various behavioral tests and raises criticism on the accuracy of the results and their interpretation. The ABR electrophysiological test we employed in the evaluation of neurodevelopmental impair- ments induced by ID seems not to be influenced by anemia. As shown in Figure 5A, all offspring of our different feeding groups developed clinically relevant anemia, yet only three out of four groups showed functional impairment in ABR patterns. This observation strongly suggests that anemia is not a determinant factor of pathological ABR patterns recorded in iron deficient animals and allows dissociation of the impairment caused by ID from effects that might be primarily due to anemia. As ABR analysis can be conducted in multiple species, including humans, this type of analysis may provide a particularly useful noninvasive tool for examining the effects of micronutrient deficiencies on CNS development. While IDA was not required to cause ABR defects, the occurrence of IDA did, however, severely impact the survival rate of the offspring. The litter survival rate was approximately 75% in the four experimental groups compared to the control groups. This decrease in survival rate may lead to an unintentional sample population bias of ‘‘survivors’’ at P45 when the functional measurements are conducted. While we cannot completely rule out that the surviving animals are on some level more robust that their littermates, it is important to note that we cannot correlate survival with absence or presence of ABR defects. The ID -2wk, ID E0 and ID E7 groups had as many animals that died during the experiments as the ID E14 group yet none of the surviving animals in the last group showed an ABR defect, while all the animals in the other three groups showed an ABR defect of various severities. Another novel observation in our study is the time-specific presence of thermoregulatory deficits in the offspring from mothers who were iron restricted two weeks prior to conception. The three other feeding groups did not develop such defects. As ID has sometimes been linked to poor thyroid function [98], which in turn can result in a thermoregulatory dysfunction [99,100,101,102,103], the decreased core body temperature in the most severe feeding regimen (ID -2wks) might involve a thyroid component. However, based on our data, we do not think that thyroid involvement is critical for the development of the ABR dysfunction, as the ID E0 and the ID E7 groups have significant ABR dysfunctions but normal core body temperatures. In summary, the analysis of developmental defects that are associated with exposure to iron restriction during different phases of fetal development emphasizes the influence of the precise window of vulnerability and the associated duration and magnitude of ID that is exerted on the developing fetal CNS. The prolonged absolute and interpeak latencies revealed by the comprehensive ABR analysis in ID animals could have important clinical implications. If ID at an early age causes hypomyelination or delayed neural maturation along the auditory pathway altering the properties of the auditory message and its transmission through the brainstem structures, it is possible that such a dysfunction could have functional significance for language acquisition and the development of other higher cognitive and emotional functions. In lieu of this information, our data not only reiterates the importance of iron supplementation as part of prenatal and gestational IDA therapeutic regimen but also as a preventive strategy. Identifying the windows of vulnerability when specifically vulnerable cell populations are generated during embryonic development will be critical in delineating the restricted window of therapeutic opportunity when iron supplementation could correct or prevent the functional deficit. Impact of Maternal Iron Restriction on Offspring Materials and Methods Animals and dietary treatment Adult female and male Sprague-Dawley rats (10–12 weeks of age) were purchased from Charles River Laboratories (Wilming- ton, MA). All animals were housed individually under controlled environmental conditions (0600–1800 h light cycle, 23uC and 32% humidity) and were provided free access to food and water. For the time mated pregnancies, the female/male pair was housed in the same cage for 24–48 hours or until the detection of vaginal plug. There are no data to support a notion of decreased fertility in any of the iron deficient (ID) diet treated groups. The litter size was comparable to controls and the newborn pups appeared healthy although their overall size is smaller in the ID - 2wk and ID E0 groups. We did, however, observe a decreased survival rate in all iron deficiency groups. By postnatal day 45, on average 10–20% of the offspring died with the most severe losses in the ID -2wk group. The lower survival rate was most likely a result of the severe anemia experienced by the offspring. Determination of gestational age For the time-mated pregnancies, the first day of gestation was determined by the appearance of vaginal plug. For exact timing of the gestational age, the crown-to-rump length (CRL) of the embryo was determined. The measurement of CRL, using a standard millimeter scale, was taken from the vertex to the rump with care taken to avoid pressure that would distort the natural curvature of the embryos. Embryonic size based on CRL in addition to vaginal plug detection allowed for a more accurate evaluation of gestational age. Experimental design (dietary regimens) The feeding protocols used in our study are depicted in Figure 4. The experimental animals were divided in four groups of specific dietary regimens, using the same iron-deficient diet containing 2–6 mg Fe/g of food (rodent TD. 80396 ID Diet – Harlan Teklad Custom Research Diets). The first diet regimen was designed to generate embryonic ID in early pregnancy. The dams were started on the iron-deficient diet two weeks before conception and the pups born to these dams represented the experimental group ‘‘ID -2wks’’. The next group of experimental animals was fed the iron- deficient diet on the day of conception or appearance of vaginal plug, designated ‘‘ID E0’’. The third experimental group of animals, designated ‘‘ID E7’’, comprised of rat pups born to dams exposed to the iron-deficient diet from the seventh gestational day onwards. The rat pups born to dams feeding on the iron-deficient diet from gestational day 14 onwards were assigned to the fourth dietary group of rats, labeled ‘‘ID E14’’. As our control (CTL), we used age-matched rats born to dams that were maintained on control iron diet throughout the duration of the experiment. The control diet contained about 240 mg Fe/g of food (Adjusted Iron Diet (240) – TD.05656 from Harlan Teklad Custom Research Diets). All other components of the diet, composition of nutrients and caloric values were identical to the ID diet except the concentration of iron. All control and experimental animals were given deionized-distilled water through glass sipper tubes. All groups received the assigned dietary treatment (iron- deficient and control diets) during lactation and after weaning until postnatal day 45, when the animals were subjected to ABR and DPOAE testing as well as evaluation of body weight, core body temperature, and iron status. Animal procedures were PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e17483