Method for constructing and evaluating an animal model of meibomian gland dysfunction

Abstract

The application relates to a kind of meibomian gland dysfunction animal model construction method and detection evaluation method, comprising the following steps: S1, 6-8 weeks old mice are preoperatively anti-infection treated 3 days before model construction;S2, the mouse is injected with intraperitoneal anesthesia, fixed on the operation platform, the meibomian gland area of eyelid is positioned under the in vivo microscope, a syringe is used to advance from the outer canthus eyelid, the needle tip is parallelly advanced to the meibomian gland area along the eyelid to establish the injection channel to the meibomian gland area, and the meibomian gland area is injected with benzalkonium chloride solution through the injection channel to induce meibomian gland injury, so as to construct the animal model.The beneficial effect is that a meibomian gland dysfunction animal model construction method with short modeling cycle, accurate operation, high repeatability and complete pathological characteristics is provided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for constructing and evaluating an animal model of meibomian gland dysfunction. Background Technology

[0002] Meibomian gland dysfunction (MGD) is the core cause of evaporative dry eye, characterized by meibomian gland duct obstruction or abnormal glandular secretion, leading to insufficient tear film lipid layer, decreased tear film stability, and chronic ocular surface inflammation. MGD has a high incidence and recurrence rate in ophthalmology and is closely related to pathological changes such as abnormal meibomian gland duct keratinization, acinar atrophy, decreased or abnormal lipid secretion, and persistent chronic inflammation, severely impairing patients' visual function and quality of life. Therefore, establishing stable, reproducible animal models that highly correlate with clinicopathological features is a crucial prerequisite for in-depth analysis of the pathogenesis of MGD, screening for effective therapeutic targets, and evaluating the efficacy of drug interventions.

[0003] Currently, MGD animal models are mainly divided into three categories: 1) Mechanical injury or burning model: This model simulates an obstruction state by physically destroying the eyelid margin or duct, but it is highly invasive, technically complex, and the inflammatory response is unstable. 2) Systemic inflammation or hormone intervention models: such as high-fat diet induction, hormone deprivation or systemic inflammatory stimulation (such as intraperitoneal injection of drugs), have a long modeling cycle (usually several weeks to several months) and non-specifically affect the systemic metabolic state, resulting in unclear inflammation localization and pathological features deviating from clinical reality; 3) Eye drop or ocular surface drug delivery model: Corneal / conjunctival damage is induced by instilling irritants into the ocular surface, but the targeting of meibomian glands is poor, and it is difficult to accurately act on the glandular tissue and stably replicate the typical pathological features of acinar atrophy, lipid metabolism disorder and inflammatory response.

[0004] The three models mentioned above cannot accurately target meibomian gland tissue, easily causing non-specific ocular surface damage; the models have poor stability and low reproducibility; they cannot simultaneously and stably present key pathological changes such as acinar atrophy, lipid metabolism disorders, and chronic inflammation, making them difficult to use for drug intervention and mechanism research. Summary of the Invention

[0005] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for constructing and evaluating an animal model of meibomian gland dysfunction, which solves the technical problems of unstable meibomian gland injury, poor model repeatability, unclear inflammation localization, and long modeling cycle.

[0006] (II) Technical Solution To achieve the above objectives, the main technical solutions adopted by the present invention include: In a first aspect, embodiments of the present invention provide a method for constructing an animal model of meibomian gland dysfunction, comprising the following steps: S1. Three days before model construction, 6-8 week old mice were given preoperative anti-infection treatment. S2. Mice were anesthetized by intraperitoneal injection and fixed on the operating platform. The meibomian gland region of the eyelid was located under a stereomicroscope. A syringe was used to insert the needle from the outer canthus of the eyelid margin, and the needle tip was advanced parallel to the eyelid to the meibomian gland region to establish an injection channel to the meibomian gland region. Benzalkonium chloride solution was injected into the meibomian gland region through the injection channel to induce meibomian gland damage, thereby constructing an animal model.

[0007] In a preferred embodiment of the present invention, in step S2, the mass concentration of the benzalkonium chloride solution is 0.1%-0.5%; and the total volume of the benzalkonium chloride solution injected into the meibomian gland region of each eye is 10-20 μL.

