Pancreatic duct ligation abrogates the trauma hemorrhage-induced gut barrier failure and the subsequent production of biologically active intestinal lymph

The studies of the mechanisms by which trauma-hemorrhagic shock leads to gut injury and dysfunction have largely ignored the nonbacterial factors contained within the lumen of the intestine. Yet, there is increasing evidence suggesting that intraluminal pancreatic proteases may be involved in this process. Thus, we tested the hypothesis that pancreatic proteases are necessary for the trauma-hemorrhagic shock-induced gut injury and the production of biologically active mesenteric lymph by determining the extent to which pancreatic duct ligation (PDL) would limit gut injury and mesenteric lymph bioactivity. To assess the effect of PDL on gut injury and dysfunction gut morphology, the mucus layer structure and the gut permeability were measured in the following four groups of male rats subjected to laparotomy (trauma) and hemorrhagic shock (pressure, 30 mmHg for 90 min): (1) rats subjected to trauma plus sham-shock (T/SS), (2) T/SS rats undergoing PDL (T/SS + PDL), (3) rats subjected to trauma and hemorrhagic shock (T/HS), and (4) rats subjected to T/HS + PDL. The ability of mesenteric lymph from these four rat groups to kill endothelial cells and activate neutrophils was tested in vitro. The PDL did not affect any of the parameters studied because there were no differences between the T/SS and the T/SS + PDL groups. However, PDL protected the gut from injury and dysfunction because PDL significantly abrogated T/HS-induced mucosal villous injury, loss of the intestinal mucus layer, and gut permeability. Likewise, PDL totally reversed the endothelial cell cytotoxic activity of T/HS lymph and reduced the ability of T/HS lymph to prime naive neutrophils for an augmented respiratory burst. Thus, it seems that intraluminal pancreatic proteases are necessary for the T/HS-induced gut injury and the production of bioactive mesenteric lymph.     

Trauma-hemorrhage-induced neutrophil priming is prevented by mesenteric lymph duct ligation

Our objective in this study was to test the hypothesis that priming of neutrophils (PMN) in vivo by trauma-hemorrhagic shock (T/HS) is mediated by factors carried in intestinal lymph that prime PMNs by enhancing their responses to inflammatory mediators. Previous studies have shown that T/HS-induced lung injury is mediated by factors contained in mesenteric lymph and that ligation of the main mesenteric lymph duct (LDL) can prevent T/HS-induced lung injury. Since T/HS-induced lung injury is associated with PMN infiltration, one mechanism underlying this protective effect may be the prevention of PMN priming and activation. Therefore, we assessed the ability of T/HS to prime PMN responses to inflammatory agonists, and the ability of mesenteric lymph duct division to protect against such T/HS-induced PMN priming in an all-rat system. PMN were collected from male rats 6 h after laparotomy (trauma) plus hemorrhagic shock (30 mmHg for 90 min; T/HS) or trauma plus sham shock (T/SS). Uninstrumented rats were used as controls (UC). In a second set of experiments, rats were subjected to T/HS with or without mesenteric lymph duct division. PMN were then stimulated with chemokine (GRO, MIP-2) and lipid (PAF) chemoattractants, and cell calcium flux was used to quantify responses to those agonists. T/SS primed PMN responses to GRO, MIP-2. and PAF in comparison to UC rats, but the addition of shock (T/HS) amplified PMN priming in a significant manner, especially in response to GRO. Mesenteric lymph duct division prior to T/HS diminished PMN priming to the levels seen in T/SS. This reversal of priming was significant for GRO and GRO/MIP-2 given sequentially, with the other agonist regimens showing similar trends. The results support the concept that trauma and hemorrhagic shock play important additive roles in inflammatory PMN priming. Entry of gut-derived inflammatory products into the circulation via mesenteric lymph seems to play a dominant role in mediating the conversion of physiologic shock insults into immunoinflammatory PMN priming. Shock-induced gut lymph priming enhances PMN responses to many important chemoattractants, most notably the chemokines, and mesenteric lymph duct division effectively reverses such priming to priming levels seen in trauma without shock.     

Trauma-shock-induced gut injury and the production of biologically active intestinal lymph is abrogated by castration in a large animal porcine model

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.     

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.     

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 Ringer's 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.     

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

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 (TLR4 mut) 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 TLR4 mut 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 TLR4 mut mice as well the TRIF mut-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.     

