Method for constructing rat xerophthalmia model
By combining scopolamine and atropine, subcutaneously injecting scopolamine hydrobromide solution and applying atropine sulfate gel to the eyes, the problems of long construction cycle and instability of dry eye model were solved, and a rapid and stable model was achieved, which is suitable for dry eye research.
Patent Information
- Application Number
- CN202410575178.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-10
- Publication Date
- 2025-11-11
AI Technical Summary
In the existing technology, the methods for constructing animal models of dry eye disease have the following drawbacks: long modeling cycle, unstable models, and susceptibility to environmental factors. There is also a lack of rapid and stable drug induction methods.
A dry eye model was induced in rats by subcutaneously injecting scopolamine hydrobromide solution and simultaneously applying atropine sulfate ophthalmic gel to the eyes.
It enables the rapid construction of stable dry eye disease models, reduces individual differences, and has strong symptom specificity, making it suitable for in-depth research on the etiology and treatment of dry eye disease.
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Abstract
Description
Technical fields:
[0001] This invention belongs to the field of animal model technology, specifically relating to a method for constructing a rat dry eye model. Background technology:
[0002] Dry eye disease (DED) is a multifactorial ocular surface disease characterized by tear film instability, accompanied by ocular symptoms caused by factors such as tear film instability and hyperosmolarity, corneal inflammation and damage, and abnormal nerve sensation. Patients with dry eye disease generally experience eye discomfort, with common symptoms including dryness, burning, foreign body sensation, stinging, itching, photophobia, redness, blurred vision, fluctuating vision, and eye pain. The etiology of DED is complex, usually involving multiple factors that interact with each other. Statistics show that the global prevalence of dry eye disease is between 5% and 50%, increasing with age, and is more pronounced in Asians. Due to the complexity of the etiology and the interaction of multiple factors, in-depth research on dry eye disease requires the establishment of clearly defined symptom types and corresponding animal models.
[0003] Animal models of dry eye disease can simulate the pathophysiological processes of dry eye disease in humans, contributing to a deeper understanding of the disease mechanisms. Choosing an appropriate animal model is a crucial step in dry eye research, directly impacting the reliability and validity of experimental results.
[0004] There are three methods for establishing animal models of dry eye: 1. Natural disease model: dry eye model is established by letting animals develop the disease naturally, which takes a long time; 2. Chemical damage model: dry eye model is established quickly by adding chemicals such as benzalkonium chloride to destroy the ocular surface epithelium; 3. Immunosuppression model: immune-related dry eye is simulated by administering immunosuppressants to suppress the immune response.
[0005] In existing technologies, methods for modeling dry eye syndrome include surgical, drug, and environmental methods. Surgical methods involve destroying the lacrimal glands or mold goblet cells to simulate dry eye symptoms; drugs are used to inhibit tear secretion or alter tear composition to induce dry eye syndrome; and environmental factors such as dryness and wind damage the ocular surface.
[0006] The known dry eye model is established by using a 6 mg / ml scopolamine hydrobromide solution. 0.2 ml is injected subcutaneously to inhibit parasympathetic nerve excitation in animals, thereby suppressing glandular secretion and reducing tear production. After 28 days of continuous administration, reduced tear production, tear mucoprotein layer disruption, decreased tear film stability, and corneal epithelial damage are observed. This method has a long modeling cycle, is limited by the environment, and the model is unstable.
[0007] Atropine sulfate ophthalmic gel is indicated for iridocyclitis, mydriasis before fundus examination, and mydriasis before refractive examination for prescription lenses.
[0008] There are currently no reports on constructing a dry eye model by subcutaneous injection of scopolamine hydrobromide solution combined with atropine ophthalmic gel. Summary of the Invention:
[0009] The technical problem this invention aims to solve is to address the shortcomings of the existing technology by providing a method for constructing a rat dry eye model induced by combined drug therapy. Our research found that scopolamine combined with atropine induces a dry eye model in animals by doubly inhibiting parasympathetic nerve excitation, leading to suppression of glandular secretion and rapidly inducing decreased tear secretion and ocular surface inflammation. Symptoms such as decreased tear secretion, reduced tear film breakup time, and corneal epithelial damage appear from the first week onwards. From the second week onwards, various dry eye indicators become significant and tend to stabilize. Furthermore, the model animals show minimal individual differences and are unaffected by environmental dryness factors. Therefore, a rapid and stable method for constructing a rat dry eye model induced by drug therapy has been established.
