Marieta Barrow Heaton

Ethanol-mediated generation of reactive oxygen species in developing rat cerebellum.

The neonatal cerebellum undergoes an early period of ethanol sensitivity in which profound neuronal loss is seen following acute exposure, while slightly later exposure produces no such loss. This study was designed to determine whether this differential susceptibility is related to differences in ethanol-induced generation of reactive oxygen species (ROS). We found that ethanol treatment on postnatal day 4 (P4), the peak period of cerebellar vulnerability, resulted in ROS increases, but slightly later exposure (on P7) produced no immediate changes in ROS, but reductions were seen at 12 and 24 h following exposure. Exposure on P14 produced consistent decreases in ROS production. Thus, differential responsiveness in oxidative processes may play a major role in the differential temporal ethanol vulnerability of developing cerebellum.

The Antioxidants Vitamin E and β-Carotene Protect Against Ethanol-Induced Neurotoxicity in Embryonic Rat Hippocampal Cultures

Fetal alcohol syndrome is characterized by numerous nervous system anomalies with the developing hippocampus being highly vulnerable. Other conditions can result from maternal ethanol consumption including oxidative stress. Critical antioxidants, such as vitamin E, can be decreased and antioxidative defenses altered. Gestational day 18 rat hippocampal cultures were exposed to ethanol ranging from 400 to 2400 mg/dl (16 h). MTT assays assessed neurotoxicity. Viability was decreased dose dependently. Supplementation with vitamin E or beta-carotene afforded neuroprotection against all ethanol concentrations. Vitamin E completely ameliorated neuronal loss following 400 and 800 mg/dl ethanol. Vitamin E increased survival to 95%, 79%, 66%, and 75% during 1600, 1800, and 2000 and 2400 mg/dl ethanol compared to nonethanol treatment. Vitamin E increased viability by 38%, 23%, 12%, and 29% at 1600, 1800, 2000, and 2400 mg/dl compared to non-vitamin E-supplemented, ethanol treatment. beta-Carotene completely ameliorated cell loss from 400 mg/dl ethanol and increased survival by 18% at 1600 mg/dl and 12% at 2000 mg/dl. This study demonstrates in vitro antioxidative neuroprotection against developmental ethanol exposure and suggests that nutritional therapies incorporating antioxidants may help protect against deleterious fetal effects from maternal alcohol abuse.

Ethanol-induced alterations in the expression of neurotrophic factors in the developing rat central nervous system.

Neonatal rats were exposed to ethanol throughout gestation, or during the early postnatal period (postnatal days 4-10 (P4-10)), and enzyme-linked immunoabsorbent assays were subsequently conducted in order to assess nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) protein content in hippocampus, septum, cortex/striatum and cerebellum. These determinations revealed that following prenatal ethanol treatment, there were significant ethanol-induced increases in NGF in P1 cortex/striatum, but no changes in any of the three neurotrophic factors (NTFs) in the other brain regions. Cortex/striatal NGF protein returned to control levels by P10. Following early postnatal exposure, BDNF was elevated in hippocampus and cortex/striatum (assessed on P10), and NGF was also enhanced in cortex/striatum at this age. Hippocampal and cortex/striatal BDNF returned to control levels by P21, but cortex/striatal NGF levels remained enhanced at this age. This NTF did not differ in ethanol and control animals by P60, however. The possible significance of elevated levels of NTFs as a function of ethanol exposure is discussed, and it is speculated that while such alterations could play a protective role, increases in these substances during critical developmental periods could also prove to be deleterious, and could even contribute to certain of the neuropathologies which have been observed following developmental ethanol exposure.

Modulation of ethanol neurotoxicity by nerve growth factor.

Dorsal root ganglion (DRG) neurons were cultured with varying concentrations of ethanol and NGF. At low concentrations of NGF (0.1 ng/ml) moderate initial ethanol levels (250 mg/dl) significantly suppressed neurite outgrowth. Higher NGF concentrations (5 ng/ml) protected against this neurotoxicity. At this higher NGF concentration, neuronal survival was not significantly affected by exposure to 0.25-4 g/dl ethanol, although survival was significantly diminished at 5 and 6 g/dl. Neurite outgrowth was a more sensitive indicator of ethanol neurotoxicity in this population, with significant decreases in process extension seen with 1 g/dl ethanol. When cultures were supplemented with 10 ng/ml NGF, however, process elaboration was significantly greater at 1 g/dl ethanol than that measured with 5 ng/ml NGF, and in fact did not differ from NGF controls. These studies indicate that NGF can provide neuroprotective effects against ethanol toxicity under these conditions. The results are discussed in relation to other recent reports of trophic factor neuroprotection.

The retrograde transport of horseradish peroxidase from the developing limb of the chick embryo

Chick embryos ranging in age from 4.0 to 18 days of incubation and 1-2-day-old hatchlings received injections of HRP solutions directly into the leg musculature. After survival periods of from 5 to 25 h motoneurons and cells in the spinal sensory ganglia were found to be stained with the HRP reaction product. It was found that the first appearance of a positive HRP reaction coincided with the time when nerve processes are first detected in the limb-bud by silver techniques (i.e. at 4.5 days of incubation). Only neurons with processes in the region of injection showed a positive reaction. The demonstration of a retrograde transport mechanism in neurons and axons which are still undergoing growth and differentiation provides a possible mechanism for the peripheral regulation of certain features of CNS neurogenesis. The application of this technique during development may also allow one to map neuroanatomical pathways during their formation.

