Impact of Maternal Iron Restriction on Offspring Figure 7. Conduction velocity is decreased throughout the brainstem along the auditory pathway in the offspring of the various dietary groups, as reflected by ABR (P4-P1) interpeak latency. 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 (P4-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 provided with ID diet from the time of mating, ID E0 group (yellow hatched bars), displayed significantly prolonged ABR (P4-P1) interpeak latencies at all frequencies examined compared to controls (CTL, black bars) (n = 35 CTL rats, n = 14 ID E0 rats,*p,0.05). B. The offspring from experimental group ID E7 (blue hatched bars) exhibited significantly prolonged (P4-P1) interpeak latencies at all frequencies tested compared to CTL (black bars) (n = 35 CTL rats; n = 27 ID E7 rats, *p,0.05). C. The offspring born to experimental group ID E14 (green hatched bars) had normal ABR (P4-P1) interpeak latencies compared to CTL (black bars) (n = 35 CTL rats; n = 29 ID E14 rats). The control and experimental dietary groups were composed of rat pups from 3 separate litters. doi:10.1371/journal.pone.0017483.g007 in adult rats without anemia [78]. These studies support our notion that anemia is not necessarily a defining predictor of neural impairments that are associated with iron deficiency. Our model of gestational ID was not only associated with persistently lower body weights as early as E15 but was also associated with functional impairments as determined by the Auditory Brainstem Responses (ABR) analysis of neural conduc- tion velocity in the offspring. The increase in P2-P1 interpeak latencies without hearing threshold changes suggested that, at least in part, impaired myelination could be one explanation for our findings. While disruptions in myelination have been demonstrated in rodents with prenatal and lactational ID [43,45,79,80,81,82,83,84] and hypomyelination is also recognized as a defining feature associated to perinatal ID in prematurely born infants [44], the greater increase in latency changes seen also by analyzing P4-P1 interpeak latencies (which reflects the entire brainstem) is consistent with findings in the literature suggesting that neuronal components could also be affected by iron deficiency. As we established a functional criterion for the ‘‘diagnosis’’ of neural impairment that could clearly be associated with the maternal dietary intake, it was possible to design a timed dietary restriction study that cannot be performed in humans but is critical for understanding the window of vulnerability. We generated maternal dietary groups that were defined by the onset of iron restriction solely based on food source. Using the ABR test as a functional readout of the neural CNS integrity of the offspring, we determined that the maternal exposure to an iron-deficient diet either prior to conception, at the start of first trimester, or at the onset of second trimester had a significant negative impact on the ABR latency response in the offspring, placing the window of vulnerability for the fetus in the first two trimesters of gestation. Interestingly, the negative CNS effects on the fetus were more severe when the ID was initiated at the beginning of pregnancy compared to ID initiated prior to the onset of pregnancy. An important result of our research was that the offspring from pregnant rats receiving the ID diet at the beginning of the third PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e17483