Zymosan-induced bacterial translocation: a study of mechanisms.

BACKGROUND AND METHODS: At nonlethal doses, zymosan induces a systemic inflammatory state and promotes bacterial translocation. This study was performed to investigate the mechanisms by which zymosan causes intestinal mucosal injury and bacterial translocation. Bacterial translocation to the mesenteric lymph node was measured 24 hrs after intraperitoneal challenge with saline or zymosan (0.1 mg) in normal (CD-1), congenitally macrophage-hyporesponsive (C3H/HeJ), complement-deficient (DBA/2), or mast cell-deficient (W/Wv) mice. Since zymosan-induced bacterial translocation may be mediated by xanthine oxidase-generated oxidants, bacterial translocation was measured in mice pretreated with the xanthine oxidase inhibitor, allopurinol. To further investigate the role of oxidants in zymosan-induced bacterial translocation, ileal and hepatic levels of xanthine oxidase, myeloperoxidase, conjugated dienes, malondialdehyde, and the antioxidants--superoxide dismutase, catalase, and glutathione peroxidase, were measured. RESULTS: Zymosan-induced mucosal injury and bacterial translocation occurred to a similar extent (p less than .05) in all four genetic strains of mice, but were reduced in the mice pretreated with allopurinol. Zymosan increased (p less than .03) ileal and hepatic xanthine oxidase activity, while reducing (p less than .01) antioxidant (catalase) activity. There was also evidence of hepatic, but not ileal, lipid peroxidation (conjugated diene) (p less than .05) and neutrophil sequestration (myeloperoxidase) (p less than .01). CONCLUSIONS: Zymosan-induced intestinal mucosal injury and bacterial translocation do not require complement activation, or the release of macrophage or mast cell products. They appear to be mediated by xanthine oxidase-generated products and associated with disruption of the normal ileal and hepatic oxidant-antioxidant balance.     

Inhibition of endotoxin-induced bacterial translocation in mice.

The primary functions of the gut are to absorb nutrients and exclude bacteria and their products. However, under certain circumstances the gut may lose its barrier function and serve as a reservoir for systemic microbial infections. These experiments were performed to determine the mechanisms whereby endotoxin causes bacteria to escape (translocate) from the gut. Bacteria translocated from the gut to the mesenteric lymph nodes of mice challenged with nonlethal doses of Escherichia coli 026:B6 or E. coli 0111:B4 endotoxin. Physical disruption of the gut mucosal barrier appears to be the primary mechanism whereby endotoxin promotes bacterial translocation. Mucosal injury and endotoxin-induced bacterial translocation were reduced by inhibition (allopurinol) or inactivation (tung-sten diet) of xanthine oxidase activity (P less than 0.01), but were not affected by the platelet-activation factor antagonists, SRI 63-441 or BN 52021. Because the inhibition or inactivation of xanthine oxidase activity reduced both the extent of mucosal injury and endotoxin-induced bacterial translocation, the effect of endotoxin on the gut appears to be mediated, at least to some degree, by xanthine oxidase-generated, oxygen-free radicals.     

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.     

Poly(adp-ribose) synthetase inhibition reduces bacterial translocation in rats after endotoxin challenge

We investigated whether 3-aminobenzamide (3-AB), a poly(ADP-ribose) synthetase (PARS) inhibitor, reduces bacterial translocation (BT) after intraperitoneal endotoxin administration. Wistar rats were randomized to receive intraperitoneal saline (control, n = 6); endotoxin (n = 8); 3-AB (n = 6); and 3-AB plus endotoxin (n = 8). Six hours later, to evaluate the endotoxin-related intestinal injury and BT, tissue and blood samples were collected. Administration of intraperitoneal endotoxin caused severe intestinal injury and BT to mesenteric lymph nodes. PARS inhibition with 3-AB completely prevented endotoxin-induced BT. No colony-forming bacteria was isolated from the samples obtained from 3-AB-pretreated animals under endotoxin challenge. Treatment with 3-AB significantly reduced the endotoxin-induced intestinal mucosal injury. The inhibition of PARS by its blocker 3-aminobenzamide during endotoxemia prevents bacterial translocation and intestinal injury in rats. PARS activation may provide a novel therapeutic approach in reducing gut barrier failure seen in endotoxemia.     

