传统中医“拖线疗法”治疗皮下瘘感染大鼠动物模型

http://www.paper.edu.cn - 1 - 中国科技论文在线 Traditional Chinese Medicine"Thread-dragging therapy"in treating a new subcutaneous infected fistula rat model Wang Chen, Lu Jinggen, Yin Haoqiang (Longhua Hospital affiliated to Shanghai Traditional Chinese Medicine University, ShangHai 200032) Foundations: Doctoral Research Foundation of Higher Education (No.20093107110005),Shanghai Science and Technology Committee Venus Project(No.10QA1406600),Shanghai Mucicipal Education Commission Innovation research project(No.10ZZ77) Brief author introduction:王琛,（1977－），女，副主任医师，擅长肛肠疾病的中医药治疗. E-mail: wangchen_longhua@yahoo.com.cn Abstract: Aim: This study was designed to develop and treat with traditional Chinese medicine method of an experimental rat model of subcutaneous fistula induced by bacteria infection. Methods: A spring-gauze was surgically implanted into 24 male Sprague-Dawley (SD) rat’s dorsal muscle, respectively. Then Staphylococcus aureus and Escherichia Coli mixed bacteria were injected into the spring-gauze. After 45days, all spring-gauzes were removed under anesthesia. X-ray and ultrasound were performed, enabling imaging fistula track assessment. Every rat was examined by a fistula probe and four rats were randomly selected for histological fistula tract assessment at the same time. Residual rats were randomly divided into in two groups. Group A treated with traditional Chinese medical method “thread-dragging therapy”. Group B treated with Western medical method “fistulotomy”. The healing time and body weights of two groups were compared.Results: At 45days imaging assessment showed a track with two openings in all rats and histological results proven lumen and surrounding granulation tissue with epithelium similar to that criteria and features of fistula in human being. The mean healing time of group A was shorter than group B with significant differences. The body weight of group A is heavier than group B at 7days after surgery and 14days after surgery respectively.Conclusions: A rat model with subcutaneous fistula induced by Staphylococcus aureus and Escherichia Coli mixed bacteria infection is easy and repeatable. Traditional Chinese medicine therapy “thread-dragging therapy” is less invasive and safe. Keywords:Subcutaneous fistula; Infected model; Traditional Chinese medicine; Thread-dragging therapy 0 Introduction A fistula is defined as an abnormal communication between any two epithelium-lined surfaces1. It can take place at any site on human body. A normal fistula will often recount a history of an abscess that has been drained either surgically or spontaneously. The primary etiology of fistula is infection. Although animal fistula model using a catheter or seton is already well described in the litarature2, we have designed this prospective study to create and evaluate an experimental rat model with subcutaneous fistula created by bacteria infection that might be more similar to the etiology and clinical features of fistula. Till now, surgery is the most effective therapy for fistula. In this study we use Chinese Medicine therapy “thread-dragging therapy” compare to West Medicine therapy “fistulotomy” to treat the newly developed rat model of subcutaneous fistula. 1 Materials and Methods 1.1 Animals and Bactertia Concentration Local ethical committee approval was obtained. All aspects of the study were performed in accordance with the Longhua Hospital Surgery of Traditional Chinese Medicine Institute for medical research. 24 male Sprague-Dawley rats (Shanghai SLAC laboratory animal Co. LTD., Shanghai, China）weighing 300 to 350g were housed four per cage with free access to tap water and food. The rats were acclimatized for one week. http://www.paper.edu.cn - 2 - 中国科技论文在线 We used 9×108 cfu/ml concentration of standard Staphylococcus aureus and 9×108 cfu/ml standard Escherichia Coli (Shanghai centre for clinic laboratory, Shanghai, China). 