Alcohol-Induced Developmental Origins of Adult-Onset Diseases.

Fetal alcohol exposure may impair growth, development, and function of multiple organ systems and is encompassed by the term fetal alcohol spectrum disorders (FASD). Research has so far focused on the mechanisms, prevention, and diagnosis of FASD, while the risk for adult-onset chronic diseases in individuals exposed to alcohol in utero is not well explored. David Barker's hypothesis on Developmental Origins of Health and Disease (DOHaD) suggests that insults to the milieu of the developing fetus program it for adult development of chronic diseases. In the 25 years since the introduction of this hypothesis, epidemiological and animal model studies have made significant advancements in identifying in utero developmental origins of chronic adult-onset diseases affecting cardiovascular, endocrine, musculoskeletal, and psychobehavioral systems. Teratogen exposure is an established programming agent for adult diseases, and recent studies suggest that prenatal alcohol exposure correlates with adult onset of neurobehavioral deficits, cardiovascular disease, endocrine dysfunction, and nutrient homeostasis instability, warranting additional investigation of alcohol-induced DOHaD, as well as patient follow-up well into adulthood for affected individuals. In utero epigenetic alterations during critical periods of methylation are a key potential mechanism for programming and susceptibility of adult-onset chronic diseases, with imprinted genes affecting metabolism being critical targets. Additional studies in epidemiology, phenotypic characterization in response to timing, dose, and duration of exposure, as well as elucidation of mechanisms underlying FASD-DOHaD inter relation, are thus needed to clinically define chronic disease associated with prenatal alcohol exposure. These studies are critical to establish interventional strategies that decrease incidence of these adult-onset diseases and promote healthier aging among individuals affected with FASD.

Acute alcohol exposure, acidemia or glutamine administration impacts amino acid homeostasis in ovine maternal and fetal plasma.

Fetal alcohol syndrome (FAS) is a significant problem in human reproductive medicine. Maternal alcohol administration alters maternal amino acid homeostasis and results in acidemia in both mother and fetus, causing fetal growth restriction. We hypothesized that administration of glutamine, which increases renal ammoniagenesis to regulate acid-base balance, may provide an intervention strategy. This hypothesis was tested using sheep as an animal model. On day 115 of gestation, ewes were anesthetized and aseptic surgery was performed to insert catheters into the fetal abdominal aorta as well as the maternal abdominal aorta and vena cava. On day 128 of gestation, ewes received intravenous administration of saline, alcohol [1.75 g/kg body weight (BW)/h], a bolus of 30 mg glutamine/kg BW, alcohol + a bolus of 30 mg glutamine/kg BW, a bolus of 100 mg glutamine/kg BW, alcohol + a bolus of 100 mg glutamine/kg BW, or received CO2 administration to induce acidemia independent of alcohol. Blood samples were obtained simultaneously from the mother and the fetus at times 0 and 60 min (the time of peak blood alcohol concentration) of the study. Administration of alcohol to pregnant ewes led to a reduction in concentrations of glutamine and related amino acids in plasma by 21-30%. An acute administration of glutamine to ewes, concurrent with alcohol administration, improved the profile of most amino acids (including citrulline and arginine) in maternal and fetal plasma. We suggest that glutamine may have a protective effect against alcohol-induced metabolic disorders and FAS in the ovine model.

Different patterns of regional Purkinje cell loss in the cerebellar vermis as a function of the timing of prenatal ethanol exposure in an ovine model.

