Intestinal ischemia occurs in a wide variety of clinical conditions including neonatal necrotising enterocolitis, transplantation and hemodynamic shock (1,2). The mechanism of ischemia/reperfusion (I/R) injury remains poorly understood, and yet there are no specific preventive methods or treatments.
Gender differences characterize the susceptibility or expression of many diseases. Men are at a relatively increased risk of cardiovascular and neurological disorders when compared premenopausal women (3,4). In general, the gender disparity has been interpreted primarily as reflecting estrogen-mediated protection against pathological conditions (5,6). Recent studies, however, suggest that male hormones may also play important roles in gender differences in disease susceptibility (7-9). The purpose of the present study was to evaluate the effects of orchiectomy or flutamide, an antagonist of the testosterone receptor, administration on I/R injury of bowel.
The experimental protocol was approved by the animal ethics committee of our institution. Thirty two Sprague-Dawley rats weighing between 260 and 290 g were divided into four groups, each containing 8 rats: group 1, sham operation; group 2, mesenteric IR; group 3, orchiectomy and mesenteric IR; group 4, flutamide and mesenteric IR. Orchiectomy was performed 14 days before mesenteric IR in group 3. Animals in the group 4 were treated with 25 mg/kg/d flutamide. Flutamide was dissolved in sesame oil as vehicle and was given via gavage throughout 14 days before mesenteric IR. Similar volumes of sesame oil were administered to the other groups of animals by the same way.
All surgical procedures were performed under ketamine (50 mg/kg) and xylazine HCl (8 mg/kg) anesthesia. The abdomen was opened through a midline incision. Simple mobilization of the superior mesenteric artery (SMA) without its clamping was performed in rats undergoing a sham operation (group 1). In I/R rats (groups 2,3 and 4), the SMA was occluded by an atraumatic microvascular clamp for 45 min followed by a reperfusion period of 3 hours. At the end of the experiments ileal tissue samples were obtained for biochemical and histopathologic analysis.
All samples were stored in deepfreeze (-20 oC) until the time of analysis. Tissue homogenates for the estimation of tissue antioxidant levels were prepared at 4°C. Ileum tissues were weighted and cut into small pieces. A 10% homogenate was prepared in ice-cold potassium phosphate buffer (PBS, pH 7.4) containing 5 mM EDTA using a glass homogenizer. The homogenate was centrifugated at 15000 rpm for 10 minutes at 4 °C. The total protein levels for homogenates was estimated by the Biuret method (10). Tissue homogenates were analyzed for MDA and NO.
Tissue homogenate MDA levels were determined by a previously described method (11). 0.5 ml of sample (plasma/ homogenate) was pipetted into a 10 ml centrifuge tube and 2.5 ml of trichloroacetic acid (20%) and 1.0ml of thiobarbituric acid (0.6%) solution was added. The tubes were heated for 30 min in a boiling water bath and the reaction mixture was then cooled in an ice-bath followed by the addition of 4.0 ml of n-butanol. The tubes were mixed with a vortex and centrifuged at 3000 rpm for 10 minutes. The absorbance of the organic layer was measured at 535 nm.
Nitric oxide determination
NO concentration in the samples were determined indirectly by measuring the nitrit levels based on Griess reaction (12). Samples were initially deproteinized with 75 mmol/L ZnSO4. After clean up, an aliquot of the sample was treated with copperized cadmium in glycine buffer at pH 9.7 to reduce nitrate to nitrite. The concentration of nitrite in this aliquot thus represented the total nitrate plus nitrite. In Griess reagent, a chromophore with a strong absorbance at 545 nm is formed by reaction of nitrite with a mixture of naphthlethylendiamine and sulphanilamide.
The ileal specimens were fixed in 10% formaldehyde, then dehydrated and embedded in paraffin wax. Samples of intestine were sectioned and stained with H&E and submitted for histopathologic evaluation by a histopathologist in a blinded fashion using light microscopy. Histological mucosal damage in each preparation was classified according to microscopic criteria described by Chiu et al (13) and modified by Oldham et al (14) as follows: grade 0, normal mucosa; grade 1, subepithelial edema, partial separation of apical cells; grade 2, epithelial cell slough from tips of villi; grade 3, progression of slough to base of villi; grade 4, partial mucosal necrosis of lamina propria; grade 5, total mucosal necrosis.
Statistical analyses were accomplished by using of SPSS computering programmes (version 13.0). All results were reported as mean ± standard deviation (SD). The comparison of the results from the various experimental groups and their corresponding controls was carried out using a one-way analysis of variance (ANOVA) followed by pairwise multiple comparison procedures (Tukey test). The differences were considered significant when P<0.05.
Table 1 shows the ileal tissue MDA, NO values and histologic injury scores for all the groups. MDA and NO levels significantly increased in I/R group animals, compared to sham operated group animals (P<0.05). However, MDA and NO levels in orchiectomy and flutamide groups were significantly lower compared to the untreated I/R group (P<0.05).
The rats in sham group had essentially normal histologic architecture (Fig 1a). The highest histologic grade (mean 4.62 ± 0.51) was determined in group 2 (Fig 1b). Samples in orchiectomy group and flutamide treatment group showed significantly decreased mean injury scores respectively grade 3.5 ± 0.75 (Fig 1c) and grade 3.25 ± 0.70 (Fig 1d) injury (P<0.05). Histopathologic injury scores were in accordance with tissue MDA and NO levels.
