动物造模,小鼠膝骨性关节炎疼痛模型 z-bio 2021-02-02 596

　　Background: Knee osteoarthritis (OA) is a common chronic degenerative disease characterized by degeneration of articular cartilage components, synovitis, subchondral bone remodeling and joint muscle atrophy. Patients with knee osteoarthritis usually suffer from knee joint pain and use a variety of treatments, including medications, intra-articular injections of hyaluronic acid and surgery. As part of our research on new pain targets for knee osteoarthritis, we will focus on pain-related molecules, including nerve growth factor (NGF). GF not only plays an important role in the maintenance and development of the nervous system, but also is the main cause of inflammation and injury. Systemic injection of nerve growth factor can cause fever and mechanical hyperalgesia. In animal models of neuropathic pain (including nerve trunk or spinal nerve ligation), systemic injection of NGF antibodies can reduce allodynia and hyperalgesia. There are several animal models available for basic research on knee osteoarthritis. This model, including the anterior cruciate ligament rupture model, is a rat medial meniscus injury. The MIA injection model causes progressive damage to the joints. Certain attributes are considered similar to OA and exhibit behaviors related to severe pain. The MIA injection model is better than the knee osteoarthritis assessment model, while the animal model (including the medical meniscus model) more closely matches the anatomical pathology of human osteoarthritis. Previous reports used the von Frey test to assess behavioral and mechanical assessments related to pain. Clinically, patients with knee osteoarthritis suffer from knee joint pain, including gait disturbance, but no mechanical hyperalgesia. Some authors use gravimetric analysis to assess knee pain behavior. For example, the rat gait analysis system can quantitatively evaluate the gait and motor function of rats and mice. This system was recently used to analyze gait dysfunction in a knee OA pain model. Regarding the pathological mechanism of knee osteoarthritis, previous immunohistochemical analysis showed that the expression of pain-related molecules in the sensory nervous system of knee osteoarthritis pain model increased. This finding demonstrates the upregulation of pain-related molecules in the sensory nervous system under pain. It is speculated that anti-NGF can effectively treat knee joint pain in mice with knee osteoarthritis. The purpose of this study is to use gait analysis system and immunohistochemical analysis to evaluate the role of NGF antibody in knee OA pain model.

　　 Method: Pain model of knee osteoarthritis: 30-week-old male C57BL/6 mice. (Control group: n = 10; untreated MIA (untreated group): n = 10; MIA + anti-NGF treatment (anti-NGF) group: n = 10) Animals were anesthetized with pentobarbital sodium (40 mg). ) During the experiment/kg, intraperitoneal injection). In the untreated and anti-NGF group, a single injection of 10 ul saline containing 0.2 mg MIA was given to the right knee joint. Use a 27G needle to inject the through bone ligament into the knee at a 90 degree angle. Three weeks after the operation, the mice were randomly divided into two treatment groups: sterile saline (10 mg/kg, intraperitoneal injection) (non-treatment group) or anti-NGF antibody (10 mg/kg, intraperitoneal injection). -NGF group). The efficacy of the drug in the treatment of neuropathic pain has been previously confirmed. In order to detect DRG neurons in the right knee joint, all 20 animals were injected with 2% retrograde neuron tracer FG into the right knee joint space. In the control group, 10 ul of sterile saline was injected into the joint space of the right knee, and 3 weeks later, 2% retrograde neuron tracer FG was injected into the joint space.

　　 Behavior evaluation (walking analysis): In short, put the mouse on a glass plate in a dark room and walk freely. The fluorescent beam passes through the glass plate. The beam is completely internally reflected. When your feet touch the glass plate, light will reflect downward. This makes the footprints clear and clear. The camera records the entire running process. As mentioned above, use pattern software to obtain, compress and analyze these data. Before the operation and 4 or 5 weeks after the operation (1 or 2 weeks after treatment) for the third time (pretreatment; pretreatment), all mice walked on the glass plate 3 times. Record 3 steps. Three variables were compared between the three groups: duty cycle, swing speed, and the ratio of ipsilateral and contralateral hindlimb movements in the printed area. Immunohistochemical analysis: all procedures, including anesthesia, perfusion, sectioning, staining, observation and evaluation of immunopositive neurons. In all three groups 4 weeks (n = 5) or 5 weeks (1 or 2 weeks after treatment) after surgery, mice were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal injection). Perfusion with 0.9% saline, then 30 ml of 4% paraformaldehyde phosphate buffer. Cut the appropriate DRG for L3-L5. The samples were fixed with 4% phosphate paraformaldehyde buffer at 4°C and stored at 4°C for 20 hours at 0.01°C BSBS and 20% sucrose. The frozen sections were immersed in a 0.3% hydrogen peroxide solution dissolved in 0.01 MPaBS. Inactivate peroxidase in tissues. Then it was treated in a 0.01 MPaS solution at room temperature for 90 minutes. Check the expression of DRG neuropeptide and CGRP. A fluorescence microscope was used to observe thirty samples in each group. We calculated the number of FG-labeled neurons and the number of FG-labeled and CGRP immunoreactive neurons. Results: Behavioral analysis: In the untreated group, compared with the control group, the contralateral duty cycle was significantly reduced before treatment, after treatment, after treatment, and two weeks after treatment. Compared with the control group, on the same side of the anti-NGF group, the duty cycle value of the contralateral hind limb was only significantly reduced before treatment. In addition, compared with the untreated group, the proportion of the anti-NGF group increased significantly. group. Before treatment, 1 week after treatment and 2 weeks after treatment, the contralateral swing rate was significantly lower than that of the control group. Compared with the control group, the anti-NGF group only significantly decreased before treatment. After 1 week of treatment, compared with the untreated group, the contralateral swing speed was significantly improved. Compared with the control group, after 1 week and 2 weeks of treatment, the ratio of printed area on the same side and the opposite side of the untreated group was significantly reduced. The anti-NGF group only decreased significantly before treatment. After 1 and 2 weeks of treatment, compared with the untreated group, the anti-NGF group improved significantly. Immunohistochemical analysis: Compared with the control group, the FG-labeled and CGRP immunolabeled neurons of the untreated group increased significantly at 1 and 2 weeks after treatment. Compared with the non-control group, the expression of CGRP in the anti-NGF group was significantly reduced 1 and 2 weeks after treatment. Conclusion: MIA injection into the knee joint can cause gait disturbance and up-regulation of CGRP expression in DRG neurons in mouse osteoarthritis pain models. In addition, intraperitoneal injection of anti-NGF antibody can inhibit the gait disturbance of DRGC neurons and the up-regulation of GRP. These results indicate that anti-NGF therapy may be useful for knee osteoarthritis.