Swiss CD-1 Mice Research Articles

In vivo reduction of skin inflammation using ferulic acid-loaded lipid vesicles derived from Brewer’s spent grain

Breweŕs spent grain (BSG) is the main by-product of the brewing industry, and due to its rapid decomposition, it generates serious environmental problems such as malodors and greenhouse gases emissions. On the other hand, this lignocellulosic compound contains a large number of antioxidants, being ferulic acid (FA) the most abundant. FA is a powerful antioxidant molecule that has demonstrated significant protective effects on key components of the skin, including keratinocytes, fibroblasts, collagen, and elastin. FA inhibits melanogenesis, promotes angiogenesis and accelerates the wound healing although its use is limited by its rapid oxidation. In this study, different hydrolysis treatments (chemical, enzymatic and hydrothermal) were performed on BSG to obtain FA. Herein FA-loaded ultradeformable liposomes (ULs) were designed to improve their stability and in vivo performance. These nanosystems allow FA permeability through human skin, as proven by an ex vivo skin permeability assay using Franz diffusion cells. The toxicity and anti-inflammatory activity of the formulation has been investigated. The free form and 100 nm FA_ULs were evaluated. Cell viability was dose-dependent and provided optimal results for the treatment of inflammatory skin conditions in an in vivo Oxazolone-induced Delayed Type Hypersensitivity model using Swiss CD1 mice, demonstrated by the reduction of the inflammatory cytokines expression, ear thickness, bioluminescence and histological evaluation. These results pave the way for FA-based treatments of skin and inflammatory conditions.

Structure-Activity Relationship of N-Ethyl-Hexedrone Analogues: Role of the α-Carbon Side-Chain Length in the Mechanism of Action, Cytotoxicity, and Behavioral Effects in Mice.

Synthetic cathinones are β-keto amphetamine derivatives whose appearance has increased dramatically in the past decades. N-Ethyl substituted cathinones have been proven to potently inhibit dopamine (DA) uptake and induce psychostimulant and rewarding effects in mice. However, little is known about the influence of the alpha-carbon side-chain length of N-ethyl cathinones on their pharmacological and toxicological effects. Thus, the aim of this study was to synthesize and investigate the in vitro and in vivo effects of five N-ethyl substituted cathinones: N-ethyl-cathinone (NEC), N-ethyl-buphedrone (NEB), N-ethyl-pentedrone, N-ethyl-hexedrone (NEH), and N-ethyl-heptedrone. HEK293 cells expressing the human DA or serotonin transporter (hDAT and hSERT) were used for uptake inhibition and binding assays. PC12 cells were used for the cytotoxicity assays. Swiss CD-1 mice were used to study the in vivo psychostimulant, anxiogenic, and rewarding properties. Our results show that all tested cathinones are able to inhibit DA uptake and are DAT-selective. The potency of DA uptake inhibitors increases with the elongation of the aliphatic side chain from methyl to propyl and decreases when increasing from butyl to pentyl, which correlates with an inverted U-shape psychostimulant response in mice at the medium dose tested. On the other hand, an increase in the α-carbon side-chain length correlates with an increase in the cytotoxic properties in PC12 cells, probably due to better membrane penetration. Moreover, all the cathinones tested have shown higher cytotoxicity than methamphetamine. Finally, our study not only demonstrated the rewarding properties of NEC and NEB but also the anxiety-like behavior induced at high doses by all the cathinones tested.

Lentinula edodes extract increases goblet cell number and Muc2 expression in an intestinal inflammatory model of Trichinella spiralis infection.

AHCC® is a standardized extract of cultured mushroom (Lentinula edodes) mycelia with a wide variety of therapeutic effects including anti-inflammatory, antitumor and antiviral effects. Trichinellosis, a food-borne parasitic zoonosis is caused by the nematode Trichinella spp. Infection with Trichinella is characterized by the induction of a Th1-type response at the beginning of the intestinal phase, followed by a dominant Th2-type response which is essential for parasite expulsion. The aim of this study was to evaluate the immunomodulatory effect of AHCC® in a murine model of Trichinella spiralis infection. Swiss CD1 mice were infected with T. spiralis larvae and treated with AHCC®. Standard treatment with albendazole (ABZ) was used as control in the assessment of parasite burden. The small intestine was taken out and the proximal segment was evaluated for several parameters: gene expression of immune and stress-reticulum mediators, histological damage score, goblet cell count and Mucin 2 (Muc2) gene expression. AHCC® modulated expression levels of both Th1 and Th2 cytokines and reduced histological damage score. In addition, AHCC® diminished the number of adults of T. spiralis in treated animals. AHCC® treatment anticipates T. spiralis expulsion and increases goblet cell number and Muc2 gene expression.

