Histological Architecture of the Intersphincteric Region of the Anal Canal: Implications for the Anatomical Basis of Anal Fistula Pathways

Histological Architecture of the Intersphincteric Region of the Anal Canal: Implications for the Anatomical Basis of Anal Fistula Pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Histological Architecture of the Intersphincteric Region of the Anal Canal: Implications for the Anatomical Basis of Anal Fistula Pathways Satoru Muro, Yasuo Nakajima, Akimoto Nimura, Keiichi Akita This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8698526/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2026 Read the published version in International Journal of Colorectal Disease → Version 1 posted 10 You are reading this latest preprint version Abstract Purpose To clarify the histological architecture of the intersphincteric region of the anal canal by delineating the layer-specific organization and spatial relationships among the anal sphincter complex and associated muscular and connective tissue components. Methods Tissue blocks containing the lateral wall of the anal canal were obtained from 11 adult human cadavers donated for anatomical research. Specimens were examined using descriptive histological and immunohistochemical analyses in both transverse and coronal planes. The internal and external anal sphincters, longitudinal muscle, levator ani, interbundle gaps, and connective tissue compartments were identified and analyzed with respect to their three-dimensional organization. Results The intersphincteric region exhibited a heterogeneous and layered architecture rather than a uniform plane. The longitudinal muscle demonstrated a mosaic organization consisting of dense and loose components. The dense component terminated near the mid-height of the internal anal sphincter, whereas the loose component expanded inferiorly and formed a spacious compartment characterized by sparse smooth muscle fibers and loose connective tissue. Inferiorly, loose longitudinal muscle fibers branched and traversed natural interbundle gaps within the external anal sphincter. In addition, two partially overlapping layers of the levator ani were consistently observed, with interposed gaps contributing to the structural complexity of the intersphincteric region. Conclusion The intersphincteric region of the anal canal is a structurally complex and compartmentalized anatomical entity. Its heterogeneous histological architecture provides an anatomical substrate that may explain the initiation and directional spread of anal fistulas, including pathways described in classical fistula classifications. Anal fistula Parks classification Intersphincteric space Longitudinal muscle Histology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Anal fistulas are frequently encountered in daily colorectal practice and remain therapeutically challenging because of their high recurrence rates and the risk of postoperative fecal incontinence [1, 2]. Successful management of anal fistulas relies on a precise understanding of the underlying anatomy of the anal canal and sphincter complex. Although several classification systems have been proposed to describe fistula pathways, their clinical utility ultimately depends on how accurately they reflect the actual anatomical structures involved. Among these systems, the Parks classification remains the most widely used framework for preoperative assessment and surgical decision-making [3, 4]. This classification categorizes fistula tracts into intersphincteric, trans-sphincteric, suprasphincteric, and extrasphincteric types according to their relationship with the internal anal sphincter (IAS) and external anal sphincter (EAS). Despite its long-standing clinical relevance, however, the Parks classification presupposes the existence of an “intersphincteric” region without providing a clear anatomical definition of this term. In the original descriptions, abscess formation was illustrated as occurring “between the sphincters,” with subsequent upward, downward, or lateral spread, yet it remained unclear whether this space includes the longitudinal muscle (LM), at what level along the anal canal it is located, or which tissue layers—such as muscle bundle clefts or connective tissue compartments—are involved [4]. Even with advances in imaging modalities such as magnetic resonance imaging (MRI) and three-dimensional endoanal ultrasonography (3D-EAUS), the detailed microanatomy of the intersphincteric region remains insufficiently understood [5, 6]. These techniques provide valuable macroscopic information but lack the resolution necessary to delineate the fine histological organization of the LM and adjacent connective tissue structures. Consequently, the intersphincteric region is often interpreted as a simple anatomical plane, despite growing evidence that it represents a more complex anatomical entity. Previous anatomical and histological studies have highlighted the multilayered architecture of the anal sphincter complex and the presence of spaces between its muscular components [7–9]. Building on these studies, our previous investigations focused on the LM coursing between the IAS and EAS and its relationship with surrounding connective and adipose tissues [10–13]. We demonstrated that the LM is not a uniform, continuous layer but instead exhibits a mosaic organization composed of dense longitudinal smooth muscle bundles and loosely arranged smooth muscle fibers intermingled with loose connective tissue [14]. These observations suggested that the region traditionally labeled as “intersphincteric” encompasses heterogeneous tissue components rather than a single, well-defined plane. The present study was designed to clarify the histological architecture of the intersphincteric region of the anal canal by systematically analyzing the layer-specific organization and spatial relationships among the IAS, LM, EAS, levator ani, and associated connective tissue compartments. By defining the structural heterogeneity of this region on a histological basis, we further sought to explore how these anatomical features may provide a substrate for the initiation and propagation of anal fistulas, including pathways described in the Parks classification. Methods Study design This was a descriptive anatomical study of the anal canal based on histological and immunohistochemical analyses of formalin-fixed cadaveric specimens. The primary objective was to delineate the histological architecture and layer-specific organization of the intersphincteric region by examining the spatial relationships among the internal anal sphincter (IAS), longitudinal muscle (LM), external anal sphincter (EAS), levator ani (LA), and intervening connective tissue compartments. Cadaver specimens Eleven adult cadavers (five males and six females; age at death, 58–91 years; mean, 80.6 years) were included in this study. All specimens were obtained through the body donation program of the Department of Anatomy at our institution in accordance with the Japanese Act on Body Donation for Medical and Dental Education. Donation was based on the donors’ documented wishes prior to death. Cadavers with a known history of anorectal disease, previous anorectal surgery, or any apparent degeneration or injury affecting the anal sphincter complex or adjacent structures were excluded. All bodies were fixed by arterial perfusion with 8% formalin and subsequently stored in 30% alcohol to prevent fungal growth while maintaining tissue pliability. Tissue sampling The pelvis was obtained en bloc from each cadaver and divided along the midsagittal plane using a diamond saw. From each hemipelvis, the lateral wall of the anal canal and surrounding tissues were excised as a single tissue block, including the IAS, LM, EAS, subcutaneous tissues, and portions of the ischioanal fossa (IAF). For histological evaluation, tissue blocks were trimmed to include the full longitudinal extent of the anal canal, from above the anorectal junction to the anal verge. Sections were prepared in both the transverse plane (perpendicular to the long axis of the anal canal) and the coronal plane (parallel to the long axis), allowing multidirectional assessment of intermuscular spaces and the course of the LM. Histological processing All tissue blocks were embedded in paraffin and serially sectioned into 5-µm-thick slices at 1-mm intervals. Coronal blocks were processed in the same manner. Sections were stained with hematoxylin and eosin, Masson’s trichrome, and Elastica van Gieson to visualize the overall architecture of muscle bundles and connective tissue components. Immunohistochemical analyses were performed when required using antibodies against smooth muscle actin (Ready-to-Use Actin, Smooth Muscle Ab-1, Clone 1A4; Thermo Fisher Scientific, Fremont, CA, USA) and skeletal muscle myosin (Ready-to-Use Myosin, Skeletal Muscle Ab-2, Clone MYSN02; Thermo Fisher Scientific) to distinguish smooth muscle fibers from skeletal muscle fibers within the LM layer and sphincter complex. Microscopic analysis Microscopic examination was performed at magnifications ranging from ×40 to ×400 to evaluate the detailed microarchitecture of the lateral wall of the anal canal. The mucosa, submucosa, and Treitz muscle were first identified, followed by assessment of the thickness, arrangement, and spatial relationships of the IAS and EAS muscle bundles. Particular attention was directed to the LM layer located between the IAS and EAS. Based on our previous findings, this layer was classified into dense and loose components composed of compact smooth muscle bundles and sparsely arranged smooth muscle fibers intermingled with loose connective tissue, respectively [14]. The Treitz muscle was identified according to established anatomical criteria [15], and the components of the levator ani muscle were evaluated based on prior detailed anatomical descriptions [16]. The distribution of intermuscular spaces, loose connective tissue, and adipose tissue around the LM and between the IAS and EAS was examined, with special emphasis on changes in these structures along the longitudinal axis of the anal canal. The branching patterns and outward course of LM fibers traversing interbundle gaps within the EAS toward the subcutaneous tissue and IAF were also documented. Observations obtained from transverse and coronal sections were integrated to construct a schematic representation of the sphincteric layers and the histological organization of the intersphincteric region. Ethical considerations This study was approved by the Institutional Review Board of the Institute of Science Tokyo (approval no. M2018-006). All cadavers were handled anonymously and in strict accordance with the donors’ wishes. Results The mucosal, submucosal, and muscular layers of the anal canal were clearly identified in transverse sections at the level of the dentate line. Within the muscular layer, the IAS, LM, and EAS were observed from the inner to outer aspects (Fig. 1 a). Adipose tissue was present in the IAF lateral to the EAS. Under high magnification, the mucosa showed a mixture of simple columnar and stratified squamous epithelium, thus confirming that the specimens represented the transitional zone adjacent to the dentate line (Fig. 1 b). The submucosa contains the anal glands and Treitz muscle. Both the IAS and EAS consisted of circularly oriented muscle bundles with discernible interbundle gaps (Fig. 1 b, red arrows). The LM occupied the intersphincteric region between the IAS and EAS and showed a longitudinal fiber orientation aligned with the anal canal axis. In transverse sections from the upper anal canal, the lamina propria and muscularis mucosae were identified beneath the simple columnar epithelium, and the Treitz muscle was located immediately inside the IAS (Fig. 2 a). From the luminal side outward, the muscular layer consists of the IAS, LM, and LA (Fig. 2 b,c). The LA appeared as two distinct layers of skeletal muscle, which were identified based on prior anatomical criteria as an inner bundle corresponding to the muscle fibers attached to the rectum (LA-re) and an outer bundle corresponding to the fibers encircling the posterior aspect of the anal canal (LA-p). The two-layered structure of LA was confirmed by immunostaining for skeletal muscle myosin (Fig. 2 c). Immunohistochemistry for smooth muscle actin showed that the LM comprised densely packed smooth muscle bundles (LM-de) and loosely arranged fibers interspersed with connective tissue (LM-lo) (Fig. 2 b). In the cross-section, dense LM bundles appeared as aligned, round profiles surrounded by loose LM fibers. Coronal sections from the upper anal canal wall showed the mucosa, submucosa, IAS, LM, EAS, LA, and ischioanal fat (Fig. 3 a). Smooth muscle immunostaining revealed focal gaps between the IAS muscle bundles (Fig. 3 b, red arrows). Both dense and loose LM regions were evident, and portions of the loose LM extended laterally and superiorly, projecting into the smooth muscle fibers (LM-ex). In contrast, skeletal muscle immunostaining delineated the layered configuration of the LA: the LA-re descended from the superior aspect, passed medially to the LA-p, and entered the intersphincteric region. Distinct gaps were present between the LA-re and LA-p, as well as between the EAS muscle bundles (Fig. 3 c, red arrows). A comparison of smooth and skeletal muscle staining showed that the projecting smooth muscle fibers (LM-ex) were enveloped and directly attached to the LA-re fibers entering the intersphincteric space from above. The IAS, LM, EAS, and ischioanal fat were identified in coronal sections of the lower anal canal (Fig. 4 a). Smooth muscle immunostaining revealed that the dense LM terminated approximately midway from the height of the IAS, below which the loose LM expanded and formed branching extensions. The loose LM fibers coursed between the EAS bundles and extended toward the subcutaneous fat (Figs. 4 b, c). The sites where the loose LM penetrated the EAS were aligned with the natural inter-bundle gaps of the EAS (Fig. 4 c, red arrows). In the inferior portion of the LM, a spacious compartment consisting of loose LM fibers and connective tissue was consistently present. Discussion This study provides a detailed histological characterization of the intersphincteric region of the anal canal based on an integrated analysis of transverse and coronal sections. As summarized schematically in Fig. 5 , the intersphincteric region was shown to be a structurally heterogeneous and compartmentalized anatomical entity rather than a simple plane. Key structural features included the presence of interbundle gaps within the internal anal sphincter (IAS), external anal sphincter (EAS), and levator ani (LA), as well as a complex spatial relationship among multiple muscular layers and intervening connective tissue components. In addition, the levator ani was composed of two partially overlapping layers, with the inner component (LA-re) extending into the intersphincteric region and separated from the outer component (LA-p) by a distinct gap. A major anatomical finding of this study was the layer-specific organization of the longitudinal muscle (LM). The dense LM component terminated at approximately the mid-height of the IAS, whereas the loose LM component expanded inferiorly, branched along the longitudinal axis of the anal canal, and traversed natural interbundle gaps within the EAS toward the subcutaneous tissue and ischioanal fossa (IAF). Furthermore, lateral–superior extensions of loose LM fibers were observed to attach around the descending LA-re fibers. Importantly, this configuration indicated that the interface between the LM and EAS does not exhibit direct anatomical continuity with the supralevator compartment. When these histological findings are interpreted in the context of the Parks classification, the region traditionally illustrated as the site of an “intersphincteric abscess” corresponds closely to the loose smooth muscle space located in the inferior portion of the LM (Fig. 6 ). The four fistula types described by Parks have been used clinically for more than five decades; however, the original descriptions did not provide a detailed definition of the histological nature of the “intersphincteric space” [4]. Earlier anatomical and histological studies primarily focused on the sphincter muscles themselves and did not sufficiently address the internal architecture of the LM situated between the IAS and EAS, thereby underestimating the structural complexity of this region [7–9, 17, 18]. From a histological perspective, the loose inferior compartment of the LM represents a low-resistance space composed of sparse smooth muscle fibers and loose connective tissue. This structural characteristic is consistent with recent evidence indicating that cryptoglandular infections preferentially spread along pre-existing low-resistance connective tissue pathways [19]. Based on the present observations, fistula pathways described in the Parks classification can be interpreted as extensions along anatomically definable structures, such as the interbundle gaps within the EAS, the gap between LA-re and LA-p, and the branching loose components of the LM. In contrast, although type IV fistulas are described as extending into the supralevator space, no direct anatomical pathway corresponding to such an extension was identified in the normal specimens examined in this study. This finding suggests that supralevator spread may require additional pathological or secondary factors beyond pre-existing anatomical continuity. The present histological redefinition of the intersphincteric region has important clinical implications. This