Establishment method and application of rat radioactive cataract model

Abstract

The invention relates to the technical field of experimental animal models, in particular to an establishment method and application of a rat radioactive cataract model. According to the invention, a rat cataract model induced by irradiation of X-rays with different doses (10 Gy, 20 Gy and 30 Gy) is constructed. Through multi-dimensional evaluation such as general situation observation, slit lamp inspection and histopathologic analysis, the influence of ionization radiation (IR) on the crystalline lens is systematically observed. The animal model successfully reproduces pathological manifestations of different radiation-damaged crystalline lenses, and an experimental basis is provided for further research on pathogenesis of radiation-induced cataract.

Description

Technical Field

[0001] This invention relates to the field of experimental animal model technology, and in particular to a method for establishing and applying a rat radiation-induced cataract model. Background Technology

[0002] Radiation-induced cataract is a common and serious complication in patients with head and neck tumors after radiotherapy. With the widespread application of radiotherapy in cancer treatment, its long-term side effects have received increasing attention. To further study the pathogenesis and dynamic development of radiation-induced cataract and to explore effective preventative and therapeutic drugs, establishing stable and reliable disease models in experimental animals is crucial. Currently, various animal models of radiation-induced cataract have been reported, including mice, rats, and rabbits. Existing techniques mainly use ionizing radiation sources such as X-rays and gamma rays to induce cataracts in experimental animals through whole-body or local irradiation. Among many experimental animals, rats are widely recognized as ideal model animals for cataract research due to their lens structure and metabolic characteristics being similar to humans, as well as their low breeding cost and short reproductive cycle. By administering single or fractional irradiation to the rat's eye with a controlled radiation source, a clinical radiotherapy scenario can be simulated, effectively replicating an animal model highly consistent with the pathological changes of human radiation-induced cataract.

[0003] The core technical challenge in establishing this model lies in the precise control of radiation dose, irradiation field, and observation period. Too low a dose is unlikely to induce cataracts, while too high a dose may cause severe damage to other ocular tissues and interfere with observation. Typically, X-rays or gamma rays are used to precisely irradiate one or both eyes of the rat. After irradiation, the degree, location, and progression of lens opacity must be observed periodically using instruments such as a slit lamp, and recorded and evaluated according to recognized grading standards. A mature and stable model should be able to demonstrate the typical progressive development process from posterior subcapsular punctate opacities to complete cataracts, thus providing an ideal experimental platform for subsequent interventional studies.

[0004] However, existing technologies have the following shortcomings: (1) Model induction is difficult, and the sensitivity of animals to radiation damage varies from person to person, resulting in insufficient model repeatability and stability. To avoid acute radiation toxicity, most studies use low-dose (e.g., 0.5-8 Gy) single or fractional irradiation. Although this improves the survival rate of animals, the incidence of cataracts is low and the progression is slow. It often takes several months to more than a year for obvious lens opacity to appear, which greatly prolongs the experimental period and reduces experimental efficiency.

[0005] (2) Inconsistent irradiation parameters: Different studies use different parameters such as radiation dose, dose rate, and irradiation field size, resulting in poor model reproducibility.

[0006] (3) The evaluation criteria are not standardized: Most studies lack a unified grading standard for lens opacity, which is highly subjective and difficult to conduct objective quantitative evaluation.

[0007] (4) Incomplete histopathological observation: Only focusing on changes in lens transparency, lacking a systematic evaluation of pathological changes such as lens fibrosis.

[0008] (5) The dose gradient is not set properly: There is a lack of systematic dose-response relationship studies and a clear dose-response relationship has not been established.

[0009] (6) Inappropriate observation time points: failed to fully reflect the dynamic development process of radiation-induced cataracts.

[0010] Therefore, there is an urgent need for a method for constructing a radiation cataract model that is highly standardized, has comprehensive and objective evaluation indicators, is easy to operate, and has good safety. Summary of the Invention

[0011] This invention provides a method for establishing a rat radiation-induced cataract model with high standardization, good reproducibility, and comprehensive evaluation indicators, overcoming the shortcomings of existing technologies such as unstable models, inconsistent evaluation standards, and incomplete observation indicators.