[0008] In a preferred embodiment of the present invention, in step S2, the injection is a single-point or multi-point injection; the injection area includes the inner, outer, and central distribution areas of the meibomian glands of the eyelid.

[0009] In a preferred embodiment of the present invention, step S1 includes preoperative anti-infection treatment, which includes instilling antibiotic eye drops onto the ocular surface of mice. The antibiotic eye drops are selected from one of the following: levofloxacin, tobramycin, and gatifloxacin eye drops.

[0010] In a preferred embodiment of the present invention, in step S2, after injecting benzalkonium chloride solution, residual solution on the skin surface is removed with a sterile cotton swab and the eye surface is rinsed with physiological saline.

[0011] In a preferred embodiment of the present invention, the mouse is a female C57BL / 6 mouse.

[0012] In a second aspect, embodiments of the present invention provide a method for detecting and evaluating an animal model of meibomian gland dysfunction, used to evaluate an animal model of meibomian gland dysfunction obtained by the construction method of the first aspect, comprising the following steps: at least two of the following assessments: ocular surface signs assessment, histological assessment, and lipid metabolism assessment.

[0013] As a preferred embodiment of the present invention, before performing histological evaluation or lipid metabolism evaluation, a meibomian gland tissue sample collection step is also included: peeling off the eyelid skin and muscles of the animal model of meibomian gland dysfunction, fully exposing and removing the meibomian gland tissue.

[0014] In a preferred embodiment of the present invention, histological evaluation includes at least one of the following: Project 1: HE staining of meibomian gland tissue to assess acinar morphology, ductal dilation, and inflammatory cell infiltration; Project 2: Immunohistochemical staining of meibomian gland tissue to detect the expression of specific inflammatory factors.

[0015] In a preferred embodiment of the present invention, lipid metabolism assessment includes at least one of the following: Project 1: Oil Red O staining of meibomian gland tissue to assess meibomian gland lipid synthesis and secretory function; Project 2: Perform lipidomics analysis on meibomian gland tissue to identify lipid molecule types and compare changes in their relative abundance.

[0016] In a preferred embodiment of the present invention, the assessment of ocular surface signs includes at least one of the following: Project 1: Observation and photography of the anterior segment of the eye using a slit-lamp microscope; Project 2: Scoring of corneal epithelial damage using sodium fluorescein staining; Project 3: Determining tear film breakup time.

[0017] (III) Beneficial Effects The beneficial effects of this invention are as follows: This invention provides a method for constructing an animal model of meibomian gland dysfunction (MGD), offering a method with a short modeling cycle, precise operation, high repeatability, and complete pathological features. By locating the model under direct visualization with a stereomicroscope and establishing an injection channel using a specific path of parallel needle insertion from the outer canthus, benzalkonium chloride solution can be accurately and directly delivered to the meibomian gland region. This method minimizes non-specific damage to other ocular surface structures such as the cornea and conjunctiva, ensuring that the lesion originates in the meibomian gland, highly simulating the core pathological origin of clinical MGD, and achieving precise and efficient induction of damage to the meibomian gland tissue. Compared to the lengthy induction cycle of several weeks to months required in existing technologies, this invention, through a single targeted injection of the potent inducing agent benzalkonium chloride solution, significantly shortens the modeling time compared to the several weeks to months required in existing technologies. Significant results are visible in approximately 7 days, and complete modeling is achieved in 14 days, stably inducing a series of typical MGD pathological changes. The constructed model can synchronously and stably present key features such as acinar atrophy, abnormal ductal keratinization, abnormal lipid secretion and composition, and local chronic inflammatory response, providing a stable in vivo research platform for in-depth exploration of the pathogenesis and pathological process of MGD and efficient screening and evaluation of therapeutic drugs.

