Cell proliferation in the embryonic mouse neocortex following acute maternal alcohol intoxication

Experiments were conducted to examine the effects of ‘binge’ or acute maternal alcohol intoxication during the early proliferative phase of embryonic brain development. Primiparous mice received ethanol as 0, 10, 15 or 20% (v/v) aqueous solutions by gavage on days 13, 14 and 15 of gestation. Mean daily doses were 0.0, 2.58, 4.03 and 5.40 ml/kg, respectively. There was no alcohol-related reduction in fetal body weight, length or fixed brain weight. Coronal sections (1 μm) of the dorsal roof of the lateral ventricles over the optic chiasma were examined from nine embryonic day 15 brains for each treatment group. The ventricular surface index of mitotic figures, the number and distribution of non-surface mitotic figures, and the depth of the cortical roof and its constituent layers were determined. There was no alcohol-related difference in any of these parameters. These results are in contrast to those of a previous experiment using the same mouse strain, in which prolonged or chronic maternal alcohol consumption in the drinking water from days 11 to 19 of pregnancy was associated with a reduction in the surface index, a reduction in the depth of the cortical roof and an increase in the non-surface mitotic figures. These latter changes, however, occurred in the presence of reduced body weight. Our observations suggest that during this particular developmental period (corresponding to the second trimester of human pregnancy) alcohol-related reductions in brain growth parallel restrictions in general body growth.     

Reductions in the plating efficiency of the fetal neural precursor cells following maternal alcohol consumption

Maternal alcohol abuse has been associated with reduced neural cell number and abnormal cell differentiation and organization in many regions of the developing mammalian brain. Experiments were conducted to determine whether changes in the neural precursor cells could be detected in primary culture following maternal alcohol consumption during the early proliferative period of the fetal brain growth spurt. Alcohol was administered to pregnant mice in the drinking water from days 11 to 19 of gestation. There was no alcohol-related increase in the incidence of fetal mortality or malformation, but fetal body and brain weights were reduced. When disaggregated cells from the day 19 fetal neopallium were grown in culture, there was a reduction in the number of astroglial colonies yielded in 42% of alcohol-exposed brains. This effect was expressed as a reduction in the absolute plating efficiency (APE) of the neural precursor cells. The APE was reduced as much as 80% in severely affected brains. There was no alcoholrelated difference in the in vitro morphogenesis of the astroglial colonies. Observations of the proliferative neural cells in situ suggest that there is an impaired recruitment of all neural cell types, but that the reduced APE reflects primarily a proportionate increase in the number of immature neurons among the cells obtained from the fetal neopallium. It appears that a prolongation of mitosis may be resulting in a general developmental delay in the fetal neocortex.     

Brain malformations in prenatal mice following acute maternal ethanol administration

Acute maternal ethanol administration (two i.p. injections of 2.9 g ethanol/kg maternal body wt) to C57B1/6J mice during gastrulation stages of embryogenesis (gestational day 7) induces a spectrum of brain and facial malformations characteristic of those seen in the human Fetal Alcohol Syndrome. Scanning electron microscopic and light microscopic analyses of the brains of embryos of gestational days 11–14 demonstrate ventro-medial forebrain deficiencies of varying degrees of severity in affected specimens. Even at the mild end of the spectrum, reductions in the size of the septal nuclei and the shape of the third ventricle are observed. As the severity of the effect increases, the septal nuclei disappear altogether, resulting in midline fusion of the corpora striata (basal ganglia). In such cases, the third ventricle is totally absent anteriorly (preoptic area) and significantly narrowed at more posterior levels, adjacent to the ventromedial nuclei. In addition, the hippocampal primordium is absent at levels which include the corpora striata, and septation of the cerebral cortex is incomplete. More posteriorly, at the level of the posterior commissure, the hippocampal primordium is present, but greatly reduced in size, and the entire brain is distinctly narrower in width. Still further posteriorly, at levels of the metencephalon which include the tectum and cerebellar plate, the cerebral aqueduct is significantly expanded, fusion of midline (raphe) structures is incomplete and the cerebellar plate does not extend as far medially as it does normally. Interestingly, these abnormalities are analogous to those observed in the holoprosencephaly series of malformations.The results of the present study support our hypothesis that severe forms of the Fetal Alcohol Syndrome mimic certain aspects of the holoprosencephaly spectrum, and indicate that special attention should be paid to possible deficiencies in the septal nuclei and basal ganglia of children born to women who abuse alcohol. The fact that gross brain malformations can be induced in this animal model at a time corresponding to the third week of human gestation (a time when most women remain unaware of pregnancy) is of significance in terms of the possible prevention of alcohol-induced birth defects and mental deficiency in man.     

