Study On Pathomorphology And Related Cytokines Of Epidural Scar By Sanqi Gel In Rats

BackgroundLaminectomy or discectomy is one of the most commonly used operation mode in spinal surgery. Coloboma caused by laminectomy is restored by fibrous connective tissue hyperplasia through inflammatiory reacting, granulation tissue producing, scar tissue producing and tissue remodeling basic pathophysiological processes. Foundation is a series of inflammatiory cell and restore cell, especially early inflammation. Epidural scar and adhesion are important causes of failed back surgery syndrome(FBSS) and influence curative effect of spinal operation seriously. Postoperative epidural fibrosis is a common occurrence after lumbar spine surgery. Fibrosis can cause compression or adhesion of the nerve root and has been implicated as one of important factors that contribute to recurrent radicular and/or low back pain after a successful laminectomy or discectomy procedure. Various materials have been tested for their ability to maintain a barrier between muscles and epidural space in order to physically and/or chemically inhibit scar tissue ingrowths, with varying results; however, neither has been used clinically after spinal surgery. In an experimental study in rats, we assessed the use of Sanqi Gel (semifluid) as a barrier to postoperative invasion of fibrous tissue into the laminectomy defect. Sanqi gel is composed of Carbopol Gel and Sanqi (RADIX ET RHIZOMA MOTOGINSENG ) concentrated solution which have the functions of promoting blood circulation to arrest pain and hemostasis. Sanqi was delayed discharge that was controlled by Carbopol Gel and has enough persistence time. Sanqi Gel has significantly preventive effect on fibrous scar formation after laminectomy, and it could be used as a good medicine to prevent epidural scar and adhesion. The purposes of this paper is to explore the mechanism and the early function of the prevention of scar formation after Laminectomy by Sanqi Gel through an experiment in rats, and supply more scientific theoretical bases and guide clinical prevention epidural scar from standpoint of modern medicine.1 Study on pathomorphology of epidural scarring and adhesion prevented by Sanqi Gel in rats.1．1 ObjectiveObserve the pathomorphology and density of collagen fiber in epidural tissue by Sanqi Gel in rats. 1．2 Methods1．2．1 Group, Model, Intervention: SD rats (♂) were used in this study, 380±20g. 72 rats were divided into 4 groups at random: Saline group, Carbopol Gel group, Zhanlp group and Sanqi Gel group with 18 rats in different group. Laminectomy was created at L3 as model and dural exposed. The naked dura mater was surrounded with Saline, Carbopol Gel,Zhanlp and Sanqi Gel in different groups. On days 7^th, 14^th and 21^st after operation, a small group which contains 6 rats were killed respectively.1．2．2 Specimens preparedness: specimens were prepared and examinated histologically in routine, such as H&E staining and Masson trichrome.1．2．3 Target and Analysis:â‘ pathomorphology evaluation of epidural tissue postoperation;â‘¡analysis the percentage of density of collagen fiber in epidural tissue(target area/total square×100%) automatically;â‘¢Epidural fibrosis in the vertebral canal was evaluated according to He-Revel system that incorporates both the extent of fibrosis and the degree of adhesions to the dura mater and nerve structures.1．3 Results1．3．1 Pathomorphology evaluationâ‘ At the end of the first week(7d) fibroblastic activity was noted at the laminectomy defect. Fibroblasts, neutrophile granulocyte, macrophage and other inflammatory cell are the major cells in the granulation tissue. Fibroblasts could be seen to follow the extensions of the haematoma. Histological analysis showed a significant difference was found between the Sanqi Gel, Zhanlp treated and other groups in terms of the inflammation, but no difference was found in terms of the extent of postoperative dural adhesion. There were unabsorbed blood cells could be found in Saline group and Carbopol Gel, but not in Sanqi Gel group.