动物造模,慢性痛瘢痕动物模型 z-bio 2021-10-11 613

　　Background: Persistent pain in the limbs and back pain after surgery can develop into chronic pain, which affects the activities of many patients and leads to a decline in the quality of life. In order to determine the histopathology and pathological mechanisms that cause persistent pain, establish an animal model of persistent painful scar, and then correlate it with the pain of the surrounding tissues and spinal nerves, and study behavior and pathological changes. Method: Rats extensively stripped the subcutaneous tissue of their hind paws to create an animal model (scar group), and examined pain-related behaviors for the next 12 weeks. After that, scar tissue staining, immunohistochemical staining of c-fos (L5 dorsal horn), and electron microscope analysis of L5 spinal nerve fibers/dorsal root were performed. Result: The mechanical pain threshold of animals injured on the ipsilateral sole was lowered. Maintain this state for 12 weeks. There are many fibroblasts in the collagen layer of scar tissue. It was found that there was no statistical difference in the c-Fos positive area of ipsilateral neurons between the scar group and the L5 level side group (sham operation) of the posterior angle of the pinhole. However, an incision of myelinated nerve fibers was observed at the dorsal root of L5 on the same side.

　　Conclusion: We have established a persistent pain scar model by extensively destroying surrounding tissues. It has been observed that the skin tissue is fibrous and thickened, which can cause sensitization and partial degradation of spinal nerves associated with painful scars. This model allows us to better understand the sensitization mechanism caused by painful scars and explore new methods for humans to treat painful scars.

　　Introduction: Scar formation is an indispensable process for repairing wounds and surgical incisions. According to reports, refractory low back pain syndrome (FBS) in scar tissue is related to peripheral nerve dysfunction. Spinal cord decompression can relieve pain in patients who usually suffer from persistent low back pain. In some cases, the pain is still there, and the nerves after decompression indicate that the peripheral nerve dysfunction in the scar site can cause chronic pain. Motor nerve innervation and muscle atrophy after back surgery were also observed, indicating that sensory nerve damage may occur. Another study reported that chronic pain in patients undergoing total hip replacement is related to intraoperative nerve damage. Several animal models of chronic pain caused by nerve damage help to understand the mechanism of neuropathic pain. However, due to the lack of specific animal models, there is currently no useful method to understand the basic mechanism of chronic pain in scar tissue. We have developed an animal model of painful scars that can be used to induce reproducible and quantifiable mechanical hyperalgesia in the subacute phase of 4 weeks. The purpose of this study is to use this animal model to determine the behavioral characteristics of pain-related scar tissue, and to study the effect of painful scars on spinal nerves and dorsal horn neurons formed over a 12-week period. In this study, behavioral and immunohistochemical techniques were used to detect changes in pain-related behaviors over time, pathological changes in surrounding tissues and spinal nerves, and sensitization of neurons in the dorsal horn of the spinal cord. Experimental animals: 26 male SD rats weighing 150 g were used. These animals are housed in plastic cages with soft bedding, where they can eat and drink freely. The rats were adapted to the environment at least 5 days before the operation and were divided into two groups (pinhole and scar group). Operation: All rats were injected intraperitoneally with sodium pentobarbital (50 mg/kg). In the scar group (n = 13, painful scar on the foot), an 18G needle was used to make a pinhole on the left heel (pinhole group). Create a scar group by inserting a steel rod into the needle hole and peeling off the subcutaneous tissue (= 3 mm). In the pinhole group (left foot, N=13 sham operation), an 18G needle on the left heel was used to create a pinhole (Figure 1). There was no surgery on the right side of any animal. Behavioral test: After the operation, the rat was returned to the original cage and kept under the same conditions as before the operation. In order to quantify the mechanical threshold of the hind paw, the response of hind paw retraction to the gradient mechanical stimulus was measured. The mechanical stimulation uses VonFrey filaments. During the test, all rats were placed on a metal mesh floor covered with a transparent plastic dome (8 * 8 * 18 cm), and the sensitive area of each hind paw was mechanically stimulated with von Frey filaments. When in use, each filament will be slightly bent, ensuring that there is sufficient force in each case. Perform 10 consecutive Freyfilament operations within 10 seconds, and then gradually conduct a series of tests starting from the weakest intensity. Observation of significant withdrawal is considered a positive reaction, while the weakest positive reaction is considered a threshold. All these behavioral tests were performed before scar formation (day 0) and continued once a week until 12 weeks after surgery. Preparation for histological and immunohistochemical analysis: 12 weeks after the operation, all rats were anesthetized with an excess of isoflurane and perfused with 4% paraformaldehyde, 0.1 M phosphate buffer, pH 7.2, and collected including spinal cord and calf Tissue samples inside. The removed tissue specimens were re-fixed with 0.1 M phosphate buffer 4% paraformaldehyde and pH 7.2. In order to maintain the shape of the sample, the tissue is immersed in a 5% to 20% gradient sucrose solution. Immediately freeze half of the spinal cord and foot samples, and cut all samples with a cryostat set to a thickness of 15 microns. After washing 3 times with TBST (Tris buffered saline containing 0.1% Tween 20), the sample sections were incubated in a blocking solution (4%) at room temperature for 2 hours. The sections were washed 3 times with washing buffer (0.4% blocking agent containing 0.1% Tween 20). Then incubate with anti-c-Fos antibody (1:1000) at 4°C for 24 hours. After washing 3 times, incubate with goat anti-rabbit IgG antibody and Alexa Fluor 546 for 2 hours (1:400) at room temperature. Finally, wash the lotion 5 times and add ProLong? with DAPI? Gold Antifad reagent, and mount the slide. All 6 rats in the scar and pinhole group were stained by immunohistochemistry. Cut the L5 horizontal spinal cord of each animal into thin slices. After all cross-sectional images are obtained, c-fos positive neurons are evaluated. Measure the protein expression area of ipsilateral or contralateral scars or pinhole meta-finger neurons, examine each rat section under a deconvolution fluorescence microscope, and use dynamic cell counting software for image analysis. In order to analyze c-Fos-IR neurons, the posterior horn of the spinal cord is divided into two parts: the superficial layer (layer I-II) and the deep layer (layer III-V).

