Method for constructing periodontitis rat model and use thereof

Abstract

A method for constructing a periodontitis rat model, which method comprises the following steps: firstly infecting a rat with Toxoplasma gondii, and then co-infecting the Toxoplasma-gondii-infected rat with Porphyromonas gingivalis and Treponema denticola so as to establish the periodontitis rat model. In the present invention, a periodontitis animal model is rapidly and stably obtained by means of the synergistic effect among Porphyromonas gingivalis, Treponema denticola and Toxoplasma gondii. Moreover, the constructed periodontitis model exhibits symptoms and signs highly consistent with those of human periodontitis, and can reflect the systemic characteristics and severity of periodontitis. The method has the advantages of rapid modeling, high success rate and good reproducibility, and can thus be used for studying the infection, development mechanism, and prevention and treatment process of periodontitis.

Description

A method for constructing a rat model of periodontitis and its application Technical Field

[0001] This invention relates to the field of periodontology, and in particular to a method for constructing an animal model for studying the pathogenesis of periodontitis and its associated infectious diseases, specifically a method for constructing a rat model of periodontitis and its application in the study of periodontitis-related diseases. Background Technology

[0002] Chronic periodontitis (CP) is a chronic infectious disease and the leading cause of tooth loss in adults, with a high incidence rate worldwide. According to data from the Fourth National Oral Health Epidemiological Survey, the detection rate of dental calculus in Chinese adults aged 35-44 is 96.7%, and the detection rate of gingival bleeding reaches 87.4%, an increase of 10.1% compared to 77.3% ten years ago. Furthermore, the prevalence and severity increase with age.

[0003] *Porphyromonas gingivalis* (P. gingivalis) is the main pathogen of plaque erosion (CP), possessing the ability to activate host inflammation and regulate immune responses. *Treponema denticola* (T. denticola), as an important bridging bacterium, can not only combine with early colonizing bacteria such as *Streptococcus* and *Actinomycetes* to colonize the tooth surface, but also co-exist with subsequent *P. gingivalis*, *Tannerella forsythia* (T. forsythia), and *Fusobacterium nucleatum* (F. nucleatum), promoting plaque biofilm formation. Simultaneously, it possesses many virulence factors that directly participate in the destruction of periodontal tissues. Studies show that *P. gingivalis* may contribute to the development and progression of CP. Gingival bleeding is a typical clinical symptom of CP, providing favorable conditions for oral infection. Toxoplasma gondii (T. gondii) is an important foodborne parasitic protozoan. After infecting the body, T. gondii can travel through the bloodstream to all parts of the body, damaging the brain, heart, and other organs, thus weakening the immune system.

[0004] Existing research has involved the construction of periodontitis models. For example, the paper "Experimental Study on the Immunomodulatory Effect of Th1 / Th2 Cell Response on Mouse Periodontitis" (CNKI: Xie Min. Experimental Study on the Immunomodulatory Effect of Th1 / Th2 Cell Response on Mouse Periodontitis [D]. Jinan University, 2008.) discloses that a mouse periodontitis animal model was first established by ligation with silk sutures and local bacterial application, and then further infected with ultraviolet-attenuated *T. gondii* to explore the relationship between the body's immune response and periodontitis. However, this method uses bacteria from periodontitis patients for infection, and the proportion and concentration of bacterial species in the tartar of different periodontitis patients are different. Therefore, using bacteria derived from patients cannot be standardized to meet the requirements of high success rate and high stability in model establishment. In addition, although many periodontitis pathogens are known in existing technologies, the properties of different bacteria are quite different, and they can also affect each other when acting on the same host. Therefore, the predictability of the effect after combined infection is poor.

