Impaired brain growth in neonatal rats exposed to ethanol.

Infant rat pups, fed through intragastric cannulas from postnatal day 4 through day 18, showed a 19 percent reduction in total brain weight when ethanol was included in their diet on days 4 through 7. This reduction in brain weight occurred even though body growth in the experimental rats was equal to that of their littermate controls. The ethanol-exposed animals were markedly hypoactive during the period of drug administration, then displayed gross body tremors for 3 to 5 days. Throughout the study, the animals treated with ethanol had poor motor coordination and were hyperresponsive. These brain and behavioral effects appear similar to those seen in fetal alcohol syndrome.

Protection from ethanol-induced limb malformations by the superoxide dismutase/catalase mimetic, EUK-134.

Based on previous in vitro studies that have illustrated prevention of ethanol-induced cell death by antioxidants, using an in vivo model, we have tested the anti-teratogenic potential of a potent synthetic superoxide dismutase plus catalase mimetic, EUK-134. The developing limb of C57BL/6J mice, which is sensitive to ethanol-induced reduction defects, served as the model system. On their ninth day of pregnancy, C57BL/6J mice were administered ethanol (two intraperitoneal doses of 2.9 g/kg given 4 h apart) alone or in combination with EUK-134 (two doses of 10 mg/kg). Pregnant control mice were similarly treated with either vehicle or EUK-134, alone. Within 15 h of the initial ethanol exposure, excessive apoptotic cell death was observed in the apical ectodermal ridge (AER) of the newly forming forelimb buds. Forelimb defects, including postaxial ectrodactyly, metacarpal, and ulnar deficiencies, occurred in 67.3% of the ethanol-exposed fetuses that were examined at 18 days of gestation. The right forelimbs were preferentially affected. No limb malformations were observed in control fetuses. Cell death in the AER of embryos concurrently exposed to ethanol and EUK-134 was notably reduced compared with that in embryos from ethanol-treated dams. Additionally, the antioxidant treatment reduced the incidence of forelimb malformations to 35.9%. This work illustrates that antioxidants can significantly improve the adverse developmental outcome that results from ethanol exposure in utero, diminishing the incidence and severity of major malformations that result from exposure to this important human teratogen.

Animal model systems for the study of alcohol teratology.

The incidence of fetal alcohol syndrome has not been declining even though alcohol has been established as a teratogen and significant efforts have been made to educate women not to abuse alcohol during pregnancy. In addition to further educational efforts, strategies to prevent or mitigate the damages of prenatal alcohol exposure are now under development. Animal models will play a significant role in the effort to develop these strategies. Because prenatal alcohol exposure causes damage by multiple mechanisms, depending on dose, pattern, and timing of exposure, and because no species of animal is the same as the human, the choice of which animal model to use is complicated. To choose the best animal model, it is necessary to consider the specific scientific question that is being addressed and which model system is best able to address the question. Animal models that are currently in use include nonhuman primates, rodents (rats, mice, guinea pigs), large animal models (pig and sheep), the chick, and simple animals, including fish, insects, and round worms. Each model system has strengths and weaknesses, depending on the question being addressed. Simple animal models are useful in exploring basic science questions that relate to molecular biology and genetics that cannot be explored in higher-order animals, whereas higher-order animal models are useful in studying complex behaviors and validating basic science findings in an animal that is more like the human. Substantial progress in this field will require the judicious use of multiple scientific approaches that use different animal model systems.

Genesis of alcohol-induced craniofacial dysmorphism.

The initial diagnosis of fetal alcohol syndrome (FAS) in the United States was made because of the facial features common to the first cohort of patients. This article reviews the development of an FAS mouse model whose craniofacial features are remarkably similar to those of affected humans. The model is based on short-term maternal treatment with a high dosage of ethanol at stages of pregnancy that are equivalent to Weeks 3 and 4 of human gestation. At these early stages of development, alcohol's insult to the developing face is concurrent with that to the brain, eyes, and inner ear. That facial and central nervous system defects consistent with FAS can be induced by more "realistic" alcohol dosages as illustrated with data from an oral alcohol intake mouse model in which maternal blood alcohol levels do not exceed 200 mg/dl. The ethanol-induced pathogenesis involves apoptosis that occurs within 12 hrs of alcohol exposure in selected cell populations of Day 7, 8, and 9 mouse embryos. Experimental evidence from other species also shows that apoptosis underlies ethanol-induced malformations. With knowledge of sensitive and resistant cell populations at specific developmental stages, studies designed to identify the basis for these differing cellular responses and, therefore, to determine the primary mechanisms of ethanol's teratogenesis are possible. For example, microarray comparisons of sensitive and resistant embryonic cell populations have been made, as have in situ studies of gene expression patterns in the populations of interest. Studies that illustrate agents that are effective in diminishing or exacerbating ethanol's teratogenesis have also been helpful in determining mechanisms. Among these agents are antioxidants, sonic hedgehog protein, retinoids, and the peptides SAL and NAP.