[0018] Using a higher concentration of benzalkonium chloride solution (0.5%), severe atrophy, significant inflammation, and lipid secretion disorders of the meibomian glands can be stably induced, successfully constructing a severe meibomian gland dysfunction model. Conversely, a lower concentration (0.3%) can induce a moderate or early-stage lesion model with well-defined pathological features but milder symptoms. Researchers can flexibly and systematically prepare specific disease models with controllable and highly reproducible pathological phenotypes according to different research objectives (such as simulating different stages of disease progression or testing the dose-dependent efficacy of drugs).

[0019] Single-point injection is suitable for simulating focal lesions, is simple to operate, and has good repeatability. Multi-point injection in the medial, lateral, and / or central regions can more comprehensively cover the meibomian gland distribution area, simulating diffuse, multifocal lesions in clinical practice, thus more realistically reproducing the pathological characteristics of MGD. This design allows the model to be specifically constructed according to different research objectives, broadening the model's scientific research application scenarios.

[0020] By administering antibiotic eye drops preoperatively for infection control, the risk of exogenous infection introduced during surgery was effectively prevented, reducing the risk of postoperative complications such as bacterial conjunctivitis or keratitis. This also avoided interference from non-specific infections and inflammations on the meibomian gland-specific injury model, ensuring that the pathological changes in the constructed model were purely induced by benzalkonium chloride solution, thus improving the accuracy of the model and experimental results. Immediately after injection, sterile cotton swabs were used to remove residual solution from the skin surface, followed by rinsing the ocular surface with saline solution, minimizing the residue and contact time of benzalkonium chloride solution in non-target tissues of the ocular surface (such as the cornea and conjunctiva).

[0021] This invention provides a method for detecting and evaluating an animal model of meibomian gland dysfunction (MGD), employing an assessment approach encompassing at least two dimensions: ocular surface signs, histology, and lipid metabolism. This comprehensive assessment system, involving multiple dimensions, indicators, and time points, overcomes the shortcomings of traditional single-method assessments, which are often one-sided and unable to fully reflect the complex pathological characteristics of the disease. By implementing this method, it is possible to verify whether the model successfully simulates the key pathological aspects of MGD from the perspectives of macroscopic functional changes, microscopic tissue structural damage, and molecular metabolic levels.

[0022] Complete dissection and removal of meibomian gland tissue allows for direct observation of the gland's pathological state. Hematoxylin and eosin (HE) staining and immunohistochemical staining enable qualitative and semi-quantitative analysis of the model's pathological structure. HE staining provides a holistic and direct assessment of core morphological changes such as acinar atrophy, ductal dilation, and abnormal keratinization, while immunohistochemical staining further locates and quantifies the infiltration of specific inflammatory factors (such as TNF-α, IL-1β, and IL-6) at the protein expression level. Used alone or in combination, these two methods comprehensively and deeply reveal the intensity of inflammatory activity and the degree of tissue damage in the model, providing crucial pathological evidence for assessing disease severity and the effectiveness of targeted anti-inflammatory therapies.

[0023] Oil Red O staining and lipidomics analysis have enabled a comprehensive analysis of lipid abnormalities, from microscopic localization to molecular hierarchy. Oil Red O staining can visually display the deposition and distribution of neutral lipids in the meibomian glands, directly assessing their synthesis and secretion functions, while lipidomics analysis can accurately identify the types, structures, and relative abundance changes of lipid molecules at the systemic level, revealing specific patterns of lipid metabolism disorders.

[0024] Slit-lamp microscopy and anterior segment observation and photography provided visual records of eyelid hyperemia, edema, secretions, and corneal transparency, offering evidence for assessing overall ocular surface inflammation and structural changes after model construction. Fluorescein staining, through standardized semi-quantitative analysis of punctate staining of the corneal epithelium, revealed secondary corneal epithelial cell damage caused by decreased tear film stability. Tear film breakup time measurement directly and quantitatively reflects the stability of the tear film lipid layer. This assay objectively measures the accelerated tear evaporation caused by meibomian gland dysfunction, validating whether the model successfully simulates the pathophysiological consequences of clinical MGD. Attached Figure Description

[0025] Figure 1 Schematic diagram of injecting benzalkonium chloride solution into the eyelid; Figure 2 This image shows the changes in the cornea of ​​mice after eyelid injection. Figure 3 A diagram showing the changes in meibomian gland openings in mice after eyelid injection. Figure 4 This image shows the changes in corneal fluorescein staining in mice after eyelid injection. Figure 5 Meibomian gland tissue of mice after eyelid injection; Figure 6 HE staining results of meibomian gland tissue from mice after eyelid injection; Figure 7 Immunohistochemical staining results of meibomian gland tissue in mice after eyelid injection; Figure 8 Oil Red O staining results of meibomian glands in mice after eyelid injection. Detailed Implementation

[0026] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art. In the following text, unless otherwise specified, the concentration of benzalkonium chloride solution refers to mass concentration.

