HSPTX protects against hemorrhagic shock resuscitation-induced tissue injury: an attractive alternative to Ringer's lactate

BACKGROUND: Conventional fluid resuscitation with Ringer's lactated (RL) activates neutrophils and causes end-organ damage. We have previously shown that HSPTX, a combination of small volume hypertonic saline (HS) and pentoxifylline (PTX), a phosphodiesterase-inhibitor, downregulates in vitro neutrophil activation and proinflammatory mediator synthesis. Herein, we hypothesized that HSPTX decreases end-organ injury when compared with RL in an animal model of hemorrhagic shock. METHODS: Sprague-Dawley rats were bled to a mean arterial pressure of 35 mm Hg for 1 hour. Animals were divided into 3 groups: sham (no shock, no resuscitation, n = 7), RL (32 mL/kg, n = 7), and HSPTX (7.5% NaCl 4 mL/kg + PTX 25 mg/kg; n = 7). Shed blood was infused after fluid resuscitation. Blood pressure was monitored until the end of resuscitation. Animals were sacrificed at 24 hour after resuscitation. Bronchoalveolar lavage fluid (BALF) was obtained for white cell count (total and differential) and TNF-alpha and IL-1beta levels were measured by ELISA. Lung and intestinal injury at 24 hour were evaluated by histopathology. Organ damage was graded by a pathologist and a score was created (0 = no injury; 3 = severe). Lung neutrophil infiltration was evaluated by MPO immune staining. RESULTS: There were no differences in mean arterial pressure between groups. At 24 hours, BALF leukocyte count was decreased by 30% in HSPTX animals (p < 0.01). TNF-alpha and IL-1beta levels were markedly decreased in HSPTX-resuscitated animals compared with their RL counterparts (p < 0.01). HSPTX-resuscitated animals (lung injury score = 1.0 +/- 0.4) had markedly decreased acute lung injury compared with RL-treated animals (2.5 +/- 0.3) (p < 0.01). RL resuscitation led to a two-fold increase in lung neutrophil infiltration whereas in HSPTX-treated animals, the number of MPO + cells was similar to sham animals (p < 0.001). Intestinal injury was markedly attenuated by HSPTX (1.1 +/- 0.3) compared with RL animals (2.6 +/- 0.4) (p < 0.001). CONCLUSIONS: HSPTX, a small volume resuscitation strategy with marked immunomodulatory potential led to a marked decrease in end-organ damage. HSPTX is an attractive alternative to RL in hemorrhagic shock resuscitation.     

Novel broad-spectrum chemokine inhibitor protects against lung ischemia-reperfusion injury.

BACKGROUND: Functional roles for chemokines have been demonstrated in several models of ischemia-reperfusion injury. The redundancy inherent to chemokine pathways makes administration of neutralizing antibodies to any single chemokine ineffective in ameliorating injury. This study was undertaken to define the pattern of chemokine expression in lung ischemia-reperfusion injury (LIRI), and to determine whether a broad-spectrum chemokine inhibitor, NR58-3.14.3, could confer significant protection against LIRI. METHODS: Left lungs of rats were rendered ischemic for 90 minutes and then reperfused for 4 hours. Chemokine secretion into the alveolar space was quantified by enzyme-linked immunoassay. Treated animals received NR58-3.14.3 prior to reperfusion. Vascular injury was measured by lung permeability index, neutrophil accumulation in lung parenchyma was determined by myeloperoxidase (MPO) activity, and alveolar leukocyte counts were quantified in bronchoalveolar lavage (BAL) effluent. A ribonuclease protection assay evaluated mRNA expression of various chemokines. RESULTS: Lavage effluent in untreated animals demonstrated significant increases in the secretion of cytokine-induced neutrophil chemoattractant (CINC), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), MIP-1alpha and monocyte chemoattractant protein-1 (MCP-1). Animals receiving NR58-3.14.3 demonstrated a 37% (p < 0.001) reduction in vascular injury and a marked reduction in lung MPO activity (p < 0.001) and alveolar cell counts (p = 0.005). Chemokine inhibition decreased mRNA expression of a number of early response cytokines when compared with positive control animals, and caused a significant decrease (p < 0.04) in the secretion of TNF-alpha. CONCLUSIONS: These studies demonstrate that chemokines are expressed after lung ischemia and reperfusion, and that broad-spectrum chemokine inhibition ameliorates reperfusion injury. mRNA expression of early response cytokines was modulated, and the secretion of TNF-alpha was decreased.     

