Construction method and application of intervertebral disc protrusion animal model

Abstract

The invention discloses a construction method of an intervertebral disc protrusion animal model, and the construction method comprises the steps: positioning the intervertebral disc of an animal through ultrasonic guidance, then inserting a syringe needle into one or more of Co4 / 5-Co9 / 10 intervertebral discs of the animal by 1.5-5mm, and then controlling the pressure acting on the intervertebral disc through the extrusion speed and the extrusion frequency, and thus, the intervertebral disc protrusion animal model is obtained. According to the intervertebral disc protrusion animal model constructed through the construction method, the intervertebral disc structure is destroyed, the stress load of the spine is considered, the main pathological process of intervertebral disc protrusion and intervertebral disc protrusion resorption research are met, the pathological process of clinical intervertebral disc protrusion can be simulated more truly, and the clinical application prospect is wide. The method is used for research on pathogenesis and treatment of intervertebral disc herniation resorption and / or intervertebral disc degeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of medical experimental models, and in particular to a method for constructing an animal model of intervertebral disc herniation and application thereof. Background Art

[0002] The prevalence of low back pain in musculoskeletal diseases worldwide is about 33%, affecting about 568 million people worldwide, and has become a global economic and medical burden. Lumbar disc herniation is the main cause of low back pain. Clinical observations have found that patients with lumbar disc herniation have reabsorption of the protruding disc after conservative treatment, and the reabsorption of the disc herniation can significantly relieve the patient's clinical symptoms. Therefore, in-depth exploration of the mechanism of disc herniation reabsorption and the adoption of active strategies to promote the progress of disc herniation reabsorption have important clinical application value. Disc herniation is characterized by disc degeneration, annulus fibrosus rupture and nucleus pulposus herniation as the main pathological manifestations. Animal models are essential for studying the pathological process of disc herniation and the reabsorption mechanism of the herniation. The ideal animal model of disc herniation should be accurate, portable, and easy to operate, and can truly simulate the pathological process of human disc herniation.

[0003] In the study of intervertebral disc herniation and herniation reabsorption, animal models are an important part of the success of basic research. In previous studies, the promotion of this animal model was limited due to the selection of model animal species, differences in modeling methods, and specificity of instruments. Currently, there are three main animal models for constructing intervertebral disc herniation or herniation reabsorption: (1) autologous intervertebral disc transplantation model: transplanting the autologous intervertebral disc nucleus pulposus of rabbits or rats into the lumbar epidural space or abdomen; (2) puncturing the intervertebral disc annulus fibrosus to induce intervertebral disc herniation model after locating the rat intervertebral disc under X-ray; (3) acupuncture of the intervertebral disc annulus fibrosus model to induce nucleus pulposus herniation in mice.

[0004] However, these three methods of constructing animal models of intervertebral disc herniation have some defects: (1) The autologous intervertebral disc transplantation model is to remove the autologous intervertebral disc nucleus pulposus tissue of rabbits or rats and transplant it into the lumbar epidural space or peritoneum. This modeling method has a large incision; secondly, since the mouse intervertebral disc is too small, it is impossible to remove the nucleus pulposus and transplant it, so this modeling method is not suitable for mice, which limits its promotion; at the same time, in this modeling method, the intervertebral disc does not experience annular fibrosis rupture, and the nucleus pulposus does not protrude from the annular fibrosis fissure, which is difficult to promote and apply in basic research on intervertebral disc herniation. (2) The second animal model construction method is to locate the rat coccygeal intervertebral disc under X-ray, and then use acupuncture to puncture the annulus fibrosus of the intervertebral disc to promote the disc protrusion; this modeling method simulates the pathological process of the intervertebral disc nucleus pulposus protruding from the annular fibrosis fissure to a certain extent, but due to the low resolution of X-ray soft tissue, it is difficult to distinguish the internal structure of the intervertebral disc and the paravertebral tissue structure, and the real-time image quality is limited by the performance of the equipment. (3) The third animal model is to induce nucleus pulposus herniation by puncturing the annulus fibrosus of the mouse caudal intervertebral disc. This method has achieved the preparation of a small animal intervertebral disc herniation model to a certain extent, but it does not use a visual instrument to locate the intervertebral disc, which makes the operation difficult and affects the use and promotion of this animal model method.

[0005] The intervertebral disc plays an important role in transmitting and dispersing stress. The extracellular matrix of the nucleus pulposus tissue of the intervertebral disc has the function of buffering the load on the spine. Long-term or repeated mechanical loads can lead to disc herniation and degeneration. Therefore, structural damage and stress load of the intervertebral disc are important factors affecting the occurrence and development of disc herniation. The influence of these two factors needs to be considered in the animal model of disc herniation. However, the current animal models of disc herniation usually cannot take these two factors into account, which makes it impossible for the constructed animal models to truly simulate the pathological process of disc herniation and cannot be effectively promoted. Therefore, there is an urgent need for a method to construct an animal model of disc herniation that can more realistically simulate the pathological process of disc herniation and promote the research and development of disc herniation and the reabsorption mechanism of disc herniation. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a method for constructing an animal model of intervertebral disc herniation and its application.

[0007] The first object of the present invention is to provide a method for constructing an animal model of intervertebral disc herniation.

[0008] The second object of the present invention is to provide an animal model of intervertebral disc herniation constructed by the above construction method.

[0009] The third object of the present invention is to provide application of the above-mentioned animal model of intervertebral disc herniation in the study of intervertebral disc degeneration.

