Biologia 69/7: 931—935, 2014
Section Zoology
DOI: 10.2478/s11756-014-0389-1
Atypical localization of myenteric ganglia in the human appendical
wall: a comparative study with animal appendix
Eliska Kubikova, Ivana Sivakova & Anna Perzelova*
Department of Anatomy, Faculty of Medicine, Commenius University, Sasinkova 2, SK-81372 Bratislava, Slovakia; e-mail:
[email protected]
Abstract: The presence of well developed appendices in some animals when compared to humans has led to speculation
that appendix is a vestigial organ. Increasing number of studies have revealed that the appendix serves as an important
organ in humans. The function of animal appendix, and the differences between species remain poorly understood. In
this study we examined human myenteric plexus and compared them with animal studies. Appendices were obtained
from five young adults in which the appendix was found to be normal after removal. Fixed appendix cryosections were
examined by immunofluorescence methods using neuronal marker antibodies to neurofilaments and beta III tubulin. Both
antibodies stained myenteric ganglia which were arranged in an apparently irregular pattern in human appendix wall. We
observed unexpected localization of myenteric ganglia in the subserosa often accompanied by rarely occurring ganglia in
the longitudinal muscle layer. These ganglia were of different sizes and shapes and unequally distributed under a thin layer
of serosa. Our findings raise many questions about the possible role of irregular and atypical myenteric ganglia localization
in relation to altered motility and subsequent pathogenesis of the appendix in inflammatory disease in humans. On the
other hand, studies of the literature have revealed simplicity in the organization of myenteric plexus, e.g., in well-developed
rabbit appendix. In addition, appendicitis in animals is restricted to in apes with similarly shaped appendix to humans.
Key words: appendicitis; myenteric ganglia; neurofilaments; beta III tubulin; neuronal markers
Introduction
Many studies have dealt with primate and mammals
appendices (Scott 1980; Fisher 2000; Smith et al. 2009).
However, the greatest attention has been focused on
well-developed rabbit appendix (e.g., Pospisil & Mage
1998; Dasso et al. 2000; Hanson & Lanning 2008).
For a long time, the human appendix was considered
a vestigial structure because the removal of the human appendix has no apparent ill effects. Many people have been found with congenital absence of the appendix without any health problems. In addition, the
well-developed appendix in rabbit when compared to
humans has been taken to support the hypothesis of
the appendix vestigiality. The histological composition
of human appendix is similar to large intestine, however, differing with concentrated lymphoid tissue in the
appendical wall. Based on these histological findings
the appendix is thought to play a role in some immune functions. Currently, more studies have revealed
that appendix serves an important role in humans as
a “safe house“ for commensal bacteria, providing support for bacterial growth and potentially facilitating reinoculation of the colon (Bollinger et al. 2007). Recent
studies have focused attention onto the role of the appendix in the pathogenesis of ulcerative colitis (Rut-
geerts et al. 1994; Andersson et al. 2001; Matsushita et
al. 2007) and acute myocardial infarction (Janszky et
al. 2011).
Gastrointestinal tissues are inervated by the enteric nervous system (ENS). The neurons of the ENS
are organized into ganglia which together with nerve
fibres form myenteric (Auerbach’s) and submucosal
(Meissner’s) plexi. Usually the myenteric plexus localization is described between the longitudinal and circular muscle layers, responsible mainly for intestinal
motility. The submucosus plexus is located in the submucosa and controls epithelial and vascular functions of
the mucosa. The aim of this study was to determine the
immunophenotype and distribution of myenteric ganglia responsible for appendix motility and to compare
our results with animal studies.
Material and methods
Specimen preparation
Fresh tissue samples of 50 human appendices were kindly
provided by the Children’s Faculty Hospital Bratislava. Experiments with human bioptic samples were approved by
the Ethical Committee of UNB Bratislava. For this study
we chose appendices from five patients of similar age (12–14
years) which were found to be normal after appendectomy.
* Corresponding author
c
2014
Institute of Zoology, Slovak Academy of Sciences
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932
E. Kubikova et al.
Fig. 1. Morphologic features of myenteric ganglia in human appendix. Indirect immunofluorescence staining for neurofilaments (A–D).