[0010] The method of the present invention has the advantages of short modeling time, no environmental restrictions during the modeling process, small individual differences among the model animals after modeling, and strong symptom specificity.
[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0012] A method for constructing a rat dry eye model, comprising: inducing dry eye syndrome with scopolamine combined with atropine, namely, subcutaneously injecting rats with scopolamine hydrobromide solution, while simultaneously applying atropine sulfate ophthalmic gel to the eyes.
[0013] Specifically, the technical solution of the present invention is as follows:
[0014] Rats were subcutaneously injected with scopolamine hydrobromide solution four times a day, 0.2 mL per rat per injection; simultaneously, atropine sulfate ophthalmic gel was instilled into the eyes once a day, 1-2 drops each time. During the experiment, subcutaneous injection of scopolamine hydrobromide solution and intraocular instillation of atropine sulfate ophthalmic gel were required daily.
[0015] The duration of medication can be 7-28 days, or longer.
[0016] The concentration of the scopolamine hydrobromide solution is 10-40 mg / mL, preferably 20-25 mg / mL.
[0017] The specifications for atropine sulfate ophthalmic gel are 2.5g:25mg.
[0018] The rats are adult male rats of the SD or Wistar strain.
[0019] The subcutaneous injection site is the subcutaneous tissue on the back, and the ocular administration site is the conjunctiva of the eyelid.
[0020] The preparation method of the scopolamine hydrobromide solution is as follows:
[0021] Accurately weigh the scopolamine hydrobromide raw material and place it in a suitable container. Add PBS solution to the container and vortex or shake at room temperature to fully suspend the raw material in the liquid. Then, sonicate for about 10 to 15 minutes to dissolve the raw material and finally prepare a scopolamine hydrobromide solution.
[0022] The mass-to-volume ratio (mg / mL) of scopolamine hydrobromide to PBS solution is 10-40:1, preferably 20-25:1.
[0023] The ultrasonic temperature should not exceed 15℃; ice cubes can be added to the water.
[0024] Compared with the prior art, the present invention has the following advantages:
[0025] 1. This invention provides a method for constructing a rat dry eye model. The method utilizes subcutaneous injection of scopolamine hydrobromide combined with ocular administration of atropine sulfate ophthalmic gel to induce a rat dry eye model. Following subcutaneous injection of scopolamine hydrobromide and atropine sulfate ophthalmic gel, differences were observed in tear secretion, tear film breakup time, and corneal staining starting from the second week. The scopolamine-atropine combination of this invention induces dry eye by doubly inhibiting parasympathetic nerve excitation, thereby suppressing glandular secretion and rapidly inducing decreased tear secretion and ocular surface inflammation, leading to the development of dry eye disease.
[0026] 2. Compared with existing methods of using scopolamine alone to induce dry eye, the scopolamine combined with atropine in this invention has the advantages of shorter modeling time, no environmental limitations in the modeling process, small individual differences among the model animals after modeling, and strong symptom specificity. It is of great significance for in-depth research on dry eye syndrome with complex etiologies and can be used to study drugs for the treatment or relief of dry eye syndrome.
[0027] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached image description:
[0028] Figure 1 This is the result of the Schirmer test, which detects phenol red cotton thread tear secretion in the model experiment of Embodiment 2 of the present invention.
[0029] Figure 2 This is the tear film breakup time (BUT) detection result in the model experiment of Embodiment 2 of the present invention.
[0030] Figure 3 This is the detection and scoring result of sodium fluorescein in Example 2 of the present invention.
[0031] Figure 4These are typical ocular surface photographs of model animals at various stages detected by sodium fluorescein in Example 2 of this invention.
[0032] Figure 5 This is the HE staining result in Example 2 of the present invention.
[0033] Figure 6 This is the TUNEL detection result in Embodiment 2 of the present invention. Detailed implementation method:
[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0035] Example 1: Preparation of scopolamine hydrobromide solution
[0036] Accurately weigh 200 mg of scopolamine hydrobromide raw material and place it in a suitable container. Add 10 mL of PBS and vortex or shake at room temperature to fully suspend the raw material in the liquid. Then, perform sonication (the sonication temperature should not exceed 15°C; ice cubes can be added to the water). During this process, the height of the sonicated water level should be the same as the height of the solvent in the container. During sonication, the experimenter needs to vortex or shake the container and observe whether the raw material is completely dissolved (do not leave the sonication table). Once dissolved, remove it immediately. Do not over-sonicate. The sonication time is approximately 10–15 minutes. After sonication dissolution, prepare a 20 mg / mL scopolamine hydrobromide solution.
