Application of cGAMP in establishing animal models of retinal vascular diseases

Abstract

The present application relates to the field of biomedical technology, and specifically to an application of cGAMP in constructing an animal model of retinal vascular disease, a model construction method and related animal models. The present application successfully constructs an animal model of retinal vascular disease for the first time by a single intravitreal injection of cGAMP, which has the advantages of modeling time period, simple method and reliable results. Compared with previous technologies, it also overcomes the problem of being unable to simulate a mature retina.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an application of cGAMP in constructing an animal model of retinal vascular disease, an animal model and a method for constructing the model. Background Art

[0002] Retinal vascular disease (RVD) is a common type of fundus vascular lesion and one of the serious blinding diseases in ophthalmology. Retinal vascular exudation, hemorrhage, and retinal vasculitis caused by retinal vascular disease seriously affect the patient's vision. Currently, the commonly used treatments for retinal vascular disease include laser treatment, anti-angiogenic drug treatment, and surgical treatment. However, laser treatment may cause complications such as vision loss and color vision abnormalities. Anti-angiogenic drugs require frequent intraocular injections and the treatment effect varies greatly from person to person. Therefore, there is currently a lack of relatively effective treatment measures for retinal vascular diseases. Therefore, finding new targets for the treatment of retinal vascular diseases is of great significance in clinical medical research.

[0003] Animal models provide a convenient and effective means to find new targets for the treatment of retinal vascular diseases. Currently, there are several relatively mature animal models for simulating retinal vascular diseases: Streptozotocin (STZ)-induced diabetic mouse model, hyperoxia-induced retinopathy of prematurity (ROP), etc., but these methods generally have the problem of unclear targets. Moreover, the modeling cycle of the STZ-induced diabetic mouse model is relatively long (8-10 weeks); and the ROP model is modeled on the seventh day after the mouse is born, at which time the retina is not fully developed, so it is impossible to simulate a mature retina. In this application, cGAMP directly binds to and activates the downstream STING signal, and the target is clear; at the same time, the modeling time is shortened, and it can be performed in newborn and adult mice.

[0004] In the classical cGAS-STING signal transduction process, double-stranded DNA abnormally exposed in the cytoplasm can be recognized by cGAS, catalyzing GTP and ATP to form the second messenger 2'-3'-cGAMP (cGAMP for short). cGAMP then binds to the receptor protein dimer located in the endoplasmic reticulum membrane, namely STING (stimulator of interferon genes), thereby initiating conformational changes in the STING dimer and thus activating it. As the STING protein is transported along the endoplasmic reticulum-endoplasmic reticulum-Golgi intermediate-Golgi body, the carboxyl terminus of STING recruits and activates TBK1 (TANK-binding kinase1, TANK binding kinase), phosphorylating IRF3 (interferon regulatory factor 3, interferon regulatory factor 3), and the phosphorylated IRF3 dimerizes and enters the cell nucleus. STING also activates the kinase Ikk, phosphorylating the IκB family of inhibitors of NF-κB (nuclear factor-kappa B). The phosphorylated IκB protein is degraded through the ubiquitin-proteasome pathway. At this time, NF-κB enters the nucleus and works with interferon regulatory factors such as IRF3 to induce the expression of type I interferon and inflammatory cytokines such as TNFβ, IL-1β and IL-6. cGAS-STING is a key innate immune signaling pathway and a natural line of defense in response to tissue damage and pathogen invasion. However, in the retina, abnormally activated cGAS-STING signals can cause a large amount of inflammatory cytokines to be released, infiltrate immune cells, and then cause neuroinflammation. It has been confirmed that abnormal activation of the cGAS-STING signaling pathway is associated with a variety of retinal diseases such as age-related macular degeneration and diabetic retinopathy. However, the prior art does not know that a mouse model of retinal vascular disease can be constructed based on the above pathway, and there is no inspiration on how to construct the model and the effect after construction.

[0005] In view of this, this application is filed. SUMMARY OF THE INVENTION

[0006] To solve the above technical problems, the present application found that after intraocular injection of cGAMP, the retina showed obvious blood exudation, new blood vessels, lymphocyte adhesion to blood vessels, and strong vasculitis, which are similar to the occurrence and development process of retinal vascular diseases. It was confirmed that an animal model of retinal vascular diseases was successfully constructed, and the model can be used as a tool for studying retinal vascular diseases. In addition, the model construction method not only shortens the modeling time, but also can be carried out in adult animals. Therefore, the present application includes at least the following purposes:

[0007] The first purpose of this application is to provide a method for constructing an animal model of retinal vascular disease;

[0008] The second purpose of this application is to provide an animal model of retinal vascular disease;

[0009] The third objective of the present application is to provide the application of cGAMP in constructing an animal model of retinal vascular disease.