[0005] Rats are widely used rodent model animals in studies of disease pathogenesis, drug metabolism, and pharmacokinetics, offering significant advantages over mice, such as larger size for easier manipulation, greater blood volume and tolerance, and a larger sample size. Given that effective prevention and treatment of periodontitis has become a hot topic for scholars both domestically and internationally, the efficient and rapid construction of rat models of periodontitis is of great significance for the research and prevention of this disease. This application, through research on the pathogenesis of periodontitis, unexpectedly discovered that co-infection of rats with *P. gingivalis*, *T. denticola*, and *T. gondii* can rapidly and stably obtain a rat model of periodontitis, with easy manipulation and a high success rate. Summary of the Invention

[0006] The purpose of this invention is to provide a method for modeling periodontitis in rats, aiming to obtain a periodontitis animal model quickly and with a high success rate for research and application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] 1. Establishment of the T. gondii infection model: Each rat was given 100 Toxoplasma gondii cysts via oral gavage to establish the T. gondii infection model.

[0009] 2. Establishment of a periodontitis model: Periodontitis was simulated by ligating the maxillary first molar 3-0 with silk suture and applying P. gingivalis and T. denticola intraorally. Specific steps included rat anesthesia, tooth localization, silk suture ligation, and inoculation with periodontal pathogens.

[0010] 3. Establishment of a rat model of periodontitis combined with T. gondii infection: After constructing a T. gondii infection model, the rats infected with T. gondii were manipulated according to the method for establishing a periodontitis model, and finally a rat model of periodontitis was constructed.

[0011] 4. Experimental design: The experimental animals were randomly divided into multiple groups.

[0012] (1) Blank control group: There was no silk ligation, periodontal pathogens, or T. gondii infection.

[0013] (2) Periodontitis group: ligation of the maxillary first molar with silk thread + periodontal pathogen infection.

[0014] (3) Toxoplasmosis group: After Toxoplasmosis infection, the maxillary first molar was ligated with silk thread.

[0015] (4) Periodontitis + Toxoplasma gondii group: After T. gondii infection, the maxillary first molar was ligated with silk thread + periodontal pathogen infection.

[0016] Compared with the prior art, the beneficial effects of the present invention are specifically reflected in:

[0017] 1) This invention successfully obtained a rat model of periodontitis in just 7 days by co-infecting rats with P. gingivalis, T. denticola, and T. gondii. The established rat model of periodontitis showed edema of the gingival papillae, failure of the gingival margin to adhere to the tooth surface, soft texture, bleeding, and periodontal pockets. As the severity increased, it led to tooth loosening and even root exposure, which are consistent with the symptoms, signs, and pathological processes of human periodontitis and can reflect the systemic nature and severity of periodontitis. In contrast, the modeling time for similar periodontitis models in the prior art is usually about one month. Therefore, the construction method of this invention greatly reduces the modeling time.

[0018] 2) This invention not only successfully obtained a rat model of periodontitis in a short time by screening different combinations of common periodontitis pathogens, but also has the advantages of high success rate, good controllability and good stability. It can be used to study the development mechanism and prevention process of periodontitis. Attached Figure Description

[0019] Figure 1. The state of rats on day 5 after intraperitoneal inoculation with Toxoplasma gondii tachyzoites (top right a: rats inoculated with Toxoplasma gondii; bottom left b: control group rats).

[0020] Figure 2. Rat immobilized on a rat board.

[0021] Figure 3. Periodontal suture ligation.

[0022] Figure 4. Intraoral inoculation with periodontal pathogens.

[0023] Figure 5 shows a rat orally gavaged with Toxoplasma gondii cysts.

[0024] Figure 6 shows the periodontal condition of a rat before ligation.

[0025] Figure 7 shows the periodontal condition of a rat in the blank group.

[0026] Figure 8 shows the periodontal condition of the periodontitis group after ligation.

[0027] Figure 9 shows the periodontal condition of the periodontitis + Toxoplasma group after ligation.

[0028] Figure 10 shows Toxoplasma gondii cysts visible in the brain homogenate of SD rats in the Toxoplasma gondii infection group.

[0029] Figure 11 shows the histopathological changes of the periodontal tissues of rats in the blank group (A), periodontitis + Toxoplasma group (B), periodontitis group (C), and Toxoplasma group (D). [[ID=@20]]

[0030] Figure 12 shows the Micro-CT scan results of the periodontal tissues of rats in the blank group (A), periodontitis + Toxoplasma group (B), periodontitis group (C), and Toxoplasma group (D). Detailed implementation manners

[0031] To better understand the present invention, the following further describes the present invention with specific examples. The terms used in the examples are for describing specific embodiments and do not limit the protection scope of the present invention.