The role of lymph factors in lung injury, bone marrow suppression, and endothelial cell dysfunction in a primate model of trauma-hemorrhagic shock

Studies in rodent models of trauma-hemorrhagic shock (T/HS) have shown that factors contained in the intestinal lymph are responsible for acute lung injury and bone marrow suppression, and that they contribute to a systemic inflammatory state. Because results observed in rodent T/HS models may not fully reflect the response of injured patients, it is necessary to determine if these results can be replicated in primates before the institution of invasive studies in humans. Thus, the three goals of this study were to determine if diversion of thoracic duct lymph reduced T/HS-induced lung injury; to compare the biologic activity of thoracic duct lymph from baboons subjected to T/HS or trauma sham-shock (T/SS); and to compare the biologic activity and composition of plasma from baboons subjected to T/SS, T/HS, and T/HS with thoracic duct lymph drainage. Three groups of baboons were studied: T/SS plus lymph diversion via a thoracic duct catheter, T/HS, and T/HS plus lymph diversion (T/HS-LD). The trauma component consisted of a neck dissection with resection of the proximal clavicle plus a laparotomy. HS was to a mean arterial pressure of 40 mmHg and was maintained at 40 mmHg until the base excess reached -5 mEq or the total shock period reached 3 h. Volume resuscitation was carried out by reinfusing the shed blood plus crystalloids. Before, during, and after the T/HS or T/SS period, blood and lymph samples were obtained for analysis, and lung samples were harvested for measurement of lung wet-to-dry ratio at 5 h after the end of the shock period. Diversion of thoracic duct lymph prevented T/HS-induced lung injury as reflected in lung wet-to-dry weight ratios (T/SS = 4.6 +/- 0.5; T/HS+LD = 4.8 +/- 0.7; T/HS = 5.4 +/- 0.6; P < 0.05). Lymph from the T/HS group collected during the early postshock period was cytotoxic for human endothelial cells (HUVECs; 16% vs. 100% survival in T/SS lymph) and increased HUVEC monolayer permeability almost 2-fold (P < 0.01). T/HS lymph and plasma also suppressed red blood cell (erythroid burst-forming unit) and white blood cell (granulocyte-monocyte colony-forming unit) progenitor cell growth of human bone marrow to approximately 50% of control, whereas T/SS lymph and plasma were not suppressive (P < 0.05). Plasma cytokine levels were increased to a similar degree in the two T/HS groups. Thus, in a primate model of T/HS, gut-derived factors carried in the lymph potentiates lung injury and endothelial dysfunction, and suppresses bone marrow progenitor cell growth.     

Bone marrow failure in male rats following trauma/hemorrhagic shock (T/HS) is mediated by mesenteric lymph and modulated by castration

Bone marrow (BM) suppression occurs following trauma/hemorrhagic shock (T/HS) in experimental animals as well as following severe injury in humans. Although the pathophysiology of BM suppression remains poorly understood, mesenteric lymph is thought to play an important role in T/HS-induced BM suppression; however, the direct effect of mesenteric lymph on BM in vitro has never been studied. In addition, recent studies in rats have also shown that female and castrated male rats are protected against T/HS-induced BM failure. We therefore hypothesized that mesenteric lymph is a source of factor(s) causing direct BM suppression and that the effects of mesenteric lymph are gender dependent. To test this hypothesis, we subjected noncastrated (NC) and castrated (C) male and proestrus female rats to T/HS or trauma sham shock (T/SS). Mesenteric lymph collected 3 h postshock was plated (4% v/v) with BM cells collected from unmanipulated male or female rats for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) colony growth. The T/HS lymph collected from NC-male rats but not from female rats caused a 50% inhibition of CFU-GM and BFU-E colony growth compared with cells cultured without lymph (P < 0.05 versus all other groups (ANOVA + Tukey). T/HS lymph collected from C-male rats also caused no significant inhibition of CFU-GM and BFU-E colony growth compared with cells cultured without lymph. Female and male BM progenitor cells had a similar response to mesenteric lymph from all groups tested. These results show that mesenteric lymph from NC-male rats suppresses CFU-GM and BFU-E progenitor growth in vitro, whereas the lymph from C-male and female rats did not. The effects of mesenteric lymph were the same regardless of whether the target BM was from male or female rats. The results therefore indicate that BM failure in male rats is directly mediated by factors present within the mesenteric lymph that appear to be modulated by castration, and protection against BM failure in female rats occurs at a systemic rather than a local level. Further studies are needed to elucidate potential therapeutic effects of lymph manipulation in hematopoiesis after injury.     

