Establishment method and application of non-human primate retinal pigment epithelium injury model

Abstract

The invention provides an establishment method and application of a non-human primate retinal pigment epithelium injury model, and belongs to the field of animal models. The method comprises the following steps: (1) performing anesthesia and mydriasis on a non-human primate; (2) attaching a full retinoscope to the cornea, and adjusting a slit lamp so that an operator can observe the macular fovea centralis, optic disc and upper and lower vascular arches of the non-human primate; and (3) carrying out photocoagulation on the photocoagulation area by using a laser photocoagulator, and ending the photocoagulation until 250-300 primary light spots appear in the photocoagulation area. The invention provides a method for constructing a non-human primate retinal pigment epithelium (RPE) injury model by using laser by taking a non-human primate as a model animal for the first time. The model forming rate of the RPE injury animal model constructed by the invention is stable, the injury caused by the RPE injury animal model is irreversible, and a stable animal disease model is provided for clinical research and development of new drugs related to dry age-related macular degeneration caused by RPE injury.

Description

Technical Field

[0001] The invention belongs to the field of animal models, and in particular relates to a method for establishing a non-human primate retinal pigment epithelium injury model and its application. Background Art

[0002] Age-related macular degeneration (AMD) refers to a disease caused by abnormal metabolism of the retinal pigment epithelium (RPE), decreased phagocytic and digestive function of the photoreceptor cell outer segment membrane disk, deposition of extracellular matrix on the basement membrane, gradual formation of drusen, and changes in the morphology and function of the macular area under the combined effects of aging and environmental factors. It is an important cause of progressive visual impairment and severe vision loss. AMD patients are mostly over 50 years old, and both eyes are affected successively or simultaneously. It is the most common blinding eye disease among the elderly population in developed countries, and it is estimated that by 2040, about 300 million people will suffer from it worldwide. Clinically, AMD can be divided into dry AMD (also known as non-neovascular AMD) and wet AMD (also known as neovascular AMD). The prevalence of dry AMD is higher, accounting for about 80% of AMD patients. Currently, wet AMD is mainly treated by intraocular injection of anti-vascular endothelial growth factor (VEGF) drugs. However, there is no clear and effective treatment for dry AMD, and there is a lack of stable animal disease models, which limits the research on treatment methods and therapeutic drugs for dry AMD. Therefore, it is urgent to develop relevant animal models and clarify relevant modeling techniques and methods to provide stable animal disease models for the clinical development of relevant new drugs.

[0003] At present, dry AMD models of different species (mice, rats, monkeys, pigs, rabbits) have been successfully established through environmental induction, drug induction and gene knockout. Among them, drug induction is through intravitreal administration, subretinal administration and other methods. Repeated administration can easily damage eye tissues and cause adverse reactions such as endophthalmitis and retinal detachment; gene knockout induction has a long modeling cycle, high cost and high technical difficulty; environmental induction, such as light-induced models, has the characteristics of short cycle, low cost and similar pathogenesis to humans, and is suitable for batch modeling.

[0004] At present, a method of using laser to induce RPE injury model in pig eyes has been reported, but the animal used in this modeling method is pigs, which do not have a macular area. The pathological manifestations of pigs are also different from those of humans, which limits their applicability in simulating human macular degeneration. The anatomical structure of the eyeball of non-human primates is most similar to that of humans, and they have a macular area, making them ideal experimental animals at present. Due to the different structures of pig eyes and non-human primate eyes, the RPE injury modeling method suitable for pigs is not suitable for non-human primates. At present, there is no report on the establishment of a simple, easy-to-repeat, stable and effective RPE injury model in non-human primates.

[0005] In order to overcome the above problems, it is urgent to develop a method for constructing a non-human primate RPE injury model with simple steps, easy to repeat, and stable model success rate. Summary of the invention

[0006] The purpose of the present invention is to provide a method for establishing a non-human primate retinal pigment epithelium injury model and its application.

