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Abstract: Background : This study aims to determine the impact and mechanism of miR-21-3p on intestinal injury and intestinal glycocalyx during fluid resuscitation in traumatic hemorrhagic shock (THS), and the different impacts of sodium lactate Ringer's solution (LRS) and sodium bicarbonate Ringer's solution (BRS) for resuscitation on intestinal damage. Methods : A rat model of THS was induced by hemorrhage from the left femur fracture. The pathological changes of intestinal tissues and glycocalyx structure were observed by hematoxylin-eosin staining and transmission electron microscope. MiR-21-3p expression in intestinal tissues was detected by real-time quantitative polymerase chain reaction. The expression of glycocalyx-, cell junction-, and PI3K/Akt/NF-κB signaling pathway-related proteins was analyzed by western blot. Results : MiR-21-3p expression was increased in THS rats, which was suppressed by resuscitation with BRS. BRS or LRS aggravated the intestinal injury and damaged intestinal glycocalyx in THS rats. The expression of SDC-1, HPA, β-catenin, MMP2, and MMP9 was upregulated, the expression of E-cad was downregulated, and the PI3K/Akt/NF-κB signaling pathway was activated in THS rats, which were further aggravated by BRS or LRS. The adverse effect of LRS was more serious than BRS. MiR-21-3p overexpression deteriorated the injury of intestinal tissues and intestinal glycocalyx; increased the expression of SDC-1, HPA, β-catenin, MMP2, and MMP9 while decreasing E-cad expression; and activated the PI3K/Akt/NF-κB signaling pathway in BRS-resuscitated THS rats. Conclusion : MiR-21-3p aggravated intestinal tissue injury and intestinal glycocalyx damage through activating PI3K/Akt/NF-κB signaling pathway in rats with THS resuscitated with BRS., (Copyright © 2024 by the Shock Society.)

Abstract: Intestinal ischemia-reperfusion injury (IIRI) is a serious disease with high morbidity and mortality. This study aims to investigate the potential regulatory mechanisms involving protein arginine methyltransferase 6 (PRMT6), Forkhead box O3a (FoxO3a), and Parkin in IIRI and elucidate their roles in mediating cell apoptosis. The IIRI animal model was established and confirmed using hematoxylin and eosin staining. Oxygen-glucose deprivation and reperfusion (OGD/R) cell model was established to mimic ischemic injury in vitro . Transient transfection was used to overexpress or knock down genes. Cell death or apoptosis was assessed by propidium iodide staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and flow cytometry. The expression of proteins was detected by western blot. The histopathology observed by hematoxylin and eosin staining suggested that the IIRI animal model was successfully established. Our findings revealed that IIRI resulted in increased Bax and decreased Bcl-2 levels. In vitro experiments showed that overexpression of Parkin decreased OGD/R injury and suppressed elevation of Bax/Bcl-2. PRMT6 regulated the methylation level of FoxO3a. Moreover, FoxO3a directly binds to Parkin, and FoxO3a overexpression reduced OGD/R-induced cell death and regulation of Parkin. Overexpression of PRMT6 can attenuate the downregulation of Parkin and elevation of Bax/Bcl-2 caused by OGD/R. Knockdown of PRMT6 promoted apoptosis in intestinal epithelial cells of OGD/R group, while PRMT6 overexpression exhibited the opposite effect. Notably, the levels of PRMT6, FoxO3a, and Parkin were decreased in IIRI mouse intestinal tissue. Knocking out PRMT6 causes a significant decrease in the lifespan of mice. Altogether, our results demonstrated that PRMT6 upregulated the expression of Parkin by regulating FoxO3a methylation level, attenuating the apoptosis induced by IIRI., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Dysfunction of the gut-blood barrier plays an important role in many diseases, such as inflammatory bowel disease, hemorrhagic shock (HS), or burn injury. However, little is known about gut barrier dysfunction after hemodynamically instable polytrauma (PT). Therefore, we aimed to evaluate the effects of PT and HS on remote intestinal damage and barrier dysfunction, especially regarding the role of zonula occludens protein 1 (ZO-1) as an important tight junction protein.Male C57BL/6 mice were subjected to either PT (thorax trauma, closed head injury, soft tissue injury, and distal femoral fracture), 60 min of pressure-controlled HS (30 ± 5 mmHg), or PT+HS, or sham procedures.Animals of all trauma groups showed an increase in abdominal girth and dilation of the intestine during the experimental period, which was largest in the PT+HS group. Increased blood-tissue permeability to albumin (assessed by Evans blue dye) was found in the HS group. Experimental groups showed a slight increase in plasma concentration of intestinal fatty acid binding protein and some intestinal damage was histologically detectable. Of note, PT+HS animals revealed significantly reduced expression of ZO-1 in intestinal epithelial cells. In an in-vitro model, stimulation of human colon epithelial cells with peptidoglycan, but not with lipopolysaccharide, resulted in elevated secretion of pro-inflammatory cytokines, reflecting inflammatory activity of the intestinal epithelium.Taken together, PT and HS lead to increased permeability of the gut-blood barrier. Bacterial components may lead to production of inflammatory and chemotactic mediators by gut epithelial cells, underlining the role of the gut as an immunologically active organ.