Prenatal alcohol exposure and fetal programming: effects on neuroendocrine and immune function.

Alcohol abuse is known to result in clinical abnormalities of endocrine function and neuroendocrine regulation. However, most studies have been conducted on males. Only recently have studies begun to investigate the influence of alcohol on endocrine function in females and, more specifically, endocrine function during pregnancy. Alcohol-induced endocrine imbalances may contribute to the etiology of fetal alcohol syndrome. Alcohol crosses the placenta and can directly affect developing fetal cells and tissues. Alcohol-induced changes in maternal endocrine function can disrupt maternal-fetal hormonal interactions and affect the female's ability to maintain a successful pregnancy, thus indirectly affecting the fetus. In this review, we focus on the adverse effects of prenatal alcohol exposure on neuroendocrine and immune function, with particular emphasis on the hypothalamic-pituitary-adrenal (HPA) axis and the concept of fetal programming. The HPA axis is highly susceptible to programming during fetal development. Early environmental experiences, including exposure to alcohol, can reprogram the HPA axis such that HPA tone is increased throughout life. We present data that demonstrate that maternal alcohol consumption increases HPA activity in both the maternal female and the offspring. Increased exposure to endogenous glucocorticoids throughout the lifespan can alter behavioral and physiologic responsiveness and increase vulnerability to illnesses or disorders later in life. Alterations in immune function may be one of the long-term consequences of fetal HPA programming. We discuss studies that demonstrate the adverse effects of alcohol on immune competence and the increased vulnerability of ethanol-exposed offspring to the immunosuppressive effects of stress. Fetal programming of HPA activity may underlie some of the long-term behavioral, cognitive, and immune deficits that are observed following prenatal alcohol exposure.

Disruption of cholesterol homeostasis in the developing brain as a potential mechanism contributing to the developmental neurotoxicity of ethanol: an hypothesis.

While excess cholesterol may have deleterious consequences, as in the case of atherosclerosis, too little cholesterol may endanger the development of the brain. Different degrees of mental retardation are often observed in inborn errors of cholesterol synthesis, such as the Smith-Lemli-Opitz syndrome or in maternal phenylketonuria, where the metabolite of accumulating phenylalanine, phenylacetate, is an inhibitor of cholesterol synthesis. Lack of cholesterol during brain development as a consequence of these genetic defects leads to severe brain damage, microencephaly and mental retardation, which are also hallmarks of the fetal alcohol syndrome (FAS). The brain relies on the in situ synthesis of cholesterol, which occurs mostly in astrocytes. Astrocyte-produced cholesterol is utilized for cell proliferation, or is released, via astrocyte-secreted high density lipoprotein-like particles containing apolipoprotein E, outside the cell, where it is taken up and utilized by neurons for dendrite outgrowth and to form synapses. We propose the hypothesis that ethanol may disrupt cholesterol homeostasis during brain development, and that this effect may be responsible, at least in part, for the central nervous system dysfunctions observed in the FAS, which include altered astrocyte proliferation, neuronal death and diminished synaptic contacts.

Muscarinic cholinergic receptor signal transduction as a potential target for the developmental neurotoxicity of ethanol.

Central nervous system dysfunctions (most notably mental retardation and microcephaly) are among the most significant effects of in utero exposure to ethanol. Ethanol has been shown to cause alterations of both neuronal and glial cells, including cell loss, and changes in their migration and maturation. Here, we propose that one of the potential targets for the developmental neurotoxicity of ethanol may be represented by the signal transduction systems activated by cholinergic muscarinic receptors. Ethanol has been shown to inhibit second messenger systems activated by various G-protein-coupled receptors, including certain subtypes of muscarinic receptors. Although the roles of muscarinic receptors in brain development have not been fully elucidated, two potentially relevant effects have been discovered in the past few years. By activating muscarinic receptors coupled to phospholipid metabolism, acetylcholine can induce proliferation of glial cells, and act as a trophic factor in developing neurons by preventing apoptotic cell death. Ethanol has been shown to inhibit both actions of acetylcholine in vitro. These effects of ethanol may lead to a decreased number of glial cells and to a loss of neurons, which have been observed following in vivo alcohol exposure. In turn, these may be the basis of microencephaly and cognitive disturbances in children diagnosed with Fetal Alcohol Syndrome.