[0007] The present invention provides a method for establishing a non-human primate retinal pigment epithelium injury model, characterized in that the method comprises the following steps:

[0008] (1) Anesthetize the non-human primate and dilate the pupil;

[0009] (2) fitting the panretinal scope to the cornea of ​​the nonhuman primate and adjusting the slit lamp microscope of the laser photocoagulator so that the operator can observe the fovea, optic disc, and superior and inferior vascular arcades of the nonhuman primate;

[0010] (3) Photocoagulation of the photocoagulation area is performed using a laser photocoagulation device until 250-300 primary light spots appear in the photocoagulation area; the photocoagulation area is the area from the optic disc to the fovea and between the upper and lower vascular arches, avoiding the fovea; the parameters of the laser photocoagulation device are set as follows: laser wavelength is 530-534 nm, spot diameter is 190-210 μm, laser power is 65-95 mW, exposure time is 0.07-0.10 s, and energy of each laser spot is 4.6-9.5 mJ.

[0011] Furthermore, the non-human primate is a monkey.

[0012] Furthermore, the monkey is a cynomolgus monkey or a rhesus monkey.

[0013] Furthermore, the parameters of the laser photocoagulation instrument are set as follows: the laser wavelength is 532 nm, the spot diameter is 200 μm, the laser power is 70-90 mW, and the exposure time is 0.08-0.09 s, that is, the energy of each laser spot is 5.6-8.1 mJ.

[0014] Furthermore, the timing for ending photocoagulation is when 280 first-order light spots appear in the photocoagulation area.

[0015] Furthermore, the method further comprises the following steps:

[0016] (4) Treat the operated eye to prevent infection.

[0017] The present invention also provides the use of the non-human primate retinal pigment epithelial injury model established by the above method for screening drugs for treating dry age-related macular degeneration caused by the retinal pigment epithelial injury model.

[0018] The present invention has achieved the following beneficial effects:

[0019] The present invention uses non-human primates as model animals for the first time and provides a method for constructing a non-human primate retinal pigment epithelium (RPE) injury model using laser. The method of the present invention can directly damage the RPE using laser, thereby causing atrophy of the outer retina. The RPE injury animal model constructed by the present invention has a stable modeling rate and the damage caused is irreversible.

[0020] The present invention uses laser photocoagulation to directly damage the RPE. The main function of the RPE is to provide nutritional support to the retinal photoreceptor cells and to phagocytose and clear the metabolic products of the retinal photoreceptor cells. Therefore, when the RPE is damaged, it cannot play a normal function, affecting the photoreceptor cells, thereby causing the retina to be damaged and the retina to present a map-like atrophy in the photocoagulation area. The fundus autofluorescence photography shows that the autofluorescence intensity is significantly weakened. Retinal optical coherence tomography (optic coherence photography, OCT) shows that the RPE layer structure is disordered and discontinuous, and the outer retina is atrophied and thinned. Fundus fluorescein angiography (FFA) shows window-like defects in the atrophic area in the early stage, and high fluorescence leakage in the late stage. Multifocal electroretinograms (mfERG) show that the retinal P1 wave amplitude density in the photocoagulation area decreases and is lower than that in the non-modeling area of ​​the same eye (indicating a decrease in visual function in the photocoagulation area).

[0021] Clinically, the onset of dry AMD is also due to environmental, age and other factors that lead to damaged RPE function, thereby affecting the metabolism of retinal photoreceptor cells. The metabolites cannot be phagocytosed and removed in time, and accumulate in the RPE layer for a long time, forming drusen, which leads to gradual atrophy of the outer retina and map-like atrophy in fundus photography; OCT shows atrophy and thinning of the retina, and disordered and discontinuous structure of the RPE layer; FFA also shows early window defects, late high fluorescence, and decreased amplitude density of the P1 wave of mfERG.

[0022] The method of the present invention for constructing a non-human primate retinal pigment epithelial injury model by using laser does not involve traditional interventional treatment methods, is easy to implement repeatedly, and has a modeling success rate of up to 100%.

[0023] The modeling method of the present invention has simple steps and provides a stable animal disease model for the clinical research and development of new drugs related to treating dry age-related macular degeneration (dAMD) caused by RPE damage.