Neurotransmitter changes in rat brain following acute alcohol intoxication

We studied the functional states of the main neurotransmitter systems in the brain following acute alcohol intoxication. Alterations in the contents of neurotransmitters, their metabolites, and neurotransmitter amino acids were the most prominent in the brainstem, thalamus, and neocortex following alcohol administration at doses of 2.5 or 5 g/kg. These alterations were observed in the key transmitters of the catecholaminergic system, such as dopamine, norepinephrine, and their metabolites. Following severe alcohol intoxication at a dose of 5 g/kg, the GABA level increased in the mentioned brain regions, showing a prevalence of inhibitory processes.     

Development of neurotransmitter systems in the mouse embryo following acute ethanol exposure: a histological and immunocytochemical study

Acute maternal ethanol administration to C57B1/6J mice on gestational day 7 (GD7) results in facial and brain abnormalities similar to those reported in human fetal alcohol syndrome (FAS). Using this model, we assessed the damage to brain structures using histological methods and changes in developing neurotransmitter systems with immunocytochemistry. Cholinergic neurons in the forebrain were stained with a monoclonal antibody to choline acetyltransferase (ChAT). Catecholaminergic neurons in the midbrain and serotoninergic neurons in the hindbrain were stained with polyclonal antisera to tyrosine hydroxylase (TH) and serotonin (5-HT), respectively. Forebrain deficiencies, including loss of midline structures (olfactory bulbs, midline septation, medial septal area) and deficits in lateral and dorsal regions (neostriatum and cerebral cortex) were found in both GD14 embryos and GD18 fetuses. In severely affected offspring, complete loss of the septal region resulted in conjoined lateral ventricles and a reduction in the thickness of the ventricular zone surrounding the single ventricle, as well as a severe loss of ChAT neurons which would normally be located in this territory. However, no consistent changes were seen in the distribution or size of TH or 5-HT neuronal cell groups in the midbrain and hindbrain. These differences in effects on specific neurotransmitter systems reflect the fact that the forebrain is most severely affected by early ethanol administration, whereas the hindbrain is relatively spared. Such differential effects could produce an imbalance in developing neurotransmitter systems in the embryonic and fetal brain, which could explain some of the functional deficits observed in children with FAS.     

Genetic differences in hypothalamic-pituitary-adrenal axis responsiveness to acute ethanol and acute ethanol withdrawal

There is a growing body of evidence suggesting that corticosteroids contribute to the increased neural excitability observed during ethanol withdrawal. In the present study, this was further investigated using mouse strains which differ in ethanol withdrawal severity. DBA/2 (DBA) mice were found to display more severe acute ethanol withdrawal seizures than C57BL/6 (C57) mice. Additionally, DBA mice showed a greater stress response than C57 mice, as measured by higher plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone, to an acute dose of ethanol. Mimicking withdrawal plasma corticosterone levels by administering corticosterone to ethanol-naive mice resulted in increases in handling-induced convulsions in the range observed during withdrawal. There did not appear to be a strain difference in sensitivity to the excitatory effects of corticosterone. In summary, the greater stress response to ethanol by DBA mice may account, in part, for the more severe ethanol withdrawal syndrome of this strain.     

Regional glucose phosphorylation rate in rat brain during acute ethanol intoxication

The effect of ethanol on regional cerebral metabolic rate for glucose (rCMRglc) was studied in rats using [6-14C]glucose. After intravenous injection, radioactivity was determined in 14 brain regions, corrected for loss of label, and divided by the integral of the arterial plasma glucose concentration measured during tracer circulation. When blood ethanol concentration was maintained at 6 g/l by intravenous infusion of ethanol for approximately 1 h, rCMRglc was found to be reduced significantly in 7 forebrain regions, compared to values of conscious control animals. In 7 further regions including brain stem regions, rCMRglc was not significantly reduced. We conclude that the effects of severe acute alcohol intoxication resemble those of global anesthesia.     

