Widespread anorectal lymphovascular networks and tissue drainage: analyses from submucosal India ink injection and indocyanine green fluorescence imaging

Abstract Aim Abdominoperineal resection is associated with poor prognosis in patients with advanced lower rectal cancer. This study aimed to analyse the functional lymphovascular network and tissue drainage in the anorectal region. Methods In this descriptive study, we performed microanatomical evaluations and intra‐operative imaging analysis in a cadaver and patients with rectal cancer. Specimens with India ink injection were collected from a cadaver and from six patients who underwent abdominoperineal resection. Intra‐operative indocyanine green fluorescence imaging was performed on four patients who underwent surgery for lower rectal cancer. India ink was injected into the submucosa at the dentate line of specimens. Tissue sections were examined by immunohistochemistry for D2‐40 and CD31. Intra‐operative indocyanine green was injected into the submucosa at the dentate line. Lymph flow was traced using a near‐infrared camera system. Results Fascia branching from the rectal longitudinal muscle layer extended to the posterior hiatal ligament and lateral endopelvic fascia connective tissue lamina on the surface of the levator ani muscle. The fascia contained veins labelled with ink in their lumina and initial lymphatics. Intra‐operative indocyanine green fluorescence imaging revealed extensive lymph flow from the muscle layer of the anal canal to the hiatal ligament and endopelvic fascia along the longitudinal muscle layer fibres. Conclusions The anorectal region contained widespread venous and lymphatic networks in proportion to its specific connective tissue framework around the longitudinal‐muscle‐layer‐extending muscle bundles, which provides extensive networks for tissue fluid and cells.


INTRODUC TI ON
Abdominoperineal resection (APR) is a standard operative procedure for advanced lower rectal cancer (RC) located within 5 cm of the anal verge. Sphincter-preserving procedures (SPPs), such as intersphincteric resection, are considered optional procedures.
The reasons for these controversies remain unclear. Thus, examining the lymphovascular network in the anorectal lesion is important as the tumour may spread beyond the APR resection area via these vasculatures. However, a limited number of studies have investigated this from a microanatomical perspective. Most microanatomical studies around the anal canal examine muscular structures [13,14]. Some studies reported anorectal lymphatic pathways using anatomical lymph node mapping [15,16]. However, this method can only investigate the location and distribution of lymph nodes and cannot investigate the functional lymphatic flow.
Some studies examined the rectal lymphatic flow, including the lateral region, using isotope methods [17][18][19], but isotopes were only detected in lymph nodes, not lymphatic ducts, rendering the conclusions of these studies speculative. Besides, the anorectal lymphatic drainage from the levator ani muscle (LAM) and its surrounding fascia has not been investigated yet. Microanatomical examinations of these areas can provide important information to adapt therapeutic strategies for lower RC.
This study aimed to analyse the detailed functional lymphovascular network in the anorectal region. We evaluated the microanatomical organization of lymphovascular communications from data obtained by immunohistochemistry after submucosal India ink injection into fresh cadavers and resected specimens, and from intraoperative indocyanine green (ICG) fluorescence imaging (FI).

India ink injection and microanatomical analyses
India ink (1 ml; Kuretake, Nara, Japan) was injected into the submucosa at the anterior, posterior and bilateral walls on the dentate line (3 cm oral side from the anal verge) of the specimens. In the study using surgical specimen tissues, lesions occupied by tumours were excluded from injection of India ink.
The cadaver was fixed in 10% formalin by arterial perfusion and routinely embalmed on the day after India ink injection. The anorectal tract and its surrounding tissues, including the internal anal sphincter, external anal sphincter (EAS) and LAM, were resected en bloc from the cadaver. The posterior and bilateral regions (up to 7 cm oral side from the anal verge) with the India ink injection were cut along the sagittal and frontal planes, respectively. The surgical specimens, excluding tissues for pathological diagnosis, were immersed for at least 1 day in 10% formalin 1 h after India ink injection and sectioned in the longitudinal plane. All samples were dehydrated in a graded ethanol series, embedded in paraffin, and cut into 5μm-thick sections for haematoxylin-eosin staining, silver impregnation staining and immunohistochemistry. The tissues excluded from pathological diagnosis, which included tumour cells not injected with India ink, were also examined.

What does this paper add to the literature?
This study provides a description of microanatomical and functional lymphovascular networks in the anorectal region, which has not been previously reported. This paper may help for developing therapeutic strategies for early and advanced lower rectal cancer. EP3095; Abcam) at room temperature overnight. After washing in phosphate-buffered saline, sections were incubated with alkalinephosphatase-conjugated anti-mouse IgG (Histofine Simple Stain AP, Mouse; Nichirei Bioscience) for 1 h at room temperature as a secondary antibody for podoplanin immunostaining. The blue immunoreaction was visualized using an alkaline phosphatase reaction (Vector blue substrate kit, Vector Laboratories). Sections were then incubated with peroxidase-conjugated anti-rabbit IgG (Histofine Simple Stain MAX-PO, Rabbit; Nichirei Bioscience) for 1 h at room temperature. The brown immunoreaction for CD31 was visualized using 3,3'-diaminobenzidine reaction (Wako Pure Chemical Industries). The stained sections were examined using a BX-60 light microscope equipped with a DP72 digital imaging system (Olympus).  software, Adobe Illustrator CC (Adobe Systems). Schemas were illustrated according to observations in our study and a previous report by Tsukada et al. [13].

