Animal model creation method

Abstract

Provided is a means to create an animal model that reproduces a state in which an abnormality has occurred in the ocular fundus due to elongation of the eye axis.　An animal model creation method according to the present invention includes a step for inducing myopia in an animal and raising the animal for a specific period of time. The specific period of time is at least 1.5 times the period of time from the induction of myopia to a plateau in reduction of refractive power.

Description

How to create an animal model

[0001] The present invention relates to a method for producing an animal model, and a method for evaluating or screening a composition for preventing or ameliorating an eye disease associated with pathological myopia or axial elongation using the animal model.

[0002] According to the latest research on myopia and high myopia, a significant increase in the myopic population is expected worldwide, with the number of people with myopia projected to reach approximately 5 billion and high myopia projected to reach approximately 1 billion by 2050 (see Non-Patent Document 1). This does not simply affect the quality of life of young people, but also means that the number of patients who will develop high myopia and go blind in the future will continue to increase.

[0003] Axial myopia, the primary type of myopia, occurs when the eye's axis elongates. As axial elongation progresses, the retina and choroid are stretched backward, thinning the retina and choroid, and mechanical stress increases, causing various abnormalities in the posterior segment of the eye. This condition in which abnormalities in the posterior segment occur due to axial elongation is called pathological myopia.

[0004] Pathological myopia is a leading cause of blindness in developed countries. According to a report by the Ministry of Health, Labor and Welfare, pathological myopia is the fourth leading cause of blindness in Japan (Report of the Ministry of Health, Labor and Welfare's Retinal and Choriooptic Atrophy Research Group, 2005). Myopic retinopathy associated with axial elongation is a leading cause of blindness in developed countries. In Japan, the Tajimi Study (a large-scale epidemiological survey) reported that myopic macular degeneration associated with high myopia is the third leading cause of visual impairment and the leading cause of blindness (Non-Patent Document 2). Excessive axial elongation has been reported to increase the risk of eye diseases such as cataracts, glaucoma, retinal detachment, retinopathy, maculopathy, choroidal neovascularization, posterior staphyloma, and optic neuropathy as complications (see Non-Patent Document 3). Thus, despite the risk of blindness from pathological myopia, there are currently no effective treatments to prevent its progression, and the establishment of such treatments is highly desirable.

[0005] It has been reported that an adult myopia induction model has been created using mice, which are easily amenable to genetic manipulation (Patent Document 1, Non-Patent Document 4). Three-week-old C57BL6J mice and a specific lens attachment device (Patent Document 1 and Figure 1 of this specification) were used. A support post 1 was surgically erected and fixed to the skull of the mouse. The support post was threaded so that an adjustment device 5, which adjusts the lens width and angle, could be secured with a nut. A minus lens of -30 diopters (D) was attached to each side of the mouse to induce myopia, and a 0D lens was attached to the other mouse as a control. A protector 4, which protrudes laterally, was attached to the frame below the lens to prevent the mouse from scratching the lens with its forepaws or other objects. The adjustment device 5 is located on the frame above the lens and adjusts the width and angle of the attached lens as the mouse grows. The adjustment device 5 is bent in a "L" shape, with a lens attached to one end and a long hole 6 attached to the support post 1 erected on the head. By passing the elongated hole 6 through the support 1 and screwing it with a nut 7, it can be fixed in place by adhering it to the skin without compressing the edges of both eyes of the mouse.

[0006] In the creation of these mice, myopia was induced for three weeks, leading to high myopia. As a result, changes in axial length and refractive index generally reached a plateau (see Fig. 3 in Non-Patent Document 4). However, these mice did not exhibit changes in electroretinogram (ERG) amplitude, which would indicate retinal dysfunction (see Fig. 6 in Non-Patent Document 4), and did not exhibit any characteristics of pathological myopia.

[0007] Japanese Patent Application Laid-Open No. 2020-023574

[0008] Brien A Holden, et.al. Ophthalmology,2016, Vol.123, No.5, pp.1036-1042Iwase A, et.al. Ophthalmology 2006, Vol.113, pp.1354-1362 Hidenari Torii, “High myopia from the perspective of aging”, 2016, Ophthalmology, Vol.58, No.6, pp.635-641Jiang X. et al., 2018, Scientific Reports, Vol.8, Article number 2026

[0009] To screen drugs for pathological myopia, an animal model that more closely resembles the state of pathological myopia in humans and that allows drug screening is needed.

[0010] Therefore, the purpose of this study is to provide a means to create an animal model that reproduces the condition in which abnormalities occur in the fundus due to axial elongation.

[0011] As a result of extensive research, the present inventors have found that inducing myopia in animals and continuing the myopia induction for a certain period after the decrease in refractive power has plateaued induces organic and functional retinal dysfunction that reflects pathological myopia. They have also found that animals with this retinal dysfunction can be used as animal models, and that these animal models can be used to evaluate and screen drugs for eye diseases related to pathological myopia and axial elongation, thereby completing this research.

