Impact of Maternal Iron Restriction on Offspring Figure 6. Increased Auditory Brainstem Response (ABR) conduction time in the offspring of various dietary groups can define a specific window of vulnerability. Auditory nerve function was analyzed by measuring the auditory brainstem responses (ABR) to tone pips at 5 log-spaced frequencies from 5.6 to 24.4 kHz (at 70 dB SPL). ABR peaks and troughs were identified using a semi-automated tool and verified by a trained observer. Latencies were calculated from onset of stimulus and the (P2-P1) interpeak latency was computed. The rats were sedated with an intraperitoneal injection of ketamine (80 mg/kg) and acepromazine (1 mg/kg). A. The offspring born to dams exposed to ID diet from the time of mating, ID E0 group (yellow bars), displayed significantly prolonged ABR (P2-P1) interwave latencies at all frequencies examined compared to controls (CTL, black bars) (n = 35 CTL rats, n = 14 ID E0 rats; Mean 6 SEM, *p,0.05). B. The offspring of dams provided with the iron-deficient diet in the second trimester, ID E7 (blue bars), exhibited significantly prolonged (P2-P1) interpeak latencies at a subset of frequencies tested compared to CTL (black bars) (n = 35 CTL rats; n = 27 ID E7 rats, *p,0.05). C. The pups born to dams provided with the iron-deficient diet in the third trimester of pregnancy, ID E14 (green bars, n = 29), had normal ABR (P2-P1) interpeak latencies compared to CTL (black bars, n = 35). The control and experimental dietary groups were composed of rat pups from 3 separate litters. doi:10.1371/journal.pone.0017483.g006 uously throughout pregnancy, confirming iron depletion. Upon more detailed examination of the maternal blood via HESKA Hema True Veterinary Hematology System, we observed minimal reductions in the measurement of mean corpuscular volume and hemoglobin compared to control values. Taken together, these results are consistent with ID and the absence of severe anemia. This dietary model thus mimics a situation that would not cause any level of clinical concern in the human population, as the absence of severe anemia would not prompt any immediate intervention and is likely to remain unnoticed. We believe that this is an important aspect of our study as it underscores the need for monitoring the status of iron beyond the level of anemia. Iron restriction to the dam two weeks prior to conception was associated with lower iron concentrations in embryonic tissues as early as embryonic day 15 (E15). The embryos maintained a very similar low iron level throughout gestation without an appreciable increase in serum iron concentrations suggesting that maternal iron stores, that apparently were not severely depleted, did not compensate for the continuously increased demand for iron by the embryo. Our observation seems to argue against the often proposed notion stating that the developing embryo is partly protected from the maternal ID through compensatory changes in the iron transport mechanisms of the placenta, which minimize the impact of ID in the fetus [68,69,70,71]. However, our data is consistent with (i) recently published data, suggesting that gestational ID might have a restrictive effect on the constitution of adequate fetal iron stores, [72,73] and with (ii) the observation by Naghii et al., indicating that fetal brain tissue can be iron depleted even in the absence of anemia in the pregnant mother [74]. Furthermore, studies from the Georgieff laboratory have shown that a transgenic model of hippocampus-specific iron deficiency without any signs of anemia also resulted in altered neuronal development as well as significant cognitive dysfunctions (reduced spatial recognition memory performance and procedural memory) [75,76,77]. Wu et al. have also shown that a short period of perinatal iron deficiency results in altered associative behavior PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17483