Alpha chemokines regulate direct lung ischemia-reperfusion injury.

BACKGROUND: Alpha chemokines function predominantly to recruit and activate neutrophils, which are important effectors of acute lung injury. This study evaluated whether blockade of 2 potent alpha chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC), is protective against lung ischemia-reperfusion injury in a warm in situ hilar clamp model. METHODS: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received antibodies to MIP-2 or CINC immediately prior to reperfusion. Lung injury was quantitated by vascular permeability to (125)I-radiolabeled bovine serum albumin, lung tissue neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) fluid. CINC and MIP-2 mRNA expression were assessed by northern blot, while ribonuclease protection assays were performed to evaluate mRNA expression for a number of early response cytokines. MIP-2 and CINC protein expression in injured lungs was determined by immunoblotting. RESULTS: Treatment with antibodies to CINC or MIP-2 was associated with significant protection against increases in vascular permeability, MPO content and alveolar leukocyte sequestration in injured lungs. Expression of CINC and MIP-2 mRNA peaked after 2 hours of reperfusion in injured lungs, and protein levels were evident on immunoblotting after 3 hours of reperfusion. Neither CINC nor MIP-2 blockade appeared to modulate cytokine mRNA expression. CONCLUSIONS: CINC and MIP-2 are important mediators involved in direct lung ischemia-reperfusion injury. They appear to function by modulating neutrophil recruitment, but not inflammatory cytokine release.     

Complement C5a receptor antagonist attenuates multiple organ injury in a model of ruptured abdominal aortic aneurysm

OBJECTIVE: Abdominal aortic aneurysm (AAA) rupture is associated with a systemic inflammatory response syndrome, characterized by increased microvascular permeability and neutrophil sequestration, leading to multiorgan dysfunction. We examined the role of a novel complement factor 5a (C5aR) receptor antagonist, the cyclic peptide AcF-(OpdChaWR), in attenuation of pathologic complement activation and tissue injury in a model of AAA rupture. METHODS: Anesthetized rats were randomized to sham (control) or shock and clamp (s+c) groups. Animals in the s+c group underwent 1 hour of hemorrhagic shock (mean arterial blood pressure < or =50 mm Hg), followed by 45 minutes of supramesenteric aortic clamping, then 2 hours of resuscitated reperfusion. Animals in the s+c group were randomized to receive an intravenous bolus of C5aR antagonist at 1 mg/kg or saline solution control at the end of hemorrhagic shock. Intestinal and pulmonary permeability to iodine 125-labeled albumin was measured as an indicator of microvascular permeability. Tissue myeloperoxidase activity, proinflammatory cytokine tissue necrosis factor-alpha (TNF-alpha) protein and mRNA, and C5aR mRNA levels were measured as indicators of neutrophil sequestration and inflammatory signaling, respectively. RESULTS: Lung permeability index was significantly increased in the s+c group compared with the sham group (4.43 +/- 0.96 vs 1.30 +/- 0.17; P <.01), and prevented with treatment with C5aR antagonist (1.74 +/- 0.50; P <.03). Lung myeloperoxidase activity was significantly increased in the the s+c group compared with the sham group (2.41 +/- 0.34 U/mg vs 1.03 +/- 0.29 U/mg; P <.009), and significantly attenuated with treatment with C5aR antagonist (1.11 +/- 0.09 U/mg; P <.006). Lung TNF-alpha protein levels were significantly elevated in both s+c groups, whereas lung TNF-alpha mRNA expression was significantly downregulated in both s+c groups compared with the sham group. Intestinal permeability index was significantly increased in animals in the s+c groups during reperfusion, compared with sham (P <.001), which was attenuated in early reperfusion with treatment with C5a receptor antagonist. Data represent mean +/- SEM, group comparisons with analysis of variance and post hoc Scheffé test. CONCLUSIONS: These results indicate that a potent antagonist of C5a receptor protects the rat intestine and lung from neutrophil-associated injury in a model of AAA rupture. These data suggest that complement-mediated inflammation can be modulated at the C5a receptor level, independent of proinflammatory TNF-alpha production, and prevent acute local and remote organ injury.     

