Frederic Chagnon

Endotoxin-induced myocardial dysfunction: effects of macrophage migration inhibitory factor neutralization.

The pathophysiology of sepsis-induced myocardial dysfunction still remains controversial. Macrophage migration inhibitory factor (MIF) has recently been identified as a cardiac-derived myocardial depressant factor in septic shock. Putative mechanisms by which MIF affects cardiac function are unknown. In an investigation of possible mechanisms of action, a rat model of endotoxin toxicity was designed using intraperitoneal (I/P) injection of lipopolysaccharides (LPS) with or without coinfusion of neutralizing anti-MIF or isotypic-matched antibodies. Echocardiographic evaluation revealed that MIF neutralization reversed endotoxin-induced myocardial dysfunction at 24 hours after injection. RNase protection assay (RPA) and Western blot established that MIF neutralization prevented LPS-induced mRNA expression and production of heart-derived inflammatory paracrine and autocrine cytokines such as IL-1s and IL-6. Moreover, MIF immunoneutralization increased heart Bcl-2/Bax protein ratio and suppressed endotoxin-induced release of mitochondrial cytochrome-c, as demonstrated by Western blotting. Inhibition of mitochondrial loss of cytochrome-c decreased in heart caspase-3 activity at 6 and 24 hours after injection. MIF neutralization also restored the LPS-induced deficient nuclear translocation of phospho-Akt and consequently the expression of the heart survival nuclear factor GATA-4. The restoration of the translocation/expression of survival factors by MIF inhibition resulted in lowered endotoxin-induced DNA fragmentation at 24 hours, a hallmark of downstream cardiomyocyte apoptosis. Our data indicate that early inactivation of MIF significantly reverses the imbalance of proapoptotic to prosurvival pathways and reduces acute inflammation of the heart thereby improving myocardial dysfunction induced by endotoxin.

The D-dopachrome tautomerase (DDT) gene product is a cytokine and functional homolog of macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor (MIF) is a pivotal regulator of the immune response. Neutralization or genetic deletion of MIF does not completely abrogate activation responses, however, and deletion of the MIF receptor, CD74, produces a more pronounced phenotype than MIF deficiency. We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a probable candidate to be the protein encoded by the homologous, D-dopachrome tautomerase (D-DT) gene. We show that recombinant D-DT protein binds CD74 with high affinity, leading to activation of ERK1/2 MAP kinase and downstream proinflammatory pathways. Circulating D-DT levels correlate with disease severity in sepsis or malignancy, and the specific immunoneutralization of D-DT protects mice from lethal endotoxemia by reducing the expression of downstream effector cytokines. These data indicate that D-DT is a MIF-like cytokine with an overlapping spectrum of activities that are important for our understanding of MIF-dependent physiology and pathology.

Endotoxin-induced heart dysfunction in rats: assessment of myocardial perfusion and permeability and the role of fluid resuscitation.

Objective:The pathophysiology of sepsis-induced myocardial dysfunction is still controversial. Whether microcirculatory hypoperfusion together with capillary leakage can occur in the heart wall also remains a matter of debate. The objective was to evaluate the impact of fluid resuscitation on endotoxin-induced myocardial dysfunction. Design:Adult rats were given intraperitoneal injection of endotoxin (lipopolysaccharide, Escherichia coli, 10 mg/kg) or phosphate-buffered solution, followed up by echocardiography and acetate micro-positron emission tomography scan imaging, together with final hemodynamic, biochemical, and pathologic evaluations up to 48 hrs. Setting:University laboratory. Subjects:Pathogen-free male Wistar rats (350 g). Interventions:Influence of isovolumic fluid infusion type (saline vs. pentastarch) on these variables was assessed in 11 groups of six animals including an unchallenged control one. Measurements and Main Results:Endotoxin injection induced a) myocardial dysfunction (decrease of ∼15–20% in left ventricular ejection fraction); b) ventricular enlargement (∼1.5- to 1.7-fold increase in left ventricular systolic volume); c) cardiac output increase (10–15%); d) myocardial hypoperfusion (∼1.5- to 2-fold decrease in acetate k1 constant rate); e) increased oxygen consumption (k2); and f) interstitial wall increase. Endotoxin injection also enhanced levels of arterial lactates and troponin I. Colloid (pentastarch) over crystalloid (saline) fluid resuscitation significantly reversed echocardiographic changes, some positron emission tomography imaging alterations, and lactate and troponin I levels without further enhancing interstitial spaces. Conclusion:Endotoxin can induce reversible myocardial alterations with evidence of coronary hypoperfusion and heart wall enlargement/damage, some of which can be prevented by fluid resuscitation. The use of crystalloids is less beneficial than pentastarch.