Neuropsychopharmacology of Emerging Drugs of Abuse: meta- and para-Halogen-Ring-Substituted α-PVP ("flakka") Derivatives.

Changes in the molecular structure of synthetic cathinones has led to an increase in the number of novel emerging drugs in the illicit drug market at an unprecedented rate. Unfortunately, little is known about the neuropsychopharmacology of recently emerged halogen-substituted α-PVP derivatives. Thus, the aim of this study was to investigate the role of para- and meta-halogen (F-, Cl-, and Br-) substitutions on the in vitro, in silico, and in vivo effects of α-pyrrolidinopentiophenone (α-PVP) derivatives. HEK293 cells expressing the human dopamine or serotonin transporter (hDAT and hSERT) were used for the uptake inhibition and transporter affinity assays. Molecular docking was used to model the interaction mechanism against DAT. Swiss CD-1 mice were used for the horizontal locomotor activity, open field test, and conditioned place preference paradigm. All compounds demonstrated potent DA uptake inhibition and higher DAT selectivity than cocaine. Meta-substituted cathinones showed higher DAT/SERT ratios than their para- analogs, which correlates with an increased psychostimulant effect in vivo and with different meta- and para-in silico interactions at DAT. Moreover, all compounds induced rewarding and acute anxiogenic effects in mice. In conclusion, the present study demonstrates the role of meta- and para-halogen substitutions in the mechanism of action and provides the first evidence of the rewarding and anxiety-like properties of halogenated α-PVP derivatives.

Preterm Delivery: Microbial Dysbiosis, Gut Inflammation and Hyperpermeability

Preterm birth is one of the main health problems encountered in the neonatal period, especially because it is also the first cause of death in the critical 1st month of life and the second in children under 5 years of age. Not only preterm birth entails short term health risks due to low weight and underdeveloped organs, but also increases the risk of suffering from non-transmissible diseases in the long term. To date, it is known that medical conditions and lifestyle factors could increase the risk of preterm birth, but the molecular mechanisms that control this process remain unclear. Luteolysis, increased inflammation or oxidative stress have been described as possible triggers for preterm birth and, in some cases, the cause of dysbiosis in preterm neonates. Several murine models have been developed to shed light into the mechanistic of preterm birth but, for the most part, are inflammation-based labor induction models and the offspring health readouts are mainly limited to survival and weight. Using a set of SWISS-CD1 mice born prematurely we analyzed inflammation and gut permeability parameters compared with term pups at weaning age. Overall, preterm mice presented higher systemic inflammation and gastrointestinal tract permeability. In this perspective article, we discuss the recent discoveries on preterm birth and the necessity of non-inflammatory murine models to really understand these phenotypes and be able to design strategies to prevent the sequels of this traumatic event in neonates.

Structure–Activity Relationship of Novel Second-Generation Synthetic Cathinones: Mechanism of Action, Locomotion, Reward, and Immediate-Early Genes