Facial midline defect in the fetal alcohol syndrome: embryogenetic considerations in two clinical cases.

We report on two unrelated patients with fetal alcohol syndrome with hypoplasia of the periocular region, resulting in a low and narrow forehead and hypotelorism. Other typical manifestations of the syndrome involving the facial midline are also present. These observations can be added to clinical and experimental evidence from other authors, supporting the concept that the facial anomalies of the fetal alcohol syndrome are the expression of a midline defect originating from the disruption of the ordered development of midline mesoderm cells during early embryogenesis.

Absence of the lateral philtral ridges: a clue to the structural basis of the philtrum.

This study compares philtral development in the normal fetus with philtral development in specimens lacking normal philtral landmarks. Distinct differences in the structure of the upper lip were discovered between the two groups using a histological comparison. A new mechanism for the structural basis of the philtrum is proposed on the basis of these differences.

Experimental fetal alcohol syndrome: proposed pathogenic basis for a variety of associated facial and brain anomalies.

Acute teratogenic exposure of C57Bl/6J mouse embryos to ethanol in vivo results, within 12 hours of initial insult, in excessive cell death in selected cell populations. The patterns of excessive cell death observed following exposure of gestational day 8 embryos (late presomite--approximately 5 somite pair stages) vary somewhat temporospatially, but primarily involve the cell populations at the rim of the anterior neural plate. The cell death patterns appear to be pathogenically correlated with subsequently observed malformations including exencephaly (anencephaly), arhinencephaly, pituitary dysplasia, bilateral or unilateral cleft lip, maxillary hypoplasia, and median facial deficiencies and clefts. The association of these brain and facial malformations in this model, and perhaps in humans, may be accounted for by early insult to the selected cell populations identified in the current investigation.

Quantitation of craniofacial anomalies in utero: fetal alcohol and Crouzon syndromes and thanatophoric dysplasia.

The study of fetal growth and development by ultrasound has been greatly facilitated in the past few years by the availability of anthropometric standards for the fetal body. Thus, the obstetrician is able to discern between normal and grossly abnormal, and even to quantitate certain fine fetal structures such as the face. This paper presents results obtained from a group of 5 patients referred to the Medical Center from private practices in Indianapolis, Indiana. Prenatal cephalometric analyses by ultrasound suggested the presence of craniofacial anomalies in all 5 cases. However, such defects were not detectable by routine ultrasonographic examination. A clinical examination after birth of each of these 5 patients suggested the following diagnoses: Fetal Alcohol Syndrome (FAS) in 2 individuals, Fetal Alcohol Effects (FAE) in one individual, Crouzon Syndrome (CS) in one patient, and Thanatophoric Dysplasia (TD) in one patient. In order to compare the craniofacial measurement values for each patient to normal standards, we developed Z-Score profiles and Pattern Variability Indexes (PVI) as described by Garn et al. [1984, 1985]. The values presented here support the idea that even mildly abnormal fetal craniofacial patterns are detectable by this relatively new application of ultrasound. At the present time, no conclusions can be made regarding the diagnostic accuracy of these patterns and profiles. However, the potential value of fetal cephalometry for documenting craniofacial dysmorphology is clearly indicated.

Fetal alcohol syndrome. Eye malformations in a mouse model.

Acute maternal ethanol administration on gestational day 7 (gastrulation stage) in C57Bl/6J mice results in a spectrum of ocular malformations. A deficiency in the anterior neural plate observable within 24 hours of exposure results in corresponding defects in the optic sulcus and subsequent optic vesicle. Deficiency in the size of the lens vesicle induced by a small optic vesicle is demonstrable as microphakia in older embryos. Delayed detachment of the lens vesicle from the surface ectoderm manifests in the live offspring as progressive corneal opacification and vascularization related to defects in corneal endothelium and Descemet's membrane. Anterior segment dysgenesis results in persistent iridocorneal adhesions, dyscoria, and abnormal formation of the anterior chamber. In contrast, ethanol exposure on day 8 of gestation did not result in eye malformations. Thus, it appears that many of the ocular abnormalities associated with fetal alcohol syndrome may result from an acute insult to the optic primordia during a very specific period that corresponds to the third week after fertilization in the human.

