A method for constructing a non-human primate model of glaucoma

Abstract

The application belongs to the technical field of biological medicine, and particularly discloses a construction method of a glaucoma non-human primate model, which comprises the following steps: injecting emulsified silicone oil into the anterior chamber of a non-human primate; and detecting after 7-8 weeks, i.e. obtaining the glaucoma non-human primate model. Compared with a traditional model, the animal model of the application does not need to repeatedly use expensive modeling instruments, nor does it need to repeatedly damage the animals by modeling multiple times. The reagent used is low in cost and easy to obtain, the modeling time is short, and the animal model can greatly save manpower, material resources and time cost, and reduce the overall preclinical development cost of new drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a method for constructing a disease model of an experimental animal, in particular to a method for constructing a non-human primate model of glaucoma. BACKGROUND

[0002] Glaucoma is a group of diseases with the common characteristics of optic disc atrophy and depression, visual field defect and visual acuity decline. Pathological intraocular pressure increase and insufficient blood supply of the optic nerve are the primary risk factors for the onset of the disease. The tolerance of the optic nerve to pressure damage is also related to the occurrence and development of glaucoma. Obstruction of any link in the aqueous humor circulation pathway can lead to pathological changes caused by elevated intraocular pressure, but some patients also present normal tension glaucoma. Glaucoma is one of the three major blinding eye diseases that lead to blindness in humans. The pathogenesis of glaucoma has not been fully elucidated so far, and there are great limitations in the study of glaucoma patients. Therefore, studying the mechanism and prevention and treatment through animal models is an effective research means.

[0003] At present, the most classic non-human primate glaucoma model is the laser trabecular meshwork-induced glaucoma model. This model has the characteristics of non-invasiveness and light inflammation, but the disadvantage is that it needs to be repeatedly modeled to achieve sustained high intraocular pressure, the experimental period is long, and the cost of experimental instruments is also high.

[0004] In the prior art, patent document CN102229959A describes a pathogenic gene animal model of primary open-angle glaucoma and a construction method thereof. The human OPTN (E50K) mutant gene is cloned into an expression vector with a retinal promoter c-kit to construct a recombinant mammalian expression vector, which is then introduced into mice to screen and identify transgenic mice. Patent document CN105943186A describes a method for establishing a chronic high eye pressure animal model. The outflow passage of aqueous humor is blocked by surgical placement of materials to cause glaucoma animal models with elevated intraocular pressure and optic nerve damage. The elevated intraocular pressure is stable, making it easier to obtain and operate. The model has the advantages of stable elevated intraocular pressure, small fluctuations, long duration of high eye pressure, and controllable target intraocular pressure. However, these methods have obvious drawbacks. First, the gene technology involved in the transgenic mouse model is complex, has high technical barriers, is expensive, difficult to operate, and has a low success rate. Except for primates, the angle structures of most animals are significantly different from those of humans, and the rodent (mouse) is more different in terms of phylogenetic development. The mouse's eyeball is too small to perform routine ophthalmic examinations, and there is a lack of data support for intraocular pressure changes and pathological progression. This model is still under research and development, and is not mature enough to be applied to the evaluation of ophthalmic drugs. Second, the glaucoma model involving surgical placement of special materials to block the trabecular meshwork requires the assistance of a professional ophthalmic microscope, which is highly specialized and difficult to operate, has a low success rate, and is risky, as it can easily cause infection and bleeding. The surgical details (fibre catheter insertion into the schlemm canal and 360 or 270 degree circumnavigation) involved in the patent are difficult to achieve, and are also prone to cause choroidal damage. The inserted polypropylene material can cause an allergic reaction, leading to anterior chamber turbidity and inflammatory exudation, which in turn affects ophthalmic examinations. Additionally, due to the small size of the animal's eye, the surgical difficulty is greater, and the success rate of the model is difficult to determine. This patent also lacks relevant intraocular pressure data and clinical ophthalmic examination data support, making it difficult to apply to drug evaluation.

[0005] The eyes of non-human primates are most similar in structure to human eyes, and due to the volume advantage of large animal eyeballs, human ophthalmic detection methods can be directly applied. Therefore, using non-human primates as models can better simulate the occurrence and development of clinical glaucoma compared to other experimental animals (rodents), and the research results are most easily applicable to humans, providing direct guidance for clinical diagnosis and treatment of human diseases.