[0037] Example 2: Construction of a rat dry eye model (scopolamine hydrobromide solution + atropine gel)
[0038] Model animals: Adult male rats of the SD or Wistar strain;
[0039] Scopolamine hydrobromide solution: concentration: 20 mg / mL, 0.2 mL / animal / dose;
[0040] Atropine sulfate ophthalmic gel (commercially available): 2.5g: 25mg.
[0041] Twenty healthy SPF-grade male SD or Wistar adult male rats were selected and subcutaneously injected with scopolamine hydrobromide solution at a concentration of 20 mg / mL, four times daily, with an interval of at least 3 hours. Each animal received 0.2 mL per injection, administered continuously throughout the experiment. Simultaneously, 1% commercially available atropine sulfate ophthalmic gel was applied to the conjunctiva of the palpebral eye, one drop once daily, continuously throughout the experiment. Both the injectable and ophthalmic administration periods lasted 28 days.
[0042] Comparative Example 1: Construction of a rat dry eye model (using scopolamine hydrobromide solution alone)
[0043] Model animals: Adult male rats of the SD or Wistar strain;
[0044] Scopolamine hydrobromide solution: concentration: 20 mg / mL, 0.2 mL / animal / dose;
[0045] Twenty healthy male SPF-grade SD or Wistar adult male rats (10 rats / dose) were selected and injected subcutaneously into the back with scopolamine hydrobromide solution at a concentration of 20 mg / mL. The injection interval was at least 3 hours, 4 times a day, with each animal receiving 0.2 mL of the solution each time. The administration was continued for 28 days during the experiment.
[0046] Comparative Example 2: Construction of a rat dry eye model (using atropine solution alone)
[0047] Model animals: Adult male rats of the SD or Wistar strain;
[0048] Atropine sulfate ophthalmic gel (commercially available): 2.5g: 25mg.
[0049] Twenty healthy male SPF-grade SD or Wistar adult male rats were selected and administered 1% commercially available atropine sulfate ophthalmic gel to the conjunctiva of the eyelids, 2 drops once daily for 28 consecutive days during the experiment.
[0050] Example 3: A rat dry eye model was established by combining different doses of scopolamine hydrobromide solution with atropine sulfate ophthalmic gel.
[0051] Model animals: Adult male rats of the SD or Wistar strain;
[0052] Scopolamine hydrobromide solution: concentrations of 10 mg / mL, 20 mg / mL, and 40 mg / mL; 0.2 mL / animal / dose.
[0053] Atropine sulfate ophthalmic gel (commercially available): 2.5g: 25mg.
[0054] Thirty healthy male SPF-grade SD or Wistar adult male rats (n=10 / dose group) were selected. Scopolamine hydrobromide solution was subcutaneously injected into the back. The concentrations of 10, 20, and 40 mg / mL of scopolamine hydrobromide solution were injected subcutaneously, with an interval of at least 3 hours, four times daily. Each animal received 0.2 mL of the solution per injection, and the administration continued throughout the experiment. Simultaneously, 1% commercially available atropine sulfate ophthalmic gel was applied to the conjunctiva of the eyelids, one drop once daily, continuously throughout the experiment. The experiment lasted 28 days.
[0055] Example 4: Comparison of indices between different types of rat dry eye models (D+A group and D group)
[0056] Data on key indicators of dry eye obtained using the methods of Example 2 (Group D+A) and Comparative Example 1 (Group D) (see Table 1) show that, compared with dry eye induced by scopolamine alone, dry eye induced by the combination of scopolamine and atropine can cause a faster decrease in tear secretion, a reduction in tear film breakup time, and an increase in fluorescence staining score (severity of corneal damage), and can maintain a more stable model effect.
[0057]
[0058] Example 5: Comparison of indices between different types of rat dry eye models (D+A group and A group)
[0059] Data on key indicators of dry eye obtained using the methods in Example 2 (Group D+A) and Comparative Example 2 (Group A) (see Table 2) show that while atropine-induced dry eye caused a faster decrease in tear secretion and tear film breakup time, the effect was short-lived (around 7 days), and the fluorescence staining score showed almost no change, indicating that this method could not significantly affect the ocular surface tissue. In contrast, the combined use of scopolamine and atropine induced more comprehensive dry eye symptoms, and the model effect remained stable and long-lasting.
