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Behavioural Brain Research 188 (2008) 263–270

Research report

Effect of perinatal iron deficiency on myelination and

associated behaviors in rat pups
Ling-ling Wu, Li Zhang, Jie Shao, Yu-feng Qin, Rong-wang Yang, Zheng-yan Zhao ∗
Department of Child Health Care, Children’s Hospital, Zhejiang University School of Medicine, 57 Zhu Gan Xiang, Hangzhou 310003, China
Received 16 September 2007; received in revised form 27 October 2007; accepted 5 November 2007
Available online 17 November 2007

Abstract
Iron deficiency in early development has been associated with irreversible alterations in brain myelination, but whether these neural changes are
mirrored in altered behaviors in rats is not known. The goals were to determine if dietary induced gestational and lactational iron deficiency alters
brain myelination and behaviors dependent on that system. Pregnant rats were randomly assigned to control (CN) or iron-deficient (ID) groups
by providing iron-sufficient (40 ppm Fe) or iron-deficient (2–6 ppm Fe) diets from gestational day 5 through to weaning of pups. Thereafter, all
offspring were fed the iron-sufficient diet. The myelination of subcortical white matter and the fimbria of hippocampus was measured by 2 ,3 -cyclic
nucleotide 3 -phosphohydrolase (CNPase, marker of oligodendrocyte) density at 25 days of age. Specific behavioral assessments were performed
at multiple time points after birth. By contrast, ID rats had significantly lower density of CNPase in the subcortical white matter but the density of
CNPase in fimbria of hippocampus was comparable to CN rats. Moreover, ID rats showed significant behavioral impairments in surface righting
reflex, negative geotaxis reflex, vibrissae-evoked forelimb placing test and novel object recognition task. In conclusion, perinatal iron deficiency
can significantly alter behavioral outcomes which may be due to delayed myelination in specific brain regions.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Iron deficiency; Rat; Brain; Behavior; Myelination

1. Introduction function of the brain myelination, which plays an important

Iron deficiency is a worldwide common nutritional disorder
among infants and women of childbearing age. The problem is
especially serious in developing countries [33]. Iron is essen-
tial for normal development of the brain because it forms an
important component of enzymes involved in neuronal oxidative
metabolism, myelin synthesis and neurotransmitter synthesis
[19].
Iron deficiency in early development is closely related
to altered behavioral outcomes. Despite of iron treatment,
children with low iron status in fetal period [20,52] or in
infancy [37–39] continue to have significantly worse motorial
and mental performance than normal children. Animal mod-
els also showed irreversible behavioral abnormalities resulting
from diet-induced iron deficiency during early development
[26,27,35]. Many of these behaviors have been linked to dys-
∗ Corresponding author. Tel.: +86 571 87061007; fax: +86 571 87033296.
E-mail address: Zhaozy@zju.edu.cn (Z.-y. Zhao).
conductive role in brain function. More directly, iron-deficient
children were reported to have longer auditory brainstem
response (ABR) and visual-evoked potentials (VEP) latencies
attributed to hypomyelination [1,48].
During rapid brain growth, the peak uptake of brain iron
coincides with the peak period of myelination which develops
rapidly during the late gestational and early postnatal periods
[14,16,22]. There were growing evidences that iron deficiency in
early development affect brain myelination [9,16,18,34,45,55],
and some of them [9,34,45] found that iron deficiency in devel-
oping rats had direct effects on myelin, including a general
decrease in myelin content and differential effects on compo-
sition of myelin. But no data were provided in previous reports
on alterations of myelination in specific brain regions or the
relationship of hypomylination with abnormal behaviors.
It is believed that iron deficiency affects developing
brain regions nonhomogeneously. One previous research [25]
reported that regional brain iron decreased heterogeneously due
to dietary iron deficiency, especially in the cortex and hippocam-
pus. Others also found the hippocampus was highly susceptible

