Dry age-related macular degeneration animal model construction method

Abstract

The invention discloses a construction method of a dry age-related macular degeneration animal model, which comprises the following steps: sodium iodate is injected into vitreous cavities above macula lutea, the concentration of the sodium iodate is 4 mg / mL-16 mg / mL, and the single injection capacity is 100 [mu] L / eye. The stable and repeatable dry age-related macular degeneration animal model is constructed by adjusting the injection range, the injection dosage and different injection positions, and the severity of the lesion of the model is obviously related to the dosage.

Description

Technical Field

[0001] The invention belongs to the field of disease models, and specifically relates to a method for constructing an animal model of dry age-related macular degeneration. Background Art

[0002] Macular degeneration is one of the causes of blindness and is related to the degeneration of retinal photoreceptor cells in the macula corresponding to the center of the fundus. AMD is mainly divided into atrophic (dry) and exudative (wet). In atrophic AMD, the retinal pigment epithelium and the photoreceptor cells in the adjacent choroidal capillaries gradually undergo degenerative atrophy. In exudative AMD, due to hemorrhage and edema, the choroidal blood vessels proliferate, leading to a relatively rapid progression of the disease. In human atrophic AMD, degeneration (thinning and reduction) of the retinal pigment epithelium (RPE) and thinning and disappearance of the outer nuclear layer (ONL) can be seen.

[0003] The light-induced retinopathy model is known as an animal model that mimics atrophic AMD. In this model, strong light is used to induce retinopathy. In fact, retinal degeneration has been shown to occur in a light-induced mouse retinopathy model. In addition, in rodent and rabbit AMD animal models, RPE degeneration can be induced by systemic administration or intravitreal injection of sodium iodate.

[0004] Therefore, when developing drugs to treat a certain disease, it is necessary to establish an animal model of the disease to study the therapeutic effect of the test substance. In order to use animal models to correctly evaluate the efficacy of drugs, the animal model used must accurately reflect the pathology of the target disease. Since non-human primates are closer to humans than rodents in genetics, neuroanatomy and pharmacokinetics, it can be expected that they can obtain actions and effects that are closer to those observed during human administration. Since the macula exists in non-human primates, AMD animal models using rodents or rabbits cannot reflect the pathology of human AMD. Therefore, in order to develop effective therapeutic drugs for humans, it is best to use non-human primates rather than rodents as animal models. Therefore, in order to develop preventive and therapeutic drugs for AMD, a non-human primate model that more accurately reflects the pathology of AMD is needed.

[0005] Before developing this model, we found through literature review that there are currently a few reports at home and abroad on the induction of dry AMD models in crab-eating macaques by intravitreal injection of sodium iodate. For this method of modeling by injection, the success of the model is directly related to the injection position and injection speed. The literature does not mention the improvement of the injection position and speed and the details of the operation. Only the dose is compared. Therefore, the results shown in different literatures show that the model is more serious and unstable, with poor repeatability, and the severity of the model varies at the same dose. Therefore, it is necessary to develop a more accurate, repeatable and stable dry AMD model, and models of different severity can simulate dry AMD patients at different stages in the clinic to meet the development needs of various drugs. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for constructing an animal model of dry age-related macular degeneration that can simulate dry AMD patients at different stages.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a method for constructing an animal model of dry age-related macular degeneration, comprising the following steps: injecting sodium iodate once or multiple times into the vitreous cavity above the macula of crab-eating monkeys, wherein the concentration of sodium iodate is 4 mg / mL to 16 mg / mL, and the single injection volume is 100 μL / eye.

[0008] The specific injection position of sodium iodate is started and ended at the position between the macula and the optic disc, and the range is controlled within the upper and lower vascular arches. The injection is made 360° clockwise or counterclockwise around the top of the macula, and the injection rate is 100μL / 30s to 100μL / 60s.

[0009] The distance between the injection needle tip and the retina is approximately 50 μm to 100 μm.

[0010] Wherein, the injection dose of sodium iodate is 0.4 mg to 1.6 mg per eye.

[0011] Among them, the severity of retinal lesions in the model is dose-dependent. When the injection dose is 0.4 mg / eye, there is no obvious change in the eye model. When the injection dose is 0.8 mg / eye, slight changes can be seen in the eye model. When the injection dose is 1.2 mg / eye, a geographic atrophy model is obtained. When the injection dose is 1.6 mg / eye, a severe geographic atrophy model is obtained.

[0012] The present invention observed macular retinopathy at a dose of 0.8 mg / eye and above 1 week after injection. The severity of retinopathy is dose-dependent, ranging from undetectable changes (0.4 mg / eye), mild changes (0.8 mg / eye), geographic atrophy (1.2 mg / eye) to severe geographic atrophy (1.6 mg / eye). The lesions of 0.8 mg / eye were reversible after the 4th week. ERG results showed that the signal amplitude of impaired retinal function decreased significantly or even disappeared. Complement C5 protein inhibitors can significantly improve retinal morphology, but do not lead to substantial improvement in retinal function. Cellular RPE destruction, photoreceptor and vascular changes were observed in all retinal lesion areas. At the same time, the severity of the model at different doses can be seen to have a significant dose correlation, and it can be seen that a stable and repeatable dry AMD model can be achieved by controlling the injection location, injection range, and injection rate.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention constructs a stable and repeatable dry age-related macular degeneration animal model by adjusting the injection method, different doses, and different injection locations. The severity of the lesions in the model shows obvious dose correlation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Sodium iodate-induced dry AMD showed a dose-dependent manner;

