Neurotrophins in the Brain: Interaction With Alcohol Exposure During Development.

Fetal alcohol spectrum disorders (FASDs) are a result of the teratogenic effects of alcohol on the developing fetus. Decades of research examining both individuals with FASDs and animal models of developmental alcohol exposure have revealed the devastating effects of alcohol on brain structure, function, behavior, and cognition. Neurotrophic factors have an important role in guiding normal brain development and cellular plasticity in the adult brain. This chapter reviews the current literature showing that alcohol exposure during the developmental period impacts neurotrophin production and proposes avenues through which alcohol exposure and neurotrophin action might interact. These areas of overlap include formation of long-term potentiation, oxidative stress processes, neuroinflammation, apoptosis and cell loss, hippocampal adult neurogenesis, dendritic morphology and spine density, vasculogenesis and angiogenesis, and behaviors related to spatial memory, anxiety, and depression. Finally, we discuss how neurotrophins have the potential to act in a compensatory manner as neuroprotective molecules that can combat the deleterious effects of in utero alcohol exposure.

Neonatal binge alcohol exposure increases microglial activation in the developing rat hippocampus.

Aberrant activation of the developing immune system can have long-term negative consequences on cognition and behavior. Teratogens, such as alcohol, activate microglia, the brain's resident immune cells, which could contribute to the lifelong deficits in learning and memory observed in humans with Fetal Alcohol Spectrum Disorders (FASD) and in rodent models of FASD. The current study investigates the microglial response of the brain 24 h following neonatal alcohol exposure (postnatal days (PDs) 4-9, 5.25 g/kg/day). On PD10, microglial cell counts and area of cell territory were assessed using unbiased stereology in the hippocampal subfields CA1, CA3 and dentate gyrus (DG), and hippocampal expression of pro- and anti-inflammatory genes was analyzed. A significant decrease in microglial cell counts in CA1 and DG was found in alcohol-exposed and sham-intubated (SI) animals compared to undisturbed suckle controls (SCs), suggesting overlapping effects of alcohol exposure and intubation alone on the neuroimmune response. Cell territory was decreased in alcohol-exposed animals in CA1, CA3, and DG compared to controls, suggesting the microglia have shifted to a more activated state following alcohol treatment. Furthermore, both alcohol-exposed and SI animals had increased levels of pro-inflammatory cytokines IL-1ß, TNF-α, CD11b, and CCL4; in addition, CCL4 was significantly increased in alcohol-exposed animals compared to SI as well. Alcohol-exposed animals also showed increased levels of anti-inflammatory cytokine TGF-ß compared to both SI and SCs. In summary, the number and activation of microglia in the neonatal hippocampus are both affected in a rat model of FASD, along with increased gene expression of pro- and anti-inflammatory cytokines. This study shows that alcohol exposure during development induces a neuroimmune response, potentially contributing to long-term alcohol-related changes to cognition, behavior and immune function.

Fetal alcohol exposure attenuates interleukin-1beta-induced fever: neuroimmune mechanisms.

Central mechanisms for the attenuating effects of fetal alcohol exposure (FAE) on interleukin-1beta (IL-1beta)-induced fever were studied in adult male offspring of dams fed a liquid diet supplemented with ethanol (E), in pair-fed (P) control and in normal (N) offspring. Hypothalamic levels of IL-1 were significantly lower in E than in N rats at 2 h, but not at 4 and 6 h, after intraperitoneal administration of lipopolysaccharide. Fever induced by intracerebroventricular (i.c.v.) IL-1 was significantly lower in E than in N and P rats. In contrast, E rats showed a normal febrile response to i.c.v. prostaglandin-E2. Thus, whereas FAE does not affect central thermoregulatory mechanisms, per se, FAE alters the kinetics of hypothalamic IL-1 production/appearance and decreases the responsiveness of central mechanisms which mediate the febrile response to IL-1.

Effects of in utero alcohol exposure on B cell development in neonatal spleen and bone marrow.

The effects of in utero alcohol exposure on neonatal lymphopoiesis were examined in a murine model of fetal alcohol syndrome. At birth, both immature and mature B cells were decreased in the spleens of neonatal animals and these subpopulations of B cells did not recover to normal levels until 3-4 weeks of life. Pre-B cells and total B cells were decreased as well in the bone marrow of ethanol-exposed animals. By 3-4 weeks of life, the number of B cells in the bone marrow recovered to normal levels, but the pre-B cells remained below normal levels through 5 weeks of age. Furthermore, a recently described early B cell progenitor was reduced in frequency in ethanol-exposed neonates. Together, these data suggest that in utero exposure to ethanol can result in abnormalities in B cell development that may initiate at an early stage of B cell development.

