Lipopolysaccharide-binding protein modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhagic shock and resuscitation cause endotoxemia and hepatocellular damage. Because lipopolysaccharide-binding protein (LBP) enhances cellular responses to endotoxin, our aim was to determine whether LBP contributes to hemorrhage/resuscitation-induced injury by comparing LBP knockout and wild-type mice. Under pentobarbital anaesthesia, wild-type and LBP-deficient mice were hemorrhaged to 30 mmHg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer solution. Serum alanine aminotransferase (ALT) necrosis, neutrophil infiltration, and 4-hydroxynonenal by histology/cytochemistry and stress kinase activation by immunoblot analysis were then determined. ALT in wild-type mice was 2,461 +/- 383 and 1,418 +/- 194 IU/l (means +/- SE), respectively, at 2 and 6 h after resuscitation versus sham ALT of 102 +/- 6 IU/l. In LBP-deficient mice, ALT was blunted at both time points to 1,108 +/- 340 and 619 +/- 171 IU/l (P < 0.05). Liver necrosis after 6 h was also attenuated from 3.5 +/- 0.8% in wild-type mice to 1.3 +/- 0.5% in LBP-deficient mice (P < 0.05). After hemorrhage/resuscitation, neutrophil infiltration increased 71% more in wild-type than LBP knockout mice. Similarly, hepatic 4-hydroxynonenal staining, indicative of lipid peroxidation, decreased from 33.8 +/- 4.5% in wild-type mice to 11.6 +/- 1.9% in knockout mice (P < 0.05). After hemorrhage/resuscitation, activation of MAPKs, JNK and ERK, occurred in wild-type mice, which was largely blocked in LBP-deficient mice. However, endotoxin in portal blood after resuscitation was not significantly different between wild-type and knockout mice. In conclusion, hemorrhagic shock and resuscitation to mice cause severe, LBP-mediated hepatocellular damage. An absence of LBP blunts hepatocellular injury with decreased neutrophil infiltration, oxidative stress, and c-Jun and ERK activation.     

Nitrosative and oxidative stresses contribute to post-ischemic liver injury following severe hemorrhagic shock: the role of hypoxemic resuscitation

Purpose

Hemorrhagic shock and resuscitation is frequently associated with liver ischemia-reperfusion injury. The aim of the study was to investigate whether hypoxemic resuscitation attenuates liver injury.

Methods

Anesthetized, mechanically ventilated New Zealand white rabbits were exsanguinated to a mean arterial pressure of 30 mmHg for 60 minutes. Resuscitation under normoxemia (Normox-Res group, n = 16, PaO₂ = 95–105 mmHg) or hypoxemia (Hypox-Res group, n = 15, PaO₂ = 35–40 mmHg) followed, modifying the FiO₂. Animals not subjected to shock constituted the sham group (n = 11, PaO₂ = 95–105 mmHg). Indices of the inflammatory, oxidative and nitrosative response were measured and histopathological and immunohistochemical studies of the liver were performed.

Results

Normox-Res group animals exhibited increased serum alanine aminotransferase, tumor necrosis factor - alpha, interleukin (IL) -1β and IL-6 levels compared with Hypox-Res and sham groups. Reactive oxygen species generation, malondialdehyde formation and myeloperoxidase activity were all elevated in Normox-Res rabbits compared with Hypox-Res and sham groups. Similarly, endothelial NO synthase and inducible NO synthase mRNA expression was up-regulated and nitrotyrosine immunostaining increased in animals resuscitated normoxemically, indicating a more intense nitrosative stress. Hypox-Res animals demonstrated a less prominent histopathologic injury which was similar to sham animals.

Conclusions

Hypoxemic resuscitation prevents liver reperfusion injury through attenuation of the inflammatory response and oxidative and nitrosative stresses.     

