A method for constructing an animal model of glaucoma, the animal model and application thereof

Abstract

The application relates to the technical field of animal models, in particular to a construction method of a glaucoma animal model, the animal model and application thereof. The construction method of the glaucoma animal model comprises the following steps: (1) injecting a suspension containing magnetic nano microspheres into the anterior chamber of an experimental animal through anterior chamber injection; (2) using a magnet to attract the magnetic nano microspheres solid, so that the magnetic nano microspheres are distributed in the angle of the anterior chamber, and then feeding the experimental animal for 35-40 days to establish the animal model. The animal model can guide the magnetic nano microspheres to the anterior chamber angle through the magnet, effectively reduces the aggregation of the magnetic nano microspheres in other positions of the anterior chamber, and solves the problem that the latex microspheres are difficult to position.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and in particular to a method for constructing a glaucoma animal model, the animal model itself, and its applications. Background Technology

[0002] Glaucoma is a progressive optic nerve disease caused by pathologically high intraocular pressure, characterized by retinal ganglion cell apoptosis and gradual loss of visual function. The pathological mechanisms of glaucoma are not fully understood, and it is difficult to conduct direct research on its pathogenesis in humans. Therefore, animal models can be used to simulate the process of glaucoma, understand its mechanisms, and develop new treatment techniques.

[0003] Common animal models of glaucoma include:

[0004] 1. Animal model of trabecular meshwork blockage

[0005] Method: This was caused by increasing the expression of fibronectin in trabecular meshwork cells, which accumulates inside and outside the cells and blocks the anterior chamber angle.

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

[0007] Disadvantages: The high intraocular pressure lasts for a short period of time, only causing mild to moderate intraocular pressure elevation; it also requires multiple long-term application of hormones, which is quite cumbersome; and it can easily lead to various hormone-related complications.

[0008] 2. Autologous blood cell anterior chamber injection model

[0009] Method: Autologous blood was extracted from experimental animals and injected into the anterior chamber. The denatured blood cells could not easily flow out of the aqueous humor like normal red blood cells, and were blocked in the trabecular meshwork, causing an increase in intraocular pressure.

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

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

[0012] 3. Latex microsphere anterior chamber injection model

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

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

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

[0016] 4. Magnetic microsphere injection model

[0017] To overcome the obstruction of fundus observation by microspheres accumulating in the anterior chamber, the simple polypropylene latex microspheres were replaced with magnetic microspheres. These were injected into the anterior chamber and guided to the anterior chamber angle using a handheld magnet. This increased the effect of blocking the anterior chamber angle and reduced the accumulation of microspheres in other parts of the anterior chamber.

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

[0019] Disadvantages: The magnetic ball in the anterior chamber can obstruct the pupil; the degree of intraocular pressure elevation and ganglion cell loss can vary depending on the animal's age and genetic background.

[0020] Therefore, this application provides a method for constructing an animal model of glaucoma, as well as the animal model and its application. Summary of the Invention

[0021] To overcome the shortcomings of the prior art, this invention provides a method for constructing a glaucoma animal model, as well as the animal model and its application. The animal model described in this application can guide magnetic nanospheres to the anterior chamber angle using a magnet, effectively reducing the aggregation of magnetic nanospheres in other locations in the anterior chamber and solving the problem of difficulty in positioning latex microspheres.

[0022] The technical solution adopted by this invention to solve its technical problem is:

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

[0024] (1) A suspension containing magnetic nanospheres was injected into the anterior chamber of the experimental animal via anterior chamber injection.

[0025] (2) Use a magnet to attract the magnetic nanosphere solid, so that the magnetic nanosphere is distributed in the corner of the room, and then feed the experimental animals for 35-40 days to establish the animal model.

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

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

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

[0029] Specifically, the preparation method of the magnetic nanospheres includes the following steps:

[0030] Fe3O4 nanoparticles were dispersed, chitosan was added for further dispersion, and a crosslinking agent was added for crosslinking and mixing. After post-treatment, magnetic nanospheres were obtained.

[0031] More specifically, the crosslinking agent is glutaraldehyde.

[0032] Specifically, the animal in question is a mammal.

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

[0034] Secondly, this application provides a glaucoma animal model, including the construction method described in the first aspect, and the resulting animal model.

