Molecular phenomics of a high-calorie diet-induced porcine model of prepubertal obesity

As obesity incidence is alarmingly rising among young individuals, we aimed to characterize an experimental model of this situation, considering the similarity between human and porcine physiology. For this reason, we fed prepubertal (63 days-old) Duroc breed females (n=20) either with a standard growth diet (3800 KCal/day) or one with a high-calorie content (5200 KCal/day) during 70 days. Computerized tomography, mass-spectrometry based metabolomics, and lipidomics, as well as peripheral blood mononuclear cell transcriptomics, were applied to define traits linked to high-calorie intake. Samples from a human cohort confirmed potential lipidomic markers. Compared to those fed a standard growth diet, pigs fed a high-calorie diet showed an increased weight gain (13%), much higher adiposity (53%), hypertriacylglyceridemia and hypercholesterolemia, in parallel to insulin resistance. This diet induced marked changes in the circulating lipidome, particularly in phosphatidylethanolamine-type molecules. Also, circulating specific diacylglycerol and monoacylglycerol contents correlated with visceral fat and intrahepatic triacylglycerol concentrations. Specific lipids associated with obesity in swine (mainly belonging to glycerophospholipid, triacylglyceride, and sterol classes) were also linked with obesity-traits in the human cohort, reinforcing the usefulness of the chosen approach. Interestingly, no overt inflammation in plasma or adipose tissue was evident in this model. The presented model is useful as a preclinical surrogate of prepubertal obesity in order to ascertain the pathophysiology interactions between energy intake and obesity development.info:eu-repo/semantics/acceptedVersio

Molecular phenomics of a high-calorie diet-induced porcine model of prepubertal obesity

As obesity incidence is alarmingly rising among young individuals, we aimed to characterize an experimental model of this situation, considering the similarity between human and porcine physiology. For this reason, we fed prepubertal (63 days old) Duroc breed females (n=21) either with a standard growth diet (3800 kcal/day) or one with a high-calorie content (5200 kcal/day) during 70 days. Computerized tomography, mass-spectrometry-based metabolomics and lipidomics, as well as peripheral blood mononuclear cell transcriptomics, were applied to define traits linked to high-calorie intake. Samples from a human cohort confirmed potential lipidomic markers. Compared to those fed a standard growth diet, pigs fed a high-calorie diet showed an increased weight gain (13%), much higher adiposity (53%), hypertriacylglyceridemia and hypercholesterolemia in parallel to insulin resistance. This diet induced marked changes in the circulating lipidome, particularly in phosphatidylethanolamine-type molecules. Also, circulating specific diacylglycerol and monoacylglycerol contents correlated with visceral fat and intrahepatic triacylglycerol concentrations. Specific lipids associated with obesity in swine (mainly belonging to glycerophospholipid, triacylglyceride and sterol classes) were also linked with obesity traits in the human cohort, reinforcing the usefulness of the chosen approach. Interestingly, no overt inflammation in plasma or adipose tissue was evident in this model. The presented model is useful as a preclinical surrogate of prepubertal obesity in order to ascertain the pathophysiology interactions between energy intake and obesity development.Supported by Centro para el Desarrollo Tecnológico e Industrial, Spain, Project reference: IPT-20111008, and Generalitat de Catalunya grants 2017SGR1719 and 2017SGR696. MJ is a "Serra Hunter" program fellow. Supported by Instituto de Salud Carlos III, Spain, Project reference: 17-00134, co-financed by FEDER Funds A way to make Europe

Evidence for Status Epilepticus and Pro-Inflammatory Changes after Intranasal Kainic Acid Administration in Mice.

