Effects of nitric oxide synthase inhibition on microvascular reactivity in septic mice.

Persistent vasodilation refractory to vasopressor agents is characteristic of septic shock. Induction of nitric oxide synthase (NOS) by sepsis-induced cytokines within the vasculature is one of the primary mediators of this refractory vasodilation. To evaluate the mechanism of vasodilation in sepsis, we used in vivo videomicroscopy to measure microvascular vasoconstrictive responses to topical suffusion of norepinephrine in mice made septic by cecal ligation and puncture, and contrasted the effects of topical superfusion of the nonselective NOS inhibitor N(G)-methyl-L-arginine (L-NMMA) and the selective inducible NOS (iNOS) inhibitor S-methyl-isothiourea (SMT). Mice with sepsis were less sensitive to the vasoconstrictive effects of norepinephrine than controls (EC50, the concentration that produces half-maximal response 2.0+/-0.6 x 10(-6) M vs. 7.9+/-2.2 x 10(-8) M, P=0.01). Selective inhibition of inducible iNOS with topical SMT (100 microM) markedly increased catecholamine reactivity in mice with sepsis but did not affect reactivity in controls (P=0.0007 for sepsis, P=0.24 for controls). Nonselective NOS inhibition with topical L-NMMA produced a similar increase in catecholamine reactivity in mice with sepsis but not controls (P=0.001 for sepsis, P=0.56 for controls). When excess (1 mM) L-arginine, the substrate for NOS, was added to the superfusion buffer along with both SMT and L-NMMA, arteriolar responsiveness to norepinephrine was decreased to the original values. These experiments demonstrate that iNOS inhibition is as effective as nonselective NOS inhibition in reversing decreased catecholamine reactivity in sepsis. This suggests a crucial role for microvascular activation of iNOS in the pathophysiology of hypotension and decreased vasopressor responsiveness in sepsis.     

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.     

Inducible nitric oxide synthase knockout mice exhibit resistance to the multiple organ failure induced by zymosan.

In the present study, by comparing the responses in wild-type mice (+/+) and mice lacking (-/-) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the role played by iNOS in the development of non-septic shock. A severe inflammatory response characterized by peritoneal exudation, high peritoneal levels of nitrate/nitrite, and leukocyte infiltration into peritoneal exudate was induced by zymosan administration in iNOS +/+ mice. This inflammatory process coincided with the damage of lung, liver, and small intestine, as assessed by histological examination. Lung, small intestine, and liver myeloperoxidase (MPO) activity, indicative of neutrophil infiltration and lipid peroxidation, were significantly increased in zymosan-treated iNOS +/+ mice. Peritoneal administration of zymosan in the iNOS +/+ mice induced also a significant increase in the plasma levels of nitrite/nitrate and in the levels of peroxynitrite at 18 h after zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine and to poly ADP-ribose synthetase (PARS) in the lung, liver, and intestine of zymosan-treated iNOS +/+ mice. The intensity and degree of nitrotyrosine and PARS were markedly reduced in tissue section from zymosan-iNOS -/- mice. Zymosan-treated iNOS -/- mice showed a significantly decreased mortality and inhibition of the development of peritonitis. In addition, iNOS -/- mice showed a significant protection on the development of organ failure since tissue injury and MPO were reduced in lung, small intestine, and liver. Furthermore, a significant reduction of suppression of mitochondrial respiration, DNA strand breakage, and reduction of cellular levels of NAD+ was observed in ex vivo macrophages harvested from the peritoneal cavity of iNOS -/- mice subjected to zymosan-induced non-septic shock. In vivo treatment with aminoguanidine (300 mg/kg 1 and 6 h after zymosan administration) significantly prevents the inflammatory process. Taken together, our results clearly demonstrate that iNOS plays an important role in zymosan-induced non-septic shock.     