RE SULTS
Our microanatomical examination of the human anorectal region  Table 1).
Tumour invasion into the stromal tissue and lymphatic ducts in the HL was observed on the pathological sections ( Figure 5). The

DISCUSS ION AND CON CLUS IONS
This study investigated the transport of tissue fluid and substances within the human anal canal by microanatomical dissection using with the cadaveric data and supported the above concept. We also found that perivascular stromal tissue and lymphatic invasion were the actual tumour-spreading pathway in patients with lower RC ( Figure 5).
The present data exhibited a dense distribution of lymphatic vessels around blood vessels in the framework, but lymphatic vessels did not contain ink deposits in their lumina. This may be attributed to the functional collapse of lymphatics in the cadaveric and resected specimens. The injection of lymphatic tracers into the submucosa of the colon in fresh cadavers is well accepted [24,25]. India ink is commonly used as a lymphatic tracer in fresh cadavers [26] but has also been injected into the submucosa of the colon in living subjects [27,28]. Thus, India ink injection is a suitable method. However, unsatisfactory tissue preservation in cadaveric and resected specimens, such as shrinkage by 10% formaldehyde fixation and degeneration of tissue, may affect the environment of the lymphatic ducts.
A previous report used low-concentration formaldehyde to fix the specimen [26], which may affect the results. Although the ICG-FI was unable to detect distinct vascular configurations in the patients in this study in whom lymphatic vessels are naturally capable of dye absorption and transport, the fascia around the LAM was thoroughly illuminated shortly after dye injection. Thus, stromal fluid, containing various substances and motile cells, may be primarily transported through the perivascular space rather than lymphatic vasculature and may subsequently flow from this prelymphatic channel into the lymphatics at certain sites in the fascial framework.
Based on our results, we discuss the therapeutic strategies for lower RC, especially in APR-indicated cases. Our anatomical findings suggest a widespread lymphovascular network and tissue drainage beyond the resection line in APR ( Figure 7A,B). Therefore, even if CRM involvement is negative based on APR, tumour cells may ports previous results suggesting that APR worsens the prognosis [2][3][4]. Based on our results, the ELAPE resection area includes the EF and HL, supporting the claims that ELAPE improves oncological outcomes. Accordingly, West et al. [6] demonstrated that ELAPE reduced CRM-positive rates compared to APR (49.6% vs. 20.3%, P < 0.001). Lehtonen et al. [8] showed that ELAPE for T3-4 lower RC reduced local recurrence rates (6.7% vs. 15.5%, P = 0.048). Shen et al. [9] reported that ELAPE improved overall survival (41.5 months vs. 29.8 months, P = 0.028) and disease-free survival (38.5 months vs. 29.3 months, P = 0.027). In contrast, Prytz et al. [11] reported that ELAPE had a high 3-year risk of local recurrence (odds ratio 4.10, 95% confidence interval 1.19-14.08). However, the report by Prytz  [29,30]. However, microanatomical ILN metastatic pathways from the anorectal lesion have not been reported. Although our study did not disclose tumour translymphatic metastatic pathways from the anal canal to ILNs and the lateral pelvic compartment, particular lymphatic channels along the HL and EF may be associated In addition, our study may provide important data regarding therapeutic strategies following local excision (LE). LE is an optional treatment for early lower RC [31,32]. Although quality of life following LE is satisfactory, several studies have reported higher recurrence rates compared to total mesorectal excision [32][33][34][35]. This can be explained by our microanatomical data which indicate that tumour cells invading the submucosal space in anorectal regions can spread widely through the HL and EF (Figure 7).
To improve recurrence rates, LEs with adjuvant radiotherapy and chemoradiotherapy have been attempted [36,37]. A study suggested the efficacy of adjuvant chemoradiotherapy for high-risk early lower RC, defined as >1000-µm submucosal invasion and the presence of lymphovascular invasion [38]. Currently, adjuvant chemoradiotherapy following LE is an optional treatment for early RC according to the guidelines [39]. This study is not without limitations. The sample size was small. In the study period, only seven participants and four participants were suitable for ink injection methods and ICG-FI, respectively. Larger studies should be conducted to verify our results. Also, the anterior portion was not analysed in this study because the relationship between the anal canal and urogenital organs cannot be analysed in surgical specimens of APR.
Discrepancies in the results of India ink injection and ICG-FI may be due to the difference in the environment of tissue between specimens and living humans. The microanatomical data from resected specimens and ICG-FI data were collected from patients with RC. Cancer induces lymphangiogenesis [40], and this might cause anatomical differences. Therefore, data collected from patients with RC might have a degree of uncertainty.
The study area was limited to the APR resection area. To discuss in more detail the therapeutic effects of ELAPE investigating the lymphatic pathways leading to lateral pelvic and inguinal lesions would be beneficial. Therefore, future studies that investigate lymphatic pathways in these areas are expected. To examine these areas, a combination of ICG-FI and microanatomical analyses with fluorescent staining using fresh cadavers may be beneficial. This

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflicts of interest.

CO N S ENT TO PA RTI CI PATE
Informed consent was obtained from all individual participants included in the study.

CO N S ENT FO R PU B LI C ATI O N
The participants have consented to the submission of the descriptive study to a journal.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.