The motivation for this study was to understand the role of iron in neural development. The study of models of limited iron availability has provided important insight but such models are often associated with the induction of anemia, the most severe form of ID. In order to disentangle the consequences of ID from those of IDA we tested various rat strains on a variety of ID diets and found that the Fisher rat on 2–6 μg/Fe/g represented a model in which dams never develop anemia while on the diet during pregnancy ensuring that the growing embryos are not affected by hypoxia. The pups, which remained on the ID diet were not significantly anemic either and manifested only a marginal transient decrease in hematocrit at P21. Critically, however, from P7 to P40, animals born to the ID dams had markedly lower brain iron levels and a decrease in iron storage capacity. This model thus provides an opportunity to determine the role of iron during development in the absence of anemia.
As expected, the offspring showed no gross abnormalities, e.g. normal weight and palor. However, detailed analysis revealed a complex pattern of abnormal ABRs characterized by a decreased latency at P14 (i,e., faster impulse conduction), no impairment at P21, and increased latency at P40 (i.e., slower impulse conduction). ABR studies are used in humans as a non-invasive test to measure brain maturation defects (Roncagliolo et al., 1998
; Amin et al., 2010
). The interpretation that altered ABRs represent maturation defects is due to the correlation between conduction velocity, the increase in axonal diameter and acquisition of myelin during development. A previous model of IDA showed increased latency of > 0.2 ms ABRs (Mihaila et al., 2011
), and increased latencies of greater than 0.1ms have been reported in human studies of IDA where such changes are considered to represent significant functional impairments (Roncagliolo et al., 1998
While the ABR analysis revealed a general impairment in brain function, which was specific to certain development ages, the challenge was to correlate this compound readout with a defined structural defect. To directly investigate structure and function relationship between ABR latency and anatomy we isolated the distal auditory nerve, which produces wave 1 of the ABR. As wave 1 of the ABR is primarily mediated by the degree of myelination and axonal diameter we reasoned that this analysis should provide insight into underlying causes of the defect we identified in ID animals. To our knowledge no such study has previously been reported.
Our analysis of MBP and PLP expression in the AN revealed no significant change in the expression of early myelin proteins (MBP isoforms 21.5 and 17 kDa) at any timepoints. We observed no changes in the number of mature oligodendrocytes at any age. In addition, our measurements revealed no significant changes in myelin thickness or g-ratio. We did, however find a transient decrease in expression of the more mature MBP isoforms as well as PLP at P21, which coincides with the peak of myelination. This transient defect seemed to have no impact on the ABR, highlighting the multifactorial nature of the ABR.
The lack of a clear correlation of ABR changes with alterations in myelination suggested that other components contributed to the impaired ABR, and our studies revealed the unexpected finding of disruption in axonal maturation. For example, at P14 in control animals, the majority of axons were 0.5–1.5 μm in diameter, while at P40 axon sizes were spread across a wide range and reached over 6μm. In contrast, P14 ID axon distribution already reached up to 4μm which was maintained until P40 without further growth. Thus, the decreased latency at P14 was correlated with the presence of larger axons, while the increased latency at P40 was correlated with a relative scarcity of large axons in ID nerves. We cannot exclude the possibility that the change in axon size distribution was due to a selective loss of specific sized axons in ID; however, this represents an unlikely explanation as no evidence was observed of axonal degeneration, excessive looping, or other defects associated with axonal degeneration (Edgar et al., 2009
). While we did not observe a measurable change in myelin sheath thicknesses of comparable sized normal and ID axons, we did identify in ID axons significant changes in phosphorylated neurofilaments (NF) without changes to MAG, a known regulator of NF phosphorylation (Dashiell et al., 2002
). ID ANs lacked the typical increase in NF phosphorylation, which is an important determinant of axonal caliber (Pant and Veeranna, 1995
) and axonal transport rate in development (Shea et al., 2004
). Our observation of normal Caspr levels in ID axons and lack of obvious impairment to overall node organization (data not shown) does not exclude but reduces concern for substantial node abnormality. While alternate explanations for the ABR defects certainly exist and include disruptions to unmeasured functions of oligodendrocytes, nodal formation, or synaptic function, the presented data strongly suggest that disrupted axonal size distribution plays a major factor in the ABR latency defects.
Impaired axonal size maturation in the ID AN raises the question of whether neuronal function is altered elsewhere, e.g. the target regions that are innervated by the potentially immature axons. The maturation of the AN is complex and not well understood but it has been shown in mice that AN axons enter the brain by E13–14 and innervate the cochlear nuclei by E16, which is well before the onset of hearing (Carney and Silver, 1983
). From E18 to approximately birth, axons enter the nuclear subdivisions forming small side branches until appropriate targets are encountered and axons cease to grow. While still controversial, the AN is believed to grow into the brainstem prior to the migration of auditory neurons, which are then attracted to the AN axons and become postmitotic at day 10–14 (Rubel and Fritzsch, 2002
). Hence, migration of most cochlear nucleus neurons coincides with the arrival of the AN axons facilitating an interaction that is controlled by both intracellular and secreted molecules and is a critical part of proper brainstem development and synaptic maturation (Brumwell et al., 2000
). As our tissue iron analysis suggests that the brain is not significantly iron deficient at the time of birth and only shows a reduction in iron content from P7 onwards, it seems likely that the origination and early innervations of AN axons would progress normally in ID and that defects will manifest at later stages, perhaps ultimately affecting the maturation of synaptic connections, which control processes like temporal processing. A paradigm for such disruption has been demonstrated through impaired dendritic arborization in the postnatal hippocampus under mild ID (Jorgenson et al., 2003
). It will be of future interest to examine dendritic complexity in regions along the auditory pathway.
The data from our nutritional model demonstrate that in utero ID does not affect the pregnant dam and produces overall healthy, non-anemic offspring, but nonetheless is sufficient to cause both CNS iron depletion and readily detectable neurological impairments. Using ABR analysis as a non-invasive tool to assess neural function, combined with a detailed morphological analysis of the auditory nerve, we did not detect signs of hypomyelination, demyelination, a decrease in mature oligodendrocytes or axonal degeneration, all of which have been observed in models of severe iron deficiency associated with anemia. In contrast, we report a novel phenotype that is characterized by the disruption of appropriate axonal maturation and continues to expand the cellular targets of iron deficiency outside of the glial compartment.