The Effect of Binge Fetal Alcohol Exposure on the Number of Vasoactive Intestinal Peptide-Producing Neurons in Fetal Sheep Brain

Abstract

Previously we demonstrated that fetal alcohol exposure attenuates hypoxic cerebral vasodilation in fetal and neonatal sheep. One mechanism may be altered expression of brain vasoactive substances. We hypothesized that early fetal alcohol exposure alters the number of fetal neurons expressing vasoactive intestinal peptide (VIP), a potent cerebral vasodilator. Thirteen pregnant ewes received daily i.v. infusions of alcohol (1.5 g/kg) or saline on days 30–54 of gestation (term = 145 days). Fourteen fetal brains (6 alcohol-exposed, 8 saline control) were obtained on gestational day 126. Using unbiased stereology, we counted immunohistochemically-labeled VIP neurons in one half of each forebrain with an optical fractionator. The total NeuN-labeled neurons were similarly counted. Alcohol-exposed fetal sheep brains had fewer VIP-immunopositive neurons per hemisphere, 14.6 × 10⁶, compared to saline controls, 19.8 × 10⁶. The total neuron number was not different, 1.19 × 10⁹ versus 1.23 × 10⁹ respectively, indicating a selective decrease in VIP neurons as a result of alcohol exposure. In sheep, alcohol exposure early in gestation is associated with fewer VIP-producing neurons later in gestation compared to saline controls; therefore, alcohol-related changes in the number of VIP-expressing neurons may be responsible in part for the attenuated hypoxic cerebral vasodilation described in fetal and neonatal sheep exposed to alcohol earlier in gestation.

Effects of maternal antenatal glucocorticoid treatment on apoptosis in the ovine fetal cerebral cortex

“…We used rhodamine‐tagged secondary antibody to mouse monoclonal primary anti‐NeuN antibody to label the apoptotic nuclei of the postmitotic neurons. Consistently with previous findings, we confirmed the suitability of the ApopTag TUNEL assay for use in ovine brain and mouse monoclonal NeuN antibody to recognize ovine NeuN‐positive neuronal nuclei (Mullen et al,1992; Sliwowska et al,2004; Anderson et al,2008). We also used a real‐time assay to measure the intensity of emissions from the cleavage products of caspase‐3 substrate to estimate the amount of caspase‐3‐related apoptosis present in the cerebral cortical tissue.…”

“…The primary mouse monoclonal antineuronal‐specific nuclear protein (NeuN) antibody has been characterized for a variety of vertebrate species (Chemicon, Temecula, CA) and has been used as a neuronal marker in sheep (Mullen et al,1992; Sliwowska et al,2004; Anderson et al,2008). We confirmed that mouse anti‐NeuN monoclonal antibody identified the sheep NeuN antigen by simultaneously examining positive and negative controls from sheep and rat cerebral cortex.…”

Binge alcohol exposure in the second trimester attenuates fetal cerebral blood flow response to hypoxia

“…In young guinea pigs exposed to alcohol as fetuses, GABA receptors are altered (10). In preliminary work, we demonstrated that alcohol exposure early in gestation was associated with fewer vasoactive intestinal peptide-producing neurons in fetal sheep (9). Decreased expression of vasoactive intestinal peptide, a potent cerebral vasodilator, might be responsible, in part, for impaired hypoxic cerebral vasodilation.…”