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Ghrelin is an important hormone involved in the control of the human appetite center. Recently, protective properties of this hormone have been recognized in various models of impairment of the gastric mucosa, including stress, ischemia and reperfusion (I/R). Ghrelin is predominantly secreted by the gastric mucosa of stomach, but there are other sources of ghrelin, for example in the hypothalamus and various parts of the central nervous system (CNS) that should be taken into consideration. This hormone exerts biological effects via the activation of growth hormone secretagogue receptor (GHSR), the presence of which was confirmed in different parts of the gastrointestinal (GI) tract and midbrain structures. Although substantial evidence of the divergent biological effects of ghrelin and the mechanism of its action has been emphasized, the precise mechanisms of ghrelin which affords GI protection is still unclear. Particularly, there is a sparse amount of evidence concerning its action on the GI system. The major aim of the present study was to evaluate the importance of peripherally and centrally administered ghrelin at different times of the ischemia and reperfusion (I/R period in the modulation of resistance of the intestinal mucosa to the injury induced by ischemia and subsequent reperfusion. Secondly, we wanted to evaluate the possible mechanism of the action of ghrelin with a particular focus on its influence on the intestinal blood flow. Male Wistar rats were divided into 4 series (A-D) of the experimental groups (n=7). In series A the importance of peripherally administered ghrelin at different time of I/R period was studied. In series B the importance of centrally administered ghrelin at different time of I/R period was evaluated. In series C and D, the mechanisms of peripherally and centrally administered hormone were examined, respectively. Two models of the I/R period were selected: short lasting (30/60 min) and long lasting (60/120 min). The following drugs were used: ghrelin (50 μg/kg i.p. or 1 nmol in 10 μl i.c.v.), 6 hydroxy dopamine (50 mg/kg i.p.), nadolol (0.5 mg/kg i.p.), calcitonin gene related peptide fragment (CGRP(8-37), 100 μg /kg i.p.), capsaicin (5-10 mg/100 ml solution s.c.). The mesenteric blood flow (MBF-ml/min), the intestinal microcirculatory blood flow (LDBF-PU), the arterio-venous oxygen difference (AVO(2)-ml/O(2)/100 ml blood), and the intestinal oxygen uptake (VO(2)) in ml O(2)/min were measured. Mucosal impairment was assessed planimetrically with the use of a digital photo analyzer (LA) and histologically with the use of the six-point Park/Chiu scale. Peripheral administration of ghrelin evoked marked increase of MBF and LDBF by 42% and 48%, respectively, with significant reduction of LA by 38%. When ghrelin was administered at the beginning of the reperfusion period during the short I/R period or prior to the long lasting I/R period, the vascular reactions and protective effects were reduced, but not completely abolished. The central administration of ghrelin before the short I/R period significantly increased the MBF and LDBF by about 32% and 35%, respectively, as well as LA reduction by about 20% in comparison to the control group. However, when ghrelin was administered prior to the long I/R period or after the onset of completed ischemia, neither vascular nor protective effects were noticed. Sensory denervation and the blockade of the CGRP1 receptors totally blocked the protective and hyperemic effects of the peripherally administered ghrelin. Selective blockade of the adrenergic system or blunting of the vagal nerves (vagotomy) significantly but not totally eliminated the effects of centrally applied ghrelin, which were abolished when both adrenergic and parasympathetic pathways were ablated. These results indicate that ghrelin applied centrally or peripherally markedly increases resistance of the intestinal tissue during the I/R period induced mucosal and hyperemic impairment evoked by I/R. Ghrelin is an important mediator of the increase in the intestinal microcirculation and elevation of the intestinal metabolism, which seems to be, at least in part, responsible for the observed protection of the intestine subjected to I/R. Impairment of this microvasculature response due to I/R seems to be responsible for a markedly observed weaker effect of ghrelin when this hormone was administered after the ischemic period. The lack of a protective effect observed after central administration of this peptide against a long lasting I/R period is probably due to damage of neural pathways caused by I/R. Finally, the peripheral activity of ghrelin in the intestine is mediated by the sensory neurons with a prominent role of CGRP released from their endings. However, this peripheral action of ghrelin depends upon the proper functioning of both the sympathetic and parasympathetic system.

