Simulated aeromedical evacuation does not affect systemic inflammation or organ injury in a murine model of hemorrhagic shock.

Hemorrhagic shock is a primary injury amongst combat casualties. Aeromedical evacuation (AE) of casualties exposes patients to a hypobaric, hypoxic environment. The effect of this environment on the host response to hemorrhagic shock is unknown. In the present study, we sought to determine the effect of simulated AE on systemic inflammation and organ injury using a murine model of hemorrhagic shock. Mice underwent femoral artery cannulation and were hemorrhaged for 60 minutes. Mice were then resuscitated with a 1:1 ratio of plasma:packed red blood cells. At 1 or 24 hours after resuscitation, mice were exposed to a 5-hour simulated AE or remained at ground level (control). Serum was analyzed for cytokine concentrations and organs were assessed for neutrophil accumulation and vascular permeability. Mice in the simulated AE groups demonstrated reduced arterial oxygen saturation compared to ground controls. Serum cytokine concentrations, neutrophil recruitment, and vascular permeability in the lung, ileum, and colon in the simulated AE groups were not different from the ground controls. Our results demonstrate that mice exposed to simulated AE following hemorrhagic shock do not exhibit worsened systemic inflammation or organ injury compared to controls. The data suggest that AE has no adverse effect on isolated hemorrhagic shock.

Hemorrhagic shock induces a proinflammatory milieu in the gut lumen.

BACKGROUND: Intestinal injury is a consequence of hemorrhagic shock and resuscitation. The intestinal mucosa has been shown to respond to ischemia/reperfusion injury with production of inflammatory mediators. Previous work in our laboratory indicates that intestinal epithelial cells secrete proinflammatory cytokines in the direction of both the lamina propria and intestinal lumen. The ability of the intestinal mucosa to transmit inflammatory signals into the gut lumen after hemorrhagic shock is unknown. We hypothesized that hemorrhagic shock results in secretion of proinflammatory cytokines into the gut lumen. METHODS: Male C57/Bl6 mice underwent femoral artery cannulation and hemorrhage to a systolic blood pressure of 20 mmHg for 1 h, then resuscitation with lactated Ringer's (LR) solution. Sham animals were cannulated only. Mice were decannulated and sacrificed at intervals. Stool and succus were removed from intestinal segments, weighed, and placed into buffer solution. Specimens were analyzed via enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with sham-injured mice, hemorrhagic shock resulted in increased intestinal luminal cytokines. At 3 h after injury, elevated levels of IL-6 were found in the cecal stool. At 6 h after injury, TNFα, IL-6, and MIP-2 were significantly elevated in the cecal stool, and IL-6 and MIP-2 were significantly elevated in the distal colonic stool. CONCLUSIONS: Hemorrhagic shock results in secretion of proinflammatory cytokines into the intestinal lumen. These findings suggest that the intestinal mucosa may transmit and receive signals in a paracrine fashion via the gut lumen.

Hypobaric hypoxia exacerbates the neuroinflammatory response to traumatic brain injury.

OBJECTIVE: To determine the inflammatory effects of time-dependent exposure to the hypobaric environment of simulated aeromedical evacuation following traumatic brain injury (TBI). METHODS: Mice were subjected to a blunt TBI or sham injury. Righting reflex response (RRR) time was assessed as an indicator of neurologic recovery. Three or 24 h (Early and Delayed groups, respectively) after TBI, mice were exposed to hypobaric flight conditions (Fly) or ground-level control (No Fly) for 5 h. Arterial blood gas samples were obtained from all groups during simulated flight. Serum and cortical brain samples were analyzed for inflammatory cytokines after flight. Neuron specific enolase (NSE) was measured as a serum biomarker of TBI severity. RESULTS: TBI resulted in prolonged RRR time compared with sham injury. After TBI alone, serum levels of interleukin-6 (IL-6) and keratinocyte-derived chemokine (KC) were increased by 6 h post-injury. Simulated flight significantly reduced arterial oxygen saturation levels in the Fly group. Post-injury altitude exposure increased cerebral levels of IL-6 and macrophage inflammatory protein-1α (MIP-1α), as well as serum NSE in the Early but not Delayed Flight group compared to ground-level controls. CONCLUSIONS: The hypobaric environment of aeromedical evacuation results in significant hypoxia. Early, but not delayed, exposure to a hypobaric environment following TBI increases the neuroinflammatory response to injury and the severity of secondary brain injury. Optimization of the post-injury time to fly using serum cytokine and biomarker levels may reduce the potential secondary cerebral injury induced by aeromedical evacuation.

