Method for establishing pathological myopia animal model without eye intervention and use thereof

Abstract

The application relates to the technical field of animal disease models, and discloses a pathological myopia animal model establishment method and application without eye intervention, which comprises the following steps: using a Hemin solution to intervene in an animal to obtain a pathological myopia animal model. The method can establish a pathological myopia animal model.

Description

A method and application for establishing an animal model of pathological myopia without ocular intervention. Technical Field

[0001] This invention relates to the field of animal disease model technology, and in particular to a method and application for establishing a pathological myopia animal model without ocular intervention. Background Technology

[0002] Myopia is the most common eye disease worldwide, and with the continuous increase in the myopic population, the incidence of high myopia and pathological myopia is also rising rapidly. High myopia and pathological myopia have numerous complications that are lifelong and progressive, such as retinal detachment, macular hemorrhage, macular lacquer cracks, choroidal neovascularization, and posterior staphyloma. Preventing low vision and blindness caused by high myopia is a public health issue related to human visual health.

[0003] The primary cause of blindness due to high myopia stems from pathological changes in the fundus, known as pathological myopia. The mechanisms and treatments for pathological myopia caused by high myopia are still under investigation. A crucial element of this research is the development of models of pathological myopia, which form the basis for further research on how to improve this condition.

[0004] In recent years, some researchers have attempted to knock out the Lrp2 gene to simulate pathological myopia, but this can only simulate eyeball enlargement and lacks the characteristic fundus changes of pathological myopia. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method and application for establishing an animal model of pathological myopia without ocular intervention. The method of this invention can establish an animal model of pathological myopia.

[0006] This invention provides a method for establishing an animal model of pathological myopia without ocular intervention, the method comprising:

[0007] Animals were treated with Hemin solution to obtain a pathological myopia animal model.

[0008] Furthermore, the Hemin solution intervention is an intraperitoneal injection of Hemin solution.

[0009] Furthermore, the mass concentration of the Hemin solution is 1.25 mg / mL.

[0010] Furthermore, when intervening in animals, the injection is administered according to the animal's weight, and the Hemin content in the animal is 25 mg / kg-50 mg / kg.

[0011] Furthermore, when intervening in animals, the injection is administered according to the animal's weight, and the Hemin content in the animal is 50 mg / kg.

[0012] Furthermore, the method for preparing the Hemin solution includes dissolving Hemin in a sterile phosphate buffer containing a polysorbate 80 solution, wherein the ratio of the polysorbate 80 solution to the sterile phosphate buffer is 1:5 by volume.

[0013] Furthermore, the mass concentration of the polysorbate 80 solution is 5%.

[0014] Furthermore, the method for preparing the polysorbate 80 solution includes dissolving polysorbate 80 in deionized water.

[0015] Furthermore, the pH value of the sterile phosphate buffer is 7.

[0016] Furthermore, the method also includes using a negative lens.

[0017] Furthermore, the negative lens induction is achieved by fixing a negative lens in front of the animal's eyes.

[0018] Furthermore, the refractive power of the negative lens is -10.0D.

[0019] Furthermore, the negative lens is made of polymethyl methacrylate with a light transmittance of 90%.

[0020] Furthermore, the diameter of the negative lens is 12.7 mm.

[0021] Furthermore, when intervening in animals, the intervention is performed once every 7 days.

[0022] Furthermore, the animals include mammals.

[0023] Furthermore, the mammal includes a male guinea pig, the guinea pig being 2-3 weeks old.

[0024] Furthermore, the pathological manifestations of the pathological myopia are leopard-spot fundus, diffuse and / or patchy choroidal atrophy around the optic disc.

[0025] The present invention also provides an animal model established by the method for establishing the pathological myopia animal model.

[0026] The present invention also provides the use of the method for establishing the animal model of pathological myopia.

[0027] Furthermore, the uses include:

[0028] (1) Its purpose in exploring the causes and mechanisms of pathological myopia;

[0029] (2) Application in screening diagnostic targets for pathological myopia;

[0030] (3) Its application in screening treatment targets for pathological myopia;

[0031] (4) Use of products for screening pathological myopia diagnosis;

[0032] (5) Use of screening products for the treatment of pathological myopia.

[0033] The embodiments of the present invention have the following technical effects:

[0034] 1. This invention uses Hemin solution to intervene in animals. Hemin solution can disrupt the tight junction structure of retinal pigment epithelium (RPE) cells, thereby obtaining a pathological myopia animal model. The pathological manifestations of the pathological myopia model obtained by the method of this invention are leopard-spot fundus, diffuse and / or patchy choroidal atrophy around the optic disc. In addition, the method of this invention does not involve ocular intervention and does not require ocular operations such as wearing glasses or intraocular injection to induce pathological myopia, and will not cause intraocular inflammation or ocular structural damage caused by the operation.

