Lionel Fizanne

The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota

Several animal studies have emphasized the role of gut microbiota in nonalcoholic fatty liver disease (NAFLD). However, data about gut dysbiosis in human NAFLD remain scarce in the literature, especially studies including the whole spectrum of NAFLD lesions. We aimed to evaluate the association between gut dysbiosis and severe NAFLD lesions, that is, nonalcoholic steatohepatitis (NASH) and fibrosis, in a well‐characterized population of adult NAFLD. Fifty‐seven patients with biopsy‐proven NAFLD were enrolled. Taxonomic composition of gut microbiota was determined using 16S ribosomal RNA gene sequencing of stool samples. Thirty patients had F0/F1 fibrosis stage at liver biopsy (10 with NASH), and 27 patients had significant F≥2 fibrosis (25 with NASH). Bacteroides abundance was significantly increased in NASH and F≥2 patients, whereas Prevotella abundance was decreased. Ruminococcus abundance was significantly higher in F≥2 patients. By multivariate analysis, Bacteroides abundance was independently associated with NASH and Ruminococcus with F≥2 fibrosis. Stratification according to the abundance of these two bacteria generated three patient subgroups with increasing severity of NAFLD lesions. Based on imputed metagenomic profiles, Kyoto Encyclopedia of Genes and Genomes pathways significantly related to NASH and fibrosis F≥2 were mostly related to carbohydrate, lipid, and amino acid metabolism. Conclusion: NAFLD severity associates with gut dysbiosis and a shift in metabolic function of the gut microbiota. We identified Bacteroides as independently associated with NASH and Ruminococcus with significant fibrosis. Thus, gut microbiota analysis adds information to classical predictors of NAFLD severity and suggests novel metabolic targets for pre‐/probiotics therapies. (Hepatology 2016;63:764–775)

Detrimental hemodynamic and inflammatory effects of microparticles originating from septic rats.

Objective:Microparticles (MPs) are membrane vesicles with procoagulant and proinflammatory properties released during cell activation and might be potentially involved in the pathophysiology of septic shock. This study was designed to assess the effects of MPs from septic origin on the systemic hemodynamics as well as on the inflammatory, oxidative, and nitrosative stresses. Design:A prospective, randomized, controlled experimental study with repeated measurements. Setting:Investigational animal laboratory. Subjects:Forty healthy rats were randomly allocated to three groups: 10 animals inoculated with MPs isolated from control rats (cMPs), 15 animals inoculated with MPs isolated from sham rats (shMPs), and 15 animals inoculated with MPs isolated from rats with peritonitis (sMPs). Interventions:Rats were anesthetized, mechanically ventilated, and infused with the same amount of cMPs, shMPs, or sMPs. We measured the heart rate, mean arterial pressure, carotid artery, and portal vein blood flows. Hemodynamic parameters were recorded during 7 hours, and then animals were killed. Aorta and heart were harvested for further in vitro tissue analyses. Measurements and Main Results:1) The cellular origin (phenotype) but not the circulating concentration of MPs was different in septic rats, characterized by a significant increase in leukocyte-derived MPs. 2) sMPs but not cMPs or shMPs decreased mean arterial pressure without any effect on carotid artery and portal vein blood flows. 3) Rats inoculated with sMPs exhibited an increase in superoxide ion production and nuclear factor kappa B activity, overexpression of inducible nitric oxide synthase with subsequent nitric oxide overproduction and decrease in endothelial nitric oxide synthase activation. Conclusions:Rats with sepsis induced by peritonitis exhibited a specific phenotype of MPs. Inoculation of sMPs in healthy rats reproduced hemodynamic, septic inflammatory patterns, associated with oxidative and nitrosative stresses.

Effects of hydrogen sulfide on hemodynamics, inflammatory response and oxidative stress during resuscitated hemorrhagic shock in rats

