raised for 35-40 days to establish an animal model.

[0026] Specifically, the magnetic nanoparticles are dispersed in deionized water to form the suspension.

[0027] Specifically, the content of magnetic nanoparticles in the suspension is 50 mg / mL, and the injection volume is 10-50 μL.

[0028] Specifically, the average particle size of the magnetic nanospheres is 100 nm.

[0029] Specifically, the method for preparing the magnetic nanospheres comprises the following steps:

[0030] The Fe3O4 nanoparticles are dispersed, chitosan is added for mixing and dispersion, a cross-linking agent is added for cross-linking and mixing, and magnetic nano-microspheres are obtained through post-treatment.

[0031] More specifically, the cross-linking agent is glutaraldehyde.

[0032] Specifically, the animal is a mammal.

[0033] More specifically, the mammal is any one of a cynomolgus monkey, a rhesus monkey or a marmoset.

[0034] In a second aspect, the present application provides a glaucoma animal model, including the animal model constructed by the construction method described in the first aspect.

[0035] In a third aspect, the present application provides an application of a glaucoma animal model, wherein the animal model constructed by the construction method described in the first aspect or the animal model described in the second aspect is applied to the evaluation of glaucoma therapeutic drugs.

[0036] The beneficial effects of the present invention are:

[0037] The construction method described in this application uses magnetic nanoparticles to construct an animal model. The magnetic nanoparticles can be accurately distributed in the anterior chamber angle under the guidance of an external magnet, thereby effectively blocking the anterior chamber angle, reducing the outflow of aqueous humor, and causing increased intraocular pressure; the stable distribution of magnetic nanoparticles in the anterior chamber angle reduces the inconsistent blocking problem caused by traditional microspheres drifting or gathering in other locations, thereby ensuring the continuous increase in intraocular pressure. Moreover, the magnetic nanoparticles are made of biocompatible materials such as chitosan, which reduces the inflammatory response after injection and improves the stability of the model and the tolerance of animals.

[0038] The construction method described in this application can accurately control the degree of increase in intraocular pressure, which is very beneficial for studying the effects of different intraocular pressure levels on optic nerve damage; compared with other methods that require multiple injections, the method using magnetic nanoparticles reduces the inflammatory response caused by repeated injections and improves the stability and reliability of the model.

[0039] In the animal model described in this application, after the drug evaluation group was treated with pilocarpine eye drops, the intraocular pressure was significantly reduced, and the cup-disc ratio and the outer diameter of the eyeball did not continue to increase and were less than the 50 μL high-dose model group in the same period. This shows that the model constructed by this method can be used for drug evaluation, providing strong support for the further development and clinical application of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0041] Figure 1 This is a result diagram of intraocular pressure detection performed in an embodiment of the present application;

[0042] Figure 2 This is a result diagram of cup-to-disc ratio in an embodiment of the present application;

[0043] Figure 3 This is a result diagram of the outer diameter of the eyeball detected in the embodiment of the present application;

[0044] Figure 4 This is the OCT scan of the right eye of the 50μL drug evaluation group of this application before modeling;

[0045] Figure 5 This is the OCT scan of the right eye after modeling in the 50μL high-dose model group of this application;

[0046] Figure 6 This is an OCT scan of the right eye of the 50 μL drug evaluation group of this application on the 44th day of drug treatment. DETAILED DESCRIPTION

[0047] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0048] As used herein, "and / or" includes the term of any and all combinations of one or more associated listed items. The terms used herein are only used to describe specific embodiments and are not intended to limit the present invention. As used herein, the singular forms "a", "an", "an" and "the" are also intended to include the plural forms, unless the context clearly indicates otherwise. It is further understood that "including", when used in this specification, specifies the stated features, integers, steps, operations, elements and / or compositions, but does not exclude the existence or addition of one or more other features, integers, steps, operations, elements, compositions and / or combinations thereof.

[0049] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It is further understood that terms, such as defined in commonly used dictionaries, are interpreted in accordance with their meanings in the context of the relevant art and are not in an idealized or overly formal sense unless explicitly so defined herein.

[0050] The exemplary invention described herein may appropriately lack any one or more element limitations that are not specifically disclosed herein. Therefore, the terms "comprise", "include", "contain", etc. should be understood broadly and non-restrictively. In addition, the terminology used herein is used as a description, not a limitation, and it is unintentional to use these terminology expressions that do not include any equivalent characteristics, but only describe some of their characteristics, but various modifications are possible within the scope of the present invention according to the rights. Therefore, although the present invention has been specifically disclosed by preferred embodiments and optional features, the modifications disclosed herein to embody the changes of the present invention may be recorded by those skilled in the art, and such modifications and changes will be considered to be within the scope of the present invention.

