Construction method of rat keratoconjunctival disease model

Abstract

The invention discloses a construction method of a rat keratoconjunctival disease model, and particularly relates to the technical field of animal models. Lipopolysaccharide (LPS) is adhered to a surgical suture and then sutured on the upper eyelid of a rat, so that inflammatory response is rapidly and continuously initiated. The method is better in modeling effect, simple and convenient to operate, short in period, high in success rate, high in repeatability, low in cost, high in safety and wide in application range.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and specifically to a method for constructing a rat corneal and conjunctival disease model. Background Technology

[0002] The cornea is a transparent, avascular tissue approximately 1 cm in diameter covering the anterior surface of the eyeball, while the conjunctiva is a mucous membrane covering the dorsum of the eyelids and the surface of the eyeball, located behind the limbus. Both the cornea and conjunctiva play crucial roles in vision. It is known that visual function is severely impaired when diseases occur in either. Corneal and conjunctival diseases are among the most common ophthalmic diseases, with complex etiologies including infectious (bacterial, viral, fungal), allergic, immune, and physical / chemical irritants. Establishing stable, reliable, and reproducible animal models is essential for a deeper understanding of their pathogenesis and for screening effective treatments. Currently, the main reported animal models include: 1. Allergic induction: Ovalbumin (OVA) or 2,4-dinitrofluorobenzene (DNFB) are commonly used as sensitizers. Sensitization is achieved through intraperitoneal injection, followed by challenge with antigen eye drops. This model can simulate type I hypersensitivity reactions well, but the modeling period is long (usually 1-2 weeks), involves many steps, and has relatively large individual differences.

[0003] 2. Bacterial / viral induction: Direct inoculation with specific pathogens (such as Staphylococcus aureus, adenovirus, etc.). This model can simulate specific infection processes, but it carries biosafety risks. Differences in pathogen virulence affect model stability, and it is susceptible to the influence of the host's immune status.

[0004] 3. Chemical induction: Acute inflammatory responses are induced by direct eye drops or injections of chemicals such as benzalkonium chloride (BAC), chloroform, and mustard oil. This type of model is relatively simple to operate and produces a rapid inflammatory response. However, it requires precise control of the concentration and duration of the irritant, can easily cause irreversible tissue damage, and has poor model specificity.

[0005] In summary, the main problems with existing technologies include: long modeling cycles, complex operations, difficulty in controlling model stability (inflammation level and duration), excessive tissue damage, and high costs. Therefore, developing a rat model for keratoconjunctival diseases that is easy to operate, has a controllable inflammatory response, good reproducibility, relatively mild tissue damage, and is suitable for various research purposes (such as drug screening and mechanism studies) has significant application value. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for constructing a rat corneal and conjunctival disease model.

[0007] On the one hand, the present invention provides a method for constructing a rat corneal and conjunctival disease model, which uses lipopolysaccharide and surgical sutures to induce modeling.

[0008] In some embodiments, the surgical suture is a non-absorbable surgical suture.

[0009] In some embodiments, the concentration of the lipopolysaccharide is 1-50 mg / mL.

[0010] In some embodiments, the concentration of the lipopolysaccharide is 1-40 mg / mL.

[0011] In some embodiments, the concentration of the lipopolysaccharide is 1-30 mg / mL, specifically 1, 5, 10, 15, 20, or 25 mg / mL.

[0012] In some embodiments, the induction method involves soaking surgical sutures in an LPS solution and then ligating them at the upper edge of the rat's upper palpebral conjunctiva.

[0013] In some embodiments, surgical sutures are used to ligate the tissue transversely at a distance of 1-3 mm at the upper margin of the rat's upper palpebral conjunctiva.

[0014] In some embodiments, the suture is cut 4-6 mm behind the ligation point, leaving a portion of the suture inside the eyelid.

[0015] In some embodiments, rats are anesthetized before the induction method, and the surgical site is then disinfected.

[0016] In some embodiments, the induced ocular symptom score in rats is ≥3.

[0017] In some embodiments, the induced ocular symptom score in rats is ≥6.

[0018] In some embodiments, the induced rat ocular symptom score is ≥7 points, preferably ≥8 points, and more preferably 9 points.

[0019] This invention provides a rat corneal and conjunctival disease model, which is constructed by the above method.

[0020] On the other hand, the present invention provides the application of a rat corneal and conjunctival disease model in screening and / or preparing pharmaceuticals and health products for the prevention and / or treatment of eye diseases.