1.2 Instruments Spring-gauze: 3cm length, 0.4cm external diameter steel spring (Shanghai Spring Corporation LTD., Shanghai, China)，0.5cm×6cm gauze (Shanghai YingJing Medical Health Materials Co. Ltd., China). The gauze was folded into 3cm and put through the spring, then high temperature sterilized. Fistula probe: 22cm length, ball shape at one top, 0.13cm diameter of the ball and 0.1cm of the probe body, 100% silver made. (Shanghai Laofengxiang Co.,Ltd. ) Threads: 20cm length,1-0 silk braided non-absorbable MERSILK suture.( ETHICON Johson& Johson Medical (China) Ltd.) 1.3 Fistula Model Creation After 12 hours of fasting, all rats had abdominal anesthesia with injection of Ketamine (100 mg/kg，Jiangsu HengRui Pharmaceutical Company Ltd., China). After anesthesia, all rats’ hair on the back was shaved by electric shaver. A 0.5cm length horizontal incision was made by scalpel on the rat’s back (about 1cm posterior to the neck) approximately 1.5cm lateral to the middle line . The second incision was performed 3cm distally with the same length. The depth of both incisions were ≥0.3cm from skin. A tunnel was created beneath the muscle by blunt dissection with hemostats to accept the spring-gauze. Two stitches were used to fix both sides of the spring -gauze to the skin (Fig 1). Immediately after recovery rats were hosted one per cage with normal diet. 图 1 在大鼠颈背部皮下植入弹簧纱条 Figure 1 Spring -gauze was implanted beneath the rat’s dorsal muscle All rats were infected by Staphylococcus aureus and Escherichia Coli mixed bacteria (0.2 ml, 9×108 cfu/ml concentration). Bacteria solutions were injected into the spring -gauze by 1ml syringe. After 45 days, all rats were reanesthetised and spring-gauzes were removed. Every rat was examined by a fistula probe and underwent fistulography and ultrasound. Four rats were randomly underwent tissue excision to obtain histology assessment. 1.3.1 Fistulography and Ultrasound All rats were examined in supine position under X-ray (Siemens R2000, Germany) at low pressure of injection with 60% Meglumine Diatrizoatis Mucilagefpa from one opening to evaluate the existence of fistula. The long axis of the fistula was identified using a high-frequency linear array ultrasound (PHILP ATL 5000, Holland) with air insufflation to identify the fistula track. Two experienced observers denoted the presence or absence of fistula on X-ray and Ultrasound respectively. http://www.paper.edu.cn - 3 - 中国科技论文在线 1.3.2 Histology Fistula Assessment Four rats were randomly choosed and an oblong incision was made by scalpel to include the margin around the fistula track. This was extended deep to the muscle, so that the two openings and fistula track were excised en bloc. Resected specimens were placed in 10% formalin for 7days for fixation. Specimens were then cut longitudinally and stained with hematoxylin and eosin. The microscope slides were analyzed by an experienced pathologist. 1.4 Treatment After fistulography and ultrasound examinations 20 rats were randomly divided into two groups. Each group has 10 rats. Group A used traditional Chinese medical surgery “thread-dragging therapy”. (Fig 2) Ten silk threads were put through the fistula track by a fistula probe, then knot loosely. From the second day after surgery, part of threads which were inside the fistula track were dragged to outside and cleaned by normal saline cotton once every day. After one week, the fistula cavity became smaller while the granulation tissue grew. When dragged threads were no more easily, five threads should be taken away. The residual threads would not be taken away until wound tissue became fresh and no discharge. By the same time, the fistula cavity would be wrapped with cotton and swab to accelerate the cavity to be healed. Group B used the West medical surgery “fistulotomy”. (Fig 3) All fistula tracks were proved by a fistula probe, then resected by scissors. From the second day after surgery all wounds were cleaned by normal saline cotton once everyday until the wounds healed. 