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   Table of Contents      
ORIGINAL ARTICLE
Year : 2018  |  Volume : 4  |  Issue : 2  |  Page : 75-81

Expression of Neuron-Specific Enolase and S-100 in the Ileum and Ileocecal Junction in the Human Fetuses at Various Gestational Ages


Department of Anatomy, Maulana Azad Medical College, University of Delhi, Delhi, India

Date of Web Publication28-Aug-2018

Correspondence Address:
Dr. Sabita Mishra
133 A, DDA, SFS, Gulabi Bagh, Delhi 110007
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mamcjms.mamcjms_16_18

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  Abstract 


Background and Objectives: The purpose of this study was to study the histogenesis and neuroanatomy of the human fetal ileum and ileocecal junction at various gestational ages (15, 18, 20, 28, and 32 weeks) using neuron-specific enolase (NSE) as a neuronal marker and S-100 as a glial marker for the enteric nervous system. Materials and Methods: For this study, five aborted normal fetuses were obtained from the fetal repository of Department of Anatomy. The ileocecal part of the small intestine of these fetuses was dissected, processed, and sectioned and stained with hematoxylin and eosin and cresyl violet. Immunohistochemistry was performed using antibodies to NSE and S-100 and observed under a BX61 Olympus microscope using a DP71 camera. Results: The myenteric and submucosal plexuses of terminal ileum and ileocecal junction show immunoreactivity for both NSE and S-100 by 20 weeks. The development of myenteric plexus is more advanced than submucosal plexus. The immature neurons at 15 weeks progressively mature with distinct cell processes at 32 weeks. The mature ileocecal junction is composed of two outer circular muscle layers and a single inner longitudinal muscle layer. At 32 weeks, ileocecal valve was visualized. Interpretation and Conclusions: Our study provides the morphological evidence of the immunoreactivity in the ganglion plexus and the development of ileum and ileocecal junction at different developmental stages. It also substantiates the concept of ileocecal junction being an intussusception of ileum into the cecum.

Keywords: Enteric nervous system, human fetus, ileocecal junction, ileum, NSE, S-100


How to cite this article:
Aggarwal V, Bakshi ST, Mishra S. Expression of Neuron-Specific Enolase and S-100 in the Ileum and Ileocecal Junction in the Human Fetuses at Various Gestational Ages. MAMC J Med Sci 2018;4:75-81

How to cite this URL:
Aggarwal V, Bakshi ST, Mishra S. Expression of Neuron-Specific Enolase and S-100 in the Ileum and Ileocecal Junction in the Human Fetuses at Various Gestational Ages. MAMC J Med Sci [serial online] 2018 [cited 2018 Sep 20];4:75-81. Available from: http://www.mamcjms.in/text.asp?2018/4/2/75/239993




  Introduction Top


The small intestine requires a highly coordinated control over muscular activity and fluid secretion to efficiently break down food particles, extract nutrients, and maintain a healthy luminal microbiome. An important role in this function is played by the ileum and the ileocecal junction, which is a transition zone regulating intestinal transit. Ileum is responsible for the absorption of vitamin B12 and the reabsorption of conjugated bile salts. Histologically, ileum is divided into four layers, which are namely (from inside to outside), mucosa, submucosa, muscularis externa, and serosa.

The enteric nervous system (ENS) is sometimes referred to as a “minibrain” because it contains all the elements of a nervous system including sensory neurons, interneurons, and motor neurons and glial cells. It contains as many neurons as the entire spinal cord. In contrast to other regions of the peripheral nervous system (PNS), the ENS is capable of mediating reflex activity in the absence of central neural input.[1]

It comprises two well-organized neural plexuses. The myenteric plexus is located between longitudinal and circular layers of muscle; it is involved in the control of digestive tract motility. The submucosal plexus is located between the circular muscle and the luminal mucosa. All the neurons and glial cells of the ENS are derived from the neural crest.[2]

The ileocecal junction is a specialized area that forms the boundary between the small and the large intestine. It has been the subject of numerous anatomical and clinical investigations. The terminal ileum joins the cecum at an acute angle, and the ileal mucosa, submucosa, and muscular layer continue through the wall of the colon and combine with corresponding layers in the cecum inferiorly and the colon superiorly to form the ileal papilla.