Systemic and microvascular responses to hemorrhagic shock and resuscitation with Hb vesicles

A phospholipid vesicle encapsulating hemoglobin (Hb vesicle, HbV) has been developed to provide O(2)-carrying capacity to plasma expanders. Its ability to restore systemic and microcirculatory conditions after hemorrhagic shock was evaluated in the dorsal skinfold window preparation of conscious hamsters. The HbV was suspended in 8% human serum albumin (HSA) at Hb concentrations of 3.8 g/dl [HbV(3.8)/HSA] and 7.6 g/dl [HbV(7.6)/HSA]. Shock was induced by 50% blood withdrawal, and mean arterial pressure (MAP) at 40 mmHg was maintained for 1 h by the additional blood withdrawal. The hamsters receiving either HbV(3.8)/HSA or HbV(7.6)/HSA suspensions restored MAP to 93 +/- 14 and 93 +/- 10 mmHg, respectively, similar with those receiving the shed blood (98 +/- 13 mmHg), which were significantly higher by comparison with resuscitation with HSA alone (62 +/- 12 mmHg). Only the HSA group tended to maintain hyperventilation and negative base excess after the resuscitation. Subcutaneous microvascular blood flow reduced to approximately 10-20% of baseline during shock, and reinfusion of shed blood restored blood flow to approximately 60-80% of baseline, an effect primarily due to the sustained constriction of small arteries A(0) (diameter 143 +/- 29 microm). The HbV(3.8)/HSA group had significantly better microvascular blood flow recovery and nonsignificantly better tissue oxygenation than of the HSA group. The recovery of base excess and improved tissue oxygenation appears to be primarily due to the increased oxygen-carrying capacity of HbV fluid resuscitation.     

Resistance training alters the response of fed state mixed muscle protein synthesis in young men

Ten healthy young men (21.0 +/- 1.5 yr, 1.79 +/- 0.1 m, 82.7 +/- 14.7 kg, means +/- SD) participated in 8 wk of intense unilateral resistance training (knee extension exercise) such that one leg was trained (T) and the other acted as an untrained (UT) control. After the 8 wk of unilateral training, infusions of L-[ring-d(5)]phenylalanine, L-[ring-(13)C(6)]phenylalanine, and d(3)-alpha-ketoisocaproic acid were used to measure mixed muscle protein synthesis in the T and UT legs by the direct incorporation method [fractional synthetic rate (FSR)]. Protein synthesis was determined at rest as well as 4 h and 28 h after an acute bout of resistance exercise performed at the same intensity relative to the gain in single repetition maximum before and after training. Training increased mean muscle fiber cross-sectional area only in the T leg (type I: 16 +/- 10%; type II: 20 +/- 19%, P < 0.05). Acute resistance exercise increased muscle protein FSR in both legs at 4 h (T: 162 +/- 76%; UT: 108 +/- 62%, P < 0.01 vs. rest) with the increase in the T leg being significantly higher than in the UT leg at this time (P < 0.01). At 28 h postexercise, FSR in the T leg had returned to resting levels; however, the rate of protein synthesis in the UT leg remained elevated above resting (70 +/- 49%, P < 0.01). We conclude that resistance training attenuates the protein synthetic response to acute resistance exercise, despite higher initial increases in FSR, by shortening the duration for which protein synthesis is elevated.     

Hypoxemic resuscitation from hemorrhagic shock prevents lung injury and attenuates oxidative response and IL-8 overexpression