[0010] In order to achieve the above object, the present invention is implemented by the following scheme:

[0011] A method for constructing an animal model of intervertebral disc herniation comprises the following steps:

[0012] Under ultrasound guidance, the intervertebral disc position of the animal is marked, and an injection needle is used to insert the needle 1.5 to 5 mm into one or more of the Co4 / 5 to Co9 / 10 intervertebral discs of the animal, and then the tail of the animal is repeatedly squeezed 3 to 20 times to obtain an animal model of intervertebral disc herniation;

[0013] The number of squeezes is calculated as follows: squeeze the tail of the animal from the distal end to the proximal end for 1 to 10 seconds, and then stretch the tail from the proximal end to the distal end for 1 to 10 seconds, which is considered as one squeeze.

[0014] During the extrusion process, the extrusion speed and the stretching speed are both 30-300 mm / min.

[0015] Preferably, before ultrasound guidance, the animal needs to be anesthetized and placed in a supine position, an ultrasound sterile coupling agent is applied to its tail, and the ultrasound probe is fixed on the ultrasound sterile coupling agent.

[0016] More preferably, the ultrasound probe is parallel to the animal's tail and is placed in the sagittal center of the animal's tail.

[0017] More preferably, the animal is a rat and / or a mouse.

[0018] Preferably, a 20-33G injection needle is used for injection.

[0019] More preferably, a 20-33G injection needle is used to penetrate 1.5-5 mm into 1-5 consecutive intervertebral discs among the Co4 / 5-Co9 / 10 intervertebral discs of the animal.

[0020] More preferably, a 20-33G injection needle is used to inject 3 mm into 1-5 consecutive intervertebral discs among the Co4 / 5-Co9 / 10 intervertebral discs of the animal.

[0021] More preferably, a 26G injection needle is used to inject 3 mm into 1 to 5 consecutive intervertebral discs among the Co4 / 5 to Co9 / 10 intervertebral discs of the animal.

[0022] More preferably, the needle is inserted 3 mm into the Co4 / 5, Co5 / 6 and Co6 / 7 intervertebral discs of the animals.

[0023] Preferably, the tail of the control animal is squeezed repeatedly 10 times.

[0024] Preferably, the extrusion speed and the stretching speed are both 150 mm / min.

[0025] Preferably, the animal's tail is repeatedly squeezed by a syringe pump.

[0026] More preferably, the repeated squeezing of the animal by the injection pump is specifically: fixing the animal at the syringe position of the injection pump, and fixing the distal end of the tail of the animal on the injection fixing plate of the injection pump, controlling the injection speed to be 30 to 300 mm / min, and controlling the injection fixing plate to cyclically inject 3 to 20 times;

[0027] The number of cycle push injections is calculated as follows: control the push injection fixing plate to push injection from the distal end to the proximal end for 1 to 10 seconds, and then control the push injection fixing plate to push injection from the proximal end to the distal end for 1 to 10 seconds, which is regarded as one cycle push injection.

[0028] More preferably, the injection speed is 150 mm / min.

[0029] Further preferably, the push injection fixing plate is controlled to cycle the push injection 10 times.

[0030] The present invention also seeks to protect the intervertebral disc herniation animal model obtained by any of the above-mentioned construction methods.

[0031] Preferably, the intervertebral disc herniation animal model is a ruptured intervertebral disc herniation animal model.

[0032] The present invention also claims to protect the use of the above-mentioned intervertebral disc herniation animal model in the study of intervertebral disc herniation reabsorption and / or intervertebral disc degeneration.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a method for constructing an animal model of intervertebral disc herniation, wherein the construction method locates the intervertebral disc of an animal by ultrasound guidance, then uses an injection needle to insert a needle 1.5 to 5 mm into one or more of the Co4 / 5 to Co9 / 10 intervertebral discs of the animal, and then controls the magnitude of the pressure acting on the intervertebral disc by the extrusion speed and the number of extrusions, thereby obtaining an animal model of intervertebral disc herniation. The animal model of intervertebral disc herniation constructed by the construction method of the present invention not only destroys the structure of the intervertebral disc, but also takes into account the stress load of the spine, conforms to the main pathological process of intervertebral disc herniation and the study of intervertebral disc herniation reabsorption, can more realistically simulate the pathological process of clinical intervertebral disc herniation, and is used for the etiology and treatment research of intervertebral disc herniation reabsorption and / or intervertebral disc degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1Schematic diagram of the construction of the mouse model of intervertebral disc herniation in Example 1; A is a schematic diagram of the use of ultrasound to locate the intervertebral disc of anesthetized mice; B is a schematic diagram of the ultrasound image of the intervertebral disc and paravertebral tissue structure of anesthetized mice; C is a schematic diagram of the intervertebral disc of the injected mouse; D is a schematic diagram of the injected mouse with the intervertebral disc pushed by a syringe pump;

[0036] Figure 2 A is an image of the stained paraffin sections of the caudal vertebrae of each group of mice in Example 2; A is an image of the stained paraffin sections of the caudal vertebrae of the control group mice; B is an image of the stained paraffin sections of the caudal vertebrae of the model group mice;

[0037] Figure 3 Figure 2 is an image of the paraffin sections of the caudal vertebrae of each group of mice in Example 2 after fluorescence staining; A is an image of the paraffin sections of the caudal vertebrae of the control group mice after fluorescence staining; B is a partial enlarged view of A; C is an image of the paraffin sections of the caudal vertebrae of the model group mice after fluorescence staining; D is a partial enlarged view of C;

[0038] Figure 4 : Figure 3 is the result of nuclear magnetic resonance detection of each group of intervertebral disc herniation mouse models; A is a T2WI image of one of the intervertebral disc herniation mouse models in each group; B is a gray value statistical result of the nucleus pulposus image of the sagittal midline section of the intervertebral disc of each group of intervertebral disc herniation mouse models;