Several new synthetic cathinones, which mimic the effect of classical psychostimulants such as cocaine or MDMA, have appeared in the global illicit drug market in the last decades. In fact, the illicit drug market is continually evolving by constantly adding small modifications to the common chemical structure of synthetic cathinones. Thus, the aim of this study was to investigate the in vitro and in vivo structure–activity relationship (SAR) of six novel synthetic cathinones currently popular as recreational drugs, pentedrone, pentylone, N-ethyl-pentedrone (NEPD), N-ethyl-pentylone (NEP), 4-methyl-pentedrone (4-MPD), and 4-methyl-ethylaminopentedrone (4-MeAP), which structurally differ in the absence or presence of different aromatic substituents and in their amino terminal group. Human embryonic kidney (HEK293) cells expressing the human isoforms of SERT and DAT were used for the uptake inhibition and release assays. Moreover, Swiss CD-1 mice were used to investigate the psychostimulant effect, rewarding properties (3, 10, and 30 mg/kg, i.p.), and the induction of immediate-early genes (IEGs), such as Arc and c-fos in the dorsal striatum (DS) and ventral striatum (VS) as well as bdnf in the medial prefrontal cortex (mPFC), of the test compounds. Our results demonstrated that all tested synthetic cathinones are potent dopamine (DA) uptake inhibitors, especially the N-ethyl analogs, while the ring-substituted cathinones tested showed higher potency as SERT inhibitors than their no ring-substituted analogs. Moreover, unlike NEP, the remaining test compounds showed clear “hybrid” properties, acting as DAT blockers but SERT substrates. Regarding the locomotion, NEP and NEPD were more efficacious (10 mg/kg) than their N-methyl analogs, which correlates with their higher potency inhibiting the DAT and an overexpression of Arc levels in the DS and VS. Furthermore, all compounds tested induced an increase in c-fos expression in the DS, except for 4-MPD, the least effective compound in inducing hyperlocomotion. Moreover, NEP induced an up-regulation of bdnf in the mPFC that correlates with its 5-HTergic properties. Finally, the present study demonstrated for the first time that NEP, 4-MPD, and 4-MeAP induce reward in mice. Altogether, this study provides valuable information about the mechanism of action and psychostimulant and rewarding properties as well as changes in the expression of IEGs related to addiction induced by novel second-generation synthetic cathinones.

In vivo bioavailability and in vitro toxicological evaluation of the new butyric acid releaser N-(1-carbamoyl-2-phenyl-ethyl) butyramide

A large body of evidence suggests that supplementation of butyric acid exerts beneficial intestinal and extra-intestinal effects. Unfortunately, unpleasant sensorial properties and unfavourable physico-chemical properties strongly limit its use in food supplements and foods for medicinal purposes. N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) is a new butyric acid releaser in solid form with neutral sensorial properties. The aim of this investigation is to provide preliminary information on its pharmacokinetic and toxicological properties through the study of a) in vivo bioavailability of FBA administered by oral gavage to male and female Swiss CD1 mice in comparison with sodium butyrate, b) the influence of digestion on FBA stability through an in vitro simulated oro-gastro-duodenal digestion process, and c) in vitro toxicological profile by means of the Ames Test and Micronucleus Test. The results reveal that FBA is a good butyric acid releaser, being able to increase butyrate serum concentration in a dose and time dependent manner in both male and female mice with a pharmacokinetic profile similar to that obtained from sodium butyrate as such. These data are confirmed by investigating the influence of digestion on FBA, which undergoes extensive hydrolysis following oro-gastro-duodenal digestion, especially in duodenal conditions, with a residual concentration of less than 10% of the initial FBA concentration. Finally, in the Ames and Micronucleus Tests, FBA does not show any in vitro genotoxicity as it is non mutagenic in the Ames Test and results to be unable to induce chromosome breaks in the Micronucleus Test. In conclusion, FBA is a new butyric acid releaser that can overcome the disadvantages of butyric acid while maintaining the same pharmacokinetic properties and safety profile, as shown by the results of the preliminary in vitro toxicological studies performed in this investigation.

Impact of the polycarbonate strippers used in assisted reproduction techniques on embryonic development.

