Establishment method of vertebral artery type cervical spondylosis animal model

Abstract

The invention provides a vertebral artery type cervical spondylosis animal model building method, and belongs to the technical field of medical experiment animal models, and the method comprises the following steps: S1, experiment animal pretreatment: carrying out anesthesia treatment on an experiment rat, and then preparing skin on the neck; s2, molding the experimental animal by injecting a molding agent, namely injecting the molding agent by taking the cervical vertebra C2 transverse process level of the rat as a needle insertion point; and S3, performing model evaluation. According to the method, a traditional hardening agent molding method is optimized and improved, the problems existing in the prior art are solved, and a more reliable experimental basis is provided for CSA research in the aspects of accuracy, safety, stability, reliability and clinics.

Description

Technical Field

[0001] The present invention relates to the establishment of an animal model, in particular to a method for establishing an animal model of cervical spondylosis, and belongs to the technical field of medical experimental animal models. Background Art

[0002] In recent years, the incidence of cervical spondylosis (CS) has been increasing annually, with approximately 900 million patients worldwide and approximately 200 million in China. Cervical spondylosis affects 10%-15% of adults, with an incidence of 80% in those over 40 years old. Among Chinese adolescents, the incidence is over 10% and continues to increase annually, showing a significant trend toward younger age groups. This presents a heavy financial and emotional burden on individuals, families, and society.

[0003] Vertebral artery type cervical spondylosis (CSA), a common type of cervical spondylosis, accounts for approximately 10%-15% of cervical spondylosis cases and has an overall incidence of approximately 17.3%. It has garnered significant attention in recent years. Its pathogenesis is complex and primarily encompasses the following: 1) Cervical mechanical imbalance: Disturbances in the dynamic and static balance of the cervical spine and imbalanced forces on the paravertebral muscles lead to altered cervical curvature, resulting in straightening, lateral curvature, or even retroflexion. This further causes traction, twisting, and compression of the vertebral arteries, leading to vertebral artery spasm and reduced blood supply. 2) Vertebral artery compression leads to insufficient vertebral-basilar artery blood supply, altered regional cerebral blood flow, and chronic ischemia and hypoxia in the corresponding brain regions, causing functional impairments and, in turn, a range of clinical symptoms, including insomnia, headache, dizziness, nausea, vomiting, tinnitus, blurred vision, memory loss, digestive and cardiovascular problems. An increasing number of studies confirm that cervical mechanical imbalance is a key factor in the development of CSA, particularly in young and middle-aged patients. A considerable number of patients suffer from transient hypertension, palpitations, chest pain and other cardiovascular symptoms caused by vertebral artery spasm. This is often due to the continuous tension of muscles caused by activities or static postures for too long (such as sitting with the head down for a long time), which in turn stimulates the abundant sympathetic nerve fibers on and around the vertebral artery wall and induces a vasoconstriction effect.

[0004] With the rapid development of the global economy and the profound changes in people's lifestyles, the incidence of CSA has shown a significant upward trend, and the age of onset is also becoming younger. In modern society, bad habits such as long hours of desk work and excessive use of electronic devices are extremely common, causing the cervical spine to be in a non-physiological posture for a long time, which greatly increases the risk of cervical spine lesions. Relevant studies have shown that in some specific occupational groups, such as office workers, IT practitioners, and students, the prevalence of CSA is much higher than that of the general population. This disease not only seriously affects the patient's physical health, but also has a negative impact on their quality of life and work efficiency, such as reduced work concentration, memory loss, fatigue, mood swings, etc. Long-term recurring attacks can easily lead to ischemic brain dysfunction, cardiovascular and cerebrovascular diseases (such as hypertension, coronary heart disease, cerebral embolism), and psychological disorders such as anxiety and depression, accelerating aging.

[0005] Currently, clinical treatment strategies for CSA include physical therapy, Chinese and Western medicine, various minimally invasive interventional treatments, and surgical treatments. However, the results are often unsatisfactory, and the recurrence rate is high, which is closely related to the lack of understanding of the disease, posing a huge challenge to clinical treatment.

[0006] Therefore, in-depth research on the pathogenesis of CSA is of great strategic significance for a deeper understanding of the disease, as well as the research and development of drug and non-drug interventions.

[0007] Currently, sclerotherapy is one of the methods used to establish animal models of CSA in animal experimental research. Its mechanism of action is as follows: Local injection of a sclerotherapy agent into the animal's cervical spine can cause mechanical imbalance in the cervical spine and compression of the vertebral artery, thereby simulating the pathological state of vertebral artery-type cervical spondylosis. Xiaozhiling Injection, a common sclerotherapy agent, whose main ingredients include gallnut and alum, triggers an inflammatory response and vasoconstriction through local stimulation, causing sclerosis of the surrounding soft tissues, resulting in compression and irritation. Because the rat's cervical spine anatomy is similar to that of humans, and with the explosive increase in CSA cases, the research and application of rats as models of vertebral artery-type cervical spondylosis has become increasingly urgent in recent years. Traditionally, the rat CSA sclerotherapy model is established by injecting 2 ml of Xiaozhiling Injection into the left C3-C5 transverse processes and surrounding tissues after successful anesthesia. The same dose of drug is then repeated once in the second week. The latest version of the vertebral artery-type cervical spondylosis modeling method in 2023, the "modified sclerosant modeling method," has an improved dosage of 1 ml / 100 g. However, based on the rat weight of approximately 200 g to 250 g used in the literature, the actual dosage of the drug has not been reduced, remaining approximately 2 ml to 2.5 ml. The injection site is between the left C3-C5 transverse processes and the surrounding tissues. However, numerous studies have found that this modeling method has significant limitations and is not as safe as claimed in the literature. The actual mortality rate of modeled animals is high, and rats are very small, with complex and delicate cervical anatomy. The blind exploration process is difficult and accuracy is difficult to guarantee. This has brought many inconveniences to related experimental research and limited in-depth exploration of the pathogenesis of CSA and the development of treatments.