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.     

低血压复苏对失血性休克鼠肠损伤及肺损伤的影响

目的 观察低血压复苏对出血未控制的失血性休克鼠肠及肺损伤的影响.方法 16只雄性出血未控制的失血性休克Sprague-Dawley(SD)大鼠随机分成正常血压复苏组(Ⅰ组)及低血压复苏组(Ⅱ组).复苏3 h后,分别比较肠损伤及肺损伤的程度.结果 Ⅰ组肠黏膜绒毛受损率为(33.13±2.17)%,而Ⅱ组为(12.38±1.69)%(P＜0.05);Ⅰ组肠道菌群移位发生率为87.5%(7/8),而Ⅱ组为25.0%(2/8)(P＜0.05);Ⅰ组及Ⅱ组伊文思蓝(EBD)漏出率分别为(3.03±0.28)%和(1.25±0.17)%(P＜0.05),肺通透指数(LPI)分别为0.140±0.018和0.085±0.004(P＜0.05),肺组织髓过氧化物酶(MPO)的浓度分别为(16.4±2.6)u/g和(33.8±2.1)u/g(P＜0.05).结论 低血压复苏可以减轻出血未控制的失血性休克鼠肠道及肺的损伤.     

Hypertonic resuscitation of hemorrhagic shock upregulates the anti-inflammatory response by alveolar macrophages

BACKGROUND: Resuscitated hemorrhagic shock predisposes patients to the development of acute respiratory distress syndrome (ARDS). Hypertonic saline (HTS) has been shown to inhibit immune cell activation in response to lipopolysaccharide (LPS) in vitro and to reduce lung damage when used for resuscitation of hemorrhagic shock in vivo. We hypothesize that HTS resuscitation of hemorrhagic shock may exert this anti-inflammatory effect by modulating alveolar macrophage function leading to an altered balance between the proinflammatory and the counter-inflammatory response. METHODS: A 2-hit rat model of shock resuscitation was used. Alveolar macrophages were harvested by bronchoalveolar lavage (BAL), and tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 were quantified in the cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Alternatively, 1 hour after resuscitation, animals received endotracheal LPS followed by endotracheal anti-IL-10 neutralizing antibody. Lung injury was determined by measuring BAL neutrophil counts 4 hours after LPS in vivo administration. RESULTS: Systemic administration of HTS significantly modulates the responsiveness of alveolar macrophages. Specifically, HTS resuscitation inhibited LPS-induced TNF-alpha production while enhancing IL-10 release in response to LPS administered ex vivo and in vivo. Anti-IL-10 antibody in vivo partially reversed the lung protective effect of HTS resuscitation. CONCLUSIONS: HTS resuscitation exerts an immunomodulatory effect on alveolar macrophages by shifting the balance of pro- and counter-inflammatory cytokine production in favor of an anti-inflammatory response. The in vivo data suggest a causal role for HTS-induced augmented IL-10 as protective. These findings suggest a novel mechanism for the in vivo salutary effect of HTS resuscitation on lung injury after resuscitated hemorrhagic shock.     

肿瘤坏死因子—α单抗对烫伤大鼠组织脂多糖结合蛋白mRNA表达 …

探讨肿瘤坏死因子－α单抗治疗对烫伤后组织ＴＮＦ－α及脂多糖结合蛋白ｍＲＮＡ表达、不同器官功能改变的影响。方法采用大鼠３５％体表面积Ⅲ度烫伤模型,检测伤后肺,肝,肠肾等组织ＴＮＦ－α及ＬＢＰｍＲＮＡ水平。结果烫伤后肺,肝,肠,肾等组织ＴＮＦ－α及ＬＢＰＭｒｎａ表达前水平均有显著升高,约为正常对照地１．７－２．５倍。     