[0035] 2. In this invention, the content of Hemin in animals needs to be further regulated. The dosage of Hemin needs to be sufficient to disrupt the tight junction structure of RPE cells, thereby establishing a pathological myopia model. On the other hand, it needs to ensure the survival rate of the model so that the model has practical significance.

[0036] 3. In this invention, the combined effect of Hemin solution and negative lens induction can further promote the development of pathological myopia. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 is a data result diagram of the axial length of guinea pigs provided in the embodiments and comparative examples of the present invention.

[0039] Figure 2 is a data result diagram of the axial length of guinea pigs provided in the embodiments and comparative examples of the present invention.

[0040] Figure 3 is a data graph provided by the embodiments and comparative examples of the present invention, wherein a in Figure 3 is the result of the posterior pole retinal thickness of guinea pigs, and b in Figure 3 is the result of the posterior pole choroidal thickness of guinea pigs.

[0041] Figure 4 is an OCT image of Example 3.

[0042] Figure 5 shows the tight junction structure of retinal RPE cells in Comparative Example 1.

[0043] Figure 6 shows the tight junction structure of retinal RPE cells in Comparative Example 2.

[0044] Figure 7 shows the tight junction structure of retinal RPE cells in Example 1.

[0045] Figure 8 shows the pathological findings of Example 1. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0047] In a first aspect, some embodiments of the present invention provide a method for establishing an animal model of pathological myopia without ocular intervention, the method comprising:

[0048] Animals were treated with Hemin solution to obtain a pathological myopia animal model.

[0049] By intervening in animals with Hemin solution, a pathological myopia animal model can be obtained by disrupting the tight junction structure of retinal pigment epithelial cells.

[0050] In some embodiments, the Hemin solution intervention is an intraperitoneal injection of Hemin solution.

[0051] In some embodiments, the mass concentration of the Hemin solution is 1.25 mg / mL.

[0052] In some embodiments, when intervening in animals, the injection is performed according to the weight of the animal, and the content of Hemin in the animal is 25 mg / kg-50 mg / kg.

[0053] Within the mass concentration range of Hemin in animals according to the present invention, a pathological myopia model in animals was established while ensuring the survival rate of the animals.

[0054] In some embodiments, when intervening in an animal, the injection is performed according to the animal's weight, and the content of Hemin in the animal is 50 mg / kg.

[0055] In some embodiments, the preparation method of the Hemin solution includes dissolving Hemin in a sterile phosphate buffer containing a polysorbate 80 solution, wherein the ratio of the polysorbate 80 solution to the sterile phosphate buffer is 1:5 by volume.

[0056] In some embodiments, the mass concentration of the polysorbate 80 solution is 5%.

[0057] In some embodiments, the preparation method of the polysorbate 80 solution includes dissolving polysorbate 80 in deionized water.

[0058] In some embodiments, the pH of the sterile phosphate buffer is 7.

[0059] In some embodiments, the method further includes employing a negative lens.

[0060] In some embodiments, the negative lens induction involves fixing a negative lens in front of the animal's eyes.

[0061] In some embodiments, the refractive power of the negative lens is -10.0D.

[0062] In some embodiments, the negative lens is made of polymethyl methacrylate with a light transmittance of 90%.

[0063] In some embodiments, the diameter of the negative lens is 12.7 mm.

[0064] In some embodiments, the intervention in the animal is performed once every 7 days.

[0065] In some embodiments, the animal includes mammals.

[0066] In some embodiments, the mammal includes a male guinea pig, the guinea pig being 2-3 weeks old.

[0067] In some embodiments, the pathological manifestations of the pathological myopia are leopard-spot fundus, diffuse and / or patchy choroidal atrophy around the optic disc.

[0068] Secondly, some embodiments of the present invention also provide an animal model established by the method for establishing a pathological myopia animal model.

[0069] Thirdly, some embodiments of the present invention also provide the use of the method for establishing the animal model of pathological myopia.

[0070] In some embodiments, the use includes:

[0071] (1) Its purpose in exploring the causes and mechanisms of pathological myopia;

[0072] (2) Application in screening diagnostic targets for pathological myopia;

[0073] (3) Its application in screening treatment targets for pathological myopia;

[0074] (4) Use of products for screening pathological myopia diagnosis;

[0075] (5) Use of screening products for the treatment of pathological myopia.

[0076] The following description uses some specific examples:

[0077] Example 1: A Hemin solution with a concentration of 1.25 mg / mL was prepared, and the Hemin content in guinea pigs was 50 mg / Kg, denoted as Hemin 50 mg / Kg.

[0078] Example 2: A Hemin solution with a concentration of 1.25 mg / mL was prepared, and the Hemin content in guinea pigs was 25 mg / Kg, denoted as Hemin 25 mg / Kg.

