动物造模,硬膜外纤维化 z-bo 2020-09-11 382

　　Background: After tissue injury, excessive extracellular matrix components will be produced and deposited during the tissue repair process, thereby forming scars. The formation of scars on the dura is called epidural fibrosis. Its existence makes reoperation more difficult, increases the operation time, and increases the risk of dural laceration and nerve root injury. Given that the incidence of lumbar reoperation is 4% to 19%, this is a common problem in spinal surgery. To prevent epidural fibrosis, free fat transplantation can cause nerve root or spinal cord compression, but insertion is a common procedure in clinical practice. In addition, various mechanical barriers and anti-inflammatory therapies are used to prevent epidural fibrosis, and their clinical success rates are different or limited. The ideal material for preventing epidural fibrosis should minimize the risk of nerve compression and not interfere with the healing of surrounding tissues. It must also be biocompatible to minimize foreign body reactions and inflammation. Transforming growth factor β (TGF-β) plays a broad role in various cells and tissues in the body. The expression of TGF-β1 in scar tissues, especially in systemic sclerosis, is increased. Compared with tissue regeneration, TGF-β1 levels are elevated in wounds that heal through scar formation. This allows the clinical use of antibodies and small molecules against TGF-β1 to prevent scar formation. In order to evaluate the role of TGF-β1 in epidural fibrosis, we applied the anti-TGF-β1 synthetic peptide p144 to a rabbit spinal surgery model. p144 showed reduction in fibrosis in animal models of liver fibrosis, bleomycin-induced skin sclerosis, and fibrosis around silicone prostheses. In other studies, AdconL and Duragen have been shown to be very effective in preventing epidural fibrosis. AdCON-1 is an absorbable gel matrix made of gelatin and carbohydrate polymer (GCP), which prevents fibroblasts from disappearing within 3 weeks. Duragen is a collagen sponge (CS), usually used as a dural implant, which can penetrate fibroblasts and use the pores of the collagen matrix as a scaffold to form new collagen 6-8. It will disappear in a week. We established a rabbit model of epidural fibrosis after pedicleectomy, and evaluated the effects of barrier materials AdconL and Duragen and TGF-β1 blocking peptide p144 on epidural fibrosis. Methods: For this study, 40 New Zealand white rabbits (weight 4-5 kg) were used for L6 vertebral resection. Four experimental groups were created: the control group, the pedicle resection site was washed with saline (n = 10). In the GCP group, AdconL was evenly distributed at the vertebral resection site, covering the vertebral resection defect (n = 10). In the CS group, the Duragen sheet was cut and placed at the vertebral resection site to accommodate the defects of the vertebral resection (n = 10). In the iTGFβ group, the p144 peptide was derived from the human TGF-β type III receptor sequence and was evenly distributed in the vertebral resection site covering the vertebral resection defect (n = 10). The cytotoxicity of iTGFβ has been evaluated in previous animal experiments. Rabbits with complications (ruptured dura mater, nerve compression, etc.) caused by anesthesia or vertebral resection are classified as unqualified and are not included in the scope of the study. Surgery: Between L6 to L6 to L7, a complete vertebral resection was performed on the yellow ligament of the animal from stem to stem. All animals were fasted 12 hours before surgery. The animals were sedated by intramuscular injection of medetomidine (0.15 mg/kg) and ketamine (10 mg/kg). Induce anesthesia by intravenous injection of 2-8 mg/kg propofol, and maintain 1.5-3% sevoflurane throughout the operation. Under anesthesia, the animal lies on a heating pad on the operating table. The waist ac is trimmed with electric scissors and disinfected with povidone iodine. L6 is determined by palpation, and a midline incision of approximately 7 cm is made in the center of the spinous process. Expose the bone plane, dissect the paraspinal muscle tissue, reach and cut the yellow ligament between L5 and L6, and then use the Kerrison-Rongeur at a 45° angle between L6 and L7 from one stem to the other. A complete vertebral resection from L6 to the yellow ligament was performed on the stem to form a defect approximately 20 mm long and approximately 7 mm wide. After the operation, the myocardium was sutured with 2/0 polylactic acid suture. Administer antibiotics (penicillin/streptomycin 0.1 ml/kg/24 hours) within 7 days. All rabbits are kept in a cage and can eat freely without being fixed. Four weeks after the operation, the rabbit was executed.

