CN118436670A - Application of cGAMP in establishing animal models of retinal vascular diseases - Google Patents

Abstract

The application relates to the technical field of biological medicines, in particular to an application of cGAMP in constructing an animal model of retinal vascular diseases, a model construction method and an animal model related to the model construction method. The application successfully constructs the retina vascular disease animal model by injecting cGAMP into the vitreous cavity in a single eye for the first time, has the advantages of simple and convenient molding time period, reliable result and the like, and also solves the problems that mature retina cannot be simulated and the like compared with the prior art.

Description

Application of cGAMP in establishing animal models of retinal vascular diseases

Technical Field

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 technique

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 RVD seriously affect patients' vision. Currently, the commonly used treatments for retinal vascular diseases include laser therapy, anti-angiogenic drug therapy, and surgical treatment. However, laser therapy may cause complications such as vision loss and color vision abnormalities. Anti-angiogenic drugs require frequent intraocular injections and the treatment effects vary 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.

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.

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.

In view of this, this application is filed.

SUMMARY OF THE INVENTION

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:

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

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

The third objective of the present application is to provide the application of cGAMP in constructing an animal model of retinal vascular diseases.

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

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.

In some embodiments, the in vivo administration is in vivo injection.

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

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

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.

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.

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.

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.

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

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

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 treating retinal vascular diseases; preferably, the drug comprises a STING inhibitor drug.

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.

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.

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.

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.

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.

In some embodiments, the in vivo administration is in vivo injection.

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

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

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.

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.

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.

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

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

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

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.

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.

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

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.

Figure 1. Schematic diagram of intraocular injection;

Figure 2. Retinal flat mount white light images showing fresh ex vivo retinal hemorrhages in mice in the control group and experimental group: A is a retinal flat mount white light image showing fresh ex vivo retinal hemorrhages in 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 fresh ex vivo retinal hemorrhages in mice in the control group (normal saline (0.9% sodium chloride aqueous solution), 1μl) and experimental group (cGAMP, 10mM, 1μl, prepared in normal saline).

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

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

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

FIG6 . Fundus photography (upper panel) and fluorescence angiography (lower panel) of living retinal blood vessels of mice in the control group (PBS) and experimental group (cGAMP, 10 mM, prepared in PBS).

Figure 7. The results of immunofluorescence staining of retinal flat mount vascular marker IB4 show the retinal blood vessels of mice in the control group (PBS) and the experimental group (cGAMP, 10 mM, prepared in PBS). The statistical graph is the quantitative results of the retinal blood vessel coverage area of the two groups of mice.

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

Figure 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). A shows the number of genes that are significantly upregulated and downregulated overall and genes related to retinal vascular diseases; B shows the upregulated and downregulated signal pathways in GO cluster analysis. C shows the enrichment of upregulated genes in cell adhesion and antigen presentation systems by Gene set enrichment analysis (GSEA).

FIG10 . Retinal fundus vascular fluorescence angiography images showing the living retinal vessels of wild-type and STING knockout mice, with a cGAMP injection dose of 10 mM.

Figure 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.

Figure 12. Immunofluorescence staining of retinal flat mounts with 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 was 10mM.

Figure 13. FITC-con A perfusion (green) shows the vascular leukocyte adhesion of wild-type and STING knockout mice. Immunofluorescence staining of 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

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.

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.

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 that commonly 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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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).

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

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

In some embodiments, the in vivo administration is in vivo injection.

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

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

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

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.

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 opening of the corneal limbus.

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.

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.

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.

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

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

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

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.

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.

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.

Purpose of this application:

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;

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

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.

In some embodiments, the in vivo administration is in vivo injection.

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

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

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 opening of the corneal limbus.

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.

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.

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

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

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

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

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.

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.

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

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).

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

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

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

The specific steps are as follows:

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; when injecting into the vitreous cavity, an insulin needle was first used to poke a small hole at the corneal margin to prevent bleeding, and then a micro-syringe was used to slowly insert the needle at an angle of about 45 degrees along the opening of the corneal margin, not too deep to avoid puncturing the retina and scratching the lens (see Figure 1 for details). The drug was slowly pushed in, and the needle stayed in the vitreous cavity for about 5 seconds after injection to prevent drug overflow, and then the needle was slowly withdrawn.

After 3 days, the mice were euthanized, the eyeballs were removed, the retinas were dissociated and spread, and the 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 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).

