Temperature-sensitive hydrogel preparation method and chronic glaucoma model construction method

Abstract

The invention discloses a temperature-sensitive hydrogel preparation method and a chronic glaucoma model construction method, and belongs to the technical field of medicine preparation and disease animal model construction. The method has the advantages that the operation is simple, the induction period is short, the intraocular pressure is continuously and stably increased, the modeling success rate is high, a disease animal model which is more in line with the pathophysiological change of chronic open-angle glaucoma is established, the problems of damage, inflammatory reaction and the like of animal eye tissues caused by short-term multiple modeling operations are solved, and the method has high conversion value.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine preparation and disease animal model construction, and particularly relates to a method for preparing a thermosensitive hydrogel and a method for constructing a chronic glaucoma model. Background Art

[0002] Glaucoma is an irreversible degenerative disease of the optic nerve and the second leading cause of blindness in the world. It is characterized by the progressive irreversible death of retinal ganglion cells and visual field loss. Open-angle glaucoma accounts for 60-70% of all glaucoma types. Its complex pathogenesis is not yet very clear, and early diagnosis is difficult. Pathological increase in intraocular pressure is still considered to be one of the most dangerous factors in the occurrence and development of glaucomatous optic nerve damage, and lowering intraocular pressure is the only controllable treatment. In the field of glaucoma research, animal models that meet the characteristics of the disease have become an important research tool, which plays an important role in better understanding the damage mechanism and treatment measures of glaucomatous optic neuropathy. With the improvement of glaucoma induction methods and the improvement of glaucoma damage to the eye measurement and quantification methods, there are many glaucoma animal models induced by different mechanisms, which can reflect the characteristics of pathological and physiological damage changes in human glaucoma.

[0003] The existing animal models of ocular hypertension glaucoma include but are not limited to the following: (1) the model of injection of specific substances into the anterior and posterior chambers. This model blocks the outflow of aqueous humor from the trabecular meshwork, causing a sharp and unstable increase in intraocular pressure, which is short-lived and requires repeated injections within a short period of time. It is also prone to cause many complications (such as anterior chamber exudation inflammation, corneal ulcers, or even corneal endothelial decompensation, lens damage, iris damage or adhesions, etc.); (2) the model of laser photocoagulation of the trabecular meshwork, limbal vessels, and scleral vessels. This model is difficult to operate, expensive, and has a short duration of high intraocular pressure. Repeated laser photocoagulation is required to maintain long-term high intraocular pressure, but it will aggravate intraocular congestion and inflammatory reactions; (3) the vitreous drug induction method, which involves the intravitreal injection of corticosteroids, tumor necrosis factor β (TGF-β), etc., which may cause retinal toxicity, and frequent intraocular operations may cause endophthalmitis; (4) transgenic animal models, such as those with GpnmbR150X and The DBA / 2J mice related to Tyrp1isa and the Tg-MYOC mice related to Tyr437 have the characteristics of chronic high intraocular pressure, but the model construction cycle is long and the economic cost is high. The above are not ideal animal models of chronic high intraocular pressure open-angle glaucoma. Therefore, in order to reveal the pathological damage mechanism of primary open-angle glaucoma in humans and explore new methods for anti-glaucoma treatment, it is very necessary to construct a new glaucoma animal model that is closer to the characteristics of human chronic open-angle glaucoma.

[0004] Interferon (IFN) is a class of biologically active polypeptides that can regulate cell functions. It has antiviral and cell regulatory properties and is mainly involved in various physiological processes such as cell proliferation, immune system, and hematopoiesis. According to the different producing cells and biological activities, IFN can be divided into three categories: Type I IFN, mainly including IFN-α and IFN-β, which have rapid antiviral effects; Type II interferon, namely IFN-γ, also known as immune interferon, has strong anti-tumor and immunoregulatory activities; Type III IFN, namely IFN-λ, can induce local antiviral immunity. Studies have reported that DDX58 gene mutations have toxic effects on trabecular meshwork cells and affect the expression of trabecular meshwork cytoskeletal proteins, accompanied by a significant increase in type I IFN production. Studies have shown that the level of IFN-γ in the aqueous humor of patients with primary open-angle glaucoma is significantly increased. After the Zika virus is injected into the anterior chamber of mice to infect the trabecular meshwork, a large amount of IFN is induced, and a small The intraocular pressure of mice increased significantly. It was found that IFN-γ inhibited the activity of trabecular meshwork cells by regulating necroptosis, while IFN-β1 activated autophagy of trabecular meshwork. Injection of IFN-γ / IFN-β1 into the anterior chamber can cause increased intraocular pressure and further produce glaucomatous optic neuropathy. Due to the physiological circulation of aqueous humor and drug metabolism, a single injection of free IFN-γ / IFN-β1 cannot maintain a stable increase in intraocular pressure. Repeated injections are required every week or even shorter to maintain the effective drug concentration of IFN-γ / IFN-β1 in the anterior chamber. Multiple operations may cause artificial damage to the anterior segment tissue and aggravate the inflammatory response. Therefore, a slow-release thermosensitive hydrogel loaded with IFN-γ / IFN-β1 is provided. After the hydrogel is injected into the anterior chamber of the animal's eye, IFN-γ and IFN-β1 can be slowly released, which can reduce the impact of frequent operations on the anterior segment tissue of the animal's eye and construct a new animal model of chronic open-angle glaucoma. Summary of the invention

[0005] The purpose of the present invention is to propose a method for preparing a thermosensitive hydrogel and a method for constructing a chronic glaucoma model. The advantages of the present invention are that the establishment of an animal model of the disease is simple in operation, short induction period, continuous and stable increase in intraocular pressure, high modeling success rate, and more consistent with the pathophysiological changes of chronic open-angle glaucoma. It solves the problems of damage to animal eye tissues and inflammatory response caused by short-term and multiple modeling operations, and has transformation value.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: A method for preparing a thermosensitive hydrogel comprises the following steps:

[0007] S1. Synthesis, dialysis and freeze-drying of HA-MA;

[0008] 1) Synthesis of HA-MA: hyaluronic acid (2 g) was dissolved in deionized water (200 mL), and then methacrylic anhydride (15 ml) was immediately added, followed by NaOH (5 M), and the pH was adjusted to between 8 and 12, and the reaction was carried out at 2 to 6 °C for 12 hours;

[0009] 2) The synthetic modified HA-MA was dialyzed using a cellulose dialysis bag (8000-10000) for 4 days, with the water changed 3 times a day;

[0010] 3) Precooling the modified HA-MA to -80°C in advance, and then placing it in a freeze dryer for freeze drying;

[0011] S2. Synthesis, dialysis and freeze-drying of thermosensitive factors;

[0012] 1) Weigh 0.2 g of HA-MA after freeze-drying in step S1 and mix it with deionized water (180 mL) to fully dissolve it in a 250 mL round-bottom flask;

[0013] 2) Add 7 mL of 10 m / v% ethyl methacrylate and 7 mL of 10 m / v% potassium persulfate into a flask, and add 7 mL of 4 v / v% N, N, N'-tetramethyleneethylenediamine into the reaction flask after deoxygenation with nitrogen, maintain positive nitrogen pressure, and react at 20°C for 24 hours;

[0014] 3), dialyze the synthesized sample using a cellulose dialysis bag (8000-10000) for 4 days, changing the water three times a day;

[0015] 4) Precooling the synthesized thermosensitive factor at -80°C in advance, and then placing it in a freeze dryer for freeze drying;

[0016] S3. Conduct gelation experiments to determine gelation conditions and achieve injectability;

[0017] S4. Observe the microstructure of hydrogel using scanning electron microscopy;

[0018] S5. Determine the Fourier transform infrared spectroscopy image of the hydrogel;

[0019] S6. Determine the hydrogel swelling ratio to detect thermal sensitivity;

[0020] S7. Detection of hydrogel degradation rate;

[0021] S8. Synthesize drug-loaded sustained-release thermosensitive hydrogels and measure the release rate.