[0012] That is, the present invention includes the following embodiments: [1] A method for producing an animal model, comprising the step of raising an animal under myopia induction for a specific period of time, wherein the specific period is at least 1.5 times the period from the induction of myopia until the decrease in refractive power reaches a plateau. [2] A method for evaluating or screening a composition for preventing or ameliorating an eye disease associated with pathological myopia or axial elongation, comprising the steps of raising an animal under myopia induction for a specific period of time, and administering a test substance to the animal, wherein the specific period of time is at least 1.5 times the period from the induction of myopia until the decrease in refractive power reaches a plateau, and the test substance is administered at the start of the specific period, during the specific period, or after the specific period.

[0013] The animal model produced by the production method of the present invention reflects the state of human pathological myopia or eye diseases associated with axial elongation, and can be used for the evaluation and screening of test substances for drugs for the state of eye diseases associated with pathological myopia or axial elongation.

[0014] 1 is an image of a set of supports and mounting devices used to create a mouse myopia induction model. 2 is a graph showing changes in refractive power in a mouse myopia induction model. After 3 weeks: p<0.0001 (n(0D)=35, n(-30D)=37), after 6 weeks: p<0.0001 (n(0D)=31, n(-30D)=35). 3 is a graph showing changes in axial length in a mouse myopia induction model. After 3 weeks: p=0.0003 (n(0D)=37, n(-30D)=39), after 6 weeks: p=0.0006 (n(0D)=32, n(-30D)=37). 4 is a graph showing changes in choroidal thickness in a mouse myopia induction model. After 3 weeks: p<0.0001 (n(0D)=36, n(-30D)=38), after 6 weeks: p<0.0001 (n(0D)=27, n(-30D)=34). These graphs show the changes in the thickness of the inner retina (Figure 5A), outer retina (Figure 5B), and total retina (Figure 5C) in a mouse myopia induction model. [Inner retina] After 3 weeks: p=0.0399 (n(0D)=35, n(-30D)=38), after 6 weeks: p=0.0102 (n(0D)=26, n(-30D)=34). [Outer retina] After 3 weeks: p=0.2078 (n(0D)=35, n(-30D)=38), after 6 weeks: p=0.4516 (n(0D)=26, n(-30D)=34). [Full retinal thickness] After 3 weeks: p=0.0794 (n(0D)=35, n(-30D)=38), after 6 weeks: p=0.0494 (n(0D)=26, n(-30D)=34). Graphs comparing the amplitude of the a-wave (FIG. 6A), b-wave (FIG. 6B), and ΣOP (FIG. 6C) of the ERG in a mouse myopia-induced model. Additionally, the implicit time of the OP wave is also shown (FIG. 6D). The 0D lens-treated group and the -30D lens-treated group each had an n=8 population.

[0015] As used herein, the term "pathological myopia" refers to myopia that has resulted in abnormalities in the fundus due to axial elongation. That is, pathological myopia is defined by the presence of (1) axial elongation and (2) fundus abnormalities.

[0016] [Method for Producing an Animal Model] The method for producing an animal model of the present invention comprises the step of raising an animal under myopia induction for a specific period of time.

[0017] Animals used to prepare animal models include birds (e.g., chickens, etc.) and mammals, with mammals having eye structures and genetic backgrounds similar to those of humans being preferred. Among mammals, animals from the order Rodentia (e.g., mice, rats, guinea pigs, gerbils, hamsters, etc.), Lagomorpha (e.g., rabbits (Oryctolagus cuniculus) and other leporidae), Eulipotyphla (e.g., shrews (Suncus murinus) and other shrews), Scandentia (e.g., tree shrews), or Primates (e.g., marmosets, rhesus monkeys, green monkeys, chimpanzees, etc.) are preferred, with Rodentia, Lagomorpha, and Eulipotyphla being more preferred due to their short life cycle and ease of use as animal models.

[0018] Among these, the animals used to prepare the animal model are preferably mice, rats, guinea pigs, gerbils, hamsters, rabbits, suncus, or chickens, with mice, rats, guinea pigs, gerbils, hamsters, rabbits, or suncus being more preferred because their eye structure and genetic background are similar to those of humans and animal models can be prepared in a short period of time, and mice are even more preferred because they are easily genetically modified.

[0019] Young animals are preferred because they are easier to induce myopia. Therefore, the timing for starting myopia induction is preferably before maturity, and more preferably shortly after weaning. For example, in mice, myopia induction is preferably started before 5 weeks of age, before 4 weeks of age, and more preferably before 3 weeks of age (e.g., 3 weeks of age).