Remote thermal injury increases LPS-induced intestinal IL-6 production.

BACKGROUND: Patients suffering from burn injury are at high risk for subsequent infection. Thermal injury followed by endotoxemia may result in a "second hit," causing an exaggerated inflammatory response with increased morbidity and mortality. The role of the intestine in this "second hit" response is unknown. We hypothesized that remote thermal injury increases the inflammatory response of intestinal mucosa to subsequent treatment with lipopolysaccharide (LPS). METHODS: Mice underwent sham or scald injury. Seven days after injury, mice were treated with LPS. Blood and bowel specimens were obtained. Serum and intestinal inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Changes in TLR-4 pathway components in intestine were measured by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and electrophoretic mobility shift assay (EMSA). Intestinal leukocyte infiltration was analyzed by myeloperoxidase assay. RESULTS: A "second hit" of injected LPS resulted in increased IL-6 in intestine of burned mice compared with sham. Similarly, jejunal IL-6 mRNA levels increased in mice with prior thermal injury, suggesting a transcriptional mechanism. Of transcription factors known to drive IL-6 expression, only AP-1 activation was significantly elevated by a "second hit" of LPS. CONCLUSION: Prior thermal injury potentiates LPS-induced IL-6 cytokine production in intestine. These results indicate a heightened inflammatory response to a second hit by intestine after burn injury.

Prior thermal injury accelerates endotoxin-induced inflammatory cytokine production and intestinal nuclear factor-κB activation in mice.

The objective of this study was to increase the understanding of the "second-hit" response in thermal injury. The authors hypothesized that prior thermal injury increases the endotoxin-induced inflammatory response of intestinal mucosa. Mice underwent sham or 25% TBSA scald injury. Seven days after injury, mice were injected with lipopolysaccharide. Blood, jejunum, and colon specimens were obtained at intervals. Serum, jejunal, and colon inflammatory cytokine levels were measured by enzyme-linked immunosorbent assay. Jejunal and colon nuclear factor (NF)-κB activation was measured by electrophoretic mobility shift assay. After remote thermal injury, lipopolysaccharide exposure led to an acute increase in serum interleukin (IL)-6, IL-10, and chemokine keratinocyte-derived chemokine (KC) levels. This correlated with lipopolysaccharide-induced increased IL-6 in colon and chemokine KC in the jejunum and colon in burned mice when compared with sham-injured mice. Lipopolysaccharide-induced NF-κB activation occurred more rapidly in jejunum and colon from burned mice compared with sham-injured mice. Prior thermal injury accelerates lipopolysaccharide-induced inflammatory cytokine production systemically in jejunum and colon. The "second hit" of lipopolysaccharide led to earlier intestinal NF-κB activation in burned mice compared with sham-injured mice. These results indicate that there is a heightened inflammatory response by jejunum and colon in response to a "second hit" of lipopolysaccharide after burn injury.

Proinflammatory chemokines in the intestinal lumen contribute to intestinal dysfunction during endotoxemia.