[0022] The present invention is further configured as follows: Step S3 specifically comprises the following steps:

[0023] 1) Using the inversion method, the prepared gel solution was placed in an incubator, and the temperature was raised from 30°C, and the heating rate was controlled at 1°C / min. The gel was inverted and tilted for observation every 30 seconds, and the angle with the horizontal plane was 60°. The absence of liquid flow within 30 seconds was used as the basis for gelation. The gelation temperature and gelation time were recorded. At the same time, the transaction temperature was determined to be 37°C. After the water bath was adjusted to 37°C, the gel was inverted and tilted for observation every 5 seconds, and the angle with the horizontal plane was 60°. The absence of liquid flow within 30 seconds was used as the basis for gelation. The gelation temperature and gelation time were recorded.

[0024] 2) The modified HA was dissolved in deionized water and prepared into different contents (1%, 3%, 5%, and the solid content was adjusted according to the actual gelation and microscopic morphology) to observe whether it can gel at 37°C and has a certain fluidity to achieve injectable properties.

[0025] The present invention is further configured as follows: Step S5 specifically comprises the following steps:

[0026] 1) The preparation of the test hydrogel sample is the same as S1 and S2. The sample is dry and does not contain water, and is larger than 0.5cm*0.5cm. After the sample preparation is completed, open the Fourier transform infrared spectroscopy platform software and connect the detection equipment, adjust the whiteboard scan parameters to meet the standard specifications, measure the Fourier transform infrared spectroscopy image of the sample, and record the results. Compare the experimental results for analysis and draw conclusions;

[0027] 2) The reaction uses potassium persulfate KPS as an initiator and TEMED as an initiator auxiliary. Due to the SO4- free radical-induced polymerization method, a thermosensitive hydrogel is prepared. The hydrogel has a characteristic peak of -OH at 3414 cm-1, which is due to the presence of -OH functional groups in HA-MA, a characteristic peak of -CH at 2910 cm-1, a strong characteristic absorption band at 1064 cm-1, which is the characteristic peak of COC, and a small absorption band at 1730 cm-1 and 1620 cm-1, which correspond to the positions of the characteristic peaks of -C=O and -NH respectively;

[0028] 3) The molecular structure and chemical groups of the chemical composition of the thermosensitive hydrogel are determined by detecting its absorption spectrum in the infrared region.

[0029] The present invention is further configured as follows: Step S6 specifically comprises the following steps:

[0030] 1) The swelling ratio of the gel was determined by weighing: the test hydrogel samples were prepared in the same way as S1 and S2, the freeze-dried samples were weighed and immersed in a phosphate buffer solution heated at 20-45°C, the measured hydrogel was swollen at the determined temperature for 24 hours, the excess water on the surface of the hydrogel was quickly wiped off with wet filter paper, and then weighed again;

[0031] 2) The equilibrium swelling ratio (ESR) of the hydrogel can be calculated using the following formula:

[0032] ESR=((Ws-Wd) / Wax100%

[0033] (Ws is the mass of the hydrogel after swelling equilibrium, and Wd is the mass of the xerogel before swelling);

[0034] 3) When the hydrogel reaches the physiological temperature of 37°C, the swelling degree of the hydrogel decreases and remains stable, which can slowly release the loaded drug.

[0035] The present invention is further configured as follows: Step S7 specifically comprises the following steps:

[0036] 1) The preparation of the test hydrogel samples was the same as S1 and S2. 500uL of hydrogel was immersed in 20mL of phosphate buffer solution (1×PBS, pH=7.4) and placed in a constant temperature water bath at 37°C. Fresh PBS solution was replaced every day. At the specified time intervals, the hydrogel was taken out and weighed;

[0037] 2) Calculation formula:

[0038] WR=(Wt / Wo)×100%

[0039] (Wherein: Wt is the weight of the gel after degradation, Wo is the initial weight of the gel);

[0040] 3) The hydrogel gradually degraded over time, reaching about 66.3% at 28 days, further demonstrating that the drug would be gradually released during the gradual degradation of the hydrogel over time.

[0041] The present invention is further configured as follows: Step S8 specifically comprises the following steps:

[0042] 1) Synthesize drug-loaded sustained-release thermosensitive hydrogels: According to the effects of IFNβ1 and IFNγ on trabecular meshwork cells, the concentrations of drugs to be embedded are 2500-5000 units / ml for IFN-β1 and 20-30000 units / ml for IFNγ, respectively, which are added to the hydrogel solution for in vitro drug release rate determination;

[0043] 2) In vitro drug release rate determination: 1 ml hydrogel samples loaded with different concentrations of IFN drugs were placed in dialysis bags, and then in 100 ml phosphate buffer solution (1×PBS, pH=7.4), placed in a 37°C constant temperature incubator, and stirred at 100 rpm. 50 μL of the released sample was drawn at specified time intervals and filled with 50 μL phosphate buffer to the original 100 mL buffer. After the samples were collected, the drug concentration was detected using an ELISA kit

[0044] 3) The release rate results showed that the release rate of 30000unit / ml IFN-γ reached 92.0% in 28 days, and the release rate of 5000unit / ml IFN-β1 reached 86.7% in 28 days, further proving that the drug-loaded hydrogel can maintain sustained drug release for about 1 month.

[0045] A method for constructing a chronic glaucoma model using a thermosensitive hydrogel comprises the following steps:

[0046] S1. Preparation before modeling;

[0047] S2. Establish animal model;

[0048] S3. The ocular changes induced by the animal model tested were consistent with the characteristics of chronic open-angle glaucoma.

[0049] The present invention is further configured as follows: Step S1 specifically comprises the following steps:

[0050] 1) Experimental animal preparation: C57BL / 6J mice, 6-8 weeks old, male, were selected as experimental animals and raised in a specific antigen-free (SPF) barrier environment with free access to food and water. The experiment began after 1 week of adaptive feeding. The feeding and operation strictly followed the ethical requirements of scientific research animals. The intraocular pressure was measured under inhalation anesthesia with a TonoLab rebound tonometer at 8-10 o'clock one day before the operation. The average intraocular pressure was recorded as the baseline intraocular pressure. All subsequent intraocular pressure measurements were performed by the same operator in the same time period.

