Introduction

Duodenal ulcer is a mucosal erosion of duodenum, due to multiple causes, including bacteria (Marshall and Warren 1984; Lykoudes 1958), chewing gum tobacco smoking, not eating properly, blood group, spice (NADDIC), chronic stress (Kim et al. 2007) and gender differences (Andes et al. 2008). Specific protection of estrogen against gastric –acid induced duodenal injury was reported (Andes et al. 2008). Peptic ulcer occurs more frequently in men than in women (Grey 1929). The sex differences are less marked after 45 years of age probably because the incidence of ulcer increases in post menopausal women (Crean 1963; Watkinson 1960). The general assumption is that the ulcer differences between sexes are related in some way to sex hormones and that the female sex hormones protect against ulceration (Crean 1963; Kyle et al. 1963). The immunity against duodenal ulcer is increase in females during pregnancy (Dey and Dey 1974). It was shown that the chance of men developing duodenal ulcer remain remarkably constant between the age of 20 years and 65 years. In contrast the chances of women developing duodenal ulcer remains relatively low throughout the whole life of her active reproductive life (Truelove 1960). Nevertheless, female sex hormones have been reported to promote ulceration (Antonsen 1955; Guerrine et al. 1967) or at best to have no effect the disease. Increased susceptibility to gastric ulceration during the late pregnancy was also reported (Kelly and Robert 1969). Duodenal ulcer sensitivity increases with age (Christensen et al. 2006). The present study was undertaken therefore, to investigate in mice the effect of ovariectomy on sensitivity of duodenal ulceration induced by using cysteamine –HCl and to determine whether estrogen administration reverse the effects of ovariectomy and aging. The study was also carried out by histological, histochemical and biochemical methods in the duodenal region of the normal, overiectomized, cyteamine injected. ovariectomized – cysteamine injected, old female mice and cysteamine injected old female mice.

Materials and methods

Experimental animals

Ovariectomy and administration of estrogen

Histology

Histochemistry

Biochemistry

Data analysis

Results

The ovariectomized mice showed little ulceration (Ulcer index 2.6) or no ulceration. The overiectomozed + estradiol- 17β injected mice showed ulceration very low compared to the overiectomized mice (ulcer index 1.64) (Table No.1) The ovariectomized + Cysteamine administered mice showed higher ulceration (Ulcer index 5.1) (Table No.1).The administration of estradiol- 17β to cysteamine injected ovariectopmized mice showed little recovery of ulceration (Ulcer index 4.2) (Table No.1). The old mice administered with cysteamine showed ulceration as high as ovariectomized + cysteamine administrated mice (Ulcer index 4.8) (Table No.2).

Figure 1

Table No.1: Ulcer severity in normal with and without Administration of estradiol

Figure 2

Table No.2. Ulcer severity in Ovariectomized with and without cysteamine and estrogen administration

Figure 3

Table 3. Carbohydrates and protein contents of soluble glycoprotein isolated from Brunner’s glands of female mice

* Values are mean ± Standard error, p< 0.05 is significant

Administration of estradiol- 17β to cysteamine treated old mice fails to recover the severity of ulceration (Table No.2). The histology of ovariectomized mice (Fig.1) showed that the pyloric glands were simple tubular and situated deep in the sub mucosa. The duodenal villi were tall, leaf like and uniformly arranged with desquamation intermittently. The crypts of Lieburkuhn and Brunner’s glands are unaffected. The cysteamine administration to the ovariectomized mice (Fig.2) cause pyloric glands with dilated lumen and picnotic nuclei and increased eosinophilia. The pyloric villi showed fissures and ramifications, and ulcer formation. The goblets cells are less in it. The Brunner’s gland acini showed reduced height, dilated lumen and nuclei with abnormal size and shape. The histochemistry showed strong PAS reaction in pyloric glands, crypts of Lieberkuhn, Brunner’s glands in ovariectomized mice but less in pyloric pit (Fig.3).