[0020] As used herein, "maturity" of an animal more specifically refers to sexual maturity, a state in which maturity and reproduction are possible. By treating an animal with a minus lens until sexual maturity, the eye axis of the animal can be elongated to a degree sufficient to cause retinal abnormalities. Specific periods until maturity are 5 to 10 weeks (preferably 7 to 9 weeks, more preferably 8 to 9 weeks, and even more preferably 9 weeks) for mice and guinea pigs, 10 to 20 weeks (preferably 14 to 18 weeks, more preferably 16 to 18 weeks) for rats, 5 to 8 months (preferably 6 to 7 months) for rabbits, 20 to 50 days (preferably 30 to 40 days) for shrews, and 5 to 7 months (preferably 5 to 6 months) for chickens.

[0021] In one embodiment, the specific period (myopia induction period) is 1.5 times or more the period from myopia induction to the plateau of the decrease in refractive power. The myopia induction period is preferably 1.6 times or more, more preferably 1.7 times or more, even more preferably 1.8 times or more, particularly preferably 1.9 times or more, particularly more preferably 2 times or more, and can be 2.2 times or more, 2.4 times or more, 2.6 times or more, 2.8 times or more, or 3 times or more the period from myopia induction to the plateau of the decrease in refractive power. The longer the myopia induction period, the higher the probability of obtaining the desired animal model. Furthermore, in order to shorten the time required for production, the myopia induction period can be 10 times or less, 9 times or less, 8 times or less, 7 times or less, 6 times or less, 5 times or less, or 4 times or less the period from myopia induction to the plateau of the decrease in refractive power. The myopia induction period can be, for example, 1.6 to 10 times, 1.7 to 8 times, 1.8 to 6 times, or 2 to 4 times.

[0022] The state in which the decrease in refractive power is plateau may be a state in which the change in the refractive power (unit: diopter (D)) of the myopia-induced eye is, for example, 20% / week or less, preferably 15% / week or less, more preferably 10% / week or less, and even more preferably 5% / week or less. The change in refractive power is measured at two specific time points t after the start of myopia induction. １ and t ２ (Unit: week, t １ <t ２ ) Refractive power A １ , A２ The rate of change of refractive index can be calculated from the following formula: ２ -A １ | / {A １ × (t ２ -t １ )}×100(% / week)

[0023] Alternatively, the plateau in the reduction of refractive power can be measured by the weekly change in refractive power of the myopia-induced eye (|A ２ -A １ | / (t ２ -t １ ) may be, for example, 2D / week or less, preferably 1.5D / week or less, more preferably 1D / week or less, even more preferably 0.5D / week or less, and even more preferably 0.25D / week or less.

[0024] The refractive power is measured by a refractometer that can be used on the animal.

[0025] The period from the induction of myopia until the decrease in refractive power reaches a plateau can be determined for the same species, strain, or individual as the animal used to prepare the animal model. In one embodiment, the method of the present invention includes a step of first determining the period from the induction of myopia until the decrease in refractive power reaches a plateau in an animal of the same strain as the animal used to prepare the animal model, and then setting a specific period required for the preparation of the animal model of the present invention based on the period.

[0026] The period from the induction of myopia until the reduction in refractive power reaches a plateau can be varied appropriately depending on the animal species and strain, but may be, for example, 2 weeks or more, 2.5 weeks or more, 3 weeks or more, 3.5 weeks or more, 4 weeks or more, 4.5 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, 10 weeks or more, 3 months or more, 4 months or more, 6 months or more, 9 months or more, 1 year or more, and may be, for example, 1 year or less, 9 months or less, 6 months or less, 4 months or less, 3 months or less, 10 weeks or less, 9 weeks or less, 8 weeks or less, 7 weeks or less, 6 weeks or less, 5 weeks or less, 4.5 weeks or less, 4 weeks or less, 3.5 weeks or less, or 3 weeks or less.

[0027] In one non-limiting embodiment, the myopia induction period can be 5 weeks or more, preferably 6 weeks or more.

[0028] When mice (e.g., premature mice, such as 3-week-old mice) are used as animals, the period from the induction of myopia to the plateau of the decrease in refractive power can be, for example, 2.5 to 5 weeks, 3 to 4.5 weeks, 3 to 4 weeks, 3 to 3.5 weeks, or 3 weeks. The specific period can be, for example, 5 to 10 weeks, 6 to 9 weeks, 6 to 8 weeks, 6 to 7 weeks, or 6 weeks.

[0029] Myopia induction can be performed on one or both eyes of an animal. When performing myopia induction on one eye of an animal, the other eye is used as a control, and the progression of myopia can be determined from the differences in the measurements of refraction, axial length, and ERG between the two eyes, without being affected by individual differences.

[0030] Myopia induction can be achieved, for example, by applying a minus lens to the animal's eye or by administering a myopia-inducing agent to the animal, or by combining the application of a minus lens and the administration of a myopia-inducing agent.