Pulmonary oxidant stress in murine sepsis is due to inflammatory cell nitric oxide

OBJECTIVE: Pulmonary oxidant stress is an important pathophysiologic feature of acute lung injury. It is unclear whether nitric oxide contributes to this oxidant stress. Thus, we examined the role of inducible nitric oxide synthase (iNOS) in pulmonary oxidant stress in murine sepsis and the differential contribution of different cellular sources of iNOS. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male iNOS+/+, iNOS-/- C57Bl/6 mice, and bone-marrow transplanted iNOS chimeric mice: +to- (wild-type iNOS+/+ donor bone-marrow transplanted into iNOS-/- recipient mice) and the reciprocal -to+ chimeras. INTERVENTIONS: Animals were randomized to sepsis (n = 264), induced by cecal ligation and perforation, vs. naive groups (n = 138). MEASUREMENTS AND MAIN RESULTS: In septic iNOS-/- vs. wild-type iNOS+/+ mice, sepsis-induced pulmonary oxidant stress (33 +/- 11 [mean +/- sem] vs. 365 +/- 48 pg 8-isoprostane/mg protein, p < .01) and nitrosative stress (0.0 +/- 0.0 vs. 0.9 +/- 0.4 micromol 3-nitrotyrosine/mmol para-tyrosine, p < .05) were abolished, despite similar septic increases in pulmonary myeloperoxidase activity in both (86 +/- 20 vs. 83 +/- 12 mU/mg protein, p = .78). In +to- iNOS chimeric mice (iNOS localized only to donor bone-marrow-derived inflammatory cells), cecal ligation and perforation resulted in significant pulmonary oxidant stress (368 +/- 81 pg 8-isoprostane/mg protein) and nitrosative stress (0.6 +/- 0.2 micromol 3-nitrotyrosine/mmol para-tyrosine), similar in degree to septic wild-type mice. In contrast, pulmonary oxidant and nitrosative stresses were absent in septic -to+ iNOS chimeras (iNOS localized only to recipient parenchymal cells), similar to iNOS-/- mice. CONCLUSIONS: In murine sepsis-induced acute lung injury, pulmonary oxidant stress is completely iNOS dependent and is associated with tyrosine nitration. Moreover, pulmonary oxidant stress and nitrosative stress were uniquely dependent on the presence of iNOS in inflammatory cells (e.g., macrophages and neutrophils), with no apparent contribution of iNOS in pulmonary parenchymal cells. iNOS inhibition targeted specifically to inflammatory cells may be an effective therapeutic approach in sepsis and acute lung injury.     

Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis.     

Role of nitric oxide in acute lung inflammation: lessons learned from the inducible nitric oxide synthase knockout mouse

OBJECTIVE: Acute lung inflammation is characterized by complex interactions among cytokines, chemokines, adhesion molecules, leukocytes, and other mediators. Proinflammatory cytokines have been implicated in the up-regulation of the inducible form of nitric oxide synthase (iNOS), which produces large amounts of nitric oxide (NO). Conversely, in some systems, NO regulates the expression of cytokines to affect leukocyte recruitment. Thus, the role of NO both exogenously administered and endogenously produced by iNOS in acute lung inflammation has not been fully elucidated. The current studies suggest a proinflammatory role for inhaled NO in a compartmentalized model of lung injury, whereas blocking of iNOS afforded protection. These results and other previous investigations have been complicated by the use of nonselective blockers of the iNOS isoform. MEASUREMENTS AND MAIN RESULTS: In an attempt to circumvent this, we examined the response of the lung to direct endotoxin challenge in mice in which iNOS had been genetically deleted (iNOS-/-). We observed a significant decrease in the inflammatory response in the iNOS-/- mice compared with wild-type mice as characterized by decreases in neutrophil accumulation and cytokine expression. Additionally, the lung cytokine response in the iNOS-/- mice was characterized by a significant increase in interleukin-12 and an inability to up-regulate interleukin-10. CONCLUSIONS: Induction of NO may be a key mediator in driving the cytokine response to endotoxin toward an increased type-2 (interleukin-10) response and a diminished type-1 (interleukin-12) response.     

IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses.

