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Among enterovirus 71 infections, brainstem encephalitis progressing abruptly to cardiac dysfunction and pulmonary edema causes rapid death within several hours. However, no currently known early indicators and treatments can monitor or prevent the unexpectedly fulminant course. We investigate the possible mechanisms and treatment of fatal enterovirus 71 infections to prevent the abrupt progression to cardiac dysfunction and pulmonary edema by using an animal model.Treatment study.Research laboratory.Sprague-Dawley rats.We microinjected 6-hydroxydopamine or vitamin C into nucleus tractus solitarii of the rat and evaluated the cardiopulmonary changes after treatment with ganglionic blocker.The time course of changes in the heart and lungs of rats with brainstem lesions were investigated. Rats were administered 6-hydroxydopamine to induce brainstem lesions, causing acute hypertension in 10 minutes and acute elevations of catecholamines accompanied by acute cardiac dysfunction and increased strong expressions of connexin 43 gap junction protein in heart and lung specimens by immunohistochemical staining within 3 hours. Severe pulmonary hemorrhagic edema was produced within 6 hours, and the rats expired rapidly within 7 hours. After hexamethonium treatment, it was found that the acute hypertension induced by 6-hydroxydopamine lesions was immediately reversed and the acute high rise of catecholamine serum level was significantly attenuated within 3 hours, accompanied by preserved cardiac output and decreased expressions of connexin 43 in the heart and lungs. No pulmonary edema occurred and the rats survived for more than 14 hours.Early hexamethonium treatment attenuates acute excessive release of catecholamines to prevent cardiac dysfunction and pulmonary edema for increasing survival rate.

Objective The molecular mechanism of major burn-induced gut damage is not clear. This study is to determine whether IkappaB-kinase (IKK)/nuclear factor-kappaB signaling in intestinal mucosa maintains gut function through the regulation of the c-Jun NH2-terminal kinase (JNK) and p38 phosphorylation. Design Prospective, experimental study. Setting Research laboratory at a university hospital. Subjects Thermal injury models in mice. Interventions Conditional intestinal epithelial cell IKKbeta knockout (Vil-Cre/Ikkbeta(F/Delta) mice and control (Ikkbeta(F/Delta) mice were subjected to 30% total body surface area third-degree burn. JNK inhibitor (SP600125) or p38 inhibitor (SB203580) was given to mice immediately after burn injury. Measurements and main results Thermal injury induced a significant increase of intestinal permeability, nuclear factor-kappaB DNA-binding activity, phosphorylated JNK, phosphorylated p38, and caspase 3 expression of intestinal mucosa in Vil-Cre/Ikkbeta(F/Delta) mice compared with those of Ikkbeta(F/Delta) mice. BCL-xL and cellular FLICE inhibitory protein, but not GADD45beta (growth arrest and DNA damage-inducing protein beta), cellular inhibitor of apoptosis 1, Bfl-1, or TRAIL, messenger RNA expression was significantly decreased in Vil-Cre/Ikkbeta(F/Delta) mice compared with that of Ikkbeta(F/Delta) mice. SP600125 decreased intestinal permeability and increased phosphorylated p38 and tumor necrosis factor receptor-associated factor 2 expression of intestinal mucosa in Vil-Cre/Ikkbeta(F/Delta) mice. SB203580 treatment enhanced thermal injury-induced gut damage in Vil-Cre/Ikkbeta(F/Delta) mice. Conclusions Thermal injury induces nuclear factor-kappaB activation of intestinal mucosa and IKK protects intestinal mucosa from thermal injury-induced gut damage. IKK blocks caspase 3 expression by up-regulating BCL-xL and cellular FLICE inhibitory protein expression. IKK inhibits JNK and p38 but not p44/42 phosphorylation of intestinal mucosa. JNK inhibition increases p38 and tumor necrosis factor receptor-associated factor 2 expression and decreases thermal injury-induced gut damage. Taken together with the enhanced thermal injury-induced gut damage by p38 inhibition, we conclude that IKK maintains gut function by inhibiting JNK phosphorylation, which suppresses p38 phosphorylation and induces gut damage.

