Abstract
Inadequate resection of the adjoining mesentery is associated with adverse outcome for colon cancer. Disruption of the integrity of the mesenteric lymphatic package has been implicated in this, though not proven. Recent studies have determined mesenteric anatomy and histology and now provide an opportunity to determine accurately the distribution of lymphatic vessels. The aim of this study was to characterise the distribution of the lymphatic vessels (LV) within the small intestinal and colonic mesentery, and in Toldt's fascia, which lies between the mesocolon and underlying retroperitoneum. Mesenteric samples were harvested from 12 human cadavers. Samples were taken from the small bowel mesentery, ascending, transverse, descending mesocolon and from both apposed and non-apposed portions of the mesosigmoid. Serial sections were stained immunohistochemically with monoclonal antibody D2-40 (podoplanin), and Masson's Trichrome. Lymphatic vessel (LV) density and radius of diffusion were determined using a stereological approach. A lymphatic network was embedded within the mesenteric connective tissue lattice throughout each mesenteric region. LV were identifiable within the submesothelial connective tissue where they measured 10.2 ± 4.1 μm in diameter and had an average radius of diffusion of 174.72 ± 97.68 μm. Unexpectedly, LV were identified in Toldt's fascia, where they measured 4.3 ± 3.1 μm in diameter and had a radius of diffusion of 165.12 ± 66.26 μm. This is the first study systematically to determine and quantify the distribution of lymphatic vessels within the mesenteric organ and to demonstrate the presence of such vessels within Toldt's fascia. A rich lymphatic network occupies all levels of the mesenteric connective tissue lattice. Within the latter, they are found within 0.1 mm of peritonealised mesenteric surfaces and are separated by an average distance of 0.17 mm and may be particularly vulnerable during surgery.
Keywords: complete mesocolic excision, lymphangiology, mesenteric organ, Toldt's fascia
Introduction
In 1909, Jamieson and Dobson described the macroscopic arrangement of lymphatic vessels draining the colon, and emphasised that the successful removal of malignant diseases depended upon the removal of any affected lymphatic area (Jamieson & Dobson, 1909). This principle underpins technical approaches to oncologic colorectal resection to the present day (Coffey, 2013). Recent reappraisals of mesocolic anatomy have provided a basis for us to re-examine the lymphangiology of the mesentery (Culligan et al. 2012, 2013, 2014; Gao et al. 2013). West et al. (2008) have demonstrated improved survival when a complete mesocolic excision (CME) is conducted compared with that observed with incomplete resection performed in the intramesocolic or muscularis propria plane. This is corroborated by the observations of Hohenberger et al. (2009), who also demonstrated improved survival with CME. It is speculated that these observations relate to complete removal of the mesenteric lymphatic package (i.e. mesentery and in situ lymphatics), similar to the benefits which were observed in rectal cancer following the introduction of total mesorectal excision (Heald et al. 1982; Enker, 1997).
The absence of studies characterising mesenteric lymphangiology is attributable to anatomical misconceptions perpetuated over the past century (Coffey, 2013; Culligan et al. 2013). Based on the work of Sir Frederick Treves in the late 19th century, the mesocolon is frequently depicted as a narrow and discontinuous structure which is largely absorbed into the retroperitoneum (Treves, 1885; Moore, 2003; Standring, 2008). Although there have been a myriad of recent surgical publications extolling the virtues of CME, they have been hindered by a failure to establish properly an anatomical basis for their approach. This problem has been recently highlighted by Mike & Kino (2014) who noted that: ‘descriptions of the anatomy of the fascial composition have mainly involved observations unrelated to fundamental embryological concepts, causing confusion regarding the explanations of the surgical procedures, with various vocabularies used without definitions’. They further draw attention to the use of the terms ‘visceral fascia’ and ‘parietal fascia’, which have not been defined.
