动物造模,慢性痛瘢痕动物模型 z-bio 2022-01-13 655

　　Methods: Rats prepared animal models (scar group) by extensively stripping the plantar subcutaneous tissue of the hind paws, and examined pain-related behaviors in the next 12 weeks. After that, we performed scar tissue staining, immunohistochemical staining of c-fos (L5 dorsal horn), and electron microscopic analysis of L5 spinal nerve fibers/dorsal root.

　　Result: The mechanical pain threshold of scarred animals on the ipsilateral plantar decreased. This state was maintained for 12 weeks. There are many fibroblasts in the collagen layer of scar tissue. It was found that the c-Fos positive area of ipsilateral neurons was not statistically different in the scar group and the pinhole dorsal horn L5 level side group (sham operation). However, the broken myelin sheathed nerve fibers were observed at the position of the dorsal root of L5 on the same side.

　　Conclusion: We have established a persistent pain scar model by extensively injuring the surrounding tissues. It is observed that the skin tissue is fibrotic and thickened, which may cause sensitization and partially degrade spinal nerves related to 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 a process that is essential for wound and surgical incision repair. It has been reported that the refractory back pain syndrome (FBS) of scar tissue is associated with peripheral nerve dysfunction. Spinal cord decompression can generally reduce the pain of patients with persistent low back pain. In some cases, the pain still exists, and the nerves after decompression suggest that peripheral nerve dysfunction at the scar site may cause chronic pain. It has also been observed that motor nerve innervation and group muscle atrophy after back surgery, which indicates 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 us understand the mechanism of neuropathic pain. However, due to the lack of specific animal models, there is currently no useful method that can be used to understand the basic mechanism of chronic pain in scar tissue. We have developed an animal model of painful scar that can be used to provoke reproducible and quantifiable mechanical hyperalgesia in the subacute phase for 4 weeks. The purpose of this study was to use this animal model to determine the behavioral characteristics of pain-related scar tissue and to explore the effects of painful scar formation on spinal cord nerves and dorsal horn neurons in a 12-week period. This study used behavioral and immunohistochemical techniques to study the changes in pain-related behaviors over time, the pathological changes of surrounding tissues and spinal cord nerves, and the sensitization of spinal dorsal horn neurons.

　　Experimental animals: 26 male SD rats weighing 150g were used. The animals are housed in plastic cages with soft bedding, and they are free to eat and drink. The rats adapt to the environment at least 5 days before surgery and are divided into two groups (pinhole and scar group).

　　Operation: All rats were intraperitoneally injected with sodium pentobarbital (50mg/kg). In the scar group (n=13, painful scar on the foot), a needle hole was punctured with an 18G needle on the left heel (pinhole group). The scar group is created by inserting a steel rod into the needle hole and peeling off the subcutaneous tissue (= 3mm). In the pinhole group (left foot, N=13 sham operation), the pinhole was made with an 18G needle on the left heel (Figure 1). There was no surgery on the right side of any animal.

　　Behavior test: After the operation, the rat was returned to the original cage and kept under the same conditions used during the preoperative period. In order to quantify the mechanical threshold of the hind paw, the response of the hind paw withdrawal to the gradient mechanical stimulus was measured. Mechanical stimulation uses Von Frey filaments. During the test, all rats were placed on a metal mesh floor covered with a transparent plastic dome (8*8*18cm), and mechanical stimulation was applied to the sensitive area of each hind paw with von Frey filaments. When applied, there is a slight bend in each filament to ensure sufficient force in each case. Perform 10 consecutive Frey filament operations within 10s, and then perform a series of tests, starting from the weakest strength and gradually increasing. Observing a significant withdrawal is considered a positive response, and the weakest positive response is considered a threshold. All of these behavioral tests are before the scar (day 0) and continue every week until the twelfth week after the operation.

　　Preparation for histological and immunohistochemical analysis: 12 weeks after surgery, all rats were anesthetized with excessive isoflurane and perfused with 4% paraformaldehyde, 0.1 M phosphate buffer, pH 7.2, and collected tissue samples, including spinal cord and double foot. The removed tissue specimens were re-fixed in 0.1 M phosphate buffer 4% paraformaldehyde, pH 7.2. In order to maintain the shape of the sample, the tissue is immersed in a gradient sucrose solution starting from 5% to 20%. The spinal cord and half of the foot samples were quickly frozen, and all the samples were sectioned with a cryostat set to a thickness of 15 microns.

