Use of polidocanol in constructing an animal model of retinal vein occlusion and a method for constructing an animal model of retinal vein occlusion

Abstract

The present invention relates to the use of polidocanol in constructing an animal model of retinal vein occlusion and a method for constructing an animal model of retinal vein occlusion, and belongs to the field of medical technology. The present invention provides a new animal model of retinal vein occlusion, which is the use of polidocanol in constructing an animal model of retinal vein occlusion and a method for constructing an animal model of retinal vein occlusion. The present invention only requires a single injection to establish a relatively stable model, and the drugs and instruments used are all available on the market. At the same time, the applicable species is the Bama miniature pig, which has an eyeball size similar to that of a human and a visual stripe area on the retina (i.e., a visually sensitive area that can correspond to the macular area of the human retina). Therefore, it can be better used to verify drug targets and the mechanism of action of candidate drugs after injection.

Description

Technical Field

[0001] The present invention relates to the use of polidocanol in constructing an animal model of retinal vein occlusion and a method for constructing an animal model of retinal vein occlusion, belonging to the field of medical technology. Background Art

[0002] Retinal vein occlusion (RVO) is the second most common vascular disease causing vision loss, second only to diabetic retinopathy in incidence. In its early stages, the disease often presents with tortuosity, congestion, dilatation, retinal hemorrhage and edema, and the formation of large areas of nonperfusion. In later stages, complications such as cystoid macular edema, vitreous hemorrhage, and neovascular glaucoma often lead to rapid vision loss and even blindness. Clinically, RVO can be divided into central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO) based on the site of vascular occlusion. The pathogenesis of RVO is not fully understood, and there are no definitive treatments for intravascular thrombosis. Currently, symptomatic treatments (such as retinal laser photocoagulation, vitrectomy, intravitreal injections of corticosteroids or anti-vascular endothelial growth factor drugs) are commonly used in ophthalmology. However, these treatments can cause retinal damage and increase the risk of endophthalmitis, cataracts, and glaucoma.

[0003] Existing methods for establishing RVO models primarily include vascular ligation, diathermy, intravitreal endothelin-1 (ET-1) injection, laser photocoagulation to occlude the retinal vein, and intravenous injection of photosensitizing drugs combined with laser photocoagulation. The vascular ligation method involves burning and ligating the central retinal vein, resulting in poor retinal venous return. This method, however, is complex, technically challenging, and highly destructive, and prone to postoperative infection and death. The diathermy method allows for precise targeting of blood vessels, using electrical heating to induce vasoconstriction, damage, and immediate occlusion. However, this method utilizes electrical heat to occlude the vessels, disrupting the endothelium and forming a mechanism distinct from that of human RVO. Its clinical relevance requires further investigation. Endothelin-1 is a potent vasoconstrictor peptide. Intravitreal injection of different concentrations of ET-1 elicits distinct vascular responses. RVO induced by moderate concentrations of ET-1 is caused by severe vascular spasm and is therefore only suitable for studying electrophysiological changes in monkeys with retinal ischemia. Laser photocoagulation is a noninvasive procedure that uses a three-sided mirror to directly direct a laser at the intended vessel, occluding it using thermal energy. However, the recanalization rate of blocked vessels is high and recanalization occurs early, requiring repeated photocoagulation. This can cause severe thermal damage to the retinal pigment epithelium and photoreceptors, often accompanied by vitreous hemorrhage due to vessel wall damage, which can impair fundus observation. Intravenous photocoagulation combined with laser photocoagulation involves injecting a photosensitizer into the vein and then irradiating the retinal vessels to be blocked. The combined effects of the photosensitizer and laser create an RVO model. This method achieves an immediate model, with recanalization of the blocked vessels occurring approximately seven days later. However, the damage to the eye caused by laser photocoagulation is related to the amount of pigment in the fundus. The higher the pigment content of the irradiated tissue, the greater its absorption of light energy, making it more susceptible to laser damage. In non-pigmented animals, due to the shielding effect of non-pigmented tissue, the choroidal vessels can directly absorb the external laser energy, resulting in a photochemical reaction and occlusion of the choroidal vessels.

