A Four-Compartment Metabolomics Analysis of the Liver,Muscle, Serum,and Urine Response to Polytrauma with Hemorrhagic Shock following Carbohydrate Prefeed

Objective

Hemorrhagic shock accompanied by injury represents a major physiologic stress. Fasted animals are often used to study hemorrhagic shock (with injury). A fasted state is not guaranteed in the general human population. The objective of this study was to determine if fed animals would exhibit a different metabolic profile in response to hemorrhagic shock with trauma when compared to fasted animals.

Methods

Proton (1H) NMR spectroscopy was used to determine concentrations of metabolites from four different compartments (liver, muscle, serum, urine) taken at defined time points throughout shock/injury and resuscitation. PLS-DA was performed and VIP lists established for baseline, shock and resuscitation (10 metabolites for each compartment at each time interval) on metabolomics data from surviving animals.

Results

Fed status prior to the occurrence of hemorrhagic shock with injury alters the metabolic course of this trauma and potentially affects mortality. The death rate for CPF animals is higher than FS animals (47 vs 28%). The majority of deaths occur post-resuscitation suggesting reperfusion injury. The metabolomics response to shock reflects priorities evident at baseline. FS animals raise the baseline degree of proteolysis to provide additional amino acids for energy production while CPF animals rely on both glucose and, to a lesser extent, amino acids. During early resuscitation levels of metabolites associated with energy production drop, suggesting diminished demand.

Conclusions

Feeding status prior to the occurrence of hemorrhagic shock with injury alters the metabolic course of this trauma and potentially affects mortality. The response to shock reflects metabolic priorities at baseline.     

Carbohydrate fed state alters the metabolomic response to hemorrhagic shock and resuscitation in liver

Hemorrhagic shock with injury results in alterations of the metabolic state of an organism, which contribute to organ dysfunction and death. Previous investigations have explored the effects of carbohydrate prefeed in murine models but few in clinically relevant large animal models. We performed carbohydrate prefeed in pigs undergoing simulated polytrauma and hemorrhagic shock with resuscitation to determine if carbohydrate prefeeding if the metabolic response to shock is dependent on fed state. Sixty-four Yorkshire pigs were divided into two experimental groups: fasted (32) and prefed (32). Experimental animals were subjected to a standardized hemorrhagic shock protocol, including pulmonary contusion and liver crush injury. To determine molecular alterations in response to trauma as a result of prefeeding, liver biopsies were obtained at set timepoints throughout the procedure. Fifty-one metabolites were profiled for each sample via proton nuclear magnetic resonance spectroscopy (¹H NMR). Partial-Least Squared Discriminant Analysis (PLS-DA) was used to examine clustering of the data with respect to fed state. Cross-validated models separated the fed from fasted animals. Metabolites contributing to the separation have known relationships to alternate carbon energy sources, amino acid metabolism, oxidative stress response, and membrane maintenance. In conclusion, metabolomic techniques allowed identification of an alternate response to shock and resuscitation, dependent upon fed state, through the use of metabolomics.     

失血性休克复苏后肠内给予特殊营养物对肠粘膜血流量的影响

为探讨失血性休克复苏后肠内营养与肠粘膜血流改变的关系 ,从SD大鼠开腹制作空肠袋 ,将激光多谱勒探头和肠粘膜张力计放置在空肠袋两端 ,根据动物分组分别向袋内注射葡萄糖、谷氨酰胺、丙氨酸及甘露醇。复制失血性休克模型 (30mmHg ,维持 6 0min) ,然后用林格氏液复苏 ,恢复灌流 6 0min。分别测定肠粘膜血流量和局部PCO2 张力 (PrCO2 )。结果显示 ,失血性休克和复苏过程中 ,谷氨酰胺和葡萄糖组粘膜血流量比甘露醇和丙氨酸组显著增加 ,PrCO2 显著降低 ;而肠内给予丙氨酸进一步降低肠粘膜血流量 ,升高PrCO2 。提示 :失血性休克复苏后 ,肠内给予丙氨酸减少肠粘膜血流量 ;而给予谷氨酰胺和葡萄糖能增加肠粘膜血流量 ,对缺血再灌流损伤的肠道提供保护作用     

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-kappaB (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-kappaB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-kappaB to DNA and the degradation of IkappaBalpha, IkappaBbeta, and IkappaBvarepsilon. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-kappaB and thus prevention of the expression of NFkappaB-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.     

