Method for constructing atrophic meibomian gland dysfunction animal model and animal model

Abstract

The invention provides a method for constructing an atrophic meibomian gland dysfunction animal model and the animal model, and belongs to the field of medicine. The problems that in an existing atrophic MGD model, a gene knockout animal model has maldevelopment of meibomian glands, and an old animal model is high in feeding cost, long in feeding period and low in model forming rate are solved. According to the method, alveolar cells of meibomian glands can be successfully damaged by liquid nitrogen frozen mouse eyelid edges, experiments prove that liquid nitrogen freezing can cause alveolar atrophy and secretion function decline within a period of time, and compared with an existing gene knockout animal model and an old animal model, the method has the advantages that unorganized specificity of a gene knockout mouse does not exist, and the gene knockout mouse can be used as an animal model. Namely, the meibomian glands and other organs have the problem of dysplasia; the feeding cost is lower than that of aged animals, the feeding period is short, and limitation on research of pathogenic mechanisms and treatment mechanisms is low; the modeling method is simpler, quicker and repeatable, and the success rate of the model in the experiment process exceeds 90%.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for constructing an animal model of atrophic meibomian gland dysfunction and the animal model. Background Art

[0002] The expert consensus on meibomian gland dysfunction in China: Definition and Classification (2023) points out that an important type of meibomian gland dysfunction is acinar atrophy meibomian gland dysfunction, which is caused by atrophy of the meibomian gland alveoli. The terminal duct may not be significantly blocked, and whether it is accompanied by duct atrophy needs further study. However, animal models of this type of atrophic meibomian gland dysfunction are still being developed and explored. In clinical practice, there are also quite a few patients with meibomian gland atrophy who do not have causes related to abnormal lipid metabolism or age. There is no suitable animal model for atrophic meibomian gland dysfunction caused by decreased acinar secretion function caused by other factors. Existing atrophic meibomian gland dysfunction MGD models mainly include gene knockout animal models and elderly animal models. For example, a patent for an autism mouse model (application number: CN201911016844.4) has been authorized, in which the sequence on the first exon of the Gabrb1 gene of the mouse is knocked out by the TALEN gene editing method. Gabrb1- / - mice can be used as a model to study the relationship between the Gabrb1 gene and autism, and can also be used to study the relationship between the Gabrb1 gene and epilepsy susceptibility. By performing behavioral screening on Gabrb1 gene knockout mice, the effect of the loss of this gene on autism and epilepsy susceptibility can be studied. This method is effective and easy.

[0003] The targeted gene is usually key to lipid metabolism or ectoderm development, such as SCD1 knockout (SCD1- / -) mice, K5-GR mice, Krox20-cKO mice, Tabby mice, etc. These models usually show dysplasia of the meibomian glands. This gene knockout mouse not only changes the function of the meibomian glands, but also causes significant changes in the morphology of the meibomian glands. Although these gene knockout models can explain the effects of the gene on the development and function of the meibomian glands at the molecular or pathway level from certain perspectives, most of them cannot simulate the pathophysiological process of patients with meibomian gland dysfunction in the clinic, because the meibomian glands of most patients are not dysplastic or even abnormal, and the changes in the morphology and function of the meibomian glands are also caused by various acquired factors. These gene knockout models do not explain how the acquired factors affect the gene, so there are still limitations in the study of the pathogenic mechanism. At the same time, gene knockout mice are systemic knockout mice, and it is impossible to specifically knock out one or several genes in the meibomian gland tissue, which will inevitably cause varying degrees of damage to the ocular surface and other tissues and organs in the body, such as corneal neovascularization, corneal epithelial keratinization, and even renal damage in mice. These side effects are not what we want, and are not conducive to our observation of the pathophysiological changes that should occur on the ocular surface under MGD.