Proven infection-related sepsis induces a differential stress response early after ICU admission

IntroductionNeuropeptides arginine-vasopressin (AVP), apelin (APL), and stromal-derived factor-1α (SDF-1α) are involved in the dysfunction of the corticotropic axis observed in septic ICU patients. Study aims were: (i) to portray a distinctive stress-related neuro-corticotropic systemic profile of early sepsis, (ii) to propose a combination data score, for aiding ICU physicians in diagnosing sepsis on admission.MethodsThis prospective one-center observational study was carried out in a medical intensive care unit (MICU), tertiary teaching hospital. Seventy-four out of 112 critically ill patients exhibiting systemic inflammatory response syndrome (SIRS) were divided into two groups: proven sepsis and non sepsis, based on post hoc analysis of microbiological criteria and final diagnosis, and compared to healthy volunteers (n = 14). A single blood sampling was performed on admission for measurements of AVP, copeptin, APL, SDF-1α, adrenocorticotropic hormone (ACTH), cortisol baseline and post-stimulation, and procalcitonin (PCT).ResultsBlood baseline ACTH/cortisol ratio was lower and copeptin higher in septic vs. nonseptic patients. SDF-1α was further increased in septic patients vs. normal patients. Cortisol baseline, ACTH, PCT, APACHE II and sepsis scores, and shock on admission, were independent predictors of sepsis diagnosis upon admission. Using the three first aforementioned categorical bio-parameters, a probability score for predicting sepsis yielded an area under the Receiver Operating Curve (ROC) curves better than sepsis score or PCT alone (0.903 vs 0.727 and 0.726: P = 0.005 and P < 0.04, respectively).ConclusionsThe stress response of early admitted ICU patients is different in septic vs. non-septic conditions. A proposed combination of variable score analyses will tentatively help in refining bedside diagnostic tools to efficiently diagnose sepsis after further validation.

In vivo intravital endoscopic confocal fluorescence microscopy of normal and acutely injured rat lungs

We present a new lung imaging technique based on endoscopic confocal fluorescence microscopy (ECFM), which is a new method that is able to provide cellular and structural assessment of living tissue using a small confocal probe in direct contact with the visceral pleura. To observe distal airspace structure and cellular condition in normal and injured lungs (hyperoxic and bleomycin challenged), we used fluorescent-specific marker contrast and ECFM. Alveolar space ECFM with spectral analyses were performed at 488-nm excitation using FITC-labeled markers or naturally fluorescent dyes. The normal lung was compared with the sick lung, where our in vivo imaging experiments correlated well with results obtained with corresponding ex vivo conventional assays. Four main elements pertaining to the acute lung injury/acute respiratory distress syndrome (ALI/ARDS) pathophysiology and established early key events were specifically studied: alveolar epithelial membrane phenotype, lung cell apoptosis, neutrophil recruitment, and edema. ECFM allowed visualization of (i) fine-tuned ultrastructural lectin (RCA-1) and sialoglycoprotein (RTI40) epithelial cell membrane expression, (ii) YO-PRO-1-related DNA linking of lung cell apoptosis, (iii) PKH2 green fluorescent cell linker-labeled neutrophil tracking in lung microcirculatory network and airspaces, (iv) FITC-dextran plasma contrast and extravasation with edema formation. ECFM provides reliable results to corresponding ex vivo fluorescent methods. ECFM, using the minimally invasive Five-1® optical instrument and specific fluorescent markers, is able to provide real-time potentially useful imaging of live unfixed normal and injured lung tissue with promising developments for improving bedside diagnostic and decision-making therapeutic strategy in patients with ALI.

Modulation of aquaporin-2/vasopressin2 receptor kidney expression and tubular injury after endotoxin (lipopolysaccharide) challenge