[0008] The limitations of traditional methods are analyzed as follows:

[0009] (1) According to the dosage in the above literature, 2.5 ml of Xiaozhiling was injected into the left side of the neck of 250 g SD rats. The injection was repeated once a week later. The drug injection dose was obviously too large, resulting in a mortality rate of about 30% of the rats. The surviving rats had a poor subsequent living condition, with significant weight loss, emaciation, severe atrophy of the bilateral neck muscles, curled up bodies, and slow reactions (such as Figure 1 This model does not conform to the clinical manifestations of chronic vertebral artery type cervical spondylosis;

[0010] (2) Traditional modeling is done by blind feel based on experience. However, rats are small and have a cramped cervical vertebrae. In practice, it is difficult to accurately locate the transverse process by touch, which can easily lead to misplacement of the spinal canal, blood vessels, or injection site deviation, resulting in animal death or model failure.

[0011] (3) Simultaneous injection of large amounts of Xiaozhiling into three transverse processes (C3-C5) that are only a few mm in size is highly irritating, causes great damage, and is time-consuming. It significantly increases the demand for anesthetics and increases the risk of death from anesthesia.

[0012] (4) It is difficult to accurately inject the drug solution into the soft tissue near the transverse process of the vertebral artery or around the transverse foramen during the blind puncture modeling process to achieve the best modeling effect. Rough injection can lead to two consequences: a) The vertebral artery and vein, jugular artery and vein, spinal cord and other important tissue structures (measured in mm) run through this area. If the blind puncture is not careful, the blood vessels and spinal cord may be damaged or the drug solution may be injected into the blood vessels and spinal cord, causing model failure, animal disability or death on the spot. b) Rough shallow injection will result in insufficient or deviation of the drug solution from the target, affecting the modeling effect or causing the modeling to be ineffective, and the homogeneity is significantly insufficient.

[0013] (5) The evaluation criteria for successful modeling of traditional models are not sufficient, and there is a lack of direct imaging indicators (such as MRI, MRA, musculoskeletal ultrasound, elastic ultrasound) and pathological results to support it. Moreover, there is a lack of further evaluation of the long-term effectiveness of the model. Therefore, the model standards are not rigorous enough, there is a lack of model stability demonstration, and the chronic disease characteristics of CSA cannot be well replicated.

[0014] Therefore, in order to further study the pathogenesis of CSA and develop more effective treatments and effective drugs, it is imperative to prepare a safer, more accurate, reliable CSA animal model that conforms to the clinical characteristics of chronic cervical spondylosis. Summary of the Invention

[0015] In response to the above problems, the present invention provides a method for establishing an animal model of vertebral artery type cervical spondylosis to improve the stability and reliability of the model.

[0016] To achieve the above-mentioned object, the technical solution of the present invention is: a method for establishing an animal model of vertebral artery type cervical spondylosis, comprising the following steps:

[0017] Step S1, experimental animal pretreatment: anesthetize the experimental rats, and then prepare the skin of the neck;

[0018] Step S2, injecting a modeling agent into the experimental animal to establish a model: the modeling agent is injected at the level of the C2 transverse process of the rat cervical vertebra;

[0019] Step S3: model evaluation.

[0020] Furthermore, during the anesthesia treatment in step S1, sevoflurane with an inhalation concentration of 2-5% is used to induce anesthesia and maintain it during the operation; and dexmedetomidine is used for intraperitoneal injection to maintain anesthesia, and the amount of the dexmedetomidine used is 0.08 μg per gram of rat body weight.

[0021] Furthermore, in step S1, after anesthesia, the rat is placed prone on the operating table, and the hair on the back and sides of the neck is cleaned.

[0022] Furthermore, in step S2, ultrasound-guided injection of a modeling agent is used to create a model. Specifically, an ultrasound diagnostic apparatus is used to first detect and record the blood flow parameters of the rat's vertebral artery. Then, the rat is placed in a prone position and ultrasound is used to locate the vertebral artery at the C2 level. Then, 0.1 ml of 0.5% lidocaine is used as the insertion point at the level of the C2 transverse process under ultrasound guidance to perform epidermal local anesthesia and analgesia. Then, an empty needle is punctured at the C2 level under ultrasound guidance until the C2 level is only 2-3 mm1 away from the vertebral artery. No blood is aspirated and the modeling agent is injected.

[0023] Furthermore, the modeling agent was Xiaozhiling stock solution, and there were four injection sites, specifically: 0.2 ml of Xiaozhiling stock solution was injected at the C2 level of the rat, only 2-3 mm away from the vertebral artery; 0.3 ml of Xiaozhiling stock solution was injected into the middle and upper segments of the erector spinae muscle on the ipsilateral side; and 0.2 ml of Xiaozhiling stock solution was injected into the longus colli muscle below the C2 vertebral artery on the ipsilateral side.

[0024] Furthermore, the modeling agent was injected once a week for three consecutive times, followed by a one-month observation and rest period, and the total modeling period was 6 weeks.

[0025] Furthermore, step S3 model evaluation includes collecting the following data before modeling, one week after the second modeling, at the completion of the modeling, and six months after modeling: musculoskeletal ultrasound images, SWE images and elastic hardness data of the erector spinae muscles, vertebral blood vessels, and longus colli muscles, vertebral artery hemodynamic indicators, MRI and vertebral artery MRA images of the whole brain and neck C1-C3 levels, open field behavioral experiments, and muscle pathology detection in the neck modeling area.

[0026] Furthermore, the evaluation indicators for successful model building include the following:

[0027] Neck ultrasound results: Inhomogeneous cord-like high echo shadows were observed around the erector spinae muscle, longus colli muscle and transverse process of the modeling side near the vertebral artery.