[0024] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0025] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 .Schematic diagram of the monkey retina structure

[0027] Figure 2 . Retinal thickness (μm) before modeling and on days 2, 6, 13, 27, 42, and 56 after modeling. The differences among the groups were statistically analyzed using the ANOVA method, and the data were described in the form of X±SD (n=8). *P≤0.05, ****P≤0.0001.

[0028] Figure 3 . Retinal OCT examination images before modeling and on days 2, 6, 13, 27, 42, and 56 after modeling. The red box is the photocoagulation area.

[0029] Figure 4 Fundus retinal photographs before modeling and on days 2, 6, 13, 27, 42, and 56 after modeling. The red box is the photocoagulation area.

[0030] Figure 5 .Autofluorescence photography of the retinal fundus before modeling and on days 2, 6, 13, 27, 42, and 56 after modeling. The red box is the photocoagulation area.

[0031] Figure 6 Fundus fluorescein angiography images before modeling and on days 2, 6, 13, 27, 42, and 56 after modeling. The red box is the photocoagulation area.

[0032] Figure 7 .mfERG results before modeling and on days 7, 28, 42, and 57 after modeling.

[0033] Figure 8 Fundus photography before modeling and on days 2, 6, 13, and 20 after modeling. The red box is the photocoagulation area.

[0034] Fig. 9 . Retinal OCT images before modeling and on days 2, 6, 13, and 20 after modeling. The red box is the photocoagulation area. DETAILED DESCRIPTION

[0035] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0036] Crab-eating macaques: Both males and females, aged 4 to 6 years old, weighing 3.0 to 6.0 kg.

[0037] Example 1: Method for constructing a non-human primate retinal pigment epithelium injury model using laser

[0038] 1. Using laser to construct a non-human primate retinal pigment epithelium injury model:

[0039] 1.1 Cynomolgus monkeys were anesthetized with 5% ketamine hydrochloride (10 mg / kg intramuscularly) and 2.5% sodium pentobarbital (25 mg / kg) by intravenous injection. After anesthesia, compound tropicamide eye drops were dripped into both eyes (1-2 drops each time. If the mydriasis effect was not obvious, it could be dripped again after 5 minutes and repeated 3-4 times). At the same time, oxybuprocaine hydrochloride eye drops were dripped (1-2 drops each time, repeated 3-4 times, with an interval of 5 minutes each time) for surface anesthesia.

[0040] 1.2 The monkey's head after mydriasis was fixed in front of an ophthalmic laser photocoagulator (laser photocoagulator brand and model: Vitra 532nm, France). The laser photocoagulator parameters were adjusted as follows: laser wavelength: 532nm, spot diameter: 200μm, laser power: 70-90mW, exposure time: 0.08-0.09s, that is, the energy of each laser spot was 5.6-8.1mJ;

[0041] 1.3 Apply the coupling agent to the panretinal lens (Volk central contact lens) that can be used for laser photocoagulation, make the panretinal lens fit the monkey cornea, observe the monkey's fundus through the slit lamp microscope that comes with the laser photocoagulation instrument, and adjust the focus of the slit lamp microscope so that the operator can clearly observe the monkey's fovea, optic disc, and upper and lower vascular arches (see the specific structural diagram for details). Figure 1 );

[0042] 1.4 Observe the fundus under a full retinal microscope, adjust the laser aiming point so that it can be clearly focused on the retina, then gently push the slit lamp forward to focus on the area from the optic disc to the fovea and the area between the upper and lower vascular arches avoiding the fovea (called the photocoagulation area, see Figure 1 ) to perform photocoagulation, so that the RPE in the photocoagulation area has a light gray spot reaction (i.e., a first-order spot), and a total of 280 effective spots in the photocoagulation area (effective spots refer to the first-order spots formed on the retina at the photocoagulation location), and then end the photocoagulation;

[0043] 1.5 After laser photocoagulation, ofloxacin ointment was added to the operated eye to prevent infection, thus obtaining a retinal pigment epithelial injury model.