Utilization of Extracorporeal Membrane Oxygenation Alleviates Intestinal Ischemia–Reperfusion Injury in Prolonged Hemorrhagic Shock Animal Model

Intestinal ischemia–reperfusion injury is one of the main factors leading to multiple organ failure after resuscitation of prolonged hemorrhagic shock; however, the current conventional fluid resuscitation still cannot effectively reduce intestinal injury caused by prolonged hemorrhagic shock. To investigate the effect of ECMO resuscitation on alleviating intestinal ischemia–reperfusion injury in a prolonged hemorrhagic shock rabbit model. Thirty New Zealand white rabbits were randomly divided into three groups: control group, conventional fluid resuscitation group, and ECMO resuscitation group. The prolonged hemorrhagic shock model was established by keeping the arterial blood pressure from 31 to 40 mmHg for 3 h through the femoral artery bleeding, and performing the resuscitation for 2 h by conventional fluid resuscitation and ECMO resuscitation, respectively. Chiu’s score of intestinal injury, serum lactate and TNF-α levels, intestinal mucosamyeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM-1), and Claudin-1expression were detected. The mean arterial blood pressure in Group 2 was significantly higher after resuscitation than in Group 1, but serum lactate and inflammatory cytokines TNF-α level were significantly lower. And Chiu’s score of intestinal injury and myeloperoxidase (MPO) activity level and ICAM-1 expression were significantly lower in the ECMO resuscitation group, in which the Claudin-1 levels were significantly increased. ECMO resuscitation for the prolonged hemorrhagic shock improves tissue perfusion and reduces the systemic inflammation, and thus alleviates intestinal damage caused by prolonged hemorrhagic shock.     

Redistribution of blood flow after thermal injury and hemorrhagic shock

Diminished mucosal mass and a diminished rate of DNA synthesis by the intestinal mucosa have been identified in the rat after thermal injury. Because these changes may be associated with ischemia, the distribution of intestinal blood flow was studied after a thermal injury and compared with the blood flow distribution after hemorrhagic shock. For the thermal injury, anesthetized animals received a standardized 20% body surface area, full-thickness injury and were given intraperitoneal saline resuscitation. By the use of 46Sc- or 141Ce-labeled microspheres, no changes in intestinal and hepatic blood flow occurred after thermal injury. In contrast, a marked redistribution of blood flow was identified after hemorrhagic shock in which a decrease in arterial blood flow was identified to the stomach and to the small and large intestine. Although clinical shock was not present, the cardiac output decreased to a comparable degree in the hemorrhagic shock and the thermal injury. These studies indicate that although physiological changes in intestinal mucosa can be demonstrated after burn injury, these changes are not due to decreases in mesenteric arterial blood flow.     

Intestinal preconditioning prevents systemic inflammatory response in hemorrhagic shock. Role of HO-1

Intestinal ischemia-reperfusion has been implicated in the systemic inflammatory response and organ injury in hemorrhagic shock, but the exact role of the intestine has never been directly demonstrated. Preconditioning (PC) with brief periods of intermittent ischemia is a known potent anti-ischemic intervention and thus can be used as a tool to assess the role of local intestinal ischemia-reperfusion injury in systemic inflammatory response. Thus rats were first subjected to sham surgery or intestinal preconditioning with four cycles of 1-min ischemia and 10 min of reperfusion 24 h before hemorrhagic shock followed by resuscitation. PC reduced fluid requirements, lung edema, and lactate and tumor necrosis factor-alpha production. These effects were abolished by the heme-oxygenase-1 (HO-1) inhibitor tin protoporphyrin (Sn-PP). PC induced more than fivefold in intestinal HO-1 expression. These results suggest that intestinal ischemia-reperfusion is a major trigger for inflammatory response and organ injury in nonseptic shock. HO-1 appears to play an important role in the protective effect of intestinal preconditioning.     