[0028] Ultrasound elastography results showed that the shear wave velocity (Cs) near the erector spinae muscle, longus colli muscle and transverse process of the model side was significantly increased compared with the control side, indicating increased tissue hardness.

[0029] Vertebral artery ultrasound blood flow measurement: blood velocity and blood volume showed a significant downward trend during modeling and 6 months after modeling, while pulsatility index and vascular resistance showed a significant upward trend;

[0030] In terms of the correlation between the model's soft tissue elasticity and vertebral artery blood flow, the vascular velocity and blood flow around the vertebral artery were negatively correlated with the elasticity and hardness of the surrounding soft tissue, while the vascular resistance and vascular index were positively correlated with the elasticity and hardness of the surrounding soft tissue.

[0031] MRI of soft tissue of neck: After modeling, T1 and T2 images showed cord-like abnormal signal shadows around the vertebral arteries at the C1-C3 level and around the neck muscles at the corresponding level;

[0032] Intracranial magnetic resonance imaging: No abnormalities were found in the whole brain MRI scans of rats in the control group and the model group;

[0033] Vertebral artery MRA-TOF blood flow signal measurement: There was no significant difference in the MRA signal intensity of the left and right vertebral arteries in the control group rats, while the signal intensity of the vertebral artery on the model side in the model group was significantly lower than that on the ipsilateral control group and the control side of the model group 6 months after modeling;

[0034] Open field test: The model group showed anxiety-like emotions, and the total movement distance and average speed, as well as the distance visited in the central area and the average speed were significantly reduced;

[0035] Pathological HE staining: significant differences were observed between the control group and the model after modeling. The muscle cells in the control group were densely arranged, the distance between the muscle bundles was compact, and there were no excessive inflammatory cells and collagen deposition; while the muscle cells in the model group were degenerated, the distance between cells was loose, more collagen and inflammatory cell infiltration appeared between the muscle bundles, and the structure was disordered and irregular.

[0036] The beneficial effects of the method for establishing an animal model of vertebral artery type cervical spondylosis of the present invention are:

[0037] The present invention optimizes and improves the traditional sclerosing agent modeling method, solves the problems existing in the prior art, and provides a more reliable experimental basis for the research of CSA from the aspects of accuracy, safety, stability, reliability and closeness to clinical practice.

[0038] The present invention uses musculoskeletal ultrasound technology to guide puncture for the first time, accurately injecting the modeling drug into the target under ultrasound visualization, effectively reducing the risk of puncture, and can dynamically observe and evaluate the ultrasound images, shear wave elastography and blood flow changes of the vertebral artery and its surrounding muscle and soft tissue before and after injection. Musculoskeletal ultrasound (MSKUS) refers to the use of high-frequency or low-frequency ultrasound probes to scan tendons, ligaments, muscles, fascia, nerves, blood vessels, joints and other related tissues to diagnose related diseases, such as tendon inflammation, ligament tears, peripheral nerve and blood vessel compression, muscle strain, fascia inflammation and joint swelling and degeneration. Shear wave elastography (SWE) is an advanced ultrasound imaging technology known as "precision electronic palpation technology". It generates shear waves in tissues by emitting acoustic radiation pulses, and converts the propagation speed of shear waves into corresponding tissue hardness according to the different propagation speeds of shear waves in tissues of different hardness, thereby performing imaging. This technology can perform elastic imaging based on two-dimensional images, providing richer and clearer diagnostic information than conventional ultrasound and color Doppler ultrasound.

[0039] The present invention locates the injection point around the level of the C2 vertebral artery, and has 4 clear points for precise injection (around the C2 vertebral artery), focusing the affected range of the vertebral artery on the C1-C3 stage, that is, the V2 end and V3 segment. Specifically: 1) All injection points are performed at the C2 level (naturally diffused to the C1-C3 range, which has been verified by nuclear magnetic resonance), reducing the affected stage and operation time of the model injection; 2) Ultrasound-guided 4-point dispersed injection beside the vertebral artery improves accuracy while reducing drug dosage, avoiding toxic side effects caused by over-concentrated injection, greatly improving the survival rate and postoperative quality of life of the model rats, making it more in line with the characteristics of clinical chronic diseases.

[0040] The present invention has made a significant improvement to the traditional rat vertebral artery type cervical spondylosis sclerosing agent (modeling agent) modeling method, and the model is more focused on imitating the vertebral artery type cervical spondylosis with sympathetic nerve symptoms that are characteristic of young and middle-aged people.

[0041] Based on the traditional method, the present invention adds shear wave imaging elasticity indicators to guide modeling and model evaluation; adds neck MRI, musculoskeletal ultrasound and cervical muscle pathology to evaluate cervical muscle lesions; adds MRI MRA-TOF blood flow imaging to evaluate long-term vertebral artery blood flow after modeling, and evaluates the long-term stability and effectiveness of the model (in line with the chronic disease characteristics of vertebral artery cervical spondylosis).

[0042] The present invention is the first to establish a vertebral artery type cervical spondylosis model with the assistance of full-process ultrasound guidance and shear wave elastography technology; for the first time, 60% of the total injection dose of traditional drugs was used to successfully and efficiently establish the model, and the single modeling dose was reduced by 60%, which is currently the lowest dose for successful and long-term modeling of this model; a single-level injection around the C2 vertebral artery is used to focus the lesion site more on the core area, making the injection simpler and easier; precise fixed-point and standardized doses are used for injection around the vertebral artery, making the model establishment standardized and more reproducible; the injection position is changed to a prone position, the ultrasound guidance field of view is wider, and the operation space for ultrasound-guided injection is larger and more convenient.

