Histomorphology of enteric neurons and enteric ganglia in different layers of human fetal colon.

Objective: Literature shows very few studies explaining morphology of enteric neurons and ganglia in humans. This study was aimed at determining the morpho-histology of enteric neurons and ganglia in human fetal colon.
Methods: Histological sections of human fetal colon were stained with hematoxylin and eosin, Bielschowsky's silver and Masson's trichrome stains to study the morpho-histology of enteric neurons and ganglia.
Results: Enteric neurons scattered in the early weeks of development and ganglionated as the fetal age progresses. Migration of enteric neurons was less and in scattered form during early weeks and as the age progresses it was more and in ganglionated form. Enteric neurons were round, oval, pyramidal and flat in all layers of colon. Enteric ganglia in serosa were oval in early weeks, oval and elongated in late weeks whereas in between the muscle layers and submucosa they were few and oval, irregular and elongated. Distance between the enteric ganglia increased in serosa but fluctuated in the remaining layers as the gestational age progressed. Number of enteric neurons and ganglia was more in serosa and less in other layers during early weeks and as the fetal age progressed they decreased in serosa but increased in other layers.
Conclusion: There are various shapes and numbers of enteric neurons and ganglia and distances between the ganglia in different layers of fetal colon.

Introduction

Literature describes mostly the morphometric studies of the enteric neurons and ganglia in several animal models and several diseased conditions in the human gastrointestinal tract. These studies have reported variations in enteric neurons and ganglia of myenteric and submucosal plexuses with respect to their shape and number. However, there are no studies found in the literature that explains arrangement, shape, migration pattern and numbers of enteric neurons and ganglia and distance between enteric ganglia in the human fetal gut at different gestational ages. The morphological study of enteric neurons and ganglia in the human fetal colon gives the basic information regarding histogenesis which can have developmental and clinical value. Therefore, the main aim of our research was to observe the developmental pattern of enteric neurons and ganglia in the fetal colon at different gestational ages.

Materials and Methods

Study design and settings

This is a quantitative, descriptive observational study. Study population is stillbirth and spontaneously aborted human fetuses (n = 36) collected by using the convenience sampling method. Fetuses with gut tube and neural tube defects were excluded. Sturge's rule “k = 1 + 3.322log10N” was used for grouping the fetuses into eight groups: 10–14, 14–18, 18–22, 22–26, 26–30, 30–34, 34–38 and >38 weeks.

Fetuses were fixed in 10% neutral buffered formalin for 2 days. Colon tissue was taken and processed in automatic rotatory tissue processor (LEICA TP 1020). Paraffin blocks were prepared by using Leuckhart's brass moulds. Hematoxylin and eosin, Masson's trichrome and Bielschowsky's silver stains were used to study the histomorphology, migration and confirmation of enteric neurons and ganglia respectively.

Observations

Sections of the colon were observed in a Labomed compound microscope under eyepiece 10x, field number (FN) 18 and objectives 10x, 40x and 100x. Number of enteric ganglia and enteric neurons and distance between the enteric ganglia in different layers of colon were observed in a Field of View (FOV)/focus by using ocular (Erma) and stage (Unilab) micrometers. Micrometers were calibrated by using the reference slide of peripheral blood smear (Table 1). Field of View was calculated as: Field number (FN)/Total magnification i.e 18/Total magnification. Total magnification (×) was calculated by multiplying the magnification of objective lens with the magnification of eyepiece. Number of enteric neurons were counted in Field of View 18 μm and area of view 254.34 μm2 of the sections under 100x (oil immersion). Number of enteric ganglia and distance between the enteric ganglia were observed in Field of View 45 μm and area of view 1.58 μm2 of the sections under 40x. Counting of neurons and ganglia was performed manually by two observers blinded to samples.

Table 1

Calibration of ocular and stage micrometers.

Objectives	Marks in ocular micrometer	Marks in stage micrometer	1 ocular mark = x stage marks (Calculation: stage marks/ocular marks)	1 stage mark = 10 μm;1 ocular mark = x μm
4×	16	50	3.125 marks	31.25 μm
10×	15	20	1.333 marks	13.33 μm
40×	16	5	0.312 marks	3.12 μm
100×	39	5	0.128 marks	1.28 μm

Statistical analysis

Data obtained were analyzed in SPSS 16.0 version. Nominal data were calculated in percentage, and their comparison was performed by using Chi-square test. Quantitative data were calculated in mean ± S.E of mean and their comparison was performed by using one-way-ANOVA test for normally distributed data and Kruskall Wallis test for non-normally distributed data in 95% confidence interval (CI) and 5% level of significance (p < 0.05). Graphs were plotted using Microsoft Excel version 2013.

Results

Enteric neurons were arranged in ganglionated form in majority of cases in serosa and in between the muscle layers whereas in submucosa (p = 0.01) they were scattered in early weeks and arranged in ganglionated form in mid and late weeks of gestation (Graph 1).

