Mesenteric lymph is responsible for post-hemorrhagic shock systemic neutrophil priming.

BACKGROUND: Hemorrhagic shock-induced splanchnic hypoperfusion has been implicated as a priming event in the two event model of multiple organ failure (MOF). We have previously shown that early postinjury neutrophil (PMN) priming identifies the injured patient at risk for MOF. Recent in vitro studies have demonstrated that postshock mesenteric lymph primes isolated human neutrophils. We hypothesize that lymphatic diversion before hemorrhagic shock abrogates systemic PMN priming and subsequent lung injury. METHODS: Sprague-Dawley rats (n >or= 5 per group) underwent hemorrhagic shock (MAP 40 mm Hg x 30 min) and resuscitation (shed blood + 2x crystalloid) with and without mesenteric lymphatic duct diversion. Sham animals underwent anesthesia and laparotomy. Whole blood was taken 2 hours after resuscitation, heparinized, and incubated for 5 min at 37 degrees C. Surface expression of CD11b (a marker for PMN priming) was determined by flow-cytometry compared with isotype controls. In addition, lung myeloperoxidase (MPO) was measured for PMN sequestration, and Evans blue lung leak was assessed in the bronchoalveolar lavage fluid in sham, and shock +/- lymph diversion animals. RESULTS: Hemorrhagic shock resulted in increased surface expression of PMN CD11b relative to sham (23.8 +/- 6.7 vs. 9.9 +/- 0.6). Mesenteric lymphatic diversion before hemorrhagic shock abrogated this effect (8.0 +/- 2.6). Lung PMN accumulation, as assessed by MPO, was greater in the lungs of nondiverted (113 +/- 14 MPO/mg lung) versus sham (55 +/- 4 MPO/mg lung, p < 0.05); lymph diversion reduced lung PMNs to control levels (71 +/- 6.5 MPO/mg lung, p < 0.05). Evans blue lung leak was 1.6 times sham in the hemorrhagic shock group; this was returned to sham levels after lymph diversion (p < 0.05). CONCLUSION: Post-hemorrhagic shock mesenteric lymph primes circulating PMNs, promotes lung PMN accumulation, and provokes acute lung injury. Lymphatic diversion abrogates these pathologic events. These observations further implicate the central role of mesenteric lymph in hemorrhagic shock-induced lung injury. Characterizing the PMN priming agents could provide insight into the pathogenesis of postinjury MOF and ultimately new therapeutic strategies.     

Hypertonic saline resuscitation abrogates neutrophil priming by mesenteric lymph

OBJECTIVE: Neutrophil (PMN) priming after hemorrhagic shock is predictive of the subsequent development of multiple organ failure, but the mechanism remains unknown. Recently, we and others have demonstrated that mesenteric lymph from shock animals resuscitated with lactated Ringer's solution (LR) is not only a potent PMN priming agent but also causes lung injury. Work by others has shown that resuscitation with hypertonic saline (HTS) protects animals from lung injury after hemorrhagic shock. Therefore, we hypothesize that resuscitation with HTS will abolish PMN priming by postshock mesenteric lymph. METHODS: After mesenteric lymph duct catheterization, male rats underwent hemorrhagic shock (mean arterial pressure of 40 mm Hg for 90 minutes) and resuscitation with shed blood plus either LR (2x volume of shed blood) or 4 mL/kg of 7% HTS (isonatremic). Priming for superoxide by PMN was measured after fMLP (1 microM) activation. RESULTS: Shock significantly decreased mesenteric lymph flow from preshock levels in both groups. LR resuscitation produced significantly more mesenteric lymph than HTS resuscitation. Mesenteric lymph from LR animals primed PMN for superoxide production, whereas, HTS eliminated this priming. CONCLUSION: HTS not only decreases postshock mesenteric lymph production, it eliminates PMN priming by mesenteric lymph, suggesting a mechanism for the beneficial effects of HTS resuscitation.     