Inducible nitric oxide synthase gene knockout mice have increased resistance to gut injury and bacterial translocation after an intestinal ischemia-reperfusion injury

OBJECTIVE: Intestinal ischemia-reperfusion after severe shock states is often associated with bacterial translocation and intestinal barrier dysfunction. Our previous studies showed that inducible nitric oxide synthase (iNOS) gene knockout mice were resistant to endotoxin-induced bacterial translocation and ileal mucosal damage. The goal of this study was to test whether iNOS mediates bacterial translocation after intestinal ischemia-reperfusion, using iNOS knockout mice (iNOS-/-) and their wild-type littermates (iNOS+/+). DESIGN: Prospective animal study with concurrent controls. SETTING: Small animal laboratory. SUBJECTS: Thirty-eight iNOS knockout mice and 51 wild-type littermates. INTERVENTIONS: iNOS+/+ mice or iNOS-/- mice were subjected to a sham operation or 30 mins of superior mesenteric artery occlusion followed by reperfusion. Twenty-four hours after reperfusion, bacterial translocation to mesenteric lymph nodes, ileal villous damage, and cecal bacterial population were evaluated. MEASUREMENTS AND MAIN RESULTS: Sham operation did not induce bacterial translocation, change cecal bacterial population levels, or cause ileal villous damage. Intestinal ischemia-reperfusion caused bacterial translocation in 72% of the iNOS+/+ mice but only 28% of the iNOS-/- mice. Both iNOS+/+ and iNOS-/- mice subjected to superior mesenteric artery occlusion (SMAO) in which bacterial translocation occurred had cecal bacterial population levels that were three logs higher than mice subjected to sham SMAO or mice subjected to SMAO in which bacterial translocation did not occur. The magnitude of villous injury was less in the iNOS-/- mice than the iNOS+/+ mice after SMAO, although the incidence of ileal villous damage was significantly higher in both the iNOS+/+ and iNOS-/- mice in which bacterial translocation occurred after SMAO than in the mice in which bacterial translocation did not occur after SMAO. iNOS+/+ mice subjected to SMAO had increased plasma concentrations of nitrite (NO2-) and nitrate (NO3-), and the plasma concentrations of NO2- and NO3- were highest in the mice in which bacterial translocation had occurred. CONCLUSION: iNOS knockout mice were more resistant to intestinal ischemia-reperfusion-induced bacterial translocation and mucosal injury than wild-type mice, suggesting that iNOS might play a role in intestinal ischemia-reperfusion-induced loss of gut barrier function.     

Increase in endotoxin-induced mucosal permeability is related to increased nitric oxide synthase activity using the Ussing chamber.

OBJECTIVE: To determine if nitric oxide production is associated with increased intestinal permeability after endotoxin challenge using the ex vivo Ussing chamber. SUBJECTS: Ileal mucosal membranes harvested from normal rats weighing 300 to 420 g. INTERVENTIONS: Endotoxin (lipopolysaccharide), 1, 10, 100 microg/ mL, or saline was placed on the serosal side of ileal mucosal membranes mounted in Ussing chambers after 10(9) Escherichia coli C-25 had been placed on the mucosal side of the ileal membranes (n = 6-7/group). In a second set of experiments, ileal membranes were exposed to 100 microg/mL lipopolysaccharide with or without the addition of the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine at a concentration of 10 mM (n = 7-8/group). MAIN OUTCOME MEASURE: Bacterial translocation of E. coli C-25 from the mucosal to the serosal side of the ileal membrane was measured every hour during the 3-hr experimental period, as were serial measurements of the potential difference and resistance values of the ileal membranes. At the conclusion of the 3-hr period, the ileal membranes were harvested and levels of inducible nitric oxide synthase and constitutive nitric oxide synthase activity were measured. RESULTS: The incidence of E. coli C-25 passage across the ileal membranes mounted in the Ussing chambers was significantly increased in the ileal membranes exposed to 10 or 100 microg/mL of lipopolysaccharide (71% and 86%, respectively) vs. the control membranes (0%) or the membranes exposed to 1 microg/mL of lipopolysaccharide (0%) (p < .05). This increase in E. coli C-25 passage in the ileal membranes exposed to 10 or 100 microg/mL of lipopolysaccharide was associated with a decrease in ileal membrane resistance and an increase in inducible nitric oxide synthase activity (p < .05). The addition of N(G)-monomethyl-L-arginine protected against lipopolysaccharide-induced bacterial translocation and prevented the lipopolysaccharide-induced increase in ileal membrane inducible nitric oxide synthase activity. CONCLUSION: These results indicate that lipopolysaccharide induction of increased ileal inducible nitric oxide synthase activity is necessary for lipopolysaccharide-induced E. coli C-25 translocation to occur in normal ileal mucosal membranes tested in the Ussing chamber system.     

