Dexmedetomidine suppresses serum syndecan-1 elevation and improves survival in a rat hemorrhagic shock model

Hemorrhagic shock causes vascular endothelial glycocalyx (EGCX) damage and systemic inflammation. Dexmedetomidine (DEX) has anti-inflammatory and EGCX-protective effects, but its effect on hemorrhagic shock has not been investigated. Therefore, we investigated whether DEX reduces inflammation and protects EGCX during hemorrhagic shock. Anesthetized Sprague-Dawley rats were randomly assigned to five groups (n=7 per group): no shock (SHAM), hemorrhagic shock (HS), hemorrhagic shock with DEX (HS+DEX), hemorrhagic shock with DEX and the α7 nicotinic type acetylcholine receptor antagonist methyllycaconitine citrate (HS+DEX/MLA), and hemorrhagic shock with MLA (HS+MLA). HS was induced by shedding blood to a mean blood pressure of 25–30 mmHg, which was maintained for 30 min, after which rats were resuscitated with Ringer’s lactate solution at three times the bleeding volume. The survival rate was assessed up to 3 h after the start of fluid resuscitation. Serum tumor necrosis factor-alpha (TNF-α) and syndecan-1 concentrations, and wet-to-dry ratio of the heart were measured 90 min after the start of fluid resuscitation. The survival rate after 3 h was significantly higher in the HS+DEX group than in the HS group. Serum TNF-α and syndecan-1 concentrations, and the wet-to-dry ratio of heart were elevated by HS, but significantly decreased by DEX. These effects were antagonized by MLA. DEX suppressed the inflammatory response and serum syndecan-1 elevation, and prolonged survival in rats with HS.     

Permeability of barriers to albumin flux in lungs of sheep resuscitated from hemorrhagic shock

We assessed pulmonary endothelial and epithelial permeability and lung lymph flow in nine adult sheep under base-line conditions and after resuscitation from profound hemorrhagic shock. Animals were mechanically ventilated and maintained on 1% halothane anesthesia while aortic pressure was held at 40 Torr for 3 h. Systemic heparin was not used. After reinfusion of shed blood, sheep recovered from anesthesia and we measured lung lymph flow (QL), lymph-to-plasma concentration ratio for proteins, and time taken to reach half-equilibrium concentration of intravenous tracer albumin in lymph (t1/2). Twenty-four hours after bolus injection of radio-albumin we lavaged subsegments of the right upper lobe and determined fractional equilibration of the tracer in the alveolar luminal-lining layer. In each sheep we had measured these parameters 7 days earlier under base-line conditions. Animals were killed, and the lungs were used for gravimetric determination of extravascular lung water (gravimetric extravascular lung water-to-dry weight ratio) 24 h after resuscitation from shock. Pulmonary endothelial injury after resuscitation was evidenced by marked increase in QL, without fall in lymph-to-plasma ratio. Time taken to reach half-equilibrium concentration fell from 169 +/- 47 (SD) min in base-line studies to 53 +/- 33 min after shock. There was no evidence of lung epithelial injury. Gravimetric extravascular lung water-to-dry weight ratio was significantly increased in these animals killed 24 h after resuscitation (4.94 +/- 0.29) compared with values in our laboratory controls (4.13 +/- 0.09, mean +/- SD). These data demonstrate a loss of lung endothelial integrity in sheep after resuscitation from profound hemorrhagic shock.     