Intestinal ischemia and reperfusion (I/R) triggers a systemic inflammatory response characterized by leukocyte mobilization from the bone marrow, release of cytokines to the circulation, and increased microvascular permeability, leading to high mortality. Females have shown attenuated inflammatory response to trauma when compared with males, indicating a role for female sex hormones in this process. Here, we have evaluated the effect of estradiol on the local gut injury induced by I/R in male rats. I/R was induced by the clamping of the superior mesenteric artery for 45 min, followed by 2 h of reperfusion. A group received 17β-estradiol (280 μg/kg, i.v., single dose) at 30 min of ischemia. Morphometric analysis of the gut showed I/R induced a reduction of villous height that was prevented by estradiol. White blood cells, notably granulocytes, were mobilized from the circulation to the intestine by I/R, which was also prevented by estradiol treatment. Groups had the intestine wrapped in a plastic bag to collect intestinal fluid, where leukocytes count, TNF-α, and IL-10 levels were increased by I/R. Serum chemokines (CINC-1, MIP-1α, MIP-2), ICAM-1 expression in the mesenteric tissue, and neutrophils spontaneous migration measured in vitro were also increased after I/R. Estradiol treatment reduced leukocytes numbers and TNF-α on intestinal fluid, serum chemokine release and also downregulated MIP-1α, MIP-2 gene expression, and spontaneous in vitro neutrophil migration. In conclusion, estradiol blunts intestinal injury induced by I/R by modulating chemokines release and leukocyte trafficking.

The intestine plays an important role in the pathogenesis of sepsis. Hydrogen gas (H2), which has anti-oxidative, anti-inflammatory, and anti-apoptotic effects, can be effectively used to treat septic mice. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive master switch that regulates the expression of antioxidant and protective enzymes. This study investigated the effects of 2% H2 on intestinal injuries and the underlying mechanisms in a mouse model of severe sepsis. Male Nrf2 knockout mice (Nrf2-KO) and wild-type (WT) mice were randomized into four groups: sham, sham+H2, cecal ligation and puncture (CLP), and CLP+H2. The survival rate was observed and recorded within 7 days, and pro-inflammatory cytokines (TNF-α, IL-6, HMGB1), anti-inflammatory cytokine (IL-10), antioxidant enzymes (superoxide dismutase, and catalase ), and oxidative products (MDA, 8-iso-PGF2α) were detected in the serum and intestine using an enzyme-linked immunosorbent assay. In addition, the protein and mRNA levels of heme oxygenase-1 (HO-1) and high mobility group box 1 (HMGB1) were measured by Western blotting and quantitative PCR, respectively. Immunofluorescence and immunohistochemistry were used to measure HMGB1 and HO-1 release into the intestine, respectively. The results showed that therapy with 2% H2 increased the survival rate, alleviated the injuries caused by oxidative stress and inflammation, reduced HMGB1 levels but increased HO-1 levels in WT septic mice, but not in Nrf2-KO mice. These data demonstrate that 2% H2 inhalation may be a promising therapeutic strategy for intestinal injuries caused by severe sepsis through the regulation of HO-1 and HMGB1 release. In addition, Nrf2 plays a key role in the protective effects of H2 against intestinal damage in this disease.