[0012] The present invention adopts the following technical solution: The first aspect of this invention provides a method for establishing a rat radiation-induced cataract model, comprising the following steps: (1) Preparation of experimental animals: Six-week-old male SPF-grade SD rats weighing 210-240g were selected. The lens was examined with a slit-lamp microscope to confirm that there were no abnormalities. They were randomly divided into a control group (0 Gy) and an irradiation group (10 Gy, 20 Gy, 30 Gy), with 10 rats in each group. (2) Preparation before irradiation: inject 0.6 mL / kg of Shutai 50 anesthetic subcutaneously into the neck and fix the rat in a prone position on the irradiation table; (3) Local irradiation: A linear accelerator was used with 6 MeV X-rays, the source-to-skin distance was set to 1 meter, the dose rate was 300 MU / min, and the irradiation field covered the area from the anterior canthus to the posterior canthus of both eyes for a single irradiation. (4) Regular examination: Slit-lamp microscopy examinations were performed at 30, 60, 75, 90, 105 and 120 days after irradiation. Compound tropicamide eye drops were used to dilate the pupils. The degree of lens opacity was graded and evaluated according to the national standard "Diagnosis of Occupational Radiation Cataract" (GBZ 95-2014). (5) Terminal treatment and sampling: 120 days after irradiation, the experimental rats were sacrificed and both eyes were removed to obtain an X-ray induced rat radiation cataract model.

[0013] Furthermore, the experimental animals underwent a 7-day acclimatization period before irradiation, with the ambient temperature controlled at 20-26℃, humidity controlled at 40-70%, and a day-night ratio of 12:12.

[0014] Further, the irradiation doses in step (3) are as follows: 0 Gy for the control group, 10 Gy for the low-dose group, 20 Gy for the medium-dose group, and 30 Gy for the high-dose group.

[0015] Furthermore, the grading standard for the degree of lens opacity in step (4) is as follows: Grade 0 - no opacity, Grade I - slight opacity, Grade II - moderate opacity, Grade III - severe opacity, Grade IV - complete opacity.

[0016] Furthermore, the eyeball was enucleated 120 days after irradiation, fixed with paraformaldehyde, and paraffin sections were prepared for H&E staining and Masson trichrome staining to assess the histopathological changes and degree of fibrosis of the lens.

[0017] Furthermore, in step (5), H&E staining is used to observe changes in the morphology of lens epithelial cells and the arrangement of fibers, while Masson trichrome staining is used to specifically detect the degree of collagen fiber deposition and fibrosis.

[0018] The second aspect of this invention provides an application of a rat radiation-induced cataract model established based on the above method in the study of the pathogenesis of radiation-induced cataracts.

[0019] A third aspect of this invention provides an application of a rat radiation-induced cataract model established based on the above method in the screening of protective drugs.

[0020] The fourth aspect of this invention provides a method for evaluating radiation-induced cataract fibrosis, which uses Masson trichrome staining to detect the degree of collagen fiber deposition in lens tissue, and evaluates the severity of fibrosis by the area and depth of the blue stained area.

[0021] Furthermore, in the Masson trichrome staining, the blue stained area represents collagen fiber deposition, and the deeper the staining and the larger the area, the more severe the degree of fibrosis.

[0022] The technical solution adopted in this invention can achieve the following beneficial effects: (1) High degree of standardization: The use of unified irradiation parameters, evaluation standards and inspection time points ensures the reproducibility and comparability of the model, and the results of different experiments are consistent.

[0023] (2) Reasonable dose gradient design: Four dose groups of 0, 10, 20 and 30 Gy were set up, and a complete dose-response relationship curve was established to meet different research needs.

[0024] (3) Comprehensive and objective evaluation indicators: Combining slit lamp examination, H&E staining and Masson trichrome staining, the changes in lens transparency, epithelial cell damage and degree of fibrosis can be evaluated simultaneously, providing multi-dimensional pathological information.