[0021] In some embodiments, the corneal and conjunctival diseases include corneal ulcers, corneal epithelial abrasions, keratitis, dry eye syndrome, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disease, superficial punctate keratitis, corneal epithelial defects, conjunctival epithelial defects, keratoconjunctivitis sicca, superior limbal keratoconjunctivitis, filamentous keratoconjunctivitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, and non-infectious conjunctivitis. Beneficial effects

[0022] 1. Applying LPS to the surgical suture line can provide long-term and stable stimulation to the eyelid area, fully leveraging the induction function of LPS. The combination of the two significantly enhanced the ocular scores in rats, and significantly increased the levels of inflammatory factors in serum, white blood cell count (WBC), and neutrophil count (NEUT) in routine blood tests. Compared with single factors, the modeling effect was better.

[0023] 2. The operation is simple, requiring only a single simple surgical procedure, without the need for complicated sensitization procedures or pathogen inoculation. The modeling cycle is short, and a stable model can be established in just 3-5 days.

[0024] 2. Non-absorbable sutures, as foreign bodies, have a clear irritant effect on the conjunctiva. When they are contaminated with LPS, they can quickly induce significant conjunctival hyperemia, edema, and inflammatory cell infiltration. The model has a high success rate, small inter-individual differences, and good reproducibility.

[0025] 3. By controlling the retention time of sutures, the severity and duration of inflammation can be controlled relatively precisely to meet different research needs (such as mild and moderate inflammation models).

[0026] 4. Early models mainly showed reversible inflammatory responses with relatively mild damage to the corneal epithelium and deep tissues. The model of this invention is more consistent with the pathological characteristics of common clinical conjunctivitis and is suitable for drug intervention research.

[0027] 5. Modeling only requires LPS and surgical sutures, making it inexpensive; in addition, it avoids the use of live pathogens, resulting in higher biosafety.

[0028] 6. The inflammation generated by this model can effectively simulate the core pathological features of conjunctivitis (congestion, edema, and inflammatory cell infiltration), making it suitable for evaluating the efficacy of various anti-inflammatory, anti-allergic, and repair drugs, as well as for studying inflammatory pathways related to conjunctivitis. Attached Figure Description

[0029] Figure 1 This is a rat weight record sheet.

[0030] Figure 2 The images show the eye conditions of the rat sham-operated group (top left), combined model group (top right), LPS model group (bottom left), and suture model group (bottom right).

[0031] Figure 3 The images show the eye conditions of the rat sham-operated group (top left), combined model group (top right), LPS model group (bottom left), and suture model group (bottom right).

[0032] Figure 4 A scoring scale for the eye condition of rats.

[0033] Figure 5 Results for serum TNF-α and IL-1β in rats.

[0034] Figure 6 HE staining images of the cornea in the rat sham-operated group (top left), combined model group (top right), LPS model group (bottom left), and suture model group (bottom right). Detailed Implementation

[0035] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention.

[0036] Example 1 1. Experimental reagents and consumables 1.1 Laboratory Animals

[0037] Forty SPF-grade male SD rats, 5 weeks old, weighing 130 ± 10 g, were used.

[0038] 1.2 Major Instruments and Consumables

[0039] Small animal anesthesia machine (Reward, R640); pathology slide cutter; blood cell analyzer; centrifuge; surgical instruments.

[0040] Tumor necrosis factor α (TNF-α) kit; interleukin 1β (IL-1β) kit; 6-0 non-absorbable sutures; lipopolysaccharide (LPS); 4% paraformaldehyde.

[0041] 3. Experimental Procedure 2.1 Experimental Preparation

[0042] After receiving the experimental rats, they were acclimatized for one week before the formal experiment began. All rats were housed in an SPF-grade animal laboratory with an ambient temperature of 23±2℃ and a humidity of 40%~60%. The environment was kept under alternating light and dark conditions for 12 hours, and the rats had free access to food and water.

[0043] 2.2 Animal Grouping

[0044] The experimental rats were randomly divided into 4 groups according to Table 1 below: sham operation group (n=10), combined model group (n=10), LPS model group (n=10), and monofilament model group (n=10).

[0045] Table 1 Experimental Groups Group Molding materials Modeling methods Sham surgery group 6-0 suture needle After anesthetizing the rats, the upper palpebral conjunctiva of the rats was punctured. Joint model group 6-0 sutures soaked in LPS After anesthetizing the rats, part of the upper palpebral conjunctiva of the rats was ligated. LPS modeling group LPS solution After anesthetizing the rats, 20 μL of 0.2 mg / mL LPS solution was administered as eye drops. Monofilament molding assembly 6-0 suture needle After anesthetizing the rats, part of the upper palpebral conjunctiva of the rats was ligated. 2.3 Model Construction 2.3.1 Experimental Preparation

[0046] Prepare a 10 mg / mL LPS solution for later use.

