Ageing and endocrine cells of human duodenum

Mechanisms of Ageing and Development 108 (1999) 39 – 48

Ageing and endocrine cells of human duodenum Olof Sandstro¨m, Magdy El-Salhy * Section for Gastroenterology and Hepatology, Department of Medicine, Uni6ersity Hospital, S-901 85 Umea˚, Sweden Received 7 October 1998; received in revised form 8 December 1998; accepted 14 December 1998

Abstract Motility and secretory disorders of the gastrointestinal tract and associated glands increase with ageing. The duodenum contains several peptide/amine producing cells that play an important role in regulating gastrointestinal motility and secretion. The present study was performed to elucidate changes in these cells that may have arisen as a result of ageing. A total of four age groups of subjects, aged 1 – 2, 20 – 29, 40 – 49 and 60 – 69 years were studied. The various endocrine cell types were identified by immunohistochemistry and quantified by computerized image analysis, and two parameters were determined; the number of cells/mm3 epithelial cells and the cell secretory index (CSI), which indicates the immunoreactive secretory granule content of the endocrine cells. Chromogranin A- and serotonin-immunoreactive (IR) cells were fewer in 1–2-year-olds than in 20 – 29-year-olds. Gastrin/CCK-IR cells were significantly more numerous in 1 – 2-year-olds and 60 – 69 years-olds than in 20 – 29-yearolds. Somatostatin-IR cells were more numerous in the 40 – 49-year-olds than in the 20 – 29 years-olds. The CSI was higher in chromogranin A-, gastric inhibitory polypeptide (GIP)-, somatostatin- and gastrin/CCK-IR cells in 1 – 2-year-olds than in 20 – 29-year-olds. There was no significant sex difference regarding the numbers and CSI of other endocrine cell types. This study established the absence of sex-related differences in all endocrine cell types investigated, regarding numbers and physiological activity. Age, on the other hand, was shown to be associated with changes in the numbers of CCK-, somatostatin- and serotoninIR, which may have some bearing on the gastrointestinal disorders of the elderly. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

* Corresponding author. Tel.: +46-90-7853867; fax: + 46-90-143986. E-mail address: [email protected] (M. El-Salhy) 0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 9 8 ) 0 0 1 5 4 - 7

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Keywords: Ageing; Cholecystokinin; Computerised image analysis; Duodenum; Endocrine cells; Immunohistochemistry; Morphometry; Serotonin; Somatostatin

1. Introduction Motility and secretory disorders of the gastrointestinal tract and associated glands increase with ageing ( Hollis and Castell, 1974; Moore et al., 1983; Bannister et al., 1987; Poston et al., 1988a; Laugier et al., 1991; Weddman et al., 1991; Madsen, 1992). Constipation and faecal incontinence (Johanson et al., 1989; Szurszewski et al., 1989; Wald, 1990), slow gastric emptying of liquid (Moore et al., 1983), increased colonic transit time (Madsen, 1992), diminished contractility of the gallbladder (Poston et al., 1988a; Weddman et al., 1991) and decreased pancreatic exocrine secretion (Laugier et al., 1991) have all been reported in elderly humans. Since the endocrine cells of the duodenum contain several peptides and amines which play an important role in regulating gastrointestinal motility and secretion (O%Doriso, 1987; Allescher and Ahmed, 1990; Ekblad et al., 1990; Rangachari, 1990), it is conceivable that changes in these cells may be related to the gut disorders described in the elderly. The present study was therefore undertaken to examine the duodenal endocrine cells from the very young to the old, using immunohistochemical and computerized image analysis methods.

2. Materials and methods

2.1. Subjects The 50 subjects included in this investigation were divided into four age groups. Group 1, aged 1 – 2 years (5 females, 5 males) were of short stature or had chronic non-specific diarrhoea. Group 2, aged 20–29 years (8 females, 5 males), and group 3, aged 40 – 49 years (9 females, 4 males), were all healthy volunteers free from gastrointestinal complaints. Group 4, aged 60–69 years (9 females and 5 males) had undergone routine standard gastroduodenal endoscopic examination because of suspected gastrointestinal bleeding, for which haemorrhoids were identified as the source. The location of these age groups on the survival curve (Official Statistics of Sweden, 1997) is illustrated in Fig. 1. The investigation was approved by the local committee on ethics at the University of Umea˚.

2.2. Gastroduodenal endoscopy and histopathology After an overnight fast, all subjects underwent a gastroduodenal endoscopy. Tissue samples (two to four) were obtained from the descending part of duodenum, distal to the papilla of Vateri, and were fixed overnight in 4% buffered formaldehyde. Following the usual procedure for wax embedding, sections were cut at 5 mm

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Fig. 1. Survival curves of women and men living in Sweden. The subjects’ ages in the present study are marked as hatched areas. Data obtained from Official Statistics of Sweden, (1997).

and stained with haematoxylin-eosin and van Gieson stain for histopathological examination.