Fetal ethanol exposure diminishes hippocampal beta-adrenergic receptor density while sparing muscarinic receptors during development.

Because of ostensible effects of fetal exposure to ethanol on cardiac and memory functions, beta-adrenergic and muscarinic receptor binding were surveyed in hippocampus and heart in 8- and 17-day-old rat pups. Pregnant, multiparous rats were intubated with either 6 g/kg ethanol or isocaloric dextrose twice daily from gestational days 10-16. At birth, offspring were fostered to untreated mothers. Pups exposed to ethanol had diminished birth weights, although there was no difference in the amount of weight gain by ethanol and control dams during gestation, nor in litter size. Ethanol pups remained smaller than control pups, but this difference was significant only until 8 days of age. At 17 days of age, ethanol pups had fewer hippocampal beta-adrenergic receptors than age-matched controls; muscarinic receptors and CA1 cell densities were not disparate. Parallel studies suggested that approximately 50% of the hippocampal beta-adrenergic receptors in 8-day-olds were of the beta 1 and beta 2 subtypes, while by 17 days of age approximately 70% of the receptors were beta 1. There was an ontogenetic increment in both beta-adrenergic and muscarinic binding from 8 to 17 days of age in hippocampus. No differences between age or drug groups were found in the binding measures in heart tissue. The present findings indicate that fetal ethanol treatment affects developmental measures and beta-adrenergic receptors in the hippocampus in a quasi-selective manner, but not hippocampal CA1-cell density.

Alcohol-induced cell death in the embryo.

Exposure to alcohol during gestation can have profound consequences, but not all cells within the embryo are affected equally. Recent advances in molecular embryology have allowed an exploration of this variation. Much of this research has focused on the embryo's vulnerability to the facial malformations characteristic of fetal alcohol syndrome. Studies using mice and chicks show that alcohol exposure at specific stages of early embryo development results in significant death among the cells destined to give rise to facial structures (i.e., cranial neural crest cells). This type of cell death is through activation of the cell's own "self-destruct" machinery (i.e., apoptosis). Researchers have advanced several theories to explain how alcohol triggers apoptosis in the neural crest cells. These theories include deficiency in a type of vitamin A compound, retinoic acid; reduced levels of antioxidant compounds (i.e., free radical scavengers) that protect against damage from toxic oxygen molecules (i.e., free radicals); and interference with the cell's normal internal communication pathways.

Circadian influence on ethanol-related intrauterine growth retardation in mice.

Circadian influences on growth and development in response to ethanol were studied in mice. On gestational day 10, pregnant animals received a single intraperitoneal injection of ethanol with the following dose levels: 1.0, 2.5 or 4.0 g/kg at one of four circadian phases (0700, 1300, 1900 or 0100 hr). 48 hrs after injection the embryonic weight and length, protein and DNA content and placental weight and protein were determined. Ethanol-related intrauterine growth retardation were shown to be dose- and circadian phase-dependent, the greatest susceptibility being seen during the dark phase. The variations observed are discussed with regard to changes in drug metabolism and tissue sensitivity.

Maternal ethanol consumption: effect on skeletal muscle development in guinea pig offspring.

The effect of ethanol on developing skeletal muscle was analyzed by examining the gastrocnemius muscle from newborn guinea pigs, exposed to ethanol during the second half of gestation. Electron microscopy revealed vacuolated sarcoplasmic reticula, enlarged lipid droplets, decreased glycogen and mitochondrial abnormalities in the skeletal muscle samples from the ethanol-exposed newborn guinea pigs. None of these abnormalities were seen in the newborn controls who were born to dams which consumed the same amount of calories as the ethanol-treated dams. The ethanol-associated abnormalities, seen in this study, are similar to those seen in ultrastructural examination of skeletal muscle from chronic alcoholics.

Craniofacial alterations in adult rats after acute prenatal alcohol exposure.