We investigated whether hypoxemic resuscitation from hemorrhagic shock prevents lung injury and explored the mechanisms involved. We subjected rabbits to hemorrhagic shock for 60 min by exsanguination to a mean arterial pressure of 40 mm Hg. By modifying the fraction of the inspired oxygen, we performed resuscitation under normoxemia (group NormoxRes, P(a)O(2)=95-105 mm Hg) or hypoxemia (group HypoxRes, P(a)O(2)=35-40 mm Hg). Animals not subjected to shock constituted the sham group (P(a)O(2)=95-105 mm Hg). We performed bronchoalveolar lavage (BAL) fluid, lung wet-to-dry weight ratio, and morphological studies. U937 monocyte-like cells were incubated with BAL fluid from each group. Cell peroxides, malondialdehyde, proteins, and cytokines in the BAL fluid were lower in sham than in shocked animals and in HypoxRes than in NormoxRes animals. The inverse was true for ascorbic acid and reduced glutathione. Lung edema, lung neutrophil infiltration, myeloperoxidase, and interleukin (IL)-8 gene expression were reduced in lungs of HypoxRes compared with NormoxRes animals. A colocalized higher expression of IL-8 and nitrotyrosine was found in lungs of NormoxRes animals compared to HypoxRes animals. The BAL fluid of NormoxRes animals compared with HypoxRes animals exerted a greater stimulation of U937 monocyte-like cells for proinflammatory cytokine release, particularly for IL-8. In the presence of p38-MAPK and Syk inhibitors and monosodium urate crystals, IL-8 release was reduced. We conclude that hypoxemic resuscitation from hemorrhagic shock ameliorates lung injury and reduces oxygen radical generation and lung IL-8 expression.     

Modulation of syndecan-1 shedding after hemorrhagic shock and resuscitation

The early use of fresh frozen plasma as a resuscitative agent after hemorrhagic shock has been associated with improved survival, but the mechanism of protection is unknown. Hemorrhagic shock causes endothelial cell dysfunction and we hypothesized that fresh frozen plasma would restore endothelial integrity and reduce syndecan-1 shedding after hemorrhagic shock. A prospective, observational study in severely injured patients in hemorrhagic shock demonstrated significantly elevated levels of syndecan-1 (554±93 ng/ml) after injury, which decreased with resuscitation (187±36 ng/ml) but was elevated compared to normal donors (27±1 ng/ml). Three pro-inflammatory cytokines, interferon-γ, fractalkine, and interleukin-1β, negatively correlated while one anti-inflammatory cytokine, IL-10, positively correlated with shed syndecan-1. These cytokines all play an important role in maintaining endothelial integrity. An in vitro model of endothelial injury then specifically examined endothelial permeability after treatment with fresh frozen plasma orlactated Ringers. Shock or endothelial injury disrupted junctional integrity and increased permeability, which was improved with fresh frozen plasma, but not lactated Ringers. Changes in endothelial cell permeability correlated with syndecan-1 shedding. These data suggest that plasma based resuscitation preserved endothelial syndecan-1 and maintained endothelial integrity, and may help to explain the protective effects of fresh frozen plasma after hemorrhagic shock.     

Urinary metabolic network analysis in trauma, hemorrhagic shock, and resuscitation

Hemorrhagic shock, often a result of traumatic injury, is a condition of reduced perfusion that results in diminished delivery of oxygen to tissues. The disruption in oxygen delivery induced by both ischemia (diminished oxygen delivery) and reperfusion (restoration of oxygen delivery) has profound consequences for cellular metabolism and the maintenance of homeostasis. The pathophysiologic state associated with traumatic injury and hemorrhagic shock was studied with a scale-invariant metabolic network. Urinary metabolic profiles were constructed from NMR spectra of urine samples collected at set timepoints in a porcine model of hemorrhagic shock that included a pulmonary contusion, a liver crush injury, and a 35 % controlled bleed. The network was constructed from these metabolic profiles. A partial least squares discriminant analysis (PLS-DA) model that discriminates by experimental timepoint was also constructed. Comparisons of the network (functional relationships among metabolites) and PLS-DA model (observable relationships to experimental time course) revealed complementary information. First, ischemia/reperfusion injury and evidence of cell death due to hemorrhage was associated with early resuscitation timepoints. Second, evidence of increased protein catabolism and traumatic injury was associated with late resuscitation timepoints. These results are concordant with generally accepted views of the metabolic progression of shock.     

