A method for constructing a non-human primate model of acute uveitis induced by cytokine IL-15

Abstract

The application discloses a method for constructing an acute uveitis model of non-human primates based on a cytokine IL-15. The method is characterized in that the cytokine IL-15 is injected into non-human primates to construct the acute uveitis model. The method for constructing the acute uveitis model is simple in operation, can cause monocular disease, has high success rate of model construction, has acute onset, has short disease course, and can be successfully constructed by single monocular vitreous cavity injection.

Description

Technical fields:

[0001] This invention relates to the field of animal model construction technology, specifically to a method for constructing a non-human primate acute uveitis model induced by the cytokine IL-15. Background technology:

[0002] Uveitis is a series of highly prevalent inflammatory eye diseases involving the uvea, retina, retinal vessels, and vitreous humor. It can cause serious complications and sequelae and is one of the leading causes of blindness. Due to various limitations in human uveitis research, and because uveitis is a dynamic disease process, it is difficult to conduct clinical studies on the microenvironment of the various components of the eye. Therefore, animal models are used to study it at the cellular and molecular levels. The ocular physiological environment and anatomical structure of non-human primates are similar to those of humans; therefore, the construction of primate models can effectively simulate human disease mechanisms. Currently, animal models used for non-human primate uveitis research include: experimental autoimmune uveitis and induction by novel derivatives of lead drugs.

[0003] However, both of the above-mentioned modeling methods still have limitations in the creation of acute uveitis models. Experimental autoimmune uveitis has an onset of 4-5 weeks, with acute onset occurring in 2-3 months, resulting in a long course. It primarily affects the retina, and the bilateral involvement leads to high animal costs. The latter method uses novel derivatives of lead drugs to induce acute uveitis for up to four weeks, requiring continuous injections into animals, which can easily cause negative emotions in the animals and interfere with the experimental results.

[0004] IL-15 is an immunomodulatory factor currently mainly used in anti-tumor immunotherapy, primarily to activate NK cells and T cells. However, there are few reports on the role of IL-15 in uveitis, and no data have been found indicating that it can be used to construct uveitis models. Summary of the Invention:

[0005] This invention solves the problems existing in the prior art and provides a method for constructing an acute uveitis model in non-human primates induced by the cytokine IL-15. The acute uveitis construction method proposed in this invention is simple to operate, can cause unilateral disease, has a high success rate in model construction, rapid onset, and short disease course.

[0006] The first objective of this invention is to provide the application of the cytokine IL-15 in constructing an acute uveitis model. This invention is the first to use IL-15 to construct a non-human primate acute uveitis model.

[0007] The second objective of this invention is to provide a method for constructing an acute uveitis model in non-human primates induced by the cytokine IL-15, wherein the acute uveitis model is constructed by injecting the cytokine IL-15 into non-human primates.

[0008] The proposed method for constructing this model involves injecting IL-15 into non-human primates to induce acute uveitis. This model construction method is characterized by its simplicity, unilateral onset, high success rate, rapid onset, and short disease course. Obvious lesions can be observed as early as the second day after IL-15 injection, with an observation period of 2-21 days.

[0009] The construction method specifically includes the following steps:

[0010] (a) Select non-human primates of appropriate weight and anesthetize them;

[0011] (b) Induction of non-human primates by injection of IL-15 solution at different concentrations;

[0012] (c) On days 2, 4, 7, 10 and 21, the anterior segment and retina of non-human primates were assessed by imaging data.

[0013] (d) Based on the imaging assessment results, the optimal concentration of IL-15 to induce acute uveitis was determined, and the acute uveitis model was constructed.

[0014] In step (a), select non-human primates of appropriate weight and anesthetize them by intramuscular injection of salbutamol. The optimal weight of the experimental animals is 3 ± 0.5 kg.

[0015] Preferably, the non-human primate is a cynomolgus monkey.

