Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling.

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation "two-pore channel" (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2-/-) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2-/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

ß1-adrenergic blockers preserve neuromuscular function by inhibiting the production of extracellular traps during systemic inflammation in mice.

Severe inflammation via innate immune system activation causes organ dysfunction. Among these, the central nervous system (CNS) is particularly affected by encephalopathies. These symptoms are associated with the activation of microglia and a potential infiltration of leukocytes. These immune cells have recently been discovered to have the ability to produce extracellular traps (ETs). While these components capture and destroy pathogens, deleterious effects occur such as reduced neuronal excitability correlated with excessive ETs production. In this study, the objectives were to determine (1) whether immune cells form ETs in the CNS during acute inflammation (2) whether ETs produce neuromuscular disorders and (3) whether an immunomodulatory treatment such as ß1-adrenergic blockers limits these effects. We observed an infiltration of neutrophils in the CNS, an activation of microglia and a production of ETs following lipopolysaccharide (LPS) administration. Atenolol, a ß1-adrenergic blocker, significantly decreased the production of ETs in both microglia and neutrophils. This treatment also preserved the gastrocnemius motoneuron excitability. Similar results were observed when the production of ETs was prevented by sivelestat, an inhibitor of ET formation. In conclusion, our results demonstrate that LPS administration increases neutrophils infiltration into the CNS, activates immune cells and produces ETs that directly impair neuromuscular function. Prevention of ETs formation by ß1-adrenergic blockers partly restores this function and could be a good target in order to reduce adverse effects in severe inflammation such as sepsis but also in other motor related pathologies linked to ETs production.

A druggable copper-signalling pathway that drives inflammation.

Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

Oligonucleotide Enhancing Compound Increases Tricyclo-DNA Mediated Exon-Skipping Efficacy in the Mdx Mouse Model.

Nucleic acid-based therapeutics hold great promise for the treatment of numerous diseases, including neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). Some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA for DMD, but the potential of this therapy is still limited by several challenges, including the poor distribution of ASOs to target tissues, but also the entrapment of ASO in the endosomal compartment. Endosomal escape is a well recognized limitation that prevents ASO from reaching their target pre-mRNA in the nucleus. Small molecules named oligonucleotide-enhancing compounds (OEC) have been shown to release ASO from endosomal entrapment, thus increasing ASO nuclear concentration and ultimately correcting more pre-mRNA targets. In this study, we evaluated the impact of a therapy combining ASO and OEC on dystrophin restoration in mdx mice. Analysis of exon-skipping levels at different time points after the co-treatment revealed improved efficacy, particularly at early time points, reaching up to 4.4-fold increase at 72 h post treatment in the heart compared to treatment with ASO alone. Significantly higher levels of dystrophin restoration were detected two weeks after the end of the combined therapy, reaching up to 2.7-fold increase in the heart compared to mice treated with ASO alone. Moreover, we demonstrated a normalization of cardiac function in mdx mice after a 12-week-long treatment with the combined ASO + OEC therapy. Altogether, these findings indicate that compounds facilitating endosomal escape can significantly improve the therapeutic potential of exon-skipping approaches offering promising perspectives for the treatment of DMD.

Extracellular traps formation following cervical spinal cord injury.

Spinal cord injuries involve a primary injury that can lead to permanent loss of function and a secondary injury associated with pathologic and inflammatory processes. Extracellular traps are extracellular structures expressed by immune cells that are primarily composed of chromatin, granular enzymes and histones. Extracellular traps are known to induce tissue damage when overexpressed and could be associated in the occurrence of secondary damage. In the present study, we used flow cytometry to demonstrate that at 1 day following a C2 spinal cord lateral hemisection in male Swiss mice, resident microglia form vital microglia extracellular traps, and infiltrating neutrophils form vital neutrophil extracellular traps. We also used immunolabelling to show that microglia near the lesion area are most likely to form these microglia extracellular traps. As expected, infiltrating neutrophils are located at the site of injury, though only some of them engage in post-injury extracellular trap formation. We also observed the formation of microglia and neutrophil extracellular traps in our sham animal models of durotomy, but formation was less frequent than following the C2 hemisection. Our results demonstrate for the first time that microglia form extracellular traps in the spinal cord following injury and durotomy. It remains however to determine the exact mechanisms and kinetics of neutrophil and microglia extracellular traps formation following spinal cord injury. This information would allow to better mitigate this inflammatory process that may contribute to secondary injury and to effectively target extracellular traps to improve functional outcomes following spinal cord injury.

