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Background and Aims: The systemic host response in sepsis is frequently accompanied by central nervous system (CNS) dysfunction. Evidence suggests that excessive formation of neutrophil extracellular traps (NETs) can increase the permeability of the blood–brain barrier (BBB) and that the evolving mitochondrial damage may contribute to the pathogenesis of sepsis-associated encephalopathy. Kynurenic acid (KYNA), a metabolite of tryptophan catabolism, exerts pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenous KYNA or its synthetic analogues SZR-72 and SZR-104 affect BBB permeability secondary to NET formation and influence cerebral mitochondrial disturbances in a clinically relevant rodent model of intraabdominal sepsis. Methods: Sprague–Dawley rats were subjected to fecal peritonitis (0.6 g kg-1 ip) or a sham operation. Septic animals were treated with saline or KYNA, SZR-72 or SZR-104 (160 µmol kg-1 each ip) 16h and 22h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic parameters to calculate rat organ failure assessment (ROFA) scores. NET components (citrullinated histone H3 (CitH3); myeloperoxidase (MPO)) and the NET inducer IL-1β, as well as IL-6 and a brain injury marker (S100B) were detected from plasma samples. After 24h, leukocyte infiltration (tissue MPO) and mitochondrial complex I- and II-linked (CI–CII) oxidative phosphorylation (OXPHOS) were evaluated. In a separate series, Evans Blue extravasation and the edema index were used to assess BBB permeability in the same regions. Results: Sepsis was characterized by significantly elevated ROFA scores, while the increased BBB permeability and plasma S100B levels demonstrated brain damage. Plasma levels of CitH3, MPO and IL-1β were elevated in sepsis but were ameliorated by KYNA and its synthetic analogues. The sepsis-induced deterioration in tissue CI–CII-linked OXPHOS and BBB parameters as well as the increase in tissue MPO content were positively affected by KYNA/KYNA analogues. Conclusion: This study is the first to report that KYNA and KYNA analogues are potential neuroprotective agents in experimental sepsis. The proposed mechanistic steps involve reduced peripheral NET formation, lowered BBB permeability changes and alleviation of mitochondrial dysfunction in the CNS. [ABSTRACT FROM AUTHOR]

Introduction: Fluid resuscitation is the cornerstone of treatment in hemorrhagic shock. Despite increasing doubts, several guidelines recommend to maintain mean arterial pressure (MAP) >65 mmHg as the most frequent indication of fluid therapy. Our aim was to investigate the effects of a MAP-guided management in a bleeding-resuscitation animal experiment. Materials and methods: After anesthesia and instrumentation (tbsl) animals were bled till the initial stroke volume index dropped by 50% (t0). Fluid replacement was performed in 4 equivalent steps (t1-4) with balanced crystalloid solution to reach the baseline values of MAP. Invasive hemodynamic measurements and blood gas analyses were performed after each step. Results: Mean arterial pressure dropped from tbsl to t0 (114±11 vs 76.9±16.9 mmHg, p<0.001) and returned to baseline by t4 (101.4±14.4 mmHg). From tbsl-t0 stroke volume index (SVI), cardiac index (CI) decreased (SVI: 40±8.6 vs 19.3±3.6 ml/m2, p<0.001; CI: 3.4±0.3 vs 1.9±0.3 l/min/m2, p<0.001), pulse pressure variation (PPV) increased (13.2±4.3 vs 22.1±4.3%, p<0.001). There was a decrease in oxygen delivery (464±45 vs 246±26.9 ml/min, p<0.001), central venous oxygen saturation (82.8±5.4 vs 53.6±12.1%, p<0.001) and increase in lactate levels (1.6±0.4 vs 3.5±1.6 mmol/l, p<0.005). SVI, CI and PPV returned to their initial values by t2. To normalize MAP fluid therapy had to be continued till t4, with the total infused volume of 4.5±0.8 l. Conclusion: In the current experiment bleeding led to hemorrhagic shock, while MAP remained higher than 65 mmHg. Furthermore, MAP was unable to indicate the normalization of SVI, CI and PPV that resulted in unnecessary fluid administration. Our data give further evidence that MAP may be an inappropriate parameter to follow during fluid resuscitation. [ABSTRACT FROM AUTHOR]

