Ethanol-treated Dams Research Articles

Original article Alcohol-induced changes in the developing cerebellum. Ultrastructural and quantitative analysis of neurons in the cerebellar cortex

Maternal ethanol consumption during pregnancy may cause foetal alcohol syndrome (FAS). Our experiments of ethanol-treated female rats were based on the FAS model in humans; therefore, the results obtained may help explain the clinical mechanism of the disease development. The ultrastructural examination of the cerebellar cortex of ten-day-old rat pups of ethanol-treated dams during pregnancy (group IA), pregnancy and lactation (group IIA), and lactation (group IIIA) revealed that alcohol administration leads to a delayed maturation of Purkinje cells. This was most strongly manifested in the pups of dams treated with ethanol during pregnancy and lactation. Moreover, this study showed degenerative changes in Purkinje cells as well as in granular layer cells in all experimental groups. There was a difference in the ultrastructural picture of both types of dying cells, which might result from different time frame of their sensitivity to ethanol administration. The quantitative analysis showed the most pronounced decrease in the density of Purkinje cells in the posterior superior fissure of cerebellar cortex in the pups of dams treated with ethanol during pregnancy.

Response of the exocrine pancreas to the CCK on offspring rats of ethanol dams. Effects of folic acid

The aim of this study was to study the reverse effect of folic acid administered during gestation and lactation to ethanol-treated dams, on cholecystokinin Cholecystokinin (CCK) stimulus-secretion coupling in pancreatic exocrine secretion in offspring rats. Animals were randomized into three groups: Control group (C) received water and basic diet during pregnancy and lactation period; ethanol-treated rats (E) received ethanol and basic diet; the ethanol+folic acid group (EF) received folic acid supplement concomitantly with ethanol administration. Body and pancreatic weight was lower in offsprings after ethanol treatment. Folic acid supplementation increased these parameters with respect to ethanol rats. After CCK stimulation, a significant decrease in amylase, lipase and chymotrypsin activities in the duodenal juice were detected in ethanol, this trend was partially corrected with folate supplementation. Ethanol exerts its action on exocrine pancreatic secretion by two pathways: 'per se' and diminishing the folic acid content, because a folic acid supplement in rats during pregnancy and lactation periods produces an advantageous effect on amylase, lipase and chymotrypsin secretion in their offspring. Although extrapolation from animal studies may be tenuous, the present findings may explain the use of folic acid in the prevention of ethanol-induced damage by increasing the enzyme levels to adequate physiological concentrations.

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.

Zinc Supplementation at the Time of Ethanol Exposure Ameliorates Teratogenicity in Mice

Background: We have previously demonstrated that ethanol teratogenicity in mice is related to the maternal expression of metallothionein (MT), a zinc (Zn)-binding protein. Ethanol induces maternal liver MT, which causes plasma Zn concentrations to decrease as Zn moves into the liver. During pregnancy it is suggested that this change decreases fetal Zn supply and contributes to abnormal development. Here we investigated whether maternal Zn supplementation at the time of ethanol exposure reduces teratogenicity. Methods: Mice were injected with 25% ethanol (0.015 ml/g intraperitoneally at 0 and 4 hr) and ZnSO4 (2.5 μgZn/g subcutaneously at 0 hr) and were killed over 16 hr to ascertain changes in plasma Zn. Plasma Zn concentrations peaked at 2 hr, where levels were 5-fold normal and then returned toward normal over 14 hr. Pregnant mice were treated in a similar manner on gestation day 8 with saline, saline + Zn, ethanol + Zn, or ethanol alone, and fetal abnormalities were assessed on gestation day 18. Results: External abnormalities were most prevalent in offspring from dams treated with ethanol. Zn treatment at the time of ethanol exposure reduced the incidence of fetal abnormalities to basal levels. Litters from dams treated with ethanol + Zn contained more fetuses and fewer fetal resorption sites compared with those from ethanol-treated dams. Conclusions: These findings demonstrate that Zn supplementation at the time of ethanol exposure significantly negates the deleterious effects of ethanol on the fetus.

