In‐vivo hemodynamic imaging of acute prenatal ethanol exposure in fetal brain by photoacoustic tomography

Prenatal ethanol exposure (PEE) can lead to structural and functional abnormalities in fetal brain. Although neural developmental deficits due to PEE have been recognized, the immediate effects of PEE on fetal brain vasculature and hemodynamics remain poorly understood. One of the major obstacles that preclude the rapid advancement of studies on fetal vascular dynamics is the limitation of the imaging techniques. Thus, a technique for noninvasive in‐vivo imaging of fetal vasculature and hemodynamics is desirable. In this study, we explored the dynamic changes of the vessel dimeter, density and oxygen saturation in fetal brain after acute maternal ethanol exposure in the second‐trimester equivalent murine model using a real‐time photoacoustic tomography system we developed for imaging embryo of small animals. The results indicate a significant decrease in fetal brain vessel diameter, perfusion and oxygen saturation. This work demonstrated that PAT can provide high‐resolution noninvasive imaging ability to monitor fetal vascular dynamics.     

Evaluating the effects of maternal alcohol consumption on murine fetal brain vasculature using optical coherence tomography

Prenatal alcohol exposure (PAE) can result in a range of anomalies including brain and behavioral dysfunctions, collectively termed fetal alcohol spectrum disorder. PAE during the 1st and 2nd trimester is common, and research in animal models has documented significant neural developmental deficits associated with PAE during this period. However, little is known about the immediate effects of PAE on fetal brain vasculature. In this study, we used in utero speckle variance optical coherence tomography, a high spatial‐ and temporal‐resolution imaging modality, to evaluate dynamic changes in microvasculature of the 2nd trimester equivalent murine fetal brain, minutes after binge‐like maternal alcohol exposure. Acute binge‐like PAE resulted in a rapid (<1 hour) and significant decrease (P < .001) in vessel diameter as compared to the sham group. The data show that a single binge‐like maternal alcohol exposure resulted in swift vasoconstriction in fetal brain vessels during the critical period of neurogenesis.      

CB1‐receptor knockout neonatal mice are protected against ethanol‐induced impairments of DNMT1, DNMT3A,and DNA methylation

The significant consequences of ethanol use during pregnancy are neurobehavioral abnormalities involving hippocampal and neocortex malfunctions that cause learning and memory deficits collectively named fetal alcohol spectrum disorder. However, the molecular mechanisms underlying these abnormalities are still poorly understood and therefore warrant systematic research. Here, we document novel epigenetic abnormalities in the mouse model of fetal alcohol spectrum disorder. Ethanol treatment of P7 mice, which induces activation of caspase 3, impaired DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) levels. Inhibition of caspase 3 activity, before ethanol treatment, rescued DNMT1, DNMT3A proteins as well as DNA methylation levels. Blockade of histone methyltransferase (G9a) activity or cannabinoid receptor type‐1 (CB1R), prior to ethanol treatment, which, respectively, inhibits or prevents activation of caspase 3, rescued the DNMT1 and DNMT3A proteins and DNA methylation. No reduction of DNMT1 and DNMT3A proteins and DNA methylation was found in P7 CB1R null mice, which exhibit no ethanol‐induced activation of caspase 3. Together, these data demonstrate that ethanol‐induced activation of caspase 3 impairs DNA methylation through DNMT1 and DNMT3A in the neonatal mouse brain, and such impairments are absent in CB1R null mice. Epigenetic events mediated by DNA methylation may be one of the essential mechanisms of ethanol teratogenesis.

     

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca²⁺]_i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca²⁺ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca²⁺]_i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca²⁺]_i was observed in Ca²⁺‐free media, suggesting intracellular Ca²⁺ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP₃R), which regulates Ca²⁺ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca²⁺]_i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca²⁺‐chelator, also inhibited the alcohol‐induced surge in [Ca²⁺]_i and prevented neuronal death. In conclusion, alcohol disrupts [Ca²⁺]_i homeostasis in CGN by releasing Ca²⁺ from intracellular stores, resulting in a sustained increase in [Ca²⁺]_i. This sustained increase in [Ca²⁺]_i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.     