[0051] 2) Preparation of drug-loaded sustained-release thermosensitive hydrogel: According to the required drug concentrations of IFN-β1 of 2500-5000unit / ml and IFNγ of 20-30000unit / ml, the in vitro degradation rate, release rate and the method of establishing an animal model in step S2 were combined, and the anterior chamber injection was performed on the animals in the model group.

[0052] The present invention is further configured as follows: Step S2 specifically comprises the following steps:

[0053] 1) Experimental animal grouping: the model group was injected with sustained-release thermosensitive hydrogel loaded with IFNγ and the group injected with sustained-release thermosensitive hydrogel loaded with IFN-β1 into the anterior chamber of the eye, and the control group was injected with thermosensitive hydrogel without drug loading into the anterior chamber of the eye;

[0054] 2) Anesthesia of experimental animals: 1% sodium pentobarbital anesthetic was injected into the abdominal cavity of mice in the model group and the control group at a dose of 40-50 mg / kg to induce general anesthesia. Sterile cotton swabs were dipped in iodine to disinfect the eyelid margin, trim the eyelashes, and expose the eyeballs. Sterile PBS or ofloxacin antibiotic eye drops were used to rinse the ocular surface, and then dry sterile cotton swabs were used to wipe off the residual liquid and other debris on the ocular surface. Before the operation, proparacaine hydrochloride eye drops were used for surface anesthesia of the eyes until the animals had no resistance to local stimulation of the whole body and the eyeballs. The anesthesia was considered satisfactory. The use of specific anesthetic drugs was not limited;

[0055] 3) Injection of hydrogel into the anterior chamber of experimental animals: fix the eyeball with micro toothless forceps, first make a lateral incision at the corneal edge with a 15° corneal puncture knife and gently press the posterior edge to release some aqueous humor, then use a micro-injector (range of 50μl) connected to a 30G needle to tilt 25° through the original corneal lateral incision to inject about 3μl of hydrogel with different drug loads, then slowly inject 1μl of air to ensure that the hydrogel is completely injected into the anterior chamber, forming a small bubble in the anterior chamber. After injection, the micro-injector needle is placed in an appropriate position in the anterior chamber for more than 30s, and then the needle is slowly withdrawn to allow the bubble to seal the corneal lateral incision by itself, thereby playing a role. To prevent the reflux of injected hydrogel, bubbles can be absorbed within a few hours. Care should be taken during the puncture and injection process to prevent the needle from piercing the iris and anterior capsule of the lens. After the operation, apply ofloxacin antibiotic eye ointment to the eyes to prevent infection. Place the mice on an animal temperature maintenance device to maintain body temperature and wait for natural recovery. After recovery, put them back into the animal breeding room. According to the degradation of the hydrogel in the anterior chamber of the eye and the fluctuation of intraocular pressure, different drug-loaded hydrogels are injected into the anterior chamber of the eye again to maintain the drug concentration in the anterior chamber of the eye. The modeling period is 3 months, and a stable chronic high intraocular pressure model is obtained.

[0056] The present invention is further configured as follows: Step S3 specifically comprises the following steps:

[0057] 1) Intraocular pressure detection: 1 day, 2 days, 3 days, 5 days, 7 days, 9 days, 11 days, 14 days, 17 days, 20 days, 24 days, and 28 days after modeling, if the intraocular pressure is stable, the intraocular pressure of mice can be measured using a TonoLab rebound tonometer twice a week at the same time period. According to the degradation of the hydrogel in the anterior chamber of the eye and the fluctuation of intraocular pressure, different drug-loaded hydrogels were injected into the anterior chamber of the eye again to maintain the drug concentration in the anterior chamber of the eye, and the measured intraocular pressure data were statistically analyzed for changes in intraocular pressure;

[0058] 2) Observation of the distribution of hydrogel in the anterior chamber: Immediately after modeling, and at 1, 2, 4, 6, 8, 10, and 12 weeks after modeling, the distribution of hydrogel in the anterior chamber of the eyes of the mice in the model group and the control group was observed using an ophthalmic ZEISS surgical microscope, and photographs were taken to analyze the hydrogel gelation state, degradation, and anterior segment inflammation-related manifestations;

[0059] 3) After the model was established, one group of experimental animals in the model group and the control group were killed at the end of the first month, the end of the second month, and the end of the third month. The intact eyeballs were taken for paraffin embedding and sectioning. After HE staining, they were photographed and observed under an optical microscope. The morphology of the cells in each layer of the cornea, the opening of the chamber angle (trabecular meshwork), the changes in the morphology and number of cells in each layer of the retina, the changes in the depth of the cup at the optic disc, and the atrophy of the retrobulbar optic nerve were compared between the model group and the control group.

[0060] 4) Retinal ganglion cell counting: After killing one group of experimental animals in the model group and the control group at the end of the first month, the second month, and the third month after modeling, the experimental animals were perfused by heart and the eyeballs were taken for retinal flat-mounting and RGCs immunostaining. The posterior pole, mid-periphery, and peripheral parts of the four quadrants of the retina (approximately 1 / 6, 1 / 2, and 5 / 6 of the retinal radius, respectively) were developed and photographed using a ZEISS microscope, and the immunolabeled RGCs were counted. The counting results were statistically analyzed to evaluate the loss of RGCs in the model group compared with the control group to understand the optic nerve damage in the high intraocular pressure model;

[0061] 5) Observation of the fine structure of the trabecular meshwork under transmission electron microscopy: After killing one group of experimental animals in the model group and the control group at the end of the first month, the second month, and the third month after modeling, 2mm*4mm iridocorneal tissue containing the trabecular meshwork was marked from the intact eyeball, fixed with 2.5% glutaraldehyde, and electron microscope specimens were prepared. The overall structure, growth state, and ultrastructural damage of the trabecular meshwork were observed under transmission electron microscopy. The comparison of the ultrastructural structure of the trabecular meshwork under transmission electron microscopy between the model group and the control group indicated that the damage and activity inhibition of IFNγ and IFN-β1 on the trabecular meshwork tissue may be involved in the pathogenesis of open-angle glaucoma;

[0062] 6) Observation of optic nerve axon degeneration by immunostaining of retrobulbar optic nerve axons: After one group of experimental animals in the model group and the control group were killed at the end of the first month, the second month, and the third month after modeling, the experimental animals were perfused with the heart and the optic nerve (ON) at 1.5-2.0 mm retrobulbar was cut, fixed in situ with 2% glutaraldehyde and 2% PFA, and sliced. After staining with 1% PPD, four slices of each ON were imaged through the 100x lens of the Zeiss M2 epifluorescence microscope to cover the entire area of ​​the ON without overlapping. Two 21.4μmX29.1μm areas were cut from the center of each image, and the surviving axons in the specified area were manually counted. After counting all images taken from a single nerve, the average number of surviving axons in each ON was calculated, and the average number of surviving neurons in the ON of the model group was compared with the average number of ON in the control group to obtain the percentage of axon survival value. The structural degeneration of ON axons between the two groups can also be compared.