Phospholipase A(2)--derived neutral lipids from posthemorrhagic shock mesenteric lymph prime the neutrophil oxidative burst

BACKGROUND: Our previous work identified posthemorrhagic shock mesenteric lymph (PHSML) lipids as key elements in polymorphonuclear neutrophil (PMN)--provoked acute lung injury. We hypothesize that gut phospholipase A(2) (PLA(2)) is responsible for the generation of proinflammatory lipids in PHSML that primes circulating PMNs for enhanced oxidative burst. METHODS: Mesenteric lymph was collected from rats (n = 5) before (preshock), during the induction of hemorrhagic shock (mean arterial pressure, 40 mm Hg x 30 minutes), and at resuscitation (shed blood + 2x lactated Ringer's solution). PLA(2) inhibition (quinacrine, 10 mg/kg, intravenously) was given before shock was induced. Extracted lipids were separated by normal phase high-pressure liquid chromatography and resuspended in albumin. PMNs were exposed to a 5% vol:vol concentration of eluted lipids and activated with N-formyl-methionyl-leucyl-phenylalanine (1 micromol/L). Superoxide production was assessed by cytochrome C reduction. RESULTS: High-pressure liquid chromatography--extracted neutral lipids of lymph collected before hemorrhagic shock did not prime the PMN oxidase, whereas isolated neutral lipids of postshock lymph primed PMNs 2.6- +/- 0.32-fold above baseline (P <.05). PLA(2) inhibition returned PHSML neutral lipid priming to baseline levels. CONCLUSIONS: PLA(2) inhibition before hemorrhagic shock abrogates the neutrophil priming effects of PHSML through reduction of the accumulation of proinflammatory neutral lipids. Identification of these PLA(2)-dependent lipids provides a mechanistic link that may have therapeutic implications for postshock acute lung injury.     

Gut-derived leukotriene B4 following hemorrhagic shock is a pivotal mediator of neutrophil priming

Introduction: The lipid fraction of mesenteric lymph collected after hemorrhagic shock (HS) and reperfusion primes the neutrophil oxidative burst in vitro. Recently, we have identified the culprit agent to be a neutral lipid consistent with Leukotriene B4 (LTB4). We hypothesized that plasma LTB4 increases after HS and that diversion of mesenteric lymph (DML) abrogates this effect. Furthermore, we posited that administration of an LTB4 antagonist blocks the priming effect of mesenteric lymph on neutrophils (PMNs). Methods: Adult male Sprague-Dawley rats (325-375 g) underwent laparotomy with cannulation of the mesenteric lymphatics for lymph diversion (DML) or laparotomy without mesenteric cannulation (Sham). Additionally, DML rats had lymph collected both pre- and post-shock. All rats underwent cannulation of the femoral artery and vein. HS to a MAP of 30 mmHg for 45 minutes was achieved via venous exsanguination. Animals were resuscitated with shed blood and with 2x the shed blood volume of NS over two hours. After resuscitation, plasma was collected for LTB4 measurement by ELISA. Lymph collected in the resuscitation phase was added to isolated PMNs that were then measured for superoxide release in the presence and absence of the LTB4 receptor antagonist CP-105,696. Results: LTB4 concentration in the lymph collected from the DML group measured 281.25 ± 53.81 ng/ml pre-shock, increasing to 692.13 ± 176.91 ng/ml post-shock (p < 0.05). LTB4 plasma concentrations after resuscitation differed based on lymph diversion. LTB4 concentration for DML animals measured 2745 ± 214.77 pg/ml vs. Sham animals, 4012 ± 628.93 pg/ml (p < 0.05). PMN pre-treatment with the LTB4 receptor antagonist completely abrogated superoxide release in response to lymph exposure compared with the untreated control, 1.39 ± 0.23 vs. 4.22 ± 0.68 nmol O₂⁻/3.75 x 10⁵ cells/mL/min respectively (p < 0.05). Conclusion: These data suggest that splanchnic ischemia secondary to HS increases concentrations of the known priming agent LTB4 in mesenteric lymph and the systemic circulation. Furthermore, selective blockade of LTB4 markedly attenuates the effect of mesenteric lymph on PMN priming, indicating a potential therapeutic location for disruption of the dysfunctional PMN priming/activation sequence.     

