determined by the Folin-Ciocalteu assay [ 19 ]. The detailed
composition of BE is given in Supplemental figure S1.
Animals and diets
Pairs of homozygous apoE-deficientmice were pur- chased from Jackson Laboratories (Charles River Labo- ratories, L’Arbresle, France) and interbred to obtain the males used for the present study. Mice were individually housed in wire-bottomed cages in a temperature-con- trolled room (22 ± 0.8C) with a 12-h light–dark cycle and a relative humidity of 55 ± 10% and had free access to food and water. All animals were maintained and handled according to the recommendations of the Insti- tutional Ethics Committee of the INRA, in accordance with decree No 87-848. They were all fed with a standard breeding diet A03 (Safe, Epinay-sur-Orge, France) before the beginning of the experiment. Eight-week-old-male mice (n = 40) were then randomly divided into two groups and fed with ad libitum for 2 weeks either a semi- purified control diet (UPAE, INRA Jouy-en-Josas, France) or the same control diet supplemented with 0.02% bil- berry (Vaccinium myrtillus L.) anthocyanin-rich extract (BE). These experimental diets were isoenergetic, and
apoEdeficientmice than COX-1 inhibition. Consequently, BM-573 could be a potential drug for prevention of atherosclerosis and this hypothesis will be tested in further investigation. This works emphases on the fact that severe selection of the mice strain chosen for the experiment allows producing reliable results with an adapted reduction in number of animals used.
PSE mice also displayed significantly increased percent- age body fat, ~20% compared to ~15% in HFC mice. To date, there has been just one study reporting significant weight gain in apo-E-deficientmice following consump- tion of a phytosterol extract . Erysipelotrichaceae, a family of bacteria previously associated with high fat con- sumption in murine models  and obesity in human co- horts , were found to be elevated in PSE when compared to HFC. Although each of the interventions ap- peared to stimulate Erysipelotrichaceae levels, PSE dem- onstrated the greatest propensity for this. This is peculiar, as it contradicts a report of Martínez et al., in which a PSE diet (5% w/w) was found to decrease Erysipelotrichaceae abundance in hamsters . However, PSE animals also presented with elevated cecum acetate production and it seems likely that a member of the Bacteroidaceae family (such as Bacteroides or Parabacteroides ) may have been responsible for the significant increase in acetate production in PSE animals. SCFA are key microbial fer- mentation metabolites which, through activation of G- protein-coupled receptors (GPCR)-41 and 43, can have important implications for host metabolic function . However, the work of Turnbaugh et al.  a decade ago highlighted an increased energy harvesting cap- acity of the obesity-associated microbiota, which was
Effects of different inulin-type fructan fractions were studied on atherosclerotic plaque formation in male apoE-deficientmice. Thirty-two mice were randomly divided into four groups and received either a semi-purified sucrose-based diet (control group), or diets in which sucrose was replaced in part by various inulin-type fructans (10 g/100 g): long-chain inulin, oligofructose, or an oligofructose-enriched inulin for 16 weeks. The presence of atherosclerotic plaques was assessed by histomorphometry in the aortic sinus. The apoE-deficientmice fed long-chain inulin or an oligofructose-enriched inulin had about 35 % and 25 % less atherosclerotic lesion area compared with the control group, respectively. Feed- ing long-chain inulin significantly reduced plasma cholesterol concentrations (P, 0·001), and the three inulin-type fructans reduced triacylglycerol (TAG) concentrations compared with the control group (P, 0·001). Both the long-chain inulin and an oligofructose-enriched inulin significantly lowered hepatic cholesterol concentrations compared with the control diet (P, 0·05). Hepatic TAG concentrations were significantly lower in all three groups fed the fructan-supplemented diets v. the control group (P, 0·0001). The results of the present study suggest that inhibition of ather- osclerotic plaque formation is more potent in the presence of long-chain inulin, either alone or in combination with oligofructose (an oligofructose- enriched inulin), and that this probably is related to changes in lipid metabolism.
These studies investigated the immune and anti-infectious response of dwarf Ghrh -/- mice presenting a severe deficiency of the GHRH/GH/IGF-1 axis.
