Preparation method of tree shrew glaucoma model

Abstract

The invention discloses a preparation method of a tree shrew glaucoma model, which comprises the following steps of: firstly, dissolving 25 micrograms of okadaic acid in 250 microliters of normal saline under a sterile condition to prepare a 0.1 microgram / microliter stock solution; the stock solution obtained in the step (1) is diluted with normal saline according to the proportion of 1: 5 and then injected to the tree shrew double-eye vaulted conjunctiva, the injection amount of each eye is 5 microliters on the first day and 3 microliters on the eighth day, and model manufacturing is completed after regular feeding is conducted for 21 days after administration is finished. According to the method, okadaic acid is injected to the vault conjunctiva of the two eyes of the tree shrew twice, and the specific anatomical-metabolic characteristics of the tree shrew are deeply coupled with a double-time-phase administration scheme, so that the three technical bottlenecks of'uncontrollable pathological process' (monkey), 'anatomical structure distortion '(rabbit) and'molecular mechanism deviation' (rodent) of a traditional model are successfully overcome; a first standardized animal model construction scheme with physiological correlation and operation economy is provided for glaucoma transformation medical research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for preparing a tree shrew glaucoma model. Background Art

[0002] Glaucoma is a complex neurodegenerative disease caused by the combined action of multiple factors. Its pathological mechanism involves multiple key links such as abnormal aqueous humor dynamics, progressive apoptosis of retinal ganglion cells (RGCs), and optic nerve axon injury. To deeply analyze the molecular pathways of glaucoma neurodegeneration and develop targeted treatment strategies, it is crucial to establish an animal model that can systematically simulate the pathological characteristics of human glaucoma and has high operability. The current mainstream glaucoma animal models have the following technical defects: (1) Non-human primate models: ① The aqueous humor production inhibition method (such as betaxolol eye drops) requires continuous administration for ≥6 months to induce RGCs loss > 20%, and the modeling period is too long; ② The laser photocoagulation method of the trabecular meshwork causes the elevation of inflammatory factor IL-6 > 300 pg / mL, leading to secondary angle closure; ③ The anterior chamber perfusion pressure method needs to maintain the intraocular pressure > 50 mmHg for 1 hour to cause optic nerve injury, which is out of line with the pathological process of human chronic glaucoma; ④ The porosity of the macaque lamina cribrosa (78 ± 5%) is significantly higher than that of humans (62 ± 4%), resulting in distortion of the axonal mechanical stress distribution. (2) Rabbit models: ① The method of injecting methylcellulose into the anterior chamber requires injecting > 20 μL of viscoelastic agent, causing the anterior chamber volume to expand to 1.5 times the normal value; ② Injection of α-chymotrypsin into the scleral venous sinus causes permanent damage to the aqueous humor outflow pathway and cannot simulate early compensatory regulation; ③ The apoptosis of RGCs induced by the retinal ischemia-reperfusion method is concentrated in the inner retina (apoptosis rate > 80%), which does not conform to the specific axonal retrograde death pattern of glaucoma. (3) Rodent models: ① The microbead injection method needs to be repeated every 14 days (cumulative > 5 times) to maintain the intraocular pressure > 25 mmHg, and the cumulative operation trauma leads to a corneal opacity rate > 30%; ② The gene modification model (such as DBA / 2J mice) has a spontaneous glaucoma process of up to 12 months, and the C57BL / 6 background strain is prone to retinal vascular compensation; ③ Although the rodent optic nerve transection model can quickly induce RGCs death (loss > 90% in 3 days), it completely loses the dose-effect relationship between intraocular pressure and nerve injury. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for preparing a tree shrew glaucoma model.

[0004] The purpose of the present invention is achieved as follows, including the following steps: (1) Preparation of okadaic acid solution: Under sterile conditions, 25 μg of okadaic acid is dissolved in 250 μL of physiological saline to prepare a 0.1 μg / μL stock solution; (2) Dilute the stock solution in step (1) with normal saline at a ratio of 1:5, and then give intrabulbar conjunctival injection to the tree shrews. The injection volume for each eye is 5 μL on the first day and 3 μL on the eighth day. After the administration, raise them routinely for 21 days to complete the model making.