[0031] As the myopia inducer, for example, endoplasmic reticulum stress inducers such as tunicamycin, thapsigargin, dithiothreitol (DTT), and A23187 can be administered to animals singly or in combination of two or more (see, for example, Patent Document 1). Among these, tunicamycin, thapsigargin, or a combination thereof is preferred as the myopia inducer. To more significantly achieve the effects of the present invention, the concentration of tunicamycin administered is preferably 0.5 μg / mL or higher, more preferably 1 μg / mL or higher, even more preferably 2 μg / mL or higher, even more preferably 5 μg / mL or higher, particularly preferably 10 μg / mL or higher, and especially preferably 20 μg / mL or higher, and can be, for example, 500 μg / mL or lower, 200 μg / mL or lower, 100 μg / mL or lower, or 50 μg / mL or lower. From the viewpoint of more significantly exhibiting the effects of the present invention, the concentration of thapsigargin to be administered is preferably 0.01 μM or more, more preferably 0.1 μM or more, even more preferably 0.2 μM or more, even more preferably 0.5 μM or more, particularly preferably 1 μM or more, and particularly preferably 2 μM or more, and can be, for example, 200 μM or less, 100 μM or less, 50 μM or less, 20 μM or less, or 10 μM or less. The number of administrations of the myopia-inducing agent can be appropriately varied depending on the type and dosage of the agent, the type and age of the animal to be administered, etc., and can be, for example, a single administration or multiple administrations, irregularly or regularly. When administered multiple times, the agent can be administered, for example, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every six months, etc.

[0032] In one embodiment, the method of the present invention comprises the step of raising a pre-adult animal with a minus lens placed in front of and spaced from the animal's eye, wherein the myopia induction is achieved by applying a minus lens to the animal.

[0033] The minus lens is preferably a colorless, transparent, spherical lens from the viewpoint of suppressing effects on vision other than myopia induction. The power of the minus lens is preferably a D (diopter) value of −15D or less, more preferably −20D or less, and even more preferably −30D or less, from the viewpoint of sufficiently inducing pathological myopia. In one embodiment, the D value of the minus lens is −30D.

[0034] The minus lens may be positioned in any manner as long as it does not physically damage the animal's eye, but is preferably positioned at a distance from the animal's eye. When the minus lens is fixed, it is preferable that it be attached to the skin in close contact with the skin so as not to compress the animal's eye or its periphery and to prevent a lens-free field of vision. It is also preferable to place the lens approximately 1 mm away from the skin to prevent hypoxia. The minus lens is positioned, for example, by attaching a lens-holding device equipped with a lens frame or the like to the animal, as shown in the Examples and FIG. 1. In one embodiment, the lens-holding device is fixed to the animal's body by screws, adhesives, or the like to prevent lens displacement. For example, as shown in the Examples, the lens may be fixed to the animal's skull using a support or the like.

[0035] The position of the eyes and the power of the minus lens suitable for efficient myopia induction change as the animal grows. Therefore, it is more preferable to further include a step of adjusting the positional relationship between the minus lens and the eyes, the power of the minus lens, or both, in accordance with the growth of the animal. More specific examples of the positional relationship between the minus lens and the eyes include the width of the lens and the angle between the eye axis and the optical axis of the lens. The frequency of adjustment is preferably at least once a week, more preferably at least once every five days, and even more preferably at least once every two to four days. The angle of the frame can be adjusted each time the lenses are cleaned and reinstalled.

[0036] From the viewpoint of facilitating the above adjustments, it is preferable that the lens mounting device further comprises an adjustment mechanism for adjusting the position of the lenses (e.g., the width or angle between the lenses, or both). Furthermore, from the viewpoint of facilitating the replacement of the minus lenses, cleaning, and measurement of the animal's eye at the midpoint of myopia induction, it is preferable that the minus lenses and the adjustment mechanism are configured to be detachable from the mounting device. Furthermore, from the viewpoint of facilitating measurement of the animal's eye, it is preferable that the mounting device is also configured to be easily detachable from the animal's body.

[0037] It is preferable that the animal be treated so that it does not touch the minus lenses while wearing them. For example, a mechanism can be provided to physically prevent the animal's legs from contacting the lenses. Examples of such mechanisms include providing a partition or the like between the animal's legs and the lenses. In one embodiment, the partition can be integrated into a wearing device that includes a lens frame, as in protector 4 in FIG. 1 . Alternatively, the animal may be fitted with a device similar to an Elizabethan collar worn by injured animals to prevent the legs from reaching the eyes or the lenses. Alternatively, the animal's legs or the like can be restrained with a restraining device or the like, but the stress on the animal may reduce the efficiency of animal model production.

[0038] From the viewpoint of minimizing any effects on the vision and health of animals other than myopia induction, it is preferable to apply methods and conditions for feeding, light / dark cycle, rearing temperature, etc. that are normally used for the animals.