[0079] Example 3: Hemin at a mass concentration of 50 mg / Kg in guinea pigs + negative lens-induced myopia (LIM), denoted as LIM + Hemin 50 mg / Kg.

[0080] Comparative Example 1: Guinea pigs were not treated and were recorded as blank.

[0081] Comparative Example 2: Lens-induced myopia, denoted as LIM.

[0082] Comparative Example 3: Hemin solution with a concentration of 1.25 mg / mL, and the content of Hemin in guinea pigs was 10 mg / Kg.

[0083] Comparative Example 4: Hemin solution with a concentration of 1.25 mg / mL, and the content of Hemin in guinea pigs was 100 mg / Kg.

[0084] Before the experiment, the animals were kept in the following conditions and environment: male 2-3 week old pigmented guinea pigs were used, with an initial weight of 150g-200g. All guinea pigs were kept in a cycle of 12 hours of light (450-500 lux) and 12 hours of darkness (0 lux). The room temperature was maintained at 24℃-26℃ and the humidity was about 60% throughout the period of the experiment. All animals had free access to food and water.

[0085] Results and Analysis:

[0086] In this invention, different volumes of Hemin solution were injected into the peritoneal cavity of guinea pigs to achieve varying Hemin concentrations: 10 mg / kg, 25 mg / kg, 50 mg / kg, and 100 mg / kg. It was found that at a Hemin concentration of 10 mg / kg, the axial length of the guinea pigs increased by only 0.5 ± 0.03 mm, and pathological myopia did not develop. At a Hemin concentration of 100 mg / kg, the axial length increased by 0.75 ± 0.04 mm, but guinea pigs in the 100 mg / kg concentration died. Therefore, the selection of the appropriate Hemin concentration in guinea pigs not only needs to ensure animal survival but also needs to establish a pathological myopia model. Based on this, blank guinea pigs were randomly divided into three groups: a blank control group (n=10), a group receiving intraperitoneal injection of 25 mg / kg Hemin (n=10), and a group receiving intraperitoneal injection of 50 mg / kg Hemin (n=10). Injections were administered weekly according to the weight of each guinea pig. The administration was repeated for 12 consecutive weeks, during which the guinea pigs were fed for a total of 12 weeks. As shown in Figure 1, after 12 weeks of continuous administration, the axial length of the eyes in mice injected with Hemin was significantly elongated. The elongation of the axial length became more significant with increasing Hemin concentration in the guinea pigs, but the mortality rate of the mice increased. Therefore, the optimal Hemin concentration in guinea pigs was selected as 25 mg / kg–50 mg / kg; further optimization yielded a Hemin concentration of 50 mg / kg.

[0087] To further verify the promoting effect of Hemin on pathological myopia in this invention, a blank control group (n=10), a LIM group (n=10), a 50 mg / Kg Hemin group (n=10), and a LIM + intraperitoneal injection of 50 mg / Kg Hemin group (n=10) were set up. Injections were administered weekly according to the weight of each guinea pig for 12 consecutive weeks. The guinea pigs were housed for a total of 12 weeks. For intraperitoneal injection, the guinea pig was first fixed, and the skin of the left lower abdomen was disinfected. Then, a 26G needle was inserted subcutaneously into the left abdomen, pushed forward approximately 0.5 cm, and then the needle was inserted into the abdominal cavity at a 45-degree angle to the skin, passing through the abdominal muscle until a feeling of emptiness was produced. After aspiration revealed no intestinal fluid, urine, or blood, the medication was slowly injected. At the start of the experiment, measurements were taken every 7 days at 2:00 PM, for a total of 13 times. Axial length of the guinea pigs was measured using acoustic A-scan mode scan (A-scan mode scan; oscillator frequency (AScan): 11MHz) under ocular surface anesthesia. For each guinea pig, eight independent and repeated measurements were performed in both eyes, and the average value was recorded, along with the standard deviation. This average value was the final axial length of the guinea pig. The results, as shown in Figure 2, indicate that after 12 weeks of continuous administration, the Hemin group significantly increased the axial length of the guinea pigs, showing a similar effect to the LIM group. Furthermore, in the LIM+Hemin group, high concentrations of Hemin in the guinea pigs did indeed promote the further development of pathological myopia.