[0051] The raw materials or reagents used in the embodiments of the present invention and the comparative examples are all purchased from mainstream manufacturers in the market. Those without indicating the manufacturer or the concentration are all analytically pure raw materials or reagents that can be routinely obtained, as long as they can play the expected role, there is no particular restriction. The instruments and equipment such as the reactor and rotary evaporator used in the present embodiment are all purchased from major manufacturers in the market, as long as they can play the expected role, there is no particular restriction. If specific techniques or conditions are not indicated in the present embodiment, the techniques or conditions described in the literature in this area or according to the product specification are carried out.

[0052] Preparation Example 1

[0053] The preparation of magnetic nanoparticles comprises the following steps:

[0054] 1. Preparation of magnetic Fe3O4 nanoparticles

[0055] The iron salt was dissolved in deionized water, and hydrogen peroxide and ammonia were added. After stirring, the mixture was transferred to a high-pressure reactor. The reaction temperature was 120°C and the reaction time was 14 hours. After the reaction, the mixture was centrifuged and dried to obtain Fe3O4 nanoparticles.

[0056] Among them, the iron salt is FeSO4·7H2O and FeCl3·6H2O mixed in a molar ratio of 1:2, and the total amount can be calculated as 1 mol. At this time, the deionized water is 30 times the mass of the iron salt, the volume of ammonia water (saturated concentrated ammonia water) is 200 mL, and the volume of hydrogen peroxide (H2O2) is 200 mL.

[0057] 2. Cross-linking reaction

[0058] Dissolving chitosan in ethyl acetate to prepare a 1 wt % chitosan solution;

[0059] F e3 O4 nanoparticles were dispersed in liquid paraffin, 1 wt% chitosan solution was added, and ultrasonic dispersion was performed for 30 min;

[0060] Then, 2.5 wt% glutaraldehyde solution was added and stirred at room temperature for 24 hours to cross-link the chitosan to form microspheres;

[0061] Filter, wash thoroughly with petroleum ether to remove unreacted substances, and then wash and dehydrate with acetone;

[0062] The product is placed in a vacuum drying oven and dried to obtain magnetic nanoparticles;

[0063] The mass ratio of Fe3O4 nanoparticles to chitosan is 1:2.

[0064] The prepared magnetic nanoparticles were tested by Fourier transform infrared spectrometer and found that: -1 and 1600cm -1 There are obvious absorption peaks at , which correspond to the stretching vibrations of -OH and -NH2 in chitosan.

[0065] At 1000-1200cm -1 The absorption peak at corresponds to the characteristic peak of Fe3O4, indicating that Fe3O4 and chitosan are successfully combined to form magnetic nanospheres.

[0066] Using a transmission electron microscope to detect, it was found that the magnetic nanoparticles were spherical, with a relatively uniform particle size distribution and an average particle size of about 100nm.

[0067] Dispersion test:

[0068] The prepared magnetic nanospheres were placed in a vial containing deionized water to form a 50 mg / mL suspension;

[0069] It was found that the magnetic nanospheres in the suspension could be dispersed in water for 60 days without precipitation, indicating that they have very good dispersibility in water.

[0070] Stability test:

[0071] The 50mg / mL suspension formed by the magnetic nanospheres was placed in a vacuum drying oven at 80°C for 24 hours; after drying, its saturation magnetization was measured to evaluate its stability. It was found that before the suspension was formed, the saturation magnetization of the magnetic nanospheres was 41emu / g, and after the stability test, the saturation magnetization of the magnetic nanospheres was 40emu / g, indicating that the magnetic stability of the magnetic nanospheres was relatively good.

[0072] Example

[0073] The method for constructing a glaucoma animal model includes the following contents:

[0074] 1. Animal Anesthesia

[0075] Four healthy cynomolgus monkeys (all male, from Guangzhou Huazhen Biotechnology Co., Ltd.) with similar age, gender, and weight and without ophthalmic diseases were selected;

[0076] Animal age: 6.45 ± 0.55 years;

[0077] Animal weight: 3.87±0.95Kg;

[0078] Intraocular pressure: 14-20 mmHg.

[0079] Healthy cynomolgus monkeys were divided into

[0080] There was 1 mouse in the low-dose model group, 1 mouse in the medium-dose model group, 1 mouse in the high-dose model group, and 1 mouse in the drug evaluation group.