Do daily manipulations of preimplantation embryos with polycarbonate (PC)-made bisphenol A (BPA)-releasing strippers influence embryo development? Compared to glass strippers, PC strippers enhance the blastocyst development rate but this does not seem to be BPA-related. PC strippers have been shown to release tiny amounts (around 0.5 ng/ml BPA) of BPA in routine human IVF procedures. A chronic exposure to BPA either in vivo or in vitro during the preimplantation period can impact post-implantation and post-natal development. BPA can act rapidly by binding to membrane receptors and inducing rapid non-genomic effects. This experimental study using mouse embryos had a balanced design and blinded evaluations of the endpoints. In vivo fertilized zygotes were obtained from outbred Swiss CD1 mice crossings after an ovarian stimulation. The zygotes were allocated to three daily handling conditions (HCs) and cultured until Day 4 in a single human commercial medium. Each day, the embryos were handled for 20 s either in a PC stripper (HC1) or in a glass stripper (HC2). In HC3, the embryos were pre-exposed to 0.5 ng/ml BPA before being handled for 20 s in a glass stripper. Handling operations were repeated on Days 1, 2 and 3. Embryo development was assessed blindly on Day 4. Expanded blastocysts were selected for a transcriptomic analysis using Agilent Sureprint G3 Mouse GE v2 microarrays and the retrotransposon LINE1-Orf2 expression was analysed using qRT-PCR, as a proxy for a global evaluation of the epigenetic status. Compared to the embryos manipulated in HC2 (n = 243), those in HC1 (n = 228) developed significantly more often to the blastocyst stage (55 vs 46%; P < 0.05). It appears the effect of these PC strippers was not BPA-related because embryos pre-exposed to BPA (HC3, n = 230) showed no difference in the blastocyst rate when compared to HC2 (43 vs 46%). When analysing same-stage blastocysts, we noticed no difference in the embryo gene expression between the three HC groups. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE148868. Our results using a mouse model designed to mimic human conditions (outbred strain, human commercial IVF dishes and a unique commercial human embryonic culture media) are reassuring since no gene was found to be differentially expressed, including LINE-1 genes, as a proxy for a global evaluation of the epigenetic status. However, no global epigenetic analysis of the genome has been performed. Furthermore, we did not evaluate post-implantation events, although BPA exposure during peri-conception could affect foeto-placental and post-natal development. Based on the precautionary principle, several European countries banned the use of BPA in baby bottles and food packaging several years before European Agencies took an official position. The question of applying this principle to plastics in closed contact with human embryos is raised. Further studies are needed for a decision to be made. This study was supported by a grant from the Agence de Biomédecine (AOR 2016). The authors declare no competing interest.

OR07-4 Preterm Birth and Transgenerational Epigenetic Programming of Hypertension in Mice

Introduction: Prematurity, a worldwide health issue, is often associated with renal tubular immaturity leading to major salt loss, whose mechanisms remain poorly understood. Moreover, these premature infants are prone to develop early hypertension in adulthood. Objective: To study ontogenesis of renal mineralocorticoid and glucocorticoid signaling pathways in preterm mice, and to evaluate their contribution to neonatal adaptation and to the emergence of hypertension in adulthood. Materials and Methods: We have developed a murine model of prematurity induced by intra-peritoneal injection of O111/B4 lipopolysaccharides (LPS) at 18 days of gestation in Swiss CD1 mice. Offspring of injected mice, when LPS did not trigger preterm birth, were used as a control to exclude intrinsic LPS effects. Pups were sacrificed at various developmental stages (D0, D7 and M6). Blood pressure and heart rate were measured in males at M6 and their plasma steroid profiles were measured using LC-MSMS. Renal mRNA and protein expression of major players of corticosteroid signaling pathways were examined using RT-qPCR and western-blot analyses. Second (F2) and third (F3) generations, established by mating prematurely born adult females with wild type males, were also analyzed. We performed Methylation DNA Immunoprecipitation-qPCR to study the methylation status of candidate corticosteroid target genes that were differentially expressed between the 2 populations. Results: As anticipated, preterm newborn mice presented with maladaptation, high neonatal mortality (35%), and a lower birth weight compared to controls (1.29±0.21 vs 1.46±0.15g, P=0.0027). Former preterm males developed hypertension at M6 (123.1±1.43 vs 114.5±0.79mmHg, P<0.0001). We found a robust activation of renal corticosteroid target gene transcription at birth in preterm mice (αENaC (+45%), Sgk1 (+132%), Gilz (+85%)), which was not related to modified expression of the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). Interestingly, a significant increased blood pressure was found in the F2 and F3 males, descendants of the preterm group, concomitantly with increased renal Gilz mRNA expression, despite similar MR or GR expression, and plasma aldosterone or corticosterone levels. Gilz promoter methylation was reduced in preterm offspring with a negative correlation between methylation and expression (P=0.008), highly suggestive of an epigenetic Gilz regulation. Conclusion: We provide first evidence for alterations of the renal corticosteroid signaling pathways induced by prematurity, with a trans-generational transmission of blood pressure dysregulation, associated with epigenetic Gilz regulation. This study should allow better understanding of prematurity-related defects, leading hopefully to better management of premature infants from birth to adulthood.