[0010] To achieve the above purpose, this application specifically adopts the following technical solutions:

[0011] According to one aspect of the present specification, the present application first provides a method for constructing an animal model of retinal vascular disease, the construction method comprising the step of administering cGAMP to the animal.

[0012] In some embodiments, the in vivo administration is in vivo injection.

[0013] In some embodiments, the in vivo injection is an intraocular injection.

[0014] In some embodiments, the intraocular injection is an intraocular vitreous cavity injection.

[0015] In some embodiments, the intraocular vitreous cavity injection comprises: after the animal is anesthetized, the animal's pupil is dilated, and the cGAMP solution is injected into the intraocular vitreous cavity along the corneal limbus opening.

[0016] In other embodiments, the intraocular vitreous cavity injection comprises: after the animal is anesthetized, the animal's pupil is dilated, an opening is made at the corneal limbus, and the cGAMP solution is injected obliquely into the intraocular vitreous cavity along the corneal limbus opening.

[0017] In other more specific embodiments, the intraocular vitreous cavity injection includes: after the animal is anesthetized, using tropicamide phenylephrine to dilate the pupil and using gel to lubricate the cornea; using a needle to poke a small hole at the corneal edge, preferably without bleeding, using a syringe to slowly insert the needle at an angle of about 45 degrees along the corneal edge opening, preferably without puncturing the retina or scratching the depth of the lens; after slowly pushing the drug in, the needle stays in the vitreous cavity for about 5 seconds.

[0018] In some embodiments, the animal is a mouse; preferably a mouse or a rat; more preferably a newborn mouse or an adult mouse;

[0019] In some embodiments, the adult mouse is, for example, a 5-8 week old adult mouse.

[0020] In some embodiments, the concentration of the cGAMP solution may be 1-25 mM;

[0021] In some embodiments, the solvent of the solution may include water, PBS or physiological saline.

[0022] According to another aspect of the present specification, the present application further provides the use of the animal model in the evaluation or screening of drugs for the treatment of retinal vascular diseases; preferably, the drug comprises a STING inhibitor drug.

[0023] According to another aspect of the specification, the present application also provides an animal model of retinal vascular disease, which is constructed by any of the above methods.

[0024] According to one aspect of the present specification, the retina of the animal model has the characteristics associated with the occurrence of retinal vascular diseases, such as obvious blood exudation, neovascularization and severe vasculitis.

[0025] In some embodiments, the animal is a mouse; preferably a mouse or a rat; more preferably a newborn mouse or an adult mouse.

[0026] In some embodiments, the adult mouse is, for example, a 5-8 week old adult mouse.

[0027] According to one aspect of the present specification, the present application further provides the use of the animal model in screening drugs for treating retinal vascular diseases; preferably, the drugs include STING inhibitor drugs.

[0028] According to one aspect of the specification, the present application also relates to the use of cGAMP in constructing an animal model of retinal vascular disease, and the construction method includes the step of administering cGAMP to the animal.

[0029] In some embodiments, the in vivo administration is in vivo injection.

[0030] In some embodiments, the in vivo injection is an intraocular injection.

[0031] In some embodiments, the intraocular injection is an intraocular vitreous cavity injection.

[0032] In some embodiments, the intravitreal injection is: after the animal is anesthetized, the animal's pupil is dilated, and the cGAMP solution is injected into the intraocular vitreous cavity along the opening of the corneal limbus.

[0033] In some embodiments, the intravitreal injection is as follows: after the animal is anesthetized, the animal's pupil is dilated, an opening is made at the limbus, and the cGAMP solution is injected obliquely into the intraocular vitreous cavity along the limbus opening.

[0034] In some embodiments, the animal is a mouse, preferably a mouse or a rat, more preferably a newborn mouse or an adult mouse; in some embodiments, the adult mouse is, for example, a 5-8 week old adult mouse.