Kainic acid (KA) is routinely used to elicit status epilepticus (SE) and epileptogenesis. Among the available KA administration protocols, intranasal instillation (IN) remains understudied. Dosages of KA were instilled IN in mice. Racine Scale and Video-EEG were used to assess and quantify SE onset. Time spent in SE and spike activity was quantified for each animal and confirmed by power spectrum analysis. Immunohistochemistry and qPCR were performed to define brain inflammation occurring after SE, including activated microglial phenotypes. Long term video-EEG recording was also performed. Titration of IN KA showed that a dose of 30 mg/kg was associated with low mortality while eliciting SE. IN KA provoked at least one behavioral and electrographic SE in the majority of the mice (>90%). Behavioral and EEG SE were accompanied by a rapid and persistent microglial-astrocytic cell activation and hippocampal neurodegeneration. Specifically, microglial modifications involved both pro- (M1) and anti-inflammatory (M2) genes. Our initial long-term video-EEG exploration conducted using a small cohort of mice indicated the appearance of spike activity or SE. Our study demonstrated that induction of SE is attainable using IN KA in mice. Typical pro-inflammatory brain changes were observed in this model after SE, supporting disease pathophysiology. Our results are in favor of the further development of IN KA as a means to study seizure disorders. A possibility for tailoring this model to drug testing or to study mechanisms of disease is offered

Neurodegeneration and neuroinflammatory gene profile after IN KA induced seizures.

<p><b>A-C)</b> Fluoro-Jade C staining is observed 24h after IN KA, diminishing at 72h. Correspondence existed between presence of FJC positive neurons and behavioral score after IN KA (B: Mouse 9, mean-score = 5.8; C: Mouse 28, mean-score = 3.8). Scale bar 50 μm. <b>D-F)</b> qPCR analysis and changes of inflammatory gene levels in mice experiencing stage 5 or higher. Analysis was performed 24h (white bars) and 72h (grey bars) after IN KA. Panels (D) and (E) represent mRNA changes for M1 and M2 microglial markers. Red bars indicate genes that are not detectable under control conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150793#sec002" target="_blank">materials and methods</a> section). Results are presented as mean ± SEM (n ≥ 7 / group). Statistical analysis was performed using a non-parametric Mann-Whitney test between control and KA-treated conditions. *: p<0.05; ** p<0.01; ***: p<0.001 compared to controls.</p

Number of mice included in the different analyses (stage≥5 Racine).

<p>Number of mice included in the different analyses (stage≥5 Racine).</p

Time-dependent microgliosis and astrogliosis following IN KA induced seizures.

<p>Analysis performed in mice experiencing stage 5 or higher. <b>A-D)</b> Representative images of IBA1 immunostaining (upper panels) and GFP fluorescence (C57BL6/J CX3CR1^+/eGFP mice; lower panels) in CA1 region in control mice (A), and 24h (B), 72h (C) and 15 days (D) after IN KA. Scale bar = 50 μm. Insets depict enlarged image of individual microglial cells. Scale bar = 10 μm <b>E-F)</b> Quantitative analysis of IBA1 immunostaining in the hippocampus 24h, 72h and 15 days after IN KA-induced SE shows significant microglial activation, including increased cell number and size. <b>G-I)</b> Representative images of GFAP immunostaining in CA1 region in control mice (G), 72h (H) and 15 days (I) after IN KA. Scale bar = 20 μm. <b>J)</b> Quantitative analysis of GFAP immunostaining in the hippocampus shows astrogliosis 72h after IN KA. Results are represented as mean ± SEM (n ≥ 6 per group). Statistical analysis was performed using a non-parametric Kruskal-Wallis one-way analysis of variance followed by Dunn’s post-test. *: p<0.05; **: p<0.01; ***: p<0.001 compared to control condition.</p

Lack of long-term microglia and astrocytes reactivity.

<p><b>A-C)</b> Representative images of IBA1 immunostaining in CA1 region in control implanted mice, IN KA mice and mouse #C (see below). Scale bar = 50 μm. <b>D-E)</b> IBA1 analysis shows no significant microglial activation in KA mice compared to control mice. Mouse #C experienced severe SE in and displayed elevated microglia density (<i>red data</i> in D). <b>F-H)</b> Representative images of GFAP immunostaining in control implanted mice, KA- mice and mouse C. Scale bar = 50 μm. <b>I)</b> Quantitative analysis of GFAP immunostaining in the hippocampus shows no difference at 49 days post KA. Results are presented as mean ± SEM. Statistical analyses were performed using a non-parametric Mann-Whitney test.</p

Video-EEG analysis during seizure progression.

<p><b>A)</b> Heat map (blue to red) and raw numbers indicate the sum of durations (in seconds) of spike activity recorded for each animal in the given recording session. <b>B-C)</b> Examples of EEG recordings during the chronic phase (<i>e</i>.<i>g</i>., SE in B and spike activity in C).</p