Involvement of brain-derived neurotrophic factor methylation in the prefrontal cortex and hippocampus induced by chronic unpredictable mild stress in male mice.

Early life stress alters brain-derived neurotrophic factor (BDNF) promoter IV methylation and BDNF expression, which is closely related to the pathophysiological process of depression. However, the role of abnormal methylation of BDNF induced by stress during adolescence due to depression has not yet been clarified. In this study, adolescent mice were exposed to chronic unpredictable mild stress (CUMS). Depression-like behaviors, BDNF promoter IV methylation, expression of DNA methyltransferases (DNMTs), demethylation machinery enzymes, BDNF protein levels, and neuronal development in the prefrontal cortex (PFC) and hippocampus (HIP) were assessed in adolescent and adult mice. The DNMT inhibitor, 5-Aza-2-deoxycytidine (5-AzaD), was used as an intervention. Stress in adolescence induces behavioral dysfunction, elevated methylation levels of BDNF promoter IV, changes in the expression of DNMT, and demethylation machinery enzymes in adolescent and adult mice. Additionally, the stress in adolescence induced lower levels of BDNF and abnormal hippocampal doublecortin (DCX) expression in adolescent and adult mice. However, DNMT inhibitor treatment in adolescent-stressed mice relieved the abnormal behaviors, normalized the methylation level of BDNF promoter IV, BDNF protein expression, expression of DNMTs, and demethylation machinery enzymes, and improved the neuronal development of adult mice. These results suggest that stress in adolescence induces short- and long-term hypermethylation of BDNF promoter IV, which is regulated by DNMTs, and leads to the development of depression.

Multiple brain regions are involved in reaction to acute restraint stress in CYLD-knockout mice.

The lysine 63 deubiquitinase cylindromatosis (CYLD) is expressed at high levels in the brain and is considered to be involved in anxious and depressive behavior, cognitive inflexibility, and autism disorders. Previous research was limited in some brain regions, including the hippocampus, striatum, and amygdala. To better understand whether CYLD plays a role in adaptation to stress and which brain regions are involved, we analyzed the behavior of CYLD-knockout mice in the elevated plus maze (EPM) and light-dark box test (LDT) after acute restraint stress (ARS) and mapped their c-Fos immunoreactivity in brain sections. Here we report that CYLD deficiency leads to an unexpected reaction to ARS in mice, and is accompanied by significant neuronal activation of brain regions including the medial prefrontal cortex (mPFC), dorsal striatum (DS), nucleus accumbens (NAc), and basal lateral amygdala (BLA), but not ventral hippocampus (vHPC). Our findings show that CYLD participates in ARS-induced anxious behavior and that this involves multiple brain regions.

Dual-energy micro-focus computed tomography based on the energy-angle correlation of inverse Compton scattering source.

BACKGROUND: Inverse Compton scattering (ICS) source can produce quasi-monoenergetic micro-focus X-rays ranging from keV to MeV level, with potential applications in the field of high-resolution computed tomography (CT) imaging. ICS source has an energy-angle correlated feature that lower photon energy is obtained at larger emission angle, thus different photon energies are inherently contained in each ICS pulse, which is especially advantageous for dual- or multi-energy CT imaging. OBJECTIVE: This study proposes a dual-energy micro-focus CT scheme based on the energy-angle correlation of ICS source and tests its function using numerical simulations. METHODS: In this scheme, high- and low-energy regions are chosen over the angular direction of each ICS pulse, and dual-energy projections of the object are obtained by an angularly-splicing scanning method. The field-of-view (FOV) of ICS source is extended simultaneously through this scanning method, thus the scale of the imaging system can be efficiently reduced. A dedicated dual-energy CT algorithm is developed to reconstruct the monoenergetic attenuation coefficients, electron density, and effective atomic number distributions of the object. RESULTS: A test object composed of different materials (carbon, aluminium, titanium, iron and copper) and line pairs with different widths (15/24/39/60 µm) is imaged by the proposed dual-energy CT scheme using numerical simulations, and high-fidelity monoenergetic attenuation coefficient, electron density, and effective atomic number distributions are obtained. All the line pairs are well identified, and the contrast ratio of the 15 µm lines is 22%, showing good accordance with the theoretical predictions. CONCLUSIONS: The proposed dual-energy CT scheme can reconstruct fine inner structures and material compositions of the object simultaneously, opening a new possibility for the application of ICS source in the field of non-destructive testing.