[0005] Compared with the prior art, the present invention has the following technical effects: In the present invention, okadaic acid is injected into the fornix conjunctiva of the eyes of tree shrews in two times. Compared with the preparation of traditional glaucoma models, the present invention uses tree shrews as glaucoma animal models for the first time. Its advantages are that the circadian rhythm of tree shrews is similar to that of humans, and the optic nerve papilla structure is highly similar to that of humans, which can accurately simulate the degenerative changes of retinal ganglion cells (RGCs) caused by increased intraocular pressure in glaucoma patients, significantly superior to rodent models; the sclera of tree shrews and humans is mainly composed of type I collagen fibers microscopically, and is more physiologically relevant in the study of glaucoma-related scleral remodeling mechanisms; in terms of retinal structure, photoreceptor cells are composed of 95% cone cells and 5% rod cells, and the most dense cone cells are located in the center of the retina, similar to the fovea centralis of the human retina. It has good color discrimination ability and stereoscopic vision, can effectively evaluate the progressive damage of glaucoma to visual function, and tree shrews show similar phenomena of vitreous cavity elongation and scleral thinning to humans when intraocular pressure increases, providing an ideal observation window for studying the axial pathological changes of glaucoma. In addition, by deeply coupling the unique anatomical-metabolic characteristics of tree shrews with a dual-phase dosing regimen, the present invention successfully overcomes the three major technical bottlenecks of traditional models, namely "uncontrollable pathological process" (monkeys), "distorted anatomical structure" (rabbits), and "deviation of molecular mechanism" (rodents), providing the first standardized animal model construction plan with both physiological relevance and operational economy for glaucoma translational medicine research. Description of the Drawings

[0006] Figure 1 It is a comparison chart of the changes in intraocular pressure (IOP) of 4 groups of tree shrews during the experimental process of this application; Figure 2 It is a comparison chart of the HE staining results of the trabecular meshwork of 4 groups of tree shrews during the experimental process of this application; Figure 3 It is a comparison chart of the HE staining results of the retinas of 4 groups of tree shrews during the experimental process of this application; (where: PEL: pigment epithelium layer; LRC: rod and cone layer; OLM: outer limiting membrane; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; LGC: ganglion cell layer; LOF: optic nerve fiber layer; ) Figure 4 It is a comparison chart of the expression of RBPMS in the retinas of 4 groups of tree shrews during the experimental process of this application; Figure 5This is the WB comparison diagram of Myocilin protein expression in the anterior segment tissues of 4 groups of tree shrews during the experiment of this application; Figure 6 This is the comparison diagram of the gray value ratio of Myocilin protein expression in the anterior segment tissues of 4 groups of tree shrews during the experiment of this application. Detailed implementation manners

[0007] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited in any way. Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0008] 1. Experimental animals 12 common male tree shrews (Tupaia belangeri chinensis) with a body weight of 120 - 150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023 - 0003] and were housed in the ordinary - grade animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021 - 0003]. They were kept in stainless - steel cages at a room temperature of (24 ± 2) °C, a relative humidity of (50 ± 5) %, and a light / dark cycle of 12 h / 12 h.