Comparative virulence of Caribbean, Brazilian and European isolates of Toxoplasma gondii

BackgroundToxoplasma gondii is a zoonotic parasite of global importance. The outcome of infection in humans can depend on a number of factors including the infecting stage of the parasite, inoculating dose and virulence of the infecting strain. Molecular epidemiological studies have demonstrated an abundance of atypical strains of T. gondii in South America, many of which have been associated with more severe sequelae of infection. The aim of this study was to compare the virulence of T. gondii strains isolated in the Caribbean to a virulent Brazilian strain and an avirulent European strain.MethodsOne hundred and twenty Swiss CD-1 mice were split into 8 groups of 15 mice and each group was inoculated with 200 tachyzoites of one of 8 isolates, comprising ToxoDB genotypes #1, #141, #265, #13, #3 and #6. Five mice per group were euthanized at day 8 post-inoculation (p.i.) and parasite burden was determined in heart, lungs and eyes using quantitative PCR. Lungs and brain were also examined by histopathology and immunohistochemistry. The remaining 10 mice per group were part of a survival experiment to assess virulence. DNA was extracted from tachyzoites of each of the 8 T. gondii isolates and genotyped at four ROP gene loci, including ROP5, ROP16, ROP17 and ROP18 to look for association with markers of virulence.ResultsInfection with ToxoDB genotype #13 from the Caribbean resulted in 100% of mice being euthanized which was comparative to infection with the virulent Brazilian strain (ToxoDB genotype #6). Significantly higher parasite burdens were recorded in the lungs and eyes of mice infected with ToxoDB genotypes #13 and #6. Genotyping of ROP loci revealed that the virulent Caribbean isolates had a different ROP18/ROP5 allelic profile (3/1) to the virulent Brazilian isolate (1/3); however, the avirulent Caribbean isolate (ToxoDB genotype #1) had the same ROP18/ROP5 profile as the avirulent European isolate (ToxoDB #3) (both 2/2). Caribbean isolates of intermediate virulence (ToxoDB #141 and #265) all had the same ROP18/ROP5 allelic profile (2/2).ConclusionsIsolates from the Caribbean with ToxoDB genotype #13 were acutely virulent for mice and comparable to a known virulent Brazilian isolate. The ROP protein allelic profile of the virulent Caribbean and Brazilian isolates differed indicating that perhaps other factors are involved in predicting virulence. Understanding virulence is important for predicting disease outcome in humans and may also aid vaccine design as well as drug discovery.

Sedative and anxiolytic effect of the methanolic extract of Erythrina edulis Triana ex Micheli in mice

The traditional use of medicinal plants and its link with pharmacological studies is essential in order to corroborate the effect scientifically. The present work was an experimental study designed to investigate the sedative and anxiolytic effect of the methanol extract of the leaves of Erythrina edulis. The extract was prepared by percolation. Different concentrations: 500, 250 and 125 mg/kg of the extract and diazepam as a controlled drug were administered intraperitoneally to Swiss CD1 mice for both, sedation and anxiety tests. The sleep enhancement test with pentobarbital and the traction test were used for the sedative activity. Results showed that the extract did not possess sedative activity in any of the administered doses. However, the possibility of an opposite effect was observed as an activation of the central nervous system probably. The anxiolytic activity was evaluated by the open field test and the elevated plus maze test, where a behavior with a low level of anxiety was observed. Altogether, the results suggest that the extract has an anxiolytic activity without sedation, which supports traditional use. But, its responses are not dose-dependent, and its mechanism of action is unknown. Besides, toxicity assay was performed in Artemia salina nauplii, resulting in a lethal concentration 50 of 16.570 ug/mL corresponding to zero toxicity. Finally, the phytochemical analysis of the extract revealed the presence of flavonoids (flavonols, flavones and phenolic carboxylic acids), anthrones, anthranols, terpenes (mono and sesquiterpenes), saponins and hydrolysable tannins.

Carcinogenicity testing of eliglustat in mice and rats

Eliglustat is a novel glucosylceramide synthase inhibitor for long-term oral treatment of type 1 Gaucher disease (GD1), an inherited metabolic disorder. The carcinogenic potential of this drug has been evaluated in lifetime carcinogenicity bioassays in mice and rats. Administration of eliglustat to Swiss CD-1 mice at 0, 10, 25 or 75 mg/kg/day for 104 weeks by dietary admixture did not influence survival or bodyweight evolution, or produce any clinical indication of poor condition. At histopathology, no increases in tumor incidence for any tumor type were attributed to treatment with eliglustat. Systemic exposure to eliglustat was confirmed by a reduction in circulating levels of glucosylceramide. Administration of eliglustat to Sprague–Dawley rats by oral gavage for 105 weeks at 0, 10, 25 or 75 mg/kg/day (males) or 103 weeks at 0, 5, 15 or 50 mg/kg/day (females) did not affect survival rates, but resulted in reduced bodyweight evolution in male rats (−18% at high dose), indicating that the MTD had been achieved. At histopathology, no increases in tumor incidence were attributed to treatment with eliglustat. Systemic exposure was confirmed by toxicokinetic analyses. In conclusion, eliglustat was not carcinogenic to mice or rats in standard lifetime bioassays.