[0043] The present invention combines for the first time multiple verification technologies including musculoskeletal, vascular, elastic ultrasound, nuclear magnetic resonance (structural images, 3D imaging of the intracranial segment of the vertebral artery and blood flow assessment), open field behavior and pathology to evaluate the model establishment in a more comprehensive and objective manner. Shear wave elastic imaging is used to dynamically record the changes in the fixed-point elasticity index of the tissue surrounding the vertebral artery of rats before and after modeling. Analysis shows that the elasticity value of the tissue beside the vertebral artery (root and below) is significantly correlated with the vertebral artery blood flow. This result makes shear wave elastic imaging a simpler and non-invasive assessment indicator of whether the model is successful during the modeling cycle, and provides a new non-invasive imaging assessment method for clinical and basic experiments. For the first time, nuclear magnetic resonance MRA-TOF vertebral artery blood flow imaging, ultrasonic vertebral artery blood flow measurement and shear wave elasticity are used to evaluate the long-term stability of the model after modeling, confirming that the model is stable in the long term and meets the chronic characteristics of vertebral artery cervical spondylosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Figure 1 It is a comparison of the rat states of the traditional animal model and the animal model of the present invention;

[0046] Figure 2 It is the anatomy and course of the human vertebral artery;

[0047] Figure 3 is the anatomy and course of the rat vertebral artery;

[0048] Figure 4 It is the anatomy of the vertebral artery in rats and humans;

[0049] Figure 5 The rat skin preparation before modeling in the embodiment of the present invention;

[0050] Figure 6 This is an embodiment of the present invention's ultrasound-guided paravertebral artery injection of Xiaozhiling;

[0051] Figure 7 This is a cross-section of the rat cervical vertebra C2 after the rat muscle-bone ultrasound modeling in the embodiment of the present invention;

[0052] Figure 8 This is the dietary status of rats after modeling in the embodiment of the present invention;

[0053] Figure 9 These are the musculoskeletal ultrasound images of the control group and the model group at various time points in the embodiment of the present invention;

[0054] Figure 10 This is a comparison of the nuclear magnetic resonance and ultrasound effects of the rat neck after modeling in the embodiment of the present invention;

[0055] Figures 11A-11BComparison of elasticity around the vertebral artery before and after modeling in the embodiment of the present invention, where detection location A is above the vertebral artery, B is the root of the vertebral artery, C is the outside of the vertebral artery, and D is below the vertebral artery; Figure 11B for Figure 11A A zoomed-in view of the histogram;

[0056] Figures 12A-12B This is the ultrasonic blood flow measurement of the vertebral artery before and after modeling in the embodiment of the present invention, wherein:

[0057] A: Schematic diagram of vertebral artery ultrasound color Doppler detection.

[0058] B: Two-dimensional ultrasound and color Doppler ultrasound images of the vertebral artery and its surrounding structures.

[0059] C: Example of vertebral artery color Doppler data acquisition,

[0060] D: Comparison of TAMAX and TAMEAN on the left and right sides of each group.

[0061] E: Comparison of PS and ED on the left and right sides of each group.

[0062] F: Comparison of PI and RI between the left and right sides of each group;

[0063] Figures 13A-13E The correlation between soft tissue elasticity and vertebral artery blood flow after modeling in the embodiment of the present invention; wherein, Figure 13B This is an enlarged view of the upper left image of 13A. Figure 13C 13A is an enlarged view from the above figure. Figure 13D For 13A, see the enlarged image below. Figure 13E This is an enlarged view of the lower left image of 13A;

[0064] Figure 14 This is the MRI of the soft tissue of the neck after modeling in an embodiment of the present invention;

[0065] Figure 15 This is a head MRI image according to an embodiment of the present invention;

[0066] Figure 16 This is the measurement of vertebral artery intracerebral blood flow signal according to an embodiment of the present invention, wherein:

[0067] A: Long-term (6 months) MRA measurement of bilateral vertebral artery intracerebral signals in the model group.

[0068] B: MRA measurement of the intracerebral signals of bilateral vertebral arteries in the control group.

[0069] C: a) Comparison of the left vertebral artery intracerebral blood flow signals in the control group and the model group in the long term (6 months).

[0070] b) Comparison of intracerebral blood flow signals of the left and right vertebral arteries in the model group in the long term (6 months).

[0071] c) Comparison of intracerebral blood flow signals of the right vertebral artery in the control group and the model group in the long term (6 months).

[0072] d) Comparison of intracerebral blood flow signals between the left and right vertebral arteries in the control group;

[0073] Figures 17A-17B This is an open field experiment according to an embodiment of the present invention, wherein Figure 17B yes Figure 17A Enlarged view of the bar graph on the right;

[0074] Figure 18 This is the HE staining of rat pathology in the embodiment of the present invention. DETAILED DESCRIPTION

[0075] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0076] Anatomy and course characteristics of human vertebral artery: combined Figure 2 As shown, the vertebral artery is divided into four stages, V1-V4. The V2 segment of the vertebral artery is also called the intervertebral foramen segment or the transverse process segment. It starts from the point where the vertebral artery enters the transverse foramen of the 6th cervical vertebra and runs upward to the transverse foramen of the axis C2 (C2-C6, about 5 cervical vertebrae). The curved course of the V3 segment of the vertebral artery after passing through the transverse foramen: The V3 segment of the vertebral artery (C1, 1 cervical vertebrae), after passing through the C2 transverse foramen, first runs posteriorly and laterally, bypasses the lateral mass of the atlas, and then turns upward and runs along the vertebral artery groove above the posterior arch of the atlas, forming an obvious curve. Combined with Figure 3 and Figure 4 As shown, the anatomy of the rat cervical spine and vertebral artery is similar to that of humans.

[0077] Example 1

[0078] A method for establishing an animal model of vertebral artery type cervical spondylosis mainly includes three parts: pretreatment of experimental animals, injection of modeling agent, and model evaluation, and specifically includes the following steps:

[0079] (1) Experimental animals

[0080] Thirty-six healthy SPF male Sprague-Dawley rats, 8 weeks old, weighing (250 ± 20) g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. They were housed at room temperature (25 ± 1)°C and humidity (45%–50%). The experiment was approved by the Experimental Animal Ethics Committee of Tianjin Medical University Cancer Hospital (AE-202501003). The animals were brought to the Tianjin Medical University Cancer Hospital laboratory and fed for 2 weeks to a weight of (250 ± 20) g before modeling.