[0035] Thirdly, this application provides an application of a glaucoma animal model, in which 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 treatment drugs.

[0036] The beneficial effects of this invention are:

[0037] The construction method described in this application uses magnetic nanospheres to construct an animal model. Guided by an external magnet, the magnetic nanospheres are precisely distributed in the anterior chamber angle, effectively blocking the angle, reducing aqueous humor outflow, and thus increasing intraocular pressure. The stable distribution of the magnetic nanospheres in the anterior chamber angle reduces the inconsistent blocking problems caused by the drift or aggregation of traditional microspheres in other locations, thereby ensuring a sustained increase in intraocular pressure. Furthermore, the magnetic nanospheres are made of biocompatible materials such as chitosan, reducing post-injection inflammatory responses and improving model stability and animal tolerance.

[0038] The construction method described in this application can precisely control the degree of intraocular pressure elevation, which is very beneficial for studying the impact of different intraocular pressure levels on optic nerve damage. Compared with other methods that require multiple injections, the method of using magnetic nanospheres 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, the drug evaluation group showed a significant decrease in intraocular pressure after treatment with pilocarpine eye drops, and the cup-to-disc ratio and outer diameter of the eyeball did not continue to increase and were smaller than those of the 50 μL high-dose model group at the same time point. This indicates that the model constructed using this method can be used for drug evaluation, providing strong support for further drug development and clinical application. Attached Figure Description

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] Figure 1 This is a diagram showing the results of intraocular pressure measurement in an embodiment of this application;

[0042] Figure 2 This is a graph showing the cup-plate ratio results from an embodiment of this application;

[0043] Figure 3 This is a diagram showing the results of eyeball outer diameter measurement according to an embodiment of this application;

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

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

[0046] Figure 6 OCT scan of the right eye on day 44 of drug treatment in the 50μL drug evaluation group of this application. Detailed Implementation

[0047] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention.

[0048] As used herein, “and / or” includes all combinations of any and one or more of the associated listed items. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the 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. Further understanding is needed; when used in this specification, “comprising” designates the stated features, integers, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof.

[0049] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further understanding is that terms, such as those defined in common dictionaries, are interpreted in accordance with their meaning in the context of the relevant field and are not idealized or overly formal, unless expressly defined herein.

[0050] The exemplary invention described herein may suitably omit any one or more limiting elements, which are not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” “containing,” etc., should be interpreted broadly and non-limitingly. Furthermore, the terminology used herein is for descriptive purposes without limitation, and it is unintentional to use terms that do not include any equivalent characteristics, but only to describe a portion of their characteristics; however, various modifications are possible within the scope of the invention according to the claims. Therefore, while the invention has been specifically disclosed through preferred embodiments and optional features, variations of the invention embodied by the modifications disclosed herein may be noted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0051] All raw materials or reagents used in the embodiments and comparative examples of this invention were purchased from mainstream manufacturers on the market. Those without specified manufacturers or concentrations are all analytical grade raw materials or reagents that are routinely available. There are no particular restrictions as long as they achieve the expected effect. The reaction vessels and rotary evaporators used in this embodiment were all purchased from major manufacturers on the market. There are no particular limitations as long as they achieve the expected effect. Where specific techniques or conditions are not specified in this embodiment, they should be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions.

[0052] Preparation Example 1

[0053] The preparation of magnetic nanospheres includes the following steps:

[0054] 1. Preparation of magnetic Fe3O4 nanoparticles

[0055] Iron salts were dissolved in deionized water, hydrogen peroxide and ammonia were added, and the mixture was stirred evenly and then transferred to a high-pressure reactor. The reaction temperature was 120℃ and the reaction time was 14 hours. After the reaction was completed, the mixture was centrifuged and dried to obtain Fe3O4 nanoparticles.

[0056] The iron salts FeSO4·7H2O and FeCl3·6H2O are 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 salts, the volume of ammonia water (saturated concentrated ammonia water) is 200 mL, and the volume of hydrogen peroxide (H2O2) is 200 mL.

[0057] 2. Crosslinking reaction

[0058] Chitosan was dissolved in ethyl acetate to prepare a 1 wt% chitosan solution;

[0059] F e3 O4 nanoparticles were dispersed in liquid paraffin, and 1 wt% chitosan solution was added and ultrasonically dispersed for 30 minutes.