不同液体复苏方式对失血性休克大鼠血流动力学的影响

目的:探讨不同液体复苏对失血性休克大鼠血流动力学的影响及机制。方法:Wistar大鼠40只,随机分为5组:假手术组、休克组、乳酸林格液(RL)复苏组、羟乙基淀粉(HES)复苏组、自身血液(BL)复苏组,每组8只,建立失血性休克大鼠模型。在失血性休克大鼠模型复制成功后1h给予对应液体复苏2h。观察并记录各组大鼠的血流动力学变化:收缩压(SBP)、舒张压(DBP)、平均动脉压(MAP)、呼吸频率(RR)和心率(HR)。结果:三组液体复苏组休克时的SBP、DBP、MAP均较休克前低,HR、RR较休克前明显加快(P<0.05)。RL复苏组各时间点SBP、DBP、MAP明显低于休克前(P<0.05)。HES组、BL组复苏各时间点SBP、DBP、MAP与休克前相近(P>0.05),但明显高于RL组。RL组各时间点RR明显快于休克前(P<0.05),HES组、BL组复苏各时间点RR与休克前相近(P>0.05),但明显低于RL组。RL组复苏60min、90min、120min的HR明显快于休克前(P<0.05),HES组、BL组苏各时间点HR同休克前相近(P>0.05),但60min、90min、120min的HR明显低于R...     

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.     

Sublethal hemorrhagic shock reduces tumor necrosis factor-alpha-producing capacity in different cell compartments

Hemorrhagic shock results in a severe impairment of the immune response. Immunological alterations after hemorrhagic shock thus appear to be responsible for reduced resistance to infectious agents commonly observed after shock and severe injury. In the present study we examined the TNF-alpha-producing capacity of immune cells derived from different organs after sublethal shock in rats. Hemorrhagic shock was established by pressure controlled bleeding to a mean arterial pressure of 35 mm Hg for 35-40 min and consecutive resuscitation in male Sprague-Dawley rats. Twenty four hours after shock, TNF-a production in response to lipopolysaccharide (LPS, Salmonella friedenau) stimulation was measured in isolated spleen, bone marrow and blood cells. TNF-a production could be induced by stimulation with 1 ng/ml, in blood, spleen and bone marrow cells collected from sham-operated animals. A maximal stimulation was observed in all cell types after stimulation with 10 ng/ml LPS and could not be further increased with LPS doses of 100 ng/ml. Hemorrhagic shock of 35 mm Hg for 35-40 min, with consecutive resuscitation did not result in mortality, in contrast to a 4 hours lasting hemorrhagic shock resulting in 80% mortality. Blood, spleen or bone marrow cells, harvested from animals 24 hours after sublethal hemorrhagic shock, showed a significantly reduced TNF-alpha production in all cell populations after LPS stimulation. Serum collected from animals 2 hours after sublethal hemorrhagic shock contained an activity not present either before or 24 hours after shock, that downregulated LPS-induced TNF-alpha production in rat whole blood cultures and the murine macrophage cell line J774. The inhibitory activity present in serum, 2 hours after shock is not IL-10 since this mediator was not detectable in any serum sample. However, in the serum samples with TNF-alpha-inhibitory activity, elevated levels of PGE2 metabolites were found, which suggests the involvement of prostaglandins in trauma-induced immunosuppression. Altered TNF-a expression might be partially explained by an inhibitory activity in the serum already present 2 hours after shock. Since adequate, but not overwhelming TNF-alpha production is essential for host response, the altered cytokine formation might explain local and systemic susceptibility to infections after hemorrhagic shock.     

Syndecan-1 is an in vivo suppressor of Gram-positive toxic shock

Heparan sulfate proteoglycans bind to and regulate many inflammatory mediators in vitro, suggesting that they serve an important role in influencing inflammatory responses in vivo. Here we evaluated the role of syndecan-1, a major heparan sulfate proteoglycan, in modulating inflammatory responses in Gram-positive toxic shock, a systemic disease that is a significant cause of morbidity and mortality. Syndecan-1-null and wild-type mice were injected intraperitoneally with staphylococcal enterotoxin B, a pyrogenic superantigen, and their inflammatory responses were assessed. Syndecan-1-null mice showed significantly increased liver injury, vascular permeability, and death in response to staphylococcal enterotoxin B challenge compared with wild-type mice. Although serum levels of systemic IL-2 and IFNgamma were similar between the two backgrounds, those of TNFalpha and IL-6 were significantly increased in syndecan-1-null mice undergoing Gram-positive toxic shock. Furthermore, syndecan-1-null mice challenged with staphylococcal enterotoxin B showed enhanced T cell accumulation in tissues, whereas immunodepletion of T cells protected syndecan-1-null mice from the magnified systemic cytokine storm, inflammatory tissue injury, and death. Importantly, syndecan-1 shedding was induced in wild-type mice injected with staphylococcal enterotoxin B, and the administration of heparan sulfate, but not syndecan-1 core protein, rescued syndecan-1-null mice from lethal toxic shock by suppressing the production of TNFalpha and IL-6, and attenuating inflammatory tissue injury. Altogether, these data suggest that syndecan-1 shedding is a key endogenous mechanism that protects the host from Gram-positive toxic shock by inhibiting the dysregulation and amplification of the inflammatory response.     