[0025] (4) Masson trichrome staining was used for the first time to evaluate radiation-induced cataract fibrosis: it can specifically show collagen fiber deposition and provides a new evaluation method for studying the fibrosis mechanism of radiation-induced cataract.

[0026] (5) Complies with national standards: The national standard GBZ 95-2014 is used to classify lens opacity. The evaluation results are objective, reliable and authoritative.

[0027] (6) Simple operation and good safety: Local irradiation reduces systemic side effects, resulting in a high animal survival rate and making it suitable for long-term observation and research.

[0028] (7) Scientifically set time window: Through dynamic observation at multiple time points, the occurrence and development process of radiation cataracts can be fully reflected. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, forming part of the present invention. The illustrative embodiments of the present invention and their descriptions explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 This is a diagram of an animal irradiation device.

[0030] Figure 2 Slit-lamp images of the lens of rats in different dose groups at different time points.

[0031] Figure 3 Pathological images of the lens of rats in each group after H&E staining.

[0032] Figure 4 Masson trichrome staining pathological images of the lenses of rats in each group.

[0033] Lens tissue sections were stained with H&E and observed under an optical microscope (scale bar = 50 μm). Images of the anterior cross-section of the lens in (a) the 0 Gy control group, (b) the 10 Gy irradiation group, (c) the 20 Gy irradiation group, and (d) the 30 Gy irradiation group. Images of the equatorial region of the lens in (e) the 0 Gy control group, (f) the 10 Gy irradiation group, (g) the 20 Gy irradiation group, and (h) the 30 Gy irradiation group. Black arrows indicate LECs, yellow arrows indicate lens fiber structures, and blue arrows indicate vacuoles. Bow: Lens arch.

[0034] Lens tissue sections were stained with Masson's trichrome and observed under an optical microscope (scale bar = 50 μm). Black arrows indicate lens optic discs (LECs), and yellow arrows indicate lens fiber structures. Blue stained areas represent collagen fiber deposits. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0036] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0037] Example 1 Example 1 of this application provides a method for establishing a rat radiation-induced cataract model, specifically including: 1. Instruments and Equipment Linear accelerator (Elekta Synergy type, UK); slit lamp microscope; pathology slide machine; optical microscope, etc.

[0038] 2. Animals and Grouping Forty male SPF-grade SD rats, weighing 210-240g and 6 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0039] Mydriasis was achieved using compound tropicamide eye drops. Slit-lamp examination confirmed the absence of abnormal lens lesions. Animals were then randomly assigned to four groups: a control group (0 Gy) and three groups with different radiation doses (10, 20, and 30 Gy), with 10 animals in each group, totaling 40 animals. They were housed in separate cages. After 7 days of acclimatization, local eye irradiation was performed. Temperature, humidity, and light were controlled in the animal laboratory. Temperature and humidity in the animal housing were automatically monitored and recorded by an environmental monitoring platform. During the experiment, the housing temperature was maintained at 20℃-26℃, and humidity at 40%-70%, with a day-night ratio of 12:12 (daytime: 7:00-19:00; nighttime: 19:00-7:00 the next day). Animals had free access to water and food.

[0040] 3. Test Methods After being anesthetized by subcutaneous injection of Sertazone 50 (0.6 mL / kg) in the neck, rats in each group were placed prone on an irradiation table and exposed to 6 MeV X-rays using an Elekta Synergy linear accelerator (UK). The irradiation field covered the line connecting the anterior and posterior canthi of both eyes, with a source-to-skin distance of 1 meter and a dose rate of 300 MU / min. Ten rats in the normal control group received sham irradiation; thirty rats in the irradiation group were randomly divided into three groups of ten each, with single irradiation doses of 10, 20, and 30 Gy, respectively.

[0041] Experimental animals were examined regularly at 30, 60, 75, 90, 105, and 120 days after irradiation. Before each examination, rats were anesthetized by subcutaneous injection of 50 ozonol (0.6 mL / kg) in the neck to dilate their pupils. After complete pupil dilation, both eyes were examined and photographed using a slit-lamp microscope. The degree of lens opacity was strictly graded according to the national standard "Diagnosis of Occupational Radiation Cataracts" (GBZ 95-2014).