IntroductionHydrogen sulfide (H2S) has been shown to improve survival in rodent models of lethal hemorrhage. Conversely, other authors have reported that inhibition of endogenous H2S production improves hemodynamics and reduces organ injury after hemorrhagic shock. Since all of these data originate from unresuscitated models and/or the use of a pre-treatment design, we therefore tested the hypothesis that the H2S donor, sodium hydrosulfide (NaHS), may improve hemodynamics in resuscitated hemorrhagic shock and attenuate oxidative and nitrosative stresses.MethodsThirty-two rats were mechanically ventilated and instrumented to measure mean arterial pressure (MAP) and carotid blood flow (CBF). Animals were bled during 60 minutes in order to maintain MAP at 40 ± 2 mm Hg. Ten minutes prior to retransfusion of shed blood, rats randomly received either an intravenous bolus of NaHS (0.2 mg/kg) or vehicle (0.9% NaCl). At the end of the experiment (T = 300 minutes), blood, aorta and heart were harvested for Western blot (inductible Nitric Oxyde Synthase (iNOS), Nuclear factor-κB (NF-κB), phosphorylated Inhibitor κB (P-IκB), Inter-Cellular Adhesion Molecule (I-CAM), Heme oxygenase 1(HO-1), Heme oxygenase 2(HO-2), as well as nuclear respiratory factor 2 (Nrf2)). Nitric oxide (NO) and superoxide anion (O2-) were also measured by electron paramagnetic resonance.ResultsAt the end of the experiment, control rats exhibited a decrease in MAP which was attenuated by NaHS (65 ± 32 versus 101 ± 17 mmHg, P < 0.05). CBF was better maintained in NaHS-treated rats (1.9 ± 1.6 versus 4.4 ± 1.9 ml/minute P < 0.05). NaHS significantly limited shock-induced metabolic acidosis. NaHS also prevented iNOS expression and NO production in the heart and aorta while significantly reducing NF-kB, P-IκB and I-CAM in the aorta. Compared to the control group, NaHS significantly increased Nrf2, HO-1 and HO-2 and limited O2- release in both aorta and heart (P < 0.05).ConclusionsNaHS is protective against the effects of ischemia reperfusion induced by controlled hemorrhage in rats. NaHS also improves hemodynamics in the early resuscitation phase after hemorrhagic shock, most likely as a result of attenuated oxidative stress. The use of NaHS hence appears promising in limiting the consequences of ischemia reperfusion (IR).

Evidence for the involvement of VPAC1 and VPAC2 receptors in pressure-induced vasodilatation in rodents

A transient increase in skin blood flow in response to an innocuous local pressure application, defined as pressure‐induced vasodilatation (PIV), delays the occurrence of ischaemia, suggesting a protective feature against applied pressure. The PIV response depends on capsaicin‐sensitive nerve fibres and calcitonin gene‐related peptide (CGRP) has been shown to be involved. In these fibres, CGRP coexists with pituitary adenylate cyclase‐activating polypeptide (PACAP). Three distinct receptors mediate the biological effects of PACAP: VPAC1 and VPAC2 receptors binding with the same affinity for PACAP and vasoactive intestinal peptide and PAC1 receptors showing high selectivity for PACAP. Because the receptors are widely expressed in the nervous system and in the skin, we hypothesized that at least one of them is involved in PIV development. To verify this hypothesis, we used [d‐p‐Cl‐Phe6,Leu17]‐VIP (nonspecific antagonist of VPAC1/VPAC2 receptors), PG 97‐269 (antagonist of VPAC1 receptors), PACAP(6–38) (antagonist of VPAC2/PAC1 receptors) and Max.d.4 (antagonist of PAC1 receptors) in anaesthetized rodents. The blockade of VPAC1/VPAC2, VPAC1 or VPAC2/PAC1 receptors eliminated the PIV response, whereas PAC1 blockade had no effect, demonstrating an involvement of VPAC1/VPAC2 receptors in PIV development. Moreover, endothelium‐independent and ‐dependent vasodilator responses were unchanged by the VPAC1/VPAC2 antagonist. Thus, the absence of a PIV response following VPAC1/VPAC2 blockade cannot be explained by any dysfunction of the vascular smooth muscle or endothelial vasodilator capacity. The involvement of VPAC1/VPAC2 receptors in the development of PIV seems to imply a series relationship in which each receptor type (CGRP, VPAC1, VPAC2) is necessary for the full transmission of the response.

Effect of isoflurane on skin-pressure-induced vasodilation

Since general anesthesia has been shown to attenuate endothelium-dependent vasodilation, it was of interest to verify whether general anesthesia would modify skin vasodilation in response to local pressure application, which is endothelium dependent. To study the effect of general anesthesia on pressure-induced vasodilation development, we examined the effects of low- and high-dose isoflurane. Skin blood flow was measured by laser Doppler flowmetry during 11.1 Pa s–1 increases in locally applied pressure in anesthetized rats treated with low or high doses of isoflurane. Following the administration of low doses of isoflurane, skin blood flow increased from baseline, with increasing local pressure application (+37 ± 10% at 2.0 kPa). The increase in skin blood flow was absent in rats treated with high doses (–20 ± 5% at 2.0 kPa), even when the anesthesia-induced hypotension was corrected by gelofusine infusion (–20 ± 10% at 2.0 kPa). Whereas sodium-nitroprusside-induced vasodilation developed following low and high doses of isoflurane, acetylcholine-induced vasodilation was impaired with high doses compared to low doses. These data show that pressure-induced vasodilation is abolished with high doses of anesthetics. It is not the anesthesia-induced hypotension, but the depth of anesthesia, which can lead to the disappearance of pressure-induced vasodilation by an alteration in endothelial function.