Th1 cytokine-induced syndecan-4 shedding by airway smooth muscle cells is dependent on mitogen-activated protein kinases

In asthma, airway smooth muscle (ASM) chemokine secretion can induce mast cell recruitment into the airways. The functions of the mast cell chemoattractant CXCL10, and other chemokines, are regulated by binding to heparan sulphates such as syndecan-4. This study is the first demonstration that airway smooth muscle cells (ASMC) from people with and without asthma express and shed syndecan-4 under basal conditions. Syndecan-4 shedding was enhanced by stimulation for 24 h with the Th1 cytokines interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α), but not interferon-γ (IFNγ), nor the Th2 cytokines IL-4 and IL-13. ASMC stimulation with IL-1β, TNF-α, and IFNγ (cytomix) induced the highest level of syndecan-4 shedding. Nonasthmatic and asthmatic ASM cell-associated syndecan-4 protein expression was also increased by TNF-α or cytomix at 4-8 h, with the highest levels detected in cytomix-stimulated asthmatic cells. Cell-associated syndecan-4 levels were decreased by 24 h, whereas shedding remained elevated at 24 h, consistent with newly synthesized syndecan-4 being shed. Inhibition of ASMC matrix metalloproteinase-2 did not prevent syndecan-4 shedding, whereas inhibition of ERK MAPK activation reduced shedding from cytomix-stimulated ASMC. Although ERK inhibition had no effect on syndecan-4 mRNA levels stimulated by cytomix, it did cause an increase in cell-associated syndecan-4 levels, consistent with the shedding being inhibited. In conclusion, ASMC produce and shed syndecan-4 and although this is increased by the Th1 cytokines, the MAPK ERK only regulates shedding. ASMC syndecan-4 production during Th1 inflammatory conditions may regulate chemokine activity and mast cell recruitment to the ASM in asthma.     

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.     

Sensitivity to endotoxin in rabbits is increased after hemorrhagic shock.

The immunoinflammatory response following trauma and hemorrhage may predispose to the development of sepsis and multiple-organ failure syndrome. Cardiac output (CO), arterial pressure, arterial PO2, and pulmonary permeability index were measured. We examined the sensitivity of rabbits to infusions of lipopolysaccharide (LPS) after hemorrhagic shock. Shock was produced by reducing CO to 40% of baseline for 90 min, followed by resuscitation with shed blood and then with lactated Ringer solution to maintain CO near baseline. Animals were assigned to three groups: 1) hemorrhagic shock only, 2) LPS only, and 3) hemorrhagic shock + LPS. Groups 1 and 3 were subjected to hemorrhagic shock on day 1. Escherichia coli LPS was infused (1.0 microgram/kg i.v.) into groups 2 and 3 on day 2. Fluid resuscitation with lactated Ringer solution was continued in an effort to maintain CO at baseline. Five hours after LPS infusion, 125I-albumin was injected intravenously, and rabbits were killed 1 h later for measurement of pulmonary permeability index. LPS infusion after shock (group 3) caused significant decreases in CO, arterial pressure, and PO2 and an increase in pulmonary permeability. These changes were not seen in the groups 1 and 2. We conclude that hemorrhagic shock and resuscitation result in a proinflammatory state, leading to increased sensitivity to subsequent exposure to LPS.     