[0081] Before surgery, the low-dose model group, medium-dose model group, high-dose model group, and drug evaluation group of cynomolgus monkeys were fasted for 12 hours, and the cynomolgus monkeys were injected with Shutai 50 injection (50 mg / ml) into the buttocks muscle for general basic anesthesia (4-6 mg / kg). After 10 minutes, the buttocks muscle was injected with Su Mianxin (3-5 mg / kg). When the animal's corneal reflex disappeared, the limbs and abdominal muscles relaxed, and the breathing was stable, the anesthesia was considered appropriate. When the animal is in the anesthesia recovery period, the environment should be kept dark and quiet, and attention should be paid to keeping the anesthetized animal warm to prevent excessive heat loss.

[0082] 2. Intracameral injection

[0083] The right eye was used as the experimental eye. The conjunctival sac was washed three times with gentamicin injection mixed with an appropriate amount of normal saline. The area around the right eye was disinfected with iodine. Sterile towels were spread and the eyelids were opened with an eyelid speculum. Under an operating microscope, a 15° puncture knife was used to pierce the cornea through the superior temporal corneal margin to release a small amount of aqueous humor until the anterior chamber became shallow.

[0084] A syringe (50 μL) pre-filled with a suspension containing 50 mg / mL of magnetic nanoparticles was inserted into the anterior chamber through the incision; the syringe was pushed to inject the suspension into the anterior chamber; the injection of the suspension was stopped, the corneal puncture was sealed with water after the needle was removed, and tobramycin dexamethasone eye ointment was applied to the eye. The magnetic beads were attracted by a ring magnet to distribute them in the angle of the chamber and the animal was awakened.

[0085] Among them, the injection volume of the low-dose model group was 10μL, the injection volume of the medium-dose model group was 25μL, the injection volume of the high-dose model group was 50μL, and the injection volume of the drug evaluation group was 50μL.

[0086] 3. Model building

[0087] After intracameral injection, the animals were awakened and fed normally for 40 days in the low-dose model group, medium-dose model group, high-dose model group, and drug evaluation group crab-eating macaques to construct glaucoma animal models, during which intraocular pressure and cup-to-disc ratio tests were performed; after 40 days of feeding, the low-dose model group, medium-dose model group, and high-dose model group continued to be fed until the 44th day, and intraocular pressure, cup-to-disc ratio, eyeball outer diameter, and OCT scans were performed;

[0088] After 40 days of normal feeding, the cynomolgus monkeys in the drug evaluation group were given pilocarpine eye drops on the experimental eye (right eye) 4 times a day, 1 drop per time on the 41st, 42nd, and 43rd days. The monkeys were fed continuously until the 44th day, and intraocular pressure, cup-disc ratio, eyeball outer diameter, and OCT scans were performed.

[0089] 4. Model testing and evaluation

[0090] ①Cup to plate ratio test

[0091] Before injecting the suspension into the anterior chamber, the cynomolgus monkeys were anesthetized according to the animal anesthesia process; when the anesthesia was moderate, tropicamide compound eye drops were instilled in both eyes to dilate the pupils to ensure that the pupils were fully dilated for easy observation of the fundus; the eyelids were opened with an eyelid speculum, and the head was fixed so that the eyeballs were facing forward; the cynomolgus monkeys were fixed on the fundus photography equipment, and the focus was adjusted to ensure a clear image; fundus photographs were taken, and the ratio of the diameter of the central depressed part of the optic nerve head (cup) to the entire optic nerve head (disc) was calculated to obtain the preoperative cup-disc ratio.

[0092] According to the above process, the cup-to-disc ratio test was performed on the high-dose model group, the medium-dose model group, the low-dose model group, and the drug evaluation group on the 1st, 5th, 7th, 40th, 41st, and 44th days. The results are detailed in Figure 2 .

[0093] ②Intraocular pressure testing

[0094] Before intracameral injection of the suspension, the cynomolgus monkeys were anesthetized according to the above animal anesthesia; when anesthesia was appropriate, the eyelids were opened with a lid speculum to ensure clear eye exposure, and the intraocular pressure was measured using a tonometer before surgery. The intraocular pressure was measured once on the 1st, 5th, 7th, 14th, 28th, 35th, 40th, 41st, and 44th days. The results are detailed in Figure 1 .

