A method for constructing a hybrid rat dry eye disease animal model

Abstract

This invention discloses a method for constructing a mixed-type rat dry eye model. The method involves administering flutamide via gavage and benzalkonium chloride eye drops to the animals to obtain the dry eye model. The aim of this invention is to establish a mixed-type dry eye model through a combined approach of flutamide gavage and benzalkonium chloride eye drops, providing new ideas and possibilities for studying the pathogenesis of dry eye and finding new treatments and drugs to prevent or delay the progression of dry eye.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for constructing a hybrid rat dry eye disease animal model. Background Technology

[0002] Dry eye syndrome affects approximately 20-30% of the global population and is one of the most common eye diseases worldwide. It not only causes eye discomfort and even vision impairment but also severely impacts vision-related quality of life. Because the exact physiological and pathological mechanisms of dry eye syndrome are not fully understood, establishing animal models of dry eye syndrome to study its pathogenesis is of great significance for its treatment.

[0003] Domestic and international ophthalmological associations agree that dry eye syndrome is a multifactorial disease, with tear film instability being one of its core causes. The pathogenesis of dry eye syndrome is complex, involving factors such as age, sex hormone levels, meibomian gland dysfunction, external environmental factors, and autoimmune diseases. It can be mainly divided into three categories: insufficient tear secretion dry eye syndrome, excessive evaporation dry eye syndrome, and mixed dry eye syndrome, which is caused by a combination of the above factors. Currently, animal models of dry eye syndrome are constructed by inducing single or mixed types through drugs, surgery, or environmental factors to simulate some of the influencing factors in the course of dry eye syndrome. Clinically, most dry eye patients present with mixed dry eye syndrome.

[0004] Androgens, a type of sex hormone, have receptors expressed in multiple locations in the eye, including the meibomian glands, cornea, conjunctiva, and retina. They can regulate lipid secretion from the lacrimal and meibomian glands, affecting the composition and stability of the tear film. Androgen deficiency can occur due to aging, autoimmune diseases, menopause, and the use of anti-androgen drugs, leading to dry eye syndrome. Flutamide, an androgen receptor antagonist, can mimic dry eye syndrome caused by hormonal imbalances. Benzalkonium chloride (BZK), a quaternary ammonium cation, is widely used as a preservative in ophthalmic preparations; it disrupts the lipid phase of the tear film, thus affecting tear film stability. The dry eye model used in this invention is closely related to clinical dry eye syndrome detection indicators: tear film stability, meibomian gland function, and tear secretion. Therefore, we hypothesize that it can effectively simulate most of the pathological conditions in dry eye syndrome. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a hybrid rat dry eye disease animal model.

[0006] The method for constructing a hybrid dry eye animal model of the present invention involves gavage of animals with flutamide and eye drops of benzalkonium chloride to obtain a dry eye animal model.

[0007] Preferably, the animal can be any kind of laboratory animal, such as a rat.

[0008] Preferably, the model is established by gavage administration of flutamide at a dose of 13 mg / kg body weight per day and eye drops of 0.2% benzalkonium chloride once a day.

[0009] Further optimization resulted in a molding time of 35 days.

[0010] This invention also provides the application of the above-described construction method in the field of dry eye disease model construction.

[0011] Preferably, it is a rat dry eye model.

[0012] This invention provides the application of the dry eye model obtained above in the study of the pathogenesis of dry eye disease.

[0013] This invention also provides the application of the dry eye model obtained above in the search for new treatments for dry eye.

[0014] The present invention also provides the application of the dry eye model obtained above in screening drugs for the prevention, treatment or delay of dry eye syndrome.

[0015] This invention aims to establish a hybrid animal model of dry eye by combining flutamide gavage with benzalkonium chloride solution eye drops, providing new ideas and possibilities for studying the pathogenesis of dry eye and finding new treatments and drugs to prevent or delay the course of dry eye. Attached Figure Description

[0016] Figure 1 This is a method for establishing a rat animal model of mixed dry eye syndrome;

[0017] Figure 2 This refers to the tear secretion volume in rats with mixed-type dry eye syndrome.

