Construction method and application of a mouse tendon injury model

Abstract

The application discloses a method for constructing a mouse tendon injury model and application, and particularly relates to the technical field of animal models. After low-dose levofloxacin injection and intense exercise, the low-dose levofloxacin injection and the intense exercise can be used in combination to model the mouse tendon injury. The low-dose levofloxacin injection avoids the systemic toxicity of high-dose drugs, and avoids the long cycle, high cost and low practical application value of the tendon injury induced by the intense exercise alone, can rapidly induce the injury within two weeks, and can be used to construct different injury degree models by adjusting the dosage or the exercise duration, and has a wide application range.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, specifically to a method for constructing and applying a mouse tendon injury model. Background Technology

[0002] Tendons are dense connective tissues that connect muscles and bones, primarily composed of type I collagen fibers and tendon cells. Their function is to transmit muscle contractile force and maintain joint stability. Tendon injuries (such as tendinitis, partial tendon rupture, or even complete tendon rupture) are common musculoskeletal disorders, frequently seen in athletes, manual laborers, and individuals taking certain medications long-term. They often lead to pain, impaired motor function, and severely impact patients' quality of life. Therefore, establishing stable, clinically relevant animal models of tendon injuries is crucial for studying injury mechanisms, developing repair drugs, and formulating prevention strategies.

[0003] Currently, existing methods for constructing tendon injury models include mechanical stimulation, chemical induction, and surgical trauma. Surgical trauma is an invasive procedure that can cause local inflammation, scar formation, and tendon adhesions, interfering with the pathological process of the injury itself. It also differs significantly from the pathogenesis of "non-traumatic tendon injuries" in clinical practice. Mechanical stimulation involves overusing the tendon through treadmill running or fatigue loading, inducing changes in the tendon's internal and external mechanisms to induce tendinopathy. However, this method is ineffective in the short term, has a long training cycle, high cost, and poor practicality. Chemical induction involves using drugs such as collagenase to reduce tendon collagen synthesis and increase tendon cell apoptosis, thereby inducing tendon injury. However, this method requires extremely high drug doses (far exceeding clinical drug levels), with dosages in the literature typically ranging from 300 mg / kg to 900 mg / kg, easily leading to systemic toxicity in animals (such as liver and kidney damage) and low model survival rates. Low-dose drug administration induces mild tendon injuries with a low incidence, making it difficult to establish stable moderate to severe injury models, and failing to meet the requirements for injury severity in drug screening or mechanism studies.

[0004] Levofloxacin is a broad-spectrum fluoroquinolone antibiotic, the levorotatory active isomer of ofloxacin, possessing stronger antibacterial activity and better pharmacokinetic properties. In preclinical toxicology and clinical studies, levofloxacin is recognized for its side effects of joint inflammation, muscle damage, rhabdomyolysis, and tendon rupture. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for constructing and applying a mouse tendon injury model.

[0006] In a first aspect, the present invention provides a method for constructing a mouse tendon injury model, wherein the model is induced by using quinolone drugs.

[0007] In some embodiments, the quinolone drug is one or more of ofloxacin, levofloxacin, norfloxacin, pefloxacin, enoxacin, ciprofloxacin, sparfloxacin, moxifloxacin, and gatifloxacin.

[0008] In some embodiments, the dosage of quinolone drugs administered to mice is ≥5 mg / kg.

[0009] In some embodiments, the dosage of quinolone drugs administered to mice is ≥10 mg / kg.

[0010] In some embodiments, the dosage of quinolone drugs administered to mice is 40-300 mg / kg, specifically 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 240, 260, 280, and 300.

[0011] In some embodiments, the induction period is ≥3 days, preferably ≥7 days, and more preferably 7-21 days.

[0012] In some embodiments, the induction method of the model includes an exercise training module following administration of a quinolone drug.

[0013] In some embodiments, the interval between administering quinolone drugs and exercise training is ≥10 min, preferably ≥30 min, and may specifically be 40, 50, 60, 70, 80, 90, 120, 150, or 180 min.

[0014] In some embodiments, the exercise training module includes, but is not limited to, one or a combination of swimming, climbing, running, and jumping.

[0015] In some embodiments, the exercise time is ≥5 min.