Background: Fluid resuscitation remains a cornerstone in the management of acute bleeding. According to Starling's 'Three-compartment model', four-times more crystalloids have the same volume effect as colloids. However, this volume-replacement ratio remains a controversial issue as it may be affected by the degradation of the endothelial glycocalyx layer, a situation often found in the critically ill. Our aim was to compare colloid and crystalloid based fluid resuscitation during an experimental stroke volume index (SVI) guided hemorrhage and resuscitation animal model. Methods: Anesthetized and mechanically ventilated pigs were randomized to receive a colloid (Voluven®,HES, n=15) or crystalloid (Ringerfundin®,RF, n=15) infusion. Animals were bled till baseline SVI (Tbsl) dropped by 50% (T0), followed by resuscitation until initial SVI was reached (T4) in four steps. Invasive hemodynamic measurements, blood gas analyses and laboratory tests were performed at each assessment points. Glycocalyx degradation markers (Syndecan-1/hematocrit ratio, Glypican/hematocrit ratio) were determined at Tbsl, T0 and T4. Results: Similar amounts of blood were shed in both groups (HES group: 506±159 mls blood, RF group: 470±127 mls blood). Hemodynamic changes followed the same pattern without significant difference between the groups. Animals received significantly less resuscitation fluid in the HES compared to the RF-group: 425 [320-665], vs 1390 [884-1585] mls, p <0.001. The volume replacement ratio was 0.92 [0.79-1.54] for HES; and 3.03 [2.00-4.23] for the RF-group ( p <0.001). There was no significant difference between the groups in the glycocalyx degradation markers. Conclusion: In this moderate bleeding-resuscitation animal model the volume-replacement ratio for crystalloids and colloids followed similar patterns as predicted by Starling's principle, and the glycocalyx remained intact. This indicates that in acute bleeding events, such as trauma or during surgery, colloids may be beneficial as hemodynamic stability may be achieved more rapidly than with crystalloids. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) is the main interface controlling molecular and cellular traffic between the central nervous system (CNS) and the periphery. It consists of cerebral endothelial cells (CECs) interconnected by continuous tight junctions, and closely associated pericytes and astrocytes. Different parts of the CNS have diverse functions and structures and may be subject of different pathologies, in which the BBB is actively involved. It is largely unknown, however, what are the cellular and molecular differences of the BBB in different regions of the brain. Using in silico, in vitro, and ex vivo techniques we compared the expression of BBB-associated genes and proteins (i.e., markers of CECs, brain pericytes, and astrocytes) in the cortical grey matter and white matter. In silico human database analysis (obtained from recalculated data of the Allen Brain Atlas), qPCR, Western blot, and immunofluorescence studies on porcine and mouse brain tissue indicated an increased expression of glial fibrillary acidic protein in astrocytes in the white matter compared with the grey matter. We have also found increased expression of genes of the junctional complex of CECs (occludin, claudin-5, and α-catenin) in the white matter compared with the cerebral cortex. Accordingly, occludin, claudin-5, and α-catenin proteins showed increased expression in CECs of the white matter compared with endothelial cells of the cortical grey matter. In parallel, barrier properties of white matter CECs were superior as well. These differences might be important in the pathogenesis of diseases differently affecting distinct regions of the brain. [ABSTRACT FROM AUTHOR]