[0035] In some embodiments, the concentration of the cGAMP solution may be 1-25 mM;

[0036] In some embodiments, the solvent of the solution may include water, PBS or saline.

[0037] Compared with the prior art, this application has at least the following technical advantages:

[0038] 1) cGAMP is a key activating molecule of the cGAS-STING immune signaling pathway. Previously, no molecule targeting cGAS-STING activation has been directly used to establish a mouse model of retinal vascular disease. This application first proposed a method for establishing a mouse model of retinal vascular disease based on cGAMP, and for the first time proposed to achieve model construction by intravitreal injection of cGAMP solution.

[0039] 2) In the present application, through a single intravitreal injection, obvious retinal blood cell exudation, neovascularization and severe vasculitis can be observed 3 days later, which greatly shortens the modeling time.

[0040] 3) The intraocular injection of the present application can be performed in both newborn and adult mice, and can be used as a tool to study developmental and mature retinal vascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 , schematic diagram of intraocular injection;

[0043] Figure 2, retinal flat mount white light images, showing the fresh ex vivo retinal hemorrhage of mice in the control group and experimental group: A is a retinal flat mount white light image showing the fresh ex vivo retinal hemorrhage of mice in the control group (PBS) and experimental group (cGAMP, 10, 25mM, prepared in PBS); B is a retinal flat mount white light image showing the control group (sterile water, 1μl) and experimental group (cGAMP, 10mM, 1μl, prepared in sterile water); C is a retinal flat mount white light image showing the fresh ex vivo retinal hemorrhage of mice in the control group (normal saline (0.9% sodium chloride aqueous solution), 1μl) and experimental group (cGAMP, 10mM, 1μl, prepared in normal saline).

[0044] Figure 3 , Immunofluorescence staining of vascular markers IB4 and TER119 in retinal flat mounts showed vascular leakage at different concentrations (0.1, 1, 10 mM).

[0045] Figure 4 , control group (PBS) and experimental group (cGAMP, 10mM, prepared in PBS) of mice retina HE staining results; Scale bar: the upper two figures are 500μm, the lower four figures are 50μm; GCL, ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, RPE: retinal pigment epithelium.

[0046] Figure 5 , OCT results show the living retinal structure of mice in the control group (PBS) and experimental group (cGAMP, 10mM, prepared in PBS). The statistical graphs are the quantification results of the central retinal thickness of mice in the control group and experimental group at 0.375mm and 0.7mm; ns indicates no statistical significance, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

[0047] Figure 6 Fundus photography (upper panel) and fluorescence angiography (lower panel) of living retinal vessels of mice in the control group (PBS) and experimental group (cGAMP, 10 mM, prepared in PBS).

[0048] Figure 7 , the results of immunofluorescence staining of retinal vascular marker IB4, showing the retinal blood vessels of mice in the control group (PBS) and experimental group (cGAMP, 10mM, prepared in PBS). The statistical graph is the quantification result of the retinal blood vessel coverage area of ​​the two groups of mice.

[0049] Figure 8, FITC-con A perfusion (green) showed the vascular leukocyte adhesion of the control and experimental group mice, and immunofluorescence staining of the leukocyte marker CD45 (red) showed the retinal vascular leukocyte infiltration and exudation of the two groups of mice.

[0050] Fig. 9 , RNA sequencing analysis shows the changes in gene expression in mice in the control group (PBS) and the experimental group (cGAMP, 10mM, prepared in PBS). Among them, A is the number of genes that are significantly upregulated and downregulated overall and genes related to retinal vascular diseases; B is GO cluster analysis showing upregulated and downregulated signal pathways. C is Gene set enrichment analysis (GSEA) analysis showing the enrichment of upregulated genes in cell adhesion and antigen presentation systems.

[0051] Fig.10 , retinal fundus vascular fluorescence angiography, showing the living retinal vessels of wild-type and STING knockout mice, with a cGAMP injection dose of 10mM.

[0052] Fig.11 , retinal flat mount white light images, showing fresh ex vivo retinal hemorrhages in wild-type and STING knockout mice. The statistical graph is the quantification of retinal hemorrhages in the two groups of mice, and the cGAMP injection volume was 10mM.

[0053] Fig.12 , Immunofluorescence staining of retinal flat mount vascular marker IB4, showing retinal blood vessels of wild-type and STING knockout mice. The statistical graph is the quantification of retinal blood vessel coverage area of ​​the two groups of mice, and the cGAMP injection volume is 10mM.

