ORIGINAL CONTRIBUTION Topographic Anatomy of the Anal Sphincter Complex and Levator Ani Muscle as It Relates to Intersphincteric Resection for Very Low Rectal Disease Yuichiro Tsukada, M.D.1,2 • Masaaki Ito, M.D., Ph.D.2 • Kentaro Watanabe, M.D.3 Kumiko Yamaguchi, M.D., Ph.D.3 • Motohiro Kojima, M.D., Ph.D.4 Ryuichi Hayashi, M.D.1,5 • Keiichi Akita, M.D., Ph.D.3 • Norio Saito, M.D., Ph.D.2 1 Advanced Clinical Research of Cancer, Juntendo University Graduate School of Medicine, Chiba, Japan 2 Department of Colorectal Surgery, National Cancer Center Hospital East, Chiba, Japan 3 Department of Clinical Anatomy, Tokyo Medical and Dental University, Tokyo, Japan 4 Exploratory Oncology Research and Clinical Trial Center and Division of Pathology, National Cancer Center, Chiba, Japan 5 Department of Head and Neck Surgery, National Cancer Center Hospital East, Chiba, Japan BACKGROUND: Intersphincteric resection has become muscle fibers that continued from the longitudinal muscle a widely used treatment for patients with rectal cancer. of the rectum. The levator ani muscle attached directly However, the detailed anatomy of the anal canal related to the lateral surface of the longitudinal smooth muscle to this procedure has remained unclear. of the rectum. The length of the attachment was longer OBJECTIVE: The purpose of this study was to clarify the in the anterolateral portion and shorter in the posterior detailed anatomy of the anal canal. portion of the anal canal. In the lateral and posterior portions, the levator ani muscle partially overlapped the DESIGN: This is a descriptive study. external anal sphincter; however, there was less overlap in SETTINGS: Histologic evaluations of paraffin-embedded the anterolateral portion. In the posterior portion, thick tissue specimens were conducted at a tertiary referral smooth muscle was present on the surface of the levator hospital. ani muscle and it continued to the longitudinal muscle of PATIENTS: Tissue specimens were obtained from the rectum. cadavers of 5 adults and from 13 patients who underwent LIMITATIONS: We observed only limited portions in abdominoperineal resection for rectal cancer. some surgical specimens because of obstruction by MAIN OUTCOME MEASURES: Sagittal sections from tumors. 9 circumferential portions of the cadaveric anal canal CONCLUSIONS: The levator ani muscle attaches directly (histologic staining) and 3 circumferential portions to the longitudinal muscle of the rectum. The spatial from patients were studied (immunohistochemistry for relationship between the smooth and skeletal muscles smooth and skeletal muscle fibers). differed in different portions of the anal canal. For RESULTS: Longitudinal fibers between the internal and intersphincteric resection, dissection must be performed external anal sphincters consisted primarily of smooth between the longitudinal muscle of the rectum and the levator ani muscle/external anal sphincter, and the appropriate surgical lines must be selected based on the Funding/Support: This work is supported by a Grant-in-Aid for Scien- specific structural characteristics of each portion. tific Research C (23590216) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. Financial Disclosure: None reported. KEY WORDS: Anal canal; Clinical anatomy; Intersphincteric resection; Levator ani muscle; Correspondence: Masaaki Ito, M.D., Ph.D., Department of Colorectal Longitudinal muscle; Low rectal cancer. Surgery, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba, 277–8577, Japan. E-mail: [email protected] hanks to advances in surgical techniques, inter- Dis Colon Rectum 2016; 59: 426–433 sphincteric resection (ISR) is now widely used as DOI: 10.1097/DCR.0000000000000565 Tan alternative treatment option that preserves the © The ASCRS 2015 anus and, thus, anal continence for patients with rectal 426 DISEASES OF THE COLON & RECTUM VOLUME 59: 5 (2016) Copyright © The American Society of Colon & Rectal Surgeons, Inc. Unauthorized reproduction of this article is prohibited. DISEASES OF THE COLON & RECTUM VOLUME 59: 5 (2016) 427 cancer.1–4 To preserve the anus with ISR, the rectum is di- lines of the 9 segments are indicated in the bottom right vided from the levator ani muscle (LAM) using an abdom- key in Figure 1. inal approach, followed by dissection between the internal anal sphincter (IAS) and the external anal sphincter (EAS) Surgical Specimens using both abdominal and perianal approaches. For this Thirteen specimens from 13 Japanese patients (9 men and procedure, it is essential that rectal surgeons have detailed 4 women; age range, 41.0–84.0 years; mean age, 68.6 years) knowledge of the anatomic relationship between the rec- who underwent APR for rectal cancer in the National tum and LAM and between the IAS and EAS. Cancer Center Hospital East without neoadjuvant chemo- It has been reported that fibers of the longitudinal radiation were used. The specimens were fixed in 10% for- muscle (LM) of the rectum blend with fibers of the LAM, malin ≈2 hours after they were resected. They were then thereby producing