Method for constructing a graded limbal injury and repair model

Abstract

A method for constructing a graded limbal injury and repair model comprises: after administering topical anesthetic drops to the operated eye of a model animal, placing a limbal injury device containing a NaOH solution in the limbal area of ​​the model animal for 30 seconds, then removing the device and irrigating the operated eye with saline; postoperatively, administering an antibacterial drug to the operated eye of the model animal several times daily; and eight days after surgery, a graded limbal injury and repair model is obtained, wherein the limbal injury device applies alkali burns of varying sizes to the limbal area, thereby achieving a graded injury effect. The construction method provided by the present invention has been shown to be simple, laboratory-friendly, inexpensive, short, and reproducible, with quantifiable evaluation methods, making it an ideal method for constructing an animal model of limbal stem cell injury.

Description

Technical Field

[0001] The present invention relates to an animal modeling technology, and in particular to a method for constructing an animal corneal limbal local injury model, so as to facilitate medical research on corneal injury and repair. Background Art

[0002] The cornea is the transparent surface tissue of the eye, performing crucial functions such as protecting the eyeball, focusing light, and maintaining visual quality. However, corneal diseases and injuries, such as inflammation, ulcers, and chemical burns, are common clinically. Corneal epithelial cells, the outermost layer of the cornea, are most susceptible to external stimuli. Their maintenance and repair depend on limbal stem cells, located in the limbus region. These stem cells possess the ability to self-renew and differentiate into epithelial cells. Common clinical injuries to the limbus, such as chemical and thermal burns, ocular surface surgical trauma, corneal inflammation, and contact lens use, can lead to limbal stem cell damage. Common clinical manifestations of limbal stem cell deficiency include corneal opacity, edema, neovascularization, corneal scarring, and ultimately, decreased vision. Clinical manifestations vary depending on the severity of limbal injury, leading to different treatment options. Generally, more severe limbal injury is associated with greater difficulty in treatment and a poorer prognosis. Limbal stem cell deficiency is a clinically intractable ocular surface disease, and finding an effective treatment remains both a challenge and a hot topic. Therefore, establishing animal models of limbal stem cell damage at varying levels is crucial to provide an effective experimental basis for clinical treatment or mechanistic research.

[0003] Currently, many papers and articles involve the application of relevant animal models. For example, a 2023 article published in the journal Cornea, titled "Novel Animal Model of Limbal Stem Cell Deficiency Induced by Forcing Eye-Open at Birth," described how limbal stem cell deficiency was induced in newborn mice by forcing them to open their eyes at birth through eyelid surgery. Another example is a 2022 article published in the journal Stem Cell Reports, titled "Plasticity of ocular surface epithelia: Using a murine model of limbal stem cell deficiency to delineate metaplasia and transdifferentiation," which described the use of the Algerbrush II corneal foreign body removal instrument to scrape the corneal epithelium from the central and limbal regions of the mouse cornea to create a model. For example, an article titled "Human limbal niche cells are a powerful regenerative source for the prevention of limbal stem cell deficiency in a rabbit model" published in the journal Scientific Reports in 2018 described the use of filter paper soaked in potassium hydroxide to burn the upper temporal area of ​​the rabbit cornea.

[0004] However, these known modeling methods have problems such as different selection of model animals, inconsistent methods of damaging the limbus, inaccurate damage range, inconsistent damage degree, or complete damage to the limbus cells, making it difficult to promote repair. These problems make it impossible to simulate the actual clinical limbus stem cell deficiency of different degrees of severity. Summary of the Invention

[0005] One object of the present invention is to provide a method for constructing a graded limbal injury and repair model, which facilitates the construction and simulation of limbal injuries of varying degrees and enables quantification of the evaluation method.

[0006] Another object of the present invention is to provide a method for constructing a graded limbal injury and repair model, so as to achieve the construction and simulation of limbal injuries of different degrees and make the scope of the injury more accurate.

