Med Mol Morphol (2011) 44:39–45
DOI 10.1007/s00795-010-0503-6© The Japanese Society for Clinical Molecular Morphology 2011

ORIGINAL PAPER
Yuichi Akashi · Tsuyoshi Noguchi · Kaoruko Nagai
Katsunobu Kawahara · Tatsuo Shimada

Cytoarchitecture of the lamina muscularis mucosae and distribution of thelymphatic vessels in the human stomach

Received: December 30, 2009 / Accepted: February 16, 2010

Abstract The aim of the present study was to clarify the
anatomical structure of the lamina muscularis mucosae(LMM) in the human stomach and to correlate it with thelymphatic spread of gastric cancer cells. Human stomachs
taken at operation or autopsy were used. The specimens
derived from these stomachs were examined by lightmicroscopy immunohistochemistry and scanning electron
microscopy (SEM). In the cardia and pyloric wall, bundlesof smooth muscle cells of the LMM were relatively loose
and thin and formed a reticular configuration. Small lymphatic capillaries (approximately 10–30 μm in diameter)
were present directly above the LMM, and relatively large
lymphatics (approximately 80–100 μm in diameter) wereobserved in the submucosal layer and within the LMM. In
contrast, the LMM in the fundus, body, and antral wall wascomposed of tight, thick bundles of smooth muscle cells thatran straight. Large lymphatics were found directly beneaththe LMM, but they were few in the lamina propria mucosae.
In addition, lymphatics adjacent to veins were also found in
the submucosa of the fundus. Structural differences in the
LMM of the stomach wall might depend on physiological
function. In this study, the relationship between the cytoarchitecture of the LMM or the distribution of lymphatic
vessels and cancer invasion is discussed.
Key words D2-40 antibody (D2-40) · Human stomach ·Lamina muscularis mucosae (LMM) · Lymphatic vessels ·Scanning electron microscopy (SEM)
Y. Akashi (*) · K. Nagai · K. Kawahara
Department of Oncological Science (Surgery II), Faculty of Medicine,
Oita University, Idaigaoka 1-1, Yufu, Oita 879-5593, Japan
Tel. +81-97-586-5853; Fax +81-97-586-5853
e-mail: akashik2@med.oita-u.ac.jp
T. Noguchi
Department of Gastroenterological Surgery, Faculty of Medicine,
Oita University, Oita, Japan
T. Shimada (*)
Department of Health Sciences, School of Nursing, Faculty of
Medicine, Oita University, Idaigaoka 1-1, Yufu, Oita 879-5593, Japan
Tel. +81-97-586-5032; Fax +81-97-586-5032
e-mail: tshimada@med.oita-u.ac.jp

Introduction
It is well known that cancer invasion depth closely correlateswith lymphatic spread. Lehnert et al.1 reported that the riskof lymph node metastasis in gastric cancer with submucosal
invasion was four times higher than that in mucosal gastriccancer. Histologically, the lamina muscularis mucosae (LMM)
separate the lamina propria mucosae from the submucosa.
So far, there have been few reports concerning the
microscopic structure of the lamina muscularis mucosae(LMM) in the human stomach. Only some textbooks onhistology (Ham,2 Rhodin,3 and Leeson and Leeson4)
described that the LMM in the human stomach was composed of a few smooth muscle layers, including collagen andelastic fibers, based on light microscopic observations froma histological or surgical point of view. It is very important
to clarify the cytoarchitecture of the LMM.
However, the LMM is not visible by scanning electron
microscopy (SEM) without chemical digestion. Recently,
the cytoarchitecture of the LMM in the human esophagus
was clearly demonstrated by SEM by utilizing the elastaseNaOH digestion method.5 Their architecture varied depending on location in the neck, thorax, and abdomen.
In addition, no studies have examined the correlation
between the lymphatic vessels and the morphological configuration of the LMM, although Lehnert et al.1 demonstrated the presence of small lymphatic vessels over the
LMM and large lymphatic vessels in the submucosal layer
in the human stomach. Furthermore, regional differences inthe distribution of lymphatic vessels in the human stomach
remain unclear, although the distribution and architectureof lymphatic vessels in the rat stomach varied in the different zones.
In the present study, the structure of the LMM and thelymphatic vessels in the human stomach were analyzed
using light microscopy and scanning electron microscopy(SEM), and its correlation with the lymphatic spread modeof gastric cancer was considered anatomically.
In this study, the three-dimensional architecture of
the LMM was visualized by SEM combined with the40elastase-NaOH digestion method. On the other hand, lymphatic vessels were identified by utilizing the immunohistochemical method,6 in addition to hematoxylin and eosin(H&E) staining.

through a graded ethanol series, dried by the t-butanol
drying method, coated with gold, and observed under an
SEM (S-800; Hitachi, Tokyo, Japan) at an acceleration
voltage of 15 kV.

