Role of xanthine oxidase-derived oxidants and leukocytes in ethanol-induced jejunal mucosal injury.

Previous reports indicate that intestinal intraluminal ethanol increases mucosal permeability (an index of mucosal injury) and histamine release by mast cells, and that the released histamine plays a role in mediating the increased permeability. In the present study, we investigated whether reactive oxygen metabolites and their major sources (xanthine oxidase and leukocytes) were involved in these ethanol effects. In rabbits, segments of the jejunum were perfused with a control solution or with 6% ethanol. In these segments, mucosal permeability was assessed by determining jejunal clearance of i.v. administered 51Cr-ethylenediaminetetraacetate (51Cr-EDTA) and 125I-bovine serum albumin (125I-BSA), and mast cell histamine release was estimated from the histamine concentration of the gut effluent. Ethanol increased 51Cr-EDTA clearance, 125I-BSA clearance, and histamine release. These ethanol effects decreased when the animals were given superoxide dismutase plus catalase (scavenger of O2- and H2O2, respectively), allopurinol, or oxypurinol (xanthine oxidase inhibitors). Administration of a monoclonal antibody (R15.7) against leukocyte adhesion molecule, CD18, inhibited completely the ethanol-induced increased 51Cr-EDTA and 125I-BSA clearances and histamine release. These and supplementary data suggest that (a) ethanol-induced mucosal injury and mast cell histamine release are mediated primarily by leukocytes, and (b) oxy radicals, especially those generated by xanthine oxidase, mediate these ethanol effects mainly by promoting leukocyte infiltration.

Phorbol myristate acetate ex vivo model of enhanced colonic epithelial permeability. Reactive oxygen metabolite and protease independence.

The initiating mechanisms involved in colonic injury are currently unknown. The goal of the current study was to examine the role of the inflammatory mediators reactive oxygen metabolites and proteases in an ex vivo model of selective epithelial permeability. Rats were prepared with exteriorized colonic chambers to which the protein kinase C (PKC) activator phorbol myristate acetate (PMA) was added in doses ranging from 5 to 800 micrograms. PMA caused a dose-dependent transient increase in epithelial permeability, but had no significant effect on microvascular permeability. There was no accumulation of neutrophils and no apparent histological changes. PMA acts via a PKC-dependent mechanism, as assessed using the PKC-inactive phorbol analog 4 alpha-phorbol didecanoate, and the response is tachyphylactic. The mechanism is independent of reactive oxygen metabolites and proteases, as shown by the lack of effect of the free radical scavengers superoxide dismutase and catalase and the general serine protease inhibitor soybean trypsin inhibitor. The classic inflammatory process does not appear to be involved in the PMA-induced epithelial permeability changes. This finding suggests that noninflammatory mechanisms may regulate the increased epithelial permeability induced by PMA. Further study to elucidate these mechanisms is of importance for understanding both normal gastrointestinal physiology and initiation of pathology.

Neutrophil-independence of the initiation of colonic injury. Comparison of results from three models of experimental colitis in the rat.

Although the role of neutrophils in the pathogenesis of several forms of gastrointestinal injury has been well demonstrated, their role in the development of experimental colonic injury is less clear. To examine whether neutrophils play a role in the development of experimental colitides, the effects of a sustained neutropenia on multiple indices of colonic injury in rats was examined 24 hr following the initiation of colitis with the intrarectal application of acetic acid, trinitrobenzene sulfonic acid (TNBS)-ethanol, or the potent proinflammatory agent, phorbol-12-myristate-13-acetate (PMA). In comparison to animals with normal neutrophil counts and colitis induced by any of the three agents, no attenuation in macroscopic damage or histopathologic injury was observed in neutropenic animals exhibiting a greater than 95% reduction in circulating neutrophils and 85% reduction in tissue-associated myeloperoxidase activity. Although the tissue edema associated with acetic acid or PMA-induced colitis was not reduced by neutropenia, the colonic edema associated with TNBS colitis was attenuated by prior neutrophil depletion with anti-neutrophil antiserum. Despite our initial hypothesis that neutrophils played a key role in the genesis of experimental colitis (especially that induced by PMA), the results demonstrated that these cells are not essential for the development of the major pathological features of colitis induced by this agent, acetic acid, or TNBS. Although these results support the proposal that in these models of colitis, inflammation develops secondary to injury (rather than the converse), further studies will be necessary to elucidate the role of inflammatory cells other than neutrophils in the genesis of experimental colitides.