Intestinal bacterial overgrowth induces the production of biologically active intestinal lymph

OBJECTIVE: We have previously documented that gut-derived lymph from rats subjected to trauma plus hemorrhagic shock (T/HS) is injurious to vascular endothelial cells and activates neutrophils (PMNs), two key events in postshock organ injury. Because T/HS leads to gut injury, intestinal bacterial overgrowth, and the loss of gut barrier function, the relative role of gut injury as opposed to intestinal bacterial overgrowth per se in the pathogenesis of biologically active intestinal lymph is unclear. We therefore studied whether mesenteric lymph can injure endothelial cells and/or active PMNs in an intestinal bacterial overgrowth model where there is no gut injury (monoassociation). METHODS: Bacterial overgrowth was established in male rats by treating the animals with 4 days of oral antibiotics followed by administration of a nonpathogenic, streptomycin-resistant strain of Escherichia coli C25. Mesenteric lymph was then collected from rats with normal flora and from E. coli C25 monoassociated rats. Its effects were tested on human umbilical vein endothelial cells (HUVECs) and human PMNs. As an additional control, lymph was collected from antibiotic-decontaminated rats that received antibiotics but were not colonized with E. coli C25. RESULTS: As compared with medium, normal flora intestinal lymph, antibiotic-decontaminated lymph, or portal plasma from the monoassociated rats, mesenteric lymph from the monoassociated rats killed HUVECs and increased the permeability of a HUVEC monolayer. In contrast to the effects on HUVECs, lymph from the monoassociated rats did not increase PMN CD11b expression or prime PMNs for an augmented respiratory burst, as compared with lymph from the rats with normal flora or from antibiotic-decontaminated rats. The effects of lymph from the monoassociated rats was not caused by bacteria, because these lymph samples were sterile. CONCLUSION: These results indicate that disruption of the normal intestinal microflora resulting in bacterial overgrowth with enteric bacilli may participate in the production of mesenteric lymph that is injurious to endothelial cells in shock, but this mechanism does not appear to be significantly involved in the activation of PMNs.     

Systemic neutrophil priming by lipid mediators in post-shock mesenteric lymph exists across species

BACKGROUND: Post-hemorrhagic shock mesenteric lymph (PHSML) has been linked with neutrophil (PMN) priming, endothelial cell (EC) activation, and acute lung injury (ALI) in rodent models. We have previously identified the lipid fraction of PHSML as containing the causative agent(s). Due to the lesson learned from the rodent gut bacterial translocation experience, we sought to confirm this phenomenon using a large animal model; hypothesizing that lymph collected from the porcine gut following ischemia/reperfusion (I/R) would cause PMN priming. METHODS: Mesenteric lymph was collected from adult pigs before, during, and for 2 hours after non-lethal hemorrhagic shock (mean arterial pressure = 30 mm Hg x 45 minutes). Whole lymph and the extracted lipid fractions of the lymph were then added to isolated human and porcine PMNs and superoxide production was measured by cytochrome C reduction. RESULTS: Hemorrhagic shock profoundly affected mesenteric lymph flow from baseline (pre-shock) flow rates of 75.63 +/- 8.86 mL/hr to 49.38 +/- 5.76 mL/hr during shock and increasing to 253.38 +/- 27.62 mL/hr after 2 hours of resuscitation. Human PMNs exposed to both whole lymph (PHSML) and its extracted lipids (PHSML Lipid) collected 2 hours after shock exhibited more than a two-fold increase in superoxide release upon activation compared with pre-shock samples: PHSML- 6.27 +/- 0.83 versus 2.56 +/- 0.60 nmolO2(-)/ 3.75 cells/mL/min, respectively (p = 0.007), PHSML Lipid- 4.93 +/- 0.34 versus 2.49 +/- 0.11 nmolO2(-)/ 3.75 cells/mL/min (p < 0.001). Similarly, porcine PMNs exhibited close to a two-fold activation when exposed to the lymph and lipid fraction: PHSML- 4.51 +/- 0.42 versus 1.06 +/- 0.28 nmolO2(-)/ 3.75 cells/mL/min (p = 0.008), PHSML Lipid-4.80 +/- 0.81 versus 1.55 +/- 0.23 nmolO2(-)/ 3.75 cells/mL/min (p = 0.002). CONCLUSION: Mesenteric lymphatics serve as the conduit for inflammatory mediators elaborated by the post-ischemic gut in both small and large animal models. Further, the causal agent(s) exist in the lipid fraction of the lymph and are active on both human and animal PMNs.     