[0081] (2) Experimental drugs and equipment

[0082] Modeling agent: Xiaozhiling injection (10 ml, 0.4 g, Jilin Province Ji'an Yisheng Pharmaceutical Co., Ltd., batch number: Z22026175)

[0083] Sevoflurane (120 ml, Shanghai Hengrui Medicine Co., Ltd., national medicine standard number H20213735).

[0084] Dexmedetomidine hydrochloride injection (2 ml, 0.2 mg, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., batch number: 24083131)

[0085] Lidocaine hydrochloride injection (5 ml, 0.1 g, Shandong Hualu Pharmaceutical Co., Ltd., National Medicine Approval No. H37022147)

[0086] 0.9% sodium chloride injection (250ml, 2.25g, Otsuka Pharmaceutical Co., Ltd., China, National Medicine Standard No. H12020025)

[0087] (3) Experimental methods

[0088] Pretreatment of experimental rats: 36 SPF-grade SD male rats were randomly divided into a control group and a model group (n=18). Ultrasound-guided modeling was used to establish the CSA rat model. Sevoflurane (2%-5%) was used for induction of anesthesia and maintenance during surgery, and dexmedetomidine 0.08ug / g was injected intraperitoneally to maintain anesthesia. After satisfactory anesthesia, the rats were placed prone on the operating table, and the hair on the back and sides of the neck was cleaned with pet electric clippers ( Figure 5 ).

[0089] Ultrasound-guided modeling: Figure 6 and Figure 7As shown, rats were first positioned in the left lateral decubitus position. A Mindray Resona 19w ultrasound system with an L20-5s high-frequency linear array probe was placed longitudinally along the body, midway between the two transverse processes, and a long-axis scan was performed. The left vertebral artery at the level of C2 (between the two transverse processes) was visualized. Vertebral artery blood flow parameters (PS, ED, TAMAX, TAMEAN, PI, and RI) were then measured and recorded. The same procedure was repeated on the right side to measure right vertebral artery blood flow parameters. The rats were then positioned in the prone position, and a short-axis scan with the L20-5s high-frequency probe was performed midway along the dorsal aspect of C2. A prominently elevated C2 spinous process was visualized as a landmark. Musculoskeletal ultrasound revealed anatomical structures such as the bilateral erector spinae muscles, bilateral vertebral arteries, bilateral carotid arteries, and the trapezius and semispinalis capitis muscles. After locating the vertebral artery at the C2 level, 0.1 ml of 0.5% lidocaine hydrochloride was used under ultrasound guidance at the level of the C2 transverse process on the left side (modeling side) for epidermal local anesthesia and analgesia. Then, a 1 ml 30GX25 mm empty needle was used under ultrasound guidance to puncture at the C2 level, and the distance from the C2 level to the vertebral artery was only 2-3 mml. No blood was aspirated, and 0.2 ml of Xiaozhiling stock solution was injected. 0.3 ml of Xiaozhiling stock solution was injected into the middle and upper segments of the left erector spinae muscle respectively, and 0.2 ml of Xiaozhiling stock solution was injected into the longus colli muscle below the left C2 vertebral artery, for a total of 1 ml ( Figure 5 ).

[0090] Detailed explanation of injection dosage (taking 250g rat as an example):

[0091] 1. 0.2ml at the root of the vertebral artery.

[0092] 2. Erector spinae muscles 0.6ml, longus colli muscles 0.2ml.

[0093] The single dose of Xiaozhiling is 0.4ml / 100g, compared to the traditional dose of 1ml / 100g. Compared with the traditional dose, the single single-site injection dose is reduced by 70%, the total single dose is reduced by 60%, and the total drug dose used in the modeling is reduced by 40%.

[0094] After a successful injection, remove the needle and press the puncture site. Observe the rats for abnormal breathing and limb movement for 5 minutes, perform elastic ultrasound and cervical C2 level vascular ultrasound at the corresponding time points, and observe changes before and after the intervention. Observe vital signs for 5 minutes after the injection if there are no abnormal signs. Return the rats to their cages for rest and continue to be closely observed for 30 minutes. Record the number of rats successfully injected and the time required. Record complications such as limited upper and lower limb movement, cardiac arrest, respiratory distress, severe hematoma, and significant weight loss. The operation usually takes 60-120 seconds to complete.

[0095] (IV) Modeling cycle and frequency

[0096] Once a week for three times in a row. After three times, observe and rest for one month. The total modeling period is 6 weeks.

[0097] Model success verification indicators (evaluation of model stability through various means such as imaging, behavior, and pathology)

[0098] (V) Model Evaluation

[0099] Musculoskeletal ultrasound images, SWE images and elastic hardness data of the erector spinae muscles, vertebral blood vessels, and longus colli muscles, vertebral artery hemodynamic indicators, whole-brain and neck (C1-C3 levels) MRI, vertebral artery MRA images, open-field behavioral experiments, and muscle pathology tests in the neck modeling area were collected from the control group and the model group before modeling, 1 week after the second modeling, after modeling, and 6 months after modeling.