[0004] In the study of aged animal models, different research teams have different reports on the sequence or causal relationship between alveolar atrophy and ocular surface changes. For example, Yoon and his collaborators reported that aged C57BL / 6 mice are valuable tools for studying ARMGD. This study analyzed aged male mice (one and two years old). They found evidence of dry eyes and related ocular surface damage at one year old, but they pointed out that significant atrophy of the meibomian glands was only clearly observed at two years old. This does not directly indicate that the damage to the ocular surface is caused by meibomian gland dysfunction, so it needs further verification as a model for studying ocular surface damage caused by atrophic meibomian gland dysfunction. In addition, the cost of raising aged animals is high and the feeding period is long. At the same time, not all aged animals will suffer from MGD, and the low modeling rate is another factor that must be considered. Summary of the invention

[0005] The technical problems to be solved by the present invention are:

[0006] In order to solve the problems of meibomian gland dysplasia in the existing atrophic MGD models, the old animal models have high breeding costs, long breeding cycles and low model success rates.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] The present invention provides a method for constructing an animal model of atrophic meibomian gland dysfunction, comprising the following steps: performing local anesthesia on the eyelid margin of the mouse after general anesthesia, and performing liquid nitrogen damage on the margins of the upper and lower eyelids of the mouse using a freezing device filled with liquid nitrogen, wherein the contact position is the center of the eyelid margin.

[0009] Furthermore, the liquid nitrogen damage time is 28s-32s.

[0010] Further, the contact position of the freezing device is 2 / 3 of the center of the eyelid margin.

[0011] Furthermore, the mice are male mice aged 6 to 10 weeks.

[0012] Furthermore, general anesthesia is performed by inhalation anesthetics, and the inhalation anesthetic may be 4% isoflurane.

[0013] Furthermore, when performing local anesthesia, 0.4% oxybuprocaine hydrochloride eye drops were used to locally anesthetize the eyelid margin.

[0014] Furthermore, the freezing device includes a contact head, which is a copper contact head, and the contact head is used to freeze the mouse eyelids with liquid nitrogen.

[0015] Furthermore, the temperature of the contact head of the freezing device when damaged by liquid nitrogen is -115°C to 85°C.

[0016] A method for constructing an animal model of atrophic meibomian gland dysfunction is used to construct the model using a method for constructing an animal model of atrophic meibomian gland dysfunction.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a method for constructing an atrophic meibomian gland dysfunction animal model and the animal model. The acinar cells of the meibomian glands can be successfully destroyed by freezing the eyelid margins of mice with liquid nitrogen. Experiments have shown that liquid nitrogen freezing can cause acinar atrophy and decreased secretory function over a period of time, and there are increased inflammatory factors caused by meibomian gland dysfunction, decreased Ki67-positive cell count reflecting proliferation ability, decreased K14-positive cell count reflecting potential differentiation ability, increased expression of hyperkeratosis index K10 and increased expression of Sprr1b, and fluctuating decrease in PPARγ expression reflecting acinar cell differentiation and lipid synthesis ability. Compared with the existing gene knockout animal model and the aged animal model, the gene knockout mouse does not have the problem of meibomian gland dysplasia, the research on the pathogenic mechanism is less limited, the feeding cost is lower than that of aged animals, and the feeding period is short. In addition, the modeling method is simpler, faster and more repeatable, and the success rate of the model exceeds 90% during the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Flow chart of the method for constructing an animal model of atrophic meibomian gland dysfunction in an embodiment of the present invention, wherein A is a schematic diagram of a freezing device, B is a state diagram of the freezing device when it is working, and C is an actual process diagram of the freezing process;

[0020] Figure 2 The figures are evaluation diagrams of tarsal acinar cells and cornea within 24 hours after liquid nitrogen injury to the eyelid margin in the embodiment of the present invention, wherein A is a 10x and 20x comparison diagram of the eyelids of the control group and mice 24 hours after liquid nitrogen injury, and the box in A indicates the specific rupture of tarsal acinar cells after liquid nitrogen treatment, and B is a comparison diagram of the cornea and conjunctiva of the control group and mice 24 hours after liquid nitrogen injury, shown by H&E staining;

[0021] Figure 3 : is an evaluation diagram of the meibomian gland and cornea after liquid nitrogen injury to the eyelid margin in an embodiment of the present invention, wherein A is a representative slit lamp image of the eyelids of the control group and the experimental group on the 7th day, the 14th day, the 21st day and the 28th day, the yellow arrow in A indicates the capillary dilation of the eyelid margin, the black arrow indicates the blockage of the meibomian gland opening, and the green arrow indicates the location of eyelid margin hypertrophy, B is a fluorescein staining image of the corneal surface of the control group and the experimental group on the 7th day, the 14th day, the 21st day and the 28th day, the red arrow in B indicates the location of punctate epithelial loss, and C is a meibomian gland scoring result table;