Neuron-specific enolase (NSE) is a brain-specific isozyme of the glycolytic enzyme enolase and is characterized by its consistent occurrence in the cytoplasm of mature neurons.[3] It is a superior marker of neuronal somata when compared to other neuronal markers such as PGP 9.5 and GAP-43.[4]

S-100 is an acidic, soluble calcium binding protein with a molecular weight of approximately 20 kDa. Immunohistochemistry and electron immunocytochemistry have demonstrated that both enteric glial cells and Schwann cells of the human gut contain densely immunoreactive S-100. This protein can therefore be regarded as a common marker for the glial components of the ENS.[3],[5]

A number of studies have been conducted previously in mammals such as pigs, cattle, aves, and mice focusing on the histogenesis of the small intestine and development of ganglionated neuronal plexuses.[6],[7],[8] Some studies have also been conducted on the human fetal intestine with regard to the basic morphology of the ileal tissue and studying the development of mucosa and smooth muscles,[2],[9],[10] migration of neural crest cells to the intestine,[1],[11],[12] functional development of ENS, and the role of interstitial cells of Cajal.[2],[13],[14]

However, most of the human studies conducted so far have either focused on the colonic development or have focused on ileal development till mid trimester. This study includes the study of fetal tissue at 15, 18, 20, 28, and 32 weeks. Moreover, there is paucity of literature on the development of glia in the human fetal ileal tissue.

The literature available on the histogenesis of ileocecal junction is sparse. Previous studies give a detailed explanation on the gross and functional anatomy of the ileocecal valve.[15],[16],[17],[18] But, there is no available literature on the detailed developmental microanatomy of the ileocecal valve in humans. Studies regarding the development of ileocecal junction are controversial with regard to its function whether a sphincter or valve.[18] Some data are available on the development of ENS in large mammals,[19],[20] but no study has been conducted regarding the development of ENS in human fetal ileocecal junction using functional neuronal and glial markers.

This is the baseline study to show the developmental microanatomy and neuroanatomy of human ileum and ileocecal junction during prenatal period and might give an understanding to diagnose and treat anomalies and pathologies related to its development.


  Materials and Methods Top


Fetal material

This was an observational study and the ethical clearance was obtained from the Institutional Ethics Committee prior to conducting the work. For this study, five aborted fetuses of gestational age 15–32 weeks were used, obtained from the fetal repository of the Department of Anatomy.

The fetuses below the gestational age of 20 weeks were obtained from abortions conducted in accordance with the Medical Termination of Pregnancy act of India, 1971, while those above 20 weeks of gestation were obtained from stillbirths. A detailed maternal history was recorded and an initial assessment of the fetus was performed to rule out any gross abnormality prior to preserving the fetuses. Only the normal fetuses were included in the study. The gestation ages of the fetuses were determined by measuring following parameters: crown-rump length, crown-heel length, bi-parietal diameter, and foot length. The fetuses were immersion fixed in 10% formalin [Table 1].
Table 1: Fetal parameters

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Dissection and tissue processing

After procuring the fetus, a median incision was given on the anterior abdominal wall for immediate fixation of the gut. The fetuses were immersed for fixation in 10% formalin. After 24 h, an abdominal incision was given and the ileal part of the small intestines and proximal part of the large intestines was dissected, giving special emphasis to have the ileocecal junction identified. The entire ileocecal part was removed and preserved in fresh fixative for 1–2 weeks. Specimens that showed any degree of autolysis were not considered for the study.

The dissected ileocecal part was preserved in the fixative for 48 h and was labeled and processed for paraffin embedding keeping the ileocecal obliquely as the cutting surface. Seven-μm thick serial sections were generated on a rotary microtome. Every 3rd and 4th section was stained with hematoxylin and eosin (H&E) stain and cresyl violet stain, respectively, to observe the layers of the developing ileum and ileocecal junction. These H&E sections would also help to identify the ileocecal junctions and the developing ileocecal valves. Stained sections were observed and photographed by a digital camera Olympus DP71.

Immunohistochemistry

Following the identification of the layers of the ileum and the ileocecal junction, the immunohistochemistry of sections of ileum and ileocecal junction was performed to localize functional markers for the developing ENS (i) NSE and (ii) S-100, at various gestational ages.