Kupffer cell activation after no-flow ischemia versus hemorrhagic shock

Kupffer cell-derived oxidant stress is critical for reperfusion injury after no-flow ischemia. However, the importance of Kupffer cells as source of reactive oxygen formation is unclear in a hemorrhagic shock model. Therefore, we evaluated Kupffer cell activation after 60 or 120 min of hemorrhage and 90 min of resuscitation (HS/RS) in pentobarbital-anesthetized male Fischer rats. Plasma glutathione disulfide (GSSG) as indicator for a vascular oxidant stress showed no significant changes after HS/RS. Plasma ALT activities were only moderately increased (100-200 U/L). Kupffer cells isolated from postischemic livers did not generate more superoxide than cells from sham controls. In contrast, the 10-fold increase of plasma GSSG and the 9-fold higher spontaneous superoxide formation of Kupffer cells after 60 min of hepatic no-flow ischemia followed by 90 min of reperfusion demonstrated the activation of Kupffer cells in this experimental model. Plasma ALT activities (1930 +/- 240 U/L) indicated severe liver injury. These results demonstrate a fundamental difference in the degree of Kupffer cell activation between the two models of warm hepatic ischemia. Our findings suggest that different therapeutic strategies are necessary to ameliorate the initial injury after low flow ischemia (hemorrhage) compared to cold (transplantation) or warm (Pringle maneuver) no-flow ischemia.     

丙酮酸钠对失血性休克大鼠缺血/再灌注损伤的保护作用

目的:研究丙酮酸钠对失血性休克后缺血/再灌注损伤的保护作用。方法:制作失血性休克大鼠模型,回输全血,同时分别给予生理盐水、谷胱甘肽和丙酮酸钠适量,于再灌注后3h处死动物。检测血浆乳酸脱氢酶(LDH)和谷草转氨酶(GOT)的活性、组织丙二醛(MDA)的含量和髓过氧化物酶(MPO)的活性,观察心、肝、肺和肾组织的病理变化。结果:丙酮酸钠组与生理盐水组相比,血浆LDH和GOT的活性降低,肝、肺和肾组织MDA的含量下降,心、肺和肾组织MPO活性降低,效果优于谷胱甘肽。心、肝、肺和肾组织形态学观察显示,丙酮酸钠使组织损伤减轻。结论:丙酮酸钠对失血性休克后再灌注损伤具有保护作用。其作用机制可能与清除氧自由基、减少中性粒细胞的浸润、减轻炎性反应有关。     

Emerging roles of microRNAs in intestinal ischemia/reperfusion–induced injury: a review

Journal of Physiology and Biochemistry - Intestinal ischemia/reperfusion (II/R) injury is a serious pathological phenomenon in underlying hemorrhagic shock, trauma, strangulated intestinal...     

Estrogen alleviates hepatocyte necroptosis depending on GPER in hepatic ischemia reperfusion injury

Journal of Physiology and Biochemistry - Hepatic ischemia reperfusion injury (IRI) occurs in liver transplantation, complex liver resection, and hemorrhagic shock, which causes donor organ shortage...     

High density lipoproteins reduce organ injury and organ dysfunction in a rat model of hemorrhagic shock.

High density lipoproteins (HDLs) inhibit the cytokine-induced expression of endothelial cell adhesion molecules both in vitro and in vivo. We examined the ability of HDLs to mediate a functional anti-inflammatory effect by measuring their ability to prevent neutrophil adhesion and transmigration in vitro. Treatment of human endothelial cell cultures with physiologic concentrations of HDLs inhibited neutrophil binding by 68 +/- 5.9% (mean and se, n=6, P<0.05) and neutrophil transmigration by 48.7 +/- 6.7% (n=8, P<0.05). We then examined the effect of HDLs on inflammatory infiltration and subsequent multiple organ dysfunction syndrome (MODS), associated with trauma in a rat model of hemorrhagic shock. Rats given human HDLs (80 mg apo A-I/kg, i.v.) 90 min after hemorrhage (which reduced mean arterial pressure to 50 mmHg) and 1 min before resuscitation showed attenuation of the increases in the serum levels of markers of MODS normally observed in this model. Severe disruption of the architecture of tissues and the extensive cellular infiltration into those tissues were also largely inhibited in animals that received HDLs. Human HDLs attenuate the MODS associated with ischemia and reperfusion injury after hemorrhagic shock in rats.     

