Aging has been defined as the progressive accumulation of changes with time which are responsible for an increasing susceptibility to diseases and death, the final event of age. The different tissues do not age at the same rate as the growth of many organs ceases with adolescence, the mucosa of gastrointestinal tract is maintained by continuous cell turn over1.
The glands of gastric mucosa are simple branched tubular structures consisting of a complex mixture of diverse epithelial cell types that undergo cell renewal2.
Stem cells, located in the isthmus of the glands, follow specific programs of proliferation and differentiation. They migrate either upward to the pit, forming surface mucous cells or downward to the base forming chief cells, parietal cells and enteroendocrine cells3. Surface mucous cells secrete neutral mucin that protects the mucosa from the Hcl, chief cells secrete a variety of pepsinogens, parietal cells produce gastric acid and enteroendocrine cells help regulation of mucosal growth and secretory activity4.
In healthy adult, maintenance of gastric mucosa integrity depends on the balance between cell proliferation and apoptosis (programmed cell death)5. Any dysfunction of these processes may accelerate or diminish the growth rate resulting in either hyperplesia, hypertrophy or atrophy of the tissues6.
Aging is associated with increased mucosal cell proliferating activity in gastrointestinal tract. These changes may occur secondary to alteration in hormones and growth factors especially tyrosine kinase7, gastrin, bombesin8, epidermal growth factors and transforming growth factor9. Age related disruption in normal cell proliferative process is translated into altered function such as malnutrition, malabsorption of particular nutrients or greater incidence of gastrointestinal diseases especially neoplasia5.
Apoptosis plays a role in maintaining cell numbers in many proliferative tissues. It takes place in all regions of gastric mucosa and more prevalent in the superficial parts and toward the base of the gastric glands. It is involved in regulation of proliferation and removal of senescent cells from the gastric mucosa10,11.
One of the most consistent pathological observation in aged animals is the increased incidence of many types of malignancies including gastric and colorectal cancers. In elderly patients, the incidence of digestive cancers increases with peak incidence occurring in seventh decade12.
Since aging was found to be associated with increased incidence rate of malignancy, the possibility that it may render gastric mucosa more susceptible to carcinogenic transformation continues to be an area of intense interest and study. So this work was designed to evaluate the age related alteration in the histological structure of rat gastric mucosa to elucidate the potential mechanisms of carcinogenesis which may be involved during aging process.
MATERIALS AND METHODS
Fifteen male albino rats were used in this study. They were kept under good hygienic conditions, allowed free water and fed ad libitum. They were classified into three equal groups (5 animals each), group A (4 months old) group B (18 months old) and group C (24 months old).
At the time of sacrifice, all the animals were anesthetized by ether inhalation. The stomach were dissected out and opened along the greater curvature. The gastric fundus was excised and prepared for light and electron microscope evaluation.
For light microscope examination, specimens were fixed in 10% formol saline and processed to prepare 5 μm thick serial paraffin sections. They were stained with haematoxylin and eosin (H & E), PAS, Masson's trichrome stains13 and immunohistochemical staining for detection of BCL-2 protein (anti-apoptotic marker)14.
Immunohistochemical reaction was carried using avidin biotin peroxidase system. The primary antibody used was a rabbit polyclonal antibody which were delivered from Sigma Laboratories (Code No. 00114386). Universal kit used avidin biotin peroxidase system produced by Nova Castra Laboratories Ltd, Uk. The same method was applied to prepare negative control sections but the primary antibody was not added. This immunohistochemical technique was carried out in Department of Pathology, Faculty of Medicine. Ain Shams University.
For electron microscope examination, small pieces (1 mm3) from fundus of stomach were immediately fixed in 2.5% glutaraldhyde, buffered in 0.1 M sodium cacodylate at 4°C and post fixed in 1% osmium tetroxide. Then, they were dehydrated in ascending grades of alcohol and embedded in resin. Ultrathin sections were cut and picked up on copper grids and stained using uranyl acetate and lead citrate15. These specimens were examined and photographed using JEOL transmission electron microscope (JEM1010) in Histology Department, Faculty of Medicine, Zagazig University.
Quantitative morphometric measurements:
Area % of the positive BCL-2 immunoreactive cells lining the fundic glands were estimated by using “Leica Quin 500C” image analyzer computer system (Leica Imaging system Ltd., Cambridge, England). The standard measuring frame of a standard area equal to 7286, 78 μm2. Measurements were done within 10 non-over lapping fields for each animal at X 400 magnification. The same method was applied for measuring the area % connective tissue in Masson's trichrome stain but the measuring frame was of area 118476-6 and an objective lens 10 (X100). The morphometric measurement was carried out in Histology Department, Faculty of Medicine, Cairo University.
