Da Zhong Xu

Hypertonic saline improves intestinal mucosa barrier function and lung injury after trauma-hemorrhagic shock.

Our objective was to test the hypotheses that small volume hypertonic saline (HTS) resuscitation protects against trauma-hemorrhagic shock (T/HS)-induced intestinal and lung injury better than standard volume resuscitation with Ringers lactate (RL), and that the degree of lung injury correlates with the degree of gut injury after therapy. Male Sprague-Dawley rats were subjected to laparotomy (trauma) and 90 min of T/HS or sham shock (T/SS), and were then resuscitated with RL or 7.5% NaCl solution at an equivalent sodium load. Intestinal and lung injury was assessed at 3 and 24 h after resuscitation. Lung permeability, pulmonary myeloperoxidase (MPO) levels, and the bronchoalveolar lavage fluid (BALF) protein to plasma protein ratio were increased after T/HS, but were significantly lower in HTS-resuscitated than RL-treated rats. The incidence of bacterial translocation (BT) was not different between the groups, but the magnitude of BT after T/HS was less after HTS than RL resuscitation. Barrier function of intestinal segments was impaired only in the T/HS rats resuscitated with RL and histological analysis demonstrated fewer injured villi in the T/HS rats resuscitated with HTS than RL. Linear regression analysis revealed direct correlations between the percent of injured villi, increased lung permeability, and pulmonary neutrophil sequestration. Resuscitation with HTS ameliorated T/HS-induced gut and lung injury seen with RL resuscitation. These results, together with the direct correlation found between gut and lung injury, suggest that lung injury after T/HS may be mediated by gut injury.

Serine proteases are involved in the pathogenesis of trauma-hemorrhagic shock-induced gut and lung injury.

The objective of this work was to test the hypothesis that intraluminal serine proteases are involved in trauma-hemorrhagic shock (T/HS)-induced intestinal and lung injury. Male Sprague-Dawley rats were administrated the serine protease inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, Nafamostat) either intraluminally into the gut or intravenously after a laparotomy (trauma) and then subjected to 90 min of hemorrhagic shock (T/HS) or sham shock (T/SS). Intestinal and lung injury was assessed at 3 h after resuscitation with Ringers lactate solution. In a second set of experiments, mesenteric lymph was collected from the groups of rats subjected to T/HS or T/SS and its ability to activate normal neutrophils was tested. Lung permeability, pulmonary myeloperoxidase levels, and the bronchoalveolar lavage fluid protein to plasma protein ratio were increased after T/HS but were significantly decreased in the T/HS rats receiving intraluminal (P < 0.05), but not intravenous, nafamostat. Likewise, T/HS-induced intestinal villus injury was less in the nafamostat-treated shock rats (P < 0.05). Last, the ability of T/HS mesenteric lymph to increase PMN CD11b expression or prime neutrophils for an augmented respiratory burst was significantly reduced by the intraluminal administration of nafamostat. Because intraluminal nafamostat reduced T/HS-induced gut and lung injury as well as the neutrophil activating ability of intestinal T/HS lymph, the presence of serine proteases in the ischemic gut may play an important role in T/HS-induced gut and hence lung injury.

HIF-1 mediates pathogenic inflammatory responses to intestinal ischemia-reperfusion injury

Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1α-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1α deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1α deficiency reduced TNF-α, IL-1β, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1β mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights.

Trauma/hemorrhagic shock mesenteric lymph upregulates adhesion molecule expression and IL-6 production in human umbilical vein endothelial cells

Trauma/hemorrhagic shock (T/HS) is associated with significant lung injury, which is mainly due to an inflammatory process, resulting from the local activation and subsequent interaction of endothelial cells and leukocytes. Adhesion molecules expressed by both cell types play a crucial role in the process of neutrophil-mediated endothelial cell injury. We have previously shown that mesenteric lymph duct ligation prevents T/HS-induced lung leukocyte infiltration and endothelial injury, suggesting that inflammatory factors originating from the gut and carried in the lymph are responsible for the lung injury observed following T/HS. Based on these observations, we hypothesized that inflammatory substances in T/HS lymph trigger lung injury by a mechanism involving the upregulation of adhesion molecules. To test this hypothesis, we examined whether T/HS mesenteric lymph induces the expression of E-selectin, P-selectin, and intracellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs). Furthermore, because the cytokine IL-6 is an important component of the endothelial inflammatory process, we investigated how T/HS lymph affects the production of IL-6 by HUVECs. Mesenteric lymph from T/HS rats increased both E- and P-selectin, as well as ICAM-1 expression on HUVECS, as compared to trauma/sham shock (T/SS) lymph or medium only groups. However, T/HS lymph failed to induce the shedding of E-selectin. In HUVECs treated with T/HS lymph, IL-6 concentrations were higher than HUVECs treated with T/SS lymph. These findings suggest that mesenteric lymph produced after hemorrhagic shock potentiates lung injury by the upregulation of endothelial cell adhesion molecule expression and IL-6 production.