Figure 4

Fig 1: Micrograph of duodenum of Ovariectomized mice

Figure 5

Fig 2: Micrograph of duodenum of Ovariectomized- cyteamine injected mice

Figure 6

Fig 3: Micrograph of duodenum of Ovariectomized mice PAS stain

Figure 7

Fig 4: Micrograph of duodenum of Ovariectomized- Cyteamine Injected Mice stained with PAS

The duodenal villi showed PAS activity but other cells are PAS negative. The cyteamine treated overiectomized mice showed reduction in PAS reactivity in all cells of pyloric glands, pyloric pits, goblet cells and Brunner’s glands (Fig 4). The biochemical studies showed that the hexose contents reduce 3 folds in cyteamine injected mice comparing to the normal (Table 3). In ovariectomized it was less than that of normal but more than that of cyteamine treated mice. But in ovariectomized cysteamine treated mice it was 6 times less than that of normal mice. The fucose contents, sialic acid contents and protein contents (Table 3) also showed the same trend as hexose.

Discussion

Peptic duodenal and gastric ulcers raise serious health problems and significant economic cost worldwide. There are approximately 500,000 new cases and 4.5 million people suffering from these diseases each year in USA (Valle et al. 2003). Duodenal ulcer is three times more common than gastric ulcer. Available evidence suggests that duodenal ulcer most likely results from an imbalance between “aggressive” factors, such as infection of Helicobacter pylori (H pylori), gastric acid and pepsin, and “defensive” factors, such as duodenal mucosal bicarbonate (HCO37 -) secretion8 (DMBS) (Valle et al. 2003; Hogan et al. 1996) a and sex hormones . It has long been observed that the ratio between men and women who develop duodenal ulcer is 1.9:1 in the US, whereas in Europe and in Asia this ratio is 2.2: 1 (Kurata et al. 1985; Rosenstock and Jorgensen 1995; Ostensen et al. 1985; Bonnevie, 1975) and 3.1: 1(Wu HC et al. 2008), respectively. These clinical observations suggest a gender difference in the incidence and severity of duodenal ulcer; however, the underlying mechanism(s) are currently unknown. As far as gender differences are concerned, sex hormones have been often evaluated as the causative factors. For example, numerous studies have suggested a protective role of estrogen in the development of various diseases including cardiovascular diseases (Gerhard and Ganz, 1995; Grodstein et al. 1996; Orshal and Khalil 2004), cerebral damage and mortality (Zhang et al., 2004; Hurn and Macrae, 2000), and osteoporosis (Gerhard and Ganz 1995; Grodstein et al. 1996; Orshal et al. 2004; Popp et al. 2006). In contrast, information regarding the protective effects of sex hormone in the gastrointestinal tract is very limited (Furner et al. 1989). The present investigation showed that cysteamine induced duodenal ulcer severity decreased on the administration of estrogen. But estrogen failed to recover the ulcer incidence in old females. This indicates that estrogen itself is not the reason for the low incidence of duodenal ulcer in female during her reproductively active period. The high incidence of duodenal ulcer in overiectomized – cysteamine injected mice may be due to the lack of estrogen in association with other factors. Anders et al. (2008) revealed the gender specific duodenal protection by estrogen in terms of HCO3 secretion and the underlying molecular mechanisms of estrogen stimulation of DMBS that is linked to ER-Ca2+-CFTR and Cl-/HCO3exchanger pathways. We observed that the incidence of duodenal ulcer is more or less identical in overiectomized –cysteamine injected females and old –cysteamine injected females. The result was reflected in histological, histochemical, and biochemical studies. Histological changes that took place in goblet cells, pyloric gland cells and Brunner’s gland cells in ovariectomized cyteamine treated mice were not observed in ovariectomized estrogen injected cysteamine treated mice. Sugars from glycoprotein of Brunner’s gland were depleted in cysteamine treated females. These findings indicate that the estrogen is able to protect the duodenal mucosa from damaging effects of cysteamine. Manekar and Namaji (1977) suggested that female sex hormones have protective effects against ulcer formation. It has been established that glycoprotein containing bicarbonate is mainly responsible for the protection of duodenal mucosa. The glycoprotein and bicarbonate are mainly secreted by Brunner’s glands. Thus it can be concluded that estrogen may be assisting in the secretion of glycoprotein from Brunner’s glands. Increase in the secretion of glycoprotein from Brunner’s gland acini and duct cells has been shown in the present investigation in estrogen treated mice. Secretion of glycoprotein of other exocrine cells of the duodenum is also influenced by the estrogen. This substantiates the role of estrogen in protecting the duodenal mucosa, but the exact mechanism is to be explored further.