[0018] Figure 3 These are representative images and statistical results of PAS and AB staining.

[0019] Figure 4 These are representative images and statistical results of the corneal fluorescein sodium staining test;

[0020] Figure 5 These are representative images and statistical results of corneal HE staining.

[0021] Figure 6 These are representative images and statistical results of corneal TUNEL staining.

[0022] Figure 7 It refers to the level of inflammatory factors in the corneal and conjunctival tissues. Detailed Implementation

[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0024] Example 1:

[0025] I. Experimental Methods

[0026] 1. Experimental apparatus

[0027] Table 1 Experimental Instruments

[0028]

[0029]

[0030] 2. Experimental reagents and chemicals

[0031] Table 2 Experimental reagents and reagents

[0032]

[0033]

[0034] 3. Solution preparation:

[0035] Ultrapure water (containing 0.5% sodium carboxymethyl cellulose powder):

[0036] Measure 900 mL of ultrapure water into a 1 L beaker using a graduated cylinder, add 5.0 g of sodium carboxymethyl cellulose powder, stir until completely dissolved, pour into a 1000 mL volumetric flask, and add ultrapure water to bring the volume to 1000 mL.

[0037] Flutamide suspension:

[0038] Accurately weigh 3.25 g of flutamide powder, add an appropriate volume of ultrapure water (containing 0.5% sodium carboxymethyl cellulose powder) and bring the volume to 1000 mL to obtain a 3.25 mg / mL flutamide suspension. Store the suspension in a separate container at -80°C and remove it immediately before use.

[0039] 0.2% benzalkonium chloride solution:

[0040] Take 1 mL of 10% benzalkonium chloride standard solution into a 50 mL centrifuge tube, add 49 mL of physiological saline to obtain 50 mL of 0.2% benzalkonium chloride solution, store it in a refrigerator at 4℃, and prepare two aliquots.

[0041] 1% sodium fluorescein solution:

[0042] Dissolve 100 mg of sodium fluorescein powder in 10 mL of physiological saline, shake to dissolve, and obtain 10 mL of 1% sodium fluorescein solution. Dispense 1.5 mL into each tube and store at -80°C.

[0043] Blinding Eye Extract Solution:

[0044] Zhangyanming extract (Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd., CA1007), the extraction process is described in Part I of the 2020 edition of the Chinese Pharmacopoeia, Zhangyanming tablet preparation method, the crude drug content is 2.48g / g;

[0045] Weigh 82.46g of the eye-brightening extract into a 500mL volumetric flask, add ultrapure water to the mark, and obtain 500mL of eye-brightening extract stock solution with a concentration of 164.92mg / mL. Mix well, dispense into 15mL tubes, and store at -80℃. Remove before use.

[0046] Low-dose group: Take 2 mL of the mother liquor of the eye-brightening extract and dilute it with 6 mL of ultrapure water to 41.23 mg / mL;

[0047] Medium-dose group: Take 4 mL of the mother liquor of the eye-brightening extract and dilute it with 4 mL of ultrapure water to 82.46 mg / mL;

[0048] High-dose group: The mother liquor of the eye-brightening extract was taken directly without dilution.

[0049] 4. Establishment of an animal model of dry eye syndrome:

[0050] Slit-lamp examination was performed on rat eyes, and rats without obvious visible eye damage or lesions were included in the study. After one week of acclimatization, in order to induce a rat model of mixed dry eye, a combined modeling regimen was adopted, which included oral administration of flutamide suspension (13 mg / kg, approximately 1.2 mL / rat) and eye drops of 0.2% benzalkonium chloride solution (one drop / time / day), once a day for 35 consecutive days. Distilled water was administered by gavage (1.2 mL / rat) and physiological saline was administered by eye drops (one drop / time / day) as controls. The interval between administration and gavage for modeling was four hours.

