Antitumor Effects of an Anthocyanin-Rich Grain Diet in a Mouse Model of Lewis Lung Carcinoma.

Functional foods enriched with plant polyphenol anthocyanins attract particular attention due to their health-promoting properties, including antitumor activity. We evaluated the effects of a grain diet rich in anthocyanins in a mouse model of Lewis lung carcinoma. Mice of the C57BL/6 strain were fed with wheat of near-isogenic lines differing in the anthocyanin content for four months prior to tumor transplantation. Although a significant decrease in the size of the tumor and the number of metastases in the lungs was revealed in the groups with both types of grain diet, the highest percentage of animals without metastases and with attenuated cell proliferation in the primary tumor were observed in the mice with the anthocyanin-rich diet. Both grain diets reduced the body weight gain and spleen weight index. The antitumor effects of the grain diets were associated with the activation of different mechanisms: immune response of the allergic type with augmented interleukin(IL)-9 and eotaxin serum levels in mice fed with control grain vs. inhibition of the IL-6/LIF system accompanied by a decrease in the tumor-associated M2 macrophage marker arginase 1 gene mRNA levels and enhanced autophagy in the tumor evaluated by the mRNA levels of Beclin 1 gene. Thus, anthocyanin-rich wheat is suggested as a promising source of functional nutrition with confirmed in vivo antitumor activity.

Combined induction of mTOR-dependent and mTOR-independent pathways of autophagy activation as an experimental therapy for Alzheimer's disease-like pathology in a mouse model.

Alzheimer's disease (AD) is associated with amyloid-ß (Aß) accumulation that might be hindered by autophagy. There are two ways to induce autophagy: through mTOR-dependent and mTOR-independent pathways (here, by means of rapamycin and trehalose, respectively). The aim of this study was to evaluate the contribution of these pathways and their combination to the treatment of experimental AD. Mice were injected bilaterally intracerebroventricularly with an Aß fragment (25-35) to set up an AD model. Treatment with rapamycin (10 mg/kg, every other day), trehalose consumption with drinking water (2 mg/mL, ad libitum), or their combination started 2 days after the surgery and lasted for 2 weeks. Open-field, plus-maze, and passive avoidance tests were used for behavioral phenotyping. Neuronal density, Aß accumulation, and the expression of autophagy marker LC3-II and neuroinflammatory marker IBA1 were measured in the frontal cortex and hippocampus. mRNA levels of autophagy genes (Atg8, Becn1, and Park2) were assessed in the hippocampus. Trehalose but not rapamycin caused pronounced prolonged autophagy induction and transcriptional activation of autophagy genes. Both drugs effectively prevented Aß deposition and microglia activation. Autophagy inhibitor 3-methyladenine significantly attenuated autophagy activation and disturbed the effect of the inducers on Aß load. The inducers substantially reversed behavioral and neuronal deficits in Aß-injected mice. In many cases, the best outcomes were achieved with the combined treatment. Thus, trehalose alone or combined autophagy activation by the two inducers may be a promising treatment approach to AD-like neurodegeneration. Some aspects of interaction between mTOR-dependent and mTOR-independent pathways of autophagy are discussed.

Neuroprotective Effects of Ceftriaxone Involve the Reduction of Aß Burden and Neuroinflammatory Response in a Mouse Model of Alzheimer's Disease.

Ceftriaxone (CEF) is a safe and multipotent antimicrobial agent that possesses neuroprotective properties. Earlier, we revealed the restoration of cognitive function in OXYS rats with signs of Alzheimer's disease (AD)-like pathology by CEF along with its modulating the expression of genes related to the system of amyloid beta (Aß) metabolism in the brain. The aim of this study was to determine the effects of CEF on behavior, Aß deposition, and associated neuroinflammation using another model of an early AD-like pathology induced by Aß. Mice were injected bilaterally i.c.v. with Aß fragment 25-35 to produce the AD model, while the CEF treatment (100 mg/kg/day, i.p., 36 days) started the next day after the surgery. The open field test, T-maze, Barnes test, IntelliCage, and passive avoidance test were used for behavioral phenotyping. Neuronal density, amyloid accumulation, and the expression of neuroinflammatory markers were measured in the frontal cortex and hippocampus. CEF exhibited beneficial effects on some cognitive features impaired by Aß neurotoxicity including complete restoration of the fear-induced memory and learning in the passive avoidance test and improved place learning in the IntelliCage. CEF significantly attenuated amyloid deposition and neuroinflammatory response. Thus, CEF could be positioned as a potent multipurpose drug as it simultaneously targets proteostasis network and neuroinflammation, as well as glutamate excitotoxicity, oxidative pathways, and neurotrophic function as reported earlier. Together with previous reports on the positive effects of CEF in AD models, the results confirm the potential of CEF as a promising treatment against cognitive decline from the early stages of AD progression.

