Imaging quantitative changes in blood-brain barrier permeability using [18F]2-fluoro-2-deoxy-sorbitol ([18F]FDS) PET in relation to glial cell recruitment in a mouse model of endotoxemia.

The quantitative relationship between the disruption of the blood-brain barrier (BBB) and the recruitment of glial cells was explored in a mouse model of endotoxemia. [18F]2-Fluoro-2-deoxy-sorbitol ([18F]FDS) PET imaging was used as a paracellular marker for quantitative monitoring of BBB permeability after i.v injection of increasing doses of lipopolysaccharide (LPS) or vehicle (saline, n = 5). The brain distribution of [18F]FDS (VT, mL.cm-3) was estimated using kinetic modeling. LPS dose-dependently increased the brain VT of [18F]FDS after injection of LPS 4 mg/kg (5.2 ± 2.4-fold, n = 4, p < 0.01) or 5 mg/kg (9.0 ± 9.1-fold, n = 4, p < 0.01) but not 3 mg/kg (p > 0.05, n = 7). In 12 individuals belonging to the different groups, changes in BBB permeability were compared with expression of markers of astrocyte (GFAP) and microglial cell (CD11b) using ex vivo immunohistochemistry. Increased expression of CD11b and GFAP expression was observed in mice injected with 3 mg/kg of LPS, which did not increase with higher LPS doses. Quantitative [18F]FDS PET imaging can capture different levels of BBB permeability in vivo. A biphasic effect was observed with the lowest dose of LPS that triggered neuroinflammation without disruptive changes in BBB permeability, and higher LPS doses that increased BBB permeability without additional recruitment of glial cells.

The Central Inflammatory Network: A Hypothalamic fMRI Study of Experimental Endotoxemia in Humans.

INTRODUCTION: Inflammation is a mechanism of the immune system that is part of the reaction to pathogens or injury. The central nervous system closely regulates inflammation via neuroendocrine or direct neuroimmune mechanisms, but our current knowledge of the underlying circuitry is limited. Therefore, we aimed to identify hypothalamic centres involved in sensing or modulating inflammation and to study their association with known large-scale brain networks. METHODS: Using high-resolution functional magnetic resonance imaging (fMRI), we recorded brain activity in healthy male subjects undergoing experimental inflammation from intravenous endotoxin. Four fMRI runs covered key phases of the developing inflammation: pre-inflammatory baseline, onset of endotoxemia, onset of pro-inflammatory cytokinemia, and peak of pro-inflammatory cytokinemia. Using masked independent component analysis, we identified functionally homogeneous subregions of the hypothalamus, which were further tested for changes in functional connectivity during inflammation and for temporal correlation with tumour necrosis factor and adrenocorticotropic hormone serum levels. We then studied the connection of these inflammation-associated hypothalamic subregions with known large-scale brain networks. RESULTS: Our results show that there are at least 6 hypothalamic subregions associated with inflammation in humans including the paraventricular nucleus, supraoptic nucleus, dorsomedial hypothalamus, bed nucleus of the stria terminalis, lateral hypothalamic area, and supramammillary nucleus. They are functionally embedded in at least 3 different large-scale brain networks, namely a medial frontoparietal network, an occipital-pericentral network, and a midcingulo-insular network. CONCLUSION: Measuring how the hypothalamus detects or modulates systemic inflammation is a first step to understand central nervous immunomodulation.

Quantitative Pulmonary Neutrophil Dynamics Using Computer-Vision Stabilized Intravital Imaging.

In vivo intravital imaging in animal models in the lung remains challenging owing to respiratory motion artifacts. Here we describe a novel intravital imaging approach based on the computer-vision stabilization algorithm, Computer-Vision Stabilized Intravital Imaging. This method corrects lung movements and deformations at submicron precision in respiring mouse lungs. The precision enables high-throughput quantitative analysis of intravital pulmonary polymorphonuclear neutrophil (PMN) dynamics in lungs. We quantified real-time PMN patrolling dynamics of microvessels in the basal state and PMN recruitment resulting from sequestration in a model of endotoxemia in mice. We focused on determining the marginated pool of PMNs in the lung. Direct visualization of marginated PMNs revealed that they are not static but highly dynamic and undergo repeated cycles of "catch and release." PMNs briefly arrest in larger diameter capillary junction (∼10 µm) and then squeeze into narrower, approximately 5-µm diameter vessels through PMN deformation. We also observed that the sequestered PMNs in lung microvessels lost their migratory capabilities in association with cell morphological change following prolonged endotoxemia. These observations underscore the value of direct visualization and quantitative analysis of PMN dynamics in lungs to study PMN physiology and pathophysiology and role in inflammatory lung injury.