[0051] 5. Treatment with eye-brightening extract:

[0052] Rats were randomly divided into the following 6 groups, with 7 rats in each group:

[0053] (1) Blank group: Distilled water was administered by gavage (1.2 mL / animal);

[0054] (2) Dry eye model group: Distilled water was administered by gavage (1.2 mL / animal);

[0055] (3) Low-dose group of Zhangyanming (164.92mg / kg, 1x clinical dose): The low-dose group of Zhangyanming extract solution (41.23mg / mL) was administered to the rats according to their body weight, about 1.2mL / rat;

[0056] (4) Medium dose of Zhangyanming extract (329.84 mg / kg, 2x clinical dose): The medium dose of Zhangyanming extract (82.46 mg / mL) was administered to each rat according to its body weight, approximately 1.2 mL per rat.

[0057] (5) High-dose group of Zhangyanming (659.68mg / kg, 4x clinical dose): The high-dose group of Zhangyanming extract solution (164.92mg / mL) was administered to the rats according to their body weight, about 1.2mL / rat;

[0058] (6) Positive control group: Distilled water was administered by gavage (1.2 mL / animal), and 0.05% cyclosporine A was administered by eye drops (one drop / time / day).

[0059] 6. Tear secretion test:

[0060] Tear secretion in rats was measured using the phenol red cotton thread test on days 0, 7, 14, 21, 28, and 35 after induction. A 3 cm length of phenol red cotton thread was carefully inserted into the rat's lower eyelid and held still for 30 seconds. Due to the capillary action of the tears, the phenol red thread turned from red to yellow. The length of the yellow portion of the thread was measured and recorded as a reference indicator of tear secretion capacity.

[0061] 7. Corneal permeability test:

[0062] On days 0, 7, 14, 21, 28, and 35 post-induction, corneal fluorescein staining was performed on rats to assess corneal epithelial barrier permeability. 5 μL of 1% fluorescein solution was instilled onto the surface of the rat's eyeball, and after assisted eye closure for 30 seconds, the corneal region was photographed using a slit-lamp microscope. The lesions were quantitatively scored using a 12-point scale based on the area of ​​the fluorescein-positive region.

[0063] 8. Histological examination:

[0064] On day 35 after model induction, rats were euthanized by overdose anesthesia, and their eyeballs and surrounding accessory ocular tissues were collected. The tissues were fixed in 4% FPA solution for 24 hours, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and cut into 5 μm paraffin sections. After dewaxing, the sections were stained with hematoxylin for 5 minutes, rinsed with running tap water, and counterstained with eosin for 30 seconds. The corneal area was photographed using an inverted microscope, and corneal thickness was quantified using ImageJ software.

[0065] The morphology and number of conjunctival goblet cells were assessed using Alcian Blue (AB) and Periodic acid-Schiff (PAS) staining methods. For AB staining, sections were stained with 1% Alcian Blue solution in 3% acetic acid for 15 minutes, followed by rinsing with running tap water. For PAS staining, sections were stained with 0.5% periodic acid for 5 minutes, rinsed with distilled water, stained with Schiff's reagent for 15 minutes, rinsed with running tap water, and finally counterstained with hematoxylin, rinsing with running tap water until the stain turned blue. Goblet cells in the conjunctival sac region were photographed using an inverted microscope; AB-positive and PAS-positive cells appeared as blue and purple cells, respectively. The number of positive cells per millimeter of conjunctival sac length represented the number of goblet cells.

[0066] 9. Apoptosis detection:

[0067] The one-step TUNEL apoptosis detection kit was used to detect apoptotic cells in corneal sections (day 35). After dewaxing 5μm paraffin sections, they were permeabilized with 20μg / mL proteinase K solution at 37°C for 30 minutes. After washing the slides with PBS, they were stained with TUNEL working solution at 37°C in the dark for 30 minutes, and the cell nuclei were counterstained with DAPI. The corneal region was photographed using a confocal laser scanning microscope. The number and quantification of TUNEL / DAPI double-positive apoptotic cells in the corneal view (235mm × 235mm) were performed using ImageJ software.

[0068] 10. Inflammatory factor detection:

[0069] The levels of cytokines (IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-18, IL-33, CXCL1, KC, CCL2, MCP-1, GM-CSF, IFN-γ, and TNF-α) were measured using a bead-based LEGENDplex multifactor analysis kit.