Graph 1

Arrangement of enteric neurons in serosa, between the muscle layers and submucosa of colon observed in H&E stained sections of different gestational weeks in 400× magnification (∗p-value = 0.01, n = 36).

Migration of enteric neurons was observed in ganglionated form in majority of cases in 14–18 weeks and 30–38 weeks from serosa to muscle layers (p = 0.01) and in 10–14 weeks and 30- >38 weeks from muscle layers to submucosa (p = 0.02) (Graph 2, Figure 1).

Graph 2

Migration of enteric neurons from serosa to muscle layers and muscle layers to submucosa in colon in H&E stained sections of different gestational weeks in 400× magnification (p-value = ∗0.01, ∗∗0.02, n = 36).

Figure 1

Masson Trichrome stained section of colon showing migration of enteric neurons and ganglia (yellow colored outline) from serosa (S) to muscle layers (ML) and muscle layers (ML) to submucosa (SM) through connective tissue fibers (blue) and muscle layers (pink) in a compound microscope in 400× magnification picture taken by One plus 9 pro, LE2121. Each mark of ocular micrometer equals 3.12 μm.

Shape of enteric ganglia was predominantly oval in serosa. In between the muscle layers, they were oval, irregular and elongated. In submucosa, neurons were in scattered form whereas a few oval and elongated ganglia were also observed (Graph 3, Figure 2). Shape of enteric neurons in serosa, between the muscle layers and submucosa were predominantly multiple in shape i.e. round, oval, pyramidal and flat (Graph 4, Figure 3, Figure 4).

Graph 3

Shape of enteric ganglia in serosa, between the muscle layers and submucosa of colon in H&E stained sections of different gestational weeks in 400× magnification (n = 26).

Figure 2

H&E stained section of colon showing different shapes of enteric ganglia (yellow outline-elongated ganglia, green outline-oval ganglia) in a compound microscope in 400× magnification picture taken by One plus 9 pro, LE2121. Each mark of ocular micrometer equals 3.12 μm. ML-muscle layers, SM-submucosa.

Graph 4

Shape of enteric neurons in serosa, between the muscle layers and submucosa of colon in H&E stained sections of different gestational weeks in 1000× magnification, oil immersion (n = 26).

Figure 3

H&E stained section of colon showing different shapes of enteric neurons (black outline-round, red outline-oval, blue outline-pyramidal, purple outline-flat) in a compound microscope in 1000× magnification, oil immersion picture taken by One plus 9 pro, LE2121. Each mark of ocular micrometer equals 1.28 μm. ML-muscle layers.

Figure 4

Bielchowsky's silver stained section of colon confirming the enteric neurons (within red colored outline) and ganglia in a compound microscope in 400× magnification picture taken by One plus 9 pro, LE2121. Each mark of ocular micrometer equals 3.12 μm. ML-muscle layers.

Distance between the enteric ganglia increased from 3.12 μm to 49.4 ± 26.58 μm in serosa and from 10.2 ± 6.29 μm to 96.1 ± 18.01 μm in between the muscle layers (p = 0.01) whereas in submucosa it fluctuated (p = 0.003) as the gestational age progressed (Table 2).

Table 2

Distance between the enteric ganglia. p-value indicates comparison between the mean distance between the enteric ganglia among different gestational age groups in different layers of colon. Asterisk denotes the obtained value is statistically significant between the gestational age groups. Observations in 400× magnification in field of view 45 μm and area of view 1.58 μm2 in H&E stained sections.

Gestational age	Serosa (Mean ± S.E) µm	Between the muscle layers (Mean ± S.E) µm	Submucosa (Mean ± S.E) µm
10–14 weeks (n = 4)	0	15.9 ± 6.4	0
14–18 weeks (n = 1)	15.6	0	43.7
18–22 weeks (n = 7)	11.7 ± 7.67	10.9 ± 7.12	9.7 ± 9.65
22–26 weeks (n = 5)	3.12 ± 3.12	10.2 ± 6.29	12.5 ± 12.48
26–30 weeks (n = 5)	13.31 ± 9.2	16.4 ± 7.89	40.1 ± 12.12
30–34 weeks (n = 5)	41.92 ± 15.17	38.4 ± 11.56	0
34–38 weeks (n = 7)	49.4 ± 26.58	56.6 ± 20.98	13.4 ± 9.27
>38 weeks (n = 2)	0	96.1 ± 18.09	88.3 ± 36.5
p-value	0.26	0.013∗	0.003∗

Number of enteric ganglia in serosa increased from 18 to 34 weeks then it decreased up to >38 weeks. In between the muscle layers, it was maximum in 14–18 weeks then it decreased up to 22–26 weeks and then increased and became constant value during 26–38 weeks. In submucosa ganglia numbers fluctuated (p = 0.01) (Table 3). Number of enteric neurons was maximum in serosa, in between the muscle layers and submucosa in 14–18 weeks. Then it decreased in serosa (p = 0.00) whereas in between the muscle layers and submucosa (p = 0.002) it fluctuated in the remaining weeks (Table 4).