Unconventional anxiety pharmacology in zebrafish: Drugs beyond traditional anxiogenic and anxiolytic spectra.

Anxiety is the most prevalent brain disorder and a common cause of human disability. Animal models are critical for understanding anxiety pathogenesis and its pharmacotherapy. The zebrafish (Danio rerio) is increasingly utilized as a powerful model organism in anxiety research and anxiolytic drug screening. High similarity between human, rodent and zebrafish molecular targets implies shared signaling pathways involved in anxiety pathogenesis. However, mounting evidence shows that zebrafish behavior can be modulated by drugs beyond conventional anxiolytics or anxiogenics. Furthermore, these effects may differ from human and/or rodent responses, as such 'unconventional' drugs may affect zebrafish behavior despite having no such profiles (or exerting opposite effects) in humans or rodents. Here, we discuss the effects of several putative unconventional anxiotropic drugs (aspirin, lysergic acid diethylamide (LSD), nicotine, naloxone and naltrexone) and their potential mechanisms of action in zebrafish. Emphasizing the growing utility of zebrafish models in CNS drug discovery, such unconventional anxiety pharmacology may provide important, evolutionarily relevant insights into complex regulation of anxiety in biological systems. Albeit seemingly complicating direct translation from zebrafish into clinical phenotypes, this knowledge may instead foster the development of novel CNS drugs, eventually facilitating innovative treatment of patients based on novel 'unconventional' targets identified in fish models.

Effects of acute and chronic arecoline in adult zebrafish: Anxiolytic-like activity, elevated brain monoamines and the potential role of microglia.

Arecoline is a naturally occurring psychoactive alkaloid with partial agonism at nicotinic and muscarinic acetylcholine receptors. Arecoline consumption is widespread, making it the fourth (after alcohol, nicotine and caffeine) most used substance by humans. However, the mechanisms of acute and chronic action of arecoline in-vivo remain poorly understood. Animal models are a valuable tool for CNS disease modeling and drug screening. Complementing rodent studies, the zebrafish (Danio rerio) emerges as a promising novel model organism for neuroscience research. Here, we assessed the effects of acute and chronic arecoline on adult zebrafish behavior and physiology. Overall, acute and chronic arecoline treatments produced overt anxiolytic-like behavior (without affecting general locomotor activity and whole-body cortisol levels), with similar effects also caused by areca nut water extracts. Acute arecoline at 10 mg/L disrupted shoaling, increased social preference, elevated brain norepinephrine and serotonin levels and reduced serotonin turnover. Acute arecoline also upregulated early protooncogenes c-fos and c-jun in the brain, whereas chronic treatment with 1 mg/L elevated brain expression of microglia-specific biomarker genes egr2 and ym1 (thus, implicating microglial mechanisms in potential effects of long-term arecoline use). Finally, acute 2-h discontinuation of chronic arecoline treatment evoked withdrawal-like anxiogenic behavior in zebrafish. In general, these findings support high sensitivity of zebrafish screens to arecoline and related compounds, and reinforce the growing utility of zebrafish for probing molecular mechanisms of CNS drugs. Our study also suggests that novel anxiolytic drugs can eventually be developed based on arecoline-like molecules, whose integrative mechanisms of CNS action may involve monoaminergic and neuro-immune modulation.

Pro-social and anxiolytic-like behavior following a single 24-h exposure to 17ß-estradiol in adult male zebrafish.

Estradiol (17ß-estradiol, E2) is a crucial estrogen hormone that regulates sexual, cognitive, social and affective behaviors in various species. However, complex central nervous system (CNS) effects of E2, including its activity in males, remain poorly understood. The zebrafish (Danio rerio) is rapidly becoming a powerful novel model system in translational neuroscience research. Here, we evaluate the effects of a single 24-h exposure to 20 µg/L of E2 on behavioral and endocrine (cortisol) responses in adult male zebrafish. Overall, E2 exerted pro-social effect in the social preference test, reduced whole-body cortisol levels, elevated exploration in the novel tank test and increased the shoal size in the shoaling test, indicative of an anxiolytic-like profile of this hormone in male zebrafish. Supporting mounting human and rodent evidence on the role of E2 in behavioral regulation, the observed pro-social and anxiolytic-like effects of E2 in male zebrafish reinforce the use of this aquatic organism in studying steroid-mediated CNS mechanisms of complex affective and social behaviors.

Evaluating the Effects of Grain of Isogenic Wheat Lines Differing in the Content of Anthocyanins in Mouse Models of Neurodegenerative Disorders.