Studies on ethanol-induced subepithelial fluid accumulation and jejunal villus bleb formation. An in vitro video microscopic approach.

Jejunal intraluminal ethanol causes morphological and mucosal microvascular injury. The purpose of the present study was to understand the mechanism of the morphological alterations caused by ethanol without the influence of ethanol's effect on the microcirculation. Therefore, we have investigated the ethanol-induced morphological changes in the absence of blood flow (i.e., in the jejunum in vitro) and compared these changes with those reported to occur in the presence of microcirculation (i.e., in the jejunum in vivo). The mucosa of jejunal segments was exposed to a control solution and to solutions containing 0.8, 1.6, and 4.8% (w/v) ethanol, using a specially designed apparatus. The morphological response of the mucosa to these solutions was continuously examined employing a video microscopic technique, and the changes were morphometrically evaluated on subsequent playback of videotapes. Ethanol caused a concentration-dependent increase in the number of villi with subepithelial fluid accumulation, i.e., blebs, and a decrease in the height of the villus core (i.e., lamina propria). With 0.8 and 1.6% ethanol, the contracted core remained partially attached to the epithelium and the total villus height (villus core plus epithelial layer) decreased. With 4.8% ethanol, the villus core contraction was so rapid that the stroma fully separated from the epithelium. Thus, among other factors, the rapidity of the villus core contractions appears to play a role in the subepithelial bleb formation and in the appearance of the bleb. The ethanol-induced changes in vitro are similar to those reported to occur in the jejunum in vivo. Therefore, we conclude that the effect of ethanol on morphology is independent of its action on the microcirculation.

Proinflammatory effects of local abdominal irradiation on rat gastrointestinal tract.

Although the role of inflammatory processes in the genesis of late changes in the gastrointestinal tract following exposure to ionizing irradiation has been extensively studied, few studies have concentrated on the presence of an acute inflammatory response in the period immediately following radiation. We therefore examined, in rats, whether the local application of 10 Gy cobalt-60 irradiation to the abdomen led to changes in the gut within the first 24 hr that were consistent with an acute inflammatory response. In stomach, small intestine, and colon, local irradiation led to a significant increase in the accumulation of plasma within the tissue by 4-8 hr following irradiation. This increase in tissue plasma volume, indicative of an increased microvascular permeability, was then sustained until the end of the 24-hr assessment period in all tissues examined. Concurrent with this was a consistent transient increase in tissue red blood cell volume, suggestive of vasodilation. Of particular note, a significant increase in the number of mucosal neutrophils was also observed between 2 and 12 hr following irradiation. This elevation in mucosal neutrophils was particularly marked in the pericryptal or deep mucosal regions of small intestine and colon and consistently preceded the vasodilation and enhanced permeability. Furthermore these pathophysiological alterations occurred at a time when histological changes in the mucosa consistent with an impaired mucosal microcirculation (ie, edema of the lamina propria and subepithelial bleb formation) were present. These results support the hypothesis that an inflammatory response occurs in the gut during the first 24 hr following abdominal irradiation. Such changes may then further exacerbate the damage initiated by the ionizing radiation.

Effects of peptide YY on intestinal blood flow distribution and motility in the dog.

Previous investigators in our laboratory have demonstrated that peptide YY (PYY), a putative gut hormone, exerts a potent emetic effect when administered intravenously to conscious dogs. The current study was carried out to examine the effects of an emetic dose of PYY on cardiovascular status, splanchnic blood flow distribution (estimated using 15 micron microspheres) and small intestinal motility in anesthetized dogs. PYY, infused i.v. at a dose of 25 pmol/kg/min led to a localized significant reduction in small intestinal muscularis externa blood flow both 15 and 30 min after the start of PYY infusion in both jejunum and ileum. This decreased muscularis perfusion was not accompanied by any significant change in whole gut wall blood flow and was explained on the basis of an observed significant redistribution of blood flow away from the muscularis towards the mucosa/submucosa. Similar, although non-significant, effects of PYY on colonic blood flow distribution were also observed. Despite the effects on jejunum and ileum, PYY exerted minimal effects on duodenal blood flow. The decrease in ileal and jejunal muscularis blood flows was accompanied by a significant increase in the amplitude of intestinal contractions in these regions. Frequency of contractions was unaltered however. These results demonstrate that PYY infusion leads to concurrent changes in small intestinal blood flow and motility.