Intestinal failure is common in patients with septic shock, with dysfunction of the gut often manifesting as both a cause and consequence of their critical illness. Most studies investigating the pathogenesis of intestinal failure focus on the systemic aspect, although few data examine the inflammatory signaling in the intestinal lumen. Having previously demonstrated apical/luminal chemokine secretion in an in vitro model of intestinal inflammation, we hypothesized that endotoxemia would induce secretion of proinflammatory chemokines into the intestinal lumen. In addition, we examined the contribution of these mediators to intestinal dysmotility. C57/BL6 male mice were injected intraperitoneally with LPS. Serum, intestinal tissue, and intestinal luminal contents were harvested for cytokine analysis. For intestinal motility studies, a transit assay was performed after oral gavage of chemokines. Caco-2 cells grown on Transwell culture inserts were used to examine the role of the intestinal epithelium in chemokine secretion. Monocyte chemoattractant protein 1 (MCP-1/CCL2) and macrophage-derived chemokine (MDC/CCL22) were secreted into the lumen of multiple segments of the gut during endotoxemia in mice. In vitro work showed that the intestinal epithelium participates in monocyte chemoattractant protein 1 and MDC secretion and expresses the CCR2 and CCR4 receptors for these chemokines. Intestinal transit studies show that oral gavage of MDC results in impaired gut motility. This study demonstrates that the intestinal lumen is an active compartment in the gut's inflammatory response. Proinflammatory chemokines are secreted into the intestinal lumen during endotoxemia. These intraluminal chemokines contribute to intestinal dysmotility, complicating intestinal failure.

Supplemental oxygen attenuates the increase in wound bacterial growth during simulated aeromedical evacuation in goats.

BACKGROUND: Bacterial growth in soft tissue and open fractures is a known risk factor for tissue loss and complications in contaminated musculoskeletal wounds. Current care for battlefield casualties with soft tissue and musculoskeletal wounds includes tactical and strategic aeromedical evacuation (AE). This exposes patients to a hypobaric, hypoxic environment. In this study, we sought to determine whether exposure to AE alters bacterial growth in contaminated complex musculoskeletal wounds and whether supplemental oxygen had any effect on wound infections during simulated AE. METHODS: A caprine model of a contaminated complex musculoskeletal wound was used. Complex musculoskeletal wounds were created and inoculated with bioluminescent Pseudomonas aeruginosa. Goats were divided into three experimental groups: ground control, simulated AE, and simulated AE with supplemental oxygen. Simulated AE was induced in a hypobaric chamber pressurized to 8,800 feet for 7 hours. Bacterial luminescence was measured using a photon counting camera at three time points: preflight (20 hours postsurgery), postflight (7 hours from preflight and 27 hours postsurgery), and necropsy (24 hours from preflight and 44 hours postsurgery). RESULTS: There was a significant increase in bacterial growth in the AE group compared with the ground control group measured postflight and at necropsy. Simulated AE induced hypoxia with oxygen saturation less than 93%. Supplemental oxygen corrected the hypoxia and significantly reduced bacterial growth in wounds at necropsy. CONCLUSIONS: Hypoxia induced during simulated AE enhances bacterial growth in complex musculoskeletal wounds which can be prevented with the application of supplemental oxygen to the host.

TNF-α induces vectorial secretion of IL-8 in Caco-2 cells.

INTRODUCTION: Intestinal epithelial cells represent an important component of innate immunity, with sophisticated responses to inflammatory stimuli. The manner in which intestinal epithelial cell polarity affects responses to inflammatory stimuli is largely unknown. We hypothesized that polarized intestinal epithelial cells exhibit a bidirectional inflammatory response dependent upon the location of the stimulus. METHODS: Caco-2 cells were grown on semi-permeable inserts in a dual-compartment culture system and treated with tumor necrosis factor-α (TNF-α; 100 ng/ml) or serum-free media in the apical or basolateral chamber. Interleukin-8 (IL-8) production in each chamber was measured by enzyme-linked immunosorbent assay. To determine receptor specificity, anti-TNF receptor antibodies were added to the apical or basolateral chamber. RESULTS: Basolateral stimulation with TNF-α resulted in increased apical and basolateral IL-8 production. Apical TNF-α stimulation resulted in increased apical, but not basolateral IL-8 production. Receptor blockade suggested TNF receptor 1 involvement on both apical and basolateral membranes, while TNF receptor 2 was only active on the apical membrane. CONCLUSION: Polarized intestinal epithelial cells respond to TNF-α stimulation with focused, directional secretion of the proinflammatory cytokine IL-8. These findings are important because they suggest that intestinal epithelial cells are capable of organizing their response to inflammatory signals and producing inflammatory mediators in a bidirectional, vectorial fashion.