[0006] Anterior chamber injection of foreign objects to induce glaucoma has been widely used in rodents and primates, and the foreign objects include chymotrypsin, methyl cellulose, compound carbomer, latex microspheres, magnetic beads, sodium hyaluronate, and hematoid bodies, etc. The main defects of the glaucoma induced by the above foreign objects are that the foreign objects adhere to the cornea, float in the anterior chamber, or cover the pupil; the anterior chamber inflammation reaction is caused, inflammatory exudates are caused, and the anterior chamber is turbid; the imaging examination is blocked, and the fundus changes cannot be clearly examined; the anterior segment lesions are caused, such as the anterior adhesion of the iris periphery and the posterior trabecular meshwork, the corneal peripheral neovascularization, the iris atrophy, the iris-lens adhesion, the pigment deposition on the corneal endothelium and the lens surface, etc. The glaucoma induced by other methods needs multiple injections, and the intraocular pressure is unstable or the duration is short, and the multiple injections can cause corneal damage and infection risk. SUMMARY

[0007] The main purpose of the present application is to provide a construction method of a glaucoma non-human primate animal model to solve the problems in the background art.

[0008] To achieve the above purpose, the present application provides the following technical solutions.

[0009] A construction method of a glaucoma non-human primate animal model, comprising the following steps:

[0010] A. injecting emulsified silicone oil into the anterior chamber of the eyeball of a non-human primate animal;

[0011] B. detecting after 7-8 weeks, that is, constructing a glaucoma non-human primate animal model.

[0012] Further, the diameter of the emulsified silicone oil is 4-10 μm, and the viscosity is 800 mPa.s.

[0013] Further, in step A, the emulsified silicone oil is composed of 4.5% dimethyl silicone oil, 0.027% oleic acid, 0.115% glycerol, 0.75% Tween 20, 0.0005% EDTA, and the balance is ultrapure water.

[0014] Further, the present application provides a preparation step of the emulsified silicone oil.

[0015] S1: dimethyl silicone oil and oleic acid are mixed to form an oil phase; glycerol, Tween 20, EDTA, and ultrapure water are mixed to form an aqueous phase;

[0016] S2: after the two phases are heated to 55-65℃ respectively, the aqueous phase is added to the oil phase for multiple times, and the mixture is stirred manually until uniform;

[0017] S3: under high-speed stirring, the mixture is high-speed sheared at 10000-20000 rpm for a total time of 10-20 minutes;

[0018] S4: Adjust the pH of the emulsified silicone oil to 7.5-8.5 with NaOH;

[0019] S5: Absorb the lower aqueous solution where the soft phospholipid is located, add an equal volume of ultrapure water, and after centrifugal separation, absorb the lower layer again; repeat 10-15 times, then absorb the free soft phospholipid, absorb the lower water layer, and leave the silicone oil layer as the initial emulsion;

[0020] S6: Observe the properties of the initial emulsion, and detect that the diameter of the silicone oil droplets is 4-10 μm.

[0021] Preferably, in step A, the emulsified silicone oil is diluted by ≥8 times, and the injection frequency is >1 time.

[0022] Preferably, in step A, when the injection frequency of the emulsified silicone oil is >1 time, the interval between each injection is ≥1 week. Preferably, the injection amount of the emulsified silicone oil each time is ≥50 μl.

[0023] Further, the non-human primates in the present application include cynomolgus monkeys, rhesus monkeys, or marmoset monkeys, etc. The glaucoma animal model constructed by the present application can be applied in the evaluation of glaucoma treatment drugs.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] The present application uses a specific emulsification method to prepare emulsified silicone oil with a suitable size to block the trabecular meshwork, thereby inducing a stable intraocular pressure glaucoma model. The emulsification method is simple, low in cost, and stable in quality control. The emulsified silicone oil has a moderate density and good dispersibility, and will not gather into a group to block the pupil or adhere to the cornea. The emulsified silicone oil has a suitable diameter size for the trabecular meshwork pores, and can completely block the trabecular meshwork with a few injections to cause stable and sustained ideal intraocular pressure. The emulsified silicone oil has low immunogenicity and will not cause anterior chamber inflammatory reaction. The single injection amount and injection frequency can control the target intraocular pressure and duration, thereby simulating acute and chronic open-angle primary glaucoma. There are sustained intraocular pressure data and clinical imaging (OCT, ERG, funduscope, ultrasound) support, and the repeatability and success rate are high.