[0007] Another object of the present invention is to provide a method for constructing a graded limbal injury and repair model, so as to achieve the construction and simulation of limbal injuries of different degrees and unify the degree of injury.

[0008] Another object of the present invention is to provide a method for constructing a graded limbal injury and repair model, so as to achieve the construction of different degrees of limbal damage, effectively simulate the clinical symptoms and pathological manifestations of patients with different degrees of limbal stem cell deficiency, and provide a better animal experimental basis for the exploration of the mechanism of limbal stem cell deficiency diseases and the research on limbal stem cell damage and repair.

[0009] A method for constructing a corneal limbus graded injury and repair model, comprising:

[0010] After applying topical anesthetic (e.g., 4 mg / ml oxybuprocaine hydrochloride eye drops) to the model animal's operated eye (e.g., 3 times), place a limbal injury device containing 1M NaOH solution on the limbus of the model animal for 30 seconds before removing it. Rinse the operated eye with normal saline, e.g., 15 ml per eye.

[0011] After surgery, administer antibiotics (e.g., 3 mg / ml levofloxacin eye drops) to the operated eyes of the model animals several times a day (e.g., 3 or 4 times);

[0012] Eight days after surgery, the animal model of corneal limbal graded injury and repair was obtained.

[0013] Model animals include mice, rats, rabbits and monkeys.

[0014] The construction method of the present invention can be achieved by coating the limbal injury device with a 1M NaOH solution, immersing the limbal injury device in a 1M NaOH solution so that the limbal injury device absorbs the 1M NaOH solution, or injecting the 1M NaOH solution into the limbal injury device and releasing it from the surface of the limbal injury device, thereby enabling the limbal injury device to deliver the 1M NaOH solution to the intended limbal area. A more convenient method for most laboratories is to soak filter paper in a 1M NaOH solution and then place it in the limbus area of ​​the model animal.

[0015] The contact surface of the limbus injury instrument with the surgical eye of the model animal is an open circular ring structure, including a first arc and a second arc. The radius of the first arc is 1mm to 2mm larger than the radius of the second arc, that is, the ring width is 1mm to 2mm. The ring width is adjusted according to the eyeball of the model animal. Taking mice as an example, the radius of the first arc is 1mm larger than the radius of the second arc. For example, the radius of the first arc is 3mm, and the radius of the second arc is 2mm. Generally, the ring width is increased accordingly for animals with large eyeballs. For example, for rats, the ring width is set to about 1.5mm. For rabbits and monkeys, the eyeballs are even larger, so the ring width is set to about 2mm.

[0016] The central angle of the limbal injury instrument is selected from 90°, 180° or 270°. When the central angle of the limbal injury instrument is large, such as 270°, the contact area between the limbal injury instrument and the operated eye of the model animal is also wide, and the damage to the limbus of the operated eye is also severe, corresponding to 3quarter Alkali Burn, simulating severe limbal injury. When the central angle of the limbal injury instrument is small, such as 90°, the contact area between the limbal injury instrument and the operated eye of the model animal is also narrow, and the damage to the limbus of the operated eye is also light, corresponding to 1quarter Alkali Burn, simulating mild limbal injury. If the central angle of the limbal injury instrument is 180°, it corresponds to 2quarter Alkali Burn, simulating moderate limbal injury.

[0017] The limbus system of the present invention damages local stem cells of the limbus of an animal, so as to facilitate clinical research on limbus repair.

[0018] The corneal transparency, corneal epithelial repair and corneal edema of the operated eyes were evaluated on the day of surgery and on the 2nd, 4th, 6th and 8th days after surgery.

[0019] The histological evaluation included eye tissue sections after the clinical evaluation follow-up of mice, HE and MASSON staining to observe the corneal and limbal tissue structure, limbal stem cell fluorescence staining to assess the density and distribution of limbal stem cells, corneal epithelial marker staining to assess corneal epithelial structure, and corneal nerve staining to assess corneal nerve damage.