region has traditionally been regarded as a homogeneous plane, an oversimplification that has been implicated in interpretive inconsistencies in endoanal ultrasonography (EAUS) and magnetic resonance imaging (MRI), as well as in discrepancies among fistula classification systems [20–22]. A shared limitation of multiple classification schemes, including more recent imaging-guided systems, is the absence of a detailed histological foundation for the intersphincteric space [23]. Recognition of the dense and loose components of the LM and their three-dimensional arrangement may help explain the heterogeneous imaging signals observed in this region, consistent with MRI-based findings suggesting that dense and loose LM components can be distinguished [14]. Moreover, sex-related differences in the course of intersphincteric fistulas reported in three-dimensional EAUS studies further support the notion that this region is anatomically complex and heterogeneous rather than uniform [24]. Several limitations of this study should be acknowledged. First, all specimens were obtained from formalin-fixed cadavers of elderly donors, and tissue characteristics such as firmness and fat content may differ from those of living individuals. Second, the analysis was limited to the lateral wall of the anal canal, and regional variations in the anterior or posterior walls were not assessed. Third, the correspondence between histological structures and the Parks classification was inferential and was not directly validated using specimens from patients with active fistulas. Finally, because this study was based on normal anatomical specimens, pathways that may arise only through pathological disruption, including supralevator extension, could not be fully evaluated. Nevertheless, the structural reinterpretation presented here is consistent with recent imaging and immunological findings, supporting its anatomical and clinical plausibility. In conclusion, this study demonstrates that the intersphincteric region of the anal canal is a structurally complex and heterogeneous anatomical entity composed of multiple muscle layers, interbundle gaps, and loose connective tissue compartments. The identification of a loose inferior compartment within the LM provides a histological substrate that allows the pathways described in the Parks classification, particularly those related to the “intersphincteric abscess,” to be interpreted as routes of least resistance within a defined anatomical framework. A clearer understanding of the three-dimensional histological architecture of this region may improve the interpretation of EAUS and MRI findings, enhance consistency among classification systems, and ultimately contribute to more informed preoperative evaluation and sphincter-preserving surgical strategies. Declarations Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Compliance with Ethical Standards Disclosure of potential conflicts of interest : None. Research involving Human Participants and/or Animals This study was approved by the Institutional Review Board of Institute of Science Tokyo (approval no. M2018-006). All cadavers were handled anonymously in accordance with the donors’ wishes. Informed consent: Donation was based on the donors’ documented wishes prior to death. Funding: This study was supported by JSPS KAKENHI (grant no. 25K18696). Author Contribution Conceptualization: Satoru Muro, Yasuo Nakajima, Keiichi Akita; Data curation: Satoru Muro; Formal analysis: Satoru Muro, Yasuo Nakajima, Akimoto Nimura, Keiichi Akita; Funding acquisition: Satoru Muro, Keiichi Akita; Investigation: Satoru Muro; Methodology: Satoru Muro, Keiichi Akita; Project administration: Satoru Muro, Keiichi Akita; Visualization: Satoru Muro, Akimoto Nimura; Writing–original draft: Satoru Muro; Writing–review & editing: Satoru Muro, Yasuo Nakajima, Akimoto Nimura, Keiichi Akita. All authors read and approved the final manuscript. Acknowledgments The authors sincerely thank the individuals who donated their bodies to science for their anatomical research. Their contributions will help increase the overall knowledge of humankind and improve patient care. We would also like to extend our gratitude to the families of the donors. This study was supported by JSPS KAKENHI (grant no. 25K18696). Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Almughamsi AM, Elhassan YH (2025) Understanding the anatomical basis of anorectal fistulas and their surgical management: exploring different types for enhanced precision and safety. Surg Today 55:457–474. https://doi.org/10.1007/s00595-025-02995-2 Tabry H, Farrands PA (2011) Update on anal fistulae: surgical perspectives for the gastroenterologist. Can J Gastroenterol 25:675–680. https://doi.org/10.1155/2011/931316 Parks AG (1961) Pathogenesis and treatment of fistuila-in-ano. Br Med J 1:463–469. https://doi.org/10.1136/bmj.1.5224.463 Parks AG, Gordon PH, Hardcastle JD (1976) A classification of fistula-in-ano. Br J Surg 63:1–12. https://doi.org/10.1002/bjs.1800630102 Abdool Z, Sultan AH, Thakar R (2012) Ultrasound imaging of the anal sphincter complex: a review. Br J Radiol 85:865–875. https://doi.org/10.1259/bjr/27314678 Schäfer A, Enck P, Fürst G, Kahn T, Frieling T, Lübke HJ (1994) Anatomy of the anal sphincters. Comparison of anal endosonography to magnetic resonance imaging. Dis Colon Rectum 37:777–781. https://doi.org/10.1007/BF02050142 Fritsch H, Brenner E, Lienemann A, Ludwikowski B (2002) Anal sphincter complex: reinterpreted morphology and its clinical relevance. Dis Colon Rectum 45:188–194. https://doi.org/10.1007/s10350-004-6144-x Lunniss PJ, Phillips RK (1992) Anatomy and function of the anal longitudinal muscle. Br J Surg 79:882–884. https://doi.org/10.1002/bjs.1800790908 Shafik A (1979) A new concept of the anatomy of the anal sphincter mechanism and the physiology of defecation. VII. Anal fistula: a simplified classification. Dis Colon Rectum 22:408–414. https://doi.org/10.1007/BF02586911 Muro S, Yamaguchi K, Nakajima Y, Watanabe K, Harada M, Nimura A, Akita K (2014) Dynamic intersection of the longitudinal muscle and external anal sphincter in the layered structure of the anal canal posterior wall. Surg Radiol Anat 36:551–559. https://doi.org/10.1007/s00276-013-1228-8 Tsukada Y, Ito M, Watanabe K, Yamaguchi K, Kojima M, Hayashi R, Akita K, Saito N (2016) Topographic anatomy of the anal sphincter complex and levator ani muscle as it relates to intersphincteric resection for very low rectal disease. Dis Colon Rectum 59:426–433. https://doi.org/10.1097/DCR.0000000000000565 Nakajima Y, Muro S, Nasu H, Harada M, Yamaguchi K, Akita K (2017) Morphology of the region anterior to the anal canal in males: visualization of the anterior bundle of the longitudinal muscle by transanal ultrasonography. Surg Radiol Anat 39:967–973. https://doi.org/10.1007/s00276-017-1832-0 Muro S, Tsukada Y, Harada M, Ito M, Akita K (2019) Anatomy of the smooth muscle structure in the female anorectal anterior wall: convergence and anterior extension of the internal anal sphincter and longitudinal muscle. Colorectal Dis 21:472–480. https://doi.org/10.1111/codi.14549 Muro S, Kagawa R, Habu M, Ka H, Harada M, Akita K (2020) Coexistence of dense and sparse areas in the longitudinal smooth muscle of the anal canal: anatomical and histological analyses inspired by magnetic resonance images. Clin Anat 33:619–626. https://doi.org/10.1002/ca.23467 Muro S, Yamaguchi K, Inoshita N, Nakajima Y, Edinam DJ, Nimura A, Akita K (2025) New anatomical insight into the muscular structure of the anal canal: revealing Treitz muscle as a directional shift of the internal anal sphincter. Ann Coloproctol 41:501–509. https://doi.org/10.3393/ac.2024.00647.0092 Muro S, Moue S, Akita K (2024) Twisted orientation of the muscle bundles in the levator ani functional parts in women: implications for pelvic floor support mechanism. J Anat 244:486–496. https://doi.org/10.1111/joa.13968 Macchi V, Porzionato A, Stecco C, Vigato E, Parenti A, De Caro R (2008) Histo-topographic study of the longitudinal anal muscle. Clin Anat 21:447–452. https://doi.org/10.1002/ca.20633 Hieda K, Cho KH, Arakawa T, Fujimiya M, Murakami G, Matsubara A (2013) Nerves in the intersphincteric space of the human anal canal with special reference to their continuation to the enteric nerve plexus of the rectum. Clin Anat 26:843–854. https://doi.org/10.1002/ca.22227 Litta F, Papait A, Lucchetti D, Farigu S, Parello A, Tenore CR, Campennì P, Silini AR, Giustiniani MC, Parolini O, Sgambato A, Ratto C (2022) The pathogenesis of cryptoglandular anal fistula: new insight into the immunological profile. Colorectal Dis 24:1567–1575. https://doi.org/10.1111/codi.16290 Emile SH, Elfeki H, El-Said M, Khafagy W, Shalaby M (2021) Modification of parks classification of cryptoglandular anal fistula. Dis Colon Rectum 64:446–458. https://doi.org/10.1097/DCR.0000000000001797 