Role of neutrophils in generalized reperfusion injury associated with resuscitation from shock

Recent studies suggest that neutrophils are an important factor in the organ injury associated with ischemia and shock. Increased neutrophil-endothelial adhesiveness is essential for neutrophil-mediated vascular injury. To examine the role of neutrophils and neutrophil adhesiveness in the development of injury after hypovolemic shock, and to determine whether this injury is a consequence of reperfusion, we used the monoclonal antibody (MAb) 60.3 (directed to the primary human neutrophil adherence glycoprotein, CD18) to block neutrophil adherence functions at the time of resuscitation in a rabbit model of hemorrhagic shock. None of the unanesthetized control animals subjected to 2 hours of shock (cardiac output, 30% of baseline) followed by resuscitation survived 5 days. All had gross and histologic evidence of injury to lungs, liver, and gastrointestinal mucosa. In contrast, 71% of the animals that received MAb 60.3 immediately before resuscitation survived 5 days (p less than 0.005), and visceral organ injury was absent or markedly attenuated. We conclude that a significant proportion of injury resulting from shock and resuscitation occurs after the ischemic insult and that increased neutrophil adhesiveness plays an important role in the development of multiple organ injury and death following shock and resuscitation (in this model). This injury may be significantly reduced by blocking neutrophil adherence functions with the MAb 60.3--even if administration is delayed until resuscitation.     

Diminished lung injury with vascular adhesion molecule-1 blockade in choline-deficient ethionine diet-induced pancreatitis

BACKGROUND: Lung injury in severe acute pancreatitis is mediated by infiltrating leukocytes. Our laboratory has previously demonstrated that acute lung injury in acute pancreatitis results in an up-regulation of vascular adhesion molecule-1 (VCAM-1) cell surface receptor expression on pulmonary vascular endothelium and neutrophil sequestration. The objective of this study was to determine whether blocking expression of VCAM-1 in acute pancreatitis would modify acute pulmonary injury. METHODS: Young female mice were fed a choline-deficient ethionine (CDE) supplemented diet to induce acute pancreatitis. After initiation of the diet, one group (acute pancreatitis treated [n = 18]) was treated with blocking doses (2.35 mg/kg) of monoclonal anti-VCAM-1 receptor antibody (Ab) at 48, 96, and 120 hours. A second group (acute pancreatitis treated control [n = 5]) was treated with a similar dose of an isotypic control for VCAM-1 (nonbinding Ab) at the same time points. A third group (acute pancreatitis untreated [n = 12]) received a CDE diet, and a fourth group (control [n = 11]) received standard food with no Ab treatment. All animals were killed at 144 hours. The dual radiolabeled monoclonal Ab method was used to quantitate VCAM-1 cell surface expression in lung tissue. Lung injury was assessed histologically, and apoptosis was detected by transferase-mediated deoxyuridine triphosphate nick end labeling assay. Pulmonary leukocyte sequestration was determined by myeloperoxidase (MPO) assay and CD18 staining. RESULTS: Pulmonary VCAM-1 cell surface expression was significantly increased in animals with acute pancreatitis when compared to controls (P <.001) and was reduced to near control levels in acute pancreatitis treated animals. On histologic examination, treated animals with acute pancreatitis exhibited significantly less lung injury and apoptosis than did untreated animals with acute pancreatitis. Leukocyte sequestration and MPO activity were significantly reduced in the treated animals with pancreatitis compared to untreated animals with pancreatitis (P <.0001) or acute pancreatitis treated controls (P <.03). CONCLUSIONS: Blocking VCAM-1 on pulmonary vascular endothelium decreases leukocyte adherence and recruitment into the lung, hence reducing lung injury in severe acute pancreatitis. Clinically, VCAM-1 antagonism may be an important adjunct to evolving therapy for distant organ injury in severe acute pancreatitis.     