[0039] Figure 5 : is a diagram showing the ultrasonic detection results of each group of intervertebral disc herniation mouse models in Example 3; A is an ultrasonic image and a 3D image of one of the intervertebral disc herniation mouse models in each group; B is a diagram showing the statistical results of paravertebral tissue swelling of each group of intervertebral disc herniation mouse models;

[0040] Figure 6 : A is an AB / OG staining test result diagram of each group of intervertebral disc herniation mouse models in Example 3; A is an AB / OG staining image of one of the intervertebral disc herniation mouse models in each group; B is a statistical result diagram of the ratio of the intervertebral disc inflammation area to the total intervertebral disc area of ​​each group of intervertebral disc herniation mouse models;

[0041] Figure 7 : A is a graph showing the results of nuclear magnetic resonance imaging of mice in each group in Example 4; A is a graph showing the T2WI images of mice in each group; B is a graph showing the statistical results of the grayscale values ​​of the nucleus pulposus images in the intervertebral discs of mice in each group; C is a graph showing the statistical results of the grayscale values ​​of the nucleus pulposus images in the paravertebral tissues of mice in each group;

[0042] Figure 8 4 are the test results of the clinical patients in Example 4; A is the VAS score result diagram of the clinical patients before and after treatment; B is the MRI image diagram before treatment; C is the MRI image diagram after treatment. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0044] The clinical sample cases shown in the embodiments of the present invention were informed to the patients and authorized by the patients before use; all animal experiments were approved by the Experimental Animal Ethics Committee of Shenzhen Zhongxun Precision Medicine Research Institute.

[0045] Example 1 A method for constructing a mouse model of intervertebral disc herniation

[0046] 1. Experimental Methods

[0047] Schematic diagram of the construction of the mouse model of intervertebral disc herniation Figure 1 As shown, the details are as follows

[0048] 1. Acupuncture of the intervertebral disc of mice under ultrasound guidance. Figure 1 A in the figure is a schematic diagram of using ultrasound to locate the intervertebral disc of anesthetized mice. Figure 1 B is a schematic diagram of the ultrasound image of the intervertebral disc and paravertebral tissue structure of anesthetized mice. Figure 1 C in the figure is a schematic diagram of the intervertebral disc of the mouse with needle inserted; the specific steps are as follows:

[0049] S1. After the mouse was anesthetized with isoflurane gas, it was placed on the ultrasound machine hot plate, and the temperature of the hot plate was set to 37°C;

[0050] S2. Place the anesthetized mouse in a prone position and disinfect the mouse tail with medical disinfectant alcohol;

[0051] S3. Turn on the ultrasound machine and select the superficial tissue mode;

[0052] S4. Apply medical ultrasound sterile coupling agent to the surface of three consecutive intervertebral discs Co4 / 5, Co5 / 6, and Co6 / 7 at the tail of the anesthetized mouse, fix the ultrasound probe on the medical ultrasound sterile coupling agent, wherein the direction of the ultrasound probe is parallel to the tail of the anesthetized mouse and is placed in the sagittal center of the tail of the anesthetized mouse, and obtain ultrasound images of the intervertebral discs and paravertebral tissues of the anesthetized mouse;

[0053] S5. Under ultrasound guidance, a 26G injection needle was used to insert the needle into three consecutive intervertebral discs (Co4 / 5, Co5 / 6, and Co6 / 7) of anesthetized mice to a depth of 3 mm to obtain intervertebral disc needled mice.

[0054] 2. Injection pump to inject mice

[0055] Figure 1D in the figure is a schematic diagram of using a syringe pump to push the needle into the intervertebral disc of a mouse;

[0056] The specific steps are as follows:

[0057] S6. The mouse with intervertebral disc injection was fixed in an animal restrainer, and the animal restrainer was fixed to the syringe position of the injection pump (Yuanhang Power, QHZS-001A). The distal end of the tail of the mouse with intervertebral disc injection was fixed to the injection fixing plate of the injection pump, and the tail of the mouse was controlled to remain straight;

[0058] S7. Turn on the syringe pump controller switch, control the injection speed of the syringe pump to 150 mm / min, and perform 10 cycles of injection to obtain a ruptured intervertebral disc herniation mouse model;

[0059] Click the back button of the injection pump to control the injection fixing plate of the injection pump to move toward the proximal end of the mouse for 10 seconds, and then click the forward button of the injection pump to control the injection fixing plate of the injection pump to move toward the distal end of the mouse for 10 seconds, which is considered as one push injection.

[0060] Example 2 Identification of a Mouse Model of Intervertebral Disc Herniation

[0061] 1. Experimental Methods

[0062] Model group: A mouse model was constructed according to the method for constructing a mouse model of intervertebral disc herniation as shown in Example 1 to obtain a mouse model of intervertebral disc herniation in the model group.

[0063] Control group: The mice in the control group were normal mice of the same age as the mice in the model group.

[0064] For the mice with intervertebral disc herniation in the model group and the mice in the control group, 24 hours after modeling, the skin of the mouse tails was peeled off, and the caudal vertebrae of the mice were placed in a formalin solution with a volume concentration of 10% and fixed for 24 hours, followed by slow decalcification using an EDTA decalcification solution with a mass concentration of 14% for 4 weeks, and then dehydrated, waxed, embedded and sectioned to obtain paraffin sections of the caudal vertebrae of the mice in the model group and the caudal vertebrae of the mice in the control group.

[0065] The caudal vertebrae paraffin sections of the model group mice and the caudal vertebrae paraffin sections of the control group mice were stained according to the AB / OG staining procedure shown in Table 1 to obtain stained caudal vertebrae paraffin sections of the model group mice and the control group mice.