Objectives: In patients undergoing cardiac surgery, both extracorporeal circulation (ECC) and intraoperative mesenterial hypoperfusion may account for increased cytokine levels and lead to postoperative gastrointestinal (GI) symptoms., Methods: We investigated levels of the intestinal damage markers intestinal fatty acid binding protein (I-FABP in plasma [n = 72] and urine [n = 37]), citrulline (in plasma [n = 35]), and claudin-3 (in urine [n = 37]) in patients undergoing aortic or mitral valve surgery with or without coronary artery bypass grafting. Furthermore, the relationship between these markers and the surgery-induced cytokine response was explored by measuring serial plasma levels of tumor necrosis factor-α, interleukin (IL)-6, IL-8, and IL-10 (n = 35). Finally, the relationship between markers of intestinal damage and GI-symptoms (abdominal pain, ileus, vomiting, diarrhea, time to first defecation) was assessed., Results: Plasma and urinary I-FABP levels, and urinary claudin-3 levels peaked at the end of surgery, while citrulline levels were not influenced by surgery. ECC duration correlated with plasma I-FABP levels (r = 0.31, P = 0.007). Plasma levels of all measured cytokines increased during surgery, with peak levels observed either at the end of surgery or on the first postoperative day. While ECC duration correlated with IL-6 and IL-8 release (r = 0.43, P = 0.01 and r = 0.36, P = 0.04 respectively), there was no direct relationship between I-FABP and claudin-3 levels and cytokine concentrations. No patients developed significant GI or non-GI complications, and I-FABP and claudin-3 release appeared not to be related to postoperative GI symptoms, although the incidence of these symptoms may have limited a reliable assessment., Conclusions: Longer duration of ECC is associated with a more pronounced release of intestinal injury markers and inflammatory cytokines, but intestinal injury markers are not directly related to the observed increase in cytokine levels or GI-symptoms. These findings indicate that ECC duration contributes to the cytokine response observed in cardiac surgery patients and that intestinal injury itself is not a causative factor for this response.

Background: How vagotomy affects host responses to gut ischemia-reperfusion (I/R) is unclear., Materials and Methods: Experiment 1: male Institute of Cancer Research mice (n = 22) were assigned to the I/R or the vago-I/R group. The I/R mice underwent 45-min superior mesenteric artery (SMA) occlusion. The vago-I/R mice received vagotomy before SMA occlusion. Survival was observed for 48 h.Experiment 2: mice (n = 55) were divided into four groups (Sham, vago, I/R, vago-I/R). Sham and vago groups did not undergo gut I/R. Mice were killed at 3 or 6 h after reperfusion, and cytokine levels in the plasma, jejunum, and ileum were evaluated. In addition, gut histology at 6 h was examined.Experiment 3: mice (n = 24) were divided into four groups as in Experiment 2. The small intestine was harvested at 3 h after reperfusion and the tissue was cultured ex vivo for 3 h. Cytokine levels of the culture supernatant were then measured., Results: Experiment 1: survival was significantly worse with vago-I/R than I/R.Experiment 2: along with severe gut injury, vago-I/R increased IL-6 and monocyte chemoattractant protein-1 (MCP-1) in plasma, IFN-γ in the jejunum and MCP-1 in the ileum, as compared with I/R. Significant positive correlations were noted between plasma and intestinal levels of pro-inflammatory cytokines (IL-6, MCP-1, and TNF-α).Experiment 3: MCP-1 in the jejunal culture medium was higher in the vago-I/R than in the I/R group., Conclusions: Vagotomy worsens survival after gut I/R, together with increases in pro-inflammatory cytokines in both plasma and the gut in association with severe intestinal tissue damage.

Both the initial trauma and the subsequent hemodynamic instability may contribute to intestinal damage, which is of great importance in (immunological) posttrauma complications. This study assesses intestinal damage using the biomarker intestinal Fatty Acid Binding Protein (iFABP) in trauma patients during the first days of their hospital admission and the risk factors involved. Plasma iFABP levels were measured in blood samples obtained from adult multiple trauma patients (n = 93) at the trauma scene by the Helicopter Emergency Medical Services, at arrival at the emergency department (ED), and at days 1, 3, 5, 7, 10, and 14 after trauma and related to injury severity and hemodynamic parameters. Plasma iFABP concentrations showed highest levels immediately after trauma at time points Helicopter Emergency Medical Services and ED. Nonsurvivors demonstrated higher iFABP levels at the ED compared with survivors. Furthermore, iFABP values at the ED correlated with Injury Severity Scores, and patients suffering from abdominal trauma demonstrated significantly higher iFABP concentrations in comparison with patients with other types of trauma or healthy controls. Also, patients presenting with a mean arterial pressure (MAP) less than 70 mmHg at the ED demonstrated significantly higher plasma iFABP concentrations in comparison with patients with a normal (70-99 mmHg) or high (>100 mmHg) MAP or healthy controls. Finally, patients with a low hemoglobin (Hb) (<80% of reference value) displayed significantly higher iFABP concentrations in comparison with patients with a normal Hb or healthy controls. Plasma iFABP levels, indicative of intestinal injury, are increased immediately after trauma in patients with abdominal trauma, low MAP, or low Hb and are related to the severity of the trauma. As intestinal injury is suggested to be related to late complications, such as multiorgan dysfunction syndrome or sepsis in trauma patients, strategies to prevent intestinal damage after trauma could be of benefit to these patients.

