Karmen L. Schmidt

Prostaglandin cytoprotection against ethanol-induced gastric injury in the rat

Abstract Using macroscopic criteria for injury, prostaglandins have been alleged to possess potent antiulcer properties despite meager histologic evidence for this cytoprotective action. This time-sequence study used light, scanning, and transmission electron microscopy to evaluate the effects of 16,16-dimethyl prostaglandin E 2 on gastric mucosal integrity after exposure to 100% ethanol. Macroscopically, virtually complete protection against injury to the glandular mucosa of the in vivo rat stomach was noted in animals receiving 10 μg/kg body wt of prostaglandin subcutaneously before oral ethanol administration when killed at 5, 20, and 60 min after ethanol exposure compared with oral ethanol after saline injection. On light microscopy the length of injured epithelium in prostaglandin/ethanol- and saline/ ethanol-treated tissues was not significantly different at all time periods studied. Although the depth of injury extended into gastric glands in both groups killed at 5 min, the deep pit surface mucus cells in prostaglandin/ethanol mucosa were less damaged and necrotic lesions were virtually absent when compared with saline/ethanol mucosa. At 20 and 60 min, cellular injury could still be identified in prostaglandin/ethanol-treated mucosa but the depth of injury became even less pronounced over time in contrast to mucosa exposed to ethanol without prostaglandin. Scanning electron microscopy and transmission electron microscopy confirmed these differences. Despite the macroscopic findings, these results indicate that prostaglandin does not prevent superficial surface mucus cell necrosis in ethanol-exposed mucosa even though it does spare cells in the pit base. The reduction in damaged cells over time in prostaglandin/ethanol-treated mucosa, in contrast to saline/ethanol-treated mucosa, supports the hypothesis that the reepithelialization of the lamina propria is initiated by spared deep-lying pit cells.

Prostaglandin prevents aspirin injury in the canine stomach under in vivo but not in vitro conditions

This study compared the ability of topical 16,16-dimethyl prostaglandin E2 in a dose range of 0.3-3.0 micrograms/ml to prevent aspirin-induced injury in the canine stomach under both in vivo and in vitro conditions. For in vitro studies, isolated strips of oxyntic mucosa were exposed to 10 or 20 mM aspirin (ASA) at pH 1-4, with and without treatment with 16,16-dimethyl prostaglandin E2. For in vivo experiments, a portion of the oxyntic stomach was mounted between the rings of a Lucite chamber, with splenic vessels intact, such that the mucosa was divided into halves. Both sides were exposed to 20 mM ASA at pH 1 or 2, and one side also received concomitant treatment with 16,16-dimethyl prostaglandin E2. After ASA exposure, tissue samples were prepared for quantitative microscopic analysis of the degree of injury. Under both experimental conditions, the magnitude of gastric injury by ASA was pH-related, being most pronounced at pH 1; this damage was worse under in vitro conditions, and both concentrations of ASA were equally damaging in this setting. 16,16-Dimethyl prostaglandin E2 failed to prevent ASA injury in vitro at any pH and ASA concentration tested, but markedly reduced the magnitude of injury in vivo. The most effective protective dose of 16,16-dimethyl prostaglandin E2 under in vivo conditions was 1.0 micrograms/ml. The diminished tolerance to ASA damage in vitro when compared with in vivo, and the inability of 16,16-dimethyl prostaglandin E2 to prevent these damaging effects in vitro, underscores the probable crucial role for blood flow, and possibly neural innervation, in mediating the protective effects of prostaglandins.

Influence of prostaglandin on repair of rat stomach damaged by absolute ethanol

This study was undertaken to determine the possible role that prostaglandins (PGs) may play in enhancing epithelial repair in ethanol-damaged gastric mucosa. Fasted rats were injected subcutaneously with 10 micrograms/kg of 16,16-dimethyl PGE2 or saline and 30 min later received an oral bolus of 1 ml of absolute ethanol or saline. At 5 min and 2, 8, and 24 hr after ethanol exposure tissues were prepared from identical regions of the glandular mucosa for microscopic evaluation. Normal, injured, and repairing tissues were differentiated and quantitated. The length of injured surface epithelium was the same in ethanol-treated tissues with and without PG pretreatment when evaluated 5 min after ethanol exposure, but the deeper epithelium was protected from injury in animals receiving PG pretreatment. Although the repair process itself was identical in the two experimental groups, in PG-treated tissues repair was initiated earlier, was more widespread, and was much more rapid than in tissues exposed to ethanol without such treatment. At the end of 24 hr of observation, only 5.5% of the surface epithelium was considered normal histologically in mucosa exposed to ethanol alone without PG pretreatment. This is in marked contrast to PG-treated tissues in which 82.7% of the gastric surface was normal at 24 hr. The mechanism responsible for our findings is unknown but may be related to PGs ability to spare the cellular pool in the gland isthmus from damage, enhancement of cellular migration from this pool to resurface the damaged epithelium, or a combination of both of these processes.

Protective effect of dimethylthiourea against mucosal injury in rat stomach. Implications for hydroxyl radical mechanism.

