A method for constructing an animal model of dry eye

Abstract

The application discloses a kind of construction methods of dry eye animal model, belong to the field of biological medicine technology.The method is induced in the way of threefold synergies of low concentration benzalkonium chloride local eye drops, controllable dry environment feeding, systemic injection of hydriodic acid anisodine, simultaneously intervenes from three core pathological links of tear fluid generation reduction, tear fluid evaporation too fast, ocular surface barrier damage, can construct stable mixed dry eye animal model for 10-21 days continuous induction.The model constructed by the application simultaneously has the typical characteristics of tear secretion reduction, tear film stability decline, corneal epithelial damage, conjunctival goblet cell loss and low chronic inflammation, and is highly matched with clinical mixed dry eye;Model success rate is high, animal tolerance is good, without surgical trauma, solve the defects of single induction model, such as single pathological feature, poor stability, excessive damage or long cycle, can be widely applied to dry eye pathogenesis research, treatment drug screening and drug efficacy evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to experimental animal model construction technology, specifically to a method for constructing an animal model of dry eye syndrome. Background Technology

[0002] Dry eye syndrome, also known as keratoconjunctivitis sicca, is a multifactorial ocular surface disease characterized by tear film homeostasis imbalance, accompanied by ocular discomfort, ocular surface inflammation, and sensory abnormalities. It is one of the most prevalent ocular surface diseases worldwide. Furthermore, with increased use of electronic devices, an aging population, and rising environmental pollutants, its incidence is increasing annually, making it a significant public health issue affecting visual quality and quality of life.

[0003] Based on the pathogenesis, dry eye syndrome is usually classified into five major categories in clinical practice: insufficient tear production, excessive evaporation, mucin deficiency, abnormal tear dynamics, and mixed type. Among them, mixed type dry eye syndrome accounts for more than 80% of clinical cases. Patients have multiple pathological changes such as reduced tear secretion, excessive tear evaporation, and damage to the ocular surface barrier. The course of the disease is chronic and progressive, accompanied by low-grade chronic inflammation of the ocular surface.

[0004] Animal models are core tools for studying the pathogenesis of dry eye disease, screening therapeutic drugs, and evaluating drug efficacy. Currently reported methods for constructing animal models of dry eye disease can be mainly divided into four categories: environmentally induced, chemically induced, surgically induced, and autoimmune.

[0005] Environmentally induced dry eye models primarily simulate dry environments that accelerate tear evaporation by artificially controlling humidity, temperature, and airflow speed, thus constructing evaporative dry eye syndrome. The advantage of this type of model is that the pathological process closely resembles clinical evaporative dry eye, without exogenous chemical or surgical trauma. However, it has significant drawbacks: first, the model construction period is long, typically requiring 4-6 weeks of continuous dry exposure to form a stable dry eye phenotype; second, there is significant individual variation in the model, significantly influenced by the animal's own tear secretion capacity, resulting in a success rate typically below 60%; and third, it can only simulate a single pathological factor of excessive evaporation and cannot reproduce the characteristics of clinically common mixed-type dry eye.

[0006] Among chemically induced models, benzalkonium chloride (BAC) eye drops are the most widely used method. Benzalkonium chloride is a quaternary ammonium cationic surfactant that can disrupt the tight junctions between corneal epithelial cells, damage the ocular surface barrier, and induce ocular surface inflammation. It can also damage conjunctival goblet cells, reducing mucin secretion. This method is simple to operate and has a short construction cycle, typically allowing observation of the dry eye phenotype within 1-2 weeks. However, it has significant limitations: First, high concentrations of benzalkonium chloride can cause acute chemical corneal damage, triggering severe acute inflammatory reactions, and even leading to corneal ulcers and perforation, which differs significantly from the low-grade inflammation and progressive damage pathological characteristics of clinical chronic dry eye. Second, it only acts locally on the ocular surface and has no significant effect on lacrimal gland secretion function; it can only simulate ocular surface-damaged dry eye and cannot reproduce the pathological changes of reduced tear secretion.

[0007] Surgically induced dry eye models primarily reduce tear production directly by removing the main lacrimal gland, destroying the meibomian glands, or blocking the lacrimal gland secretion ducts, thus creating a tear-deficiency dry eye. While these models demonstrate a clear tear reduction effect and good stability, they involve significant surgical trauma, high animal mortality, and, being acute tear loss, cannot simulate the clinical process of chronic, progressive tear function decline. Furthermore, the surgical procedures are highly complex, demanding advanced skills from the experimenters, and individual model variations are significantly influenced by the surgical technique.