Resveratrol protects against organ injury caused by trauma-hemorrhage, although the mechanism remains unknown. We have previously shown that it exerts protective effects in the liver via estrogen receptors and their signaling. Thus, we set out to determine whether resveratrol-mediated estrogen receptor-dependent p38 mitogen-activated protein kinase (MAPK)/heme oxygenase 1 activation protects the intestine after trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure, ~ 40 mmHg for 90 min) followed by fluid resuscitation. Animals were pretreated with an estrogen receptor antagonist (ICI 182,780), a specific p38 MAPK inhibitor (SB-203580), or a heme oxygenase enzyme antagonist (chromium-mesoporphyrin) 30 min before vehicle or resveratrol (30 mg/kg) administration, followed by resuscitation, and were killed 2 h thereafter. Intestinal water content, myeloperoxidase activity, and TNF-α, IL-6, intercellular adhesion molecule 1, cytokine-induced neutrophil chemoattractant (CINC) 1, and CINC-3 levels and edema of the lung were measured. Mean arterial blood pressure, cardiac output, positive maximal pressure of left ventricular increase (+dP/dtmax), and negative maximal pressure of left ventricular decrease (-dP/dtmax) were also determined. Intestinal p38 MAPK activity and heme oxygenase 1 expression were also determined. Trauma-hemorrhage led to an increase in intestinal water content, myeloperoxidase activity, and TNF-α, IL-6, intercellular adhesion molecule 1, CINC-1, and CINC-3 levels. This was accompanied by a decrease in intestinal p38 MAPK activity. Administration of resveratrol improved all of the above parameters. Resveratrol treatment also increased intestinal heme oxygenase 1 expression as compared with vehicle-treated trauma-hemorrhaged rats. Administration of ICI 182,780, SB-203850, or chromium-mesoporphyrin with resveratrol abolished the resveratrol-mediated improvement of the above parameters. Resveratrol administration also attenuated trauma-hemorrhage-induced cardiac dysfunction and edema of the lung. These results suggest that estrogen receptor-dependent upregulation of the p38 MAPK/heme oxygenase 1 pathway plays a critical role in mediating the salutary effects of resveratrol on shock-induced intestinal injury.

Intestinal ischemia/reperfusion (I/R) injury has been shown to cause intestinal mucosal injury and adversely affect function. Ischemic preconditioning (IPC) has been shown to protect against intestinal I/R injury by reducing polymorphonuclear leukocyte infiltration, intestinal mucosal injury, and liver injury, and preserve intestinal transit. Bone morphogenetic protein 7 (BMP-7) has been shown to protect against I/R injury in the kidney and brain. Recently, microarray analysis has been used to examine the possible IPC candidate pathways. This work revealed that IPC may work through upregulation of BMP-7. The purpose of this study was to examine if pretreatment with BMP-7 would replicate the effects seen with IPC in the intestine and liver after intestinal I/R. Rats were randomized to six groups: sham, I/R (30 min of superior mesenteric artery occlusion and 6 h of R), IPC+R (three cycles of superior mesenteric artery occlusion for 4 min and R for 10 min), IPC+I/R, BMP-7+R (100 microm/kg recombinant human BMP-7), or BMP-7+I/R. A duodenal catheter was placed, and 30 min before sacrifice, fluorescein isothiocyanate-Dextran was injected. At sacrifice, dye concentrations were measured to determine intestinal transit. Ileal mucosal injury was determined by histology and myeloperoxidase activity was used as a marker of polymorphonuclear leukocyte infiltration. Serum levels of aspartate aminotransferase were measured at sacrifice to determine liver injury. Pretreatment with BMP-7 significantly improved intestinal transit and significantly decreased intestinal mucosal injury and serum aspartate aminotransferase levels, comparable to animals undergoing IPC. In conclusion, BMP-7 protected against intestinal I/R-induced intestinal and liver injury. Bone morphogenetic protein 7 may be a more logical surrogate to IPC in the prevention of injury in the setting of intestinal I/R.

Although small animal rodent studies indicate that there is a sexual dimorphism in the resistance to organ injury after trauma-hemorrhagic shock (T/HS), confirmatory studies are largely lacking in more clinically relevant large animal species. Thus, we tested the hypothesis that castration would reduce the susceptibility of adult minipigs to gut injury and abrogate the production of biologically active intestinal (mesenteric) lymph after T/HS. The hemodynamic response to T/HS was similar between castrated and noncastrated minipigs. Mesenteric lymph collected during the preshock period and in the trauma-sham shock (T/SS) animals did not have increased biological activity. However, T/HS-lymph from the noncastrated males increased the respiratory burst of normal neutrophils, increased endothelial cell monolayer permeability, and was cytotoxic for endothelial cells. Castration abrogated the T/HS-induced neutrophil-activating and endothelial-injurious activities of mesenteric lymph, and the biological activity of the T/HS-lymph from the castrated minipigs was not different from the T/SS animals. As compared with the T/SS minipigs, T/HS increased ileal mucosal injury and intestinal permeability. This increase in gut permeability after T/HS was manifest by in vivo bacterial translocation and by the increased passage of bacteria as well as permeability probes across intestinal segments when tested in the Ussing chamber system. In contrast, neither mucosal injury nor increased intestinal permeability was observed in the castrated minipigs subjected to T/HS. In summary, this large animal porcine study validates the notion that castration limits gut injury and the production of biologically active intestinal lymph after T/HS.