[0039] The production method of the present invention preferably further comprises a step of confirming that fundus abnormalities have been induced in the animal. Fundus abnormalities include, for example, organic or functional disorders of the retina or choroid. Organic disorders can be confirmed, for example, by measuring the thickness of the retina or choroid and finding that thinning has occurred. Optical coherence tomography (OCT), which allows non-destructive testing, and spectral domain OCT (SD-OCT) in particular, are suitable for measuring the thickness of the retina or choroid. Functional disorders can be confirmed, for example, by measuring the thickness of the retina or choroid at a level of 0.5 to 10 cd·s / m ２This can be confirmed by a decrease in the amplitude of the a-wave, b-wave, or OP-wave of the ERG due to the light stimulus of the above. In particular, it is preferable that the amplitude of the b-wave, which is attenuated in pathological myopia in humans, is attenuated.

[0040] Preferably, the production method of the present invention further comprises a step of selecting individuals in which the amplitude of the a-wave, b-wave, or c-wave of the ERG of the eye in which myopia has been induced is reduced compared to that of the eye in which myopia has not been induced.

[0041] In this specification, the amplitude of each component of the ERG of the eye in which myopia has been induced is reduced, for example, from 0.5 to 10 cd·s / m ２ This refers to a state in which the amplitude of each component of the ERG obtained by the light stimulus is, for example, 90% or less, preferably 80% or less, more preferably 70% or less, and even more preferably 60% or less, compared to the amplitude of an eye in which myopia is not induced.

[0042] The animal model obtained by the production method of the present invention can be used as an animal model for pathological myopia or eye diseases associated with axial elongation.

[0043] Examples of eye diseases associated with axial elongation include, but are not limited to, complications due to pathological myopia, cataracts, glaucoma, retinal detachment, retinopathy, retinal and choroidal atrophy, macular hemorrhage, macular hole, maculopathy, macular degeneration (e.g., myopic macular degeneration), foveoschisis, choroidal neovascularization, posterior staphyloma, and optic neuropathy (e.g., myopic optic neuropathy).

[0044] The myopia-induced eye of the animal model preferably exhibits a decrease in refractive power, axial elongation, or both. Axial elongation is preferably confirmed by measuring axial length using, for example, optical coherence tomography (OCT), which allows non-destructive testing, particularly spectral-domain OCT (SD-OCT).

[0045] Preferably, the myopia-induced eyes of the animal models exhibit thinning of the choroid, retina, or both.

[0046] When the animal is a mouse, the myopia-induced eye of the model mouse produced by the production method of the present invention preferably has at least one property selected from the group consisting of the following (i) to (iii), from the viewpoint of being a model of human pathological myopia or eye diseases related to axial elongation: (i) 0.5 to 10 cd·s / m ２ (ii) The amplitude of the a-wave of the ERG obtained by a light stimulus of 0.5 to 10 cd·s / m is 80% or less, more preferably 70% or less, and even more preferably 60% or less of the value of an eye in which myopia has not been induced. ２ (iii) The amplitude of the b-wave of the ERG obtained by the light stimulus of 0.5 to 10 cd·s / m is 80% or less, more preferably 70% or less, and even more preferably 60% or less of the value of an eye in which myopia has not been induced. ２ The sum of the amplitudes of OP1 to OP4 of the ERG obtained by the light stimulus (ΣOP) is 85% or less, more preferably 75% or less, and even more preferably 65% ​​or less of the value of an eye without myopia induction.

[0047] When the animal is a mouse, the myopia-induced eye of the model mouse produced by the production method of the present invention preferably further satisfies the following (iv) or (v), and more preferably satisfies both (iv) and (v): (iv) The refractive power is -2D or less, preferably -3D or less, more preferably -4D or less, even more preferably -5D or less, and even more preferably -6D or less, compared to an eye in which myopia has not been induced. (v) The axial length is shortened by 0.01 mm or more, preferably 0.02 mm or more, and even more preferably 0.03 mm or more, compared to an eye in which myopia has not been induced. As an example, and not limited to this example, in the case of a mouse model, if the axial length is about 0.28 mm in the control group, it can be about 0.31 mm or more.

[0048] When the animal is a mouse, the myopia-induced eye of the model mouse produced by the production method of the present invention preferably further satisfies the following (vi) or (vii), and more preferably satisfies both (vi) and (vii): (vi) The choroidal thickness is thinner by 1.5 μm or more, more preferably 2 μm or more, compared to before myopia induction. (vii) The thickness of the entire retina is thinner, i.e., -8 μm or more, more preferably -10 μm or more, compared to before myopia induction.

[0049] In this specification, each of the evaluation items (i) to (vii) is measured by the method described in the Examples.