[0004] In general, the existing RVO model has a short vascular recanalization time and cannot be maintained for a long time. In addition, it may cause damage to the retinal pigment epithelium and photoreceptor cells in addition to vascular occlusion. Therefore, based on the above situation, it is very necessary to establish an animal model with good reproducibility and that can present a pathological process similar to that of the disease to conduct research on etiology, pathology and therapy.

[0005] Application number: CN202011557373.0, invention name: Method for constructing a novel retinal vein occlusion mouse model, relating to a method for constructing a novel retinal vein occlusion mouse model. The method for constructing the novel retinal vein occlusion mouse model includes the steps of using a photodynamic method to intervene in the mouse and then injecting a Muller cell metabolic inhibitor into the vitreous. The retinal vein occlusion mouse model prepared by this construction method is stable and can achieve long-lasting retinal edema, which is convenient for verifying drug targets and the mechanism of action of candidate drugs after injection. This method requires two operations to establish the model, and the steps are relatively complicated. At the same time, the applicable animal species is mice, and the retinal structure of mice is still quite different from that of humans.

[0006] Selecting appropriate experimental animals and constructing animal models close to human diseases are the foundation and premise of medical research. Currently, the choice of experimental animals is mainly mammals, among which rodents are the most used. However, there is still a certain gap between them and humans in terms of tissue structure, metabolic characteristics, pathogenesis, etc. In recent years, more and more scholars at home and abroad have begun to use miniature pigs to construct disease models. Due to their unique advantages in physiological and biochemical indicators, anatomical structure, dietary characteristics, drug metabolism and disease development, they are considered to be ideal animal species for medical research. At present, Bama miniature pigs have been used in cardiovascular, skin plastic surgery, endocrinology and metabolism, digestion, oral medicine, and organ transplantation. However, there are few studies on the use of miniature pigs in the field of ophthalmology. (Zou Disha, et al., Research progress of Bama miniature pig animal models in the medical field, Chinese Journal of Animal Husbandry and Veterinary Medicine 2017, 44 (4): 1128-1134).

[0007] Clinically, foam sclerosant is a safe and effective drug for treating vascular diseases, mainly used to treat varicose veins, hemangiomas and other diseases. Polydocanol injection is a foam sclerosant that is clinically used to treat central veins, reticular veins and small varicose veins of spider veins. Polydocanol mainly achieves the purpose of clinical treatment of varicose veins by destroying the surfactant molecules on the cell membrane of vascular endothelial cells, destroying the cell membrane structure, lysing vascular endothelial cells, causing vascular fibrosis, occlusion and thrombosis. Varicose veins and retinal vein occlusion are two different diseases that affect different parts of the vascular system. Varicose veins mainly affect the superficial veins of the lower limbs, while retinal vein occlusion affects the retinal blood vessels of the eyes. There are currently no relevant literature reports on the use of polidocanol injection in the construction of an animal model of retinal vein occlusion. Summary of the Invention

[0008] The present invention provides a new animal model of retinal vein occlusion, which is the use of polidocanol in constructing an animal model of retinal vein occlusion and a method for constructing an animal model of retinal vein occlusion.

[0009] The present invention provides the use of polidocanol in constructing an animal model of retinal vein occlusion.

[0010] Wherein, the animal is a mammal.

[0011] Wherein, the polydocanol is applied to the retinal vein in an amount of 0.4-0.5 mg / eye / time.

[0012] The present invention provides a method for constructing an animal model of retinal vein occlusion, which comprises the following steps:

[0013] a. Prepare the modeling agent: Mix polidocanol injection with air and repeatedly beat to form a dense foam. The volume ratio of the polidocanol injection to air is 1:3-4 (v / v). The concentration of the polidocanol injection is 1% w / v. After mixing with air, each ml contains 2-2.5 mg of polidocanol.