2.3. Immunohistochemistry Sections were also immunostained with the avidin-biotin complex (ABC) method (DAKO A/S, Glostrup, Denmark) as described earlier in detail (El-Salhy et al., 1993). A detailed account of the antisera used is presented in Table 1. Specificity controls were the same as those described previously (El-Salhy et al., 1993). Table 1 Detailed account of the antisera used Antibody against

Working dilution

Code no.

Source

N-terminal of purified chromogranin A (M)a Porcine secretin (P)b

1:1500

M869

1:1000

R-787502

Bovine gastric inhibitory peptide (GIP), (P) Synthetic human gastrin-17 (P)c Synthetic human somatostatin (P) Porcine pancreatic glucagond (P) Porcine motilin Serotonin (M)

1:1600

R-786403-B2

Dakopatts, Glostrup, Denmark Eurodiagnostika, Malmo¨, Sweden Eurodiagnostika

R-783511 A566 B-31 R-842206 M758

Eurodiagnostika Dakopatts Eurodiagnostika Eurodiagnostika Dakopatts

a

1:10 000 1:1600 1:2500 1:5000 1:1600

(M), monoclonal antibodies. (P), polyclonal antibodies. c Specific for CCK/gastrin C-terminus. d Specific for glucagon N-terminus and cross-reacts with both pancreatic and enteroglucagon. b

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2.4. Computerized image analysis Morphometric measurements were performed with the Quantimet 500 MC image analysis system (Leica, Cambridge, England), linked to an Olympus microscope type BX50. The parameters measured were: the number of cells, the area covered by immunoreactive granules, and the area covered by epithelial cells. The program used in this system is QWIN (Leica’s Windows-based image analysis tool kit). The measurements were standardized by setting up a routine using the interactive programming system ‘QUIPS’ as described in detail earlier (El-Salhy et al., 1997). The sections were coded, sex and age being unknown to the examiner. A × 20 objective and a screen frame representing a tissue area of 0.047 mm2 were used. A total of 40 randomly chosen fields were quantified: 20 in the villi and 20 in the crypts, from five sections at least 50 mm apart. The cell secretory index (CSI) was calculated as previously described (El-Salhy et al., 1997). To measure the number of villi/mm and crypts/mm baseline, a line was drawn and aligned with the base of the crypts in three perpendicularly cut sections from each individual, using interactive measurements in the manual menu and a × 4 objective. The height of 20 villi in three sections was measured using the interactive measurement menu and distance parameter.

2.5. Statistical analysis The 1 – 2, 40 – 49 and 60 – 69-year-old groups were compared with the 20–29-yearold group, using the Mann – Whitney non-parametric U-test. P-values less than 0.05 were considered significant.

3. Results

3.1. Gastroduodenal endoscopy and histopathology In all the subjects included in this study, stomach and duodenum were macroscopically normal. Histopathological examination of the descending duodenum revealed a normal structure in all subjects.

3.2. Immunohistochemistry Chromogranin A-, secretin-, gastric inhibitory polypeptide (GIP) -, cholecystokinin (CCK)/gastrin- (Fig. 2), somatostatin- and serotonin-immunoreactive (IR) cells were detected in all age groups. A few motilin-IR cells were encountered, but no glucagon-IR cells. As described previously (El-Salhy et al., 1994), chromogranin A-, CCK/gastrin- and serotonin-IR cells were found in both villi and crypts. Secretin-IR cells were found mostly in the villi, while GIP- and somatostatin-IR cells occurred predominantly in the crypts. These cells were basket- or flask-shaped.

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Fig. 2. Cholecystokinin/gastrin C-terminus-immunoreactive cells in the crypts of duodenum of a 24-year-old subject (A) and of a 67-year-old subject (B). ×250.

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3.3. Computerized image analysis The results of the morphometric measurements are presented in Fig. 3. The chromogranin A- and serotonin-IR cells were fewer in the 1–2-year-old than in the 20– 29-year-old group. Gastrin/CCK-IR cells were significantly more numerous in the 1 – 2-year-old and 60 – 69-year-old than in 20–29-year-old group. SomatostatinIR cells were more numerous in the 40–49-year-old than in the 20–29 years-old group. The CSI of chromogranin A-, GIP-, somatostatin- and gastrin/CCK-IR cells was higher in the 1 – 2-year-old than the 20–29 year-old group. There was no significant difference between men and women regarding the number of cells or their CSI (Table 2). The numbers of villi/mm baseline in the 1–2, 20–29, 40–49 and 60–69-year-old groups were 5.8, 7.3, 5.8 and 5.7, respectively. The corresponding figures for crypts were 23.9, 44.6, 36 and 40, respectively. The lengths of the villi in these groups were 340, 355, 405, and 381 mm. Crypts were fewer (P= 0.04) in the 1–2-year-old than Table 2 Numbers cells per mm3 of epithelial cells and cell secretory index (mm3 per cell) of various endocrine cell types in women and mena Number of endocrine cells