[0066] Table 1 AB / OG staining procedure

[0067] step Reagents Volume (mL) time 1 Xylene 200 5min / time, 3 times 2 100% ethanol (volume concentration) 200 5min 3 95% ethanol (volume concentration) 200 5min 4 85% ethanol (volume concentration) 200 5min 5 water 200 5min / time, 3 times 6 1% hydrochloric acid ethanol solution (volume concentration) 200 30s 7 ABH solution 200 40min 8 water 200 5min / time, 3 times 9 1% hydrochloric acid ethanol solution (volume concentration) 200 5s 10 water 200 5min / time, 3 times 11 0.5% ammonia water (volume concentration) 200 15s 12 water 200 5min / time, 3 times 13 95% ethanol (volume concentration) 200 5s 14 OG Solution 200 2.5min 15 water 200 2min 16 95% ethanol (volume concentration) 200 2min 17 100% ethanol (volume concentration) 200 2min / time, 2 times 18 Xylene 200 2min / time, 2 times 19 Neutral gum 0.01 Mounting

[0068] Then, a microscope (ZEISS Axlo Imager M2) was used to collect images of the stained paraffin sections of the caudal vertebrae of the model group mice and the control group mice in bright field mode.

[0069] For the paraffin sections of the caudal vertebrae of the mice in the model group and the caudal vertebrae of the mice in the control group, tissue immunofluorescence staining was performed according to the tissue immunofluorescence staining procedure shown in Table 2 to obtain the caudal vertebrae paraffin sections of the mice in the model group and the mice in the control group after fluorescence staining, respectively.

[0070] Table 2 Tissue immunofluorescence staining procedures

[0071]

[0072]

[0073] Then, images were collected using the fluorescence mode of a microscope (ZEISS Axlo Imager M2); the primary antibody was KRT19, labeled with Alexa Fluor 488 (green fluorescence channel), and the cell nuclei were stained with DAPI (blue fluorescence channel). The above two channels were used to collect images of paraffin sections of the caudal vertebrae after fluorescence staining of the model group mice and the control group mice.

[0074] 2. Experimental Results

[0075] The images of paraffin sections of the caudal vertebrae of each group of mice after pathological staining are shown in the figure. Figure 2 As shown, Figure 2 A in the figure is the paraffin section image of the tail vertebrae of the control group mice after staining, where NP represents nucleus pulposus and AF represents annulus fibrosus; Figure 2 B is the stained paraffin section image of the tail vertebrae of mice in the model group.

[0076] The results showed that after AB / OG staining, in the paraffin section images of the coccygeal vertebrae of the control group mouse model, the mouse intervertebral disc morphology was intact, the annulus fibrosus was neatly arranged, the nucleus pulposus was centered and the extracellular matrix was abundant; while in the paraffin section images of the coccygeal vertebrae of the model group mouse model of intervertebral disc herniation, the mouse intervertebral disc morphology was destroyed, the intervertebral space was reduced, obvious cracks appeared in the annulus fibrosus, and the boundary between the annulus fibrosus and the nucleus pulposus was unclear, the extracellular matrix was reduced, and the paravertebral tissue was swollen.

[0077] The images of the paraffin sections of the tail vertebrae of each group of mice after fluorescent staining are shown in the figure. Figure 3 As shown, Figure 3 A in the figure is the paraffin section image of the tail vertebrae of the control group mice after fluorescent staining; Figure 3 B in Figure 3 A partial enlarged view of A in FIG. Figure 3 C in the figure is the paraffin section image of the tail vertebrae of the model group mice after fluorescence staining; Figure 3 D in Figure 3 A partial enlarged view of C in FIG.

[0078] The results showed that after tissue immunofluorescence staining, in the paraffin section images of the caudal vertebrae of the control group mouse model, the nucleus pulposus cells labeled by KRT19 were located in the center of the intervertebral disc, and no nucleus pulposus cells were found in the paravertebral tissue, indicating that the intervertebral disc structure of the control group mice was intact; while in the paraffin section images of the caudal vertebrae of the model group mouse model with intervertebral disc herniation, the nucleus pulposus cells were distributed in the paravertebral tissue, indicating that the intervertebral disc structure of the model group mice was destroyed and the nucleus pulposus protruded outside the intervertebral disc.

[0079] Results show that the intervertebral disc herniation mouse model constructed according to the construction method shown in Example 1 can destroy the intervertebral disc structure of the constructed intervertebral disc herniation mouse model, which is more consistent with the pathological process of intervertebral disc herniation. The constructed mouse model can be more realistically used to study clinical intervertebral disc herniation.

[0080] Example 3 Stability test of a method for constructing a mouse model of intervertebral disc herniation

[0081] 1. Experimental Methods

[0082] The intervertebral disc herniation mouse model of model group 1, normal mouse model of control group 1 and intervertebral disc herniation mouse model of comparison group were constructed respectively. Three intervertebral disc herniation mouse models were constructed in parallel in each group. The specific construction methods are as follows:

[0083] Model group 1: A mouse model of intervertebral disc herniation was constructed according to the construction method shown in Example 1 to obtain a mouse model of intervertebral disc herniation in model group 1.

[0084] The control group 1 consisted of normal mice of the same age as the model group 1.

[0085] The construction method of the intervertebral disc herniation mouse model shown in the control group is different from the construction method shown in Example 1 in that the step of "injecting mice with a syringe pump in a circular manner" is not performed to obtain the intervertebral disc herniation mouse model of the control group.