Lymph from a primate baboon trauma hemorrhagic shock model activates human neutrophils

We have reported that toxic factors in intestinal lymph are responsible for acute lung injury and bone marrow suppression and that they contribute to a systemic inflammatory state based on studies in rodent models of trauma-hemorrhagic shock. Rodent models may not completely reflect the responses of injured patients. Thus, it is important to confirm these findings in primates before applying them to injured human patients with trauma. Thus, we have recently established baboon trauma-hemorrhagic shock (T/HS) and trauma-sham shock (T/SS) models that showed that gut-derived factors carried in the lymph potentiates lung injury and causes human endothelial dysfunction and suppresses human bone marrow progenitor cell growth. Here, we further investigated the effects of these primate lymph samples on human neutrophils. We hypothesized that toxic factors in baboon lymph may prime and/or activate human polymorphonuclear leukocyte (PMN) leading to overproduction of superoxide, thereby contributing to the development of adult respiratory distress syndrome and multiple organ failure. To this effect, we have examined the priming effect of baboon T/HS and T/SS lymph on PMN respiratory burst and expression of adhesion molecule in human neutrophils. The results of these studies indicate that PMN treated with baboon T/HS lymph showed significantly induced respiratory burst responses compared with PMN treated with T/SS lymph or medium when phorbol myristate acetate PMA was applied after lymph pretreatment. Secondly, we found that the expression of CD11b adhesion molecule was increased by incubation with T/HS lymph. These results suggest that baboon lymph from T/HS models can increase respiratory burst and adhesion molecule expression in human PMN, thereby potentially contributing to PMN-mediated organ injury.     

Anticoagulants influence the in vitro activity and composition of shock lymph but not its in vivo activity

Many models of trauma-hemorrhagic shock (T/HS) involve the reinfusion of anticoagulated shed blood. Our recent observation that the anticoagulant heparin induces increased mesenteric lymph lipase activity and consequent in vitro endothelial cell cytotoxicity prompted us to investigate the effect of heparin-induced lipase activity on organ injury in vivo as well as the effects of other anticoagulants on mesenteric lymph bioactivity in vitro and in vivo. To investigate this issue, rats subjected to trauma-hemorrhage had their shed blood anticoagulated with heparin, the synthetic anticoagulant arixtra (fondaparinux sodium), or citrate. Arixtra, in contrast to heparin, did not increase lymph lipase activity or result in high levels of endothelial cytotoxicity. Yet, the arixtra-treated rats subjected to T/HS still manifested lung injury, neutrophil priming, and red blood cell dysfunction, which was totally abrogated by lymph duct ligation. Furthermore, the injection of T/HS mesenteric lymph, but not sham-shock lymph, collected from the arixtra rats into control mice recreated the pattern of lung injury, polymorphonucleocyte (PMN) priming, and red blood cell dysfunction observed after actual shock. Consistent with these observations, citrate-anticoagulated rats subjected to T/HS developed lung injury, and the injection of mesenteric lymph from the citrate-anticoagulated T/HS rats into control mice also resulted in lung injury. Based on these results, several conclusions can be drawn. First, heparin-induced increased mesenteric lymph lipase activity is not responsible for the in vivo effects of T/HS mesenteric lymph. Second, heparin should be avoided as an anticoagulant when studying the biology or composition of mesenteric lymph because of its ability to cause increases in lymph lipase activity that increase the in vitro cytotoxicity of these lymph samples.     