[0026] Compared with the traditional model, the animal model of the present application does not need to repeatedly use expensive modeling instruments, nor does it need to repeatedly damage the animals for modeling, the reagent cost is low and easy to obtain, the modeling time is short, and the human, material and time costs can be greatly saved, and the overall preclinical stage research and development cost of new drugs is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Intraocular pressure change graph of right eye anterior chamber of different cynomolgus monkeys (animals 1, 2, 3) after three injections of 10 times diluted emulsified silicone oil 50 μl and left eye;

[0028] Figure 2 Effect of timolol on intraocular pressure of model after use;

[0029] Figure 3 Intraocular pressure changes of cynomolgus monkey right eye after four times injection of 20 times diluted emulsified silicone oil 50 μl in the anterior chamber and left eye;

[0030] Figure 4 Intraocular pressure changes of cynomolgus monkey right eye after four times injection of 7 times diluted emulsified silicone oil 50 μl in the anterior chamber and left eye;

[0031] Figure 5 Optical coherence tomography (OCT) results of cynomolgus monkey No. 1 model after intraocular pressure rise;

[0032] Figure 6 Optical coherence tomography (OCT) results of cynomolgus monkey No. 2 model after intraocular pressure rise;

[0033] Figure 7 Optical coherence tomography (OCT) results of cynomolgus monkey No. 3 model after intraocular pressure rise;

[0034] Figure 8 Fundus mirror scanning results of cynomolgus monkey No. 1 model after intraocular pressure rise;

[0035] Figure 9 Fundus mirror scanning results of cynomolgus monkey No. 2 model after intraocular pressure rise;

[0036] Figure 10 Fundus mirror scanning results of cynomolgus monkey No. 3 model after intraocular pressure rise;

[0037] Figure 11 Electrophysiology results of cynomolgus monkey model after intraocular pressure rise; (A represents the change of oscillatory potential of right and left eyes after injection of 10 times diluted emulsified silicone oil in the anterior chamber of right eye, compared with normal left eye, the oscillatory potential of right eye; B represents the right and left eyes after injection of 10 times diluted emulsified silicone oil in the anterior chamber of right eye, compared with normal left eye, the amplitude of right eye is significantly reduced) ;

[0038] Figure 12 Trabecular meshwork HE staining after injection of 10 times diluted emulsified silicone oil in the anterior chamber of right eye;

[0039] Figure 13 Optic disc HE staining after injection of 10 times diluted emulsified silicone oil in the anterior chamber of right eye;

[0040] Figure 14 Nerve fiber layer HE staining after injection of 10 times diluted emulsified silicone oil in the anterior chamber of right eye.

[0041] Figure 15 Intraocular pressure changes of cynomolgus monkey right eye after four times injection of 10 times diluted emulsified silicone oil 20 μl in the anterior chamber and left eye; DETAILED DESCRIPTION

[0042] The application will be described in greater detail with reference to specific embodiments and drawings. The following examples are helpful for further understanding of the application by those skilled in the art, but do not limit the application in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the application. These are within the scope of the application.

[0043] The term "dimethicone": Polydimethylsiloxane is a hydrophobic organic silicone material, also known as dimethicone;

[0044] The term "oleic acid": Oleic acid is a monounsaturated Omega-9 fatty acid, which exists in animals and plants, and its chemical formula is C 18 H 34 O2.

[0045] Example 1

[0046] A method for constructing a non-human primate model of glaucoma, constructing a cynomolgus monkey model, comprising the following steps:

[0047] 1. According to the mass percentage: 4.5% dimethicone, 0.027% oleic acid, 0.115% glycerol, 0.75% Tween 20, 0.0005% EDTA, and the rest is ultrapure water, prepare the corresponding quality of emulsified silicone oil preparation raw materials.