L-Cysteine attenuates osteopontin-mediated neuroinflammation following hypoxia-ischemia insult in neonatal mice by inducing S-sulfhydration of Stat3.

We previously show that L-Cysteine administration significantly suppresses hypoxia-ischemia (HI)-induced neuroinflammation in neonatal mice through releasing H2S. In this study we conducted proteomics analysis to explore the potential biomarkers or molecular therapeutic targets associated with anti-inflammatory effect of L-Cysteine in neonatal mice following HI insult. HI brain injury was induced in postnatal day 7 (P7) neonatal mice. The pups were administered L-Cysteine (5 mg/kg) at 24, 48, and 72 h post-HI. By conducting TMT-based proteomics analysis, we confirmed that osteopontin (OPN) was the most upregulated protein in ipsilateral cortex 72 h following HI insult. Moreover, OPN was expressed in CD11b+/CD45low cells and infiltrating CD11b+/CD45high cells after HI exposure. Intracerebroventricular injection of OPN antibody blocked OPN expression, significantly attenuated brain damage, reduced pro-inflammatory cytokine levels and suppressed cerebral recruitment of CD11b+/CD45high immune cells following HI insult. L-Cysteine administration reduced OPN expression in CD11b+/CD45high immune cells, concomitant with improving the behavior in Y-maze test and suppressing cerebral recruitment of CD11b+/CD45high immune cells post-HI insult. Moreover, L-Cysteine administration suppressed the Stat3 activation by inducing S-sulfhydration of Stat3. Intracerebroventricular injection of Stat3 siRNA not only decreased OPN expression, but also reversed HI brain damage. Our data demonstrate that L-Cysteine administration effectively attenuates the OPN-mediated neuroinflammation by inducing S-sulfhydration of Stat3, which contributes to its anti-inflammatory effect following HI insult in neonatal mice. Blocking OPN expression may serve as a new target for therapeutic intervention for perinatal HI brain injury.

Inhibiting Brd4 alleviated PTSD-like behaviors and fear memory through regulating immediate early genes expression and neuroinflammation in rats.

Post-traumatic stress disorder (PTSD) is characterized by depression/anxiety and memory failure, primarily fear memory. According to the reports, neuroinflammation and synaptic plasticity can play a role in the neurophysiological mechanisms underlying PTSD. Bromodomain-containing protein 4 (Brd4) intriguingly affects regulating of inflammatory responses and learning and memory. This study aimed to explore the effect of inhibiting Brd4 on depression/anxiety-like behaviors, spatial and fear memory, and underlying mechanisms in a model of PTSD. Inescapable foot shocks (IFS) with a sound reminder in 6 days were used to induce PTSD-like behaviors which were tested using contextual and cue fear tests, sucrose preference test, open-field test, elevated plus maze test, and Y-maze test. Meanwhile, the Brd4 inhibitor JQ1 was used as an intervention. The results found that IFS induced PTSD-like behaviors and indicated obvious Brd4 expression in microglia of the prefrontal cortex (PFC), hippocampus, and amygdala, pro-inflammatory cytokines over-expression, microglial activation, and nuclear factor-kappa B over-expression in PFC and hippocampus but not in amygdala. Meanwhile, the alterations of immediate early genes (IEGs) were found in PFC, hippocampus, and amygdala. Besides, dendritic spine density was reduced in PFC and hippocampus but was elevated in amygdala of rats with IFS. In addition, treatment with JQ1 significantly reduced freezing time in the contextual and cue fear test, reversed the behavioral impairment, decreased the elevated neuroinflammation, and normalized the alteration in IEGs and dendritic spine densities. The results suggested that Brd4 was involved in IFS-induced PTSD-like behaviors through regulating neuroinflammation, dynamics of IEGs, and synaptic plasticity.