Folic acid intestinal absorption in newborn rats at 21 day postpartum: Effects of maternal ethanol consumption

This study was designed to examine the effects of prenatal and postnatal exposure of ethanol in the in vivo absorption of free folic acid in the small intestine in pups rats at the 21 st day after birth. The rats were accustomed to increasing amounts of ethanol (5 to 20%, vol/vol) in tap water for 1 month. During pregnancy and suckling period, ethanol-fed dams were assigned again to ethanol 20% in drinking water. Two sets of experiments were performed. In the first set, jejunal free folic acid absorption in control group and litters nursed by dams receiving ethanol showed a gradual increase along with the increase of perfusion time at all the assayed concentrations. In general, in litters of ethanol-fed dams, jejunal free folic acid absorption expressed as nmol/intestinal surface, nmol/g tissue wet weight and nmol/g tissue dry weight were higher than in control animals. In the second set of experiments, in distal ileum loops, free folic acid absorption did not occur in control pups, but appeared in litters exposed to ethanol. Milk folic acid levels are significantly decreased in ethanol-treated dams. However, only a slight decrease in the serum folic acid levels occurs in litters of ethanol-fed dams. In conclusion, the results obtained in the present work suggested a different pattern of free folic acid absorption in distal ileum for the two groups. The exposure of rats to ethanol during the pregnancy and suckling period, can affect postnatal development of intestinal functions and could play a role in the genesis of malnutrition observed in the infant.

CHRONIC PRENATAL ETHANOL EXPOSURE DOES NOT AFFECT THE EXPRESSION OF SELECTED GENES IN RAT BRAIN DEVELOPMENT

To investigate possible mechanisms contributing to the teratogenic effects of ethanol on the nervous system, we examined the effects of in utero exposure to ethanol on gene expression in the postnatal brain. Pregnant rats were exposed to ethanol by constant inhalation of ethanol vapor during gestational days 8-21, generating a mean blood alcohol level of approximately 150 mg/100 ml during the last week of gestation. Particular care was given to ensure proper nutrition and weight gain in both control and EtOH-treated groups. At different times after birth, the brains of pups from ethanol-treated dams and from a parallel series of untreated dams were dissected into major regions. Cytoplasmic RNA was assayed for different mRNAs encoding proteins representative of both glial and neuronal cell types, by Northern blot and solution hybridization. In situ hybridization was performed with T alpha-1 tubulin and PLP. There were no significant differences between pups from ethanol-treated and control dams in the amount of mRNAs encoding the myelin protein proteolipid protein (PLP), the astrocyte glial fibrillary acidic protein (GFAP), the neurally enriched T alpha-1 isotype of alpha-tubulin, the nerve terminal components synaptophysin p38 and SNAP 25. The mRNA encoding the alpha 1-subunit of the receptor for gamma-amino butyric acid (GABA) was slightly decreased in expression in the hindbrain but not cortex of pups from EtOH-treated dams. These results suggest that, when weight gain is controlled, chronic exposure of rats to ethanol during the last 2 weeks of gestation has little influence on the postnatal expression of these cell type specific genes.

Effects of maternal alcohol consumption on milk and blood lymphocytes and IgG antibody levels from Trichinella spiralis-infected rats.

Immunity to the intestinal parasite Trichinella spiralis can be transferred from the mother to the neonate during lactation. Previous studies in our laboratory showed that the passage of immunity to pups from ethanol-treated dams was depressed. This study examined the effect of ethanol consumption during pregnancy and lactation on the T. spiralis-associated immune components in milk and blood. Groups of female rats were fed either ethanol-containing or isocaloric liquid diets for 30 days before T. spiralis infection, mated and maintained on corresponding diets through pregnancy and lactation. Two-color flow cytometric analysis was performed for lymphocyte populations, enzyme-linked immunoabsorbent assay for specific IgG, and radial immunodiffusion assay for total IgG. The percentage of total T cells and their subsets, T helper cells and T cytotoxic/suppressor cells in milk and those in blood were similar between pair-fed and ethanol-treated animals. However, the percentage of natural killer cells in milk from ethanol animals was significantly reduced compared with the pair-fed group (33% vs. 54%). The percentage of activated or memory type T helper cell subset (OX2-W3/25+) was significantly increased in the blood of the ethanol-treated group. Pair-fed animals showed higher T. spiralis-specific IgG antibody levels in both in milk and blood compared with ethanol-treated animals. In ethanol-treated animals, specific IgG levels and total IgG concentration in milk were significantly lower than those in blood, whereas in pair-fed animals, only total IgG concentration in milk was lower than that in blood. This study indicates that ethanol consumption during pregnancy and lactation alters the maternal immune system.(ABSTRACT TRUNCATED AT 250 WORDS)