[0048] 2. Preparation of emulsified silicone oil: dimethicone and oleic acid are mixed to form an oil phase; glycerol, Tween 20, EDTA, and ultrapure water are mixed to form an aqueous phase; after heating the two phases to 55-65°C, the aqueous phase is added to the oil phase in small amounts and stirred manually; under high-speed stirring, mix and shear at 10,000-20,000 rpm for a total of 10-20 minutes; adjust the pH of the emulsified silicone oil to 7.5-8.5 using NaOH; remove the lower aqueous solution containing soft phospholipids, add an equal volume of ultrapure water, and centrifuge to separate the layers; remove the free soft phospholipids after repeating 10-15 times, remove the lower water layer, and leave the silicone oil layer as the initial emulsion; observe the properties of the initial emulsion, and detect that the silicone oil droplet diameter is 4-10 μm; after ultraviolet sterilization, it is ready for use.

[0049] 3. The emulsified silicone oil prepared above is diluted 10 times with water to obtain emulsified silicone oil with a viscosity of 800 mPa.s, which is injected into the anterior chamber of the eyes of cynomolgus monkeys. The injection frequency of the emulsified silicone oil is 1-3 times, and the injection frequency is greater than 1 time. The interval between each injection is 1-2 weeks, and the injection amount of the emulsified silicone oil is 50-200 μl. Preferably, the emulsified silicone oil is injected 3 times, and the injection amount of each injection is 50 μl. The glaucoma non-human primate animal model is constructed after 7-8 weeks of detection.

[0050] Comparative Example 1

[0051] The other steps are the same as those in Example 1, except that the injection frequency is 4 times and the injection amount is 20 ul.

[0052] Comparative Example 2

[0053] The other steps are the same as those in Example 1, except that the emulsified silicone oil prepared is diluted 7 times to obtain emulsified silicone oil with a viscosity of 625 mPa.s. The injection frequency of the emulsified silicone oil is 4 times, and the injection amount is 50 ul.

[0054] Comparative Example 3

[0055] The other steps are the same as those in Example 1, except that the emulsified silicone oil prepared is diluted 20 times to obtain emulsified silicone oil with a viscosity of 432 mPa.s. The injection frequency of the emulsified silicone oil is 4 times, and the injection amount is 50 ul.

[0056] Table 1 Modeling effects of different examples and comparative examples

[0057]

[0058] Test Example

[0059] 1. Preparation of emulsified silicone oil

[0060] a) Avoid direct sunlight and air mixing as much as possible during the whole operation;

[0061] b) Dimethyl silicone oil (45 g) and oleic acid (0.27 g) are mixed to form an oil phase; glycerol (1.15 g), Tween 20 (7.5 g), EDTA (0.005 g), and ultrapure water (100 mL) are mixed to form an aqueous phase;

[0062] c) After heating the dimethyl silicone oil and oleic acid at 55-65 °C, the glycerol, Tween 20, EDTA, and ultrapure water are mixed to form an aqueous phase. The aqueous phase is added to the oil phase in small amounts and stirred manually until uniform (after adding a small amount of water and stirring uniformly, the next amount of water is added, and the process is repeated at least 5 times. Stir for 10-20 minutes to ensure uniformity);

[0063] d) mixing under high speed shearing (10000-20000 rpm) with high speed stirring, total time 10-20 minutes; e) adjusting the pH of the emulsion to 7.5-8.5 with NaOH;

[0064] f) aspirating the lower aqueous solution in which the soft phospholipid is located, adding an equal volume of ultrapure water, and after centrifugal separation, aspirating the lower layer again;

[0065] After repeating 10-15 times, aspirate the free soft phospholipid, aspirate the lower aqueous layer, and leave the silicone oil layer as the initial emulsion; g) observe the properties of the initial emulsion and confirm the dispersibility; observe the size of the silicone oil droplets under a microscope to obtain a silicone oil droplet diameter of 4-10 μm;

[0066] h) sterilize the initial emulsion with ultraviolet radiation, and store at 4°C before use.

[0067] 2. Anterior chamber injection:

[0068] a) after general anesthesia with Alvedon, topical anesthesia with lidocaine and anesthesia with isoflurane, the eyelid is propped open;

[0069] b) disinfect the eyeball and conjunctiva with polyvidone iodine, and clean with physiological saline;

[0070] c) pass a 20G needle through the corneal layer at the junction of the temporal corneal margin and the sclera to reach the anterior chamber;

[0071] d) inject 50 μl of 10-fold diluted emulsified silicone oil into the anterior chamber;

[0072] e) fix the syringe in place, stabilize the needle, loosen the needle seat to determine that there is no significant rebound, and then remove the syringe, and press the injection site with a cotton swab to reduce silicone oil leakage;

[0073] f) apply antibiotic ointment topically to the eye to prevent infection;

[0074] g) injection frequency: once every 1-2 weeks before the intraocular pressure increases, and continue the silicone oil anterior chamber injection when the intraocular pressure decreases to normal.