[0032] For the experimental methods without specific conditions noted in the following examples, they are usually in accordance with conventional conditions or the conditions recommended by the manufacturers. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources. Unless otherwise stated, the substances described in the present invention are calculated by mass percentage and mass fraction.

[0033] Porphyromonas gingivalis: No.: ATCC 33277, provided by the laboratory of Stomatological College of Guangzhou Medical University.

[0034] Treponema denticola: No.: DSM 14222, provided by the laboratory of Stomatological College of Guangzhou Medical University.

[0035] Toxoplasma gondii: No.: Toxoplasma gondii (T.gondii) Me49, provided by the Parasitology Laboratory of the College of Veterinary Medicine, South China Agricultural University.

[0036] SD rats: Purchased from the Animal Experiment Center of Southern Medical University, production experimental animal license number: SCXK(Guangdong)2021 - 0041.

[0037] This experiment was conducted in the animal laboratory of the College of Veterinary Medicine, South China Agricultural University. The license number for the use of experimental animals is: SYXK(Guangdong)2024-0136.

[0038] The present invention provides a method for establishing a rat model of P. gingivalis and T. denticola co-infecting with T. gondii. The specific implementation steps of this method will be described in detail below.

[0039] 1. Preparation of experimental materials

[0040] 1.1 Experimental animals: SPF-grade 4-week-old male SD rats with a body weight of 200±20 g were selected. The animals were housed in a clean animal room at the specific pathogen-free (SPF) center, with an environmental temperature of 22±2°C and a humidity of 50%±10%.

[0041] 1.2 Bacteria: Porphyromonas gingivalis ATCC 33277 standard strain and Treponema denticola DSM 14222 standard strain.

[0042] 1.3 Toxoplasma: Toxoplasma gondii Me49 strain.

[0043] 1.4 Main materials and reagents: Brain heart infusion (BHI) medium, hemin, paraformaldehyde, vitamin K1.

[0044] 1.5 Main experimental instruments: including anaerobic incubator, automatic dehydrator, paraffin embedding machine, paraffin tissue slicer, etc.

[0045] 1.6 Preparation of main reagents: Prepare BHI solid medium and liquid medium according to the reagent instructions.

[0046] 2. Experimental methods

[0047] 2.1 Periodontitis group: Periodontitis was induced by ligating and applying P. gingivalis and T. denticola on the first maxillary molar of rats.

[0048] Toxoplasma group: Rats were infected by oral gavage with Toxoplasma cysts.

[0049] Periodontitis + Toxoplasma group: Combining the above two methods, periodontitis and T. gondii infection were induced simultaneously.

[0050] Blank group: Normal saline was used for each infection step, and the rats were housed in the same environment and under the same conditions.

[0051] Control group 1: F. nucleatum was used to replace T. denticola for infection, and the other conditions were the same as those in the periodontitis + Toxoplasma group.

[0052] Control group 2: T. forsythia was used instead of T. denticola for infection, and all other conditions were the same as those in the periodontitis + toxoplasmosis group.

[0053] Control group 3-4: Periodontitis was induced by ligating the first maxillary molar of rats and applying P. gingivalis and T. denticola. The difference from the periodontitis + toxoplasmosis group was that the ratio of P. gingivalis and T. denticola was adjusted.

[0054] The *F. nucleatum* ATCC 2558 and *T. forsythia* ATCC 700198 were provided by the Oral Microbiology Laboratory of Guangzhou Medical University.

[0055] 2.2 Sample collection and testing methods: including periodontal symptom examination, imaging examination, pathological examination, etc.

[0056] Example 1: Periodontitis + Toxoplasmosis Group

[0057] Establishing a rat model of periodontitis:

[0058] (1) Select clean-grade SD rats and acclimatize them for 7 days before infection;

[0059] (2) Kunming rats chronically infected with T. gondii were sacrificed, and the whole brain was removed, ground into a homogenate and diluted with physiological saline to a dose of 100 brain cysts / mL to obtain the infection solution. After the rats were acclimatized for 7 days, each rat was given 1 mL of the infection solution by gavage.

