Supplemental methodology:
Supplemental Figure 1 and legend
Flow
diagram
illustrating
workflow process: Phase one
(left): surgical mobilisation of the
colon
and
mesocolon
was
recorded, edited, audio included,
and a three dimensional model of
the mesenteric organ was rendered
using
Blender
©.
An
accompanying
detailed
text
commentary was generated (see
below in supplementary methods
“surgical
mobilisation”).
2.5
dimension
illustrations
were
extracted from the model and utilised to demonstrate the sites of sample harvest. Phase two
(right): Samples were harvested pre and post-mobilisation from sites indicated in Figure 1
(see manuscript). These were prepared as described, stained as indicated, then interpreted via
consensus by KC, FQ, PD and JCC.
Sample Processing:
To stain with H&E sections were first dewaxed with xylene, and then bathed in a series of
ethanol solutions of decreasing concentration. Staining was then performed with Mayer’s
haematoxylin for 6 minutes, a rinse in running water for 4 minutes and counterstaining with
eosin for 2 minutes. Following staining, specimens were then dehydrated through a series of
ethanol solutions of increasing concentration, and cleared with xylene prior to mounting
media and a cover slide being applied.
To apply Masson’s Trichrome with Gomori’s Aldehyde Fuchsin, specimens were brought
to water via xylene and a series of ethanol solutions of decreasing concentration. Specimens
were then oxidised in equal parts 0.5% KMnO4/0.5% H2SO4 for 2 minutes, then rinsed in tap
water and bleach in 2% Sodium Metabisulphite for another 2 minutes, before being washed
in water 30 seconds, followed by 70% ethanol for 1 minute. Following this, specimens were
sequentially stained in Gomori’s Aldehyde Fuchsin for 1 min, Celestine blue for 4 minutes,
and Mayer’s Haemalum for 4 minutes, with appropriate rinses. Next, specimens were
differentiated in acid alcohol for 20 seconds, and then rinsed with running tap water for 4
minutes before being stained in Masson’s Cytoplasmic Stain for one minute. After this step
they were rinsed briefly in water and differentiated in 1% dodeca-Molybdophosphoric Acid
for 2 minutes. Next they were rinsed in water and counterstained in fastgreen or light green
for 1 min. Finally, specimens were differentiated in 1% acetic acid, dehydrated through a
series of ethanol solutions of increasing concentration, and then cleared with xylene.
Following this, mounting media and a cover slide were applied.
Samples for immunohistochemisty were initially dewaxed in xylene and then brought to
TBS through a series of ethanol solutions of decreasing concentration. 3x 5 minutes rinses in
TBS were then carried out. Samples were then incubated in 3% H2O2/methanol for 30
minutes, followed by a further 3 rinses in TBS. Antigen retrieval step was then carried out;
slides were placed in a sodium citrate buffer, in a water steamer, for 20 minutes, followed by
a further 3x 5 minute rinses in TBS. Samples were then blocked with 5% goat serum solution
for 2 hours, at room temperature. The primary antibody was then applied, (a 1:200 dilution of
podoplanin in the blocking solution), with slides incubating overnight at 4oC. The following
day slides were rinsed 3x 5 minutes in TBS prior to the secondary antibody being applied for
30 minutes. Slides were again rinsed in TBS for 3x minute cycles. Following this the slides
are developed with AEC chromogen for up to 30 minutes. Once satisfactory staining has been
confirmed by brief light microscopy, slides were rinsed in running water for 5 minutes,
before being counterstained with 20% Mayer’s haematoxylin for 30 seconds. Finally slides
were rinsed, wiped carefully and coverslipped using an aqueous mounting media.
Surgical mobilisation:
Surgical mobilisation:
In one cadaver, the entire colon and mesocolon were surgically mobilised as previously
described.2 A video recording of the entire process is also included in the supplementary
methodology.
Figures 2A-D (see over) are axial views of
the mesocolon (A) and mesosigmoid (B-D)
that depict the relationship with the
underlying connective tissue fascia and
retroperitoneum.
These
schematic
representations thus depict the structures
forming the planes that are utilised during
surgical mobilisation. Similar anatomic
relationships occur on the right side and
thus are not included in this image. Figure
2B demonstrates the mesosigmoid just
distal to the junction with the descending
(left) colon. Figure 2C demonstrates the
mesosigmoid at approximately the mid-point of the sigmoid colon. Figure 2D demonstrates
the mesosigmoid and sigmoid just proximal to the junction with the rectum
Figure 3A-E (left) are schematic and colored
illustrations of (A) the entire mesocolon, (B) the
right mesocolon and contiguity with the small
intestinal mesentery, (C) the hepatic flexure with
the colonic component conceptually removed, (D)
the splenic flexure with the colonic component
conceptually removed and (E) the left mesocolon
with the sigmoid colon removed. In each the grey
area represents connective tissue present between
the colon/mesocolon and the retroperitoneum.
This is the connective tissue that must be
separated in mesofascial separation in order to
achieve mobilisation. In B-E the hepatocolic and
splenocolic condensations, and greater omentum, are not included in order to simplify the
illustration. As part of flexural mobilisation these “ligaments” (i.e. condensations of greater
omentum) must be divided to provide access to the plane required for mobilisation of the
colonic and mesenteric components of the flexures.