[0020] The corneal transparency evaluation method is: slit lamp observation of corneal transparency and corneal transparency scoring:

[0021] 0 points: the cornea is completely transparent;

[0022] 1 point: The cornea is slightly cloudy, but the iris and pupil are clearly visible;

[0023] 2 points: The cornea is slightly cloudy, but the iris and pupil can still be seen;

[0024] 3 points: The cornea is cloudy and the pupil is difficult to see clearly;

[0025] 4 points: The cornea is completely cloudy and the pupil cannot be seen.

[0026] The corneal epithelial repair was evaluated by taking anterior segment photographs after staining with 1% sodium fluorescein and measuring the fluorescent staining area using ImageJ.

[0027] The degree of corneal edema was evaluated by optical coherence tomography (OCT) and corneal thickness was measured using the scanner's built-in image processing software analysis system.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The construction method provided by the present invention is simple, easy to use in the laboratory, inexpensive, short in duration, easily reproducible, and has a quantifiable evaluation method. It can be used as an ideal method for constructing an animal model of limbal stem cell damage.

[0030] During the construction process provided by the present invention, the central angle of the corneal injury instrument is controlled to damage the corneal limbus of the animal's operated eye to varying areas, thereby constructing a graded model of limbal injury. Postoperative evaluation clearly demonstrated differences in clinical and histological evaluations between the different grades of limbal injury models. This graded model can partially simulate the symptoms of limbal stem cell deficiency, which encompasses mild, moderate, and severe limbal injury, and is consistent with the pathological process and characteristics of clinical limbal stem cell injury.

[0031] The animal model of graded limbal injury obtained by the construction method provided by the present invention can directly understand the role of limbal stem cells in repairing corneal epithelial cells, and provide an experimental basis for exploring the mechanism of limbal stem cell deficiency and studying corneal injury repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the process of constructing an animal model of graded corneal limbal injury and repair according to the present invention; A is a schematic diagram of the modeling process, B is a photograph of the corneal injury instrument (filter paper ring) at different central angles, and C is the placement of the corneal injury instrument in the mouse corneal limbus during animal surgery;

[0033] Figure 2Figure 1 is a graph showing the clinical evaluation results of the mouse model of graded limbal injury; A shows the slit lamp observation of the animals in the control (normal mice), 1quarter Alkali Burn (simulating mild 1 / 4 limbal injury), 2quarter Alkali Burn (simulating moderate 1 / 2 limbal injury) and 3quarter Alkali Burn (simulating severe 3 / 4 limbal injury) groups on the day of modeling and 2, 4, 6 and 8 days after surgery; B shows the state of ocular surface defect repair of the mice after treatment using fluorescein sodium staining, which was carefully observed under a slit lamp on the day of modeling and 2, 4, 6 and 8 days after surgery; C shows the state of ocular surface OCT detection of the animals in each group; D shows the control (normal mice), 1quarter Alkali Burn (simulating mild 1 / 4 limbal injury), 2quarter Alkali Burn (simulating moderate 1 / 2 limbal injury) and 3quarter Alkali Burn (simulating severe 3 / 4 limbal injury) groups on the day of modeling and 2, 4, 6 and 8 days after surgery; Burn (simulating severe 3 / 4 limbal injury) animals in each group were scored for corneal transparency on the day of modeling and 2, 4, 6, and 8 days after surgery. E is the statistical result of calculating the corneal injury area of ​​each group of animals based on the results of sodium fluorescein staining. F is the statistical result of regularly observing the corneal thickness of the three groups of mice on the day of modeling and 2, 4, 6, and 8 days after surgery. In each figure, *P<0.05; **P<0.01; ***P<0.001;