[0063] In summary, the present invention has the following beneficial effects:

[0064] The establishment of an animal model of the disease has simple operation, short induction period, continuous and stable increase in intraocular pressure, high modeling success rate, and is more consistent with the pathophysiological changes of chronic open-angle glaucoma. It solves the problems of damage to animal eye tissue and inflammatory response caused by multiple short-term modeling operations, and has the advantage of high translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 is a schematic diagram of the characterization results of the sustained-release thermosensitive hydrogel of the present invention;

[0066] Figure 2 It is a schematic diagram of anterior segment photography after the hydrogel is injected into the anterior chamber of the eye of a mouse according to the present invention. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0068] Embodiment 1:

[0069] refer to Figure 1 , a method for preparing a thermosensitive hydrogel, comprising the following steps:

[0070] S1. Synthesis, dialysis and freeze-drying of HA-MA

[0071] 1) Synthesis of HA-MA (methacrylic anhydride modified hyaluronic acid), hyaluronic acid (2 g) was dissolved in deionized water (200 mL), and then methacrylic anhydride (15 ml) was immediately added, followed by NaOH (5 M), the pH was adjusted to between 8 and 12, and the reaction was carried out at 2-6°C for 12 hours;

[0072] 2) The synthetic modified HA-MA was dialyzed using a cellulose dialysis bag (8000-10000) for 4 days, with the water changed 3 times a day;

[0073] 3) The modified HA-MA was pre-cooled at -80°C and then placed in a freeze dryer for freeze drying.

[0074] S2. Synthesis, dialysis and freeze-drying of thermosensitive factors

[0075] 1) Weigh 0.2 g of HA-MA after freeze-drying in step S1 and mix it with deionized water (180 mL) to fully dissolve it in a 250 mL round-bottom flask;

[0076] 2) Add 7 mL of 10 m / v% ethyl methacrylate and 7 mL of 10 m / v% potassium persulfate into a flask, and add 7 mL of 4 v / v% N, N, N'-tetramethyleneethylenediamine into the reaction flask after deoxygenation with nitrogen, maintain positive nitrogen pressure, and react at 20°C for 24 hours;

[0077] 3) The synthesized sample was dialyzed using a cellulose dialysis bag (8000-10000) for 4 days, with the water changed 3 times a day.

[0078] 4) The synthesized thermosensitive factor is pre-cooled at -80°C and then placed in a freeze dryer for freeze drying.

[0079] S3. Conduct gelation experiments to determine gelation conditions and achieve injectability

[0080] 1) Using the inversion method, the prepared gel solution was placed in an incubator, and the temperature was raised from 30°C. The heating rate was controlled at 1°C / min. The solution was inverted and tilted for observation every 30 seconds, with an angle of 60° to the horizontal plane. The absence of liquid flow within 30 seconds was used as the basis for gelation. The gelation temperature and gelation time were recorded. At the same time, the transaction temperature was determined to be 37°C. After the water bath was adjusted to 37°C, the solution was inverted and tilted for observation every 5 seconds, with an angle of 60° to the horizontal plane. The absence of liquid flow within 30 seconds was used as the basis for gelation. The gelation temperature and gelation time were recorded. The results showed that the hydrogel could form a gel in 20 seconds at 37°C in vitro.

[0081] 2) The modified HA was dissolved in deionized water and prepared into different contents (1%, 3%, 5%, and the solid content was adjusted according to the actual gelation and micromorphology), and observed whether it could gel at 37°C and have a certain fluidity to achieve injectable properties. The results showed that 1% HA hydrogel did not gel, and 5% HA hydrogel could gel but had poor fluidity. The final gelation content was determined to be 3% HA.

[0082] S4. Observation of hydrogel microstructure using scanning electron microscopy

[0083] In this test, the preparation of hydrogel samples is the same as S1. The hydrogel cross-section samples are obtained by cutting with scissors or breaking with liquid nitrogen. The samples are directly glued to the sample stage with conductive glue for testing. The vacuum is drawn after degassing. When a certain vacuum degree is reached, the voltage is increased and the sample stage is raised to a suitable height. The sample position is found and the image is scanned according to the shooting magnification and image scale. The results are shown in the figure: the hydrogel shows a network structure, which is consistent with the loose and porous structural characteristics of the hydrogel. The drug can shuttle through the carrier by diffusion to achieve drug release.

[0084] S5. Determination of Fourier transform infrared spectroscopy images of hydrogels

[0085] 1) The preparation of the hydrogel sample in this test is the same as S1 and S2. The sample is dry and does not contain water, and is larger than 0.5cm*0.5cm. After the sample preparation is completed, open the Fourier transform infrared spectroscopy imaging platform software and connect the detection equipment, adjust the whiteboard scanning parameters to meet the standard specifications, measure the Fourier transform infrared spectroscopy image of the sample, and record the results. Compare the experimental results for analysis and draw conclusions;

[0086] 2) The reaction uses potassium persulfate KPS as an initiator and TEMED as an initiator auxiliary. The thermosensitive hydrogel is prepared by SO4- free radical-induced polymerization. The infrared results are as follows Figure 1 As shown in C, the hydrogel has a characteristic peak of -OH at 3414 cm-1, which is due to the existence of -OH functional groups in HA-MA, a characteristic peak of -CH at 2910 cm-1, a strong characteristic absorption band at 1064 cm-1, which is the characteristic peak of COC, and a small absorption band at 1730 cm-1 and 1620 cm-1, which correspond to the positions of the characteristic peaks of -C=O and -NH, respectively;

[0087] 3) By detecting the absorption spectrum of the thermosensitive hydrogel in the infrared region to determine its molecular structure and chemical groups of chemical composition, it can be shown that the addition of the thermosensitive factor to the hydrogel does not affect the infrared light spectrum waveform of the hydrogel, proving that the overall chemical bonds in the system have not changed.

[0088] S6. Determination of hydrogel swelling ratio to detect thermal sensitivity

[0089] 1) The swelling ratio of the gel was determined by weighing: In this test, the hydrogel samples were prepared in the same way as S1 and S2. The freeze-dried samples were weighed and immersed in a phosphate buffer solution heated at 20-45°C. After the measured hydrogel was swollen at the determined temperature for 24 hours, the excess water on the surface of the hydrogel was quickly wiped off with wet filter paper and weighed again;

[0090] 2) The equilibrium swelling ratio (ESR) of the hydrogel can be calculated using the following formula:

[0091] ESR=((Ws-Wd) / Wax100%

[0092] (Ws is the mass of the hydrogel after swelling equilibrium, and Wd is the mass of the xerogel before swelling);

[0093] 3) The swelling curve is shown in the figure: when the hydrogel reaches the physiological temperature of 37°C, the swelling degree of the hydrogel decreases and remains stable, which can play a role in slowly releasing the loaded drug.