We tested the hypothesis that the female intestine is more resistant to gut I/R injury than the male intestine by comparing the effects of the isolated pure gut I/R superior mesenteric artery occlusion (SMAO) model on gut morphology and whether SMAO-induced distant organ injury (lung, bone marrow [BM], neutrophils, and red blood cells [RBCs]) would differ between male and proestrus female rats. At 6 or 24 h after SMAO or sham SMAO, gut injury, lung permeability, pulmonary neutrophil sequestration, RBC deformability, and BM RBC and white blood cell progenitor growth were measured, as was the ability of the plasma from these rats to activate naive rat neutrophils. At both 6 and 24 h after SMAO, the female rats had significantly less intestinal injury and reduced gut-induced lung injury, BM suppression, RBC dysfunction, and neutrophil activation than male rats subjected to SMAO. These results indicate that the resistance of proestrus female rats to gut injury and gut-induced distant organ injury is greater than that observed in male rats.

The development of sepsis and multiple organ failure are important determinants of the outcome in critically ill patients. Hepatosplanchnic hypoperfusion and resulting intestinal and hepatic cell damage have been implicated as central events in the development of sepsis and multiple organ failure. Our aim was to study (1) the relation between intramucosal perfusion and intestinal and hepatic cell damage in an early phase of sepsis and (2) the correlation of these parameters with mortality. Two groups of patients were consecutively selected after intensive care unit admission: patients with postoperative abdominal sepsis (n = 19) and patients with pneumonia-induced sepsis (n = 9). Intramucosal perfusion was assessed by gastric tonometry (Pr-aCO2 gap, Pico2). Circulating levels of intestinal fatty acid binding protein (I-FABP) and liver (L)-FABP were used as markers for intestinal and hepatic cellular damage, respectively. Outcome was determined on day 28. Pr-aCO2 gap correlated with I-FABP (Pearson r = 0.56; P < 0.001) in all patients, and gastric mucosal Pico2 correlated significantly with I-FABP (r = 0.57; P = 0.001) in patients with abdominal sepsis. At intensive care unit admission, nonsurvivors had significantly higher I-FABP and L-FABP values than survivors (I-FABP: 325 vs. 76 pg/mL, P < 0.04; L-FABP: 104 vs. 31 ng/mL, P < 0.04). Patients with abdominal sepsis was especially responsible for high-admission I-FABP and L-FABP levels in nonsurvivors (I-FABP: 405 vs. 85 pg/mL, P < 0.04; L-FABP: 121 vs. 59 ng/mL, P < 0.04). This study shows that splanchnic hypoperfusion correlates with intestinal mucosal damage, and that elevated plasma levels of I-FABP and L-FABP are associated with a poor outcome in critically ill patients with abdominal sepsis.

We have shown that acute edema impairs intestinal transit and we wanted to know whether this could be from changes in the physical characteristics of the intestine. Our hypothesis was that acute edema will change the physical characteristics of the intestine, which were measured by standardized engineering measures of elastic modulus, to determine stiffness and opening angle, and to determine residual stress. Rats were randomized to sham, mild edema (80 mL/ kg of normal saline resuscitation), and severe edema groups (80 mL/kg of normal saline resuscitation with intestinal venous hypertension). Segments of distal ileum were hung to a fixed point in a tissue bath and to a tensiometer and were stretched in increments of 1 mm, recording the new length and the corresponding force from the tensiometer to determine elastic modulus. Next, two transverse cuts were made yielding a 1- to 2-mm-thick ring-shaped segment of tissue and were then cut radially to open the ring. The opening angle was measured. Acute intestinal edema led to a decrease in transit, elastic modulus, and opening angle of the intestine in the absence of ischemic injury. Acute intestinal edema leads to a significant loss in stiffness and residual stress and is a plausible explanation for how acute edema impairs intestinal transit.