[0034] Figure 3Figure 3 is a histopathological section and a fluorescent staining section to observe the repair of limbal defects; A is the HE staining image of the normal cornea, mild 1 / 4 limbal injury group, moderate 1 / 2 limbal injury group, and severe 3 / 4 limbal injury group (scale bar in the figure: 100 μm); B is the MASSON staining image of the normal cornea, mild 1 / 4 limbal injury group, moderate 1 / 2 limbal injury group, and severe 3 / 4 limbal injury group (scale bar in the figure: 100 μm); C is the P63α green immunofluorescence staining image of the normal cornea, mild 1 / 4 limbal injury group, moderate 1 / 2 limbal injury group, and severe 3 / 4 limbal injury group (scale bar in the figure: 50 μm); D is the normal cornea, mild 1 / 4 limbal injury group, moderate 1 / 2 limbal injury group, and severe 3 / 4 limbal injury group. Figure 4: TUNEL red immunofluorescence staining of apoptosis in the normal cornea, mild 1 / 4 limbal injury group, moderate 1 / 2 limbal injury group, and severe 3 / 4 limbal injury group (scale bar in the figure: 100 μm). E is the fluorescence intensity statistics of the limbal stem cell marker P63α green immunofluorescence staining results in the normal cornea, mild 1 / 4 limbal injury group, moderate 1 / 2 limbal injury group, and severe 3 / 4 limbal injury group. F is the TUNEL red immunofluorescence staining results of apoptosis in the normal cornea, mild 1 / 4 limbal injury group, moderate 1 / 2 limbal injury group, and severe 3 / 4 limbal injury group. In each figure, *P<0.05, **P<0.01, ***P<0.001.

[0035] Figure 4 Figure 2 is a graph showing the results of corneal nerve recovery; A is the βIII Tubulin red staining results for the normal cornea, mild 1 / 4 limbal injury group, moderate 1 / 2 limbal injury group, and severe 3 / 4 limbal injury group (scale: 100 μm); B is a statistical graph showing the percentage of nerve area, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are intended only to illustrate the technical solution of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solution of the present invention, and all such modifications or equivalents should be included in the scope of the claims of the present invention.

[0037] The sources of the main materials used in the following examples of the present invention are as follows:

[0038] Paraformaldehyde was purchased from Sigma-Adrich.

[0039] Donkey serum was purchased from Gibco.

[0040] Anti-P63α antibody was purchased from Santa Cruz Company.

[0041] TUNEL kit was purchased from Shanghai Biotech Co., Ltd., China.

[0042] Anti-MMP9 antibody, anti-IL-6 antibody and anti-TNFα antibody were purchased from BD Biosciences.

[0043] Dapi was purchased from Invitrogen.

[0044] Alexa Fluor Cy3-fluorescent secondary antibody and Alexa Fluor 488-fluorescent secondary antibody were purchased from Jackson ImmunoResearch.

[0045] Anti-βIII Tubulin antibody was purchased from abcam, USA.

[0046] Mice were purchased from Shanghai JST Laboratory Animal Co., Ltd. (Shanghai, China). They were housed in an SPF-grade animal room maintained at a temperature of 20–24°C and a relative humidity of 55–75%. The protocol was approved by the Animal Ethics Committee of the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine.

[0047] The various test methods used in the following examples of the present invention are specifically described as follows:

[0048] 1) H&E staining:

[0049] Dewax the paraffin sections in xylene for 8-10 minutes. Next, dewax the sections in anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, 70% ethanol for 2 minutes, and finally distilled water for 2 minutes. Stain the tissue in hematoxylin for 10 minutes, then rinse the sections in water for 10 minutes and stain with eosin for 2 minutes. Finally, place the sections in 95% alcohol, 100% alcohol, and xylene, in that order. Remove the sections from the xylene, mount them in a neutral resin, and photograph them under a microscope.