Trimethyltin intoxication induces marked changes in neuropeptide expression in the rat hippocampus

In situ hybridization and immunocytochemistry were applied to investigate changes in the expression of somatostatin, neuropeptide Y, neurokinin B, cholecystokinin, dynorphin, and Met-enkephalin in the rat hippocampus after administration of a single peroral dose of trimethyltin hydroxide (9 mg/kg). Two time intervals were investigated: 5 days after trimethyltin treatment, when CA3 damage becomes manifest and is associated with increased aggression, seizure susceptibility, and memory deficit, and 16 days after trimethyltin, when neuronal damage is almost maximal and seizure susceptibility is declining. Robust but transient increases of neuropeptide Y, neurokinin B, and Met-enkephalin mRNA levels were revealed in the granule cell layer of the dentate gyrus and increased neuropeptide Y and neurokinin B immunoreactivities were found in mossy fibers. In reverse, dynorphin mRNA and immunoreactivity were decreased transiently in the dentate gyrus and mossy fibers, respectively. Strong over-expression of NPY mRNA was also observed in hilar interneurons and in CA1 and CA3 pyramidal cells as well as in the cortex at 5 days postdosing. Cholecystokinin- or neurokinin B-containing basket cells were preserved, while somatostatin-bearing interneurons were damaged by trimethyltin exposure. These neurochemical changes induced by trimethyltin intoxication strikingly parallel to those observed in animal models of temporal lobe epilepsy and may reflect activation of endogenous protective mechanisms. It is also suggested that hilar interneurons respond differently to trimethyltin exposure, for which neuropeptides are valuable markers. Synapse 29:333–342, 1998. © 1998 Wiley-Liss, Inc.     

Brain marker protein changes after short- and long-term ethanol intoxication and withdrawal in the rat

The brain marker proteins, D1, D2, and D3, localised to neuronal membranes, and mitochondrial and cytoplasmic marker proteins (MM and CM), were studied during 1-6 days (short term) intragastrically-induced severe ethanol intoxication and during 1 month (long-term) ethanol intoxication established by a liquid diet regimen. The concentrations of the same brain proteins were also measured during withdrawal from the ethanol intoxication periods. Three categories of effect were encountered: decreased concentration of brain marker proteins during severe short-term intoxication the effect being most marked for D3, possibly indicating degradation of mature synapses; increased concentration of proteins D2 and MM during withdrawal, the D2 changes possibly indicating formation of new synapses; increased concentration of D1 protein and MM during long-term intoxication. We suggest that the changes in brain marker proteins reflect dynamic changes of subcellular neuronal structures which may form a part of the basis of functional tolerance to and physical dependence upon ethanol or the reversion of these states after withdrawal of ethanol.     

Complex plastic changes in the neuropeptide Y system during ethanol intoxication and withdrawal in the rat brain

Previous studies show that chronic ethanol treatment induces prominent changes in brain neuropeptide Y (NPY). The purpose of the present study was to explore ethanol effects at a deeper NPY‐system level, measuring expression of NPY and its receptors (Y1, Y2, Y5) as well as NPY receptor binding and NPY‐stimulated [³⁵S]GTPγS functional binding. Rats received intragastric ethanol repeatedly for 4 days, and the NPY system was studied in the hippocampal dentate gyrus (DG), CA3, CA1, and piriform cortex (PirCx) and neocortex (NeoCx) during intoxication, peak withdrawal (16 hr), late withdrawal (3 days), and 1 week after last ethanol administration. NPY mRNA levels decreased during intoxication and at 16 hr in hippocampal regions but increased in the PirCx and NeoCx at 16 hr. NPY mRNA levels were increased at 3 days and returned to control levels in most regions at 1 week. Substantial changes also occurred at the receptor level. Thus Y1, Y2, and Y5 mRNA labelling decreased at 16 hr in most regions, returning to control levels at 3 days, except for PirCx Y2 mRNA, which increased at 3 days and 1 week. Conversely, increases in NPY receptor binding occurred in hippocampal regions during intoxication and in functional binding in the DG and NeoCx during intoxication and at 16 hr and in PirCx during intoxication and at 1 week. Thus this study shows that ethanol intoxication and withdrawal induce complex plastic changes in the NPY system, with decreased/increased gene expression or binding occurring in a time‐ and region‐specific manner. These changes may play an important role in mediating ethanol‐induced changes in neuronal excitability. © 2009 Wiley‐Liss, Inc.     