[0075] 3. IOP (intraocular pressure) detection:

[0076] a) according to the product instructions, use the iCare tonometer to monitor the intraocular pressure of both eyes, and each time requires repeated measurement 3 times, and take the average value (all values need to be recorded); if there is a significantly higher / lower intraocular pressure value (+-30% or more) at one time, increase the measurement by 1 time;

[0077] b) pay attention to the number of times and the frequency of replacement of the iCare tonometer measurement probe;

[0078] c) fixed time point in the morning (8-9 am), after general anesthesia with Alvedon, topical anesthesia with lidocaine and deep anesthesia with isoflurane, the eyelid is propped open;

[0079] d) IOP detection: intraocular pressure was measured three times per week.

[0080] 4. Imaging

[0081] a) Optical coherence tomography (OCT) examination: retinal tomography (OCT): horizontal, radial, and circular scanning were used to examine the contrast of the optic disc depth, disc margin area, cup / disc area ratio, optic cup formation measurement index, retinal nerve fiber layer thickness, visual field detection, and the degree of vascular distortion within the optic disc.

[0082] b) Ophthalmoscope: retinal artery damage.

[0083] c) Slit lamp: corneal and anterior chamber structure.

[0084] 5. Neuroelectrophysiology: flash electroretinogram (FERG)

[0085] Flash electroretinogram (FERG) is a group of potential changes recorded at the corneal end when retinal neurons are stimulated by light. The waveform is the result of the combined action of various retinal neurons.

[0086] 6. Pathology:

[0087] After modeling (elevated intraocular pressure) for 6 weeks, the animals were enucleated and fixed with formalin solution and stained with HE.

[0088] Primary open-angle glaucoma is characterized by elevated intraocular pressure, with the anterior chamber angle always open. The outflow of aqueous humor is blocked in the trabecular meshwork-Schlemm canal system. Histological examination suggests that the trabecular meshwork thickens, the meshwork narrows or occludes, and the endothelial cells in the Schlemm canal wall decrease. The main clinical manifestations are high eye pressure, gradually increasing eye pressure with disease progression, normal or deeper anterior chamber depth, open anterior chamber angle, retinal nerve fiber layer loss, and other pathological changes.

[0089] The modeling results are as follows:

[0090] Figure 1Figure 1 is a graph of intraocular pressure changes in three cynomolgus monkeys during modeling, the first animal intraocular pressure change graph, the right eye of the animal was injected with 10 times diluted silicone oil three times, and the intraocular pressure slowly increased to 50 mmHg, then slowly decreased to normal intraocular pressure, and the fourth time the anterior chamber was given 10 times diluted silicone oil, and the intraocular pressure slowly increased to about 40 mmHg; the second animal intraocular pressure change graph, the right eye of the animal was injected with 10 times diluted silicone oil three times, and the intraocular pressure slowly increased to 60 mmHg, then slowly decreased to normal intraocular pressure, and the fourth time the anterior chamber was given 10 times diluted silicone oil, and the intraocular pressure slowly increased to about 35 mmHg; the third animal intraocular pressure change graph, the right eye of the animal was injected with 10 times diluted silicone oil twice, and the intraocular pressure slowly increased to 55 mmHg, and then the intraocular pressure was always maintained above 30 mmHg.

[0091] Figure 2 Figure 4 is a graph of intraocular pressure changes and the effect of the intraocular pressure lowering drug timolol on the seventh cynomolgus monkey, the right eye of the animal was injected with 10 times diluted silicone oil twice, and the intraocular pressure slowly increased and maintained at 33 mmHg; 29 days after injection, the intraocular pressure lowering drug was given (one drop at a time, twice a day), and the intraocular pressure slowly decreased to normal after drug administration, indicating that the drug has good pressure-lowering effect.