Dawka S, Yagnik VD (2021) Comparison between the modified Parks and Garg classifications of cryptoglandular anal fistulas. Dis Colon Rectum 64:e589. https://doi.org/10.1097/DCR.0000000000002208 Teymouri A, Keshvari A, Khorasanizadeh F, Kazemeini A, Behboudi B, Fazeli MS, Keramati MR, Ashjaei A, Tafti SMA, Naseri A (2025) External validation of the modified Parks classification of cryptoglandular anal fistula to predict failure of healing: the protocol for a retrospective analysis. Int J Surg Protoc 29:118–121. https://doi.org/10.1097/SP9.0000000000000055 Brillantino A, Iacobellis F, Marano L, Renzi A, Talento P, Brusciano L, Gambardella C, Favetta U, Schiano Di Visconte M, Monaco L, Grillo M, Maglio MN, Foroni F, Palumbo A, Sotelo MLS, Vicenzo L, Palladino E, Frezza G, Menna MP, Mauro P, Picardi S, Mensorio MM, Mosca V, Bottino V, Ioia G, Rispoli C, Serafino MD, Caruso M, Ronza R, Frittoli B, Schettini D, Stoppino L, Iafrate F, Lombardi G, Antropoli C, Cappabianca S, Docimo L, Grassi R, Reginelli A (2025) Validation of a novel imaging-guided and anatomy-based classification system for anorectal fistulas: a retrospective clinical evaluation study. Ann Coloproctol 41:207–220. https://doi.org/10.3393/ac.2024.00675.0096 da Silva Fernandes GO, Maciel MQ, Lima Barreto RG, Oliveira Cajazeiras MT, da Mota NC, Pavan Y, Travassos Pinto M, Freitas de Aquino L, Pinheiro Barreto JB (2024) Correlation of the anatomy of the intersphincteric anal fistula with sex: an analysis through anorectal three-dimensional ultrasound. J Coloproctol 44:e229–e233. https://doi.org/10.1055/s-0044-1793855 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2026 Read the published version in International Journal of Colorectal Disease → Version 1 posted Editorial decision: Revision requested 08 Feb, 2026 Reviews received at journal 06 Feb, 2026 Reviews received at journal 05 Feb, 2026 Reviewers agreed at journal 31 Jan, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers invited by journal 27 Jan, 2026 Editor assigned by journal 26 Jan, 2026 Submission checks completed at journal 26 Jan, 2026 First submitted to journal 26 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8698526","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581615496,"identity":"dec5808c-02ab-4557-8255-162da0bc5c5c","order_by":0,"name":"Satoru Muro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYNACGyBmb2Bg4AHzDhCjJQ2IeQ6TrEUiGaaFANCdkfvwAUOCXR7/zPdHN7xhsJNnYDyL3xqzG+nGBgwJycUSt5PZbs5hSDZsYDiXQEBLGpsE4w/mxAaglts8DMxA5WcMCGthSKhPnH/zMEhLPdFaDiduuMEM0nKYCC1nnjEbJCQcT9x4Jtns5hyD44ZtBP1yPI3xwYeE6sR5xw8+u/GmolqeX4JAiIEBwlSgk9gkzhDWgQb4e0jWMgpGwSgYBcMbAABod0WBISj6QgAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Muro","suffix":""},{"id":581615497,"identity":"6502d80d-2573-44e0-923f-40d4b5d7abd3","order_by":1,"name":"Yasuo Nakajima","email":"","orcid":"","institution":"Moriyama Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yasuo","middleName":"","lastName":"Nakajima","suffix":""},{"id":581615498,"identity":"44687a3b-6541-43c2-8895-62c73212fc5a","order_by":2,"name":"Akimoto Nimura","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Akimoto","middleName":"","lastName":"Nimura","suffix":""},{"id":581615499,"identity":"8e7ca64b-fa9e-482f-9196-6ae9dabfeee7","order_by":3,"name":"Keiichi Akita","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Keiichi","middleName":"","lastName":"Akita","suffix":""}],"badges":[],"createdAt":"2026-01-26 09:09:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8698526/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8698526/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00384-026-05123-9","type":"published","date":"2026-03-27T16:08:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101417164,"identity":"23782055-d48d-44ff-9f27-b3fbe4ce412e","added_by":"auto","created_at":"2026-01-29 12:52:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2257318,"visible":true,"origin":"","legend":"\u003cp\u003eTransverse histological sections at the dentate line level. (a) Low-magnification transverse section demonstrating the mucosa (M), submucosa (SM), and muscular layers composed of the internal anal sphincter (IAS), longitudinal muscle (LM), and external anal sphincter (EAS). The fat of the ischioanal fossa (IAF) was visible lateral to the EAS. (b) High-magnification view of the transitional epithelium showing a mixture of simple columnar epithelium (SCE) and stratified squamous epithelium (SSE). The SM contains anal glands (AGs) and the Treitz muscle (TM). Red arrows highlight the gaps in the muscle bundles within the IAS and EAS. The LM occupies the intersphincteric region, with its fibers oriented longitudinally along the anal canal axis\u003c/p\u003e","description":"","filename":"figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8698526/v1/3532b0b7d2998f3b26fb92d9.jpg"},{"id":101417166,"identity":"db226807-b895-4772-85c8-3e9928f475e5","added_by":"auto","created_at":"2026-01-29 12:52:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2568279,"visible":true,"origin":"","legend":"\u003cp\u003eTransverse histological sections of the upper anal canal. (a) Simple columnar epithelium with underlying lamina propria and muscularis mucosae (MM). The Treitz muscle (TM) lies immediately internal to the internal anal sphincter (IAS). The muscular layer consists of IAS, longitudinal muscle (LM), and the levator ani (LA). Based on established anatomical criteria, the LA was identified as an inner bundle attached to the rectum (LA-re) and an outer bundle encircling the posterior anal canal (LA-p). (b) Smooth muscle immunostaining showing dense LM (LM-de) and loose LM (LM-lo). LM-de appeared as aligned round bundles surrounded by LM-lo fibers. (c) Skeletal muscle immunostaining confirmed the two-layered configuration of the LA (LA-re and LA-p)\u003c/p\u003e","description":"","filename":"figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8698526/v1/fe5f9320087a0e97e8e44d59.jpg"},{"id":101417165,"identity":"806ef5b9-635c-449f-ac22-19284ea1e56d","added_by":"auto","created_at":"2026-01-29 12:52:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2036240,"visible":true,"origin":"","legend":"\u003cp\u003eCoronal histological sections of the upper anal canal wall. (a) Low-magnification coronal sections identifying mucosa (M), submucosa (SM), internal anal sphincter (IAS), longitudinal muscle (LM), external anal sphincter (EAS), levator ani (LA), and ischioanal fossa (IAF) fat. (b) Smooth muscle immunostaining showing focal interbundle gaps within the IAS (red arrows). Dense and loose LM components are present, with portions of loose LM projecting superolaterally as extension fibers (LM-ex). (c) Skeletal muscle immunostaining demonstrating the layered arrangement of the LA: the LA-re descends medially to the LA-p and enters the intersphincteric region. Red arrows indicate gaps between the LA-re and LA-p and within EAS bundles. A comparison of the stains shows LM-ex fibers enveloping and directly attaching to LA-re as they enter the intersphincteric space\u003c/p\u003e","description":"","filename":"figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8698526/v1/235eb2ca14462d59f46552fc.jpg"},{"id":101751416,"identity":"4d8a691b-a0c2-48b6-bd2f-772513bd163f","added_by":"auto","created_at":"2026-02-03 10:20:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1629125,"visible":true,"origin":"","legend":"\u003cp\u003eCoronal histological sections of the lower anal canal. (a) Identification of the internal anal sphincter (IAS), longitudinal muscle (LM), external anal sphincter (EAS), and ischioanal fossa (IAF) fat. A prominent loose compartment composed of loose LM (LM-lo) fibers and loose connective tissue occupies the inferior portion of the LM. (b) Smooth muscle immunostaining demonstrates termination of the dense LM at approximately mid-IAS height. Below this level, the loose LM expands into a branching network that traverses the gaps between the EAS bundles and extends toward the subcutaneous fat. (c) Skeletal muscle immunostaining showing the EAS muscle bundles. Red arrows indicate gaps within the EAS bundles\u003c/p\u003e","description":"","filename":"figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8698526/v1/12858c24af7ff4af480df7e5.jpg"},{"id":101417167,"identity":"31371dd7-2b28-4f72-bedd-6a3ac316a610","added_by":"auto","created_at":"2026-01-29 12:52:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1594931,"visible":true,"origin":"","legend":"\u003cp\u003eIntegrated schematic reconstruction of the intersphincteric region.The key anatomical features illustrated include the following: (1) Interbundle gaps within the internal anal sphincter (IAS), external anal sphincter (EAS), and levator ani (LA); (2) Loose space in the inferior longitudinal muscle (LM); (3) Overlapping configuration of the LA inner bundle attached to the rectum (LA-re) and the LA outer bundle encircling the posterior anal canal (LA-p), with LA-re extending into the intersphincteric space\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8698526/v1/6f655702f8b9087ce6d05b4f.jpg"},{"id":101751558,"identity":"c269567c-280a-4eef-83e8-d327a9a04492","added_by":"auto","created_at":"2026-02-03 10:21:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3078157,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic interpretation of the histological architecture of the intersphincteric region in relation to the Parks classification of anal fistulas. This diagram integrates the histologically identified structures of the internal anal sphincter (IAS), longitudinal muscle (LM), external anal sphincter (EAS), and levator ani (LA), with particular emphasis on the loose smooth muscle compartment in the inferior portion of the LM. The region traditionally illustrated by Parks as the site of an “intersphincteric abscess” is shown to correspond anatomically to this inferior loose LM compartment. Natural interbundle gaps within the IAS and EAS, as well as the gap between the inner (LA-re) and outer (LA-p) components of the levator ani, are depicted as structural continuities that may account for the directional extension patterns described in the Parks classification.