Assessing long-term neuroinflammatory responses to encephalopathy using MRI approaches in a rat endotoxemia model.

Sepsis-associated encephalopathy (SAE) induces neuroinflammation, which is associated with cognitive impairment (CI). CI is also correlated with aging. We used contrast-enhanced magnetic resonance imaging (MRI), perfusion MRI, and MR spectroscopy to assess long-term alterations in BBB permeability, microvascularity, and metabolism, respectively, in a rat lipopolysaccharide-induced SAE model. Free radical-targeted molecular MRI was used to detect brain radical levels at 24 h and 1 week post-LPS injection. CE-MRI showed increased Gd-DTPA uptake in LPS rat brains at 24 h in cerebral cortex, hippocampus, thalamus, and perirhinal cortex regions. Increased MRI signal intensities were observed in LPS rat brains in cerebral cortex, perirhinal cortex, and hippocampus regions 1 week post-LPS. Long-term BBB dysfunction was detected in the cerebral cortex at 6 weeks post-LPS. Increased relative cerebral blood flow (rCBF) in cortex and thalamus regions at 24 h, decreased cortical and hippocampal rCBF at 6 weeks, decreased cortical rCBF at 3 and 12 weeks, and increased thalamus rCBF at 6 weeks post-LPS, were detected. MRS indicated that LPS-exposed rat brains had decreased: NAA/Cho metabolite ratios at 1, 3, 6, and 12 weeks; Cr/Cho at 1, 3, and 12 weeks; and Myo-Ins/Cho at 1, 3, and 6 weeks post-LPS. Free radical imaging detected increased radical levels in LPS rat brains at 24 h and 1 week post-LPS. LPS-exposed rats were compared to saline-treated controls. We clearly demonstrated BBB dysfunction, impaired vascularity, and decreased brain metabolites, as measures of long-term neuroinflammatory indicators, as well as increased free radicals in a LPS-induced rat SAE model.

Myocardial mechanical dysfunction following endotoxemia: role of changes in energy substrate metabolism.

Cardiovascular depression due to endotoxemia remains a major cause of mortality in intensive care patients. To determine whether drug-induced alterations in cardiac metabolism may be a viable strategy to reduce endotoxemia-mediated cardiac dysfunction, we assessed endotoxemia-induced changes in glucose and fatty acid metabolism under aerobic and post-ischemic conditions. Endotoxemia was induced in male Sprague-Dawley rats by lipopolysaccharide (Escherichia coli 0111:B4c, 4 mg/kg, i.p.) 6 h prior to heart removal for ex vivo assessment of left ventricular (LV) work and rates of glucose metabolism (glucose uptake, glycogen synthesis, glycolysis and glucose oxidation) and palmitate oxidation. Under aerobic conditions, endotoxemic hearts had impaired LV function as judged by echocardiography in vivo (% ejection fraction, 66.0 ± 3.2 vs 78.0 ± 2.1, p < 0.05) or by LV work ex vivo (2.14 ± 0.16 vs 3.28 ± 0.16, Joules min(-1) g dry wt(-1), p < 0.05). However, rates of glucose uptake, glycogen synthesis, glycolysis, and glucose oxidation were not altered. Palmitate oxidation was lower in endotoxemic hearts in proportion to the decreased workload, thus metabolic efficiency was unaffected. In hearts reperfused following global ischemia, untreated hearts had impaired recovery of LV work (52.3 ± 9.4 %) whereas endotoxemic hearts had significantly higher recovery (105.6 ± 11.3 %, p < 0.05). During reperfusion, fatty acid oxidation, acetyl CoA production and metabolic efficiency were similar in both groups. As impaired cardiac function appeared unrelated to depression of energy substrate oxidation, it is unlikely that drug-induced acceleration of fatty acid oxidation will improve mechanical function. The beneficial repartitioning of glucose metabolism in reperfused endotoxemic hearts may contribute to the cardioprotected phenotype.

EFFECTS OF EXPERIMENTAL HUMAN ENDOTOXEMIA ON DIAPHRAGM FUNCTION.