[0016] Preferably, the cytokine IL-15 injection method is a single intravitreal injection in one eye, without the need for repeated injections.

[0017] Further preferably, the injection dose of the cytokine IL-15 is 0.045-4.5 μg. The preferred cynomolgus monkey used in this invention is an adult cynomolgus monkey.

[0018] Further preferred, the injection dose of the cytokine IL-15 is 0.45-4.5 μg.

[0019] Preferably, the following steps are required when injecting IL-15: anesthesia is administered via intramuscular injection of salbutamol, the conjunctival sac is rinsed with diluted povidone-iodine, the eyeball is rinsed with saline, the needle is inserted vertically at 2.5-3 mm from the temporal limbus, and IL-15 solution is injected into one eye. After the injection, Tobradex eye ointment is applied to the eye.

[0020] At the same volume of IL-15 solution (e.g., 90 μL), the symptoms of acute uveitis in cynomolgus monkeys worsened with increasing concentration, while 0.5 μg / mL (i.e., 0.45 μg) did not cause symptoms. Preferably, in this invention, the concentration of the IL-15 solution is 0.5–50 μg / mL, the injection dose is 90 μL, and the injection volume is based on the mass of IL-15 in the IL-15 solution. For example, if the injection volume is 0.45 μg, a single injection dose for adult cynomolgus monkeys can be 90 μL of an IL-15 solution with a concentration of 5 μg / mL, or 45 μL of an IL-15 solution with a concentration of 10 μg / mL.

[0021] The specific steps for injecting IL-15 into cynomolgus monkeys are as follows: anesthesia is administered via intramuscular injection of salbutamol, the conjunctival sac is rinsed with diluted povidone-iodine for 60 seconds, the eyeball is then rinsed with saline, and the needle is inserted vertically at the temporal limbus at 2.5-3 mm. 90 μL of IL-15 solution is injected into one eye, and Tobradex eye ointment is applied after the injection.

[0022] Imaging studies for uveitis include slit-lamp photography to assess inflammation of the anterior segment cornea, anterior chamber, and iris; fundus photography and OCT to assess changes in fundus morphology and structure; and retinal angiography via intravenous injection of sodium fluorescein (15 mg / kg) in the hindlimb to assess fundus vascular inflammation and leakage.

[0023] Different IL-15 concentrations cause different degrees of symptom severity, which worsens with increasing IL-15 concentration, and the course of the disease is also prolonged with increasing IL-15 concentration.

[0024] A third objective of this invention is to provide the application of the acute uveitis model constructed by the method in screening or preparing drugs for treating uveitis.

[0025] A fourth objective of this invention is to provide a method for screening candidate drugs for the prevention / treatment of uveitis, the method comprising applying a test drug to a obtained uveitis model and selecting a test drug that can improve uveitis symptoms.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] 1. This invention is the first to propose that IL-15 can be used to construct an animal model of acute uveitis, which expands the medical research application of IL-15 and also highlights the potential side effects of clinical use of IL-15.

[0028] 2. The method for constructing acute uveitis proposed in this invention is simple to operate, can be used for unilateral cases, has a high success rate in model construction, rapid onset, and short course of disease. It only requires a single intravitreal injection in one eye to successfully construct the model, and anterior segment uveitis lesions can be observed on the second day after injection.

[0029] 3. The severity of symptoms in IL-15-induced acute uveitis can increase with rising IL-15 concentrations, and the course of the disease can also be prolonged with rising IL-15 concentrations.

[0030] 4. The non-human primate model of uveitis constructed in this invention is closer to the human retinal anatomy, physiology and pathology than other animal models, and is more suitable for the development of treatment methods and drugs related to human uveitis. Attached image description:

[0031] Figure 1 Imaging data of all groups of cynomolgus monkeys before IL-15 injection, including slit-lamp examination, fundus photography, OCT and FFA (retinal angiography).

[0032] Figure 2 Slit-lamp imaging results of four groups of cynomolgus monkeys on the second day after injection.