Limitation of the damage to the organs such as heart, liver, intestine, stomach and brain by an earlier brief complete occlusion of their arteries is defined as ischemic preconditioning (IP). No study so for has been undertaken to check whether brain-gut axis is involved in the gastroprotection exhibited by gastric IP or in that induced by repeated brief episodes of ischemia of remote organs such as heart and liver. This study was designed to determine the possible involvement of vagal and sensory afferent nerves, in the mechanism of gastric and remote organ IP on the gastric mucosa in rats exposed to prolonged ischemia-reperfusion with or without functional ablation of sensory nerves by capsaicin or in those with removed vagal innervation by vagotomy. This gastric IP was induced by short ischemia episodes (occlusion of celiac artery 1-5 times for 5 min) applied 30 min before subsequent ischemia followed by 3 h of reperfusion (I/R) and compared with remote IP induced by occlusion of left descending coronary artery or hepatic artery plus portal vein. The area of gastric lesions was determined by planimetry, gastric blood flow (GBF) was measured by H(2)-gas clearance method and mucosal biopsy samples were taken for the assessment of calcitonin gene-related peptide (CGRP) by RIA. Exposure of gastric mucosa to standard 3 h of I/R produced numerous gastric lesions and significant fall in the GBF and mucosal CGRP content. Two 5 min short ischemic episodes by occlusion of coronary or hepatic arteries, significantly reduced gastric damage induced by I/R with the extent similar to that exhibited by two short (5 min) episodes of gastric ischemia. These protective effects of gastric and remote IPs were accompanied by a restoration of the fall in the CGRP content caused by I/R alone. Protection and hyperemia induced by gastric IP were significantly attenuated in capsaicin-denervated or vagotomized animals and completely removed in those exposed to the combination of vagotomy and capsaicin-denervation. The IP-induced protection and hyperemia were restored by the administration of exogenous CGRP to gastric IP in capsaicin-treated animals. Gastroprotective and hyperemic actions of remote IP were markedly diminished in capsaicin-denervated rats and in those subjected to vagotomy. We conclude that brief ischemia in remote organs such as heart and liver protects gastric mucosa against gastric injury induced by I/R as effectively as gastric IP via mechanism involving both vagal and sensory nerves releasing vasodilatatory mediators such as CGRP.

Gastric mucosa subjected to repeated brief episodes of ischemia exhibits an increased resistance to damage caused by a subsequent prolonged ischemic insult and this is called gastric preconditioning. In this study, L-NNA, a non-selective NO-synthase inhibitor, and aminoguanidine, a relative inhibitor of inducible NO-synthase (iNOS), were applied prior to short ischemia (occlusion of celiac artery 1-5 times for 5 min) followed by a subsequent exposure to 0.5 h of ischemia and 3 h of reperfusion (I/R).Male Wistar rats were used in all studies.Short ischemia reduced significantly I/R-induced lesions while raising significantly the GBF and luminal NO content. These effects were attenuated by L-NNA and aminoguanidine and restored by addition of L-arginine and SNAP to L-NNA and aminoguanidine. Pretreatment of with adenosine (10 mg/kg i.p.) significantly reduced I/R lesions and accompanying fall in the GBF induced by I/R. These protective and hyperemic effects of standard preconditioning and adenosine were significantly attenuated by pretreatment with 8-phenyl theophylline (SPT, 10 mg/kg i.g.), an antagonist of adenosine A1 and A2 receptors.We conclude that gastric ischemic preconditioning is considered as one of the major protective mechanism in the stomach that involves key vasodilatory mediators such as NO and adenosine.