Action of the ciliary neurotrophic factor in mouse brainstem

Systemic administration of ciliary neurotrophic factor (CNTF) determines sustained weight loss in rodents and humans. The anorectic effect of CNTF is due, at least in part, to activation of the signal transducer and activator of transcription (STAT) 3, STAT1 and STAT5 in neuronal and glial cells of the tuberal hypothalamus (Severi et al., 2012, 2013 and unpublished data). To assess whether CNTF also exerts an action on the brainstem feeding centres, male Swiss CD-1 mice were intraperitoneally treated with rat recombinant CNTF (0.3 mg/kg of body weight; CNTF-treated mice) or with saline (control mice) for 45 minutes (min) to perform P-STAT3, P-STAT1 and P-STAT5 immunohistochemistry, and for 120 min to detect c-Fos expression. Double staining experiments with neuronal and glial markers and confocal microscopy analyses were performed to assess the phenotype of the CNTF-responsive cells. P-STAT3-, P-STAT1- or P-STAT5-positive cells were not detected in brainstem coronal sections from control mice. In contrast, CNTF administration led to a strong activation of the transcription factors STAT3, STAT1 and STAT5 in CNTF receptor α-bearing glial cells of the area postrema, a circumventricular organ devoid of blood-brain barrier, that allows circulating molecules to gain access to the adjacent brain parenchyma. In 120 min CNTF-treated mice specific nuclear staining for c-Fos was detected not only in cells of the area postrema but also in numerous neurons widely distributed in the nucleus of the solitary tract (Sol), the dorsal motor nucleus of vagus (DMV) and parabrachial nuclei. Ongoing experiments on capsaicin-treated (deafferented) mice will disclose a possible vagal contribution to the Sol and DMV neuronal activation due to a peripheral action of CNTF in the gastro-intestinal tract. Collectively, these data show for the first time that CNTF similarly to other satiety factor, like leptin, has redundant actions on both hypothalamic and brainstem energy balance neuronal networks. Unravelling the phenotype of brainstem neurons responsive to CNTF will clarify its role in distinctive aspects of feeding behaviour.

Repeated doses of methylone, a new drug of abuse, induce changes in serotonin and dopamine systems in the mouse

Methylone, a new drug of abuse sold as "bath salts," has similar effects to ecstasy or cocaine. We have investigated changes in dopaminergic and serotoninergic markers, indicative of neuronal damage induced by methylone in the frontal cortex, hippocampus, and striatum of mice, according to two different treatment schedules. Methylone was given subcutaneously to male Swiss CD1 mice at an ambient temperature of 26 °C. Treatment A consisted of three doses of 25 mg/kg at 3.5-h intervals between doses for two consecutive days, and treatment B consisted of four doses of 25 mg/kg at 3-h intervals in 1 day. Repeated methylone administration induced hyperthermia and a significant loss in body weight. Following treatment A, methylone induced transient dopaminergic (frontal cortex) and serotoninergic (hippocampus) impairment. Following treatment B, transient dopaminergic (frontal cortex) and serotonergic (frontal cortex and hippocampus) changes 7 days after treatment were found. We found evidence of astrogliosis in the CA1 and the dentate gyrus of the hippocampus following treatment B. The animals also showed an increase in immobility time in the forced swim test, pointing to a depressive-like behavior. In cultured cortical neurons, methylone (for 24 and 48 h) did not induce a remarkable cytotoxic effect. The neural effects of methylone differ depending upon the treatment schedule. Neurochemical changes elicited by methylone are apparent when administered at an elevated ambient temperature, four times per day at 3-h intervals, which is in accordance with its short half-life.