0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2007.11.003
264 L.-l. Wu et al. / Behavioural Brain Research 188 (2008) 263–270
to iron deficiency during the early developmental period [12,47].
In addition, iron deficiency may induce fetal hypoxia and they
could coexist in infants of diabetic mothers and intrauterine
growth restricted infants [3]. Hypoxia–ischemia in newborn
could lead to selective injury to the subcortical white matter and
altered neurobehaviors. Thus, we hypothesized that perinatal
iron deficiency may selectively affect myelination of subcortical
white matter and the myelination of hippocampus.
In current study, we used a perinatal model to examine the
effects of iron deficiency on brain myelination and associated
behaviors. Based on previous studies, we hypothesized that early
iron deficiency alters myelination of specific brain regions and
accounts for abnormalities in myelination-dependent behaviors
in rats.
2. Materials and methods
2.1. Animals and dietary treatment
Male and female Sprague–Dawley rats weighing 250–300 and 180–200 g,
respectively, were purchased from the Center of Animal Experiment at Zhe-
jiang University. On arrival, they were fed a 40 mg/kg Fe diet (TD70481,
Harlan Teklad Nutritionals, Madison, WI, USA) for 2 weeks prior to mat-
ing. Pregnant dams were randomly assigned to iron-deficient (ID) and control
(CN) groups at gestational day (G) 5. Control dams continued the 40 mg/kg
Fe diet throughout gestation and lactation. ID dams received a 2–6 mg/kg Fe
diet (TD80936, Harlan Teklad Nutritionals) from G 5 to PND 21. At PND
1–2, pups were culled to not more than 10 pups per litter, retaining a balance
of males and females as able. After weaning at PND 21, all pups received
the 40 mg/kg iron diet until sacrifice. Animals were housed in a temperature-
controlled animal facility with a reversed, 12-h light:12-h dark cycle. All animals
were given deionized-distilled water through glass sipper tubes. The experi-
mental protocol was approved by the Center of Animal Experiment at Zhejiang
University.
2.2. Determination of iron deficiency
The dams’ iron status was closely followed by measuring hemoglobin (Hb)
and hematocrit (Hct) at G 5 and 20. The blood samples were collected via tail
puncture. Iron deficiency of rat pups was determined by the physical landmarks
including body weight and muscle strength. The body weights of rat pups were
measured at PND 6, 9, 12, 15, 18 and 21. Muscle strength was measured by
bar-hanging test, which is a test for forelimb grip strength and endurance [35].
In this test, the rat was held by body so that its forelimbs touched a 20-cm long,
0.5-cm diameter bar elevated 50 cm above the padded floor and allowed to grasp.
The hanging time spent on the bar by grasping with open forelimbs was recorded
for up to 180 s with a stopwatch. The rat pups were tested at PND 6, 9, 12, 15,
18 and 21.
2.3. Immunohistochemistry
At 21 days of age, the pups were weaned, and then fed iron-sufficient diets.
Brain was examined at 25 days of age. Ten CN and 10 ID (PND 25) were used for
immunohistochemical studies. After euthanasia with intraperitoneal pentobar-
bital (150 mg/kg), animals were perfused with 4% paraformaldehyde and their
brains were removed and then processed for paraffin embedded tissues. Brain
coronal sections were cut at 4 ␮m and then were immunostained for CNPase with
EnVisionTM two-step strategy and high-temperature antigen retrival (Pressure
Cooker, Supor Co., China). Sections were deparaffinized twice, 10 min each
in 100% xylene, and then hydrated with 100% ethanol for 5 min twice, 95%
ethanol for 3 min and 80% ethanol for 5 min. After 2–5 min soakings in distilled
water, the slides were put into the pressure cooker filled with 1000 mL of boil-
ing sodium citrate buffer (pH 6.0) and heated under pressure. Two minutes after
steaming, the pressure cooker was removed from the heating source and cooled
down to room temperature with tap water. The container was opened, and the
slides were rinsed 2 × 3 min with phosphate-buffered saline (PBS, 0.01 M pH
7.2–7.4). Then the brain sections were incubated with 3% hydrogen peroxide in
phosphate-buffered saline for 10 min to block endogeneous peroxidase activity