[0054] Fig.13 , FITC-con A perfusion (green) shows the vascular leukocyte adhesion of wild-type and STING knockout mice, and immunofluorescence staining of the leukocyte marker CD45 (red) shows the retinal vascular leukocyte infiltration and exudation of the two groups of mice. The cGAMP injection volume is 10mM. DETAILED DESCRIPTION OF THE INVENTION

[0055] Although the present application can be implemented in many different forms, what is disclosed here is its specific illustrative implementation that verifies the principle of the present application. It should be emphasized that the present application is not limited to the specific implementations illustrated. In addition, any section headings used herein are only used for organizational purposes and are not to be interpreted as limiting the subject matter described.

[0056] The following terms or definitions are provided only to help understand the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0057] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0058] The terms "comprise", "include", "have", "contain" or "involve" are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0059] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0060] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0061] The term "and / or" is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0062] The terms "such as" and "ie" are used merely as examples and are not intended to be limiting, and should not be construed as referring only to those items explicitly listed in the specification.

[0063] 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more of the above values. Also included are any larger numbers or fractions therebetween.

[0064] Conversely, the term "no more than" includes every value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

[0065] The terms "plurality", "at least two", "two or more", "at least a second", etc., should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also includes any larger number or fraction therebetween.

[0066] The terms "approximately" and "substantially" represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the characteristic in question. The term usually represents ±10%, preferably ±5%, of the indicated value.

[0067] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers).

[0068] Various aspects of the present disclosure are further described in detail:

[0069] The method for constructing a retinal vascular disease mouse model of the present application comprises the step of administering cGAMP to the animal.

[0070] In some embodiments, the in vivo administration is in vivo injection.

[0071] In some embodiments, the in vivo injection is an intraocular injection.

[0072] In some embodiments, the intraocular injection is an intraocular vitreous cavity injection.

[0073] The "cGAMP" in this application is 2'-3'-cyclic GMP-AMP, and its structural formula is:

[0074]

[0075] Prior to this application, although it was known in the prior art that cGAMP was mechanistically related to retinal vascular diseases, it was not known that cGAMP could be directly used for intraocular injection to achieve the construction of a disease mouse model. This application discovered for the first time that a disease mouse model could be successfully constructed based on intraocular injection of cGAMP, and not only proposed a new application of cGAMP, but also a new injection method. Therefore, any method based on "intravitreal injection of cGAMP" falls within the scope of protection of this application, and those skilled in the art can actually adjust the parameters during the injection process according to specific needs.

[0076] In some specific embodiments, the intraocular vitreous cavity injection comprises: after the animal is anesthetized, the animal's pupil is dilated, and the cGAMP solution is injected into the intraocular vitreous cavity along the corneal limbus opening.

[0077] In other specific embodiments, the intraocular vitreous cavity injection comprises: after the animal is anesthetized, the animal's pupil is dilated, an opening is made at the corneal limbus, and the cGAMP solution is injected obliquely into the intraocular vitreous cavity along the corneal limbus opening.

[0078] In some further specific embodiments, the intraocular vitreous cavity injection includes: after the animal is anesthetized, using tropicamide phenylephrine to dilate the animal's pupil and using gel to lubricate the cornea; using a needle to poke a small hole at the corneal edge, preferably without bleeding, using a syringe to slowly insert the needle at an angle of about 45 degrees along the corneal edge opening, preferably without puncturing the retina and scratching the depth of the lens; after slowly pushing the drug in, the needle stays in the vitreous cavity for about 5 seconds.

[0079] In some embodiments, the animal is a mouse. In some preferred embodiments, the mouse is a mouse or a rat. It is known in the art that there is sufficient fundus photography and histological evidence of living animals in the prior art to show that the vascular structure and function in the eyes of mice and rats show a high degree of similarity. (For example, see the document Transnational Vision Science and Technology, 2022, 1; 11 (8): 11. doi: 10.1167 / tvst.11.8.11.), so it can be understood that the method of the present application is also applicable to rats.

[0080] In some embodiments, the mouse or rat is a newborn mouse or an adult mouse;

[0081] In some embodiments, the adult mouse is a 5-8 week old adult mouse.