[0008] Autoimmune models are mainly used to simulate dry eye caused by autoimmune diseases such as Sjögren's syndrome. They are usually constructed through immune induction or gene editing. The models have a high degree of matching with pathological features, but the construction cycle is as long as several months, the success rate is low, and the operation is complicated, making them unsuitable for large-scale drug screening studies.

[0009] To address the shortcomings of single-induction models, there are a few reports on combined induction models in the existing technology, such as benzalkonium chloride combined with dry environment induction and partial lacrimal gland resection combined with benzalkonium chloride induction. However, existing combined models still have significant deficiencies: First, using only two factors combined cannot fully simulate the triple pathological characteristics of mixed dry eye; second, the parameter settings have not been systematically optimized, resulting in either excessive damage deviating from the chronic disease course or mild phenotypes leading to model instability; third, there is a lack of systematic evaluation of core pathological indicators such as inflammatory characteristics and goblet cell changes in the model, resulting in insufficient validation of model effectiveness.

[0010] Therefore, developing a method for constructing an animal model of dry eye that can highly simulate the pathological characteristics of clinical mixed dry eye disease, has a moderate construction cycle, high model stability, is easy to operate, and does not require gene editing or surgical procedures has important scientific research value and application prospects. Summary of the Invention

[0011] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for constructing an animal model of dry eye syndrome. This method employs a triple synergistic induction approach, which combines topical application of low-concentration benzalkonium chloride, controlled dry environment feeding, and systemic intervention with anticholinergic drugs. This approach constructs a mixed-type animal model of dry eye syndrome that can simultaneously simulate reduced tear secretion, excessive tear evaporation, ocular surface barrier damage, and chronic inflammation. It has the advantages of typical pathological features, high model stability, short construction period, simple operation, and good animal tolerance.

[0012] The objective of this invention can be achieved through the following technical solutions:

[0013] A method for constructing an animal model of dry eye disease includes the following steps:

[0014] S1 Selection and Adaptation of Laboratory Animals: SPF-grade healthy adult rodents were selected and adapted to living in a standard environment for 3-7 days, excluding individuals with underlying eye diseases.

[0015] S2 triple synergistic induction intervention: Simultaneous use of three methods: local eye drops of low-concentration benzalkonium chloride solution, controlled parameter dry environment feeding intervention, and systemic administration of anticholinergic drugs, for 10-21 consecutive days;

[0016] S3 Model Validation and Evaluation: After induction, the model was validated using tear fluid parameters, ocular surface morphology parameters, histopathological parameters, and inflammatory factor parameters to obtain a stable animal model of dry eye.

[0017] As a preferred technical solution of the present invention, the experimental animals in step S1 are 6-8 week old male BALB / c mice with a weight of 18-22g; the adaptive feeding conditions are: temperature 22-26℃, relative humidity 45%-55%, 12h light-dark cycle, and free access to food and water.

[0018] As a preferred embodiment of the present invention, the mass-volume concentration of the low-concentration benzalkonium chloride solution in step S2 is 0.08%-0.15%; the eye drops are administered 3-5 times daily, with 5-10 μL per eye each time, and the interval between two eye drops is at least 3 hours.

[0019] As a preferred embodiment of the present invention, the benzalkonium chloride solution has a mass-volume concentration of 0.1%, and is administered as 8 μL of eye drops four times a day for 14 consecutive days.

[0020] As a preferred technical solution of the present invention, the feeding conditions of the controllable parameter drying environment in step S2 are as follows: ambient temperature 22-26℃, relative humidity 25%-35%, horizontal airflow speed 1.5-2.5m / s, continuous dry exposure for 12-18 hours per day, and feeding in a standard environment for the remaining time.

[0021] As a preferred embodiment of the present invention, the drying environment parameters are: temperature 24±1℃, relative humidity 30%±2%, wind speed 2.0m / s, and daily drying exposure for 16 hours.

[0022] As a preferred embodiment of the present invention, the anticholinergic drug in step S2 is scopolamine hydrobromide; the administration method is subcutaneous injection in the neck and back, the dosage is 0.3-0.8 mg / kg body weight, twice a day, with an interval of 8-10 hours between the two administrations.