Hypertonic saline and pentoxifylline attenuates gut injury after hemorrhagic shock: the kinder, gentler resuscitation

BACKGROUND: We have previously demonstrated that postshock resuscitation with Hypertonic saline and Pentoxifylline (HSPTX) attenuates pulmonary and histologic gut injury when compared with Ringer's lactate (RL). In this study, we hypothesized that the decrease in gut injury observed with HSPTX is associated with the attenuation of inducible nitric oxide synthase (iNOS) activity and production of ileal proinflammatory mediators after hemorrhagic shock. METHODS: In a rat model of hemorrhagic shock, resuscitation was conducted with RL (32 mL/kg; n = 7) or HSPTX (4 mL/kg 7.5% NaCl + PTX 25 mg/kg; n = 7). Sham animals that did not undergo shock were also studied. Four hours after resuscitation, the terminal ileum was collected for evaluation of nitrite, tumor necrosis factor (TNF)-alpha, Interleukin (IL)-6, and cytokine-induced neutrophil chemoattractant (CINC) by enzyme immunoassay. Heme oxygenase-1 (HO-1), iNOS, cytoplasmic inhibitor of kappa B (Ikappa B) phosphorylation, and nuclear factor (NF)kappa B p65 nuclear translocation were determined by Western blot. RESULTS: HSPTX resuscitation resulted in a 49% decrease in iNOS when compared with RL (p < 0.05). Similar results were obtained when examining nitrite (882 +/- 59 vs. 1,435 +/- 177 micromol/L; p < 0.01), and HO-1 content (p < 0.05). RL resuscitation resulted in markedly higher levels of TNF-alpha (83 +/- 27 vs. 9 +/- 5 pg/mL; p < 0.01), IL-6 (329 +/- 58 vs. 118 +/- 43 pg/mL; p < 0.05), and CINC (0.43 +/- .06 vs. 0.19 +/- .08 ng/mL; p < 0.05) than HSPTX. The increase in cytokines observed with RL was also associated with an increase in I-kappaB phosphorylation (p < 0.01) and NF-kappaB p65 nuclear translocation (p < 0.001). CONCLUSION: The attenuation in gut injury after postshock resuscitation with HSPTX is associated with downregulation of iNOS activity and subsequent proinflammatory mediator synthesis. HSPTX has the potential to be a superior resuscitation fluid with significant immunomodulatory properties.     

Sex hormones modulate distant organ injury in both a trauma/hemorrhagic shock model and a burn model

BACKGROUND: Emerging data suggest a gender dimorphism in resistance and susceptibility to distant organ injury after mechanical and thermal trauma. The aim of this study was to determine the role that testosterone and estradiol play in modulating resistance or susceptibility to distant organ injury, and whether their effects were associated with differences in the production of nitric oxide. METHODS: Adult male, female, castrated male, and ovariectomized female Sprague-Dawley rats were given intraperitoneal pentobarbital sodium anesthesia and subjected to trauma/sham shock or trauma/hemorrhagic shock (T/HS). A second set of animals were subjected to a 40% total body surface area, third-degree burn or sham burn. At 3 hours after resuscitation, plasma levels of nitrite/nitrate were measured, and the extent of lung injury (permeability to Evans Blue dye and neutrophil sequestration by myeloperoxidase) and intestinal injury (morphology) were determined. RESULTS: Proestrus females showed resistance to lung and gut injury after both T/HS and burns, and had low levels of nitrite/nitrate production. This resistance to injury was abrogated by ovariectomy with an associated increase in nitric oxide production. Males showed increased lung and gut injury after both T/HS and burns associated with increased production of nitrite/nitrate. Castration decreased susceptibility to both lung and gut injury, and decreased production of nitrite/nitrate. A correlation was noted between intestinal and lung injury, and both intestinal and lung injury correlated with plasma nitrite/nitrate levels. CONCLUSIONS: Male sex hormones potentiate, while female hormones reduce T/HS and burn-induced lung and gut injury. Production of nitric oxide is associated with increased lung and gut injury after T/HS and burns.     