[0082] In some embodiments, the concentration of the cGAMP solution may be 1-25 mM;

[0083] In some embodiments, the solvent of the solution may include water, PBS or physiological saline, etc. It can be understood that these conventional solvents will neither excessively affect the physiological activity of the mouse body nor excessively change the cGAMP activity, and therefore are suitable for this application.

[0084] The retinal vascular disease animal model of the present application is a mouse with retinal vascular disease-related characteristics, which can be constructed by any of the above-mentioned methods.

[0085] According to one aspect of the present specification, the characteristics related to retinal vascular diseases include: having features related to the occurrence of retinal vascular diseases, such as obvious blood exudation, new blood vessels and severe vasculitis.

[0086] Purpose of this application:

[0087] On the one hand, the application is the basic application of the animal model of the present application. It can be understood that the animal model described in the present application can be used to evaluate or screen drugs for treating retinal vascular diseases; the types of drugs are not limited, and as a disease model, it can be used for the evaluation and screening of various related drugs;

[0088] In some embodiments, the drug may be a STING inhibitor drug, such as demonstrated in Example 5 of the present application.

[0089] Another application is the use of cGAMP in constructing an animal model of retinal vascular disease, wherein the construction method comprises the step of administering cGAMP to the animal.

[0090] In some embodiments, the in vivo administration is in vivo injection.

[0091] In some embodiments, the in vivo injection is an intraocular injection.

[0092] In some embodiments, the intraocular injection is an intraocular vitreous cavity injection.

[0093] In some specific embodiments, the intraocular vitreous cavity injection comprises: after the animal is anesthetized, the animal's pupil is dilated, and the cGAMP solution is injected into the intraocular vitreous cavity along the corneal limbus opening.

[0094] In other specific embodiments, the intraocular vitreous cavity injection comprises: after the animal is anesthetized, the animal's pupil is dilated, an opening is made at the corneal limbus, and the cGAMP solution is injected obliquely into the intraocular vitreous cavity along the corneal limbus opening.

[0095] In some further specific embodiments, the intraocular vitreous cavity injection includes: after the animal is anesthetized, using tropicamide phenylephrine to dilate the animal's pupil and using gel to lubricate the cornea; using a needle to poke a small hole at the corneal edge, preferably without bleeding, using a syringe to slowly insert the needle at an angle of about 45 degrees along the corneal edge opening, preferably without puncturing the retina and scratching the depth of the lens; after slowly pushing the drug in, the needle stays in the vitreous cavity for about 5 seconds.

[0096] In some embodiments, the animal is a mouse, preferably a mouse or a rat;

[0097] In some specific embodiments, the mouse is a newborn mouse or an adult mouse;

[0098] In some more specific embodiments, the adult mouse is, for example, a 5-8 week old adult mouse.

[0099] In some embodiments, the concentration of the cGAMP solution may be 1-25 mM;

[0100] In some embodiments, the solvent of the solution may include water, PBS or physiological saline, etc. It can be understood that these conventional solvents will neither excessively affect the physiological activity of the mouse body nor excessively change the cGAMP activity, and therefore are suitable for the present application.

[0101] The present application is further described by the accompanying drawings and the following examples. The accompanying drawings and examples are only intended to illustrate specific embodiments of the present application and should not be construed as limiting the scope of the present application in any way.

[0102] Example 1: Preparation of disease mouse model by intraocular administration of cGAMP

[0103] In this example, mice were injected with a single intravitreal injection of cGAMP (purchased from selleck, item number: S7904). The solvents for preparing cGAMP were 1x sterile phosphate buffered saline (PBS), sterile water or normal saline. The concentration of cGAMP was set to a gradient of 0.1 mM, 1 mM, 10 mM or 25 mM. The mice were preferably 5-8 week old c57BL / 6J mice (Experimental Animal Center of Sun Yat-sen University, Guangzhou).

[0104] The experimental group and the control group were set up for drug administration:

[0105] 1) Experimental group: intravitreal injection of cGAMP (solvent: 1xPBS);

[0106] 2) Control group: an equal volume of 1xPBS was injected into the vitreous cavity.

[0107] The specific steps are as follows:

[0108] After the mice were anesthetized with 1% sodium pentobarbital, tropicamide phenylephrine eye drops were first used to dilate the pupils of the mice, and the cornea was lubricated with gel; for intravitreal injection, an insulin needle was first used to poke a small hole at the corneal margin to avoid bleeding, and then a micro-syringe was used to slowly insert the needle along the corneal margin opening at an angle of about 45 degrees. It should not be too deep to avoid puncturing the retina and scratching the lens (see for details). Figure 1 Slowly push the drug in, and after injection, the needle stays in the vitreous cavity for about 5 seconds to prevent the drug from spilling out, and then slowly withdraw the needle.