Objective: To determine whether inhibition of inducible nitric oxide synthase to stabilize endothelial permeability and to retain hypertonic saline in the vascular space will ameliorate burn-induced gut barrier dysfunction. Design: Prospective, experimental study. Setting: Research laboratory at a university hospital. Subjects: Thermal injury models in the rat. Interventions: In experiment 1, specific pathogen free rats underwent 3% total body surface area burn or sham burn and were given 7.5 mL/kg hypertonic saline (7.5% NaCl), 7.5 mg/kg saline, or 50 mL/kg saline (nearly equal sodium load with hypertonic saline) in the right femoral vein for 15 mins for fluid resuscitation at 0, 4, or 8 hrs after burn. In experiment 2, S-methylisothiourea (7.5 mg/kg, intraperitoneally), a specific inducible nitric oxide synthase inhibitor, was given immediately after burn to rats from different groups as in experiment 1. At 24 hrs after burn, the intestinal mucosa was assayed for myeloperoxidase activity and lipid peroxidation, the distribution of fluorescein isothiocyanate-dextran across the lumen of the small intestine was determined, and bacterial translocation to the mesenteric lymph nodes and ileum histology were also examined. Measurements and Main Results: Burn induced significant increases in intestinal mucosa inducible nitric oxide synthase expression, myeloperoxidase activity, lipid peroxidation, intestinal permeability, bacterial translocation to mesenteric lymph nodes, and villi sloughing in rats. Hypertonic saline administration at 0 or 4 hrs after burn worsened intestinal mucosa lipid peroxidation, neutrophil sequestration, intestinal permeability, and villi sloughing compared with those of burn + 7.5 mg/kg saline and burn + 50 mL/kg saline rats. To the contrary, burn + S-methylisothiourea rats with hypertonic saline injection at 4 or 8 hrs after burn showed an improvement of gut barrier function compared with burn + S-methylisothiourea + 7.5 mg/kg saline and burn + S-methylisothiourea + 50 mL/kg saline rats. Administration of hypertonic saline at 8 hrs after burn and S-methylisothiourea injection also significantly attenuated the bacterial translocation to mesenteric lymph nodes and villi sloughing. Conclusions: Using hypertonic saline as a resuscitation fluid in early burn shock markedly augmented the thermal injury-induced intestinal mucosa neutrophil deposition, lipid peroxidation, and intestinal hyperpermeability. Inhibition of inducible nitric oxide synthase not only significantly attenuated neutrophil deposition and mucosa lipid peroxidation but also reversed the deteriorating effects of hypertonic saline on thermal injury-induced gut barrier dysfunction and bacterial translocation.

Objective: The molecular mechanisms of lung damage following thermal injury are not clear. The purpose of this study was to determine whether interleukin (IL)-1 mediates burn-induced inducible nitric oxide synthase (iNOS) expression, peroxynitrite production, and lung damage through c-Jun NH 2 -terminal kinase (JNK) signaling. Design: Prospective, experimental study. Setting: Research laboratory at a university hospital. Subjects: Thermal injury models in the mice. Interventions: IL-1 receptor type 1 (lL-1R1) -/- mice, Tnfrsf1a -/- mice, and wild-type (WT) mice were subjected to 30% total body surface area third-degree bum. The JNK inhibitor, SP600125, was given to mice to study the involvement of the JNK pathway in thermal injury-induced lung damage. WT → WT, WT → IL-1R1 -/- , and lL-1R -/- → WT chimeric mice were generated to determine the role of hematopoietic cells in IL-1-mediated lung damage. Neutrophils were harvested and treated in vitro with N-Momiyl-methionylleucyl-phenylalanine (fMLP). Measurements and Main Results: IL-1R1 -/- mice rather than Tnfrsf1a -/- mice showed less thermal injury-induced lung damage. IL-1R1 -/- mice displayed less lung JNK activity; intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), chemokine receptor 2 (CXCR2), and macrophage inflammatory protein-2 (MIP2), messenger RNA expression; myeloperoxidase activity; and neutrophil p38 mitogen-activated protein kinase (MAPK) phosphorylation after thermal injury. SP600125 significantly reduced thermal injury-induced blood dihydrorhodamine (DHR) 123 oxidation, iNOS expression, and lung permeability in WT mice but not in lL-1R1 -/- mice. IL-1R1 -/- → WT chimeric mice rather than WT → IL-1R1 -/- chimeric mice showed less thermal injury-induced lung damage. fMLP increased reactive oxygen species (ROS) production of neutrophils in WT mice but not in IL-1R1 -/- mice. SP600125 decreased ROS production of neutrophils in WT mice but not in IL-1R1 -/- mice. Conclusions: Thermal injury-induced lung JNK activation; lung ICAM, VCAM, CXCR2, and MIP2 expression; and DHR 123 oxidation are IL-1 dependent. JNK inhibition decreases IL-1-mediated thermal injury-induced lung damage. Given that the IL-1 receptor is critical in thermal injury-induced p38 MAPK phosphorylation and ROS production of neutrophils, we conclude that IL-1 mediates thermal injury-induced iNOS expression and lung damage through the JNK signaling pathway.