IL-6 is induced often together with the proinflammatory cytokines TNFalpha and IL-1 in many alarm conditions, and circulating IL-6 plays an important role in the induction of acute phase reactions. However, whether this endogenous IL-6 plays any additional pro- or antiinflammatory roles in local or systemic responses remains unclear. In this study, the role of IL-6 in acute inflammatory responses was investigated in animal models of endotoxic lung or endotoxemia by using IL-6+/+ and IL-6-/- mice. Aerosol exposure of endotoxin induced increased IL-6 and proinflammatory cytokines TNFalpha and MIP-2 and a neutrophilic response in the lung of IL-6+/+ mice. However, the levels of TNFalpha and MIP-2 and neutrophilia were significantly higher in the lung of IL-6-/- mice. The rate of neutrophil apoptosis in these mice was similar to that in IL-6+/+ mice. A low constitutive level of antiinflammatory cytokine IL-10 was not enhanced by endotoxin and remained similar in the lung in both IL-6+/+ and IL-6-/- mice. Systemically, intraperitoneal delivery of endotoxin resulted in much more pronounced circulating levels of TNFalpha, MIP-2, GM-CSF, and IFNgamma in IL-6-/- mice than in IL-6+/+ mice, and administration of recombinant IL-6 to IL-6-/- mice abolished these differences. In contrast, circulating IL-10 levels were induced to a similar degree in both IL-6+/+ and IL-6-/- mice. Thus, our studies reveal that endogenous IL-6 plays a crucial antiinflammatory role in both local and systemic acute inflammatory responses by controlling the level of proinflammatory, but not antiinflammatory, cytokines, and that these antiinflammatory activities by IL-6 cannot be compensated for by IL-10 or other IL-6 family members.     

Antithrombin reduces mesenteric venular leukocyte interactions and small intestine injury in endotoxemic rats

We examined the hypothesis that recombinant human antithrombin would reduce mesenteric venule leukocyte adhesion and small intestine injury in endotoxemic rats. Endotoxemic (endotoxin 10 mg/kg, intravenously) rats were treated either with saline or recombinant human antithrombin (250 and 500 U/kg). In some rats, indomethacin (100 mg/kg, intraperitoneally) was injected 60 min prior to endotoxin and recominant human antithrombin (500 U/kg) treatment. Compared to controls, intravital videomicroscopy of the mesentric venule showed an increase of leukocyte rolling (55+/-17 versus 70+/-19 leukocytes/min; P < 0.05) and firm adhesion (1.1+/-0.3 versus 5.8+/-0.8 leukocytes/100 microm; P < 0.05) in endotoxemic rats. Recombinant human antithrombin attenuated endotoxin-induced venular endothelium leukocyte adhesive cascade. The beneficial effects of recombinant human antithrombin on leukocyte adhesion were inhibited by indomethacin (100 mg/kg, intraperitoneally) in endotoxemic rats. Endotoxin treatment increased fluorescein isothiocyanate (FITC)-labeled dextran 4,000 (FD4) gut lumen to plasma ratio and wet weight/dry weight ratio. Recombinant human antithrombin (500 U/kg) attenuated endotoxin-induced gut injury. These observations suggest that recombinant human antithrombin reduces endothelium-leukocyte interactions in endotoxemic rats by interacting with local prostacyclin production.     

Mechanisms of inducible nitric oxide synthase (iNOS) inhibition-related improvement of gut mucosal acidosis during hyperdynamic porcine endotoxemia

OBJECTIVE: To determine the mechanisms of improved gut mucosal acidosis associated with selective inducible nitric oxide synthase (iNOS) inhibition. DESIGN: Prospective, controlled experimental study. SETTING: Animal research laboratory. ANIMALS: Fourteen domestic pigs. INTERVENTIONS: Anesthetized and mechanically ventilated pigs received continuous i.v. endotoxin for 24 h. A selective iNOS-inhibitor (1400 W, n=8) or vehicle (control, n=6) was started at 12 h of endotoxin and infused until the end of the experiment. MEASUREMENTS AND RESULTS: Before as well as at 12 and 24 h of endotoxin, portal venous flow (ultrasound probe), intestinal oxygen (O(2)) extraction, portal venous-arterial carbon dioxide (CO(2)) content difference and ileal mucosal-arterial PCO(2) gap (fiberoptic sensor) were assessed together with video recordings of the villous microcirculation (number of perfused/unperfused villi) using orthogonal polarization spectral imaging via an ileostomy. The gut wall microvascular blood flow (units) and hemoglobin O(2) saturation ( micro Hb-O(2)) were assessed with a combined laser Doppler flow and remission spectrophotometry probe. 1400 W blunted the otherwise progressive rise in the PCO(2) gap without affecting portal venous flow, regional O(2) and CO(2) exchange or the number of unperfused villi. While endotoxin markedly aggravated the heterogeneity of the microvascular blood flow and oxygenation, 1400 W had no further effect. CONCLUSIONS: Given the uninfluenced parameters of the ileal mucosal microcirculation in our model of long-term porcine endotoxemia, selective iNOS inhibition probably improved the PCO(2) gap due to a redistribution of the microvascular perfusion within the gut wall and/or an amelioration of the cellular respiration.     