图 2 治疗组大鼠采用“拖线疗法” Figure 2 Thread-dragging therapy on model 图 3 对照组大鼠采用瘘管切开 Figure 3 Fistulotomy on model 1.5 Statistics Statistical analysis was performed by using SPSS for Windows version 11.5 (SPSS, Inc,. Chicago, IL). Quantitative results are given as means (±standard error) for cure time. ANOVA was used to compare the body weight between two groups at different periods. http://www.paper.edu.cn - 4 - 中国科技论文在线 2 RESULTS 2.1 Spring-gauze representation All spring-gauze were removed and found with little pus on the spring-gauze (Fig 4). 图 4 造模后 45 天取出的弹簧纱条表面有少量黄色脓液 Figure 4 Little yellow pus can be found at the surface of spring-gauze at 45days. 2.2 Fistulography and Ultrasound All fistulas were satisfactorily represented at 45days by fistulography and ultrasound examinations. 2.3 Fistula presentation All rats were examined and proven by a probe at 45days. 2.4 Histology The specimens were found lumens with minimal epithelium and fibroblast, the thickness of inner layer was uneven. (Fig 5) 图 5 造模后 45 天组织病理学证实有管腔和少量上皮及纤维组织（×50） Figure 5 Histology of fistulas at 45days found lumens with minimal epithelium and fibroblas. (×50) 2.5 Healing time and wound scar The mean healing time of group A is 16.2±0.79d, while group B is 21.7±0.82d. The difference is statistical significant. And group B had more severe scar than group A at healing time. (Fig6, 7) http://www.paper.edu.cn - 5 - 中国科技论文在线 图6 拖线疗法大鼠治愈后背部有二个小的疤痕 Figure 6 Two tiny scar on rats with thread-dragging therapy 图 7 瘘管切开大鼠治愈后背部创面较大 Figure 7 Severe scar on rat with fistulotomy 2.6 Body weight Body weights of each group were tested at four periods: before surgery, 7day after surgery, 14day after surgery and when totally healed. Body weight of group A is heavier then group B at 7day after surgery and 14days after surgery with statistic difference. (Fig 8) 图 8 两组大鼠治疗不同时间的体重变化。时间 2 和时间 3 对应治疗第 7 天和第 14 天，治疗组大鼠平均体 重大于对照组大鼠平均体重。 Figure 8 Body weights of two groups at different periods. Time 2 and Time 3 display to 7days and 14days after surgery, the body weights of group A is heavier then group B. 3 DISCUSSION Fistula is a common disease in human being. Surgery is the most effective therapy. Fistulotomy and fistulectomy are easy for subcutaneous fistula. But when fistula locates on patients’ important structures like face，breast or near the main joints then lay open procedures may have some complications as ugly scar, long healing time and postoperative dysfunction.3 More and more surgeons begin to look for less invasive therapies as sealant, fistula plug or http://www.paper.edu.cn - 6 - 中国科技论文在线 thread-dragging therapy4,5.The mechanism of these therapies should be proven. This study is designed to develop an experimental animal subcutaneous fistula model which is not only similar to clinical features but also to histological characters.6 In primary research we put gauze through the muscle and inject bacteria into the gauze to create acute infection. When we removed the gauze, the tissue around the gauze had adhesion to the gauze and taken away as well. So we have modified the gauze using spring encircles the gauze to prevent adhesion. The bacteria can then be injected into the gauze and contact the surrounding tissue entirely. When we pull out the spring, the surrounding tissue will not be damaged. Abscesses and fistula represent different stages along the continuum of a common pathogenic spectrum. The abscess represents the acute inflammatory event whereas the fistula is representative of the chronic process. Previous researches found the most common Gram-positive bacteria and Gram-negative bacteria in abscess and fistulae were Staphylococcus aureus and Escherichia coli, respectively7,8,12.Because there is no existing research about bacterial concentration for subcutaneous fistula, in this study we use 9×108 cfu/ml concentration of bacteria based on intraabdominal infections model.9 Whether other concentrations of bacteria might interfere the formation of fistula has not been answered in this study. Fistulae at other sites often closed spontaneously when there is no gross residual sepsis, no continuing underlying disease process and no distal obstruction.10 Whether the fistula model is successful or not depends on the duration of the fistula. As