[0028] Example 1 This embodiment provides a method for constructing an animal model of meibomian gland dysfunction, including the following steps: (1) Three days before the model was constructed, several 8-week-old SPF-grade C57BL / 6 female mice were taken and levofloxacin eye drops were instilled into the ocular surface of all mice 4 times a day for preoperative anti-infection treatment. (2) Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg), fixed on the operating platform, and the meibomian gland region of the eyelid was located under a stereomicroscope. A 1 mL syringe was used to insert the needle from the outer canthus of the eyelid margin, and the needle tip was advanced parallel to the eyelid to the meibomian gland region to establish an injection channel to the meibomian gland region. A micro-injection needle was then used to inject a 0.3% concentration and a volume of 10 μL of benzalkonium chloride solution into the meibomian gland region through the injection channel to induce meibomian gland damage. The injection method was single-point injection, and the injection areas were the inner, outer and central areas of the eyelid. After injecting the benzalkonium chloride solution, the residual solution on the skin surface was removed with a sterile cotton swab, and the ocular surface was rinsed with physiological saline to finally construct the animal model.

[0029] Example 2 This embodiment provides a method for constructing an animal model of meibomian gland dysfunction. The difference between this embodiment and embodiment 1 is that: in step (2), the concentration of benzalkonium chloride solution is 0.5%, the volume is 10 μL, and the injection method is multi-point injection, which is performed on the inner, outer and central areas of the eyelid respectively. The remaining steps are the same.