Acute lung injury after hemorrhagic shock is dependent on gut injury and sex

Recent studies have established gut-derived lymph rather than portal blood as the major source of toxic mediators after hemorrhagic shock that causes distant organ injury. Similarly, emerging data have identified sex as a major modifier of the response to injury and illness. Thus we tested the hypothesis that female rats would be more resistant to shock-induced lung injury than male rats because females are more resistant to shock-induced gut injury and produce mesenteric lymph that is less toxic to endothelial cells. Male and female rats were subjected to sham or hemorrhagic shock and lung permeability was quantitated by Evans blue dye and protein extravasation into the alveolar space. Next, mesenteric lymph collected from shocked and sham-shocked rats of both sexes was incubated with human umbilical vein endothelial cells (HUVECs) and assayed for toxicity. Trypan blue dye exclusion and the release of lactate dehydrogenase assessed HUVEC viability and injury respectively. Lastly, sections of the terminal ileum were histologically examined for evidence of shock-induced mucosal injury. Male rats but not female rats subjected to hemorrhagic shock had evidence of increased lung permeability and produced mesenteric lymph that was cytotoxic to HUVECs. Shock caused gut injury in the male rats whereas histological evidence of gut injury was not observed in the female rats. Hemorrhagic shock-induced lung injury depends on gut injury and mesenteric lymph appears to be the route by which gut-derived toxic factors exit the gut to cause lung injury. The resistance of female rats to shock-induced lung injury appears to be secondary to their resistance to shock-induced gut injury.     

Amiloride moderates increased gut permeability and diminishes mesenteric lymph-mediated priming of neutrophils in trauma/hemorrhagic shock

BACKGROUND: Amiloride, an inhibitor of Na+/H+ exchangers and Na+ channels has been shown recently to ameliorate both gut and lung injury in rats subjected to a combined insult of trauma and hemorrhagic shock (T/HS). We have shown previously that mesenteric lymph duct ligation prevents T/HS-induced lung endothelial injury and neutrophil activation, suggesting that toxic inflammatory factors originating from the gut and carried in the lymph are responsible for the lung injury observed after T/HS. This study investigates whether the protective effect of amiloride against T/HS-induced lung injury was associated with decreased lymph toxicity and gut permeability. METHODS: Male rats subjected to trauma (laparotomy) plus hemorrhagic shock (mean arterial pressure, 30 mm Hgx90 min) (T/HS) or trauma plus sham shock (T/SS) and treated with amiloride or its vehicle had their mesenteric lymph duct catheterized. Mesenteric lymph collected before and after shock was assayed for biologic activity on endothelial cells (cytotoxicity and permeability) and neutrophils (respiratory burst activity). Gut permeability was assessed by monitoring plasma concentrations of the fluorescent dye FITC-dextran after its injection into the ileum. RESULTS: Amiloride administration reduced the capacity of post-shock mesenteric lymph to prime neutrophils for an increased respiratory burst. Amiloride failed to decrease the ability of mesenteric lymph to kill endothelial cells or increase their permeability. Amiloride decreased gut permeability. CONCLUSIONS: The mechanisms of the lung protective effect of amiloride in rats undergoing T/HS may be secondary to decreased neutrophil activation, diminished gut permeability, or an effect on the end organ.     

Postresuscitation tissue neutrophil infiltration is time-dependent and organ-specific

BACKGROUND: Hemorrhagic shock with conventional resuscitation (CR) primes circulating neutrophils and activates vascular endothelium for increased systemic inflammation, superoxide release, and end-organ damage. Adjunctive direct peritoneal resuscitation (DPR) with intraperitoneal instillation of a clinical peritoneal dialysis solution decreases systemic inflammation and edema formation by enhancing tissue perfusion. The aim of this study is to determine the effect of adjunctive DPR on neutrophil and fluid sequestration. METHODS: Anesthetized rats were hemorrhaged to 40% mean arterial pressure for 60 min. Animals were randomized for CR with the return of the shed blood plus two volumes of saline, or CR plus adjunctive DPR with 30 mL of intraperitoneal injection of a clinical peritoneal dialysis solution. Tissue myeloperoxidase (MPO) level, a marker of neutrophil sequestration, and total water content were assessed in the gut, lung, and liver in sham animals and at time-points 1, 2, 4, and 24 h postresuscitation. RESULTS: Resuscitation from hemorrhagic shock increases MPO level in all tissues in a near-linear fashion during the first 4 h following resuscitation. This occurs irrespective of the resuscitation regimen used. Tissue MPO level returned to baseline at 24 h following resuscitation except in the liver where CR and not adjunctive DPR caused a significant rebound increase. Adjunctive DPR prevented the CR-mediated obligatory fluid sequestration in the gut and lung and maintained a relative normal tissue water in these organs compared with CR alone (n = 7, F = 10.1, P < 0.01). CONCLUSION: Hemorrhagic shock and resuscitation produces time-dependent organ-specific trends of neutrophil sequestration as measured with tissue levels of myeloperoxidase, a marker of neutrophil infiltration. Modulation of the splanchnic blood flow by direct peritoneal resuscitation did not alter the time-dependent neutrophil infiltration in end-organs, suggesting a subordinate role of blood rheology in the hemorrhage-induced neutrophil sequestration. Vulnerable window for neutrophil-mediated tissue damage exists during the first 4 h following resuscitation from hemorrhagic shock in rats. Direct peritoneal resuscitation prevents the early obligatory fluid sequestration and promotes early fluid mobilization.     