[0022] Figure 4 : is an evaluation diagram of meibomian gland loss and morphological changes after liquid nitrogen injury to the eyelid margin in an embodiment of the present invention, wherein A is a diagram of meibomian gland loss and gross morphological changes of meibomian glands on days 7, 14, 21 and 28 in the control group and the experimental group, the red rectangular box in A represents the area of ​​meibomian gland damage, and B is a meibomian gland loss area analysis table;

[0023] Figure 5 : This is an evaluation diagram of the meibomian gland meibum changes and lipid synthesis after liquid nitrogen injury to the eyelid margin in an embodiment of the present invention, wherein A is a diagram of histological changes and lipid synthesis changes after Oil Red O staining in the control group and the experimental group on the 7th day, the 14th day, the 21st day and the 28th day, B is a diagram of immunofluorescence changes of PPARγ in the control group and the experimental group on the 7th day, the 14th day, the 21st day and the 28th day, C is a statistical diagram of fluorescence intensity in Figure B, D is a result diagram of western blot of PPARγ in the control group and the experimental group on the 7th day, the 14th day, the 21st day and the 28th day, and E is a statistical diagram of Western blot of PPARγ;

[0024] Figure 6: is an inflammation assessment graph of meibomian gland acinar cells after liquid nitrogen injury to the eyelid margin in an embodiment of the present invention, wherein A is a PCR result graph of inflammatory factors IL-1β and IL-18, B is a graph showing changes in TNFα after immunofluorescence staining in the control group and the experimental group on the 7th, 14th, 21st and 28th day, C is a graph showing changes in K14 after immunofluorescence staining in the control group and the experimental group on the 7th, 14th, 21st and 28th day, D is a statistical graph of the immunofluorescence intensity of the inflammatory factor TNFα, and E is a statistical graph of the immunofluorescence intensity of K14;

[0025] Figure 7 : is an evaluation diagram of cell proliferation and apoptosis of meibomian gland acinar cells after liquid nitrogen injury to the eyelid margin in an embodiment of the present invention, wherein A is a change diagram of Ki67 after immunofluorescence staining of the control group and the experimental group on the 7th day, the 14th day, the 21st day and the 28th day, B is a Ki67 change result analysis table, C is a change diagram of TUNEL after immunofluorescence staining of the control group and the experimental group on the 7th day, the 14th day, the 21st day and the 28th day, and D is a TUNEL change result analysis table;

[0026] Figure 8 : This is an evaluation diagram of keratinization of meibomian gland acinar cells after liquid nitrogen injury to the eyelid margin in an embodiment of the present invention, wherein A is a diagram showing changes in K10 after immunofluorescence staining in the control group and the experimental group on days 7, 14, 21, and 28, and D is a table showing the analysis of changes in Sprr1β gene expression. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0028] Specific implementation plan 1: Combine Figures 1 to 8 As shown, the present invention provides a method for constructing an animal model of atrophic meibomian gland dysfunction, comprising the following steps: performing local anesthesia on the eyelid margin of mice, and using a freezing device filled with liquid nitrogen to damage the edges of the upper and lower eyelids of the mice with liquid nitrogen, the damage time is 28s-32s, preferably 30s, and the contact position is 2 / 3 of the center of the eyelid margin.

[0029] Preferably, the mice are 6-10 weeks old, have free access to standard rodent food and water, and are maintained in a standard pathogen-free environment at 25°C ± 1°C, 60% ± 10% relative humidity, with an alternating 12-hour light-dark cycle (from 8:00 am to 8:00 pm).

[0030] Preferably, general anesthesia is performed by inhalation anesthetics before local anesthesia, and the inhalation anesthetic may be 4% isoflurane (Sinopharm Chemical Reagent Co., Ltd., China).

[0031] Preferably, when performing local anesthesia, 0.4% oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical, Japan) can be used to locally anesthetize the eyelid margin.