â‘¡At the end of the second week(14d) fibrocytes and fibroblasts were both noted at the laminectomy defect. Granulation tissue has been fibrosing and collagening. Disordered collagen fiber has been found in other groups but not in Sanqi Gel group. There was also smooth dura mater and light adhesion in Sanqi Gel treated site.â‘¢At the end of the third week(21d) fibroblasts were all taken place by fibrocytes and loose scar tissue was appeard. Collagen fiber growing up and mature. Grossly and disordered collagen fiber has been found in other groups but not in Sanqi Gel group.The same as dura adhesion.1．3．2 Density of collagen fiberCollagen fiber was not matured until the end of the second week(14d). There was a significant difference in density of collagen fiber between the Sanqi Gel-treated and saline-treated or Carbopol Gel-treated rats in terms of the extent of epidural tissue along the dura was observed(p<0．01) , Zhanlp-treated was few than Carbopol Gel-treated(p<0．05) . Collagen fiber has all matured at the end of the third week(21d). There was a significant difference in density of collagen fiber between the Sanqi Gel-treated and the other treated rats in terms of the extent of epidural tissue along the dura was observed(p<0．01 or p<0．05) , Zhanlp-treated was few than Carbopol Gel-treated(p<0．05) . Graph1-1 Collagen density in peridural tissue on14d,21d postoperation1．3．3 Evaluation of adhesionsIn axial sections either stained with H&E or Masson trichrome, a significant difference in adhesion between saline-treated and the other treated rats in terms of epidural adhesion along the dura was observed (p<0．01 or p<0．05) at the end of the second week(14d), and there a tend of to be better in the Sanqi Gel-treated rats. There was also a significant difference in adhesion between the Sanqi Gel-treated and the other treated rats in terms of the extent of epidural adhesion along the dura was observed(p<0．01 or p<0．05) at the end of the third week(21d). Carbopol Gel-treated was worse than Zhanlp-treated rats(p<0．05) at the end of the third week(21d).2 Study on the expression of related cytokines of epidural scar and adhesion by Sanqi Gel in rats.2．1 ObjectiveTo observe the impact of Sanqi Gel prevent epidural scaring and adhesion on the expression of COX-2, TGF-β, TIMP-1 and MMP-1 during the first three week in SD rats. The purposes of this study is to explore the mechanism of the prevention of scar formation after Laminectomy by Sanqi Gel.2．2 Methods2．2．1 Group, Model, Intervention: the same as Experiment 1．2．2．2 Target and Analysis: the expression of COX-2, TGF-β, TIMP-1 and MMP-1 were detected by immunohistochemistry (method of S-P) and quantificational analysed by computer image analysis system; Count positive cells and measure mean optical density(MOD) automatically.2．3 Results 2．3．1 The expression of COX-2 in the epidural tissue.2．3．1．1 Position of COX-2 express: positive cells have been found in macrophages, vascular endothelial cells, fibroblasts, other inflammatory cells and some myocytes. There are the most positive cells on 16 than 14d,21d postoperation; it was wear off at the end of the third week(21d).2．3．1．2 MOD of COX-2 express:â‘ MOD value in Saline-treated or Carbopol Gel-treated was significantly higher than that in Zhanlp-treated(p<0．05) or Sanqi Gel-treated(p<0．05) rats 7d postoperation. There was no significant difference in the MOD of COX-2 between Zhanlp-treated and Sanqi Gel-treated(p>0．05) rats 7d postoperation. Those relation between four groups could simple expressed as: Saline group, Carbopol Gel group>Sanqi Gel group, Zhanlp group.â‘¡MOD value in Saline-treated, Carbopol Gel-treated and Zhanlp-treated was significantly higher than that in Sanqi Gel-treated rats(p<0．05) 14d postoperation. MOD value in Zhanlp-treated was significantly lower than that in Saline-treated and Carbopol Gel-treated (P<0．05) rats 14d postoperation, but higher than Sanqi Gel-treated(p<0．05) rats. Those relation between four groups could simple expressed as: Saline group, Carbopol Gel group>Zhanlp group>Sanqi Gel group.2．3．1．3 Number of positive cells: generally, change tendency of the number of positive cells of COX-2 expression during the first three weeks postoperation was generally consistent with that of MOD value among different groups.2．