Hypertonic saline attenuates end-organ damage in an experimental model of acute pancreatitis

BACKGROUND: Hypertonic saline (HTS) has been noted previously to reduce neutrophil activation. The aim of this study was to elucidate the effect of hypertonic resuscitation on the development of end-organ damage in an animal model of pancreatitis. METHODS: Pancreatitis was induced in Sprague-Dawley rats by intraperitoneal injection of 20 per cent L-arginine. Animals were randomized into four groups (each n = 8): controls; pancreatitis without intervention; pancreatitis plus intravenous resuscitation with normal saline (0.9 per cent sodium chloride 2 ml/kg) at 24 and 48 h; or HTS (7.5 per cent sodium chloride 2 ml/kg) at these time points. Pulmonary endothelial leakage was assessed by measurement of lung wet : dry ratios, bronchoalveolar lavage protein and myeloperoxidase activity. RESULTS: Animals that received HTS showed less pancreatic damage than those resuscitated with normal saline (1.0 versus 3.0; P = 0.04). Lung injury scores were also significantly diminished in the HTS group (1.0 versus 3.5; P = 0.03). Pulmonary neutrophil sequestration (myeloperoxidase activity 1.80 units/g) and increased endothelial permeability (bronchoalveolar lavage protein content 1287 microgram/ml) were evident in animals resuscitated with normal saline compared with HTS (1.22 units/g and 277 microgram/ml respectively; P < 0.02). CONCLUSION: HTS resuscitation results in a significant attenuation of end-organ injury following a systemic inflammatory response to severe pancreatitis.     

Inhibition of matrix metalloproteinases reduces local and distant organ injury following experimental acute pancreatitis

BACKGROUND: Pulmonary complications from pancreatitis involve parenchymal destruction via proteolytic enzymes. Matrix metalloproteinases (MMPs) may play an important role in pulmonary injury following acute severe pancreatitis. We hypothesized that local and distant organ injury would be decreased by the presence of an MMP inhibitor (Batimistat; BB-94) following severe acute pancreatitis (AP). METHODS: Eighteen male rats were randomized into two groups: BB-94 (AP + 40 mg/kg/24 h BB-94 ip x three doses) or control (AP + 20 ml/kg/24 h normal saline ip x three doses). Necrotizing AP was induced by retrograde infusion of 5% sodium taurocholate (1.5 ml/kg) into the pancreatic duct. Twenty additional animals were randomized into BB-94 and control groups for the survival study. Serum was evaluated for amylase and MMP activity. Pancreatic sections were graded for edema, necrosis, neutrophil infiltrate, and hemorrhage. Myloperoxidase (MPO) activity was used to determine PMN infiltration in the lung. Evan's Blue dye extravasation was used to quantify vascular permeability. RESULTS: Animals in the BB-94 group had decreased amylase levels (1086.0 +/- 61.7 U/L vs 2232.7 +/- 309.9 U/L; P < 0.05), decreased cellular infiltrate (1.4 +/- 0.2 vs 2.3 +/- 0.2; P < 0.02), and decreased necrosis (4.1 +/- 0.3 vs 6.1 +/- 0.4; P < 0.005) compared to the control group. Lung tissue following pancreatitis in the BB-94 group demonstrated decreased MPO activity (41.5 +/- 2.4 units vs 57.3 +/- 2.9 units; P < 0.05) and decreased vascular permeability (18.3 +/- 2.8 mg/100 g vs 30.1 +/- 4.6 mg/100 g; P < 0.05). Animals treated with BB-94 had 100% survival compared to 50% survival in control at 72 h. CONCLUSIONS: Pancreatitis results in increased local and distant MMP activity. Pulmonary and pancreatic injury following AP can be abrogated by treatment with an MMP inhibitor (Batimistat; BB-94) which may result in decreased morbidity and mortality.     