Multisystem organ failure represents a major cause of mortality in intestinal ischemia and reperfusion (I/R), and oxidative stress plays a key role in its pathogenesis. Hypothermia is beneficial in I/R injury, but its effects on systemic oxidative stress have not been elucidated. The aim of this study was to evaluate the effects of moderate hypothermia on systemic oxidative stress after intestinal I/R injury. Anaesthetized adult rats (n = 10 per group) underwent 60 min of intestinal ischemia followed by 120 min of reperfusion or sham operation at normothermia (36 degrees C-38 degrees C) or moderate hypothermia (30 degrees C-32 degrees C). At sacrifice, ileum, liver, lungs, and kidneys were removed to determine the concentration of malondialdehyde (a marker of lipid peroxidation), reduced and oxidized glutathione (a major endogenous antioxidant), and glutathione redox state. Plasma malondialdehyde and nitrate plus nitrite (reflecting nitric oxide production) were also analyzed. A marked elevation of malondialdehyde was observed after I/R at normothermia in plasma, ileum, and lungs; however, hypothermia during I/R prevented this increase. I/R at normothermia caused a profound decrease in reduced glutathione and glutathione redox state in the ileum, but this was not observed in I/R at hypothermia. Interestingly, hypothermia increased glutathione content of control intestine. Nitric oxide production was increased only in normothermic I/R animals. Moderate hypothermia attenuates systemic oxidative stress associated with experimental intestinal I/R in an animal model by decreasing lipid peroxidation in plasma, ileum, lungs, and kidneys, by preventing the depletion of gut glutathione, and by reducing systemic nitric oxide production. However, whether these effects persist after rewarming is unknown.

The insult from severe hemorrhage is a multifactorial injury involving ischemia/reperfusion with inflammatory dysfunction. Our laboratories and others have demonstrated that the administration of exogenous carbon monoxide (CO) at low concentrations provides cytoprotection in vivo and in vitro. The purpose of these investigations was to test the hypothesis that CO protects against hemorrhagic shock- and resuscitation-induced systemic inflammation and end-organ damage. C57BL/6 mice underwent anesthesia and arterial cannulation. Mice were bled to reach a mean arterial pressure (MAP) of 25 mmHg and were maintained at this pressure for 2.5 h. Mice were then resuscitated with shed blood plus two times the volume of shed blood with Ringer's lactate. Sham animals were not bled. Additionally, mice were maintained in room air or in an environment of CO (250 parts per million). Primary mouse hepatocytes were harvested and used for in vitro cell viability and ATP measurement. These data demonstrate that delivery of a low concentration of inhaled CO protects against the development of end-organ injury decreases serum levels of inflammatory cytokines and increases serum levels of the anti-inflammatory cytokine IL-10. Additionally, CO paradoxically abrogates hemorrhage-induced hepatic cellular hypoxia. Furthermore, CO protected mouse hepatocytes from hypoxia-induced death while maintaining normal ATP levels. CO protects against systemic effects of hemorrhagic shock and resuscitation. The precise cellular mechanisms involved require further elucidation. CO may prove to be an adjunctive therapy that could be instituted rapidly and with ease as an out-of-hospital therapeutic modality for severe blood loss after trauma.

Reactive oxygen species contribute to the multiple organ dysfunction syndrome in hemorrhagic shock. Here, we investigate the effects of two chemically distinct inhibitors of NADPH oxidase on the circulatory failure and the organ dysfunction and injury associated with hemorrhagic shock in the anesthetized rat. Hemorrhage (sufficient to lower mean arterial blood pressure of 45 mmHg for 90 min) and subsequent resuscitation with shed blood resulted (within 4 h after resuscitation) in a delayed fall in blood pressure and in renal dysfunction and liver injury. Treatment of rats upon resuscitation with the NADPH oxidase inhibitors diphenylene iodonium (DPI, 1 mg/kg i.v.) reduced renal dysfunction and liver injury, whereas apocynin (3 mg/kg i.p.) did reduce the liver injury, but not the renal dysfunction caused by hemorrhagic shock. DPI and apocynin also attenuated the lung and intestinal injury (determined by histology) caused by hemorrhage and resuscitation. In the liver, DPI and apocynin abolished the increase in the formation of superoxide anions associated with hemorrhagic shock. However, neither DPI nor apocynin had a significant effect on the delayed circulatory failure caused by hemorrhage and resuscitation. In addition, DPI and apocynin did not reduce the increase in nitric oxide synthesis caused by hemorrhagic shock. Moreover, DPI reduced the activation of the transcription factor activator protein-1 caused by severe hemorrhage and resuscitation in the liver. Thus, we propose that an enhanced formation of superoxide anions by NADPH oxidase contributes to the liver injury caused by hemorrhagic shock, and that inhibitors of NADPH oxidase may represent a novel therapeutic approach for the therapy of hemorrhagic shock.