[0070] 20 mg of corneal and conjunctival tissue (day 35) was added to 0.5 mL of pre-chilled PBS containing a mixture of protease inhibitors. Homogenization was performed until no obvious particulate matter was observed, followed by centrifugation at 10000 g for 10 minutes at 4°C. The supernatant was collected for total protein quantification (BCA method), with 25 μL of each supernatant used for multifactor kit detection. Multifactor analysis was performed according to the manufacturer's instructions. 25 μL of a bead mixture and 25 μL of detection buffer were added to a filter plate, followed by 25 μL of the sample solution. The plate was sealed and incubated in the dark with shaking for 2 hours. After thorough washing, 25 μL of detection antibody was added, and the plate was incubated for 1 hour with shaking and closed. Then, 25 μL of LSA-PE was added, and the plate was incubated for another 30 minutes. After thorough washing, 150 μL of wash buffer was added, and analysis was performed using CyExpert 2.4.0.28. Quantification of the detected factors was expressed as mean fluorescence intensity (MFI), calculated according to a standard curve, and normalized to the total protein concentration using ng of protein per mg.

[0071] II. Experimental Results

[0072] This study used male Sprague-Dawley rats weighing 300-350g to induce a mixed-type dry eye model by intragastric administration of flutamide (13mg / kg) once daily, combined with eye drops of 0.2% benzalkonium chloride solution for 35 consecutive days. The "Zhangyanming" treatment groups were treated by intragastric administration of 1x clinical dose (165mg / kg), 2x clinical dose (330mg / kg), and 4x clinical dose (660mg / kg), respectively. Simultaneously, the positive control group was treated with 0.05% cyclosporine A eye drops once daily as a positive control. Figure 1 As shown.

[0073] Tear secretion and corneal barrier permeability were measured on days 0, 7, 14, 21, 28, and 35. A cotton thread soaked in tear phenol red was placed on the lower eyelid of the rats at the 1 / 3 mark, held still for 30 seconds, and the length of the thread soaked in tear filament was recorded to directly characterize the total tear secretion. At day 0, there was no significant difference in tear secretion between the model group and the control group. Later in the modeling process, the total tear secretion in the model group gradually decreased, especially on days 28 and 35, where tear secretion was significantly reduced. Figure 2 As shown.

[0074] The mucus layer is the innermost of the three layers of the tear film, primarily produced by goblet cells located in the conjunctiva. These mucin-rich goblet cells play a crucial role in eye health. In the goblet cells of the conjunctival sac, PAS staining makes acidic mucin appear purple, while AB staining makes neutral mucin appear blue. Compared to the control group, the model group showed a significant decrease in the number of goblet cells, changes in cell morphology, and instances of shrinkage or loss, such as… Figure 3As shown, this suggests that in this mixed-type dry eye animal model, the normal mucus secretion function of goblet cells in the conjunctival region is impaired, and the tear film structure may be damaged.

[0075] Besides reduced total tear production and a decrease in goblet cell count, dry eye syndrome often causes increased corneal and conjunctival epithelial permeability and even shedding. Corneal permeability is assessed using a fluorescein staining test, followed by slit-lamp microscopy imaging and a 12-point scoring system to quantify the severity of corneal damage. Before modeling, almost all rats had no corneal epithelial damage, and fluorescein-positive spots were negligible. However, after modeling, increased corneal epithelial permeability occurred, resulting in punctate or even patchy areas of positive fluorescein staining. Figure 4 As shown, in the first two weeks, the corneal fluorescein staining score of rats in the model group was greater than 6.0, reaching a peak of 7.0 on day 14, which was significantly different from the blank group. This means that under this mixed dry eye animal model, the corneal permeability of rats increased and the corneal damage was significant. This observation may also indicate that, compared with other days, day 14 is the best time point to assess the changes in corneal permeability under this mixed dry eye model.

[0076] In the later stages of the experiment, corneal damage in the model group rats was alleviated, which may be due to the cornea's self-repairing ability, meaning that the model may simulate early-stage mild dry eye syndrome. To further investigate the reasons, H&E staining and TUNEL staining were used to reveal changes in corneal structure and morphology after drug treatment.