Ethanol-induced vascular permeability changes in the jejunal mucosa of the dog.

We have demonstrated previously that ethanol causes alterations in jejunal morphology and fluid secretion, and that these changes are accompanied by an increase in mucosal arteriolar blood flow, shunting of blood through noncapillary microvessels, hyperemia, hemoconcentration, and an enhanced loss of plasma protein into the gut lumen. All the microcirculatory changes are compatible with an ethanol-induced mucosal microvascular stasis. The present study was undertaken to examine whether these changes are accompanied by an enhanced mucosal microvascular permeability. Using a colloidal carbon vascular labeling technique, we demonstrated that 6% wt/vol intraluminal ethanol caused marked accumulation of carbon particles in the walls of the capillaries and microvenules of the villus tips. The effect of 3% ethanol was much less pronounced, and the extent of carbon entrapment did not significantly differ from microvessels in villi perfused with Krebs-Ringer solution without ethanol. Electron microscopy indicated that the carbon was localized between the endothelial cell and the basal lamina of the microvessels. Because the carbon accumulation was always localized at the vicinity of endothelial cell junctions, the escape of carbon particles from the lumen probably occurred through these cell junctions. This could have been the result of a transient retraction of endothelial cells. Electron microscopic examination also demonstrated that the lumina of some capillaries and venules of the villus tip of jejunal segments perfused with 6% ethanol were clogged with carbon particles. This latter finding indicates that there was stasis in these vessels at the time of carbon injection. Similar intraluminal carbon accumulation was never seen in control jejunum or jejunum perfused with 3% ethanol. Thus the results of this study indicate that 6% (wt/vol) ethanol causes microvascular injury and stasis. The findings with 6% ethanol are reminiscent of those described by others in the early phases of acute inflammation, and thus it is possible that the microvascular changes caused by ethanol may be related to the liberation of some of the chemical mediators of the immediate response to acute injury.

Ethanol-induced mucosal microvascular stasis and enhanced plasma protein loss in the dog jejunum.

On the basis of previous studies in our laboratory, we have proposed that ethanol-induced alterations in the mucosal microcirculation, particularly vascular stasis, may be responsible for the genesis of the mucosal morphologic lesions and enhanced fluid secretion observed during acute perfusion of the jejunum with ethanol. In an attempt to substantiate this in the present study, we examined (a) the effects of acute intraluminal alcohol perfusion (3.0% and 6.0% wt/vol) on canine mucosal arteriolar blood flow, mucosal red blood cell volume, and mucosal "plasma volume" (as reflected by total mucosal albumin volume), and (b) whether ethanol induces an enhanced secretory filtration of plasma from the jejunal mucosal microvasculature into the gut lumen. These studies indicate that intraluminal perfusion of the jejunum with 6% ethanol leads to a significant (p less than 0.05) increase in both mucosal arteriolar blood flow and red blood cell volume. Despite these changes, mucosal "plasma volume" (total albumin volume) remained unaltered, indicating that hemoconcentration had occurred in the mucosa of the ethanol-perfused segments. These events could result in congestion or stasis within the blood vessels of the mucosa. Perfusion with 6% ethanol also caused a significant increase (p less than 0.05) in the loss of plasma proteins from the mucosal microvasculature into the gut lumen. These results are consistent with our previous proposal that ethanol leads to mucosal microvascular stasis and plasma transudation and that these conditions may in turn lead to the mucosal morphologic lesions and altered fluid transport induced by ethanol.

Effect of ethanol on morphology and total, capillary, and shunted blood flow of different anatomical layers of dog jejunum.