　　Histological and histomorphological analysis: For histological evaluation, the spine was collected, fixed in 4% formalin for 1 week, and within 8 weeks of decalcification solution (10% EDTA, 7.5% PVP) with 10 mM Trishcl , PH 6.95 fixed). ) Decalcification, dehydrated with ethanol step by step, and embedded in paraffin. 4μm thick serial sections were taken from the center and both ends of each treatment group and stained with hematoxylin and eosin (H&E), Mason tricolor and siliused to evaluate scar tissue. Digital images were collected using Zeiss AXIOCAMI CC3 camera and AXIO IMAGERM1 microscope. The cell density, new bone formation and collagen content of epidural scar tissue were analyzed by histomorphology. For cell density, the number of cells is the unit of cells per square millimeter, using IMAGEJ, 15 areas per slide, and 3 slides per animal. The area of new bone formation and the distance covered by vertebral resection are expressed as the area (mm2) and distance (mm) from the Mason triple staining site, respectively. Two experimental pathologists blindly scored scar density, epidural adhesion and inflammatory cell infiltration. The expansion of the adhesion between the dura and fibrous tissue is He et al. It will be graded according to the classification described. The adhesion density is graded according to the classification used by Preul et al. The level of inflammatory cell infiltration is semi-quantitative: 0 (no inflammatory cell infiltration), 1 (less than 30% of the area is infiltrated by inflammatory cells, magnification 100x), 2 (scar tissue) 30-70% infiltration) Inflammatory cell infiltration, magnification of 100x) and 3 (severe inflammatory cell infiltration distributed in 70% of the scar tissue, magnification of 40x).

　　Statistical information: Graphpadprism 5.0 software is used for all statistical analysis. The statistical significance level is set to P\u003c0.05, and Kolmogorov-Smirnov is used to test the normality of continuous variables. Unit dispersion analysis was used to analyze differences in average cell density between treatment groups. The Dunnett multiple comparison test was used to detect the difference in cell density between each group and the control group. The Clascal Wallis test is used to analyze fibrous adhesions, scar density, inflammatory infiltration, collagen content and new bone formation. We used Dunn's multiple comparison test to compare the differences between each treatment group and the control group. The histological scores of all groups were analyzed by weighted κ coefficient. Finally, the Spearman coefficient was used to evaluate the correlation between collagen content and cell number. Conclusion: Two experimental pathologists recorded scar density, dural adhesions and inflammatory cell infiltration to determine the effectiveness of various treatments for epidural fibrosis. The control group and iTGFβ group showed dense defects in the angiogenic connective tissue, resulting in similar average scar density scores. In the CS group, the tissue density of the defect was low, and the scar density score was significantly reduced. The scar density score of the GCP group was the lowest, which was statistically significant compared with the control group. The observer's agreement on scar density within the group was 74.6%. Comparing the degree of adhesion between the dura mater and scar tissue, the score of the GCP group decreased, but it was not statistically significant compared with the control group. There was no significant difference between the CS and iTGFβ groups and the control group. The degree of adhesion between observers reached a consensus in class of 74.6%. Compared with the control group, the inflammatory cell infiltration scores of the iTGFβ and CS groups were significantly higher. There was no significant difference between the GCP group and the control group. The class agreement among observers of inflammation invasion was 85.7%. Compared with the control group, CS, iTGFβ and GCP in the treatment group had no significant differences in cell density and new bone formation. The collagen content was determined by the Silius red staining method. Compared with the control group, there was no significant difference in the proportion of Sirius red-stained area at the surgical site in the iTGFβ or CS treatment group. On the other hand, the collagen density of the GCP group was significantly lower than that of the control group.

　　Conclusion: Animal models indicate that epidural scars will form after pedicle resection. GCP scaffold can reduce the amount of collagen tissue and cells in the epidural space after pedicleectomy. Other treatments (CS and iTGFβ) have not been shown to reduce the incidence of epidural fibrosis or adhesions. The data in this study indicate that iTGFβ administered at this dose cannot reduce epidural fibrosis in a rabbit spinal surgery model.