[0094] S7. Detection of hydrogel degradation rate

[0095] 1) The preparation of hydrogel samples in this test is the same as S1 and S2. 500uL of hydrogel is immersed in 20mL of phosphate buffer solution ((1×PBS, pH=7.4)), placed in a constant temperature water bath at 37°C, and fresh PBS solution is replaced every day. At the specified time interval, the hydrogel is taken out and weighed;

[0096] 2) Calculation formula:

[0097] WR=(Wt / Wo)×100%

[0098] (Wherein: Wt is the weight of the gel after degradation, Wo is the initial weight of the gel);

[0099] 3) The hydrogel gradually degraded over time, reaching about 66.3% at 28 days, further demonstrating that the drug would be gradually released during the gradual degradation of the hydrogel over time.

[0100] S8. Synthesis of drug-loaded sustained-release thermosensitive hydrogels and determination of release rates

[0101] 1) Synthesize drug-loaded sustained-release thermosensitive hydrogel: Based on the previous studies conducted by this project team, the effects of IFNβ1 and IFNγ on trabecular meshwork cells have been explored. The concentrations of drugs to be embedded are 2500-5000unit / ml for IFN-β1 and 20-30000unit / ml for IFNγ, respectively. They are added to the hydrogel solution for in vitro drug release rate determination;

[0102] 2) In vitro drug release rate determination: 1 ml hydrogel samples loaded with different concentrations of IFN drugs were placed in dialysis bags, and then in 100 ml phosphate buffer solution (1×PBS, pH=7.4), placed in a constant temperature incubator at 37°C, and stirred at 100 rpm. 50 μL of the released sample was drawn at specified time intervals and filled with 50 μL phosphate buffer to the original 100 mL buffer. After the samples were collected, the drug concentration was detected using an ELISA kit (the samples were stored at -20°C after collection, and the ELISA test samples were processed at 4°C to avoid IFN degradation affecting the experimental results);

[0103] 3) The release rate results showed that the release rate of 30000unit / ml IFN-γ reached 92.0% in 28 days, and the release rate of 5000unit / ml IFN-β1 reached 86.7% in 28 days, further proving that the drug-loaded hydrogel can maintain sustained drug release for about 1 month.

[0104] Embodiment 2:

[0105] A method for constructing a chronic glaucoma model using a thermosensitive hydrogel comprises the following steps:

[0106] S1. Preparation before modeling

[0107] 1) Experimental animal preparation: C57BL / 6J mice, 6-8 weeks old, male, were selected as experimental animals and raised in a specific antigen-free (SPF) barrier environment with free access to food and water. The experiment began after 1 week of adaptive feeding. The feeding and operation strictly followed the ethical requirements of scientific research animals. The intraocular pressure was measured under inhalation anesthesia with a TonoLab rebound tonometer at 8-10 o'clock one day before the operation. The average intraocular pressure was recorded as the baseline intraocular pressure. All subsequent intraocular pressure measurements were performed by the same operator in the same time period.

[0108] 2) Preparation of drug-loaded sustained-release thermosensitive hydrogel: The preparation method is the same as S1, S2, and S8 in Example 1. According to the required loaded drug concentrations, IFN-β1 is 2500-5000unit / ml, and IFNγ is 20-30000unit / ml. Combined with the in vitro degradation rate, release rate and the S2 animal model establishment method in Example 2, the model group animals were injected into the anterior chamber of the eye.

[0109] S2. Animal model establishment method

[0110] 1) Experimental animal grouping: the model group was injected with sustained-release thermosensitive hydrogel loaded with IFNγ and the group injected with sustained-release thermosensitive hydrogel loaded with IFN-β1 into the anterior chamber of the eye, and the control group was injected with thermosensitive hydrogel without drug loading into the anterior chamber of the eye;

[0111] 2) Anesthesia of experimental animals: 1% sodium pentobarbital anesthetic was injected into the abdominal cavity of mice in the model group and the control group at a dose of 40-50 mg / kg to induce general anesthesia. Sterile cotton swabs were dipped in iodine to disinfect the eyelid margin, trim the eyelashes, and expose the eyeballs. Sterile PBS or ofloxacin antibiotic eye drops were used to rinse the ocular surface, and then dry sterile cotton swabs were used to wipe off the residual liquid and other debris on the ocular surface. Before the operation, proparacaine hydrochloride eye drops were used for surface anesthesia of the eyes until the animals had no resistance to local stimulation of the whole body and the eyeballs. The anesthesia was considered satisfactory. The use of specific anesthetic drugs was not limited;

[0112] 3) Injection of hydrogel into the anterior chamber of experimental animals: fix the eyeball with micro toothless forceps, first make a lateral incision at the corneal edge with a 15° corneal puncture knife and gently press the posterior edge to release some aqueous humor, then use a micro-injector (range of 50μl) connected to a 30G needle to inject about 3μl of hydrogel with different drug loads through the original corneal lateral incision at an angle of 25°, then slowly inject 1μl of air to ensure that the hydrogel is completely injected into the anterior chamber, forming a small bubble in the anterior chamber. After injection, the micro-injector needle is placed in an appropriate position in the anterior chamber for more than 30s, and then the needle is slowly withdrawn to allow the bubble to seal the corneal lateral incision by itself. The incision prevents the injected hydrogel from flowing back. The bubbles can be absorbed within a few hours. Care should be taken during the puncture and injection process to prevent the needle from piercing the iris and anterior capsule of the lens. After the operation, ofloxacin antibiotic eye ointment is applied to the eyes to prevent infection. The mice are placed on an animal temperature maintenance device to maintain body temperature and wait for natural recovery. After recovery, they are returned to the animal breeding room. According to the degradation of the hydrogel in the anterior chamber of the eye and the fluctuation of intraocular pressure, different drug-loaded hydrogels are injected into the anterior chamber of the eye again to maintain the drug concentration in the anterior chamber of the eye. The modeling period is 3 months, and a stable chronic high intraocular pressure model is obtained.