Rifaximin-mediated gut microbiota regulation modulates the function of microglia and protects against CUMS-induced depression-like behaviors in adolescent rat.

BACKGROUND: Chronic unpredictable mild stress (CUMS) can not only lead to depression-like behavior but also change the composition of the gut microbiome. Regulating the gut microbiome can have an antidepressant effect, but the mechanism by which it improves depressive symptoms is not clear. Short-chain fatty acids (SCFAs) are small molecular compounds produced by the fermentation of non-digestible carbohydrates. SFCAs are ubiquitous in intestinal endocrine and immune cells, making them important mediators of gut microbiome-regulated body functions. The balance between the pro- and anti-inflammatory microglia plays an important role in the occurrence and treatment of depression caused by chronic stress. Non-absorbable antibiotic rifaximin can regulate the structure of the gut microbiome. We hypothesized that rifaximin protects against stress-induced inflammation and depression-like behaviors by regulating the abundance of fecal microbial metabolites and the microglial functions. METHODS: We administered 150 mg/kg rifaximin intragastrically to rats exposed to CUMS for 4 weeks and investigated the composition of the fecal microbiome, the content of short-chain fatty acids in the serum and brain, the functional profiles of microglia and hippocampal neurogenesis. RESULTS: Our results show that rifaximin ameliorated depressive-like behavior induced by CUMS, as reflected by sucrose preference, the open field test and the Morris water maze. Rifaximin increased the relative abundance of Ruminococcaceae and Lachnospiraceae, which were significantly positively correlated with the high level of butyrate in the brain. Rifaximin increased the content of anti-inflammatory factors released by microglia, and prevented the neurogenic abnormalities caused by CUMS. CONCLUSIONS: These results suggest that rifaximin can regulate the inflammatory function of microglia and play a protective role in pubertal neurodevelopment during CUMS by regulating the gut microbiome and short-chain fatty acids.

MSCs-extracellular vesicles attenuated neuroinflammation, synapse damage and microglial phagocytosis after hypoxia-ischemia injury by preventing osteopontin expression.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) significantly suppressed hypoxia-ischemia (HI)-induced neuroinflammation in neonatal mice. However, its underlying mechanism is still unknown. Osteopontin (OPN) is one of the key molecules involved in neuroinflammation. We demonstrate here for the first time a key role of OPN in EVs-mediated neuroinflammation following HI. Firstly, HI exposure upregulated OPN expression in Iba-1+/ TMEM119+ microglia and Iba-1+/TMEM119- monocytes/macrophages. Blocking OPN mRNA expression with LV-shOPN attenuated edema, infarct volumes, and the levels of inflammatory cytokines following HI exposure. MSCs-EVs treatment remarkably restored synaptic reorganization and up-regulated synaptic protein expression post-HI, concomitant with reducing OPN levels. Moreover, MSCs-EVs treatment rescued microglial phagocytosis of viable neurons following HI, concomitant with decreasing OPN expression. In addition, blocking NF-κB activation with pyrrolidine dithiocarbamate (PDTC, NF-κB inhibitor) or MSCs-EVs attenuated HI-induced OPN expression in the ipsilateral cortex. This study demonstrates that upregulation of OPN expression in cerebral immune cells aggravated brain damage and inflammation following HI insult. MSCs-EVs suppressed neuroinflammation, synaptic damage and microglial phagocytosis after HI injury by preventing NF-κB-mediated OPN expression in neonate mice.

Hydrogen-rich saline promotes microglia M2 polarization and complement-mediated synapse loss to restore behavioral deficits following hypoxia-ischemic in neonatal mice via AMPK activation.