and were rinsed 2 × 3 min in PBS. Serum from nonimmune animals was used
as blocking buffer, and the slides were incubated for 15 min. Then the primary
antibody (mouse anti-rat CNPase monoclonal antibody, NeoMarkers, MS-349-
PO, dilution 1:50) was applied for overnight at 4 ◦ C. After being washed three
times with PBS, the slides were incubated with goat anti-mouse IgG Poly-HRP
for 40 min at 37 ◦ C. With DAB chromagen added, the slides were observed and
examined for color change in light microscope. This was followed by counter-
stain with hematoxylin for 1 min and rinsing in tap water for 1 min. In a manner
of casting lots, 2 slides were picked out in which the primary antibody was
omitted and was substituted with PBS as the blank control.
Each brain section for CNPase was served for quantitative analysis of inte-
grated optical density (IOD) using NIH image analysis (Scion Image) under
identical light conditions. The regions of white matter analysed included: sub-
cortical white matter and fimbria of the hippocampus. Three slides from each
brain were analyzed and a mean IOD of CNPase was determined.
2.4. Behavioral assessments
The behavioral assessments were chosen to systematically assess hypotheses
about effects of perinatal iron deficiency on specific neurobiological systems
based on previous studies. Behavioral testing was performed between 9 and 14 h
(during the light cycle). Individual rats from each litter received 2–3 behavioral
tests. Testers were blind to diet group status at the time of testing.
2.4.1. Neurological reflex development
2.4.1.1. Surface righting reflex [29,40]. For this test, the rat was placed on a
flat surface in a supine position and released. The time required to regain all four
paws in contact with the surface was recorded on a stopwatch. The maximum
time allowed for righting was 2 s. Each animal was given one trial per testing
day. Scores for this test were given as follows: Score 1, the righting latency was
longer than 2 s; Score 2, the righting latency was 2 s or shorter, but longer than
1 s; Score 3, the righting latency was 1 s or shorter. The higher the score was,
the shorter the righting latency was. The rat pups were tested at PND 6 and 9.
2.4.1.2. Negative geotaxis reflex [29,40]. For this test, the rat was placed on
a 45◦ inclined plywood surface in a head-down position and evaluated as to
whether it rotated 180◦ to a head-up position within 30 s. The time taken to
complete the 180◦ turn was recorded on a stopwatch. Each animal was given
one trial per experiment day. The rat pups were tested at PND 6, 9, 12, 15 and
18.
2.4.2. Sensorimotor function
Upper extremity sensorimotor function was assessed by vibrissae-evoked
forelimb placing test and damage to the forelimb region of the brain sensorimotor
cortex causes chronic deficits in this function [13,44,49]. For this test, the rat was
held by its torso allowing the ipsilateral forelimb to hang free. While holding the
animal, the experimenter made gentle up and down movements in space prior to
place testing, which facilitated muscle relaxation and eliminated any struggling
movements. Independent testing of each forelimb was induced by brushing the
respective vibrissa on the edge of a table top once per trial, for 10 trials. Normal
animals place the ipsilateral forelimb quickly onto the countertop in response to
the vibrissae stimulation. The percentage of ipsilateral forelimb placements out
of 10 trials per side was averaged for each animal. This sensorimotor function
was assessed at PND 24 and 29.
2.4.3. Novel object recognition (NOR) task
This visual memory task is based on the differential exploration of famil-
iar and novel objects and it is a test for visual cortical and hippocampal
functions [10,23]. The task began with habituating rats to the testing box
(60 cm × 40 cm × 30 cm) for 3 days. In the first trial, two identical objects (sam-
ples) were placed into the box and the rat was allowed to explore for 3 min.
After a half-hour delay, the second trial began. In the second trial, the rat was
placed back into the same box which contained a familiar (the sample) and a
 L.-l. Wu et al. / Behavioural Brain Research 188 (2008) 263–270 265

