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

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 (Rutgeerts 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
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 X-100. 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 because 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.

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.

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 astrocytes (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 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.

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.

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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