[0032] Preferably, the freezing device comprises a detachably connected nitrogen storage bottle and a bottle cap, the bottle cap is provided with a copper rod seat, the copper rod seat is provided with a thermal insulation protection sleeve, the copper rod is arranged in the thermal insulation protection sleeve, and one end of the copper rod extends out of the thermal insulation protection sleeve and is connected to the thin copper rod, the other end of the copper rod penetrates the bottle cap and extends into the nitrogen storage bottle, the side wall of the thermal insulation protection sleeve is provided with a wire opening, the wire of the thermometer extends into the wire opening and contacts the copper rod, the wire opening is fixed by a limit clamp, and the thermometer is an electronic ultra-low temperature thermometer. The thin copper rod of the freezing device is a contact head, and the contact head is a copper contact head with good temperature conduction performance. The temperature of the contact head of the freezing device when damaged by liquid nitrogen is -115℃--85℃.

[0033] Preferably, mice are euthanized on the 7th, 14th, 21st and 28th day after liquid nitrogen injury for comparison with a control group that was not subjected to liquid nitrogen injury and was euthanized on the 28th day.

[0034] Preferably, the comparison items include changes in eyelid tissue structure at all levels, cornea and conjunctiva 24 hours after liquid nitrogen injury, comparison of eyelid margin and ocular surface damage between the experimental group and the control group within one month, comparison of meibomian gland loss and morphological changes between the experimental group and the control group within one month, comparison of histological changes and lipid synthesis changes of MGs between the experimental group and the control group within one month, comparison of changes in meibomian gland tissue inflammation between the experimental group and the control group within one month, comparison of changes in meibomian gland acinar cell proliferation and apoptosis between the experimental group and the control group within one month, and comparison of changes in keratinization of meibomian gland acinar cells between the experimental group and the control group within one month. The statistical methods for the above comparisons are all single-factor analysis methods.

[0035] The other combinations and connection relationships of this embodiment are the same as those of the first embodiment.

[0036] Example

[0037] Animal Materials:

[0038] Eight-week-old male C57 / BL6 mice were obtained from Changchun Biotechnology (China). Experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Ethics Committee of Harbin Medical University. Animals had free access to standard rodent chow and water and were maintained in a standard pathogen-free environment at 25°C ± 1°C, relative humidity 60% ± 10%, with alternating 12-h light-dark cycles (from 8:00 am to 8:00 pm).

[0039] Reagent materials:

[0040] Rabbit anti-cytokeratin 10 (K10, ab76318), anti-cytokeratin 14 (K14, ab181595), anti-Ki67 (ab16667), anti-PPAR-γ (ab45036). Alexa Fluor 488-conjugated IgG (A11055, A21206) and Alexa Fluor 594-conjugated IgG (A11058) were from Wuhan Tri-Eagle Company. 4,0,6-diamidino-2-phenylindole (DAPI; Bio-Tech).

[0041] Model building method:

[0042] Under general anesthesia, 4% isoflurane (Sinopharm Chemical Reagent Co., Ltd., China) was inhaled in oxygen, and then 0.4% oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical, Japan) were applied topically for local anesthesia of the eyelid margin. Liquid nitrogen was placed in a thermos bottle, and then a special pure copper device was used for temperature conduction, and a special freezing device was applied to the upper and lower eyelid margins. The temperature of the device in contact with the eyelid can be maintained at around -110 degrees Celsius. By applying the above device in contact with the eyelid margin for 30 seconds (such as Figure 1 The contact site was approximately the central two-thirds of the eyelid margin. In the control group, mice were not treated and housed in the same environment as mice that received liquid nitrogen cryotherapy. The experimental group mice were euthanized 7, 14, 21, and 28 days after eyelid margin cryotherapy. The control group mice were euthanized after 28 days.

[0043] Experimental structure:

[0044] (1) Changes in the meibomian glands and other ocular surface tissues after freezing the eyelid margin with liquid nitrogen

[0045] Combination Figure 2 As shown, 24 hours after the eyelid margin was damaged by liquid nitrogen, the meibomian gland tissue was obtained from the mouse for hematoxylin and eosin (H&E) staining observation. Only the acinar structure of the meibomian gland was found to be destroyed, and no changes were observed in other structures such as the palpebral muscle tissue, conjunctiva, and cornea. This suggests that the application of liquid nitrogen to the eyelid margin can specifically damage the acinar cells of the meibomian gland.