[0077] like Figure 5 As shown in the representative HE-stained images, the corneal tissue in the model group was thinner than that in the control group, indicating a change in tissue morphology and structure. Statistical analysis using ImageJ showed that the corneal tissue thickness in the model group was significantly reduced compared to the control group. The statistical analysis results are as follows: Figure 5 As shown on the right.

[0078] To further investigate the reasons for corneal thinning in rats after combined modeling, TUNEL staining was used to label fragmented DNA in apoptotic cells and examine the apoptotic level of corneal epithelial cells. Representative fluorescent staining images are shown below. Figure 6 As shown in the figure. Compared with the control group, most corneal epithelial cells in the model group were marked as TUNEL / DAPI double-positive cells, indicating that epithelial cell apoptosis was significantly increased and corneal barrier function was severely impaired in this mixed dry eye animal model. The count / view of TUNEL / DAPI double-positive cells was statistically analyzed using ImageJ, and the results are shown in the figure. Figure 6 As shown on the right.

[0079] Inflammation is a core factor in the development of dry eye syndrome. The levels of inflammatory factors in rat corneal and conjunctival tissue were detected using the Biolegend multifactor kit. The quantification of these factors was expressed as mean fluorescence intensity (MFI), calculated using a standard curve, and normalized to the total protein concentration using ng content per mg of protein. Experimental results showed that ( Figure 7 In this mixed dry eye animal model, the levels of inflammatory factors TNF-α and IL12p70 in the corneal and conjunctival tissues of rats were significantly increased, indicating the occurrence of inflammation.

[0080] III. Advantages of combined flutamide gavage and benzalkonium chloride eye drops in modeling

[0081] 1. Advantages of combined modeling compared to flutamide modeling alone

[0082] Hormonal imbalance is one of the influencing factors of dry eye syndrome, and androgen deficiency is clinically considered a high-risk factor for dry eye. Flutamide, administered orally, can competitively bind to androgen receptors with its active form, mimicking the physiological state of androgen deficiency. While flutamide alone can simulate dry eye syndrome caused by hormonal imbalance, it has drawbacks such as a long modeling period and milder severity of some key phenotypes.

[0083] SIt (tear secretion): Four weeks after modeling, there was no significant difference in tear secretion between the low-concentration group and the control group. The tear secretion of the medium- and high-concentration groups was significantly lower than that of the control group, but the decrease was smaller. By the eighth week after modeling, the tear secretion of the medium- and high-concentration groups had decreased to the level of the fourth week after combined modeling. The specific data of tear secretion in each group of rats are shown in the table below. 1 .

[0084] Table 1-3 Tear secretion test of rats in each group at different time points ( n=8l / mm)

[0085] Table 1-3 The St of rats at different time points of each group

[0086]

[0087] Note: Compared with the control group # P<0.05; compared with the high concentration group Δ P<0.05; compared with the medium concentration group ◆ P<0.05VSControl group # P<0.05; VS Highconcentration group ΔP<0.05;VSMediumconcentrationgroup ◆ P<0.05

[0088] Corneal fluorescein staining: Under slit-lamp cobalt blue light, yellow-green fluorescent areas are considered positive for fluorescein staining, indicating corneal barrier damage. The corneal fluorescein staining score is positively correlated with corneal barrier damage. Figure 3 As shown in the table below. Two weeks after drug administration to establish the model, there was no significant change in the fluorescein sodium staining score between the low, medium, and high concentration groups and the control group. Four weeks after drug administration to establish the model, the staining score of the medium and high concentration groups was significantly increased compared with the control group, but the staining score was lower, indicating a milder degree of corneal damage. By the eighth week after drug administration to establish the model, the staining score of the high concentration group was comparable to that of the second week after combined modeling. The fluorescein sodium staining data of rats in each group are shown in the table below.