[0109] After 3 days, the mice were euthanized, the eyeballs were removed, the retinas were dissociated and spread, and images were taken under a microscope. For immunostaining, the eyeballs were removed after the mice were euthanized and fixed in 4% paraformaldehyde (PFA) for 10 minutes. After fixation, the eyeballs were dissected, and the neural retina was separated. After the neural retina was separated, it was continued to be fixed in 4% paraformaldehyde (PFA) for 5 minutes. After fixation, it was washed 3 times with PBS to remove excess PFA. The retina was permeabilized with 0.3% PBST (PBS containing 0.3% Triton X-100) at room temperature for 20 minutes and blocked with 2.5% BSA (prepared in PBST) at room temperature for 30 minutes. The retina was then incubated with the primary antibody at 4°C overnight and incubated with the secondary antibody at room temperature for 2 hours the next day. After washing with PBS, the slides were sealed with antifade sealing agent. Images were acquired using a LSM980 confocal microscope (Carl Zeiss, Germany).

[0110] The results are as follows Figure 2 As shown: Figure 2 A is a retinal flat-mount white light image showing the fresh ex vivo retinal hemorrhage of mice in the control group (PBS) and the experimental group (cGAMP, 10, 25mM, prepared in PBS). Compared with the intravitreal injection of PBS, the retinas of the corresponding mice injected with 105mM or 25mM cGAMP have obvious hemorrhage spots; 2B is a retinal flat-mount white light image showing the control group (sterile water, 1μl) and the experimental group (cGAMP, 10mM, 1μl, prepared in sterile water); 2C is a retinal flat-mount white light image showing the fresh ex vivo retinal hemorrhage of mice in the control group (normal saline, 1μl) and the experimental group (cGAMP, 10mM, 1μl, prepared in normal saline), with 5 mice in each group. It can be seen that compared with the normal control group mice such as intravitreal injection of PBS, sterile water and normal saline, the retinas of the corresponding mice injected with cGAMP have obvious hemorrhage spots.

[0111] In order to further test the minimum concentration of cGAMP, this example also injected different concentrations (0.1, 1, 10 mM) of cGAMP into the vitreous cavity of mice to observe the leakage of blood vessels in mice. The results of retinal flat mount fluorescence staining showed that ( Figure 3 ), slight red blood cell leakage occurred in the retinal blood vessels of mice injected with 1mM cGAMP, and obvious red blood cell leakage occurred in the retinal blood vessels of mice injected with 10mM cGAMP, which shows that the purpose of this application can be achieved on the basis of greater than 1mM cGAMP. Based on the bleeding results, this application selected 10mM as the concentration basis for subsequent experiments.

[0112] Example 2: Intravitreal injection of cGAMP induces pathological changes such as retinal edema, exudation and neovascularization in mice

[0113] Based on the method similar to Example 1, intravitreal injection was performed on mice, and a control group and an experimental group were designed, with 3-5 mice in each group.

[0114] 1) Experimental group: intravitreal injection of cGAMP (solvent: 1xPBS, sterile water or normal saline);

[0115] 2) Control group: Intravitreal injection of an equal volume of solvent: 1xPBS, sterile water or normal saline.

[0116] The following analyses were performed on mice after intravitreal injection of cGAMP:

[0117] HE staining analysis of eyeball tissue: Mice were injected into the vitreous cavity, and samples were collected 3 days later for HE staining to analyze the retinal structure.

[0118] Retinal morphology analysis: OCT (Optical coherence tomography) is a non-invasive method that can perform high-resolution tomographic imaging of the retina in vivo; its non-invasive characteristics provide good technical support for the dynamic recording of the diagnosis of retinal lesions and the evaluation of drug efficacy. On the third day after intravitreal injection of cGAMP, mice were anesthetized with 1% sodium pentobarbital (70μl / 10g) and mydriasis was performed with tropicamide. Small animal OCT (Heidelberg Engineering) was used to observe and compare the retinal morphology of each group of mice in vivo.