[0046] Mice were evaluated at specific intervals of 7, 14, 21, and 28 days after the liquid nitrogen injury was applied to the eyelid margin. Compared to controls that did not receive liquid nitrogen injury, changes in the smoothness and thickness of the eyelid margin were observed, and corneal fluorescent staining increased over time (e.g., Figure 3A and B show that the eyelid margin changes were the most significant in the 21st day group, the eyelid margin was the roughest, and the corneal fluorescence staining was the strongest. The meibomian gland scoring standard indicates the pathological changes in the eyelid margin morphology after cryotherapy.

[0047] The absence of meibomian glands is an important diagnostic indicator of meibomian gland dysfunction (MGD). After liquid nitrogen injury is applied to the eyelid margin, progressive morphological changes of the meibomian glands are observed, reflecting glandular atrophy and loss of the main ducts (eg, Figure 4 It was further demonstrated that the atrophic MGD model was established by damaging the eyelid margin of mice with liquid nitrogen.

[0048] (2) Changes in lipid synthesis after liquid nitrogen injury to the eyelid margin

[0049] Oil red O staining showed that the staining intensity of meibomian gland acinar cells became lighter and less after liquid nitrogen injury. The morphology of meibomian gland acinar cells showed varying degrees of atrophy as the observation time prolonged. Oil red O staining in the ducts showed incomplete filling (e.g. Figure 5 As shown in A). PPAR-γ (peroxisome proliferator-activated receptor γ) plays an important role in maintaining the morphology of the meibomian glands (meibomian gland acinar cells) and the differentiation of acinar cells, and is also a key factor in lipid synthesis in meibomian gland acinar cells. PPAR-γ immunofluorescence staining and western blot analysis showed that PPAR-γ expression decreased after liquid nitrogen injury was applied to the eyelid margin, and the decrease in expression was particularly significant 7 and 21 days after treatment. The decrease in PPAR-γ expression on the 7th day may be the result of massive destruction of acinar cells, but the significant decrease on the 21st day can rule out acute injury. Although a slight increase in PPAR-γ expression was observed 14 and 28 days after injury, the expression level was still below the normal range. This indicates that the application of liquid nitrogen to the eyelid margin leads to a decrease in lipid synthesis capacity in MG and suggests that acinar cell differentiation is impaired (such as Figure 5 BE). From the changes in lipid synthesis, it can be seen that low temperature injury can cause the ability of acinar cells to synthesize lipids to decrease, and this effect can last for a period of time. The lesions are the same as those caused by meibomian gland damage, which further proves that the present invention can be used as an atrophic MGD model.

[0050] (3) Inflammation of meibomian gland tissue after liquid nitrogen injury to the eyelid margin

[0051] Inflammatory infiltration is a characteristic feature of meibomian gland dysfunction (MGD). The expression levels of IL-1β and IL-18 and immunofluorescence observations of TNFα showed that inflammatory cytokines peaked on the 7th day, which may be due to the acute phase of injury. However, as the experimental observation period prolonged, the inflammation did not show a significant decrease, but remained at a high level. The results of observations on the 21st and 28th days showed that inflammatory cytokines were still higher than those in the control group. On the 21st day, the three inflammatory factors detected were all in a high range and the expression of PPARγ was the weakest at this time. Studies have pointed out that IL-1β can inhibit the proliferation of meibomian gland acinar cells, inhibit lipid accumulation and PPARγ expression, and promote cell apoptosis while activating the p38-MAPK signaling pathway. This may be the reason for the further decline and deterioration of the lipid synthesis function of the meibomian gland (such as Figure 6 A, B). This demonstrates that liquid nitrogen injury can lead to subsequent symptoms consistent with inflammatory infiltration caused by meibomian gland dysfunction.

[0052] (4) Cell proliferation and apoptosis in MG after liquid nitrogen injury to the eyelid margin

[0053] Ki67 serves as a marker for actively proliferating cells, including those within the meibomian glands and various other tissues. Under normal physiological conditions, Ki67 is expressed in the basal and ductal cells of the meibomian gland alveoli, indicative of cell proliferation. In the present model, a decrease in Ki67 expression was observed. At 14 days post-treatment, expression was confined to a portion of the basal region of the alveoli. By day 21, Ki67 expression was significantly reduced, and minimal expression was detected when assessed on day 28. This continuous decrease in Ki67 expression may reflect the response of the alveolar cells to cryo-induced injury, indicating that the proliferation function of cells within the meibomian glands continues to decrease and remain at a low level. The decrease in Ki67-positive cells may also indicate the presence of meibomian gland dysfunction and / or impaired regenerative capacity of the alveolar cells (e.g., Figure 7 (as shown in A and B).