In the first formal appraisal of mesenteric anatomy, our group examined the structure of the mesocolon as seen during surgery; through a series of 109 total abdominal colectomies, the mesocolon was mobilised utilising the techniques of planar colonic surgery. This approach allowed characterisation of the in situ shape of the mesocolon, a shape which is lost once the mesocolon has been excised. This work demonstrated several important anatomical features of the mesocolon, including: (i) the mesocolon was continuous from ileocaecal to rectosigmoid level; (ii) a mesenteric confluence occurred at the ileocaecal and rectosigmoid junction as well as at hepatic and splenic flexure; (iii) each flexure (and ileocaecal junction) was a complex of peritoneal and omental attachments to the colon centred on a mesenteric confluence; (iv) the proximal rectum originated at the confluence of the mesorectum and mesosigmoid; and (v) Toldt's fascia separated all apposed portions of the mesocolon from the underlying retroperitoneum. Thus, rather than being ‘absorbed’ into the retroperitoneum, all portions of the mesocolon remain separated from the true retroperitoneum by Toldt's fascia. The fascia arises whenever mesocolic and retroperitoneal mesothelial surfaces come into constant contact (e.g. posterior to the right and left mesocolon and interposed between these and the retroperitoneum in these regions). In addition, where the fascia coalesces with peritoneal folds (i.e. the right lateral peritoneal fold) a distinctive white line is apparent (i.e. the White line of Toldt). These findings directly reconcile current anatomical and surgical descriptions of the mesocolon, thereby providing an anatomic basis and justification for the technique of CME (Culligan et al. 2012).
Several other anatomical observations were made which clarified mesenteric anatomy and collectively provided an opportunity to determine the histologic structure of the mesenteric organ (i.e. the small intestinal mesentery, mesocolon, mesosigmoid and mesorectum; Culligan et al. 2014). We identified a connective tissue lattice within the mesentery, at all levels. The lattice comprises submesothelial connective tissue layers from which septations arise that separate mesenteric adipocytes into lobules. Intriguingly, preliminary data demonstrated the presence of lymphatic channels in several locations within the lattice.
Given the improved understanding of mesocolic surgical anatomy and histology it is now possible to formally characterise the distribution of lymphatic channels within the small intestinal and colonic mesentery (i.e. the mesenteric organ). Any such study should focus on the relationship between lymphatic channels and the histologic components of the mesentery (i.e. the connective tissue lattice). Based on this, we aimed to determine the distribution of lymphatic channels and their relationships to mesenteric structures, and then to compare this across mesenteric regions. Collectively, this would generate a roadmap of mesenteric lymphangiology.
Methods
The experimental workflow process is summarised in Fig. 1. The specific locations from which tissue samples were harvested are illustrated in a three-dimensional model (3D) of the adult human mesocolon. The model was generated using a 3D digital sculpting tool, ZBrush 4R6 (Pixologic Inc., Los Angeles, CA, USA) with the images then captured in a 2.5-dimension format (Fig. 1A). This model is not a precise replica, it is a collation of histological features and thus is representative of the generalised mesocolic micro-architecture.
Fig. 1.
(A) A 2.5-dimensional (2.5D) image of the mesocolon. This image was generated from a 3D model developed in Zbrush. The locations of sample harvest are indicated. (B) Series of images demonstrating the process of sample orientation for embedding, sectioning and mounting. An expanded methodology is contained within the supplementary material of Culligan et al. (2014) (C) Overview of the general structure of the mesocolon including (i) section stained with Masson's Trichrome (left panel) and (ii) 2.5D depiction generated using Zbrush (right panel).
Harvesting mesocolic samples
Mesenteric samples were harvested from 12 adult human cadavers with no prior medical history of colorectal cancer, inflammatory bowel disease or diverticulitis. All cadaveric material used was bequeathed to the Medical School, National University of Ireland Galway for further advancement of medical knowledge. This is covered by legislation governing the practice of Anatomy in the Republic of Ireland (Medical Practitioners Act 2007). Five of the 12 cadavers were male, and the mean age at death was 78 years (range: 64–96 years). Cadavers were previously embalmed using a standard mixture containing formalin, glycerine, phenol, and methanol (12 L water + 2.4 L of a 37–41% formalin solution + 2 L phenol + 6 L glycerine + 6 L methanol).
