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 ± 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 ± 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 ± 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 ± 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 +/− SEM, * p<0.05).

To determine the restricted developmental window in which maternal iron restriction impacts fetal development, we established one control (CTL) group and four experimental groups of iron deficient animals: CTL group: animals received control iron diet (240 µg Fe/g food) throughout the experiment; ID-2wks group: animals began the iron-deficient diet (2–6 µg Fe/g food) two weeks before conception; ID E0 group: the iron-deficient diet was introduced the day of plug detection (E0); ID E7 group: animals were placed on the iron-deficient diet on embryonic day 7 (E7); ID E14 group: animals were fed the iron-deficient diet starting on embryonic day 14 (E14); All pregnant Sprague–Dawley female rats in different diet regimens groups were maintained on the respective diet from the assigned start point throughout pregnancy and lactation. After weaning, the pups were maintained on iron-deficient diet until the day of auditory testing (postnatal day 45).

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 ± 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.

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.

A. Hematocrit levels of pregnant rats exposed to an iron-deficient diet two weeks prior to conception (ID -2wks) (red bars) were evaluated at gestational day E15, E17, E19, and E21 and compared to time matched pregnant rats that received a control diet (black bars). CTL pregnant dams: n = 6 for each gestational age; ID -2wks pregnant dams: n = 6 for each gestational age; expressed values are mean ± SEM. B. The serum iron concentrations in iron-deficient diet treated animals (ID -2wks) and control animals (CTL) were determined via atomic absorption spectroscopy (AAS) at E15, E17, E19, and E21 (CTL pregnant rats: n = 3 for each time point; ID -2wks pregnant rats: n = 3 for each time point; Mean ± SEM, * p<0.001). While there were no significant changes in the hematocrit values between the two groups, iron restricted dams showed significantly lower serum iron concentrations compared to control dams, confirming their iron deficiency (ID) status.

Female Sprague-Dawley rats were provided with the iron-deficient diet two weeks before conception and remained on this diet throughout pregnancy and lactation. Rat pups born to these dams were maintained on iron-deficient diets until testing at postnatal day 45 (P45). Cochlear function was also assessed for all animals (CTL and ID -2wks groups) at ∼P45. A. The ABR (P2-P1) interpeak latencies recorded from ID -2wks (red bars) animals showed a highly significant increase compared to CTL (black bars) at all frequencies tested. 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 sound pressure level-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; Mean ± SEM, p<0.0001). B. The ABR interpeak latencies displayed increased variability in the ID -2wks animals (red circles) compared to CTL (open circles) and the mean value was increased in iron deficient animals. C. The ABR (P4-P1) interpeak latencies recorded from ID -2wks (red hatched bars) animals were significantly prolonged compared to CTL (black bars) at all frequencies tested (Mean ± SEM, p<0.0001). D. The ID dietary regimen was not associated with outer hair cell damage or cochlear dysfunction as revealed by Distortion-Product of Otoacoustic Emissions (DPOAE) analysis. There was no significant difference between control (black bars) and iron-deficient rats (red bars) in DPOAE amplitudes. (For A–D: n = 35 CTL rats from 5 separate litters and n = 15 ID -2wks rats from 3 separate litters; Mean ± SEM).

The hematological status, weight, and core temperature were evaluated in each animal after the ABR test completion on postnatal day 45 in the various dietary groups (depicted in Figure 4). A. The table summarizes the results of the hematological tests. All groups of experimental iron deficient animals (ID -2wks, ID E0, ID E7, and ID E14) presented with significantly decreased hematocrit (Ht) values, significantly lower hemoglobin (Hb) levels, profoundly decreased red blood cells (RBC) counts and markedly reduced mean corpuscular volume (MVC) compared to controls, demonstrating the presence of anemia in dietary iron restricted rats. Serum iron concentrations in iron-deficient diet treated animals were significantly lower compared to control animals, confirming the anemic condition of experimental animals. (Ht measurement: n = 11 CTL rats, n = 9 ID -2wks rats, n = 11 ID E0 rats, n = 9 ID E7 rats, and n =  9 ID E14 rats; Hb, RBC, and MCV determination: n = 6 CTL rats, n = 4 ID -2wks rats, n = 4 ID E0 rats, n = 4 ID E7 rats, and n = 6 ID E14 rats; Mean ± SEM, * p<0.0001; serum iron analysis: n = 5 rats for all groups, Mean ± SEM, * p<0.01; each CTL and experimental group included rats from 3 separate litters). B. Experimental iron deficient animals exhibited marked growth retardation compared with controls. (n = 35 CTL rats, n = 15 ID -2wks rats, n = 14 ID E0 rats, n = 27 ID E7 rats, and n = 29 ID E14 rats; all CTL and experimental groups contained rat pups from at least 3 separate litters; Mean ± SEM, * p<0.0001). C. Experimental group ID -2wks (n = 18 rats from 3 separate litters) offspring exhibit significantly reduced core body temperature in comparison to CTL animals (n = 17 rats from 3 different litters; * p<0.05). These changes are not seen in the other three experimental groups (n = 13 ID E0 rats, n = 13 ID E7 rats, and n = 14 ID E14 rats). All groups are composed of animals from 3 separate litters.

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.