[0086] 1. Nuclear magnetic resonance imaging

[0087] One week after the construction of each group of mouse models, the Bruker PharmaScan 7T small animal MRI scanner was used to perform sagittal T2WI scanning of the caudal intervertebral disc of each group of mice to obtain the T2WI scanning images of each group of mouse models; the parameters in the MRI scanning process were set as: sequence repetition time 4200ms, echo time 58ms, layer thickness 0.75mm, and layer spacing 0.75mm.

[0088] Based on the T2WI scanning images of each group of mice, the grayscale values ​​of the nucleus pulposus images of the sagittal midline sections of the intervertebral discs of each group of mice were calculated using ImageJ software and homogenized to obtain the statistical result graphs of the grayscale values ​​of the nucleus pulposus images of each group of mice. The maximum, minimum, mean, standard deviation, coefficient of variation and range ratio (maximum value / minimum value) of the grayscale values ​​of the nucleus pulposus images of each group of mice were calculated respectively.

[0089] 2. Ultrasonic testing

[0090] One week after the mouse models in each group were constructed, ultrasonic detection was performed using a small animal ultrasound instrument (Vevo3100 Fuji Film Visual Sonics). The parameters during the ultrasonic detection process were set as follows: scanning thickness 0.05 mm, scanning time 8 s, and ultrasonic detection from left to right.

[0091] After the ultrasonic detection was completed, Vevo LAB 3.2.0 software was used for three-dimensional reconstruction and volume calculation to obtain the ultrasonic images and 3D images of the paravertebral tissue of each group of mice, as well as the statistical results of the paravertebral tissue swelling of each group of mice. The maximum value, minimum value, mean value, standard deviation, coefficient of variation and range ratio (maximum value / minimum value) of the paravertebral tissue swelling volume of each group of mice were calculated.

[0092] 3. AB / OG staining test

[0093] One week after the modeling of each group of mice was completed, the tail skin of each group of mice was peeled off, and the caudal vertebrae of the mice were placed in a formalin solution with a volume concentration of 10% and fixed for 24 h, followed by slow decalcification using an EDTA decalcification solution with a mass concentration of 14% for 4 weeks. The samples were then dehydrated, waxed, embedded, and sectioned to obtain paraffin sections of the caudal vertebrae of the model group mice and paraffin sections of the caudal vertebrae of the control group mice.

[0094] The caudal vertebrae paraffin sections of each group of mice were AB / OG stained according to the AB / OG staining procedure shown in Table 1 in Example 2. After staining, images were collected using a microscope (ZEISS Axlo Imager M2), and ImageJ software was used to quantify the intervertebral disc inflammation area of ​​each group of mice, and the ratio of the intervertebral disc inflammation area to the total intervertebral disc area of ​​each group of mice was calculated. The maximum, minimum, mean, standard deviation, coefficient of variation and range ratio (maximum / minimum) of the ratio of the intervertebral disc inflammation area to the total intervertebral disc area of ​​each group of mice were calculated.

[0095] 2. Experimental Results

[0096] The results of nuclear magnetic resonance imaging of each group of mouse models are shown in the figure Figure 4 As shown, Figure 4 A in the figure is a T2WI image of one mouse model in each group. Figure 4B in the figure is the statistical result of gray value of nucleus pulposus image of sagittal midline section of intervertebral disc of each group of mice.

[0097] Figure 4 Figure A shows: the nucleus pulposus of the intervertebral disc of the mice in the control group 1 showed high signal, and the intervertebral height was basically the same; the nucleus pulposus signal of the intervertebral disc of the mice with intervertebral disc herniation in the comparison group was reduced, the signal was uneven in three consecutive modeling intervertebral discs (i.e., three consecutive needle-inserted intervertebral discs), the intervertebral height was reduced, the paravertebral tissue had local high signal, and the boundary between the intervertebral disc fiber ring and the nucleus pulposus was unclear; the nucleus pulposus signal of the intervertebral disc in the mice with intervertebral disc herniation in the model group 1 was significantly reduced, the nucleus pulposus signal loss in three consecutive modeling intervertebral discs (i.e., three consecutive needle-inserted intervertebral discs) was basically the same, the intervertebral height was reduced, the high signal in the paravertebral tissue was relatively uniform, and the intervertebral disc fiber and the boundary with the nucleus pulposus were lost.

[0098] Figure 4 Figure B shows that compared with the control group 1 mice, the nucleus pulposus in the model group 1 and the control group of intervertebral disc herniation mice were reduced (P < 0.01), while there was no significant difference in the nucleus pulposus between the model group 1 intervertebral disc herniation mice and the control group of intervertebral disc herniation mice (P > 0.01).

[0099] The grayscale value calculation results of the nucleus pulposus images of the intervertebral discs of mice in each group are shown in Table 3.

[0100] Table 3 Calculation results of grayscale values ​​of nucleus pulposus images

[0101] Group Mean Maximum Minimum Maximum value / minimum value (range ratio) Standard Deviation Coefficient of variation (%) Control group 1 1.000 1.130 0.829 1.363 0.106 10.6 Comparison group 0.433 0.648 0.285 2.274 0.140 32.3 Model Group 1 0.482 0.623 0.348 1.790 0.096 19.9

[0102] The results showed that the coefficient of variation of the grayscale value of the nucleus pulposus image of the mouse model of intervertebral disc herniation in the control group was 32.3%, and the range ratio was 2.274; the coefficient of variation of the grayscale value of the nucleus pulposus image of the mouse model of intervertebral disc herniation in model group 1 was 19.9%, and the range ratio was 1.790.

[0103] This indicates that although both model group 1 and control group successfully constructed mice with intervertebral disc herniation, the differences between mice with intervertebral disc herniation within model group 1 were smaller, the modeling method was more stable, and the constructed intervertebral disc herniation model was more reliable.