[0060] Add 2.5 wt% glutaraldehyde solution and continue stirring at room temperature for 24 hours to allow chitosan to crosslink and form microspheres;

[0061] Filter the solution, wash thoroughly with petroleum ether to remove unreacted substances, and then wash with acetone to dehydrate it.

[0062] The product was dried in a vacuum drying oven to obtain magnetic nanospheres;

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

[0064] The prepared magnetic nanospheres were analyzed using Fourier transform infrared spectroscopy, and it was found that the magnetic nanospheres exhibited a magnetic flux density of 3400 cm⁻¹. -1 and 1600cm -1 There are obvious absorption peaks at these locations, corresponding to the stretching vibrations of -OH and -NH2 in chitosan, respectively.

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

[0066] Detection using a transmission electron microscope revealed that the magnetic nanospheres were spherical with a relatively uniform particle size distribution, and an average particle size of approximately 100 nm.

[0067] Dispersion test:

[0068] The prepared magnetic nanospheres were placed in a small bottle containing deionized water to form a suspension of 50 mg / mL.

[0069] The discovery that magnetic nanospheres in suspension can be dispersed in water for 60 days without precipitation indicates that they have excellent dispersibility in water.

[0070] Stability test:

[0071] A 50 mg / mL suspension of magnetic nanospheres was dried 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 the saturation magnetization of the magnetic nanospheres was 41 emu / g before suspension formation, and 40 emu / g after the stability test, indicating that the magnetic nanospheres have good magnetic stability.

[0072] Example

[0073] The methods for constructing animal models of glaucoma include the following:

[0074] 1. Animal anesthesia

[0075] Four healthy cynomolgus monkeys (all male, from Guangzhou Huazhen Biotechnology Co., Ltd.) of similar age, sex, weight and no eye diseases were selected.

[0076] Animal age: 6.45 ± 0.55 years;

[0077] Animal body weight: 3.87±0.95Kg;

[0078] Intraocular pressure: 14-20 mmHg.

[0079] Healthy crab-eating macaques were divided into

[0080] One animal was placed in the low-dose model group, one animal in the medium-dose model group, one animal in the high-dose model group, and one animal in the drug evaluation group.

[0081] Before surgery, cynomolgus monkeys in the low-dose, medium-dose, high-dose, and drug evaluation groups were fasted for 12 hours. General anesthesia (4-6 mg / kg) was administered via intramuscular injection of Sutacetin 50 injection (50 mg / ml) into the buttocks. Ten minutes later, Sutacetin (3-5 mg / kg) was injected intramuscularly into the buttocks. Anesthesia was considered adequate when the corneal reflex disappeared, the limb and abdominal muscles relaxed, and breathing became stable. During the anesthesia recovery period, the environment should be kept dark and quiet, and care should be taken to keep the anesthetized animals warm to prevent excessive heat loss.

[0082] 2. Anterior chamber injection

[0083] Using the right eye as the experimental eye, the conjunctival sac was rinsed three times with gentamicin injection mixed with an appropriate amount of physiological saline. The area around the right eye was disinfected with povidone-iodine, a sterile drape was laid, and the eyelid was opened with an eyelid opener. Under the operating microscope, a 15° puncture knife was first used to puncture the cornea through the upper temporal limbus to release a small amount of aqueous humor until the anterior chamber became shallow.

[0084] A syringe (50 μL capacity) pre-filled with a suspension containing 50 mg / mL of magnetic nanospheres was inserted into the anterior chamber through an incision. The syringe was then pushed to inject the suspension into the anterior chamber. After stopping the injection, the corneal puncture site was sealed with water, and tobramycin-dexamethasone eye ointment was applied. Magnetic beads were attracted using a ring magnet to distribute them at the anterior chamber angle, and the animal was revived.

[0085] The injection volumes were as follows: low-dose model group: 10 μL; medium-dose model group: 25 μL; high-dose model group: 50 μL; and drug evaluation group: 50 μL.