The data obtained from image analyzer were analyzed on an IBM. PC, using the statistical soft ware “Statistical for windows”. The parameters were tested using student “t” test and the results were considered significant when P <0.0516.
Light microscope examination of the gastric fundus of adult male rat (4 months old) revealed mucosa, submucosa and muscularis externa. The fundic mucosa contained simple branched tubular glands lines by mucous neck cells, parietal cells and chief cells lamina propria was observed (Fig. 1). Parietal cells appeared as large cells with central pale nuclei, prominent nucleoli and eosinophilic cytoplasm. Small chief cells with basal nuclei and apical acidophilic cytoplasm were also seen. (Fig. 2). Collagen fibers were seen in the thin lamina propria and between the basal part of the gastric glands (Fig. 3). Strong positive PAS reaction appeared in the surface mucous cells and mucous neck cells (Fig. 4). Brown positive BCL-2 immunoreaction were seen in few fundic gland cells (Fig. 5).
Ultrastructurally, the fundic glands of rats (4 months old) showed parietal cells appeared with mitochondria and wide intracellular canaliculi containing many microvilli (Fig. 6). The chief cells showed central nuclei with prominent nucleoli, mitochondria, basal rough endoplasmic reticulum and apical large electron dense granules (Fig. 7). Enteroendocrine cells were seen with apical large euchromatic nuclei and small electron dense granules (Fig. 8).
Light microscopic examination of fundic mucosa of rats (18 months old) showed irregular fundic glands widely separated by many connective tissue and blood vessels. Some glands appeared atrophied and other were distorted (Fig. 9). Many collagen fibers were seen in between the fundic glands and in the lamina propria which contained congested blood vessels (Fig. 10). Strong PAS reaction was observed in the surface mucous cells and mucous neck cells (Fig. 11). Positive BCL-2 immunoreaction appeared in many fundic gland cells (Fig. 12). Ultrastracturally, the funcic glands revealed parietal cells with many mitochondria, narrow intracellular canaliculi containing few microvilli and many tubulovesicles (Fig. 13). Chief cells appeared with dilated rough endoplasmic reticulum, loss of their secretory granules and many apical vacuoles. Enteroendocrine cells contained nuclei with peripheral heterochromatin condensation, many basal vacuoles and loss of their granules (Figs. 14,15).
Light microscopic examination of gastric mucosa of rats (24 months old) showed disorganization of fundic glands which appeared as scattered cells separated by excessive connective tissue. Some parietal cells appeared with pale cytoplasm and dark nuclei (Fig. 16). Moreover, gastric ulcer was seen with atrophy of the surface epithelium, flattening of their nuclei and widening of glandular pits. Some glands appeared dilated, distorted or atrophied separated by wide spaces (Fig. 17). Excessive collagen fibers were seen in the thick lamina propria between the atrophic glands (Fig. 18) and at the base of the ulcer (Fig. 19).
Decreased PAS reaction was observed in the mucous neck cells (Fig. 20). Furthermore, many positive BCL-2 immunoreactive cells were seen (Fig. 21). Ultrastructurally, the fundic gland of rats (24 months old) appeared disorganized with scattered cells separated by blood vessels and fibroblasts (Fig. 22). Parietal cells contained irregular nuclei with peripheral chromatin condensation, distorted intra cellular canaliculi, microvilli and tubulovesicles (Figs. 22,23). Chief cells appeared with small pyknotic nuclei, many vacuoles and few apical electron dense granules (Fig. 24). Enteroendocrine cells contained irregular nuclei with condenced peripheral chromatin and many vacuoles (Fig. 25).
The area % of collagen fibers in rat gastric mucosa was significantly increased in group B as compared to group A and in group C as compared to group B (Table 1, Fig. 26).
Also, the area % of positive BCL-2 immunoreactive cells significantly increased in fundic glands of group B as compared to group A and in group C as compared to group B (Table 2, Fig. 27).
The increase in the aging population has led to a growing interest in achieving a better understanding of the aging process and of diseases that are predominantly expressed during advancing age such as malignancy1.