[0016] In some embodiments, the exercise time is ≥10 min, preferably 10-50 min, and more preferably 20-40 min.

[0017] In some embodiments, mouse fatigue tests and / or tendon injury scores are used to evaluate the success of the modeling.

[0018] In some embodiments, the specific steps for constructing a mouse tendon injury model are as follows:

[0019] Mice in the model group were given quinolone drugs by gavage, followed by corresponding strenuous exercise training; after training, they continued to be fed; after a period of feeding, the time spent in the rotarod experiment was measured before the end point, and the mice were euthanized at the final end point, and the tendon damage of the mice was recorded.

[0020] The present invention also provides a mouse tendon injury model, characterized in that it is constructed by the method described in any one of the above-mentioned methods.

[0021] On the other hand, the present invention provides the application of a mouse tendon injury disease model in any of the following:

[0022] (1) Research on the pathogenesis of tendinopathy;

[0023] (2) Screening and / or preparing medicines and health products for the prevention and / or treatment of musculoskeletal diseases related to tendon injury.

[0024] In some embodiments, the tendon injury includes, but is not limited to, rotator cuff injury, patellar tendon injury, Achilles tendon injury, and flexor tendon injury.

[0025] In some embodiments, the tendon injury induction model can be other rodents such as mice, rats, or rabbits.

[0026] Beneficial effects:

[0027] 1. Low-dose levofloxacin can inhibit collagen synthesis and tendon cell apoptosis, making tendons vulnerable and reducing their ability to repair subsequent injuries. If strenuous exercise is performed on this basis, on the one hand, cyclic stress is generated on the tendons, and the tendons are easily torn in a vulnerable state. Furthermore, the tendon cell repair ability has been inhibited by the drug, so damage accumulates rapidly within 30 minutes.

[0028] In addition, low-dose levofloxacin can activate matrix metalloproteinases (MMP-1, MMP-3). Strenuous exercise increases anaerobic respiration in tendon cells, leading to a large accumulation of lactic acid. The acidic environment further activates matrix metalloproteinases, which in turn reduces collagen fiber synthesis and decreases tensile strength.

[0029] The peak absorption period of the drug is 1-2 hours after administration. At this time, tendon cells are in a state of functional inhibition, and exercise load can maximize the damage effect.

[0030] 2. Low doses avoid the systemic toxicity of high-dose drugs and can specifically weaken tendons; the combination of the two can avoid the disadvantages of long cycle, high cost and low practical application value of tendon injury induced by strenuous exercise alone. It can rapidly induce injury within two weeks, and different injury models can be constructed by adjusting the dosage or exercise duration, making it widely applicable. Attached Figure Description

[0031] Figure 1 These are schematic diagrams of mouse swimming training (left) and mouse pole climbing training (right).

[0032] Figure 2This is a schematic diagram of a mouse rotarod fatigue test experiment.

[0033] Figure 3 This is a table recording the weight of mice.

[0034] Figure 4 This is a record of the time when the mouse fell onto the stick.

[0035] Figure 5 This is a gross diagram of the gastrocnemius muscle and tendon of a mouse.

[0036] Figure 6 Anatomical diagram of the gastrocnemius muscle and tendon in a mouse. Detailed Implementation

[0037] 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.

[0038] Example 1

[0039] 1. Experimental reagents and consumables

[0040] 1.1 Laboratory Animals

[0041] Thirty SPF-grade male ICR mice, 6 weeks old, weighing 25±2g, were used.

[0042] 1.2 Major Instruments and Consumables

[0043] Pole climbing device; rotating bar fatigue tester (Shanghai Xinruan, XR-6C); surgical instruments.

[0044] Levofloxacin tablets (Nanjing Zhengke), 4% paraformaldehyde.

[0045] 2. Experimental Procedure

[0046] 2.1 Experimental Preparation

[0047] After receiving the experimental mice, they were acclimatized for one week before the formal experiment began. All mice 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 in alternating light and dark conditions for 12 hours, and the mice had free access to food and water.

[0048] 2.2 Animal Grouping

[0049] The experimental mice were randomly divided into 6 groups according to Table 1 below: a normal group (n=5), a single-drug group (n=5), a swimming group (n=5), a drug-drug + swimming group (n=5), a pole-climbing group (n=5), and a drug-drug + pole-climbing group (n=5).