Protective effect of albumin on lung injury in traumatic/hemorrhagic shock in rats

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Protective effect of albumin on lung injury in traumatic/ hemorrhagic shock rats

Objective. To determine the effect of albumin administration on lung injury in traumatic/hemorrhagic shock （T/HS） rats. Methods： Forty-eight adult Sprague-Dawley rats were divided into three groups randomly （ n = 16 in each group） ： Group A, Group B, Group C. In Group A, rats underwent laparotomy without shock. In Group B, rats undergoing T/HS were resuscitated with their blood plus lactated Ringer＇s （twice the volume of shed blood ）. In Group C, rats undergoing T/HS were resuscitated with their shed blood plus additional 3 ml of 5% human albumin. The expression of polymorphonuclear neutrophils CD18/CD11b in jugular vein blood was evaluated. The main lung injury indexes （the activity of myeloperoxidase and lung injury score） were measured. Results： Significant differences of the expression of CD18/11b and the severity degree of lung injury were found between the three groups. （P〈0.05）. The expression of CD18/CD11b and the main lung injury indexes in Group B and Goup C incresed significantly compared with those in Group A （P 〈0.05）. At the same time, the expression of CD18/CD11b and the main lung injury indexes in Group C decreased dramatically, compared with those in Group B （ P 〈0.05 ）. Conclusions ： The infusion of albumin during resuscitation period can protect lungs from injury and decrease the expression of CD18/CD11b in T/HS rats.     

Gelsolin is depleted in post-shock mesenteric lymph

BACKGROUND: Gelsolin is a plasma protein that functions to depolymerize actin filaments preventing capillary plug formation following tissue injury. It also functions to mediate the inflammatory response by binding proinflammatory lipids such as lysophosphatidic acid, sphingosine-1-phosphate and phosphoinositides. Clinically, reduced gelsolin concentrations have been associated with increased mortality in critically ill, trauma, and burn patients. We have previously shown that following hemorrhagic shock with splanchnic hypoperfusion, mesenteric lymph contains lipid components that cause neutrophil and EC activation and that protein concentrations are severely diluted due to resuscitation. We hypothesized that lipid binding proteins such as gelsolin may be depleted after trauma/hemorrhagic shock leading to increased lipid bioactivity. METHODS: Shock was induced in SD rats by controlled hemorrhage and the mesenteric duct cannulated for lymph collection. Resuscitation was performed by infusing 2x SB volume in NS over 30 min, followed by 1/2 SB volume over 30 min, then 2x SB volume in NS over 60 min. Pre and post-shock lymph was loaded at equal protein concentrations on 2D-gels, followed by trypsin digestion and identification with mass spectrometry (MS-MS). Proteomics data were confirmed with Western blotting then quantitated by densitometry. Analysis of variance was used evaluate statistical data. RESULTS: Gelsolin decreased in mesenteric lymph following hemorrhagic shock. CONCLUSIONS: Gelsolin is found at high levels (comparable to plasma) in mesenteric lymph. Following hemorrhagic shock, gelsolin levels decrease significantly, possibly due to consumption by the actin scavenging system. The magnitude of this change in concentration could release lipid bioactivity and predispose the lung and other organs to capillary injury.     