Effects of C2 hemisection on respiratory and cardiovascular functions in rats.

High cervical spinal cord injuries induce permanent neuromotor and autonomic deficits. These injuries impact both central respiratory and cardiovascular functions through modulation of the sympathetic nervous system. So far, cardiovascular studies have focused on models of complete contusion or transection at the lower cervical and thoracic levels and diaphragm activity evaluations using invasive methods. The present study aimed to evaluate the impact of C2 hemisection on different parameters representing vital functions (i.e., respiratory function, cardiovascular, and renal filtration parameters) at the moment of injury and 7 days post-injury in rats. No ventilatory parameters evaluated by plethysmography were impacted during quiet breathing after 7 days post-injury, whereas permanent diaphragm hemiplegia was observed by ultrasound and confirmed by diaphragmatic electromyography in anesthetized rats. Interestingly, the mean arterial pressure was reduced immediately after C2 hemisection, with complete compensation at 7 days post-injury. Renal filtration was unaffected at 7 days post-injury; however, remnant systolic dysfunction characterized by a reduced left ventricular ejection fraction persisted at 7 days post-injury. Taken together, these results demonstrated that following C2 hemisection, diaphragm activity and systolic function are impacted up to 7 days post-injury, whereas the respiratory and cardiovascular systems display vast adaptation to maintain ventilatory parameters and blood pressure homeostasis, with the latter likely sustained by the remaining descending sympathetic inputs spared by the initial injury. A better broad characterization of the physiopathology of high cervical spinal cord injuries covering a longer time period post-injury could be beneficial for understanding evaluations of putative therapeutics to further increase cardiorespiratory recovery.

Evaluation of Gastrocnemius Motor Evoked Potentials Induced by Trans-Spinal Magnetic Stimulation Following Tibial Nerve Crush in Rats.

Peripheral nerve injuries induce long-lasting physiological and severe functional impairment due to motor, sensory, and autonomic denervation. Preclinical models allow us to study the process of nerve damage, evaluate the capacity of the peripheral nervous system for spontaneous recovery, and test diagnostic tools to assess the damage and subsequent recovery. Methods: In this study on Sprague-Dawley rats, we: (1) compared the use of two different anesthetics (isoflurane and urethane) for the evaluation of motor evoked potentials (MEPs) induced by trans-spinal magnetic stimulation (TSMS) in gastrocnemius and brachioradialis muscles; (2) monitored the evolution of gastrocnemius MEPs by applying paired-pulse stimulation to evaluate the neuromuscular junction activity; and (3) evaluated the MEP amplitude before and after left tibialis nerve crush (up to 7 days post-injury under isoflurane anesthesia). The results showed that muscle MEPs had higher amplitudes under isoflurane anesthesia, as compared with urethane anesthesia in the rats, demonstrating higher motoneuronal excitability under isoflurane anesthesia evaluated by TSMS. Following tibial nerve crush, a significant reduction in gastrocnemius MEP amplitude was observed on the injured side, mainly due to axonal damage from the initial crush. No spontaneous recovery of MEP amplitude in gastrocnemius muscles was observed up to 7 days post-crush; even a nerve section did not induce any variation in residual MEP amplitude, suggesting that the initial crush effectively severed the axonal fibers. These observations were confirmed histologically by a drastic reduction in the remaining myelinated fibers in the crushed tibial nerve. These data demonstrate that TSMS can be reliably used to noninvasively evaluate peripheral nerve function in rats. This method could therefore readily be applied to evaluate nerve conductance in the clinical environment.

Effect of an enteral amino acid blend on muscle and gut functionality in critically ill patients: a proof-of-concept randomized controlled trial.