[0113] S3. The ocular changes induced by the animal model were consistent with the characteristics of chronic open-angle glaucoma

[0114] 1) Intraocular pressure detection: 1 day, 2 days, 3 days, 5 days, 7 days, 9 days, 11 days, 14 days, 17 days, 20 days, 24 days, and 28 days after modeling in the model group and control group mice, if the intraocular pressure is stable, the mouse intraocular pressure can be measured using a TonoLab rebound tonometer twice a week at the same time period. According to the degradation of the hydrogel in the anterior chamber of the eye and the fluctuation of intraocular pressure, different drug-loaded hydrogels are injected into the anterior chamber of the eye again to maintain the drug concentration in the anterior chamber of the eye, and the measured intraocular pressure data are statistically analyzed for changes in intraocular pressure. The results showed that after modeling, the intraocular pressure of the model group mice increased steadily and maintained a high value, while the intraocular pressure of the control group mice remained at the baseline intraocular pressure fluctuation without a significant increase. There was a statistical difference between the two groups, indicating that this modeling method can cause chronic high intraocular pressure;

[0115] 2) Observation of the distribution of hydrogel in the anterior chamber: Immediately after modeling, and at 1, 2, 4, 6, 8, 10, and 12 weeks after modeling, the distribution of hydrogel in the anterior chamber of the mice in the model group and the control group was observed using an ophthalmic ZEISS surgical microscope, and photographs were taken to analyze the hydrogel gelation state, degradation, and anterior segment inflammation-related manifestations. The results showed that the hydrogel was evenly distributed in the anterior chamber of the mice in the model group and the control group after modeling (e.g. Figure 2 a, b), and then gelled. Four weeks after modeling, some of the hydrogel remained in the anterior chamber of the eye (e.g. Figure 2c, d), indicating that the hydrogel slowly degrades over time and the embedded drug can slowly release the drug. In addition, no obvious inflammatory infiltration was found in the anterior segment tissues of the mice including the cornea, conjunctiva, iris, anterior chamber, and lens at various time points;

[0116] 3) After the modeling was established, one group of experimental animals in the model group and the control group were killed at the end of the first month, the end of the second month, and the end of the third month, and the complete eyeballs were taken for paraffin embedding and sectioning. After HE staining, they were photographed and observed using an optical microscope. The morphology of cells in each layer of the cornea, the openness of the chamber angle (trabecular meshwork), the changes in the morphology and number of cells in each layer of the retina, the changes in the depth of the cup at the optic disc, and the atrophy of the retrobulbar optic nerve were compared between the model group and the control group. The results showed that there was no significant difference in the morphology of each layer of the cornea between the model group and the control group. The trabecular meshwork structure could be observed at the chamber angle, which could be judged as an open chamber angle. Compared with the control group, the thickness of the entire retina in the model group became thinner, the number of retinal ganglion cells decreased, and the cup at the optic disc deepened and the retrobulbar optic nerve atrophy could be observed in some cases. The above performance indicated that the model was consistent with the eyeball changes of open-angle glaucoma.

[0117] 4) Retinal ganglion cell counting: After killing one group of experimental animals in the model group and the control group at the end of the first month, the second month, and the third month after modeling, the experimental animals were perfused by heart and the eyeballs were taken for retinal flat-mounting and RGCs immunostaining. The posterior pole, mid-periphery, and peripheral parts of the four quadrants of the retina (approximately 1 / 6, 1 / 2, and 5 / 6 of the retinal radius, respectively) were developed and photographed using a ZEISS microscope, and the immunolabeled RGCs were counted. The counting results were statistically analyzed to evaluate the loss of RGCs in the model group compared with the control group to understand the optic nerve damage in the high intraocular pressure model;

[0118] 5) Observation of the fine structure of the trabecular meshwork under transmission electron microscopy: After killing one group of experimental animals in the model group and the control group at the end of the first month, the second month, and the third month after modeling, 2mm*4mm iridocorneal tissue containing the trabecular meshwork was marked from the intact eyeball, fixed with 2.5% glutaraldehyde, and electron microscope specimens were prepared. The overall structure, growth state, and ultrastructural damage of the trabecular meshwork were observed under transmission electron microscopy. The comparison of the ultrastructural structure of the trabecular meshwork under transmission electron microscopy between the model group and the control group indicated that the damage and activity inhibition of IFNγ and IFN-β1 on the trabecular meshwork tissue may be involved in the pathogenesis of open-angle glaucoma;

[0119] 6) Observation of optic nerve axon degeneration by immunostaining of retrobulbar optic nerve axons: After one group of experimental animals in the model group and the control group were killed at the end of the first month, the second month, and the third month after modeling, the experimental animals were perfused with the heart and the optic nerve (ON) at 1.5-2.0 mm retrobulbar was cut, fixed in situ with 2% glutaraldehyde and 2% PFA, and sliced. After staining with 1% PPD, four slices of each ON were imaged through the 100x lens of the Zeiss M2 epifluorescence microscope to cover the entire area of ​​the ON without overlapping. Two 21.4μmX29.1μm areas were cut from the center of each image, and the surviving axons in the specified area were manually counted. After counting all images taken from a single nerve, the average number of surviving axons in each ON was calculated, and the average number of surviving neurons in the ON of the model group was compared with the average number of ON in the control group to obtain the percentage of axon survival value. The structural degeneration of ON axons between the two groups can also be compared.

[0120] The present invention also provides an innovative anterior chamber drug delivery method, which utilizes methacrylic anhydride-modified hyaluronic acid (HA-MA) to embed IFN-γ (20-30000 Unit / ml) and IFN-β1 (2500-5000 Unit / ml) in a thermosensitive hydrogel to achieve a single local injection and maintain long-term release. The drug-loaded thermosensitive hydrogel can rapidly gel, stably swell, slowly degrade, and gradually release drugs at a physiological temperature of 37°C, and this process can be maintained for at least 28 days. Among them, IFN-γ inhibits the activity of trabecular meshwork cells by regulating necroptosis of trabecular meshwork, IFN-β1 inhibits the activity of trabecular meshwork cells by activating autophagy of trabecular meshwork, and neither IFN-γ nor IFN-β1 causes obstruction of aqueous humor outflow and increased intraocular pressure by causing changes in the anatomical structure of the aqueous humor outflow tract. The hydrogel does not block the anatomical structure of the chamber angle after gelation. After the drug-loaded thermosensitive hydrogel is injected into the anterior chamber of the animal's eye, it can slowly release IFN-γ and IFN-β1, reduce the effects of frequent operations on the anterior segment tissues of the mouse cornea, iris, lens, etc., and prepare a model with a significant increase in intraocular pressure and a stable maintenance, an open chamber angle, which is consistent with glaucomatous optic nerve changes and gradually progresses over time, thereby constructing a new animal model of chronic open-angle glaucoma.

[0121] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for preparing a thermosensitive hydrogel, characterized in that: The following steps are involved: S1. Synthesis, dialysis and freeze-drying of HA-MA; 1) Synthesis of HA-MA: hyaluronic acid (2 g) was dissolved in deionized water (200 mL), and then methacrylic anhydride (15 ml) was immediately added, followed by NaOH (5 M), and the pH was adjusted to between 8 and 12, and the reaction was carried out at 2 to 6 °C for 12 hours; 2) The synthetic modified HA-MA was dialyzed using a cellulose dialysis bag (8000-10000) for 4 days, with the water changed 3 times a day; 3) Precooling the modified HA-MA to -80°C in advance, and then placing it in a freeze dryer for freeze drying; S2. Synthesis, dialysis and freeze-drying of thermosensitive factors; 1) Weigh 0.2 g of HA-MA after freeze-drying in step S1 and mix it with deionized water (180 mL) to fully dissolve it in a 250 mL round-bottom flask; 2) Add 7 mL of 10 m / v% ethyl methacrylate and 7 mL of 10 m / v% potassium persulfate into a flask, and add 7 mL of 4 v / v% N, N, N'-tetramethyleneethylenediamine into the reaction flask after deoxygenation with nitrogen, maintain positive nitrogen pressure, and react at 20°C for 24 hours; 3), dialyze the synthesized sample using a cellulose dialysis bag (8000-10000) for 4 days, changing the water three times a day; 4) Precooling the synthesized thermosensitive factor at -80°C in advance, and then placing it in a freeze dryer for freeze drying; S3. Conduct gelation experiments to determine gelation conditions and achieve injectability; S4. Observe the microstructure of hydrogel using scanning electron microscopy; S5. Determine the Fourier transform infrared spectroscopy image of the hydrogel; S6. Determine the hydrogel swelling ratio to detect thermal sensitivity; S7. Detection of hydrogel degradation rate; S8. Synthesize drug-loaded sustained-release thermosensitive hydrogels and measure the release rate.

