Impact of Maternal Iron Restriction on Offspring Figure 8. Proposed model displaying the impact of gestational iron restriction on characterized neural cell populations along a developmental time line. At embryonic day 10.5 (E10.5), multipotent neural stem cells can be isolated from the spinal cord. These cells give rise to glial restricted (GRP) and neuron restricted precursor cells (NRP) that can be isolated at around E13.5. Committed oligodendrocyte progenitor cells (OPC) are generated at around E18.5 and are thought to persist through early adulthood. The first mature myelin-producing oligodendrocytes arise around the day of birth (postnatal day 1). Blue arrows indicate the time of initiation of the iron-deficient diet that is associated with a disruption in the ABR conduction velocity at postnatal day 45 and mark the window of vulnerability. Red arrows represent the time of initiation of the iron-deficient diet that is not associated with impaired auditory nerve conduction. doi:10.1371/journal.pone.0017483.g008 trimester exhibited normal auditory nerve conduction velocities as reflected by ABR recordings despite IDA being present in all experimental rats at the time of testing. This result might shed some light on seemingly contradictory results found in humans; some investigators reported no alterations of ABR latencies in ID children with or without anemia [85,86], while others found that IDA in 6 month old babies produced abnormally prolonged ABR latencies that persisted years after the children’s iron status was corrected with iron therapy [43,81]. Our study indicates that there is a precise window of vulnerability during which ID can affect the fetal CNS leading to postnatal neural impairments. The lack of defect seen in some studies could be a consequence of the insult occurring outside the window of vulnerability, rather than a perceived general insensitivity of newborns to iron restriction. Without the establishment of the maternal iron status during pregnancy or at least cord blood iron levels, it cannot be resolved whether the initiation of ID and the possible onset of a later apparent pathology happened in utero. Therefore, any conclusions about the importance and/or need of pre- and postnatal iron supplementation have to be made with caution. The window of vulnerability, which we have defined in our studies, also offers an important clue for the underlying cellular impairment and seems to be consistent with our previous work on the study of cellular targets of ID [53]. As illustrated in Figure 8, ID during embryonic CNS development seems to affect neural precursor cell populations leading to an imbalance of specific precursor populations, which might be ultimately responsible for reduced oligodendrocyte numbers, abnormal myelination, and neural pathologies. We have shown previously that the differen- tiation and proliferation of embryonic glial precursor cells can be modulated by exogenous iron concentrations [87]. Interestingly, the timing of the generation, expansion and differentiation of these precursor cells coincides with the window of vulnerability we have defined in our diet regimens. In the rat embryo, gliogenesis starts around day E13.5 with the generation of embryonic glial restricted precursor cells (GRPs), which shortly before birth give rise to oligodendrocyte precursors (OPCs) that persist postnatally [88]. Even slight alterations of proliferation and/or of differentiation abilities of precursor cells (GRPs and OPCs) occurring before the time of oligodendrocyte generation could translate into a myelination defect. The notion of early embryonic cells being especially responsive to suboptimal iron levels is also supported by studies from Badarocco et al., whose interesting experiments using intracranial apotransferrin injections suggest that iron may affect oligodendrocyte development at early stages of embryogenesis rather than during late development [89]. The defects that are associated with iron deficiency are, however, unlikely to be restricted to the glial population. As shown in Figure 8, neuronal progenitor cells develop in a similar time window and could equally well be targets of iron deficiency. The cognitive impairment seen in the offspring is most likely a combination of an impact on both neuronal and glial embryonic progenitor cell pools. The absence of a functional defect in late onset ID suggests that, if the diet is introduced at embryonic day 14 (red arrow) and maintained, the time point of iron depletion is past the period when the most vulnerable embryonic populations expand and generate their progeny. Under these conditions, the impact of ID on glial cell generation and/or neuronal maturation does not seem to be severe enough to result in detectable auditory dysfunction. It should be pointed out that despite the lack of an ABR latency defect in our study at the late gestational time point (ID E14), we cannot exclude the presence of other abnormalities. For instance, a number of studies in rodent and primate models of late gestational and postnatal ID reported significant and persistent behavioral abnormalities and neurochemical distur- bances [19,90,91,92,93,94,95,96,97]. As development occurs along a temporal continuum, it would be expected that deficiencies occurring at different times cause different types of outcomes. The timed maternal iron restriction coupled with a persistently deficient diet used in our model, led to another surprising finding that sheds some new light on the role of anemia in iron deficiency. Due to the associated fatigue, muscle weakness, and dyspnea during exertion, anemia is considered a confounding factor in PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e17483