In the present study, examination of fundic mucosa of rats (18 months old) revealed irregular fundic glands widely separated by many fibrous tissue and thin walled blood vessels. Some glands appeared atrophied and others distorted. Abundant collagen fibers were seen between the atrophic glands and around the congested blood vessels. This results were confirmed by significant increase in the mean area percent of the collagen fibers between the fundic glands as compared to that of adult rats (4 months old rats). Ultrastructurally, degenerative changes were seen in the fundic glands cells. Parietal cells appeared inactive as they contained narrow intracellular canaliculi with few microvilli. Some chief cells showed dilated endoplasmic reticulum with absence of their secretory granules. Some enteroendocrine cells showed absence of their secretory granules with appearance of many basal vacuoles. These observations were in accordance with the results obtained by Hollander et al.17 and Majumdar et al.18. They reported that aging is associated with atrophy of fundic mucosal glands and marked deposition of connective tissue in the lamina propria especially between the base of the glands. Furthermore, the fundic mucosal cells showed evidence of decrease secretory activity with decrease number of intracytoplasmic secretory granules.
Epidemiological studies reported an increase prevalence of atrophic gastritis in elderly patient. A series of studies has focused on the long term effects of Helicobacter pylori infection and its role in the development of atrophic gastritis that occur with aging19. As a result of atrophic gastritis, inflammatory cytokines such as IL IB and TNF-α inhibit parietal cells and lead to decline in fundic acid secretion20. Hypochlorhydria may lead to bacterial over growth21 and malabsorption of food bound cobalamin22, vitamin B12, calcium and Ferric iron23.
Furthermore, Lenaz et al.24 and Marmol et al.25 suggested that chronic infection may lead to chronic inflammation with increased production of reactive oxygen species which are generated by mitochondria. There is an increasing evidence that reactive oxygen species may lead to a heightened sensitivity of the aging stomach to proinflammatory stimuli, damage of DNA, mutation and carcenogenesis26. Chronic inflammation in fundic mucosa may also affect expression of fundic satiety inducible peptides such as leptin and ghrelin which play a role in the physiopathology of anorexia in elderly patients27,28.
On the other hand, the present study revealed that the fundic mucosa of rats (24 months old) showed marked disorganization of fundic glands which appeared as scattered cells separated by excessive connective tissue. Some parietal cells appeared with pale cytoplasm and dark nuclei. Fundic ulcer was also seen with atrophy of its surface epithelium, flattening of their nuclei and widening of the gland pits. Some fundic glands appeared dilated, distorted and atrophied. Also decreased mucin appeared in mucous neck cells. Excessive collagen fibers were observed in thick lamina propria and between the atrophic glands. These results were confirmed by significant increase in the area % of collagen fibers in fundic mucosa of rat of group C as compared to rats of group B. These findings were correlated with the work carried out by Fligiel et al.29 who declared that increased incidence of fundic ulcer observed in the aged could partly attributed to increased susptibility of the mucosa to various damaging agents together with impediment of the repair process. Wyle et al.30 and Fujiwara et al.31 reported that the prevalence of fundic and duodenal ulcers is high in old patients with Helicobacter pylori infection. Although the precise mechanism is not fully understood, the cytotoxic Vac A produced by Helicobacter pylori may inhibit proliferation and migration of epithelial cells. It also interferes with epidermal growth factor binding to its receptors thereby inhibiting proliferation and migration of epithelial cells during ulcer healing.
Ultrastructurally, the present study revealed that the fundic mucosa of rats (24 months) showed excessive degenerative changes in parietal cells, chief cells and enteroendocrine cells. They have small indented nuclei with marked peripheral heterochromatin condensation. Parietal cells showed distorted intracellular canaliculi. The chief cells and enteroendocrine cells appeared with dilated rough endoplasmic reticulum, many vacuoles and few secretory granules. These results were in accordance with that of Jaszewski et al.32 who reported that atrophic gastritis and its associated intestinal metaplasia were age- related lesions and were considered to be precancerous. Majumdar1 and Salles12 suggested that the incidence of digestive cancers increased in elderly patients. Carcinogenesis results from the accumulation of mutations during progression from normal epithelium to carcinoma, long term exposure to cancer-causing agents and high incidence of mutation in several tumor suppressor genes, especially APC (adenomatous polyposis coli) and P53 in the fundic mucosa of older patients.
A striking findings of this study was a significant increase in the area % of positive BCL2 immunoreactive cells in the fundic glands of rats of group B as compared to rats of group A and in rats of group C as compared to that of group B. This indicated that the aging is associated with attenuation of apoptosis. This data agreed with Komatsuk et al.33 who reported that aberrant BCL-2 expression is a frequent occurance in chronic gastritis, fundic epithelial dysplesia and fundic cancer. Inhibition of apoptosis through BCL-2 expression appears to be associated with promotion of fundic adenocarcinoma. In addition, Xiao et al.34 reported that the aging is accompanied by a concomitant stimulation (200%) of anti-apoptotic BCL-XL and reduction (75%) in proapoptotic bck levels. This indicated that aging enhances proliferation but attenuates apoptosis. These changes may be responsible for age related rise in malignancy. Moreover, Majumdar and Jaszewski35 have found that the age-related rise in fundic and colonic mucosal proliferation was also accompanied by reduction in apoptosis. This finding raises the possibility whether aberrant survival of a group of cells could increase the susceptibility of the gastrointestinal mucosa to certain carcinogens or tumor promoters that initiate the process of carcinogenesis of gastrointestinal tract.