BACKGROUND: Hypoxia-ischemia (HI) during the perinatal period is one of the most common causes of acute mortality and chronic neurologic morbidity. Hydrogen-rich saline (HS) treatment in neonatal mice has been reported to alleviate brain injury following HI, but the mechanisms involved are not known. METHODS: A modified version of the Rice-Vannucci method for the induction of neonatal HI brain injury was performed on postnatal day 7 mouse pups. Animals or BV2-cells received HS and an AMPK inhibitor at indicative time post-injury. RESULTS: In the current study, we show that HS treatment attenuated the accumulation of CD11b+/CD45high cells, suppressed HI-induced neuro-inflammation, induced microglial anti-inflammatory M2 polarization, was associated with promoting AMPK activation, and inhibited nuclear factor-κB activation as demonstrated both in vivo and in vitro. In addition, HS treatment reversed HI-induced neurological disabilities, was associated with improving damaged synapses, and restored the expression levels of synaptophysin and postsynaptic density protein 95 following HI insult. Furthermore, HI insult which increased levels of complement component C1q, C3, and C3aR1 was observed. Importantly, C1q deposited in the infarct core and lesion boundary zone following HI injury, was found to co-localize within regions of synapse loss, whereas HS treatment reversed these effects of HI on synapse loss and complement component levels. Notably, the AMPK inhibitor reversed the beneficial effects of HS as described above. CONCLUSIONS: These results demonstrate that HS restored behavioral deficits after HI in neonatal mice. These beneficial effects, in part, involve promoting microglia M2 polarization and complement-mediated synapse loss via AMPK activation.

CEST MRI monitoring of tumor response to vascular disrupting therapy using high molecular weight dextrans.

PURPOSE: Vascular disrupting therapy of cancer has become a promising approach not only to regress tumor growth directly but also to boost the delivery of chemotherapeutics in the tumor. An imaging approach to monitor the changes in tumor vascular permeability, therefore, has important applications for monitoring of vascular disrupting therapies. METHODS: Mice bearing CT26 subcutaneous colon tumors were injected intravenously with 150 kD dextran (Dex150, diameter, d~ 20 nm, 375 mg/kg), tumor necrosis factor-alpha (TNF-α; 1 µg per mouse), or both (n = 3 in each group). The Z-spectra were acquired before and 2 h after the injection, and the chemical exchange saturation transfer (CEST) signals in the tumors as quantified by asymmetric magnetization transfer ratio (MTRasym ) at 1 ppm were compared. RESULTS: The results showed a significantly stronger CEST contrast enhancement at 1 ppm (∆MTRasym = 0.042 ± 0.002) in the TNF-α-treated tumors than those by Dex150 alone (∆MTRasym = 0.000 ± 0.005, P = 0.0229) or TNF-α alone (∆MTRasym = 0.002 ± 0.004, P = 0.0264), indicating that the TNF-α treatment strongly augmented the tumor uptake of 150 kD dextran. The MRI findings were verified by fluorescence imaging and immunofluorescence microscopy. CONCLUSIONS: High molecular weight dextrans can be used as safe and sensitive CEST MRI contrast agents for monitoring tumor response to vascular disrupting therapy and, potentially, for developing dextran-based theranostic drug delivery systems.

Ethanol Exposure Induces Microglia Activation and Neuroinflammation through TLR4 Activation and SENP6 Modulation in the Adolescent Rat Hippocampus.

The ethanol-induced toll-like receptor 4 (TLR4) signal activation of microglia and neuroinflammation are observed in both adolescent and adult rat brains, but the regulatory mechanisms of some TLR4 signaling-related factors in this process are still unclear. SUMO-specific protease 6 (SENP6) inhibits neuroinflammation by dampening nuclear factor kappa-B (NF-κB) activation via the de-SUMOylation of NF kappa-B essential modulator (NEMO). This study investigates the effects of long-term ethanol consumption on neuroinflammation in the hippocampus of adolescent rats and the regulatory roles of TLR4 and SENP6. Twenty-one days of ethanol exposure in adolescent rats were used to develop an animal model. The number of microglia, microglial activation, and the expression of TLR4 in the hippocampus of adolescent rats were examined by immunoreactivity. The levels of TLR4, activation of NF-κB including IkB-α and p-NF-κB-p65, and SENP6 were measured by western blotting. Proinflammatory cytokines including TNF-α, IL-1ß, and IL-6 were measured by enzyme-linked immunosorbent assay. The NF-κB activation and proinflammatory cytokines released in overexpressed SENP6 and siRNA targeting SENP6 microglial cells after treatment with ethanol were estimated in vitro. This study found that alcohol exposure increased the number of activated microglia and the levels of p-NF-κB-p65 and proinflammatory cytokines, while it decreased the SENP6 level in wild-type rats, but not in TLR4 knockout rats. The ethanol-induced increases of p-NF-κB-p65, TNF-α, and IL-1ß were dampened by overxpression of SENP6 and enhanced in SENP6-siRNA microglia. Our data suggest that ethanol exposure during adolescence induces the microglia-mediated neuroinflammation via TLR4 activation, and SENP6 plays an essential role in dampening NF-κB activation and neuroinflammation.