As previously described, a 1-cm2 area of mesocolon was sharply excised at each of the following levels: (i) ascending mesocolon (midway between the caecum and hepatic flexure); (ii) transverse mesocolon (midway between hepatic and splenic flexure); (iii) descending mesocolon (midway between the splenic flexure and commencement of the mesosigmoid); (iv) mobile mesosigmoid (i.e. the lateral and freely mobile region of the mesosigmoid); (v) apposed mesosigmoid (i.e. the medial and non-mobile region of the mesosigmoid); and (vi) mesentery of the terminal ileum (5 cm proximal to the ileocaecal junction) (Culligan et al. 2014). For each location, samples were taken perpendicular to the longitudinal axis of the mesentery and to a depth that ensured inclusion of the mesocolon, intervening fascia and the underlying retroperitoneum. The location of sample excision is indicated in Fig. 1(A).
Tissue orientation and processing
Tissue specimens were pinned to a cork board following excision to maintain correct orientation throughout the evaluation process (i.e. the top edge of the specimen corresponded to the surface facing the peritoneal cavity and the bottom edge corresponded to the surface closer to the retroperitoneum; Fig. 1B). Specimens initially were fixed and stored in the same embalming fluid mixture outlined above. Following fixation, specimens were dehydrated though a series of ethanol solutions of increasing concentration, before being embedded in paraffin wax and sectioned to 5 μm. Sections were then stained using the lymphatic endothelium-specific monoclonal antibody D2-40, with serial sections being stained with Masson's Trichrome, a collagen-enhancing stain.
Staining procedures
Masson's Trichrome. Samples were dewaxed in xylene and rehydrated through a series of ethanol solutions of decreasing concentrations. Sequential staining was performed through baths of potassium permanganate, sodium metabisulphite, Gomori's aldehyde fuschin, Celestine blue, Mayer's Haemalum, Masson's cytoplasmic stain and fast green. Slides were differentiated in 1% acetic acid, dehydrated through a series of ethanol solutions of increasing concentration, and finally mounted in a mixture of distyrene, plasticizer and xylene (DPX; Sigma-Aldrich Corporation, St. Louis, MO, USA) and a coverslip applied.
Immunohistochemistry. Samples were dewaxed in xylene and washed in a series of ethanol solutions of decreasing concentration to yield Tris buffered solution (TBS). Samples were rinsed in TBS and incubated in 3% H2O2/methanol for 30 min at room temperature. Antigen retrieval was performed by placing slides in sodium citrate buffer and submerging in a waterbath for 20 min at 98 °C. Samples were then blocked with 5% goat serum solution for 2 h at room temperature. The primary antibody was applied to the slides using a 1 : 200 dilution of the lymphatic endothelium-specific monoclonal antibody D2-40 (Dako Diagnostics Ireland, Ltd.) and incubated overnight at 4 °C.
Slides were then rinsed in TBS prior to the secondary antibody being applied for 30 min (Envision+ System/HRP Mo (AEC+), Dako Diagnostics Ireland, Ltd.). Slides were developed with AEC chromogen for up to 30 min. Staining was confirmed by light microscopy before being counterstained with 20% Mayer's haematoxylin for 30 s. Finally, slides were rinsed, wiped carefully and coverslipped using an aqueous mounting media (Aquatex; VWR International Ltd, Lutterworth, Leics, UK). Sections of cadaveric human tonsil were utilised as positive and negative controls.
Slide review
All slides were reviewed using a Leica DM750 light microscope, with an ICC50 HD camera attachment (Leica Microsystems Limited, Switzerland). Slide review and interpretation were conducted via consensus, by K.C. in conjunction with two anatomists (F.Q. and P.D.) and the principal investigator (J.C.C.). Review focused on lymphatic channels within (i) submesothelial connective tissue layers, (ii) interlobular connective tissue septations, and (iii) Toldt's fascia, in each of the mesenteric regions sampled. Two serial sections (Masson's Trichrome and D2-40-staining, respectively) were examined from each of the six mesenteric areas. As this was conducted across all 12 cadavers, a total of 144 slides were reviewed.