Hypertonic saline infusion for pulmonary injury due to ischemia-reperfusion

HYPOTHESIS: Inhibition of neutrophil-endothelial cell interactions by hypertonic saline (HTS) may confer protection against organ injury in states of immunologic disarray. This study tested the hypothesis that infusion of HTS modulates the development of end-organ injury in a model of lower-torso ischemia-reperfusion injury. DESIGN: Ischemia-reperfusion injury was induced in 30 male Sprague-Dawley rats by infrarenal aortic cross-clamp for 30 minutes, followed by reperfusion for 2 hours. At 0 and 60 minutes of reperfusion, intravenous HTS (7.5% sodium chloride, 4 mL/kg) was administered to 6 rats each, and another 12 received either 4 or 30 mL/kg of isotonic sodium chloride solution. Six rats received HTS, 4 mL/kg, before ischemia. At 2 hours, we assessed liver function, pulmonary injury, neutrophil infiltration (myeloperoxidase activity), endothelial permeability (bronchoalveolar lavage and wet-dry weight ratios), and proinflammatory cytokine levels (tumor necrosis factor alpha and interleukin 6). RESULTS: Infusion with HTS before or after ischemia significantly reduced end-organ injury. Histopathologic pulmonary injury scores were markedly attenuated in the HTS group (5.82 +/- 1.3) and the HTS pretreated group (4.91 +/- 1.6) compared with the isotonic sodium chloride solution groups (8.54 +/- 1.1) (P =.04). Pulmonary neutrophil sequestration (2.07 +/- 0.23) and increased endothelial permeability (4.68 +/- 0.44) were manifest in animals resuscitated with isotonic sodium chloride solution compared with HTS treatment (1.54 +/- 0.19 [P =.04] and 2.06 +/- 0.26 [P =.02]) and pretreatment (1.18 +/- 0.12 [P =.04] and 1.25 +/- 0.07 [P =.002]). In addition, a significant reduction in serum tumor necrosis factor alpha (P =.04) and interleukin 6 (P =.048) levels was observed, whereas HTS resuscitation attenuated the upsurge in aspartate transaminase (P =.03) and alanine transaminase levels (P =.047). CONCLUSIONS: Resuscitation with HTS attenuates the pulmonary edema and tissue injury due to lower-torso ischemia-reperfusion and maintains a more benign immunologic profile.     

Pancreatitis-induced acute lung injury. An ARDS model.

Cerulein-induced acute pancreatitis in rats is associated with acute lung injury characterized by increased pulmonary microvascular permeability, increased wet lung weights, and histologic features of alveolar capillary endothelial cell and pulmonary parenchymal injury. The alveolar capillary permeability index is increased 1.8-fold after a 3-hour injury (0.30 to 0.54, p less than 0.05). Gravimetric analysis shows a similar 1.5-fold increase in wet lung weights at 3 hours (0.35% vs. 0.51% of total body weight, p less than 0.05). Histologic features assessed by quantitative morphometric analysis include significant intra-alveolar hemorrhage (0.57 +/- 0.08 vs. 0.12 +/- 0.02 RBC/alveolus at 6 hours, p less than 0.001); endothelial cell disruption (28.11% vs. 4.3%, p less than 0.001); and marked, early neutrophil infiltration (7.45 +/- 0.53 vs. 0.83 +/- 0.18 PMN/hpf at 3 hours, p less than 0.001). The cerulein peptide itself, a cholecystokinin (CCK) analog, is naturally occurring and is not toxic and in several in vitro settings including exposure to pulmonary artery endothelial cells, Type II epithelial cells, and an ex vivo perfused lung preparation. The occurrence of this ARDS-like acute lung injury with acute pancreatitis provides an excellent experimental model to investigate mechanisms and mediators involved in the pathogenesis of ARDS.     

In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury

OBJECTIVE: Nitric oxide regulates vascular tone, inhibits platelet aggregation, and inhibits leukocyte adhesion, all of which are important modulators of ischemia-reperfusion injury. This study aimed to determine the effects of endothelial constitutive nitric oxide synthase gene transfer on ischemia-reperfusion injury in a rat lung transplant model. METHODS: In group I, donor animals were injected intravenously with 5 x 10(9) pfu of adenovirus-encoding endothelial constitutive nitric oxide synthase. Groups II and III served as controls, whereby donor animals were injected with either 5 x 10(9) pfu of adenovirus encoding beta-galactosidase or saline solution, respectively. Twenty-four hours after injection, left lungs were harvested and preserved for 18 hours at 4 degrees C, then implanted into isogeneic recipients, which were put to death 24 hours later. Recombinant endothelial constitutive nitric oxide synthase gene expression was evaluated by Western blotting and immunohistochemistry. Lung grafts were assessed by measuring arterial oxygenation, myeloperoxidase activity, and wet/dry weight ratios. RESULTS: Western blotting confirmed the overexpression of endothelial constitutive nitric oxide synthase in lungs so transfected compared with controls. Twenty-four hours after reperfusion, mean arterial oxygenation was significantly improved in group I compared with group II and III controls (189.4 +/- 47.1 mm Hg vs 71.7 +/- 8.9 mm Hg and 67.8 +/- 12.2 mm Hg, P =.02, P =.01, respectively). Myeloperoxidase activity, a reflection of tissue neutrophil sequestration, was also significantly reduced in group I compared with groups II and III (0.136 +/- 0.038 DeltaOD/mg/min vs 0. 587 +/- 0.077 and 0.489 +/- 0.126 DeltaOD/mg/min, P =.001, P =.01, respectively). CONCLUSION: Adenovirus-mediated gene transfer with endothelial constitutive nitric oxide synthase ameliorates ischemia-reperfusion injury as manifested by significantly improved oxygenation and decreased neutrophil sequestration in transplanted lung isografts. Endothelial constitutive nitric oxide synthase gene transfer may reduce acute lung dysfunction after lung transplantation.     