[0050] 2) Masson staining:

[0051] Prepare Weigert's Iron Hematoxylin Staining Solution by mixing equal amounts of Weigert's Iron Hematoxylin Solution A and Weigert's Iron Hematoxylin Solution B. After treating the sections with Weigert's Iron Hematoxylin Staining Solution, rinse gently with water for 3 minutes. Differentiate with Acidic Alcohol Differentiation Solution for 20 seconds and rinse gently with water for 3 minutes. Bluing Solution for 20 seconds and rinse with water for 3 minutes. Stain with Ponceau Fuchsin Solution for 5 minutes and rinse with water for 3 minutes. Wash the sections gently with acetic acid working solution to prevent detachment. After treatment with phosphomolybdic acid solution, remove excess phosphomolybdic acid. Counterstain with aniline blue and treat the sections with acetic acid working solution until no blue color is lost. Dehydrate rapidly with 95% ethanol and then dehydrate three times with anhydrous ethanol. Clear the sections with xylene and mount with neutral resin. Finally, the sections can be examined under a microscope.

[0052] 3) Immunofluorescence:

[0053] Place the frozen sections in a 4°C wet box; add PBS and wash for 10 minutes each time, for a total of 3 times to remove residual paraformaldehyde; prepare blocking solution (10% donkey serum + 0.3% Triton X-100 + PBS), add to the culture dish, cover the slide, and place it at room temperature for 1 hour; add PBS and rinse for 10 minutes each time, for a total of 3 times; after absorbing all the liquid, add the primary antibody (the antibody dilution is prepared according to the antibody instructions) and incubate at 4°C overnight; the next day, recover the primary antibody, and then rinse repeatedly with PBS + 0.1% Tweeen for 10 minutes each time, for a total of 3 times; after absorbing all the liquid, add the corresponding fluorescent secondary antibody (the dilution ratio needs to be prepared according to the antibody instructions) and incubate at room temperature for 1 hour; discard the secondary antibody, add PBS and wash repeatedly for 10 minutes each time, for a total of 3 times; add Dapi to stain the cell nucleus; use sterile tweezers to remove the cell slide, place the slide on a slide, seal it with mounting medium, and observe under a fluorescence microscope.

[0054] 4) Whole corneal nerve staining

[0055] The mouse eyeballs were fixed in 4% paraformaldehyde solution and fixed at 4°C overnight. The next day, the cornea was cut along the limbus and fixed on ice for 1 hour. After washing with PBS 3 to 5 times, it was placed in a 96-well plate and fixed with blocking solution (10% donkey serum + 0.3% Triton X-100 + PBS). The primary antibody dilution was prepared in proportion, 200 μL of antibody was added to each well, and it was incubated at 4°C overnight. The cornea was removed the next day, washed several times, and the fluorescent secondary antibody was added. It was incubated at room temperature in the dark for 1 hour. After washing several times, Dapi was added, the slides were sealed, and the slides were placed under a confocal microscope for observation.

[0056] Example 1

[0057] The animal model of corneal limbal graded injury and repair was constructed in this example. Figure 1 As shown, the specific method is:

[0058] S1. Prepare 1M NaOH solution and an injury device with a large circle diameter of 6 mm, a small circle diameter of 4 mm, and an open ring contact area with the corneal limbus. The ring width of the device is 1 mm, and the central angles of the filter paper rings are 90°, 180°, and 270°, respectively. Use them for each group of model animals;

[0059] S2. Use oxybuprocaine hydrochloride eye drops (4 mg / ml) to instill the mouse's operated eye three times for ocular surface anesthesia;

[0060] S3. Soak the filter paper ring in 1M NaOH solution, remove it and remove excess alkali solution, then place the filter paper ring in the mouse corneal limbus area for 30 seconds;

[0061] S4. Remove the filter paper ring and rinse the operated eye with normal saline, using 15 ml for each eye;

[0062] S5. Administer antibiotic eye drops to the operated eye of the animal three times a day.

[0063] S6. The corneal transparency, corneal epithelial repair, and corneal edema of the operated eyes of mice were evaluated on the day of surgery and on days 2, 4, 6, and 8 after surgery.