The p38 mitogen-activated protein kinase (MAPK) pathway is a proinflammatory signal transduction pathway for the production of cytokines and cellular response to stress, such as bacterial LPS or ischemia. We examined the effects of FR167653, a specific inhibitor of p38 MAPK, to explore the relationship between intestinal barrier damage and remote renal dysfunction. Immunohistochemical data showed the accumulation of neutrophils in the intestine after burn, and a horseradish peroxidase (HRP) tracer experiment showed burn-induced intestinal barrier damage. Our quantitative bacterial culture data demonstrated that viable bacteria reached the remote organs after burn and prevented the invading viable bacteria from using FR167653. Western blotting identified increased phosphorylation of p38 MAPK in the kidney after burn, and it may also have shown the possibility that endotoxin associated with the bacterial translocation enhances the activation of the p38 MAPK pathway. We blocked the intestinal barrier damage using FR167653, which resulted in reduced neutrophils in the intestine. FR167653 also prevented the increased phosphorylation of p38 MAPK in the kidney, which resulted in reduced neutrophils in the glomerulus and the reduction of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta mRNA in the kidneys, and, finally, prevented burn-induced renal failure. This study provides evidence for the hypothesis that the p38 MAPK pathway controls inflammatory mediators and not only improves intestinal function but also reduces remote renal failure after burn. We identified the pathophysiologic role of the p38 MAPK pathway in the development of renal failure after burn.

Although several studies have reported that splenectomy increases susceptibility to bacterial infections, other reports have indicated that splenectomy does not induce such susceptibility. To clarify this discrepancy, we studied the effects of splenectomy in the models of lipopolysaccharide (LPS) or Escherichia coli challenge and intestinal obstruction, focusing on cytokine production and bacterial clearance in the liver. Using C57BL/6 mice at 4 weeks after splenectomy or a sham operation, either LPS or E. coli was injected or an intestinal obstruction was made to examine the mortality, serum cytokine levels, cytokine production of the liver mononuclear cells (MNCs), and bacterial clearance in the liver. As a result, no differences were observed in survival rates after LPS or E. coli challenge between the mice with and without splenectomy. However, in a model of intestinal obstruction, splenectomized mice survived significantly longer than the sham-operated mice. Liver MNCs from splenectomized mice produced a significantly larger amount of interferon-gamma compared with those from sham-operated mice. Furthermore, bacterial counts in the liver at 2 h after E. coli injection and at 24 h after intestinal obstruction were significantly decreased in splenectomized mice compared with sham-operated mice. In conclusion, splenectomy does not impair host defense against bacteria infection provided that recovery is sufficient to allow compensatory processes in the liver to occur.

P-selectin is an adhesion molecule expressed on activated endothelial and platelet membranes containing 9 short consensus repeats (SCRs) similar to the composition of complement regulatory proteins. In our murine model of intestinal ischemia and reperfusion where local injury is mediated by the classical complement pathway we hypothesized the SCRs would moderate the complement response. Confirmatory data were sought following hindlimb ischemia and reperfusion where injury is both complement- and neutrophil-mediated. Mice deficient in P-selectin (P-/-) were found to have similar intestinal and hindlimb permeability compared to normal wild types mice (P+/+). When reconstituted with P+/+ platelets, but not P-/- platelets, P-/- mice subjected to intestinal ischemia had a significant 29% decrease in permeability (P < 0.05) and after hindlimb ischemia the decrease was 33% (P<0.05). Reperfusion after intestinal ischemia led to a 76% fall in CH50 in P-/- compared to sham animals (P < 0.05) indicating complement activation and consumption, but only a 36% fall in animals reconstituted with P+/+ platelets (P < 0.05). Full-length, soluble P-selectin (sPsel) derived from processed platelets, but not the truncated version of sPsel has been shown to adhere to a heat labile fraction of serum and sensitized red blood cells thereby reducing Clq adherence to the sensitized red cell. From these data we conclude that sPsel moderates complement activation by competing with C1q binding to antibody, thereby limiting activation of the classical pathway that mediates murine reperfusion injury.

Prostaglandins (PGs), which play a pivotal role in the cytokine network, have been implicated in the pathophysiology of circulatory shock, but the precise role of cyclooxygenase-2 (COX-2) in circulatory shock is still not known. This study investigated whether or not COX-2 affects liver and bowel injury during hemorrhagic shock (HS). Male Sprague-Dawley rats were subjected to decompensated HS followed by resuscitation. Besides the time course of tissue injury and mRNA expression of COX-2 in the liver and bowel during HS, we investigated the effect of N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide (NS398), a specific inhibitor of COX-2, on injury to these organs. The liver injury, evaluated by plasma aminotransferase levels and histology, was evident at the end of the shock period (Shock-0h) and had significantly increased 1 h after the start of resuscitation (Shock-1h) (P < 0.01). The bowel injury, assessed by histological findings in the terminal ileum, was scarcely detectable at Shock-0h but became noticeable at Shock-1h (P < 0.01). COX-2 mRNA expression was up-regulated in the liver during HS whereas it did not change in the bowel. NS398 treatment significantly exacerbated both liver and bowel injury. These lines of evidence suggest that COX-2-derived PGs provide protection against HS-induced liver and bowel injury.

