大鼠椎间盘退变疼痛模型 z-bo 2020-12-03 351

　　Introduction: The intervertebral disc is a unique structure composed of a tough outer ring, the annulus fibrosis (AF), and a gel-like inner core, the nucleus pulposus (NP). Although there have been in-depth studies on the process of disc degeneration, the exact mechanism of discogenic low back pain has not yet been elucidated. Many functions of discogenic pain have not been explained, including severe disc degeneration in a few patients, severe clinical infections, and chronic back pain.

　　 Animal pain models are essential for understanding the complexity of pain and the development and testing of new therapies. Clinical assessment of joint pain is performed by observing human movement and reflex contact, and by asking patients to report the quality and intensity of their pain. Animal pain can be observed: 1. Pain-related behaviors, such as pain or hissing from licking or biting, shaking of the affected limb; 2. Response to thermal or mechanical stimulation. The rabbit disc puncture model is useful in the study of the biological mechanism of disc degeneration and experimental therapy of disc regeneration. After the circular needle puncture, the rabbit's lumbar intervertebral disc gradually degenerated. Quantitative assessment of degeneration through conventional radiography, magnetic resonance imaging (magnetic resonance imaging) and histological examination. However, rabbits often exhibit minimal pain behavior during disc degeneration in this model. When rabbits feel pain, they restrict their activities and cannot thrive. Rabbits do not show pain behavior; therefore, they are not suitable for the study of discogenic low back pain. Due to the sensitive behavioral responses of rats (for example, vocalization), rat models have been widely used to study chronic inflammation and neuropathic limb pain and the pharmacokinetics of analgesics. Most studies on disc degeneration are in the tail of the rat, not the lumbar disc. Part of the reason is due to its access to anatomy and minimal surgical morbidity. Damage to the intervertebral disc in the tail of the rat may cause a painful response. However, unlike the vertebral disc, the tail disc is not weight-bearing, and the neuroanatomy or the involved components are still unclear. Therefore, it is quite difficult to explain the harmful pathways produced. A recent study showed that a behavioral model that measures pain in rats may be suitable for studying discogenic low back pain. They claim that rats induced by the disc puncture needle (0.4 mm in diameter) can increase the grooming and general pain behavior ("wet dog") shaking. In our current research, we are trying to establish a new correlation model between pain and pathological changes in the structure of the disc. The chronic discogenic low back pain we developed is an evaluation of a behavioral hyperalgesia animal model. We studied the biological connection between the changes in the cells and structures within the disc components and the development of symptoms of chronic low back pain. We also describe the symptoms associated with pain modulators in the dorsal root ganglion (DRG) and spinal back pain. We conducted pharmacological trials to explore the potential for therapeutic analgesia to modulate chronic back pain.

　　Materials and methods: Induction of intervertebral disc degeneration: Adult SD rats, (250 to 300g), fasted for 24 hours before surgery, it is easier to perform abdominal punctures. The animal is anesthetized with oxygen (1 L/min) and 1.5% halothane through a mask to maintain anesthesia. Use aseptic technique for surgery. In the supine position of the animal, the hair on the abdomen was shaved, disinfected with a local disinfectant (chlorhexidine gluconate) and alcohol three times, and the abdominal cavity was opened with a surgical blade. The midline incision of the abdomen is about 1.5 to 2 cm, and the abdominal organs are gently moved to observe the disk space of the spine and lumbar spine. The viscera is irrigated and preheated with sterile saline and covered with sterile gauze to keep the tissue moist. Induce intervertebral disc degeneration, and insert a disc puncture microdrill (0.5 mm in diameter and 0.25 square mm in area, equivalent to a 25-gauge needle) into the intervertebral disc (L4, L5 and L5, L6) to a depth of 2 mm. Cut 2 mm shorter than the length of the drill bit by using a polyethylene plug sleeve. The muscle is sutured with silk thread, and all the skin edges are sutured with 4-0 nylon thread. Take off the anesthesia mask and put it back in a clean cage. Two days after the operation, all animals were injected with buprenorphine 0.1 mg/kg twice a day for postoperative analgesia. The animals are monitored to ensure that there are no serious injuries. Another group of animals used a larger drill to puncture the disc. For the sham operation control group, the disc was exposed in the same way as the experimental group, but without puncture (12 in each group). The biochemical, histological, and imaging analysis of disc degeneration is described in the following sections for evaluation. The animal model of neuropathic pain caused by L5 spinal nerve ligation was used to compare the tissue protein and RNA levels of the intervertebral disc degeneration model.

