Robert D. Specian

Hemorrhagic shock induces bacterial translocation from the gut.

Sepsis and multiple organ failure are common after hemorrhagic shock. The goal of the current experiments was to determine whether hemorrhagic shock would promote the translocation of bacteria from the gut to visceral organs. Twenty-four hours after being subjected to sham shock, or 30, 60, or 90 minutes of shock (30 mm Hg), rats were sacrificed and their organs quantitatively cultured for translocating bacteria. There was a direct relationship between the duration of hemorrhagic shock and the 24-hour mortality rate (p = 0.02). Bacteria did not translocate from the gut in the sham-shock rats, but did translocate to the mesenteric lymph nodes, livers, and spleens of the rats subjected to hemorrhagic shock (p less than 0.01). Rats subjected to 90 minutes of shock shock exhibited a greater degree of bacterial translocation than rats receiving 30 or 60 minutes of shock (p less than 0.05). The most common translocating bacteria were Escherichia coli and Enterococcus. Hemorrhagic shock injured the gut mucosa and caused subepithelial edema and focal areas of necrosis. Thus hemorrhagic shock followed by reinfusion of shed blood disrupts the gut barrier and allows indigenous bacteria normally contained within the gut to cause systemic infections.

Obstructive jaundice promotes bacterial translocation from the gut

Experiments were performed to determine if obstructive jaundice promotes the translocation of bacteria from the gastrointestinal tract to visceral organs. Three groups of mice were studied: control (n = 20), sham ligated (n = 28), and bile duct ligated (n = 33). The sham-ligated group underwent laparotomy and manipulation of the portal region, whereas the ligated group had their common bile ducts ligated. Seven days later, the mice were killed, their organs cultured, and the gastrointestinal tract examined histologically. The bilirubin levels of the ligated group (18.7 mg/dL) were elevated compared with the other groups (0.5 mg/dL) (p less than 0.05). The incidence of bacterial translocation was higher in the ligated (33%) than in the control (5%) or sham-ligated (7%) groups (p less than 0.05). Since bile is important in binding endotoxin and maintaining a normal intestinal microflora, cecal bacterial populations were quantitated. The cecal levels of gram-negative, enteric bacilli were 100-fold higher in the bile duct-ligated mice in which bacterial translocation occurred (p less than 0.05), indicating that intestinal bacterial overgrowth was a major factor responsible for bacterial translocation. The mucosal appearance of the intestines from the control and sham-ligated groups was normal. In contrast, subepithelial edema involving the ileal villi was present in the ligated group. In conclusion, the absence of bile within the gastrointestinal tract allows intestinal overgrowth with enteric bacilli and the combination of bacterial overgrowth and mucosal injury appears to promote bacterial translocation.

Mechanisms of acute and chronic intestinal inflammation induced by indomethacin

The objective of this study was to characterize the mechanisms of acute and chronic intestinal mucosal injury and inflammation induced by subcutaneously injected indomethacin (Indo). One injection of Indo (7.5 mg/kg) produced acute injury and inflammation in the distal jejunum and proximal ileum that were maximal at three days and completely resolved within one week. Two daily subcutaneous injections of Indo produced a more extensive and chronic inflammation that lasted in an active form in more than 75% of the rats for at least two weeks. Epithelial injury, as measured by enhanced mucosal permeability, was significantly elevated only at one day in the acute model (one injection) but was persistently elevated in the chronic model (two injections). Bile duct ligation completely attenuated increased mucosal permeability in the acute model, however, depletion of circulating neutrophils had no effect. Neither Indo (0–0.1 mg/ml) nor normal bile was cytotoxic to cultured rat intestinal epithelial cells; however, they synergistically promoted significant cytotoxicity. Bile collected from rats treated with Indo was cytotoxic towards the epithelial cells in a dose-dependent manner. Sulfasalazine and metronidazole (100 mg/kg/day, both) attenuated enhanced mucosal permeability in the chronic model. Massive bacterial translocation into the mesenteric lymph nodes, liver, and spleen following two injections of Indo was significantly attenuated by metronidazole. We conclude that: (1) a single injection of Indo produces acute intestinal mucosal injury and inflammation that resolve completely within three to seven days, whereas two daily injections of Indo produce both acute and chronic injury and inflammation, (2) enterohepatic circulation of Indo is important in promoting the acute phases of injury and inflammation, (3) circulating neutrophils do not play a role in the pathogenesis of this model, and (4) endogenous bacteria play an important role in exacerbating and/or perpetuating the chronic phases of injury and inflammation.

A comparative analysis of two models of colitis in rats.