　　After washing three times with TBST (Tris buffered saline containing 0.1% Tween 20), the sample slices were incubated in blocking solution (4%) for 2 hours at room temperature. The sections were washed three times with washing buffer (0.4% blocking agent with 0.1% Tween 20). Subsequently, incubate with anti-c-Fos antibody (1:1000) for 24 hours at 4°C. After washing three times, incubate with goat anti-rabbit IgG antibody and Alexa Fluor 546 for 2 hours (1:400) at room temperature. Finally, wash the lotion five times and add ProLong® Gold Antifad reagent containing DAPI to mount the slide.

　　Six rats in the scar and pinhole groups were stained by immunohistochemistry. The L5 horizontal spinal cord of each animal was sliced. After obtaining all the cross-sectional images, evaluate the c-fos positive neurons. Measure the protein expression area of the metatarsophalangeal joint dorsal horn neurons of the ipsilateral or contralateral scar or pinhole, examine each rat section with a deconvolution fluorescence microscope, and use dynamic cell counting software for image analysis. To analyze the c-Fos-IR neurons, the dorsal horn of the spinal cord is divided into two parts: the superficial layer (layer I-II), and the deep layer (layer III-V).

　　Histopathology: The foot was embedded in paraffin and cut into sections with a thickness of 5-10 microns. All tissues were sliced serially, and the scar tissue formed on the left hind limb was stained with hematoxylin-eosin (HE) and Masson trichrome. Five rats in the scar and pinhole group were subjected to histopathological examination.

　　Electron microscope analysis: L5 spinal nerve and proximal spinal ganglion were immediately removed, and cut into small pieces with a razor blade. Spinal nerves were fixed with 2% glutaraldehyde and then fixed with 1% OsO4. The sections were stained with uranyl acetate and lead citrate and examined under a microscope.

　　Result: Pain-related behavior: mechanical stimulation with von Frey, with the passage of time, the pain threshold produced is shown in the figure. Compared with the preoperative control value, there is no obvious threshold change at any time point in the contralateral 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 limbs decreased at 5, 9, and 10 weeks after the operation. In addition, compared with the contralateral 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 was significantly lower. The comparison between the pinhole and scar groups showed that the ipsilateral hind paw and the contralateral hind paw had a reduced withdrawal threshold at all time points after surgery.

　　Histopathological examination of cicatricial plantar: Compared with the pinhole group, the dermis and epidermis of the scar group became thicker and contained more collagen fibers. It was found that the size of collagen fiber bundles changed, and the boundary between the dermis and the subcutaneous area was unclear. Many migrating cells with round or oval nuclei were observed between collagen fibers. Compared with the pinhole group, the scar group showed aggregation of collagen fibers in the dermis and subcutaneous layer. The directions of these collagen fibers are random. Five rats in the scar and pinhole groups had similar histological results.

　　Immunohistochemical examination of the spinal dorsal horn: There was no statistical difference in the protein expression of the C-Fos-IR neuron regions on the ipsilateral and contralateral sides of the spinal dorsal horn L5 horizontal plate I-II between the scar and pinhole groups. Similarly, there was no statistically significant difference in the protein expression of C-Fos-IR neuron regions on the ipsilateral and contralateral sides of the III-V layer at the L5 level of the spinal dorsal horn between these groups.

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

　　Discussion: A scar on the skin is defined as "a macroscopic skin structure that interferes with the normal structure and function due to wound healing." Therefore, a scar is an abnormal skin structure, a failed regeneration, not a biomechanical problem. In addition, scars are 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 the unpleasant sensory symptoms of these wound healing. People who suffer from chronic pain after laparotomy are found to have adhesion of substance P and calcitonin gene-related peptide to the peritoneum. It has been determined that there is regulation of substance P and calcitonin gene-related peptide in the dorsal horn of the ipsilateral spinal cord. Pathological examination of the foot on the scarred side showed a hyperplastic scar, which was considered painful.

　　Several types of chronic pain animal models have been developed and used to study the development and maintenance of chronic pain mechanisms. Some chronic pain models, such as neuropathic pain, muscular pain models, arthralgia and joint fixation animal models of bilateral spinal cord, have shown hyperalgesia in previous behavioral and immunohistochemical studies. In this study, the withdrawal threshold of the ipsilateral hind paw was lower than the value before surgery. Compared with the pinhole group (including the same side and the contralateral side) at the same time point, the contralateral withdrawal threshold of the scar group decreased.

　　Conclusion: We have developed an animal model of painful scars and postoperative hyperalgesia 12 weeks after surgery. It may be the result of changes in the plasticity of the central nervous system caused by persistent pain. Our model can better understand the sensitization mechanism caused by painful scars, and in the future, it will help to study new methods for treating human painful scars.