Nitric oxide directly impairs intestinal barrier function

Excess production of nitric oxide (NO) has been implicated in endotoxin-induced loss of gut barrier function in vivo. Thus, we tested the direct effect of NO on the barrier function of intestinal mucosal membranes suspended ex vivo in Ussing chambers and on IEC-6 enterocyte monolayers. In these experiments, ex vivo-mounted ileal membranes or IEC-6 cell enterocyte monolayers were exposed to the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) over a dose range (10 microm to 2 mM) or medium. SNAP at concentrations of 1 or 2 mM, but not 10 or 100 microM, increased the rates of bacterial translocation (BT) across both the ileal membranes and the IEC-6 monolayers by >1 log (P < 0.05), as well as the permeability of the IEC-6 monolayers to phenol red (P < 0.05). The ileal membranes exposed to 1 or 2 mM SNAP for 3 h manifested histologic evidence of mucosal injury and decreases in electrical resistance and potential difference values (P < 0.05), while the IEC-6 cells exposed to SNAP for 18 h had increased levels of cell death (P < 0.05). Since NO produced locally by stimulated enterocytes could contribute to barrier dysfunction, NO production, iNOS mRNA levels, and monolayer permeability were measured in enterocytes (IEC-6 and Caco-2) exposed to medium, endotoxin (lipopolysaccharide [25 microg/mL]) or a cytokine mixture (IL-1beta 10 ng/mL, TNF-alpha 10 ng/mL, and INF-gamma 250 U/mL) for 6 or 24 h. Endotoxin increased NO production, iNOS mRNA expression, and monolayer permeability in the IEC-6, but not the Caco-2 cells, while exposure to the cytokine mixture increased both NO production, iNOS mRNA expression, and monolayer permeability in both the IEC-6 and Caco-2 cell lines. Based on the results of these studies it appears that NO can directly increase ileal mucosal membrane and enterocyte monolayer permeability and BT and that increased NO production and iNOS mRNA expression is associated with endotoxin- and/or cytokine-induced loss of enterocyte monolayer barrier function.     

Changes in gut mucosal nitric oxide synthase (NOS) activity after thermal injury and its relation with barrier failure

This study was designed to investigate changes in mucosal NOS activity after burns and its relation to barrier failure. In Experiment 1, female specific pathogen free (SPF) Sprague-Dawley rats underwent 35% total body surface area (TBSA) burn. One to six days after burn, intestinal permeability was determined from the plasma leakage of fluorescein isothiocyanate (FITC)-dextran 4400, intestinal mucosal cNOS and iNOS activity were assayed using Griess' reagent, and the cellular localization of iNOS was examined using immunostaining. In Experiment 2, S-methylisothiourea (SMT) was given (5 mg/kg, i.p. every 12 h) for 2 days to suppress inducible NOS (iNOS) activity after thermal injury. On postburn Day 2, the effect of SMT on gut mucosal NOS activity, intestinal permeability, and barrier function were evaluated. The activity of iNOS increased 24 h after the injury and up to a maximum of twofold on postburn Day 2, and decreased thereafter. The increase in iNOS activity in gut mucosa correlated well with the increase in intestinal permeability, an index for barrier failure (r = .776, p = .0002). Results from iNOS immunostaining showed that changes in mucosal iNOS activity after the burn occurred mainly in the enterocytes rather than in the macrophages. Administration of SMT decreased mucosal iNOS activity, intestinal permeability, and bacterial translocation incidence to mesenteric lymph node concurrently. In conclusion, thermal injury induces intestinal mucosal iNOS, which is principally in the enterocytes. The increased intestinal iNOS activity was closely related to barrier failure. SMT inhibited intestinal mucosal iNOS activity and prevented barrier failure as demonstrated by a decrease in BT occurrence and intestinal permeability.     