Functional foods enriched with plant polyphenols and anthocyanins in particular attract special attention due to multiple beneficial bioactive properties of the latter. We evaluated the effects of a grain diet rich in anthocyanins in a mouse model of Alzheimer's disease induced by amyloid-beta (Aß) and a transgenic mouse model of Parkinson's disease (PD) with overexpression of human alpha-synuclein. The mice were kept at a diet that consisted of the wheat grain of near isogenic lines differing in anthocyanin content for five-six months. The anthocyanin-rich diet was safe and possessed positive effects on cognitive function. Anthocyanins prevented deficits in working memory induced by Aß or a long-term grain mono-diet; they partially reversed episodic memory alterations. Both types of grain diets prolonged memory extinction and rescued its facilitation in the PD model. The dynamics of the extinction in the group fed with the anthocyanin-rich wheat was closer to that in a group of wild-type mice given standard chow. The anthocyanin-rich diet reduced alpha-synuclein accumulation and modulated microglial response in the brain of the transgenic mice including the elevated expression of arginase1 that marks M2 microglia. Thus, anthocyanin-rich wheat is suggested as a promising source of functional nutrition at the early stages of neurodegenerative disorders.

Effects of Grape Polyphenols on the Life Span and Neuroinflammatory Alterations Related to Neurodegenerative Parkinson Disease-Like Disturbances in Mice.

Functional nutrition is a valuable supplementation to dietary therapy. Functional foods are enriched with biologically active substances. Plant polyphenols attract particular attention due to multiple beneficial properties attributed to their high antioxidant and other biological activities. We assessed the effect of grape polyphenols on the life span of C57BL/6 mice and on behavioral and neuroinflammatory alterations in a transgenic mouse model of Parkinson disease (PD) with overexpression of the A53T-mutant human α-synuclein. C57BL/6 mice were given a dietary supplement containing grape polyphenol concentrate (GPC-1.5 mL/kg/day) with drinking water from the age of 6-8 weeks for life. Transgenic PD mice received GPC beginning at the age of 10 weeks for four months. GPC significantly influenced the cumulative proportion of surviving and substantially augmented the average life span in mice. In the transgenic PD model, the grape polyphenol (GP) diet enhanced memory reconsolidation and diminished memory extinction in a passive avoidance test. Behavioral effects of GP treatment were accompanied by a decrease in α-synuclein accumulation in the frontal cortex and a reduction in the expression of neuroinflammatory markers (IBA1 and CD54) in the frontal cortex and hippocampus. Thus, a GP-rich diet is recommended as promising functional nutrition for aging people and patients with neurodegenerative disorders.

Sham surgical embryo transfer affects offspring neurodevelopment and manifestation of hypertensive phenotype in ISIAH rats.

The study investigates how surgery during pregnancy, i.e., sham operation associated with embryo transfer, affects hypertensive phenotype in ISIAH rats genetically predisposed to hypertension. ISIAH rats born after maternal surgery at fourth day of pregnancy were compared with naturally conceived controls. Surgery during pregnancy in ISIAH rats caused acceleration of neurodevelopment in young offspring, as well as aggravating hypertension, suppressing exploratory activity, reducing hippocampal BDNF expression, and compensatory increasing of hippocampal neuronal density in adult ISIAH offspring. Maternal surgery during early pregnancy caused alterations in offspring phenotype in hypertensive ISIAH rat model.

Behavioral and physiological effects of acute and chronic kava exposure in adult zebrafish.

Kava kava (Piper methysticum) is a medicinal plant containing kavalactones that exert potent sedative, analgesic and anti-stress action. However, their pharmacological effects and molecular targets remain poorly understood. The zebrafish (Danio rerio) has recently emerged as a powerful new model organism for neuroscience research and drug discovery. Here, we evaluate the effects of acute and chronic exposure to kava and kavalactones on adult zebrafish anxiety, aggression and sociality, as well as on their neurochemical, neuroendocrine and genomic responses. Supporting evolutionarily conserved molecular targets, acute kava and kavalactones evoked dose-dependent behavioral inhibition, upregulated brain expression of early protooncogenes c-fos and c-jun, elevated brain monoamines and lowered whole-body cortisol. Chronic 7-day kava exposure evoked similar behavioral effects, did not alter cortisol levels, and failed to evoke withdrawal-like states upon discontinuation. However, chronic kava upregulated several microglial (iNOS, Egr-2, CD11b), astrocytal (C3, C4B, S100a), epigenetic (ncoa-1) and pro-inflammatory (IL-1ß, IL-6, TNFa) biomarker genes, downregulated CD206 and IL-4, and did not affect major apoptotic genes in the brain. Collectively, this study supports robust, evolutionarily conserved behavioral and physiological effects of kava and kavalactones in zebrafish, implicates brain monoamines in their acute effects, and provides novel important insights into potential role of neuroglial and epigenetic mechanisms in long-term kava use.