Chronic and acute prenatal and postnatal ethanol exposure on lymphocyte subsets from offspring thymic, splenic, and intestinal intraepithelial sources.

The overall objective of this study was to analyze the effects of a combined prenatal and postnatal (entire gestational human chronic drinking model) ethanol exposure on T-cell development in mice. Specifically, this study evaluated the effects of chronic exposure to prenatal ethanol on lymphocyte makeup and proliferative capabilities of postnatal offspring's (4 and 12 weeks) peripheral lymphoid tissues. Chronic exposure regimens were conducted over the entire gestational period and through postnatal day 14 or 21. Thymus, spleen, and intestinal intraepithelial lymphocytes were harvested and analyzed by flow cytometry for percentages of T-cell subsets. Splenic lymphocytes were also analyzed for their ability to proliferate in response to a T-cell mitogen. Limited effects of chronic ethanol exposure were seen.

Chronic ethanol exposure alters leukocyte subsets in repopulating spleens, but does not alter negative selection in thymuses of sublethally irradiated mice.

Results from previous in vitro experiments in this laboratory suggested that ethanol may affect selection processes in the thymus. To determine whether ethanol allows escape of potentially autoreactive T-cell clones from negative selection, we fed ethanol to sublethally irradiated, young, adult C57BR mice during the time of thymic and splenic repopulation as a new model of human third trimester fetal alcohol exposure. The mice received a whole-body, sublethal dose (6 Gy) of gamma irradiation at 5 to 6 weeks of age. Feeding of a liquid diet providing 25% of calories as ethanol (EDC) or an isocaloric control liquid diet was begun 3 days after irradiation and was continued for 5 weeks. Each EDC mouse had 2 weight- and age-matched controls, 1 pair-fed (PF), and 1 fed ad libitum (AD LIB). Average blood alcohol concentrations (90 to 440 mg/100 ml) were higher than those reported previously for neonatal mice exposed to ethanol through lactation. At 5 weeks after irradiation, the EDC mice had lower total thymocyte numbers (p < 0.05) and a higher proportion of CD4-CD8-thymocytes than either the PF or AD LIB mice (p < 0.05), which is consistent with findings using in utero models of ethanol exposure. Ethanol exposure also altered the proportion of leukocyte subsets in repopulating spleens. B cells were the most sensitive to the detrimental effects of ethanol and, as a percentage of total nucleated cells in the spleen, B cells were decreased in the EDC group, compared with both the PF and AD LIB groups (p < 0.05). C57BR mice normally delete by negative selection thymocytes bearing v beta 17 T-cell receptors. There was no discernible effect of ethanol exposure during thymic and splenic repopulation on the expression of V beta 17a on thymocytes and splenic T lymphocytes, indicating that ethanol does not affect negative selection.

Fetal alcohol exposure augments the blunting of tumor necrosis factor production in vitro resulting from in vivo priming with lipopolysaccharide in young adult male but not female rats.

We previously reported altered responses of thymocytes and splenocytes to mitogen stimulation in fetal alcohol-exposed (FAE) male Sprague-Dawley rats. We also reported enhanced neuroendocrine responses to stressful stimuli in these animals. The experiments we describe herein aimed at testing whether young adult FAE rats manifest a notable dysregulation in the neuroendocrine-immune response to pathogen administration. We tested the effect of in vivo priming of the animal with a low dose of endotoxin [lipopolysaccharide (LPS), 5 micrograms/kg], considered to be suboptimal from the perspective of mounting detectable levels of circulating monokines several hours after administration, upon the production of immunoreactive tumor necrosis factor (TNF-alpha) in response to a further in vitro challenge of peripheral blood mononuclear cells with 2.5 micrograms/ml of LPS 90 min after priming. We show that the response to the LPS pathogen in vitro after priming is significantly blunted (p < 0.01) in male rats exposed prenatally to alcohol, compared with control male animals. FAE female rats and FAE ovariectomized female rats do not show significant differences in the priming response, compared with control animals. We also show that there is no correspondence between plasma corticosterone levels and TNF-alpha production after priming in any of the groups tested.

Effects of maternal ethanol consumption on the subsequent development of immunity to Trichinella spiralis in rat neonates.