\u003c/p\u003e","description":"","filename":"figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8698526/v1/d977191b2ade6e8a7407d249.jpg"},{"id":105755513,"identity":"323e0a2c-2f6f-4403-a425-237f438acbeb","added_by":"auto","created_at":"2026-03-30 16:27:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13668259,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8698526/v1/06d8d9f6-a88a-472d-91b8-a5e1965a1207.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Histological Architecture of the Intersphincteric Region of the Anal Canal: Implications for the Anatomical Basis of Anal Fistula Pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnal fistulas are frequently encountered in daily colorectal practice and remain therapeutically challenging because of their high recurrence rates and the risk of postoperative fecal incontinence [1, 2]. Successful management of anal fistulas relies on a precise understanding of the underlying anatomy of the anal canal and sphincter complex. Although several classification systems have been proposed to describe fistula pathways, their clinical utility ultimately depends on how accurately they reflect the actual anatomical structures involved.\u003c/p\u003e \u003cp\u003eAmong these systems, the Parks classification remains the most widely used framework for preoperative assessment and surgical decision-making [3, 4]. This classification categorizes fistula tracts into intersphincteric, trans-sphincteric, suprasphincteric, and extrasphincteric types according to their relationship with the internal anal sphincter (IAS) and external anal sphincter (EAS). Despite its long-standing clinical relevance, however, the Parks classification presupposes the existence of an \u0026ldquo;intersphincteric\u0026rdquo; region without providing a clear anatomical definition of this term. In the original descriptions, abscess formation was illustrated as occurring \u0026ldquo;between the sphincters,\u0026rdquo; with subsequent upward, downward, or lateral spread, yet it remained unclear whether this space includes the longitudinal muscle (LM), at what level along the anal canal it is located, or which tissue layers\u0026mdash;such as muscle bundle clefts or connective tissue compartments\u0026mdash;are involved [4].\u003c/p\u003e \u003cp\u003eEven with advances in imaging modalities such as magnetic resonance imaging (MRI) and three-dimensional endoanal ultrasonography (3D-EAUS), the detailed microanatomy of the intersphincteric region remains insufficiently understood [5, 6]. These techniques provide valuable macroscopic information but lack the resolution necessary to delineate the fine histological organization of the LM and adjacent connective tissue structures. Consequently, the intersphincteric region is often interpreted as a simple anatomical plane, despite growing evidence that it represents a more complex anatomical entity.\u003c/p\u003e \u003cp\u003ePrevious anatomical and histological studies have highlighted the multilayered architecture of the anal sphincter complex and the presence of spaces between its muscular components [7\u0026ndash;9]. Building on these studies, our previous investigations focused on the LM coursing between the IAS and EAS and its relationship with surrounding connective and adipose tissues [10\u0026ndash;13]. We demonstrated that the LM is not a uniform, continuous layer but instead exhibits a mosaic organization composed of dense longitudinal smooth muscle bundles and loosely arranged smooth muscle fibers intermingled with loose connective tissue [14]. These observations suggested that the region traditionally labeled as \u0026ldquo;intersphincteric\u0026rdquo; encompasses heterogeneous tissue components rather than a single, well-defined plane.\u003c/p\u003e \u003cp\u003eThe present study was designed to clarify the histological architecture of the intersphincteric region of the anal canal by systematically analyzing the layer-specific organization and spatial relationships among the IAS, LM, EAS, levator ani, and associated connective tissue compartments. By defining the structural heterogeneity of this region on a histological basis, we further sought to explore how these anatomical features may provide a substrate for the initiation and propagation of anal fistulas, including pathways described in the Parks classification.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis was a descriptive anatomical study of the anal canal based on histological and immunohistochemical analyses of formalin-fixed cadaveric specimens. The primary objective was to delineate the histological architecture and layer-specific organization of the intersphincteric region by examining the spatial relationships among the internal anal sphincter (IAS), longitudinal muscle (LM), external anal sphincter (EAS), levator ani (LA), and intervening connective tissue compartments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCadaver specimens\u003c/h3\u003e\n\u003cp\u003eEleven adult cadavers (five males and six females; age at death, 58\u0026ndash;91 years; mean, 80.6 years) were included in this study. All specimens were obtained through the body donation program of the Department of Anatomy at our institution in accordance with the Japanese Act on Body Donation for Medical and Dental Education. Donation was based on the donors\u0026rsquo; documented wishes prior to death.\u003c/p\u003e \u003cp\u003eCadavers with a known history of anorectal disease, previous anorectal surgery, or any apparent degeneration or injury affecting the anal sphincter complex or adjacent structures were excluded. All bodies were fixed by arterial perfusion with 8% formalin and subsequently stored in 30% alcohol to prevent fungal growth while maintaining tissue pliability.\u003c/p\u003e\n\u003ch3\u003eTissue sampling\u003c/h3\u003e\n\u003cp\u003eThe pelvis was obtained en bloc from each cadaver and divided along the midsagittal plane using a diamond saw. From each hemipelvis, the lateral wall of the anal canal and surrounding tissues were excised as a single tissue block, including the IAS, LM, EAS, subcutaneous tissues, and portions of the ischioanal fossa (IAF).\u003c/p\u003e \u003cp\u003eFor histological evaluation, tissue blocks were trimmed to include the full longitudinal extent of the anal canal, from above the anorectal junction to the anal verge. Sections were prepared in both the transverse plane (perpendicular to the long axis of the anal canal) and the coronal plane (parallel to the long axis), allowing multidirectional assessment of intermuscular spaces and the course of the LM.\u003c/p\u003e\n\u003ch3\u003eHistological processing\u003c/h3\u003e\n\u003cp\u003eAll tissue blocks were embedded in paraffin and serially sectioned into 5-\u0026micro;m-thick slices at 1-mm intervals. Coronal blocks were processed in the same manner. Sections were stained with hematoxylin and eosin, Masson\u0026rsquo;s trichrome, and Elastica van Gieson to visualize the overall architecture of muscle bundles and connective tissue components.\u003c/p\u003e \u003cp\u003eImmunohistochemical analyses were performed when required using antibodies against smooth muscle actin (Ready-to-Use Actin, Smooth Muscle Ab-1, Clone 1A4; Thermo Fisher Scientific, Fremont, CA, USA) and skeletal muscle myosin (Ready-to-Use Myosin, Skeletal Muscle Ab-2, Clone MYSN02; Thermo Fisher Scientific) to distinguish smooth muscle fibers from skeletal muscle fibers within the LM layer and sphincter complex.