[0050] Table 1

[0051] Group quantity Dosage time Dosage Training methods normal group 5 / / / Single-drug group 5 14 days 65 mg / kg / Swimming Group 5 14 days / Swim for 30 minutes Drug administration + swimming group 5 14 days 65 mg / kg Swim for 30 minutes Pole climbing group 5 14 days / 30 minutes of pole climbing Drug administration + pole climbing group 5 14 days 65 mg / kg 30 minutes of pole climbing

[0052] 2.3 Model Construction

[0053] 2.3.1 Drug administration model

[0054] (1) Mice in the normal group, swimming group and pole climbing group were not given gavage.

[0055] (2) Mice in the single-drug group, the drug-drug + swimming group, and the drug-drug + pole climbing group were administered levofloxacin solution by gavage at a dose of 65 mg / kg.

[0056] (3) After the administration of the drug, put the mouse back into the cage and feed it normally.

[0057] 2.3.2 Training and Model Building

[0058] (1) Training and modeling began 1 hour after the mice were given oral gavage.

[0059] (2) Mice in the swimming group and the drug-treated + swimming group were placed in a 30℃ water tank for free swimming training for 30 min. When the mice floated still, they were gently nudged with a net to continue swimming, as follows. Figure 1 Left.

[0060] (3) Mice in the pole climbing group and the drug-treated + pole climbing group were placed at the top of the pole climbing device, and their turning and reaching the bottom of the pole were observed. After the mice reached the bottom of the pole, they were placed back at the top, and the training continued for 30 minutes, as follows. Figure 1 right.

[0061] 2.4 Data collection, endpoint sampling, and indicator testing

[0062] 2.4.1 Process Data Collection

[0063] (1) Mouse body weight observation:

[0064] (2) Weigh and record the weight of the mice twice a week.

[0065] Mouse rotarod fatigue test:

[0066] The fatigue test of the rotating bar began 3 days before the final sampling point, as follows: Figure 2 On day 1, mice were placed on a rotarod fatigue tester to adapt. On day 2, mice were placed on a rotarod fatigue tester to adapt at a low speed. On day 3, mice were placed on a rotarod fatigue tester to start the accelerated test. Each mouse was measured 3 times with a measurement interval of 15 minutes. The fall time of the mice was collected and the average fall time was calculated.

[0067] (3) Tendon injury score in mice:

[0068] The degree of tendon injury in each group of mice was scored according to Table 2 below.

[0069] Table 2

[0070] 0 degrees No damage, normal condition. Grade I (Mild) Mild muscle fiber damage, no loss of strength, mild pain. Grade II (Moderate) Some tendon fibers were ruptured, resulting in significant pain, decreased strength, and mild functional impairment. Grade III (Severe) Complete fracture, severe pain, obvious deformity, and complete loss of function.

[0071] 2.4.2 Sampling at the endpoint

[0072] Fifteen days after the mouse model was established, the mice in each group were euthanized, and the right gastrocnemius muscle and tendon of the mice were harvested and photographed for record-keeping.

[0073] 2.4.3 Data Processing and Result Determination

[0074] 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.

[0075] 3 Results Analysis

[0076] 3.1 Changes in mouse body weight

[0077] as follows Figure 3 As shown, the body weight of mice in each group continued to increase 15 days after modeling, and there was no significant difference between the groups.

[0078] 3.2 Mouse rotarod experiment

[0079] as follows Figure 4 As shown, compared with the normal group, there were no significant differences in the landing time of mice in the single-drug group, swimming group, and pole-climbing group. Compared with the normal group, the landing time of mice in the drug-treated + swimming group and the drug-treated + pole-climbing group was significantly reduced, with the drug-treated + pole-climbing group showing the greatest reduction in landing time.

[0080] 3.3 Tendon Injury Scoring in Mice

[0081] Table 3 shows the tendon damage in mice. Swimming and pole climbing training alone did not affect the mice's athletic performance, and the tendons remained normal. Levofloxacin administration alone without training had little impact on athletic performance and had no significant effect on the gastrocnemius muscle and tendons. Administration of levofloxacin combined with swimming and administration of levofloxacin combined with pole climbing significantly reduced the mice's athletic ability and had a greater impact on the gastrocnemius muscle and tendons, with the most significant tendon damage observed in the administration of levofloxacin combined with pole climbing group.