[0009] 2. Main reagents and consumables Reagent Name Company Okadaic Acid, Ammonium Salt Sigma FAS Eye Fixative Seville Paraformaldehyde Guangfu Hematoxylin and Eosin (HE) Staining Kit Solarbio Blocking Goat Serum Zhongshan Golden Bridge RBPMS Antibody Abcam Goat Anti-Rabbit IgG H&L (DyLight® 488) Abcam myocilin Antibody Abcam GAPDH Antibody Abcam Anti-rabbit IgG CST RIPA Lysis Buffer (Strong) Beyotime Prestained Protein Marker Thermo Fisher Bovine Serum Albumin (BSA) Sigma SurperECLPlus Ultra-sensitive Luminescence Pulilai 3. Main instruments Instrument Name Company Automatic Tissue Dehydrator Wuhan Junjie Rotary Microtome Leica Tissue Spreader Leica Paraffin Embedding Machine Leica Microscope OLYMPUS BX53 Nanophotometer Ultra-micro Spectrophotometer IMPLEN Protein Electrophoresis Cell, Electrotransfer Cell, Power Supply Bio-Rad ECL Luminescence Imager Tanon 4. Preparation of reagents Preparation of okadaic acid solution: Under sterile conditions, 25 μg of okadaic acid was dissolved in 250 μL of physiological saline to prepare a 0.1 μg / μL stock solution. When in use, the okadaic acid stock solution was diluted at ratios of 1:20, 1:10, and 1:5 according to the experimental groups, and the concentrations of the diluted okadaic acid working solutions were 5 ng / μL, 10 ng / μL, and 20 ng / μL respectively.

[0010] 5. Experimental methods 5.1 Preparation of animal models Experimental groups: ① Sham operation group (sham), ② Okadaic acid (25 ng) group, ③ Okadaic acid (50 ng) group, ④ Okadaic acid (100 ng) group The tree shrews were administered drugs in both eyes. In group ①, each tree shrew received an injection of sterile physiological saline (5 μL on the 1st day and 3 μL on the 8th day) into the fornix conjunctiva of each eye. In groups ② - ④, each tree shrew received an injection of okadaic acid solutions with concentrations of 5 ng / μL, 10 ng / μL, and 20 ng / μL into the fornix conjunctiva of each eye according to the grouping (5 μL on the 1st day and 3 μL on the 8th day). After the drug administration, the animals were continued to be raised for 21 days.

[0011] 5.2 Administration method of okadaic acid The tree shrews were anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 2 μl / g body weight. After complete anesthesia, topical anesthesia of the eyes was performed with oxybuprocaine hydrochloride eye drops. The periorbital area was disinfected with povidone-iodine solution and repeatedly rinsed with sterile normal saline. The tree shrews were fixed under a stereomicroscope, and the lower eyelids were separated to expose the inferior bulbar conjunctiva and fornix conjunctiva below. A 10-μL microsyringe with a 33-gauge needle was inserted into the fornix conjunctiva to inject the drug into the conjunctiva. The control group of tree shrews was injected with the same volume of normal saline at the same site. After injection, the needle was slowly withdrawn, and tobramycin eye ointment was applied to the injection site and the surrounding area before returning the tree shrews to the cage.

[0012] 5.3 Intraocular pressure measurement Before and on the 1st, 7th, 14th, 21st, and 28th days after modeling, intraocular pressure of the tree shrews was measured. The specific operation was as follows: After the tree shrews were anesthetized, topical anesthesia of both eyes was given with oxybuprocaine hydrochloride eye drops. The IOP of both eyes was measured with an Icare TonoLab rebound tonometer. The probe of the tonometer needed to be perpendicular to the corneal plane and aligned with the center of the cornea. Each eye was measured 5 times and the average value was taken. If the error was large, the measurement was repeated and the average value was taken and recorded.

[0013] 5.4 HE staining After the administration of drugs to the tree shrews, an excessive amount of 3% sodium pentobarbital was intraperitoneally injected, and the animals were euthanized. The eyeballs of the tree shrews were carefully removed using an ophthalmic microscope and placed in an eyeball fixative solution for fixation at 4°C for 24 h. The eyeballs were dehydrated in a gradient of 75%, 85%, 90%, 95%, and 100% ethanol in sequence, and then the eyeballs were placed in xylene for clarification; the dehydrated and clarified eyeballs were infiltrated with paraffin to replace the clarifying agent xylene in the eyeballs, followed by paraffin embedding, sectioning, and subsequent HE staining.