[0015] Figure 2 Treatment with C5 complement inhibitors in a sodium iodate-induced dry AMD model;

[0016] Figure 3 Adaptive optics scanning laser ophthalmoscope (AOSLO) was used to observe the changes of photoreceptor cells. Figure 4 ERG result graph with dark adaptation of 0.01; Figure 5 Dark adaptation 3.0 ERG result graph; Figure 6 ERG result graph of dark-adapted oscillatory potential; Figure 7 Dark-adapted ERG result graph 10.0; Figure 8 ERG result diagram of light adaptation 3.0; Fig. 9 Figure 3. Light-adapted 30Hz flicker ERG results. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0018] Example 1

[0019] The method for establishing the dry AMD animal model of the present invention comprises injecting sodium iodate into the vitreous cavity above the macula of a cynomolgus monkey (provided by Jiangsu Dingtai Pharmaceutical Research (Group) Co., Ltd.). Sodium iodate is used for monocular intravitreal injection of cynomolgus monkeys (0.4 mg / eye, 0.8 mg / eye, 1.2 mg / eye, 1.6 mg / eye). The injection starts and ends at the position between the macula and the optic disc, the needle tip of the injection needle is about 50 μm to 100 μm away from the retina (between the posterior limiting membrane of the vitreous and the inner limiting membrane of the retina), the range is controlled within the upper and lower vascular arches, and the injection is performed clockwise or counterclockwise around the macula 360° in a circle, and the injection rate is 100 uL / 60s. Fundus photography (FP), optical coherence tomography (OCT), fundus fluorescein angiography (FFA), electroretinogram (ERG) and fundus autofluorescence (FAF) were performed before injection (Pre) and 1, 2, 4, 6, 8, and 12 weeks after injection (W1, W2, W4, W8, and W12).

[0020] By injecting sodium iodate into the vitreous body through the above-mentioned injection technique, the injection position, injection range and injection rate can be controlled to induce dry age-related macular degeneration, and obtain consistency of modeling under the same modeling dose in different eyes of different animals. That is, ONL thinning, RPE degeneration and retinal geographic atrophy are close to human dry AMD, or even human atrophic AMD. The experimental results show that the severity of retinal lesions is dose-dependent, such as Figure 1 The dose of sodium iodate was 0.4 mg / eye to obtain an eye model with no obvious changes, the dose of sodium iodate was 0.8 mg / eye to obtain an eye model with slight changes, the dose of sodium iodate was 1.2 mg / eye to obtain an eye model with geographic atrophy, and the dose of sodium iodate was 1.6 mg / eye to obtain an eye model with severe geographic atrophy. Figure 4-Figure 9 , ERG results showed that the signal amplitude of retinal dysfunction was significantly reduced or even disappeared.

[0021] One week after modeling, complement C5 protein inhibitor (1.2 mg / eye) was administered to one eye treated with sodium iodate (1.2 mg / eye) for verification. Figure 2 It can be seen that complement C5 protein inhibitor can significantly improve retinal morphology, but does not lead to substantial improvement in retinal function.

[0022] The changes in RPE and photoreceptor cells were further quantitatively analyzed by using the reflectivity and autofluorescence mode of adaptive optical scanning laser ophthalmoscope (AOSLO) in model animals injected with different doses of sodium iodate (0.8 mg / eye and 1.2 mg / eye). Figure 3It can be seen that RPE destruction, photoreceptor and vascular changes at the cellular level were observed in all retinal lesion areas, demonstrating that a slightly changed eye model and a geographic atrophy eye model were obtained, respectively.

Claims

1. A method for constructing an animal model of dry age-related macular degeneration, characterized in that: The following steps are involved: Sodium iodate is injected once or multiple times into the vitreous cavity above the macula of cynomolgus monkeys, wherein the concentration of sodium iodate is 4 mg / mL to 16 mg / mL and the single injection volume is 100 μL / eye.

2. The method for constructing an animal model of dry age-related macular degeneration according to claim 1, characterized in that: The specific injection position of sodium iodate is to start and end at the position between the macula and the optic disc, within the upper and lower vascular arches, and inject 360° clockwise or counterclockwise around the top of the macula, with an injection rate of 100μL / 30s~100μL / 60s.

3. The method for constructing an animal model of dry age-related macular degeneration according to claim 1, characterized in that: The tip of the injection needle is approximately 50 μm~100 μm away from the retina.

4. The method for constructing an animal model of dry age-related macular degeneration according to claim 1, characterized in that: The injection dose of sodium iodate is 0.4 mg to 1.6 mg per eye.

5. The method for constructing an animal model of dry age-related macular degeneration according to claim 1, characterized in that: The severity of retinal lesions in the model is dose-dependent. When the injection dose is 0.4 mg / eye, no obvious changes are observed in the eye model. When the injection dose is 0.8 mg / eye, slight changes are observed in the eye model. When the injection dose is 1.2 mg / eye, a geographic atrophy model is obtained. When the injection dose is 1.6 mg / eye, a severe geographic atrophy model is obtained.

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