[0044] According to the above method, four (eight eyes) cynomolgus monkey retinal pigment epithelial injury models were constructed.

[0045] Table 1. Laser power, exposure time, and energy per laser spot when constructing 4 (8 eyes) cynomolgus monkey retinal pigment epithelial injury models

[0046]

[0047] 2. Model testing data:

[0048] 2.1 Detection Method

[0049] 2.1.1 Retinal optical coherence tomography (OCT) examination

[0050] The monkeys to be tested were anesthetized with 5% ketamine hydrochloride (intramuscular injection, 10 mg / kg) and 2.5% sodium pentobarbital (intravenous injection, 25 mg / kg). The anesthetic dose can be adjusted appropriately according to the animal's condition. Compound topiramate was used to dilate the pupil (eye drops, 1-2 drops / eye). Laser ophthalmology diagnostic instrument (optical coherence tomography) was used to perform OCT scanning of the monkey's fundus retina. The image was obtained by rapid linear scanning with the fovea as the center, showing the structure of each layer of the retina. After the operation was completed, 1-2 drops of ofloxacin eye ointment were applied to prevent corneal drying and infection.

[0051] 2.1.2 Fundus photography and fundus fluorescein angiography (FFA) examination

[0052] The monkeys to be examined were anesthetized with 5% ketamine hydrochloride (intramuscular injection, 10 mg / kg) and 2.5% sodium pentobarbital (intravenous injection, 25 mg / kg). The anesthetic dose can be adjusted appropriately according to the animal's condition. Compound tobicarbamide was used to dilate the pupil (eye drops, 1-2 drops / eye). After fundus photography was performed using an ophthalmic laser diagnostic instrument (fundus photography and angiography), sodium fluorescein injection (20 mg / kg) was rapidly injected from the monkey's limb vein to take photos of the fundus images in the early (within 1 minute) and late (after 5 minutes) after angiography. After the operation was completed, 1-2 drops of ofloxacin eye ointment were applied to prevent corneal drying and infection.

[0053] 2.1.3 Fundus autofluorescence photography

[0054] The monkeys to be examined were anesthetized with 5% ketamine hydrochloride (intramuscular injection, 10 mg / kg) and 2.5% sodium pentobarbital (intravenous injection, 25 mg / kg). The anesthetic dose can be adjusted appropriately according to the animal's condition. Compound topiramate was used to dilate the pupil (eye drops, 1-2 drops / eye), and fundus autofluorescence photography was performed using an ophthalmic laser diagnostic instrument (fundus photography and angiography). After the operation was completed, 1-2 drops of ofloxacin eye ointment were applied to prevent corneal dryness and infection.

[0055] 2.1.4 Multifocal electroretinogram (mf-ERG) examination

[0056] The monkeys to be tested were anesthetized with 5% ketamine hydrochloride (intramuscular injection, 10 mg / kg) and 2.5% sodium pentobarbital (intravenous injection, 25 mg / kg). The anesthetic dose can be adjusted appropriately according to the animal's condition. Compound topiramate was used for mydriasis (eye drops, 1-2 drops / eye), and multifocal ERG detection (mfERG) was performed using an electrophysiological eye diagnosis system. 36 hexagonal stimulation patterns were used to stimulate the photocoagulation area, and the waveform amplitude was recorded.

[0057] 2.2 Test data

[0058] 2.2.1 Retinal OCT examination

[0059] Before modeling, the boundaries of the retinal layers in both eyes of all animals were recognizable, the structure was continuous, the thickness was moderate, the reflection was normal, and the macular structure was intact.

[0060] From the 2nd to the 56th day after modeling, the RPE structure in the photocoagulation area was discontinuous, the outer retina was atrophied, the structure was disordered, and the layer boundaries were unclear, while the layers of the other retina were clear. On the 2nd day after modeling, the retina in the photocoagulation area of ​​all animals was significantly thickened, and from the 6th to the 56th day, the retinal thickness gradually decreased.