3．2 The expression of TGF-β₁ in the epidural tissue.2．3．2．1 Position of TGF-β₁ express: positive cells have been found in fibroblasts, vascular endothelial cells and macrophages early period postoperation. However, positive cells just have been found arounding collagen fiber at the end of the third week(21d) postoperation.2．3．2．2 MOD of TGF-β₁ express: MOD value in Saline-treated, Carbopol Gel-treated and Zhanlp-treated was significantly higher than that in Sanqi Gel-treated(p<0．05) rats 7d,14d and 21d postoperation. There was no difference among Saline-treated, Carbopol Gel-treated and Zhanlp-treated rats 7d,14d and 21d postoperation(p>0．05) . MOD value on 7d postoperation was higher than that on 14d and 21d postoperation in different groups, and it was wear off on past time.2．3．2．3 Number of positive cells: generally,change tendency of the number of positive cells of TGF-β₁ expression during the first three weeks postoperation was generally consistent with that of MOD value among different groups. However, there was no significantly difference between each group 7d postoperation(p>0．05) .2．3．3 The expression of TIMP-1 in the epidural tissue.2．3．3．1 Position of TIMP-1 express: positive cells have been found in fibroblasts and vascular endothelial cells 7d period postoperation and its around collagen fiber 14d,21d postoperation. The number of positive cells decreased gradually but with increased MOD value.2．3．3．2 MOD of TIMP-1 express: MOD value in different groups was weak 7d postoperation. There was no difference between each groups at the end of the first week(p>0．05) . The expression of TIMP-1 increased gradually on 14d and 21d postoperation and Sanqi Gel-treated was lower than the other treat in rats(p<0．05) ; Carbopol Gel-treated took the highest place(p<0．05) . Those relation between four groups could simple expressed as: Sanqi Gel group 0．05) . There was a difference between Sanqi Gel-treated and the other treats in rats at the end of third week(21d)(p<0．05) postoperation, and Sanqi Gel group was higher expressed than the others.2．3．4．3 Number of positive cells: generally,change tendency of the number of positive cells of MMP-1 expression during the first three weeks postoperation was generally consistent with that of MOD value among different groups, but the number of positive cells sharp decreased from 14d to 21d postoperation.3 Conclusions3．1 Sanqi Gel is composed of Sanqi concentrated solution and Carbopol Gel. Sanqi Gel is biocompatible, with no hypersensitivity reaction. Sanqi gel is the ideal material would be one with viscous semifluid properties, which would allow it to be injected into the region of the surgical dissection without risk of neurologic compromise, It also must be biocompatible and ideally be slowly absorbed over time. 3．2 Sanqi Gel has a significant preventive effection on fibrous scar formation after laminectomy, and it could be used as a good medicine to prevent postoperative epidural adhesion, but Carbopol Gel couldnot. Sanqi is the active ingredient.3．3 Sanqi Gel reduce inflammatory reactions by inhibit the expression of COX-2 in the region of the surgical dissection. Meanwhile,Sanqi Gel can reduce hematoma.3．4 Sanqi Gel will keep the balance of MMP-1/TIMP-1 during the granulation time and wouldnot delay the first heal time.3．5 The possible mechanism of preventing epidural scar after laminectomy by use of Sanqi Gel could be reducing the expression of TGF-β₁ and TIMP-1 in epidural tissue. Furthermore,reduce the inhibition to the activity of MMP-1 is a possible way. On the other hand, Sanqi Gel may stimulate the expression of MMP-1 directly and biodegrade collagenous extxacellular matrix,prevent the epidural scarring after all.3．6 Why are the collagen fiber ordered and regulated been found in Sanqi Gel-treated rats but not other rats? It was relatived with the low expression of TGF-β₁, and cicatricial contracture sotomy has been decreased. This is just a speculate on the subject, and these findings support further investigation of Sanqi Gel as an interpositional material to prevent post-laminectomy epidural scar.