Ethanol Alters Proliferation and Differentiation of Normal and Chromosomally Abnormal Human Embryonic Stem Cell‐Derived Neurospheres

Ethanol is a powerful substance and, when consumed during pregnancy, has significant psychoactive and developmental effects on the developing fetus. These abnormalities include growth retardation, neurological deficits, and behavioral and cognitive deficiencies, commonly referred to as fetal alcohol spectrum disorder. The effect of ethanol has been reported to affect cellular development on the embryonic level, however, not much is known about mutations contributing to the influence of ethanol. The purpose of our study was to determine if mutation contribute to changes in differentiation patterning, cell‐cycle regulatory gene expression, and DNA methylation in human embryonic stem cells after ethanol exposure. We exposed human embryonic stem cells (with and without know DNA mutations) to a low concentration (20 mM) of ethanol and measured neurosphere proliferation and differentiation, glial protein levels, expression of various cell‐cycle genes, and DNA methylation. Ethanol altered cell‐cycle gene expression between the two cell lines; however, gene methylation was not affected in ether lines.. Birth Defects Res (Part B) 98:283–295, 2013. © 2013 Wiley Periodicals, Inc.     

Postnatal ethanol exposure alters levels of 2‐arachidonylglycerol‐metabolizing enzymes and pharmacological inhibition of monoacylglycerol lipase does not cause neurodegeneration in neonatal mice

The consumption of ethanol by pregnant women may cause neurological abnormalities, affecting learning and memory processes in children, and are collectively described as fetal alcohol spectrum disorders. However, the molecular mechanisms underlying these changes are still poorly understood. In our previous studies, we found that ethanol treatment of postnatal day 7 (P7) mice significantly enhances the anandamide levels but not the 2‐arachidonylglycerol (2‐AG) levels and induces widespread neurodegeneration, but the reason for the lack of significant effects of ethanol on the 2‐AG level is unknown. In this study, we examined developmental changes in diacylglycerol lipase‐α, β (DAGL‐α and β) and monoacylglycerol lipase (MAGL). We found that the levels of these proteins were significantly higher in adult brains compared to those detected early in brain development. Next, we examined the influence of P7 ethanol treatment on these enzymes, finding that it differentially altered the DAGL‐α protein and mRNA levels but consistently enhanced those of the DAGL‐β. Interestingly, the ethanol treatment enhanced MAGL protein and mRNA levels. Inhibition of MAGL with KML29 failed to induce neurodegeneration in P7 mice. Collectively, these findings suggest that ethanol significantly activates DAGL‐β and MAGL in the neonatal brain, resulting in no net change in 2‐AG levels.

     

Examination of Age‐dependent effects of fetal ethanol exposure on behavior,hippocampal cell counts,and doublecortin immunoreactivity in rats

Ethanol is known as a potent teratogen having adverse effects on brain and behavior. However, some of the behavioral deficits caused by fetal alcohol exposure and well expressed in juveniles ameliorate with maturation may suggest some kind of functional recovery occurring during postnatal development. The aim of this study was to reexamine age‐dependent behavioral impairments in fetal‐alcohol rats and to investigate the changes in neurogenesis and gross morphology of the hippocampus during a protracted postnatal period searching for developmental deficits and/or delays that would correlate with behavioral impairments in juveniles and for potential compensatory processes responsible for their amelioration in adults. Ethanol was delivered to the pregnant dams by intragastric intubation throughout 7–21 gestation days at daily dose of 6 g/kg. Isocaloric intubation and intact control groups were included. Locomotor activity, anxiety, and spatial learning tasks were applied to juvenile and young‐adult rats from all groups. Unbiased stereological estimates of hippocampal volumes, the total number of pyramidal and granular cells, and double cortin expressing neurons were carried out for postnatal days (PDs) PD1, PD10, PD30, and PD60. Alcohol insult during second trimester equivalent caused significant deficits in the spatial learning in juvenile rats; however, its effect on hippocampal morphology was limited to a marginally lower number of granular cells in dentate gyrus (DG) on PD30. Thus, initial behavioral deficits and the following functional recovery in fetal‐alcohol subjects may be due to more subtle plastic changes within the hippocampal formation but also in other structures of the extended hippocampal circuit. Further investigation is required. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 498–513, 2014     

The brain‐derived neurotrophic factor VAL68MET polymorphism modulates how developmental ethanol exposure impacts the hippocampus