[0014] The paraffin sections were dewaxed to water, stained with hematoxylin at room temperature for 3 - 5 min; washed with tap water for 10 min to remove the floating color, differentiated with 1% hydrochloric acid alcohol for 2 s; rinsed with tap water for 10 min, and after observing that the cell nuclei were blue-violet under the microscope, stained with eosin at room temperature for 5 min; dehydrated in gradient alcohol, clarified in xylene, and sealed with neutral gum. The color development results were observed and photographed under an ordinary optical microscope.

[0015] 5.5 Immunofluorescence staining of retinal flat mounts After anesthetizing the tree shrews to death with an overdose, the eyeballs were quickly removed and placed in a 4% paraformaldehyde solution for fixation on ice for half an hour. Under a stereomicroscope, the redundant adipose and fascial tissues, cornea, iris, lens, and vitreous around the globe were removed with fiber instruments. The optic nerve was transected, and the intact retinal tissue was carefully isolated and cut into a four-lobed petal shape, then placed in 4% paraformaldehyde for continued fixation for 2 hours. The retina was transferred to an EP tube, rinsed with PBST, blocked with 5% goat serum for 90 minutes, and then incubated with the primary antibody (RBPMS, 1:300) at 4°C overnight. After rinsing with PBST, it was fixed again with 4% paraformaldehyde at room temperature for 10 minutes. After rinsing with PBST, the secondary antibody solution diluted with the blocking solution (Goat Anti-Rabbit IgG H&L DyLight® 488, 1:1000) was added and incubated in the dark at room temperature for 90 minutes. After the incubation was completed, the retinal tissue was adsorbed and flattened on the retina after rinsing with PBS, and an anti-fluorescence quencher was added for mounting. The experimental results were observed and photographed under a fluorescence microscope.

[0016] 5.6 Western blot detection After anesthetizing the tree shrews to death with an overdose, the eyeballs were quickly removed and placed on ice. The anterior segment tissues were separated and placed in a centrifuge tube containing RIPA lysis buffer (containing 50 μL protease inhibitor). After thoroughly cutting the tissue with a sterile ophthalmic scissors, it was homogenized in an ice bath environment with an ultrasonic cell disruptor for 20 s and then left to lyse on ice for 20 min. Centrifuge at 12,000 rpm at 4°C for 10 min, collect the supernatant. Approximately 360 μL of the supernatant was aspirated from each tube, and 90 μL of 5× loading buffer (1 / 4 of the supernatant volume) was added to each tube. After thorough mixing, it was centrifuged instantaneously for 10 s and then boiled in a 100°C metal bath for 10 min to fully denature the protein. After the sample cooled, the protein concentration was measured. Then SDS-PAGE protein electrophoresis was performed. Electrophoresis was carried out at a constant voltage of 80 V until the lower edge of the sample contacted the separating gel (about 30 min), and then continued at a constant voltage of 130 V for nearly 2 h until the bromophenol blue just overflowed from the lower edge of the separating gel and then the electrophoresis was stopped; Wet transfer was carried out at 280 mA / 1 h for membrane transfer. It was blocked with 5% skim milk at room temperature for 2 h, incubated with the primary antibody (rabbit anti-myocilin antibody 1:1000) in a 4°C refrigerator with a silent mixer overnight, washed with TBST buffer 3 times, incubated with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000) at room temperature for 2 h, washed with TBST buffer 3 times, and developed with an ECL reagent in a gel imaging system using chemiluminescence method.

[0017] 6. Experimental results 6.1 Intraocular pressure changes in tree shrews The intraocular pressure (IOP) of the tree shrews in the Sham group remained basically the same before and after modeling, with little change. Compared with the Sham group, the IOP of the Okadaic acid group showed an increasing trend on the 1st, 7th, 14th, 21st, and 28th days after modeling, and it was dose-dependent.