　　 Animal behavior test: stress test vocalization threshold: press the 0.5-cm2 device prompt to directly puncture the back skin to measure the hissing threshold based on the force of the stress gauge, and compare it with the threshold obtained from the sham operation control group. At 100g/s, the force is slowly increased until you hear a hiss. Use a cutting force of 1000g to prevent tissue damage. Repeat the test, and the average value is the injury threshold. Postoperative detection was delayed until one week after surgery, allowing abdominal tissue to heal. The pain test is clinically relevant because the patient’s pain is given to the mechanical pressure test on the disc to assess the degree of their back pain.

　　 Mechanical hyperalgesia: Allow the rat to adapt to a wire mesh covered with a transparent plastic cage for 15 minutes, and a calibrated von Freys is applied from the sole of the foot to determine the 50% force pain threshold. The filament strength ranges from 0.04 to 15 grams, starting with 2 grams. The filament is applied to the skin with sufficient pressure and held for up to six seconds. A brisk lifting of the foot was recorded as a positive response.

　　 Detect the mechanical pain threshold of the cervical spondylopathy model as an indicator of secondary allergies in both legs. Calculate the average value of the left and right sides from all leg pain test data.

　　 Movement incoordination: The rat is placed on a rotating rod (10rpm rotation performance test), and its coordination and balance ability is recorded after the fall time is delayed. Drug therapy: The drug treatment experiment was started seven weeks after the induction of intervertebral disc degeneration. The rats were given 0.5ml of drug solution intraperitoneally (ip) or intragastrically (po). The test drug was α2δ1 subtype calcium channel blocker. Rebalin 20mg/kg. Mixed cyclooxygenase (COX-1 and COX-2) inhibitors, ip ketorolac tromethamine 20mg/kg. COX-2 selective inhibitor, celecoxib 50 mg/kg orally. μ-opioid receptor agonist, morphine sulfate 6.7mg/kg, ip and excipients. Each medicine has an interval of three to four days to minimize the impact of drug residues. Due to the repeated noxious stimulation itself, pressure hyperalgesia drugs are injected at most four time points to limit local tissue hypersensitivity. No drug dose caused sedation or uncoordinated movement of rats.

　　Histochemistry and data analysis: The animals were sacrificed, and each vertebral lumbar motion segment was aseptically dissected. The tissue was fixed in 4% paraformaldehyde, decalcified ethylenediaminetetraacetic acid solution (replaced every five days). The decalcified disc was cut and embedded in paraffin. Cut into 5 micron slices. Safranin O-fast green staining was used to observe gross morphology and loss of cartilage proteoglycan. We compare all the obtained disc grading scales, with scores ranging from 4 to 12: Type I = AF, Type II = the boundary between AF and NP, Type III = cells in NP and Type IV = NP matrix. Because each category has a maximum of 3 points, a total score of 12 (level 12) represents severe degradation. Non-blind researchers read the pictures in groups of animals. Then randomly grade. Toluidine blue staining to observe the general morphology of mast cells. Immunohistochemistry was performed using anti-CD11b antibodies according to established methods. In short, to block endogenous peroxidase, tissue sections were incubated overnight for primary rat anti-mouse macrophage CD11b antibody binding. Tissue sections were then incubated with secondary biotin antibody, followed by culture with streptavidin-horseradish peroxidase conjugate and 0.5 mg/mL benzidine.

　　 Intervertebral disc imaging analysis: Use mx-20 specimen X-ray photography system to measure the height of the intervertebral disc. In the final stage of the experiment, the lumbar motion segments (L4, L5, L5, L6) were removed and filmed. The images are digitized, evaluated and graded. Double-blind independently measured disk height. Results: Disc puncture causes chronic back pain symptoms: We have previously tried to use No. 21 drill (0.8 mm diameter and 0.64 square mm area) to induce progressive back pain, but compared with the sham operation group, it gradually developed within a week Traumatic pressure hyperalgesia. By using a smaller micro-drill (0.5 mm in diameter and 0.25 square mm in area, equivalent to level 25), compared to the sham-operated control group, applying force stimulation beside the L4/L5 panel induces the slow development of pressure hyperalgesia The neighing threshold. The model showed a period of persistent painful behavior (more than seven weeks after surgery) with strong discogenic back pain symptoms. There was no secondary hyperalgesia in the hind paws of the animals in the mechanical hyperalgesia test group and the sham operation group by Frey test. The use of a disk drill to puncture animals is often shown in the straight leg elevation test (often used to diagnose pain caused by compressed nerves) and often no pain response, such as caused by a herniated disc. There is no change in the performance of the animals tested with rotation.