Mast cell stabilization improves cardiac contractile function following hemorrhagic shock and resuscitation

Hemorrhagic shock (HS) is associated with cardiac contractile dysfunction. Mast cell (MC) degranulation is hypothesized to mediate the cardiodepressant effect. Cardiac function was assessed after HS and resuscitation (HS/R) with the administration of the MC stabilizers to prevent MC degranulation. Anesthetized male Sprague-Dawley rats were randomized to sham-operated control or HS/R groups and underwent 60 min of HS followed by 2 h of resuscitated reperfusion. Animals in the HS/R groups were randomized to receive cromolyn (5 mg/kg), ketotifen (1 mg/kg), or saline 15 min before shock. Hearts were excised following HS or 2 h of reperfusion, and function was assessed on a Langendorff apparatus. A second group of randomized animals had serial blood samples taken to assess MC degranulation by quantifying levels of serum beta-hexosaminidase. Hearts were excised at 0 min (before HS) and following 60 min of HS (before resuscitation) for a histological evaluation of MC density and degranulation. In vivo MC stabilization using ketotifen and cromolyn improved cardiac peak systolic pressure (P < 0.05), contractility (P < 0.05), and relaxation (P < 0.05) compared with that of HS controls. Serum beta-hexosaminidase increased during HS/R and was inhibited by MC stabilization (P < 0.05). Degranulation was inhibited when assessed by histochemistry and immune fluorescence. The inhibition of MC degranulation can significantly improve cardiac function following HS/R.     

不同液体复苏对失血性休克大鼠肠粘膜的影响

目的：观察不同液体复苏对失血性休克大鼠肠粘膜的影响以及肠粘膜的变化。方法：利用大鼠失血性休克模型以及不同的补液方式,在复苏后120分钟时处死大鼠,取回肠4cm,做病理切片并根据Chiu等方法评估回肠黏膜上皮损伤指数。结果：液体复苏组的肠粘膜损伤程度小于休克不补液组（p〈0.05）,而限制型液体复苏组的肠粘膜损伤程度小于充分液体复苏组（p〈0.05）。结论：通过本实验对肠粘膜的观察可以得出,对于失血性休克,液体复苏时有效的抗休克方式,而对于复苏的方式来说,从肠黏膜的保护方面来说,限制型液体复苏是优于传统的充分液体复苏的。     

不同液体复苏对失血性休克大鼠肠粘膜的影响

目的:观察不同液体复苏对失血性休克大鼠肠粘膜的影响以及肠粘膜的变化。方法:利用大鼠失血性休克模型以及不同的补液方式,在复苏后120分钟时处死大鼠,取回肠4cm,做病理切片并根据Chiu等方法评估回肠黏膜上皮损伤指数。结果:液体复苏组的肠粘膜损伤程度小于休克不补液组(p<0.05),而限制型液体复苏组的肠粘膜损伤程度小于充分液体复苏组(p<0.05)。结论:通过本实验对肠粘膜的观察可以得出,对于失血性休克,液体复苏时有效的抗休克方式,而对于复苏的方式来说,从肠黏膜的保护方面来说,限制型液体复苏是优于传统的充分液体复苏的。     

严重多发创伤失血性休克患者限制性液体复苏的临床应用评价

目的:评价限制性液体复苏在严重多发失血性休克救治中的作用.方法:对88例多发创伤合并失血性休克的患者随机分为两组,观察组(n=48)在创伤失血性休克出血未控制前行限制性液体复苏,对照组(n=40)则进行常规行充分液体复苏.比较两组的液体摄入量、输血量、收缩压、术前复苏时间;住院期间急性呼吸窘迫综合征(ARDS)、急性肾功能衰竭(ARF)、弥漫性血管内凝血(DIC)、脓毒血症的发生率和总死亡率.结果:观察组的液体输入量和术前复苏时间均低于对照组,差异有显著性(P＜0.05);两组输血量和收缩压差异无显著性.观察组ARDS、脓毒血症的发生率和总死亡率均明显低于对照组,差异有显著性(P＜0.05).结论:在创伤性休克术前未控制性出血条件下,限制性液体复苏可缩短术前复苏时间,明显降低并发症的发生率和死亡率.     