From each age group, 10 sections were immunostained for each of the antibodies on 7-μm thick paraffin embedded tissue sections. Sections after washing in phosphate buffer were treated with methanol and hydrogen peroxide to block the endogenous peroxidase activity. Thereafter, non-specific binding was reduced by incubating in normal horse serum. These were then incubated in the primary antibody at required dilutions. The primary antibody was visualized by incubating in a biotinylated secondary antibody and using diaminobenzidine as a chromogen. The sections were observed under a BX61 Olympus microscope and analyzed by image proplus software and digital photography was performed using a DP71 camera attached to the microscope [Table 2].
Table 2: Primary antibodies

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  Results Top


Ileum

Histology on hematoxylin and eosin stain and cresyl violet stain

At 15 weeks, the ileal lumen is completely covered with villi. The muscularis externa is primitive, because there is incomplete differentiation of circular and longitudinal muscle layers [[Figure 1]A]. At 18 weeks, the villus epithelia appear more developed and there is a relative decrease in luminal occupancy. Blood vessels appear in the lamina propria [[Figure 1]B]. At 20 weeks, mucosa is lined by tall columnar epithelium and the villi have receded away from the lumen [[Figure 1]C]. Lymphoid aggregations are seen in lamina propria. At 28 weeks, peyer patches are well formed in lamina propria. Serosa is well formed at this stage [[Figure 1]D]. At 32 weeks, there is advanced maturation of the complete intestinal wall. Muscularis mucosa is seen [[Figure 1]E].
Figure 1: (A) Histology of ileum on H&E and CV staining. A, micrograph (10×) at 15 weeks showing the transverse section stained with cresyl violet. The lumen is completely occupied with villi. Muscularis externa is not well differentiated into longitudinal and circular muscle layers (arrow). (B) Histology of ileum on H&E and CV staining. B, (10×) transverse section at 18 weeks stained with H&E. Blood vessels seen in lamina propria (arrow). (C) Histology of ileum on H&E and CV staining. C, (10×) transverse section at 20 weeks stained with H&E. A well-developed mucosa can be clearly seen. (D) Histology of ileum on H&E and CV staining. D, (10×) transverse section at 28 weeks stained with H&E. Peyer patches are seen in lamina propria (arrow). Serosa is well formed (arrow head). (E) Histology of ileum on H&E and CV staining. E, (10×) transverse section at 32 weeks stained with H&E. Muscularis mucosa is seen (arrow). L − lumen of the ileum; v − villi; LM − longitudinal muscle layer; CM − circular muscle layer; H&E − hematoxylin and eosin; CV − cresyl violet stain. Scale bar represents 20 μm

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Development of neuronal tissue showing neuron-specific enolase and S-100 immunoreactivity

At 15 weeks, the myenteric neurons are small and round immature cells which are present in chains but not demarcated into distinct ganglia [[Figure 2]A]. There is no NSE immunoreactivity present but some myenteric glial cells are weakly positive for S-100. The submucosal neurons are rudimentary. At 18 weeks, the myenteric neurons increased in number and the neuronal processes appear longer. The submucosal neurons are present but appear primitive than myenteric neurons. Some myenteric and submucosal neurons show immunoreactivity for NSE [[Figure 2]B] but submucosal glia are negative for S-100. At 20 weeks, the myenteric plexus is immunoreactive for both NSE and S-100 [[Figure 2]C]. The submucosal glia appear elongated and are weakly positive for S-100. At 28 weeks, myenteric neurons are triangular and are segmented into ganglia. Submucosal and myenteric plexuses are strongly positive for both NSE and S-100 [[Figure 2]D]. At 32 weeks, the ileocecal leaflet is identified covered by villi with a core of lamina propria. Distinct circularly arranged myenteric plexus is seen [[Figure 2]E]. Neurons are mature with processes.
Figure 2: Immunoreactivity of ileum for NSE and S-100. (A) micrograph (40×) at 15 weeks immunostained with anti S-100 antibodies. The myenteric neurons are small and round and not arranged into distinct ganglia (arrow). (B) (10×) at 18 weeks immunostained with anti NSE antibodies. Submucosal neurons show weak positivity (arrow). (C) (40×) at 20 weeks immunostained with anti S-100 antibodies. The myenteric glia is immunopositive for S-100 (arrow). (D) (10×) at 28 weeks immunostained with anti S-100 antibodies. The myenteric and submucosal plexuses show very strong immunoreactivity for S-100 (arrow heads). L – lumen of the ileum; v – villi; LM – longitudinal muscle layer; CM – circular muscle layer. Scale bars 10 µm (A & C) and 20 µm (B & D)

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Development of ileocecal junction