[0060]
[0061] Example 6: Indicators of the rat dry eye model constructed in Example 3
[0062] Data on key indicators of dry eye obtained using different scopolamine solutions (10 mg / mL, 20 mg / mL, and 40 mg / mL) combined with atropine in Example 3 (see Table 3) show that, compared to the 10 mg / mL dose, the 20 mg / mL group induced a faster decrease in tear secretion, a shorter tear film breakup time, and an increased fluorescence staining score (severity of corneal damage), while maintaining a more stable model effect. Compared to the 40 mg / mL dose, the 20 mg / mL group showed no significant difference in model establishment progress or data, indicating that it is unnecessary to use scopolamine concentrations to achieve better model effects, and also avoiding the probability of drug tolerance and induced side effects caused by higher concentrations.
[0063]
[0064] Example 7: Schirmer test for phenol red cotton thread tear secretion in a rat dry eye model of Example 2.
[0065] Experimental Methods: Dry eye model rats were anesthetized. Phenolic red cotton thread was used to measure the tear secretion of all rats. The cotton thread was placed in the outer 1 / 3 of the conjunctival sac of the lower eyelid of the rat, removed after 2 minutes, and the wetted length of the test strip was quickly measured (accurate to 0.5 mm). Each animal was tested three times, and the results were recorded and statistically analyzed.
[0066] Results: Subcutaneous injection of scopolamine hydrobromide solution combined with ophthalmic atropine sulfate gel resulted in a significant decrease in tear secretion in the model group compared to the normal group on the third day after modeling (*P<0.05). From the seventh day after modeling, the tear secretion in the model group showed a significant decrease compared to the normal group (***P<0.001), and this change remained stable for more than a month. See the appendix for detailed results. Figure 1 .
[0067] Conclusion: The method of the present invention can significantly reduce tear secretion in model animals in a short period of time, and this change is sustained and stable.
[0068] Example 8: Tear film breakup time (BUT) detection in a rat dry eye model of Example 2
[0069] Experimental methods: Dry eye model rats were anesthetized. One drop of physiological saline was used to moisten sodium fluorescein test paper. The tip of the test paper was gently touched to the inner side of the lower eyelid of the experimental rat and held for a moment. The experimental rat was assisted to blink 3 times to make the fluorescein evenly distributed. The time when the first black dry spot appeared on the cornea was observed under cobalt blue light of a slit lamp. Each animal was tested 3 times, and the results were recorded and statistically analyzed.
[0070] Results: Subcutaneous injection of scopolamine hydrobromide solution combined with ophthalmic atropine sulfate gel significantly reduced tear film breakup time in the model group compared to the normal group on the third day after modeling (*P<0.05). Starting on the seventh day after modeling, the tear film breakup time in the model group was significantly reduced compared to the normal group (**P<0.01), and this change remained stable for more than a month. See the appendix for detailed results. Figure 2 .
[0071] Conclusion: The method of the present invention can significantly reduce the tear film breakup time in model animals in a short period of time and maintain a stable effect for more than a month.
[0072] Example 9: Detection of sodium fluorescein in a rat dry eye model of Example 2
[0073] Experimental methods: Dry eye model rats were anesthetized. Fluoresin sodium test paper was moistened with 1 drop of physiological saline. The tip of the test paper was gently touched to the inner side of the lower eyelid of the experimental rat and held for a moment. The experimental rat was assisted to blink so that the fluoresin sodium was evenly diffused throughout the entire ocular surface. After 5-10 minutes, the corneal fluorescence staining was observed under cobalt blue diffused light of a slit lamp. The results were recorded and statistically analyzed.
[0074] Results: Following subcutaneous injection of scopolamine hydrobromide solution combined with ocular atropine sulfate gel, on the third day after modeling, the model group showed a significant reduction in fluorescein sodium staining scores compared to the normal group (*P<0.001). Starting on the seventh day after modeling, this significant change tended to stabilize, and the difference remained significant (*P<0.001). This stable change persisted for more than one month. See the appendix for detailed results. Figure 3 .
[0075] Conclusion: The method of the present invention can aggravate and expand the degree and range of corneal epithelial staining in model animals in a short period of time, and maintain it continuously and stably for more than a month.
[0076] Example 10: Imaging results of sodium fluorescein detection in a rat dry eye model of Example 2
[0077] Experimental method: Same as in Example 7.