Gut injury and gut-induced lung injury after trauma hemorrhagic shock is gender and estrus cycle specific in the rat

BACKGROUND: The purpose of this study was to test the hypothesis that trauma-hemorrhagic shock (T/HS)-induced gut and lung injury is modulated by gender and the stage of the estrus cycle at the time of injury. METHODS: We compared the incidence and magnitude of gut and lung injury in male and female rats subjected to a laparotomy (trauma) followed by 90 minutes of shock (mean arterial pressure, 30 mm Hg) (T/HS) or sham shock. RESULTS: Lung injury and pulmonary neutrophil sequestration as well as gut injury were increased after T/HS in the diestrus female and the male rats, but not in the estrus or proestrus female rats. Although T/HS caused gut and lung injury in the male and the female diestrus rats, the magnitude of injury was less in the female diestrus than in the male rats (p < 0.05). A strong correlation was found between intestinal villous injury and lung injury as well as between gut injury and pulmonary leukosequestration in the female rats subjected to T/HS (p < 0.0001). Plasma nitric oxide levels were approximately two- to threefold higher in the male and the diestrus female rats subjected to T/HS than in other groups (p < 0.05), and a high degree of correlation (r2 = 0.68, p < 0.0001) was found between villous injury and plasma nitric oxide levels. Ileal constitutive nitric oxide synthase (NOS) and inducible NOS (iNOS) activity was measured. Ileal constitutive NOS activity was similar between the groups, but iNOS activity was three- to fourfold higher in the T/HS male rats than in the sham shock or the T/HS proestrus groups (p < 0.01). CONCLUSION: Gender and estrus cycle stage influence susceptibility to T/HS-induced gut and lung injury. This difference in susceptibility to organ injury was associated with increased plasma nitric oxide levels and increased ileal iNOS activity.     

Evidence for neutrophil-related acute lung injury after intestinal ischemia-reperfusion

Intestinal ischemia-reperfusion injury is a common and important clinical event associated with the activation of an endogenous inflammatory response. Some of the mediators of this response may be involved in the pathogenesis of multiple organ system failure. The purpose of this study was to determine whether remote organ dysfunction--specifically, acute lung injury--occurs after intestinal ischemia-reperfusion injury. After an ischemia-reperfusion event in rat intestine, whole lungs were obtained for measurement of tissue adenosine triphosphate (ATP) and myeloperoxidase values, and evaluation of histologic condition. In addition, lung microvascular permeability was assessed by determination of the rate at which iodine 125-labeled bovine serum albumin sequestration in the extravascular compartment occurred. Lung tissue ATP levels were no different in sham-operated animals than in those that had undergone 120 minutes of intestinal ischemia. Within 15 minutes of gut reperfusion, however, lung ATP decreased from 3.82 +/- 0.27 to 1.53 +/- 0.90 x 10(-7) moles/50 mg tissue, p less than 0.05. Neutrophil accumulation in the lungs, estimated by tissue myeloperoxidase determination, increased sevenfold (0.13 +/- 0.02 to 0.97 +/- 0.25 units/gm, p less than 0.05) after 120 minutes of ischemia and 15 minutes of reperfusion. Lung microvascular permeability increased threefold after 120 minutes of intestinal ischemia and 120 minutes of reperfusion (0.10 +/- 0.01 vs. 0.35 +/- 0.05 [lung/blood counts per minute], p less than 0.05). Intestinal ischemia followed by reperfusion is associated with acute lung injury characterized by increased microvascular permeability, histologic evidence of alveolar capillary endothelial cell injury, reduced lung tissue ATP levels, and the pulmonary sequestration of neutrophils. These data confirm an acute lung injury associated with intestinal ischemia-reperfusion and suggest a possible pathogenic role for the neutrophil.     