Application of High-Resolution CUBE Sequence in Exploring Stroke Mechanisms of Atherosclerotic Stenosis of Middle Cerebral Artery.

BACKGROUND: This study aimed to analyze the vascular wall and atherosclerotic plaques of the middle cerebral artery (MCA) and compare their differences between patients with cerebral infarction and transient ischemic attack (TIA) using 3-dimensional fast-spin-echo T1-weighted sequence (namely CUBE). METHODS: Forty-seven patients with atherosclerotic stenosis of the MCA were included in this study. They received magnetic resonance examinations with routine T1WI, T2WI, 3-dimensional time-of-flight magnetic resonance angiography and diffusion-weighted imaging, as well as high-resolution CUBE T1WI sequence. Two physicians independently observed the location and degree of enhancement of the atheromatous plaques. The vessel area and lumen area at the maximal-lumen-narrowing and reference site were measured to calculate the plaque area, rate of stenosis, and remodeling index of the MCA. The chi-squared test was used to compare the differences of degree of enhancement between the cerebral infarction and TIA groups. The differences of rate of stenosis and remodeling index were compared by independent sample t test. RESULTS: Twenty-five lesion vessels in the infarction group and 22 in the TIA group were analyzed. The difference of stenosis rate between the groups was not statistically significant. The lesion vessels of infarction group had a significantly larger remodeling index and plaque area, and the plaques had a significantly higher degree of enhancement, compared to the TIA group. CONCLUSIONS: CUBE T1WI can be used to characterize the MCA vessel wall and atherosclerotic plaque. Positive remodeling and enhanced plaques are closely correlated with the occurrence of brain stroke.

l-Cysteine suppresses hypoxia-ischemia injury in neonatal mice by reducing glial activation, promoting autophagic flux and mediating synaptic modification via H2S formation.

We previously reported that l-Cysteine, an H2S donor, significantly alleviated brain injury after hypoxia-ischemic (HI) injury in neonatal mice. However, the mechanisms underlying this neuroprotective effect of l-Cysteine against HI insult remain unknown. In the present study, we tested the hypothesis that the protective effects of l-Cysteine are associated with glial responses and autophagy, and l-Cysteine attenuates synaptic injury as well as behavioral deficits resulting from HI. Consistent with our previous findings, we found that treatment with l-Cysteine after HI reduced early brain injury, improved behavioral deficits and synaptic damage, effects which were associated with an up-regulation of synaptophysin and postsynaptic density protein 95 expression in the lesioned cortex. l-Cysteine attenuated the accumulation of CD11b+/CD45high cells, activation of microglia and astrocytes and diminished HI-induced increases in reactive oxygen species and malondialdehyde within the lesioned cortex. In addition, l-Cysteine increased microtubule associated protein 1 light chain 3-II and Beclin1 expression, decreased p62 expression and phosphor-mammalian target of rapamycin and phosphor-signal transducer and activator of transcription 3. Further support for a critical role of l-Cysteine was revealed from results demonstrating that treatment with an inhibitor of the H2S-producing enzyme, amino-oxyacetic acid, reversed the beneficial effects of l-Cysteine described above. These results demonstrate that l-Cysteine effectively alleviates HI injury and improves behavioral outcomes by inhibiting reactive glial responses and synaptic damage and an accompanying triggering of autophagic flux. Accordingly, l-Cysteine may provide a new a therapeutic approach for the treatment of HI via the formation of H2S.