[0030] Example 3 This embodiment provides a method for detecting and evaluating an animal model of meibomian gland dysfunction, which is used to evaluate the animal model and includes the following steps: Step 1, Assessment of ocular surface signs: Ocular surface signs of mice were assessed 3, 7 and 14 days after modeling. 1) The anterior segment of the mouse was observed and photographed using a slit-lamp microscope. Changes in eyelid congestion, edema, secretions, and corneal transparency were observed and recorded. 2) Fluorescein sodium staining: 1 μL of 1% fluorescein sodium solution was added into the conjunctival sac of mice, and the corneal staining was observed under cobalt blue light. The degree of corneal epithelial damage was semi-quantitatively scored using the Oxford corneal fluorescence staining scoring system. 3) Tear film breakup time (BUT) detection: After fluorescein staining, the time from the last blink to the appearance of the first dry spot on the cornea was recorded. The average value was taken after three consecutive measurements, and the changes in ocular surface damage and tear film stability at each time point were recorded. Step 2, Meibomian gland tissue sampling: The mice were anesthetized with sodium pentobarbital (40 mg / kg). After anesthesia, the mice were fixed on the platform. Microforceps and scissors were used to dissect the eyelid skin and muscle tissue of the mice to fully expose the meibomian glands, avoiding damage to the glands and surrounding tissues. The meibomian gland tissue was picked up with microforceps and the entire meibomian gland tissue was cut off. The changes in the meibomian gland tissue were observed. Step 3, Histological assessment: 1) HE staining: The removed mouse meibomian gland tissue was immediately fixed in 4% paraformaldehyde for 24 hours, routinely dehydrated, and embedded in paraffin. The section thickness was 4 μm. After dewaxing with xylene and rehydration with graded ethanol, the sections were stained with hematoxylin and eosin (HE). The structure of meibomian gland acini, duct morphology, and changes in eyelid epithelium were observed under a light microscope. Acini atrophy, duct dilation, abnormal keratinization, and inflammatory cell infiltration were assessed, and a semi-quantitative pathological score was performed. 2) Immunohistochemical staining: Mouse eyelid margins and meibomian gland tissues fixed in 4% paraformaldehyde were routinely dehydrated, embedded in paraffin, and serially sectioned to a thickness of 4 μm. After dewaxing with xylene and rehydration with graded ethanol, antigen retrieval was performed using citrate buffer (95-98℃, 10-15 min). After cooling to room temperature, the sections were incubated with 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase activity. After washing with PBS, the sections were blocked with 5% bovine serum albumin (BSA) and incubated at room temperature for 30 min. Primary antibodies against inflammatory factors (TNF-α, IL-1β, IL-6) were added, and the sections were incubated overnight at 4℃. The next day, the sections were warmed to room temperature, washed with PBS, and the corresponding HR antibodies were added. P-labeled secondary antibody was incubated at room temperature for 30 min, and then stained with DAB chromogenic reagent. The staining time was controlled under a microscope. The cell nuclei were counterstained with hematoxylin, dehydrated, cleared, and mounted. The images were observed and acquired under an optical microscope to assess the localization and expression of inflammatory factors in the eyelid margin epithelium, meibomian gland acini, and ducts. ImageJ software was used to perform semi-quantitative analysis of the positively stained areas. The average optical density (IOD / Area) or the percentage of positive area was used as the evaluation index to compare the differences in the expression levels of inflammatory factors in the mouse eyelid margin and meibomian gland tissue. Step 4, Lipid Metabolism Assessment: 1) Oil Red O staining: Fresh meibomian gland tissue was quick-frozen in OCT embedding medium, with a section thickness of 10 μm. After the sections were air-dried, they were stained with Oil Red O staining solution to show the distribution of neutral lipids in the meibomian glands. After counterstaining the cell nuclei with hematoxylin, the sections were mounted, observed and photographed under a microscope, and the lipid staining area and staining intensity were quantitatively analyzed using image analysis software to assess changes in the lipid synthesis and secretion functions of the meibomian glands. 2) Lipidomics analysis: Meibomian gland tissue was collected, and lipids were extracted using the methanol-chloroform method. After centrifugation, drying, and reconstitution, lipid components of the meibomian gland were analyzed by liquid chromatography-mass spectrometry (LC-MS). The types and relative abundance changes of lipid molecules in mouse meibomian glands were detected and compared. Differential lipid molecules were screened by multivariate statistical analysis to explore the characteristics of meibomian gland lipid metabolism disorder in the benzalkonium chloride solution-induced MGD model.

[0031] Comparative Example 1 This comparative example provides a method for constructing an animal model of meibomian gland dysfunction. The difference between this comparative example and Example 1 is that the concentration of benzalkonium chloride solution injected in step (2) is 0.1% and the volume is 10 μL; the other steps are the same.

[0032] Comparative Example 2 This comparative example provides a method for constructing an animal model of meibomian gland dysfunction. The difference between this comparative example and Example 1 is that step (2) involves injecting a 0.9% sodium chloride solution; the remaining steps are the same.

[0033] The animal models of Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were evaluated, and the evaluation results are as follows: Figures 2 to 8 As shown, the following analytical conclusions can be drawn: Figure 2 For changes in the mouse cornea, compared with Comparative Examples 1 and 2, the corneal epithelium in Examples 1 and 2 showed varying degrees of loss 3, 7 and 14 days after eyelid injection.

[0034] Figure 3 The changes in the meibomian gland openings in mice were compared with those in Comparative Example 2. After injection into the eyelids of Examples 1, 2, and 14, the meibum gradually thickened, and varying degrees of obstruction, lipid plugs, keratinization, and rounding appeared at the meibomian gland openings.

[0035] Figure 4 The changes in corneal fluorescein staining in mice were compared with those in Comparative Example 2. After eyelid injection in Examples 1, 2, and 14, the corneal epithelium in the lower central region showed varying degrees of staining.

[0036] Figure 5 The changes in the meibomian gland tissue of mice were compared with those in Comparative Example 2. After eyelid injection, the meibomian gland ducts in the upper eyelids of Examples 1, 2 and 1 were uneven in thickness, and the meibomian glands in the lower eyelids were locally atrophied and absent.