[0033] Figure 3 Slit-lamp imaging results of cynomolgus monkeys in the Saline group, IL-15 group (50 μg / mL), and IL-15 group (5 μg / mL) at different time points after intravitreal injection.

[0034] Figure 4 Fundus photography results of cynomolgus monkeys at different time points in the IL-15 group (5μg / mL).

[0035] Figure 5 Results of OCT and retinal angiography (FFA) on day 21 after intravitreal injection of IL-15 in cynomolgus monkeys in the IL-15 group (5 μg / mL).

[0036] Figure 6 Retinal angiography results of cynomolgus monkeys in the IL-15 group (50 μg / mL) 6 weeks after intravitreal injection of IL-15. Detailed implementation method:

[0037] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0038] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental materials and reagents used herein are commercially available products conventionally available in this technical field.

[0039] The reagents and instruments used in the following examples are: anesthetic (Shutai), iodine tincture, normal saline, IL-15 group (50 μg / mL), IL-15 group (5 μg / mL), IL-15 group (0.5 μg / mL), eyelid speculum, intraocular gauge, 1 mL syringe, and "30G" BD insulin needle. The sources and catalog numbers of some reagents and materials are shown in Table 1 below.

[0040] Table 1

[0041]

[0042] Example 1

[0043] 1. Laboratory animals: Crab-eating macaques

[0044] 2. Experimental Procedure

[0045] (1) Six cynomolgus monkeys weighing 3±0.5kg were included, with each eye counted as an independent sample. Two eyes were included in each group and divided into Saline group, IL-15 group (50μg / mL), IL-15 group (5μg / mL), and IL-15 group (0.5μg / mL). The Saline group received 90μL of physiological saline intravitreal injection, while the other groups received 90μL of IL-15 solution of the corresponding concentration via intravitreal injection.

[0046] (2) After anesthesia with 5 mg / kg of salbutamol, the cynomolgus monkey was fixed on the examination table. Preoperative imaging data was collected, including the following examinations: the physiological state of the anterior segment of the cynomolgus monkey was recorded by slit-lamp photography, the fundus morphology and physiological structure of the cynomolgus monkey were recorded by fundus photography and OCT, and the retinal angiography was performed by intravenous injection of sodium fluorescein (15 mg / kg) in the hind limb to record the physiological structure of the fundus blood vessels of the cynomolgus monkey before surgery.

[0047] (3) After the preoperative examinations were completed, the cynomolgus monkeys in each group were transferred to the operating room and fixed on the operating table. Under a microscope, the eyelids were opened with an eyelid speculum, and the conjunctival sac was rinsed with 5% dilute povidone-iodine. Then, the eyeballs were rinsed with physiological saline. Then, 90 μL of physiological saline, 90 μL of 50 μg / mL IL-15 solution, 90 μL of 5 μg / mL IL-15 solution, and 90 μL of 0.5 μg / mL IL-15 solution were drawn with a "30G" BD insulin needle. The air in the syringe was purged and the solution was ready for use. The intraocular gauge was used for positioning, and a mark was made at 2.5 mm from the temporal limbus. The insulin needle was inserted vertically for about 4 mm, and then 90 μL of IL-15 solution from each group was injected. After the injection, Tobradex eye ointment was applied to the eyes.

[0048] (4) On the second day after the injection of IL-15 into cynomolgus monkeys, the inflammation of the anterior segment was assessed by slit-lamp photography. It was preliminarily determined that the IL-15 group (0.5 μg / mL) would not cause acute uveitis in cynomolgus monkeys.

[0049] (5) On the 4th and 7th days thereafter, the remaining two groups of cynomolgus monkeys were assessed by slit-lamp photography to further confirm that the IL-15 group (5μg / mL) had induced acute uveitis in the cynomolgus monkeys, and the inflammation worsened with increasing concentration.