[0042] 120 days after irradiation, the patient underwent cervical dislocation euthanasia under deep anesthesia with Sertazone 50 (0.6 mL / kg), and the eyeballs were completely enucleated. The enucleated eyeballs were immediately fixed in 4% paraformaldehyde for 24 hours, and paraffin sections were prepared for H&E staining and Masson's trichrome staining.

[0043] 4. Test Results 4.1 General Condition Observation In the control group, rats showed normal diet, excretion, lifestyle, and fur color. In the 10 Gy irradiation group, rats experienced slight hair loss around the eyes and jaw, decreased appetite, and slowed movement in the fourth week after irradiation; these symptoms gradually improved after one week. In the 20 Gy irradiation group, rats experienced hair loss around the eyes and jaw in the second week after irradiation, with a persistent decrease in appetite; these symptoms gradually improved after two weeks. In the 30 Gy irradiation group, rats experienced decreased appetite, tremors, and slowed movement in the first week after irradiation; the symptoms were most severe between 7-14 days, and they fully recovered after 5 weeks.

[0044] 4.2 Results of slit-lamp microscopy examination X-ray irradiation can induce lens opacity in SD rats, showing obvious dose-response relationship and time-dependent characteristics. Figure 2(Tables 2 and 3). The lenses of the control group (0 Gy) rats remained transparent throughout the observation period. The 10 Gy group showed almost no opacity in the early stages (within 60 days after irradiation), but some animals developed mild grade I-II opacity in the later stages, indicating a certain cumulative damage effect. The 20 Gy group showed opacity from day 30 onwards, with the degree of opacity gradually increasing over time. By day 105, all animals reached grade II or higher opacity, and by day 120, 80% of the animals had grade III-IV opacity. The 30 Gy group showed the most rapid and severe progression; scattered punctate opacities were visible in the posterior capsule of the lens as early as day 30; approximately 75% of the animals reached grade II-III opacity by day 60; approximately 65% ​​of the animals reached grade III or higher by day 75; most animals had grade III-IV opacity after day 90; and by day 120, most animals exhibited complete diffuse opacity (grade IV).

[0045] The Kruskal-Wallis H test results at each time point all indicated that there were statistically significant differences in the degree of lens opacity among the different dose groups (p < 0.001).

[0046] Further pairwise comparisons showed that the 0 Gy group was significantly different from the 20 Gy and 30 Gy groups at most time points (p<0.05 or p<0.001), but the difference with the 20 Gy group was not statistically significant at 30 days (p=0.371); while there was no statistically significant difference with the 10 Gy group at all time points (p>0.05), even at the end of the period only a few 10 Gy animals showed mild turbidity. The difference between the 20 Gy and 30 Gy groups was significant at 60 days (p=0.004), but the difference decreased without statistical significance from 75 days onwards (p>0.05), suggesting that the turbidity of the 20 Gy group gradually worsened in the later stages. The difference between the 20 Gy and 10 Gy groups was not significant at 30 days (p=0.371), but reached statistical significance from 60 days onwards (p=0.007), and continued to increase in the later stages.

[0047] Table 1. Degree of lens opacity in rats at different time points

[0048] Note: Lens opacity is classified into grades 0-IV, where grade 0 is no opacity and grades I-IV are progressively more opaque.

[0049] Table 2. Rank-sum test results of lens opacity levels in different dosage groups

[0050] Note: n is the number of eyes per dose group; T1 and T2 are the rank sums of the two groups, respectively; p The value is a two-sided probability value adjusted by the Bonferroni correction method.

[0051] 4.3 H&E staining Different doses of X-ray irradiation produced significant dose-dependent changes in the structure of the rat lens. In the control group, the lens epithelial cells were arranged in a single layer in a regular manner, and the lens fibers were neatly arranged. Figure 3 In the 10 Gy irradiation group, the number of epithelial cells decreased, and some cells showed microvacuoles in their cytoplasm. In the 20 Gy irradiation group, the epithelial cells and lens fibers were disorganized. The lesions were most pronounced in the 30 Gy irradiation group, with significantly disorganized anterior capsule epithelial cells and extensively disorganized lens fibers.