Adenosine A2A receptor activation reduces lung injury in trauma/hemorrhagic shock.

Objective:Hemorrhagic shock and resuscitation trigger a global ischemia/reperfusion phenomenon, in which various inflammatory processes critically contribute to the ensuing tissue damage. Adenosine is an endogenous nucleoside that is released during shock. Activation of adenosine A2A receptors can broadly inactivate inflammatory cascades. The current study was designed to evaluate the effect of A2A receptor activation on organ injury and inflammation in the setting of global ischemia/reperfusion elicited by trauma/hemorrhagic shock and resuscitation. Design:Prospective animal study with concurrent control. Setting:Small animal laboratory. Subjects:Adult male Sprague-Dawley rats. Interventions:The rats were subjected to a laparotomy (trauma) and 90 mins of hemorrhagic shock or trauma/sham shock. The selective A2A receptor agonist CGS-21680 (2-p-(2-carboxyethyl) phenethylamino-5′-N-ethyl-carboxamidoadenosine; 0.5 mg/kg) or its vehicle was injected 30 mins before shock or immediately after resuscitation. At 3 hrs following resuscitation, animals were killed and tissue was harvested for analysis. Lung permeability and pulmonary myeloperoxidase levels were used to quantitate lung injury. Intestinal injury was determined by histologic analysis of terminal ileum. Red blood cell deformability was measured by a laser-assisted ektacytometer. In this assay, a decrease in the elongation index is a marker of decreased red blood cell deformability. Measurements and Main Results:Pretreatment with CGS-21680 protected the lung but not the gut against shock-induced injury and prevented the shock-induced decrease in red blood cell deformability. Posttreatment with CGS-21680 ameliorated shock-induced lung injury but failed to prevent gut injury and preserve red blood cell deformability. Conclusion:A2A receptor agonists may represent a novel therapeutic approach in preventing organ injury following trauma/hemorrhagic shock.

Trauma-Hemorrhagic Shock Mesenteric Lymph Induces Endothelial Apoptosis That Involves Both Caspase-Dependent and Caspase-Independent Mechanisms

Objective:To determine the mechanism by which gut-derived factors present in mesenteric lymph from rats subjected to trauma-hemorrhagic shock (T/HS) induce endothelial cell death. Summary Background Data:Intestinal ischemia after hemorrhagic shock results in gut barrier dysfunction and the subsequent production of biologically active and tissue injurious factors by the ischemic gut. These factors are carried in the mesenteric lymph and reach the systemic circulation via the mesenteric lymph, thereby ultimately resulting in distant organ injury. Although studies have established that trauma-hemorrhagic (T/HS) shock but not trauma-sham-shock (T/SS) mesenteric lymph is cytotoxic to endothelial cells, whether T/HS lymph-induced endothelial cell death occurs via an apoptotic or a necrotic pathway is unknown. The mechanisms underlying T/HS lymph-induced cytotoxicity are likewise unknown. Methods:Human umbilical vein endothelial cell (HUVEC) monolayers were incubated with medium, sham-shock, or post shock mesenteric lymph (5%) for 4 hours, after which the mode of cell death (ie, apoptosis versus necrosis) was determined using morphologic (confocal microscopy), biochemical (nucleosomal release), and DNA-based (gel electrophoresis) assays. To clarify the cellular pathways involved in T/HS lymph-induced HUVEC cell death, caspase-3, caspase-9, caspase-8, and BID activity was measured as was the ability of the pan-caspase inhibitor z-VAD-fmk to prevent T/HS lymph-induced cell death. Results:T/HS, but not T/SS, mesenteric lymph or medium was cytotoxic and caused the appearance of the classic morphologic signs of apoptosis, including membrane blebbing, cell shrinkage, and apoptotic body formation. Nucleosomal release and a DNA laddering pattern was also observed in the HUVECs incubated with T/HS lymph. These signs of apoptosis were associated with increased caspase activity as reflected in activation of the pro-apoptotic caspases, caspase-8, -9, and -3, as well as the pro-apoptotic bcl-2-related protein BID. However, since the broad-spectrum caspase inhibitor z-VAD-fmk delayed T/HS lymph-induced HUVEC cell death, but did not prevent it fully, it appears that other factors besides caspases are involved in the endothelial cell toxicity of T/HS lymph. Conclusions:Gut-derived factors in T/HS, but not T/SS, mesenteric lymph cause endothelial cell death via an apoptotic mechanism that involves both caspase-dependent and caspase-independent pathways.