2. A method for preparing a thermosensitive hydrogel according to claim 1, characterized in that: The specific steps of step S3 are as follows: 1) Using the inversion method, the prepared gel solution was placed in an incubator, and the temperature was raised from 30°C, and the heating rate was controlled at 1°C / min. The gel was inverted and tilted for observation every 30 seconds, and the angle with the horizontal plane was 60°. The absence of liquid flow within 30 seconds was used as the basis for gelation. The gelation temperature and gelation time were recorded. At the same time, the transaction temperature was determined to be 37°C. After the water bath was adjusted to 37°C, the gel was inverted and tilted for observation every 5 seconds, and the angle with the horizontal plane was 60°. The absence of liquid flow within 30 seconds was used as the basis for gelation. The gelation temperature and gelation time were recorded. 2) The modified HA was dissolved in deionized water and prepared into different contents (1%, 3%, 5%, and the solid content was adjusted according to the actual gelation and microscopic morphology) to observe whether it can gel at 37°C and has a certain fluidity to achieve injectable properties.

3. The method for preparing a thermosensitive hydrogel according to claim 1, characterized in that: The specific steps of step S5 are as follows: 1) The preparation of the test hydrogel sample is the same as S1 and S2. The sample is dry and does not contain water, and is larger than 0.5cm*0.5cm. After the sample preparation is completed, open the Fourier transform infrared spectroscopy platform software and connect the detection equipment, adjust the whiteboard scan parameters to meet the standard specifications, measure the Fourier transform infrared spectroscopy image of the sample, and record the results. Compare the experimental results for analysis and draw conclusions; 2) The reaction uses potassium persulfate KPS as an initiator and TEMED as an initiator auxiliary. Due to the SO4- free radical-induced polymerization method, a thermosensitive hydrogel is prepared. The hydrogel has a characteristic peak of -OH at 3414 cm-1, which is due to the presence of -OH functional groups in HA-MA, a characteristic peak of -CH at 2910 cm-1, a strong characteristic absorption band at 1064 cm-1, which is the characteristic peak of COC, and a small absorption band at 1730 cm-1 and 1620 cm-1, which correspond to the positions of the characteristic peaks of -C=O and -NH respectively; 3) The molecular structure and chemical groups of the chemical composition of the thermosensitive hydrogel are determined by detecting its absorption spectrum in the infrared region.

4. The method for preparing a thermosensitive hydrogel according to claim 1, characterized in that: The specific steps of step S6 are as follows: 1) The swelling ratio of the gel was determined by weighing: the test hydrogel samples were prepared in the same way as S1 and S2, the freeze-dried samples were weighed and immersed in a phosphate buffer solution heated at 20-45°C, the measured hydrogel was swollen at the determined temperature for 24 hours, the excess water on the surface of the hydrogel was quickly wiped off with wet filter paper, and then weighed again; 2) The equilibrium swelling ratio (ESR) of the hydrogel can be calculated using the following formula: ESR=((Ws-Wd) / Wax100% (Ws is the mass of the hydrogel after swelling equilibrium, and Wd is the mass of the xerogel before swelling); 3) When the hydrogel reaches the physiological temperature of 37°C, the swelling degree of the hydrogel decreases and remains stable, which can slowly release the loaded drug.

5. The method for preparing a thermosensitive hydrogel according to claim 1, characterized in that: The specific steps of step S7 are as follows: 1) The preparation of the test hydrogel samples was the same as S1 and S2. 500uL of hydrogel was immersed in 20mL of phosphate buffer solution (1×PBS, pH=7.4) and placed in a constant temperature water bath at 37°C. Fresh PBS solution was replaced every day. At the specified time intervals, the hydrogel was taken out and weighed; 2) Calculation formula: WR=(Wt / Wo)×100% (Wherein: Wt is the weight of the gel after degradation, Wo is the initial weight of the gel); 3) The hydrogel gradually degraded over time, reaching about 66.3% at 28 days, further demonstrating that the drug would be gradually released during the gradual degradation of the hydrogel over time.

6. The method for preparing a thermosensitive hydrogel according to claim 1, characterized in that: The specific steps of step S8 are as follows: 1) Synthesize drug-loaded sustained-release thermosensitive hydrogels: According to the effects of IFNβ1 and IFNγ on trabecular meshwork cells, the concentrations of drugs to be embedded are 2500-5000 units / ml for IFN-β1 and 20-30000 units / ml for IFNγ, respectively, which are added to the hydrogel solution for in vitro drug release rate determination; 2) In vitro drug release rate determination: 1 ml hydrogel samples loaded with different concentrations of IFN drugs were placed in dialysis bags, and then in 100 ml phosphate buffer solution (1×PBS, pH=7.4), and placed in a constant temperature incubator at 37°C and stirred at 100 rpm. 50 μL of the released sample was drawn at specified time intervals and filled with 50 μL phosphate buffer to the original 100 mL buffer. After the samples were collected, the drug concentration was detected using an ELISA kit; 3) The release rate results showed that the release rate of 30000unit / ml IFN-γ reached 92.0% in 28 days, and the release rate of 5000unit / ml IFN-β1 reached 86.7% in 28 days, further proving that the drug-loaded hydrogel can maintain sustained drug release for about 1 month.

7. A method for constructing a chronic glaucoma model using a thermosensitive hydrogel according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. Preparation before modeling; S2. Establish animal model; S3. The ocular changes induced by the animal model tested were consistent with the characteristics of chronic open-angle glaucoma.

8. The method for constructing a chronic glaucoma model using a thermosensitive hydrogel according to claim 7, characterized in that: The specific steps of step S1 are as follows: 1) Experimental animal preparation: C57BL / 6J mice, 6-8 weeks old, male, were selected as experimental animals and raised in a specific antigen-free (SPF) barrier environment with free access to food and water. The experiment began after 1 week of adaptive feeding. The feeding and operation strictly followed the ethical requirements of scientific research animals. The intraocular pressure was measured under inhalation anesthesia with a TonoLab rebound tonometer at 8-10 o'clock one day before the operation. The average intraocular pressure was recorded as the baseline intraocular pressure. All subsequent intraocular pressure measurements were performed by the same operator in the same time period. 2) Preparation of drug-loaded sustained-release thermosensitive hydrogel: According to the required drug concentrations of IFN-β1 of 2500-5000unit / ml and IFNγ of 20-30000unit / ml, the in vitro degradation rate, release rate and the method of establishing an animal model in step S2 were combined, and the anterior chamber injection was performed on the animals in the model group.