The present study was undertaken to determine whether dimethylthiourea (DMTU), a hydroxyl radical scavenger, could prevent gastric injury in the rat stomach induced by various noxious agents. Fasted rats (N=6–8/group) were given a 1-ml oral bolus of saline or DMTU over the dose range 10–500 mg/kg. After 30 min, animals received 1 ml of 100% ethanol orally and were sacrificed 5 min later. At sacrifice, stomachs were harvested and the degree of macroscopic damage was assessed by planimetry. In selected animals, specimens of gastric mucosa were also processed for histology. Saline pretreatment prior to ethanol exposure resulted in 22.5% injury to the glandular epithelium when assessed macroscopically. DMTU pretreatment prevented such injury in a dose-related fashion with only 2% of the mucosa showing injury with a 500 mg/kg dose (P<0.01 vs control). Although the superficial injury involving surface mucous cells induced by ethanol was not altered by DMTU, the deep damage to gastric glands was almost completely prevented. Other experiments in which DMTU was given intraperitoneally demonstrated similar protective effects against ethanol injury. Additional studies showed that indomethacin did not prevent the protective effects of oral or intraperitoneal DMTU, excluding a role for endogenous prostaglandins, and that DMTU was equally protective when administered within minutes or as long as 2 hr prior to ethanol exposure. Furthermore, DMTU was also shown to be protective against gastric injury induced by concentrated acid or base. Inin vitro studies in which hydroxyl radicals were actually generated, DMTU was noted to scavenge the hydroxyl radical in a dose-related fashion. The ability of DMTU to prevent gastric injury by three different damaging agents suggests that the hydroxyl radical may play a major role in the pathogenesis of such injury and that DMTU mediated its protective action by scavenging this radical species.

Gender differences in ethanol oxidation and injury in the rat stomach.

A significant fraction of orally consumed ethanol is metabolized by the alcohol dehydrogenase (ADH) enzyme present in the gastric mucosa. Human studies have shown that this first pass metabolism of ethanol correlates quite closely with gastric ADH activity which has been demonstrated to be greater in men than women. The present study was undertaken to determine if gender influences the magnitude of ethanol-induced injury in rat gastric mucosa and whether any differences can be linked to altered levels of ADH activity. Since prostaglandins (PGs) have been shown to markedly attenuate the severity of gastric injury induced by ethanol in the rat stomach, a further goal of this study was to determine whether the efficiency of PGs protective action was in any way influenced by gender. Accordingly, both male and female rats were pretreated subcutaneously with 16,16-dimethyl PGE2 (10 micrograms/kg) or saline 30 minutes prior to administering an oral dose of 50% ethanol in saline or saline alone. They were then sacrificed 5 minutes later. In a portion of animals (n = 6 per group), samples of mucosa from the glandular stomach were obtained and kinetic activity of ADH determined. In another portion of animals (n = 6 per group), gastric tissue samples from the glandular mucosa were examined by light microscopy and the magnitude of mucosal injury quantified. Alcohol-treated females showed significantly (p less than 0.05) less superficial and more deep mucosal injury than male counterparts. Further, ADH kinetic activity in female rats was significantly less than that observed in male counterparts of similar weight (83% of males; p less than 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological Characteristics of Prostaglandin Cytoprotection

The term “cytoprotection” originally described the ability of prostaglandins, independent of their known antisecretory activity, to prevent macroscopic evidence of gastric injury induced in different animal models under various experimental conditions. Several recent reports have challenged this concept because the apparent macroscopic protection could not be confirmed microscopically. To determine whether prostaglandins do indeed possess cytoprotective properties, and if such effects are dependent on the dose and the route of prostaglandin administration, studies were performed using 16,16 dimethyl prostaglandin E2 (PGE2) and the known gastric damaging agent, ethanol. Fasted rats received either oral or subcutaneous PGE2 in doses of 10 or 20 μg/kg or equal volumes of saline. Thirty minutes later, animals were given a 1-ml oral bolus of 50% or 100% ethanol or an equal volume of saline. At 5 minutes to 24 hours following ethanol, animals were sacrificed, and tissues from the glandular portion of the stomach were removed for histologic quantification of injury. At 5 minutes following ethanol, PGE2 reduced the depth of injury, but had no protective effects against surface cell damage when compared with control animals. By 24 hours after ethanol, most of the PGE2-treated mucosa was repaired. Oral administration of 10 μg/kg PGE2 was more cytoprotective at 5 minutes after ethanol than when administered by subcutaneous injection. This relationship was not true for the 20 μg/kg dose. We conclude that cytoprotection can be confirmed histologically, but is limited primarily to the deep mucosa. The ability of PGE2 to enhance healing is probably related to the prevention of deep mucosal injury, which thereby allows epithelial reconstitution to occur. Lastly, the effectiveness of PGE2 as a cytoprotective agent is dose- and route-dependent.