限制性液体复苏对多发性骨折合并创伤失血性休克患者凝血功能、心肌损害指标及预后的影响

目的:探讨限制性液体复苏对多发性骨折合并创伤失血性休克患者凝血功能、心肌损害指标及预后的影响。方法:选取我院收治的多发性骨折合并创伤失血性休克患者77例,分为研究组(n=39)、对照组(n=38),对照组给予常规液体复苏,研究组给予限制性液体复苏,比较两组患者凝血功能、心肌损害指标、输液量、失血量、输血量、并发症发生率及病死率。结果:研究组的输液量、失血量、输血量均少于对照组(P0.05)。与复苏前相比,两组患者复苏1 h后凝血酶原时间(PT)、凝血活酶时间(APTT)、凝血酶时间(TT)均延长,且研究组长于对照组(P0.05);两组患者复苏1 h后肌酸激酶(CK)、肌酸激酶-同工酶(CK-MB)、肌钙蛋白T(CTnT)水平均下降,且研究组低于对照组(P0.05)。研究组复苏期间并发症发生率、病死率均低于对照组(P0.05)。结论:限制性液体复苏治疗多发性骨折合并创伤失血性休克患者,可改善患者凝血功能和预后,降低并发症发生率,同时还可减轻心肌损害。     

Resuscitative Effect of Hyperoxia Fluid on High-Altitude Hemorrhagic Shock in Rats and Antishock Mechanisms

Pathophysiological characteristics of hemorrhagic shock at high altitude are different from that at plain which involve severe injury, high mortality, difficult treatment and compromised liquid tolerance. High-altitude pulmonary/cerebral edema and multiple-organ dysfunction render the conventional treatment ineffective. Herein, we evaluated the resuscitation effects of hyperoxia solution on high-altitude hemorrhagic shock in rats. For this purpose, a rat model of high-altitude (3,658 m) hemorrhagic shock was established on the plateau and hyperoxia solution (4 ml/kg) was infused through external jugular vein for resuscitation at 60 min post-hemorrhage. Blood pressure, blood gas, left and right ventricular pressure, lung and brain water content, survival time, survival rate at 2 h, levels of inflammatory cytokines and free oxygen radicals in blood and tissue were determined. After resuscitation with hyperoxia solution, blood pressure, arterial oxygen partial pressure, left and right ventricular systolic pressure, ±dp/dt max, survival time and rate were significantly increased. Lung and brain water content were unchanged, malondialdehyde activity in lung, brain and plasma and levels of TNF-α, IL-1, IL-6, and endothelin were significantly decreased. Besides, CGRP was elevated with reduced injury and improved lung and kidney functions. Concludingly, resuscitation with hyperoxia solution is feasible and more effective than other classical liquids, making it the first choice of treatment for high-altitude hemorrhagic shock.     

Hemorrhagic shock primes the hepatic portal circulation for the vasoconstrictive effects of endothelin-1

To test whether hemorrhagic shock and resuscitation (HSR) alters the vascular responsiveness of the portohepatic circulation to endothelins (ETs), we studied the macro- and microcirculatory effects of the preferential ET(A) receptor agonist ET-1 and of the selective ET(B) receptor agonist sarafotoxin 6c (S6c) after 1 h of hemorrhagic hypotension and 5 h of volume resuscitation in the isolated perfused rat liver ex vivo using portal pressure-flow relationships and epifluorescence microscopy. Although HSR did not cause major disturbances of hepatic perfusion per se, the response to ET-1 (0.5 x 10(-9) M) was enhanced, leading to greater increases in portal driving pressure, total portal resistance, and zero-flow pressures and more pronounced decreases in portal flow, sinusoidal diameters, and hepatic oxygen delivery compared with time-matched sham shock controls. In sharp contrast, the constrictive response to S6c (0.25 x 10(-9) M) remained unchanged. Thus HSR primes the portohepatic circulation for the vasoconstrictive effects of ET-1 but does not alter the effects of the ET(B) receptor agonist S6c. The enhanced sinusoidal response may contribute to the subsequent development of hepatic microcirculatory failure after secondary insults that are associated with increased generation of ET-1.     