[0014] b. Apply the modeling agent into the retinal vein to prepare a retinal vein occlusion animal model.

[0015] Wherein, the animal is a mammal.

[0016] Wherein, the animal is Bama miniature pig.

[0017] Wherein, the volume ratio of the polydocanol injection to air is: 1:3 (v / v).

[0018] Among them, the device for preparing the modeling agent is: containing: two threaded syringes and a three-way valve; one threaded syringe draws out the polydocanol injection, and the other threaded syringe draws out the air, the two threaded syringes are respectively connected to the two ends of the three-way valve, and the three-way valve switch is turned to connect the two ends of the syringe, and the syringes at both ends are pushed successively to fully mix the polydocanol injection and the air, and quickly pumped until dense foam is formed.

[0019] The present invention also provides use of the animal model of retinal vein occlusion constructed by the construction method in screening drugs for preventing and / or treating retinal vein occlusion.

[0020] The present invention provides a method for screening candidate drugs for preventing and / or treating retinal vein occlusion, wherein the candidate drugs are administered to the animal model of retinal vein occlusion constructed by the construction method.

[0021] Clinically, foam sclerosant is a safe and effective treatment for vascular diseases, primarily for varicose veins and other conditions. Foam sclerosant is a liquid formulation that forms a dense foam when mixed with a certain proportion of air. When injected into a vein, the dense foam does not flow back with the blood, but instead irritates and damages vascular endothelial cells at the injection site, thereby blocking blood flow. Therefore, leveraging this characteristic of foam sclerosant, the present invention utilizes a 48G needle to inject it into the retinal vein, aiming to create a stable and long-lasting RVO model by occluding the blood vessels, thus providing a new and reliable animal model for preclinical research of related drugs.

[0022] The beneficial effects of the present invention are:

[0023] The present invention only requires a single injection to establish a relatively stable model, and the drugs and instruments used are all commercially available. The applicable species is the Bama miniature pig, whose eyeballs are similar in size to humans and whose retina has a visual stripe area (i.e., a visually sensitive area that corresponds to the macular area of the human retina). Therefore, the model can be better used to verify drug targets and the mechanism of action of candidate drugs after injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A device for preparing a modeling agent;

[0025] Figure 2 This is the expansion of the distal end of the retinal vein after injection of polidocanol injection (the red box is the injected vein, and the blood vessel can be seen to be significantly dilated and thickened);

[0026] Figure 3 Fundus photography of miniature pigs in the 0.5% polidocanol injection group (black arrows indicate retinal hemorrhages; red arrows indicate tortuous and dilated retinal blood vessels; yellow arrows indicate large retinal hemorrhages; and green arrows indicate areas of retinal atrophy);

[0027] Figure 4 Fundus angiography of miniature pigs in the 0.5% polidocanol injection group (the red arrows indicate the dark shadows caused by retinal hemorrhage; the yellow arrows indicate the hyperfluorescent leakage of blood vessels; the green arrows indicate retinal atrophy foci and local retinal hyperfluorescent areas);

[0028] Figure 5 This is the OCT image of the miniature pig retina in the 0.5% polidocanol injection group (the area indicated by the red arrow is subretinal fluid; the area indicated by the yellow arrow shows medium-high reflective signals in the retina, indicating retinal hemorrhage; the area indicated by the green arrow is retinal atrophy);

[0029] Figure 6This is the retinal histopathological examination picture of miniature pigs in the 0.5% polidocanol injection group (the black box area shows atrophy of the lower retina);

[0030] Figure 7 Fundus photography of miniature pigs in the 1% polidocanol injection group (black arrows indicate tortuosity and dilation of retinal vessels; red arrows indicate retinal hemorrhage);