Activated protein C attenuates microvascular injury during systemic hypoxia

In response to hypoxia, an inflammatory cascade is initiated and microvascular injury ensues. Specifically, within 10 min, leukocyte adherence to the endothelium begins, and leukocyte emigration and vascular leak soon follow. Activated protein C (APC) has been reported to have both anticoagulant and anti-inflammatory properties. Activated protein C is best described in its role as a treatment for sepsis. However, it has been used, with some success, in experimental models of hypoxic injury. We hypothesized that APC would be protective against microvascular injury during systemic hypoxia. Randomized prospective animal study. Adult male Sprague-Dawley rats. To characterize the microvascular response to APC exposure during hypoxia, four rat groups were used: saline control, APC infusion alone (100 mg/kg bolus), hypoxia alone (10% O2), and simultaneous hypoxia + APC infusion. Measurements of leukocyte adherence (no. per 100-microm venule), leukocyte emigration (no. per 4,000 microm(2)), and venular leak by fluorescein isothiacyanate-labeled albumin (Fo/Fi) were performed during intravital microscopy of the intact venular bed. Leukocyte adherence decreased from 14.5 (+/-1.2) cells/100-microm venule in hypoxic rats to 4.4 (+/-1.5) cells/100-microm venule in those treated with both hypoxic gas and APC infusion (P < 0.001). Similarly, leukocyte emigration in hypoxic rats reached 12.3 (+/- 2.2) cells/4,000-microm(2) venule, but was reduced to 3.5 (+/-0.3) cells/4,000-microm(2) venule (P <.001). Venular permeability to protein was also significantly decreased in the APC-treated group from 0.82 (+/-0.14) to 0.25 (+/-0.14) (P < 0.001). The infusion of APC attenuates the inflammatory response during systemic hypoxia at the microvascular level, as evidenced by measurements of leukocyte adherence, emigration, and venular permeability. Further investigation is needed to examine the potential role of APC in the treatment of hypoxic injury.     

P-Selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice

The expression of leukocyte and endothelial cell adhesion molecules (CAMs) is essential for the emigration of leukocytes during an inflammatory response. The importance of the inflammatory response in the development of atherosclerosis is indicated by the increased expression of adhesion molecules, proinflammatory cytokines, and growth factors in lesions and lesion-prone areas and by protection in mice deficient in various aspects of the inflammatory response. We have quantitated the effect of deficiency for intercellular adhesion molecule (ICAM)-1, P-selectin, or E-selectin on atherosclerotic lesion formation at 20 wk of age in apolipoprotein (apo) E(-/-) (deficient) mice fed a normal chow diet. All mice were apo E(-/-) and CAM(+/+) or CAM(-/-) littermates, and no differences were found in body weight or cholesterol levels among the various genotypes during the study. ICAM-1(-/-) mice had significantly less lesion area than their ICAM-1(+/+) littermates: 4.08 +/- 0.70 mm(2) for -/- males vs. 5.87 +/- 0.66 mm(2) for +/+ males, and 3.95 +/- 0. 65 mm(2) for -/- females vs. 5.59 +/- 1.131 mm(2) for +/+ females, combined P < 0.0001. An even greater reduction in lesion area was observed in P-selectin(-/-) mice: 3.06 +/- 1.04 mm(2) for -/- males vs. 5.09 +/- 1.22 mm(2) for +/+ males, and 2.85 +/- 1.26 mm(2) for -/- females compared with 5.60 +/- 1.19 mm(2) for +/+ females, combined P < 0.001. The reduction in lesion area for the E-selectin null mice, although less than that seen for ICAM-1 or P-selectin, was still significant (4.54 +/- 2.14 mm(2) for -/- males vs. 5.92 +/- 0.63 mm(2) for +/+ males, and 4.38 +/- 0.85 mm(2) for -/- females compared with 5.94 +/- 1.44 mm(2) for +/+ females, combined P < 0.01). These results, coupled with the closely controlled genetics of this study, indicate that reductions in the expression of P-selectin, ICAM-1, or E-selectin provide direct protection from atherosclerotic lesion formation in this model.     

Effects of iloprost, a stable prostacyclin analog, on intestinal leukocyte adherence and microvascular blood flow in rat experimental endotoxemia.