[0100] Example 2

[0101] This embodiment uses traditional modeling to compare with the modeling method of the present invention to evaluate the modeling establishment of Example 1 of the present invention and its advantages over traditional modeling. Traditional modeling is carried out according to the method disclosed in the background technology section, that is, after successful anesthesia, the Xiaozhiling injection is injected into the left C3-C5 transverse process and surrounding tissues, and the injection of equal doses of drugs is repeated once in the second week. The dose of Xiaozhiling injection is 1ml / 100g rat body weight for injection into the left neck, and repeated once after one week (for details, please refer to "Experimental Study on the Establishment of Vertebral Artery Cervical Spondylosis Model in Rats by Modified Sclerosant Injection Method", China Orthopedics, Volume 36, Issue 2, February 2023). The specific evaluation observation content and results are as follows:

[0102] (1) Observation content and results after traditional modeling:

[0103] Recombination Figure 1 As shown, the traditional modeling method has a mortality rate of about 30% in rats due to a significantly excessive drug injection dose. The surviving rats have a poor subsequent living condition, with significant weight loss, emaciation, severe atrophy of the bilateral neck muscles, curled-up bodies, and slow reactions. This model does not conform to the clinical manifestations of chronic vertebral artery type cervical spondylosis. Although the above-mentioned prior art document records a zero mortality rate for the modeling, the inventors of this application did not obtain the success rate recorded in the document when conducting experiments according to the prior art method. Due to the high mortality rate and the poor condition of the rats after modeling, subsequent experiments were not continued.

[0104] Traditional modeling relies on blind feel and experience. However, rats are small and have a cramped cervical vertebrae. Accurately locating the transverse process by touch is difficult in practice, and can easily lead to misplaced injection into the spinal canal, blood vessels, or deviation from the injection site, resulting in animal death or model failure.

[0105] (2) Observation contents and results of the modeling of the present invention:

[0106] 1. Modeling observation content of the present invention

[0107] 1) Dietary conditions: Dietary conditions of rats before and after modeling;

[0108] 2) Comparison of musculoskeletal ultrasound images: Comparison of cervical musculoskeletal ultrasound images of the control group and the model group after modeling and six months after modeling;

[0109] 3) Shear wave elastography (SWE) examination: Shear wave elastography (SWE) examination of the vertebral artery was performed in the control group and the model group before modeling, one week after the second modeling, after modeling, and six months after modeling;

[0110] 4) Vertebral artery ultrasound examination: Vertebral artery ultrasound examination was performed in the control group and the model group before modeling, one week after the second modeling, after modeling, and six months after modeling;

[0111] 5) MRI manifestations of C1-C3 of the neck: T1 and T2 MRI manifestations of C1-C3 of the neck of rats in the control group and model group after modeling;

[0112] 6) Whole-brain MRI imaging: After the modeling, rats in the control and model groups were subjected to whole-brain MRI to observe whether there were any cerebrovascular complications after modeling to verify the safety of the model;

[0113] 7) MRA-TOF blood flow imaging evaluation: Magnetic resonance angiography (MRA) (TOF) was performed on the neck of rats in the control group and 6 months after modeling to observe the long-term changes in vertebral artery blood flow after modeling;

[0114] 8) Open field test: The control group and the model group completed the open field test on rats;

[0115] 9) Muscle fascia pathology detection: Muscle fascia pathology detection in the neck modeling area of the control group and the modeling group

[0116] 2. Observation results after modeling of the present invention

[0117] 1) Rat diet

[0118] Combine Figure 8 As shown in the figure, the food intake of rats decreased slightly in the first two days after model establishment, and gradually increased after the third day and gradually approached normal.

[0119] 2) Ultrasound and MRI imaging of the model neck

[0120] Combine Figure 9 Figure 10 shows MRI images of the cervical spine at the C2 level and corresponding ultrasound images at the C2 plane. The T2-weighted images on the modeling side show linear high signals, with secondary fasciitis and muscle fibrosis near the splenius capitis and semispinalis capitis muscles. The ultrasound images show a mixed pattern of high and low echoes. Both the erector spinae side and the erector spinae side show high signals and high echoes.

[0121] like Figure 9 Figure 10 shows a musculoskeletal ultrasound section of the rat cervical spine at C2. The median spinous process and the lamina on both sides are visible. The left side (L) is the modeling side, where the erector spinae, longus colli, and transverse processes surrounding the vertebral artery show hyperechoic areas. The right side of the control side (R) shows normal tissue echogenicity.

[0122] 3) Ultrasound elastography analysis

[0123] like Figures 11A-11B As shown: Four sets of elastic ultrasound images show the changes in tissue elasticity caused by the four injection points of the invention, indicating that the Cs shear wave velocity on the left side increased significantly after modeling, indicating that the hardness of the tissue around the vertebral artery increased significantly after modeling, which is consistent with the characteristics of vertebral artery type cervical spondylosis with sclerosis of the tissue around the vertebral artery.

[0124] 4) Vertebral artery ultrasound blood flow measurement

[0125] like Figures 12A-12B As shown, after skin preparation, rats were placed in the lateral decubitus position and scanned along the lateral neck long axis using an L20-5s high-frequency probe. Ultrasound images of the vertebral artery and transverse process were visualized, and vascular ultrasound was used to measure vertebral artery blood flow parameters to assess the modeling effect. The results showed that blood flow in the model group showed a significant downward trend during and 6 months after modeling, while vascular resistance showed a significant upward trend, suggesting that the vertebral vessels were compressed or stimulated by surrounding tissues, resulting in a certain degree of spasm or compression, leading to insufficient blood supply.

[0126] 5) Correlation detection between model soft tissue elasticity and vertebral artery blood flow

[0127] like Figures 13A-13E As shown, vascular velocity and blood flow around the vertebral artery were negatively correlated with the elastic stiffness of the surrounding tissue, while vascular resistance and vascular index were positively correlated with the elastic stiffness of the surrounding tissue. This suggests that tissue sclerosis and inflammatory response after modeling affected the blood flow and vascular resistance of rats, similar to the pathological characteristics of patients with vertebral artery-type cervical spondylosis, such as muscle stiffness, mechanical imbalance, vascular spasm, or compression. At the same time, the results showed that elastic imaging technology can be used as an important noninvasive reference indicator for assessing blood flow and vascular resistance, verifying that clinical release treatment after muscle and soft tissue sclerosis and tension is of great significance for improving chronic head and neck microcirculatory disorders and insufficient blood supply caused by cervical spine-related diseases.