[0018] Table 1 Results of intraocular pressure (IOP) of the four groups of tree shrews during the experiment ( , n = 6) The trabecular meshwork tissue morphology of the tree shrews in the normal group was composed of irregular, porous lamellar substances, similar to a porous sponge structure, and aqueous humor could flow out from the porous trabecular meshwork tissue; after giving okadaic acid, the trabecular meshwork structure changed. After injecting 25 ng of okadaic acid, the trabecular meshwork gradually aggregated, and the gaps between the trabecular beams decreased; after injecting 50 ng of okadaic acid, this change was more obvious, some trabecular beams aggregated, and the gaps disappeared; after injecting 100 ng of okadaic acid, the gaps between the trabecular beams almost completely disappeared, and the trabecular meshwork became thickened.

[0019] 6.3 Retinal morphological changes The retinal morphology of the tree shrews in the normal group was normal, with clear stratification, neat cell arrangement, and a large number of cell nuclei in the LGC layer; after giving okadaic acid, the retinal structure changed. After injecting 25 ng of okadaic acid, the retinal structure was basically the same as that of the normal group, and only a slight decrease in the number of cells in the LGC layer was observed; after injecting 50 ng of okadaic acid, the structures of the LGC layer, INL layer, ONL layer, and LRC layer were significantly damaged, the cell arrangement was chaotic, and cell necrosis and cavities appeared; after injecting 100 ng of okadaic acid, the retinal thickness was significantly thinned, the number of cells in the LGC layer, INL layer, and ONL layer decreased significantly, cell necrosis occurred, and the structural changes of the retina were more obvious than those of the group.

[0020] 6.4 Retinal flat mounts To further verify the damage of okadaic acid to the retina, we performed immunofluorescence staining on the retinal flat mounts of the four groups of tree shrews and used the RNA-binding protein with multiple splicing (RBPMS) to label the retinal ganglion cells (RGCs). The results showed that compared with the normal group, the number of RGCs in the retina of tree shrews decreased significantly after giving okadaic acid, and it was dose-dependent. As the dosage of okadaic acid increased, the reduction of RGCs became more and more serious, and the most RGCs died after injecting 100 ng of okadaic acid.

[0021] 6.5 Western blot detection Western blot was used to measure the protein level of Myocilin in the anterior segment tissues of tree shrews. The results showed that, compared with the sham group, the protein expression level of Myocilin in the anterior segment tissues of tree shrews increased significantly after intravitreal injection of okadaic acid, and showed a dose-dependent manner. After injection of 100 ng okadaic acid solution, the expression level of Myocilin in the anterior segment tissues of tree shrews was the highest. The abnormal expression of Myocilin protein is closely related to glaucoma, and its high expression will lead to the dysfunction of trabecular meshwork cells, thereby affecting aqueous humor outflow and intraocular pressure regulation.

[0022] Table 2 Changes in the expression of Myocilin protein in 4 groups of tree shrews during the experiment Experimental Grouping Myocilin / GAPDH Sham 0.584±0.204 25 ng 0.705±0.183 50 ng 0.954±0.081 100 ng 1.164±0.096 The technical principle of the present invention is as follows: The circadian rhythm of tree shrews is similar to that of humans, and the structure of the optic nerve head is highly similar to that of humans. It can accurately simulate the degenerative changes of retinal ganglion cells (RGCs) caused by increased intraocular pressure in glaucoma patients, which is significantly better than rodent models; the sclera of tree shrews and humans is mainly composed of type I collagen fibers microscopically, and is more physiologically relevant in the study of glaucoma-related scleral remodeling mechanisms; in terms of retinal structure, photoreceptor cells are composed of 95% cone cells and 5% rod cells, and the most densely packed cone cells are located in the center of the retina, similar to the fovea centralis of the human retina. It has good color discrimination ability and stereoscopic vision, can effectively evaluate the progressive damage of glaucoma to visual function, and tree shrews show similar phenomena of vitreous cavity elongation and scleral thinning to humans when intraocular pressure increases, providing an ideal observation window for studying the axial pathological changes of glaucoma.

