An animal model of high oxygen-induced retinal neovascularization and a preparation method thereof

Abstract

The application provides an animal model of high-oxygen-induced retinal neovascular disease and a preparation method thereof. The method comprises the following steps: placing a mouse born for 3-7 days in an environment with an oxygen concentration of 75-85%, and maintaining for 10-30 days. The method can successfully induce a long-acting retinal neovascular disease model, has a high modeling rate, fewer complications, more persistent retinal neovascular lesions, and a model duration of 55 days. The animal model provides a more optimal model for exploring long-acting anti-retinal neovascular related drugs.

Description

Technical Field

[0001] This patent belongs to the field of biomedicine and relates to an animal model of hyperoxia-induced retinal neovascularization and its preparation method. Background Technology

[0002] Fundus diseases encompass inflammation and tumors of the retina, choroid, optic nerve, and vitreous body; vascular diseases; and ocular lesions caused by various degenerative diseases and multi-systemic diseases. Fundus diseases are diverse and significantly impair visual function, making them a challenging area to treat. Common fundus diseases include vascular diseases such as diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion, among other retinal neovascularization diseases. Preventing retinal neovascularization and safely and effectively treating existing retinal neovascularization have become research hotspots in recent years. Currently, the mechanisms of retinopathy are not fully understood. Establishing animal models for retinopathy research is time-consuming and costly, hindering early screening of related drugs. Therefore, establishing reliable, stable, high-film-forming, and long-lasting animal models of retinal neovascularization can lay a model foundation for exploring the mechanisms and treatments of retinal neovascularization diseases.

[0003] Oxygen-induced retinopathy (OIR) is currently the most commonly used animal model for studying retinal neovascularization. Hyperoxia leads to retinopathy primarily because the retinal vessels in premature infants are not fully developed. Hyperoxia exposure inhibits the growth of normal retinal vessels, resulting in oxygen-deficient aperfusion areas in the retina. This induces the release of neovascularization factors, leading to pathological neovascularization. All these abnormalities severely affect retinal development and function, ultimately resulting in retinopathy. Pathological angiogenesis is a hallmark of ischemic retinopathy, and OIR, a purely hypoxia-driven angiogenesis model, is widely used for ischemic retinopathy. Currently, the OIR animal model has become a commonly used experimental model for studying pathological neovascularization, providing significant assistance in the research of ischemic retinopathy and the treatment of ocular neovascularization. However, the hyperoxia volume fraction used in most OIR animal models does not fully match clinical applications, and the model duration is short with a high mortality rate. Mice are only kept in a hyperoxia environment for 5 days, resulting in a short duration of retinal neovascularization modeling and a window of only 13 days available for drug efficacy studies. This makes them unsuitable for preclinical studies of long-acting anti-retinal neovascularization drugs.

[0004] Therefore, in order to meet the clinical need for long-acting treatment, new preclinical models of retinal neovascularization are needed to provide longer and more durable treatment. Summary of the Invention

[0005] The present invention provides an animal model of hyperoxia-induced retinal neovascularization and its preparation method. The method includes placing mice aged 3-7 days in an environment with an oxygen concentration of 75-85% for 10-30 days. The method provided by the present invention is simple, rapid, efficient, has a low animal mortality rate, and induces retinal neovascularization over a long period. This animal model can well meet the needs of models requiring long-acting anti-retinal neovascularization drugs.

[0006] The effectiveness of OIR mouse models is influenced by various factors, including induction time, mouse breed and age, and the concentration and duration of hyperoxia. Excessive induction time may lead to excessive damage to the mouse retina, affecting the stability and reliability of the model. Before angiogenesis reaches a certain stage, longer induction times may indeed enhance the modeling effect. However, the timing of this stage is not fixed and is influenced by multiple factors, making it impossible to define a precise point in time. Some mouse breeds may be more sensitive to hyperoxia, while others may have stronger tolerance. If the mice are too young, their retinal vessels are not fully developed, and the resulting immature retinal vessels may lead to a more severe response to hyperoxia, resulting in more severe lesions. Conversely, if the mice are too old, their response to hyperoxia is too weak, failing to achieve the desired modeling effect.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides a method for preparing an animal model of hyperoxia-induced retinal neovascularization, wherein mice aged 3-7 days are placed in an environment with an oxygen concentration of 75-85% for 10-30 days.

[0009] In some embodiments of the present invention, the oxygen concentration is 78%, 80%, or 82%; and / or, the environment is a sealed oxygen chamber.