Cardiovascular magnetic resonance black-blood thrombus imaging for the diagnosis of acute deep vein thrombosis at 1.5 Tesla.

BACKGROUND: The aim was to investigate the feasibility of a cardiovascular magnetic resonance (CMR) black-blood thrombus imaging (BBTI) technique, based on delay alternating with nutation for tailored excitation black-blood preparation and a variable flip angle turbo-spin-echo readout, for the diagnosis of acute deep vein thrombosis (DVT) at 1.5 T. METHODS: BBTI was conducted in 15 healthy subjects and 30 acute DVT patients. Contrast-enhanced CMR venography (CE-CMRV) was conducted for comparison and only performed in the patients. Apparent contrast-to-noise ratios between the thrombus and the muscle/lumen were calculated to determine whether BBTI could provide an adequate thrombus signal for diagnosis. Two blinded readers assessed the randomized BBTI images from all participants and made independent decisions on the presence or absence of thrombus at the segment level. Images obtained by CE-CMRV were also randomized and assessed by the two readers. Using the consensus CE-CMRV as a reference, the sensitivity, specificity, positive and negative predictive values, and accuracy of BBTI, as well as its diagnostic agreement with CE-CMRV, were calculated. Additionally, diagnostic confidence and interobserver diagnostic agreement were evaluated. RESULTS: The thrombi in the acute phase exhibited iso- or hyperintense signals on the BBTI images. All the healthy subjects were correctly identified from the participants based on the segment level. The diagnostic confidence of BBTI was comparable to that of CE-CMRV (3.69 ± 0.52 vs. 3.70 ± 0.47). High overall sensitivity (95.2%), SP (98.6%), positive predictive value (96.0%), negative predictive value (98.3%), and accuracy (97.7%), as well as excellent diagnostic and interobserver agreements, were achieved using BBTI. CONCLUSION: BBTI is a reliable, contrast-free technique for the diagnosis of acute DVT at 1.5 T.

Quantitative measurement of cerebral blood volume using velocity-selective pulse trains.

PURPOSE: To develop a non-contrast-enhanced MRI method for cerebral blood volume (CBV) mapping using velocity-selective (VS) pulse trains. METHODS: The new pulse sequence applied velocity-sensitive gradient waveforms in the VS label modules and velocity-compensated ones in the control scans. Sensitivities to the gradient imperfections (e.g., eddy currents) were evaluated through phantom studies. CBV quantification procedures based on simulated labeling efficiencies for arteriolar, capillary, and venular blood as a function of cutoff velocity (Vc) are presented. Experiments were conducted on healthy volunteers at 3T to examine the effects of unbalanced diffusion weighting, cerebrospinal (CSF) contamination and variation of Vc. RESULTS: Phantom results of the used VS pulse trains demonstrated robustness to eddy currents. The mean CBV values of gray matter and white matter for the experiments using Vc = 3.5 mm/s and velocity-compensated control with CSF-nulling were 5.1 ± 0.6 mL/100 g and 2.4 ± 0.2 mL/100 g, respectively, which were 23% and 32% lower than results from the experiment with velocity-insensitive control, corresponding to 29% and 25% lower in averaged temporal signal-to-noise ratio values. CONCLUSION: A novel technique using VS pulse trains was demonstrated for CBV mapping. The results were both qualitatively and quantitatively close to those from existing methods. Magn Reson Med 77:92-101, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Early adolescent stress-induced changes in prefrontal cortex miRNA-135a and hippocampal miRNA-16 in male rats.