[0061] The retinal thickness before modeling, 2, 6, 13, 27, 41, and 56 days after modeling were 275.0±14.47μm, 471.5±50.31μm, 340.3±21.75μm, 303.5±16.68μm, 299.5±18.27μm, 281.5±13.92μm, and 274.5±12.87μm, respectively. On the second day after modeling, due to edema after modeling, the retinal thickness increased significantly, and the retinal thickness showed a downward trend from the 13th to the 56th day of the model period. For details, see Figures 2-3 .

[0062] 2.2.2 Fundus photography examination

[0063] Fundus photography from the 2nd to 56th day after modeling showed that a large laser photocoagulation area was visible between the temporal side of the optic disc and the upper and lower vascular arches at the edge of the fovea of ​​all animals. On the 2nd day after modeling, obvious edema was observed in the retina of the photocoagulation area of ​​all animals. From the 6th to 56th day after modeling, the edema gradually subsided, and the retina in the photocoagulation area was atrophied to varying degrees. Figure 4 .

[0064] 2.2.3 Fundus autofluorescence examination

[0065] Fundus autofluorescence images from the 2nd to 56th day after modeling showed that patches of autofluorescence lesions were observed in the retinal laser photocoagulation area of ​​all animals, and the degree of attenuation gradually increased, indicating that RPE atrophy occurred in the laser photocoagulation area. Figure 5 .

[0066] 2.2.4 Fundus fluorescein angiography (FFA) examination

[0067] Before modeling, the retinal arteries and veins of all animals were well filled, the vascular morphology and diameter were normal, and the choroidal background fluorescence was evenly distributed, with no obvious abnormalities.

[0068] On the third day after modeling, high fluorescence was observed in the retinal photocoagulation area of ​​both eyes of all animals in the early stage of angiography. Due to the severe degree of retinal edema, the entire photocoagulation area showed strong fluorescence in the late stage of angiography. From the 6th to the 56th day after modeling, retinal edema in the photocoagulation area gradually subsided. Multiple small or large window-like defects were observed in the early stage of angiography, and high fluorescence was observed in the late stage. Figure 6 .

[0069] 2.2.5 mfERG examination

[0070] The amplitude density of the P1 wave refers to the ratio of the amplitude of the P1 wave to its corresponding spatial area (in degrees) in the mfERG, usually expressed in nV / deg. 2 (Nanovolt per degree squared). In medical research, the amplitude density of the P1 wave is used to assess retinal function.

[0071] Before modeling and on days 7, 28, 42, and 57 after modeling, the P1 wave amplitude density in the non-modeling area was 23.03±3.67nV / deg, respectively. 2 、20.18±2.87nV / deg 2 、22.10±5.33nV / deg 2 、28.53±5.95nV / deg 2 、26.60±6.79nV / deg 2 ; The amplitude density of P1 wave in the modeling area was 19.55±3.68nV / deg 2 、11.61±3.85nV / deg 2 、12.33±1.60nV / deg 2 、13.18±4.37nV / deg 2 、15.60±0.67nV / deg 2 .

[0072] On days 7, 28, 42, and 57 after modeling, the amplitude density of the P1 wave in the modeling area was significantly decreased compared with that in the non-modeling area, and the difference was statistically significant (P≤0.05), indicating that retinal function was significantly decreased. Figure 7 .

[0073] In summary, the retinal pigment epithelial injury model was repeatedly established in 4 cynomolgus monkeys (8 eyes) according to the method of Example 1, and the model establishment success rate was 100%.

[0074] The following is a control method for constructing a non-human primate retinal pigment epithelial injury model using laser.

[0075] Comparative Example 1:

[0076] 1. Building the Model

[0077] The RPE injury model was constructed by referring to the method of Example 1, with the only difference being that the parameters of the laser photocoagulation instrument were adjusted as follows: laser wavelength: 532 nm, spot diameter: 200 μm, laser power: 50 mW, exposure time: 0.07 s, i.e., the energy of each laser spot was 3.5 mJ; the laser photocoagulation area was located in the inferior temporal macular area.