成年大鼠脑创伤后神经前体细胞的增殖及迁移

目的 研究液压冲击性脑损伤后成年大鼠神经前体细胞的增殖及迁移。方法 制作液压冲击性脑损伤模型，免疫组织化学方法动态检测巢蛋白（Nestin）和5溴脱氧尿苷（BrdU）的表达。BrdU标记方法确定增列殖的前体细胞；Nestin的表达用于确定神经前体细胞。结果 同正常对照组相比较，伤侧皮层、海马及室下区的Nestin阳性细胞数于伤后1d明显增多，7d达高峰,30d消失；BrdU阳性细胞数于作后3d达高峰，而7d以后逐渐减小，室下区BrdU阳性细胞及Nestin阳性细胞经胼胝体向对侧迁移。结论 液压冲击性脑损伤可激发成年大鼠神经前体细胞增殖及迁移。     

Changes in calcium currents during ethanol withdrawal in a genetic mouse model

Changes in voltage dependent calcium currents in the dentate gyrus of the hippocampal slice during ethanol withdrawal were studied using an alcohol withdrawal seizure prone mouse strain (WSP) and compared to a withdrawal-resistant strain (WSR). There was a significant increase in the high voltage activated calcium currents during the withdrawal period in the WSP strain, while those of the resistant strain showed no significant enhancement. These results suggest that an increase in calcium currents is one factor involved in the alcohol withdrawal hyperexcitability of the prone strain observed in vivo.     

Mapping of a putative genetic locus determining ethanol intake in the mouse

In the mouse, there is evidence that a single genetic locus is a major determinant of differences in ethanol intake between some preferring and non-preferring inbred strains. In this report, we present evidence from two independent experiments indicating that this locus maps to chromosome 1 and that its expressed product is the abundant protein LTW-4 (a 28 kDa, pI 5.6 protein expressed in brain, liver and kidney). The genetic association was found using a panel of 14 polypeptides of mouse brain which were visualized by two-dimensional electrophoresis and which exhibit genetic variation in isoelectric point. Fifteen BXD recombinant inbred strains and the two parental strains were typed for these loci and also tested for ethanol acceptance. Strains exhibiting the basic allele showed significantly higher ethanol acceptance. When 19 distantly related inbred mouse strains were tested for ethanol acceptance and typed for LTW-4, it was again found that strains exhibiting the basic allele showed significantly higher ethanol acceptance.     

Neuromediator changes in different rat brain regions after acute morphine intoxication

We investigated the effects of acute morphine intoxication on the functions of the dopaminergic, noradrenergic, and serotonergic systems, and the GABA level in the cerebral cortex, thalamus, and striatum of the rat brain. It was shown that a single morphine administration resulted in changes in neurotransmitter systems, which depended on the morphine dosage and the brain regions examined. Activation of both dopamine and noradrenaline release and catabolism were mostly expressed in the thalamus and striatum. The changes in the GABA level revealed in the cerebral cortex after different acute morphine intoxication were related to the level of behavioral activity of the animals. Changes in the serotonergic system were less pronounced.     

The effect of acute ethanol intoxication and chronic ethanol consumption on vascular permeability around cerebral stab wounds in mice

Summary It has been suggested that prolongation of a breach in the blood-brain barrier to protein might promote the regeneration of injured axons in the brain and spinal cord. Other workers have found that ethanol given up to 24h after injury will exacerbate exudation of protein from damaged blood vessels in the brains of rats. This raised the possibility that ethanol might be used to maintain cerebral blood vessels in a state of permeability to protein after injury and thus to encourage regeneration of severed central axons.Stab wounds were made in the brains of mice, and the animals were treated with ethanol for up to 4 weeks. Exudation of protein into the neuropil was detected by use of fluorescently labelled albumin. Single intoxicating doses of ethanol did not increase leakage of the tracer from blood vessels adjacent to lesions that were 3h old. This contrasts with its effect in rats and may be due to a difference in the sensitivity to ethanol of the two species. Ethanol, in the drinking water, increased the extent of exudation of protein around lesions in animals treated for 1 week but had no effect in animals treated for 2, 3 or 4 weeks. Ethanol did not significantly lengthen the period after injury during which the cerebral blood vessels were permeable to protein, so treatment with ethanol would not be expected to support regeneration of damaged axons in the central nervous system (CNS).This work was supported by a grant from the Multiple Sclerosis Society of Canada to Dr. J. A. Kiernan, Department of Anatomy, University of Western Ontario     