[0031] Figure 8 Fundus angiography of miniature pigs in the 1% polidocanol injection group (the dark shadow indicated by the red arrow is fluorescence obscuration caused by retinal hemorrhage; the yellow arrow indicates hyperfluorescent leakage at the vascular terminal);

[0032] Figure 9 This is the OCT image of the miniature pig retina in the 1% polidocanol injection group (the red arrow points to retinal neuroepithelial edema; the yellow arrow points to retinal hemorrhage; the green arrow points to retinal atrophy);

[0033] Figure 10 This is the retinal histopathological examination image of miniature pigs in the 1% polidocanol injection group (A represents retinal vascular dilation and hemorrhage / congestion, and B represents retinal atrophy (the black box area represents retinal atrophy));

[0034] Figure 11 Fundus photography, FFA, and retinal OCT images of miniature pigs in the 3% polidocanol injection group (A is fundus photography, B is fundus fluorescein angiography, and C is retinal OCT image);

[0035] Figure 12 HE staining of the retina in the 3% polidocanol injection group (A: black arrows indicate retinal vascular dilation and hemorrhage / congestion; B: black box area indicates retinal atrophy; C: lens degeneration). DETAILED DESCRIPTION

[0036] Example 1 Method for constructing an animal model of retinal vein occlusion according to the present invention

[0037] This method was performed on Bama miniature pigs and has been approved by the Experimental Animal Use and Management Committee of Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd. The foam sclerosant used was polidocanol injection, with concentrations of 0.5%, 1%, and 3%, respectively, that is, 0.5g, 1g, and 3g of polidocanol per 100mL.

[0038] Bama miniature pigs were divided into three groups, namely, 0.5% polidocanol injection group, 1% polidocanol injection group and 3% polidocanol injection group.

[0039] Before modeling, Bama miniature pigs were anesthetized with compound tropicamide eye drops for pupil dilation and a 0.22 mL / kg intramuscular injection of 50 mg / kg of Zotai 50 (22.73 mg / mL of Zotai 50, 9.09 mg / mL of xylazine, and 18.18 mg / mL of ketamine hydrochloride) (0.22 mL / kg intramuscular injection). After anesthesia, the animals were intubated and maintained on a ventilator. Oxybuprocaine eye drops were administered periodically for topical anesthesia.

[0040] The specific modeling method is as follows: After disinfecting the eye skin, eyelid margin, and conjunctival sac with povidone-iodine, a scleral tunnel needle is used to puncture the eyeball at the nasal and temporal ciliary body flat parts to create a scleral tunnel. Using a threaded syringe and a three-way valve, a mixture of polidocanol injection and air in a ratio of 1:3 (v / v) is repeatedly pushed and pumped to form a dense foam (using two threaded syringes, one to extract an appropriate amount of polidocanol injection, and the other to extract air three times the volume of polidocanol injection. The two threaded syringes are connected to the two ends of the three-way valve respectively. The three-way valve switch is turned to connect the two ends of the syringes. The two syringes are pushed in sequence to fully mix the polidocanol injection and air, and then quickly pumped until a dense foam is formed). An appropriate amount of modeling agent mixed with air was withdrawn. An illumination fiber was inserted into one scleral tunnel, and a 48-gauge injection needle was inserted into the other. Using the push-in system of a phacovitrectomy device (Constellation, Alcon), a sclerosant (200 μL) was injected into the retinal vein distal to the retinal vein near the edge of the optic disc. The dose of polidocanol was 0.4–0.5 mg / eye / time. After the injection, the injection site was compressed with an illumination fiber for approximately 5 minutes. After the procedure, the fiber and injection needle were removed, the scleral tunnel needle was removed, and ofloxacin eye ointment was applied to prevent infection.

[0041] The device for preparing the modeling agent is as follows Figure 1 shown.

[0042] Among them, 1 is syringe 1, 2 is syringe 2, and 3 is a three-way valve. The three-way valve can be an ordinary ball valve.