[0060] (3) The rats treated in step (3) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital;

[0061] (4) After anesthetizing the rats, ligation was performed on the cervical region of the bilateral maxillary first molars using 3-0 silk thread, and then 1*10 9 CFU / mLP. gingivalis bacterial culture with 2*10 9 T. denticola bacterial suspension at CFU / mL was inoculated at a dose of 0.1 mL / tooth into the ligature line and gingival sulcus of rat teeth; inoculation frequency was once a day for 7 consecutive days;

[0062] (5) The modeling success is determined by measuring the periodontal symptoms of rats treated in step (4) and measuring the alveolar bone resorption. The alveolar bone resorption rate is ≥20%, and the periodontal pocket probing depth is ≥1mm. If both conditions are met, the modeling is successful.

[0063] (6) The rats treated in step (4) were examined for oral cavity, imaging and pathological conditions.

[0064] Example 2: Periodontitis group:

[0065] Establishing a rat model of periodontitis:

[0066] (1) Select clean-grade SD rats and acclimatize them for 7 days before infection;

[0067] (2) Each rat was administered 1 mL of physiological saline by gavage;

[0068] (3) The rats treated in step (2) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital;

[0069] (4) After anesthetizing the rats, ligation was performed on the cervical region of the bilateral maxillary first molars using 3-0 silk thread, and then 1*10 9 CFU / mLP. gingivalis bacterial culture with 2*10 9 T. denticola bacterial suspension at CFU / mL was inoculated at a dose of 0.1 mL / tooth into the ligature line and gingival sulcus of rat teeth; inoculation frequency was once a day for 7 consecutive days;

[0070] (5) The modeling success is determined by measuring the periodontal symptoms of rats treated in step (4) and measuring the alveolar bone resorption. The alveolar bone resorption rate is ≥20%, and the periodontal pocket probing depth is ≥1mm. If both conditions are met, the modeling is successful.

[0071] (6) Oral, imaging and pathological examinations were performed on the rats after treatment in step (4).

[0072] Example 3: Toxoplasma gondii group:

[0073] (1) Select clean-grade SD rats and acclimatize them for 7 days before infection;

[0074] (2) Kunming rats chronically infected with T. gondii were sacrificed, and the whole brain was removed, ground into a homogenate and diluted with physiological saline to a dose of 100 brain cysts / mL to obtain the infection solution. After the rats were acclimatized for 7 days, each rat was given 1 mL of the infection solution by gavage.

[0075] (3) The rats treated in step (2) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital;

[0076] (4) After anesthetizing the rats, use 3-0 silk thread to ligate the neck of the first molars on both sides of the maxilla, and then inject physiological saline at a dose of 0.1 mL / tooth into the ligation thread and the gingival sulcus of the rat's teeth; the injection frequency is once a day; the injection is carried out for 7 consecutive days.

[0077] (5) The modeling success is determined by measuring the periodontal symptoms of rats treated in step (4) and measuring the alveolar bone resorption. The alveolar bone resorption rate is ≥20%, and the periodontal pocket probing depth is ≥1mm. If both conditions are met, the modeling is successful.

[0078] (6) The rats treated in step (4) were examined for oral cavity, imaging and pathological conditions.

[0079] Example 4: Blank group:

[0080] (1) Select clean-grade SD rats and acclimatize them for 7 days before infection;

[0081] (2) Each rat was administered 1 mL of physiological saline by gavage;

[0082] (3) The rats treated in step (2) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital;

[0083] (4) After anesthetizing the rats, use 3-0 silk thread to ligate the neck of the first molars on both sides of the maxilla, and then inject physiological saline at a dose of 0.1 mL / tooth into the ligation thread and the gingival sulcus of the rat's teeth; the injection frequency is once a day; the injection is carried out for 7 consecutive days.

