| BackgroundEpidural scar after laminectomy is one important reason for the secondary spinal stenosis, therefore, the prevention of epidural scar adhesion which is to prevent secondary spinal stenosis, is of great significance to the improvement of symptoms and the rehabilitation of functions after operation.Previous research has confirmed Traditional Chinese Medicine could reduce scar formation through promoting blood circulation and enhancing Qi to arrest pain and hemostasis.In an experimental study in rats,we assessed Sanqi Gel had significantly preventive effect on fibrous scar formaton after laminectomy. Sanqi has the functions of promoting blood circulation,enhancing Qi for hemostasis and anti-fibrosis,sodium hyaluronate is an acid mucopolysaccharide in the organism,with good biocompatibility,and has some role in prevention of postoperative scar adhesions.As a new surgical embedded materials,Sanqi sodium hyaluronate Gel (called Sanqi Gel for short) is composed of Sanqi and sodium hyaluronate in this research.The purposes of the paper is to explore the mechanism about collagen metabolism and scar contraction.Objective1 Observe the pathomorphology of the epidural scar tissues,the growth and density of Collagen-I and Collagen-III fiber in epidural tissue caused by Sanqi Gel in rabbits.2 Study the effects of Sanqi Gel on the expression of Collagen-ⅠCollagen-Ⅲ,TIMP-1,MMP-1,α-SMA in the process of rabbits' epidural scar formation. The purpose of this study is to explore the mechanism of collagen metabolizability and the contraction of scar in the prevention of scar formation after Laminectomy caused by Sanqi Gel.Methods1 Preparation of Sanqi GelSanqi crude drugs were extracted with 60% ethanol. Alcohol was recovered from the alcohol extract, which was concentrated and it added water to solve. Then it took the supernatant and sodium hyaluronate was added. Standing and making it swelling enough, it was stirred uniformly, added water for injection to adjust volume and sterilized for 30 minutes. Then that is the gel(contain Notoginseng crude drugs 0.5g/ml). 2 Experiment methodIn this study,there were ninety-six rabbits which were white and two to two point five kilogrammes,six-month old and half of which were male. The rabbits were divided into saline group,Sanqi group,sodium hyaluronate group and Sanqi Gel group randomly. The laminectomy of rabbit was performed, and the duras were surrounded with saline,Sanqi liquid, sodium hyaluronate and Sanqi Gel respectively. Animals of each group were killed at one,two,four,eight weeks after operation. Use HE staining for pathomorphology observation of epidural scar tissues, Sirius red staining for observing the growth of Collagen-Ⅰand Collagen-Ⅲ, In situ hybridrzation histochemistry staining for the analysis of Collagen-ⅠmRNA,Collagen-ⅢmRNA,TIMP-1mRNA,MMP-1mRNA expression,and Immunohistochemistry staining for the analysis of a-SMA expression.Result1 Pathomorphology evaluation1.1 HE staining①At the end of the first week, a little unabsorbed hematoma was noted at the laminectomy defect. Inflammatory cell could be seen to follow the extensions of the haematoma, a bit of extracellular collagen fibers appeared.The epidural scar integrated with granulation tissue closely in saline group,Sanqi group and sodium hyaluronate group, but not in Sanqi Gel group. There was light adhesion and latent lacuna in Sanqi Gel group. Unabsorbed blood cells and inflammation cells could be found less in Sanqi Gel group than that of the other three groups (Fig.1-1～1-4).②At the end of the second week, granulation tissue was noted at the laminectomy defect,with no hematoma.More extracellular collagen fibers appeared.There was little adhesion in Sanqi Gel group,but much adhesion in saline group. Sanqi group and sodium hyaluronate group were better than saline group,but inferior to Sanqi Gel group (Fig.1-5～1-8).③At the end of the fourth week, granulation tissue became fibrotic,and collagen fibers matured, There was some lacuna in Sanqi Gel group. Adhesion was widely in saline group, and compared with saline group,there was less adhesion in Sanqi group and sodium hyaluronate group (Fig.1-9～1-12).④At the end of the eighth week, scar contracture presented itself and a portion of tissue ossified.Epidural space could be seen clearly in Sanqi Gel group. Adhesion was tight in saline group.Sanqi group and sodium hyaluronate group were better than saline group,but inferior to Sanqi Gel group (Fig.1-13～1-16).1.2 Sirius red stainingCollagen fibers was pink under the ordinary microscope.By using polarization microscope, type I collagen was red or yellow fiber that was thick, type III collagen was green fiber that was meshy or punctiform.