Models of hemorrhagic shock: Differences in the physiological and inflammatory response

IntroductionThe hemorrhagic shock (HS) model is commonly used to initiate a systemic post-traumatic inflammatory response. Numerous experimental protocols exist and it is unclear how differences in these models affect the immune response making it difficult to compare results between studies. The aim of this study was to compare the inflammatory response of different established protocols for volume-controlled shock in a murine model.MethodsMale C57/BL6 mice 6–10 weeks and weighing 20–25 g were subjected to volume-controlled or pressure-controlled hemorrhagic shock. In the volume-controlled group 300 μl, 500 μl, or 700 μl blood was collected over 15 min and mean arterial pressure was continuously monitored during the period of shock. In the pressure-controlled hemorrhagic shock group, blood volume was depleted with a goal mean arterial pressure of 35 mmHg for 90 min. Following hemorrhage, mice from all groups were resuscitated with the extracted blood and an equal volume of lactated ringer solution. Six hours from the initiation of hemorrhagic shock, serum IL-6, KC, MCP-1 and MPO activity within the lung and liver tissue were assessed.ResultsIn the volume-controlled group, the mice were able to compensate the initial blood loss within 30 min. Approximately 800 μl of blood volume was removed to achieve a MAP of 35 mmHg (p < 0.001). No difference in the pro-inflammatory cytokine (IL-6 and KC) profile was measured between the volume-controlled groups (300 μl, 500 μl, or 700 μl). The pressure-controlled group demonstrated significantly higher cytokine levels (IL-6 and KC) than all volume-controlled groups. Pulmonary MPO activity increased with the severity of the HS (p < 0.05). This relationship could not be observed in the liver.ConclusionVolume-controlled hemorrhagic shock performed following current literature recommendations may be insufficient to produce a profound post-traumatic inflammatory response. A decrease in the MAP following blood withdrawal (300 μl, 500 μl or 700 μl) was usually compensated within 30 min. Pressure-controlled hemorrhagic shock is a more reliable for induction of a systemic inflammatory response.     

牛磺酸对失血性休克复苏家兔血浆和心肌NOS活性影响

目的:探讨牛磺酸对失血性休克复苏后血浆和心肌一氧化氮合酶(NOS)活性、一氧化氮(NO)含量变化的影响.方法:新西兰种兔24只随机分为3组(n=8):对照组、休克组、牛磺酸治疗组.采用失血性休克复苏动物模型.连续观察休克前、休克1.5 h、复苏后1 h、2 h、3 h血浆一氧化氮合酶(NOS)活性、一氧化氮代谢产物(NO-2/NO-3)含量、乳酸脱氢酶(LDH)活性的动态变化.测定复苏后3 h心肌一氧化氮合酶(NOS)活性、一氧化氮代谢产物(NO-2/NO-3)含量的变化,并常规留取心肌标本观察形态学改变.结果:①休克组复苏后各时限血浆NOS活性、NO-2/NO-3含量、LDH活性显著高于休克前及休克1.5 h;②休克组复苏后3 h心肌NOS活性、NO-2/NO-3含量显著高于对照组,心肌出现明显水肿和脂肪变性;③牛磺酸(40 mg·kg-1 iv)可显著缓解上述变化.结论:失血性休克复苏后NOS的激活和NO的大量释放,可能介导了休克复苏所致心肌损伤,牛磺酸可减少NO的生成使心肌损伤减轻.     

Granulocyte colony-stimulating factor alters the systemic metabolomic profile in healthy donors

Introduction

Peripheral blood stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) from healthy donors are commonly used for allogeneic stem cell transplantation. The effect of G-CSF administration on global serum metabolite profiles has not been investigated before.

Objectives

This study aims to examine the systemic metabolomic profiles prior to and following administration of G-CSF in healthy adults.

Methods

Blood samples were collected from 15 healthy stem cell donors prior to and after administration of G-CSF 10 µg/kg/day for 4 days. Using a non-targeted metabolomics approach, metabolite levels in serum were determined using ultrahigh performance liquid chromatography-tandem mass spectrometry and gas chromatography/mass spectrometry.