Trauma-shock-induced gut injury and the production of biologically active intestinal lymph is abrogated by castration in a large animal porcine model

Although small animal rodent studies indicate that there is a sexual dimorphism in the resistance to organ injury after trauma-hemorrhagic shock (T/HS), confirmatory studies are largely lacking in more clinically relevant large animal species. Thus, we tested the hypothesis that castration would reduce the susceptibility of adult minipigs to gut injury and abrogate the production of biologically active intestinal (mesenteric) lymph after T/HS. The hemodynamic response to T/HS was similar between castrated and noncastrated minipigs. Mesenteric lymph collected during the preshock period and in the trauma-sham shock (T/SS) animals did not have increased biological activity. However, T/HS-lymph from the noncastrated males increased the respiratory burst of normal neutrophils, increased endothelial cell monolayer permeability, and was cytotoxic for endothelial cells. Castration abrogated the T/HS-induced neutrophil-activating and endothelial-injurious activities of mesenteric lymph, and the biological activity of the T/HS-lymph from the castrated minipigs was not different from the T/SS animals. As compared with the T/SS minipigs, T/HS increased ileal mucosal injury and intestinal permeability. This increase in gut permeability after T/HS was manifest by in vivo bacterial translocation and by the increased passage of bacteria as well as permeability probes across intestinal segments when tested in the Ussing chamber system. In contrast, neither mucosal injury nor increased intestinal permeability was observed in the castrated minipigs subjected to T/HS. In summary, this large animal porcine study validates the notion that castration limits gut injury and the production of biologically active intestinal lymph after T/HS.     

Bile modulates intestinal epithelial barrier function via an extracellular signal related kinase 1/2 dependent mechanism

Objective Obstructive jaundice is frequently complicated by infections and has been associated with increased bacterial translocation and gut mucosal hyperpermeability in animal models. Proper expression of the tight junction (TJ) proteins ZO-1 and occludin is important for normal gut barrier function. We tested whether bile modulates intestinal epithelial ZO-1 and occludin expression.Animals (a) Male C57BL/6 mice; (b) male Sprague-Dawley rats.Interventions (a) Mice were subjected to common bile duct ligation (CBDL) or a sham procedure, and 96 h later all surviving animals were killed for measurement of ileal mucosal permeability to FITC-labeled dextran (everted gut sac technique), bacterial translocation to mesenteric lymph nodes, and ileal epithelial ZO-1 and occludin expression (western blots). (b) Rat IEC-6 enterocytic monolayers were incubated in the presence or absence of graded concentrations of rat bile and/or U0126, an inhibitor of extracellular signal related kinase (ERK) 1/2 activation.Results (a) Compared to sham-treated controls, CBDL significantly increased gut mucosal permeability and bacterial translocation and markedly decreased ileal epithelial expression of ZO-1 and occludin. In a follow-up in vivo experiment, gavaging mice with fresh rat bile twice daily significantly ameliorated the deleterious effects of CBDL on gut barrier function. (b) Addition of 1% (v/v) bile to media enhanced phosphorylation of ERK1/2, increased the expression of ZO-1 and occludin and decreased permeability to FITC-dextran. All of these bile-mediated effects were blocked by 10 µM U0126.Conclusions These data support the view that the presence of bile in the intestinal lumen is essential for normal gut barrier function, possibly because compounds present in bile initiate ERK1/2-dependent signaling that is essential for normal expression of key TJ proteins.This revised version was published online in April 2005 with a corrected section title.Grant support: 5R01 GM 37631-18 from the National Institutes of Health     

Indomethacin disrupts the protective effect of phosphatidylcholine against bile salt-induced ileal mucosa injury