[0095] ③Eyeball outer diameter detection

[0096] After 44 days of feeding, the high-dose model group, medium-dose model group, low-dose model group, and drug evaluation group cynomolgus monkeys were euthanized, and the eyeballs of both eyes were removed. The fat and connective tissue around the eyes were removed under a surgical microscope, and the eyes were rinsed three times with HBS buffer. Then, the anterior-posterior diameter and left-right diameter of the eyeball were measured and recorded using a vernier caliper (recorded 3 times respectively and the average value was taken). For details, see Figure 3 .

[0097] from Figure 1-Figure 3 It can be seen that in terms of intraocular pressure test results,

[0098] 10μL low-dose model group: intraocular pressure increased to a certain extent after injection, but the increase was small.

[0099] 25μL medium-dose model group: IOP increased significantly after injection and remained at a high level at subsequent time points.

[0100] 50μL high-dose model group: IOP increased significantly after injection, and the IOP levels were higher and lasted longer at subsequent time points.

[0101] It is currently believed that the crab-eating macaque animal model, in which the intraocular pressure of the experimental eye is maintained stably and greater than 35 mmHg for more than one month, can be used as a mild intraocular pressure model.

[0102] And from Figure 1 It can be seen that the 25μL medium-dose model group reached 35mmHg on the 5th day and maintained it for more than 30 days, the 50μL high-dose model group reached 42mmHg on the 5th day and maintained it for more than 30 days, and the 50μL drug evaluation group reached 40mmHg on the 5th day and maintained it for more than 30 days. It can be considered that an animal model of glaucoma caused by high intraocular pressure has been established.

[0103] Therefore, in the glaucoma animal model, the greater the amount of magnetic nanoparticles injected, the more obvious the effect of blocking the chamber angle, resulting in a greater increase in intraocular pressure and a longer duration. In the 10μL low-dose model group, due to the small amount of magnetic nanoparticles injected, the blocking effect was limited, so the increase in intraocular pressure was small, and the intraocular pressure gradually decreased over time; in the 25μL medium-dose model group and the 50μL high-dose model group, due to the large amount of magnetic nanoparticles injected, the blocking effect was stronger, resulting in a significant increase in intraocular pressure, and it remained at a high level at subsequent time points.

[0104] In terms of cup-to-disc ratio testing,

[0105] 10μL low-dose model group: The cup-to-disc ratio increased to a certain extent after injection, but the increase was small, and the cup-to-disc ratio gradually stabilized at subsequent time points.

[0106] 25μL medium-dose model group: The cup-to-disc ratio increased significantly after injection and continued to increase at subsequent time points.

[0107] 50μL high-dose model group: The cup-to-disc ratio increased significantly after injection, and the increase in the cup-to-disc ratio was more obvious at subsequent time points.

[0108] Cause Analysis:

[0109] Dose effect: The increase in cup-to-disc ratio reflects the damage to the optic nerve. In the 10μL low-dose model group, the increase in cup-to-disc ratio was smaller because the intraocular pressure increased less and the degree of optic nerve damage was less severe. In the 25μL medium-dose model group, the intraocular pressure increased significantly and the degree of optic nerve damage was more severe, so the cup-to-disc ratio increased more and lasted longer. In the 50μL high-dose model group, the cup-to-disc ratio increased the most because the dose was larger, the intraocular pressure was higher, and the optic nerve damage was more severe.

[0110] In terms of eyeball outer diameter detection:

[0111] 10μL low-dose model group: The outer diameter of the eyeball increased to a certain extent after injection, but the increase was small.

[0112] 25μL medium-dose model group: The outer diameter of the eyeball increased significantly after injection.

[0113] 50μL high-dose model group: The outer diameter of the eyeball increased significantly after injection, and the increase was greater.

[0114] By comparing the 50μL high-dose model group and the 50μL drug evaluation group, we found that:

[0115] Before drug instillation, the intraocular pressure and cup-to-disc ratio of the 50μL drug evaluation group remained consistent with those of the 50μL high-dose model group.

[0116] In the 50μL high-dose model group, intraocular pressure increased significantly after injection and remained at a high level at subsequent time points; in the 50μL drug evaluation group, intraocular pressure decreased significantly after 3 days of drug treatment.

[0117] In the 50μL high-dose model group, the cup-to-disc ratio increased significantly after injection and continued to increase at subsequent time points; in the 50μL drug evaluation group, the cup-to-disc ratio was smaller than that of the 50μL high-dose model group at the same stage after 3 days of drug treatment, indicating that after drug instillation, the cup-to-disc ratio of the experimental eye did not continue to increase and was smaller than that of the 50μL high-dose model group during the same period.