[0023] As a preferred technical solution of the present invention, the three intervention methods are started simultaneously in step S2, the daily eye drop operation and subcutaneous injection operation are spaced at least 30 minutes apart, and there is no time conflict between dry environment exposure and eye drop operation.

[0024] As a preferred technical solution of the present invention, the model validation indicators in step S3 specifically include: tear secretion volume, tear film breakup time, corneal fluorescein staining score, conjunctival goblet cell count, corneal epithelial pathological score, and the content of IL-1β and TNF-α in corneal tissue.

[0025] Application of an animal model of dry eye syndrome prepared according to the above construction method in the screening and pharmacodynamic evaluation of drugs for the treatment of dry eye syndrome

[0026] As a preferred technical solution of the present invention,

[0027] The beneficial effects of this invention are:

[0028] (1) This invention combines the ocular surface barrier damage effect of low concentration benzalkonium chloride, the tear evaporation enhancement effect of dry environment, and the tear secretion inhibition effect of scopolamine. It simultaneously intervenes in the three core pathological links of reduced tear production, excessive tear evaporation, and ocular surface barrier damage, and successfully constructs a mixed dry eye model that simultaneously has insufficient tear secretion, excessive evaporation, ocular surface damage and chronic inflammation, which makes up for the deficiency of the single or dual induction model with single pathological characteristics.

[0029] (2) This invention uses a low concentration of benzalkonium chloride of 0.08%-0.15%, combined with a dry environment and systemic drug intervention, to avoid the acute chemical damage and severe inflammatory response caused by high concentration of benzalkonium chloride. The model inflammatory response is in a low-grade chronic state, and the corneal damage progressively worsens, which is highly similar to the chronic course of clinical dry eye disease. It is more suitable for mechanism research and long-term efficacy evaluation of chronic dry eye.

[0030] (3) This invention reduces the influence of individual animal differences on a single factor through the synergistic effect of three factors, and the success rate of model construction can reach more than 95%. The coefficient of variation of each indicator within individuals is less than 10%, which is significantly better than the existing single induction model. A stable dry eye phenotype can be formed after 14 days of induction, and the phenotype can be stably maintained for at least 7 days after the induction is stopped, which meets the time requirements of various experiments.

[0031] (4) This invention does not require surgical operation. The model can be constructed by eye drops, environmental control and subcutaneous injection. The operation is simple and suitable for batch construction. Animals have no surgical trauma and the mortality rate is low. It is significantly better than surgical induction model and high concentration chemical induction model and meets the ethical requirements of experimental animals. Detailed Implementation

[0032] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0033] 1. Experimental Materials

[0034] 1.1 Laboratory Animals

[0035] SPF-grade male BALB / c mice, 6-8 weeks old, weighing 18-22g. All animals were housed in an SPF-grade animal facility under standard conditions: temperature 24±1℃, relative humidity 50%±5%, 12h / 12h light / dark cycle, and free access to sterile feed and sterilized drinking water. Animals were acclimatized for 3 days prior to the experiment, and individuals with eye abnormalities were excluded by slit-lamp microscopy examination.

[0036] 1.2 Some reagents

[0037] Periodic acid-Schiff (PAS) staining kit (Beijing Solarbio Science & Technology Co., Ltd.); Hematoxylin-eosin (HE) staining kit (Beijing Solarbio Science & Technology Co., Ltd.); Mouse IL-1β ELISA kit (Wuhan Boster Biological Engineering Co., Ltd.); Mouse TNF-α ELISA kit (Wuhan Boster Biological Engineering Co., Ltd.); Sodium pentobarbital (Merck, Germany).

[0038] 2. General testing methods

[0039] All experimental animals underwent the following index tests after induction. All tests were performed by the same experimenter to reduce operational errors. Mice were allowed to acclimatize in a dark room for 10 minutes before the tests.

[0040] 2.1 Measurement of tear secretion (Schirmer I test)

[0041] Ocular surface anesthesia was performed using 0.4% oxybuprocaine hydrochloride eye drops. After the anesthesia took effect, the Schirmer test strip was held with sterile forceps, one end folded 1 mm, and gently placed in the outer 1 / 3 of the conjunctival sac of the lower eyelid of the mouse, allowing the test strip to hang naturally outside the eyelid. The eyelid was gently closed to prevent the test strip from falling off. After accurately timing for 5 minutes, the test strip was removed, and the length from the fold to the tear-wetted end was measured and recorded as the tear secretion volume (unit: mm / 5min). Both eyes of each mouse were tested, and each eye was measured three times. The average value was used for statistical analysis. During the measurement, the cornea and eyelashes were avoided.