[0089] Table 1-2 Corneal fluorescein staining scores of rats in different time points ( n = 8Score)

[0090] Table1-2Fluorescein staining score of rats at different time points of each group

[0091]

[0092] Note: Compared with the control group # P<0.05; compared with the high concentration group △ P<0.05; compared with the medium concentration group ◆ P<0.05VSControl group # P<0.05; VS High concentration group Δ P<0.05; VS Mediumconcentration group ◆ P<0.05

[0093] Corneal epithelial cell apoptosis: When tear production is insufficient and the number and function of goblet cells secreting mucin are reduced or lost, the aqueous and mucin layers of the tear film are damaged, the tear film structure is disrupted and imbalanced, tear evaporation is further accelerated, creating a hypertonic environment and inducing inflammation. Simultaneously, the corneal epithelium loses the nutritional support and barrier protection provided by the tear film, and comes into direct contact with the external environment or foreign bodies on the ocular surface, thus exacerbating the irritation to the corneal epithelium, potentially leading to damage to the corneal barrier function and epithelial cells. The combined model significantly increased the level of epithelial cell apoptosis, which was not detected when flutamide was used alone. Experimental results are shown in […]. Figure 6 .

[0094] 2. Advantages of combined molding compared to benzalkonium chloride molding alone

[0095] Benzalkonium chloride is a commonly used preservative in ophthalmic preparations. Long-term use can cause serious damage to the ocular surface. It is suitable for short-term modeling of severe dry eye, but it has limitations such as short model duration but severe symptoms. [2] .

[0096] Model maintenance time: After modeling with benzalkonium chloride alone, indicators such as tear secretion decreased significantly in the first two weeks after modeling, and recovered in the fourth week after modeling, with no significant difference from the control group. Specific experimental results are shown in the table below. 2 The combined model maintained tear secretion levels for a longer period, showing a significant decrease compared to the control group in both the fourth and fifth weeks after modeling. Specific experimental results can be found in [link to experimental results]. Figure 2 .

[0097] Table 1. Changes in tear secretion parameters in the two groups of mice.

[0098]

[0099] Note: Compared with the control group, * P < 0.05

[0100] Inflammation: The hypertonic environment of the ocular surface caused by insufficient tear production can trigger inflammation. After combined modeling, the levels of inflammatory factors TNF-α and IL12p70 in the corneal and conjunctival tissues of rats were significantly increased, indicating the occurrence of inflammation. The increase in IL12p70 was not detected by either flutamide or benzalkonium chloride models alone. The experimental results are shown below. Figure 7 .

[0101] Serum testosterone concentration: After gavage administration of flutamide, its active form competitively binds to androgen receptors with the androgen active form, triggering a compensatory increase in testosterone (the main active form of androgens) levels, mimicking the hormonal imbalance among the influencing factors of dry eye syndrome. This phenomenon was not detected when benzalkonium chloride was used alone to establish the model. For specific experimental results, please refer to the literature. 3 .

[0102] References

[0103] [1] Peng Ling. Experimental study on flutamide-induced dry eye model in rats [D]. Southern Medical University, 2016.

[0104] [2] Du Jing, Li Yong, Gao Jinrong, et al. Expression of interleukin-1 receptor-associated kinase 1 (IRAK1) and NF-κB in corneal and conjunctival tissues of benzalkonium chloride-induced dry eye mice [J]. New Advances in Ophthalmology, 2019, 39(03):223-228.

[0105] [3] Peng Ling, Cai Weihao, Lu Xiaohe, et al. Pathological changes in corneal and lacrimal gland tissues and expression of TNF-α and IL-1α in flutamide-induced dry eye model rats [J]. New Advances in Ophthalmology, 2016, 36(06):506-511.

Claims

1. A method for constructing a mixed type dry eye animal model, characterized by, The model of dry eye is obtained by intragastrically administering flutamide at 13 mg / kg body weight per day and eye dropping 0.2% benzalkonium chloride once a day, and the animals are rats.

2. The construction method of claim 1, wherein, The modeling time is 35 days.

3. The dry eye model obtained by the construction method of any one of claims 1-2 is applied to the study of the pathogenesis of dry eye.

4. The dry eye model obtained by the construction method of any one of claims 1-2 is applied to the screening of drugs for preventing or delaying dry eye.

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