Studies in rat models of fetal alcohol spectrum disorders have indicated that the cerebellum is particularly vulnerable to ethanol-induced Purkinje cell loss during the third trimester-equivalent, with striking regional differences in vulnerability in which early-maturing regions in the vermis show significantly more loss than the late-maturing regions. The current study tested the hypothesis that the sheep model will show similar regional differences in fetal cerebellar Purkinje cell loss when prenatal binge ethanol exposure is restricted to the prenatal period of brain development equivalent to the third trimester and also compared the pattern of loss to that produced by exposure during the first trimester-equivalent. Pregnant Suffolk sheep were assigned to four groups: first trimester-equivalent saline control group, first trimester-equivalent ethanol group (1.75 g/kg/day), third trimester-equivalent saline control group, and third trimester-equivalent ethanol group (1.75 g/kg/day). Ethanol was administered as an intravenous infusion on 3 consecutive days followed by a 4-day ethanol-free interval, to mimic a weekend binge drinking pattern. Animals from all four groups were sacrificed and fetal brains were harvested on gestation day 133. Fetal cerebellar Purkinje cell counts were performed in an early-maturing region (lobules I-X) and a late-maturing region (lobules VIc-VII) from mid-sagittal sections of the cerebellar vermis. As predicted, the third trimester-equivalent ethanol exposure caused a significant reduction in the fetal cerebellar Purkinje cell volume density and Purkinje cell number in the early-maturing region, but not in the late-maturing region. In contrast, the first trimester-equivalent ethanol exposure resulted in significant reductions in both the early and late-maturing regions. These data confirmed that the previous findings in rat models that third trimester-equivalent prenatal ethanol exposure resulted in regionally-specific Purkinje cell loss in the early-maturing region of the vermis, and further demonstrated that first trimester ethanol exposure caused more generalized fetal cerebellar Purkinje cell loss, independent of the cerebellar vermal region. These findings support the idea that prenatal ethanol exposure in the first trimester interferes with the genesis of Purkinje cells in an unselective manner, whereas exposure during the third trimester selectively kills post-mitotic Purkinje cells in specific vermal regions during a vulnerable period of differentiation and synaptogenesis.

The role of cortisol in chronic binge alcohol-induced cerebellar injury: Ovine model.

Women who drink alcohol during pregnancy are at high risk of giving birth to children with neurodevelopmental disorders. Previous reports from our laboratory have shown that third trimester equivalent binge alcohol exposure at a dose of 1.75 g/kg/day results in significant fetal cerebellar Purkinje cell loss in fetal sheep and that both maternal and fetal adrenocorticotropin (ACTH) and cortisol levels are elevated in response to alcohol treatment. In this study, we hypothesized that repeated elevations in cortisol from chronic binge alcohol are responsible at least in part for fetal neuronal deficits. Animals were divided into four treatment groups: normal control, pair-fed saline control, alcohol and cortisol. The magnitude of elevation in cortisol in response to alcohol was mimicked in the cortisol group by infusing pregnant ewes with hydrocortisone for 6 h on each day of the experiment, and administering saline during the first hour in lieu of alcohol. The experiment was conducted on three consecutive days followed by four days without treatment beginning on gestational day (GD) 109 until GD 132. Peak maternal blood alcohol concentration in the alcohol group was 239 ± 7 mg/dl. The fetal brains were collected and processed for stereological cell counting on GD 133. The estimated total number of fetal cerebellar Purkinje cells, the reference volume and the Purkinje cell density were not altered in response to glucocorticoid infusion in the absence of alcohol. These results suggest that glucocorticoids independently during the third trimester equivalent may not produce fetal cerebellar Purkinje cell loss. However, the elevations in cortisol along with other changes induced by alcohol could together lead to brain injury seen in the fetal alcohol spectrum disorders.

Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss.

Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258+/-10 mg/dl peak blood ethanol concentration) or saline in a "3 days/wk binge" pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis.

Temporal vulnerability of fetal cerebellar Purkinje cells to chronic binge alcohol exposure: ovine model.