Hypertonic saline resuscitation limits neutrophil activation after trauma-hemorrhagic shock

There is evidence suggesting that the ischemic gut is a major source of factors that lead to neutrophil activation, and that neutrophil activation can be reduced by hypertonic saline resuscitation. Thus, we tested whether trauma-hemorrhagic shock-induced neutrophil activation can be reduced by hypertonic saline resuscitation, as well as whether hypertonic saline reduces the ability of mesenteric lymph from shocked animals to activate neutrophils. Male Sprague-Dawley rats subjected to trauma (laparotomy), plus 90 min of shock [mean arterial pressure (MAP) MAP = 30 mmHg] or sham shock were resuscitated with Ringer's lactate or 7.5% hypertonic saline at an equivalent sodium load. Whole blood samples were collected before shock as well as at 1 and 2 h after the end of the shock period for neutrophil CD11b and CD18 expression measurements. In a second set of experiments, mesenteric lymph samples collected from rats subjected to trauma plus hemorrhagic shock (T/HS) or trauma plus sham-shock (T/SS) and resuscitated with Ringer's lactate or hypertonic saline were tested for their ability to modulate PMN CD11b, CD18, or L-selectin expression, as well as prime PMN for an augmented respiratory burst. To avoid confounding results due to interspecies differences, while at the same time looking at potential human responses, both naive rat and human PMN were tested. Both CD11b and CD18 expression were increased in PMN harvested from rats subjected to T/HS and resuscitated with Ringer's lactate solution, but not in T/HS rats resuscitated with hypertonic saline. These results indicate that PMN activation is increased to a greater extent in Ringer's lactate-resuscitated than hypertonic saline-resuscitated animals. Likewise, mesenteric lymph from the T/HS rats resuscitated with Ringer's lactate increased naive rat and human PMN CD11b and CD18 expression to a greater extent than did T/HS lymph from the hypertonic saline-treated rats. Additionally, T/HS lymph from the Ringer's lactate- but not the hypertonic saline-treated rats induced PMN L-selectin shedding. Lastly, T/HS lymph from the Ringer's lactate-treated rats induced the greatest PMN respiratory burst. These results indicate that resuscitation from T/HS with hypertonic saline is associated with less PMN activation than resuscitation with Ringer's lactate, and that factors produced or released by the postischemic intestine and carried in the mesenteric lymph contribute to neutrophil activation after an episode of T/HS.     

Mesenteric lymph from rats subjected to trauma-hemorrhagic shock are injurious to rat pulmonary microvascular endothelial cells as well as human umbilical vein endothelial cells

Previously, we have documented that gut-derived lymph from rats subjected to trauma/hemorrhagic shock (T/HS) is injurious to human umbilical vein endothelial cells (HUVEC). To verify these findings in an all rat systems, the ability of T/HS lymph to increase rat pulmonary microvascular endothelial cell (RPMVEC) monolayer permeability and kill RPMVEC was compared with that observed with HUVECs. RPMVEcs isolated from male rats or HUVECs were grown in 24-well plates for the cytotoxicity assays or on permeable filters in a two-chamber system for permeability assays. Mesenteric lymph was collected from male rats subjected to trauma (laparotomy) plus hemorrhagic shock (T/HS group) or to a laparotomy plus sham-shock (T/SS group). The T/HS group had their mean arterial pressure decreased to 30 mmHg and kept there for 90 min. Lymph samples centrifuged to remove the cellular component were incubated with the RPMVECs or HUVECs at a 10% concentration. Neither T/SS lymph nor post-T/HS portal vein plasma was toxic to or increased the permeability of the RPMVECs or HUVECs. The pattern of cytotoxicity observed in the HUVECs incubated with T/HS mesenteric lymph was similar to that observed in the RPMVECs, as reflected by trypan blue dye exclusion, with more than 95% of the HUVECs and RPMVECs being killed after a 16-h incubation with T/HS mesenteric lymph. However, at earlier time points the amount of LDH released from the HUVEC cells incubated with T/HS lymph was greater than that observed with the PRMVEC, although trypan blue dye exclusion was similar. Similarly, incubation with 10% T/HS lymph increased the permeability of both HUVEC and RPMVEC monolayers more than 2-fold, even with an incubation period as short as 1 h. In conclusion, these results provide further evidence that T/HS lymph, but not T/SS lymph or post-T/HS portal vein plasma, is injurious to endothelial cells and that RPMVECs are as susceptible to injury as HUVECs. Additionally, these studies support the emerging concept that gut-induced distant organ injury is mediated by factors contained in mesenteric lymph.     

Entry of gut lymph into the circulation primes rat neutrophil respiratory burst in hemorrhagic shock.