Objective To determine whether burn-induced peroxynitrite production and expression of lung inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CXCR2, macrophage inflammatory protein (MIP)-2, and neutrophil chemokine (KC) are mediated by the c-Jun NH2-terminal kinase (JNK). Design Prospective, experimental study. Setting Research laboratory at a university hospital. Subjects Thermal injury models in the mice. Interventions In experiment 1, specific pathogen-free C57/BL6 mice were subjected to 30% total body surface area third-degree burn over shaved back. At 0 hr, 2 hrs, 4 hrs, and 6 hrs after burn, lung tissues of those mice were harvested for JNK activity assay, AP-1 DNA-binding activity, and pJNK immunohistochemistry. In experiment 2, a specific JNK inhibitor, SP600125, was given (30 mg/kg intraperitoneally) to mice immediately postburn to suppress the JNK activity. At 8 hrs after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine (DHR) 123 oxidation. Lung tissues were harvested for myeloperoxidase (MPO) determination, ICAM-1, VCAM-1, CXCR2, KC, MIP-2, interleukin-1beta, and interleukin-6 messenger RNA expression; iNOS immunohistochemical staining; and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. Measurements and main results The JNK activity and AP-1 DNA-binding activity of lung tissue significantly increased to a peak at 2 hrs and 4 hrs, respectively, after thermal injury. Immunohistochemical study demonstrated that the increase of the pJNK was mostly from the bronchiole epithelial cells. This increase of MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased six-fold, nine-fold, and four-fold after burn. SP600125 administration obliterated the thermal injury-induced JNK activity, AP-1 DNA-binding activity, and iNOS expression in lung tissue. SP600125 treatment also significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 54%, 8%, and 47%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. Furthermore, SP600125 abolished thermal injury-induced ICAM-1, VCAM-1, CXCR2, MIP-2, and KC but not interleukin-1beta and interleukin-6 messenger RNA levels of lung tissues. Conclusions Thermal injury induces lung tissue JNK activation and AP-1 DNA-binding activity mainly from airway epithelial cells. Thermal injury-induced peroxynitrite production and lung iNOS, ICAM-1, and VCAM-1 expression are mediated by the JNK signaling. JNK inhibition decreases thermal injury-induced lung neutrophil infiltration and subsequently pulmonary hyperpermeability.

Intestinal ischemia and reperfusion injury was known to cause postinjury multiple organ failure by neutrophil and unclear nonneutrophil factors. Peroxynitrite formed by the rapid reaction between superoxide and nitric oxide, is a toxic substance that contributes to tissue injury in a number of biological systems. In this study, the role of nitric oxide and neutrophils on lung damage after burn was investigated.Prospective, experimental study.Research laboratory at a university hospital.Thermal injury models in the rat.In experiment 1, specific pathogen-free Sprague-Dawley rats underwent 35% total body surface area burn. At 4, 8, 16, and 24 hrs after burn, intestinal mucosa and lung tissue were harvested for myeloperoxidase (MPO) assay, blood was collected for measurement of peroxynitrite-mediated oxidation of dihydrorhodamine 123, and pulmonary microvascular dysfunction was quantified by measuring the extravasation of Evans blue dye. In experiment 2, polymorphonuclear granulocyte antibody (0.12 mL/100 g administered intraperitoneally 16 hrs before burn), S-methylisothiourea (7.5 mg/kg, intraperitoneally, immediately after burn), a specific inducible nitric oxide synthase inhibitor, and sterile saline (15 mL/kg, intraperitoneally, immediately after burn) were given to different groups of thermally injured animals individually. The plasma dihydrorhodamine 123 oxidation level, intestinal and lung MPO activity, lung permeability, and lung histology were evaluated at 8 hrs after burn. The cellular localization of nitrotyrosine, a marker for peroxynitrite reactivity, was also examined by immunostaining. In experiment 3, 3-morpholinosydnonimine (10 mM, intraperitoneally), a peroxynitrite donor, was given to nonburned rats to examine the peroxynitrite effect on lung inducible nitric oxide synthase expression.The level of MPO activity in intestine and lung, blood dihydrorhodamine 123 oxidation, and lung permeability were increased up to 2-fold, 2.5-fold, 2-fold, and 2-fold of normal, respectively, at 8 hrs after burn. S-methylisothiourea injection significantly decreased (p.05) 31% of the lung MPO activity, 41% of the blood peroxynitrite level, 54% of the lung permeability, and the lung peroxynitrite production in burned rats. Polymorphonuclear granulocyte antibody pretreatment significantly decreased 60% of the intestinal MPO, 92% of the blood peroxynitrite level, and 56% the lung MPO activity in burned rats, but the lung permeability was only slightly decreased by polymorphonuclear granulocyte antibody pretreatment. Furthermore, 3-morpholinosydnonimine increased the lung inducible nitric oxide synthase messenger RNA levels.Thermal injury induces blood dihydrorhodamine 123 oxidation, intestinal and lung neutrophil deposition, lung nitrotyrosine production, and lung damage. Both specific inhibition of inducible nitric oxide synthase and polymorphonuclear granulocyte antibody pretreatment decrease blood dihydrorhodamine 123 oxidation and intestinal and lung neutrophil deposition, but only inducible nitric oxide synthase inhibition with S-methylisothiourea reduces lung peroxynitrite production and thermal injury-induced lung damage. Nitric oxide and the ensuing peroxynitrite production in lung play a more important role than neutrophil in contributing to thermal injury-induced lung damage.