[0064] S7. After the clinical evaluation and follow-up of mice, eye tissue sections were taken, and HE and MASSON staining were used to observe the corneal and limbal tissue structure, limbal stem cell fluorescence staining was used to evaluate the density and distribution of limbal stem cells, corneal epithelial TUNEL staining was used to evaluate corneal epithelial cell apoptosis, and corneal nerve staining was used to evaluate corneal nerve repair.

[0065] Example 2 Analysis of clinical evaluation results of the graded limbal injury mouse model

[0066] like Figure 2As shown in Figures A and 2D, the animals in each group of this example: control (normal mice), 1quarter AlkaliBurn (simulating mild 1 / 4 limbal injury), 2quarter AlkaliBurn (simulating moderate 1 / 2 limbal injury), and 3quarter AlkaliBurn (simulating severe 3 / 4 limbal injury) were regularly observed under slit lamp on the day of modeling and 2, 4, 6, and 8 days after surgery. The statistical results showed that: transient corneal opacity in the alkali burn area occurred in the 1quarter AlkaliBurn (simulating mild 1 / 4 limbal injury) and 2quarter AlkaliBurn (simulating moderate 1 / 2 limbal injury) injury groups, and began to recover after 4 days, while the corneal opacity in the 3quarter AlkaliBurn (simulating severe 3 / 4 limbal injury) group did not improve significantly. The corneas of mice in the 1quarter Alkali Burn group (simulating mild 1 / 4 limbal injury) had essentially regained transparency after 8 days, with only slight turbidity in the peripheral area. The corneas of mice in the 2quarter Alkali Burn group (simulating moderate 1 / 2 limbal injury) had essentially regained transparency on the uninjured side after 8 days, but some turbidity remained on the injured side. The corneas of mice in the 3quarter Alkali Burn group (simulating severe 3 / 4 limbal injury) experienced persistent corneal edema starting on day 4. Corneal transparency scores showed statistically significant differences among the three groups. This suggests that limbal stem cell damage can lead to corneal turbidity, and that greater the degree of damage, the higher the turbidity, and the slower the repair of the damage.

[0067] like Figure 2As shown in Figures B and 2E, the fluorescein staining and statistical results of the animals in each group of this example: control (normal mice), 1quarter AlkaliBurn (simulating mild 1 / 4 limbal injury), 2quarter AlkaliBurn (simulating moderate 1 / 2 limbal injury), and 3quarter AlkaliBurn (simulating severe 3 / 4 limbal injury) groups showed that the fluorescein-stained areas of the 1quarter AlkaliBurn (simulating mild 1 / 4 limbal injury) and 2quarter AlkaliBurn (simulating moderate 1 / 2 limbal injury) groups were basically consistent with the filter paper-covered limbal alkali burn area. In the 3quarter AlkaliBurn (simulating severe 3 / 4 limbal injury) group, fluorescein-stained areas were present in the central cornea in addition to the limbus, suggesting that limbal stem cell deficiency can also cause cell damage in the central cornea. The corneas of all three groups of mice were able to partially heal. Early on, the 1-quarter Alkali Burn group (simulating mild 1 / 4 limbal damage) showed significantly faster corneal repair than the other two groups, with only slight staining on day 8. On day 8, the 2-quarter Alkali Burn group (simulating moderate 1 / 2 limbal damage) also showed faster corneal repair than the 3-quarter Alkali Burn group (simulating severe 3 / 4 limbal damage). There were statistically significant differences in the speed of repair of the damaged area among the three groups. This suggests that limbal stem cell damage can also lead to epithelial damage in the central cornea, while remaining healthy limbal stem cells can help regenerate and repair epithelial cells. The more limbal stem cells there are, the faster the repair.