BACKGROUND: A defining feature of prolonged critical illness is muscle wasting, leading to impaired recovery. Supplementation with a tailored blend of amino acids may bolster the innate gut defence, promote intestinal mucosa repair and limit muscle loss. METHODS: This was a monocentric, randomized, double-blind, placebo-controlled study that included patients with sepsis or acute respiratory distress syndrome. Patients received a specific combination of five amino acids or placebo mixed with enteral feeding for 21 days. Markers of renal function, gut barrier structure and functionality were collected at baseline and 1, 2, 3 and 8 weeks after randomization. Muscle structure and function were assessed through MRI measurements of the anterior quadriceps volume and by twitch airway pressure. Data were compared between groups relative to the baseline. RESULTS: Thirty-five critically ill patients were randomized. The amino acid blend did not impair urine output, blood creatinine levels or creatinine clearance. Plasma citrulline levels increased significantly along the treatment period in the amino acid group (difference in means [95% CI] 5.86 [1.72; 10.00] nmol/mL P = 0.007). Alanine aminotransferase and alkaline phosphatase concentrations were lower in the amino acid group than in the placebo group at one week (ratio of means 0.5 [0.29; 0.86] (P = 0.015) and 0.73 [0.57; 0.94] (P = 0.015), respectively). Twitch airway pressure and volume of the anterior quadriceps were greater in the amino acid group than in the placebo group 3 weeks after randomization (difference in means 10.6 [0.99; 20.20] cmH20 (P = 0.035) and 3.12 [0.5; 5.73] cm3/kg (P = 0.022), respectively). CONCLUSIONS: Amino acid supplementation increased plasma citrulline levels, reduced alanine aminotransferase and alkaline phosphatase levels, and improved twitch airway pressure and anterior quadriceps volume. Trial registration ClinicalTrials.gov, NCT02968836. Registered November 21, 2016.

Effects of Chronic High-Frequency rTMS Protocol on Respiratory Neuroplasticity Following C2 Spinal Cord Hemisection in Rats.

High spinal cord injuries (SCIs) lead to permanent diaphragmatic paralysis. The search for therapeutics to induce functional motor recovery is essential. One promising noninvasive therapeutic tool that could harness plasticity in a spared descending respiratory circuit is repetitive transcranial magnetic stimulation (rTMS). Here, we tested the effect of chronic high-frequency (10 Hz) rTMS above the cortical areas in C2 hemisected rats when applied for 7 days, 1 month, or 2 months. An increase in intact hemidiaphragm electromyogram (EMG) activity and excitability (diaphragm motor evoked potentials) was observed after 1 month of rTMS application. Interestingly, despite no real functional effects of rTMS treatment on the injured hemidiaphragm activity during eupnea, 2 months of rTMS treatment strengthened the existing crossed phrenic pathways, allowing the injured hemidiaphragm to increase its activity during the respiratory challenge (i.e., asphyxia). This effect could be explained by a strengthening of respiratory descending fibers in the ventrolateral funiculi (an increase in GAP-43 positive fibers), sustained by a reduction in inflammation in the C1-C3 spinal cord (reduction in CD68 and Iba1 labeling), and acceleration of intracellular plasticity processes in phrenic motoneurons after chronic rTMS treatment. These results suggest that chronic high-frequency rTMS can ameliorate respiratory dysfunction and elicit neuronal plasticity with a reduction in deleterious post-traumatic inflammatory processes in the cervical spinal cord post-SCI. Thus, this therapeutic tool could be adopted and/or combined with other therapeutic interventions in order to further enhance beneficial outcomes.

CD38-NADase is a new major contributor to Duchenne muscular dystrophic phenotype.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration. Two important deleterious features are a Ca2+ dysregulation linked to Ca2+ influxes associated with ryanodine receptor hyperactivation, and a muscular nicotinamide adenine dinucleotide (NAD+ ) deficit. Here, we identified that deletion in mdx mice of CD38, a NAD+ glycohydrolase-producing modulators of Ca2+ signaling, led to a fully restored heart function and structure, with skeletal muscle performance improvements, associated with a reduction in inflammation and senescence markers. Muscle NAD+ levels were also fully restored, while the levels of the two main products of CD38, nicotinamide and ADP-ribose, were reduced, in heart, diaphragm, and limb. In cardiomyocytes from mdx/CD38-/- mice, the pathological spontaneous Ca2+ activity was reduced, as well as in myotubes from DMD patients treated with isatuximab (SARCLISA® ) a monoclonal anti-CD38 antibody. Finally, treatment of mdx and utrophin-dystrophin-deficient (mdx/utr-/- ) mice with CD38 inhibitors resulted in improved skeletal muscle performances. Thus, we demonstrate that CD38 actively contributes to DMD physiopathology. We propose that a selective anti-CD38 therapeutic intervention could be highly relevant to develop for DMD patients.

High frequency repetitive Transcranial Magnetic Stimulation promotes long lasting phrenic motoneuron excitability via GABAergic networks.

Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABAA and GABAB receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.

Analysis of inspiratory and expiratory muscles using ultrasound in rats: A reproducible and non-invasive tool to study respiratory function.