Nuclei stained with Hoechst. Scale 50 µm.
The appendices were cut along their longitudinal axes. Specimens for cryosections were embedded with OCT and cut
into 10 µm thick sections.
Immunohistochemistry
Cryosections were fixed in methanol-acetone (1:1) solution
for 15 min at (–15 ◦C) or with 4% p-formaldehyde for 15 min
at room temperature. Myenteric ganglia were detected by
indirect immunofluorescence staining using neuronal marker
antibodies to neurofilament (1:100 dilution, clone NF-01,
Exbio, Prague) and two antibodies against beta III tubulin,
monoclonal (1:100, TUJ 1, Abcam), and polyclonal sera developed in rabbit (1:100, Sigma). Fixed cryosections were
incubated for 1 h with primary and for 30 min with 1:50 diluted secondary antibodies (Sigma). Double labelling with
NF-01 and polyclonal sera to beta III tubulin was performed
with primary, and afterwards with appropriate mixtures of
secondary antibodies for 1 h and 30 min, respectively. Staining of cryosections fixed with p-formaldehyde was performed
by permeabilization of cell membrane with 0.5% Triton X100. Nuclei were stained with Hoechst 33342 (5 µg ml−1 in
PBS, Sigma) for 1 min.
Results
Morphological analysis
Morphological features of myenteric ganglia were studied by indirect immunofluorescence of neurofilaments
which are expressed only in neuronal cells. The antibodies recognize an epitope on heavy neurofilament protein
(210 kDa) of various species (Lukas et al. 1993). Immunofluorescence showed a strong staining of ganglia.
These were of different size and shape (Figs 1A–D).
Three main pictures of myenteric ganglia distribution
in appendical wall were observed in all appendix samples examined. Myenteric ganglia were distributed as
follows: mainly within the circular layer (Figs 2A, B),
within the circular layer and rarely in the subserosa
(Figs 2C, D), and within both muscle layers (Figs 2E,
F). Most commonly, ganglia were located within the
circular muscle layer. On the other hand, we observed
unexpected localization of myenteric ganglia in the subserosa. These subserosal ganglia of different sizes and
shapes were found but rarely and were unequally distributed under a thin layer of serosa.
Co-expression of neurofilaments and beta III tubulin
Alfa and beta tubulin, the major components of microtubules are present in almost all eukaryotic cells. Six
isotypes of beta-tubulin have been identified. Beta III
tubulin was found in central and peripheral nervous
system and appears to be specific for neurons. (Ferreira & Caceres 1992; Menezes & Luskin 1994). Monoclonal antibodies and polyclonal sera stained myenteric ganglia at similar intensities and localization. Both
above-mentioned fixations were applicable for detection
of beta III tubulin. However, the staining with monoclonal antibodies to neurofilaments was more intense
on cryosections fixed with methanol-acetone. It is beUnauthenticated
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Arrangement of myenteric ganglia in human and animal appendix
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Fig. 2. Localization of myenteric ganglia in human appendical wall. Indirect immunofluorescence staining for neurofilaments (A–F)
and nuclei with Hoechst (B, D, F). Myenteric ganglia on longitudinal cryosections were distributed within the circular layer (A, B),
within the circular layer and in the subserosa (C, D), and within longitudinal and circular muscle layers (E, F). Note the irregular
distribution of myenteric ganglia. Scale 50 µm.
cause for double labelling we used this fixation. The
positively stained meynteric ganglia for neurofilaments
and beta III tubulin were found at the similar localization (Figs 3A–D). Staining intensities were slightly
stronger (brighter) with neurofilaments than with beta
III tubulin.