[0042] 2.2 Tear film breakup time (BUT) measurement

[0043] After topical anesthesia, 1 μL of 1% sodium fluorescein solution was instilled into the conjunctival sac of the mouse using a micropipette. The mouse's eyelids were gently moved to induce blinking three times, ensuring even distribution of fluorescein across the entire tear film. The tear film breakup time was measured under a slit-lamp microscope in cobalt blue light mode. The time from the last blink to the appearance of the first dark spot or irregular tear rupture point on the tear film surface was recorded as the tear film breakup time, measured in seconds (s). Each eye was measured three times, with at least 5 minutes between each measurement. The average value was used for statistical analysis.

[0044] 2.3 Corneal fluorescein staining score

[0045] After the BUT measurement, the corneal epithelium was continuously irradiated with cobalt blue light to observe the fluorescein staining. A double-blind scoring system using the internationally recognized 12-point scale was employed: the cornea was divided into four quadrants: superior temporal, inferior temporal, superior nasal, and inferior nasal. Each quadrant was scored from 0 to 3 points based on the degree of staining: 0 points = no staining puncta; 1 point = scattered punctate staining, number <10; 2 points = diffuse punctate staining, number ≥10 but without fusion; 3 points = patchy staining with fusion or corneal epithelial defects. The scores from the four quadrants were summed to obtain the total score, ranging from 0 to 12 points. A higher score indicates more severe corneal epithelial damage. Each eye was scored by two independent experimenters, and the average score was taken.

[0046] 2.4 Conjunctival goblet cell count

[0047] After all in vivo parameters were measured, mice were euthanized by intraperitoneal overdose with 2% sodium pentobarbital (50 mg / kg). Intact eyeballs were rapidly enucleated and fixed in pre-cooled 4% paraformaldehyde solution for 24 hours, maintaining the sagittal orientation during fixation. The eyes were routinely dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. 4 μm thick serial sections were prepared along the sagittal plane of the eyeball. Intact sections containing both the bulbar and fornix conjunctiva were selected for PAS staining. Under an optical microscope, five non-overlapping high-power fields (×400) were randomly selected, and the number of purplish-red PAS-positive goblet cells in each field was counted. The average number of cells per high-power field (cells / HPF) was calculated.

[0048] 2.5 Observation and scoring of corneal histopathology

[0049] Serial paraffin sections of the above-mentioned eyeballs were subjected to routine HE staining. The integrity of the corneal epithelial structure, the degree of stromal edema, and the presence of inflammatory cell infiltration were observed under an optical microscope. A 5-point scale was used for pathological scoring: 0 points = intact corneal epithelium, clear layers, no stromal edema, and no inflammatory cell infiltration; 1 point = slightly irregular epithelial surface, mild stromal edema, and occasional scattered inflammatory cells; 2 points = partial epithelial defect, involving 1-2 layers of cells, moderate stromal edema, and a small amount of inflammatory cell infiltration; 3 points = large area of ​​epithelial defect, involving more than 3 layers of cells, severe stromal edema, and a large number of inflammatory cell infiltrations; 4 points = full-thickness corneal damage, stromal necrosis and dissolution, and diffuse infiltration of a large number of inflammatory cells.

[0050] 2.6 Measurement of corneal tissue inflammatory factor content

[0051] After euthanasia, corneal tissue was rapidly dissected on ice, and iris and conjunctival tissue were removed. Corneas from every two mice were combined into one sample, and 150 μL of pre-chilled RIPA lysis buffer (containing 1% PMSF) was added. The sample was thoroughly homogenized on ice. Centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected. The total protein concentration was determined using a BCA protein quantification kit. Following the ELISA kit instructions, the levels of IL-1β and TNF-α were detected. Standards were also replicated simultaneously. Results are expressed as pg / mg total protein.

[0052] Example 1

[0053] Experimental animal grouping

[0054] Twenty healthy male BALB / c mice, weighing 19-21g, were randomly divided into Example 1 group, consisting of 10 mice and 20 eyes.

[0055] Model building methods

[0056] (1) Adaptation feeding for 3 days, daily slit lamp examination to confirm no eye abnormalities, and initial weight recorded.