Stereological assessment of the lymphatic vessels
Stereology combines geometry and statistics to extrapolate three-dimensional information from a two-dimensional section (Gundersun et al. 1988). Briefly, the density of the lymphatic vessels (VD) and length density (Lv) (i.e. an estimate of the length of a vessel) were calculated with the following formulae (Dockery & Fraher, 2007). An estimation of the average number of vessels was obtained by placing a 60-mm2 grid over five randomly chosen fields per region. A count was made of all immunopositive vessels lying wholly within two reference squares on the grid. Only vessels which were wholly within these squares were counted, those that cross the grid or lie outside the bounds of these squares were not counted. By repeating this procedure at multiple randomly chosen sites, an estimation of the average number of vessels in a given layer may be obtained; this was then divided by the area of the reference grid to obtain an estimation of vessel density as follows:
 |
Length density was calculated as follows:
 |
By utilising length density (Lv), the cylindrical radius of diffusion surrounding a vessel was then determined with the following formula (Nyengaard et al. 2000). The radius of diffusion refers to the cylindrical area surrounding a vessel which it supplies.
 |
Stereological and statistical analyses, including anova testing, were carried out using Microsoft Office excel 2007 (data reported as mean ± SD) with significance set as P < 0.05.
Results
The histological structure of the small intestinal and colonic mesentery was similar throughout each area examined. A schematic outline of the typical histological structure is illustrated in Fig. 1(C). A vessel was designated as lymphatic if there was strong uptake of the monoclonal antibody D2-40, in either a luminal or a linear shape (representing a collapsed vessel). The distribution of lymphatic vessels was similar across each mesenteric area. Immunopositive vessels were identifiable within (i) the submesothelial connective tissue layer beneath the mesothelial surfaces of the small intestinal and colonic mesentery, (ii) the connective tissue septations within the body of the mesentery, and (iii) Toldt's fascia, located between apposed portions of the mesocolon and underlying retroperitoneum.
Lymphatic vessels associated with the anterior mesothelial surface
Immunopositive lymphatic vessels were consistently identified across all regions within the submesothelial monolayer on the anterior (upper or ‘peritoneal’) surface of the mesocolon. These measured 10.2 ± 4.1 μm in diameter (1–20 μm), and were typically located within 100 μm of the mesothelial surface (Fig. 2). Cumulative vessel density for the anterior submesothelial connective tissue layer was 10.7 ± 8.3 mm−2 (1.2–26.5 mm−2). The mean radius of diffusion was therefore 175 ± 98 μm (77–363 μm). In other words, one can expect to encounter a lymphatic vessel on average every 175 μm (i.e. every 0.17 mm) within the submesothelial connective tissue underlying the anterior peritoneal surface. A summary of the stereological results can be found in Table 1, with a breakdown by anatomical area in Table 2.
Fig. 2.
(A) Photomicrograph of the mobile portion of the mesosigmoid. D2-40 immunopositivity is evident in lymphatic vessels immediately beneath the mesothelial surface. Scale bar: 100 μm. (B) Photomicrograph demonstrating staining with Masson's Trichrome to identify collagen. The region depicted is immediately adjacent to that demonstrated in (A) (i.e. serial sections). The collagenous nature of the submesothelial connective tissue layer is apparent. Scale bar: 100 μm.
Table 1.
Summary of stereological measurements for each tissue layer sampled
| Vessel diameter (μm) | Vessel density (mm−2) | Radius of diffusion (μm) | |
|---|---|---|---|
| Anterior mesothelium | 10.2 ± 4.1 (1–20) | 10.7 ± 8.3 (1.2–26.5) | 175 ± 98 (77–363) |
| Posterior mesothelium | 9.6 ± 4.8 (1–20) | 10.9 ± 9.1 (2.4–33.7) | 149 ± 53 (69–257) |
| Toldt's fascia | 4.3 ± 3.1 (1–15) | 8.3 ± 5.1 (2.4–14.5) | 165 ± 66 (105–257) |
| Perivascular | 24.7 ± 9.3 (1–40) |
All figures reported in mean ± SD (range).