[0050] The animal model thus prepared can be used, for example, to evaluate or screen compositions for preventing or ameliorating eye diseases associated with pathological myopia or axial elongation, as described below, and can also be used for research into eye diseases associated with pathological myopia or axial elongation.

[0051] [Method for evaluating or screening a composition] The method for evaluating or screening a composition for preventing or ameliorating eye diseases associated with pathological myopia or axial elongation of the present invention (hereinafter sometimes referred to as the "method of the present invention") comprises the steps of raising an animal under myopia induction for a specific period of time, and administering a test substance to the animal, wherein the specific period is at least 1.5 times the period from the induction of myopia until the reduction in refractive power reaches a plateau, and the test substance is administered at the start of the specific period, during the specific period, or after the specific period.

[0052] The above-mentioned eye diseases associated with axial elongation, animals, the step of raising animals for a specific period under myopia induction, and specific aspects of the specific period are the same as those described in the above [Method for producing an animal model].

[0053] The test substance is not particularly limited and may be, for example, a low molecular weight compound, a polypeptide, a nucleic acid, a lipid, a carbohydrate, a high molecular weight compound, etc., and may be a single substance or a combination (mixture) of multiple substances. The test substance may be one type or two or more types. When two or more types of test substances are administered to different animals, screening can be performed based on a comparison of the evaluations between the test substances.

[0054] Screening may be performed by varying the concentration of the test substance at two or more different levels. In this manner, the concentration-dependent effect of preventing or improving eye diseases associated with pathological myopia or axial elongation can be verified.

[0055] The timing of initiating administration of the test substance can be appropriately selected depending on the purpose of use of the test substance, the type of animal, etc. In one embodiment, the test substance is administered after a specific period has elapsed under myopia induction. In another embodiment, the test substance is administered from a specific period under myopia induction. In a more specific embodiment, the test substance is administered from the time when the decrease in refractive power reaches a plateau until the end of the specific period. According to this aspect, by administering the test substance from a stage before irreversible fundus abnormalities occur, it is possible to more likely identify a test substance that is effective.

[0056] The administration period of the test substance can be appropriately selected depending on the purpose of use of the test substance, the magnitude of the effect, the type of animal, etc. The administration period of the test substance can be, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 1 month or more, 2 months or more, 3 months or more, 6 months or more, or 12 months or more, and can be, for example, 12 months or less, 6 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or 2 weeks or less.

[0057] The number of times the test substance is administered can be selected appropriately depending on the purpose of use of the test substance, the magnitude of the effect, the type of animal, etc. The test substance may be administered once, or multiple times, either irregularly or regularly.

[0058] The administration route of the test substance can be appropriately selected depending on the purpose of use of the test substance, the magnitude of the effect, the type of animal, etc. Examples of administration routes of the test substance include oral administration, local ocular administration (e.g., eye drop administration, intravitreal administration, subconjunctival administration, etc.), intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intrathecal administration, enteral administration, vaginal administration, nasal administration, and inhalation administration.

[0059] As used herein, "amelioration" of a condition, symptom, or disease refers to curing, improving, or alleviating the condition, symptom, or disease; preventing or slowing the worsening of the condition, symptom, or disease; or reversing, preventing, or slowing the progression of the condition, symptom, or disease.

[0060] In one embodiment, the method of the present invention further comprises a step of evaluating the suppression of axial elongation, fundus abnormalities, or both after the administration. In the evaluation, for example, whether axial elongation, fundus abnormalities, or both have been suppressed can be determined by comparing with pre-administration or a control. Axial elongation and fundus abnormalities can be confirmed by the method described in the above section "Method for producing an animal model."

[0061] In one embodiment, the method of the present invention further comprises a step of screening test substances based on the above evaluation, and is used for screening test substances. Screening of test substances is preferably carried out by selecting test substances that exhibit a greater degree of suppression of axial elongation, fundus abnormalities, or both.

[0062] The present invention includes the following aspects. [1] A method for producing an animal model, comprising a step of raising an animal under myopia induction for a specific period of time, wherein the specific period is at least 1.5 times the period from the induction of myopia until the reduction in refractive power reaches a plateau. [2] The method according to [1], wherein the animal is a rodent, a shrew, or a lagomorph. [3] The method according to [1] or [2], further comprising a step of confirming that a fundus abnormality has been induced in the animal. [4] The method according to any one of [1] to [3], wherein the myopia induction is performed by applying a minus lens to the animal, and comprising a step of raising the animal with the minus lens positioned in front of but spaced apart from the animal's eyes, and a step of adjusting the positional relationship between the minus lens and the eyes and / or the power of the minus lens according to the growth of the animal. [5] The method according to [4], further comprising a means for preventing contact of a part of the animal's body with the minus lens and / or the eyes during the raising. [6] The method according to any one of [1] to [3], wherein the myopia induction is performed by administering a myopia-inducing agent. [7] A myopia animal model obtained by the method according to any one of [1] to [6]. [8] A method for evaluating or screening a composition for preventing or ameliorating an eye disease associated with pathological myopia or axial elongation, comprising the steps of raising an animal under myopia induction for a specific period of time, and administering a test substance to the animal, wherein the specific period is at least 1.5 times the period from the induction of myopia until the decrease in refractive power reaches a plateau, and the test substance is administered at the start of the specific period, during the specific period, or after the specific period. [9] The method according to [8], wherein the test substance is administered after the decrease in refractive power reaches a plateau after the induction of myopia.