Two models of colitis produced in rats that have received significant attention over the past few years are the acetic acid and trinitrobenzene sulfonic acid (TNBS) models. The objective of this study was to quantify and compare the temporal relationship among mucosal permeability, epithelial injury, and inflammation induced by acetic acid, ethanol (vehicle), ethanol plus TNBS (unbuffered, pH 1.0), and ethanol plus TNBS (pH 7.4). Data obtained show that the inflammation induced by these four irritants results from caustic injury to the colonic epithelium and interstitium as measured by the rapid and dramatic increases in mucosal permeability and tissue water content as well as by histological analysis. The injurious nature of TNBS was confirmed in a separate series of studies showing that buffered TNBS (pH 7.4), in the absence of ethanol, is toxic to cultured rat intestinal epithelial cell monolayers. Only after 1-2 days of the initial insult, were signs of classical inflammation observed, including increases in colonic myeloperoxidase activity (neutrophil infiltration) and colon weight as well as hyperemia and mucosal ulcerations. Although ethanol plus TNBS (pH 1.0 or 7.4) tended to produce higher mucosal permeabilities (epithelial cell injury) at 1-2 weeks after the enemas than acetic acid or ethanol groups, only the ethanol plus TNBS (pH 7.4) permeabilities were found to be significantly enhanced. In addition, all four groups showed significant elevations in colonic myeloperoxidase activity and colon weight at 1-2 weeks after enema. It is suggested that these models of colitis are useful to study events that occur at the time of inflammation and repair. However, these models may have significant limitations in understanding events that initiate inflammation of the intestine in human inflammatory bowel disease.

Effect of hemorrhagic shock on bacterial translocation, intestinal morphology, and intestinal permeability in conventional and antibiotic-decontaminated rats.

Bacterial translocation and ileal and cecal injury have been shown to occur 24 h after limited periods of hemorrhagic shock. The present studies were performed to determine the temporal sequence of mucosal injury, permeability, and bacterial translocation after hemorrhagic shock. The results indicated that bacterial translocation and mucosal injury have occurred by 2 h after a 30-min episode of shock (mean arterial pressure 30 mm Hg). Although the histologic extent of the intestinal mucosal injury was less at 2 h postshock than at 24 h postshock, at both times intestinal barrier function was lost as measured by permeability to horseradish peroxidase. Since the role of translocating bacteria in potentiating the loss of intestinal barrier function after shock is unclear, the second goal was to determine whether the extent of shock-induced mucosal injury and permeability could be reduced or abrogated by antibiotic decontamination of the gut. The extent of shock-induced mucosal injury and intestinal permeability was similar between rats with a normal gut flora (greater than 10(6) bacteria/g cecum) and antibiotic-decontaminated rats (less than 10(3) bacteria/g cecum) 2 h postshock, although the incidences of bacterial translocation were 67% and 0, respectively. Thus, shock-induced mucosal permeability and injury appear not to be directly related to the presence of translocating bacteria.

Mucosal injury and inflammation in a model of chronic granulomatous colitis in rats

BACKGROUND The objective of this study was to characterize the acute and chronic inflammation induced by the intramural injection of peptidoglycan-polysaccharide (PG-PS) into the distal colon of genetically susceptible rats. METHODS Blood-to-lumen clearance of 51Cr-ethylenediaminetetraacetic acid, colonic myeloperoxidase activity, colon weight, and plasma nitrite and nitrate levels were determined to quantitate colonic mucosal injury, inflammation, and nitric oxide (NO) production, respectively. RESULTS Intramural injection of PG-PS into the distal colon produced a local biphasic inflammatory response composed of an acute episode 3 days after injection; this was followed by a spontaneous reactivation of chronic granulomatous colitis manifested by colonic thickening, adhesions, and infiltration of the submucosa and muscularis propria with macrophages, neutrophils, and lymphocytes at 3-4 weeks. Mucosal ulcers were evident only at 3 weeks, but hepatic nodules, splenic necrosis, and arthritis were evident at both 3 and 4 weeks after PG-PS injection. PG-PS produced significant increases in colonic mucosal permeability, myeloperoxidase activity, and plasma nitrite and nitrate levels at 3 weeks postinjection compared with controls. PG-PS stimulated the production of nitrite by elicited peritoneal macrophages and neutrophils in vitro. CONCLUSIONS PG-PS produces a chronic granulomatous colitis in rats; this colitis is characterized by enhanced NO production.

Ethanol-Induced Injury to the Rat Gastric Mucosa Role of Neutrophils and Xanthine Oxidase-Derived Radicals

Ethanol-induced gastric mucosal injury closely resembles an inflammatory response. Thus, in vivo and in vitro experimental models were used to assess whether ethanol is proinflammatory in concentrations likely to be encountered by the gastric mucosa during acute intoxication. Perfusing the rat gastric lumen with progressively increasing concentrations of ethanol (10%, 20%, and 30%) resulted in a dose-dependent increase in 51Cr-ethylenediaminetetraacetic acid clearance from blood-to-gastric lumen. Rendering the animals neutropenic (with antineutrophil serum) ameliorated the ethanol-induced mucosal injury; the degree of protection was directly related to the severity of neutropenia. Neither superoxide dismutase, catalase, nor sodium benzoate offered any protection against ethanol-induced injury, indicating that neither superoxide anion, hydrogen peroxide, nor the hydroxyl radical is involved. To assess further whether ethanol could exert proinflammatory effects an in vitro model consisting of cultured bovine microvascular endothelial cells and isolated human neutrophils was used. Ethanol at concentrations of 1.0%-4.0% (but not at 0.1%-0.5%) increased neutrophil adherence to endothelial cells and enhanced neutrophil-mediated endothelial cell injury. We conclude that ethanol is proinflammatory at concentrations that may be achieved in the gastric mucosa during acute intoxication. The ethanol-induced, neutrophil-mediated cell injury does not appear to involve oxy radicals.