Bumetanide blocks the acquisition of conditioned fear in adult rats.

BACKGROUND AND PURPOSE: Bumetanide has anxiolytic effects in rat models of conditioned fear. As a loop diuretic, bumetanide blocks cation-chloride co-transport and this property may allow bumetanide to act as an anxiolytic by modulating GABAergic synaptic transmission in the CNS. Its potential for the treatment of anxiety disorders deserves further investigation. In this study, we evaluated the possible involvement of the basolateral nucleus of the amygdala in the anxiolytic effect of bumetanide. EXPERIMENTAL APPROACH: Brain slices were prepared from Wistar rats. extracellular recording, stereotaxic surgery, fear-potentiated startle response, locomotor activity monitoring and Western blotting were applied in this study. KEY RESULTS: Systemic administration of bumetanide (15.2 mg·kg-1 , i.v.), 30 min prior to fear conditioning, significantly inhibited the acquisition of the fear-potentiated startle response. Phosphorylation of ERK in the basolateral nucleus of amygdala was reduced after bumetanide administration. In addition, suprafusion of bumetanide (5 or 10 µM) attenuated long-term potentiation in the amygdala in a dose-dependent manner. Intra-amygdala infusion of bumetanide, 15 min prior to fear conditioning, also blocked the acquisition of the fear-potentiated startle response. Finally, the possible off-target effect of bumetanide on conditioned fear was excluded by side-by-side control experiments. CONCLUSIONS AND IMPLICATIONS: These results suggest the basolateral nucleus of amygdala plays a critical role in the anxiolytic effects of bumetanide.

NKCC1 mediates traumatic brain injury-induced hippocampal neurogenesis through CREB phosphorylation and HIF-1α expression.

Traumatic brain injury (TBI) is one of the most prevalent causes of worldwide mortality and morbidity. We previously had evidenced that TBI induced Na-K-2Cl co-transporter (NKCC1) upregulation in hippocampus. Here, we aim to investigate the role of NKCC1 in TBI-induced neurogenesis and the detailed mechanisms. The TBI-associated alternations in the expression of NKCC1, HIF-1α, VEGF, MAPK cascade, and CREB phosphorylation were analyzed by Western blot. TBI-induced neurogenesis was determined by immuno-fluorescence labeling. Chromatin immunoprecipitation was used to elucidate whether HIF-1α would activate VEGF gene after TBI. We found that the level of hippocampal NKCC1 and VEGF began to rise 8 h after TBI, and both of them reached maxima at day 7. Along with the upregulation of NKCC1 and VEGF, MAPK cascade was activated and hippocampal neurogenesis was promoted. Administration of CREB antisense oligonucleotide significantly attenuated the expression of HIF-1α, while HIF-1α antisense oligonucleotide exhibited little effect on the expression of CREB. However, HIF-1α antisense oligonucleotide administration did effectively suppress the expression of VEGF. Our results of the chromosome immunoprecipitation also indicated that HIF-1α could directly act on the VEGF promoter and presumably would elevate the VEGF expression after TBI. All these results have illustrated the correlation between NKCC1 upregulation and TBI-associated neurogenesis. The pathway involves the activation of Raf/MEK/ERK cascade, CREB phosphorylation, and HIF-1α upregulation, and finally leads to the stimulation of VEGF expression and the induction of neurogenesis.

Reproductive abnormalities in adult male mice following preimplantation exposures to estradiol or pesticide methoxychlor.

Adult females of ICR strain of mice were bred, separated into different experimental groups, and treated as follows. On Days 2-4 of pregnancy, the mice received daily subcutaneous injections of either 0.05 ml sesame oil (vehicle) or same volume of 5.0mg of purified methoxychlor (MXC) suspended in the vehicle. Another group received a single subcutaneous injection of 1.0 microg of estradiol-17beta (E) on Day 2 of pregnancy only. Male offspring were tested at 3 and 6 months of age. At 3 months, E or MXC did not alter the weights of seminal vesicles, preputial glands, or testes, although after exposure for 30 min to a female in estrus behind a partition, testosterone levels were significantly reduced in treated males in comparison to control males exposed to the same partition test. At 6 months, the preputial glands and testes weight remained unchanged, while the seminal vesicles were significantly heavier in E- and MXC-treated males. Same partition tests again revealed that in E and MXC groups, testosterone levels remained significantly lower in comparison to control males. MXC or E exposures during preimplantation appear to induce long-term effects on the sexual development in 3 and 6 month-old-males by compromising their sexual arousal and altering seminal vesicles weights in the older group.