[0082] Table 3

[0083] Group: Degree of damage: Damage manifestations: normal group 0 degrees (none) No damage, normal condition. Single-drug group 0 degrees (none) No damage, normal condition. Swimming Group 0 degrees (none) No damage, normal condition. Drug administration + swimming group Grade I (Mild) Mild muscle fiber damage, no loss of strength, mild pain. Pole climbing group 0 degrees (none) No damage, normal condition. Drug administration + pole climbing group Grade II (Moderate) Some tendon fibers were ruptured, resulting in significant pain, decreased strength, and mild functional impairment.

[0084] 3.4 Gross diagram of mouse tendon

[0085] as follows Figure 5 and Figure 6As shown, the gastrocnemius muscles of mice in the normal group had normal color and intact tendons. In the single-drug group, the gastrocnemius muscles of mice showed slightly pale color and slightly shortened tendons. The gastrocnemius muscles of mice in the swimming and pole-climbing groups also showed slightly pale color, but the tendons were normal. In the drug-treated + swimming group, the tendons of mice were dark in color, with mild congestion of the small saphenous vein, short and thick tendons, a rough surface, and a loss of luster. In the drug-treated + pole-climbing group, the tendons were bright red, with obvious congestion of the small saphenous vein, short and thick tendons, swelling, congestion, and adhesions.

[0086] Example 2

[0087] As shown in Table 4 below, changing the administration time, dosage, or training method and time can still successfully induce a mouse tendon injury model.

[0088] Table 4

[0089] Group quantity Dosage time Dosage Training methods Drug administration + pole climbing group 5 14 days 50 mg / kg 40 minutes of pole climbing Drug administration + pole climbing group 5 14 days 80 mg / kg 20 minutes of pole climbing Drug administration + pole climbing group 5 21 days 50 mg / kg 20 minutes of pole climbing Drug administration + pole climbing group 5 7 days 80 mg / kg 40 minutes of pole climbing Drug administration + swimming group 5 14 days 50 mg / kg Swim for 40 minutes Drug administration + swimming group 5 14 days 80 mg / kg Swim for 20 minutes Drug administration + swimming group 5 21 days 50 mg / kg Swim for 20 minutes Drug administration + swimming group 5 7 days 80 mg / kg Swim for 40 minutes

[0090] 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 mouse tendon injury model, characterized in that, The model was induced using quinolone drugs.

2. The construction method according to claim 1, characterized in that, Quinolone drugs are one or more of ofloxacin, levofloxacin, norfloxacin, pefloxacin, enoxacin, ciprofloxacin, sparfloxacin, moxifloxacin, and gatifloxacin.

3. The construction method according to claim 1, characterized in that, The dosage of quinolone drugs in mice is ≥5 mg / kg.

4. The construction method according to claim 1, characterized in that, The induction period is ≥3 days.

5. The construction method according to claim 1, characterized in that, The model was induced by administering quinolone drugs followed by an exercise training module.

6. The construction method according to claim 5, characterized in that, The interval between administering quinolone medications and exercise training should be ≥10 minutes.

7. The construction method according to claim 5, characterized in that, The sports training module includes one or more of the following: swimming, climbing, running, and jumping.

8. The construction method according to claim 5, characterized in that, The exercise time is ≥5 minutes.

9. The construction method according to any one of claims 1-8, characterized in that, The success of the model was evaluated using mouse fatigue tests and / or tendon injury scores.

10. A mouse tendon injury model, characterized in that, It is constructed by the method described in any one of claims 1-9.

11. The use of the mouse tendon injury disease model of claim 10 in any of the following: (1) Research on the pathogenesis of tendon injury; (2) Screening and / or preparing drugs and health products for the prevention and / or treatment of tendon injury-related tendinopathy.

12. The application according to claim 11, characterized in that, The tendon injuries include rotator cuff injuries, patellar tendon injuries, Achilles tendon injuries, or flexor tendon injuries.

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