Background: Methanogenesis can indicate the fermentation activity of the gastrointestinal anaerobic flora. Methane also has a demonstrated anti-inflammatory potential. We hypothesized that enriched methane inhalation can influence the respiratory activity of the liver mitochondria after an ischemia-reperfusion (IR) challenge. Methods: The activity of oxidative phosphorylation system complexes was determined after in vitro methane treatment of intact liver mitochondria. Anesthetized Sprague-Dawley rats subjected to standardized 60-min warm hepatic ischemia inhaled normoxic air (n = 6) or normoxic air containing 2.2% methane, from 50 min of ischemia and throughout the 60-min reperfusion period (n = 6). Measurement data were compared with those on sham-operated animals (n = 6 each). Liver biopsy samples were subjected to high-resolution respirometry; whole-blood superoxide and hydrogen peroxide production was measured; hepatocyte apoptosis was detected with TUNEL staining and in vivo fluorescence laser scanning microscopy. Results: Significantly decreased complex II-linked basal respiration was found in the normoxic IR group at 55 min of ischemia and a lower respiratory capacity (~60%) and after 5 min of reperfusion. Methane inhalation preserved the maximal respiratory capacity at 55 min of ischemia and significantly improved the basal respiration during the first 30 min of reperfusion. The IR-induced cytochrome c activity, reactive oxygen species (ROS) production and hepatocyte apoptosis were also significantly reduced. Conclusions: The normoxic IR injury was accompanied by significant functional damage of the inner mitochondrial membrane, increased cytochrome c activity, enhanced ROS production and apoptosis. An elevated methane intake confers significant protection against mitochondrial dysfunction and reduces the oxidative damage of the hepatocytes. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare stroke volume (SVI) to cardiac index (CI) guided resuscitation in a bleeding-resuscitation experiment. Twenty six pigs were randomized and bled in both groups till baseline SVI (Tbsl) dropped by 50% (T0), followed by resuscitation with crystalloid solution until initial SVI or CI was reached (T4). Similar amount of blood was shed but animals received significantly less fluid in the CI-group as in the SVI-group: median = 900 (interquartile range: 850–1780) versus 1965 (1584–2165) mL, p=0.02, respectively. In the SVI-group all variables returned to their baseline values, but in the CI-group animals remained underresuscitated as indicated by SVI, heart rate (HR) and stroke volume variation (SVV), and central venous oxygen saturation (ScvO2) at T4 as compared to Tbsl: SVI = 23.8 ± 5.9 versus 31.4 ± 4.7 mL, HR: 117 ± 35 versus 89 ± 11/min SVV: 17.4 ± 7.6 versus 11.5 ± 5.3%, and ScvO2: 64.1 ± 11.6 versus 79.2 ± 8.1%, p<0.05, respectively. Our results indicate that CI-based goal-directed resuscitation may result in residual hypovolaemia, as bleeding caused stress induced tachycardia “normalizes” CI, without restoring adequate SVI. As the SVI-guided approach normalized most hemodynamic variables, we recommend using SVI instead of CI as the primary goal of resuscitation during acute bleeding. [ABSTRACT FROM AUTHOR]

Tissue capnometry may be suitable for the indirect evaluation of regional hypoperfusion. We tested the performance of a new sublingual capillary tonometer in experimental hemorrhage. Thirty-six anesthetized, ventilated mini pigs were divided into sham-operated (n = 9) and shock groups (n = 27). Hemorrhagic shock was induced by reducing mean arterial pressure (MAP) to 40 mmHg for 60 min, after which fluid resuscitation started aiming to increase MAP to 75% of the baseline value (60-180 min). Sublingual carbon-dioxide partial pressure was measured by tonometry, using a specially coiled silicone rubber tube. Mucosal red blood cell velocity (RBCV) and capillary perfusion rate (CPR) were assessed by orthogonal polarization spectral (OPS) imaging. In the 60 min shock phase a significant drop in cardiac index was accompanied by reduction in sublingual RBCV and CPR and significant increase in the sublingual mucosal-to-arterial PCO2 gap (PSLCO2 gap), which significantly improved during the 120 min resuscitation phase. There was significant correlation between PSLCO2 gap and sublingual RBCV (r = -0.65, p < 0.0001), CPR (r = -0.64, p < 0.0001), central venous oxygen saturation (r = -0.50, p < 0.0001), and central venous-to-arterial PCO2 difference (r = 0.62, p < 0.0001). This new sublingual tonometer may be an appropriate tool for the indirect evaluation of circulatory changes in shock. [ABSTRACT FROM AUTHOR]

Background and Purpose: To provide evidence for the protective role of inorganic nitrite against acute ischaemia and reperfusion-induced ventricular arrhythmias in a large animal model. Experimental Approach: Dogs, anaesthetized with chloralose and urethane, were administered intravenously with sodium nitrite (0.2 µmolkg-1min-1) in two protocols. In protocol 1 nitrite was infused 10 min prior to and during a 25 min occlusion of the left anterior descending (LAD) coronary artery (NaNO2-PO; n = 14), whereas in protocol 2 the infusion was started 10 min prior to reperfusion of the occluded vessel (NaNO2-PR; n = 12). Control dogs (n = 15) were infused with saline and subjected to the same period of ischaemia and reperfusion. Severities of ischaemia and ventricular arrhythmias, as well as changes in plasma nitrate/nitrite (NOx) levels in the coronary sinus blood, were assessed throughout the experiment. Myocardial superoxide and nitrotyrosine (NT) levels were determined during reperfusion. Changes in protein S-nitrosylation (SNO) and S-glutathionylation were also examined. Key Results: Compared with controls, sodium nitrite administered either pre-occlusion or pre-reperfusion markedly suppressed the number and severity of ventricular arrhythmias during occlusion and increased survival (0% vs. 50 and 92%) upon reperfusion. There were also significant decreases in superoxide and NT levels in the nitrite treated dogs. Compared with controls, increased SNO was found only in NaNO2-PR dogs, whereas S-glutathionylation occurred primarily in NaNO2-PO dogs. Conclusions: Intravenous infusion of nitrite profoundly reduced the severity of ventricular arrhythmias resulting from acute ischaemia and reperfusion in anaesthetized dogs. This effect, among several others, may result from an NO-mediated reduction in oxidative stress, perhaps through protein SNO and/or S-glutathionylation. [ABSTRACT FROM AUTHOR]