Vitamin E and β-carotene protect against ethanol combined with ischemia in an embryonic rat hippocampal culture model of fetal alcohol syndrome

Abstract Neurodevelopmental damage can occur as a result of in utero exposure to alcohol. Oxidative stress processes are one of many proposed mechanisms thought to contribute to nervous system dysfunction characterized in fetal alcohol syndrome (FAS). Therefore, this study examined neuroprotective effects of antioxidant supplementation during ethanol (EtOH) treatment (0, 200, 400, 800 or 1600 mg/dl) combined with concomitants of EtOH exposure: acute (2-h) ischemia (aISCH) and chronic (16-h) hypoglycemia (cHG). The antioxidants vitamin E and β -carotene protected embryonic hippocampal cultures against 0–1600 mg/dl EtOH/aISCH/cHG treatments. In addition, neuronal viability, as measured by MTT ((3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; 5 mg/ml)), was equal to untreated cultures when supplemented with vitamin E or β -carotene at 0–800 mg/dl or 0–200 mg/dl EtOH/aISCH/cHG, respectively. These in vitro studies mirror potential in utero ethanol-exposed CNS conditions and may lead to therapeutic strategies targeted at attenuating neurodevelopmental FAS-related deficits.

Mitochondrially targeted vitamin E and vitamin E mitigate ethanol-mediated effects on cerebellar granule cell antioxidant defense systems

Ethanol (EtOH) disrupts the structure and function of the developing nervous system, sometimes leading to birth defects associated with fetal alcohol syndrome (FAS). Animal FAS models indicate that cellular membrane peroxidation, intracellular oxidant accumulation, and suppression of endogenous antioxidant enzymes contribute to the toxic effects of EtOH. Mitochondrially targeted vitamin E (MitoVit E), a chemically engineered form of vitamin E (VE) designed to accumulate in the mitochondria, has been shown to inhibit intracellular oxidant accumulation and cell death more effectively than VE. In previous investigations, we have shown that, in vivo, VE reduces neuronal death in the developing cerebellum of EtOH-exposed animals, and, in vitro, VE prevents apoptotic and necrotic death of EtOH-exposed cerebellar granule cells (CGCs). The present investigation shows that, in a FAS CGC model, 1 nM MitoVit E renders significant neuroprotection against EtOH concentrations as high as 1600 mg/dL. The present study also demonstrates that, in this same model, MitoVit E mitigates EtOH-induced accumulation of intracellular oxidants and counteracts suppression of glutathione peroxidase/glutathione reductase (GSH-Px/GSSG-R) functions, protein expression of gamma-glutamylcysteine synthetase (gamma-GCS), and total cellular glutathione (GSH) levels. In the presence and absence of EtOH, VE amplifies the protein expression levels of gamma-GCS, an enzyme that performs the rate-limiting step for GSH synthesis, and total GSH levels. These results suggest that MitoVit E and VE ameliorate EtOH toxicity through non-oxidant mechanisms-modulations of endogenous cellular proteins-and antioxidant means.

The effects of chronic ethanol consumption on neurotrophins and their receptors in the rat hippocampus and basal forebrain

Damage to the basal forebrain frequently results in deficits in learning and memory. Mnenonic dysfunction also occurs following prolonged ethanol consumption in humans and in animal models of chronic ethanol intake, accompanied by specific abnormalities in synaptic transmission between the basal forebrain and hippocampus. The integrity of at least some of the reciprocal neuronal connections between these brain regions is influenced by target-derived neurotrophic factors. We used a semiquantitative reverse transcription polymerase chain reaction technique to measure the messenger RNA for neurotrophins BDNF and NGF, and for their receptors trkB, trkA, and the low affinity receptor, p75(NTR) in the hippocampus and basal forebrain of rats after 28 weeks of alcohol consumption without malnutrition. This chronic ethanol treatment (CET) resulted in a marked and selective reduction in basal forebrain trkA mRNA. Western blotting revealed a similar reduction of basal forebrain trkA protein. CET effects on basal forebrain trkA may reflect impaired NGF signaling that could compromise septohippocampal synaptic connections, cholinergic differentiation, and emergent functional abilities dependent on these properties.

Oculomotor neuroblast migration in the chick embryo in the absence of tecto-tegmental fibers.

Abstract This study investigated a hypothesized relationship between the migration of the oculomotor complex, particularly the ventromedial subnucleus, and the presence of the tecto-tegmental fiber system in the chick embryo. It has been suggested that the presence of these fibers at the time of the initiation and continuation of neuroblast migration in this system might serve as some sort of stimulus influencing or guiding this migration. In order to examine this hypothesis, the dorsal midbrain was ablated in stage 12 embryos, thus removing the source of the tecto-tegmental fibers prior to the development of the oculomotor complex. The results indicated that in the complete absence of the tecto-tegmental fibers, all oculomotor subnuclei migrated normally, attained their normal terminal locations, and appeared to differentiate normally. These results are discussed in relation to other studies of possible extracellular influences during early neurogenesis and the possible importance of within-system versus without-system influences is considered.

Bcl-2 overexpression protects the neonatal cerebellum from ethanol neurotoxicity

The developing nervous system is extremely sensitive to ethanol, and exposure often produces a condition known as the fetal alcohol syndrome. Although mechanisms underlying developmental ethanol toxicity have long been sought, they remain poorly understood. In this study, we examined the ability of the cell death repressor gene bcl-2 to protect against ethanol neurotoxicity. Transgenic mice overexpressing bcl-2 in neurons were exposed to ethanol vapor on postnatal days 4 and 5, which is the peak period of vulnerability of cerebellar Purkinje cells to ethanol. While exposure of wild-type animals to ethanol resulted in significant loss of Purkinje cells by P5, similar exposure of homozygous and heterozygous transgenics had no effect on the number of these neurons. This study suggests that bcl-2 can protect neurons from ethanol neurotoxicity and that modulation of cell death effector or repressor gene products may play a significant role in developmental ethanol neurotoxicity.