Aging is associated with dramatic progressive changes in the histological structure of the fundic mucosa in the form of distorsion, disorganization, fibrosis and ulceration of fundic glands. Also parietal cells, chief cells and enteroendocrine cells showed degenerative changes. At the same time, these histological alterations were accompanied by attenuation of apoptosis. So, aging may predispose the stomach to malignant transformation.
Majumdar AP. (2003):
Regulation of gastrointestinal mucosal growth during aging
. J.Physiol.Pharmacol.;54(Suppl 4):143-154.
Karam SM and Leblond CP. (1992):
Identifying and counting epithelial cell types in the “corpus” of the mouse stomach. Anat. Rec. Feb;232(2):231-246.
Karam SM and Leblond CP. (1993):
Dynamics of epithelial cells in the corpus of the mouse stomach. I. Identification of proliferative cell types and pinpointing of the stem cell. Anat. Rec. Jun; 236(2):259-279.
Lipkin M. (1987):
Proliferation and differentiation of normal and diseased gastrointestinal cells. In: Johnson LR, editor. Physiology of the gastrointestinal tract. 2nd
ed.: Raven Press, New York. p. 255-284.
Metel'skii ST. (2004):
Vliianie vozrasta na vsasyvanie i membrannoe pishchevarenie v tonkoi kishke krys. [Effect of age on absorption and membrane digestion in the rat small intestine]. Ross.Fiziol.Zh.Im.I.M.Sechenova. Jan;90(1):98-105.
Konturek PC, Brzozowski T, Konturek SJ, Pajdo R, Konturek JE, Kwiecien S, Taut A and Hahn EG. (1999):
Apoptosis in gastric mucosa with stress-induced gastric ulcers. J. Physiol. Pharmacol. Jun; 50(2):211-225.
Yarden Y. (1985):
Growth factor receptor tyrosine kinases. Ann. Rev. Biochem.;54:897-930.
Majumdar AP. (1990):
Growth and maturation of the gastric mucosa. In: Morisset J, Solomon S, editors. Growth of the gastrointestinal tract: Gastrointestinal hormones and growth factor: CRC Press, New York. p. 119-130.
Turner JR, Liu L, Fligiel SE, Jaszewski R and Majumdar AP. (2000): Aging
alters gastric mucosal responses to epidermal growth factor and transforming growth factor-alpha. Am. J. Physiol.Gastrointest. Liver Physiol. May;278(5):G805-G810.
Neu B, Herrmuth H, Ernst F, Vaupel W, Reindl W, Hutzler P, Atkinson MJ, Classen M and Schepp W. (2001):
Differential expression of CD95, Bcl-2 and Bax in rat gastric chief and parietal cells. Microsc.Res.Tech. Jun 1;53(5):377-388.
Tarnawski A, Pai R, Deng X, Ahluwalia A, Khomenko T, Tanigawa T, Akahoshi T, Sandor Z and Szabo S. (2007): Aging
gastropathy-novel mechanisms: Hypoxia, up-regulation of multifunctional phosphatase PTEN and proapoptotic factors. Gastroenterology;133(6):1938-1947.
Salles N. (2007):
Basic mechanisms of the aging
gastrointestinal tract. Dig. Dis.;25(2):112-117.
Bancroft JD and Gamble M. (2002):
Theory and practice of histological techniques. 5th
ed. Churchill Livingstone.
Kiernan JA. (2000):
Histological and histochemical methods: Theory and practice. 3rd
ed. Butterworth Heinemann: Oxford.
Glauert AM and Lewis PR. (1998):
Biological specimen preparation for transmission electron microscopy. 1st ed. Princeton University Press.
Armitage P and Berry G. (1994):
Statistical methods in medical research. 3rd
ed. Blackwell Science: Oxford, UK.
Hollander D, Tarnawski A, Stachura J and Gergely H. (1989):
Morphologic changes in gastric mucosa of aging rats
. Dig. Dis. Sci. Nov; 34(11):1692-1700.