OBJECTIVE: To investigate the effects of iloprost, a stable prostacyclin analog, on leukocyte adherence in intestinal venules and intestinal microvascular blood flow in experimental endotoxemia. DESIGN: Prospective, randomized, controlled animal study. SETTING: Experimental laboratory. SUBJECTS: Twenty-one male Wistar rats weighing 190 +/- 40 g. INTERVENTIONS: The rats were divided equally into three groups: the first was a control group; the second received endotoxin (20 mg/kg lipopolysaccharide from Escherichia coli O55:B5 intravenously); and the third received endotoxin and intravenous iloprost infusion (2 ng.kg-1.min-1). MEASUREMENTS AND MAIN RESULTS: The distal small intestine of the animals was examined by using intravital fluorescence videomicroscopy 2 hrs after endotoxin challenge. Leukocytes were stained in vivo by means of rhodamine 6G. Intestinal microvascular blood flow was measured by laser Doppler flowmetry in the terminal ileum. Iloprost treatment significantly attenuated the count of adherent leukocytes in collecting venules (control, 61 +/- 10 n/mm2; lipopolysaccharide, 364 +/- 60 n/mm2; iloprost, 232 +/- 29 n/mm2; p <.05) and in postcapillary venules (control, 96 +/- 14 n/mm2; lipopolysaccharide, 470 +/- 21 n/mm2; iloprost 390 +/- 41 n/mm2; p <.05). Intestinal microvascular blood flow was decreased significantly in the lipopolysaccharide group (-49%), whereas iloprost-treated animals showed no significant difference compared with the control group. CONCLUSIONS: The study demonstrated that administration of iloprost attenuated leukocyte adherence in postcapillary and collecting intestinal venules and improved intestinal microvascular blood flow. Thus, iloprost treatment may impact endotoxin-induced intestinal injury.     

Pretreatment with high-fat enteral nutrition reduces endotoxin and tumor necrosis factor-alpha and preserves gut barrier function early after hemorrhagic shock

Gram-negative sepsis is a potentially fatal clinical syndrome characterized by a proinflammatory response (tumor necrosis factor-alpha) to bacterial (endo)toxins and gut barrier function loss. Recently, we found that high-fat enteral nutrition protects against late bacterial translocation in a model of hemorrhagic shock in rats. However, the basis for this protection is unknown. We hypothesized that the observed protection is the result of an early inhibition of endotoxin and the subsequent inflammatory response resulting in a preserved gut barrier function. Sprague-Dawley rats were divided into a group that was starved overnight (HS-S), fed with a low-fat enteral diet (HS-LF) or fed wih a high-fat enteral diet (HS-HF), and subsequently subjected to a nonlethal hemorrhagic shock. Ninety minutes after hemorrhage, arterial endotoxin significantly decreased in HS-HF rats (4.0 +/- 0.6 pg/mL) compared with HS-LF rats (10.7 +/- 0.9 pg/mL, P = 0.002) and HS-S rats (15.2 +/- 2.2 pg/mL P = 0.001). Interestingly, arterial tumor necrosis factor-alpha was also decreased in HS-HF rats (17.9 +/- 10.4 pg/mL) compared with HS-LF (83.5 +/- 16.7 pg/mL, P < 0.01) and HS-S rats (180.9 +/- 67.9 pg/mL, P < 0.02). Loss of tight junction structure (ZO-1) observed in ileum and colon of control hemorrhagic shock rats was prevented in HS-HF rats. In parallel, intestinal barrier function was preserved in HS-HF rats, evidenced by a reduced permeability to horseradish peroxidase (P < 0.05), less bacterial invasion, and a 10-fold reduction of bacterial translocation early after hemorrhagic shock. This report describes a new strategy to nutritionally prevent endotoxemia, the subsequent inflammatory response and gut barrier failure following hemorrhagic shock. High-fat enteral nutrition requires further evaluation as an intervention to prevent a potentially fatal systemic inflammatory response in patients at risk for sepsis.     