BACKGROUND: Human magnetic resonance imaging (MRI) and autopsy studies reveal abnormal cerebellar development in children who had been exposed to alcohol prenatally, independent of the exposure period. Animal studies conducted utilizing the rat model similarly demonstrate a broad period of vulnerability, albeit the third trimester-equivalent of human brain development is reported to be the most vulnerable period, and the first trimester-equivalent exposure produces cerebellar Purkinje cell loss only at high doses of alcohol. However, in the rat model, all 3 trimester-equivalents do not occur prenatally, requiring the assumption that intrauterine environment, placenta, maternal interactions, and parturition do not play an important role in mediating the damage. In this study, we utilized the ovine model, where all 3 trimester-equivalents occur in utero, to determine the critical window of vulnerability of fetal cerebellar Purkinje cells. METHODS: Four groups of pregnant sheep were used: first trimester-equivalent pair-fed saline control group, first trimester-equivalent alcohol group (1.75 g/kg), third trimester-equivalent pair-fed saline control group, and third trimester-equivalent alcohol group (1.75 g/kg). The alcohol exposure regimen was designed to mimic a human binge pattern. Alcohol was administered intravenously on 3 consecutive days beginning on day 4 and day 109 of gestation in the first and third trimester-equivalent groups, respectively, and the alcohol treatment was followed by a 4-day inter-treatment interval when the animals were not exposed to alcohol. Such treatment episodes were replicated until gestational day 41 and 132 in the first and third trimester-equivalent groups, respectively. All fetal brains were harvested on day 133 and processed for stereological cerebellar Purkinje cell counting. RESULTS: Significant deficits were found in the fetal cerebellar Purkinje cell number and density in the first and third trimester-equivalent alcohol exposed fetuses compared with those in the saline controls. However, there was no difference between the first and third trimester-equivalent alcohol administered groups. When comparing the present findings to those from a previous study where the duration of alcohol exposure was all 3 trimester-equivalents of gestation, we did not detect a difference in fetal cerebellar Purkinje cell number. CONCLUSIONS: We conclude that the fetal cerebellar Purkinje cells are sensitive to alcohol exposure at any time during gestation and that women who engage in binge drinking during the first trimester are at a high risk of giving birth to children with cerebellar damage even if drinking ceases after the first trimester. Our findings also support the hypothesis that only a certain population of Purkinje cells are vulnerable to alcohol-induced depletion irrespective of the timing or duration of alcohol exposure.

All three trimester binge alcohol exposure causes fetal cerebellar purkinje cell loss in the presence of maternal hypercapnea, acidemia, and normoxemia: ovine model.

BACKGROUND: The third trimester equivalent has been identified, both in rat and sheep models, as a period of cerebellar vulnerability to alcohol-mediated injury. We wished to determine whether alcohol exposure throughout gestation results in greater injury compared with exposure limited to the third trimester equivalent. While this question has previously been addressed in the rat model, where the third trimester equivalent occurs postnatally, it has not yet been addressed in an animal model where all 3 trimester equivalents occur prenatally, as in the ovine. We also wished to correlate cerebellar Purkinje cell loss to alcohol-mediated alterations in maternal arterial pH and blood gases as these responses might be important mechanistically in mediating the damage. METHODS: Three groups of pregnant sheep were used: an untreated normal control group, a saline control group, and an alcohol group (1.75 g/kg of the body weight). The alcohol exposure regimen was designed to mimic a human binge pattern; alcohol was administered intravenously on 3 consecutive days, followed by 4 days without alcohol, beginning day 4 of gestation, continuing to the end of the third trimester equivalent of human brain growth, day 132 of gestation. RESULTS: All 3 trimester alcohol-exposed fetal brains exhibited significant deficits in cerebellar volume and Purkinje cell number compared with those of control subjects. We did not detect a difference in the reduction of Purkinje cell number when comparing between all 3 trimester and third trimester alcohol exposure studies. The neuronal loss was accompanied by maternal hypercapnea, acidemia, and normoxemia. CONCLUSIONS: These findings demonstrate in an ovine model where all 3 trimester equivalent of brain growth occur in utero that the fetal cerebellar Purkinje cells are more sensitive to the timing of alcohol exposure and less so to the duration of exposure. Decreases in maternal P(a)O(2) were not detected, suggesting that maternal hypoxia does not play a role in fetal Purkinje cell loss. And finally, we conclude that alcohol-induced changes in maternal arterial pH may play a role in alcohol-mediated developmental brain injury.