Activation of toll-like receptor 4 is necessary for trauma hemorrhagic shock-induced gut injury and polymorphonuclear neutrophil priming.

ABSTRACT Interactions of toll-like receptors (TLRs) with nonmicrobial factors play a major role in the pathogenesis of early trauma-hemorrhagic shock (T/HS)–induced organ injury and inflammation. Thus, we tested the hypothesis that TLR4 mutant (TLR4mut) mice would be more resistant to T/HS-induced gut injury and polymorphonuclear neutrophil (PMN) priming than their wild-type littermates and found that both were significantly reduced in the TLR4mut mice. In addition, the in vivo and ex vivo PMN priming effect of T/HS intestinal lymph observed in the wild-type mice was abrogated in TLR4mut mice as well the TRIFmut-deficient mice and partially attenuated in Myd88−/− mice, suggesting that TRIF activation played a more predominant role than MyD88 in T/HS lymph–induced PMN priming. Polymorphonuclear neutrophil depletion studies showed that T/HS lymph–induced acute lung injury was PMN dependent, because lung injury was totally abrogated in PMN-depleted animals. Because the lymph samples were sterile and devoid of endotoxin or bacterial DNA, we investigated whether the effects of T/HS lymph was related to endogenous nonmicrobial TLR4 ligands. High-mobility group box 1 protein 1, heat shock protein 70, heat shock protein 27, and hyaluronic acid all have been implicated in ischemia-reperfusion-induced tissue injury. None of these “danger” proteins appeared to be involved, because their levels were similar between the sham and shock lymph samples. In conclusion, TLR4 activation is important in T/HS-induced gut injury and in T/HS lymph–induced PMN priming and lung injury. However, the T/HS-associated effects of TLR4 on gut barrier dysfunction can be uncoupled from the T/HS lymph–associated effects of TLR4 on PMN priming.

Hemorrhagic shock induces endothelial cell apoptosis, which is mediated by factors contained in mesenteric lymph

Objective:Trauma-hemorrhagic shock is one of the leading causes of acute respiratory distress syndrome. This syndrome is associated with disruption of the alveolar barrier consisting of both epithelial and endothelial cells, which leads to a major increase in epithelial and microvascular permeability in the lungs. Although alveolar epithelial cell apoptosis has been documented as a contributing factor to this increase in permeability, it is unclear whether endothelial cell apoptosis occurs following trauma-hemorrhagic shock and, if so, the source of factors leading to this process. Design:Prospective animal study with concurrent control. Setting:Small-animal laboratory. Subjects:Adult male Sprague-Dawley rats. Interventions:Trauma-hemorrhagic shock in rats was induced by laparotomy followed by blood withdrawal to achieve a mean arterial blood pressure of 30 mm Hg for 90 mins. At the end of the shock period, the rats were resuscitated, and 3 hrs later lungs were taken for histologic analysis. In other experiments, mesenteric lymph was collected from trauma-hemorrhagic shock and trauma-sham shock rats, and the biological activity of these lymph samples was tested for their ability to kill cultured endothelial cells or endothelial cells of isolated femoral veins. Measurements and Main Results:Trauma-hemorrhagic shock triggered endothelial cell apoptosis in the lung as assessed using the Tunnel assay as well as by light and electron microscopic analysis. Since our previous studies have documented that mesenteric lymph is a major contributor to lung injury following shock, we also tested the hypothesis that factors in the mesenteric lymph were responsible for the endothelial cell apoptosis-inducing effect of shock. Preventing the mesenteric lymph from reaching the lung by mesenteric lymph duct ligation decreased endothelial cell apoptosis. Mesenteric lymph obtained from rats subjected to trauma-hemorrhagic shock elicited apoptosis in cultured endothelial cells and when placed into isolated femoral vein as well as increased endothelial cell monolayer permeability. Conclusions:Trauma-hemorrhagic shock induces endothelial as well as epithelial cell apoptosis in the lung via factors contained in the mesenteric lymph, thereby contributing to the pathophysiology of the acute respiratory distress syndrome.