Materials and methods

Results

Human stomachs taken at operation or autopsy were usedas materials. Normal stomach tissues observed with the
naked eye were also obtained with informed consent from45 adult patients who underwent surgery for gastric cancer
at Oita University. Specimens were longitudinally cut into
5-mm-thick slices with razor blades from the cardia, fundus,
corpus, pyloric part, and pylorus, and processed for lightmicroscopic and scanning electron microscopic examination according to the procedures described below. The
gastric epithelium in all stomachs examined by us had histologically a normal appearance.

No individual variations of morphology in either the LMM
or lymphatic vessels were found.
Light microscopic findings of the lamina muscularis
mucosae (LMM)

Tissue blocks were fixed with 10% formalin, dehydrated inan ethanol series, cleaned by xylene, and embedded in paraffin. The sections (about 5 μm thick) were cut and stained with
H&E. The formalin-fixed paraffin sections in five stomachs
were also stained immunohistochemically with monoclonal
mouse anti-human D2-40 antibody (D2-40) (Dako North
America, Carpinteria, CA, USA)7 to identify the lymphatic
vessels. D2-40 is a new selective marker of lymphatic epithelium. D2-40 to a O-linked sialoglycoprotein (MW, 40 kDa)
was purified from ascitic fluid, as previously described.8 Afterepitope retrieval with citrate buffer (pH 6.0), sections weretreated with 0.3% hydrogen peroxide and incubated sequentially with D2-40 (1:2000) for 12–18 h at room temperature.
Then, they were subjected to Dako Envision System-HRP
Labelled Polymer Anti-mouse (Dako North America) for
30 min. Immunoactivity was visualized with a DAB solution;sections were counterstained with Mayer’s hematoxylin andexamined under a light microscope.

Figure 1 a–e shows light micrographs of H&E-stained longitudinal sections of the human stomach. The LMM, separating the lamina propria mucosa from the submucosa, hada thickness of 50–300 μm and consisted of slender, spindleshaped smooth muscle cells. The smooth muscle cells formed
three to seven muscle bundles. The thickness and cytoarchitecture of the LMM varied considerably at different sites inthe stomach.
In the cardia, the muscle bundles, consisting of three tosix smooth muscle cells, were separated by wide spaces,
were relatively sparse, and had a reticular arrangement (Fig.
1a). In the fundus, nearer to the fundic cardia, the spaces
between the muscle bundles were narrower, with a more
dense reticular arrangement (Fig. 1b). More distally, themuscle bundles had a linear arrangement. In the corpus, theLMM differed in structure between the lesser curvature andthe greater curvature. In the lesser curvature, the LMM wasabout 100 μm thick, and the muscle bundles had a linear
arrangement (Fig. 1c). In the greater curvature, the LMM
was about 200 μm thick (Fig. 1d). In the pylorus, the structure of the LMM was similar to that in the cardia, namely,
sparse muscle bundles with wide separation and a reticular
structure. The LMM was about 200 μm thick (Fig. 1e). In allareas of the stomach, blood vessels and lymphatic ducts
passing through the muscle bundles of the LMM wereobserved.

Scanning electron microscopy (SEM)

SEM findings of the lamina muscularis mucosae (LMM)

The tissue blocks fixed in Karnovsky’s fixative in ten stomachs were used for SEM. The blocks were treated with 6 N
NaOH at 60°C for 10–15 min9 or with 8 N HCl at 37°C for
3 h10 for the digestion of collagen fibers. After being rinsed
thoroughly in physiological saline, the blocks (about 2 cm ×2 cm) were placed in an elastase solution (1–5 mg/ml; Tohri,
Tokyo, Japan) at 37°C for 12–18 h. The remaining paraffinblocks were used for SEM. They were deparaffined withxylene, hydrated, fixed again in Karnovsky’s fixative, andimmersed in 2 N NaOH at 37°C for 3 h to digest proteoglycan.11 All specimens were then washed thoroughly in distilled water and placed in cacodylated-buffered 1% osmium
tetroxide, 1% tannic acid solution, and 1% osmium tetroxide for 1 h each. The specimens were then dehydrated

Treatment of the gastric tissue with NaOH or HCl at 60°C
for 10 min followed by elastase treatment resulted in theremoval of collagen and elastic fibers, thus exposing thestromal surface of smooth muscle cells in the LMM.
SEM observation of the cardia LMM from the side of
the submucosa showed aggregates of groups of smoothmuscle cells at random or in a reticular pattern (Fig. 2). The
smooth muscle cells were slender, with spaces of 20–30 μm
between the cells. In the fundus and corpus LMM, thesmooth muscle cells were dense and generally arranged
longitudinally. The LMM was composed of smooth muscle
cells with a linear arrangement, with almost no space
between the cells (Fig. 3a,b). The antral LMM was basicallysimilar to that of the corpus. Slender smooth muscle cells