[0063] Next, the present invention will be specifically explained with reference to examples and test examples, but the present invention is not limited to the following examples and test examples.

[0064] In the figures herein, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0065] Test Example 1: Preparation and Evaluation of a Mouse Myopia Induction Model (Preparation Method) When a mouse is fitted with a minus lens, the eye axis elongates to the position where an image is formed when wearing the minus lens, i.e., to the state where the image is clearly visible when wearing the minus lens. As a result, the eye axis elongates, and an eye condition similar to that of axial myopia can be created.

[0066] Here, pathological myopia was induced in 3-week-old C57BL / 6J mice shortly after weaning using the following method. First, the mice were anesthetized with a triple-anesthesia mixture consisting of Domitor (Nippon Zenyaku Kogyo Co., Ltd.), Betolfar (Meiji Seika Pharma Co., Ltd.), and midazolam (Sandoz Corporation), and the skull was exposed with scissors. A support post 1 was erected on the skull and fixed with dental cement (Super-Bond, Sun Medical Co., Ltd.). The support post was provided with a screw thread so that the adjustment device described below could be fixed with a nut.

[0067] The mice were divided into two groups: a myopia induction group (sometimes referred to as the "-30D" group) and a control group (sometimes referred to as the "0D" group). To induce myopia, minus lenses of -30 diopters (Rainbow Contact, Rainbow Optical Laboratory Co., Ltd.)2 were worn on both eyes for 6 weeks using the wearing device shown in Figure 1. Similarly, 0D lenses were worn on the control group. A protector 4 protruding laterally was attached to the frame at the bottom of the lens to prevent the mouse from scratching the lens with its forepaws or the like when wearing the lens. The protector 4 prevented the mouse from touching the lens, preventing it from being scratched.

[0068] An adjustment device 5 was attached to the frame above the lens, allowing the width and angle of the attached lens to be adjusted as the mouse grew. The adjustment device 5 was bent in an L-shape, with the lens attached to one end and an elongated hole 6 on the other end so that it could be attached to a support 1 erected on the head. By passing the elongated hole 6 through the support 1 and screwing it in with a nut 7, the lens could be fixed in place by adhering it to the skin without compressing the edges of both eyes of the mouse. It is preferable to leave a gap of about 1 mm when it is in close contact with the skin.

[0069] The adjustment mechanism, consisting of a support 1, a nut 7, and an adjustment tool 5, allowed the lens width and angle to be adjusted as the mouse grew, ensuring that the lens was positioned properly within the mouse's eye. During breeding, the lens was removed from the attachment and cleaned every three days, and the lens position was adjusted to ensure the appropriate angle and width relative to the mouse's eye axis. Because the lens can be easily removed from this myopia induction model, measurements of the mouse's axial length and refractive index can be easily performed, allowing for more detailed analysis of myopia progression. Since long-term myopia induction is required for the induction of pathological myopia, lens replacement may be necessary depending on the individual, strain, or species used. However, the attachment used in this method allows for easy lens replacement, making it suitable for creating an animal model using myopia induction.

[0070] (Evaluation Method) Before wearing the lenses, and after 3 and 6 weeks of wearing them, the refraction, axial length, thickness of the choroid and retina (inner layer, outer layer, and total layer) of the mouse eyes were measured by the following methods (1) and (2), and the difference from before wearing was calculated. The results were analyzed by t-test. (1) Refraction was measured using a refractometer (Infrared photorefractor for mice, manufactured by Professor Schaeffel of the University of Tübingen). (2) Axial length, and choroidal and retinal thickness were measured by whole-eye imaging using SD-OCT (Spectral-Domain OCT, Envisu R4310, Bioptigen Inc.). A circular region with a radius of 0.5 mm from the optic disc was selected, and the posterior choroidal region was measured and analyzed using ImageJ. The area was then divided by the circumference to determine the average choroidal thickness. The retinal thickness was measured using the same original image as for the choroidal thickness. The retina was divided into inner and outer layers at the outer plexiform layer (OPL) as the boundary, and the inner and outer posterior retinal regions were measured and analyzed using ImageJ. The area was then divided by the circumference to determine the average retinal thickness.