Role of Erk1/2, p70s6K, and eNOS in isofluraneinduced cardioprotection during early reperfusionin vivo

PURPOSE: Administration of isoflurane during early reperfusion after prolonged coronary artery occlusion decreases myocardial infarct size by activating phosphatidylinositol-3-kinase (PI3K) signal transduction. The extracellular signal-related kinases (Erk1/2) represent a redundant mechanism by which signaling elements downstream from PI3K, including 70-kDA ribosomal protein s6 kinase (p70s6K) and endothelial nitric oxide synthase (eNOS), may be activated to reduce reperfusion injury. We tested the hypothesis Erk1/2, p70s6K, and eNOS mediate isoflurane-induced postconditioning in rabbit myocardium in vivo. METHODS: Barbiturate-anesthetized rabbits (n = 78) instrumented for measurement of systemic hemodynamics were subjected to a 30-min coronary occlusion followed by three hours reperfusion. Rabbits were randomly assigned to receive 0.9% saline (control), the Erk1/2 inhibitor PD 098059 (2 mg x kg(-1)), the p70s6K inhibitor rapamycin (0.25 mg x kg(-1)), the nonselective nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME; 10 mg x kg(-1)), the selective inducible NOS antagonist aminoguanidine hydrochloride (AG, 300 mg x kg(-1)), or the selective neuronal NOS inhibitor 7-nitroindazole (7-NI, 50 mg x kg(-1)) in the presence or absence of 1.0 minimum alveolar concentration isoflurane administered for three minutes before and two minutes after reperfusion. RESULTS: Brief exposure to 1.0 minimum alveolar concentration isoflurane reduced (P < 0.05) infarct size (21 +/- 4% [mean +/- SD] of left ventricle area at risk, respectively; triphenyltetrazolium staining) as compared to control (41 +/- 5%). PD 098059, rapamycin, and L-NAME, but not AG nor 7-NI, abolished the protection produced by isoflurane. CONCLUSION: The results suggest that the protective effects of isoflurane against infarction during early reperfusion are mediated by Erk1/2, p70s6K, and eNOS in vivo.     

Evidence for tumor necrosis factor-induced pulmonary microvascular injury after intestinal ischemia-reperfusion injury.

Acute lung injury characterized by increased microvascular permeability is one feature of multiple-organ system failure and the adult respiratory distress syndrome. Intestinal ischemia-reperfusion injury has been linked to this type of acute lung injury. The purpose of these experiments was to examine the pathogenic mediators that link the two processes, with particular emphasis on the roles of endotoxin and tumor necrosis factor alpha (TNF alpha). Previously described characteristics of the acute lung injury in this rat model of intestinal ischemia-reperfusion include pulmonary neutrophil sequestration, depletion of lung tissue ATP, alveolar endothelial cell disruption, and increased microvascular permeability. Plasma levels of TNF in the systemic circulation of sham-operated animals and those with intestinal ischemic injury less than 60 minutes in duration were very low or undetectable. Intestinal ischemia for 120 minutes was associated with TNF elevation to 1.19 +/- 0.50 U/mL. Reperfusion for periods of 15 and 30 minutes generated 5- to 10-fold increases in circulating TNF levels (6.61 +/- 3.11 U/mL, p greater than 0.05 and 10.41 +/- 5.41 U/mL, p = 0.004 compared to sham); however this increase in circulating TNF was transient and largely cleared within 60 minutes after initiating reperfusion. Portal vein endotoxin levels were found to increase significantly before the appearance of TNF in systemic plasma, suggesting that gut-derived endotoxin may induce TNF release from hepatic macrophages into the systemic circulation. Anti-TNF antibody attenuated the increase in pulmonary microvascular permeability in this preparation but did not prevent pulmonary neutrophil sequestration. These observations suggest that endotoxin and TNF have pathogenic roles in this acute lung injury, but that mechanisms of adherence of neutrophils to endothelial cells independent of TNF may be involved. The accumulation of neutrophils in the lung but the prevention of a vascular permeability increase in the presence of antibody to TNF may imply an in vivo role for TNF in the process of neutrophil activation. These studies provide additional evidence of the importance of the endogenous inflammatory mediators in the development of systemic injury in response to local tissue injury.     