一氧化碳释放分子对重症急性胰腺炎肺损伤保护作用的实验研究

目的 探讨一氧化碳释放分子(CORM-2)对大鼠重症急性胰腺炎(SAP)肺损伤的保护作用及机制.方法 30只雄性Wistar大鼠随机分为3组(n=10):sham组、SAP组,CORM-2组.以3.5%牛磺胆酸钠逆行注射胰胆管的方法制作SAP模型.CORM-2组于SAP造模0.5 h后经阴茎背动脉注射CORM-2(8 mg/kg).各组均于造模6 h后取材,测定血清肿瘤坏死因子-α(TNF-α)含量;采用RT-PCR法检测肺组织细胞因子诱导的中性粒细胞趋化因子(CINC)及细胞间黏附分子-1(ICAM-1)mRNA的表达;同时检测肺组织髓过氧化物酶(MPO)活性、湿/干重比及对肺脏进行病理学评分.结果 与SAP组相比,CORM-2组血清TNF-α水平、肺组织病理损伤程度、湿/干重比、MPO活性、CINC及ICAM-1 mRNA的表达均显著降低(P＜0.05).结论 应用CORM-2能够降低血清TNF-α水平、下调肺组织CINC及ICAM-1 mRNA的表达,进而有效地抑制肺组织中性粒细胞的大量浸润,从而对SAP肺损伤起到明显的保护作用.     

Hemorrhagic shock induced up-regulation of P-selectin expression is mediated by factors in mesenteric lymph and blunted by mesenteric lymph duct interruption

BACKGROUND: Previous studies have shown that mesenteric lymph duct interruption prevents lung injury and decreases lung neutrophil sequestration after hemorrhagic shock (HS). Since endothelial cells rapidly express P-selectin after ischemia/reperfusion injury and HS-induced lung injury appears to involve neutrophil-endothelial cell interactions, we tested the following two hypotheses. First, that HS increases endothelial cell P-selectin expression and that interruption of mesenteric lymph flow in vivo would diminish this expression. Second, that incubation of human umbilical vein endothelial cells with post-HS mesenteric lymph but not sham shock (SS) lymph or postshock portal vein plasma would up-regulate P-selectin expression. METHODS: Pulmonary microvascular P-selectin expression was measured in male rats subjected to 90 minutes of HS (30 mm Hg), SS, or HS with lymphatic ligation, with a dual radiolabeled monoclonal antibody technique. The lungs from these animals were subsequently harvested and P-selectin expression was expressed as mean +/- SEM nanograms of monoclonal antibody per gram of tissue. RESULTS: Pulmonary P-selectin expression was 2.0 +/- 0.4 after SS, 9.7 +/- 3.0 after HS, but decreased to 2.3 +/- 0.3 after HS with lymph interruption (p < 0.05 HS vs. SS or HS plus lymph ligation). Incubation of human umbilical vein endothelial cells with shock lymph collected 3 to 4 hours after shock resulted in a nearly fivefold increase in P-selectin expression (p < 0.001) as compared with SS lymph, lymph collected 6 hours after shock, or postshock portal vein plasma. CONCLUSION: These results support the concept that gut-derived lymph promotes HS-induced lung injury through up-regulation of microvascular adhesion molecules and that intestinal lymph duct interruption may prevent distant organ injury by blunting the expression of these molecules.     

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.     

Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock.