[0057] (2) Simultaneously initiate a triple induction program and intervene continuously for 14 days:

[0058] ① Benzalkonium chloride eye drops: Prepare a 0.1% (w / v) benzalkonium chloride working solution using sterile physiological saline, and use immediately after preparation. Administer 8 μL of the solution to each eye four times daily at 8:00, 12:00, 16:00, and 20:00. When administering the drops, gently pull open the lower eyelid of the mouse to form a conjunctival sac using your thumb and forefinger, and slowly instill the solution. After instilling the solution, gently close the eyelid for 10 seconds to prevent spillage.

[0059] ② Dry environment rearing: At 8:00 AM daily, mice were moved into an artificial climate chamber with the following parameters set: temperature 24±1℃, relative humidity 30%±2%, and horizontal wind speed 2.0 m / s. Sterile feed and sterilized drinking water were provided in the chamber to ensure free access to food. The mice were continuously exposed to dry conditions for 16 hours each day. At 12:00 AM, the mice were removed from the climate chamber and placed in a standard rearing environment overnight.

[0060] ③ Scopolamine injection: Dilute scopolamine hydrobromide injection solution with physiological saline to a working solution of 0.05 mg / mL. Administer subcutaneously via the neck and back at 8:30 and 16:30 daily. The dosage is 0.5 mg / kg body weight, and the injection volume should be precisely adjusted according to the mouse's body weight on that day.

[0061] (3) During the induction period, observe the mice’s mental state, food and water intake, and record weight changes and abnormal eye manifestations. If serious adverse reactions such as corneal ulcers or eyelid adhesions occur, terminate the experiment in time.

[0062] Indicator Testing

[0063] After 14 days of continuous induction, all in vivo and in vitro indicators were tested according to the general testing methods described above.

[0064] Example 2

[0065] Experimental animal grouping

[0066] Twenty healthy male BALB / c mice, weighing 18-22g, were randomly divided into two groups: Example 2, consisting of 10 mice and 20 eyes.

[0067] Model building methods

[0068] (1) Adapt to feeding for 3 days and confirm that there are no eye abnormalities.

[0069] (2) Simultaneously initiate a triple induction program and intervene continuously for 10 days:

[0070] ① Benzalkonium chloride eye drops: Prepare a 0.15% benzalkonium chloride saline solution and administer 8 μL of the solution to each eye 4 times a day at 8:00, 12:00, 16:00 and 20:00.

[0071] ② Dry environment rearing: At 8:00 a.m. every day, the mice were moved into an artificial climate chamber with the following parameters: temperature 24±1℃, relative humidity 25%±2%, horizontal wind speed 1.5m / s, and continuous dry exposure for 12 hours every day. At 20:00, the mice were removed and placed in a standard rearing environment overnight.

[0072] ③ Scopolamine injection: Prepare a scopolamine hydrobromide working solution with a concentration of 0.06 mg / mL, and administer subcutaneously at 8:30 and 16:30 daily, with a dosage of 0.6 mg / kg body weight.

[0073] (3) Observe the general condition of the mice daily during the induction period.

[0074] Indicator Testing

[0075] After 10 days of continuous induction, all indicators were tested according to the general testing method.

[0076] Example 3

[0077] Experimental animal grouping

[0078] Twenty healthy male BALB / c mice, weighing 18-22g, were randomly divided into three groups (Example 3), with a total of 10 mice and 20 eyes.

[0079] Model building methods

[0080] (1) Adapt to feeding for 3 days and confirm that there are no eye abnormalities.

[0081] (2) Simultaneously initiate a triple induction program and intervene continuously for 21 days:

[0082] ① Benzalkonium chloride eye drops: Prepare a 0.08% benzalkonium chloride saline solution and administer 10 μL of the solution to each eye three times a day at 8:00, 14:00 and 20:00.

[0083] ② Dry environment rearing: At 6:00 a.m. every day, the mice were moved into an artificial climate chamber with the following parameters: temperature 24±1℃, relative humidity 35%±2%, horizontal wind speed 2.5m / s. The mice were continuously exposed to dryness for 18 hours each day. At 24:00, the mice were removed and placed in a standard rearing environment overnight.

[0084] ③ Scopolamine injection: Prepare a scopolamine hydrobromide working solution with a concentration of 0.04 mg / mL, and administer subcutaneously at 8:30 and 16:30 daily, with a dosage of 0.4 mg / kg body weight.