[0071] Furthermore, electroretinogram (ERG) evaluation was performed on the eyes of mice with myopia induction for 3 and 6 weeks (evaluation was performed 3 weeks after the end of myopia induction). ２ ERGs were measured in both eyes by applying a light stimulus of 1000 Hz. After dark adaptation for more than 8 hours, tropicamide and phenylephrine hydrochloride were administered, and both eyes were dilated before applying the light stimulus. The active electrode was recorded using a contact electrode. The reference electrode was placed in the mouth. A clip electrode on the mouse's tail was used as ground. The low-pass filter of the amplifier was set to 30 Hz. Statistical analysis was performed using an unpaired t-test.

[0072] (Results) As shown in Figure 2, treatment with -30D lenses significantly reduced the refraction of the mice three weeks after myopia induction, and the change then plateaued, but myopia was maintained even after six weeks. Furthermore, as shown in Figure 3, treatment with -30D lenses significantly reduced axial length after three weeks compared to treatment with 0D lenses, and this difference further increased until six weeks later. This demonstrates that axial elongation continues even six weeks after myopia induction.

[0073] As shown in Figure 4, the choroid thinned progressively from 3 to 6 weeks after treatment with the -30D lens, whereas the choroid thickened with growth after treatment with the -0D lens.

[0074] As shown in Figure 5, the retina showed progressive thinning of the inner and entire layers from 3 to 6 weeks after treatment with the -30D lens compared to treatment with the -0D lens. The inner retinal layer was significantly thinner after 3 weeks of treatment with the -30D lens than with the -0D lens, and damage was observed at an early stage. Furthermore, as a result of the progressive thinning of the inner retinal layer, significant thinning of the entire retinal layer was also observed after 6 weeks. No significant changes were observed in the outer retinal layer.

[0075] As shown in Figure 6, mice treated with -30D lenses for 6 weeks showed significant attenuation in all ERG components, including a-wave amplitude (A), b-wave amplitude (B), and ΣOP (sum of the amplitudes of OP1 to OP4) (C), compared to mice treated with 0D lenses. It is known that b-wave amplitude is attenuated in highly myopic human eyes, and a similar attenuation was confirmed in myopia-induced mice. Meanwhile, it is known that the use of minus lenses with the same refractive index does not affect ERG after 3 weeks of myopia induction (Non-Patent Document 4).

[0076] Therefore, it was confirmed for the first time that myopia induction using -30D lenses for 6 weeks induces functional damage in the retina of mouse eyes. As described above, considering that organic damage progressed from the inner retinal layer to the entire retinal layer and choroid, these organic and functional damages in the fundus were brought about by the elongation of the axial length due to myopia induction, and the resulting mice exhibited fundus abnormalities caused by pathological myopia.

[0077] The changes in ocular parameters after 6 weeks of myopia induction were as follows:

[0078] Previously reported myopia induction models have not shown the organic and functional changes in the retina in addition to the changes in refraction and axial length reported in humans. In contrast, the myopia induction model created in this example possesses all the characteristics of human pathological myopia, demonstrating its potential as an excellent model. This is likely due to the fact that the lens position can be finely adjusted as the animal grows during the creation of this myopia induction model, and the inclusion of a lens protector prevents scratches on the lens, allowing for more pronounced induction of pathological myopia, making it possible to induce myopia over a long period of six weeks.

[0079] 1...Support, 2...Minus lens, 3...Frame, 4...Protector, 5...Adjustment tool, 6...Slot, 7...Nut

Claims

1. A method for producing an animal model, comprising a step of raising an animal under myopia induction for a specific period of time, wherein the specific period is at least 1.5 times the period from the induction of myopia until the decrease in refractive power reaches a plateau.

2. The method of claim 1, wherein the animal is a rodent, shrew, or lagomorph.

3. The method of claim 1, further comprising the step of confirming that fundus abnormalities have been induced in the animal.

4. The method according to claim 1, wherein the myopia induction is carried out by applying a minus lens to the animal, and the method comprises the steps of: raising the animal with the minus lens placed in front of the animal's eyes at a distance from the eyes; and adjusting the positional relationship between the minus lens and the eyes and / or the power of the minus lens according to the growth of the animal.

5. The method according to claim 4, further comprising applying a means to prevent any part of the animal's body from coming into contact with minus lenses and / or eyes during rearing.

6. The method of claim 1, wherein the myopia induction is performed by administering a myopia-inducing agent.

7. A method for evaluating or screening a composition for preventing or ameliorating an eye disease associated with pathological myopia or axial elongation, comprising the steps of: raising an animal under myopia induction for a specific period of time; and administering a test substance to the animal, wherein the specific period is at least 1.5 times the period from the induction of myopia until the reduction in refractive power reaches a plateau, and the test substance is administered at the start of, during, or after the specific period.

8. The method of claim 7, wherein the test substance is administered after the reduction in refractive power has plateaued following the induction of myopia.

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