Although gut lipid absorptive capacity is depressed after trauma and hemorrhagic shock, it remains unknown whether this is associated with an impairment in the enterocyte triglyceride (TG) synthesis, and if so, whether villus or crypt cells participate in the lipid absorption under such conditions. To study this, midline laparotomy (i.e., trauma induction) was performed in the rats, and then the animals were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of shed blood volume was returned in the form of Ringer's lactate. The rats were then resuscitated with four times the volume of maximal bleedout with Ringer's lactate over 60 min. For lipid loading test, 1 mL of olive oil was given intraduodenally as a bolus injection upon the completion of resuscitation. The enterocytes along the crypt-villus axis from the proximal small intestine were isolated at either 0, 1.5, or 5 h after fat feeding. The intracellular and pre-chylomicron TG contents as well as the microsomal enzyme activities associated with TG synthesis were determined thereafter. The results indicated that intracellular and pre-chylomicron TG contents and lipid esterifying enzyme (i.e., acyl-coenzyme A:monoglyceride acyltransferase) activity decreased significantly in villus tip cells after trauma-hemorrhage and resuscitation. The intracellular TG content and lipid esterifying enzyme activity in crypt cells after trauma and hemorrhagic shock were also reduced. However, there was no significant decrease in pre-chylomicron TG content in the crypt cells isolated from trauma-hemorrhaged rats. It is unlikely that crypt cells participate in lipid transport after trauma and hemorrhagic shock. In contrast, the TG synthesis in villus tip cells decreased significantly following trauma-hemorrhage and resuscitation, which may contribute, at least in part, to the impaired gut lipid absorptive capacity observed under such conditions.

Although studies have indicated that the gut lipid absorptive capacity is impaired after trauma and hemorrhagic shock, the mechanism responsible for this remains unknown. The aim of this study, therefore, was to determine whether intestinal alkaline phosphatase (IAP) plays any role in the depressed gut lipid absorptive function observed under such conditions. To determine this, 5 cm midline laparotomy (i.e., trauma induced) was performed in the rats, and animals were then bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of shed blood volume was returned in the form of Ringer's lactate. The rats were resuscitated with four times the volume of maximal bleedout with Ringer's lactate over 60 min. For in vivo lipid loading test, 1 mL olive oil was given intraduodenally upon the completion of resuscitation. The enterocytes were isolated at either 0, 1.5, or 5 h after fat feeding, and the soluble, membranous and pre-chylomicron (pre-CM) IAP activities as well as triglyceride contents were determined thereafter. The results indicated that the soluble IAP activity in the villus tip cells isolated from trauma-hemorrhaged rats increased significantly at 0, 1.5, or 5 h after fat feeding. The pre-CMIAP activity, however, decreased markedly in villus tip cells at 1.5 or 5 h after lipid administration, which was associated with the decreased pre-CM triglyceride contents in villus tip cells under such conditions. The altered IAP activity in villus tip cells, therefore, appears to play a role in the depressed gut lipid absorption and transport following trauma-hemorrhage and resuscitation.

This study examines the hypothesis that hydroxyl radical (OH.) generation during intestinal reperfusion activates the complement system forming the potent chemotaxin C5a. Anesthetized Sprague-Dawley rats underwent 120 min of intestinal ischemia and 60 min of reperfusion (IIR). Complement (C) activation was assessed by measuring total plasma C activity and C5a-related chemotaxis and leukoaggregation. Dimethylthiourea and the iron chelator deferoxamine were utilized to assess the role of the OH. in the activation of C in this model. Sham-operated animals served as controls. Total plasma C activity of animals sustaining IIR was 64% of controls (p < .05). Plasma of animals sustaining IIR induced greater chemotaxis and leukoaggregation than plasma from sham-operated groups (p < .05). Treatment of IIR plasma with anti-C5a antibody ameliorated the enhanced leukoaggregation characteristic of IIR plasma. Pretreatment with dimethylthiorea and deferoxamine prevented reperfusion-induced activation of complement and inhibited the chemotactic activity of plasma from IIR animals. These data are consistent with the hypothesis that IIR activates complement and that the OH. generated during reperfusion may be one mechanism by which C is activated in this injury model.