The transverse section at the junction of ileum and cecum revealed that there are three distinct muscle layers which constitute the ileocecal junction. On one side, there is the circular muscle layer which is continuous with the circular muscle layer of the ileum and on the other side circular muscle layer which is continuous with circular muscle layer of the cecum. In between these two circular muscle layers, there is a single longitudinal muscle layer. These muscle layers increase in thickness and become more differentiated as the age of the fetus increases [[Figure 3]A and [Figure 3]C]. These observations give us the impression that the ileocecal junction is formed as a result of intussusception of ileum into the cecum. The neuronal plexuses are present inner to both the circular muscle layers and in between the circular and longitudinal muscle layers [[Figure 3]B]. These plexuses show immunoreactivity for NSE and S-100 at 20 weeks. In one of our specimens at 32 weeks of gestation, an ileocecal leaflet is observed. It is lined by a well-developed mucosa resembling the mucosa of the cecum. The base of the leaflet has a thick circular muscle coat and a thin longitudinal muscle layer. Myenteric plexuses are seen embedded between these two layers [[Figure 3]E].
Figure 3: (A & B) transverse section of human ileocecal junction (10×) at 20 weeks. A, stained with cresyl violet. B, immunostained with anti S-100 antibodies. Myenteric plexus is seen outer to both circular muscles (arrow) and submucosal plexus seen inner to the circular muscle layers (star). (C, D) Ileocecal junction (10×) at 32 weeks. (C) Hematoxylin & eosin stained. (D) immunostained with anti NSE antibodies. (E) Ileocecal leaflet (White arrow) at 32 weeks of gestation (10×). Hematoxylin & eosin stained. The mucosa is well developed and resembles cecal mucosa. Myenteric plexus seen embedded in between the circular and longitudinal muscle layers (arrow). CM - circular muscle layer, LM - longitudinal muscle layer. Scale bar 20  µm

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  Discussion Top


The present study has demonstrated the histogenesis and neuroanatomy of ileum and ileocecal junction at various gestational ages (15, 18, 20, 28, and 32 weeks). The ileal mucosa is immature and the villi occupy the entire lumen at 15 weeks. There is a progressive decrease in luminal occupancy and increasing maturity as the age of the fetus increases. This occurs in conjunction with the development and differentiation of muscularis externa into circular and longitudinal muscle layers. This period is also marked by the development of blood vessels and the lymphoid tissue in the lamina propria. The shape and size of neurons are round and small at 15 weeks. They progressively turn into large and polygonal cells having secondary processes on them at 32 weeks. At 32 weeks, the appearance of the ileum resembles that of the infant ileum.

Our study also shows that the neurons and glia at both the myenteric and submucosal plexuses in the fetal ileum become immunoreactive for S-100 and NSE by the end of 20 weeks which is consistent with the studies conducted earlier.[12],[21] Earlier studies[21] indicate that the development of ENS in ileum is least advanced when compared to pylorus and colon. This may be the reason for the appearance of immunoreactivity late in the second trimester in the ileum. We also found that the development of myenteric plexus precedes the development of submucosal plexus which is supported by the previous observations.[2] The total number of cells per myenteric ganglion was higher than the submucosal ganglion.

Our study substantiates the concept of ileocecal junction being an intussusception of ileum into the cecum because of the presence of single longitudinal muscle layer in between the two circular muscle layers along with the continuity in nerve plexus. This view is shared by the previous study conducted in piglets.[19] Previous studies report vaguely that there is a higher concentration of ganglia in the adult human ileocecal junction when compared to ileum,[15] but this relation was not found in our study of the fetal ileocecal junction. The ganglia present at ileocecal junction (ICJ) were found to be immunoreactive for NSE and S-100 at 20 weeks that closely resembles the immunoreactivity of the ileum.Our study provides a morphological evidence of the development of ileum and ileocecal valve at different time points. This might help to understand and correlate pathologies associated with this region. Knowledge gained from such studies might also help surgeons to operate on and construct a more anatomically functional valve.

This study has been conducted in the five human fetuses, which are in scattered developmental stages. Detailed statistical data are needed to further analyze the results. More extensive studies would be needed including a number of fetuses in close and consecutive developmental time points in second and third trimester to determine the more detailed and accurate development microanatomy of the ileocecal valve.

In conclusion, we have demonstrated the developmental microanatomy and neuroanatomy of the ileum and ileocecal region. To get into greater insights, it would be of interest to examine the expression of more neuronal and glia markers at the ICJ and correlate the findings with the functional anatomy of the ICJ in vivo.

Acknowledgements

All the chemicals used in the study and the fetal material are the courtesy of Department of Anatomy, Maulana Azad Medical College and the research was conducted as a part of ICMR STS program.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

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