Light microscopy

41
Fig. 1. a In the cardia, themuscle bundles of the lamina
muscularis mucosae (LMM),
consisting of three to six smoothmuscle cells, are separated bywide spaces, are relatively
sparse, and have a reticular
structure. b Nearer to the cardia,
the spaces between the muscle
bundles are narrower, with amore dense reticular structure.
c More distal to the cardia, themuscle bundles have a parallel
structure. In the gastric corpus,
the LMM differs in structure
between the lesser curvatureand the greater curvature. In thelesser curvature, the LMM isabout 100 μm thick, and themuscle bundles have a parallel
structure. d In the greater
curvature, the LMM is about
200 μm thick, with a reticular
structure. L, lymphatics orlymphatic capillaries. e In thepylorus, the structure of the
LMM is similar to the cardia,
namely, sparse muscle bundles
with wide separation and a
reticular structure. Thickness isabout 200 μm

with a linear arrangement extended to the pylorus (Fig. 4a).
In the LMM from this area, longitudinal lymphatic vessels
were also observed (Fig. 4b). The pylorus LMM closely
resembled that of the cardia. Smooth muscle cells had areticular arrangement, with spaces between the cells.

Lymphatic vessels
Lymphatic capillaries distributed in the gastric tissue wererelatively easily identified on light microscopy with H&E:
these were identified by their size, somewhat larger thanthat of blood capillaries, irregular shape, and a thin layer ofepithelial cells. The connective tissue around the lymphatic
capillaries was sparse. To clarify more clearly the identification of lymphatic vessels and their distribution, we immunostained specimens with D2-40, which specifically reactedwith endothelial cells of lymphatic capillaries and lymphat-

ics. In the cardia, lymphatic capillaries were abundant above(lamina propria) and below (submucosa) the LMM (Fig.
5a). In the submucosa, the lymphatics had a relatively large
diameter. Lymphatics were identified by the presence ofvalves. Lymphatic capillaries were also distributed justbelow the esophageal stratified squamous epithelium of the
cardia (Fig. 5b). No lymphatic capillaries were seen justbelow the gastric simple columnar epithelium, but they
were seen around the gastric glands.
In areas of the stomach other than the cardia, relatively
small lymphatic capillaries (approximately 10–30 μm in
diameter) were observed above the LMM in the lamina
propria, and fairly large lymphatic capillaries or lymphatics(approximately 80–100 μm in diameter) were observed justbelow the LMM in the submucosa (Fig. 6a–c). In addition,in the fundus, large lymphatic capillaries or lymphatics adjacent to veins coursing through the submucosa were noted(Fig. 6a). In the gastric angle, the lymphatic capillaries in the

42

LMM ran parallel to the smooth muscle in a longitudinal
direction (Fig. 6c). In the pylorus, as in the cardia, the lymphatic capillaries traversed the LMM in a nearly perpendicular direction (Fig. 6d).
The most interesting finding in the present SEM study
was that the lymphatics ran not only within the LMM but
also just below the LMM in all regions (Figs. 4b, 7a,b).

Fig. 2. Scanning electron micrograph (SEM) of the cardia LMM fromthe side of the submucosa shows aggregates of smooth muscle cells at
random or in a reticular pattern. The smooth muscle cells are slender,
with spaces of 20–30 μm between the cells

Fig. 3. a SEM of the corpus
LMM. The smooth muscle cells
are dense and generally
arranged longitudinally. The
LMM is composed of smoothmuscle cells with a parallel
structure, with almost no space
between the cells. b SEM of the
corpus LMM from the side of
the submucosa. The smoothmuscle runs relatively parallel