血清降钙素原在感染性休克患者中的表达及与APACHEⅡ评分的相关性分析

目的:探讨血清降钙素原在感染性休克患者中的表达及与APACHEⅡ评分的相关性分析。方法:选取2012年1月至2013年9月我院收治的58例感染性休克患者,设为感染性休克组,早期采取液体复苏的方法进行救治。另选取同期于我院体检的年龄、性别与之相匹配的52例健康人设为对照组。测定两组不同时间的血清PCT含量,对APACHEⅡ评分进行评定,并进行统计学分析。结果:感染行休克组患者的复苏液体量最少为4332mL,最多为10645mL,平均为(7602.85±1628.47)mL,复苏达标的平均时间为(17.67±3.75)h。感染性休克组患者血清PCT含量治疗后24h为(8.77±4.66)μg/L,明显低于治疗前的(17.78±5.89)μg/L;感染性休克组患者治疗前与治疗后24h血清PCT含量相比于对照组受试者的(0.03±0.01)μg/L均显著提高;感染性休克组患者治疗前与治疗后24hAPACHEⅡ评分分别为(16.68±3.27)分、(10.46±3.18)分,相比于对照组的(7.22±1.15)分均显著提高;感染性休克组患者APACHEⅡ评分治疗后24h明显低于治疗前。差异有统计学意义(P0.05)。感染性休克组治疗前后PCT含量与APACHEⅡ评分均呈一定的正相关(P0.05);PCT含量变化与快速复苏的液体量呈一定的负相关(P0.05);而与APACHEⅡ评分不存在明显的相关性(P0.05)。结论:将血清降钙素原应用于感染性休克患者的诊断及治疗过程中评估病情变化,能够为液体复苏提供有效的指导。血清降钙素原含量与APACHEⅡ评分呈正相关,但其变化与APACHEⅡ评分不存在相关性,而与复苏液体量呈负相关。     

A nonerythropoietic peptide that mimics the 3D structure of erythropoietin reduces organ injury/dysfunction and inflammation in experimental hemorrhagic shock

Recent studies have shown that erythropoietin, critical for the differentiation and survival of erythrocytes, has cytoprotective effects in a wide variety of tissues, including the kidney and lung. However, erythropoietin has been shown to have a serious side effect-an increase in thrombovascular effects. We investigated whether pyroglutamate helix B-surface peptide (pHBSP), a nonerythropoietic tissue-protective peptide mimicking the 3D structure of erythropoietin, protects against the organ injury/ dysfunction and inflammation in rats subjected to severe hemorrhagic shock (HS). Mean arterial blood pressure was reduced to 35 ± 5 mmHg for 90 min followed by resuscitation with 20 mL/kg Ringer Lactate for 10 min and 50% of the shed blood for 50 min. Rats were euthanized 4 h after the onset of resuscitation. pHBSP was administered 30 min or 60 min into resuscitation. HS resulted in significant organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung). In rats subjected to HS, pHBSP significantly attenuated (i) organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung), (ii) increased the phosphorylation of Akt, glycogen synthase kinase-3β and endothelial nitric oxide synthase, (iii) attenuated the activation of nuclear factor (NF)-κB and (iv) attenuated the increase in p38 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. pHBSP protects against multiple organ injury/dysfunction and inflammation caused by severe hemorrhagic shock by a mechanism that may involve activation of Akt and endothelial nitric oxide synthase, and inhibition of glycogen synthase kinase-3β and NF-κB.     

Detrimental effects of complement activation in hemorrhagic shock.

The complement system has been implicated in early inflammatory events and a variety of shock states. In rats, we measured complement activation after hemorrhage and examined the hemodynamic and metabolic effects of complement depletion before injury and worsening of complement activation after hemorrhage and resuscitation [with a carboxypeptidase N inhibitor (CPNI), which blocks the clearance of C5a]. Rats were bled to a mean arterial pressure of 30 mmHg for 50 min and were then resuscitated for 2 h. Shock resulted in significant evidence of complement consumption, with serum hemolytic activity being reduced by 33% (P < 0.05). Complement depletion before injury did not affect hemorrhage volume (complement depleted = 28 +/- 1 ml/kg, complement intact = 29 +/- 1 ml/kg, P = 0.74) but improved postresuscitation mean arterial pressure by 37 mmHg (P < 0.05) and serum bicarbonate levels (complement depleted = 22 +/- 3 meq/ml, complement intact = 13 +/- 8 meq/ml, P < 0.05). Pretreatment with CPNI was lethal in 80% of treated animals vs. the untreated hemorrhaged group in which no deaths occurred (P < 0.05). In this model of hemorrhagic shock, complement activation appeared to contribute to progressive hypotension and metabolic acidosis seen after resuscitation. The lethality of CPNI during acute blood loss suggests that the anaphylatoxins are important in the pathophysiological events involved in hemorrhagic shock.     

