Intramucosal nerve cells in human small intestine

Journal of the Autonomic Nerc,ous System, 44 (1993) 129-136

129

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00

JANS 01414

Intramucosal nerve cells in human small intestine Shengyun Fang, Ruiwei Wu and James Christensen Department of Histology and Embryology, Anhui Medical University, Hefei, China, and Diuision of Gastroenterology, Department of lnternal Medicine, University of lowa College of Medicine, Iowa City, Iowa, USA (Received 23 November 1992) (Revision received 12 February 1993) (Accepted 12 February 1993)

Key words: Neuron; Histochemistry; Morphometry; Mucosa; Small Intestine; Human Abstract We investigated the intramucosal nerve cells of the h u m a n small intestine with histochemical m e t h o d s to demonstrate nicotinamide adenine dinucleotide diaphorase and acetylcholinesterase and with morphometry. Intramucosal neurons appeared as solitary cells or in small groups, especially in the ileum. Most intramucosal nerve cell bodies were round or oval; some were flat or spindle-shaped. They mostly lay close to the muscularis mucosae, but some were located within the muscularis mucosae and others were some distance away from it. The processes of some mucosal neurons projected towards the submucosa. Most mucosal nerve cells showed acetylcholinesterase activity. The frequency distribution of nerve cell profile areas in the intramucosal cells in the d u o d e n u m differed from that of cells in the ileum ( P < 0.001). There were more large mucosal nerve cells in the mucosa of the d u o d e n u m than in the ileum. T h e r e was no significant difference between the frequency distributions of cell profile areas of cells of the mucosa and cells of Meissner's and Henle's plexuses in the same region. We conclude that intramucosal nerve cells, similar to those of the submucosal plexus, exist in the h u m a n small intestine. The size of intramucosal nerve cell profiles differs between the d u o d e n u m and ileum. This is consistent with their possible different functions.

Introduction

The enteric nervous system consists of five laminar intramural plexuses. The myenteric and submucosal plexuses are the two major ganglionated plexuses. The subserosal and deep muscular plexuses contain a few ganglionic neurons. According to the conventional view, the mucosal plexus is a mere extension of the submucosal plexus and does not contain any nerve cell bodies [12]. But intramucosal nerve cells do exist Correspondence to: J. Christensen, D e p a r t m e n t of Internal Medicine, Division of Gastroenterology-Hepatology, University of Iowa Hospitals, Iowa City, IA 52242, USA.

in the rat gut [19]. Studies of the innervation of the mucosa of the gut indicate that processes from the myenteric plexus and nerves of extrinsic origin also participate in the constitution of the mucosal plexus [4,18]. In man, occasional nerve cell bodies exist in the neuroendocrine complex in the lamina propria of the appendix [20]. Neuron specific enolase-immunoreactive cells in the lamina propria of human gut have been reported, but it was difficult to tell whether these were neurons or endocrine cells [1]. In this study, the intramucosal nerve cells of the human small intestine were studied by means of conventional histological and histochemical techniques.

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Materials and Methods

Tissue S p e c i m e n s of h u m a n small i n t e s t i n e (initial p a r t o f d u o d e n u m n = 5, distal p a r t of d u o d e n u m n = 2, p r o x i m a l j e j u n u m n = 1, distal i l e u m n = 4) w e r e o b t a i n e d fresh (within h a l f an h o u r of o p e r ation) from s e g m e n t s o f i n t e s t i n e r e s e c t e d for c a r c i n o m a (from 12 p a t i e n t s o f e i t h e r sex, m e a n age 50 years, r a n g e 3 6 - 6 6 years). In each case, t h r e e full-thickness s a m p l e s w e r e t a k e n f r o m a m a c r o s c o p i c a l l y n o r m a l a r e a at least 5 cm away from t h e t u m o r margin. O n e of t h e t h r e e s a m p l e s

was p r o c e s s e d for p a r a f f i n sections, to be s t a i n e d by h e m a t o x y l i n - e o s i n , in o r d e r to exclude n e o plastic infiltration or o t h e r a b n o r m a l i t i e s a n d to o b s e r v e the i n t r a m u c o s a l n e u r o n s . T h e o t h e r two s a m p l e s w e r e p r o c e s s e d for histochemistry.

Tissue processing Two tissue s a m p l e s in each case w e r e fixed in 4 % p a r a f o r m a l d e h y d e in 0.1 m o l / l p h o s p h a t e b u f f e r e d saline (PBS) at p H 7.3 for 1 8 - 2 0 h at 4°C. T h e s e w e r e rings cut from the gut a n d imm e r s e d in t h e fixative w i t h o u t stretch. A f t e r samples w e r e w a s h e d in PBS in 7 % sucrose for 12 h

Fig. 1. The shapes and locations of intramucosal neurons or neuron groups (arrows). (a) Duodenum. (b) Jejunum. (e, d) Ileum. A, Brunner's gland; above • is Intestinal gland; m, muscularis mucosae. (Hematoxylin and eosin, original magnification, × 400.) Bars in a-d show 50 ~m.