[0104] The ultrasound test results of each group of mouse models are shown in the figure Figure 5 As shown, Figure 5 A in the figure is the ultrasound image and 3D image of one mouse model in each group. Figure 5 B in the figure is the statistical result of paravertebral tissue swelling in each group of mouse models.

[0105] The results showed that in the control group 1 mouse model, the boundary between the intervertebral space and the vertebral body was clear, and the paravertebral tissue was thin and flat; in the comparison group intervertebral disc herniation mouse model, the intervertebral space was blurred, the boundary with the vertebral body was unclear, the paravertebral tissue was swollen, and the degree of paravertebral tissue swelling of the three consecutive modeled intervertebral discs (i.e., three consecutive needle-inserted intervertebral discs) was different; in the model group 1 intervertebral disc herniation mouse model, the boundary between the intervertebral disc and the vertebral body was unclear, the paravertebral tissue was swollen, and the degree of paravertebral swelling of the three consecutive modeled intervertebral discs (i.e., three consecutive needle-inserted intervertebral discs) was relatively uniform.

[0106] The statistical results of paravertebral tissue swelling in each group showed that compared with the mice in the control group 1, the swelling degree of the paravertebral tissue in the mice with intervertebral disc herniation in the comparison group and the model group 1 was significantly increased (P < 0.01), while there was no statistical difference in the swelling degree of the paravertebral tissue between the comparison group and the model group 1 (P > 0.01).

[0107] The calculation results of the swelling volume of paravertebral tissues in each group of mice are shown in Table 4.

[0108] Table 4 Calculation results of paravertebral tissue swelling volume

[0109] Group Mean Maximum Minimum Maximum value / minimum value (range ratio) Standard Deviation Coefficient of variation (%) Control group 1 0.028 0.032 0.023 1.391 0.003 9.8 Comparison group 1.226 2.593 0.531 4.883 0.681 55.5 Model Group 1 1.229 1.562 0.964 1.620 0.230 18.7

[0110] The results showed that the coefficient of variation of the swelling volume of the paravertebral tissue in the mouse model of intervertebral disc herniation in the control group was 55.5%, and the range ratio was 4.883; while the coefficient of variation of the swelling volume of the paravertebral tissue in the mouse model of intervertebral disc herniation in the model group 1 was 18.7%, and the range ratio was 1.620.

[0111] The results showed that the degree of intervertebral disc swelling between the intervertebral disc herniation mouse models in model group 1 was significantly closer, and the dispersion of swelling volume was significantly smaller, indicating that the method of constructing intervertebral disc herniation mice in model group 1 had better repeatability and the constructed intervertebral disc herniation model was more reliable.

[0112] The AB / OG staining test results of each group of mouse models are shown in the figure Figure 6 As shown, Figure 6 A in the figure is the AB / OG staining image of one of the mouse models in each group. Figure 6 B is the statistical result of the ratio of the intervertebral disc inflammation area to the total intervertebral disc area in each group of mouse models.

[0113] The results showed that the intervertebral disc morphology of the mice in the control group 1 was intact, the annulus fibrosus was neatly arranged, the nucleus pulposus was centered and the extracellular matrix was rich; the annulus fibrosus of the intervertebral disc of the mice with intervertebral disc herniation in the comparison group was disordered, the boundary between the annulus fibrosus and the nucleus pulposus disappeared, the extracellular matrix was significantly lost, the paravertebral tissue of the intervertebral disc was inflammatory infiltrated, and the degree of inflammatory infiltration of the three consecutive modeled intervertebral discs (i.e., three consecutive needle-inserted intervertebral discs) was different; the intervertebral disc morphology of the mice with intervertebral disc herniation in the model group 1 was obviously destroyed, the intervertebral space was reduced, the annulus fibrosus had obvious cracks, the boundary between the annulus fibrosus and the nucleus pulposus was lost, the extracellular matrix was significantly reduced, the nucleus pulposus protruded along with the annulus fibrosus cracks, and the intervertebral disc and paravertebral tissue showed obvious inflammatory infiltration, and the degree of inflammatory infiltration of the three consecutive modeled intervertebral discs (i.e., three consecutive needle-inserted intervertebral discs) was basically consistent.

[0114] Statistical results of the ratio of the intervertebral disc inflammation area to the total intervertebral disc area of ​​each group of mice ( Figure 6 B) shows that compared with control group 1, the intervertebral disc and paravertebral tissues of the mice with intervertebral disc herniation in the comparison group and model group 1 showed significant inflammatory infiltration (P < 0.01); while compared with the mice with intervertebral disc herniation in the comparison group and model group 1, there was no significant difference in the inflammatory infiltration of the intervertebral disc and paravertebral tissues (P > 0.01), but the dispersion of the proportion of the inflammatory area of ​​the intervertebral disc to the total area of ​​the intervertebral disc in each mouse with intervertebral disc herniation in the comparison group was greater.

[0115] The statistical results of the ratio of the inflammatory area of ​​the intervertebral disc to the total area of ​​the intervertebral disc of each group of mice are shown in Table 5.

[0116] Table 5 Statistical results of the ratio of the intervertebral disc inflammation area to the total intervertebral disc area of ​​each group of mice

[0117] Group Mean Maximum Minimum Maximum value / minimum value (range ratio) Standard Deviation Coefficient of variation (%) Control group 1 - - - - - - Comparison group 0.200 0.362 0.087 4.161 0.092 46.0 Model Group 1 0.204 0.263 0.167 1.575 0.030 14.7

[0118] The results showed that the coefficient of variation of the ratio of the intervertebral disc inflammation area to the total intervertebral disc area in the control group of intervertebral disc herniation mouse model was 46.0%, and the range ratio was 4.161; while the coefficient of variation of the ratio of the intervertebral disc inflammation area to the total intervertebral disc area in the model group 1 intervertebral disc herniation mouse model was 14.7%, and the range ratio was 1.575.