[0118] In the 50μL high-dose model group, the outer diameter of the eyeball increased significantly after injection; in the 50μL drug evaluation group, after drug treatment, the outer diameter of the eyeball did not continue to increase and was smaller than that of the 50μL high-dose model group in the same period.

[0119] It can be seen that after the successful modeling, the intraocular pressure of the 50 μL drug evaluation group was significantly reduced through drug treatment, and the cup-disc ratio and the outer diameter of the eyeball did not continue to increase and were smaller than the 50 μL high-dose model group in the same period. Therefore, the model constructed by the construction method described in this application can be used for drug evaluation, which provides strong support for the further development and clinical application of drugs.

[0120] At the same time, when conducting the above-mentioned animal test, the slit lamp microscope was used to observe whether there was inflammation such as corneal edema and fundus vascular edema. No inflammation was found in the 10μL low-dose model group, the 25μL medium-dose model group, the 50μL high-dose model group, and the 50μL drug evaluation group, indicating that the model constructed by the magnetic nanoparticles of the present application has good biocompatibility. It was also found that the magnetic nanoparticles could be dispersed relatively well in the chamber angle. During the experiment, only a small amount of magnetic nanoparticles dispersed to other positions were used to adsorb to the chamber angle with a magnet on the 21st day.

[0121] 50μL drug evaluation group, before modeling, Figure 4 As shown, the RNFL thickness of the right eye was within the normal range, and the optic nerve fiber layer structure was intact with no obvious abnormalities;

[0122] The 50 μL high-dose model group was fed for 44 days. Figure 5 As shown: The thickness of the RNFL in the right eye was significantly reduced, and the thinning of the optic nerve fiber layer in the upper and lower areas was more obvious, indicating that the model was successful.

[0123] 50μL drug evaluation group, 3 days after the drug was dripped, such as Figure 6 As shown, the RNFL thickness of the right eye was uneven, and the fiber layer was thinned in some areas, but it was slightly improved compared with the 50μL high-dose model group. This may be because after the drug was instilled, the intraocular pressure of the experimental eye was reduced, and the RNFL thickness would not be further reduced and the optic nerve fiber layer would not be further thinned.

[0124] It can be seen that the effectiveness of the constructed glaucoma animal model can be verified through the experiment of the 50μL drug evaluation group. The experimental results showed that the intraocular pressure, cup-disc ratio and outer diameter of the eyeball of the 50μL drug evaluation group after modeling were consistent with the 50μL high-dose model group, indicating that the model can successfully simulate the pathological characteristics of glaucoma.

[0125] The 50 μL drug evaluation group showed significant improvement in intraocular pressure after treatment with pilocarpine eye drops, indicating that this model can be used for drug evaluation.

[0126] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention may also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of glaucoma, characterized in that: The following steps are involved: (1) Injecting a suspension containing magnetic nanoparticles into the anterior chamber of the eye of the experimental animal by intracameral injection; (2) The magnetic nano-microsphere solid is attracted by a magnet so that the magnetic nano-microsphere is distributed in the anterior chamber angle, and then the experimental animal is raised for 35-40 days to establish an animal model.

2. The construction method according to claim 1, characterized in that: The magnetic nanospheres are dispersed in deionized water to form the suspension.

3. The method according to claim 2, characterized in that The content of magnetic nano-microspheres in the suspension is 50 mg / mL, and the injection volume is 10-50 μL.

4. The construction method according to claim 1, characterized in that: The average particle size of the magnetic nano-microspheres is 100 nm.

5. The construction method according to claim 1, characterized in that: The method for preparing the magnetic nanospheres comprises the following steps: The Fe3O4 nanoparticles are dispersed, chitosan is added for mixing and dispersion, a cross-linking agent is added for cross-linking and mixing, and magnetic nano-microspheres are obtained through post-treatment.

6. The construction method according to claim 5, characterized in that: The cross-linking agent is glutaraldehyde.

7. The construction method according to claim 1, characterized in that: The animal is a mammal.

8. The construction method according to claim 7, characterized in that: The mammal is any one of a cynomolgus monkey, a rhesus monkey or a marmoset.

9. A glaucoma animal model, characterized in that: The animal model is constructed by the construction method according to any one of claims 1 to 8.

10. An application of a glaucoma animal model, characterized in that: The method comprises applying the animal model constructed by the construction method according to any one of claims 1 to 8 or the animal model according to claim 9 to the evaluation of glaucoma treatment drugs.

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