Despite blood transfusions are administered to restore adequate tissue oxygenation, transfusion guidelines consider only hemoglobin as trigger value, which gives little information about the balance between oxygen delivery and consumption. Central venous oxygen saturation is an alternative, however its changes reflect systemic metabolism and fail to detect regional hypoxia. A complementary parameter to ScvO2 may be central venous-to-arterial carbon dioxide difference (CO2-gap). Our aim was to investigate the change of alternative transfusion trigger values in experimental isovolemic anemia. After splenectomy, anesthetized Vietnamese mini pigs (n = 13, weight range: 18–30 kg) underwent controlled bleeding in five stages (T1–T5). During each stage approximately 10% of the estimated starting total blood volume was removed and immediately replaced with an equal volume of colloid. Hemodynamic measurements and blood gas analysis were then performed. Each stage of bleeding resulted in a significant fall in hemoglobin, the O2-extraction increased significantly from T3 and ScvO2 showed a similar pattern and dropped below the physiological threshold of 70% at T4. By T4 CO2-gap increased significantly and well correlated with VO2/DO2 and ScvO2. To our knowledge, this is the first study to show that anemia caused altered oxygen extraction may have an effect on CO2-gap. [ABSTRACT FROM AUTHOR]

Purpose: To detect the possible biochemical signs of inflammatory activation in the peripheral circulation in a rodent model of hippocampus irradiation, and to examine the effects of L-alpha-glycerylphosphorylcholine (GPC) in this experimental protocol. Materials and methods: Anesthetized Sprague-Dawley rats were subjected to 40 Gy cobalt irradiation of both hemispheres of the hippocampus, with or without GPC treatment (50 mg/kg intravenously (i.v.), 5 min before the irradiation, n = 6, each). A third group ( n = 6) served as saline-treated control. Blood samples were obtained 3 h after the end of irradiation in order to examine the changes in plasma histamine, tumor necrosis factor-alpha (TNF-α), interleukin 1-beta, interleukin 6 (IL-6) and interleukin 10 (IL-10); liver tissue samples were taken to determine adenosine triphosphate (ATP) concentrations. Results: The hepatic ATP levels were significantly declined, while plasma concentrations of circulating TNF-α, IL-6, IL-10 and histamine were significantly increased after hippocampus irradiation. GPC treatment significantly reduced the irradiation-induced release of cytokines and histamine, and the liver ATP level was maintained at the control value. Conclusions: Targeted brain irradiation produced measurable pro- and anti-inflammatory cytokine changes in the systemic circulation. GPC supplementation provides significant protection against irradiation-induced peripheral pro-inflammatory activation and ATP depletion. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated methane generation in aerobic cells. Our aims were to investigate the methanogenic features of sodium azide (NaN3)-induced chemical hypoxia in the whole animal and to study the effects of L-α-glycerylphosphorylcholine (GPC) on endogenous methane production and inflammatory events as indicators of a NaN3-elicited mitochondrial dysfunction. Group 1 of Sprague-Dawley rats served as the sham-operated control; in group 2, the animals were treated with NaN3 (14 mg·kg-1·day-1 sc) for 8 days. In group 3, the chronic NaN3 administration was supplemented with daily oral GPC treatment. Group 4 served as an oral antibiotic-treated control (rifaximin, 10 mg·kg-1·day-1) targeting the intestinal bacterial flora, while group 5 received this antibiotic in parallel with NaN3 treatment. The whole body methane production of the rats was measured by means of a newly developed method based on photoacoustic spectroscopy, the microcirculation of the liver was observed by intravital videomicroscopy, and structural changes were assessed via in vivo fluorescent confocal laser-scanning microscopy. NaN3 administration induced a significant inflammatory reaction and methane generation independently of the methanogenic flora. After 8 days, the hepatic microcirculation was disturbed and the ATP content was decreased, without major structural damage. Methane generation, the hepatic microcirculatory changes, and the increased tissue myeloperoxidase and xanthine oxidoreductase activities were reduced by GPC treatment. In conclusion, the results suggest that methane production in mammals is connected with hypoxic events associated with a mitochondrial dysfunction. GPC is protective against the inflammatory consequences of a hypoxic reaction that might involve cellular or mitochondrial methane generation. [ABSTRACT FROM AUTHOR]