[0084] (5) The modeling success is determined by measuring the periodontal symptoms of rats treated in step (4) and measuring the alveolar bone resorption. The alveolar bone resorption rate is ≥20%, and the periodontal pocket probing depth is ≥1mm. If both conditions are met, the modeling is successful.

[0085] (6) The rats treated in step (4) were examined for oral cavity, imaging and pathological conditions.

[0086] The preparation methods of control group 1 and control group 2 are the same as those in Example 1. The only difference from Example 1 is that control group 1 and control group 2 are respectively limited to specific bacterial strains.

[0087] The preparation method of control group 3 differs from that of example 1 in that the concentration of P. gingivalis bacterial culture in step (4) is 1.5*10. 9 CFU / mL, T. denticola bacterial concentration is 1.5*10 9 The CFU / mL ratio is the same, and the remaining steps and parameters are exactly the same.

[0088] The preparation method of control group 4 differs from that of Example 1 in that the concentration of P. gingivalis bacterial culture in step (4) is 2*10. 9 CFU / mL, T. denticola bacterial concentration is 1*10 9 The CFU / mL ratio is the same, and the remaining steps and parameters are exactly the same.

[0089] There were eight experimental groups, with 20 rats in each group.

[0090] 3. Sample collection and intraoral examination, imaging and pathological examination:

[0091] Intraoral examination: Examine changes in gingival color and texture, probing depth, and tooth mobility in rats. One week after modeling, observe the sample rats for changes in the color of the free gingiva and gingival papillae (whether they are bright red or dark red, and whether the attached gingiva is involved); whether the gingival tissue is swollen and the extent of swelling; changes in the glossy spots on the gingival surface; whether the gingival margin is thickened and whether it adheres well to the tooth surface; whether there is purulent discharge or erosion at the ligation site; and the texture of the gingiva (whether it lacks elasticity or is soft). During probing, observe for bleeding from the gingiva, whether periodontal pockets can be reached and their depth; and whether the experimental teeth are mobile and the extent of mobility.

[0092] Criteria for successful intraoral examination of the model: The free gingiva and gingival papillae are bright red or dark red and extend to the attached gingiva; the gingival tissue is swollen, the gingival margin is thickened and does not adhere to the tooth surface; there is purulent discharge or erosion at the ligation site; the gingiva is soft or lacks elasticity; there is bleeding during probing; there is a certain periodontal pocket; the experimental tooth is loose.

[0093] Preparation of specimens required for imaging and pathological examination: Maxillary bone samples and periodontal tissue structure specimens were collected from rats that met the criteria for imaging and pathological examination after processing in step (5). To preserve the structure of the periodontal tissue of the first molar to the greatest extent, the cardiac perfusion method was used to fix the tissue. The cardiac perfusion method is as follows: 1 mL of 1.5% sodium pentobarbital solution was injected intraperitoneally to anesthetize the rats, and the rats' breathing and heartbeat were observed at any time. Rubber bands were wrapped around the incisors of the limbs and head to fix the rats on the rat board in a supine position, and cardiac perfusion fixation was started. After the rats were anesthetized, the chest wall was cut along the anterior axillary line on the body surface below the ribs, and a 5 cm lateral incision was made. The liver and diaphragm were carefully separated with scissors. After cutting, the anterior chest wall was turned upward to expose the heart. The ventricle was gently clamped and fixed with hemostatic forceps. Then, the tip of the syringe needle (the tip of the needle should be blunt) was gently rotated and inserted into the ascending aorta about 1 mm at the apex of the heart. The right atrial appendage was cut open with scissors. Inject 50 mL of physiological saline. After a clear liquid flows out from the right atrial appendage and the rat's liver turns white, continue to perfuse with 4% paraformaldehyde fixative, equivalent to the rat's body weight. Dissect and harvest the rat's maxilla, collect periodontal tissue, and preserve it in 4% paraformaldehyde.

[0094] Imaging examination: Micro-CT scans and three-dimensional reconstructions were performed on the maxillary bone specimens to measure alveolar bone resorption.