Early-life stress increases susceptibility to post-traumatic stress disorders (PTSD), in which the dysfunction of 5-hydroxytryptamine plays an important role. miRNA-135a in the prefrontal cortex (PFC) and miRNA-16 in the hippocampus (HIP) are closely related to the 5-HT neurotransmitter system. Here, we investigated behavior, miRNA-135a in the PFC, miRNA-16 in the HIP, and 5-HT1AR expression in both brain regions in adolescent and adult rats that were exposed to inescapable stress during their adolescence. Paroxetine hydrochloride and corticotropin-releasing factor antagonist (CP-154,526) were used as intervening measures. Our study demonstrated that early adolescent stress induced anxiety-like behaviors and spatial memory damage, a reduction in miRNA-135a expression was associated with increased 5-HT1AR expression in PFC, and increased miRNA-16 expression in the HIP of stressed rats. Drug treatments alleviated behaviors and reversed the miRNA-135a, miRNA-16, and 5-HT1AR expression in stressed rats.

Oxytocin inhibits lipopolysaccharide-induced inflammation in microglial cells and attenuates microglial activation in lipopolysaccharide-treated mice.

BACKGROUND: Overactivated microglia is involved in various kinds of neurodegenerative diseases. Suppression of microglial overactivation has emerged as a novel strategy for treatment of neuroinflammation-based neurodegeneration. In the current study, anti-inflammatory effects of oxytocin (OT), which is a highly conserved nonapeptide with hormone and neurotransmitter properties, were investigated in vitro and in vivo. METHODS: BV-2 cells and primary microglia were pre-treated with OT (0.1, 1, and 10 µM) for 2 h followed by LPS treatment (500 ng/ml); microglial activation and pro-inflammatory mediators were measured by Western blot, RT-PCR, and immunofluorescence. The MAPK and NF-κB pathway proteins were assessed by Western blot. The intracellular calcium concentration ([Ca(2+)]i) was determined using Fluo2-/AM assay. Intranasal application of OT was pre-treated in BALB/C mice (adult male) followed by injected intraperitoneally with LPS (5 mg/kg). The effect of OT on LPS-induced microglial activation and pro-inflammatory mediators was measured by Western blot, RT-PCR, and immunofluorescence in vivo. RESULTS: Using the BV-2 microglial cell line and primary microglia, we found that OT pre-treatment significantly inhibited LPS-induced microglial activation and reduced subsequent release of pro-inflammatory factors. In addition, OT inhibited phosphorylation of ERK and p38 but not JNK MAPK in LPS-induced microglia. OT remarkably reduced the elevation of [Ca(2+)]i in LPS-stimulated BV-2 cells. Furthermore, a systemic LPS-treated acute inflammation murine brain model was used to study the suppressive effects of OT against neuroinflammation in vivo. We found that pre-treatment with OT showed marked attenuation of microglial activation and pro-inflammatory factor levels. CONCLUSIONS: Taken together, the present study demonstrated that OT possesses anti-neuroinflammatory activity and might serve as a potential therapeutic agent for treating neuroinflammatory diseases.

Dynamic Alterations of miR-34c Expression in the Hypothalamus of Male Rats after Early Adolescent Traumatic Stress.

Several types of microRNA (miRNA) overexpression in the brain are associated with stress. One of the targets of miR-34c is the stress-related corticotrophin releasing factor receptor 1 mRNA (CRFR1 mRNA). Here we will probe into the short-term effect and long-term effect of early adolescent traumatic stress on the expression of miR-34c and CRFR1 mRNA. Traumatic stress was established by electric foot shock for six consecutive days using 28-day rats. The anxiety-like behaviors, memory damage, CRFR1 protein, CRFR1 mRNA, and miR-34c expression were detected in our study. The results of our study proved that exposure to acute traumatic stress in early adolescent can cause permanent changes in neural network, resulting in dysregulation of CRFR1 expression and CRFR1 mRNA and miR-34c expression in hypothalamus, anxiety-like behavior, and memory impairment, suggesting that the miR-34c expression in hypothalamus may be an important factor involved in susceptibility to PTSD.

Palmitic acid affects proliferation and differentiation of neural stem cells in vitro.