Signal processing methodology to study the cutaneous vasodilator response to a local external pressure application detected by laser Doppler flowmetry

The existence of a cutaneous pressure-induced vasodilation (PIV) has recently been reported. This paper proposes a signal processing methodology to improve PIV knowledge. Temporal variations of laser Doppler signals rhythmic activities are first analyzed on anesthetized rats. The results lead to a method that provides a better PIV understanding.

The combination of everolimus and zoledronic acid increase the efficacy of gemcitabine in a mouse model of pancreatic adenocarcinoma

Background Gemcitabine is a standard treatment for pancreatic adenocarcinoma. Many mechanisms are involved in gemcitabine resistance, such as reduced expression of the human equilibrative nucleoside transporter 1 (hENT1) membrane transporter, deoxycytidine kinase deficiency, and changes in the signal transmission of mitogen-activity protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) pathways. Aim To evaluate the anti-tumor efficiency of blocking signaling pathways using combined action of gemcitabine, everolimus and zoledronic acid versus gemcitabine alone in a mouse subcutaneous xenograft. Methods Implantations of two human pancreatic adenocarcinoma cells lines (PANC1, K-ras mutated and gemcitabine-resistant; and BxPc3, wild-type K-ras and gemcitabine-sensitive) were performed on male athymic nude mice. The mice received different treatments: gemcitabine, gemcitabine plus everolimus, everolimus, gemcitabine plus zoledronic acid, everolimus plus zoledronic acid, or gemcitabine plus everolimus and zoledronic acid, for 28 days. We measured the tumor volume and researched the expression of the biomarkers involved in the signaling pathways or in gemcitabine resistance. Results In wild-type K-ras tumors, the combinations of gemcitabine plus everolimus; zoledronic acid plus everolimus; and gemcitabine plus zoledronic acid and everolimus slowed tumor growth, probably due to caspase-3 overexpression and reduced Annexin II expression. In mutated K-ras tumors, gemcitabine plus everolimus and zoledronic acid, and the combination of zoledronic acid and everolimus, decreased tumor volume as compared to gemcitabine alone, inhibiting the ERK feedback loop induced by everolimus. Conclusion The combination of zoledronic acid and everolimus has an antitumor effect and could increase gemcitabine efficacy.

Laser Doppler flowmetry signals to quantify effects of isoflurane on the peripheral cardiovascular system of healthy rats

The optical Doppler effect resulting from interactions between laser light photons and red blood cells of the microcirculation is used to characterize the influence of isoflurane, an halogenated volatile anesthetic, on the peripheral cardiovascular system. After having recorded laser Doppler flowmetry blood perfusion signals on isoflurane-induced anesthetized healthy rats, wavelet analyses show a significant decrease of the myogenic and neurogenic activities when isoflurane dose increases from 1.5% to 3%. Moreover, the approximate entropy shows a weak decrease of signal irregularity when dose of isoflurane increases. These findings demonstrate the usefulness of the optical Doppler effect in physiological and pharmacological applications.

Laser Doppler flowmetry in healthy rats: impact of isoflurane anesthetic on signal complexity

Laser Doppler flowmetry (LDF) technique is an optical tool used in clinical investigations to monitor microvascular blood flow. Recent preliminary works have shown that LDF signals recorded in young healthy human subjects are weakly multifractal. Such an information is important as it could lead to a better knowledge of the underlying optical processes giving rise to the signals. In the present work, our goal is to analyze the behavior of LDF signals in anesthesia conditions. For this purpose, we herein study the possible modifications brought by isoflurane, an anesthetic commonly used in clinical practice, on the complexity of LDF signals. In order to conduct our work, twenty LDF signals from anesthetized healthy rats are processed. Anesthesia is performed by using doses of isoflurane varying between 1.5% and 3%, which leads to very light and very deep anesthesia, respectively. The signal processing approach is carried out with two different methods, a parametric generalized quadratic variation based estimation method and a Hurst rescaled range analysis. The results show that extreme doses of isoflurane lead to no distinguishable modification on the characterization of LDF signals based on the two above approaches. These findings infer that, if isoflurane changes the microvascular tissue optical properties, these modifications have no influence on LDF signals complexity measured by the two signal processing approaches used herein.