The exposure of gastric mucosa to damaging factors, such as ethanol, water restraint stress, or ischemia followed by reperfusion, produces pathological changes: inflammatory process, hemorrhagic erosions, even acute ulcers. The base of these changes is a disturbance of protective mechanisms and disrupture of gastric mucosal barrier. Previous studies pointed out the role of disturbances of gastric blood flow, mucus secretion and involvement of prostaglandins and nitric oxide formation in the pathomechanism of gastric mucosa lesions. The role of reactive oxygen species (ROS) in these processes has been little studied.The purpose of our present investigations is to explain the participation of ROS in acute gastric mucosal damage by various irritants.Experiments were carrying out on 80 male Wistar rats. To assess gastric blood flow (GBF) laser Doppler flowmeter was used. The area of gastric lesions was established by planimetry. The levels of proinflammatory cytokines were measured by ELISA technique. The colorimetric assays were used to determine of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) as well as superoxide dismutase (SOD) activity.We demonstrated that 3.5 h of water immersion and restraint stress (WRS), 30 min of gastric ischemia followed by 60 min of reperfusion or intragastric administration of 100% ethanol, all resulted in appearance of acute gastric mucosal lesions accompanied by a significant decrease of gastric blood flow. These lesions are also accompanied by the significant increase of proinflammatory cytokines including interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) plasma level. Biological effects of ROS were estimated by measuring tissue level of MDA and 4-HNE, the products of lipid peroxydation by ROS, as well as the activity of SOD, the scavanger of ROS. It was established that 3.5 h of WRS, ischemia-reperfusion and 100% ethanol lead to significant increase of MDA and 4-HNE mucosal level, accompanied by a decrease of SOD activity (significant in WRS and ethanol application).The pathogenesis of experimental mucosal damage in rat stomach includes the generation of ROS that seem to play an important role, namely due to generation of lipid peroxides, accompanied by impairment of antioxidative enzyme activity of cells.

Leptin, a product of ob gene controlling food intake, has recently been detected in the stomach and shown to be released by CCK and implicated in gastroprotection against various noxious agents but it is unknown whether centrally applied leptin influences ischemia-reperfusion (I/R)-induced gastric erosions that progress into deeper gastric ulcerations. In this study we compared the effects of leptin and CCK-8 applied intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) on gastric mucosal lesions induced by I/R and topical application of 75% ethanol. Several major series of Wistar rats were used to examine the effects of leptin and CCK applied centrally on gastroprotection against I/R and ethanol in rats with A) vagotomy by cutting of vagal nerves, B) suppression of NO-synthase with L-NNA (20 mg/kg i.p.), C) inactivation of sensory nerves by capsaicin (125 mg/kg s.c.) and D) inhibition of CGRP receptors with CGR(8-37) (100 microg/kg i.p.) applied with or without the i.c.v. pretreatment with leptin or CCK-8. Rats were anesthetized 1 h after ethanol administration or at 3 h and 3 days upon the end of ischemia to measure the gastric blood flow (GBF) and then to determine the area of gastric lesions by planimetry. Blood was withdrawn for the measurement of plasma leptin and gastrin levels by radioimmunoassay (RIA). Leptin (0.1-20 microg/kg i.p.) dose-dependently attenuated gastric lesions induced by 75% ethanol and I/R; the dose reducing these lesions by 50% (ED50) was 8 microg/kg and 6 microg/kg, respectively and this protective effect was similar to that obtained with CCK-8 applied in a standard dose of 10 microg/kg i.p. This protective effect of leptin was accompanied by a significant increase in GBF and plasma gastrin levels whereas CCK-8 increased plasma leptin levels but failed to affect plasma gastrin levels. Leptin and CCK-8 applied i.c.v. in a dose of 625 ng/rat reduced significantly the area of I/R induced gastric lesions and raised the GBF and plasma leptin levels with the extent similar to those achieved with peripheral administration of leptin or CCK-8 (10 microg/kg i.p.). The protective and hyperemic effects of centrally administered leptin or CCK-8 (625 ng/rat i.c.v.) were completely abolished by vagotomy and significantly attenuated by sensory denervation with capsaicin or by CGRP antagonist, CGRP(8-37). The pretreatment with L-NNA to inhibit NO-synthase activity attenuated significantly the protective and hyperemic effects of CCK but not those of leptin while capsaicin denervation counteracted leptin-induced protection and rise in the GBF but attenuated significantly those of CCK. We conclude that: 1) central leptin exerts a potent gastroprotective activity against I/R-induced gastric erosions that progress into deeper gastric lesions and this protection depends upon vagal activity and sensory nerves and involves hyperemia probably mediated by NO and 2) leptin mimics the gastroprotective effect of CCK and may be implicated in the protective and hyperemic actions of this peptide against mucosal damage evoked by I/R.