In basal conditions, thymic parameters and T-cell responses of Ghrh -/- mice were not severely affected but a constant B-cell lymphopaenia was observed. Thus, we investigated vaccine and anti-infectious responses of Ghrh -/- mice toward Streptococcus pneumonia, a B-dependent pathogen,
2 ways of vaccination against S.pneumoniae
Characteristics of Ghrh -/- immune system in basal conditions: ↗ TREC, ↗ naive T cells, ↙↙ B cells .
Therefore, we investigated the B-dependent vaccine and immune responses of Ghrh -/- mice to 2 anti-pneumococcal vaccines and to a
sublethal infection by S.pneumoniae.
2.2. Water avoidance stress
The water avoidance stress (WAS) is a murine model of chronic stress that induces several symptoms associated to IBS. In particular, WAS increased visceral sensibility, colonic motility, intestinal permeability, pro-inflammatory cytokine production and mast cell count. 21,23–25 After one week of acclimatization at 23°C (12-hour light-dark cycle; dark phase: 8:00 PM - 8:00 AM), WAS model was performed during 1 hour per day during 10 consecutive days as previously described. 21 After the last WAS procedure, mice were euthanized. Colonic samples were either taken, washed with ice-cold PBS and immediately frozen until further analysis or immediately used to evaluate paracellular permeability.
2.2 | Spontaneous thrombosis following silencing of antithrombin and protein C
Female Slc44a2 −/− and Slc44a2 +/+ mice 6 weeks of age were intra-
venously injected with siRNAs targeting antithrombin (siSerpinc1: #S62673; Ambion) and protein C (siProc: #S72192) complexed with invivofectamine 3.0 (Invitrogen) as previously described. 8 A dose of 80 nmol of siSerpinc1 and siProc per kg of body weight in study one and 60 nmol in study two was used. The endpoint was reached once 50% of all mice displayed previously described typical clinical fea- tures. 8 Blood was collected 24 hours pre-injection via tail cut using dipotassium ethylenediaminetetraacetic (K 2 EDTA) acid coated vials (Sarstedt). Blood was also collected from the IVC with 11 µmol/L so- dium citrate upon sacrifice and under anesthesia induced by subcuta- neous injection of ketamine (100 mg/kg), xylazine (12.5 mg/kg), and atropine (125 µg/kg). Cell counts were assessed by SysmexXT-2000iV (Sysmex Europe GMBH).
Drusen, the white yellowish deposits that can be seen in funduscopy, are a hallmark of age related macular disease (AMD). Histologically, drusen are believed to be dome-shaped or more confluent lipid accumulations between the retinal pigment epithelium and the choroicapillaries. Recent advances in mouse fundoscopy have revealed the presence of drusen-like structures in chemokine knockout animals in the absence of sizeable dome-shaped material below the RPE. We show that aged CX3CR1 -/- mice present with drusen-like
is 2 times higher in plantaris muscle compared to soleus (Fletcher et al., 2017), which may explain while it function is more easily put in evidence in this muscle.
Considering the emerging role of skeletal muscle in autocrine and paracrine signaling, we also analyzed the expression of several myokines known to be activated by exercise. Among them, IL-15 is a cytokine secreted by skeletal muscle that promotes endurance adaptations via a stimulation of the oxidative energy metabolism and the Sirt1/PGC1a axis in young mice (Quinn et al., 2013). IL-15 levels are increased by exercise in humans (Rinnov et al., 2014). On the other skeletal muscle aging and sarcopenia are associated with low levels of IL-15 (Yalcin et al., 2018). Here, we show that exercise reduced IL-15 mRNA level in aged control and Nmrk2 −/− mice. On the other hand, we showed that the IL-15 gene expression is upregulated at baseline in the soleus of Nmrk2 −/− mice. Since we observed
PAI-1 is a multifunctional protein. In addition to its serpin function, it also binds to vitronectin, thereby regulating cell adhesion and migration (Czekay and Loskutoff, 2004). This protein also has a LRP binding site involved in controlling cell migration via uPA/uPAR internalization (Herz and Strickland 2001) and in regulating cell proliferation and apoptosis via the PI3K/AKT pathway (Balsara et al., 2006). To dissect functional properties of PAI-1, we and others have used mutated forms of rPAI-1 that unraveled novel PAI-1 activities unrelated to serine protease-inhibitory activity (Bajou et al., 2001; Lambert et al., 2003; Praus et al., 2002; Stefansson et al., 2001; Balsara et al., 2006). However, this strategy is hampered in the present model, by the occurrence of tumor transformation and progression in utero and the impossibility of using adenovirus-mediated gene transfer or recombinant protein injection at early stages. In addition, the anatomy of the eye hinders the access of pharmacological compounds to intraocular environment in neonatal or adult mice (Bouquet et al., 2003).