I/R injury to the small intestine can occur in many clinical conditions, such as necrotising enterocolitis, mesenteric vascular occlusion, incarserated hernia, midgut volvulus, hemodynamic shock and transplantation of the small intestine (1,2). Tissue damage and impaired function occur after temporary ischaemia, not only during the hypoxic period, but also after reoxygenation. Oxidative stress associated with the overproduction of reactive oxygen species (ROS) plays a key role in the pathophysiology of I/R injury (15). ROS are potent oxidizing and reducing agents that can directly damage cellular membranes by lipid peroxidation (16) and result in cellular structural destroy. Overproduction of free radicals and/or inadequacy of the antioxidant defense system lead to oxidant injury (1,16).
Gender appears to influence systemic and organ-specific inflammatory sequelae of ischemia-reperfusion. Men are at a relatively increased risk of ischemic cardiovascular and neurological disorders when compared premenopausal women (4,5). Indeed, population studies indicate that men have higher mortality rate after myocardial infarction (17). Clinical evidence has demonstrated striking sex differences in the incidence and outcome of stroke (18). In addition, clinical studies have shown that female patients survived better than males after liver transplantation or hepatocellular carcinoma resection (19). Although many studies have emphasized the beneficial effects of estrogen in gender differences in I/R injury (5,6), some others have implicated male sexual hormones (7-9). Androgen hormone deficiency and blockade improves survival in male rodents in systemic inflammatory conditions (3,20). Park et al shown that the presence of testosterone, rather than the absence of estrogen, appears to be more relevant to the gender difference in susceptibility to kidney I/R injury. Orchiectomy protects the kidney from ischemical reperfusion injury (21).
There are few studies that investigate effects of sex hormons in intestinal I/R injury. Xiao et al demonstrated protective effect of estrogen treatment intestinal I/R injury in ovariectomised female rats (22). Estradiol and flutamide decrease hypoxia plus acidosis-induced gut permeability and mucosal injury in male rats (23). Szabo et al reported that in small intestinal I/R the leukocytic inflammatory response and microcirculatory dysfunction develop more rapidly and are initially more pronounced in male murines (24). In our study we aimed to investigate the effect of testosterone depletion (castration) or tertosterone receptor blockade (flutamide) on mesenteric I/R model in male rats.
MDA is the end-product of lipid peroxidation and is a well-known parameter for determining the increased free radical formation and tissue damage in bowel (25). In our study, level of MDA significantly increased in group 2 when compared to the sham group (P<0.05). Pretreatment with flutamide or orchiectomy significantly decreased MDA levels in ileal tissue (P<0.05). MDA levels was correlated with histopathologic injury scores.
NO is an endogenously produced reactive molecule with important biological functions such as regulation of vascular tone as well as mucosal barrier function (26). In addition to these regulatory functions, it have detrimental effects. Overproduction of NO can lead to cellular damage through combination with superoxide to produce potently cytotoxic peroxynitrite, or by its end products such as nitrate and nitrite (26,27).
There is controversy whether NO is a mediator for gastrointestinal mucosal defence or demage. Naito et all demonstrated that overproduction of NO and NO-derived reactive species from iNOS plays a major role in oxidative stress of reperfusion-induced intestinal injury (28). Takada et al showed that intraluminal NO level increase after mesenteric I/R and L-NAME, a potent inhibitor of NO synthase, reduce the degree of intestinal tissue injury (29). On the contrary there are data that NO generators decrease mucosal injury and L-NAME greatly exacerbates intestinal injury after I/R (26).
Our study showed that I/R injury of the bowel elevated NO production in ileal tissue. Orchiectomy or treatment with flutamide significantly decreased the concentration of intestinal NO levels when compared with the nontreated I/R group. NO levels was correlated with MDA levels and histogic injury scores. This result parallels those in other reports that indicate the overproduction of NO as a detrimental factor responsible for the pathogenesis of bowel in inflammatory response in I/R (30,31).
The result of this study demontrated that pretreatment with flutamide or orchiectomy plays a role in attenuating I/R injury of the small intestine as it was shown by the decrease of tissue MDA, NO levels and histopathological injury scores.
It has been demonstrated that endogenous testosterone have some physiological properties such as proinflammatory, proapoptotic and vasoconstrictor (32-34), which may play a role in producing organ dysfunction following ischemia-reperfusion. Castration or treatment with testosterone receptor blockers can reduce this testosterone related injury. Studies have shown that testosterone receptor blockade with flutamide in male mice restores the depressed immune (35), cardiovascular and hepatocellular functions following hemorrhagic shock (36). Although the precise mechanism responsible for the salutary effects of flutamide on organ prevention is not known, several effects could be considered. It is possible that flutamide administration inhibits the vasoconstrictive actions of thromboxane A2 and/or enhances the release of the vasodilator prostacyclin leading to better organ perfusion (32). Furthermore, testosterone receptor blockade may be enhanced by specific cellular effects of estrogen, which is known to produce beneficial effects after ischemia-reperfusion (22,28). Studies demonstrated that pretreatment of males with flutamide increases estrogen level and estrogen receptor expression (37,38). Yu et al showed that the reduction of neutrophil accumulation and intestinal injury following trauma-hemorrhage occures via the upregulation of estrogen receptor dependent hemeoxygenase-1 expression by flutamide (39).
In conclusion, these results suggest that castration or testosterone receptor blockade decreases the level of intestinal I/R injury in male rats and we can say that intestinal I/R injury is an another example for different variations of the same diseases depending on gender differences.