[0092] Figure 3 Figure 5 is a graph of intraocular pressure changes in the fifth cynomolgus monkey after injection of 50ul of 20 times diluted silicone oil, the right eye of the animal was injected with 20 times diluted silicone oil four times at the same time point, and the intraocular pressure was still maintained in the normal range, indicating that 20 times diluted silicone oil cannot induce high intraocular pressure.

[0093] Figure 4 Figure 6 is a graph of intraocular pressure changes in the sixth cynomolgus monkey after injection of 50ul of 7 times diluted silicone oil, the right eye of the animal was injected with 7 times diluted silicone oil four times at the same time point, and the intraocular pressure was still maintained in the normal range, indicating that 7 times diluted silicone oil cannot induce high intraocular pressure.

[0094] Figure 15 Figure 7 is a graph of intraocular pressure changes in the seventh cynomolgus monkey after injection of 20ul of 10 times diluted silicone oil, the right eye of the animal was injected with 10 times diluted silicone oil four times at the same time point, and the intraocular pressure was still maintained in the normal range, indicating that 10 times diluted silicone oil injection of 20ul also cannot induce high intraocular pressure.

[0095] The above intraocular pressure results of the seven animals show that an appropriate amount of 10 times diluted emulsified silicone oil can induce high intraocular pressure, while 7 times and 20 times diluted emulsified silicone oil have no effect on intraocular pressure, and at the same time, it is shown that timolol has a significant pressure-lowering effect, and also shows that the glaucoma model induced by 10 times diluted emulsified silicone oil can be used for clinical drug evaluation.

[0096] Figure 5 、 6, 7 are OCT results of the above-mentioned first to third cynomolgus monkeys, wherein the No. 1 animal is the change of the retinal nerve fiber layer thickness after the right eye anterior chamber injection of 10-fold diluted emulsified silicone oil, and the results show that the right eye nasal retinal nerve fiber layer is obviously thinned; the results of the No. 2 cynomolgus monkey show that the right eye nasal, nasal superior and temporal superior retinal nerve fiber layers are obviously thinned; the results of the No. 3 cynomolgus monkey show that the retinal nerve fiber layer is obviously thinned except the right eye nasal inferior, and the above-mentioned OCT results of the three animals show that the 10-fold diluted emulsified silicone oil can induce a glaucoma model with high intraocular pressure and retinal nerve layer change.

[0097] Figure 8 、 9 , 10 are fundus mirror results of the above-mentioned first to third cynomolgus monkeys, wherein the No. 1 cynomolgus monkey is the change of the anterior chamber, iris, cornea and vitreous body after the right eye anterior chamber injection of 10-fold diluted emulsified silicone oil, and the results show that compared with the normal left eye, the right eye anterior chamber angle is widely blocked by the silicone oil, but the anterior chamber is clear without inflammatory exudation, the corneal refraction is normal, and the iris and vitreous body have no inflammatory change; the results of the No. 2 cynomolgus monkey show that compared with the normal left eye, the right eye anterior chamber angle is widely blocked by the silicone oil, but the anterior chamber is clear without inflammatory exudation, the corneal refraction is normal, and the iris and vitreous body have no inflammatory change; the results of the No. 3 cynomolgus monkey show that compared with the normal left eye, the right eye anterior chamber angle is widely blocked by the silicone oil, but the anterior chamber is clear without inflammatory exudation, the corneal refraction is normal, and the iris and vitreous body have no inflammatory change; and the above-mentioned fundus mirror results of the three animals show that the 10-fold diluted emulsified silicone oil can induce a glaucoma model with high intraocular pressure, but will not cause adverse reactions such as inflammation.

[0098] Figure 11 Fig. 10A is a diagram of the change of the left and right eye oscillatory potentials after the right eye anterior chamber injection of 10-fold diluted emulsified silicone oil, and compared with the normal left eye, the right eye oscillatory potential, Figure 11 Fig. 10B is a diagram of the left and right eye photopic electroretinogram after the right eye anterior chamber injection of 10-fold diluted emulsified silicone oil, and compared with the normal left eye, the right eye amplitude is obviously reduced; and the above-mentioned electrophysiological results show that the 10-fold diluted emulsified silicone oil can induce a glaucoma model with high intraocular pressure and electrophysiological change