[0119] This indicates that the differences among the mice with intervertebral disc herniation in model group 1 are small, and the intervertebral disc herniation mouse model constructed using the construction method shown in model group 1 is more stable and uniform, and the construction method shown in model group 1 has better repeatability.

[0120] Example 4 Application of Ruptured Disc Herniation Mouse Model

[0121] 1. Experimental Methods

[0122] 1. Detection of Huoxue Zhitong Decoction in the intervention of ruptured intervertebral disc mouse model

[0123] The mice in the blood circulation group, control group 2 and model group 2 were set up respectively, each group contained 6 mice, and the specific treatments were as follows:

[0124] Blood circulation group: According to the construction method shown in Example 1, mice with ruptured intervertebral disc herniation were constructed, and then the mice with ruptured intervertebral disc herniation were gavaged with a concentrated solution of Huoxue Zhitong Fang with a human equivalent dose of 7.8 g / kg (gavage concentration 0.5 g / mL, 1 time / day); wherein the Huoxue Zhitong Fang was composed of 10g of Curcuma, 10g of Pheretima, 10g of Taraxacum, 10g of Corydalis, 10g of Angelica dahurica and 10g of Achyranthes bidentata; preparation of Huoxue Zhitong Fang concentrated solution: adding 10 times the mass of water of the raw medicinal materials to the Huoxue Zhitong Fang, soaking for 30min, then decocting for 45min, collecting the filtrate after filtering, adding 10 times the volume of water to the filter residue and continuing to decoct for 45min, filtering with gauze, combining the filtrate, and concentrating with a rotary evaporator, concentrating the Huoxue Zhitong Fang to 0.5g / mL, then packaging, and then storing at -20°C to obtain Huoxue Zhitong Fang concentrated solution.

[0125] Control group 2: Normal mice of the same age as the mice in the blood circulation group were intragastrically treated with an equal dose of physiological saline (once a day).

[0126] The only difference between model group 2 and the Huoxue group was that the Huoxue Zhitong prescription concentrate was replaced with an equal dose of normal saline, and the rest of the treatments were exactly the same.

[0127] After continuous gavage for 2 weeks, each group of mice was subjected to sagittal T2WI scanning of the caudal intervertebral disc using a Bruker PharmaScan 7T small animal MRI scanner to obtain T2WI scanning images of each group of mice; the parameters during the MRI scanning process were set as: sequence repetition time 4200ms, echo time 58ms, layer thickness 0.75mm, and layer spacing 0.75mm.

[0128] Based on the T2WI images of each group of mice, the grayscale values ​​of the nucleus pulposus images of the sagittal midline sections of each intervertebral disc of each group of mice (including the grayscale values ​​of the nucleus pulposus images in the intervertebral disc and paravertebral tissue) were calculated using ImageJ and homogenized (with the control group 2 as the benchmark).

[0129] 2. Huoxue Zhitong Decoction for the treatment of patients with clinical ruptured lumbar disc herniation

[0130] The clinical patient information and administration method are as follows:

[0131] Lan, male, 32 years old, chief complaint: low back pain for 3 days. Medical history: The patient had low back pain 3 days ago after carrying heavy objects, mainly tingling and radiating pain, limited activity, occasional pain in the left lower limb, and no obvious numbness. Specialist examination: The range of motion of the waist is significantly limited, the range of motion of the waist is significantly limited, the flexion range of motion is 30°, the extension range of motion is 10°, and the rotation range of motion is 15°. The pain is aggravated during flexion, extension and rotation, especially during flexion. Positive tenderness of L5 and S1 spinous processes, positive left straight leg raising test, left extensor muscle strength IV, and normal Achilles tendon reflex on both sides. The tongue is dark red, the fur is thin and white, and the pulse is stringy. VAS score: 8 points, JOA score: 14 points, ODI index: 36%. Imaging examination: MRI shows L5 / S1 intervertebral disc herniation with nerve root compression.

[0132] The diagnosis of traditional Chinese medicine was low back pain, with the syndrome of qi stagnation and blood stasis; the diagnosis of Western medicine was ruptured lumbar disc herniation.

[0133] The treatment plan is: use the prescription for promoting blood circulation and relieving pain, prescribing 10g of Curcuma zedoaria, 10g of earthworm, 10g of dandelion, 10g of Corydalis yanhusuo, 10g of Achyranthes bidentata, 7 doses, 1 dose per day, decocted in water, taken in the morning and evening; the decoction is to add 10 times the volume of water of the raw medicinal materials, soak for 30 minutes, then boil for 45 minutes, collect the filtrate after filtering, add 10 times the volume of water to the residue and continue to boil for 45 minutes, filter with gauze, and combine the filtrate.

[0134] The patients continued to take the Huoxue Zhitong prescription for 1 week, and their sensory organs were recorded. The patients then continued to take the Huoxue Zhitong prescription for 2 weeks. A follow-up visit was conducted 1 month after the initial diagnosis (after treatment), and the VAS score, JOA score and ODI index were recorded. At the same time, MRI imaging examinations were performed on the patients.

[0135] 2. Experimental Results

[0136] 1. The results of nuclear magnetic resonance imaging of mice in each group are shown in the figure below. Figure 7 As shown, Figure 7 A in the figure is the T2WI image of mice in each group. Figure 7 B in the figure is the statistical result of the gray value of the nucleus pulposus image in the intervertebral disc of each group of mice. Figure 7 C in the figure is the statistical result of gray value of nucleus pulposus image in paraspinal tissue of each group of mice.