OBJECTIVE: Endothelial cell injury by polymorphonuclear neutrophil (neutrophil [PMN]) respiratory burst after trauma and hemorrhagic shock (T/HS) predisposes subjects to acute respiratory distress syndrome and multiple organ failure. T/HS mesenteric lymph injures endothelial cell and lymph duct ligation (LDL) before T/HS prevents pulmonary injury. We investigated the role of mesenteric lymph in PMN priming by T/HS. DESIGN: Prospective experiment in rats. SETTING: University hospital laboratory. SUBJECTS: Adult male rats. INTERVENTIONS: Mesenteric lymph was obtained from rats undergoing T/HS (30 mm Hg, 90 mins) or sham shock (T/SS). Plasma was harvested from uninstrumented control (UC), T/HS, T/SS, and T/HS+LDL rats. PMNs were isolated from UC, T/HS, and T/HS+LDL rats. MEASUREMENTS AND MAIN RESULTS: PMNs from UC rats were incubated in buffer, 1% T/HS lymph, and 1% T/SS lymph. PMNs from UC rats were incubated in UC, T/HS, T/SS, and T/HS+LDL plasma. PMN respiratory burst was initiated by using macrophage inflammatory protein (MIP)-2/platelet-aggregating factor (PAF) or phorbol myristate acetate. Cytosolic calcium ([Ca2+]i) responses to MIP-2/PAF were assayed in PMN from UC, T/HS, and T/HS+LDL rats. PMN preincubated in T/HS lymph showed significant elevations in MIP/PAF-elicited respiratory burst compared with T/HS lymph or buffer only (p <.05; analysis of variance/Tukey's test). T/HS lymph incubation also increased (p <.05) phorbol myristate acetate elicited respiratory burst compared with buffer or T/SS. Preincubation in T/HS plasma increased MIP-2/PAF-elicited respiratory burst (p <.05) compared with UC or T/SS plasma. LDL blocked T/HS priming of respiratory burst. Control PMN [Ca2+]i responses to MIP-2 and PAF were low. T/SS PMN were significantly more responsive, but the T/HS PMN showed still higher responses (p <.01). LDL reversed the priming of [Ca2+]i responses by T/HS (p <.01). CONCLUSIONS: PMNs are primed by T/HS lymph but not T/SS lymph and by T/HS plasma but not T/SS plasma. LDL before shock prevents T/HS plasma from priming PMN. The magnitude of respiratory burst found here paralleled the [Ca2+]i responses seen to receptor dependent initiating agonists. Mesenteric lymph is both necessary and sufficient to prime PMN after T/HS in the rat, and it primes PMN in part by enhancing [Ca2+]i responses to G-protein coupled chemoattractants. Mesenteric lymph mediates postshock PMN dysfunction.     

Molecular signatures of trauma-hemorrhagic shock-induced lung injury: hemorrhage- and injury-associated genes

The etiology of trauma-hemorrhagic shock (T/HS)-induced acute lung injury has been difficult to elucidate because of, at least in part, the inability of in vivo studies to separate the noninjurious pulmonary effects of trauma-hemorrhage from the tissue-injurious ones. To circumvent this in vivo limitation, we used a model of T/HS in which T/HS lung injury was abrogated by dividing the mesenteric lymph duct. In this way, it was possible to separate the pulmonary injurious response from the noninjurious systemic response to T/HS by comparing the pulmonary molecular responses of rats subjected to T/HS, which did and did not develop lung injury, with those of nonshocked rats. Using high-density oligonucleotide arrays and treatment group comparisons of whole lung tissue collected at 3 h after the end of the shock or sham-shock period, 139 of 8,799 assessed genes were identified by significant analysis of microarrays. Hemorrhage without the secondary effects of lung injury modulated the expression of 21 genes such as interleukin 1beta, metallothionein-2, and myeloctomatosis oncogene (c-myc). In response to injury, 42 genes were identified to be differentially expressed. Upregulated genes included the L1 retroposon and guanine deaminase, whereas downregulated genes included catalase and superoxide dismutase 1. Real-time polymerase chain reaction confirmed the differential expression for selected genes. PathwayAssist analysis identified interleukin 1beta as a central regulator of two subpathways of stress response-related genes (c-myc and superoxide dismutase 1/catalase) as well as several unrelated genes such as lipoprotein lipase. Our model system provided a unique opportunity to distinguish the molecular changes associated with T/HS-induced acute lung injury from the systemic molecular response to T/HS.     