Cell secretory index

Women

Men

Women

Men

3791894514 1070491400 656529 7382

31020 93918 9664 91478 51449 96715

1459 10 131 911 145 9 11

126 9 12 105 98 129 9 13

Secretin Villous Crypts

46519428 –

6392 9604 –

114 99 –

123 913 –

GIP Villous Crypts

– 107519 1610

– 6666 9 1180

– 153 912

– 125 9 12

Gastrin 9 CCK Total Villous Crypts

151529 1496 82609 657 259609 4388

11509 9 1565 7186 9 1161 17892 9 2131

150 9 12 168 918 141 9 13

147 9 15 166 9 23 142 9 17

– 13039 1849

– 14021 9 1915

– 121 9 6

– 143 9 17

3637294966 1070593628 3588495697

45813 9 26828 6820 9 1010 32993 9 6358

129 910 127 9 9 120 9 11

142 9 17 136 9 21 157 9 21

Chromogranin A Total Villous Crypts

Somatostatin Villous Crypts Serotonin Total Villous Crypts a

The values were pooled from different age groups.

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Fig. 3. Number and cell secretory index of various endocrine cell types in different age groups. *P B 0.05, **PB 0.01, ***PB 0.001.

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in the 20 – 29-year-old group. There was no statistical difference between the 40–49 and 60 – 69-year-old groups and the 20–29-year-old group regarding the number of crypts/mm base line. Nor was there any statistical difference in the number of villi/mm baseline in any of the age groups, or regarding the villous length.

4. Discussion In the present investigation, the endocrine cells were studied by two morphometric parameters, namely their number and their CSI. The former gives information about the anatomical peptide/amine-producing units, while the latter indicates the immunoreactive secretory granule content of these anatomical units (El-Salhy et al., 1997). CSI constitutes a summation of peptide/amine synthesis and peptide/amine release. In order to determine the exact nature of the changes in this index, the cellular content of the complementary mRNA as a parameter of peptide/amine synthesis is required (El-Salhy et al., 1997). It is well known, however, that endocrine cells with a high physiological activity (high rates of peptide synthesis and release) are degranulated and have few cytoplasmic secretory granules (ElSalhy et al., 1997). It was assumed in the present investigation, therefore, that an increase in the CSI represents a low physiological activity in the endocrine cells, and vice versa. It has been reported that the chromogranin immunoreactivity of peptide-producing cells varies among species, among gastrointestinal segments and even in one and the same endocrine cell type (Ceting et al., 1989). It would, therefore, seem that chromogranin-IR cells are not representative of the whole population of endocrine cells, which might in turn explain why in the present study the chromogranin A-IR cells were less numerous than all endocrine cells combined. In children, chromogranin A-IR cells were fewer than in young adults, due to a decrease in the number of these cells in both the villi and crypts. On the other hand, the CSI of these cells was higher in children than in young adults, possibly indicating low physiological activity of these cells. The antiserum against gastrin used in this study is directed against the gastrin/ CCK C-terminus, cross-reacting with both gastrin and CCK. It is well known, however, that human duodenum contains few gastrin cells and is the main location for CCK cells (Sjo¨lund et al., 1983). Thus, the cells identified by this antiserum are mostly CCK cells. In children, the total number of CCK cells was higher than young adults. Since the increase in cell number occurred in crypts, the increase in the total number may due to the reduced number of crypts observed here in children rather than being a real increase. In the 60–69-year-old group, CCK-IR cells were more numerous than in young adults, which is consistent with an earlier observation in guinea-pigs that the concentration of CCK in duodenal mucosa increased with age (Poston et al., 1988b). This increase may compensate for a reduction in CCK receptors in pancreas and gall bladder. This assumption gains support from the previous observation that CCK receptors were reduced in pancreas and gall bladder muscle cells in old guinea-pigs (Poston et al., 1988a,b).

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Somatostatin inhibits the secretion of acid, pepsinogen and gastrin as well as endocrine cell secretion (Chiba and Yamada, 1994). The increase in somatostatinIR cell number observed in middle-aged individuals may compensate for a decrease in the receptors in target tissues. In children, serotonin-IR cells were fewer than in young adults. Serotonin is known to stimulate pyloric contraction and motility in small intestine and colon, as well as accelerating transit through both small and large intestines (Lidberg, 1985; Tally, 1992; Oosterbosch et al., 1993; Goard et al., 1994; von der Ohe et al., 1994). The physiological implication of the paucity of serotonin cells observed in children is rather unclear. The CSI of all endocrine cell types (except serotonin-IR cells) was higher than that of the young adults, possibly reflecting low physiological activities of these cells. It is noteworthy, however, that the children group suffered from short stature or chronic non-specific diarrhoea. The possibility that this diarrhoea affects the endocrine cells in the duodenum can not be rolled out. The present study has shown that there is no sex-related difference in any of the duodenal endocrine cells as regards the numbers and physiological activities. It also showed that CCK-, somatostatin- and serotonin-IR cell numbers change with age, an observation which may have some bearing on the gastrointestinal disorders observed in the elderly.

Acknowledgements This study was supported by grants from the Medical Faculty, Umea˚ University.

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