Prenatal exposure to alcohol causes a wide range of deficits known as fetal alcohol spectrum disorders (FASDs). Many factors determine vulnerability to developmental alcohol exposure including timing and pattern of exposure, nutrition and genetics. Here, we characterized how a prevalent single nucleotide polymorphism in the human brain‐derived neurotrophic factor (BDNF) gene (val66met) modulates FASDs severity. This polymorphism disrupts BDNF's intracellular trafficking and activity‐dependent secretion, and has been linked to increased incidence of neuropsychiatric disorders such as depression and anxiety. We hypothesized that developmental ethanol (EtOH) exposure more severely affects mice carrying this polymorphism. We used transgenic mice homozygous for either valine (BDNF^val/val) or methionine (BDNF^met/met) in residue 68, equivalent to residue 66 in humans. To model EtOH exposure during the second and third trimesters of human pregnancy, we exposed mice to EtOH in vapor chambers during gestational days 12 to 19 and postnatal days 2 to 9. We found that EtOH exposure reduces cell layer volume in the dentate gyrus and the CA1 hippocampal regions of BDNF^met/met but not BDNF^val/val mice during the juvenile period (postnatal day 15). During adulthood, EtOH exposure reduced anxiety‐like behavior and disrupted trace fear conditioning in BDNF^met/met mice, with most effects observed in males. EtOH exposure reduced adult neurogenesis only in the ventral hippocampus of BDNF^val/val male mice. These studies show that the BDNF val66met polymorphism modulates, in a complex manner, the effects of developmental EtOH exposure, and identify a novel genetic risk factor that may regulate FASDs severity in humans.     

Intrauterine hyperexposure to dexamethasone of the common marmoset monkey revealed normal cerebral metabolite concentrations in adulthood as assessed by quantitative proton magnetic resonance spectroscopy in vivo

Background  Animal models of human brain disorders often have to rely on non-human primates because of their immunological, physiological, and cognitive similarities to humans.
Methods  Localized proton magnetic resonance spectroscopy was performed to assess cerebral metabolite profiles of male common marmoset monkeys in vivo and to determine putative alterations of adult brain metabolism in response to intrauterine hyperexposure to the synthetic glucocorticoid hormone dexamethasone.
Results  Excellent spectral quality allowed for absolute quantification of the concentrations of major metabolites in predominantly white matter, gray matter, and thalamus. Marmoset monkeys intrauterinely hyperexposed to dexamethasone revealed normal neurochemical profiles at adulthood.
Conclusions  Prenatally applied dexamethasone does not lead to persistent metabolic alterations affecting adult brain integrity.     

Magnetic resonance image and blood manganese concentration as indices for manganese content in the brain of rats

Neurological disorders similar to parkinsonian syndrome and signal hyperintensity in brain on T₁-weighted magnetic resonance (MR) images have been reported in patients receiving long-term total parenteral nutrition (TPN). These symptoms have been associated with manganese (Mn) depositions in brain. Although alterations of signal intensity on T₁-weighted MR images in brain and of Mn concentration in blood are theoretically considered good indices for estimating Mn deposition in brain, precise correlations between these parameters have not been demonstrated as yet. Male Sprague-Dawley rats received TPN with 10-fold the clinical dose of the trace element preparation (TE-5) for 7 d. At 0, 2, 4, 6, and 8 wk post-TPN, the cortex, striatum, midbrain, and cerebellum were evaluated by MR images, and Mn concentration in blood and Mn content in these brain sites were measured by atomic absorption spectrometry. Immediately after TPN termination, signal hyperintensity in brain sites and elevated Mn content in blood and brain sites were observed. These values recovered at 4 wk post-TPN. A positive correlation was observed between either the signal intensity in certain brain sites or Mn content in blood and the relevant brain sites. Our observations suggest that the Mn concentration in blood and signal intensity in the brain sites on T₁-weighted MR images are reliable indices for monitoring Mn contents in brain.     

Comparison of the deleterious effects of binge drinking‐like alcohol exposure in adolescent and adult mice

A major cause of alcohol toxicity is the production of reactive oxygen species generated during ethanol metabolism. The aim of this study was to compare the effect of binge drinking‐like alcohol exposure on a panel of genes implicated in oxidative mechanisms in adolescent and adult mice. In adolescent animals, alcohol decreased the expression of genes involved in the repair and protection of oxidative DNA damage such as atr, gpx7, or nudt15 and increased the expression of proapoptotic genes such as casp3. In contrast, in the adult brain, genes activated by alcohol were mainly associated with protective mechanisms that prevent cells from oxidative damage. Whatever the age, iterative binge‐like episodes provoked the same deleterious effects as those observed after a single binge episode. In adolescent mice, multiple binge ethanol exposure substantially reduced neurogenesis in the dentate gyrus and impaired short‐term memory in the novel object and passive avoidance tests. Taken together, our results indicate that alcohol causes deleterious effects in the adolescent brain which are distinct from those observed in adults. These data contribute to explain the greater sensitivity of the adolescent brain to alcohol toxicity.