Amiloride combined with small-volume resuscitation with hypertonic saline is superior in ameliorating trauma-hemorrhagic shock-induced lung injury in rats to the administration of either agent alone

OBJECTIVE: Recognition of the limitations of standard crystalloid resuscitation has led to exploration for alternative resuscitation strategies that might better prevent the development of trauma-hemorrhage-induced organ dysfunction and systemic inflammation. Thus, the goal of this study was to compare the effects of two resuscitation strategies alone and in combination with that of standard resuscitation with Ringer's lactate. These two strategies were intravenous injection of amiloride, an inhibitor of Na/H exchange and epithelial Na channels, and resuscitation with hypertonic saline. DESIGN: Prospective animal study with concurrent control. SETTING: Small animal laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Rats injected with amiloride or its vehicle were subjected to trauma-hemorrhagic shock (T/HS) or trauma sham-shock (T/SS) and resuscitated with Ringer's lactate or hypertonic saline. The T/HS model consisted of a laparotomy plus 90 mins of shock (mean arterial pressure 30 mm Hg). Three hours after the end of the shock or sham-shock period, lung permeability, lung histology, pulmonary neutrophil sequestration, neutrophil CD11b expression, gut injury, and red blood cell rigidification were assessed. MEASUREMENTS AND MAIN RESULTS: Both amiloride and hypertonic saline reduced T/HS-induced pulmonary permeability and neutrophil sequestration, and coadministration of these two agents was more efficacious than administration of the individual agents. In contrast, whereas gut injury was attenuated by both amiloride and hypertonic saline, combined administration of amiloride and hypertonic saline failed to further protect the gut. Additionally, hypertonic saline reduced both neutrophil CD11b expression and red blood cell rigidification, whereas amiloride was without effect. CONCLUSIONS: Combined administration of amiloride and small-volume resuscitation with hypertonic saline may be a strategy worthy of further evaluation in the therapy of shock-induced distant organ injury.     

Resistance of the female, as opposed to the male, intestine to I/R-mediated injury is associated with increased resistance to gut-induced distant organ injury

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.     

Mesenteric lymph duct ligation provides long term protection against hemorrhagic shock-induced lung injury

Recently we have shown that ligation of the main mesenteric lymph (MLN) duct prior to an episode of hemorrhagic shock (HS) prevents shock-induced lung injury. Yet, ligation or diversion of intestinal lymph immediately prior to injury is not clinically feasible. Diversion of intestinally derived lymph after injury to protect against secondary insults is possible, but it is not known how long the protective effects of lymph ligation would last. Thus, we tested whether ligation of the MLN duct seven days prior to HS would still be protective. Male Sprague-Dawley rats were subjected to laparotomy with or without MLN duct ligation. Seven days later, half of the sham and actual MLN duct ligated animals randomly were selected to undergo HS (30 mmHG for 90 min). The other half of the animals was subjected to sham shock. Lung permeability, pulmonary myeloperoxidase (MPO) activity, and bronchoalveolar fluid (BALF) protein content were used to determine lung injury. Lymphatic division 7 days prior to HS continued to prevent shock induced lung injury as assessed by a lower Evans Blue dye concentration, BALF protein and MPO activity. In addition, there was no evidence of Patent Blue dye in the previously ligated MLN duct. Since ligation of the main mesenteric lymphatic duct continues to protect against shock-induced lung injury 1 week after duct ligation, it is feasible that lymphatic ligation performed after an injury remains protective against certain secondary insults for at least 1 week.     

Role of the gut lymphatic system in multiple organ failure

The central concept of this review is that gut-derived factors contained primarily in the mesenteric lymph rather than the portal blood contribute to distant organ injury. This hypothesis is supported by recent studies indicating that division of the mesenteric lymphatic ducts prevents lung injury after hemorrhagic shock and significantly ameliorates lung injury after thermal injury. The mechanism of hemorrhagic shock-induced lung injury appears to be through mesenteric lymph-induced activation of neutrophils and activation/injury of endothelial cells. This notion is supported by in vitro studies indicating that mesenteric lymph, but not portal vein plasma, collected after a nonlethal episode of hemorrhagic shock activates neutrophils, increases endothelial cell monolayer permeability, and can even cause endothelial cell death. This concept that gut-derived factors contained primarily in the mesenteric lymph rather than the portal system potentiate the development of distant organ (lung) injury, if correct, would help clarify several important issues. First, because the lung is the first organ exposed to mesenteric lymph (i.e., mesenteric lymph enters the subclavian via the thoracic duct), it would help explain the clinical observation of why the lung is generally the first organ to fail in severely injured patients. Second, this gut lymphatic hypothesis would provide new information on the pathophysiology of gut-induced lung injury. Finally, it would help explain the discordant results between experimental and some clinical studies on the role of gut injury and loss of gut barrier function in the development of a systemic inflammatory state and distant organ injury.     