Ultrasound imaging is a non-invasive technique to assess organ function. Its potential application in rodents to evaluate respiratory function remains poorly investigated. We aimed to assess and validate ultrasound technique in rats to analyze inspiratory and expiratory muscles. We measured respiratory parameters to provide normal eupneic values. Histological studies and plethysmography were used to validate the technique and assess the physiological implications. A linear relationship was observed between ultrasound and histological data for diaphragm and rectus abdominis (RA) measurement. The tidal volume was significantly correlated with the right + left RA area (r = 0.76, p < 0.001), and the rapid shallow breathing index was significantly and inversely correlated with the right + left RA area (r=-0.53, p < 0.05). In the supine position, the right and left diaphragm expiratory thickness were not associated with tidal volume obtained in the physiological position. Ultrasound imaging is highly accurate and reproducible to assess and follow up diaphragm and RA structure and function in rats.

Permanent diaphragmatic deficits and spontaneous respiratory plasticity in a mouse model of incomplete cervical spinal cord injury.

High spinal cord injuries (SCI) lead to permanent respiratory insufficiency, and the search for new therapeutics to restore this function is essential. To date, the most documented preclinical model for high SCI is the rat cervical C2 hemisection. However, molecular studies with this SCI model are limited due to the poor availability of genetically modified specimens. The aim of this work was to evaluate the pathophysiology of respiratory activity following a cervical C2 injury at different times post-injury in a C57BL/6 mouse model. No significant spontaneous recovery of diaphragmatic activity was observed up to 30 days post-injury in eupneic condition. However, during a respiratory challenge, i.e. mild asphyxia, a partial restoration of the injured diaphragm was observed at 7 days post-injury, corresponding to the crossed phrenic phenomenon. Interestingly, the diaphragmatic recording between 2 respiratory bursts on the injured side showed an amplitude increase between 1-7 days post-injury, reflecting a change in phrenic motoneuronal excitability. This increase in inter-burst excitability returned to pre-injured values when the crossed phrenic phenomenon started to be effective at 7 days post-injury. Taken together, these results demonstrate the ability of the mouse respiratory system to express long-lasting plasticity following a C2 cervical hemisection and genetically modified animals can be used to study the pathophysiological effects on these plasticity phenomena.

Diaphragm: Pathophysiology and Ultrasound Imaging in Neuromuscular Disorders.

Respiratory muscles are classically involved in neuromuscular disorders, leading to a restrictive respiratory pattern. The diaphragm is the main respiratory muscle involved during inspiration. Ultrasound imaging is a noninvasive, radiation-free, accurate and safe technique allowing assessment of diaphragm anatomy and function. The authors review the pathophysiology of diaphragm in neuromuscular disorders, the methodology and indications of diaphragm ultrasound imaging as well as possible pitfalls in the interpretation of results.

Aldosterone and Vascular Mineralocorticoid Receptors in Murine Endotoxic and Human Septic Shock.

OBJECTIVES: Vascular mineralocorticoid receptors play a role in vascular tone and blood pressure regulation, might participate in the pathophysiology of circulatory failure during sepsis, and represent a potential therapeutic target in this disease. We aimed to study the effects of mineralocorticoids and the involvement of vascular mineralocorticoid receptors in murine endotoxic and human septic shock. DESIGN: Experimental study. SETTING: Translational investigation including animal research and in vitro experiments using human vascular cells and plasma from septic patients. SUBJECTS: Adult male C57Black 6 mice, adult patients with septic shock. INTERVENTIONS: Mice were injected with lipopolysaccharide and/or aldosterone. Human endothelial and smooth muscle cells were treated with pro-inflammatory cytokines with or without aldosterone, nuclear factor-κB inhibitor BAY 11-7082, or plasma from septic patients. MEASUREMENTS AND MAIN RESULTS: Aldosterone improved 5-day survival, invasive arterial pressure, and in vivo and ex vivo arterial response to phenylephrine at 18 hours after induction of murine endotoxic shock. Both α1-adrenoceptor and mineralocorticoid receptor expressions studied in mouse aortas were down-regulated at 6 and 18 hours in endotoxemic mice and restored in aldosterone-treated mice. Furthermore, tumor necrosis factor-α decreased both mineralocorticoid receptor and α1-adrenoceptor expressions within 5 hours in human vascular cells in a nuclear factor-κB pathway-dependent manner. Mineralocorticoid receptor expression was also blunted in human cells treated with plasma from septic patients. CONCLUSION: We found a beneficial effect of mineralocorticoids on survival, blood pressure, and vascular reactivity, associated with a restoration of α1-adrenoceptor expression in endotoxic shock. Furthermore, blunted vascular mineralocorticoid receptor expression might participate in hemodynamic failure during sepsis.