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Fig. 2. The shapes and locations of intramucosal neurons (arrows). (a, b) D u o d e n u m ; h contains a spindle-shaped neuron or a small ganglion (arrow) with an extension projecting towards the submucosa. (e) Jejunum. (d, e, f) Ileum; f shows a flattened neuron (arrow) closely associated with the muscularis mucosae; e shows an oval neuron with an extension projecting towards the submucosa; m, muscularis mucosae. A, Intestinal gland (to the right) (a, b). zx, Brunner's gland (above) (a, b). (Stained by N A D H - d i a p h o r a s e histochemistry; original magnification, ×400.) Bars in a-f show 50/zm.

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at 4°C, consecutive cryostat sections (10/xm thick) were cut perpendicular to the outer surface of the intestine.

Results Typical intramucosal nerve cell bodies were demonstrated on paraffin sections stained by hematoxylin-eosin. The cell bodies were large in size, and round or oval in shape (Fig. 1). In the detection of N A D H - d i a p h o r a s e on cryostat sections, the intramucosal nerve cells (which were similar to those of the myenteric and submucosal plexuses) were intensely positive [12], but the endocrine cells and the epithelial cells, including those of intestinal glands, were less strongly positive. No other cells except the intramucosal neurons in the lamina propria were intensely positive (Fig. 2). In the initial part of the duodenum, the Brunner's glands in the submucosa and lamina propria near the muscularis mucosae were poorly visualized with the NADH-diaphorase histochemical stain (Fig. 2a, b). The shapes of the intramucosal nerve cell bodies were the same in the N A D H - d i a p h o r a s e stained sections as in the paraffin sections. Round, oval and spindle-shaped cells were visualized (Fig. 2). We believe that some mucosal nerve cells are flat (see Fig. 2D because their cell bodies could be observed in three or more serial sections (10 Izm thick each). That is, the diameters of these ceils are more than 3 0 / z m , and these cells are intensely positive for N A D H - d i a p h o r a s e as are other intramural ganglionic neurons. The intramucosal nerve cells in the duodenum were larger than those in the ileum, but fewer. There were about four nerve cells per m m 2 of outer surface area of the initial part of duodenum and 11 per m m 2 of outer surface area in the ileum.

Histochemistry The N A D H - d i a p h o r a s e histochemical method demonstrates all the enteric neurons [11]. We therefore used that method to seek mucosal neurons. The sections were incubated for 50 min at 37°C in a medium made up of 25 parts of a 0.5 m g / m l stock solution of Nitro blue tetrazolium in distilled water, 25 parts of 0.1 M PBS at p H 7.3, 50 parts of H 2 0 , and 0.5 m g / m l of the reduced form of N A D H . The sections were mounted in glycerinated gelatin [12]. After some sections were incubated in the above medium for about 30 min at 37°C, until the N A D H - d i a p h o r a s e positive mucosal nerve cells appeared, we used the 'direct coloring' method for demonstration of acetylcholinesterase (ACHE) [17]. Tetra-isopropyl pyrophosphoramide (isoO M P A ) was used to inhibit the activity of nonspecific cholinesterase.

Measurement of cell profile The largest profile area of each neuron from the adjacent sections was measured by using a graticule fitted into the eyepiece of the microscope [14]. The unit square was 16.83/zm 2. Neurons were measured according to the number of squares they occupied [14].

Stat&tical analyses Frequency distributions of cell profile areas were compared using the chi-square test.

TABLE I

Locations of mucosal neurons

Duodenum Ileum

Numb er

Number of

Locations

of cases

neurons counted

Lamina propria a

Associated with M b

number

%

number

%

number

%

7 4

118 200

21 19

17.8 9.5

59 157

50.0 78.5

38 24

32.2 12.0

neurons were in the lamina propria, not closely associated with the muscularis mucosae. b M, muscularis mucosa.

Within M

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Some nerve cells were very close to the muscularis mucosae (Fig. 2), and others lay either within or some distance away from the muscularis mucosae (Table I). The nerve cells most often appeared singly, but a few lay in small groups (Table II). By combining the N A D H - d i a p h o r a s e and A C h E histochemical staining, we found that the majority of the intramucosal nerve cells were AChE-positive (Fig. 3). The frequency distribution of nerve cell profile areas in the mucosa of the duodenum was remarkably different ( P < 0.001) from that of the ileum (Figs. 4 and 5). To compare intramucosal nerve cells with those of the submucosal plexus ganglia, the distribution of ganglia across the submucosa was first observed in the segments of the first part of the duodenum. Normalizing the thickness of the submucosa to 100%, the ganglia of the submucosa lay predominantly within 10% either of the mus-

Fig. 4. Frequency distributions of sizes of nerve cell bodies (cell profile area) in mucosa and submucosa of human duodenum. The areas of 86, 88 and 51 nerve cell bodies were measured in neurons of the mucosa, of Meissner's plexus, and of Henle's plexus respectively. There was no significant difference between the three areas in the distribution of mucosal neurons by size (chi-square test).