Blockade of complement activation prevents local and pulmonary albumin leak after lower torso ischemia-reperfusion.

Lower torso ischemia and reperfusion leads to both local and remote tissue injuries. The purpose of this study was to assess the role of complement in mediating the local and remote microvascular permeability after bilateral hind limb tourniquet ischemia. Four hours of ischemia and 4 hours of reperfusion produced an increased skeletal muscle permeability index (muscle/blood 125I albumin ratio) of 2.90 +/- 0.35 compared with the index in nonischemic muscle of 0.25 +/- 0.02 (p < 0.01). Muscle wet-to-dry-weight ratio increased from 3.93 +/- 0.04 in sham to 5.55 +/- 0.09 in ischemic muscle (p < 0.0001). Lung permeability rose at 4 hours as indicated by the increased bronchoalveolar lavage (BAL)/blood 125I albumin ratio 4.36 +/- 0.41 x 10(-3) versus sham 2.64 +/- 0.28 x 10(-3) (p < 0.05) and neutrophil sequestration 0.28 +/- 0.02 U/g myeloperoxidase (MPO) versus sham 0.14 +/- 0.02 U/g (p < 0.001). Serum lytic activity of the classical but not the alternate complement pathway was reduced. The soluble complement receptor (sCR1) was used to inhibit complement activity and attenuated the increase in the permeability index after reperfusion in ischemic muscle 1.11 +/- 0.08 (p < 0.01) and reduced the lung BAL/blood 125I albumin ratio to sham levels 2.46 +/- 0.39 x 10(-3) (p < 0.05) at 6 mg/animal, without reducing the lung neutrophil sequestration, 0.24 +/- 0.02 U/g. The authors conclude that complement activation occurred during tourniquet ischemia and mediated permeability changes in the ischemic muscle and the lungs during reperfusion.     

Systemic thermal injury in anesthetized rabbits causes early pulmonary vascular injury that is not ablated by lazaroids

PURPOSE: To study the effects of a systemic thermal injury on the pulmonary vasculature with and without inhibitors of lipid peroxidation (U74389G). METHODS: In a prospective, placebo control, randomized, and blinded multi-group study, burn shock was induced by scalding thermal injury (65C) to 35% body surface area in rabbits (n = 28). Hemodynamics and gas exchange were followed for 240 min post burn in four groups: No Burn, Burn-Control, Burn-U74 (10 mg.Kg-1 U74389G), No Burn-U74 (10 mg.Kg-1 U74389G). RESULTS: Scald resulted in early pulmonary injury as measured by increased pulmonary vascular resistance in the pooled Burn group compared with the No Burn groups (942 +/- 358 vs 605 +/- 255 dynes.sec-1.cm-5 respectively, P < 0.05). These pulmonary changes were associated with alveolar sequestration of leukocytes (4.8 +/- 2.9 vs 17.7 +/- 6.0 cells x 10(9).L-1, P < 0.05) in the No Burn and Burn groups respectively. Histological evidence of decreased neutrophil sequestration after scald injury was present in U74 treated animals (3+ vs 2+, P < 0.05 in the Burn and No Burn groups respectively and 2+ vs 2+, P > 0.05 in the Burn-U74 and No Burn-U74 groups respectively) although bronchial alveolar lavage still demonstrated neutrophil sequestration (5.3 +/- 2.5 vs 12.2 +/- 3.3 cell 10(9).L-1, P < 0.05 in No Burn-U74 and Burn-U74 groups respectively). Similarly, circulating white blood cells were increased in the Burn group but not Burn-U74 group four hours post burn. The increase in pulmonary vascular resistance after burn was not altered by administration of U74. CONCLUSIONS: Systemic burn results in early pulmonary vascular changes associated with leukocyte sequestration. After scald injury administration of lazaroids (U744389G) did not lessen pulmonary vascular resistance changes but did reduce neutrophil sequestration.     