BACKGROUND: Indomethacin (Indo) exerts local toxic effects on small intestinal mucosa, possibly in association with hydrophobic bile salts. We investigated the potential toxic effects of Indo on ileal mucosa and the role of phosphatidylcholine (PC). MATERIALS AND METHODS: Transmucosal resistance and Na-fluorescein permeability of ileal mucosa segments from female Wistar rats were determined in Ussing chambers during a 30-min incubation with model systems containing: control-buffer, taurodeoxycholate (TDC), Indo, TDC-Indo, TDC-PC, or TDC-PC-Indo. Decrease of resistance and increase of permeability were considered as parameters for mucosal injury. After incubation in Ussing chambers, the histopathology was examined to quantify the extent of mucosal injury. Also, in CaCo-2 cells, LDH-release was determined as a measure of cytotoxicity, after incubation with various model systems. RESULTS: Decrease of resistance and increase of permeability were highest in systems containing TDC-Indo (P < 0.01). Phosphatidylcholine protected against the cytotoxic effects of TDC in absence of Indo only. Extent of mucosal injury by histological examination was also highest in systems containing TDC-Indo (P = 0.006). Again, PC exhibited protective effects in absence of Indo only. The LDH-release by CaCo2-cells was strongest in TDC-Indo systems (P < 0.001). CONCLUSIONS: Indomethacin disrupts protective effects of PC against bile salt-induced ileal mucosa injury. This finding is relevant for small intestinal injury induced by non-steroidal anti-inflammatory drugs.     

A potential role of hyperbaric oxygen exposure through intestinal nuclear factor-kappaB

OBJECTIVE: Recent studies have demonstrated the therapeutic effectiveness and pharmacologic mechanisms of hyperbaric oxygen therapy (HBOT) in the treatment of a systemic shock state. To elucidate the in vivo role of HBOT during sepsis, we evaluated the effects of HBOT on intestinal mucosal injury and bacterial translocation after lipopolysaccharide challenge. DESIGN: Experimental study. SETTING: First Department of Surgery and Division of Emergency Care, Kagoshima University School of Medicine, Kagoshima, Japan. SUBJECTS:: Male rats were treated with lipopolysaccharide by an intraperitoneal route or with lipopolysaccharide and HBOT. INTERVENTIONS: The survival rate, small intestinal tissue damage, and bacterial translocation in the HBOT-treated group were compared with those in the untreated group. Moreover, plasma tumor necrosis factor-alpha and nitrite/nitrate concentrations, inducible nitric oxide synthase and myeloperoxidase activities, and nuclear factor-kappaB in ileal mucosa were investigated. HBOT was initiated 3 hrs after lipopolysaccharide challenge and administered as 100% oxygen, at 2.53 x 10 kPa (2.5 atm absolute), for 60 mins. MEASUREMENTS AND MAIN RESULTS: When a sublethal dose of lipopolysaccharide (24 mg/kg) was given, the survival rate was much better in the HBOT-treated group (75%) than in the untreated group (33%). HBOT given 3 hrs after lipopolysaccharide injection (10 mg/kg) also lessened the histologic tissue damage of the terminal ileum and the incidence and magnitude of bacterial translocation to mesenteric lymph nodes at 24 hrs after the lipopolysaccharide injection. Moreover, HBOT was able to reduce mucosal inducible nitric oxide synthase and myeloperoxidase activities and plasma nitrite/nitrate concentrations but not serum tumor necrosis factor-alpha concentrations. Immunohistochemical examination revealed that HBOT specifically modified the mucosal nuclear factor-kappaB activation within 4-6 hrs after the injection. CONCLUSIONS: HBOT performed 3 hrs after lipopolysaccharide challenge alleviates intestinal barrier dysfunction and improves survival rates. Herein, we propose one possible mechanism for these beneficial effects: HBOT can modify the nuclear factor-kappaB activation in the intestinal mucosa and attenuate the sequential nitric oxide overproduction and myeloperoxidase activation. Consequently, bacterial translocation could be potentially decreased. We believe that the present study should lead to an improved understanding of HBOT's potential role in sepsis.     