[0085] (3) Observe the general condition of the mice daily during the induction period.

[0086] Indicator Testing

[0087] After 21 days of continuous induction, all indicators were tested according to the general testing method.

[0088] Comparative Example 1: Benzalkonium Chloride Induction

[0089] Use only 0.1% benzalkonium chloride solution for eye drops, 8 μL each time, 4 times a day for 14 consecutive days; keep the animals in a standard feeding environment throughout the process, without exposure to a dry environment, and do not inject scopolamine. The remaining procedures are the same as in Example 1.

[0090] Comparative Example 2: Induction by a Single Dry Environment

[0091] They were raised in a dry environment with the same temperature, humidity, wind speed, and daily exposure time as in Example 1 for 14 consecutive days; no benzalkonium chloride eye drops or scopolamine injections were administered. All other procedures were the same as in Example 1.

[0092] Comparative Example 3: Scopolamine-induced induction

[0093] Scopolamine hydrobromide was administered subcutaneously at a dose of 0.5 mg / kg twice daily for 14 consecutive days. The animals were kept in a standard rearing environment throughout the process, without benzalkonium chloride eye drops or exposure to a dry environment. All other procedures were the same as in Example 1.

[0094] Comparative Example 4: Benzalkonium Chloride + Dry Environment Dual Induction

[0095] The animals were fed with 0.1% benzalkonium chloride eye drops in a dry environment, with the same parameters as in Example 1, for 14 consecutive days; scopolamine was not injected. All other procedures were the same as in Example 1.

[0096] Comparative Example 5: Benzalkonium Chloride + Scopolamine Dual Induction

[0097] The patients were treated with 0.1% benzalkonium chloride eye drops and scopolamine subcutaneously, with the same parameters as in Example 1, for 14 consecutive days. They were kept in a standard rearing environment throughout the process and were not exposed to a dry environment. All other procedures were the same as in Example 1.

[0098] Comparative Example 6: Dry Environment + Scopolamine Double Induction

[0099] Animals were kept in a dry environment and administered scopolamine subcutaneously for 14 consecutive days, with the same parameters as in Example 1; benzalkonium chloride eye drops were not administered. All other procedures were the same as in Example 1.

[0100] Comparative Example 7: High-concentration benzalkonium chloride triple induction

[0101] The concentration of benzalkonium chloride solution was adjusted to 0.3%, while the remaining drying environment parameters, scopolamine dosage, and induction period were consistent with those in Example 1.

[0102] Comparative Example 8: Low-concentration benzalkonium chloride triple induction

[0103] The concentration of benzalkonium chloride solution was adjusted to 0.05%, and the remaining drying environment parameters, scopolamine dosage, and induction period were consistent with those in Example 1.

[0104] Comparative Example 9: Short-cycle triple induction

[0105] The induction period was adjusted to 7 days, while the remaining benzalkonium chloride concentration, drying environment parameters, and scopolamine dosage were consistent with those in Example 1.

[0106] Comparative Example 10: Triple Induction in High Humidity Environment

[0107] The relative humidity of the dry environment was adjusted to 50%±2%, while the other parameters, such as temperature, wind speed, benzalkonium chloride concentration, scopolamine dosage, and induction period, were the same as in Example 1.

[0108] Comparative Example 11: Atropine Replacement of Scopolamine Triple Induction

[0109] Scopolamine hydrobromide was replaced with an equal dose of atropine sulfate, with the administration method and frequency remaining unchanged. The remaining benzalkonium chloride concentration, drying environment parameters, and induction period were consistent with those in Example 1.

[0110] In addition, a normal control group was set up: 10 healthy male BALB / c mice were selected, raised in a standard environment without any intervention, and tested for the same indicators at the same time as a baseline control.

[0111] Performance test results and analysis

[0112] tear fluid parameters test results

[0113] The results of tear secretion and tear film breakup time in each group of mice are shown in Table 1.

[0114] Table 1 Comparison of tear secretion and tear film breakup time in mice of different groups ( (n=20)

[0115]

[0116] Results of ocular surface morphology and histopathological indicators

[0117] The results of corneal fluorescein staining scores, conjunctival goblet cell counts, and corneal pathological scores for each group of mice are shown in Table 2.