FIGURE 4 Local production of endogenous opioids in inflamed intestine of piroxicam-
treated IL10 -/- mice inhibits abdominal pain. (A) Colonic sensitivity of IL-10 -/- mice fed with standard chow diet containing (black circles) or not (white circles) piroxicam was measured by colorectal distension. Abdominal muscle contraction was recorded in response to distension pressure of 15, 30, 45 and 60 mmHg. (B) Piroxicam-treated IL10 -/- mice were injected with PBS (white circles) or naloxone-methiodide (NLX-meth) (black circles) 30 min before pain assessment. Data are expressed as means ± SEM (n = 10-14 animals). Statistical analysis was performed using repeated-measures two-way ANOVA and subsequent post hoc tests as described in Materials and Methods. *p < 0.05. (C) Colitis severity assessed by wall thickness (left panel) and macroscopic tissue damage (right panel) 30 min after intra- peritoneal administration of either PBS (white histogram) or NLX-meth (black histogram) (same groups of animals as above). Data are expressed as means ± SEM. Statistical analysis was performed using Mann-Whitney U test.
There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson’s disease (PD) begin many years before the
appearance of the characteristic motor symptoms. Neuropsychiatric, sensorial and cognitive deficits are recognized as early non-motor manifestations of PD, and are not attenuated by the current anti-parkinsonian therapy. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, Parkin-deficientmice do not display spontaneous degeneration of the nigrostriatal pathway or enhanced vulnerability to dopaminergic neurotoxins such as 6-OHDA and MPTP. Here, we employed adult homozygous C57BL/6 mice with parkin gene deletion on exon 3 (parkin 2/2 ) to further investigate the relevance of Parkin in the regulation of non- motor features, namely olfactory, emotional, cognitive and hippocampal synaptic plasticity. Parkin 2/2 mice displayed normal performance on behavioral tests evaluating olfaction (olfactory discrimination), anxiety (elevated plus-maze), depressive-like behavior (forced swimming and tail suspension) and motor function (rotarod, grasping strength and pole). However, parkin 2/2 mice displayed a poor performance in the open field habituation, object location and modified Y-maze tasks suggestive of procedural and short-term spatial memory deficits. These behavioral impairments were accompanied by impaired hippocampal long-term potentiation (LTP). These findings indicate that the genetic deletion of parkin causes deficiencies in hippocampal synaptic plasticity, resulting in memory deficits with no major olfactory, emotional or motor impairments. Therefore, parkin 2/2 mice
SDS) between analyses. Electropherograms were acquired at 15 Hz using P/ACE TM MDQ software .