Ileocaecal junction and right colon/mesocolon: In brief, the peritoneal fold inferolateral to
the ileocaecal mesenteric confluence was sharply divided in a process that was continued
proximally along the root of the small intestinal mesentery. This exposed the interface
between fascia and overlying mesentery/mesocolon from duodenal to ileocaecal level. By
separating the mesentery/mesocolon from the fascia and thus retroperitoneum (i.e.
mesofascial separation) the entire small intestinal mesentery was fully mobilised. Next, the
right-sided lateral peritoneal fold was sharply divided exposing the interface between right
mesocolon and underlying fascia. In separating both these structures (i.e. mesofascial
separation), the right mesocolon was now fully mobilised and observed as contiguous with
the small intestinal mesentery.
Hepatic flexure and transverse mesocolon: The hepatocolic ligament (a lateral condensation
of the greater omentum) was sharply divided thereby exposing the cephalad surface of the
hepatic mesenteric confluence. Continuing this process medially across the transverse colon,
the greater omentum was fully separated from the transverse mesocolon and the latter could
now be observed as being contiguous with the hepatic mesenteric confluence. Using
mesofascial separation the mesenteric confluence was then separated from the second part of
the duodenum and the head of the pancreas as far medially as the origin of the transverse
mesocolon (i.e. at the origin of the middle colic artery). Without disrupting the middle colic
adipovascular pedicle, mesofascial separation continued laterally along the body and tail of
pancreas towards the splenocolic ligament (also a lateral condensation of the greater
omentum) which was then sharply divided.
Splenic flexure and left colon/mesocolon: Next, the left lateral peritoneal fold was sharply
divided exposing the interface between left mesocolon and underlying fascia. These were
separated to mobilise the left mesocolon off the retroperitoneum. By extending this process
towards the splenic flexure, and onto the splenocolic ligament, the splenic mesenteric
confluence (i.e. the splenic flexure) was now fully mobilised. The left mesocolon could then
be fully medialised.
Sigmoid and mesosigmoid: The lateral peritoneal fold associated with the apposed
mesosigmoid (i.e. at the junction of both apposed and mobile components of the
mesosigmoid) was sharply divided exposing the interface between apposed mesosigmoid and
underlying fascia (mesofascial separation). Both were again separated and the apposed
mesosigmoid was fully medialised. To aid this, the upper mesorectum was separated from
associated fascia. This completed the mesocolic mobilisation in the mesofascial plane and the
mesocolon was evident as a contiguous structure from small intestinal mesentery to
mesorectal level.
Slide review:
This was conducted by consensus rather than via independent observations. This process
involved slide review by four investigators (KC, FQ, PD and JCC). We felt this approach
was necessary as both FQ and PD are both anatomists trained in the traditional mode of
mesocolic and mesenteric appraisal (i.e. that the mesocolon is fragmented rather than
contiguous). KC is a surgical trainee whilst JCC is a practicing colorectal and general
surgeon. Thus both KC and JCC appraised the mesocolon as contiguous (in keeping with the
surgical approach to the same). Prior to review, both FQ and PD were appraised of mesocolic
contiguity from small bowel mesenteric to mesorectal level. This was demonstrated to both
via surgical mobilisation of the mesenteric organ from duodenum to rectal level.
Supplementary results:
Figure 4 A-G: Schematic colored illustrations
of the mesosigmoid and intersigmoidal fossa.
4(A) represents the sigmoid colon and
mesocolon in the undisturbed state. The dotted
line represents the lateral border of the apposed
mesosigmoid. 4(B) Here the mobile sigmoid
has been partially deflected medially to
demonstrate the peritoneal adhesions (i.e.
congenital attachments) between the lateral
surface of the mesosigmoid and the peritoneum of
the left iliac fossa. Once these are divided then the
sigmoid can be fully deflected medially and the
true intersigmoidal fossa becomes apparent as
indicated. Figures 4D-G represent axial views of
the sigmoid and mesosigmoid as one progresses
distally from the descending colon to the junction with the rectum. The levels correspond to
the lines a-d depicted in Figure 4A. The intersigmoid fossa is depicted in each illustration.
Supplementary
Figure
5:
(A)
Photomicrograph (Massom`s Trichrome)
of fascia and retroperitoneum following
mobilisation of the ascending mesocolon.
The superficial layers of the fascia were
fragmented whilst in deeper layers
lamellae of collagen remained intact. (B)
Photomicrograph
(haematoxylin
and
eosin,
(H&E))
demonstrating
the
mesothelial monolayer evident between
connective tissue and retroperitoneum
(Scale 100 µm). (C) Photomicrograph
(H&E) demonstrating the underlying
fascia, mesothelial monolayer and
retroperitoneum following mobilisation of
the descending mesocolon.
Superficial
layers of the connective tissue were
fragmented whilst deeper layers remained
contiguous. Scale 100 µm.
Figure 6: Immunohistochemical
evaluation of lymphatic channels as
identified using podoplanin.
(A)
Positive control taken from tonsil
tissue and counterstained with eosin.
(B) Podoplanin positivity (arrow) in
submesothelial connective tissue in
ascending mesocolon. (C) Podoplanin
immunopositivity in vessel located
within Toldt`s fascia (further example
shown in inset). (D) Podoplanin
positivity within a fibrous connective
tissue septum within the transverse
mesocolon.
Acknowledgements:
The authors would like to acknowledge Mr Joe Pangrace in the Medical Illustration Department of the
Cleveland Clinic (Ohio) for the schematic illustrations provided in the supplementary methodology
and results.