[0010] In some embodiments of the present invention, the method further includes: exposing the young mice to a normoxic environment for 20-40 minutes per day; the normoxic environment refers to an oxygen concentration of 19.5% to 23.5%.

[0011] In some embodiments of the present invention, the duration is 10-15, 10-20, or 21-30 days.

[0012] In some embodiments of the present invention, the duration is 20 days.

[0013] In some embodiments of the present invention, the mouse is a 7-day-old mouse.

[0014] In some embodiments of the present invention, the mouse is a C57BL / 6N or C57BL / 6J mouse, for example, a C57BL / 6J mouse.

[0015] In some embodiments of the present invention, the exposure time is 30 minutes.

[0016] A second aspect of the present invention provides an animal model of hyperoxia-induced retinal neovascularization, said animal model being prepared by the method described in the first aspect of the present invention.

[0017] The third aspect of the present invention provides a method for detecting retinal vascular distribution, wherein the method comprises performing FFA detection and / or lectin GS-IB4 staining on the retina of an animal model prepared by the method described in the first aspect of the present invention or an animal model described in the second aspect of the present invention.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] This invention prolongs the duration of hyperoxia-induced retinal neovascularization in mice, successfully establishing a long-lasting retinal neovascularization model. Compared to previous short-term oxygen-induced retinopathy models, this invention extends the duration of retinal neovascularization formation and increases the survival rate of young mice in a hyperoxia environment. This invention tracks and records the development of the model at various time points, evaluates the model's effectiveness and stability, and establishes a more scientific long-lasting hyperoxia-induced retinal neovascularization model. This model is more suitable for research on long-acting drugs against retinal neovascularization.

[0020] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0021] The reagents and raw materials used in this invention are all commercially available.

[0022] The positive and progressive effects of this invention are as follows: the retinal neovascularization disease model provided by this invention lasts for 55 days, and the window period for drug efficacy research is 28 days. The method for preparing the retinal neovascularization disease model provided by this invention is simple, fast, efficient, has a low animal mortality rate, and a long duration of retinal neovascularization lesions, which can well meet the model requirements for long-acting anti-retinal neovascularization drugs. Attached Figure Description

[0023] Figure 1 The results of FFA examination at various time points in the blank group and the OIR group (hyperoxygen-induced for 20 days) of young mice in Example 1 of the present invention are shown.

[0024] Figure 2The results of retinal patch staining at various time points in the blank group and the OIR group (hyperoxygen induced for 20 days) of young mice in Example 1 of the present invention are shown.

[0025] Figure 3 The results of FFA examination at various time points in young mice 10 days after hyperoxia induction in Example 2 of this invention.

[0026] Figure 4 The results of FFA examination at various time points in young mice after 30 days of hyperoxia induction in Example 3 of this invention.

[0027] Figure 5 The results of FFA examination on day 17 of young mice after 5 days of hyperoxia induction are shown in Comparative Example 1 of this invention. Detailed Implementation

[0028] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0029] In the embodiments of the present invention, a long-lasting hyperoxia-induced retinal neovascularization model was established, which has a longer duration and a higher survival rate in young mice compared to the classic hyperoxia-induced retinopathy model.

[0030] The experimental animals used in the embodiments of this invention were 7-day-old SPF-grade C57BL / 6J juvenile mice, of any sex, housed together with their mothers. Eye examinations showed no abnormalities. They were provided with 12 hours of light daily, appropriate amounts of feed, and free access to water. Animal experimental procedures followed the 3R principle.

[0031] The reagents used in the embodiments of the present invention are as follows:

[0032] Compound Tropicamide Eye Drops (Santen Pharmaceutical China Co., Ltd.); Ofloxacin Eye Ointment (Shenyang Xingqi Eye Drops Co., Ltd.); Levofloxacin Eye Drops (Santen Pharmaceutical China Co., Ltd.); Shutai® 50 (Victal Ltd., France); Xylazine Hydrochloride Injection (Jilin Huamu Animal Health Products Co., Ltd.); Sodium Fluorescein Injection (Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd.); Lectin GS-IB4 (Thermo Fisher).