Discussion
The myenteric plexi of five apparently normal human appendices were studied with neuronal marker
antibodies. Based on our previous studies on human
glial cells we used antibodies against neurofilaments
which are not co-expressed with glial markers. We also
demonstrated meyenteric ganglia immunophenotypes
with further antibodies against beta III tubulin which
is widely used as a neuronal marker. However, we recently observed co-expression of beta III tubulin with
glial fibrillary acidic protein (GFAP) in cultured adult
human astrocytes (unpublished data). In other studies, beta III tubulin was found in human fetal astro-
cytes (Draberova et al. 2008). Indirect immunofluorescence with neurofilaments antibodies on appendix
cryosections revealed strong staining of nerve fibres inside as well as outside myenteric ganglia. Double labelling showed the similar co-localization of neurofilaments and beta III tubulin. Both antibodies are useful for the study of myenteric ganglia morphology and
distribution in human appendical wall. We observed
the unexpected localization of myenteric ganglia in the
subserosa. They were of different sizes and shapes unequally distributed under a thin layer of serosa often
accompanied by the rare occurrence of ganglia in the
longitudinal muscle layer. Our study showed irregular
distribution of myenteric ganglia as well as the majority of them were found in the circular muscle layer.
Similarly the study of Emery & Underwood (1970)
described irregular localization of myenteric ganglia
in the human appendix. On the other hand, Hanani
(2004) revealed that in most cases, the inervation of
the external muscles of the appendix consisted of three
concentric networks of ganglia located both between
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E. Kubikova et al.
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Fig. 3. Myenteric ganglia in human appendix stained with neuronal markers, nuclei with Hoechst. Double labeling for neurofilaments
(A, C) and beta III tubulin (B, D) demonstrate the co-expression of both neuronal markers. Scale 50 µm.
the circular and longitudinal muscle layers and within
them.
Acute appendicitis is most common between ages
10 to 20 but can occur at any ages (Addis et al. 1990;
Anderson et al. 2012). Usually appendicitis is caused
by obstruction of the lumen (Piper et al. 1982). Increased risks for appendicitis were described mainly as
low-fibre diet (Burkitt 1971; Adamis et al. 2000), infections due to edema causing obstruction of appendix
lumen and hereditary (Andersson et al. 1979). Familiar
history of appendicitis is considered to be due a particular position of the appendix, which predisposes it
to infection (Shperber et al. 1986). However, irregular
myenteric ganglia distribution with currently unknown
relation to appendix position may also be hereditary.
We suppose that the direct cause for obstruction
of appendix lumen may by insufficient inervation of appendical muscle layers which results in flaccid appendix
emptying. One factor for altered inervation may be
atypical localization of myenteric ganglia. Mentioned
risks for appendicitis may only develop this disease.
This hypothesis is supported by the increased prevalence of appendicitis in children and young adults whose
experience of inappropriate diet or gastrointestinal infections is accordingly shorter than adults.
Comparative analysis of human and animal myenteric
plexuses
The most important question about the human appendix is its pathology and poorly understood etiology of appendicitis in children and young adults. In
animals, appendicitis was described only in anthropoid
apes (Scott 1980). However, the vermiform appendix is
restricted to humans and apes. Fisher (2000) indicated
large gaps in our knowledge of the primate cecum and
its appendages. The appendix is absent in some primates (Scott 1980). Recent studies shown, that in animals lacking an appendix the terminal part of the cecum is rich in lymphoid tissue (Smith et al. 2009; Mala
2003). In many primates and mammals the appendix is
much more open and sack-shaped. We tried to compare
the arrangement of animal and human myenteric plexi.
However, only rabbit plexus has been well examined.
For example Hanani (2004) described rabbit myenteric
plexus as a three dimensional network but less extensive
than in the human appendix.
In conclusion, morphological similarities of narrow
worm-shaped appendix are related with appendicitis in
humans and apes. The myenteric plexus plays an important role in narrow appendix emptying, whereas the
easy empting of large open appendix present in many
primates and mammals is probably protective against
appendicitis.
Acknowledgements
We would like to thank Dr. MacLeod for critical reading
and linguistic correction of the manuscript and Mrs.Hillova,
Mrs. Skopekova, Mrs. Galfyova and Dr. Weismann for technical assistance. This study was supported by VEGA grant
No. 1/3439/06 and ITMS: 26240120023, co-financed by the
European Regional Development.
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Received December 5, 2013
Accepted March 4, 2014
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