Table 2.
Breakdown of stereological measurements of vessel density (VD), and radius of diffusion (R.Diff) by anatomical location
| Location (N) | Asc (3) | Trans (3) | Desc (4) | Mob Sig (8) | App Sig (3) | TI (7) | Overall (28) | |
|---|---|---|---|---|---|---|---|---|
| Anterior (28) | VD (mm−2) | 7.4 ± 8.3 | 13.2 ± 10.8 | 13.8 ± 7.5 | 9.3 ± 8.8 | 3.8 ± 3.1 | 13.6 ± 8.9 | 10.7 ± 8.3 |
| R.Diff (μm) | 220 ± 134 | 149 ± 94 | 121 ± 43 | 202 ± 115 | 247 ± 109 | 150 ± 102 | 175 ± 98 |
| Asc (3) | Trans (5) | Desc (2) | Mob Sig (4) | App Sig (3) | TI (6) | Overall (23) | ||
|---|---|---|---|---|---|---|---|---|
| Posterior (23) | VD (mm−2) | 8.4 ± 2 | 20.5 ± 14.5 | 12 ± 7.6 | 6.6 ± 4.2 | 10.2 ± 7.8 | 6.9 ± 4.5 | 10.9 ± 9.1 |
| R.Diff (μm) | 140 ± 20 | 95 ± 45 | 125 ± 41 | 180 ± 65 | 142 ± 44 | 169 ± 51 | 148 ± 53 |
| Asc (2) | Desc (4) | App Sig (1) | Overall (7) | ||
|---|---|---|---|---|---|
| Toldt's fascia (7) | VD (mm−2) | 6.6 ± 5.9 | 10.5 ± 4.5 | 2.4 | 8.3 ± 5.1 |
| R.Diff (μm) | 189 ± 96 | 130 ± 29 | 257 | 165 ± 66 |
All figures reported as mean ± SD.
The anatomical locations where the specimen was obtained are as follows: Asc, ascending mesocolon; Trans, transverse mesocolon; Desc, descending mesocolon; Mob Sig, mobile portion of the mesosigmoid; App Sig, apposed portion of the mesosigmoid; TI, small bowel mesentery adjacent to the terminal portion of the ileum.
Lymphatic vessels located within the body of the mesocolon
Within the mesocolon, lymphatic vessels were identified accompanying blood vessels travelling within the connective tissue lattice. Average vessel diameter measured 24.7 ± 9.3 μm (1–40 μm). Within the body of the mesocolon, blood and lymphatic vessels were found to run together within a connective tissue lattice. Septae of connective tissue arose from the submesothelial connective tissue and traversed the mesocolon, thereby compartmentalising the adipocytes of the mesocolon. An example of a lymphatic vessel travelling in one such septation is illustrated in serial Fig. 3(A,B). A typical lymphovascular bundle is demonstrated in Fig. 3(C,D).
Fig. 3.
(A) Photomicrograph of the ascending mesocolon demonstrating D2-40 immunopositivity in several lymphatic vessels immediately below the mesothelial surface, travelling within a connective tissue septation which arises from the submesothelial connective tissue. Scale bar: 25 μm. (B) Photomicrograph demonstrating staining with Masson's Trichrome to identify collagen. The region depicted is immediately adjacent to that demonstrated in Fig. 2(C) (i.e. serial sections). Scale bar: 25 μm. (C) Lymphovascular bundle from within the mesosigmoid, lying between connective tissue septae dividing the surrounding adipocyte compartments. Immunopositivity is demonstrated in the lymphatic vessels, with no uptake in the accompanying capillaries. Scale bar: 100 μm. (D) Serial section of (C) stained with Masson's Trichrome. Scale bar: 100 μm.