[0119] Fundus photography: On the third day after intravitreal injection of cGAMP, mice were anesthetized with 1% sodium pentobarbital (70 μl / 10 g), and fundus images were obtained using a Micron IV retinal imaging system (Phoenix Research Laboratories, Pleasanton, CA) after mydriasis with tropicamide. Mice were injected with 2% sodium fluorescein solution (Alcon laboratories, TX, USA) (5 μl / g), and fluorescein angiography images were immediately recorded.

[0120] Retinal flat mount and immunofluorescence staining: After euthanasia of mice, the eyeballs were removed and fixed in 4% paraformaldehyde (PFA) for 10 minutes. After fixation, the eyeballs were dissected and the neural retina was isolated. After the neural retina was isolated, it was fixed in 4% paraformaldehyde (PFA) for 5 minutes. After fixation, it was washed three times with PBS to remove excess PFA. For immunostaining, the retina was permeabilized with 0.3% PBST (PBS containing 0.3% Triton X-100) at room temperature for 20 minutes and blocked with 2.5% BSA (prepared in PBST) at room temperature for 30 minutes. The retina was then incubated with the primary antibody at 4°C overnight and incubated with the secondary antibody at room temperature for 2 hours the next day. After washing with PBS, the slides were mounted with antifade mounting medium. Images were acquired using a LSM980 confocal microscope (CarlZeiss, Germany).

[0121] Data statistics: Data is presented as average value + Standard variance, data analysis was performed using the student t test method. *: P < 0.05, **: P < 0.001, ***: P < 0.0001.

[0122] Experimental results:

[0123] Three days after intravitreal injection of cGAMP or PBS, the retinal tissues were collected and analyzed by HE staining. Figure 4 As shown, the retinal structures of all layers of normal control mice injected with PBS into the vitreous cavity were intact, while the cGAMP-injected mice showed overall retinal thickening and edema, enlarged retinal blood vessel lumen, and congestion in some blood vessels.

[0124] OCT results Figure 5 As shown, OCT can be used to perform high-resolution observation of the various layers of retinal tissue in living mice. Compared with the normal control group mice injected with PBS into the vitreous cavity, the mice injected with cGAMP showed overall thickening and edema of the retina, and a large amount of highly reflective exudates in the vitreous cavity.

[0125] Fundus photography and fluorescein angiography are used to observe the structure of fundus blood vessels. Figure 6 As shown: Compared with the normal control group mice injected with PBS into the vitreous cavity, the retinal blood vessels of the cGAMP-injected mice became larger and fluorescent leakage occurred.

[0126] Results of immunofluorescence staining of retinal vascular marker IB4 ( Figure 7 ) Obvious leakage and new blood vessel formation can be seen in the retina of mice injected with cGAMP.

[0127] Example 3: Increase in perivascular leukocytosis after intravitreal injection of cGAMP

[0128] Retinal vascular perfusion fluorescence staining was performed on mice after intravitreal injection of cGAMP. Specifically, the mice were anesthetized and perfused through the left ventricle with a saline solution containing 40 μg / ml FITC-Con A. The eyeballs were collected and the neural retina was dissociated and fixed with 4% paraformaldehyde (PFA), and then immunofluorescence staining was performed using anti-CD45 antibody.

[0129] Data statistics: Data were presented in the form of mean + standard deviation, and data were analyzed using the student t test method. *: P < 0.05, **: P < 0.001, ***: P < 0.0001.

[0130] Vascular inflammation is an important factor in the pathological changes and disease progression of many retinal vascular diseases such as diabetic retinopathy, age-related macular degeneration, and glaucoma. Figure 8 As shown in Figure 3, compared with the normal control mice injected with PBS into the vitreous cavity, there were a large number of CD45-positive leukocytes infiltrating around the retinal blood vessels in the mice injected with cGAMP.

[0131] Example 4: cGAMP causes a significant increase in retinal vascular disease-related genes

[0132] The retina was collected on the 3rd day after intravitreal injection of cGAMP, and total RNA was extracted using TRIZOL method and sent for sequencing after purification.

[0133] Trim Galore v1.18 was used to delete Aadapters from the sequencing data. HISAT2 v2.2.1 was used to locate the raw sequencing data to the GRCm39 genome. FeatureCounts v2.0.1 was used to calculate the fragments per kilobase of exon (FPKM). DESeq2 was used to identify the differentially expressed genes (DEGs) between the control group and the light-damaged group (fold change ≥ 2 and FDR ≤ 0.05). The R package “cluster analyzer” was used to perform functional enrichment analysis on the genes.