Mesenteric lymph from burned rats induces endothelial cell injury and activates neutrophils

ObjectiveOur previous studies indicated that mesenteric lymph duct ligation prevented burn-induced lung injury. Thus, the goal of the present study was to begin to investigate potential mechanisms of this protective effect. DesignProspective animal study with concurrent control. SettingSmall animal laboratory. SubjectsAdult male Sprague-Dawley rats. InterventionsMesenteric lymph and portal vein plasma were collected from male rats subjected to a 40% third-degree scald burn or sham burn. The biological effects of these lymph and plasma samples were tested for their ability to kill human umbilical vein endothelial cells (HUVECs), increase HUVEC monolayer permeability, and activate polymorphonuclear leukocytes (PMNs), as reflected in CD11b adhesion molecule expression and superoxide production. Additionally, ileal specimens were harvested at the end of the experiment (6 hrs postburn) for histologic analysis. Measurements and Main ResultsPostburn mesenteric lymph produced during the first 2 hrs after burn injury and tested at a 5% concentration, but not sham-burn lymph or portal plasma from burned rats, was toxic for HUVECs resulting in cell death after an 18-hr incubation period. Similarly, only postburn lymph increased HUVEC monolayer permeability. Postburn lymph activated both rat and human PMNs as reflected in increased CD11b expression and augmentation of the phorbol myristate acetate-induced superoxide response. Neither sham-burn lymph nor postburn portal vein plasma activated PMNs. Both the burn and sham-burn lymph samples were sterile, indicating that the effects of burn lymph on the HUVECs or PMNs were not due to translocating bacteria. Last, an association was found between burn-induced gut injury and the production of toxic burn lymph. ConclusionsBurn-induced gut injury results in the production of biologically active factors that are carried in the mesenteric lymph, but not the portal plasma, which injure endothelial cells and activate PMNs and thus could contribute to distant organ injury.

Pancreatic Duct Ligation Reduces Lung Injury Following Trauma and Hemorrhagic Shock

Objective:To determine whether pancreatic digestive enzymes released into the ischemic gut during an episode of T/HS are involved in the generation of distant organ injury. This hypothesis was tested by examining the effect of PDL on T/HS-induced intestinal injury, lung injury, and RBC deformability. Summary Background Data:The effect of pancreatic duct ligation (PDL) on distant organ injury following trauma/hemorrhagic shock (T/HS) was examined. PDL before T/HS decreases lung and red blood cell (RBC) injury and exerts a limited protective effect on the gut. Pancreatic proteases in the ischemic gut appear to be involved in gut-induced lung and RBC injury. Based on recent work, it appears that proinflammatory and/or toxic factors, which are generated by the ischemic intestine, play an important role in the pathogenesis of multiple organ failure. The process by which these toxic factors are generated remains unknown. Previous experimental work has clearly documented that intraluminal inhibition of pancreatic proteases decreases the degree of T/HS-induced lung injury and neutrophil activation. One possible explanation for this observation is that the toxic factors present in intestinal lymph are byproducts of interactions between pancreatic proteases and the ischemic gut. Methods:Male Sprague-Dawley rats were subjected to a laparotomy (trauma) and 90 minutes of sham (T/SS) or T/HS with or without PDL. At 3 and 24 hours following resuscitation, animals were killed and samples of gut, lung, and blood were collected for analysis. Lung permeability, pulmonary myeloperoxidase levels, and bronchoalveolar fluid protein content were used to quantitate lung injury. Intestinal injury was determined by histologic analysis of terminal ileum (% villi injured). To assess RBC injury, RBC deformability was measured, as the RBC elongation index (RBC-EI), using a LORCA device. Results:At 3 and 24 hours following resuscitation, PDL prevented shock-induced increases in lung permeability to both Evans blue dye and protein in addition to preventing an increase in pulmonary myeloperoxidase levels. T/HS-induced impairments in RBC deformability were significantly reduced at both time points in the PDL + T/HS group, but deformability did not return to T/SS levels. PDL did reduce the magnitude of ileal injury at 3 hours after T/HS, but the protective effect was lost at 24 hours after T/HS. Conclusions:PDL prior to T/HS decreases lung injury and improves RBC deformability but exerts a limited protective effect on the gut. Thus, the presence of pancreatic digestive enzymes in the ischemic gut appears to be involved in gut-induced lung and RBC injury.