[0118] Table 2 Comparison of ocular surface morphology and histopathological indicators among different groups of mice ( (n=20)

[0119]

[0120] Results of ocular surface inflammatory factor detection

[0121] The results of detecting the levels of IL-1β and TNF-α in the corneal tissue of mice in each group are shown in Table 3.

[0122] Table 3 Comparison of inflammatory factor content in corneal tissue of mice in each group ( (n=10)

[0123]

[0124] Model success rate and animal tolerance evaluation

[0125] The criteria for successful model construction were "tear secretion <4mm / 5min, tear film breakup time <5s, and corneal fluorescein staining score >5". The success rate of each model and the mortality rate of animals were statistically analyzed, and the results are shown in Table 4.

[0126] Table 4 Comparison of model success rate and animal mortality rate in each group

[0127]

[0128] Model stability verification

[0129] To verify the sustained stability of the model, 19 mice successfully constructed in Example 1 were selected. All induction interventions were stopped, and the mice were kept in a standard environment for 7 days. The core indicators were tested again on the 3rd and 7th days after the induction was stopped. The results are shown in Table 5.

[0130] Table 5. Model stability test results after induction was stopped ( (n=19)

[0131] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing an animal model of dry eye syndrome, characterized in that, Includes the following steps: S1 Selection and Adaptation of Laboratory Animals: SPF-grade healthy adult rodents were selected and adapted to living in a standard environment for 3-7 days, excluding individuals with underlying eye diseases. S2 triple synergistic induction intervention: Simultaneous use of three methods: local eye drops of low-concentration benzalkonium chloride solution, controlled parameter dry environment feeding intervention, and systemic administration of anticholinergic drugs, for 10-21 consecutive days; S3 Model Validation and Evaluation: After induction, the model was validated using tear fluid parameters, ocular surface morphology parameters, histopathological parameters, and inflammatory factor parameters to obtain a stable animal model of dry eye.

2. The method for constructing an animal model of dry eye according to claim 1, characterized in that, The experimental animals described in step S1 are 6-8 week old male BALB / c mice, weighing 18-22g; the acclimatization conditions are: temperature 22-26℃, relative humidity 45%-55%, 12h light-dark cycle, and free access to food and water.

3. The method for constructing an animal model of dry eye according to claim 1, characterized in that, The low concentration benzalkonium chloride solution mentioned in step S2 has a mass-volume concentration of 0.08%-0.15%; the eye drops are administered 3-5 times daily, with 5-10 μL administered to each eye each time, and the interval between two eye drops is at least 3 hours.

4. The method for constructing an animal model of dry eye according to claim 3, characterized in that, The benzalkonium chloride solution had a mass-volume concentration of 0.1%, and was administered as 8 μL of eye drops four times daily for 14 consecutive days.

5. The method for constructing an animal model of dry eye according to claim 1, characterized in that, The controllable parameters of the dry environment in step S2 are as follows: ambient temperature 22-26℃, relative humidity 25%-35%, horizontal airflow speed 1.5-2.5m / s, continuous dry exposure for 12-18 hours per day, and the remaining time is spent in a standard environment.

6. The method for constructing an animal model of dry eye according to claim 5, characterized in that, The drying environment parameters are: temperature 24±1℃, relative humidity 30%±2%, wind speed 2.0m / s, and daily drying exposure for 16 hours.

7. The method for constructing an animal model of dry eye according to claim 1, characterized in that, The anticholinergic drug mentioned in step S2 is scopolamine hydrobromide; the administration method is subcutaneous injection in the neck and back, the dosage is 0.3-0.8 mg / kg body weight, twice a day, with an interval of 8-10 hours between the two administrations.

8. The method for constructing an animal model of dry eye according to claim 1, characterized in that, In step S2, the three intervention methods are started simultaneously, with a minimum interval of 30 minutes between daily eye drop and subcutaneous injection, and no time conflict between dry environment exposure and eye drop.

9. The method for constructing an animal model of dry eye according to claim 1, characterized in that, The model validation indicators mentioned in step S3 specifically include: tear secretion volume, tear film breakup time, corneal fluorescein staining score, conjunctival goblet cell count, corneal epithelial pathology score, and the content of IL-1β and TNF-α in corneal tissue.

10. The application of the dry eye animal model prepared by the construction method according to any one of claims 1-9 in the screening and pharmacodynamic evaluation of drugs for the treatment of dry eye.

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