4-Hydroxynonenal as a second messenger of free radicals and growth modifying factor

Immunohistochemical analysis of the distribution of the lipid peroxidation product 4-hydroxynonenal (HNE) in the brain of baboons exposed to experimental hemorrhagic traumatic shock or sepsis showed that systemic oxidative stress and the thereby generated HNE affect the blood:brain barrier and the regulation of cerebral blood flow determining secondary brain damage. Similarly, HNE was determined during ischemia in the brain blood vessels of rats exposed to ischemia/reperfusion injury of the brain. After reperfusion, HNE disappeared from the blood vessels but remained in neurones and in glial cells. Since HNE modulates cell proliferation and differentiation (including proto-oncogene expression), it is postulated that HNE might have prominent local and systemic effects that are not only harmful but beneficial, too, determining the outcome of various pathophysiological conditions based on oxidative stress.     

Geldanamycin treatment inhibits hemorrhage-induced increases in KLF6 and iNOS expression in unresuscitated mouse organs: role of inducible HSP70.

The aim of this study was to determine whether hemorrhage affects the levels of a variety of stress-related proteins and whether changes can be inhibited by drugs reported to provide protection from ischemia and reperfusion injury. Male Swiss Webster mice were subjected to a 40% hemorrhage without resuscitation. Western blot analysis indicated that c-Jun (an AP-1 protein), Kruppel-like factor 6 (KFL6), and inducible nitric oxide synthase (iNOS) were upregulated sequentially in that order. Pretreatment of mice with geldanamycin (GA) 16 h before hemorrhage effectively inhibited the expression of the proteins KLF6 and iNOS, whereas caffeic acid phenethyl ester did not. GA pretreatment increased inducible heat shock protein (HSP) 70 but not HSP90 in both sham and hemorrhagic tissues. The overexpressed inducible HSP70 formed complexes with KLF6 and iNOS. These results suggest that GA may be therapeutically useful for reducing hemorrhage-induced injury when used as a presurgical treatment or when added to resuscitation fluids.     

Analyses of changes in myocardial long non-coding RNA and mRNA profiles after severe hemorrhagic shock and resuscitation via RNA sequencing in a rat model

Background

Ischemia–reperfusion injury has been proven to induce organ dysfunction and death, although the mechanism is not fully understood. Long non-coding RNAs (lncRNAs) have drawn wide attention with their important roles in the gene expression of some biological processes and diseases, including myocardial ischemia–reperfusion (I/R) injury. In this paper, a total of 26 Sprague–Dawley (SD) rats were randomized into two groups: sham and ischemia–reperfusion (I/R) injury. Hemorrhagic shock was induced by removing 45% of the estimated total blood volume followed by reinfusion of shed blood. High-throughput RNA sequencing was used to analyze differentially expressed (DE) lncRNAs and messenger RNAs (mRNAs) in the heart tissue 4 h after reperfusion. Myocardial function was also evaluated.

Results

After resuscitation, the decline of myocardial function of shocked animals, expressed by cardiac output, ejection fraction, and myocardial performance index (MPI), was significant (p?<?0.05). DE lncRNAs and mRNAs were identified by absolute value of fold change?≥?2 and the false discovery rate ≤?0.001. In rats from the I/R injury group, 851 lncRNAs and 1015 mRNAs were significantly up-regulated while 1533 lncRNAs and 1702 m RNAs were significantly down-regulated when compared to the sham group. Among the DE lncRNAs, we found 12 location-associated with some known apoptosis-related protein-coding genes which were up-regulated or down-regulated accordingly, including STAT3 and Il1r1. Real time PCR assays confirmed that the expression levels of five location-associated lncRNAs (NONRATT006032.2, NONRATT006033.2, NONRATT006034.2, NONRATT006035.2 and NONRATT029969.2) and their location-associated mRNAs (STAT3 and Il1r1) in the rats from the I/R injury group were all significantly up-regulated versus the sham group.

Conclusions

The DE lncRNAs (NONRATT006032.2, NONRATT006033.2, NONRATT006034.2 and NONRATT006035.2) could be compatible with their role in myocardial protection by stimulating their co-located gene (STAT3) after hemorrhagic shock and resuscitation. The final prognosis of I/R injury might be regulated by different genes, which is regarded as a complex network.