[0099] Figure 12 、 13 , 14 is a diagram of the pathological change of the trabecular meshwork, optic disc and nerve fiber layer after the intraocular pressure of the glaucoma model of the cynomolgus monkey is increased, Figure 12 Fig. 14A1 is a HE staining diagram after the right eye anterior chamber injection of 10-fold diluted emulsified silicone oil, and the 10-fold enlarged diagram shows that the silicone oil widely blocks the trabecular meshwork of the right eye anterior chamber angle, causes the outflow of aqueous humor to be blocked and further causes the intraocular pressure to be increased; Figure 12 Fig. 14A2 is a HE staining diagram of the optic disc after the right eye anterior chamber injection of 10-fold diluted emulsified silicone oil, and the results show that the right eye peripheral retinal nerve fiber layer is obviously disappeared; Figure 13 the results show that the right eye ganglion cell layer cell number is obviously reduced, Figure 14 the results show that the right eye ganglion cell layer cell degeneration.

[0100] The above intraocular pressure and pathological data results show that the trabecular meshwork can be widely blocked by injecting 10 times diluted emulsified silicone oil into the right anterior chamber for several times, which causes the outflow passage of aqueous humor to be blocked and in turn can cause persistent high intraocular pressure, while the fundus mirror results show that the anterior chamber angle is normal, the anterior chamber is clear without turbidity, and the cornea, iris, and vitreous body have no inflammatory reaction. The pathological, OCT, and electroretinogram results show that the optic nerve fiber layer is obviously thinned and degenerated and is accompanied by visual field defects, and the open-angle glaucoma model meets the clinical characteristics. Moreover, three weeks of continuous treatment with the adrenergic receptor blocker timolol can significantly inhibit aqueous humor production and reduce intraocular pressure. Therefore, the model has the advantage of being close to the clinical manifestations.

[0101] The present application has many specific application approaches, and the above description is only the preferred embodiment of the present application. It should be noted that the above examples are only used to illustrate the present application, and are not used to limit the protection scope of the present application. For ordinary skilled persons in the art, several improvements can be made without departing from the principles of the present application, and these improvements should also be considered as the protection scope of the present application.

Claims

1. A method for constructing a non-human primate model of glaucoma, characterized by, The method comprises the following steps: A. Injecting emulsified silicone oil into the anterior chamber of the eye of a non-human primate; B. Detecting after 7-8 weeks, i.e. constructing a non-human primate model of glaucoma; In step A, the emulsified silicone oil has a diameter of 4-10 μm and a viscosity of 800 mPa·s; In step A, the emulsified silicone oil is composed of 4.5% dimethyl silicone oil, 0.027% oleic acid, 0.115% glycerol, 0.75% Tween 20, 0.0005% EDTA, and the balance of ultrapure water; The preparation steps of the emulsified silicone oil are as follows: S1: mixing dimethyl silicone oil and oleic acid to form an oil phase; mixing glycerol, Tween 20, EDTA, and ultrapure water to form an aqueous phase; S2: after heating the two phases to 55-65℃, the aqueous phase is added to the oil phase in small amounts and stirred manually; S3: mixing under high-speed stirring and high-speed shearing at 10000-20000 rpm for a total of 10-20 minutes; S4: adjusting the pH of the emulsified silicone oil to 7.5-8.5 using NaOH; S5: absorbing the lower aqueous solution containing soft phospholipids, adding an equal volume of ultrapure water, centrifuging and separating the layers, and then absorbing the lower layer; repeating 10-15 times, then absorbing the free soft phospholipids, absorbing the lower water layer, and leaving the silicone oil layer as the initial emulsion; S6: observing the properties of the initial emulsion and detecting that the diameter of the silicone oil droplets is 4-10 μm; The injection amount of emulsified silicone oil is ≥50 μl each time, the emulsified silicone oil is diluted by ≥8 times for use, and the injection frequency is >1 time. In step A, the injection frequency of the emulsified silicone oil is 2. The construction method according to claim 1, characterized in that, When the injection frequency is >1 time, the interval between each injection is ≥1 week. The non-human primate includes a cynomolgus monkey, a rhesus monkey, or a marmoset.

3. The construction method of claim 2, wherein, 4. Use of the animal model obtained by the construction method of claim 3 in the evaluation of glaucoma treatment drugs. ​ ​

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