Impact of Maternal Iron Restriction on Offspring Figure 2. Maternal iron restriction prior to conception is associated with embryonic iron deficiency, postnatal anemia, and reduced postnatal central nervous system iron levels. The ID -2wks and CTL pregnant dams were sacrificed at different time points during gestation (E13, E15, E17, and E19) and embryos were characterized in respect to total iron content by AAS analysis. A. The nutritional ID -2wks regimen generated embryonic ID as early as E15 (Mean 6 SEM, * p,0.001). B. The embryos of ID -2wks dams were significantly smaller than CTL embryos at all gestational ages after E13. (For A and B: at E13, n = 12 CTL embryos and n = 10 ID-2wks embryos; at E15, n = 6 CTL embryos and n = 6 ID -2wks embryos; at E17, n = 11 CTL embryos and n = 13 ID -2wks embryos; at E19, n = 6 CTL embryos and n = 6 ID -2wks embryos; for all gestational ages, each CTL and ID -2wks group was composed of embryos from 3 separate litters; Mean 6 SEM, * p,0.05). C. Hematocrit measurements in ID -2wks pups (red bars) revealed values significantly lower than in CTL pups (black bars) at postnatal day 7 (P7) (Mean 6 SEM, * p,0.0001). D. Serum iron concentrations measured at P7 were significantly decreased in blood samples collected from ID -2wks rat pups (red bars) compared to CTL pups (black bars)(For C and D: CTL and ID-2wks each had n = 8 pups from 3 separate litters, Mean 6 SEM, * p,0.0001). E. AAS at postnatal day 45 showed reduced iron level in ID spinal cord and cerebellum compared to CTL samples (spinal cord: n = 8 CTL and n = 5 ID -2wks samples; cerebellum: n = 5 CTL and n = 5 ID -2wks samples; each CTL and ID -2wks group contained tissue samples harvested from 3 separate litters, Mean +/2 SEM, * p,0.05). doi:10.1371/journal.pone.0017483.g002 postnatal animals suggested a change in overall iron homeostasis, our functional readout was focused on the impact of iron deficiency on brain development. We were, therefore, particularly interested in the status of brain iron levels in postnatal animals. AAS analysis of two central nervous system (CNS) regions that are particularly easy to harvest and allow for a good volumetric normalization (spinal cord and cerebellum) confirmed that there was a significant decrease in the concentration of iron in these tissues (Figure 2E). The decrease in iron concentration was more pronounced in the cerebellum, which had a much higher base-line level than the spinal cord. Embryonic iron deficiency leads to changes in auditory nerve conduction velocity Having shown that the early maternal ID severely impairs the iron homeostasis and weight of the offspring, we next asked whether this gestational insult also results in impaired CNS development in the offspring. Studies in humans, monkeys, and rodent animal models have suggested functional impair- ments in neural signaling as measured by impaired conduction velocity in the auditory system. This measurement of nerve conductivity is particularly useful as it allows for a unified and non-invasive analysis of a stereotypical neural response profile that results in a wave diagram with each wave representing the neural activity along the ascending pathway (see for review [54,55,56,57]). Specific ABR wave parameters like interpeak latencies between P2 and P1 are thought to be a reflection of the myelination status [58,59], while changes in P3 and P4 interpeak latencies are also suggestive of impairments in synapse maturation [60,61,62,63]. Measurements of Distortion-Product of Otoacoustic Emissions (DPOAE) can be used to exclude impairments that are due to a peripheral loss of hair cell PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17483