High-lipid diet composed of saturated fatty acids (SFAs) has significant detrimental effects on brain homeostasis, and deleterious effects of SFAs on various cells have been well documented. However, the effects of SFAs on neural stem Cells (NSCs) function have not been fully explored. The aim of this study was to determine whether palmitic acid (PA) affected the proliferation and differentiation of murine-derived NSCs. The results showed that PA dose dependently suppressed viability of NSCs and was cytotoxic at high concentrations. The toxic levels of PA inhibited the proliferation of NSCs as shown by reduced bromodeoxyuridine labeling of NSCs, which is correlated with reactive oxygen species generation. Pretreatment of the cells with the antioxidant N-acetyl-L-cysteine inhibitor significantly attenuated the effects of PA on the proliferation of NSCs. Furthermore, nontoxic levels of PA promoted astrocytogenesis in the differentiated NSCs, associated with Stat3 activation and altered expression of serial of basic helix-loop-helix transcription factor genes. Altogether, our data have demonstrated that PA has a significant impact on proliferation and differentiation of NSCs in vitro and may be useful for elucidating the role of SFAs in regulating NSCs fate in physiological and pathological settings.

Hydrogen sulfide attenuates hypoxia-induced neurotoxicity through inhibiting microglial activation.

Endogenously produced hydrogen sulfide (H2S) may have multiple functions in the brain including potent anti-inflammatory effects. Activated microglia can secrete various pro-inflammatory cytokines and neurotoxic mediators, which may contribute to hypoxic injuries in the developing brain. The aim of this study is to investigate the potential role of H2S in altering hypoxia-induced neurotoxicity via its anti-inflammatory actions as examined in vitro and in vivo models. Using the BV-2 microglial cell line, we found that sodium hydrosulfide (NaHS), a H2S donor, significantly inhibited hypoxia-induced microglial activation and suppressed subsequent pro-inflammatory factor release. In addition, treating murine primary cortical neurons with conditioned medium (CM) from hypoxia-stimulated microglia induced neuronal apoptosis, an effect that was reversed by CM treated with NaHS. Further, NaHS inhibited phosphorylation of the p65 subunit of NF-κB, phosphorylation of ERK and p38 but not JNK MAPK in these hypoxia-induced microglia. When administered in vivo to neonatal mice subjected to hypoxia, NaHS was found to attenuate neuron death, an effect that was associated with suppressed microglial activation, pro-inflammatory cytokines and NO levels. Taken together, H2S exerts neuroprotection against hypoxia-induced neurotoxicity through its anti-inflammatory effect in microglia. This effect appears to be attributable to inhibition of iNOS, NF-κB, ERK and p38 MAPK signaling pathways. Our results suggest a potential therapeutic application of H2S releasing drugs in hypoxic brain damage treatment.

Chronic ketamine exposure induces permanent impairment of brain functions in adolescent cynomolgus monkeys.

Ketamine, a non-competitive N-methyl-D-aspartic acid receptor antagonist, has emerged as an increasingly popular drug among young drug abusers worldwide. Available evidence suggests that ketamine produces acute impairments of working, episodic and semantic memory along with psychotogenic and dissociative effects when a single dose is given to healthy volunteers. However, understanding of the possible chronic effects of ketamine on behavior, cognitive anomalies and neurochemical homeostasis is still incomplete. Although previous human studies demonstrate that ketamine could impair a range of cognitive skills, investigation using non-human models would permit more precise exploration of the neurochemical mechanisms which may underlie the detrimental effects. The current study examined the abnormalities in behavior (move, walk, jump and climb) and apoptosis of the prefrontal cortex using terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) and apoptotic markers, including Bax, Bcl-2 and caspase-3 in adolescent male cynomolgus monkeys (Macaca fascicularis) after 1 or 6 months of sub-anesthetic ketamine administration (1 mg/kg, i.v.). Results showed that ketamine decreased locomotor activity and increased cell death in the prefrontal cortex of monkeys with 6 months of ketamine treatment when compared with the control monkeys. Such decreases were not found in the 1-month ketamine-treated group. Our study suggested that ketamine administration of recreational dose in monkeys might produce permanent and irreversible deficits in brain functions due to neurotoxic effects, involving the activation of apoptotic pathways in the prefrontal cortex.