[0137] The results showed that in the control group 2, the central nucleus pulposus of the intervertebral disc of mice showed a high signal, and the intervertebral height was basically the same, and no obvious high signal expression was found in the paravertebral tissue (i.e., the nucleus pulposus did not protrude from the annulus fibrosus into the paravertebral tissue); compared with the control group 2, the central nucleus pulposus signal of the intervertebral disc of mice in the model group 2 was significantly reduced, and a high expression signal was shown in the paravertebral tissue, indicating that the central nucleus pulposus of the intervertebral disc protruded into the paravertebral tissue; compared with the model group 2, the central nucleus pulposus signal of the intervertebral disc of mice in the blood circulation group did not show a significant change, but the high expression signal in the paravertebral tissue was significantly reduced, indicating that the protruding nucleus pulposus in the blood circulation group was reabsorbed.

[0138] The statistical results of the gray value of the nucleus pulposus of each group of mice ( Figure 7 B and C) showed that compared with control group 2, the gray value of the nucleus pulposus in the central part of the intervertebral disc in the model group was significantly decreased, while the gray value of the nucleus pulposus in the paravertebral tissue was significantly increased (P < 0.01); compared with model group 2, the gray value of the nucleus pulposus in the central part of the intervertebral disc of mice in the blood circulation group did not change significantly (P > 0.01), while the gray value of the nucleus pulposus in the paravertebral tissue was significantly reduced (P < 0.05).

[0139] This indicates that the herniated nucleus pulposus of the mice with ruptured disc herniation in the blood circulation group showed reabsorption, and the ruptured disc herniation mouse model constructed by the construction method shown in Example 1 can be used for drug efficacy evaluation, especially for drug efficacy evaluation for promoting reabsorption of disc herniation.

[0140] 2. Clinical patient test results Figure 8 As shown, Figure 8 A in the figure is the VAS score result chart of clinical patients before and after treatment. Figure 8 B in the figure is the MRI image of the clinical patient before treatment. Figure 8 C in the figure is the MRI image of the clinical patient after treatment.

[0141] After taking the Huoxue Zhitong prescription for one week, the patient's low back pain was significantly relieved, there was no obvious pain in the left lower limb, and the patient could move freely, but he still felt pain after fatigue. The MRI images at the follow-up visit (i.e., the MRI images after treatment) showed that the intervertebral disc herniation had been reduced, the nerve root compression symptoms were significantly alleviated, and the absorption rate of the nucleus pulposus herniation was close to 20%. At the same time, the VAS score of the clinical sample after treatment was 2 points, the JOA score was 23 points, and the ODI index was 11%, all of which showed that the Huoxue Zhitong prescription promoted the reabsorption of ruptured disc herniation.

[0142] The results after administering the Huoxue Zhitong prescription to clinical patients were consistent with the results of the medication in the ruptured disc herniation mouse model constructed by the construction method shown in Example 1, indicating that the ruptured disc herniation mouse model constructed according to the construction method shown in Example 1 can be effectively used to evaluate the effect of drugs in ruptured disc herniation.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for constructing an animal model of intervertebral disc herniation, characterized in that: The following steps are involved: Under ultrasound guidance, the intervertebral disc position of the animal is marked, and an injection needle is used to insert the needle 1.5 to 5 mm into one or more of the Co4 / 5 to Co9 / 10 intervertebral discs of the animal, and then the tail of the animal is repeatedly squeezed 3 to 20 times to obtain an animal model of intervertebral disc herniation; The number of squeezes is calculated as follows: squeeze the tail of the animal from the distal end to the proximal end for 1 to 10 seconds, and then stretch the tail from the proximal end to the distal end for 1 to 10 seconds, which is considered as one squeeze. During the extrusion process, the extrusion speed and the stretching speed are both 30-300 mm / min.

2. The construction method according to claim 1, characterized in that: Use a 20-33G injection needle to insert the needle.

3. The construction method according to claim 2, characterized in that: Use a 20-33G injection needle to insert the needle 1.5-5 mm into 1-5 consecutive intervertebral discs among the Co4 / 5-Co9 / 10 intervertebral discs of the animal.

4. The construction method according to claim 3, characterized in that: Use a 20-33G injection needle to insert the needle 3 mm into 1-5 consecutive intervertebral discs among the Co4 / 5-Co9 / 10 intervertebral discs of the animals.

5. The construction method according to claim 1, characterized in that: The tail of the control animals was squeezed repeatedly 10 times.

6. The construction method according to claim 1, characterized in that: The extrusion speed and the stretching speed are both 150 mm / min.

7. The construction method according to claim 1, characterized in that: The animal's tail was repeatedly squeezed by a syringe pump.

8. The construction method according to claim 7, characterized in that: The method of controlling the repeated squeezing of the tail of the animal by the injection pump is specifically as follows: the animal is fixed at the syringe position of the injection pump, and the distal end of the tail of the animal is fixed on the injection fixing plate of the injection pump, the injection speed is controlled to be 30 to 300 mm / min, and the injection fixing plate is controlled to cyclically push for 3 to 20 times; wherein the number of cyclic pushes is calculated as follows: the injection fixing plate is controlled to push from the distal end to the proximal end for 1 to 10 seconds, and then the injection fixing plate is controlled to push from the proximal end to the distal end for 1 to 10 seconds, which is regarded as one cyclic push.

9. An animal model of intervertebral disc herniation obtained by the construction method according to any one of claims 1 to 8.

10. Use of the intervertebral disc herniation animal model according to claim 9 in the study of intervertebral disc herniation reabsorption and / or intervertebral disc degeneration.

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