[0078] Results: After subcutaneous injection of scopolamine hydrobromide solution combined with ophthalmic atropine sulfate gel, on the seventh day of modeling, the model group showed localized small-scale fluorescent dotting or filamentous fluorescence phenomena in corneal fluorescein staining, with the staining area exceeding two quadrants. The corneal staining score was significantly higher than that of the normal group. Starting on the 14th day of modeling, large-area patchy staining appeared in corneal fluorescein staining, with the staining area exceeding three quadrants. This significant change tended to stabilize and the difference remained significant (*P<0.001), and this stable change lasted for more than one month. For detailed results, please refer to the appendix. Figure 4 .
[0079] Conclusion: The method of the present invention can aggravate and expand the degree and range of corneal epithelial staining in model animals in a short period of time, and maintain it continuously and stably for more than a month.
[0080] Example 11 HE staining experiment in the rat dry eye model of Example 2
[0081] Experimental methods: The test animals were euthanized, and the eyeballs of the upper and lower eyelids were collected intact. The eyeballs were fixed in 10% neutral buffered formalin, dehydrated with graded ethanol, embedded in paraffin blocks, and sectioned in arrow shape. Serial sections of the anterior segment (3 μm each) were examined under a microscope, and photographs were taken at 20× magnification to record the morphological effects on the cornea and observe changes.
[0082] Results: HE staining at the tissue level confirmed that the corneal surface of the model animals was rough, and significant edema was observed in the epithelium and stroma. See the appendix for detailed results. Figure 5 .
[0083] Conclusion: The method of the present invention can induce typical dry eye-related symptoms in key tissues such as the cornea of model animals.
[0084] Example 12: TUNEL detection in a rat dry eye model of Example 2
[0085] Experimental Methods: After euthanasia of the animals, eyeballs from each group were paraffin-embedded. Paraffin sections from the anterior segment of each eyeball were stained with terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) using a fluorescein in situ cell death assay kit. Following staining, images were taken under a fluorescence microscope. Each section was photographed at the same optical parameters at an appropriate magnification. These non-overlapping, low-magnification images, covering the entire corneal section, were used to quantify the expression level of TUNEL-positive cells in each section.
[0086] Results: TUNEL staining showed that the model group had a significantly higher number of TUNEL-positive cells in the cornea compared to the normal group (P<0.01). See the appendix for detailed results. Figure 6 .
[0087] Conclusion: The method of the present invention can enhance the apoptosis process of animal corneal cells and increase the number of TUNEL-positive cells.
Claims
1. A method for constructing a rat dry eye model, characterized in that, Scopolamine combined with atropine induces dry eye syndrome.
2. The method for constructing a rat dry eye model according to claim 1, characterized in that, Rats were subcutaneously injected with scopolamine hydrobromide solution, and simultaneously 1% atropine sulfate ophthalmic gel was instilled into their eyes.
3. The method for constructing a rat dry eye model according to claim 2, characterized in that, Rats were subcutaneously injected with scopolamine hydrobromide solution, 0.2 mL each time, four times a day. The concentration of the scopolamine hydrobromide solution was 10-40 mg / mL. At the same time, atropine sulfate ophthalmic gel was instilled into the eyes once a day, 1-2 drops each time. Subcutaneous injection of scopolamine hydrobromide solution and ophthalmic instillation of atropine sulfate ophthalmic gel were performed continuously every day.
4. A method for constructing a rat dry eye model according to any one of claims 1-3, characterized in that, The rats are adult male rats of the SD or Wistar strain.
5. A method for constructing a rat dry eye model according to claim 2 or 3, characterized in that, The subcutaneous injection site was the back of the rat, and the ocular administration site was the conjunctival sac of the eyelid.
6. A method for constructing a rat dry eye model according to claim 2 or 3, characterized in that, The concentration of scopolamine hydrobromide solution is 10-40 mg / mL.
7. The method for constructing a rat dry eye model according to claim 1, characterized in that, The preparation method of the scopolamine hydrobromide solution is as follows: accurately weigh scopolamine hydrobromide and place it in a suitable container, add PBS solution, vortex or shake at room temperature to fully suspend the raw drug in the liquid, and then sonicate for 10-15 minutes to dissolve it and prepare the scopolamine hydrobromide solution.
8. The method for constructing a rat dry eye model according to claim 7, characterized in that, The mass-to-volume ratio of scopolamine hydrobromide to PBS solution is 20-25:
1.
9. The use of the dry eye model obtained by the method for constructing a rat dry eye model according to any one of claims 1-8 in evaluating the efficacy of drugs for treating or alleviating dry eye syndrome.
10. The application of the dry eye model obtained by the method for constructing a rat dry eye model according to any one of claims 1-8 in the study of the pathogenesis of dry eye.