Inducible nitric oxide production is an adaptation to cardiopulmonary bypass-induced inflammatory response

BACKGROUND: Cardiopulmonary bypass (CPB) increases nitric oxide (NO) production by the activation of NO synthases (NOS). However, the role of NO from inducible NOS (iNOS) in CPB-induced inflammatory response remains unclear. We examined the effect of a selective iNOS inhibitor, aminoguanidine, on CPB-induced inflammatory response in a rat-CPB model. METHODS: Adult Sprague-Dawley rats underwent 60 minutes of CPB (100 mL x kg(-1) x min(-1), 34 degrees C). Group A (n = 10) received 100 mg/kg of aminoguanidine intraperitoneally 30 minutes before the initiation of CPB, and group B (n = 10) served as controls. RESULTS: There were significant time-dependent changes in plasma interleukin (IL)-6, IL-8, nitrate + nitrite, the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr, an indicator of peroxynitrite formation), and respiratory index (RI). Three hours after CPB termination, IL-6, IL-8, and RI were significantly higher in group A (IL-6, 397.5+/-80.6 pg/mL; IL-8, 26.99+/-6.57 ng/mL; RI, 1.87+/-0.31) than in group B (IL-6, 316.5+/-73.9 pg/mL, p <0.05; IL-8, 17.21+/-3.12 ng/mL, p < 0.01; RI, 1.57+/-0.24, p < 0.05) although nitrate + nitrite (31.8+/-4.1 micromol/L) and %NO2-Tyr (1.15%+/-0.20%) were significantly lower in group A than in group B (nitrate + nitrite, 50.2+/-5.0 micromol/L, p < 0.01; %NO2-Tyr, 1.46%+/-0.21%, p < 0.01). Western immunoblot analysis from lung tissue of group A identified marked iNOS inhibition without inhibiting endothelial-constitutive NOS, and neutrophil accumulation in the lung specimens was significantly greater in group A (6.5+/-0.7/alveoli) than in group B (4.4+/-0.4/alveoli, p < 0.01). CONCLUSIONS: These results suggest that NO production from iNOS may be an adaptive response for attenuating the CPB-induced inflammatory response.     

The outcome of non-selective vs selective nitric oxide synthase inhibition in lipopolysaccharide treated rats.

Nitric oxide (NO), generated by inducible nitric oxide synthase (NOS) following lipopolysaccharide (LPS) administration, produces renal failure through autoinhibition of glomerular endothelial NOS activity. Preadministration of selective iNOS inhibitors abolishes this effect. Although nonselective NOS inhibitors further decrease GFR, current clinical trials investigate the effect of nonselective NOS inhibition in septic patients. The goals of our study were to determine whether treatment with selective NOS inhibitors can reverse the decrease in GFR in LPS treated rats with already established renal failure and to define the outcome of LPS treated rats following nonselective NOS inhibition. Four hours following the administration of LPS (4 mg/kg), we measured creatinine clearance (CrCl) before and after the administration of either L-NIL (selective iNOS inhibitor, 3 mg every 20 minutes) or saline. Selective iNOS inhibition attenuated the decrease in blood pressure [Controls: 105 +/- 6 to 98 +/- 5, LPS: 92 +/- 5* to 83 +/- 4*, LPS + L-NIL: 88 +/- 6* to 94 +/- 6 mm Hg; *p < 0.05, vs controls (n = 6)], and reversed the decrease in GFR after LPS [Controls: 2.21 +/- 0.13 to 2.07 +/- 0.11, LPS: 0.82 +/- 0.18* to 0.66 +/- 0.22*, LPS + L-NIL: 0.76 +/- 0.15* to 1.86 +/- 0.15 ml/min; *p < 0.05 vs controls (n = 6)]. We next studied the effect of complete non-selective NOS inhibition (L-NAME 200 mg, 2 hours after LPS) on LPS treated rats. All (6/6) animals treated with both LPS and L-NAME died within 2 hours following LPS, while rats treated with either LPS, L-NAME, or LPS + L-NIL survived. Histologic studies performed in all experimental groups were unremarkable. Overnight mortality was studied using smaller doses of L-NAME. All LPS + L-NAME (10/10) and 1/10 LPS treated rats died. L-NAME, control, and LPS + L-NIL animals survived. The characteristic histologic findings in LPS + L-NAME rats were diffuse ischemic changes, most importantly acute myocardial infarction. In conclusion: Selective iN-OS inhibition might prove to have clinical application as it prevents the decrease in GFR following LPS, even after renal failure is established. Treatment with a non selective NOS inhibitor in septic patients should be reconsidered.     