Increased gut permeability and bacterial translocation in Pseudomonas pneumonia-induced sepsis

OBJECTIVE: Gut injury and barrier dysfunction may contribute to the pathogenesis of sepsis and multiple organ dysfunction syndrome. The objective of this study was to determine whether gut injury could be demonstrated in hyperdynamic, normotensive sepsis induced by Pseudomonas pneumonia. DESIGN: Randomized animal study. SETTING: University laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Sepsis was induced by intratracheal instillation of Pseudomonas aeruginosa. MEASUREMENTS AND MAIN RESULTS: We measured gut mucosal and microvascular injury. In the first experiment, gut mucosal permeability was measured by 51Cr-EDTA uptake in control (n = 6), pneumonia 20-hr (n = 4), and pneumonia 40-hr (n = 4) groups. In the second experiment, microvascular permeability was measured by albumin extravasation, and morphologic abnormalities were scored in control (n = 6), pneumonia 20-hr (n = 9), and pneumonia 40-hr (n = 11) groups. Bacterial translocation to mesenteric lymph nodes was determined in both experiments. Cardiac index increased significantly in the pneumonia compared with control rats (64+/-2.1, 68+/-1.3, vs. 46+/-2 mL/min/100 g, p < .05; all results are listed in the order of pneumonia 20-hr, pneumonia 40-hr, and control groups as mean +/- SEM). Mean blood pressure was normal and was not different between groups (112+/-3, 111+/-2, vs. 118+/-2 mm Hg). 51Cr-EDTA recovery in urine 6 hrs after gavage increased significantly in both pneumonia groups vs. controls (17.5+/-2.2%, 17.9+/-7%, vs. 4+/-0.7%; p < .05). Albumin leak (tissue/plasma ratio) increased significantly in the middle and distal small intestine in the pneumonia 40-hr group vs. controls (0.68+/-0.05, 0.76+/-0.07, vs. 0.45+/-0.04, p < .05 in the middle small gut; 0.75+/-0.09, 0.85+/-0.07, vs. 0.51+/-0.05, p < .05 in the distal small gut). Bacterial translocation to mesenteric lymph nodes increased significantly in pneumonia 40-hr rats vs. controls (positive culture 67% vs. 8%; p < .05). CONCLUSIONS: This study demonstrates gut mucosal and microvascular injury and gut barrier dysfunction in normotensive sepsis secondary to bacterial pneumonia. The mechanism and significance of the injury need to be determined.     

Dopexamine attenuates flow motion in ileal mucosal arterioles in normotensive sepsis

OBJECTIVES: Injury to the small intestine is thought to play a crucial role in the development and propagation of sepsis. Cellular hypoxia, caused by hypoperfusion, may result in increased mucosal permeability, thus allowing the translocation of bacteria and endotoxin to the circulation. The purpose of this study was to assess the effect of the synthetic catecholamine, dopexamine, on the mucosal microcirculation of the septic rat ileum. DESIGN: Randomized, crossover study. SETTING: Teaching hospital animal laboratory. SUBJECTS: Sprague-Dawley male rats. INTERVENTIONS: Sepsis was induced by cecal ligation and perforation in 11 male Sprague-Dawley rats. Six sham animals were also studied. At 24 hrs, rats were anaesthetized, intubated, ventilated, and prepared for intravital microscopy of the mucosal surface of the ileum. Dopexamine (8 microg/kg/min) and saline were infused intravenously into each rat using a randomized crossover design. MEASUREMENTS AND MAIN RESULTS: Observations were videotaped for later analysis of arteriolar flow patterns, red cell velocity, arteriolar diameter, and intercapillary area. All values are expressed as mean +/- SEM. The main effect of dopexamine infusion in the sepsis group was the attenuation of the rhythmic blood flow patterns (flow motion) observed during saline infusion. In each subject, dopexamine decreased the absolute number of arterioles exhibiting flow motion by 35.93+/-6.81% (p<.001, paired t-test). Dopexamine decreased the amount of time red cell flow was stopped in marginal and central arterioles by 11.83+/-2.49% (p<.001, paired t-test). Dopexamine did not alter significantly the diameter of the marginal arterioles, the intercapillary area, or the red cell velocity compared with saline in the sepsis group. The sham group displayed marked microvascular differences compared with the sepsis group with respect to arteriolar diameter (13.32+/-0.05 vs. 9.46+/-0.24 mm, p<.001), intercapillary area (975.93+/-60.60 vs. 1256.03+/-43.88 mm2, p<.05 ), red cell velocity (611.40+/-38.77 vs. 289.15+/-36.45, p<.001), and blood flow patterns (% displaying flow motion, 15.89+/-6.09 vs. 58.22+/-9.63, p<.01; % time stopped flow, 1.96+/-0.89 vs. 20.21+/-3.92, p<.005). CONCLUSIONS: These results indicate that dopexamine increased overall blood flow and possibly oxygen delivery to the mucosa by altering patterns of blood flow within the villi. The observation that the diameter of the marginal arterioles is not affected by dopexamine indicates that dopexamine influences the mucosal microcirculation at the level of higher order arterioles. We conclude that sepsis results in abnormal microvascular villus blood flow and that dopexamine can partially restore these changes towards normal.     