Objective:Sepsis-induced organ dysfunctions remain prevalent and account for >50% of intensive care unit admissions for acute renal failure with a mortality rate nearing 75%. In addition to the fact that the mechanisms underlying the pathophysiology of sepsis-related acute renal failure are unclear, the impact on septic-induced acute renal failure of either norepinephrine, a gold-standard vasopressor, and arginine vasopressin, a candidate alternative, are not well understood. Design:Randomized and controlled in vivo study. Setting:Research laboratory and animal facilities. Subjects:Adult rats treated with endotoxin (lipopolysaccharide) and/or vasopressors. Interventions:Rats were intraperitoneally injected with lipopolysaccharide (12 mg/kg) or saline and then infused with either saline, 0.375 &mgr;g/&mgr;L arginine vasopressin, or 32.5 &mgr;g/&mgr;L norepinephrine for 18 hrs. These vasopressor rates yielded respective targeted blood levels observed in human septic shock. Measurements and Main Results:Renal function, including glomerular filtration rate and fraction, renal blood flow, aquaporin-2, and arginine vasopressin-2 (V2 receptor) networking, water and salt handling, and urinary protein excretion, were evaluated. After lipopolysaccharide challenge arginine vasopressin infusion: 1) impaired creatinine clearance without affecting renal blood flow, glomerular filtration rate, and fraction but reduced free-water clearance, both of which being partially restored by the V2 receptor antagonist SR-121463B; 2) decreased the recognized ability of arginine vasopressin alone to recruit aquaporin-2 to the apical membrane increase its mRNA expression and urinary release; 3) increased urinary protein content but decreased specific kidney injury molecule-1, and Clara cell protein-16 release (p < 0.05 vs. lipopolysaccharide alone). Conversely, norepinephrine infusion did not add to lipopolysaccharide-induced alteration of urine biochemistry, except for improved creatinine clearance and increased microalbuminuria. Conclusion:In this endotoxic model, dose-targeted arginine vasopressin infusion increased lipopolysaccharide-induced renal dysfunction without affecting renal blood flow and glomerular function, but with particular disruption of aquaporin-2/V2 receptor networking, consecutive decreased salt and water handling ability. This is in clear contrast with norepinephrine infusion and suggests specific arginine vasopressin-induced “tubular epithelial dysfunction.”

Resuscitation fluids and endotoxin-induced myocardial dysfunction: is selection a load-independent differential issue?

ABSTRACT Along with redistributive shock, myocardial dysfunction is now recognized as highly prevalent in early severe sepsis. Indeed, aside from their distinct loading potency, resuscitation fluids have been poorly investigated as to their specific molecular impact on myocardial dysfunction. The objective of this study was to evaluate the load-independent biological impact of different resuscitation fluids on endotoxin-induced myocardial dysfunction. Adult rats implanted with a central venous catheter were given an intraperitoneal injection of endotoxin (lipopolysaccharides [LPSs], Escherichia coli, 10 mg/kg) or normal saline (sham) and subsequently infused or not with similar “fluid potency” loading resuscitation fluid (normal saline, albumin [Alb], or hypertonic saline solution) for 6 to 24 h, followed by echocardiographic and hemodynamic monitoring together with biochemical and histopathologic evaluation. Intervention was to assess the selective influence of load-independent fluid infusion on the aforementioned parameters in groups of animals challenged or not with LPS. At comparative plasma volumes, Alb improved myocardial homeostasis after LPS challenge by (i) reducing left ventricular relative wall diastolic thickness, interstitial space enlargement, and endogenous Alb content; (ii) limiting cardiac apoptosis and sustaining extracellular signal–regulated mitogen-activated protein kinase activation; and (iii) enhancing the expression pattern of heme-oxygenase 1/inducible nitric oxide synthase. Hypertonic saline solution was also cardioprotective by early prevention of myocardial dysfunction and by reducing cardiac apoptosis. Fluid infusions have distinct load-independent structural/biological impacts on endotoxin-induced myocardial dysfunction. Albumin and hypertonic saline solution are the most pleiotropic fluids in protecting the heart after a “sepsis” hit. ABBREVIATIONSLPSs — lipopolysaccharidesNS — normal salineAlb — albuminHO-1 — heme oxygenase 1iNOS — inducible nitric oxide synthaseNO — nitric oxideLVEDd — left ventricular end-diastolic diameterLVESd — left ventricular end-systolic diameterFS — fractional shorteningIVSD — end-diastolic interventricular septumLVRWD — end-diastolic left ventricular relative wallI-R – ischemia-reperfusionCO — cardiac outputPVR — peripheral vascular resistance

Tracking of Mesenchymal Stem Cells with Fluorescence Endomicroscopy Imaging in Radiotherapy-Induced Lung Injury

Mesenchymal stem cells (MSCs) have potential for reducing inflammation and promoting organ repair. However, limitations in available techniques to track them and assess this potential for lung repair have hindered their applicability. In this work, we proposed, implemented and evaluated the use of fluorescence endomicroscopy as a novel imaging tool to track MSCs in vivo. MSCs were fluorescently labeled and injected into a rat model of radiation-induced lung injury via endotracheal (ET) or intravascular (IV) administration. Our results show that MSCs were visible in the lungs with fluorescence endomicroscopy. Moreover, we developed an automatic cell counting algorithm to quantify the number of detected cells in each condition. We observed a significantly higher number of detected cells in ET injection compared to IV and a slight increase in the mean number of detected cells in irradiated lungs compared to control, although the latter did not reach statistical significance. Fluorescence endomicroscopy imaging is a powerful new minimally invasive and translatable tool that can be used to track and quantify MSCs in the lungs and help assess their potential in organ repair.