The results are shown in Figure 2: Figure 2A 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). It can be seen that compared with the intravitreal injection of PBS, the corresponding mice injected with 105mM or 25mM cGAMP have obvious hemorrhage spots in the retina; 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), and the number of mice in each group is 5. It can be seen that compared with the normal control group mice such as intravitreal injection of PBS, sterile water and normal saline, the corresponding mice injected with cGAMP have obvious hemorrhage spots in the retina.

In order to further test the minimum concentration of cGAMP, this embodiment also injected different concentrations (0.1, 1, 10mM) of cGAMP into the vitreous cavity of mice to observe the leakage of mouse blood vessels. It can be observed from the results of retinal flat-mount fluorescence staining (Figure 3) that there was slight red blood cell leakage in the retinal blood vessels of mice injected with 1mM cGAMP, and obvious red blood cell leakage in the retinal blood vessels of mice injected with 10mM cGAMP. It can be seen 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.

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

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.

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

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

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

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.

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.

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.

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 3 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 overnight at 4°C 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).

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.

Experimental results:

Three days after intravitreal injection of cGAMP or PBS, samples were collected from mice and retinal structure was analyzed by HE staining. The results are shown in Figure 4. The retinal structures of all layers of the normal control group mice injected with PBS were intact, while the cGAMP-injected mice showed overall retinal thickening and edema, enlarged retinal blood vessel lumen, and visible congestion in some blood vessels.

The OCT results are shown in Figure 5. 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 retinal thickening and edema, and a large amount of highly reflective exudates in the vitreous cavity.

The fundus blood vessel structure was observed by fundus photography and fluorescein angiography as shown in Figure 6: It can be seen that 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 in diameter and had fluorescence leakage.

The results of immunofluorescence staining of the vascular marker IB4 in retinal flat mounts (Figure 7) showed that obvious leakage and new blood vessel formation occurred in the retina of cGAMP-injected mice.

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

Retinal vascular perfusion fluorescence staining was performed on mice injected with cGAMP intravitreally. 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.

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.

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, glaucoma, etc. Through the results of retinal vascular perfusion fluorescence staining (as shown in Figure 8), it can be observed that compared with the normal control group mice injected with PBS in the vitreous cavity, there are a large number of CD45-positive leukocytes infiltrating around the retinal blood vessels in the mice injected with cGAMP.

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

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.

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 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.

The analysis results are shown in Figure 9, which show 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) (Figure 9A); Gene Ontology (GO) analysis also showed that cGAMP-upregulated genes were concentrated in innate immunity, inflammatory response, cell adhesion, and antigen presentation and delivery pathways (Figure 9B-C). In addition, genes such as Ccr1, Tlr8, Icam1, and Icam2 have been confirmed to be highly expressed in diabetic retinopathy, age-related macular degeneration, glaucoma, foveal telangiectasia, and macular edema, and are associated with disease progression.

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

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.

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.

The results are shown in Figure 10. Compared with STING knockout mice, the retinal blood vessels of wild-type mice have larger diameters and fluorescent leakage. From the fresh retinal flat mounts isolated from the body (Figure 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 the vascular marker IB4 in the retinal flat mounts (Figure 12), it can be seen that the retina of wild-type mice has obvious leakage and neovascularization, while STING knockout mice do not. Through the results of retinal vascular perfusion fluorescent staining (Figure 13), it can be observed that the number of CD45-positive leukocytes around the retinal blood vessels of STING knockout mice is significantly reduced.

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.

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 (10)

1. A method of constructing an animal model of retinal vascular disease, comprising the step of administering cGAMP to an animal.

2. The method of claim 1, wherein the in vivo administration is in vivo injection.

3. The method of claim 2, wherein the in vivo injection is an intraocular injection.

4. A method of construction according to claim 3, wherein the intraocular injection is an intraocular vitreous cavity injection.

5. The method of claim 4, wherein the intravitreal injection is: after the animals were anesthetized, the pupils of the animals were dispersed and cGAMP solution was injected into the vitreous cavity of the eye along the limbal opening.

6. The method of claim 1-5, wherein the animal is a mouse.

7. An animal model of retinal vascular disease, characterized in that the animal model is constructed by the method of any one of claims 1-6.

8. Use of the animal model of claim 7 in the evaluation or screening of a medicament for the treatment of retinal vascular diseases.

Use of cGAMP in the construction of an animal model of retinal vascular disease, comprising the step of administering cGAMP to the animal in vivo.

10. The use according to claim 9, wherein said in vivo administration is an in vivo injection. Preferably, the in vivo injection is an intraocular injection; more preferably, the intraocular injection is an intraocular vitreous cavity injection.