RNA extraction and mRNA QPCR
P35 (n = 4 WT; n = 4 Mecp2-deficient) and P55 (n = 4 WT; n = 4 Mecp2-deficient) mice were killed by cervical dislocation, and their brain areas were dissected out. The caudate-putamen and hippocampal tissues were dissected as previously described for biochemical analysis. The ventral midbrain area (SNpr+substantia nigra pars compacta) was dissected instead of the SNpr alone in order to increase total RNA yield. All samples were kept at 280uC until analysis. Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA samples were treated with 3 U/ m L of DNase I, RNase-free (Qiagen) at 37 uC during 30 min followed by enzyme inactivation at 65uC during 5 min. The purity of the RNA samples was analysed using the ND-1000 spectrophotometer (NanoDrop, Thermo Scientific) and quality was assessed by electrophoresis on a denaturing agarose gel. Reverse transcription of 2 m g of total RNA was performed in a 20 m l reaction containing 4 m l of FS super script 56(Invitrogen), 12,5 nM of Random primers, 10 mM dNTP, 0,1 M DTT (Invitrogen), 40 U Rnase out (invitrogen), and 200 U of Superscript II reverse transcriptase (Invitrogen). Quan- titative PCR reaction was performed using a LightCycler 480 system (Roche) in a 20 m L reaction containing 10 uL of SYBR Green I Master kit (Roche), 2 m l of cDNA (1/10 dilution of the first-strand reaction) and 200 nM of each primer. Each reaction was performed in triplicate. We quantified the glutamate decarboxylase 1 (GAD1); glutamate decarboxylase 2 (GAD2); sodium/potassium/chloride cotransporter 1 (Nkcc1), potassium/ chloride cotransporter 2 (Kcc2); vesicular glutamate transporter 1 and 2 (Vglut1/2); Vesicular inhibitory amino acid transporter (Viaat1, also known as Slc32a1 or Vgat) expression using specific pairs of primers. Primer sequences are available upon request. The reference gene, Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used to account for procedural loss. To normalize our results and quantify mRNA levels, we used the DD C t method
Animals and treatment with genistein
Four-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were housed in a room maintained on a 12-h light, 12-h dark cycle under constant temperature (22–25 C) with ad libitum access to food and water. The protocol of this study was approved by the Institutional Animal Care and Use Committee at Virginia Polytechnic Institute and State University. Mice were initially fed a modified AIN-93G rodent diet with corn oil substituted for soybean oil (Dyet, Inc., Bethlehem, PA) for 2 wk and then were randomly divided into three groups with 12 mice per group and fed a diet containing either 0 g (groups 1 and 2) or 0.25 g (group 3) genistein per kilogram. This genistein dosage was used (approximately a human intake of 25–200 mg/d) because it is within the range that humans can realistically consume through taking supplements (39). We confirmed by performing HPLC analysis that the basal diet is free of genistein. After 2 wk, diabetic mice were induced with ip injection of strep- tozotocin (STZ) dissolved in 0.1 m M cold sodium citrate buffer (pH 4.5) at 40 mg/kg daily for 5 consecutive days. Control mice received ip citrate buffer. After this procedure, mice were con- tinually treated with the control or genistein diet. Body weight and feed intake were recorded weekly throughout the study. To confirm the results, we repeated this animal experiment using the same study protocols.
Whether BMP6 and HJV may also signal to hepcidin independently of each other is still a topic of discussion 11 . To provide direct evidence that BMP6 and HJV can separately stimulate hepcidin, we intercrossed Hjv and Bmp6 knockout mice and looked whether deletion of both Bmp6 and Hjv in mice of the F2 progeny was aggravating the phenotype of single knockout animals. Whether BMP6 and HJV are both required for the upregulation of hepcidin by inflammatory stimuli is another unresolved issue. We took the opportunity of having genetically comparable single and double knockout animals to challenge them with LPS and examine the impact of Bmp6 and/or Hjv deletion on Smad signaling and hepcidin expression after stimulation.
Quantification of hepatic hepcidin 1 mRNA
The expression level of hepcidin 1 mRNA transcripts was determined in the liver of carbonyl iron, iron dextran, Hfe -/- mice models and their controls by real time quantitative polymerase chain reaction (RT-PCR). Total liver RNAs were isolated using the Nucleospin® 8 RNA (Macherey-Nagel). The mRNAs were reverse transcribed with the M-MLV reverse transcriptase (Promega ® ). The following primers were used to amplify hepcidin 1 (forward: 5’-TTCCCAGTGTGGTATCTGTTGC-3’ and reverse: 5’-GGTCAGGATGTGGCTCTAG GC-3’), Mfsd5 (forward 5’- tgttgggtgtcatacaagc-3’ and Reverse 5’- ggtctagcacaggtgtcc-3’) and Tbp ‘TATA-binding protein) (forward: 5’-AAACTCTGACCACTGCACCG-3’ and reverse: 5’-GTGTGGCAGGAGTGATAGGG-3’) as references. Real-time quantitative PCR assays were performed using the qPCR MasterMix Plus for SYBR ® Green I (Eurogentec ® ) and the system StepOne Plus (Real-Time PCR System – Applied Biosystems ® ). All results were analyzed by StepOne Software v2.1 (Applied Biosystems ® ). For each cDNA sample, the difference between the threshold cycle for hepcidin 1 amplification and the threshold cycle for TBP was calculated. This enabled normalization of the amount of target to the endogenous reference, TBP.