[0095] Pathological examination: Periodontal tissue specimens were stained with hematoxylin and eosin (HE) to observe the pathological morphological changes of periodontal tissues. The specific HE staining method was as described in the HE staining kit instructions. The HE staining kit was purchased from Solarbio Science & Technology Co., Ltd., catalog number: G1120.

[0096] Success rate of modeling = (Number of mice successfully modeled / Total number of mice in the group) × 100%

[0097] Average alveolar bone resorption rate = (Sum of alveolar bone resorption rates in this group of mice / Total number of mice in this group) × 100%

[0098] Average periodontal pocket probing depth = Sum of periodontal pocket probing depths in this group of mice / Total number of mice in this group

[0099] All results above should be rounded to two significant figures.

[0100] 4. Experimental Results

[0101] Table 1. Detection of rat samples used in modeling.

[0102]

[0103] Note: "*" indicates that the experimental group is significantly different from the control group; "#" indicates that the experimental group is significantly different from the periodontitis + toxoplasmosis group; "*" indicates p < 0.05; "**" indicates p < 0.01; "***" indicates p < 0.001; "#" indicates p < 0.05; "ns" indicates no significant difference compared with the control group or the periodontitis + toxoplasmosis group.

[0104] The above experimental results show that the rat model of periodontitis constructed in this application, which involves co-infecting rats with *P. gingivalis*, *T. denticola*, and *T. gondii*, achieves a modeling success rate of over 95% within 7 days, with a short modeling time and high success rate. Furthermore, intraoral examination, imaging, and pathological examinations were performed on 19 successfully modeled rats. Intraoral examination results showed that all 19 successfully modeled rats in the periodontitis + toxoplasmosis group exhibited dark red gingival papillae, swollen gingival tissue, erosion at the ligation site, soft gingival texture, and bleeding upon probing.

[0105] HE staining revealed that in the blank group (A in Figure 11), the junctional epithelium showed no loss of attachment and remained located at the cementoenamel junction, with intact tooth structure. In the periodontitis group, a small portion of the epithelium proliferated into the connective tissue, with minor resorption of the alveolar ridge crest and alveolar bone proper, and fewer osteoclasts in the alveolar bone. In contrast, the periodontal tissue of the periodontitis group (C in Figure 11) showed loss of junctional epithelium attachment, alveolar bone destruction, and visible osteoclasts, with an increased number compared to the Toxoplasma gondii group (D in Figure 11). The periodontal tissue of the periodontitis + Toxoplasma gondii group (B in Figure 11) showed significant loss of junctional epithelium attachment, significant alveolar bone destruction, and a large number of osteoclasts, significantly more than in the periodontitis group (C in Figure 11) and the Toxoplasma gondii group (D in Figure 11).

[0106] Micro-CT results showed that, compared with the blank group (A in Figure 12), the periodontitis group (C in Figure 12), and the toxoplasmosis group (D in Figure 12), the alveolar bone height, integrity, and fullness of the periodontitis + toxoplasmosis group (B in Figure 12) were significantly reduced.

[0107] Specifically, a comparison of the results of the rat model with periodontitis and Toxoplasma gondii with those of the periodontitis group and the Toxoplasma gondii group shows that:

[0108] (1) It is almost impossible to obtain a rat model of periodontitis 7 days after infection by using periodontitis pathogens or T. gondii alone. Only 1 out of 20 rats in the periodontitis group had obvious periodontitis symptoms, specifically redness of the periodontal tissue, slight bleeding of the gingiva when probed, and obvious swelling of the gingival tissue. No obvious symptoms were observed in the other 19 rats in the group and in the Toxoplasma gondii group.

[0109] (2) Toxoplasmosis infection can aggravate periodontitis, manifested as worsening of clinical symptoms and increased alveolar bone resorption on radiographs. In rat models, this aggravating effect was significant (P < 0.001).

[0110] The results of the rat model with periodontitis and Toxoplasma gondii were compared with those of control groups 1-2. It was found that the infection effects of different periodontitis pathogens on rats varied greatly. The combination of P. gingivalis and T. denticola was significantly more effective than the combination of P. gingivalis and F. nucleatum or P. gingivalis and T. forsythia. This indicates that infection with P. gingivalis and T. denticola is more suitable for synergistic effect with T. gondii infection to rapidly obtain a rat model of periodontitis, and it has a significant impact on the success rate of model establishment.