The effect of hypertonic saline resuscitation on bacterial translocation after hemorrhagic shock in rats

Translocation of enteric bacteria occurs in rats after hemorrhagic shock. A proposed mechanism involves intestinal mucosal injury by hypoperfusion. Recent work suggests that moderate hypovolemia causes gut arteriolar constriction, which is ameliorated by hypertonic saline resuscitation. Bacterial translocation should, therefore, be reduced when hypertonic saline (HS) is used as the resuscitative fluid. Seventy-eight Sprague-Dawley rats were anesthetized and subjected to 30 minutes of hemorrhagic shock (systolic blood pressure 30 to 50 mm Hg) through a modified Wigger's model. Resuscitation was performed with either shed blood (B), 3% HS + 1/2B (1:1), or with 7.5% HS + 1/2B (1:1). Spleen, liver, and mesenteric lymph nodes were sent for quantitative culture 24 hours later. Translocation occurred if enteric organisms were cultured from at least one organ. Statistical analysis used the Fisher exact test. Compared to autotransfusion, hemodilutional resuscitation from hemorrhagic shock with hypertonic saline resulted in a significant reduction in bacterial translocation (p values were 0.03 and 0.04 for 3% and 7.5% hypertonic saline, respectively). The reduction in translocation after hypertonic saline resuscitation may be the consequence of microcirculatory alterations preventing gut hypoperfusion.     

Mesenteric lymph duct ligation against renal injury in rats after hemorrhagic shock

Background: The kidney is a common target in multiple organ dysfunction syndrome (MODS). The aim of this study is to determine the role of intestinal lymphatic pathway on renal injury in hemorrhagic shock rats. Methods: Wistar rats were divided into sham, shock, and ligation groups. The hemorrhagic shock model was induced in the shock and ligation groups. After resuscitation, the mesenteric lymph ducts were ligated in the ligation group. Blood from the carotid artery was taken to determine renal functional indices. The kidneys were used to observe histomorphological changes at 6 h after resuscitation. In addition, kidney homogenate was used to determine malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and myeloperoxidase (MPO) levels at 90 min after shock and 0, 1, 3, 6, 12, and 24 h after resuscitation. And the survival rate of 24 h was recorded. Results: The survival rate in shock group was obviously lower than sham and ligation groups. The urea and creatinine contents in the serum of shock and ligation groups were significantly higher than the sham group; the indices in the ligation group were significantly lower than the shock group. Histological studies showed various degrees of renal injury in the shock and ligation groups with a lesser severity in the ligation group. MDA, TNFα, IL-6, and MPO in renal homogenate of the shock group were raised, and the activity of SOD was lower in comparison to the sham group. Further, MDA, TNFα, IL-6, and MPO in renal homogenate of the ligation group at 6, 12, and 24 h were lower, and the SOD activity was higher than that of the shock group at the same time points. Conclusion: The mesenteric lymph duct ligation could be used to attenuate renal injury in shock rats. Its mechanism might be related to reducing the polymorph nuclear (PMN) and decreasing inflammatory mediator and free radical.     

Post-hemorrhagic shock mesenteric lymph activates human pulmonary microvascular endothelium for in vitro neutrophil-mediated injury: the role of intercellular adhesion molecule-1

BACKGROUND: Splanchnic hypoperfusion is believed to be central in the pathogenesis of hemorrhagic shock-induced acute respiratory distress syndrome and multiple organ failure. Our previous work focused on the portal circulation as the conduit for gut-derived mediators of acute respiratory distress syndrome. Our current focus is the proinflammatory effects of postshock mesenteric lymph. We hypothesize that postshock lymph induces neutrophil (PMN)-mediated endothelial cell damage in an intercellular adhesion molecule-1 (ICAM-1)-dependent fashion, and devised a two-insult model to test this hypothesis. METHODS: Rats (n > or = 5) underwent hemorrhagic shock (mean arterial pressure, 40 mm Hg for 30 minutes) and resuscitation (shed blood plus two times crystalloid) with lymph collection. Human pulmonary microvascular endothelial cells (HMVECs) were divided into three groups and grown to near confluence. Group 1 was incubated for 6 hours in 1% preshock or postshock lymph and ICAM-1 was measured by flow cytometry. Group 2 consisted of coculture of HMVECs and PMNs after endothelial cell activation to determine whether postshock lymph would stimulate PMN adherence. Group 3 was incubated under identical conditions, but PMNs were added for 30 minutes, and then activated with 4.5 micromol/L lysophosphatidylcholine (lyso-PC) for 1 hour to ascertain cytotoxicity. HMVEC density was measured using microscopy and recorded as HMVECs per millimeter squared. ICAM-1-blocking antibody and isotype control were used to assess the effects of ICAM-1 on PMN cytotoxicity. A buffer control was used for comparison using analysis of variance with Tukey's correction. RESULTS: Postshock lymph activated HMVECs for increased surface expression of ICAM-1 and stimulated PMNs to adhere to endothelial cell monolayers. Activation of PMNs with lyso-PC in the presence of postshock lymph resulted in marked HMVEC death. The addition of an ICAM-1-blocking antibody abrogated this effect. Neither postshock lymph alone (758 +/- 35 HMVECs/mm(2)), nor postshock lymph in the presence of quiescent PMNs alone (734 +/- 28 HMVECs/mm(2)), nor lymph plus lyso-PC (834 +/- 21 HMVECs/mm(2)) provoked endothelial cell damage. CONCLUSION: Postshock mesenteric lymph activates endothelial cells for increased ICAM-1 expression and PMN adherence. Furthermore, postshock lymph acts as an inciting event in a two-event in vitro model of PMN-mediated endothelial cell injury. These findings further substantiate the key mechanistic role of mesenteric lymph in hemorrhagic shock-induced acute lung injury and suggest that ICAM-1 expression is pivotal in the two-event model of multiple organ failure.     