[0054] TUNEL staining showed that cell apoptosis persisted after liquid nitrogen injury, with an increasing trend from the 7th to the 14th day of observation. From the 14th to the 28th day, cell apoptosis was basically stable and maintained at a relatively high level (e.g. Figure 7 C and D).

[0055] Basal epithelial cells showed K14 staining, indicating the presence of intermediate filaments in the epithelial cell skeleton, which is one of the markers of basal cells. The data showed that K14 staining decreased after local cryoinjury, indicating that the meibomian gland alveoli atrophy (e.g. Figure 6 C and E). The symptoms are the same as those caused by meibomian gland dysfunction.

[0056] (5) Keratinization of MG after liquid nitrogen injury to the eyelid margin

[0057] Hyperkeratosis is a clear pathological sign of obstructive meibomian gland dysfunction, resulting in ductal dilatation and alveolar atrophy, which may be seen in atrophic meibomian gland dysfunction. K10 (keratin 10) is a biomarker of terminally differentiated keratinized epithelium, which is expressed at a relatively low level in the central duct of normal meibomian glands. Starting from the seventh day after cryotherapy of the eyelid margin with liquid nitrogen, immunofluorescence showed that K10 was expressed in the alveoli, and the expression of K10 in the duct increased as the observation period prolonged. In addition, areas of meibomian gland atrophy and loss showed high levels of K10 expression. This suggests that keratinization is significantly increased after liquid nitrogen treatment of the eyelid margin, and this hyperkeratotic state increases over time (e.g., Figure 8 A).

[0058] Sprr1b (small proline-rich protein 1B) is considered one of the hallmarks of keratinization. To further verify the occurrence of keratinization, we evaluated the mRNA expression level of Sprr1b and observed that its expression increased after treatment with liquid nitrogen. It is worth noting that Sprr1b expression was particularly significantly increased in the groups 7 and 30 days after treatment. Although it decreased slightly in the groups 14 and 21 days, the levels were still higher than those in the control group (such as Figure 8 B). The same symptoms as those caused by meibomian gland dysfunction.

[0059] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of atrophic meibomian gland dysfunction, characterized in that: The following steps are involved: The mice were generally anesthetized and then locally anesthetized at the eyelid margins. Liquid nitrogen injury was performed on the margins of the upper and lower eyelids of the mice using a freezing device filled with liquid nitrogen, with the contact position being the center of the eyelid margins.

2. The method for constructing an animal model of atrophic meibomian gland dysfunction according to claim 1, characterized in that: The liquid nitrogen damage time is 28s-32s.

3. The method for constructing an animal model of atrophic meibomian gland dysfunction according to claim 2, characterized in that: The contact point of the freezing device is 2 / 3 of the way along the middle of the eyelid margin.

4. The method for constructing an animal model of atrophic meibomian gland dysfunction according to claim 3, characterized in that: The mice are male mice aged 6 to 10 weeks.

5. The method for constructing an animal model of atrophic meibomian gland dysfunction according to claim 4, characterized in that: General anesthesia is administered by inhalation anesthetic, which may be 4% isoflurane.

6. The method for constructing an animal model of atrophic meibomian gland dysfunction according to claim 5, characterized in that: For local anesthesia, 0.4% oxybuprocaine hydrochloride eye drops were used to locally anesthetize the eyelid margins.

7. The method for constructing an animal model of atrophic meibomian gland dysfunction according to claim 6, characterized in that: The freezing device comprises a contact head, which is a copper contact head, and the contact head is used to perform liquid nitrogen freezing on the mouse eyelids.

8. The method for constructing an animal model of atrophic meibomian gland dysfunction according to claim 7, characterized in that: The temperature of the contact head of the freezing device when damaged by liquid nitrogen is -115°C to 85°C.

9. A method for constructing an animal model of atrophic meibomian gland dysfunction, characterized in that: The model is constructed using the method for constructing an animal model of atrophic meibomian gland dysfunction described in claims 1-8.

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