OBJECTIVE: To determine whether gut-derived factors leading to organ injury and increased endothelial cell permeability would be present in the mesenteric lymph at higher levels than in the portal blood of rats subjected to hemorrhagic shock. This hypothesis was tested by examining the effect of portal blood plasma and mesenteric lymph on endothelial cell monolayers and the interruption of mesenteric lymph flow on shock-induced lung injury. SUMMARY BACKGROUND DATA: The absence of detectable bacteremia or endotoxemia in the portal blood of trauma victims casts doubt on the role of the gut in the generation of multiple organ failure. Nevertheless, previous experimental work has clearly documented the connection between shock and gut injury as well as the concept of gut-induced sepsis and distant organ failure. One explanation for this apparent paradox would be that gut-derived inflammatory factors are reaching the lung and systemic circulation via the gut lymphatics rather than the portal circulation. METHODS: Human umbilical vein endothelial cell monolayers, grown in two-compartment systems, were exposed to media, sham-shock, or postshock portal blood plasma or lymph, and permeability to rhodamine (10K) was measured. Sprague-Dawley rats were subjected to 90 minutes of sham or actual shock and shock plus lymphatic division (before and after shock). Lung permeability, pulmonary myeloperoxidase levels, alveolar apoptosis, and bronchoalveolar fluid protein content were used to quantitate lung injury. RESULTS: Postshock lymph increased endothelial cell monolayer permeability but not postshock plasma, sham-shock lymph/plasma, or medium. Lymphatic division before hemorrhagic shock prevented shock-induced increases in lung permeability to Evans blue dye and alveolar apoptosis and reduced pulmonary MPO levels. In contrast, division of the mesenteric lymphatics at the end of the shock period but before reperfusion ameliorated but failed to prevent increased lung permeability, alveolar apoptosis, and MPO accumulation. CONCLUSIONS: Gut barrier failure after hemorrhagic shock may be involved in the pathogenesis of shock-induced distant organ injury via gut-derived factors carried in the mesenteric lymph rather than the portal circulation.     

Effects of two fluid resuscitations on the bacterial translocation and inflammatory response of small intestine in rats with hemorrhagic shock

Objective： To investigate the effects of two fluid resuscitations on the bacterial translocation and the inflammatory factors of small intestine in rats with hemorrhagic shock. Methods： Fifty SD healthy male rats were randomly divided into 5 groups （ n = 10 per group） ： Group A （ Sham group）, Group B （ Ringer＇ s solution for 1 h ）, Group C （Ringer＇ s solution for 24 h ）, Group D （ hydroxyethyl starch for 1 h ） and Group E （（ hydroxyethyl starch for 24 h）. A model of rats with hemorrhagic shock was established. The bacterial translocation in liver, content of tumor necrosis factor-α （TNF-α） and changes of myeloperoxidase enzyme （MPO） activities in small intestine were pathologically investigated after these two fluid resuscitations, respectively. Results ： The bacterial translocation and the expression of TNF-α in the small intestine were detected at 1 h and 24 h after fluid resuscitation. There were significant increase in the number of translocated bacteria, TNF-α and MPO activities in Group C compared with Group B, significant decrease in Group E compared with Group D and in Group B compared with Group D. The number of translocated bacteria and TNF-α expression significantly decreased in Group E as compared with Group C. Conclusions： The bacterial translocation and the expression of TNF-α in the small intestine exist 24 h after fluid resuscitation. 6 % hydroxyethyl starch can improve the intestinal mucosa barrier function better than the Ringer＇ s solution.     

Hypertonic saline resuscitation attenuates neutrophil lung sequestration and transmigration by diminishing leukocyte-endothelial interactions in a two-hit model of hemorrhagic shock and infection

BACKGROUND: Hypertonic saline (HTS) attenuates polymorphonuclear neutrophil (PMN)-mediated tissue injury after hemorrhagic shock. We hypothesized that HTS resuscitation reduces early in vivo endothelial cell (EC)-PMN interactions and late lung PMN sequestration in a two-hit model of hemorrhagic shock followed by mimicked infection. METHODS: Thirty-two mice were hemorrhaged (40 mm Hg) for 60 minutes and then given intratracheal lipopolysaccharide (10 microg) 1 hour after resuscitation with shed blood and either HTS (4 mL/kg 7.5% NaCl) or Ringer's lactate (RL) (twice shed blood volume). Eleven controls were not manipulated. Cremaster intravital microscopy quantified 5-hour EC-PMN adherence, myeloperoxidase assay assessed lung PMN content (2 1/2 and 24 hours), and lung histology determined 24-hour PMN transmigration. RESULTS: Compared with RL, HTS animals displayed 55% less 5-hour EC-PMN adherence (p = 0.01), 61% lower 24-hour lung myeloperoxidase ( p= 0.007), and 57% lower mean 24-hour lung histologic score ( p= 0.027). CONCLUSION: Compared with RL, HTS resuscitation attenuates early EC-PMN adhesion and late lung PMN accumulation in hemorrhagic shock followed by inflammation. HTS resuscitation may attenuate PMN-mediated organ damage.