大鼠脑梗死后神经前体细胞的增殖及电针作用的实验研究

目的　研究脑梗死病灶周围及海马处神经前体细胞增殖水平的动态变化及电针治疗对其的影响。方法　采用易卒中型肾性高血压大鼠 (RHRSP) ,电凝法凝闭大脑中动脉 (MCAO)。用Garcia等的综合评分法评定大鼠的神经行为学功能 ,免疫组化观察梗死灶边缘、对侧镜区及双侧海马 5 溴脱氧尿核苷 (Bromodeoxyuridine,BrdU)标记细胞的变化。结果　MCAO后大鼠轻偏瘫 ,5天时神经行为学功能恢复正常。MCAO后梗死灶边缘、双侧镜区及双侧海马均有BrdU阳性细胞分布 ,且病灶侧多于病灶对侧 ,病灶周围分布密集。电针治疗促使梗死灶边缘BrdU阳性细胞增多 ,随着治疗时间增加细胞增多更明显。结论　脑梗死可诱导病灶周边及海马神经前体细胞增殖水平上调 ,2周内神经前体细胞随着电针治疗时间的增加而增多。神经前体细胞可能是脑梗死康复的重要物质基础。     

猫视神经慢性受压后视网膜神经节细胞的动态观察

目的 观察猫视神经慢性受压后视网膜神经节细胞(RGCs)的动态变化.方法 30只成年家猫按随机数字表法等分为正常对照组、假手术组、压迫1周组、压迫2周组、压迫4周组和压迫8周组,每组5只.利用球囊植入法建立慢性视神经损伤模型.所有动物在术前2周用Dil逆行标记RGCs.取各组动物视网膜进行光镜、电镜观察,并在荧光显微镜下对RGCs进行计数.结果 正常成年猫视网膜HE染色可见三层细胞核,各层间界限较为清楚,从玻璃体腔向巩膜依次为神经节细胞层、双极细胞层和感光细胞层;视神经慢性受压4周时视网膜变化仍不明显:受压8周时可见RGCs核明显稀疏,大而染色浅的细胞核基本消失,视网膜总厚度变薄,胶质细胞增生.电镜下正常猫RGCs核呈卵圆形、核大、核质均匀,有时可见核仁,胞浆占细胞比例小,但细胞器丰富;受压2周时视网膜无明显改变;受压4周和8周时可见RGCs胞浆成分疏松、内质网扩张、线粒体肿胀、质膜下出现空泡、核膜皱缩内陷、胞浆空化、染色质边聚.正常组DiI逆行标记的RGCs的密度在406～527个/mm2之间,平均为(465±38)个/mm2,中央区密度高于周边区;视神经受压后前4周RGCs数量无明显改变;受压8周时RGCs数量有显著下降,平均密度为(293±32)个/mm2.下降约37%.结论 视神经慢性受压后RCJCs将出现延迟的变性.为视神经受压后RGCs的保护提供了时机.     

Sex-dependent changes in blood-brain barrier permeability and brain NA(+),K(+) ATPase activity in rats following acute water intoxication

To understand the increased susceptibility of the development of serious complications to hypoosmotic hyponatremia in young females, we examined the resistance of blood brain barrier (BBB) permeability to water along with the synaptosomal Na(+),K(+)ATPase activity in both sexes of rats during acute water intoxication. Four groups of rats were used: Group I and II were normal female and male rats injected with only Evans-blue. Group III and IV were water intoxicated female and male rats respectively. BBB permeability in female rats was found to be increased following acute water intoxication. In contrast, synaptosomal Na(+),K(+)ATPase activities in both water intoxicated male and female rats were found significantly lower than those in control rats. But inhibition in enzyme activity in synaptosomes from water intoxicated female rats was more pronounced than those of corresponding male rats. Our results concluded that female sex steroids may be responsible for the highly significant decrease in synaptosomal Na(+),K(+)ATPase activity and increased BBB permeability in female rats following water intoxication.     

脑梗死大鼠神经前体细胞增殖水平的研究

目的研究脑梗死病灶周围及海马处神经前体细胞增殖水平的动态变化。方法采用易卒中型肾性高血压大鼠(RHRSP)，电凝大脑中动脉(MCA)主干制成脑梗死(MCAO)模型。行大鼠神经功能评定，免疫组化观察并计数梗死灶边缘、对侧镜区及双侧海马5-溴脱氧尿核苷(Bromodeoxyuridine，BrdU)标记的细胞。结果MCAO后大鼠神经功能评分减低，5d时恢复正常。MCAO后梗死灶边缘、对侧镜区及双侧海马均有BrdU阳性细胞分布，且病灶侧多于病灶对侧，集中分布于病灶周围。结论脑缺血可诱导神经前体细胞增殖并移向病灶，可能成为脑梗死恢复的重要物质基础。