Inducible nitric oxide synthase is not required in the development of endotoxin tolerance in mice

We investigated the role of inducible nitric oxide synthase (iNOS) in endotoxin tolerance, which was induced in mice genetically deficient of iNOS (iNOS-/-) and in wild-type littermates. In non-tolerant wild-type mice, endotoxin induced high mortality, elevation of plasma levels of nitrite and nitrate, tumor necrosis factor a (TNFalpha), and interleukin 10 (IL-10). These events were preceded by degradation of inhibitors kappaBalpha (IkappaBalpha) and kappaBI (IkappaBbeta), and activation of nuclear factor-kappaB (NF-kappaB) in the lung. Pretreatment of wild-type mice with a sublethal dose of endotoxin prior to lethal endotoxin administration ameliorated lethality and blunted TNFalpha production, whereas IL-10, nitrite, and nitrate production was maintained. These events were associated with reduction of IKBa degradation and NF-kappaB activation in the lung. The kinetics of degradation of IkappaBbeta were also altered. In parallel experiments, nontolerant iNOS-/- mice experienced similar mortality after endotoxin as nontolerant wild-type mice. Plasma levels of nitrite and nitrate were not elevated after lethal endotoxin administration. IL-10 levels were significantly reduced in comparison to nontolerant wild-type mice, whereas TNFalpha levels were similarly increased. These events were preceded by lesser degradation of IkappaBalpha and reduced NF-kappaB activation in the lung. Pretreatment of iNOS-/- mice with a sublethal endotoxin ameliorated lethality. TNFalpha production was significantly reduced, whereas IL-10 production was significantly increased when compared to nontolerant iNOS-/- mice. Degradation of IkappaBalpha and activation of NF-kappaB in the lung were not altered by endotoxin tolerance, whereas kinetics of IkappaBbeta degradation was only delayed. Our data suggests that iNOS is not required for the development of endotoxin tolerance, and that other signal transduction pathways, rather than NF-kappaB, may regulate induction of endotoxin tolerance in the absence of iNOS.     

Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia

OBJECTIVE: To characterize microcirculatory actions of activated protein C in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction. DESIGN: Animal study using intravital microscopy. SETTING: Animal research facility. SUBJECTS: Male Syrian golden hamsters, 6-8 wks old with a body weight of 60-80 g. INTERVENTIONS: In skinfold preparations, endotoxemia was induced by intravenous administration of 2 mg/kg endotoxin (lipopolysaccharide, Escherichia coli). Intravital microscopy allowed quantitative analysis of arteriolar and venular leukocyte adhesion and functional capillary density (cm) that served as a measure of microvascular perfusion failure. Activated protein C (APC group, n = 8, 24 microg/kg intravenously) was substituted continuously during 8 hrs after lipopolysaccharide, whereas endotoxemic buffer-treated animals (control, n = 7) served as controls. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide increased leukocyte adhesion and decreased functional capillary density to 50% of baseline values (p <.01 vs. baseline). Activated protein C treatment inhibited (p <.05) lipopolysaccharide-mediated leukocytic response and attenuated (p <.05) endotoxic perfusion failure in nutritive capillaries. CONCLUSIONS: Activated protein C-induced protection from lipopolysaccharide-mediated microcirculatory dysfunction was characterized in vivo for the first time. The impressive modification of leukocyte cross-talk indicates systemic anti-inflammatory activated protein C effects on leukocytes and the endothelium, subsequently improving capillary perfusion. These actions could represent the in vivo mechanism of activated protein C interactions observed in patients with severe sepsis.     

