Adequacy of Maternal Iron Status Protects against Behavioral, Neuroanatomical, and Growth Deficits in Fetal Alcohol Spectrum Disorders

Fetal alcohol spectrum disorders (FASD) are the leading non-genetic cause of neurodevelopmental disability in children. Although alcohol is clearly teratogenic, environmental factors such as gravidity and socioeconomic status significantly modify individual FASD risk despite equivalent alcohol intake. An explanation for this variability could inform FASD prevention. Here we show that the most common nutritional deficiency of pregnancy, iron deficiency without anemia (ID), is a potent and synergistic modifier of FASD risk. Using an established rat model of third trimester-equivalent binge drinking, we show that ID significantly interacts with alcohol to impair postnatal somatic growth, associative learning, and white matter formation, as compared with either insult separately. For the associative learning and myelination deficits, the ID-alcohol interaction was synergistic and the deficits persisted even after the offsprings’ iron status had normalized. Importantly, the observed deficits in the ID-alcohol animals comprise key diagnostic criteria of FASD. Other neurobehaviors were normal, showing the ID-alcohol interaction was selective and did not reflect a generalized malnutrition. Importantly ID worsened FASD outcome even though the mothers lacked overt anemia; thus diagnostics that emphasize hematological markers will not identify pregnancies at-risk. This is the first direct demonstration that, as suggested by clinical studies, maternal iron status has a unique influence upon FASD outcome. While alcohol is unquestionably teratogenic, this ID-alcohol interaction likely represents a significant portion of FASD diagnoses because ID is more common in alcohol-abusing pregnancies than generally appreciated. Iron status may also underlie the associations between FASD and parity or socioeconomic status. We propose that increased attention to normalizing maternal iron status will substantially improve FASD outcome, even if maternal alcohol abuse continues. These findings offer novel insights into how alcohol damages the developing brain

ID-alcohol exposure impairs myelination.

<p><b>A–L,</b> Immunostain for myelin basic protein (MBP) of P35 cerebellum following the indicated treatments. <b>B, E, H, K</b> are the corresponding enlargements of Lobule I, and <b>C, F, I, L</b> are enlargements of Lobule VIa. There were fewer myelin tracts within the granule cell layer (arrows) in ID-alcohol cerebellum (<b>K, L</b> ) compared with controls (<b>B, C</b>) or alcohol-only (<b>H, I</b> ) or ID (<b>E, F</b>), and confirmed by quantifying the MBP⁺ area within the granule cell layer for lobule I (<b>M</b>) and lobule VIa (<b>N</b>). N = 6–8 rats per group. <b>*</b> Significantly different from IS pups at same alcohol dose, <b>†</b> significantly different from 0 g/kg alcohol within same iron status.</p

Maternal ID increases alcohol-induced cerebellar apoptosis at P10.

<p>(<b>A</b>) There were no significant main effects of Iron status or Alcohol dose on proliferation, quantified using phosphorylated histone-H3 immunoreactivity. (<b>B</b>) Apoptosis, assessed using cleaved-caspase-3 immunoreactivity, was decreased in the P10 cerebellum. Both ID (<i>P</i> = 004) and alcohol (<i>P</i> = 0.001) increased apoptosis and their effects were additive. N≥5 rats per treatment group. <b>*</b>, significantly different from IS pups within the same alcohol dose; <b>†</b>, significantly different from 0 g/kg alcohol within the same Iron status.</p

Maternal ID and alcohol interact to adversely affect somatic growth in males but not females.

<p>Body weight on P1–10 in male (A, C, E) and female (B, D, F) pups treated with 0 (A, B), 3.5 (C, D), or 5 g/kg alcohol (E, F). N≥22 rats per treatment group per sex. ID-only reduced growth in both males and females. Alcohol-only did not affect growth in either sex, but interacted with ID to reduce growth in males and not females. Markers of significance were omitted for clarity purposes.</p

Maternal ID modulates cued and contextual fear conditioning in alcohol-exposed pups.

<p>Percent freezing to cue (<b>A</b>) and context (<b>B</b>) in male and female pups. N = 7–11 rats per treatment group per sex. There was a main effect of Alcohol within ID males on cued (F_(2,16) =6.1, <i>P</i> = 0.011) and within ID males (F_(2,6) =7.4, <i>P</i> = 0.024) and females (F_(2,14) =6.9, <i>P</i> = 0.008) on contextual fear conditioning. <b>†</b>, significantly different from 0 g/kg alcohol within the same Iron status.</p

Maternal IDAA profoundly exacerbates alcohol-induced deficits in offspring’s delay ECC performance.

<p>Percent acquisition and amplitude of conditioned responses (CR) in IS and ID P35 offspring receiving 0 (<b>A, B</b>), 3.5 (<b>C, D</b>), or 5 (<b>E, F</b>) g/kg alcohol per day during the brain growth spurt. N = 9–16 rats per treatment group. There were significant main effects of Iron status, Alcohol dose, and an Iron status×Alcohol dose interaction. The interactive effect of Iron status and Alcohol was observed in ID rats that received 5 g/kg alcohol (<b>E, F</b>), where they were more impaired in acquiring CRs compared to IS rats that received 5 g/kg alcohol. Markers of significance omitted for clarity purposes.</p

Rat dams fed an ID diet have moderate ID without anemia.

<p>Hematocrit (<b>A</b>), hemoglobin (<b>B</b>), red-cell distribution width (<b>C</b>), and liver iron (<b>D</b>) in rat dams on P5 and/or P22 fed IS or ID diets. Dashed lines indicate the normal reference range for non-pregnant adult rats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047499#pone.0047499-Car1" target="_blank">[19]</a>. N = 6–8 rats per group at each time point. <b>*</b>, significantly different from IS rats at the same time point as determined by linear mixed modeling.</p