HSPTX protects against hemorrhagic shock resuscitation-induced tissue injury: an attractive alternative to Ringer's lactate

BACKGROUND: Conventional fluid resuscitation with Ringer's lactated (RL) activates neutrophils and causes end-organ damage. We have previously shown that HSPTX, a combination of small volume hypertonic saline (HS) and pentoxifylline (PTX), a phosphodiesterase-inhibitor, downregulates in vitro neutrophil activation and proinflammatory mediator synthesis. Herein, we hypothesized that HSPTX decreases end-organ injury when compared with RL in an animal model of hemorrhagic shock. METHODS: Sprague-Dawley rats were bled to a mean arterial pressure of 35 mm Hg for 1 hour. Animals were divided into 3 groups: sham (no shock, no resuscitation, n = 7), RL (32 mL/kg, n = 7), and HSPTX (7.5% NaCl 4 mL/kg + PTX 25 mg/kg; n = 7). Shed blood was infused after fluid resuscitation. Blood pressure was monitored until the end of resuscitation. Animals were sacrificed at 24 hour after resuscitation. Bronchoalveolar lavage fluid (BALF) was obtained for white cell count (total and differential) and TNF-alpha and IL-1beta levels were measured by ELISA. Lung and intestinal injury at 24 hour were evaluated by histopathology. Organ damage was graded by a pathologist and a score was created (0 = no injury; 3 = severe). Lung neutrophil infiltration was evaluated by MPO immune staining. RESULTS: There were no differences in mean arterial pressure between groups. At 24 hours, BALF leukocyte count was decreased by 30% in HSPTX animals (p < 0.01). TNF-alpha and IL-1beta levels were markedly decreased in HSPTX-resuscitated animals compared with their RL counterparts (p < 0.01). HSPTX-resuscitated animals (lung injury score = 1.0 +/- 0.4) had markedly decreased acute lung injury compared with RL-treated animals (2.5 +/- 0.3) (p < 0.01). RL resuscitation led to a two-fold increase in lung neutrophil infiltration whereas in HSPTX-treated animals, the number of MPO + cells was similar to sham animals (p < 0.001). Intestinal injury was markedly attenuated by HSPTX (1.1 +/- 0.3) compared with RL animals (2.6 +/- 0.4) (p < 0.001). CONCLUSIONS: HSPTX, a small volume resuscitation strategy with marked immunomodulatory potential led to a marked decrease in end-organ damage. HSPTX is an attractive alternative to RL in hemorrhagic shock resuscitation.     

Selective iNOS Inhibition Attenuates Acetylcholine- and Bradykinin-induced Vasoconstriction in Lipopolysaccharide-exposed Rat Lungs

Background: Nonselective nitric oxide synthase (NOS) inhibition has detrimental effects in sepsis because of inhibition of the physiologically important endothelial NOS (eNOS). The authors hypothesized that selective inducible NOS (iNOS) inhibition would maintain eNOS vasodilation but prevent acetylcholine- and bradykinin-mediated vasoconstriction caused by lipopolysaccharide-induced endothelial dysfunction.

Methods: Rats were administered intraperitoneal lipopolysaccharide (15 mg/kg) with and without the selective iNOS inhibitors L-N 6-(1-iminoethyl)-lysine (L-NIL, 3 mg/kg), dexamethasone (1 mg/kg), or the nonselective NOS inhibitor N[omega]-nitro-L-arginine methylester (L-NAME, 5 mg/kg). Six hours later, the lungs were isolated and pulmonary vasoreactivity was assessed with hypoxic vasoconstrictions (3% O2), acetylcholine (1 [mu]g), Biochemical Engineering, and bradykinin (3 [mu]g). In additional lipopolysaccharide experiments, L-NIL (10 [mu]M) or 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 100 [mu]M), a selective muscarinic M3 antagonist, was added into the perfusate.

Results: Exhaled nitric oxide was higher in the lipopolysaccharide group (37.7 +/- 17.8 ppb) compared with the control group (0.4 +/- 0.7 ppb). L-NIL and dexamethasone decreased exhaled nitric oxide in lipopolysaccharide rats by 83 and 79%, respectively, whereas L-NAME had no effect. In control lungs, L-NAME significantly decreased acetylcholine- and bradykinin-induced vasodilation by 75% and increased hypoxic vasoconstrictions, whereas L-NIL and dexamethasone had no effect. In lipopolysaccharide lungs, acetylcholine and bradykinin both transiently increased the pulmonary artery pressure by 8.4 +/- 2.0 mmHg and 35.3 +/- 11.7 mmHg, respectively, immediately after vasodilation. L-NIL and dexamethasone both attenuated this vasoconstriction by 70%, whereas L-NAME did not. The acetylcholine vasoconstriction was dose-dependent (0.01-1.0 [mu]g), unaffected by L-NIL added to the perfusate, and abolished by 4-DAMP.     