Endotoxin-induced bacterial translocation and mucosal permeability: role of xanthine oxidase, complement activation, and macrophage products.

Background and MethodsPreviously, we documented that nonlethal doses of endotoxin injure the intestinal mucosal barrier and promote bacterial translocation from the gut to systemic organs. The current study was performed to determine the role of cytokines and complement activation in the pathogenesis of endotoxin-induced mucosal injury and bacterial translocation, as well as to quantify the magnitude of endotoxin-induced intestinal mucosal permeability. ResultsThe frequency of endotoxin-induced bacterial translocation was similar between normal outbred (88%), complement deficient (67%), and macrophage-hyporesponsive (55%) mice, indicating that neither complement nor macrophage activation is necessary for endotoxin-induced bacterial translocation to occur. As early as 2 hrs after endotoxin challenge, there was evidence of a greater than two-fold increase in ileal (p = .008) but not jejunal (p = .11) permeability as measured by the clearance of 51Cr EDTA. Both the increase in endotoxin-induced ileal permeability and the occurrence of bacterial translocation were largely prevented by pretreatment with allopurinol, a competitive inhibitor of xanthine oxidase. ConclusionsThese results suggest that endotoxin-induced bacterial translocation, mucosal injury, and ileal permeability are mediated via activation of xanthine oxidase, and not through complement activation or the liberation of macrophage products.

Hemorrhagic shock-induced bacterial translocation: the role of neutrophils and hydroxyl radicals.

We previously documented a relationship between xanthine oxidase activation, intestinal injury, and bacterial translocation (BT) in rats subjected to hemorrhagic shock. The current experiments were performed to determine the relative roles of hydroxyl radicals and neutrophils in the pathogenesis of shock-induced mucosal injury and BT. The incidence of BT was higher in the shocked rats (30 mm Hg for 30 min) than the sham-shock controls (87% vs 12.5%; p less than 0.01). Administration of the hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), or the iron chelator, deferoxamine, reduced the incidence of BT from 87% to 20% and 40%, respectively (p less than 0.05). DMSO and deferoxamine appear to prevent shock-induced BT by blunting the magnitude of shock-induced mucosal injury. In contrast, neutrophil depletion did not prevent BT or protect the intestinal mucosa in shocked rats. Instead, the incidence of systemic spread of translocating bacteria past the mesenteric lymph nodes to the livers and spleens of the shocked rats was higher in the neutrophil-depleted rats (56%) than in any other group (p less than 0.01). Thus, shock-induced BT and intestinal injury appear to be mediated by oxidants (.OH) derived from xanthine oxidase, rather than granulocytes.

Microvascular Responses to Inhibition of Nitric Oxide Production: Role of Active Oxidants

The objective of this study was to assess the potential contribution of hydrogen peroxide (H2O2) to the leukocyte-endothelial cell adhesion and increased microvascular permeability observed in rat mesenteric venules after inhibition of nitric oxide synthesis with NG-nitro-L-arginine methyl ester (L-NAME). Leukocyte adherence and emigration and leakage of fluorescein isothiocyanate-labeled albumin were monitored in postcapillary venules before and after exposure of the tissue to L-NAME. H2O2 production in mesenteric tissue was monitored by using dihydrorhodamine 123 (DHR), the H2O2-sensitive fluorochrome. L-NAME elicited a rapid increase in both the rate of albumin extravasation and oxidation of DHR, which was followed by an increased adherence and emigration of leukocytes in postcapillary venules. Treatment with either catalase or dimethylthiourea attenuated the L-NAME-induced oxidative stress, albumin leakage, and leukocyte-endothelial cell adhesion. Oxidation of DHR was enhanced in animals treated with either 3-amino-1,2,4-triazole (ATZ), an inhibitor of endogenous catalase, or a combination of ATZ and maleic acid diethyl ester, which depletes intracellular glutathione. Animals receiving a CD11/CD18-specific antibody to prevent leukocyte adhesion/emigration exhibited a reduced oxidation of DHR in response to L-NAME. These findings indicate that most of the H2O2 (and secondarily derived oxidants) generated in mesenteric tissue exposed to an inhibitor of nitric oxide production is due to accumulation of activated leukocytes.