[0134] The analysis results are as follows Fig. 9 As shown in Figure 2, the results indicate that cGAMP treatment activated the innate immune response and also led to the upregulation of genes involved in antigen presentation (H2-Q6, Cd74, Sh2d1b1), chemokine production (Ccr1, Ccr2), and T cell activation (Gimap3, Cd3e) ( Fig. 9 A); Gene Ontology (GO) analysis also showed that the genes upregulated by cGAMP were concentrated in innate immunity, inflammatory response, cell adhesion, and antigen presentation and delivery pathways ( Fig. 9 BC). In addition, genes such as Ccr1, Tlr8, Icam1, and Icam2 have been shown to be highly expressed in diabetic retinopathy, age-related macular degeneration, glaucoma, juxtafoveal telangiectasia, and macular edema, and are associated with disease progression.

[0135] Example 5. Specificity of cGAMP-induced retinal vascular response in mice

[0136] The cGAS-cGAMP-STING signaling pathway is a key innate immune signaling pathway that senses cytoplasmic DNA and was first discovered in 2013. Its main function is to detect DNA in the cytoplasm and then trigger the expression of pro-inflammatory genes such as interferon and interleukin. Among them, after exogenous or endogenous (nuclear or mitochondrial) DNA leaks into the cytoplasm, it can be recognized by cGAS and produce the second messenger cGAMP, which in turn activates downstream STING and TBK1 and ultimately leads to the activation of transcription factors such as IRF3 and NF-κB, inducing the transcriptional expression of pro-inflammatory factors such as Interferonβ, IL6, and IL1β.

[0137] The downstream of cGAMP molecule is STING. In order to verify the specificity of cGAMP-induced retinal vascular response, this example injected 10mM cGAMP into the vitreous cavity of wild-type and STING knockout mice respectively to observe the retinal vascular response. Fundus photography and fluorescein angiography were used to observe the fundus blood vessels.

[0138] The results are as follows Fig.10 As shown, compared with STING knockout mice, wild-type mice have larger retinal blood vessels and more fluorescence leakage. Fig.11 ) It can be observed that compared with STING knockout mice, the wild-type mouse retina has obvious hemorrhage spots. From the results of immunofluorescence staining of vascular marker IB4 in retinal flat mounts ( Fig.12 ) It can be seen that there is obvious leakage and new blood vessel formation in the retina of wild-type mice, but not in STING knockout mice. The results of retinal vascular perfusion fluorescence staining ( Fig.13) It can be observed that the number of CD45-positive leukocytes around the retinal blood vessels in STING knockout mice was significantly reduced.

[0139] In summary, the experimental results show that the retina of the retinal vascular disease model mouse prepared by the method of the present application shows obvious exudation, edema, neovascularization and vascular inflammation, and can be used as a tool for studying retinal vascular diseases and a drug screening platform. The above results prove that the method of the present invention can obtain an animal disease model of retinal vascular diseases in a relatively short period of time.

[0140] The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and illustration. These descriptions are not intended to limit the present application to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical application, so that those skilled in the art can realize and utilize the various exemplary embodiments of the present application and various selections and changes. The scope of the present application is intended to be limited by the claims and their equivalents.

Claims

1. A method for constructing an animal model of retinal vascular disease, characterized in that: The construction method comprises the step of administering cGAMP into an animal; the administration in vivo is intravitreal injection; the animal is a mouse; the intravitreal injection is: after the animal is anesthetized, the animal's pupil is dilated, and a cGAMP solution is injected into the intravitreal cavity along the opening of the corneal limbus; the concentration of the cGAMP is >1 mM.

2. Use of an animal model in the evaluation or screening of drugs for treating retinal vascular diseases; the animal model is constructed by the construction method described in claim 1.

3. Application of cGAMP in the construction of animal models of retinal vascular diseases, characterized in that: The construction comprises the step of administering cGAMP into an animal; the administration in vivo is intravitreal injection; the animal is a mouse; the intravitreal injection is: after the animal is anesthetized, the animal pupil is dilated, and a cGAMP solution is injected into the intravitreal cavity along the opening of the corneal limbus; the concentration of the cGAMP is >1 mM.

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