[0052] 4.4 Masson's trichrome staining The control group had dense and orderly arranged lens fibers with a uniform light blue coloration. Figure 4 In the 10 Gy irradiation group, a slight increase in blue staining was observed in localized areas, suggesting early collagen deposition. In the 20 Gy irradiation group, the blue-stained areas significantly expanded and deepened, indicating increased collagen deposition and a marked increase in fibrosis. In the 30 Gy irradiation group, large areas showed deep blue staining, indicating severe collagen deposition and fibrotic lesions.

[0053] This model successfully established a dose-dependent rat radiation-induced cataract model, providing a reliable experimental basis for further research.

[0054] This invention established rat cataract models induced by X-ray irradiation at different doses (10 Gy, 20 Gy, 30 Gy). The effects of radiation exposure (IR) on the lens were systematically observed through multi-dimensional evaluation, including general observation, slit-lamp examination, and histopathological analysis. The results showed that the occurrence and progression of radiation-induced cataracts exhibited clear dose- and time-dependent characteristics, with the high-dose group showing earlier and more severe lens opacity. H&E staining revealed damage to lens optic arteries (LECs) and disordered fibrous arrangement, while Masson's trichrome staining demonstrated that the radiation-induced lens fibrosis process worsened with increasing dose. This animal model successfully reproduced the pathological manifestations of lens damage from different radiation exposures, providing an experimental basis for further research into the pathogenesis of radiation-induced cataracts.

[0055] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A method for establishing a rat model of radioactive cataract, characterized in that, Comprising the following steps: (1) Select 6-week-old male SPF SD rats as experimental animals, weighing 210-240 g, and check the lens for abnormalities with a slit lamp before randomly dividing them into control and irradiation groups; (2) Subcutaneous injection of anesthetic in the neck, and fix the rats in a prone position on the irradiation table; (3) Use a linear accelerator 6 MeV X-ray for bilateral local irradiation, with a source-skin distance of 1 meter and a dose rate of 300 MU / min, and the irradiation field covers the area from the anterior to the posterior commissure of the eye; (4) Perform slit lamp examination and lens opacity grading at 30, 60, 75, 90, 105, and 120 days after irradiation; (5) Sacrifice the experimental rats at 120 days after irradiation, and remove the eyeballs to obtain an X-ray-induced rat radiation cataract model.

2. The method of claim 1, wherein, The irradiation group is divided into three different dose groups of 10 Gy, 20 Gy, and 30 Gy.

3. The method of claim 1, wherein, The lens opacity grading standard is as follows: 0 grade-no turbidity, I grade-mild turbidity, II grade-moderate turbidity, III grade-severe turbidity, and IV grade-complete turbidity.

4. The method of claim 1, wherein, The eyeballs are removed at 120 days after irradiation, fixed with paraformaldehyde, and paraffin sections are prepared for H&E staining and Masson's trichrome staining to evaluate the histopathological changes and fibrosis degree of the lens tissue.

5. The method of claim 1, wherein, The experimental animals are adaptively fed for 7 days before irradiation, with an environmental temperature of 20-26°C, humidity of 40-70%, and a day-night ratio of 12:

12.

6. The method of claim 5, wherein, The lens turbidity rate of the 30 Gy irradiation group reached 100% at 120 days after irradiation, of which 70% was complete turbidity of grade IV.

7. A rat radiation cataract model established based on the method of any one of claims 1-6 for use in the study of the pathogenesis of radiation cataract.

8. A rat radiation cataract model established based on the method of any one of claims 1-6 for use in the screening of protective drugs.

9. A method of assessing radiation cataract fibrosis, characterized by, The degree of collagen fiber deposition in the lens tissue is detected by Masson's trichrome staining, and the severity of fibrosis is evaluated by the area and depth of blue staining.

10. The method of claim 9, wherein, In the Masson's trichrome staining, the blue staining area represents collagen fiber deposition, and the deeper and larger the area, the more severe the fibrosis.

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