we know fistulas at other sites in the body do not heal because they become epithelialized.9 Persistence of idiopathic anal fistula may be related to epithelialization.12,13 In this study, at 45days morphology of the fistulas maybe due to the implantation of spring-gauze. But the histology showed epithelialization at both two openings. We think spring-gauze is probably a medium and not the main reason for fistula creation. Epithelialization from either or both ends of the fistula track might be the explanation for the existence of chronic fistula. Thread-dragging therapy as one of the traditional Chinese Medicine therapies for fistula can protect the normal muscle and skin above the fistula track. Compare to fistulotomy there are usually no severe wounds, less pain、energy consumption and scarring . The body weight of fistulotomy group is lighter than thread-dragging group after surgery because of large wound. But there is no difference between two groups when wound healed because fistulotomy group spent nealy 5days longer than thread-dragging group. Thread-dragging therapy can not only shorten the healing time but also minimized the scar tissue. 4 CONCLUSIONS A rat subcutaneous fistula model with infection of Staphylococcus aureus and Escherichia coli is feasible. Morphologic and histological assessments demonstrate the track between two openings and granulation tissue around the fistula lumen. “Thread-dragging therapy” is microinvasive and safe. It can be used as alternative therapy for fistula. REFERENCES [1] Bruce G. Wolff, James W. Fleshman, David E. Beck, et al. The ASCRS Textbook of Colon and Rectal Surgery [M]. 2007 [2] Gordon N, Buchanan. M.Sc, Paul Sibbons, et al. Experimental Model of Fistula-In-Ano. Diseases of the Colon & Rectum [J]. 2005; 48: 353~358. [3] Jin-Gen LU, Hua-Fa Que, Hong-Feng Chen.et al. Superiority of thread-dragging therapy in treatment of refractory sinus tracts of fistulae. Journal of Chinese Integrative Medicine [J]. 2008; 6: 991~994. [4] Eric K. Johnson, M.D., Janette U. Gaw, M.D. et al. Efficacy of Anal Fistula Plug vs. Fibrin Glue in Closure http://www.paper.edu.cn - 7 - 中国科技论文在线 of Anorectal Fistulas. Diseases of the Colon & Rectum [J]. 2006; 49: 371~376. [5] Jin-Gen LU , Yong-Qing CAO, Chun-Mei HE. et al. Clinical research of thread-drainage through fistula method in treating patients with simple anorectal fistula. Journal of Chinese Integrative Medicine [J]. 2006; 4: 140~146 [6] Michael T Fitch, David E Manthey, Henderson D McGinnis, et al. A skin abscess model for teaching incision and drainage procedures. BMC Medical Education [M].2008 [7] Grace RH, Harper IA, Thompson RG. Anorectal sepsis: microbiology in relation to fistula-in-ano. Br J Surg [J]. 1982; 69: 401~ 403. [8] Lunniss PJ, Fairs B, Rees HG, et al. Histological and microbiological assessment of the role of microorganisms in chronic anal fistula. Br J Surg [J]. 1993; 80: 1072 [9] Ao Yang, L I You-sheng, L I Jie-shou, et al. A laboratory model of intraabdominal infections by intestinal fistula. Chin J Exp Surg [J]. 2004; 21: 99~100. [10] F. Seow-Choen, A.J.Hay, S. Heard, et al. Bacteriology of anal fistulae. Br J Surg [J].1992; 79: 27~28. [11] Harding Rains AJ, Mann CV, eds. Bailey and Love's Short Practice of Surgery. 20th ed. London: HK Lewis[M], 1988 [12] Lunniss PJ, Sheffield JP, Talbot IC, et al. Persistence of idiopathic anal fistula may be related to epithelialization. Br J Surg [J].1995; 82: 32~33. [13] Hofstädter, J Schölmerich, H Herfarth, et al. Morphological characterisation of Crohn’s disease fistulae. Gut [J].2004; 53: 1314~1321. 传统中医“拖线疗法”治疗皮下瘘感染 大鼠动物模型 王琛，陆金根，银浩强 （上海中医药大学附属龙华医院，上海 200032） 摘要：目的：本研究的目的在于建立一种皮下瘘感染动物模型，并采用中医“拖线疗法”进 行治疗。方法：24 只雄性 SD 大鼠颈背部皮下植入弹簧纱条，并注入金黄色葡萄球菌和大肠 杆菌混合菌液。45 天后在麻醉下取出弹簧纱条，经 X 线，超声，探针及病理检测验证模型 成功。将模型大鼠随机分成两组，治疗组采用“拖线疗法”对照组采用“瘘管切开”治疗， 并对两组大鼠的创面愈合时间和大鼠体重变化进行比较分析。结果：造模后 45 天影像检查 证实有瘘管管道和外口存在，组织病理学检查显示有管腔及周围肉芽组织填充，与人类瘘管 结构相似。治疗组创面平均愈合时间明显短于对照组且有统计学差异，术后第 7天和 14 天 治疗组大鼠的体重较对照组大鼠重。结论：将金黄色葡萄球菌和大肠杆菌混合菌感染制做皮 下瘘大鼠动物模型具是简单可重复的。传统中医“拖线疗法”治疗皮下瘘大鼠损伤较小且安 全。 关键词：皮下瘘；感染动物模型；传统中医；拖线疗法