The immune response of rat pups to the intestinal parasite Trichinella spiralis was studied to determine if maternal pre- and/or postnatal ethanol consumption affected neonatal immune responses. Female rats were fed ethanol-containing (36% of calories) or pair-fed control liquid diets and include groups that were maintained on ethanol as follows: group 1, from day 1 of pregnancy through weaning and whose pups were then placed on ethanol to sacrifice; group 2, from day 1 of pregnancy through lactation; group 3, from day 1 of pregnancy through pup delivery; and group 4, from day 1 of lactation through weaning. A parallel group of animals was pair-fed isocaloric control diet until sacrifice. The pups of all litters were immunized orally with 500 L1 (T. spiralis) larva 5 days after weaning. To examine the effects of maternal ethanol on primary immune responses, one-fourth of the pups from each litter were sacrificed on days 10 and 20 after immunization. To examine the effects on neonatal secondary immune responses, the remaining pups were challenged with 1,000 larva 30 days after the initial immunization and sacrificed either 3 or 8 days after challenge. At the time of sacrifice, blood samples were collected, the intestine removed to determine T. spiralis worm burdens, and suspensions of mesenteric lymph node (MLN) cells prepared. Intestinal worm counts and serum levels of anti-T. spiralis IgM and IgG antibodies, interleukin-2 (IL-2), and tumor necrosis factor (TNF) were determined. In vitro proliferation responses of MLN cells to T. spiralis antigen and to the mitogen concanavalin A (Con A) were also examined. Pups from groups 1 to 3 demonstrated significantly higher intestinal worm counts (decreased immunity) than the pair-fed controls at the day 20 primary immune response sacrifice, and pups from group 1 had significantly higher worm counts at day 3 after a secondary immune challenge. Pups of dams from groups 1, 3, and 4 had significantly lower IgM antibody titers at the day 20 primary immune response sacrifice. All experimental ethanol groups (1 to 4) demonstrated significantly lower IgG antibody titers than that observed in pair-fed control pups at the 20-day sacrifice. IgM antibody titers showed significant reductions for ethanol-treated groups at 3 and 8 days after T. spiralis secondary challenge. In addition, IgG antibody titers were also significantly reduced for all alcohol groups at 3 and 8 days during the secondary immune response. Serum IL-2 and TNF levels were significantly lower in all experimental ethanol groups (1 to 4) relative to pair-fed controls at day 20 during a primary immune response, and IL-2 levels at 3 days postchallenge were lower in groups 2 to 4 after a secondary immune challenge. MLN proliferation responses to antigen and Con A were significantly reduced in ethanol groups 1 to 3 and to Con A in group 4 at day 10 after a primary immune challenge. Ethanol group 3 pups also demonstrated a reduced response to antigen at day 20. For animals undergoing a secondary immune response, ethanol group 2 demonstrated a reduced response to antigen at day 3, whereas groups 2 and 4 showed increased reactivity to antigen at days 3 and 8 postchallenge. These results show that maternal ethanol consumption diminishes the capacity of neonates to respond to T. spiralis antigen and that the depressed immune response involves T- and B-cell-mediated reactions and also affects the production of certain cytokines. These results also suggest that the diminished immune responses are increased with longer periods of maternal and neonatal exposure to ethanol.

In utero exposure to ethanol affects postnatal development of T- and B-lymphocytes, but not natural killer cells.

The effect of intrauterine exposure to ethanol on lymphocyte development in the neonatal period was studied in C57BI/6J mice. Mice were bred, and then the female mice were assigned to 1 of 3 diet groups, 25% ethanol-derived calories (EDC), pair-fed control, or ad libitum laboratory chow. At birth, all offspring were cross-fostered to surrogate mothers who had been fed laboratory chow. At weekly intervals, the neonatal mice were weighed, and 4 mice from each group were used to assess the development of splenic lymphocytes. The total number of splenocytes was similar in all three groups at each sampling. The number of T-cells, B-cells, and natural killer (NK) cells was measured by flow cytometry. T-cells and NK cells did not vary significantly among the three diet groups. However, the total number of B-cells was decreased for the first 3 weeks of life in the ethanol-exposed animals. The function of the T-cells and B-cells was determined by assessing the response to lipopolysaccharide, pokeweed mitogen, phytohemagglutinin, and concanavalin A. The response to all four mitogens was significantly reduced in the ethanol-exposed animals and did not recover to control levels until 4-5 weeks of life. Ethanol exposure had no significant effect on the kinetics of acquisition of NK lytic function, as assessed by determining the killing of chromium-51 labeled YAC-1 tumor target cells. These data show that prenatal exposure to ethanol causes a transient immunodeficiency in some, but not all compartments of the immune system.

Effect of maternal ethanol consumption on in vitro tumor necrosis factor, interleukin-6 and interleukin-2 production by rat milk and blood leukocytes.