[0111] The results of the rat model with periodontitis and Toxoplasma gondii were compared with those of control group 3-4. It can be seen that different ratios of the same bacteria can affect the modeling effect to a certain extent. The ratio of P. gingivalis and T. denticola bacterial solution of 1:2 used in this application has the best infection effect and can further improve the success rate of modeling.

[0112] Conclusion: This invention successfully established a rat model of periodontitis. The experimental results show a synergistic effect between *T. gondii* infection and the combined infection of *P. gingivalis* and *T. denticola*. Furthermore, the model is simple to construct, has a short construction time, high reproducibility, and can be mass-produced. This model can effectively simulate the pathological process of human *P. gingivalis* + *T. denticola* with *T. gondii* infection, providing a powerful tool for studying the impact and mechanism of *T. gondii* infection on the course of periodontitis.

[0113] The above are merely embodiments of the present invention, described in a relatively specific and detailed manner, but should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for establishing a rat model of periodontitis, characterized in that, Includes the following steps: (1) Select clean-grade Sprague-Dawley (SD) rats and acclimatize them for 7 days before infection; (2) Kunming rats chronically infected with Toxoplasma gondii were sacrificed, and the whole brain was removed, ground into a homogenate and diluted with physiological saline to a dose of 100 cells / mL brain cysts to obtain the infection solution. After the rats were acclimatized for 7 days, each rat was given 1 mL of the infection solution by gavage. (3) The rats treated in step (2) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital; (4) After anesthetizing the rats, ligation was performed on the cervical region of the bilateral maxillary first molars using 3-0 silk thread, and then 1×10 9 CFU / mL *Porphyromonas gingivalis* bacterial suspension with 2×10 9 CFU / mL of Treponema pallidum bacterial suspension was inoculated into the ligature line and gingival sulcus of rat teeth at a dose of 0.1 mL / tooth; the inoculation frequency was once a day. Vaccination for 7 consecutive days; (5) Seven days later, measure and observe the alveolar bone and periodontal pockets of the rats after treatment in step (4) to determine whether the modeling was successful. The alveolar bone and periodontal pocket conditions described in step (5) are as follows: alveolar bone resorption rate ≥20% and periodontal pocket probing depth ≥1mm. If both conditions are met, it indicates that the rat model of periodontitis has been successfully established.

2. The method for establishing a rat model of periodontitis according to claim 1, characterized in that, The weight of the rats was in the range of 200±20g.

3. The method for establishing a rat model of periodontitis according to claim 1, characterized in that, The method further includes the following steps: after successful modeling, performing oral, imaging and pathological examinations on the rats treated in step (4).

4. The method for establishing a rat model of periodontitis according to claim 3, characterized in that, The procedures for the oral cavity, imaging, and pathological examinations are as follows: A1: General anesthesia was achieved by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital solution; A2: Wrap rubber bands around the incisors of the limbs and head, fix them to the mouse board and place them in a supine position; A3: After anesthetizing the rats, cut open the chest wall along the anterior axillary line on the body surface below the ribs, make a 5-6 cm lateral incision, separate the liver and diaphragm, and expose the heart after cutting. A4: Fix the ventricle, and then rotate the tip of the syringe needle at the apex of the heart and insert it into the ascending aorta 1 mm. A5: Cut open the right atrial appendage with scissors and inject 50mL of physiological saline. After the liquid flows out of the right atrial appendage and the rat's liver turns white, continue to perfuse the rat with 4% paraformaldehyde fixative solution equal to the rat's body weight. After the perfusing is completed, dissect and cut the rat's maxilla, collect periodontal tissue, and store it in 4% paraformaldehyde. A6: Micro-CT scanning and three-dimensional reconstruction of the maxillary bone specimen; A7: Use HE staining on periodontal tissue specimens to observe the pathological morphological changes in periodontal tissues.

5. The rat model obtained by the method described in any one of claims 1-4 can be used in the study of the mechanism of periodontitis.

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