BACKGROUND: Exposure during pregnancy to alcohol (ethanol) produces a number of adverse effects. One of them is fetal alcohol syndrome. The hallmark of fetal alcohol syndrome (FAS) is craniofacial dysmorphism and the changes in craniofacial measurement are dependent on the alcoholic dose and its time of exposure. Since prenatal ethanol exposure can alter craniofacial development in rodents and reliably produce long-term behavioral effect in them, the present study was designed to extend the same changes in the Sprague Dawley species. METHODS: The albino rat was studied to determine whether gestational exposure to alcohol (Ethanol) produces permanent craniofacial effect. On gestational day (GD7-10) 25% ethanol was injected intraperitoneally to pregnant rats. Various dimensions for skull and face of adult male rats were taken. RESULTS: Both vertical and coronal dimensions were altered in the exposed animals. CONCLUSIONS: This study demonstrates that exposure to ethanol on a critical gestational period produces permanent craniofacial defects.

[Maternal environment and developmental brain damages].

To estimate and to prevent the developmental brain damage caused by maternal environmental agents, the results were compared between epidemiological and experimental studies. I. Ethanol. The features of CNS involvement in human FAS (fetal alcohol syndrome) and FAE (fetal alcohol effects) were developmental delay and intellectual impairment. In rat FAS or FAE, a beneficial effect of supplementary zinc on the fetal cerebrum was observed, but was limited. The consistent dysformation of synapses during early brain development may be associated with the functional impairment of CNS in FAS and FAE II. Caffeine. A reduction in the fetal cerebral weight was observed with caffeine ingestion during pregnancy at levels of 1.5-3.0 micrograms caffeine/ml or g wet wt. in dams and fetuses. Maternal caffeine disturbs the neonatal cerebrum and produces behavioral abnormalities in developing rats. III. X-irradiation. Our study provides evidence of the protection by vitamin E of neuronal development in X-irradiated rat fetuses, through its antioxidant properties, against attacks by free radicals and/or lipid peroxide. IV. Low copper level in brain. A teratogenic effect of triethylene tetramine dihydrochloride, a chelating drug for copper, on the mouse brain was noted, both grossly and microscopically, with a dose of higher than 6,000 micrograms/l, which is twenty times as high as the clinical treatment dose. V. Tobacco. Thirty-three percent of human FTS (fetal tobacco syndrome) cases had CNS involvement, which was characterized by developmental delay. Fifty percent or more of FTS children of women who smoked more than 20 cigarettes per day had CNS involvement. In conclusion, the concomitant effects of maternal environmental agents on human brain development should be studied.

Fetal alcohol syndrome: diagnosis, management, and prevention.

Fetal alcohol syndrome (FAS) and fetal alcohol effects (FAE) encompass a pattern of birth defects in persons whose mother ingested alcohol during pregnancy. Persons with FAE display fewer of the FAS traits. The hallmarks of FAS are pre- and postnatal growth retardation, central nervous system dysfunction, and characteristic facial dysmorphology. However, its effects can be multi-systemic--encompassing the cardiac, skeletal, and muscular systems, as well as presenting as lack of coordination, hyperactivity, diminished or distorted sense of danger, and lack of ability to function as an independent adult. The frequent incidence of this constellation of symptoms has a far-reaching impact (familial, medical, educational, and societal) because a myriad of professionals and large amounts of funding are used to help manage FAS/FAE children and adults. This article identifies, for a primary care provider, the essential characteristics of FAS/FAE and discusses available management options. Early diagnosis and continued education are advantageous at all levels, benefiting the individual and all of society.

[Neural tube dysraphia at the level of the midbrain in alcoholic embryopathy]. / Dizrafiia nervnoi trubki na urovne srednego mozga pri alkogol'noi émbriopatii.

Forty-four 5-12-week embryos obtained from mothers who abused alcohol during pregnancy were examined for brain development. Six cases of dysraphia of the neural tube at the midbrain level were revealed. In two cases, it was defined as primary. This was confirmed by the impairment of neurulation in the neural tube, by the growth of the ends of the neural plate, not yet grown together (hyperplasia), and derangement of the mesoderm (partial cranioschisis). The use of alcohol might be the most probable cause that gave rise to dysraphia. In these cases, the teratogenic termination period was referred to the first 4 weeks of pregnancy. In 4 cases, the secondary rupture of the walls of the midbrain, i.e. secondary dysraphia of the traumatic genesis was established.