　　Histopathology: Embed the foot in paraffin and cut into 5-10 micron thick sections. All tissues were sliced serially, and the scar tissue formed on the bottom of the left hind limb was stained with hematoxylin-eosin (HE) and Masson's trichrome. Five rats in the scar and pinhole group underwent histopathological examination.

　　Electron microscopic analysis: immediately remove the L5 spinal nerve and dorsal root ganglia, and chop them with a razor. Spinal nerves were first fixed with 2% glutaraldehyde, and then with 1% OsO4. The sections were stained with uranyl acetate and lead citrate and examined under a microscope. Results: Pain-related behavior: von Frey performed mechanical stimulation, and the pain threshold over time is shown in the figure. Compared with the preoperative control value, there was no obvious threshold change at any time on the opposite side of the hind paw in the pinhole group or the hind paw in the scar group. However, in the scar group, the withdrawal threshold of the ipsilateral hind limb was lowered at 5, 9 and 10 weeks postoperatively. In addition, compared with the other side of the same animal, the withdrawal threshold of the same side (treatment side) at 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, and 12 weeks after surgery is significantly lower Comparing the pinhole and scar groups, we found that the ipsilateral hind paw and the contralateral hind paw had lower exit thresholds at all time points after surgery. Histopathological examination of scar pemphigoid: Compared with the pinhole group, the dermis and epidermis of the scar group were thicker and contained more collagen fibers. It was found that the size of the collagen fiber bundles changed and the boundary between the dermis and the subcutaneous area was not clear. Many migrating cells with round or oval nuclei were observed between collagen fibers. Compared with the pinhole group, the scar group showed collagen fibers aggregation in the dermis and subcutaneous layer. The orientation of these collagen fibers is random. Five rats in the scar and pinhole group showed similar histological results. Immunohistochemical examination of the dorsal horn of the spinal cord: there was no statistically significant difference in protein expression between the ipsilateral and contralateral C-Fos-IR neuron regions of the L5 horizontal plate I-II of the dorsal horn. Pinhole group. Similarly, between these groups, there was no statistically significant difference in protein expression at the L5 level of the dorsal horn of the spinal cord, the ipsilateral and contralateral C-Fos-IR neuron regions of the III-V layer.

　　Electron microscope analysis of nerve fibers: The nerve fibers on the control side are normal. No axon or myelin degeneration was observed. The plasma membrane of the axon is in close contact with the myelin sheath. In contrast, injured nerve fibers showed fragments of myelin sheath, while Schwann sheath and axons were intact. There is no close contact between the axon plasma membrane and myelin sheath.

　　Discussion: Skin scars are defined as "visually visible skin structures that interfere with normal structure and function through wound healing". Therefore, scarring is an abnormal skin structure and regeneration failure, not a biomechanical problem. Scars are also considered to be one of the factors related to chronic pain. The increased density of substance P and calcitonin gene-related peptide (CGRP) in wound healing may be related to these unpleasant sensory symptoms of wound healing. People suffering from chronic pain after open surgery have discovered substance P and calcitonin gene-related peptide that are connected to the peritoneum. It has been confirmed that the posterior horn of the ipsilateral spinal cord has regulation of substance P and calcitonin gene-related peptide. Pathological examination of the scarred foot shows that hyperplastic scars are considered painful. Several types of chronic pain animal models have been developed and used to study the development and maintenance of chronic pain mechanisms. Several chronic pain models, such as neuropathic pain, myalgia models, arthralgia and bilateral spinal arthralgia models, have shown hyperalgesia in previous behavioral and immunohistochemical studies. In this study, the withdrawal threshold of the ipsilateral hind paw was lower than the preoperative value. Compared with the pinhole group at the same time point (including the same side and the opposite side), the contralateral withdrawal threshold of the scar group was reduced.

　　Conclusion: Twelve weeks after the operation, we developed an animal model of painful scar formation and postoperative hyperalgesia. This may be the result of changes in the plasticity of the central nervous system due to persistent pain. Our model allows us to better understand the sensitization mechanism caused by painful scars and will help us in the future to study new methods for treating human painful scars.