[0050] (6) On days 2, 4, 10 and 21 after IL-15 injection in cynomolgus monkeys, fundus photography was used to evaluate changes in fundus morphology and structure in the IL-15 group (5 μg / mL). On day 21 after IL-15 injection, OCT and retinal angiography were used to evaluate retinal structure and fundus vascular inflammation and leakage in the IL-15 group (5 μg / mL).

[0051] (7) Six weeks after IL-15 injection, retinal angiography was used to assess fundus and vascular inflammation in cynomolgus monkeys in the IL-15 group (50 μg / mL).

[0052] 3. Results

[0053] All groups of cynomolgus monkeys had normal physiological states in the anterior and posterior segments of the eyeballs before injection. Figure 1 ).

[0054] Slit-lamp radiography revealed that on the second day after intravitreal injection of IL-15, the IL-15 group (50 μg / mL) and the IL-15 group (5 μg / mL) showed more severe anterior segment inflammation compared to the Saline group, with significant pupillary margin exudation, KP (+++), Tyn (+++), and pupillary adhesion. However, no obvious anterior segment lesions were observed in the IL-15 group (0.5 μg / mL). Figure 2Preliminary results indicate that acute uveitis did not occur in the cynomolgus monkeys in the IL-15 group (0.5 μg / mL).

[0055] Slit-lamp radiography revealed that cynomolgus monkeys in the IL-15 group (50 μg / mL) and the IL-15 group (5 μg / mL) still exhibited severe anterior segment inflammation on days 4 and 7 after intravitreal injection. The symptoms were more severe than on day 2, and worsened with increasing concentration. This further confirms that cynomolgus monkeys can develop acute uveitis at an IL-15 concentration of 5 μg / mL. Figure 3 ).

[0056] Fundus photography revealed fundus lesions in cynomolgus monkeys treated with IL-15 (5 μg / mL) on days 2, 4, 10, and 21 after intravitreal injection. Fundus examination was impossible on day 2 due to severe anterior segment inflammation. Subretinal hemorrhages were observed on day 4, and optic nerve edema was observed on day 10. Imaging data on days 14 and 21 showed regression of IL-15-induced fundus lesions. Figure 4 Furthermore, OCT and retinal angiography results on day 21 also showed spontaneous remission of uveitis in cynomolgus monkeys in the IL-15 group (5 μg / mL). Figure 5 ).

[0057] Retinal angiography revealed that, 6 weeks after intravitreal injection of IL-15 (50 μg / mL), cynomolgus monkeys in the IL-15 group still exhibited blurred fundus images, and FFA showed retinal vascular leakage and optic disc edema, indicating that uveitis symptoms persisted. Figure 6 ).

[0058] 4. Conclusion

[0059] IL-15 can be used to establish an acute uveitis model in cynomolgus monkeys. This method is simple to operate, has a high success rate, and the symptoms and course of uveitis are affected by the concentration of IL-15. Furthermore, the concentration of IL-15 ranges from 0.5 to 50 μg / mL, with increasing concentration leading to more severe acute uveitis symptoms in cynomolgus monkeys; however, a concentration of 0.5 μg / mL does not induce symptoms.

[0060] The above description of the embodiments is only for the purpose of helping to understand the technical solution and core idea of ​​the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a non-human primate model of acute uveitis induced by cytokine IL-15, characterized by, An acute uveitis model was established by injecting the cytokine IL-15 into a non-human primate, namely a cynomolgus monkey. The IL-15 injection method was a single intravitreal injection in one eye, and the injection dose of IL-15 was 0.45-4.5 μg.

2. The construction method according to claim 1, characterized in that, The following steps are required when injecting IL-15: anesthesia is administered via intramuscular injection of salbutamol, the conjunctival sac is rinsed with diluted povidone-iodine, and the eyeball is rinsed with saline. The needle is inserted vertically at the temporal limbus 2.5-3 mm away, and the IL-15 solution is injected into one eye. After the injection, Tobradex eye ointment is applied to the eye.

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