Adherent neutrophils mediate permeability after atelectasis.

Re-expansion of atelectatic lung is associated with increased permeability. This study tests whether neutrophils mediate this event. Right middle lobar atelectasis was induced in anesthesized rabbits (n = 18) by intraluminal obstruction of the bronchus after a 20-minute ventilation with 100% O2. After 1 hour of bronchial obstruction and 20 minutes after lobar re-expansion, leukopenia was noted, 2870 +/- 210 white blood cells (WBC)/mm3, relative to control animals treated with a noninflated balloon catheter, 6500 +/- 410 WBC/mm3 (p less than 0.05). Three hours after re-expansion, neutrophils were sequestered in the previously atelectatic region 78 +/- 7 polymorphonuclear leukocytes (PMN)/10 high-power field (HPF), as well as in nonatelectatic areas, 40 +/- 3 PMN/10 HPF, higher than control values of 26 +/- 3 PMN/10 HPF (p less than 0.05). In the atelectatic region, neutrophil sequestration was associated with increased protein concentration in lobar bronchoalveolar lavage (BAL) of 1370 +/- 100 micrograms/mL, higher than control values of 270 +/- 20 micrograms/mL (p less than 0.05). Reexpansion also induced increases in lung wet-to-dry weight ratio (W/d) of 6.2 +/- 0.2, higher than control values of 4.3 +/- 0.1 (p less than 0.05). Rendering rabbits neutropenic (n = 18) (0 to 4 PMN/mm3) limited the atelectasis-induced protein accumulations in BAL (520 +/- 60 micrograms/mL) and increase in lung W/d (5.2 +/- 0.1) (both p less than 0.05). Intravenous (I.V.; treatment of another group (n = 18) with an anti-CD 18 monoclonal antibody (R 15.7, 1 mg/kg) before balloon deflation prevented leukopenia (6550 +/- 560 WBC/mm3), minimized neutrophil sequestration (36 +/- 2 PMN/10 HPF), and attenuated protein leak (710 +/- 95 micrograms/mL) and the increased lung W/d (5.6 +/- 0.1) (all p less than 0.05). A final atelectatic group (n = 9) was treated I.V. with the anti-intercellular adhesion molecule-1 monoclonal antibody (RR 1/1, 1 mg/kg), which also prevented leukopenia and showed similar protection of microvascular barrier function. These data indicate that adherent neutrophils in large part mediate lung permeability and edema after atelectasis and re-expansion. Adhesion receptors of both neutrophils and endothelial cells regulate this event.     

Hemorrhagic shock induced up-regulation of P-selectin expression is mediated by factors in mesenteric lymph and blunted by mesenteric lymph duct interruption

BACKGROUND: Previous studies have shown that mesenteric lymph duct interruption prevents lung injury and decreases lung neutrophil sequestration after hemorrhagic shock (HS). Since endothelial cells rapidly express P-selectin after ischemia/reperfusion injury and HS-induced lung injury appears to involve neutrophil-endothelial cell interactions, we tested the following two hypotheses. First, that HS increases endothelial cell P-selectin expression and that interruption of mesenteric lymph flow in vivo would diminish this expression. Second, that incubation of human umbilical vein endothelial cells with post-HS mesenteric lymph but not sham shock (SS) lymph or postshock portal vein plasma would up-regulate P-selectin expression. METHODS: Pulmonary microvascular P-selectin expression was measured in male rats subjected to 90 minutes of HS (30 mm Hg), SS, or HS with lymphatic ligation, with a dual radiolabeled monoclonal antibody technique. The lungs from these animals were subsequently harvested and P-selectin expression was expressed as mean +/- SEM nanograms of monoclonal antibody per gram of tissue. RESULTS: Pulmonary P-selectin expression was 2.0 +/- 0.4 after SS, 9.7 +/- 3.0 after HS, but decreased to 2.3 +/- 0.3 after HS with lymph interruption (p < 0.05 HS vs. SS or HS plus lymph ligation). Incubation of human umbilical vein endothelial cells with shock lymph collected 3 to 4 hours after shock resulted in a nearly fivefold increase in P-selectin expression (p < 0.001) as compared with SS lymph, lymph collected 6 hours after shock, or postshock portal vein plasma. CONCLUSION: These results support the concept that gut-derived lymph promotes HS-induced lung injury through up-regulation of microvascular adhesion molecules and that intestinal lymph duct interruption may prevent distant organ injury by blunting the expression of these molecules.