Table 3

Number of enteric ganglia. p-value indicates comparison between the mean number of enteric ganglia among different gestational age groups in different layers of colon. Asterisk denotes the obtained value is statistically significant between the gestational age groups. Observations in 400× magnification in field of view 45 μm and area of view 1.58 μm2 in H&E stained sections.

Gestational age	Serosa (Mean ± S.E)	Between the muscle layers (Mean ± S.E)	Submucosa (Mean ± S.E)
10–14 weeks (n = 4)	0	1 ± 1	0
14–18 weeks (n = 1)	1	4	2
18–22 weeks (n = 7)	0.1 ± 0.14	1.3 ± 0.47	0.14 ± 0.14
22–26 weeks (n = 5)	0.4 ± 0.24	1.2 ± 0.58	0.6 ± 0.4
26–30 weeks (n = 5)	0.4 ± 0.24	2 ± 0.7	0.8 ± 0.37
30–34 weeks (n = 5)	1 ± 0.31	2 ± 0.54	0
34–38 weeks (n = 7)	0.57 ± 0.2	2 ± 0.43	0.28 ± 0.18
>38 weeks (n = 2)	0.4	1.7 ± 1	1
p-value	0.06	0.4	0.01∗

Table 4

Number of enteric neurons. p-value indicates comparison between the mean number of enteric neurons among different gestational age groups in different layers of colon. Asterisk denotes the obtained value is statistically significant between the gestational age groups. Observation in 1000× magnification in field of view 18 μm and area of view 254.34 μm2 in H&E stained sections.

Gestational age	Serosa (Mean ± S.E)	Between the muscle layers (Mean ± S.E)	Submucosa (Mean ± S.E)
10–14 weeks (n = 4)	0	11 ± 1.58	4.5 ± 1.19
14–18 weeks (n = 1)	35	31	18
18–22 weeks (n = 7)	2.28 ± 1.53	14 ± 2.97	4.14 ± 0.98
22–26 weeks (n = 5)	3 ± 2	10 ± 3.01	2.8 ± 0.91
26–30 weeks (n = 5)	3.8 ± 1.74	12.6 ± 3.72	4.8 ± 1.2
30–34 weeks (n = 1)	0	5	4
34–38 weeks (n = 3)	0	4.67 ± 0.88	3.33 ± 1.45
p-value	0.00∗	0.07	0.002∗

Discussion

Animal studies described that enteric neuroblasts at first arrange in serosa, migrate through muscle layers to submucosa, and second wave of migration occurs back from submucosa to muscle layers to form myenteric plexus and remigrate to submucosa to form submucosal plexus. Whereas in our study, enteric neurons were scattered in early weeks (18–22 weeks) and as the age progresses (22–38 weeks) they were arranged in the ganglionated form in all layers of colon. In late weeks (34–38 weeks) serosa has no neurons and ganglia. This implied that enteric neuroblasts were scattered in the beginning in serosa but as age progresses they form ganglia and undergo migration into the muscle layers forming myenteric plexus and into submucosa forming submucosal plexus. There was no evidence of second wave of migration from submucosa back to muscle layers in this study.

Shape of enteric ganglia are elongated, oval and polygonal in rodents, ovoid, oval, elongated, polygonal, round and irregular in cattle,, circular, oval, egg-shaped, polygonal and rectangular in adult rats, and enteric neurons are arranged in rows without forming ganglia in embryonic rats. Whereas in our human fetal study showed, in early weeks, they were oval in serosa, oval, irregular and elongated in between the muscle layers and scattered neurons and absence of ganglia in submucosa. As the gestational age progressed, they were oval and elongated in serosa, oval, irregular and elongated in between the muscle layers and oval and elongated in submucosa.

Shape of enteric neurons are round, oval, pyramidal, fusiform and stellate in myenteric plexus and round, oval and elliptical in the submucosal plexus in rats. Guinea-pigs study showed elliptical and round neurons, whereas cattle study showed round, oval, elongated and irregular neurons in myenteric plexus. Our study showed round, oval, pyramidal and flat enteric neurons in the early weeks and predominantly round as the gestational age progresses. The shape of neurons observed in this human fetal study was similar to the animal studies.