On the basis of previous studies in our laboratory we postulated that the ethanol-induced alteration in jejunal morphology was the result of its effect on the microcirculation. The present study was undertaken to examine the validity of this hypothesis. Accordingly, the effects of intraluminal ethanol perfusion (3.0 and 6.0% w/v) on mucosal morphology; water, glucose, and sodium transport; and regional blood flow were examined in in vivo jejunal segments of pentobarbital-anesthetized dogs. Compared to control segments, those perfused with ethanol exhibited a significant increase in the prevalence of morphological alterations of the mucosa, consisting of subepithelial fluid accumulation (bleb formation) and exfoliation. Those villi with epithelial damage exhibited villus cores significantly shorter than those with a normal, undamaged epithelium. Segments perfused with ethanol exhibited a depressed net water absorption, to the point that net secretion occurred in the segments perfused with 6% ethanol. Net absorption of glucose was similarly depressed by intraluminal perfusion with ethanol, whereas net absorption of sodium was unaffected. Regional jejunal blood flows were estimated using a dual, radiolabeled microsphere technique. Both total jejunal wall and total mucosal blood flow (in ml/min/100 g dry tissue) in the ethanol-perfused segments were significantly increased over control. Similarly, jejunal wall and mucosal capillary blood flows were increased by ethanol perfusion. Neither submucosal nor muscularis blood flows were affected by intraluminal perfusion with ethanol. Compared to control, shunting or nonentrapment of 9-micron microspheres was increased in the mucosa of the ethanol-perfused segments. In contrast to this, shunting of 9-micron microspheres in the submucosa and muscularis was unaffected by intraluminal perfusion with ethanol. It therefore appears that the ethanol-induced mucosal morphological alterations are accompanied by a localized mucosal hyperemia, and an increased shunting of blood through the mucosa. Based on the results of this and other studies, a microvascular mechanism was tentatively proposed to explain the pathogenesis of the ethanol-induced morphological changes.

Simultaneous estimation of arteriolar, capillary, and shunt blood flow of the gut mucosa.

Microspheres of 15 and 9 microns diameter were injected simultaneously into the left ventricle of the dog, and the entrapment of these microspheres in different layers of the gut wall was measured under resting conditions, vasoconstriction, and subsequent vasodilation. Results show that some of the 9-microns spheres passed into the portal blood through all layers of the gut wall, whereas 15-microns spheres were completely entrapped in the tissue. The pattern of entrapment of 15-microns spheres during vasoconstriction and during subsequent vasodilation suggests that these microspheres measure adequately the arteriolar inflow of the muscularis, submucosa, and mucosa (i.e., villus plus crypt) but, due to series arrangement between the arterioles of the villus and the crypt, cannot measure the arteriolar flow of the villus and crypt separately. The entrapment of 9-microns microspheres in the muscularis, submucosa, crypt, and villus that occurred during vasoconstriction did not change during subsequent vasodilation. This suggests that these microspheres became lodged in the precapillary sphincter or capillary and therefore measured the capillary flow of these layers. Accordingly, the difference between the arteriolar (measured by 15 microns) and the capillary (measured by 9 microns) flow of the muscularis, submucosa, and mucosa may provide an estimate of the noncapillary (shunt) flow of these layers.

Effect of ethanol on jejunal regional blood flow in the rabbit.

The effects of intraluminal ethanol perfusion (3.0% and 6.0% vt/vol) on mucosal morphology, water transport, and regional blood flow were examined in in vivo jejunal segments of pentobarbital-anesthetized rabbits. Compared with control segments, ethanol-perfused segments exhibited morphological alterations of the mucosa consisting of subepithelial fluid accumulation (bleb formation), exfoliation of enterocytes, and vascular congestion. The prevalence of epithelial damage was significantly increased in the segments perfused with 6% ethanol. Net water transport was significant (p less than 0.025) depressed in segments perfused with 3.0% and 6.0% wt/vol ethanol. In animals in which the control segment was absorbing water, ethanol led to a depression in net water absorption or to the reversal of absorption to net secretion. In animals in which the control segment exhibited secretion, ethanol led to an enhanced net secretion. Blood flow through the total jejunal wall and through the luminal layer (consisting of mucosa plus submucosa) was significantly (p less than 0.05) increased by the presence of 3.0% and 6.0% wt/vol ethanol in the intestinal lumen. Blood flow in the external layer of the jejunum (consisting of muscularis plus serosa) did not change significantly. It therefore appears that the ethanol-induced alterations in jejunal mucosal morphology and water transport are accompanied by a localized mucosal or submucosal hyperemia, or both. However, a direct cause and effect relationship between these remains to be established.