Majumdar AP, Jasti S, Hatfield JS, Tureaud J and Fligiel SE. (1990):
Morphological and biochemical changes in gastric mucosa of aging rats
. Dig. Dis. Sci. Nov; 35(11):1364-1370.
Pilotto A and Salles N. (2002):
Helicobacter pylori infection in geriatrics. Helicobacter;7 Suppl 1:56-62.
Haruma K, Kamada T, Kawaguchi H, Okamoto S, Yoshihara M, Sumii K, Inoue M, Kishimoto S, Kajiyama G and Miyoshi A. (2000):
Effect of age and Helicobacter pylori infection on gastric acid secretion. J. Gastroenterol. Hepatol.;15:277-283.
Parlesak A, Klein B, Schecher K, Bode JC and Bode C. (2003):
Prevalence of small bowel bacterial overgrowth and its association with nutrition intake in nonhospitalized older adults. J. Am. Geriatr.Soc. Jun; 51(6):768-773.
Van Asselt DZ, Van den Broek WJ, Lamers CB, Corstens FH and Hoefnagels WH. (1996):
Free and protein-bound cobalamin absorption in healthy middle-aged and older subjects. J. Am. Geriatr. Soc. Aug;44(8):949-953.
Kaptan K, Beyan C, Ural AU, Cetin T, Avcu F, Gulsen M, Finci R and Yalcin A. (2000):
Helicobacter pylori—is it a novel causative agent in Vitamin B12 deficiency? Arch. Intern. Med. May 8;160(9):1349-1353.
Lenaz G, Bovina C, D'Aurelio M, Fato R, Formiggini G, Genova ML, Giuliano G, Merlo Pich M, Paolucci U, Parenti Castelli G and Ventura B. (2002):
Role of mitochondria in oxidative stress and aging
. Ann. N.Y. Acad. Sci. Apr;959:199-213.
Mármol F, Sánchez J, López D, Martínez N, Mitjavila MT and Puig Parellada P. (2009):
Oxidative stress, nitric oxide and prostaglandin E2 levels in the gastrointestinal tract of aging rats
. J. Pharm. Pharmacol.;61(2):201-206.
Teshima S, Kutsumi H, Kawahara T, Kishi K and Rokutan K. (2000):
Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1. Am. J. Physiol. Gastrointest. Liver Physiol. Dec;279(6):G1169-G1176.
Azuma T, Suto H, Ito Y, Ohtani M, Dojo M, Kuriyama M and Kato T. (2001):
Gastric leptin and Helicobacter pylori infection. Gut Sep; 49(3):324-329.
Zhao Z and Sakai T. (2008):
Characteristic features of ghrelin cells in the gastrointestinal tract and the regulation of stomach ghrelin expression and production. World J. Gastroenterol.; 14(41):6306-6311.
Fligiel SE, Relan NK, Dutta S, Tureaud J, Hatfield J and Majumdar AP. (1994): Aging
diminishes gastric mucosal regeneration: Relationship to tyrosine kinases. Lab. Invest. May;70(5):764-774.
Wyle FA, Chang KJ, Stachura J and Tarnawski A. (1993):
Helicobacter pylori cytotoxin and the healing of experimental gastric ulcers. Eur. J. Gastroenterol. Hepatol.;5(Suppl 3):S75-S79.
Fujiwara Y, Wyle F, Arakawa T, Domek MJ, Fukuda T, Kobayashi K and Tarnawski A. (1997):
Helicobacter pylori culture supernatant inhibits binding and proliferative response of human gastric cells to epidermal growth factor: Implications for H. pylori interference with ulcer healing? Digestion;58(3):299-303.
Jaszewski R, Ehrinpreis MN and Majumdar AP. (1999): Aging
and cancer of the stomach and colon. Front. Biosci. Mar. 15;4: D322-D328.
Komatsu K, Suzuki S, Ohara S, Asaki S, Toyota T and Suzuki H. (1996):
[Expression of Bcl-2 and Bax in human gastric cancer tissue]. Nippon Rinsho Jul;54(7):1929-1934.
Xiao ZQ, Moragoda L, Jaszewski R, Hatfield JA, Fligiel SE and Majumdar AP. (2001): Aging
is associated with increased proliferation and decreased apoptosis in the colonic mucosa. Mech. Ageing Dev. Oct;122(15):1849-1864.
Majumdar AP and Jaszewski R. (2003): Aging
of the esophagus and stomach. In: Pilotto A, Malfertheiner P and Holt PR, editors. Aging
and the gastrointestinal tract. Karger: Basel. p. 40-56.
Keywords:© 2010 The Egyptian Journal of Histology
Fundus of stomach; aging; rats; histology; ultrastructure.