9. The method for constructing a chronic glaucoma model using a thermosensitive hydrogel according to claim 7, characterized in that: The specific steps of step S2 are as follows: 1) Experimental animal grouping: the model group was injected with sustained-release thermosensitive hydrogel loaded with IFNγ and the group injected with sustained-release thermosensitive hydrogel loaded with IFN-β1 into the anterior chamber of the eye, and the control group was injected with thermosensitive hydrogel without drug loading into the anterior chamber of the eye; 2) Anesthesia of experimental animals: 1% sodium pentobarbital anesthetic was injected into the abdominal cavity of mice in the model group and the control group at a dose of 40-50 mg / kg to induce general anesthesia. Sterile cotton swabs were dipped in iodine to disinfect the eyelid margin, trim the eyelashes, and expose the eyeballs. Sterile PBS or ofloxacin antibiotic eye drops were used to rinse the ocular surface, and then dry sterile cotton swabs were used to wipe off the residual liquid and other debris on the ocular surface. Before the operation, proparacaine hydrochloride eye drops were used for surface anesthesia of the eyes until the animals had no resistance to local stimulation of the whole body and the eyeballs. The anesthesia was considered satisfactory. The use of specific anesthetic drugs was not limited; 3) Injection of hydrogel into the anterior chamber of experimental animals: fix the eyeball with micro toothless forceps, first make a lateral incision at the corneal edge with a 15° corneal puncture knife and gently press the posterior edge to release some aqueous humor, then use a micro-injector (range of 50μl) connected to a 30G needle to inject about 3μl of hydrogel with different drug loads through the original corneal lateral incision at an angle of 25°, then slowly inject 1μl of air to ensure that the hydrogel is completely injected into the anterior chamber, forming a small bubble in the anterior chamber. After injection, the micro-injector needle is placed in an appropriate position in the anterior chamber for more than 30s, and then the needle is slowly withdrawn to allow the bubble to seal the corneal lateral incision by itself. The incision prevents the injected hydrogel from flowing back. The bubbles can be absorbed within a few hours. Care should be taken during the puncture and injection process to prevent the needle from piercing the iris and anterior capsule of the lens. After the operation, ofloxacin antibiotic eye ointment is applied to the eyes to prevent infection. The mice are placed on an animal temperature maintenance device to maintain body temperature and wait for natural recovery. After recovery, they are returned to the animal breeding room. According to the degradation of the hydrogel in the anterior chamber of the eye and the fluctuation of intraocular pressure, different drug-loaded hydrogels are injected into the anterior chamber of the eye again to maintain the drug concentration in the anterior chamber of the eye. The modeling period is 3 months, and a stable chronic high intraocular pressure model is obtained.

10. The method for constructing a chronic glaucoma model using a thermosensitive hydrogel according to claim 7, characterized in that: The specific steps of step S3 are as follows: 1) Intraocular pressure detection: 1 day, 2 days, 3 days, 5 days, 7 days, 9 days, 11 days, 14 days, 17 days, 20 days, 24 days, and 28 days after modeling, if the intraocular pressure is stable, the intraocular pressure of mice can be measured using a TonoLab rebound tonometer twice a week at the same time period. According to the degradation of the hydrogel in the anterior chamber of the eye and the fluctuation of intraocular pressure, different drug-loaded hydrogels were injected into the anterior chamber of the eye again to maintain the drug concentration in the anterior chamber of the eye, and the measured intraocular pressure data were statistically analyzed for changes in intraocular pressure; 2) Observation of the distribution of hydrogel in the anterior chamber: Immediately after modeling, and at 1, 2, 4, 6, 8, 10, and 12 weeks after modeling, the distribution of hydrogel in the anterior chamber of the eyes of the mice in the model group and the control group was observed using an ophthalmic ZEISS surgical microscope, and photographs were taken to analyze the hydrogel gelation state, degradation, and anterior segment inflammation-related manifestations; 3) After the model was established, one group of experimental animals in the model group and the control group were killed at the end of the first month, the end of the second month, and the end of the third month, and the intact eyeballs were taken for paraffin embedding and sectioning. After HE staining, the eyes were photographed and observed using an optical microscope. The morphology of the cells in each layer of the cornea, the opening of the chamber angle (trabecular meshwork), the changes in the morphology and quantity of the cells in each layer of the retina, the changes in the depth of the cup at the optic disc, and the atrophy of the retrobulbar optic nerve were compared between the mice in the model group and the control group; 4) Retinal ganglion cell counting: After killing one group of experimental animals in the model group and the control group at the end of the first month, the second month, and the third month after modeling, the experimental animals were perfused by heart and the eyeballs were taken for retinal flat-mounting and RGCs immunostaining. The posterior pole, mid-periphery, and peripheral parts of the four quadrants of the retina (approximately 1 / 6, 1 / 2, and 5 / 6 of the retinal radius, respectively) were developed and photographed using a ZEISS microscope, and the immunolabeled RGCs were counted. The counting results were statistically analyzed to evaluate the loss of RGCs in the model group compared with the control group to understand the optic nerve damage in the high intraocular pressure model; 5) Observation of the fine structure of the trabecular meshwork under transmission electron microscopy: After killing one group of experimental animals in the model group and the control group at the end of the first month, the second month, and the third month after modeling, 2mm*4mm iridocorneal tissue containing the trabecular meshwork was marked from the intact eyeball, fixed with 2.5% glutaraldehyde, and electron microscope specimens were prepared. The overall structure, growth state, and ultrastructural damage of the trabecular meshwork were observed under transmission electron microscopy. The comparison of the ultrastructural structure of the trabecular meshwork under transmission electron microscopy between the model group and the control group indicated that the damage and activity inhibition of IFNγ and IFN-β1 on the trabecular meshwork tissue may be involved in the pathogenesis of open-angle glaucoma; 6) Observation of optic nerve axon degeneration by immunostaining of retrobulbar optic nerve axons: After one group of experimental animals in the model group and the control group were killed at the end of the first month, the second month, and the third month after modeling, the experimental animals were perfused with the heart and the optic nerve (ON) at 1.5-2.0 mm retrobulbar was cut, fixed in situ with 2% glutaraldehyde and 2% PFA, and sliced. After staining with 1% PPD, four slices of each ON were imaged through the 100x lens of the Zeiss M2 epifluorescence microscope to cover the entire area of ​​the ON without overlapping. Two 21.4μmX29.1μm areas were cut from the center of each image, and the surviving axons in the specified area were manually counted. After counting all images taken from a single nerve, the average number of surviving axons in each ON was calculated, and the average number of surviving neurons in the ON of the model group was compared with the average number of ON in the control group to obtain the percentage of axon survival value. The structural degeneration of ON axons between the two groups can also be compared.

Images