INTRODUCTION: Systemic inflammation is a well-known risk factor for respiratory muscle weakness. Studies using animal models of inflammation have shown that endotoxin administration induces diaphragm dysfunction. However, the effects of in vivo endotoxin administration on diaphragm function in humans have not been studied. Our aim was to evaluate diaphragm function in a model of systemic inflammation in healthy subjects. METHODS: Two groups of 12 male volunteers received an intravenous bolus of 2 ng/kg of Escherichia coli lipopolysaccharide (LPS) and were monitored until 8 h after LPS administration. In the first group, the twitch transdiaphragmatic pressure (Pditw) and compound muscle action potential of the diaphragm (CMAPdi) were measured. In addition, plasma levels of cytokines, heart rate, and arterial blood pressure were measured. In the second group, catecholamines as well as respiratory rate and blood gas values were measured. Diaphragm ultrasonography was performed in four subjects with severe shivering. RESULTS: Lipopolysaccharide administration resulted in flulike symptoms, hemodynamic alterations, and increased plasma levels of cytokines. The Pditw increased after LPS administration from 31.2 ±â€Š2.0 cmH2O (baseline) to 38.8 ±â€Š2.0 cmH2O (t = 1 h) and 35.4 ±â€Š2.0 cmH2O (t = 1.5 h). There was no correlation between cytokine plasma levels and the Pditw. We found a trend toward a gradual decrease in the CMAPdi from 0.78 ±â€Š0.07 mV (baseline) to 0.58 ±â€Š0.05 mV (t = 2 h). Respiratory rate increased after LPS administration from 16.8 ±â€Š0.5 breaths/min (baseline) to 20.3 ±â€Š0.6 breaths/min (t = 4 h), with a resulting decrease in PaCO2 of 0.5 ±â€Š0.1 kPa. Plasma levels of epinephrine peaked at t = 1.5 h, with an increase of 1.3 ±â€Š0.3 nmol/L from baseline. Rapid diaphragm contractions consistent with shivering were observed. CONCLUSIONS: This study shows that, in contrast to diaphragm dysfunction observed in animal models of inflammation, in vivo diaphragm contractility is augmented in the early phase after low-dose endotoxin administration in humans.

Depletion of myocardial glucose is observed during endotoxemic but not hemorrhagic shock in a porcine model.

INTRODUCTION: Metabolic dysfunction is one of the hallmarks of sepsis yet little is known about local changes in key organs such as the heart. The aim of this study was to compare myocardial metabolic changes by direct measurements of substrates, such as glucose, lactate and pyruvate, using microdialysis (MD) in in-vivo porcine endotoxemic and hemorrhagic shock. To assess whether these changes were specific to the heart, we simultaneously investigated substrate levels in skeletal muscle. METHODS: Twenty-six female pigs were randomized to three groups: control (C) n = 8, endotoxemic shock (E) n = 9 and hemorrhagic shock (H) n = 9. Interstitial myocardial pyruvate, lactate and glucose were measured using MD. Skeletal muscle MD was also performed in all three groups. RESULTS: Marked decreases in myocardial glucose were observed in the E group but not in the H group compared to controls (mean difference (CI) in mmol/L: C versus E -1.5(-2.2 to -0.8), P <0.001; H versus E -1.1(-1.8 to -0.4), P = 0.004; C versus H -0.4(-1.1 to 0.3), P = 0.282). Up to four-fold increases in myocardial pyruvate and three-fold increases in lactate were seen in both shock groups with no differences between the two types of shock. There was no evidence of myocardial anaerobic metabolism, with normal lactate:pyruvate (L:P) ratios seen in all animals regardless of the type of shock. CONCLUSIONS: Endotoxemia, but not hemorrhage, induces a rapid decrease of myocardial glucose levels. Despite the decrease in glucose, myocardial lactate and pyruvate concentrations were elevated and not different than in hemorrhagic shock. In skeletal muscle, substrate patterns during endotoxemic shock mimicked those seen in myocardium. During hemorrhagic shock the skeletal muscle response was characterized by a lack of increase in pyruvate and higher L:P ratios. Hence, metabolic patterns in the myocardium during endotoxemic shock are different than those seen during hemorrhagic shock. Skeletal muscle and myocardium displayed similar substrate patterns during endotoxemic shock but differed during hemorrhagic shock.

Erythropoietin attenuates cardiac dysfunction in experimental sepsis in mice via activation of the ß-common receptor.

