动物造模,膝骨性关节炎 z-bo 2023-03-04 607

　　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 a variety of treatment methods are used, including drug treatment, intra-articular injection of hyaluronic acid, and surgery. As part of studying new targets for knee osteoarthritis pain, we are focusing on molecules related to pain, including nerve growth factor (NGF). NGF not only plays an important role in maintaining and developing the nervous system, it is also the main cause of inflammation and injury. Systemic injection of nerve growth factor induces thermal pain and mechanical hyperalgesia. Systemic injection of NGF antibody reduces allodynia and hyperalgesia in animal models of neuropathic pain, including nerve trunk or spinal nerve ligation. There have been several animal models for basic research on knee osteoarthritis. Including anterior cruciate ligament rupture model, this model rat medial meniscus tear. The MIA injection model causes progressive damage to the joints, and certain characteristics may be considered similar to OA and have significant pain-related behaviors. The MIA injection model is better than the knee joint pain assessment model, and animal models including the medical meniscus model better approximate the anatomical pathology of human osteoarthritis. Previous reports used the von Frey test to assess pain-related behavior, which was a mechanical evaluation. Clinically, patients with knee osteoarthritis suffer from knee joint pain, including gait disturbances 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 impaired gait function in knee OA pain models. Regarding the pathological mechanism of knee OA pain, previous immunohistochemical analysis showed that the expression of pain-related molecules in the sensory nervous system in the knee OA pain model increased. This finding proves the upregulation of pain-related molecules in the sensory nervous system under pain. It is hypothesized that anti-NGF is effective in treating knee joint pain in mice with knee osteoarthritis. The purpose of this study is to use the gait analysis system and immunohistochemical analysis to evaluate the role of NGF antibody in knee OA pain model.

　　Method: Knee osteoarthritis pain model: 30-week-old male C57BL/6 mice. (Control group: n = 10; MIA without treatment (non-treatment group): n = 10; MIA + anti-NGF treatment (anti-NGF) group: n = 10). During the experiment, the animals were anesthetized with sodium pentobarbital (40 mg /Kg, intraperitoneal injection). In the non-treatment group and anti-NGF group, a single injection of 10ul normal saline containing 0.2mg MIA was given to the right knee joint. The knee is injected through the patellar ligament with a 27G needle at a 90 degree angle. Three weeks after the operation, the mice were randomly divided into two treatment groups to receive sterile normal saline (10 mg/kg, intraperitoneal injection) (non-treatment group) or anti-NGF antibody (10 mg/kg, intraperitoneal injection) (anti-NGF group) . The efficacy of this drug in the treatment of neuropathic pain has been previously confirmed. In order to detect the right knee joint dorsal root ganglion (DRG) neurons, all 20 animals were injected with 2% retrograde neuron tracer FG into the right knee joint cavity. The control group was injected with 10ul of sterile normal saline into the joint cavity of the right knee, and 3 weeks later, 2% retrograde neuron tracer FG was injected into the joint cavity.

　　Behavior evaluation (gait analysis): Simply put, the mouse is placed on a glass plate in a dark room and can walk freely. The light beam of the fluorescent lamp passes through the glass plate. The beam is completely internally reflected. When a paw touches the glass plate, the light beam reflects downward. This resulted in a sharp and bright image of the paw print. The entire running process is recorded with a camera. These data are acquired, compressed, and analyzed using pattern software according to the previous description. Before operation and 3 (pretreatment; pretreatment) 4 weeks or 5 weeks after operation (1 or 2 weeks after treatment), all mice walked on the glass plate 3 times. Record 3 gaits. We compared the ratio of ipsilateral and contralateral hindlimb movements in three variables between the three groups, namely duty cycle; swing speed; and printing area.

　　Immunohistochemical analysis: all procedures, including anesthesia, perfusion, sectioning, staining, observation and evaluation of immunopositive neurons. In all three groups, four (n = 5) or five (n = 5) weeks after surgery (one or two weeks after treatment), mice were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal injection) . 0.9% saline perfusion followed by 30ml perfusion of 4% paraformaldehyde phosphate buffer. Cut the right DRGs of L3-L5. The samples were fixed in 4% paraformaldehyde phosphate buffer at 4 degrees, and then stored at 4°C in 0.01 M PBS containing 20% sucrose for 20 hours. Frozen sections were soaked in 0.3% hydrogen peroxide solution dissolved in 0.01MPBS. Inactivate peroxidase in tissues. Then it was treated in 0.01M PBS solution at room temperature for 90 minutes. Explore the expression of DRGs neuropeptide and CGRP. 30 samples in each group were observed using a fluorescence microscope. The number of FG-labeled neurons and FG-labeled and CGRP immunoreactive neurons was calculated.

　　Result: Behavior analysis: Compared with the control group, the non-treatment group, the contralateral duty cycle of the pretreatment group, 1 week after treatment, and 2 weeks after treatment, was significantly reduced. Compared with the control group, on the same side of the anti-NGF group, the contralateral hindlimb duty cycle value was significantly reduced only before treatment. In addition, the ratio of the anti-NGF group was significantly increased compared with the non-treatment group. Pretreatment, 1 week after treatment, and 2 weeks after treatment, the contralateral swing speed was significantly lower than that of the control group. Compared with the control group, the anti-NGF group only significantly decreased before treatment. One week after treatment, the contralateral swing speed was significantly improved compared with the non-treatment group. Compared with the control group, the ratio of printed areas on the ipsilateral and contralateral side of the non-treatment group 1 week and 2 weeks after treatment decreased significantly. The anti-NGF group only decreased significantly before treatment. 1 week and 2 weeks after treatment, compared with the non-treatment group, the anti-NGF group was significantly improved.

　　Immunohistochemical analysis: Compared with the control group, FG-labeled and CGRP immunolabeled neurons increased significantly in the non-treatment group 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 week and 2 weeks after treatment.

　　Conclusion: MIA injection into the knee joint induces gait disturbance and up-regulation of CGRP expression in DRG neurons in a mouse knee osteoarthritis pain model. In addition, intraperitoneal injection of anti-NGF antibody inhibits gait disturbance and up-regulation of GRP in DRGC neurons. These results indicate that anti-NGF therapy may be valuable for knee osteoarthritis.