[0043] The specific usage of the device is:

[0044] Use threaded syringe 1 to draw out the polidocanol injection solution, and another threaded syringe 2 to draw out air. Connect the two threaded syringes to the ends of the three-way valve. Turn the three-way valve switch to connect the two ends of the syringe. Push the two syringes in sequence to fully mix the polidocanol injection solution and air. Quickly pump until dense foam is formed. After repeated pumping and mixing, turn the three-way valve until the two ends of the syringe are connected.

[0045] The purpose of the above-mentioned device is bubbling, which is the process of introducing gas (usually air) into the liquid. Therefore, the present invention is not limited to the above-mentioned device, and currently commonly used stirring methods, mechanical bubbling methods (bubblers, bubble pumps), gas injection methods, vacuum bubbling methods, etc. can all be applied.

[0046] Example 2 Detection of the animal model of retinal vein occlusion prepared by the present invention and screening test of different doses of polidocanol injection

[0047] 1. Retinal optical coherence tomography (OCT)

[0048] Examinations were performed before administration and on days 2, 7, 14, and 28 after administration. Bama miniature pigs were anesthetized (the anesthesia method was the same as in Example 1) and mydriasis was administered with compound tropicamide eye drops (1-2 drops / eye). Rapid linear OCT scans of the pig fundus retina were performed using a laser ophthalmology diagnostic device (Spectralis OCT, Heidelberg, Germany) to visualize the structure of each retinal layer.

[0049] 2. Fundus photography and fluorescein angiography (FFA)

[0050] Examinations were performed before administration and on days 2, 7, 14, and 28 after administration. Bama miniature pigs were anesthetized (the anesthesia method was the same as in Example 1) and mydriatic with compound tropicamide eye drops (1-2 drops / eye). Fundus photography was performed using an ophthalmic laser diagnostic instrument (SpectralisHRA, Heidelberg, Germany). Fluorescein sodium injection (0.1 mL / kg) was then rapidly injected into the pig's ear vein. Fundus images were taken early (within 1 minute) and late (after 5 minutes) after angiography.

[0051] 3. Histopathological examination

[0052] On day 28 after administration, Bama miniature pigs were anesthetized according to body weight (anesthesia method was the same as in Example 1) and euthanized by exsanguination via the abdominal aorta. The eyeballs were removed and fixed in a modified Davidson's fixative. Paraffin sections were prepared according to standard histopathological techniques, and hematoxylin-eosin (HE) staining was performed and microscopic examination was performed.

[0053] A few minutes after the injection of polidocanol into the retinal vein during the operation, the distal end of the vein was significantly dilated, indicating that the vascular endothelium was damaged and the blood vessel was gradually blocked. Figure 2 .

[0054] Fundus photography of miniature pigs in the 0.5% polidocanol injection group on the second day after injection ( Figure 3 ) Scattered hemorrhages can be seen around the injected vein and the retinal area where the vein is responsible for venous return, and the vein is tortuous and dilated. FFA examination ( Figure 4) can be seen in local fluorescence shielding and high fluorescence leakage at the vascular terminals. On the 7th to 14th day after injection, fundus photography can show large retinal hemorrhages, the degree of venous tortuosity and dilation is reduced, and FFA can show large retinal hemorrhages shielding fluorescence. On the 28th day after injection, retinal hemorrhages gradually absorbed, retinal atrophy foci can be seen, FFA can show local retinal high fluorescence, and no fluorescence leakage is seen at the vascular terminals. On the 2nd day after drug injection, OCT examination ( Figure 5 ) Subretinal fluid was observed in the injected area, which was caused by the drug being injected into the subretina during administration. OCT showed retinal hemorrhage on the 7th day after injection. From 14 to 28 days after administration, the retina in the hemorrhage area gradually atrophied. No obvious retinal edema was observed at the end of observation. Histopathological examination on the 28th day after injection showed retinal atrophy in the injected eye ( Figure 6 ).