There is limited evidence that the tissue-protective effects of erythropoietin are mediated by a heterocomplex of the erythropoietin receptor and the ß-common receptor ('tissue-protective receptor'), which is pharmacologically distinct from the 'classical' erythropoietin receptor homodimer that is responsible for erythropoiesis. However, the role of the ß-common receptor and/or erythropoietin in sepsis-induced cardiac dysfunction (a well known, serious complication of sepsis) is unknown. Here we report for the first time that the ß-common receptor is essential for the improvements in the impaired systolic contractility afforded by erythropoietin in experimental sepsis. Cardiac function was assessed in vivo (echocardiography) and ex vivo (Langendorff-perfused heart) in wild-type and ß-common receptor knockout mice, that were subjected to lipopolysaccharide (9 mg/kg body weight; young mice) for 16-18 hours or cecal ligation and puncture (aged mice) for 24 hours. Mice received erythropoietin (1000 IU/kg body weight) 1 hour after lipopolysaccharide or cecal ligation and puncture. Erythropoietin reduced the impaired systolic contractility (in vivo and ex vivo) caused by endotoxemia or sepsis in young as well as old wild-type mice in a ß-common-receptor-dependent fashion. Activation by erythropoietin of the ß-common receptor also resulted in the activation of well-known survival pathways (Akt and endothelial nitric oxide synthase) and inhibition of pro-inflammatory pathways (glycogen synthase kinase-3ß, nuclear factor-κB and interleukin-1ß). All the above pleiotropic effects of erythropoietin were lost in ß-common receptor knockout mice. Erythropoietin attenuates the impaired systolic contractility associated with sepsis by activation of the ß-common receptor, which, in turn, results in activation of survival pathways and inhibition of inflammation.

Postoperative splanchnic blood flow redistribution in response to fluid challenges in the presence and absence of endotoxemia in a porcine model.

We hypothesized that fluid administration may increase regional splanchnic perfusion after abdominal surgery-even in the absence of a cardiac stroke volume (SV) increase and independent of accompanying endotoxemia. Sixteen anesthetized pigs underwent abdominal surgery with flow probe fitting around splanchnic vessels and carotid arteries. They were randomized to continuous placebo or endotoxin infusion, and when clinical signs of hypovolemia (mean arterial pressure, <60 mmHg; heart rate, >100 beats · min(-1); urine production, <0.5 mL · kg(-1) · h(-1); arterial lactate concentration, >2 mmol · L(-1)) and/or low pulmonary artery occlusion pressure (target 5-8 mmHg) were present, they received repeated boli of colloids (50 mL) as long as SV increased 10% or greater. Stroke volume and regional blood flows were monitored 2 min before and 30 min after fluid challenges. Of 132 fluid challenges, 45 (34%) resulted in an SV increase of 10% or greater, whereas 82 (62%) resulted in an increase of 10% or greater in one or more of the abdominal flows (P < 0.001). During blood flow redistribution, celiac trunk (19% of all measurements) and hepatic artery flow (15%) most often decreased, whereas portal vein (10%) and carotid artery (7%) flow decreased less frequently (P = 0.015, between regions). In control animals, celiac trunk (30% vs. 9%, P = 0.004) and hepatic artery (25% vs. 11%, P = 0.040) flow decreased more often than in endotoxin-infused pigs. Accordingly, blood flow redistribution is a common phenomenon in the postoperative period and is only marginally influenced by endotoxemia. Fluid management based on SV changes may not be useful for improving regional abdominal perfusion.

Mild endotoxemia during mechanical ventilation produces spatially heterogeneous pulmonary neutrophilic inflammation in sheep.

BACKGROUND: There is limited information on the regional inflammatory effects of mechanical ventilation and endotoxemia on the production of acute lung injury. Measurement of F-fluorodeoxyglucose (F-FDG) uptake with positron emission tomography allows for the regional, in vivo and noninvasive, assessment of neutrophilic inflammation. The authors tested whether mild endotoxemia combined with large tidal volume mechanical ventilation bounded by pressures within clinically acceptable limits could yield measurable and anatomically localized neutrophilic inflammation. METHODS: Sheep were mechanically ventilated with plateau pressures = 30-32 cm H2O and positive end-expiratory pressure = 0 for 2 h. Six sheep received intravenous endotoxin (10 ng x kg x min), whereas six did not (controls), in sequentially performed studies. The authors imaged with positron emission tomography the intrapulmonary kinetics of infused N-nitrogen and F-FDG to compute regional perfusion and F-FDG uptake. Transmission scans were used to assess aeration. RESULTS: Mean gas fraction and perfusion distribution were similar between groups. In contrast, a significant increase in F-FDG uptake was observed in all lung regions of the endotoxin group. In this group, F-FDG uptake in the middle and dorsal regions was significantly larger than that in the ventral regions. Multivariate analysis showed that the F-FDG uptake was associated with regional aeration (P < 0.01) and perfusion (P < 0.01). CONCLUSIONS: Mild short-term endotoxemia in the presence of heterogeneous lung aeration and mechanical ventilation with pressures within clinically acceptable limits produces marked spatially heterogeneous increases in pulmonary neutrophilic inflammation. The dependence of inflammation on aeration and perfusion suggests a multifactorial basis for that finding. F-FDG uptake may be a sensitive marker of pulmonary neutrophilic inflammation in the studied conditions.