[0128] 6) MRI of soft tissue of neck

[0129] like Figure 14As shown in Figure 2, both T1 and T2 images of the healthy control group (A) and the model group (B) clearly demonstrate the C1-C3 vertebrae and surrounding structures, including the transverse foramen, vertebral artery, erector spinae muscles, splenius capitis, and trapezius muscles. Three ultrasound-guided injections of Xiaozhiling at C2 in the model group resulted in the development of cord-like abnormal signal intensity around the vertebral artery and C1-C3 neck muscles on T1 and T2 images. This intensity was high on T2-weighted images and low on T1-weighted images, accompanied by signs of fascial thickening and edema. Further observation revealed that this cord-like abnormal signal intensity extended to the transverse process, the transverse foramen, and the tissue surrounding the vertebral artery. This suggests that soft tissue lesions in the neck have spread to surrounding critical structures, potentially affecting structures such as the vertebral artery, potentially leading to spasm, compression, or stenosis of the vertebral artery, thereby impairing blood supply to the brain. This is consistent with the characteristics of vertebral artery-type cervical spondylosis, characterized by muscle strain and fascial lesions leading to cervical stress imbalance or physical compression due to sclerosis of surrounding tissues.

[0130] 7) Intracranial magnetic resonance imaging (MRI)

[0131] like Figure 15 As shown, no abnormalities were found in the whole brain MRI scans of the healthy control group rats and the vertebral artery model rats, indicating that the invention is safe for the brain and does not cause organic damage to the brain. It is also consistent with the characteristics of vertebral artery type cervical spondylosis. After patients have head symptoms such as headache, dizziness, tinnitus, blurred vision, etc., the head MRI results are often negative, that is, there are no obvious organic lesions, but the patients often have very severe clinical symptoms. This is a feature and difficulty in the diagnosis of vertebral artery type cervical spondylosis.

[0132] 8) Vertebral artery MRA-TOF blood flow signal measurement

[0133] like Figure 16 Figure 2: Blood flow signal measurements of the superior cervical and intracranial segments of the vertebral arteries. While there was no significant difference in MRA signal intensity between the left and right vertebral arteries in the control group, the signal intensity of the left vertebral artery in the model group was significantly lower than that of the ipsilateral control group and the right vertebral artery in the model group six months after modeling. This indicates that the model induces a long-term, sustained decrease in vertebral artery blood flow and demonstrates strong model stability. This characteristic creates a reliable model for studying the pathogenesis and the effects of chronic vertebral artery blood supply insufficiency on brain function, and also aligns with the clinical characteristics of vertebral artery blood supply insufficiency in vertebral artery-type cervical spondylosis.

[0134] 9) Open field test

[0135] like Figures 17A-17B As shown, the model group showed anxiety-like emotions compared to the control group, with significantly decreased activity, distance visited in the central area, and speed. The group also showed extreme caution, suggesting significant anxiety-like behavior. This is consistent with the anxiety-like state experienced by patients with vertebral artery-type cervical spondylosis due to chronic ischemia and sympathetic nerve stimulation.

[0136] 10) Pathological HE staining

[0137] like Figure 18 As shown in the figure, significant differences were observed between the control group and the model after modeling. The control group showed densely packed muscle cells, compact distances between muscle bundles, and no excessive inflammatory cells or collagen deposition. In contrast, the model group showed degeneration of muscle cells, loose distances between cells, and more collagen and inflammatory cell infiltration between muscle bundles, resulting in a disordered and irregular structure.

[0138] In summary, the present invention has the following advantages:

[0139] 1. High operation efficiency and short operation time: The whole process is visualized, replacing the traditional blind puncture, with high efficiency and short operation time.

[0140] 2. High safety: Ultrasound-guided operation avoids accidental penetration of blood vessels, nerves, spinal cord, intervertebral discs and non-related muscles, which is highly safe.

[0141] 3. Innovative four-point injection method: Four-point injection around the vertebral artery, with clear and standardized position and injection dose, and strong replicability.

[0142] 4. High precision and controllable lesion coverage: Ultrasound guidance and elastography assessment ensure precise injection. Traditional modeling methods use blind injections at C3-C5, but the actual lesion coverage is C2-C6. This method only requires injection at four points at the C2 level, with a smaller upper and lower lesion coverage (C1-C3), which is closer to clinical characteristics. This has been verified by MRI.

[0143] 5. Low medication dosage and minimal animal damage: Precise injection reduces single-dose medication dosage by 60% and total injection dosage by 40%. This avoids the severe muscle damage, atrophy, poor animal quality of life, and death often associated with high-dose Xiaozhiling injections. The improved model is more consistent with clinical chronic disease characteristics.

[0144] 6. Low animal mortality: The model survival rate is 100%, the quality of life after surgery is high, and it complies with animal ethics requirements.

[0145] 7. Elastic imaging-assisted tracking and evaluation of modeling effects: Elastic imaging measures the changing trend of elasticity values at specific locations around the vertebral artery and analyzes its correlation with vertebral artery blood flow indicators. It suggests that the elasticity values at the root of the vertebral artery and below the vertebral artery are most significantly correlated with vertebral artery blood flow parameters, which can indicate the modeling progress and effects during the modeling process.

[0146] 8. The model effect is long-term, stable and reliable: Six months after modeling, the differences in rat vertebral artery MRA, elastic ultrasound and vascular ultrasound were still significant, indicating that the model is highly stable and closer to the chronic disease characteristics of clinical cervical spondylosis with a long course of disease.

[0147] 9. The evaluation method is objective, comprehensive, and has good modeling effect: multi-time point musculoskeletal, vascular, elastic ultrasound, head and neck magnetic resonance imaging (MRI, MRA), and open field behavioral verification are added to verify the cutting-edge technology and the results are more stable and reliable.