[0033] Example 1

[0034] Animal models:

[0035] Day 7 (P7) OIR group C57BL / 6J pups and their lactating mothers were placed in cages within a closed oxygen chamber, with the oxygen concentration controlled at (80±2)%. The oxygen chamber was ventilated daily, bedding was changed, and food and water were provided, ensuring the mice were exposed to a normoxic environment for (30±10) min. Daily ventilation of (30±10) min effectively released harmful gases and reduced the adverse effects of hyperoxia on the mothers. After 20 days of hyperoxia (P27), the OIR group pups were returned to a normoxic environment, with a 12 h / 12 ​​h light / dark cycle during model induction. Seven-day-old control group pups and their lactating mothers were housed in a normoxic environment.

[0036] Inspection method:

[0037] FFA examinations were performed on days 7 (P34), 14 (P41), 21 (P48), and 28 (P55) after modeling to observe changes in the area of ​​the retinal non-perfusion zone caused by hypoxia and the recovery of blood vessels at each time point. Mydriasis was achieved three times with compound tropicamide eye drops before the examination to ensure adequate pupil dilation. Mice were anesthetized with Sutacetin® 50 and xylazine, and intraperitoneally injected with 10% sodium fluorescein solution for fundus image acquisition.

[0038] On day 7 (P34) and day 14 (P41) after modeling, OIR group mice were euthanized, and their retinas were harvested for stained slides. The lectin GS-IB4 was used as the dye, and the non-perfusion areas and neovascularization of the retina were observed under a fluorescence microscope and photographed.

[0039] Experimental results:

[0040] (1) FFA results:

[0041] After the modeling was completed, FFA was performed on the young mice in the OIR group and the control group. The results showed that ( Figure 1 After 20 days of hyperoxia, young mice were returned to normoxia. On day 7 (P34), the large retinal vessels of these mice showed irregular dilation and tortuous course, with large areas of non-perfusion around the optic disc, indicating that neovascularization was being induced in the retina. On day 14 (P41), the area of ​​non-perfusion around the optic disc decreased, and numerous clusters of neovascularization were visible in the mid-peripheral retina, accompanied by strong fluorescence leakage. On day 21 (P48), the area of ​​non-perfusion gradually decreased, retinal neovascularization increased, and fluorescence leakage intensified. On day 28 (P55), the non-perfusion areas disappeared, no neovascularization occurred, and fluorescence leakage lessened. In contrast, the control group of young mice, which had been kept in a normoxia environment, showed uniform retinal blood vessels with no fluorescence leakage or pathological neovascularization.

[0042] (2) Results of retinal patch staining:

[0043] Retinas of pup rats in the experimental group were collected from P34 and P41 and prepared as slides. After staining the retina with lectin GS-IB4, it was found that ( Figure 2 In the control group, large areas of non-perfusion and tortuous, dilated, and disordered neovascularization were visible in the retina, with strong fluorescence leakage. The changes in the area of ​​non-perfusion and the trend of angiogenesis were consistent with the FFA results. In the control group, the blood vessels of the young mice were radially distributed, basically fully developed, and there was no strong fluorescence leakage.

[0044] Example 2

[0045] Animal models:

[0046] C57BL / 6J pups and their lactating mothers, aged 7 days (P7), were placed in cages and placed in a closed oxygen chamber with an oxygen concentration controlled at (80±2)%. The oxygen chamber was opened daily for ventilation, bedding was changed, and food and water were added to ensure that the mice were exposed to a normoxic environment for (30±10) min. After 10 days of hyperoxic environment, the pups were returned to a normoxic environment (P17). The light / dark cycle during the model induction period was 12 h / 12 ​​h.

[0047] Inspection method:

[0048] On day 7 (P24) and day 14 (P31) after modeling, FFA examination was performed to observe the changes in the area of ​​the non-perfusion zone in the retina caused by hypoxia.

[0049] Experimental results:

[0050] FFA examination was performed on young mice 10 days after hyperoxia induction, and the results showed that ( Figure 3 After being kept in a hyperoxic environment for 10 days, the young mice were returned to a normoxic environment on day 7 (P24). Numerous new blood vessels were visible in the retina of the young mice, accompanied by strong fluorescent leakage, and the area of ​​non-perfusion was small. On day 14 (P31), the retina had basically returned to normal.

[0051] Example 3

[0052] Animal models:

[0053] C57BL / 6J pups, aged 7 days (P7), and their lactating mothers were placed in cages within a closed oxygen chamber, with the oxygen concentration controlled at (80±2)%. The oxygen chamber was ventilated daily, bedding was changed, and food and water were provided to ensure the mice were exposed to a normoxic environment for (30±10) min. After 30 days of hyperoxic rearing (P37), the pups were returned to a normoxic environment. During the model induction period, the light / dark cycle was 12h / 12h.