Lymphatic vessels in association with the posterior mesothelial surface
Lymphatic vessels were observed within the submesothelial connective tissue layer deep to the mesothelium of the posterior surface of the mesocolon (i.e. the deep surface overlying Toldt's fascia). Lymphatic vessels here measured 9.6 ± 4.8 μm in diameter (1–20 μm) and typically were located within 100 μm of the mesothelium (Fig. 4A,B). The average cumulative vessel density for the posterior submesothelial connective tissue layer was 10.9 ± 9.1 mm−2 (2.4–33.7 mm−2), and the radius of diffusion was 149 ± 53 μm (69–257 μm). Therefore one can expect to encounter a lymphatic vessel on average every 149 μm (i.e. every 0.15 mm) within the submesothelial connective tissue associated with the posterior peritoneal surface. A summary of the stereological results can be found in Table 1, with a breakdown by anatomical area in Table 2.
Fig. 4.
(A) Photomicrographs taken from the mobile mesosigmoid. A lymphatic vessel has stained positive for D2-40; it is located immediately adjacent to the lateral (corresponding to deep) mesothelial layer of the mesocolon. Scale bar: 25 μm. (B) Serial section, adjacent to (A) stained with Masson's Trichrome. The collagenous nature of the layer is highlighted. Scale bar: 25 μm. (C) Photomicrograph of the descending portion of the mesocolon. Immunopositivity for D2-40 can be seen in a longitudinal section of a thin lymphatic vessel within Toldt's fascia. Scale bar: 25 μm. (D) Section adjacent to (C) stained with Masson's Trichrome to highlight the collagenous nature of this fascial layer.
Toldt's fascia
Toldt's fascia occurred beneath the non-mobile portions of the mesocolon, namely the ascending/descending mesocolon, and the medial portion of the mesosigmoid. Lymphatic vessels were identifiable in Toldt's fascia in almost one-third of sections which contained this layer (i.e. seven of 24 sections where Toldt's fascia was identifiable; Fig. 4C,D). These were narrow in calibre, with a diameter of 4.3 ± 3.1 μm (1–15 μm). The average cumulative vessel density was 8.3 ± 5.1 mm−2 (2.4–14.5 mm−2). The radius of diffusion was 165 ± 66 μm (105–257 μm). Although there was a lower lymphatic vessel density within Toldt's fascia, there was no statistically significant difference between the anterior mesothelium, posterior mesothelium or Toldt's fascia for vessel density (P = 0.758) or the radius of diffusion (P = 0.504). A summary of the stereological results can be found in Table 1, with a breakdown by anatomical area in Table 2.
Connective tissue lattice
To illustrate the relationship between the lymphatic network and the connective tissue lattice we have digitally sculpted both together (Fig. 5A) and in isolation (Fig. 5B). Figure 5(A) is a pictorial representation of the mesenteric connective tissue lattice; the adipocytes have been conceptually removed for illustrative purposes. Lymphatic channels and nodes are evident within the lattice. In Fig. 5(B) the connective tissue has been digitally subtracted, leaving the lymphatic network. Clearly, a complex network of lymphatic vessels is in evidence, with channels close to the mesothelial surface and traversing the body of the mesocolon.
Fig. 5.
(A) A 2.5D image of the mesocolon generated using Zbrush. The adipocytes have been removed better to demonstrate the connective tissue lattice found within the mesocolon. Lymphatic vessels and nodes can be appreciated within the connective tissue lattice. (B) The connective tissue lattice has been conceptually subtracted from this model, leaving the lymphatic network in place. Lymphatic vessels are identifiable immediately within the surface mesothelium as well as traversing the body of the mesocolon.
Discussion
The present study adopted an immunohistochemical approach to determine accurately the distribution of lymphatic vessels within the mesenteric organ (i.e. within the small intestinal and colonic mesentery). Our previous work demonstrated that the latter contains a complex connective tissue lattice comprising submesothelial monolayers and interlobular septations (Culligan et al. 2014). Herein, we found that lymphatic vessels occurred within both submesothelial connective tissue monolayers and septations, in all mesocolic regions (Figs 5). Based on the stereological appraisal, vessels occurred on average every 0.14 mm, and within 0.1 mm from the mesocolic surfaces (anterior and posterior). In addition, and unexpectedly, lymphatic channels were also identified within the fascial layer interposed between the mesocolon and retroperitoneum (i.e. Toldt's fascia, Fig. 4C,D).