Effects of esmolol on systemic and pulmonary hemodynamics and on oxygenation in pigs with hypodynamic endotoxin shock.

PURPOSE: The aim of this experimental study is to investigate cardiovascular tolerance of blockade of beta-adrenergic receptors in an endotoxin model. DESIGN: Prospective, randomized, controlled study. SETTING: Animal laboratory in a university medical center. METHODS: Ten anesthetized, mechanically ventilated pigs were challenged with intravenous lipopolysaccharide (LPS) to achieve a status of profound hypodynamic shock. Systemic and pulmonary hemodynamics and cardiac output were continuously monitored throughout the 5-h study period, and blood samples were taken at baseline (T - 30 min), 1 h from the beginning of LPS infusion (T + 60 min), and every 2 h (T + 180 min and T + 300 min). Animals were randomly assigned to continuous intravenous esmolol infusion titrated to decrease heart rate by 20% or isotonic saline. RESULTS: Esmolol decreased heart rate by 20%, while in the saline group, heart rate increased by 7% and 22% at T + 180 min and T + 300 min, respectively (p < 0.001). In esmolol-treated animals, cardiac index decreased by 9% at T + 180 min and by 2% at T + 300 min, and in controls by 14% at T + 180 min and by 27% at T + 300 min (p = 0.870). In esmolol-treated animals, median (interquartile range, IQR) stroke index was 31 (6) and 47 (11) ml/min/m(2) at T + 180 min and T + 300 min, respectively, and decreased steadily from 45 (20) to 18 (13) ml/min/m(2) in controls (p = 0.030). There were no significant differences between groups for any other hemodynamics variables, except for systemic vascular resistance (SVR) (p = 0.017). CONCLUSIONS: In large animals with endotoxemic shock, continuous infusion of esmolol, a selective beta-1 adrenergic blocker, titrated to decrease heart rate by 20%, was well tolerated and may offset LPS-induced cardiac dysfunction by a preload positive effect.

Human and experimental septic shock are characterized by depletion of lipid droplets in the adrenals.

RATIONALE: Cholesteryl ester deficiency which results in adrenal lipid store depletion has been proposed as a potential mechanism of sepsis associated adrenal insufficiency. OBJECTIVE: We investigated histological abnormalities associated with sepsis in human and mice adrenals. METHODS: From January 2006 to 2008, seven patients who died of septic shock and seven patients with rapidly fatal nonseptic illness were included. Adrenals were sampled within 12 h from death. Adrenals were also taken from 13 lipopolysaccharide (LPS)-challenged mice, 5 cecal ligation and puncture (CLP) mice and 5 controls. We semi-quantitatively analysed intensity of inflammation, necrosis, haemorrhage and lipid depletion. MEASUREMENTS AND MAIN RESULTS: In patients, lipid depletion scores were significantly higher in septic shock than in controls (p = 0.011). In animals, lipid depletion was higher following LPS or CLP than in controls (p = 0.003). In adrenal cortex, in patients and not in animals, global scores for inflammation (p = 0.002), necrosis (p = 0.009) and haemorrhage (p = 0.009) were significantly higher in septic shock than in controls. Similarly, in zona fasciculata, in patients and not in animals, scores for inflammation (p = 0.007), necrosis (p = 0.023) and haemorrhage (p = 0.023) were significantly higher in septic shock than in controls. CONCLUSIONS: This study shows that diffuse lipid depletion in zona fasciculata is a hallmark of human septic shock, experimental endotoxaemia and sepsis. In patients, sepsis was associated with inflammation, necrosis and haemorrhage predominantly in zona fasciculata.

Constriction of rat extra-splenic veins to lipopolysaccharide involves endothelin-1.

The spleen has an important role in blood volume regulation and increased resistance of post-capillary hilar veins (in mesentery adjoining the spleen) can regulate this. This study investigated whether venular constriction to lipopolysaccharide (LPS) involved endothelin-1 (ET-1). Pressure myography was used to study isolated extra-splenic (hilar) vessels from male Wistar rats (n = 111). Arteries and veins were treated with LPS (50 microg ml(-1)) for 4 h. Extra-splenic veins constricted to LPS (p < 0.05), but there was no effect on arteries. Denudation did not abolish venular constriction to LPS, indicating an endothelial independent mechanism. However, the dual ET-1 receptor antagonist bosentan (10(-5) M) and specific ET(A) and ET(B) antagonists ABT-627 (atrasentan, 6.3 x 10(-6) M) and A-192621(1.45 x 10(-6) M) completely abolished constriction of LPS-treated veins. ET-1 alone also constricted the extra-splenic arteries and veins (p < 0.05), with a greater response observed in veins (p < 0.05). ELISA also confirmed that serum and spleen levels of ET-1 increased in response to LPS (p < 0.05). That LPS-induced constriction of extra-splenic veins is mediated by ET-1. Greater constriction of post- versus pre-capillary extra-splenic vessels to LPS would result in increased intra-splenic fluid extravasation and hypovolaemia in vivo.