Ventilation-induced lung injury is associated with an increase in gut permeability

Mechanical ventilation is associated with several harmful effects mainly related to high tidal volumes (Vt). Ventilator-induced lung injury can be responsible for an increased production of inflammatory mediators. We evaluated remote consequences on the gut of lung triggered inflammatory response, neutralizing anti-tumor necrosis factor (TNF) antibody was administered to assess the role of TNF in lung and gut permeability changes. Rats were anesthetized and ventilated for 2 h. A control group (Con: Vt = 10 mL/kg) was compared with a high Vt group (HV: Vt = 30 ml/kg). One microCi of I125-labeled human serum albumin was injected to measure extravascular albumin space. Gut permeability was evaluated by plasma-to-lumen ratio leakage of I125 human serum albumin. Extravascular albumin space increased in the HV group from 446 +/- 50 microL to 2783 +/- 887 microL. Gut index of permeability increased from 5.1 +/- 1.2 to 14.2 +/- 4.9. Anti-TNF antibody prevented both lung and gut increase in permeability. High tidal volume ventilation resulted in an increase in lung edema and gut permeability, antagonism of TNF with neutralizing antibodies abrogated the increase in gut permeability as well as lung edema.     

The thrombin antagonist hirudin fails to inhibit endotoxin-induced leukocyte/endothelial cell interaction and microvascular perfusion failure

Besides its central role in coagulatory pathways, thrombin is known to be a key mediator of macrophage and granulocyte activation in vitro. During recent years the concept of thrombin inhibition by the specific thrombin inhibitor, hirudin, has been established to treat septic disorders. Since basic mechanisms of sepsis include leukocyte/endothelial cell interaction and deterioration of capillary perfusion, we hypothesized that hirudin modulates leukocyte activation and microvascular injury. Severe endotoxemia was induced in Syrian hamsters by intravenous administration of endotoxin (lipopolysaccharide [LPS], E. coli, 2mg/kg) at 0 h. Hirudin (0.25 mg/kg/h) was substituted intravenously during the 4 h after the induction of endotoxemia (n = 7, hirudin). In control animals (n = 6, control) LPS was given without hirudin substitution. In skinfold chamber preparations leukocyte/endothelial cell interaction and functional capillary density (FCD, measure of capillary perfusion) were analyzed during a 24-h period after LPS injection using intravital fluorescence microscopy. Hirudin effectively normalized thromboplastin time and antithrombin activity when compared to controls (P < 0.05, ANOVA). However, hirudin did not attenuate LPS-induced arteriolar and venular leukocyte adherence, and even tended to increase leukocyte adherence after 24 h (P > 0.05, MANOVA). In parallel, addition of hirudin led to a significant deterioration of FCD over time when compared to controls (hirudin: baseline = 171 +/- 19 cm(-1) versus 16 +/- 9 at 24 h; control: baseline = 150 +/- 20 cm(-1) versus 62 +/- 18 at 24 h; P < 0.05). The fall in FCD in hirudin animals was associated with a significant increase of wet-to-dry weight ratios in lung, kidney, muscle, and small intestine (P < 0.05 versus control, ANOVA). Thus our study does not indicate a protective effect of hirudin on microcirculation during endotoxemia, despite an improvement of coagulatory parameters. This result may at least in part explain the lack of efficacy of hirudin on lethality during endotoxemia and sepsis.     

Mast cell mediators regulate vascular permeability changes in Arthus reaction.

Plasma exudation characterizes the early phase of acute inflammation. The possible role of mast cells and their mediators in this event in immune complex-induced injury was studied. Dye exudation was assessed from 5 min to 2 hr after initiating reverse passive Arthus reaction in mast cell-deficient mice, WBB6F1-W/Wv (W/Wv), and their normal congenic controls, WBB6F1-+/+ (+/+). The response to antibody (10, 30 and 100 micrograms/site, i.d.) was dose- and time-dependent in both groups of mice. At the lower doses of antibody, 10 and 30 micrograms/site, exudation was significantly less (30% and 40%, respectively) in W/Wv as compared to +/+ mice between 15 to 45 min. With 100 micrograms of antibody/site, significant differences between W/Wv and +/+ mice were noted only at 15 and 30 min. The deficit in permeability changes in W/Wv mice was reversed by local mast cell reconstitution. In +/+ mice, pyrilamine and methysergide pretreatment reduced vascular permeability to the same extent by 70, 60 and 35% when stimulated for 30 min with 10, 30 and 100 micrograms of antibody/site, respectively. An equivalent inhibition was observed with the 5-lipoxygenase inhibitor A-63162. None of the inhibitors decreased plasma permeation in W/Wv mice. These results indicate that the mast cell mediators histamine and serotonin regulate vascular permeability early during an immune complex-mediated inflammation. The data also suggest the involvement of leukotrienes and the importance of mast cells in their synthesis. The profile of inhibition in +/+ mice agrees well with the difference in exudation observed between normal and mast cell-deficient mice.