     

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B‐GFP transgenic mice

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B‐GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (?540 to +31). Using the fresh brain sections of F1B‐GFP transgenic mice, we found that the F1B‐GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B‐GFP⁺ cells existed in the brains of F1B‐GFP transgenic mice. We demonstrated that one population of F1B‐GFP⁺ cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B‐GFP⁺ cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B‐GFP⁺ cells and tyrosine hydroxylase indicated that a subpopulation of F1B‐GFP⁺‐neuronal cells was dopaminergic neurons. Importantly, these F1B‐GFP⁺/TH⁺ cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B‐GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 232–248, 2015     

NMDA receptor‐deficient mice display sexual dimorphism in the onset and severity of behavioural abnormalities

N‐methyl‐d ‐aspartate (NMDA) receptor‐deficient mice can be used to understand the role that NMDA receptors (NMDARs) play in the pathophysiology of neurodevelopmental disorders such as schizophrenia. Genetically modified mice with low levels of NR1 subunit (NR1 knockdown mice) have reduced receptor levels throughout development, and have robust abnormalities in behaviours that are relevant to schizophrenia. We traced the onset and severity of these behaviours at three developmental stages to understand when in development the underlying circuits depend on intact NMDAR function. We examined social behaviour, working memory, executive function, locomotor activity and stereotypy at 3, 6 and 12 weeks of age in NR1 knockdown mice and their wild‐type littermates. We discovered that each of these behaviours had a unique developmental trajectory in mutant mice, and males showed an earlier onset and severity than females in several behaviours. Hyperlocomotion was most substantial in juvenile mice and plateaued in adult mice, whereas stereotypy progressively worsened with age. Impairments in working memory and sociability were sexually dimorphic, with deficits first detected in peri‐adolescent males but only detected in adult females. Interestingly, executive function was most impaired in peri‐adolescent mice of either sex. Furthermore, while juvenile mutant mice had some ability to problem solve in the puzzle box test, the same mice lost this ability when tested 4 weeks later. Our studies highlight key developmental periods for males and females in the expression of behaviours that are relevant to psychiatric disorders.     

Study on the resonance Rayleigh scattering and resonance non‐linear scattering spectra of ion‐association complexes of [PdI4]2−‐protein systems and their analytical applications

In an acid medium solution, proteins such as bovine serum albumin, human serum albumin, ovalbumin, hemoglobin, lysozyme, γ‐globulin, α‐chymotrypsin and papain could react with [PdI₄]^2? by virtue of electrostatic attraction and hydrophobic force to form ion‐association complexes. As a result, the resonance Rayleigh scattering (RRS) and resonance nonlinear scattering such as second‐order scattering (SOS) and frequency doubling scattering (FDS) intensities were enhanced greatly and new scattering spectra appeared. The maximum scattering peaks of RRS, SOS and FDS were at 367, 720 and 370 nm, respectively. The enhanced RRS, SOS and FDS intensities were directly proportional to the concentrations of proteins. The detection limits for the different proteins were 2.4–11.8 ng/mL for RRS method, 9.5–47.9 ng/mL for SOS method and 4.6–18.5 ng/mL for FDS method. In this work, the influences of the interaction of [PdI₄]^2? with proteins on spectral characteristics of RRS, SOS and FDS were investigated and the optimum conditions were tested. Meanwhile, the effects of coexisting substances were tested and the results showed that the method exhibited a good selectivity. Based on the above research, a highly sensitive, simple and rapid method for the determination of trace amounts of proteins by resonance light scattering technique has been developed. It can be applied to the determination of proteins in tablet, human serum and urine samples. Copyright © 2009 John Wiley & Sons, Ltd.     

A novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers for the enrichment and determination of polychlorinated biphenyls in fish samples

The novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers (rGO@m‐MIPs) were successfully synthesized as adsorbents for six kinds of polychlorinated biphenyls (PCBs) in fish samples. rGO@m‐MIPs was prepared by surface molecular imprinting technique. Besides, Fe₃O₄ nanoparticles (NPs) were employed as magnetic supporters, and rGO@Fe₃O₄ was in situ synthesis. Different from functional monomer and cross‐linker in traditional molecularly imprinted polymer, here, 3,4‐dichlorobenzidine was employed as dummy molecular and poly(ethylene‐co‐vinyl alcohol) was adopted as the imprinted polymers. After morphology and inner structure of the magnetic adsorbent were characterized, the adsorbent was employed for disperse solid phase extraction toward PCBs and exhibited great selectivity and high adsorption efficiency. This material was verified by determination of PCBs in fish samples combined with gas chromatography‐mass spectrometry (GC‐MS) method. According to the detection, the low detection limits (LODs) of PCBs were 0.0035–0.0070 µg l⁻¹ and spiked recoveries ranged between 79.90 and 94.23%. The prepared adsorbent can be renewable for at least 16 times and expected to be a new material for the enrichment and determination of PCBs from contaminated fish samples. Copyright © 2015 John Wiley & Sons, Ltd.