Albumin protects against gut-induced lung injury in vitro and in vivo

OBJECTIVE: Since albumin has the ability to detoxify, we assessed whether low-dose albumin could protect against trauma/hemorrhagic shock (T/HS)-induced endothelial cell, lung, gut, and red blood cell (RBC) injury in vivo and endothelial cell injury in vitro. SUMMARY BACKGROUND DATA: T/HS cause ischemic insult to the gut, resulting in the release of biologically active factors into the mesenteric lymph, which then cause injury to multiple distant organs. METHODS: In vitro experiments tested the ability of albumin to reduce the cytotoxicity of mesenteric lymph from male rats subjected to T/HS (laparotomy + MAP 30 mm Hg for 90 minutes) for human umbilical vein endothelial cell (HUVEC). In subsequent in vivo experiments, the ability of albumin given as part of the resuscitation regimen to protect against T/HS-induced injury was tested by comparing the magnitude of injury in T/HS rats receiving human albumin (shed blood + 0.12, 0.24, or 0.36 g/kg) or lactated Ringer's solution (shed blood + 2 x volume of shed blood as LR) with that observed in rats subjected to trauma/sham shock. Rats were killed after a 3-hour recovery period and had lung permeability evaluated by bronchoalveolar lavage and myeloperoxidase assays, intestinal microvillous injury by histology, and RBC deformability using ektacytometry. RESULTS: Both bovine and human albumin prevented T/HS lymph-induced HUVEC cytotoxicity in vitro, even when added 30 minutes after the lymph (viability 15 +/- 4% to 88 +/- 3%, P < 0.01). In vivo RBC deformability was better preserved by blood plus albumin than blood plus lactated Ringer's solution (P < 0.01). Likewise, albumin administration reduced T/HS-induced lung permeability and neutrophil sequestration in a dose-dependent fashion, with 0.36 g/kg of albumin effecting total lung protection (P < 0.01). In contrast, albumin treatment did not prevent T/HS-induced gut injury. CONCLUSIONS: Low-dose albumin protects against gut lymph-induced lung, HUVEC, and RBC injury by neutralizing T/HS lymph toxicity.     

Influence of gut microflora on mesenteric lymph cytokine production in rats with hemorrhagic shock

OBJECTIVE: The aim of the present study was to test the hypothesis that the resident gut microflora play a role in modulating gut cytokine production under normal circumstances and in response to tissue injury with or without hemorrhagic shock. METHODS: The postnodal lymph was collected from the main mesenteric lymphatic channel 1 hour before, during (1.5 hours), and hourly for 6 hours after 90 minutes of sham or actual hemorrhagic shock (30 mm Hg) in the following three groups of rats, all of which had laparotomies and vascular instrumentation: rats with a normal gut flora (NF), rats whose gut flora had been decontaminated with oral antibiotics (AD), and rats with Escherichia coli C25 intestinal overgrowth (MA). Interleukin (IL)-6 and TNF levels in the mesenteric lymph were measured using cytokine-dependent cellular assays. Endotoxin levels and endotoxin-neutralizing capacity in the lymph were also measured. RESULTS: Mesenteric lymph IL-6 levels in the laparotomized MA-sham animals were significantly elevated compared with NF-sham animals at 2 to 4 hours (p < 0.05) and at 5 and 6 hours after sham shock (p < 0.01). Similarly, IL-6 levels in laparotomized AD-shock animals were increased when compared with NF-shock animals 3 hours after shock (p < 0.001). Lymph tumor necrosis factor bioactivity, although present in all surgically manipulated groups, was scarcely detectable in untouched animals. Endotoxin-neutralizing capacity was significantly impaired in shocked animals compared with untouched animals. CONCLUSION: Changes in the gut microflora modulate the gut cytokine production after tissue injury with or without hemorrhagic shock, with intestinal bacterial overgrowth leading to the greatest increase in mesenteric lymph IL-6 levels.     