Effects of ethanol and prostaglandin on rat gastric mucosal tight junctions

The effect of 16,16 dimethyl prostaglandin E2 (PG) upon tight junctions (TJs) of adjacent surface mucous cells (SMCs) as a possible mechanism by which PGs mediate their protective effects was studied using transmission electron microscopy (TEM) and freeze fracture (FF) techniques. Fasted rats were subcutaneously injected with 10 micrograms/kg of PG or an equal volume of saline, followed 30 min later by 1 ml of oral 100% ethanol or saline. Ten or sixty minutes later, animals were sacrificed and stomach blocks were prepared for TEM or FF using standard techniques. Electron micrographs (X60,000) were obtained and the distance between SMC inner membrane leaflets was measured with a micrometer and expressed as TJ width. Stomach blocks for FF were stored at 4 degrees C, cryoprotected, freeze fractured, and photographed by TEM (X30,000). At 0.5-micron intervals, measurements of TJ strand number and depth were made. No statistical differences were found in TJ width or strand number of SMCs among the various experimental groups when compared with controls at each sacrifice time. At the 60 but not 10 min sacrifice time, TJ depth was greatly increased in cells treated with 10 micrograms/kg PG prior to ethanol exposure (P less than 0.025) in contrast to control mucosae. We conclude that 16,16-dimethyl PGE2 induces no changes in the structural composition of TJs as a possible explanation for its protective properties. The increase in TJ depth observed in ethanol exposed mucosa following PG pretreatment at the 60 min sacrifice time is most likely related to the architectural restructuring that occurs during reconstitution of damaged surface epithelium.

Prevention of the inhibitory effects of aspirin on sodium transport in canine gastric mucosa by prostaglandin: Correlation with mucosal morphology

Using an in vitro canine gastric mucosal preparation, this study evaluated the effects of 1 mM aspirin in a buffered Ringer solution (pH = 7.4), with and without concomitant prostaglandin (PG) treatment, on net sodium transport (mucosa to serosa) across gastric epithelium. Administration of aspirin to the mucosal bathing solution for 2 hr significantly decreased the potential difference (PD), short circuit current (Isc), and net sodium transport (net J-Na+) when compared with untreated control mucosa. In mucosa treated with 16,16-dimethyl PGE2 (8 X 10(-6) M) in the serosal bathing solution 40 min after aspirin exposure and for 80 min thereafter, the initial inhibitory effects on PD, Isc, and net J-Na+ induced by aspirin were completely reversed within 40 min of PG treatment, having returned to control values. Histologically, mucosa exposed to aspirin alone showed evidence of diffuse cellular injury involving 50-60% of the surface epithelium. In contrast, mucosa treated with prostaglandin in conjunction with aspirin exposure demonstrated damage involving only 20-30% of the epithelium. These findings suggest that stimulation of sodium transport by PG may play a role in mediating the cytoprotective effects of PGs against aspirin-induced gastric mucosal injury.

Ultrastructural Aspects of Prostaglandin Cytoprotection in an Alcohol Injury Model

The effects of absolute ethanol (EtOH) and 16, 16-dimethyl-prostaglandin E2 (PGE2) on the ultrastructure of rat gastric mucosa were assessed using standard electron microscopic techniques. Fasted rats were injected sub-cutaneously with 10 μg/kg body weight of PGE2 or saline. After 30 min, animals received orally 100% EtOH or an equal volume of saline. Gastric mucosa was sampled at 5 min and 1,2,8, and 24 h post-EtOH and tissues procured for scanning and transmission electron microscopy. Both mucosal epithelial and connective tissue compartments were assessed. At 5 min after EtOH, in saline-pretreated animals, tissues showed extensive destruction of epithelium and connective tissue; major breaks in the basal laminae were coincident with hemorrhage. Deep-lying epithelial cells and most of the basal lamina were spared in tissues exposed to PGE2 prior to EtOH, permitting nearly complete repair within 24 h. Restitution proceeded over small breaks or pleats of basal lamina but failed in regions of total destruction of this structure. These data indicate that PGE2 protects not only deep-lying epithelial cells from EtOH damage but associated connective tissue as well and that sparing of both compartments leads to orderly and rapid epithelial restitution.

The Role of Vagal Innervation in Adaptive Cytoprotection

In an earlier study1 we observed that alcohol-induced gastric damage in the rat was exacerbated by truncal vagotomy. We further noted that the mild irritant, 30% ethanol, which significantly reduced the magnitude of injury when mucosa was subsequently exposed to 100% alcohol, was no longer capable of eliciting this protective effect under conditions of vagal denervation. These observations suggested that vagal integrity played an important role in mucosal defense and was necessary for adaptive cytoprotection to occur, at least under conditions of injury induced by alcohol. While the mechanism(s) responsible for these findings has remained elusive, the possibility that capsaicin-sensitive fibers may play a role in mediating these vagotomy effects must be considered. This contention is based on the fact that as many as 80% of fibers comprising the gastric vagus are afferent in nature, being composed of unmyelinated and thinly myelinated afferent neurons2, and capsaicin, the pungent ingredient of red peppers, is able to impede the conducting activity of such primary afferent neurons when administered in high doses3,4,5. Further, functional ablation of capsaicin-sensitive fibers has been shown to enhance gastric injury and ulcer formation in a number of experimental models including those in which gastric damage was induced by pylorus ligation6, acid distension of the stomach6, and in response to various noxious agents such as indomethacin7 and ethanol7.