\u003c/p\u003e\n\u003ch3\u003eMicroscopic analysis\u003c/h3\u003e\n\u003cp\u003eMicroscopic examination was performed at magnifications ranging from \u0026times;40 to \u0026times;400 to evaluate the detailed microarchitecture of the lateral wall of the anal canal. The mucosa, submucosa, and Treitz muscle were first identified, followed by assessment of the thickness, arrangement, and spatial relationships of the IAS and EAS muscle bundles.\u003c/p\u003e \u003cp\u003eParticular attention was directed to the LM layer located between the IAS and EAS. Based on our previous findings, this layer was classified into dense and loose components composed of compact smooth muscle bundles and sparsely arranged smooth muscle fibers intermingled with loose connective tissue, respectively [14]. The Treitz muscle was identified according to established anatomical criteria [15], and the components of the levator ani muscle were evaluated based on prior detailed anatomical descriptions [16].\u003c/p\u003e \u003cp\u003eThe distribution of intermuscular spaces, loose connective tissue, and adipose tissue around the LM and between the IAS and EAS was examined, with special emphasis on changes in these structures along the longitudinal axis of the anal canal. The branching patterns and outward course of LM fibers traversing interbundle gaps within the EAS toward the subcutaneous tissue and IAF were also documented.\u003c/p\u003e \u003cp\u003eObservations obtained from transverse and coronal sections were integrated to construct a schematic representation of the sphincteric layers and the histological organization of the intersphincteric region.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEthical considerations\u003c/h2\u003e \u003cp\u003eThis study was approved by the Institutional Review Board of the Institute of Science Tokyo (approval no. M2018-006). All cadavers were handled anonymously and in strict accordance with the donors\u0026rsquo; wishes.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe mucosal, submucosal, and muscular layers of the anal canal were clearly identified in transverse sections at the level of the dentate line. Within the muscular layer, the IAS, LM, and EAS were observed from the inner to outer aspects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Adipose tissue was present in the IAF lateral to the EAS. Under high magnification, the mucosa showed a mixture of simple columnar and stratified squamous epithelium, thus confirming that the specimens represented the transitional zone adjacent to the dentate line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The submucosa contains the anal glands and Treitz muscle. Both the IAS and EAS consisted of circularly oriented muscle bundles with discernible interbundle gaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, red arrows). The LM occupied the intersphincteric region between the IAS and EAS and showed a longitudinal fiber orientation aligned with the anal canal axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn transverse sections from the upper anal canal, the lamina propria and muscularis mucosae were identified beneath the simple columnar epithelium, and the Treitz muscle was located immediately inside the IAS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). From the luminal side outward, the muscular layer consists of the IAS, LM, and LA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c). The LA appeared as two distinct layers of skeletal muscle, which were identified based on prior anatomical criteria as an inner bundle corresponding to the muscle fibers attached to the rectum (LA-re) and an outer bundle corresponding to the fibers encircling the posterior aspect of the anal canal (LA-p). The two-layered structure of LA was confirmed by immunostaining for skeletal muscle myosin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Immunohistochemistry for smooth muscle actin showed that the LM comprised densely packed smooth muscle bundles (LM-de) and loosely arranged fibers interspersed with connective tissue (LM-lo) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In the cross-section, dense LM bundles appeared as aligned, round profiles surrounded by loose LM fibers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCoronal sections from the upper anal canal wall showed the mucosa, submucosa, IAS, LM, EAS, LA, and ischioanal fat (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Smooth muscle immunostaining revealed focal gaps between the IAS muscle bundles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, red arrows). Both dense and loose LM regions were evident, and portions of the loose LM extended laterally and superiorly, projecting into the smooth muscle fibers (LM-ex). In contrast, skeletal muscle immunostaining delineated the layered configuration of the LA: the LA-re descended from the superior aspect, passed medially to the LA-p, and entered the intersphincteric region. Distinct gaps were present between the LA-re and LA-p, as well as between the EAS muscle bundles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, red arrows). A comparison of smooth and skeletal muscle staining showed that the projecting smooth muscle fibers (LM-ex) were enveloped and directly attached to the LA-re fibers entering the intersphincteric space from above.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe IAS, LM, EAS, and ischioanal fat were identified in coronal sections of the lower anal canal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Smooth muscle immunostaining revealed that the dense LM terminated approximately midway from the height of the IAS, below which the loose LM expanded and formed branching extensions. The loose LM fibers coursed between the EAS bundles and extended toward the subcutaneous fat (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). The sites where the loose LM penetrated the EAS were aligned with the natural inter-bundle gaps of the EAS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, red arrows). In the inferior portion of the LM, a spacious compartment consisting of loose LM fibers and connective tissue was consistently present.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides a detailed histological characterization of the intersphincteric region of the anal canal based on an integrated analysis of transverse and coronal sections. As summarized schematically in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the intersphincteric region was shown to be a structurally heterogeneous and compartmentalized anatomical entity rather than a simple plane. Key structural features included the presence of interbundle gaps within the internal anal sphincter (IAS), external anal sphincter (EAS), and levator ani (LA), as well as a complex spatial relationship among multiple muscular layers and intervening connective tissue components. In addition, the levator ani was composed of two partially overlapping layers, with the inner component (LA-re) extending into the intersphincteric region and separated from the outer component (LA-p) by a distinct gap.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA major anatomical finding of this study was the layer-specific organization of the longitudinal muscle (LM). The dense LM component terminated at approximately the mid-height of the IAS, whereas the loose LM component expanded inferiorly, branched along the longitudinal axis of the anal canal, and traversed natural interbundle gaps within the EAS toward the subcutaneous tissue and ischioanal fossa (IAF). Furthermore, lateral\u0026ndash;superior extensions of loose LM fibers were observed to attach around the descending LA-re fibers. Importantly, this configuration indicated that the interface between the LM and EAS does not exhibit direct anatomical continuity with the supralevator compartment.\u003c/p\u003e \u003cp\u003eWhen these histological findings are interpreted in the context of the Parks classification, the region traditionally illustrated as the site of an \u0026ldquo;intersphincteric abscess\u0026rdquo; corresponds closely to the loose smooth muscle space located in the inferior portion of the LM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The four fistula types described by Parks have been used clinically for more than five decades; however, the original descriptions did not provide a detailed definition of the histological nature of the \u0026ldquo;intersphincteric space\u0026rdquo; [4]. Earlier anatomical and histological studies primarily focused on the sphincter muscles themselves and did not sufficiently address the internal architecture of the LM situated between the IAS and EAS, thereby underestimating the structural complexity of this region [7\u0026ndash;9, 17, 18].