Detection of P-glycoprotein activity in endotoxemic rats by 99mTc-sestamibi imaging.

UNLABELLED: (99m)Tc-sestamibi is a widely used radiopharmaceutical agent for myocardial and oncologic imaging. Because of its unique role as a P-glycoprotein (Pgp)-specific substrate, this compound can be used to examine Pgp functional activity in vitro and in vivo under pathologic conditions. Our objective was to use (99m)Tc-sestamibi as a tool to investigate whether systemic inflammation induced by Escherichia coli lipopolysaccharide (LPS) would affect in vivo Pgp function in the brain, heart, liver, and kidneys of rats. Moreover, we also wanted to examine LPS-mediated effects in the placenta of pregnant rats because of the limited amount of in vivo data on this tissue. METHODS: Rats were injected intraperitoneally with LPS or an equal volume of saline as controls. After certain time periods (6 or 24 h), animals were administered 20 MBq of (99m)Tc-sestamibi intravenously, and then images were taken at 0.5, 1, 2, and 3 h. Tissues of rats were excised for (99m)Tc-sestamibi biodistribution analysis by gamma-counting and messenger RNA (mRNA) analysis by reverse transcription-polymerase chain reaction. Western blot analysis with antibody C-219 was used to detect Pgp levels. RESULTS: LPS treatment for 6 h caused a significant downregulation of mdr1a mRNA levels in the brain, heart, and liver, whereas 24 h of LPS treatment significantly reduced mdr1a mRNA levels only in the liver. A significant downregulation of mdr1a mRNA was seen in the brain, heart, and liver within 6 h after LPS administration. Imaging and biodistribution studies demonstrated a higher accumulation of (99m)Tc-sestamibi in the brain, heart, and liver of LPS-treated rats. In the brain, LPS-imposed downregulation of mdr1a mRNA levels was transient, with significant suppression at 4, 6, and 12 h, and the levels recovered to nearly normal by 24 h. This time-dependent downregulation of mRNA correlated with protein levels determined by Western blot analysis. Biodistribution studies of pregnant rats demonstrated a 3.5-fold-higher accumulation of (99m)Tc-sestamibi in the fetal tissues of LPS-treated pregnant rats than in saline-treated control rats. Furthermore, placental mdr1a and mdr1b mRNA levels were also significantly downregulated by LPS treatment. CONCLUSION: Our results indicate that LPS-induced systemic inflammation caused an increased retention of (99m)Tc-sestamibi in the brain, heart, liver, and fetal tissues. These results correlated with a reduction in mdr1a mRNA levels in each organ.

Endotoxemia alters splanchnic capacitance.

The splanchnic circulation constitutes a major portion of the total capacitance vasculature and may affect venous return and subsequently cardiac output during low output states. This study assessed the effects of rapid (10 microg/kg over 5 min) and slow (10 microg/kg over 60 min) induction of endotoxin (Escherichia coli) shock on splanchnic blood volume in 8 farm swine. Blood volume was measured by using Tc99m-labeled erythrocytes and radionuclide imaging. Baseline arterial pressure (MAP), central venous pressure (CVP), and liver, splenic, mesenteric and total splanchnic volumes were stable during the 30-min baseline. Approximately 30 min after the rapid endotoxin infusion, splenic volume decreased by 45%, whereas liver volume increased by 40% and MAP decreased by 60% (P < 0.01). The reduction in splenic volume occurred within 10 min of the endotoxin infusion, whereas liver volume changes occurred after MAP reduction. The slow endotoxin infusion also reduced splenic volume by approximately 50% (P = 0.05), whereas MAP declined by 30% (P < 0.05). However, the slow endotoxin infusion lowered liver volume (P < 0.05). Mesenteric volume was unaffected by the fast or slow endotoxin infusion. Total splanchnic volume was unaffected by the fast infusion but decreased by 37% in the slow infusion group (P < 0.05). In summary, E. coli endotoxin reduces splenic blood volume and increases liver blood volume after acute hypotension ensues. Endotoxin does not increase total splanchnic blood volume and may actually decrease total splanchnic volume in the absence of circulatory collapse. This endotoxin shock model is not associated with blood volume pooling in the splanchnic capacitance circulation.