[0037] Figure 6 HE staining results of mouse meibomian gland tissue show that different concentrations of benzalkonium chloride solution injected into the eyelids resulted in varying degrees of histological changes in the mouse meibomian gland tissue at different time points. In Comparative Example 2, clear meibomian gland acinar structures, relatively regular cell arrangement, and intact acinar outlines were observed at 3, 7, and 14 days post-injection, with no obvious abnormal pathological changes. In Comparative Example 1, the overall structure of the meibomian gland tissue was basically intact, with only mild acinar structural changes and loose cytoplasm in some acinar cells, indicating mild tissue damage. In contrast, the meibomian gland tissues of Examples 1 and 2 showed more pronounced pathological changes at all time points. Disordered acinar structure appeared as early as 3 days post-injection, with obvious vacuolar changes in the acinar cell cytoplasm and enlargement of some acinar cavities. By 7 and 14 days post-injection, the vacuolar changes were further aggravated, the acinar arrangement became more loose, and some acinar structures were incomplete, indicating progressive damage to the meibomian gland tissue.

[0038] Figure 7 The results show the immunohistochemical staining of mouse meibomian gland tissue. According to the TNF-α immunohistochemical staining results, the expression level of TNF-α in mouse meibomian gland tissue differed significantly at different time points after eyelid injection of different concentrations of benzalkonium chloride solution. In Comparative Example 2, only a small amount of TNF-α positive staining was observed at 3, 7, and 14 days after injection, mainly in a scattered distribution, suggesting a low level of inflammation in normal meibomian gland tissue. In Comparative Example 1, a slight increase in TNF-α positive cells was observed at 3 and 7 days after injection, with a slightly enhanced staining intensity compared to the control group. By day 14, the expression level tended to stabilize, indicating a mild overall inflammatory response. In contrast, Examples 1 and 2 showed significantly enhanced TNF-α positive staining at all time points. A significant increase in TNF-α positive cells was observed 3 days after injection, mainly distributed around the acini and in the acini stroma. By day 7 after injection, TNF-α expression further increased, with a significant increase in both the number of positive cells and the staining intensity. At day 14 after injection, TNF-α positive expression remained at a high level, suggesting a certain degree of persistence in the inflammatory response. Semi-quantitative analysis of each group showed that, compared with Comparative Example 2, the proportion of TNF-α positive cells in Examples 1 and 2 was significantly increased at each time point (P<0.05 or P<0.01), and the overall expression level of Example 1 was higher than that of Example 2, showing a certain dose-dependent change.

[0039] Figure 8The results of Oil Red O staining of mouse meibomian glands showed significant differences in lipid distribution among the groups 14 days after injection. In Comparative Example 2, the meibomian gland acini were structurally intact, with relatively uniform lipid distribution and no obvious abnormal lipid accumulation. Compared to Comparative Example 2, Comparative Example 1 showed enhanced lipid staining and a slight increase in the number of lipid droplets within the acini. In Examples 1 and 2, Oil Red O staining was significantly enhanced, indicating a large accumulation of red lipid droplets within the acini, partial acini dilation, and obstructed lipid excretion. This suggests that injection of higher concentrations of benzalkonium chloride solution can lead to abnormal lipid accumulation in the meibomian glands. These results indicate that eyelid injection of benzalkonium chloride solution can cause lipid metabolism disorders in the meibomian glands, and the degree of lipid deposition increases with increasing benzalkonium chloride solution concentration.