Results

Comparison of the metabolite profiles of donors before and after G-CSF treatment revealed 239 metabolites that were significantly altered. The major changes of the metabolite profiles following G-CSF administration included alteration of several fatty acids, including increased levels of several medium and long-chain fatty acids, as well as polyunsaturated fatty acids; while there were lower levels of other lipid metabolites such as phospholipids, lysolipids, sphingolipids. Furthermore, there were significantly lower levels of several amino acids and/or their metabolites, including several amino acids with known immunoregulatory functions (methionine, tryptophan, valine). Lastly, the levels of several nucleotides and nucleotide metabolites (guanosine, adenosine, inosine) were also decreased after G-CSF administration, while methylated products were increased. Some of these altered products/metabolites may potentially have angioregulatory effects whereas others may suggest altered intracellular epigenetic regulation.

Conclusion

Our results show that G-CSF treatment alters biochemical serum profiles, in particular amino acid, lipid and nucleotide metabolism. Additional studies are needed to further evaluate the relevance of these changes in healthy donors.

     

两种液体复苏方案对创伤后非控制性失血性休克家兔血流动力学的影响

目的:建立非控制性失血性休克的家兔模型,研究两种不同复苏方案对家兔血流动力学的影响.方法:24只家兔复制成非控制性失血性休克模型,随即分成四组,假手术组(SHAM组)、休克未治疗组(SWT组)、积极性复苏组(PT组)、限制性复苏组(LT组).在实验设定的时间段内,采用两种复苏方案输入的液体有:7.5%高渗氯化钠溶液(7.5%Hypertonie saline,HTS).乳酸林格氏液(Lactated Ringers solution,LRS),羟乙基淀粉溶液(Hydroxyethyl Starch 130/0.4,HES).在实验的Omin、30min、60min、90min,监测家兔的左心室压力上升或下降的最大速率(themaximal change rateofleftintraventricularpressure.±dp/dtrnax)以及左心室收缩压(left intraventricular systolic pressure,LVSP),并在90min实验结束时,处死家免,统计出血量,补液量.结果:在监测指标±dp/dtmax和LVSP上,PT组和LT组分别与SWT组比较时,60min、90min的指标均有统计学意义(P＜0.05);60min时,PT组与LT组相比较,各指标数值虽略大,但差异无统计学意义(P＞0.05);90min时,PT组与LT组相比较各项指标均有统计学意义(P＜0.05).补体量上,30-60min、60-90min两个阶段,PT组与LT组比较所用LRS、HES的量均有统计学意义(P＜0.05);失血量上,PT组分别与LT组、SWT组比较后,均有统计学意义(P＜0.05),LT组与SWT组比较无统计学意义(P＞0.05).结论:在非控制性失血性休克的早期救治中,采用限制性液体复苏方案较积极性液体复苏,补液量少,并且对家兔心功能的维持作用效果比较明显;尤其在控制活动性失血上,与积极性复苏相比,限制性复苏优势明显.     

IL-6 is essential for development of gut barrier dysfunction after hemorrhagic shock and resuscitation in mice

We sought to determine the role of IL-6 as a mediator of the alterations in gut barrier function that occur after hemorrhagic shock and resuscitation (HS/R). C57Bl/6 wild-type (WT) and IL-6 knockout (KO) mice on a C57Bl/6 background were subjected to either a sham procedure or HS/R. Organ and tissue samples were obtained 4 h after resuscitation. In WT mice, HS/R significantly increased ileal mucosal permeability to fluorescein isothiocyanate-labeled dextran (average molecular mass, 4 kDa) and bacterial translocation to mesenteric lymph nodes. These alterations in gut barrier function were not observed in IL-6 KO animals. HS/R increased ileal steady-state mRNA levels for IL-6, TNF, and IL-10 in WT but not in IL-6 KO mice. Ileal mucosal expression of the tight junction protein, ZO-1, decreased after HS/R in WT but not IL-6 KO mice. Collectively, these data support the view that expression of IL-6 is essential for the development of gut barrier dysfunction after HS/R.