[0086] 3. Model Establishment

[0087] After anterior chamber injection and animal resuscitation, cynomolgus monkeys in the low-dose, medium-dose, high-dose, and drug evaluation groups were fed normally for 40 days to establish glaucoma animal models. Intraocular pressure and cup-to-disc ratio were measured during this period. After 40 days of feeding, the low-dose, medium-dose, and high-dose model groups continued to be fed until day 44, and intraocular pressure, cup-to-disc ratio, outer diameter of the eyeball, and OCT scans were performed.

[0088] After 40 days of normal feeding and evaluation, the cynomolgus monkeys in the normal feeding and evaluation group were given pilocarpine eye drops in the experimental eye (right eye) on days 41, 42 and 43, 4 times a day, 1 drop each time; feeding continued until day 44, when intraocular pressure, cup-to-disc ratio, outer diameter of the eyeball and OCT scan were performed.

[0089] 4. Model Detection and Evaluation

[0090] ① Cup-to-plate ratio test

[0091] Before injecting the suspension into the anterior chamber, the cynomolgus monkey was anesthetized according to the animal anesthesia procedure. When the anesthesia was adequate, compound tropicamide eye drops were instilled into both eyes to dilate the pupils and ensure that the pupils were fully dilated to facilitate observation of the fundus. The eyelids were opened using an eyelid speculum, and the head was fixed so that the eyeballs were facing forward. The cynomolgus monkey was fixed on the fundus photography equipment, and the focus was adjusted to ensure that the image was clear. Fundus photographs were taken, and the diameter ratio of the central fossa (cup) of the optic nerve head to the entire optic nerve head (disc) was calculated to obtain the preoperative cup-disc ratio.

[0092] Following the above procedure, cup-to-plate ratio measurements were performed on days 1, 5, 7, 40, 41, and 44 in the high-dose model group, medium-dose model group, low-dose model group, and drug evaluation group. The results are detailed in [link to results]. Figure 2 .

[0093] ② Intraocular pressure measurement

[0094] Before injecting the suspension into the anterior chamber, the cynomolgus monkeys were anesthetized according to the animal anesthesia method described above. When the anesthesia was adequate, the eyelids were opened using an eyelid speculum to ensure clear eye exposure. Preoperative intraocular pressure was measured using a tonometer, and intraocular pressure was measured again on days 1, 5, 7, 14, 28, 35, 40, 41, and 44. Results are detailed in [link to relevant documentation]. Figure 1 .

[0095] ③ Ocular outer diameter measurement

[0096] On day 44 after feeding, the cynomolgus monkeys in the high-dose model group, medium-dose model group, low-dose model group, and drug evaluation group were euthanized. Both eyeballs were removed, and the periorbital fat and connective tissue were removed under a surgical microscope. The eyes were rinsed three times with HBS buffer, and the anteroposterior and lateral diameters of the eyeballs were measured and recorded using calipers (three times each, averaged). See details for further information. Figure 3 .

[0097] from Figures 1-3 It can be seen that, regarding the results of intraocular pressure measurement,

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

[0099] In the 25μL medium-dose model group, intraocular pressure increased significantly after injection and remained at a high level at subsequent time points.

[0100] In the 50μL high-dose model group, intraocular pressure increased significantly after injection, and the intraocular pressure level was higher and lasted longer at subsequent time points.

[0101] Currently, it is believed that the cynomolgus monkey animal model, in which the intraocular pressure of the experimental eye is stably maintained at more than 35 mmHg for more than one month, can be used as a model of low intraocular pressure.

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

[0103] Therefore, in animal models of glaucoma, the larger the amount of magnetic nanospheres injected, the more pronounced the angle-blocking effect, resulting in a greater and longer-lasting increase in intraocular pressure. The 10μL low-dose group, with fewer injected magnetic nanospheres, had a limited blocking effect, resulting in a smaller increase in intraocular pressure, which gradually decreased over time. The 25μL medium-dose and 50μL high-dose groups, with more injected magnetic nanospheres, had a stronger blocking effect, leading to a significant increase in intraocular pressure that remained at a high level at subsequent time points.