Atrial natriuretic peptide induces shedding of endothelial glycocalyx in coronary vascular bed of guinea pig hearts

Atrial natriuretic peptide (ANP) is reported to enhance vascular permeability in vivo. Our aim was to evaluate the impact of ANP on coronary extravasation of fluids and macromolecules and on the integrity of the endothelial glycocalyx. Isolated guinea pig hearts (n = 6/group) were perfused with Krebs-Henseleit buffer in a Langendorff mode. A 6% hydroxyethyl starch (HES) solution was infused into the coronary system for 20 min without (Control group) and simultaneously with (ANP group) ANP at 10(-9) M. In two further series, the glycocalyx was enzymatically degraded by means of heparinase (Hep) application (10 IU over 15 min), followed again by the infusion of HES in the absence (Hep group) and presence (ANP+Hep group) of ANP. Net fluid filtration, extravasation of HES, electron microscopic visualization of the glycocalyx, and quantification of shedding of syndecan-1, a component of the glycocalyx, were determined. An increase in fluid leak was observed in ANP, ANP+Hep, and Hep hearts [+29%, +31%, +14%, respectively; a decrease was observed in Control hearts (-13%)]. Similarly, an accelerated extravasation of colloid was observed in these three groups. Coronary release of syndecan-1 increased 9- to 18-fold during infusion of ANP. Electron microscopy revealed a dramatic degradation of the glycocalyx after ANP. These results indicate that the endothelial glycocalyx serves as a barrier to transmural exchange of fluid and colloid in the coronary vascular system. ANP causes rapid shedding of individual components of the glycocalyx and histologically detectable degradation. Thus the permeability-increasing effect of ANP may be at least partially related to changes in the integrity of the endothelial glycocalyx.     

外源性硫化氢对创伤失血性休克大鼠血浆炎症因子的影响

目的：研究外源性硫化氢（H2S）对创伤失血性休克大鼠炎症反应的影响。方法：选择健康成年雄性SD大鼠随机分为四组：假手术组（Sham）,模型组（HTS）,生理盐水组（NS）,NaHS处理组（NaHS）,采用创伤失血性休克模型,Sham组完成所有手术操作,但不放血和复苏,HTS组完成所有手术操作放血后给予Ringer＇s液复苏,NS组放血后在Ringer＇s液复苏前腹腔注射与NaHS组等容量的生理盐水,NaHS组在复苏前给与NaHS28μmol/kg（生理盐水稀释至0.5ml）腹腔注射。持续监测各组平均动脉压（MAP）及心律（HR）,并通过测定血浆中TNF-α、IL-1β、IL-6和IL-10浓度的变化,观察外源性硫化氢对创伤失血性休克大鼠血浆炎症因子的影响。结果：①与HTS组及NS组比较,NaHS组复苏后MAP明显改善（P〈0.05）。②与HTS组及NS组比较,复苏后1小时NaHS组血浆TNFα、IL-1β、IL-6浓度明显降低（P〈0.05）;而IL-10浓度四组间差异不明显（P〉0.05）。结论：外源性硫化氢可改善创伤失血性休克大鼠复苏后平均动脉压及抑制复苏后早期炎症反应。     

Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin

Background

Increased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity.