[0047] Soak the 6-0 non-absorbable surgical sutures in a 10 mg / mL LPS solution for later use.

[0048] 2.3.2 Experimental Modeling

[0049] The rats were fasted but allowed free water the night before the model was built.

[0050] The surgical procedure is as follows: ① After inhalation anesthesia, the left whiskers of the rats in the model group were cut off, and the area around the eyes was disinfected.

[0051] ② Place the rat in a prone position on the operating tablecloth, and tilt the rat's head slightly to the right.

[0052] ③ Gently lift the left upper eyelid of the rat with tissue forceps, and gently flip the left upper eyelid conjunctiva upward to expose the conjunctiva on the upper side of the rat's left eye.

[0053] ④ Using 6-0 non-absorbable surgical sutures soaked in LPS solution, a transverse ligation was made at a distance of 2 mm along the upper margin of the rat's upper palpebral conjunctiva. The suture was then cut 5 mm posterior to the ligation point, leaving a portion of the suture inside the eyelid. This facilitates the inspection of any suture detachment and enhances the model's effectiveness.

[0054] ⑤ After observing that there was no obvious bleeding in the upper eyelid conjunctiva, the rat's upper eyelid was flipped downwards to restore its original position.

[0055] ⑥ Stop the anesthesia and wait for the rat to wake up before putting it back in its cage for normal feeding.

[0056] The monofilament model group maintained the same operation, except that the 6-0 non-absorbable surgical suture was used to directly ligate the upper edge of the upper palpebral conjunctiva of rats without picking up LPS.

[0057] The LPS model group directly used LPS to stimulate the upper palpebral conjunctiva of rats.

[0058] The sham-operated group of rats underwent the same procedure, but with the use of a standard suture needle and without ligation.

[0059] 2.4 Data collection, endpoint sampling, and indicator testing 2.4.1 Process Data Collection

[0060] (1) Observation of rat body weight: Weigh and record the rats' weight every other day.

[0061] (2) Observation of the appearance of rat eyeballs: After modeling, the rats' eyeballs were observed daily, and photos were taken every other day to record the progression of conjunctivitis.

[0062] 2.4.2 Sampling at the endpoint

[0063] Five days after the rat model was established, the rats were euthanized, and the left eyeball of the rat was collected and fixed in 4% paraformaldehyde solution.

[0064] Blood samples of 0.5 ml were collected from the right orbit of rats and treated with EDTA-2K anticoagulation before routine blood tests. Whole blood was then collected from the abdominal aorta of the rats, allowed to stand for 30 min, and then centrifuged at 3000 rpm / min for 10 min. The serum from the supernatant was collected and stored at -80℃.

[0065] 2.4.3 Indicator Testing 2.4.3.1 Observation and scoring methods for ocular symptoms in rats

[0066] On the day of final sampling, after anesthetizing the rats, photographs were taken of the eyeballs of each group of rats, and the appearance symptoms and severity of conjunctivitis in the rats were recorded according to the scores in Table 2 below.

[0067] Table 2 symptom 0 points 1 point 2 points 3 points Conjunctival edema none Mild edema Moderate edema Severe edema Conjunctival congestion none Mild congestion Moderate congestion Severe congestion Conjunctival secretions none Small amount of secretions Moderate discharge Large amount of secretions The total score is the sum of the scores for each of the four categories: those with severe conjunctival congestion, edema, and discharge have a maximum score of 9 points, those with moderate conjunctival congestion have a maximum score of 6 points, and those with mild conjunctival congestion have a maximum score of 3 points.

[0068] 2.4.3.2 Method for determining the levels of TNF-α and IL-1β in rat serum

[0069] The levels of TNF-α and IL-1β in rat serum were determined by ELISA. The procedure was strictly followed according to the instructions of the ELISA kit. The specific experimental steps were the same as those for the determination of TNF-α and IL-1β levels in rat serum.

[0070] 2.4.3.3 Methods for histological examination a. Perform transparent dewaxing with xylene dewaxing solution for 15 min, followed by anhydrous ethanol for 1.5 min, 95% ethanol for 1.5 min, 80% ethanol for 1.5 min, and then wash with water for 5 min; b. Stain with hematoxylin for 10 min, then rinse with running water twice, rinsing for 5 min each time; c. Hydrochloric acid-alcohol differentiation for 30 seconds; d. Rinse with water for 5 minutes; e. Stain with eosin for 3-5 minutes; f. Routine dehydration, clearing and mounting: 95% ethanol for 1.5 min, anhydrous ethanol for 1.5 min, and mounting with neutral resin.