双歧杆菌对烫伤大鼠肠道黏膜机械及生物屏障的改善作用

目的 :探讨严重烫伤后补充外源性双歧杆菌对肠黏膜机械及生物屏障的保护作用及其对肠道细菌或内毒素移位的影响。方法 :70只 Wistar大鼠按随机数字表法分为烫伤对照组 ( BC)、烫伤治疗组 ( BT)和假伤对照组 ( NC)。BT组伤后灌饲双歧杆菌悬液 ( 5× 10 1 2 CFU/ L) 1.5 ml,2次 / d。观察细菌或内毒素移位、肠黏膜菌群和肠黏膜损伤情况等。结果 :1伤后 3 d,BC、BT组细菌移位率分别为 42 %和 16 % ( P=0 .0 0 40 ) ,5 d分别为30 %和 6 % ( P=0 .0 0 2 0 ) ;伤后 1d,BC组内毒素水平显著高于 BT组。 2 BC组回盲部黏膜菌群中双歧杆菌量仅为 NC组的 1/ 10～ 1/ 6 0 ,BT组双歧杆菌量明显增多 ;BC组大肠杆菌量在 1、3 d增加约 2 0～ 30倍 ,但 3 d后BT组大肠杆菌量显著减少并接近正常水平。 3回肠黏膜损伤以 1、3d最为明显 ,BC组伤后 5 d损伤评分仍明显高于 NC组 ( P<0 .0 5 ) ;BT组 3d时已明显修复 ,5 d已与 NC组无明显差异。结论 :补充外源性双歧杆菌能促进烫伤后受损肠黏膜屏障和生物屏障修复 ,有效防治肠道细菌或内毒素移位。     

Peritoneal lavage with oxygenated perfluorochemical preserves intestinal mucosal barrier function after ischemia-reperfusion and ameliorates lung injury

OBJECTIVE: To evaluate the effect of peritoneal lavage with an oxygenated perfluorochemical (PFC) on intestinal ischemia-reperfusion injury (IIR), we assessed intestinal barrier function in terms of bacterial translocation and endotoxemia, morphologic changes, and changes of intestinal luminal pH in rats subjected to IIR. We also examined lung injury after IIR to test the effect of oxygenated PFC lavage on remote organ failure. DESIGN: Prospective, randomized, and controlled animal study. SETTING: Laboratory of a university hospital. SUBJECT: Male Sprague-Dawley rats. INTERVENTIONS: Rats were subjected to ischemia by clipping the superior mesenteric artery. Reperfusion was achieved by release of the clip. Lavage of the abdominal cavity was performed by inflow and outflow of oxygenated PFC solution during ischemia. RESULTS: Rats undergoing peritoneal lavage with oxygenated PFC (PFC group) showed significantly better survival after IIR. The frequency of bacterial translocation and the endotoxin concentration in superior mesenteric venous blood were significantly lower in the PFC group. Luminal acidosis also was alleviated in the PFC group. Furthermore, PFC lavage preserved the intestinal mucosal architecture and inhibited interstitial edema and infiltration of inflammatory cells in the lungs. CONCLUSION: We conclude that peritoneal lavage with oxygenated PFC protects the intestinal mucosa and maintains mucosal barrier function after IIR. Preservation of the intestinal mucosa ameliorates lung injury after IIR.     

Neutrophil depletion in rats reduces burn-injury induced intestinal bacterial translocation

OBJECTIVE: To determine whether neutrophil depletion could eradicate intestinal bacterial translocation in bum-injured rats. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: The rats were intravenously administered a rabbit anti-rat neutrophil antibody causing profound neutropenia and subjected to a 30% total body surface area scald burn. MEASUREMENTS AND MAIN RESULTS: The depletion of neutrophils from the intestine was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates. In addition, the presence of activated/extravasated neutrophils in intact intestines was determined via immunohistochemical localization of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component protein p47phox. Bacterial translocation was measured using agar cultures and by determining Escherichia coli beta-galactosidase gene via polymerase chain reaction/Southern blot analyses of mesenteric lymph node and spleen, liver, lung, and blood. MPO measurements demonstrated a six-fold increase above the control value in the intestinal tissue in rats on day 1 postburn. The presence of activated neutrophils (expression of p47phox protein) was also markedly increased in the intestines of these rats. The increased MPO activity and p47phox expression accompanied a translocation of indigenous E. coli into the mesenteric lymph node without a spread to other organs. The administration of anti-neutrophil antibody to burn animals prevented an increase in MPO activity and bacterial translocation. CONCLUSION: These studies indicate that enhanced intestinal bacterial translocation caused by burn injury could be related to the increased infiltration of activated neutrophils into the intestinal tissue after bum. The release of neutrophil products such as superoxide anion may effect intestinal tissue damage leading to bacterial translocation of indigenous E. coli.     

Diamine oxidase (histaminase). A circulating marker for rat intestinal mucosal maturation and integrity.