①At the end of the first week, collagen fibers was less visible at the modeling area. Porous and fascicularis typeⅠcollagen could be seen,with punctiform type III collagen spreading around. Collagen fiber was lesser in each group and there was no obvious regularity (Fig.2-1～2-4).②At the end of the second week, collagen fiber increased significantly compared with the first week at the modeling area. Pyknotic and fascicularis typeⅠcollagen could be seen. Punctiform typeⅢcollagen was distributed around typeⅠcollagen. TypeⅠcollagen arranged indiscriminately in a crisscross pattern and typeⅢcollagen was relatively small in saline groupn Sanqi group and sodium hyaluronate group. While in Sanqi Gel group, typeⅠcollagen was regular and typeⅢcollagen was relatively conferted(Fig.2-5-2-8).③At the end of the fourth week, collagen fiber was more and grosser than that of the second week at the modeling area. TypeⅠcollagen got coarser and typeⅢcollagen was around it reticulately. TypeⅠcollagen arranged indiscriminately in a crisscross pattern in saline group,Sanqi group and sodium hyaluronate group. Comparatively in Sanqi Gel group typeⅠcollagen which arranged in order was parcer and typeⅢcollagen was more (Fig.2-9～2-12).④At the end of the eighth week, collagen fiber was less than that of the fourth week at the modeling area. TypeⅠcollagen was girdle-shaped and typeⅢcollagen was around it. TypeⅠcollagen arranged indiscriminately in a crisscross pattern in saline group,Sanqi group and sodium hyaluronate group. Nevertheless in Sanqi Gel group typeⅠcollagen which arranged in order was parcer and typeⅢcollagen was more (Fig.2-13～2-16). 2 Area density of collagen fiberThe area density of TypeⅠcollagen in Sanqi Gel group was significantly lower than that in saline group,Sanqi group and sodium hyaluronate group 4w and 8w postoperation (P<0.01). Sanqi group and sodium hyaluronate group was lower than that in saline group 1w,2w and 4w postoperation(P<0.01orP< 0.05); Area density of TypeⅢcollagen in Sanqi Gel group was significantly higher than that in saline group,Sanqi group and sodium hyaluronate group 4w and 8w postoperation (P< 0.01). Sanqi group and sodium hyaluronate group was higher than that in saline group (P<0.01orP<0.05).(Tab.1 and Tab.2) 3 The expression of correlated cytokines and collagenases3.1 The expression of TypeⅠcollagen mRNA and TypeⅢcollagen mRNA The expression of TypeⅠcollagen mRNA in Sanqi Gel group was significantly lower than that in saline group,Sanqi group and sodium hyaluronate group 4w postoperation (P<0.01). Sanqi group and sodium hyaluronate group was lower than that in saline group 1w and 2w postoperation (P<0.01orP<0.05); The expression of TypeⅢcollagen mRNA in Sanqi Gel group was significantly higher than that in saline group,Sanqi group and sodium hyaluronate group 4w postoperation(P<0.01). (Tab.3 and Tab.4)3.2 The expression of TIMP-1 mRNA and MMP-1 mRNAThe expression of TIMP-1 mRNA in Sanqi Gel group was significantly lower than that in saline group,Sanqi group and sodium hyaluronate group 2w and 4w postoperation (P<0.01orP<0.05). Sanqi group and sodium hyaluronate group was significantly lower than that in saline group 2w postoperation (P<0.01orP<0.05); The expression of MMP-1 mRNA in Sanqi Gel group was significantly higher than that in saline group,Sanqi group and sodium hyaluronate group 2w and 4w postoperation (P<0.01orP<0.05). Sanqi group and sodium hyaluronate group was higher than that in saline group 2w postoperation (P<0.01orP<0.05).(Tab.5 and Tab.6)3.3 The expression of a-SMA antibodyAt the end of the first and second week,the expression of a-SMA antibody in Sanqi Gel group,Sanqi group and sodium hyaluronate group was significantly lower than that in saline group (P<0.01orP<0.05),and there was no significantly difference between Sanqi Gel group> Sanqi group and sodium hyaluronate group (P > 0.05). At the end of the fourth and eighth week, the expression of a-SMA antibody in Sanqi Gel group was significantly lower than that in saline group, Sanqi group and sodium hyaluronate group (P<0.01 orP< 0.05),and there was no significantly difference between Sanqi group,sodium hyaluronate group and saline group (P> 0.05).(Tab.7)Conclusion1 Sanqi Gel could lessen peridural adhesion,and improve collagen's arrangement of the rabbits after lamineetomy.2 Sanqi Gel could stimulate the synthesization of typeⅢcollagen,inhibit the synthesization of typeⅠcollagen,reduce the rigidity of scar tissue and increase it's flexibility.3 Sanqi Gel could inhibit the activeness of TIMP-1, stimulate the expression of MMP-1,and reduce the scar fibrosis after all.4 Sanqi Gel could inhibit the expression ofα-SMA,thus reduce the contraction of scar. |
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