a conjoined,5–7 combined,8 or conjoint9 dehydrated and embedded in paraffin, after which 4-μm– LM layer between the IAS and EAS. However, the detailed thick sagittal sections were prepared. Sections that were anatomy of the region between the IAS and EAS, the so- not affected by the tumors were observed. This pathologi- called intersphincteric space, and the precise surgical line cal study was performed in accordance with the provisions within this space have not yet been clarified. of the Declaration of Helsinki, and the protocol for this Furthermore, with the progress of laparoscopic tech- study was approved by the ethics committees of the Na- niques, some surgeons now perform ISR endoscopical- tional Cancer Center Hospital East (2013–143). ly.10–13 During endoscopic surgery, structures in the deep pelvic region can be observed more clearly and at greater Histological and Immunohistochemical Staining magnification than they can during open surgery. During Sections were stained with hematoxylin and eosin and endoscopic ISR, the attachment of the LAM to the rectum Elastica van Gieson. The distributions of smooth and skel- and the intersphincteric space can be seen very clearly etal muscle were confirmed through immunohistochemi- from the abdominal approach, and it becomes possible to cal staining of surgical specimens. For antigen retrieval, more precisely select a surgical line; however, the optimal the slides were microwaved in 10 mM sodium citrate buf- surgical line in this area remains undefined. fer (pH 6.0). Endogenous peroxidase activity was inacti- The purpose of this study was to describe in detail the vated by incubating the tissues for 30 minutes in methanol anatomy of the LAM, EAS, IAS and rectum so as to enable containing 0.3% H2O2. Nonspecific binding was blocked improved radial margins while reducing the incidence of by incubating the tissues for 30 minutes at room tem- postoperative incontinence in patients with rectal cancer. perature in phosphate-buffered saline containing 0.05% We used cadaveric specimens to study specifically intended Tween 20 and 5% goat serum. Once blocked, the sections sections, as well as surgical specimens from patients who were incubated overnight at room temperature with anti- underwent abdominoperineal resections (APRs), which smooth muscle actin (ready-to-use, Actin, Smooth Muscle offer better consistency and morphologic quality than Ab-1, Clone 1A4; Thermo Fisher Scientific, Fremont, CA) muscle tissue from formalin-fixed postmortem material. and antiskeletal muscle myosin (ready-to-use, Myosin, Skeletal Muscle Ab-2, Clone MYSN02, Thermo Fisher Scientific) as primary antibodies. The sections were then MATERIALS AND METHODS washed and incubated for 30 minutes at room tempera- Cadaveric Specimens ture with biotinylated goat antimouse IgG (1:200 dilution, Five pelvises from 5 Japanese cadavers (1 man and Vector Laboratories, Burlingame, CA) as the secondary 4 ­women; age range, 84.0–100.0 years; mean age, 90.4 years) antibody. The signal was amplified using a VECTASTAIN obtained from the dissecting room of the School of Medi- ABC kit (Vector Laboratories) and TSA Biotin System cine, Tokyo Medical and Dental University, were used for (PerkinElmer, Tokyo, Japan) according to the manufactur- cadaveric studies. The cadavers were donated to the Tokyo er instructions, and the immunocomplexes were detected Medical and Dental University Department of Anatomy using 3,3′-diaminobenzidine (Wako, Osaka, Japan) as the for use in clinical studies, within the guidelines of the Act chromogen. Finally, the sections were counterstained us- on Body Donation for Medical and Dental Education law ing hematoxylin. of Japan. Cadavers were fixed by arterial perfusion with 8% formalin and preserved in 30% alcohol to resist fungus Statistical Analysis and to maintain the softness of the tissues. The rectum, We used the Wilcoxon test with the Bonferroni method anus, and circumjacent muscles and connective tissue to compare the continuous variables of 3 cohorts (lateral, were removed en bloc, hewn out radially into blocks, fixed anterolateral, and posterior). The level of significance was in 10% formalin, dehydrated, and embedded in paraffin. A set at p < 0.017 by dividing 0.05 by 3. All of the statistical total of 9 segments of the anal canal, from anterolateral to analyses were performed using JMP version 8.0 (SAS Insti- posterior, were sectioned in the sagittal plane. The cutting tute Inc, Cary, NC). All of the measurements were made by Copyright © The American Society of Colon & Rectal Surgeons, Inc. Unauthorized reproduction of this article is prohibited. 428 TSukada ET Al: ANATOMY OF THE ANAL CANAL A B C D E FGH I FIGURE 1. Cadaveric specimens. Sections were stained with Elastica van Gieson. The sections are labeled as shown in the bottom right key. The longitudinal fibers that continued from the LM are indicated by the asterisks in A–I. The longitudinal fibers that ran between the skeletal muscles of the inferior part of the EAS are indicated by arrowheads in A, B, and E–I.