Bench-to-bedside review: Beta-adrenergic modulation in sepsis.

Sepsis, despite recent therapeutic progress, still carries unacceptably high mortality rates. The adrenergic system, a key modulator of organ function and cardiovascular homeostasis, could be an interesting new therapeutic target for septic shock. Beta-adrenergic regulation of the immune function in sepsis is complex and is time dependent. However, beta2 activation as well as beta1 blockade seems to downregulate proinflammatory response by modulating the cytokine production profile. beta1 blockade improves cardiovascular homeostasis in septic animals, by lowering myocardial oxygen consumption without altering organ perfusion, and perhaps by restoring normal cardiovascular variability. Beta-blockers could also be of interest in the systemic catabolic response to sepsis, as they oppose epinephrine which is known to promote hyperglycemia, lipid and protein catabolism. The role of beta-blockers in coagulation is less clear cut. They could have a favorable role in the septic pro-coagulant state, as beta1 blockade may reduce platelet aggregation and normalize the depressed fibrinolytic status induced by adrenergic stimulation. Therefore, beta1 blockade as well as beta2 activation improves sepsis-induced immune, cardiovascular and coagulation dysfunctions. Beta2 blocking, however, seems beneficial in the metabolic field. Enough evidence has been accumulated in the literature to propose beta-adrenergic modulation, beta1 blockade and beta2 activation in particular, as new promising therapeutic targets for septic dyshomeostasis, modulating favorably immune, cardiovascular, metabolic and coagulation systems.

Macromolecular leak from extrasplenic lymphatics during endotoxemia.

BACKGROUND: The spleen has an important physiological role in maintaining blood volume; this study aimed to determine whether during pathophysiological circumstances, namely endotoxemia, the extrasplenic pathway is dysfunctional. We hypothesize that increased 'leakiness' of lymphatics in response to lipopolysaccharide (LPS) provides a route for loss of protein-rich fluid into third spaces and prevents the spleen from maintaining blood volume homeostasis. METHODS AND RESULTS: Male Wistar rats (200-280 g, n = 24) were anesthetized with thiopental (40-90 mg x kg(-1) x hr(-1), i.v.) to study the extrasplenic (vessels in mesentery adjoining the spleen) and ileal mesenteric microcirculation using fluorescently labeled albumin (66 KDa FITC-BSA, 0.02 g.100 g(-1), i.v.) with intravital microscopy. LPS (150 microg x kg(-1) x hr(-1) i.v.) induced constriction of rat extrasplenic venules (-14 +/- 2.4% from 40.4 +/- 7.8 microm, p < 0.05) and no change in arteriolar diameter (-4.6 +/- 4.7% from 32.6 +/- 4.3 microm). As the spleen is freely permeable to protein, a greater increase in venular versus arteriolar extrasplenic resistance increases intrasplenic capillary hydrostatic pressure, leading to fluid efflux into the lymphatics, draining the spleen. In agreement we report here increased extrasplenic venular resistance with LPS and lymphatic dilation to accommodate this fluid (13.5 +/- 6% from 18.5 +/- 4.8 microm, p < 0.05). However, the extrasplenic pathway then appeared to dysfunction, with macromolecular leak from extrasplenic venules (24.6 +/- 6.4%, p < 0.05) and lymphatics (12.1 +/- 3.4%, p < 0.05), indicated by increased interstitial FITC-BSA fluorescence. This was less than from ileal mesenteric venules (324 +/- 32%, p < 0.05). There was a concurrent decrease in mean arterial pressure (T(180): -15.1 +/- 6.9% from MAP of 130.3 +/- 8.8 mmHg at T(0), p < 0.05). CONCLUSION: Lymphatics are generally considered to demonstrate unidirectional and inward uptake of large molecules. However, during endotoxemia, we have demonstrated that extrasplenic lymphatics also allow the leakage of large protein molecules out into interstitial spaces. Fluid losses from extrasplenic lymphatics could therefore contribute to hypovolemia and hypotension associated with sepsis.