[0023] Specifically: 1. Anatomical adaptability of double-phase injection of fornix conjunctiva (1) Targeted delivery: The volume of the subfornix conjunctival space in tree shrews (8.3 ± 1.2 μL) is precisely matched with the injection dose (5 μL for the first time + 3 μL for the second time) (verified by MRI contrast), avoiding drug reflux (the incidence rate < 5%, significantly lower than 23% in rats); (2) Temporal control: High-dose impact (50 ng - 100 ng) on the first day induces the depolymerization of the trabecular meshwork cytoskeleton (the fluorescence intensity of F-actin drops to 52 ± 6% of the baseline), and the supplementary dose (30 ng - 60 ng) on the eighth day maintains the contractile phenotype of TM cells by inhibiting PP2A (the expression of α-SMA is up-regulated 3.1 times), simulating the progressive increase in aqueous humor outflow resistance in human glaucoma in two stages.

[0024] 2. Dose-site specificity advantage of okadaic acid (1)Metabolic advantage: The elimination half-life of okadaic acid by CYP3A4 in the tree shrew liver (t1 / 2 = 9.2 h) is significantly longer than that in rats (t1 / 2 = 4.1 h, p < 0.01), allowing administration at 7-day intervals without causing systemic toxicity (serum ALT level maintained at 25 ± 3 U / L, normal range 20 - 40 U / L); (2)Local effect: The injection site in the upper temporal quadrant corresponds to the area of mechanical stress concentration on the lamina cribrosa (finite element analysis shows that the von Mises stress in this area reaches 1.8 MPa), making the distribution of RGCs axonal damage highly consistent with the characteristic arcuate defect of human glaucoma (similarity index of HRT-III optic disc analysis is 0.87).

[0025] 3. Breakthrough in controllability of pathological process (1)Dynamic regulation window: The time window for the second injection (day 8) precisely corresponds to the conversion node from the intraocular pressure compensation period to the decompensation period in tree shrews (the turning point of the decreasing slope of aqueous humor outflow facility appears at 6.5 ± 0.8 days); (2)Molecular regulation network: RNA-seq shows that the down-regulation amplitude of miR-29b induced by this protocol (to 34 ± 5% of the baseline level) is completely synchronized with the up-regulation trend of the fibrosis markers (COL1A1 / COL3A1) in the aqueous humor outflow pathway of human glaucoma (Pearson r = 0.93 in Cluster analysis).

[0026] 4. Comparison of technical effects (based on cross-species Meta-analysis) Evaluation Index Non-human Primate Model Rabbit Model Rodent Model The Tree Shrew Model of the Present Invention Modeling Period (days) 180-360 28-42 14-60 21 Specificity of RGCs Loss Diffuse Full-thickness Necrosis Perivascular Concentration Arcuate Defect Standard Deviation of Intraocular Pressure Fluctuation 4.2 mmHg 6.8 mmHg 5.5 mmHg 1.9 mmHg Cost of Histological Verification $12,000 / sample $3,500 / sample $800 / sample $1,200 / sample Repeatability Index 0.61 0.43 0.72 0.89

Claims

1. A method for preparing a tree shrew glaucoma model, characterized in that, It includes the following steps: (1) Preparation of okadaic acid solution: Under sterile conditions, 25 μg of okadaic acid is dissolved in 250 μL of physiological saline to prepare a stock solution of 0.1 μg / μL; (2) Dilute the stock solution in step (1) with physiological saline at a ratio of 1:5 and then give intrascleral injection to tree shrews. The injection volume for each eye is 5 μL on the first day and 3 μL on the eighth day. After the administration, the tree shrews are routinely bred for 21 days to complete the model production.

2. The preparation method of a tree shrew glaucoma model according to claim 1, characterized in that, The tree shrews used are common-grade male Tupaia belangeri chinensis with a body weight of 120-150 g.

3. The preparation method of a tree shrew glaucoma model according to claim 1, characterized in that, The tree shrews are housed in stainless steel cages at a room temperature of 24±2°C, a relative humidity of 50±5%, and a light / dark cycle of 12 h / 12 h.

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