[0040] Lipomics analysis was performed on meibomian gland tissue from mice. The results showed significant differences in the composition and expression levels of multiple lipid subclasses between the normal group (i.e., healthy mice that did not receive any drugs) and Example 1. Hierarchical clustering heatmaps clearly distinguished the two groups in the overall lipid profile, suggesting a systemic alteration in the lipid composition of the meibomian glands in Example 1. In ceramide (Cer) lipids, Example 1 showed a significantly different distribution compared to the normal group, with multiple ceramide molecules showing significant changes, indicating impaired meibomian gland lipid metabolic homeostasis. Similarly, in cholesterol ester (CE) lipids, there was a clear separation between Example 1 and the normal group, with multiple cholesterol ester molecules exhibiting abnormal changes, reflecting impaired meibomian gland lipid storage and secretion functions. In fatty acid (FA) lipids, the differences between Example 1 and the normal group were particularly significant, with some fatty acid molecules showing marked changes, suggesting that abnormal fatty acid composition may affect the stability of the tear film lipid layer. Overall, the lipidomics results showed that injection of benzalkonium chloride solution significantly altered key lipid categories such as ceramides, cholesterol esters, and fatty acids in the meibomian glands of mice. The lipid profile characteristics were highly consistent with the lipidomics alteration trends reported in clinical MGD patients, further validating the reliability and clinical relevance of this MGD animal model from the perspective of lipid metabolism.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an animal model of meibomian gland dysfunction, characterized in that, Includes the following steps: S1. Three days before model construction, 6-8 week old mice were given preoperative anti-infection treatment. S2. Mice were anesthetized by intraperitoneal injection and fixed on the operating platform. The meibomian gland region of the eyelid was located under a stereomicroscope. A syringe was used to insert the needle from the outer canthus of the eyelid margin, and the needle tip was advanced parallel to the eyelid to the meibomian gland region to establish an injection channel to the meibomian gland region. Benzalkonium chloride solution was injected into the meibomian gland region through the injection channel to induce meibomian gland damage, thereby constructing an animal model.

2. The method for constructing an animal model of meibomian gland dysfunction as described in claim 1, characterized in that, In step S2, the mass concentration of the benzalkonium chloride solution is 0.1%-0.5%; the total volume of the benzalkonium chloride solution injected into the meibomian gland region of each eye is 10-20 μL.

3. The method for constructing an animal model of meibomian gland dysfunction as described in claim 2, characterized in that, In step S2, the injection may be a single-point or multi-point injection; The injection areas include the inner, outer, and central distribution areas of the meibomian glands in the eyelids.

4. The method for constructing an animal model of meibomian gland dysfunction as described in claim 1, characterized in that, In step S1, preoperative anti-infection treatment includes instilling antibiotic eye drops onto the ocular surface of the mice; The antibiotic eye drops are selected from one of levofloxacin, tobramycin, and gatifloxacin eye drops.

5. The method for constructing an animal model of meibomian gland dysfunction as described in claim 1, characterized in that, In step S2, after injecting the benzalkonium chloride solution, the residual solution on the skin surface is removed with a sterile cotton swab, and the eye surface is rinsed with physiological saline.

6. The method for constructing an animal model of meibomian gland dysfunction as described in any one of claims 1-5, characterized in that, The mice were female C57BL / 6 mice.

7. A method for detecting and evaluating an animal model of meibomian gland dysfunction, characterized in that, The method for evaluating an animal model of meibomian gland dysfunction obtained by the construction method according to any one of claims 1-6 includes the following steps: at least two of the following assessments: ocular surface signs assessment, histological assessment, and lipid metabolism assessment.

8. The method for detecting and evaluating an animal model of meibomian gland dysfunction as described in claim 7, characterized in that, Prior to the histological assessment or the lipid metabolism assessment, a meibomian gland tissue sample collection step is also included: peeling off the eyelid skin and muscle of the animal model of meibomian gland dysfunction, fully exposing and removing the meibomian gland tissue.

9. The method for detecting and evaluating an animal model of meibomian gland dysfunction as described in claim 8, characterized in that, The histological assessment includes at least one of the following: Project 1: HE staining of meibomian gland tissue to assess acinar morphology, ductal dilation, and inflammatory cell infiltration; Project 2: Immunohistochemical staining of meibomian gland tissue to detect the expression of specific inflammatory factors.

10. The method for detecting and evaluating an animal model of meibomian gland dysfunction as described in claim 8, characterized in that, The lipid metabolism assessment includes at least one of the following: Project 1: Oil Red O staining of meibomian gland tissue to assess meibomian gland lipid synthesis and secretory function; Project 2: Perform lipidomics analysis on meibomian gland tissue to identify lipid molecule types and compare changes in their relative abundance.

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