　　 Hyperalgesia caused by intervertebral disc degeneration is related to disc stenosis: at the end of the ninth week after surgery, the lumbar motion segments are dissected, and the total height of the intervertebral disc is determined on the basis of X-ray imaging. The disc obtained by a rat is compared with the discs of its own and all other rats. Compared with the control group, significant disc stenosis was observed in the damaged spinal canal, and disc height decreased in about 55%. In order to rule out normal changes, the damage of the disc due to aging and/or altered movement may affect the adjacent disc, we compare the disc (injury and preoperative) in a single animal. Our results show that the height of the disc in the preoperative group is consistent with that of the sham operation control group. In the adjacent disks (L2 and L3 and L3, L4), we did not observe significant changes that may be due to the impact of the injured interdisk. The disc height was measured twice at a three-week interval to verify the repeatability of the disc height index. The error of the intervertebral height index measuring instrument is estimated to be the smallest (within standard deviation (SW) = 0.003, coefficient of variation is 8.5%). Hyperalgesia caused by disc puncture is related to changes in disc morphology: histological evaluation of disc specimens was made nine weeks after surgery. The morphological changes of the discs in the sham control group (n = 6) and the 25 gauge needle puncture experimental group (n = 6) were compared in the equivalent part. A semi-quantitative histological analysis was performed using a grading system, with safranin O staining and toluidine blue staining as described above. The control group showed a Masuda score of 4 points for the normal disc. A complete AF and normal fibrocartilage plate pattern, a clear boundary between AF and NP, and preserves the proteoglycan content. On the contrary, the experimental group showed severe damage to the disc, significant proteoglycan consumption, massive loss of NP and/or collapse of NPS. At 9 weeks postoperatively, the highest Masuda score was 12 points after the disc injury. Importantly, at nine weeks postoperatively, the pressure threshold for vocalization was 410±25 G in the disc puncture group and 958±26 g in the control group (P<0.001), indicating the correlation between disc degeneration and persistent pain. Mast cells (shown by strong toluidine blue staining) play a key role in wound healing and connective tissue inflammatory response. A large number of mast cells have been observed in the interdiscal injury. On the basis of immunohistochemical analysis, CD11b further validated our observation through specific antibodies of immune cells. The highly increased CD11b antibody immune response indicates a high degree of inflammation in the injured area of the disc.

　　 The pain pathway caused by intervertebral disc degeneration may overlap with neuropathic pain: neuropathic pain model, a group of pain modifiers in DRGs and spinal dorsal horn neurons are up-regulated (↑) or down-regulated (↓). These pathological molecules include calcitonin gene-related peptide (↓), substance P (↓) and brain-derived neurotrophic factor (↑), neuropeptide Y (↑), galanin (↑) through astrocytes The activation of TNFα, MCP-1 and TLR4 channels. In our animal model, symptomatic behavioral chronic back pain after intervertebral disc degeneration, we found the expression of TNFα, MCP-1 and TLR4 in DRG (L4/L5) and spinal dorsal horn. We observed a significant increase in the expression of α2δ1. The voltage-gated calcium channel of a subunit of α2δ1 is involved in the pathway of neuropathic pain. In animals, the dorsal horn of the spinal cord and disc-induced pain increase in mRNA and protein levels. These results indicate that the pathway of chronic pain caused by disc degeneration at least partially overlaps with neuropathic pain. The comprehensive comparison of gene expression profiles between neurogenic pain and intervertebral disc degeneration is shown in the table.

　　 Drug test for the therapeutic adjustment of chronic low back pain caused by intervertebral disc degeneration: To study the therapeutic modulation of drugs to treat chronic pain in animal models. Because the fake abdominal incision observed that the animal did not show any hyperalgesia. This indicated that their painful experience was not due to abdominal incisions, and they chose to use rat disc injuries (but not sham-operated animals) to test drugs, including morphine, celecoxib, ketorolac and pregabalin. Including morphine as a prototype control drug that is effective for certain types of pain (for example, nociceptive, inflammation, and postoperative pain). The effectiveness of morphine to reduce the primary pressure hyperalgesia of the disc indicates the noxious process caused by the injury. Celecoxib is used as a drug for inflammatory pain, but it is not effective for nociceptive (acute) pain. Pregabalin is effective for neuropathic pain and is not effective for inflammation or nociceptive pain.

　　 Conclusion: Our research results provide a basis for future research on new therapeutic interventions, which may improve patients' back pain caused by intervertebral disc degeneration.