Diamine oxidase (histaminase) is an enzyme found in high concentrations in the intestinal mucosa of humans and other mammalian species. We investigated whether plasma and mucosal levels of diamine oxidase activity reflect both the maturational status of the mucosa during its development in the newborn rate and the degree of mucosal damage during its injury in the adult rat. Litter mates were reared under identical conditions and killed at different ages from day 0 to day 40 after birth. Diamine oxidase in the small intestine was low at birth, increased gradually with age, reached a peak at 22 d, and then remained at normal adult levels, similar to the developmental patterns of maltase and sucrase. Plasma diamine oxidase rose in parallel with intestinal levels (n = 500, r = 0.84, P less than 0.001), reached a peak at 24 d, and then remained at normal adult levels. Diamine oxidase activity in 15 nonintestinal tissues was less than 5% of ileal mucosal activity, and no nonintestinal activities showed increase with age. Adult rat intestinal loops were perfused with hyperosmolar sodium sulfate solutions to produce selective damage to villus mucosa. With increasing mucosal damage, there was a progressive decrease in the enzyme activities studied; first, lactase levels fell, then maltase and sucrase, and finally mucosal and plasma diamine oxidase activity levels fell. The decrease in plasma diamine oxidase reflected the degree of mucosal damage (n = 29, P less than 0.04). Diamine oxidase activity is thus unique among intestinal mucosal enzymes studied to date in that circulating levels can serve as a marker of mucosal maturation and integrity.     

Effects of levosimendan, a novel inotropic calcium-sensitizing drug, in experimental septic shock.

OBJECTIVE: Levosimendan is a novel inodilator that improves cardiac contractility by sensitizing troponin C to calcium. This drug has proved to be effective in treating advanced congestive heart failure but has not been evaluated in septic settings. The purpose of the present study was to study the effects of this drug in a porcine model of endotoxemia. DESIGN: Prospective experimental study. SUBJECTS: Fourteen landrace pigs. INTERVENTIONS: All animals were anesthetized and catheterized for measurement of central and pulmonary hemodynamics. Ultrasonic flow probes were placed around the renal artery and portal vein to measure blood flow. A tonometer was placed in the ileum to measure mucosal pH. Levosimendan was given to six animals as a bolus (200 microg x kg(-1)) followed by a continuous infusion (200 microg x kg(-1) x hr(-1)). Thirty minutes after onset of levosimendan treatment, all animals received endotoxin (20 microg x kg(-1) x hr(-1) for 3 hrs). MEASUREMENTS AND MAIN RESULTS: At baseline, levosimendan induced a systemic vasodilation with a reduction in blood pressure and an increase in heart rate. A tendency to an increase in cardiac index did not reach statistical significance (p =.055). Cardiac index and systemic oxygen delivery were markedly improved in the levosimendan group during endotoxemia. Systemic vascular resistance and blood pressure were reduced in the levosimendan group. The latter parameter, however, was only different from the control group during the initial phase of endotoxin shock but not at the late, most pronounced phase of shock. Levosimendan also efficiently attenuated endotoxin-induced pulmonary hypertension. Portal venous blood flow and gut oxygen delivery were improved, but no concomitant reduction in endotoxin-induced intestinal mucosal acidosis was observed. Renal blood flow was unaffected, as was the endotoxin-induced increase in plasma endothelin-1-like immunoreactivity. These findings support previous reports of calcium desensitization as a potential component in septic myocardial depression. Furthermore, the vasodilatory properties of this drug were well tolerated in the current model of hypodynamic endotoxin shock, and they may have contributed to improved regional blood flow as seen in the gut as well as improved systemic perfusion by means of reduced biventricular afterload. CONCLUSION: Pretreatment with levosimendan in pigs subjected to endotoxin shock improved cardiac output and systemic and gut oxygen delivery. In addition, pulmonary hypertension largely was attenuated without any adverse effects on gas exchange. These results are promising in several aspects, but the role of levosimendan in the treating circulatory failure in sepsis remains to be established.     