[0055] On the second day after injection, fundus photography of the miniature pigs in the 1% polidocanol injection group showed scattered bleeding spots and large hemorrhages around the injected vein and the retinal area where the vein was responsible for venous return. The veins were tortuous and dilated. In the early stage of FFA examination, large areas of fluorescence obscuration were observed, and in the late stage, high fluorescence leakage of the vascular terminals was observed ( Figure 7 、 Figure 8 ). By the 28th day after injection, the retinal hemorrhage was gradually absorbed, and FFA still showed fluorescence shielding and high fluorescence. On the second day after drug injection, OCT images showed low reflection signals between the retinal neuroepithelial layers, indicating edema of the retinal neuroepithelial layer. At the same time, local retinal thickening was accompanied by enhanced reflection signals, indicating retinal hemorrhage. By the 14th day after injection, the degree of edema and hemorrhage of the retinal neuroepithelium was gradually reduced. On the 28th day after injection, the thickness of the retinal neuroepithelial layer was significantly decreased, and the structures of the various layers of the retina were disordered and unrecognizable, indicating retinal atrophy ( Figure 9 Histopathological examination revealed retinal vascular dilation, hemorrhage / congestion, and retinal atrophy in the injected eye ( Figure 10 ).

[0056] Fundus photography, FFA, and retinal OCT examinations failed to obtain fundus images in the miniature pigs in the 3% polidocanol injection group on days 2, 7, 14, and 28 after injection. Slit lamp examination revealed large retinal hemorrhages on day 2 after administration, and large white deposits in the posterior lens capsule appeared on days 7 to 28 after administration, which appeared ground-glass-like and made fundus observation impossible (see Figure 11 ).

[0057] In the 3% polidocanol injection group, vasodilation, hemorrhage / congestion, retinal atrophy, and lens degeneration were observed in the injected eye. Figure 12 ).

[0058] The experimental results of the above-mentioned different concentrations of polydocanol injection showed that no obvious retinal edema occurred at the 0.5% concentration, edema occurred at the 1% concentration and could last for 2 weeks, and the 3% concentration caused more severe damage and the fundus could not be observed after injection, so the 1% concentration was the best.

[0059] In summary, injection of 1% polidocanol into the retinal vein using a 48G needle can significantly induce retinal vein occlusion and retinal edema formation, which lasts for at least two weeks and can provide a new animal model for the pharmacodynamic study of related drugs.

Claims

1. A method for constructing an animal model of retinal vein occlusion, characterized by: It includes the following steps: a. Prepare a modeling agent: Mix polidocanol injection with air and repeatedly beat to form a dense foam. The volume ratio of the polidocanol injection to air is 1:

3. The concentration of the polidocanol injection is 1% w / v. After mixing with air, each ml contains 2-2.5 mg of polidocanol. b. administering the modeling agent into the retinal vein to prepare an animal model of retinal vein occlusion; The polidocanol is applied to the retinal vein at a dosage of 0.4-0.5 mg / eye / time; The animal is a Bama miniature pig; the device for preparing the modeling agent is: containing: two threaded syringes and a three-way valve; one threaded syringe draws out the polydocanol injection, and the other threaded syringe draws out the air, the two threaded syringes are respectively connected to the two ends of the three-way valve, the three-way valve switch is turned to connect the two ends of the syringe, and the syringes at both ends are pushed successively to fully mix the polydocanol injection and the air, and quickly pumped until dense foam is formed.

2. Use of the animal model of retinal vein occlusion constructed by the construction method of claim 1 in screening drugs for preventing and / or treating retinal vein occlusion.

3. A method for screening candidate drugs for preventing and / or treating retinal vein occlusion, characterized by: The candidate drug is administered to the animal model of retinal vein occlusion constructed by the construction method of claim 1.

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