Acute exercises induce disorders of the gastrointestinal integrity in a murine model

Many endurance athletes complain about gastrointestinal (GI) symptoms. It is assumed that exercise-induced shift of perfusion with consecutive hypoperfusion of the enteral vascular system leads to an increased GI permeability and tissue damage. Therefore, the aim of the study was to investigate permeability, apoptosis, electrogenic ion transport (Isc), and tissue conductance (Gt) of the small intestine in a murine exercise model. After spirometry, male Swiss CD-1 mice were subjected to an intensive treadmill exercise (80% VO2max). Sedentary mice served as controls. The small intestine was removed at several time intervals post-exercise. Apoptotic cells were determined by the TUNEL method, while fluorescein isothiocyanate dextran permeation indicated intestinal permeability. The Gt and Isc measurements were carried out in a modified Ussing chamber. Apoptosis of epithelial cells increased continuously until 24 h post exercise (0.8 ± 0.42 versus 39.2 ± 26.0%; p < 0.05). Compared with the control group the permeability increased 2 h after exercise (0.47 ± 0.07 versus 0.67 ± 0.14 FU/min; p < 0.05). Isc measurements of the ileum were augmented after 24 h (3.33 ± 0.56 versus 5.77 ± 1.16 μEq/h/cm(2); p < 0.05). At this time the Gt increased as well (28.8 ± 3.37 versus 32.5 ± 2.59 mS/cm(2); p < 0.05). In the murine exercise model there is evidence that after intense endurance exercise repair processes occur in small intestinal epithelial cells, which affect permeability, Gt, and Isc. The formation of lamellipodia to close the "leaky" tight junctions caused by apoptosis might be an underlying mechanism.

BDNF-endocannabinoid interactions at neocortical inhibitory synapses require phospholipase C signaling

Endogenous cannabinoids (endocannabinoids) and neurotrophins, particularly brain-derived neurotrophic factor (BDNF), are potent synaptic modulators that are expressed throughout the forebrain and play critical roles in many behavioral processes. Although the effects of BDNF at excitatory synapses have been well characterized, the mechanisms of action of BDNF at inhibitory synapses are not well understood. Previously we have found that BDNF suppresses presynaptic GABA release in layer 2/3 of the neocortex via postsynaptic tropomyosin-related kinase receptor B (trkB) receptor-induced release of endocannabinoids. To examine the intracellular signaling pathways that underlie this effect, we used pharmacological approaches and whole cell patch-clamp techniques in layer 2/3 pyramidal neurons of somatosensory cortex in brain slices from juvenile Swiss CD1 mice. Our results indicated that phospholipase Cγ (PLCγ) is involved in the CB1 receptor-mediated synaptic effect of BDNF, because the BDNF effect was blocked in the presence of the broad-spectrum PLC inhibitors U-73122 and edelfosine, whereas the inactive analog U-73343 did not alter the suppressive effect of BDNF at inhibitory synapses. Endocannabinoid release can also be triggered by metabotropic glutamate receptor (mGluR)-mediated activation of PLCβ, and BDNF has been shown to enhance spontaneous glutamate release. An mGluR antagonist, E4CPG, however, did not block the BDNF effect. In addition, the effect of BDNF was independent of other signaling pathways downstream of trkB receptor activation, namely, mitogen-activated protein kinase and phosphoinositide 3-kinase pathways, as well as protein kinase C signaling.

Preference for high-fat diet is developed by young Swiss CD1 mice after short-term feeding and is prevented by NMDA receptor antagonists

Obesity is a worldwide epidemic that is increasing at an alarming rate. One of its causes is the increased availability and consumption of diets rich in fat. In the present study, we investigated the effects of short-term consumption of a high fat diet (HFD) on dietary preferences in Swiss CD1 mice and its relation in time to specific metabolic effects. Mice that were weaned 21days postpartum and fed a chow diet for one week were afterward subjected to a diet preference test for 5days, exposed to both a regular diet (RD) and HFD. We found that mice did not show any preferences. In a second experiment, two groups of mice that were weaned 21days postpartum and subjected to a chow diet for one week were fed either RD or HFD for 18days, and a diet preference test was performed for 5days. After this short-term consumption of HFD, mice preferred HFD, while mice subjected to RD did not show any preference. Importantly, no differences in blood glucose levels were found between the groups prior to and after the experiments. The results support our hypothesis that the preference for HFD is not a spontaneous behavior in CD1 mice, but it can be observed after short-term consumption; additionally, this preference develops before metabolic effects appear. Finally, this preference for HFD could not be observed when the mice were i.p. injected daily with low doses of the NMDA receptor antagonists, ketamine, ifenprodil or MK-801 during the HFD feeding period. These data suggest that acquisition of dietary preference for HFD is a NMDA receptor-dependent learning process.