Rats exposed prenatally to alcohol exhibit impairment in spatial navigation test

Prenatal exposure to ethanol causes learning disabilities and low I.Q. scores. The objective of the present studies was to investigate whether exposure of rats to ethanol in utero, would induce a deficit in spatial memory in adult life. Pregnant rats were fed with an ethanol diet from day 1 of pregnancy till parturition. Control rats were either pair-fed with an isocaloric sucrose diet or were fed with lab-chow ad libitum. On the first day of birth, offspring exposed to ethanol in utero were placed with a control mother fed with lab-chow, while offspring of the lab-chow fed dams were placed with ethanol-treated dams. At 40, 60 and 90 days postnatally, behavioral testing was performed using the Morris swim maze, a test of spatial memory. Results indicated that the offspring exposed to ethanol in utero presented deficits in spatial memory processes. Ethanol did not completely block the learning of the swim maze task but the alcohol-exposed offspring exhibited longer latencies to perform the task, swam longer distances prior to locating and climbing onto the platform, and when the platform was removed, searched for it in all 4 quadrants of the pool. Restricted caloric intake during gestation and maternal behavior in early postnatal life also induced deficits in the performance on the swim maze task. However, these deficits were mild and short-lasting being absent at 60 and 90 days of age. In contrast, the deficits induced by ethanol were more severe and longer-lasting, being present in adult life.

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.

Effect of fetal exposure to ethanol on complex maze acquisition in rats tested as adults.

Holtzman rat dams were maintained on a liquid diet containing 35% ethanol-derived calories from Days 5 to 18 of gestation. Beginning on Day 19, the dams were given 5 days of choice between the ethanol diet and lab chow and water, which allowed the animals to decrease their alcohol consumption slowly. 9 male offspring of the ethanol-treated dams were compared to 9 male offspring of pair-fed dams on the acquisition of a LRRL sequence in a four-choice maze, with training beginning at 116 days of age. While the alcohol group had higher means on all acquisition measures and higher variances on all error measures, the group means were not significantly different on any of the acquisition measures.

Long-term effects of fetal ethanol exposure on pituitary-adrenal response to stress

Pregnant female rats were fed either a 5.0–5.5% w/v ethanol-containing liquid diet ad lib or pair-fed the isocaloric control diet during gestation weeks 2 and 3. At 75–105 days of age, female offspring of the ethanol-treated dams showed significantly greater corticosterone responses than pair-fed- or normally-derived offspring to the stress of cardiac puncture or of noise and shaking, while pituitary-adrenal responses to exposure to a novel environment, cold or 2–3 days of fasting were normal. Adrenal sensitivity to ACTH in dexamethasone-suppressed adult offspring was unaffected by the prenatal treatment. The results demonstrate that fetal ethanol exposure enhances adult pituitary-adrenal responses to certain stressors, including alcohol as demonstrated previously, and suggest that the long-term effects may be mediated by developmental actions of alcohol on central neural mechanisms involved in the regulation of this neuroendocrine system.

Effect of acute maternal ethanol exposure upon fetal development in the rat

Two studies on the effect of acute ethanol administration during Days 9-12 of gestation showed significant fetal weight reduction (6%-8%) in rats from ethanol-treated dams compared to water controls. Maternal weight gains could not account for the differences. These effects of acute doses (3-4 g ethanol/kg body weight), given only during organogenesis, suggest that chronic ethanol exposure during gestation is not required for some developmental retardation to occur. What effect this retardation may have upon subsequent behavioral development remains to be determined.