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom a histological perspective, the loose inferior compartment of the LM represents a low-resistance space composed of sparse smooth muscle fibers and loose connective tissue. This structural characteristic is consistent with recent evidence indicating that cryptoglandular infections preferentially spread along pre-existing low-resistance connective tissue pathways [19]. Based on the present observations, fistula pathways described in the Parks classification can be interpreted as extensions along anatomically definable structures, such as the interbundle gaps within the EAS, the gap between LA-re and LA-p, and the branching loose components of the LM. In contrast, although type IV fistulas are described as extending into the supralevator space, no direct anatomical pathway corresponding to such an extension was identified in the normal specimens examined in this study. This finding suggests that supralevator spread may require additional pathological or secondary factors beyond pre-existing anatomical continuity.\u003c/p\u003e \u003cp\u003eThe present histological redefinition of the intersphincteric region has important clinical implications. This region has traditionally been regarded as a homogeneous plane, an oversimplification that has been implicated in interpretive inconsistencies in endoanal ultrasonography (EAUS) and magnetic resonance imaging (MRI), as well as in discrepancies among fistula classification systems [20\u0026ndash;22]. A shared limitation of multiple classification schemes, including more recent imaging-guided systems, is the absence of a detailed histological foundation for the intersphincteric space [23]. Recognition of the dense and loose components of the LM and their three-dimensional arrangement may help explain the heterogeneous imaging signals observed in this region, consistent with MRI-based findings suggesting that dense and loose LM components can be distinguished [14]. Moreover, sex-related differences in the course of intersphincteric fistulas reported in three-dimensional EAUS studies further support the notion that this region is anatomically complex and heterogeneous rather than uniform [24].\u003c/p\u003e \u003cp\u003eSeveral limitations of this study should be acknowledged. First, all specimens were obtained from formalin-fixed cadavers of elderly donors, and tissue characteristics such as firmness and fat content may differ from those of living individuals. Second, the analysis was limited to the lateral wall of the anal canal, and regional variations in the anterior or posterior walls were not assessed. Third, the correspondence between histological structures and the Parks classification was inferential and was not directly validated using specimens from patients with active fistulas. Finally, because this study was based on normal anatomical specimens, pathways that may arise only through pathological disruption, including supralevator extension, could not be fully evaluated. Nevertheless, the structural reinterpretation presented here is consistent with recent imaging and immunological findings, supporting its anatomical and clinical plausibility.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that the intersphincteric region of the anal canal is a structurally complex and heterogeneous anatomical entity composed of multiple muscle layers, interbundle gaps, and loose connective tissue compartments. The identification of a loose inferior compartment within the LM provides a histological substrate that allows the pathways described in the Parks classification, particularly those related to the \u0026ldquo;intersphincteric abscess,\u0026rdquo; to be interpreted as routes of least resistance within a defined anatomical framework. A clearer understanding of the three-dimensional histological architecture of this region may improve the interpretation of EAUS and MRI findings, enhance consistency among classification systems, and ultimately contribute to more informed preoperative evaluation and sphincter-preserving surgical strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e \u003cp\u003e \u003cb\u003eDisclosure of potential conflicts of interest\u003c/b\u003e: None.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eResearch involving Human Participants and/or Animals\u003c/strong\u003e \u003cp\u003eThis study was approved by the Institutional Review Board of Institute of Science Tokyo (approval no. M2018-006). All cadavers were handled anonymously in accordance with the donors\u0026rsquo; wishes.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed consent:\u003c/strong\u003e \u003cp\u003eDonation was based on the donors\u0026rsquo; documented wishes prior to death.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported by JSPS KAKENHI (grant no. 25K18696).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Satoru Muro, Yasuo Nakajima, Keiichi Akita; Data curation: Satoru Muro; Formal analysis: Satoru Muro, Yasuo Nakajima, Akimoto Nimura, Keiichi Akita; Funding acquisition: Satoru Muro, Keiichi Akita; Investigation: Satoru Muro; Methodology: Satoru Muro, Keiichi Akita; Project administration: Satoru Muro, Keiichi Akita; Visualization: Satoru Muro, Akimoto Nimura; Writing\u0026ndash;original draft: Satoru Muro; Writing\u0026ndash;review \u0026amp; editing: Satoru Muro, Yasuo Nakajima, Akimoto Nimura, Keiichi Akita. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors sincerely thank the individuals who donated their bodies to science for their anatomical research. Their contributions will help increase the overall knowledge of humankind and improve patient care. We would also like to extend our gratitude to the families of the donors. This study was supported by JSPS KAKENHI (grant no. 25K18696).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlmughamsi AM, Elhassan YH (2025) Understanding the anatomical basis of anorectal fistulas and their surgical management: exploring different types for enhanced precision and safety. Surg Today 55:457\u0026ndash;474. https://doi.org/10.1007/s00595-025-02995-2\u003c/li\u003e\n\u003cli\u003eTabry H, Farrands PA (2011) Update on anal fistulae: surgical perspectives for the gastroenterologist. Can J Gastroenterol 25:675\u0026ndash;680. https://doi.org/10.1155/2011/931316\u003c/li\u003e\n\u003cli\u003eParks AG (1961) Pathogenesis and treatment of fistuila-in-ano. Br Med J 1:463\u0026ndash;469. https://doi.org/10.1136/bmj.1.5224.463\u003c/li\u003e\n\u003cli\u003eParks AG, Gordon PH, Hardcastle JD (1976) A classification of fistula-in-ano. Br J Surg 63:1\u0026ndash;12. https://doi.org/10.1002/bjs.1800630102\u003c/li\u003e\n\u003cli\u003eAbdool Z, Sultan AH, Thakar R (2012) Ultrasound imaging of the anal sphincter complex: a review. Br J Radiol 85:865\u0026ndash;875. https://doi.org/10.1259/bjr/27314678\u003c/li\u003e\n\u003cli\u003eSch\u0026auml;fer A, Enck P, F\u0026uuml;rst G, Kahn T, Frieling T, L\u0026uuml;bke HJ (1994) Anatomy of the anal sphincters. Comparison of anal endosonography to magnetic resonance imaging. Dis Colon Rectum 37:777\u0026ndash;781. https://doi.org/10.1007/BF02050142\u003c/li\u003e\n\u003cli\u003eFritsch H, Brenner E, Lienemann A, Ludwikowski B (2002) Anal sphincter complex: reinterpreted morphology and its clinical relevance. Dis Colon Rectum 45:188\u0026ndash;194. https://doi.org/10.1007/s10350-004-6144-x\u003c/li\u003e\n\u003cli\u003eLunniss PJ, Phillips RK (1992) Anatomy and function of the anal longitudinal muscle. Br J Surg 79:882\u0026ndash;884. https://doi.org/10.1002/bjs.1800790908\u003c/li\u003e\n\u003cli\u003eShafik A (1979) A new concept of the anatomy of the anal sphincter mechanism and the physiology of defecation. VII. Anal fistula: a simplified classification. Dis Colon Rectum 22:408\u0026ndash;414. https://doi.org/10.1007/BF02586911\u003c/li\u003e\n\u003cli\u003eMuro S, Yamaguchi K, Nakajima Y, Watanabe K, Harada M, Nimura A, Akita K (2014) Dynamic intersection of the longitudinal muscle and external anal sphincter in the layered structure of the anal canal posterior wall. Surg Radiol Anat 36:551\u0026ndash;559. https://doi.org/10.1007/s00276-013-1228-8\u003c/li\u003e\n\u003cli\u003eTsukada Y, Ito M, Watanabe K, Yamaguchi K, Kojima M, Hayashi R, Akita K, Saito N (2016) Topographic anatomy of the anal sphincter complex and levator ani muscle as it relates to intersphincteric resection for very low rectal disease. Dis Colon Rectum 59:426\u0026ndash;433. https://doi.org/10.1097/DCR.0000000000000565\u003c/li\u003e\n\u003cli\u003eNakajima Y, Muro S, Nasu H, Harada M, Yamaguchi K, Akita K (2017) Morphology of the region anterior to the anal canal in males: visualization of the anterior bundle of the longitudinal muscle by transanal ultrasonography. Surg Radiol Anat 39:967\u0026ndash;973. https://doi.org/10.1007/s00276-017-1832-0\u003c/li\u003e\n\u003cli\u003eMuro S, Tsukada Y, Harada M, Ito M, Akita K (2019) Anatomy of the smooth muscle structure in the female anorectal anterior wall: convergence and anterior extension of the internal anal sphincter and longitudinal muscle. Colorectal Dis 21:472\u0026ndash;480. https://doi.org/10.1111/codi.14549\u003c/li\u003e\n\u003cli\u003eMuro S, Kagawa R, Habu M, Ka H, Harada M, Akita K (2020) Coexistence of dense and sparse areas in the longitudinal smooth muscle of the anal canal: anatomical and histological analyses inspired by magnetic resonance images. Clin Anat 33:619\u0026ndash;626. https://doi.org/10.1002/ca.23467\u003c/li\u003e\n\u003cli\u003eMuro S, Yamaguchi K, Inoshita N, Nakajima Y, Edinam DJ, Nimura A, Akita K (2025) New anatomical insight into the muscular structure of the anal canal: revealing Treitz muscle as a directional shift of the internal anal sphincter. Ann Coloproctol 41:501\u0026ndash;509. https://doi.org/10.3393/ac.2024.00647.0092\u003c/li\u003e\n\u003cli\u003eMuro S, Moue S, Akita K (2024) Twisted orientation of the muscle bundles in the levator ani functional parts in women: implications for pelvic floor support mechanism. J Anat 244:486\u0026ndash;496. https://doi.org/10.1111/joa.13968\u003c/li\u003e\n\u003cli\u003eMacchi V, Porzionato A, Stecco C, Vigato E, Parenti A, De Caro R (2008) Histo-topographic study of the longitudinal anal muscle. Clin Anat 21:447\u0026ndash;452. https://doi.org/10.1002/ca.20633\u003c/li\u003e\n\u003cli\u003eHieda K, Cho KH, Arakawa T, Fujimiya M, Murakami G, Matsubara A (2013) Nerves in the intersphincteric space of the human anal canal with special reference to their continuation to the enteric nerve plexus of the rectum. Clin Anat 26:843\u0026ndash;854. https://doi.org/10.1002/ca.22227\u003c/li\u003e\n\u003cli\u003eLitta F, Papait A, Lucchetti D, Farigu S, Parello A, Tenore CR, Campenn\u0026igrave; P, Silini AR, Giustiniani MC, Parolini O, Sgambato A, Ratto C (2022) The pathogenesis of cryptoglandular anal fistula: new insight into the immunological profile. Colorectal Dis 24:1567\u0026ndash;1575. https://doi.org/10.1111/codi.16290\u003c/li\u003e\n\u003cli\u003eEmile SH, Elfeki H, El-Said M, Khafagy W, Shalaby M (2021) Modification of parks classification of cryptoglandular anal fistula. Dis Colon Rectum 64:446\u0026ndash;458. https://doi.org/10.1097/DCR.0000000000001797\u003c/li\u003e\n\u003cli\u003eDawka S, Yagnik VD (2021) Comparison between the modified Parks and Garg classifications of cryptoglandular anal fistulas. Dis Colon Rectum 64:e589. https://doi.org/10.1097/DCR.0000000000002208\u003c/li\u003e\n\u003cli\u003eTeymouri A, Keshvari A, Khorasanizadeh F, Kazemeini A, Behboudi B, Fazeli MS, Keramati MR, Ashjaei A, Tafti SMA, Naseri A (2025) External validation of the modified Parks classification of cryptoglandular anal fistula to predict failure of healing: the protocol for a retrospective analysis. Int J Surg Protoc 29:118\u0026ndash;121. https://doi.org/10.1097/SP9.0000000000000055\u003c/li\u003e\n\u003cli\u003eBrillantino A, Iacobellis F, Marano L, Renzi A, Talento P, Brusciano L, Gambardella C, Favetta U, Schiano Di Visconte M, Monaco L, Grillo M, Maglio MN, Foroni F, Palumbo A, Sotelo MLS, Vicenzo L, Palladino E, Frezza G, Menna MP, Mauro P, Picardi S, Mensorio MM, Mosca V, Bottino V, Ioia G, Rispoli C, Serafino MD, Caruso M, Ronza R, Frittoli B, Schettini D, Stoppino L, Iafrate F, Lombardi G, Antropoli C, Cappabianca S, Docimo L, Grassi R, Reginelli A (2025) Validation of a novel imaging-guided and anatomy-based classification system for anorectal fistulas: a retrospective clinical evaluation study. Ann Coloproctol 41:207\u0026ndash;220. https://doi.org/10.3393/ac.2024.00675.0096\u003c/li\u003e\n\u003cli\u003eda Silva Fernandes GO, Maciel MQ, Lima Barreto RG, Oliveira Cajazeiras MT, da Mota NC, Pavan Y, Travassos Pinto M, Freitas de Aquino L, Pinheiro Barreto JB (2024) Correlation of the anatomy of the intersphincteric anal fistula with sex: an analysis through anorectal three-dimensional ultrasound. J Coloproctol 44:e229\u0026ndash;e233. https://doi.org/10.1055/s-0044-1793855\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-colorectal-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijcd","sideBox":"Learn more about [International Journal of Colorectal Disease](http://link.springer.com/journal/384)","snPcode":"384","submissionUrl":"https://submission.nature.com/new-submission/384/3","title":"International Journal of Colorectal Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Anal fistula, Parks classification, Intersphincteric space, Longitudinal muscle, Histology","lastPublishedDoi":"10.21203/rs.3.rs-8698526/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8698526/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo clarify the histological architecture of the intersphincteric region of the anal canal by delineating the layer-specific organization and spatial relationships among the anal sphincter complex and associated muscular and connective tissue components.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTissue blocks containing the lateral wall of the anal canal were obtained from 11 adult human cadavers donated for anatomical research. Specimens were examined using descriptive histological and immunohistochemical analyses in both transverse and coronal planes. The internal and external anal sphincters, longitudinal muscle, levator ani, interbundle gaps, and connective tissue compartments were identified and analyzed with respect to their three-dimensional organization.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe intersphincteric region exhibited a heterogeneous and layered architecture rather than a uniform plane. The longitudinal muscle demonstrated a mosaic organization consisting of dense and loose components. The dense component terminated near the mid-height of the internal anal sphincter, whereas the loose component expanded inferiorly and formed a spacious compartment characterized by sparse smooth muscle fibers and loose connective tissue. Inferiorly, loose longitudinal muscle fibers branched and traversed natural interbundle gaps within the external anal sphincter. In addition, two partially overlapping layers of the levator ani were consistently observed, with interposed gaps contributing to the structural complexity of the intersphincteric region.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe intersphincteric region of the anal canal is a structurally complex and compartmentalized anatomical entity. Its heterogeneous histological architecture provides an anatomical substrate that may explain the initiation and directional spread of anal fistulas, including pathways described in classical fistula classifications.\u003c/p\u003e","manuscriptTitle":"Histological Architecture of the Intersphincteric Region of the Anal Canal: Implications for the Anatomical Basis of Anal Fistula Pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 12:52:18","doi":"10.21203/rs.3.rs-8698526/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-08T17:51:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-06T08:12:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-05T14:30:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39967309520047840604053825230287858525","date":"2026-01-31T10:09:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67421270686147340715885595104948384820","date":"2026-01-28T07:52:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213161180746929608270465573661702949644","date":"2026-01-27T12:11:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T11:13:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-27T01:04:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-27T01:04:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Colorectal Disease","date":"2026-01-26T08:53:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-colorectal-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijcd","sideBox":"Learn more about [International Journal of Colorectal Disease](http://link.springer.com/journal/384)","snPcode":"384","submissionUrl":"https://submission.nature.com/new-submission/384/3","title":"International Journal of Colorectal Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a6b2f1ad-5d20-417a-8e8a-cf1a47eeb553","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:20:37+00:00","versionOfRecord":{"articleIdentity":"rs-8698526","link":"https://doi.org/10.1007/s00384-026-05123-9","journal":{"identity":"international-journal-of-colorectal-disease","isVorOnly":false,"title":"International Journal of Colorectal Disease"},"publishedOn":"2026-03-27 16:08:53","publishedOnDateReadable":"March 27th, 2026"},"versionCreatedAt":"2026-01-29 12:52:18","video":"","vorDoi":"10.1007/s00384-026-05123-9","vorDoiUrl":"https://doi.org/10.1007/s00384-026-05123-9","workflowStages":[]},"version":"v1","identity":"rs-8698526","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8698526","identity":"rs-8698526","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}