An Intradermal Inoculation Model of Scrub Typhus in Swiss CD-1 Mice Demonstrates More Rapid Dissemination of Virulent Strains of Orientia tsutsugamushi

Scrub typhus is an important endemic disease of the Asia-Pacific region caused by Orientia tsutsugamushi. To develop an effective vaccine to prevent scrub typhus infection, a better understanding of the initial host-pathogen interaction is needed. The objective of this study was to investigate early bacterial dissemination in a CD-1 Swiss outbred mouse model after intradermal injection of O. tsutsugamushi. Three human pathogenic strains of O. tsutsugamushi (Karp, Gilliam, and Woods) were chosen to investigate the early infection characteristics associated with bacterial virulence. Tissue biopsies of the intradermal injection site and draining lymph nodes were examined using histology and immunohistochemistry to characterize bacterial dissemination, and correlated with quantitative real-time PCR for O. tsutsugamushi in blood and tissue from major organs. Soluble adhesion molecules were measured to examine cellular activation in response to infection. No eschar formation was seen at the inoculation site and no clinical disease developed within the 7 day period of observation. However, O. tsutsugamushi was localized at the injection site and in the draining lymph nodes by day 7 post inoculation. Evidence of leukocyte and endothelial activation was present by day 7 with significantly raised levels of sL-selectin, sICAM-1 and sVCAM-1. Infection with the Karp strain was associated with earlier and higher bacterial loads and more extensive dissemination in various tissues than the less pathogenic Gilliam and Woods strains. The bacterial loads of O. tsutsugamushi were highest in the lungs and spleens of mice inoculated with Karp and Gilliam, but not Woods strains. Strains of higher virulence resulted in more rapid systemic infection and dissemination in this model. The CD-1 mouse intradermal inoculation model demonstrates features relevant to early scrub typhus infection in humans, including the development of regional lymphadenopathy, leukocyte activation and distant organ dissemination after low-dose intradermal injection with O. tsutsugamushi.

Effect of Aging on Superovulation Efficiency, Aneuploidy Rates, and Sister Chromatid Cohesion in Mice Aged Up to 15 Months1

Human eggs are highly aneuploid, with female age being the only known risk factor. Here this aging phenomenon was further studied in Swiss CD1 mice aged between 1 and 15 mo. The mean number of eggs ± SEM recovered from mice following superovulation peaked at 22.5 ± 3.8 eggs/oviduct in 3-mo-old females, decreasing markedly between 6 and 9 mo old, and was only 2.1 ± 0.2 eggs/oviduct by 15 mo. Measurement of aneuploidy in these eggs revealed a low rate, ∼3-4%, in mice aged 1 and 3 mo, rising to 12.5% by 9 mo old and to 37.5% at 12 mo. Fifteen-month-old mice had the highest rate of aneuploidy, peaking at 60%. The in situ chromosome counting technique used here allowed us to measure with accuracy the distance between the kinetochores in the sister chromatids of the eggs analyzed for aneuploidy. We observed that this distance increased in eggs from older females, from 0.38 ± 0.01 μm at 1 mo old to 0.82 ± 0.03 μm by 15 mo. Furthermore, in 3- to 12-mo-old females, aneuploid eggs had significantly larger interkinetochore distances than euploid eggs from the same age, and measurements were similar to eggs from the oldest mice. However, the association between aneuploidy and interkinetochore distance was not observed at the oldest, 15-mo age, despite such measurements being maximal. We conclude that in aging CD1 mice, a reduction in the ovulated egg number precedes a rise in aneuploidy and, furthermore, except at very advanced ages, increased interkinetochore distance is associated with aneuploidy.

Targets and Effects of Yessotoxin, Okadaic Acid and Palytoxin: A Differential Review

In this review, we focus on processes, organs and systems targeted by the marine toxins yessotoxin (YTX), okadaic acid (OA) and palytoxin (PTX). The effects of YTX and their basis are analyzed from data collected in the mollusc Mytilus galloprovincialis, the annelid Enchytraeus crypticus, Swiss CD1 mice and invertebrate and vertebrate cell cultures. OA and PTX, two toxins with a better established mode of action, are analyzed with regard to their effects on development. The amphibian Xenopus laevis is used as a model, and the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) as the experimental protocol.