Mechanical strain induces a persistent upregulation of syndecan-1 expression in smooth muscle cells

Syndecan-1 belongs to a family of transmembrane proteoglycans, acts as a coreceptor for growth factor binding, as well as cell-matrix and cell-cell interactions, and is induced in smooth muscle cells (SMCs) following balloon catheter injury. In this report, we investigated syndecan-1 expression in SMCs in response to several distinct biomechanical force profiles and the related syndecan shedding response. Syndecan-1 mRNA expression increased in response to 5% and 10% cyclic strain (24 h: 206 +/- 40% and 278 +/- 33%, respectively, P < 0.05) when compared to unstrained controls. When subjected to 10% cyclic strain for periods of up to 48 h, syndecan-1 mRNA levels remained elevated at 294 +/- 31%. Notably, the SMC mechanosensor mechanism remained responsive after an initial 24 h "preconditioning" period, as evident by a fivefold increase in syndecan-1 gene expression following a change in cyclic stress from 10% to 20% (48 h: 516 +/- 55%, P < 0.05). Of note, similar behavior was not observed in an analysis of syndecan-2 mRNA levels. Commensurate with mRNA responses, mechanical stress induced an increase in cell-associated syndecan-1 protein levels with an associated increase in protein shedding. Given the varied functions of syndecan-1, stress-induced effects on SMC syndecan-1 expression and shedding may represent an additional component of the pro-inflammatory, growth-stimulating pathways that are activated in response to changes in the mechanical microenvironment of the vascular wall. Syndecan-1 expression is uniquely influenced by changes in the phase and magnitude of the local stress field.     

Evaluation of gilthead sea bream, Sparus aurata, sperm quality after cryopreservation in 5 ml macrotubes

Cryopreservation produces several types of damage in spermatozoa, leading to fertility impairment. The reduction arises both from a lower viability post-thaw and from sublethal dysfunctions in some of the surviving cells. In the present study, we have analysed the effect of cryopreservation in 5 ml macrotubes on the quality of post-thawed gilthead sea bream sperm. Several standard sperm quality parameters were determined: pH and osmolarity of seminal plasma, sperm concentration, and motility. An exhaustive determination of sperm quality before and after cryopreservation was investigated. Several parameters related with spermatozoal status were determined: ATP content, plasma membrane integrity and functionality, mitochondrial functionality, and sperm fertility. Our results demonstrated that gilthead sea bream spermatozoa suffer several types of damage after freezing/thawing. The percentage of viable cells slightly decreased after cryopreservation, however plasma membrane was affected by cryopreservation, since cells could not resist the hyperosmotic shock. Mitochondrial status was affected by cryopreservation since there was a decrease in the parameters of sperm motility, ATP content (3.17 nmol ATP/10(5) spermatozoa to 1.7 nmol ATP/10(5) spermatozoa in 1:20 frozen samples) and an increase of the percentage of cells with mitochondrial depolarized membranes (11% for fresh and 27% for 1:20 frozen samples). Fertility rate was similar either using fresh or frozen/thawed sperm (77 and 75% hatched larvae, respectively).     

Human vasoactive hormone adrenomedullin and its binding protein rescue experimental animals from shock

We recently discovered that vascular responsiveness to adrenomedullin (AM), a vasoactive hormone, decreases after hemorrhage, which is markedly improved by the addition of its binding protein AMBP-1. One obstacle hampering the development of AM/AMBP-1 as resuscitation agents in trauma victims is the potential immunogenicity of rat proteins in humans. Although less potent than rat AM, human AM has been shown to increase organ perfusion in rats. We therefore hypothesized that administration of human AM/AMBP-1 improves organ function and survival after severe blood loss in rats. To test this, male Sprague-Dawley rats were bled to and maintained at an MAP of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. At 15 min after the beginning of resuscitation, human AM/AMBP-1 (12/40 or 48/160 microg/kg BW) were administered intravenously over 45 min. Various pathophysiological parameters were measured 4h after resuscitation. In additional groups of animals, a 12-day survival study was conducted. Our result showed that tissue injury as evidenced by increased levels of transaminases, lactate, and creatinine, was present at 4h after hemorrhage and resuscitation. Moreover, pro-inflammatory cytokines TNF-alpha and IL-6 were also significantly elevated. Administration of AM/AMBP-1 markedly attenuated tissue injury, reduced cytokine levels, and improved the survival rate from 29% (vehicle) to 62% (low-dose) or 70% (high-dose). However, neither human AM alone nor human AMBP-1 alone prevented the significant increase in ALT, AST, lactate and creatinine at 4h after the completion of hemorrhage and resuscitation. Moreover, the half-life of human AM and human AMBP-1 in rats was 35.8 min and 1.68 h, respectively. Thus, administration of human AM/AMBP-1 may be a useful approach for attenuating organ injury, and reducing mortality after hemorrhagic shock.     