cularis mucosae or of the circular muscle, implying that the submucosal plexus can be divided into two layers, Meissner's plexus (closer to the mucosa), and Henle's plexus (closer to the circular muscle) [2,14] (Fig. 6). The distributions of submucosal plexus ganglia in the distal duodenum and in the ileum were similar to that of the initial part of the duodenum. It was difficult to construct a histogram of the locations of submucous ganglia in ileum and distal duodenum because of the plicae. Accordingly, when measuring the cell profile areas in the submucosa, those close to or near the muscularis mucosae were

TABLE I1 Distribution of mucosal neuron groups

Duodenum Ileum

Number of cases

Neuron or neuron groups counted

7 4

101 142

Neuron or neuron groups 1 neuron

2 neurons

3-5 neurons

number

%

number

%

number

%

83 123

82.2 86.6

14 13

13.9 9.2

4 6

4.0 4.2

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400

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150

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Fig. 5. Frequency distribution of sizes of nerve cell bodies (cell profile area) in mucosa and submucosa of human ileum. The areas of 82, 93 and 127 nerve cell bodies were measured in neurons of the mucosa, of Meissner's plexus, and of Henle's plexus respectively. There was no significant difference between the three areas in the distribution of mucosal neurons by size (chi-square test).

assigned to Meissner's plexus, and those close to or near the circular muscle were assigned to Henle's plexus. T h e r e were no statistically signifi-

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Fig. 6. Distribution of ganglia across the submucosa in the first part of the duodenum (n = 5). Abscissa shows the distance across the submucosa normalized to 100% with 0% representing the border with the muscularis mucosae and 100% the border with the circular muscle. This figure shows that the submucosal ganglia mainly lie near either the muscularis mucosae or the circular muscle.

cant differences between the frequency distributions of cell profile areas of the intramucosal neurons and those of Meissner's and Henle's plexuses in the same region (Figs. 4 and 5). Eighty-four percent (of 82 cells) of intramucosal nerve cell profiles had an area of 100-250 /xm 2 and the largest neurons had an area of 4 0 0 / x m 2 in the ileum. Seventy percent (of 86 cells) of mucosal nerve cell profiles had an area of 150-350 /xm 2 and the largest neurons had an area of 650 /xm 2 in the duodenum.

Discussion By using hematoxylin-eosin, N A D H - d i a p h o r ase, and A C h E histochemical staining, we found many nerve cells within the mucosa of the human small intestine. The innervation of the gut mucosa has been widely studied [3-10,16,21-23], but the intramucosal nerve cells have not yet been described, except for occasional neurons found in the neuroendocrine complex of the human appendix [20], and one report from rat ileum [19]. It is unknown whether the intramucosal nerve cells of the human small intestine participate in the constitution of a neuroendocrine complex. The characters of certain cells in the gut mucosa, such as the neuron specific enolase (NSE) immunoreactive cells [1] and the neuroendocrine-like cells [24], have not been fully explored, but they seem to have some characteristics of nerve cells. NSE immunoreactive cells were not demonstrated by A C h E histochemical staining [1], but A C h E activity could be detected in most of the intramucosal nerve cells. The interstitial cells of Cajal have been reported in the mucosa and in the submucous and myenteric plexuses [10], but NADH-diaphorase histochemistry does not demonstrate them (10,12,14]. So, in detecting intramucosal neurons, N A D H - d i a p h o r a s e histochemistry seems reliable. This was supported by our observation that N A D H - d i a p h o r a s e positive cells are also A C h E positive. N A D H - d i a p h o r a s e histochemistry is considered as a method for selective demonstration of the myenteric and submucous plexus nerve cells [11]. In the present study, by using this method,

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we found that the intramucosal nerve cells are also intensely positive to the stain for NADH-diaphorase. No other cells (notably endocrine cells) were so reactive. This result indicates that the intramucosal nerve cells are also selectively demonstrated. The extensions of some intramucosal neurons appeared to project towards the submucosa (Fig. 2b, e). This structure suggests that mucosal neurons might be connected to the submucosal ganglia. The submucosal plexus of the human small intestine can be divided into Meissner's plexus and Henle's plexus as described in the colonic submucosal plexus of opossum [2] and human [14]. Although different populations of intramucosal neurons, based on the size of the nerve cell body, were identified in duodenum and ileum, no such differences were seen between intramucosal neurons and those of Meissner's and Henle's plexuses. Apart from their different anatomical locations, intramucosal neurons and Meissner's and Henle's plexus neurons appear to constitute a homogeneous population. In the human sigmoid colon, Hoyle and Burnstock [15] observed that the frequency distribution of the cell body areas of galanin-containing nerve cells was similar for Henle's plexus as for Meissner's plexus, but these two plexuses seemed to contain different populations of neurons based on neuronal size when they were stained for NADH-diaphorase activity. This suggests that the size populations of neurons correlate with the neurotransmitter they contained. We have given what we believe to be the first description of intramucosal neurons in the human intestine. They resemble the submucosal plexus neurons. The existence of neurons in the mucosa has broad implications in the understanding of mucosal neurobiology.

Acknowledgement This work was supported in part by Research Grant A M 11242 from the National Institutes of Health.

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