ELABELA Improves Cardio-Renal Outcome in Fatal Experimental Septic Shock

Objectives: Apelin-13 was recently proposed as an alternative to the recommended &bgr;-adrenergic drugs for supporting endotoxin-induced myocardial dysfunction. Since Apelin-13 signals through its receptor (Apelin peptide jejunum) to exert singular inotropic/vasotropic actions and to optimize body fluid balance, this candidate pathway might benefit septic shock management. Whether the newly discovered ELABELA (ELA), a second endogenous ligand of the Apelin peptide jejunum receptor highly expressed in the kidney, further improves cardio-renal impairment remains unknown. Design, Setting, and Subjects: Interventional study in a rat model of septic shock (128 adult males) to assess the effects of ELA and Apelin-13 on vascular and cardio-renal function. Experiments were performed in a tertiary care University-based research institute. Interventions: Polymicrobial sepsis-induced cardiac dysfunction was produced by cecal ligation puncture to assess hemodynamic efficacy, cardioprotection, and biomechanics under acute or continuous infusions of the apelinergic agonists ELA or Apelin-13 (39 and 15 µg/kg/hr, respectively) versus normal saline. Measurements and Main Results: Apelinergic agonists improved 72-hour survival after sepsis induction, with ELA providing the best clinical outcome after 24 hours. Apelinergic agonist infusion counteracted cecal ligation puncture–induced myocardial dysfunction by improving left ventricular pressure-volume relationship. ELA-treated cecal ligation puncture rats were the only group to 1) display a significant improvement in left ventricular filling as shown by increased E-wave velocity and left ventricular end-diastolic volume, 2) exhibit a higher plasma volume, and 3) limit kidney injury and free-water clearance. These beneficial renal effects were superior to Apelin-13, likely because full-length ELA enabled a distinctive regulation of pituitary vasopressin release. Conclusions: Activation of the apelinergic system by exogenous ELA or Apelin-13 infusion improves cardiovascular function and survival after cecal ligation puncture–induced sepsis. However, ELA proved better than Apelin-13 by improving fluid homeostasis, cardiovascular hemodynamics recovery, and limiting kidney dysfunction in a vasopressinergic-dependent manner.

Apelin Compared With Dobutamine Exerts Cardioprotection and Extends Survival in a Rat Model of Endotoxin-Induced Myocardial Dysfunction.

Objective: Dobutamine is the currently recommended &bgr;-adrenergic inotropic drug for supporting sepsis-induced myocardial dysfunction when cardiac output index remains low after preload correction. Better and safer therapies are nonetheless mandatory because responsiveness to dobutamine is limited with numerous side effects. Apelin-13 is a powerful inotropic candidate that could be considered as an alternative noncatecholaminergic support in the setting of inflammatory cardiovascular dysfunction. Design: Interventional controlled experimental animal study. Setting: Tertiary care university-based research institute. Subjects: One hundred ninety-eight adult male rats. Interventions: Using a rat model of “systemic inflammation–induced cardiac dysfunction” induced by intraperitoneal lipopolysaccharide injection (10 mg/kg), hemodynamic efficacy, cardioprotection, and biomechanics were assessed under IV osmotic pump infusions of apelin-13 (0.25 &mgr;g/kg/min) or dobutamine (7.5 &mgr;g/kg/min). Measurements and Main Results: In this model and in both in vivo and ex vivo studies, apelin-13 compared with dobutamine provoked distinctive effects on cardiac function: 1) optimized cardiac energy–dependent workload with improved cardiac index and lower vascular resistance, 2) upgraded hearts’ apelinergic responsiveness, and 3) consecutive downstream advantages, including increased urine output, enhanced plasma volume, reduced weight loss, and substantially improved overall outcomes. In vitro studies confirmed that these apelin-13–driven processes encompassed a significant and rapid reduction in systemic cytokine release with dampening of myocardial inflammation, injury, and apoptosis and resolution of associated molecular pathways. Conclusions: In this inflammatory cardiovascular dysfunction, apelin-13 infusion delivers distinct and optimized hemodynamic support (including positive fluid balance), along with cardioprotective effects, modulation of circulatory inflammation and extended survival.