[0078] According to the above method, three (six eyes) cynomolgus monkey models were constructed.

[0079] 2. Model testing data:

[0080] The detection method is the same as in Example 1.

[0081] 1. Fundus photography

[0082] Fundus photography on the 2nd to 20th day after modeling showed that the retina in the photocoagulation area of ​​the temporal macula of all animals became lighter in color, and no obvious laser spots were observed. On the 2nd day after modeling, obvious edema was observed in the retina of the photocoagulation area of ​​all animals. From the 6th to 20th day after modeling, the edema gradually subsided, and no obvious retinal atrophy was observed. Figure 8 .

[0083] 2. Retinal OCT examination

[0084] Before modeling, the boundaries of the retinal layers in both eyes of all animals were recognizable, the structure was continuous, the thickness was moderate, the reflection was normal, and the macular structure was intact.

[0085] On the second day after modeling, retinal edema and thickening occurred in the photocoagulation area of ​​both eyes of all animals. On the 6th to 20th day, RPE structure discontinuity, outer retinal atrophy, structural disorder, unclear layer boundaries, and other layers of retina were clear in both eyes of all animals. Fig. 9 .

[0086] 3. mfERG examination

[0087] Before modeling and on the 2nd, 6th, 13th and 20th days after modeling, the amplitude density of P1 wave in the modeling area was 20.55±2.37nV / deg respectively. 2 、18.73±3.27nV / deg 2 、19.76±4.61nV / deg 2 、24.61±4.67nV / deg 2 、17.52±4.89nV / deg 2 There was no significant change in the amplitude density of P1 wave at each time point after modeling compared with the adaptation period.

[0088] In summary, three crab-eating macaques (six eyes) were modeled according to the method of Comparative Example 1. The obtained crab-eating macaque model showed disordered structure of the outer retina and RPE layer in the photocoagulation area, and mild atrophy and thinning of the outer retina. However, the visual function reflected by the mfERG test showed no significant change compared with before modeling, indicating that the macular area in the temporal inferior part of the retina was selected for modeling under the laser parameters, and the retinal pigment epithelial injury model of crab-eating macaques could not be successfully constructed.

Claims

1. A method for establishing a non-human primate retinal pigment epithelium injury model, characterized in that: The method comprises the following steps: (1) Anesthetize the non-human primate and dilate the pupil; (2) fitting the panretinal scope to the cornea of ​​the nonhuman primate and adjusting the slit lamp microscope of the laser photocoagulator so that the operator can observe the fovea, optic disc, and superior and inferior vascular arcades of the nonhuman primate; (3) Photocoagulation of the photocoagulation area is performed using a laser photocoagulation device until 250-300 primary light spots appear in the photocoagulation area; the photocoagulation area is the area from the optic disc to the fovea and between the upper and lower vascular arches, avoiding the fovea; the parameters of the laser photocoagulation device are set as follows: laser wavelength is 530-534 nm, spot diameter is 190-210 μm, laser power is 65-95 mW, exposure time is 0.07-0.10 s, and energy of each laser spot is 4.6-9.5 mJ.

2. The method according to claim 1, characterized in that The non-human primate is a monkey.

3. The method according to claim 2, characterized in that The monkey is a cynomolgus monkey or a rhesus monkey.

4. The method according to claim 1, characterized in that: The parameters of the laser photocoagulation instrument are set as follows: the laser wavelength is 532 nm, the spot diameter is 200 μm, the laser power is 70-90 mW, the exposure time is 0.08-0.09 s, and the energy of each laser spot is 5.6-8.1 mJ.

5. The method according to claim 4, characterized in that The timing for ending photocoagulation is when 280 first-order light spots appear in the photocoagulation area.

6. The method according to any one of claims 1 to 5, characterized in that: The method further comprises the following steps: (4) Treat the operated eye to prevent infection.

7. Use of the non-human primate retinal pigment epithelial injury model established by the method described in any one of claims 1 to 6 for screening drugs for treating dry age-related macular degeneration caused by the retinal pigment epithelial injury model.

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