[0071] 2.4.4 Data Processing and Result Determination

[0072] The experimental data are expressed as mean ± standard error (Mean ± SEM). All data were processed and statistically analyzed using GraphPad Prism 8.0. The t-test was used to compare the means of two groups, and the ANOVA test was used to compare the means of multiple groups. p < 0.05 was considered statistically significant.

[0073] 3. Results Analysis 3.1 Rat body weight

[0074] like Figure 1 As shown, within 5 days of model establishment, the body weight of rats in each group showed an increasing trend, and there was no significant difference between the groups.

[0075] 3.2 Observation and scoring of ocular symptoms in rats

[0076] like Figure 2 As shown, the conjunctiva of rats in the sham-operated group was free of congestion, edema, and secretions, and the area around the eyes was clean and dry; rats in each model group had conjunctival congestion, edema, and more secretions, and the area around the eyes was red, swollen, and moist.

[0077] like Figure 3 As shown, the conjunctival surface of rats in the sham-operated group was smooth and without blood streaks; the conjunctival surface of rats in each model group was rough and had obvious blood streaks.

[0078] like Figure 4 As shown, the ocular symptom scores of rats in each model group were significantly higher than those in the sham surgery group. The LPS group scored 2-3, the monofilament group scored 3-4, and the combined model group scored the highest, reaching 8-9, which was significantly higher than the monofilament model group and the LPS model group.

[0079] 3.3 Serum TNF-α and IL-1β levels

[0080] as follows Figure 5 As shown, compared with the sham-operated group, the serum levels of TNF-α and IL-1β in each model group were increased, with the combined model group showing the greatest increase.

[0081] 3.4 Complete Blood Count (CBC)

[0082] As shown in Table 3 below, compared with the sham-operated group, the white blood cell count (WBC) and neutrophil count (NEUT) in the whole blood of rats in each model group were increased, and the combined model group had the highest values.

[0083] Table 3 Group WBC NEUT Sham surgery group 7145±2296 21.9%±3.4% Joint model group 14532±1578** 27.9%±2.3% LPS modeling group 7667±381 22.9%±0.9% Monofilament molding assembly 10417±1066** 26.5%±1.3%** Note: ** indicates comparison with the sham surgery group, p<0.01; * indicates comparison with the sham surgery group, p<0.05.

[0084] 3.5 Ocular pathology H&E staining The results of corneal HE staining are as follows Figure 6 As shown, in the sham-operated group, the corneal epithelial cell layer of rats was normal and flat, with a tight and orderly arrangement; the surface cells were not keratinized, the tissue structure was clear and transparent, without congestion and swelling, and without inflammatory infiltration, with occasional lymphocyte phagocytosis. The corneal surface of rats in each model group showed varying degrees of roughness and unevenness, with the combined modeling group showing the most significant modeling effect. Defects and new cells were visible in the epithelial cell layer, and the stromal layer showed disordered fiber arrangement, with a large number of eosinophils and neovascularization.

[0085] 4. Experiment Summary

[0086] In this experiment, there was no significant difference in body weight between the sham-operated group and the model group. Compared with the sham-operated group, the combined model group showed significantly increased ocular symptom scores, serum TNF-α and IL-1β levels, and whole blood WBC and NEUT levels. The conjunctival pathological staining results showed obvious lesions, indicating that the induction effect of LPS was significantly enhanced under the long-term stimulation of the suture. The combination of the two successfully and efficiently induced a rat keratitis model. Example 2

[0087] 2.3 In the model construction section, changing the LPS solution concentration to 5 mg / mL or 15 mg / mL, while keeping other conditions the same as in Example 1, also resulted in successful model construction.

[0088] It should be understood that the above detailed description of the technical solutions of the present invention with reference to preferred embodiments is illustrative and not restrictive. Those skilled in the art can modify the technical solutions described in the embodiments or make equivalent substitutions for some of the technical features based on reading this specification; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a rat model of corneal and conjunctival disease, characterized in that, This method uses lipopolysaccharide and surgical sutures to induce modeling.

2. The method as described in claim 1, characterized in that, The surgical sutures are non-absorbable.

3. The method as described in claim 2, characterized in that, The concentration of the lipopolysaccharide is 1-50 mg / mL.

4. The method as described in claim 3, characterized in that, The induction method involves soaking surgical sutures in LPS solution and then ligating them at the upper edge of the rat's upper palpebral conjunctiva.

5. The method according to any one of claims 1-4, characterized in that, The induced ocular symptom score in rats was ≥3.

6. A rat model of corneal and conjunctival disease, characterized in that, It is constructed by the method described in any one of claims 1-5.

7. The use of the rat corneal and conjunctival disease model according to claim 6 in screening and / or preparing pharmaceuticals and health products for the prevention and / or treatment of eye diseases.

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