Estrogen protects renal endothelial barrier function from ischemia-reperfusion in vitro and in vivo

Emerging evidence suggests that renal endothelial function may be altered in ischemia-reperfusion injury. Acute kidney injury is sexually dimorphic, and estrogen protects renal tubular function after experimental ischemic injury. This study tested the hypothesis that during ischemia-reperfusion, estrogen alters glomerular endothelial function to prevent hyperpermeability. Glomerular endothelial cells were exposed to 8-h oxygen-glucose deprivation (OGD) followed by 4- and 8-h reoxygenation-glucose repletion. After 4-h reoxygenation-glucose repletion, transendothelial permeability to Ficoll-70 was reduced, and transendothelial resistance increased, by 17β-estradiol vs. vehicle treatment during OGD (OGD-vehicle: 91.0 ± 11.8%, OGD-estrogen: 102.6 ± 10.8%, P < 0.05). This effect was reversed by coadministration of G protein-coupled receptor 30 (GPR30) antagonist G15 with 17β-estradiol (OGD-estrogen-G15: 89.5 ± 6.9, P < 0.05 compared with 17β-estradiol). To provide preliminary confirmation of this result in vivo, Ficoll-70 was administered to mice 24 h after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Blood urea nitrogen (BUN) and serum creatinine (SCr) in these mice were elevated within 12 h following CA/CPR and reduced at 24 h by pretreatment with 17β-estradiol (BUN/SCr 17β-estradiol: 34 ± 19/0.2 ± 0.1 vehicle: 92 ± 49/0.5 ± 0.3, n = 8-12, P < 0.05). Glomerular sieving of Ficoll 70 was increased by CA/CPR within 2 h of injury and 17β-estradiol treatment (θ; 17β-estradiol: 0.74 ± 0.26 vs. vehicle: 1.05 ± 0.53, n = 14-15, P < 0.05). These results suggest that estrogen reduces postischemic glomerular endothelial hyperpermeability at least in part through GPR30 and that estrogen may regulate post CA/CPR glomerular permeability in a similar fashion in vivo.     

IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock

In hemorrhagic shock and trauma, patients are prone to develop systemic inflammation with remote organ dysfunction, which is thought to be caused by pro-inflammatory mediators. This study investigates the role of the immuno-modulatory cytokine IL-10 in the development of organ dysfunction following hemorrhagic shock. Male C57/BL6 and IL-10 KO mice were subjected to volume controlled hemorrhagic shock for 3 h followed by resuscitation. Animals were either sacrificed 3 or 24 h after resuscitation. To assess systemic inflammation, serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with the Luminex? multiplexing platform; acute lung injury (ALI) was assessed by pulmonary myeloperoxidase (MPO) activity and lung histology and acute liver injury was assessed by hepatic MPO activity, hepatic IL-6 levels, and serum ALT levels. There was a trend towards increased IL-6 and KC serum levels 3 h after resuscitation in IL-10 KO as compared to C57/BL6 mice; however this did not reach statistical significance. Serum MCP-1 levels were significantly increased in IL-10 KO mice 3 and 24 h following resuscitation as compared to C57/BL6 mice. In IL-10 KO mice, pulmonary MPO activity was significantly increased 3 h following resuscitation and after 24 h histological signs of acute lung injury were more apparent than in C57/BL6 mice. In contrast, no significant differences in any liver parameters were detected between IL-10 KO and C57/BL6 mice. Our data indicate that an endogenous IL-10 deficiency augments acute lung but not liver injury following hemorrhagic shock.