[0068] like Figure 2 As shown in Figures C and 2F, the OCT and corneal thickness statistical results of the animals in each group of this example: control (normal mice), 1-quarter Alkali Burn (simulating mild 1 / 4 limbal injury), 2-quarter Alkali Burn (simulating moderate 1 / 2 limbal injury), and 3-quarter Alkali Burn (simulating severe 3 / 4 limbal injury) show that all groups experienced transient corneal edema, with corneal edema being most pronounced 2 and 4 days after modeling. The degree of corneal edema increased with the severity of the injury, and the corneal thickness of the 3-quarter Alkali Burn (simulating severe 3 / 4 limbal injury) group was significantly higher than that of the other two groups. On day 8, the corneal edema of some mice in the 1-quarter Alkali Burn (simulating mild 1 / 4 limbal injury) group had recovered to a near-normal level. Corneal thickness measurements showed statistically significant differences among the three groups.

[0069] Example 3 Histopathological sections and fluorescence staining results

[0070] Eyeball tissue sections were sectioned and HE and MASSON staining were used to observe the corneal and limbal tissue structure, limbal stem cell fluorescence staining was used to evaluate the density and distribution of limbal stem cells, corneal epithelial TUNEL staining was used to evaluate corneal epithelial cell apoptosis, and corneal nerve staining was used to evaluate corneal nerve repair.

[0071] Figure 3 Figure A shows the corneal healing of each group of mice under HE staining. It can be observed that the epithelium in the central region of the cornea of ​​the control (normal mice) mice is intact, with uniform thickness and tightly arranged cells. The cells in the limbus are continuous and neat, and the structures of the retina, iris, and ciliary body are clear. However, the central region of the corneal epithelium in the 1quarter Alkali Burn (simulating mild 1 / 4 limbal damage), 2quarter Alkali Burn (simulating moderate 1 / 2 limbal damage), and 3quarter Alkali Burn (simulating severe 3 / 4 limbal damage) groups all exhibit varying degrees of structural abnormalities: as the severity of damage increases, the epithelium becomes thinner, the number of layers decreases, and the arrangement becomes more disorganized. Discontinuous cell arrangement is observed in the limbus, indicating limbal cell damage. Figure 3 B shows the arrangement of collagen in the central and limbal regions of the mouse cornea under MASSON staining. It can be observed that the corneal collagen of the control (normal) mice is tightly and orderly arranged, while the corneal collagen of the 1quarter Alkali Burn (simulating mild 1 / 4 limbal injury), 2quarter Alkali Burn (simulating moderate 1 / 2 limbal injury), and 3quarter Alkali Burn (simulating severe 3 / 4 limbal injury) groups exhibit varying degrees of looseness and edema. The collagen arrangement of the 1quarter Alkali Burn (simulating mild 1 / 4 limbal injury) group is closest to that of the normal group, while the corneal collagen arrangement of the 2quarter Alkali Burn (simulating moderate 1 / 2 limbal injury) and 3quarter Alkali Burn (simulating severe 3 / 4 limbal injury) groups is significantly disordered, with significant stromal edema. Figure 3C. Limbal epithelial cells were stained with green fluorescence for the limbal stem cell marker P63α. In the control (normal mouse) group, P63α-positive limbal stem cells were distributed throughout the epithelium. In the 1quarter Alkali Burn (simulating mild 1 / 4 limbal damage) group, some positive cells remained. In the 2quarter Alkali Burn (simulating moderate 1 / 2 limbal damage) group, a few positive cells were observed. In the 3quarter Alkali Burn (simulating severe 3 / 4 limbal damage) group, almost no positive cells were observed. This suggests that the model successfully caused varying degrees of damage to limbal stem cells, and that postoperative recovery slowed with increasing damage severity. Figure 3 D. TUNEL red fluorescence staining of epithelial cells in the central area of ​​the cornea for apoptosis showed that there were basically no positive cells in the control (normal mouse) group, a few positive cells were seen in the 1quarter Alkali Burn (simulating mild 1 / 4 limbal damage) group, but only in the epithelium, some positive cells were seen in the epithelium and the upper half of the stroma in the 2quarter Alkali Burn (simulating moderate 1 / 2 limbal damage) group, and positive cells were seen in the entire epithelium and stroma in the 3quarter Alkali Burn (simulating severe 3 / 4 limbal damage) group, indicating that the number of apoptotic cells in the central cornea increased with the severity of damage. Figure 3 E and Figure 3 F represents the P63α fluorescence intensity and the number of TUNEL-positive cells, respectively, and the results are statistically significant. The H&E and Masson staining of pathological sections, as well as the P63α and TUNEL fluorescence staining of frozen sections, confirm from both histological and cellular perspectives that this model successfully damages limbal stem cells. It also confirms that limbal stem cell deficiency damages the corneal epithelium, with more severe damage leading to slower repair.