[0148] In summary, this invention significantly improves the traditional model in terms of operation method, drug dosage, and model verification, and the observation follow-up period reaches 6 months. It has significantly improved safety, accuracy, effectiveness, reliability, and stability. As a common and difficult-to-treat disease, vertebral artery-type cervical spondylosis is a clinical pain point. The emergence of this invention will make a significant contribution to the research and development of the pathogenesis, clinical manifestations, diagnostic criteria, and prevention and treatment strategies of CSA.

[0149] Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

Claims

1. A method for establishing an animal model of vertebral artery type cervical spondylosis, characterized in that: The steps include: Step S1, experimental animal pretreatment: anesthetize the experimental rats, and then prepare the skin of the neck; Step S2, injecting a modeling agent into the experimental animal to establish a model: the modeling agent is injected at the level of the C2 transverse process of the rat cervical vertebra; Step S3: model evaluation.

2. The method for establishing an animal model of vertebral artery type cervical spondylosis according to claim 1, characterized in that: In step S1, sevoflurane with an inhalation concentration of 2-5% is used to induce anesthesia and maintain it during the operation; dexmedetomidine is used for intraperitoneal injection to maintain anesthesia, and the dosage of the dexmedetomidine is 0.08 μg per gram of rat body weight.

3. The method for establishing an animal model of vertebral artery type cervical spondylosis according to claim 1 or 2, characterized in that: In step S1, after anesthesia, the rat is placed prone on the operating table, and the hair on the back and sides of the neck is cleaned.

4. The method for establishing an animal model of vertebral artery type cervical spondylosis according to claim 1, characterized in that: In step S2, ultrasound-guided injection of the modeling agent is used to create the model. Specifically, an ultrasound diagnostic instrument is used to first detect and record the blood flow parameters of the rat's vertebral artery. Then, the rat is placed in a prone position and ultrasound is used to locate the vertebral artery at the C2 level. Then, 0.1 ml of 0.5% lidocaine is used as the needle insertion point at the level of the C2 transverse process under ultrasound guidance to perform epidermal local anesthesia and analgesia. Then, an empty needle is punctured at the C2 level under ultrasound guidance until the C2 level is only 2-3 mm1 away from the vertebral artery. No blood is aspirated, and the modeling agent is injected.

5. The method for establishing an animal model of vertebral artery type cervical spondylosis according to claim 4, characterized in that: The modeling agent was Xiaozhiling stock solution, and there were four injection sites, specifically: 0.2 ml of Xiaozhiling stock solution was injected at the C2 level of the rat, only 2-3 mm away from the vertebral artery; 0.3 ml of Xiaozhiling stock solution was injected into the middle and upper segments of the erector spinae muscle on the ipsilateral side; and 0.2 ml of Xiaozhiling stock solution was injected into the longus colli muscle below the C2 vertebral artery on the ipsilateral side.

6. The method for establishing an animal model of vertebral artery type cervical spondylosis according to claim 5, characterized in that: The modeling agent was injected once a week for three consecutive times, followed by a one-month observation and rest period. The total modeling period was 6 weeks.

7. The method for establishing an animal model of vertebral artery type cervical spondylosis according to claim 1, characterized in that: Step S3 model evaluation includes collecting the following data before modeling, one week after the second modeling, at the completion of the modeling, and six months after modeling: musculoskeletal ultrasound images, SWE images and elastic hardness data of the erector spinae muscles, vertebral blood vessels, and longus colli muscles, vertebral artery hemodynamic indicators, MRI and vertebral artery MRA images of the whole brain and neck C1-C3 levels, open field behavioral experiments, and muscle pathology detection in the neck modeling area.

8. The method for establishing an animal model of vertebral artery type cervical spondylosis according to claim 7, characterized in that: The evaluation indicators for successful model building include the following: Neck ultrasound results: The erector spinae muscle, longus colli muscle and transverse process on the modeling side showed hyperechoic areas near the vertebral artery; Ultrasound elastography results showed that the shear wave velocity near the erector spinae muscle, longus colli muscle and transverse process of the model side was significantly increased compared with the control side, indicating that the tissue hardness increased; Vertebral artery ultrasound blood flow measurement: blood velocity and blood volume showed a significant downward trend during modeling and 6 months after modeling, while pulse index and vascular resistance showed a significant upward trend; In terms of the correlation between the model's soft tissue elasticity and vertebral artery blood flow, the vascular velocity and blood flow around the vertebral artery were negatively correlated with the elasticity and hardness of the surrounding soft tissue, while the vascular resistance and pulsatility index were positively correlated with the elasticity and hardness of the surrounding soft tissue. MRI of soft tissue of neck: After modeling, T1 and T2 images showed cord-like abnormal signal shadows around the vertebral arteries at the C1-C3 level and around the neck muscles at the corresponding level; Intracranial magnetic resonance imaging: No abnormalities were found in the whole brain MRI scans of rats in the control group and the model group; Vertebral artery MRA-TOF blood flow signal measurement: There was no significant difference in the MRA signal intensity of the left and right vertebral arteries in the control group rats, while the signal intensity of the vertebral artery on the model side in the model group was significantly lower than that on the ipsilateral control group and the control side of the model group 6 months after modeling; Open field test: The model group showed anxiety-like emotions, and the total movement distance, movement speed, and the distance and speed visited in the central area were significantly reduced; Pathological HE staining: significant differences were observed between the control group and the model after modeling. The muscle cells in the control group were densely arranged, the distance between the muscle bundles was compact, and there were no excessive inflammatory cells and collagen deposition; while the muscle cells in the model group were degenerated, the distance between cells was loose, more collagen and inflammatory cell infiltration appeared between the muscle bundles, and the structure was disordered and irregular.

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