Recombinant human activated protein C attenuates endotoxin-induced lung injury in awake sheep

Introduction  

Acute lung injury often complicates severe sepsis. In Gram-negative sepsis, bacterial endotoxin activates both coagulation and inflammation. Enhanced lung vascular pressures and permeability, increased extravascular lung water content and deteriorated gas exchange characterize ovine endotoxin-induced lung injury, a frequently used model of acute lung injury. Recombinant human activated protein C (rhAPC), with its anticoagulant, anti-inflammatory, fibrinolytic and antiapoptotic effects, reportedly reduces the respirator-dependent days and the mortality of patients with severe sepsis. We speculate whether rhAPC antagonizes endotoxin-induced lung injury in sheep.     

Nitric oxide synthase inhibitor ameliorates oral total parenteral nutrition-induced barrier dysfunction

The expression of inducible nitric oxide synthase (iNOS) is increased in the intestine and results in mucosal damage after endotoxin challenge. Although the oral administration of total parenteral nutrition (TPN) solution promotes bacterial translocation (BT) and increases the intestinal permeability, the role of NO in the nutrition-induced loss of mucosal barrier function remains unclear. The distribution of fluorescein isothiocyanate-dextran (FITC-dextran, 4400) across the lumen of small intestine in rat was examined to investigate the role of NOS activity on the intestinal permeability under oral TPN feeding. Fifty-one rats were randomly divided into 4 groups. Group I (control group) was fed with rat chow, group II received TPN solution orally. Groups III and IV received TPN solution supplemented with NOS inhibitors. On day 9, FITC-dextran was injected into the intestinal lumen. After 30 min, blood samples were taken from portal vein and analyzed for plasma FITC-dextran level by fluorescence spectrophotometry. Samples of small intestine were frozen and sectioned in a cryostat for morphological and NOS histochemical studies. Homogenates of small intestine were used for NOS activity measurement. The plasma level of FITC-dextran showed a significant increase (P < 0.05) in rats fed with oral TPN compared with the control ones. Supplement with NOS inhibitors significantly decreased the intestinal permeability in groups III and IV compared with group II. Similarly, the total NOS activities showed a significant 2-fold increase (P< 0.05) in group II, and NOS inhibitors decreased the elevated NOS activity. These data suggest that oral TPN feeding for 9 days leads to an increase in permeability to dextran and the total NOS activity of small intestine, and both induction of the intestinal permeability and NOS activity were inhibited by treatment with NOS inhibitors. Addition of S-methylisothiourea (SMT), an iNOS selective inhibitor, profoundly inhibited 66% of the induced iNOS activity (P < 0.05) and reduced 74% of the diet-induced increase in intestinal permeability (P < 0.05) in group II. The induced permeability change in rats receiving oral TPN is mainly due to the activity of intestinal mucosal iNOS. The induction of iNOS is an important mediator for intestinal barrier dysfunction. Administration of SMT, which specifically decreases iNOS activity, is useful in the prevention of diet-induced barrier failure.     

Hypoxia combined with Escherichia coli produces irreversible gut mucosal injury characterized by increased intestinal cytokine production and DNA degradation.

The objective of the present study was to determine whether hypoxia/reoxygenation in the absence or presence of intestinal bacteria would affect the integrity of the gut mucosal epithelium (as evidenced by histologic changes) and increase the local production of cytokines (interleukin 6 [IL-6] and tumor necrosis factor [TNF]). Rat ileal mucosal membranes were harvested and their electrophysiologic properties and barrier function were measured ex vivo in the Ussing chamber system. Membranes were exposed to normoxia, normoxia + Escherichia coli, hypoxia for 40 min followed by normoxia, or hypoxia for 40 min + E. coli followed by normoxia for 3 h. IL-6 and TNF levels were measured using cytokine-dependent cellular assays. Morphological changes and the degree of DNA fragmentation were used as quantitative markers of gut mucosal injury. Mucosal integrity was maintained in the normoxia group. The addition of bacteria increased the IL-6 response and reduced mucosal integrity. During the hypoxic period, a transient decline in resistance (R) occurred and cytokine production was reduced. In the hypoxic ileal membranes not exposed to E coli, reoxygenation reversed the change in R and increased IL-6 production. The combination of hypoxia/reoxygenation plus E. coli bacterial challenge resulted in the greatest extent of gut mucosal injury and increase in TNF production. The results of this study support the hypothesis that the combination of increased intestinal bacterial levels superimposed on an ischemia/reperfusion injury increases the magnitude of gut mucosal injury and the production and subsequent release of proinflammatory cytokines.