[0072] By whole corneal immunofluorescence nerve staining (βIII Tubulin), the arrangement of corneal nerves in the control (normal mice), 1quarter Alkali Burn (simulating mild 1 / 4 corneal limbal damage), 2quarter Alkali Burn (simulating moderate 1 / 2 corneal limbal damage) and 3quarter Alkali Burn (simulating severe 3 / 4 corneal limbal damage) groups were observed. Figure 4A. Alkali burn can cause corneal nerve damage in mice. Eight days after modeling, compared to the control (normal mice) group, the nerves in the 1 / 4 limbal burn (simulating mild 1 / 4 limbal injury), 2 / 4 limbal burn (simulating moderate 1 / 2 limbal injury), and 3 / 4 limbal burn (simulating severe 3 / 4 limbal injury) groups were sparsely and disorganized. As the severity of the injury increased, the corneal nerve arrangement became more sparse. Figure 4 B. Statistical analysis of the percentage of corneal nerve area. The degree of damage increased with the degree of injury, and the degree of corneal nerve damage was significantly different, and the results were statistically significant. The above results confirm that the limbal stem cell graded injury model can also cause graded damage to the corneal nerves. This shows that the method of this embodiment successfully constructed an animal model of limbal stem cell (partial / local) injury, which is consistent with the pathological process and injury characteristics after clinical limbal stem cell injury, and provides an experimental basis for the exploration of the mechanism of limbal stem cell deficiency and the study of corneal damage repair.

Claims

1. A method for constructing a corneal limbus graded injury and repair model, characterized in that include: After the model animal's eye was anesthetically instilled, a limbal injury device containing NaOH solution was placed on the limbus of the model animal for 30 seconds before being removed and the eye was rinsed with normal saline. After surgery, antibiotics were dropped into the operated eyes of the model animals 3-4 times a day; The limbal graded injury and repair model was obtained 8 days after surgery; The limbal injury device performs alkali burns of different areas on the limbal region to achieve a graded injury effect; The contact surface between the limbus injury device and the model animal's surgical eye is an open circular structure, including a first circular arc and a second circular arc, which injures the local stem cells of the animal's limbus. The radius of the first circular arc is 3 mm, and the radius of the second circular arc is 2 mm. The central angle of the limbal injury device is 90° to simulate mild limbal injury, 180° to simulate moderate limbal injury, and 270° to simulate severe limbal injury; The modeling animal is a mouse; The concentration of NaOH solution is 1M.

2. The method for constructing a graded limbal injury and repair model according to claim 1, characterized in that: The corneal transparency, corneal epithelial repair and corneal edema of the operated eyes were evaluated on the day of surgery and on the 2nd, 4th, 6th and 8th days after surgery. The corneal transparency evaluation method comprises: observing corneal transparency with a slit lamp and scoring corneal transparency; The corneal epithelial repair evaluation method is as follows: after staining with 1% sodium fluorescein, the anterior segment of the eye is photographed, and the fluorescent staining area is measured using ImageJ to evaluate the corneal epithelial repair; The method for evaluating the degree of corneal edema is as follows: an optical coherence tomography scanner is used to evaluate the degree of corneal edema, and a built-in image processing software analysis system of the scanner is used to measure the corneal thickness.

Images