It has been shown that the transfer of immunity via lactation plays an important role in providing early protection to the neonate. Maternal ethanol consumption also results in a reduced transfer of immunity to their neonates against a Trichinella spiralis infection. Because of the known presence of cytokines in milk and their important role in inflammation, we tested the effects of maternal ethanol consumption on cytokine production by milk and blood cells from T. spiralis-infected rats. With T. spiralis antigen, Concanavalin A (Con A) or lipopolysaccharide stimulation, milk cells from both ethanol-treated and pair-fed groups were capable of producing tumor necrosis factor (TNF), interleukin (IL)-6 and IL-2. There were no differences between groups for TNF or IL-6 production by milk cells. Milk cells from the ethanol group produced a significantly higher amount of IL-2 upon Con A stimulation, as compared with that from the pair-fed group (16 +/- 4 units/10(6) cells vs. 4 +/- 1 units/10(6) cells, p < 0.05). After stimulation with Con A, blood cells from the ethanol group produced significantly lower amounts of TNF (17 +/- 15 units/10(6) cells) than that from the pair-fed group (102 +/- 64 units/10(6) cells, p < 0.05). The amount of TNF and IL-6 produced by milk cells was significantly lower, as compared with that produced by blood cells. This study suggests that ethanol consumption has some modulatory effects on cytokine production by milk and blood cells.

Effect of postnatal ethanol exposure on expression of differentiation antigens of murine splenic lymphocytes.

Ethanol is a recognized immunosuppressive agent in the chronic alcoholic. However, the effects of ethanol exposure on the developing immune system have not been extensively investigated. This study evaluated the effects of early postnatal ethanol exposure, via breast milk, on splenic lymphocyte differentiation antigen expression in offspring reared by ethanol-fed mice. Maternal mice were fed a liquid diet containing 20% ethanol-derived calories during pregnancy (E-P), pregnancy and lactation (E-PL), or lactation (E-L). Ad libitum-fed (C) and pair-fed (PF) control groups, fed a control liquid diet, were included. Expression of differentiation antigens on splenic lymphocytes from 21-day-old offspring reared by females in 1 of the 3 ethanol exposure conditions was evaluated by flow cytometry. Offspring reared by E-P females had similar numbers of splenic lymphocytes as offspring reared by C and pair-fed during pregnancy (PF-P) females. In contrast, offspring reared by E-PL and E-L females had fewer splenic lymphocytes than both PF-PL and PF-L (respectively), and C offspring. The number of Thy 1.2+, CD4+, CD8+, and IgG+ (B-cell) splenic lymphocytes was reduced in E-PL and E-L offspring compared with PF and C offspring. E-P offspring had fewer CD4+ and IgG+ splenic lymphocytes than C, but not PF-P, offspring. The percentage of Thy 1.2+ splenic lymphocytes was significantly reduced among E-PL and E-L offspring compared with PF-PL and PF-L (respectively), and C offspring.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of alcohol on immunity and neoplasia in fetal alcohol exposed and adult rats.

Alterations of immune function can result not only from alcohol consumption by the adult human or animal, but also from fetal alcohol exposure (FAE). We have demonstrated long-lasting effects of FAE on T cell function and effects of adult ethanol (EtOH) consumption on tumorigenesis. Here, we present recent data that demonstrate that 1) FAE alters the biphasic pattern of thymocyte activation during peripubertal development probably due to effects other than the CD3 pathway; and 2) the long-lasting impaired proliferative response of splenocytes from FAE rats is not due to loss of their ability to express interleukin-2 receptors (IL2R), thus reflecting interference with events following the IL2-IL2R interaction. We also provide direct evidence that acute in vivo administration of EtOH to adult Fisher 344 rats can suppress blood natural killer (NK) cytotoxicity and that such suppression mediates the observed enhanced metastatic growth of a syngeneic mammary tumor.

Increased rate of E-rosette formation by T lymphocytes of pregnant women who drink ethanol.

Ethanol use by pregnant women increased, in a dose-dependent manner, the rate of sheep erythrocyte rosette (E-rosette) formation with T lymphocytes. The time curve for E-rosette formation by T cells from nondrinking subjects was biphasic, with a rapid formation of half the E-rosettes within the first 16 min, followed by a much slower rate for E-rosette formation until the maximal T-cell percentage was reached overnight. For pregnant drinkers, greater than 85% of the E-rosettes formed during the initial rate period, with a concomitant smaller number forming during the overnight incubation. Despite the faster initial rate of E-rosette formation in the drinking subjects, the total percentage T cells was the same for both groups. Other demographic factors, like tobacco or marijuana use, or trimester, did not significantly contribute to the observed differences. An increase in the rate of E rosetting was also obtained by incubating lymphocytes from nondrinkers overnight in physiologically attainable concentrations of ethanol (less than or equal to 0.1%). These results demonstrate that drinking by pregnant women, even at relatively moderate levels (2 oz/week absolute ethanol), causes alterations in their cellular immune systems. With the ability of ethanol to cross the placental barrier and persist in utero, it is apparent that these levels of ethanol have the potential to affect the developing fetal immune system.