Background: In the assessment of hypovolemia the value of functional hemodynamic monitoring during spontaneous breathing is debated. The aim of our study was to investigate in spontaneously breathing subjects the changes in hemodynamic parameters during graded central hypovolemia and to test whether slow patterned breathing improved the discriminative value of stroke volume (SV), pulse pressure (PP), and their variations (SVV, PPV). In addition, we tested the alterations in labial microcirculation. Methods: 20 healthy volunteers participated in our study. Central hypovolemia was induced by lower body negative pressure (LBNP). Continuous signals of ECG, non-invasive blood pressure and central venous pressure were recorded. During baseline and each stage of LBNP the labial microcirculation was investigated by orthogonal polarization spectral imaging, 3 minute periods of patterned breathing at 6 and 15/min respiratory rate were performed, and central venous blood gas analysis was done. Data from baseline and those of different LBNP levels were compared by analysis of variance and those of different breathing rates by t-test. Finally, we performed ROC analysis to assess the discriminative values of SV, PP, SVV and PPV. Results: Moderate central hypovolemia induced by LBNP caused significant, clinically relevant falls in PP (p < 0.05) and SV and central venous oxygen saturation (ScvO2) (p < 0.001). The proportion of perfused vessels (p < 0.001) and microvascular flow index decreased (p < 0.05). PPV increased (p < 0.001), however the magnitude of fluctuations was greater during slow patterned breathing (p < 0.001). SVV increased only during slow patterned breathing (p < 0.001). ROC analysis confirmed the best predictive value for SV (at 56 ml cut-off AUC 0.97, sensitivity 94%, specificity 95%). Slow patterned breathing improved the discriminative value of SVV (p = 0.0023). Conclusions: Functional hemodynamic monitoring with slow patterned breathing to control spontaneous respiration may be worthy for further study in different populations for the assessment of hypovolemia and the prediction of volume responsiveness. [ABSTRACT FROM AUTHOR]

Abstract: The role of endogenous nitric oxide (NO) in modulating myocardial contractility is still unclear, in part because of unknown, secondary effects of blocking NO release. We hypothesized that the nonspecific inhibition of nitric oxide synthase (NOS) enhances endothelin-1 (ET-1) effects, which can play a role in ET-A receptor-dependent myocardial contractile responses. The myocardial contractility was estimated from the slope of the left ventricular end-systolic pressure–diameter relationship in closed-chest, pentobarbital-anesthetized dogs. Group 1 (n =7) was the saline-treated control, while in groups 2 (n =7) and 3 (n =7) N-nitro-l-arginine (NNA, 4mgkg−1), a nonselective NOS blocker, was administered with or without pretreatment with the ET-A receptor antagonist ETR-P1/fl peptide (100nmolkg−1 iv). Plasma ET-1, nitrite/nitrate (NO x ) and blood superoxide levels were measured, and myocardial ET-1 content and xanthine oxidoreductase (XOR) activity were determined from myocardial biopsies. The infusion of NNA over 120min decreased the plasma NO x , significantly elevated the plasma ET-1 and blood superoxide levels, and in parallel greatly increased the left ventricular contractility as compared with the untreated controls [47.5 vs 30mmHgmm−1]. The myocardial ET-1 content decreased simultaneously, while the XOR activity and blood superoxide level were significantly elevated. These effects, including NNA-induced positive inotropy, were significantly suppressed by pretreatment with ETR-P1/fl peptide. These results demonstrate that a diminished NO synthesis leads to a preponderant ET-1 effect, which increases myocardial contractility through an ET-A receptor-dependent mechanism. [Copyright &y& Elsevier]