Mesenteric lymph return is an important contributor to vascular hyporeactivity and calcium desensitization after hemorrhagic shock

Vascular hyporeactivity is an important factor in irreversible shock, whereas calcium desensitization is one of the mechanisms of vascular hyporeactivity, and the intestinal lymphatic pathway plays an important role in multiple organ injury after severe hemorrhagic shock (HS). In this study, our aims were to determine the effects of mesenteric lymph on vascular reactivity during HS and the mechanisms involved. First, the in vivo pressor response was observed by intravenous injection of norepinephrine (3 μg/kg) at different time points after HS. We found that mesenteric lymph duct ligation (MLDL) and mesenteric lymph drainage (MLD) enhanced the pressor response at multiple time points after shock. Next, vascular reactivity and calcium sensitivity in superior mesenteric artery (SMA) vascular rings were examined using an isolated organ perfusion system. Vascular reactivity and calcium sensitivity were higher for SMA rings from rats that had undergone HS plus MLDL or MLD that those from rats that had undergone only HS. The effects of MLDL and MLD on vascular reactivity and calcium sensitivity were significantly increased following incubation with the calcium sensitizer angiotensin II and were reduced after incubation with the calcium sensitivity inhibitor insulin. When SMA rings from normal rats were incubated with mesenteric lymph from rats subjected to HS, lymph obtained 0 to 0.5 h after shock enhanced vascular reactivity and calcium sensitivity, whereas lymph obtained 1 to 3 h after shock blunted these effects. We finally examined vascular reactivity and calcium sensitivity in HS rats subjected to MLD at 0 to 3 h or 1 to 3 h after shock. We found that contractile activity of SMAs in response to norepinephrine or Ca was higher in HS rats subjected to MLD at 1 to 3 h after shock compared with rats subjected to MLD at 0 to 3 h after shock. These results indicate that mesenteric lymph return plays an important role in biphasic changes in vascular reactivity during HS. Even more importantly, mesenteric lymph 1 h after shock was an important contributor to vascular hyporeactivity, and its mechanism of action was related to calcium desensitization. Targeting lymph may therefore have therapeutic potential in the treatment of severe shock-induced hypotension.     

Prevention of hemorrhagic shock-induced intestinal tissue injury by glutamine via heme oxygenase-1 induction

Hemorrhagic shock (HS) is an oxidative stress that causes intestinal tissue injury. Heme oxygenase 1 (HO-1) is induced by oxidative stress and is thought to play an important role in the protection of tissues from oxidative injury. We previously reported the ileum to be the most susceptible to HS-induced tissue injury site in the intestine because HO-1 induction is the lowest at this site. We also previously demonstrated that glutamine (GLN) significantly induced HO-1 in the lower intestinal tract. In the present study, we investigated whether GLN pretreatment improves HS-induced intestinal tissue injury in the ileum by HO-1 induction. Treatment of rats with GLN (0.75 g/kg, i.v.) markedly induced functional HO-1 protein in mucosal epithelial cells in the ileum. Glutamine treatment before HS (MAP of 30 mmHg for 60 min) significantly ameliorated HS-induced mucosal inflammation and apoptotic cell death in the ileum, as judged by significant decreases in gene expression of TNF-alpha, iNOS, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1, myeloperoxidase activity, the number of infiltrated neutrophils, DNA fragmentation by in situ oligo ligation assay, and activated caspase-3 expression, and by increases in gene expression of IL-10 and Bcl-2. In contrast, treatment with tin mesoporphyrin, a specific inhibitor of HO activity, abolished the beneficial effect of GLN pretreatment. These findings indicate that GLN pretreatment significantly ameliorated tissue injury in the ileum after HS by inducing HO-1. Glutamine treatment may thus protect mucosal cells from HS-induced oxidative damage via the anti-inflammatory and antiapoptotic properties of HO-1.