[0054] Inspection method:

[0055] On day 7 (P44) and day 14 (P51) after modeling, FFA examination was performed to observe the changes in the area of ​​the non-perfusion zone in the retina caused by hypoxia.

[0056] Experimental results:

[0057] FFA examination was performed on young mice induced by hyperoxia for 30 days, and the results showed that ( Figure 4 After being kept in a hyperoxic environment for 30 days, the young mice were returned to a normoxic environment on day 7 (P44). Numerous new blood vessels were visible in the retina, and the area of ​​non-perfusion zone was even smaller. On day 14 (P51), the retina had basically returned to normal.

[0058] Comparative Example 1

[0059] Animal models:

[0060] C57BL / 6J pups and their lactating mothers were placed in cages on day 7 (P7) and placed in a closed oxygen chamber with an oxygen concentration of 75%. After being raised in a hyperoxic environment for 5 days (P12), the pups were returned to a normoxic environment. The light / dark cycle during the model induction period was 12 h / 12 ​​h.

[0061] Inspection method:

[0062] On day 5 after modeling (P17), an FFA examination was performed to observe the changes in the area of ​​the non-perfusion zone in the retina caused by hypoxia.

[0063] Experimental results:

[0064] FFA was performed on young mice 5 days after hyperoxia induction, and the results showed that ( Figure 5 After being kept in a high-oxygen environment for 5 days, the mice were returned to a normal oxygen environment on day 5 (P17). Numerous new blood vessels were visible in the retinas of the young mice, accompanied by strong fluorescent leakage, and the area of ​​non-perfusion zone was small.

[0065] Experimental conclusion:

[0066] In Example 1, after 20 days of hyperoxia induction in P7 C57BL / 6J juvenile mice, FFA results showed that: large areas of non-perfusion were visible in the retina at P34; numerous neovascularizations appeared in the retina at P41; neovascularization increased in the retina at P48; and neovascularization gradually diminished and returned to normal at P55. The model duration was 55 days, with a 28-day window for drug efficacy studies. In the classic hyperoxia-induced retinopathy model in juvenile mice (Comparative Example 1), after 5 days of induction in a hyperoxia environment, neovascularization peaked at P17, and was almost completely regressed at P25. The model duration was 25 days, with a 13-day window for drug efficacy studies. In summary, the model duration in Example 1 was longer than that in Comparative Example 1, and the area of ​​the non-perfusion area was larger. The models in Examples 2 and 3 were longer than those in Comparative Example 1, but there was no significant difference in the area of ​​the non-perfusion area.

[0067] This invention features stability, high efficiency, higher model formation rate, and longer duration of retinal neovascularization. It can serve as a hyperoxia-induced long-acting retinal neovascularization model, providing a better model selection for the efficacy evaluation of preclinical long-acting anti-retinal neovascularization drugs such as AAV.

Claims

1. A method for preparing an animal model of hyperoxia-induced retinal neovascularization, characterized in that, The method includes placing mice aged 3-7 days in an environment with an oxygen concentration of 75-85% for 10-30 days.

2. The method as described in claim 1, characterized in that, The oxygen concentration is 78%, 80%, or 82%; and / or the environment is a sealed oxygen chamber.

3. The method as described in claim 1 or 2, characterized in that, The method further includes exposing the young mice to a normoxic environment for 20-40 minutes each day; normoxic refers to an oxygen concentration of 19.5% to 23.5%.

4. The method according to any one of claims 1-3, characterized in that, The duration is 10-15, 10-20, or 21-30 days.

5. The method according to any one of claims 1-4, characterized in that, The duration is 20 days.

6. The method according to any one of claims 1-5, characterized in that, The mice in question were 7 days old.

7. The method according to any one of claims 1-6, characterized in that, The mice were C57BL / 6N or C57BL / 6J mice.

8. The method according to any one of claims 1-7, characterized in that, The exposure time was 30 minutes.

9. An animal model of hyperoxia-induced retinal neovascularization, characterized in that, The animal model is prepared by the method described in any one of claims 1-8.

10. A method for detecting retinal blood vessel distribution, characterized in that, The method involves performing FFA detection and / or lectin GS-IB4 staining on the retina of an animal model prepared by the method described in any one of claims 1-8 or an animal model as described in claim 9.

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