In their study, Kim HK et al. found 90.77 ± 36.07 μm distance in between the enteric ganglia in thirteen-week human fetuses, whereas in our study in 10 to >38 weeks human fetuses distance between the ganglia was less in serosa in all weeks but it was closer to the observation of Kim HK et al. in between the muscle layers (96.1 ± 18.09 μm) and submucosa (88.3 ± 36.5 μm) in >38 weeks fetuses (Table 2). In our study, distance between the enteric ganglia in serosa was 15.6 μm in the early weeks (14–18 weeks). As the gestational age progresses, the distance decreased in 18–26 weeks and increased in 26–38 weeks. In between the muscle layers, it was 15.9 ± 6.4 μm in early weeks (10–14 weeks), decreased in 18–26 weeks and increased in 26->38 weeks (p = 0.013). In submucosa, it was 43.7 μm in early weeks (14–18 weeks) and fluctuated as the age progressed (p = 0.003). It was observed that as the gestational age progresses the distance between ganglia increases.

Human study showed 174.5 ± 93/100 mm myenteric ganglia and 123 ± 98.50/100 mm submucosal ganglia and animal studies showed 19.21 ± 0.99/mm2, 25.54 ± 3.03/mm2 and 6.48 ± 0.33/mm2 enteric ganglia in histochemical techniques (Giemsa stain, NADH-diaphorase and NADPH-diaphorase) and 51 ± 5 myenteric and 31 ± 2 submucosal ganglia per circumferential tissue section in immunohistochemistry. Whereas, in our study in 10 to > 38 weeks human fetuses in 1.58 μm2 area of colon, number of ganglia was observed more (Table 3) in comparison to the given human and animal studies. In our study, in serosa number of enteric ganglia was decreased in early weeks, increased in midweeks and decreased in late weeks, in between the muscle layers they were increased in early and midweeks and decreased in late weeks whereas in submucosa (p = 0.01) they were decreased in early weeks and increased in mid and late weeks. Our study showed that there is fluctuation in the number of enteric ganglia as the gestational age progresses but age-related studies described that there are empty ganglia with less number of neurons, and studies in gastrointestinal diseases described that there is decrease in the number of ganglia.

Animal studies showed that enteric neurons are 11.78 ± 2.89/mm2 in mesenteric border, 12.06 ± 2.69/mm2 in intermediate part and 10.67 ± 1.89/mm2 in antimesenteric border of jejunum in rats and 1271 ± 227.54 neurons/6.64 mm2, 530 ± 92.97 neurons/6.64 mm2, and 417 ± 34.42 neurons/6.64 mm2 in different stains in rats and 1600 neurons/mm2 in guinea pigs. Whereas, our study in human fetuses showed more number of the enteric neurons counted in 254.34 μm2 area of colon in 10 to >38 gestational weeks (Table 4) as compared to the animal studies. In our study, in serosa it was 35/254.34 μm2 in 14–18 weeks, decreased in 18–22 weeks, increased in 18–30 weeks (p = 0.00), in between the muscle layers it was 11 ± 1.58/254.34 μm2 in 10–14 weeks, increased in 14–18 weeks, decreased in 18–26 weeks, increased in 26–30 weeks and decreased in 30–38 week whereas in submucosa (p = 0.01) it was 4.5 ± 1.19/254.34 μm2 in 10–14 weeks then it fluctuated as the age progresses. Our study observed that there is fluctuation in the number of enteric neurons as the gestational age progresses but studies in adult animals and humans described that there will be decreases in number of enteric neurons as age increases.

Outcomes of this study described the arrangement, migration and shape of enteric neurons and ganglia and distance between the enteric ganglia in human fetal colon to give a standard view of morpho-histology of enteric neurons and ganglia in human during development and it can be further studied in large sample groups.

Conclusion

As the gestational age progresses, various shapes of scattered enteric neurons arrange in various forms of ganglia. These at first lie in serosa, then penetrate the muscle layers to form the myenteric plexus and finally migrate into the submucosa to form the submucosal plexus in the colon with variation in the number of enteric neurons, ganglia and distance between the ganglia.

Recommendations

The knowledge regarding the morphology of fetal enteric neurons and ganglia of colon may provide information to clinicians regarding their role in the peristalsis and to understand the associated gut abnormalities.

Source of funding

This study was supported by the (DST), India by providing India Science and Research Fellowship (ISRF) with grant number DO/CCSTDS/197/2019 and the (UGC), Nepal by providing a partial Ph.D. fellowship/2021.

Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

Ethical approval was taken from the Institutional Review Committee of Manipal College of Medical Sciences, Nepal with registration code MEMG/IRC/GA (i) on 1 March 2017 and from the Institutional Ethics Committee (IEC) of Kasturba Medical College and Kasturba Hospital (KMC & KH), India with registration number IEC: 192/2020 on 11 March 2020.

Authors contributions

CB conceived and designed the study, conducted research, provided research material, collected and organized data, wrote an initial draft of the article. PPP, AG analyzed and interpreted data. SGK wrote the final drafts of the manuscript and critically reviewed it, and provided logistic support. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.