[0104] Regarding the cup-to-plate ratio detection,

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

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

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

[0108] Cause analysis:

[0109] Dose-response: An increase in the cup-to-disc ratio reflects optic nerve damage. In the 10μL low-dose model group, due to a smaller increase in intraocular pressure and milder optic nerve damage, the increase in cup-to-disc ratio was smaller. In the 25μL medium-dose model group, due to a significantly increased intraocular pressure and more severe optic nerve damage, the increase in cup-to-disc ratio was larger and lasted longer. In the 50μL high-dose model group, due to the larger dose, higher intraocular pressure, and more severe optic nerve damage, the increase in cup-to-disc ratio was the largest.

[0110] Regarding the measurement of the outer diameter of the eyeball:

[0111] The 10μL low-dose model group showed a certain degree of increase in the outer diameter of the eyeball 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 even greater.

[0114] A comparison between the 50μL high-dose model group and the 50μL drug evaluation group revealed the following:

[0115] Before the medication was instilled, the intraocular pressure and cup-to-disc ratio in the 50μL drug evaluation group remained consistent with those in 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 lower than that of the 50μL high-dose model group at the same stage after 3 days of drug treatment, indicating that the cup-to-disc ratio of the experimental eye did not continue to increase after drug instillation and was lower than that of the 50μL high-dose model group at the same time.

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

[0119] As can be seen, after successful model establishment, the 50μL drug evaluation group, through drug treatment, showed a significant reduction in intraocular pressure, and the cup-to-disc ratio and outer diameter of the eyeball did not continue to increase and were smaller than those of the 50μL high-dose model group at the same time point. Therefore, the model constructed using the method described in this application can be used for drug evaluation, providing strong support for further drug development and clinical application.

[0120] Simultaneously, during the aforementioned animal experiments, slit-lamp microscopy was used to observe for inflammation such as corneal edema and retinal vascular edema. No inflammation was observed in the 10μL low-dose model group, 25μL medium-dose model group, 50μL high-dose model group, and 50μL drug evaluation group, indicating that the model constructed using magnetic nanospheres in this application has good biocompatibility. Furthermore, it was found that the magnetic nanospheres could be well dispersed in the iridocorneal angle. During the experiment, only a small amount of the dispersed magnetic nanospheres needed to be re-adsorbed to the iridocorneal angle using a magnet on day 21.

[0121] 50μL drug evaluation group, before modeling, such as Figure 4 As shown, the thickness of the right RNFL is within the normal range, the optic nerve fiber layer structure is intact, and no obvious abnormalities are observed.

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

[0123] In the 50μL drug evaluation group, after 3 days of drug infusion, if... Figure 6 As shown, the RNFL thickness in the right eye was uneven, with thinning of the fiber layer in some areas. However, it was slightly improved compared to the 50μL high-dose model group. This may be because the intraocular pressure in the experimental eye was reduced after the drug was instilled, and the RNFL thickness did not decrease further or the optic nerve fiber layer became thinner.

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

[0125] The 50μL 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 implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of glaucoma, characterized in that, Includes the following steps: (1) A suspension containing magnetic nanospheres was injected into the anterior chamber of the experimental animal via anterior chamber injection. The magnetic nanospheres are dispersed in deionized water to form the suspension; The content of magnetic nanospheres in the suspension is 50 mg / mL, and the injection volume is 10-50 μL; The method for preparing the magnetic nanospheres includes the following steps: dispersing Fe3O4 nanoparticles, adding chitosan for mixing and dispersion, and then adding a crosslinking agent for crosslinking and mixing to obtain magnetic nanospheres; the crosslinking agent is glutaraldehyde; (2) Use a magnet to attract the magnetic nanospheres so that the magnetic nanospheres are distributed in the corner of the room. Then, after feeding the experimental animals for 35-40 days, the animal model is established. On the 21st day, use a magnet again to attract the magnetic nanospheres that have been dispersed to the remaining positions to the corner of the room. The experimental animals are mammals; the mammals are any one of cynomolgus monkeys, rhesus monkeys, or marmosets; The animal model is a glaucoma animal model with changes in intraocular pressure, cup-to-disc ratio, and outer diameter of the eyeball.

2. The construction method according to claim 1, characterized in that, The magnetic nanospheres have an average particle size of 100 nm.

3. An application of an animal model of glaucoma, characterized in that, The animal model constructed by the construction method described in any one of claims 1-2 is applied to the evaluation of glaucoma treatment drugs.

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