Recently, West et al. (2008) demonstrated poorer overall survival associated with surgical dissection close to the muscularis propria or within the mesocolon, compared with dissection conducted in the mesocolic plane (i.e. not disrupting the mesocolic package). Hohenberger et al. (2009) showed a clear survival benefit for patients undergoing complete vs. incomplete mesocolic excision of the colon. The present results may partially explain these observations. As mentioned, lymphatic vessels occur within 0.1 mm of the mesocolic surface and, in the mesocolon proper, occur every 0.14–0.17 mm. Collectively, this generates a rich lymphatic network within the mesenteric connective tissue lattice (Fig. 5). It is thus highly likely that surgical dissection that breaches the mesocolic surface (i.e. muscularis propria or intramesocolic plane surgery) extensively disrupts the lymphatic network. In complete mesocolic resection (i.e. where disruption of mesocolic integrity is limited), lymphatic disruption may in turn be reduced.
The findings of this study thus prompt several additional studies. For example, in patients undergoing an oncologic colorectal resection, it is important to determine the density of lymphatic vessels at the cut margins of the mesocolon. The density of mesenteric lymphatic vessels should be mapped throughout the mesentery with a view to identifying regions (adipovascular, pedicular and interpedicular areas) that may be safer to breach (Culligan et al. 2012). It is important to characterise the features of the mesenteric connective tissue lattice and lymphatic network in oncologic and non-oncologic disease states.
This is also the first study to identify lymphatic vessels within Toldt's fascia. There are two technical approaches to dealing with this fascia in resectional colorectal surgery (Culligan et al. 2013). In the first, a mesofascial separation is conducted to separate both, thereby leaving the fascia in situ. In the second, a retrofascial separation is conducted, thereby removing the fascia along with the mesocolon. We identified lymphatic vessels in approximately one-third of specimens that contained Toldt's fascia. This observation prompts the question as to whether the fascia should be removed, as routine, in all oncologic colorectal resections. No direct communication was seen between the lymphatics of the mesocolon and those in Toldt's fascia. Further work will be needed to interrogate the exact territory drained by the vessels in Toldt's fascia and to establish their true clinical relevance.
The current study is limited by the use of cadaveric tissue. However, several studies, including our own previous work, have demonstrated that the use of cadaveric tissue can be a viable alternative in a variety of tissues (Nicholson et al. 2005; Gupta & Gauba, 2011). Podoplanin, a mucin-type transmembrane glycoprotein, is well established as a lymphatic-specific marker (Cîmpean et al. 2007; Kono et al. 2007). In addition, the D2-40 monoclonal antibody has been successfully utilised in several studies to identify lymphatic vessels (Walgenbach-Bruenagel et al. 2006; Kenney & Jain, 2008). However, there is some cross-reactivity between mesothelial cells and future studies could utilise alternative lymphatic markers to control for this property. Finally, the cohort size meant that conclusions about the possible influence of demographic features (i.e. age and sex) on lymphatic vessel density were not appropriate. These questions should be addressed in future studies.
In summary, a lymphatic network is housed with the connective tissue lattice of the mesenteric organ. Throughout the length of the mesocolic component of this organ, lymphatic vessels are found within 0.1 mm of anterior and posterior surfaces, and are distributed at an average distance of 0.14 mm. Lymphatic channels are present in Toldt's fascia.
Acknowledgments
The authors would like to thank Dara Walsh for his assistance with the illustrations and the 3D model of the connective tissue lattice, Mark Canney for his assistance with the immunohistochemical staining, and John O'Sullivan for his assistance with the statistical analysis. The authors would like to acknowledge Prof. Jörg Wilting of the University of Göttingen, for helpful advice.
Conflict of interest
The authors have no conflicts of interest to declare.
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