[0088] The thickness of the retina and choroid in guinea pigs was measured under non-anesthesia conditions using fundus color imaging combined with optical coherence tomography (OCT). The instrument used in this invention was an SS-OCT system (VG200D, SVisionImaging, Ltd., Henan, China), with a working wavelength close to 1050 nm and an ultra-fast scanning speed of 200,000 AScans per second. After pupil dilation in both eyes of the guinea pigs, fundus color imaging and OCT images were acquired centered on the optic disc. After acquiring qualified images, horizontal and vertical scan images were exported. For each OCT image, the thickness of the retina and choroid at the 3 and 9 o'clock horizontal points and the 0 and 6 o'clock vertical points were extracted. The average thickness of the guinea pig choroid was measured using ImageJ software, repeated three times, and the average value and standard deviation were recorded. As shown in Figure 3, compared to the LIM group, the Hemin group exhibited significantly reduced posterior retinal and choroidal thickness, indicating that the Hemin group demonstrated better technical efficacy in the posterior retina and choroid. Furthermore, the LIM+Hemin group confirmed that high concentrations of Hemin in guinea pigs do indeed promote the development of pathological myopia. OCT image analysis in Figure 4 further validated that the LIM+Hemin group showed retinal and choroidal atrophy and reduced retinal and choroidal thickness. Therefore, this further confirms that high concentrations of Hemin in guinea pigs do indeed promote the development of pathological myopia, with effects similar to those induced by LIM.

[0089] For transmission electron microscopy (TEM) analysis, the enucleated eyeballs were placed in 2.5% glutaraldehyde fixative (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). After 10 minutes, the eyeballs were removed, cut open at the posterior equator, and the cornea, lens, and vitreous tissue were removed. A 1-2 mm² tissue sample was taken from the posterior pole of the resulting eyeball wall and pre-fixed in the fixative solution, then placed at room temperature in the dark for 2 hours. The specimens were then stored at 4°C and embedded, sectioned, and photographed under TEM within one week. The results, shown in Figures 5-7, reveal that the tight junction structure of the retinal RPE cells in the Hemin group changed compared to other groups. This confirms that the tight junctions of the retinal RPE cells in guinea pigs treated with Hemin were disrupted, leading to pathological myopia in the guinea pigs.

[0090] Guinea pigs injected intraperitoneally with 50 mg / kg Hemin all exhibited significant leopard-spot-like fundus (M1 stage pathological myopia), and 30% of the guinea pigs showed diffuse, patchy choroidal atrophy around the optic disc, characteristic of pathological myopia (M2-M3 stage pathological myopia), as shown in Figure 8. Simultaneously, OCT images revealed retinal and choroidal atrophy in the guinea pigs, and transmission electron microscopy images showed disruption of tight junctions in the RPE cells of the guinea pig retina after Hemin intervention, with the disruption increasing with increasing agonist concentration. This can serve as a marker of pathological myopia and as a novel animal model for exploring the etiology and mechanisms of pathological myopia's occurrence and development.

[0091] In summary, the method of the present invention can establish a pathological myopia animal model while ensuring animal survival rate.

[0092] It should be noted that the terminology used in this invention is for describing specific embodiments only and is not intended to limit the scope of this application. As shown in this specification, unless the context clearly indicates otherwise, words such as "a," "an," "an," and / or "the" do not specifically refer to the singular and may include the plural. The terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, or apparatus that includes said element.

[0093] It should also be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. Unless otherwise expressly specified and limited, the terms "installed," "connected," "linked," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components. For those skilled in the art, the specific meaning of the above terms in the present invention can be understood according to the specific circumstances.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing an animal model of pathological myopia without ocular intervention, characterized in that, The method includes: intervening in animals by intraperitoneal injection of Hemin solution to obtain a pathological myopia animal model; the mass concentration of the Hemin solution is 1.25 mg / mL; when intervening in the animals, the injection is performed according to the weight of the animals, and the content of Hemin in the animals is 25 mg / kg-50 mg / kg; the intervention is performed once every 7 days; the animals are male guinea pigs, and the guinea pigs are 2-3 weeks old.

2. The method according to claim 1, characterized in that, When intervening in animals, the injection is administered according to the animal's weight, and the Hemin content in the animal is 50 mg / kg.

3. The method according to claim 1, characterized in that, The method further includes using a negative lens; the negative lens is used to induce the negative lens to be fixed in front of the animal's eyes; the refractive power of the negative lens is -10.0D; the material of the negative lens is polymethyl methacrylate with a light transmittance of 90%; the diameter of the negative lens is 12.7mm.

4. The method according to claim 1, characterized in that, The pathological manifestations of the pathological myopia are leopard-spot fundus, diffuse and / or patchy choroidal atrophy around the optic disc.

5. Use of the method for establishing an animal model of pathological myopia according to any one of claims 1-4 for non-diagnostic or therapeutic purposes.

6. The use according to claim 5, characterized in that, The uses include: investigating the causes and mechanisms of pathological myopia occurrence and development; screening diagnostic targets for pathological myopia; screening therapeutic targets for pathological myopia; screening diagnostic products for pathological myopia; and screening therapeutic products for pathological myopia.

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