Discussion
The Japanese Research Society for Gastric Cancer12 defined
gastric cancer confined to the mucosa and/or the submucosa
as early gastric cancer, regardless of the presence of lymph
node metastasis. Lehnert et al.1 suggested that lymphatic
spread was closely related to cancer invasion into the submucosal layer in early gastric cancer. Thus, studying the
microscopic structure of the LMM and the distribution of
the lymphatic vessels of the stomach wall might provide aclue to the mechanism of gastric cancer spread in the earlyphase.
The structural variation of the LMM in the humanesophagus was reported by Nagai et al.5 Based on lightmicroscopic findings, a few authors3,4 reported that the
LMM in the human stomach consisted essentially of two or
three layers of smooth muscle cells, but they did not describethat the structure of the LMM varied depending on thelocation in the stomach. The present light microscopic andscanning electron microscopic studies in the human stomach
revealed that the thickness and arrangement of the LMM
varied considerably in different regions. Especially, thecardia of the stomach is located next to the esophagus, and
it is of interest to clarify the structure to analyze the modeof cancer spread. In fact, the classification and the surgicalapproach for cardia cancer and Barrett’s esophageal cancer
have become increasingly controversial because of theircomplicated mode of lymphatic spread, despite the Siewert13
classification as to cardia cancer. Our morphological results
demonstrated that the cytoarchitecture of the lamina muscularis mucosae depended on the location in the humanstomach, and a few smooth muscle bundle layers showed areticular arrangement in the cardia or pylorus and a linear
arrangement in the other parts. In addition, the smoothmuscle bundles showed not a regular arrangement but acomplicated reticular one. Intracardiac pressure has beenreported to be 15–40 mmHg, which is higher than in the

43Fig. 4. a The antral LMM isbasically similar to that of the
corpus. Slender smooth muscle
cells with a parallel structure
extend to the pylorus. bLongitudinal lymphatics (L) in
the LMM are also observed

Fig. 5. a In the cardia, lymphatic(L) capillaries are abundant justabove (lamina propria) andbelow (submucosa) the LMM.
b Lymphatic capillaries are
distributed just below theesophageal stratified squamous
epithelium of the cardia.
Lymphatic capillaries are notseen just below the gastricsimple columnar epithelium, butthey are seen around the gastricglandsFig. 6. a In the fundus, fairly
large lymphatic capillaries orlymphatics are observed below
the LMM in the submucosa. V,
vein. b In the gastric angle,
lymphatic capillaries are seen
above and below the LMM. c In
the gastric angle, lymphatic
capillaries in the LMM andsubmucosa run parallel to thesmooth muscle in a longitudinal
direction. d In the pylorus, as in
the cardia, the lymphatic
capillaries traverse the LMM ina nearly perpendicular direction

44
Fig. 7. a In the greater curvatureof the corpus, SEM shows aninteresting finding, namely,
lymphatics (L) runninglongitudinally through the
LMM. Some of these branches
extend into the submucosa.
b Magnified view of aother parts.14 Nagai et al.5 reported that the reticulararrangement of the LMM in the cardia might be related to
the need to maintain higher pressure. Furthermore, its relatively thin structure and large spaces between reticular lymphatic vessels might correlate with the high incidence oflymphatic spread of cancer cells. It is likely that the reticulararrangement of the LMM in the pylorus is essentially similar
to that of the cardia.
In contrast to the reports by Nagai et al.5 and Hashimoto
et al.15 concerning the distribution of lymphatic vessels in
the lamina propria of the esophagus, several studies16–20
reported on the lymphatic flow in the stomach. Because thegastric epithelium in the present specimens showed anormal appearance, it appeared that the structure and distribution of lymphatic vessels were not influenced by thedisease. Our immunohistochemical results agreed with theprevious report by Lehnert et al.1 that lymphatic vessels
could be seen in the lamina propria mucosae and the submucosal layer in the human stomach. It is worthy of noticethat lymphatic capillaries in the human cardia were abundantly distributed beneath the stratified squamous epithelium, although those in the rat forestomach were not present
beneath stratified squamous epithelium.21 Recent advances
have made it possible to observe lymphatic capillaries
directly above the LMM and relatively large lymphatics inthe submucosal layer of the cardia and pylorus immunohistochemically using D2-40. Especially, the present SEM
study clearly demonstrated that large lymphatics were distributed within and directly beneath the LMM in all theregions in the human stomach. These findings might suggestthat gastric cancer spreads easily via the lymphatic route in
the relatively early stage, when cancer cells invade into the
LMM in the stomach. Thus, the LMM might function as akind of critical barrier against direct invasion of cancer cells.
The SEM findings of the present study, showing that thelymphatic vessels above the LMM passed through to thesubmucosal lymphatic vessels, strongly support the previous

report by Yoffey and Courtice.22 In addition, the presenceof a lymphatic network, which is in close proximity to veins,stimulates interest in the relationship with invasion ofcancer cells.
In conclusion, the microscopic observations of thepresent study might provide a clue to explain the mechanism of lymphatic spread of gastric cancer in all parts of thestomach.
Acknowledgments The authors express their gratitude to Mr. Hiroaki
Kawazato and Miss Aiko Yasuda (Division of Biomolecular Medicine
and Medical Imaging, Department of Life Science, Institute of Scientific Research, Oita University, Japan) for their kind assistance.

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