Hypertonic saline resuscitation of hemorrhagic shock diminishes neutrophil rolling and adherence to endothelium and reduces in vivo vascular leakage

OBJECTIVE: To evaluate the in vivo effects of hypertonic saline (HTS) resuscitation on the interactions of endothelial cells (ECs) and polymorphonuclear neutrophils (PMNs) and vascular permeability after hemorrhagic shock. SUMMARY BACKGROUND DATA: The PMN has been implicated in the pathogenesis of EC damage and organ injury following hemorrhagic shock. Compared to Ringer's lactate (RL), HTS resuscitation diminishes PMN and EC adhesion molecule expression and organ sequestration of PMNs. METHODS: In a murine model of hemorrhagic shock (50 mmHg for 45 minutes followed by resuscitation) using intravital microscopy on cremaster muscle, the authors studied PMN-EC interactions and vascular leakage (epifluorescence after 50 mg/kg fluorescent albumin) in three resuscitation groups: HTS (shed blood + 4 cc/kg 7.5% HTS, n = 12), RL (shed blood + RL [2x shed blood volume], n = 12), and sham (no hemorrhage or resuscitation, n = 9). EC ICAM-1 expression was evaluated by immunohistochemistry. Data, presented as mean +/- SEM, were evaluated by analysis of variance with Bonferroni correction. RESULTS: There were no differences between groups in flow mechanics. Compared to RL, HTS animals (t = 90 minutes) displayed diminished PMN rolling and PMN adhesion to EC at time intervals beyond t = 0. There were no differences between the sham and HTS groups. Vascular leakage was 45% lower in HTS than in RL-resuscitated animals. Cremaster EC ICAM-1 expression was similar in the two groups. CONCLUSIONS: Using HTS instead of RL to resuscitate hemorrhagic shock diminishes vascular permeability in vivo by altering PMN-EC interactions. HTS could serve as a novel means of immunomodulation in hemorrhagic shock victims, potentially reducing PMN-mediated tissue injury.     

A study of the biologic activity of trauma-hemorrhagic shock mesenteric lymph over time and the relative role of cytokines

BACKGROUND: Gut-derived factors in intestinal lymph have been recently shown to cause lung injury, activate neutrophils, and injure endothelial cells in rats subjected to hemorrhagic shock (T/HS). However, the time course of the appearance and disappearance of these factors in intestinal lymph is unclear. Thus the goal of this study was to characterize the biologic activity of T/HS lymph collected at various times during and after shock. METHODS: Male rats subjected to trauma (laparotomy) plus hemorrhagic shock (mean arterial pressure, 90 mm Hg x 90 min) (T/HS) or trauma plus sham shock (T/SS) had their mesenteric lymph duct catheterized. Mesenteric lymph collected before shock, during shock, and hourly for 6 hours after shock was assayed for cytokine levels (tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, interleukin-1, and transforming growth factor-beta) as well as biologic activity on endothelial cells (cytotoxicity and permeability) and neutrophils (CD11b adhesion molecule expression and respiratory burst activity). RESULTS: T/HS, but not T/SS, lymph injured endothelial cells and activated neutrophils, although the cytokine levels did not differ between the T/HS and T/SS lymph samples. The biologic activity of T/HS lymph appeared during the shock (gut ischemic) period. The temporal pattern of activity varied on the basis of the biologic activity being tested, with the neutrophil-activating properties of the T/HS lymph persisting longest. CONCLUSIONS: These results suggest that gut ischemia itself is sufficient to induce the production of biologically active T/HS lymph and that the temporal pattern of biologic activity varies over time on the basis of the property being tested. Consequently, studies directed at identifying the active factors in T/HS lymph must take these temporal patterns of activity into account.