Table 1
Hemaglobin and hematocrit variables of the control (CN) and iron-deficient (ID) rat dams at gestational day (G) 5 and G 20
Group CN (n = 8) ID (n = 8) t P

Hematological indices at G 5
Hemoglobin (g/L) 144.0 ± 9.41 143.8 ± 7.81 0.029 0.977
Hematocrit 0.40 ± 0.026 0.41 ± 0.028 −0.941 0.363
Hematological indices at G 20
Hemoglobin (g/L) 148.0 ± 9.89 94.5 ± 10.89 10.289 0.000
Hematocrit 0.38 ± 0.040 0.18 ± 0.030 11.574 0.000

Values are means ± S.D. for hemoglobin and hematocrit variables.

novel object, and allowed to explore freely for 3 min. Behavior was considered
exploratory when an animal was touching or directed towards the object at a
distance ≤1 cm with its nose; while behavior was not considered exploratory
when an animal was turning around or sitting on the object. The time spent for
exploring each object was recorded. The pecent time spent exploring the novel
object served as the measure of recognition memory for the familiar object.
Normal rats discriminate between the two objects in the second trial and they
spend more time in exploring the new object than the familiar one. After each
experiment, both the objects and the arena were scraped by 10% ethanol to avoid
interference effect of the olfactory cue due. In the present study, rat pups were
tested at PND 25 and 35.

2.5. Statistical analysis

Data were analyzed by using SPSS software. All biological data were
examined for normal distributions. Normal distributed dada were expressed
as mean ± S.D. Differences were analyzed by group comparisons t-test. Data
of negative geotaxis reflex had skewed distribution, hence median time and
interquartile ranges were reported. Significance of inter-group difference was Fig. 1. Body weights of control rats and iron-deficient rats at multiple time point
determined using the non-parametric Mann–Whitney test. One-sample t-test during the pre-weaning period. Values are mean ± S.D. for each group and age.
were used to determine whether the exploration percentages were significantly Significant difference from the control group (* P < 0.01). Significant difference
different from chance levels (50%). P-values <0.05 were considered significant. from the control group (** P < 0.001).

3. Results
3.3. Brain myelination
3.1. Maternal iron status
To explore the effect of perinatal iron deficiency on CNPase
At gestational day 5, no difference was found in hematologi- concentrations in specific brain regions, we performed an
cal indices (hemoglobin and hematocrit variables) of two groups immunohistochemical examination of CNPase in brain sections
of dams (P > 0.05). At gestational day 20, the hemoglobin and
hematocrit variables were significantly lower in iron-deficient
dams than in control dams (Table 1), confirming that profound
iron-deficient anemia existed in the iron-deficient dams at late
gestational days.

3.2. Physical landmarks of pups

3.2.1. Body weights

Fig. 1 shows changes in the body weights of ID rats during
the pre-weaning period. The mean body weights of iron-deficient
rat pups were significantly lower, about 75–85% those of con-
trol animals. Our findings indicate that iron deficiency result in
significant growth failures in body weights.

3.2.2. Bar hanging test

As shown in Fig. 2, ID rats showed significant shortened Fig. 2. Bar-hanging time of control rats and iron-deficient rats at multiple time
duration of grasping a bar, which indicated a deficit in muscle point during the pre-weaning period. Significant difference from the control
strength and endurance at every experiment day. group (* P < 0.05). Significant difference from the control group (** P < 0.001).
266 L.-l. Wu et al. / Behavioural Brain Research 188 (2008) 263–270

Fig. 3. (A–D) Medium-power brightfield photomicrographs (magnification factors: 100) depicting immunohistochemistry of CNPase in subcortical white matter
(A and B) and the fimbria of hippocampus (C and D) of 25-day-old CN (A and C) and ID (B and D) rats. (A) Subcortical white matter above the hippocampus of
CN rats, (B) subcortical white matter above the hippocampus of ID rats, (C) fimbria of hippocampus of CN rats and (D) fimbria of hippocampus of ID rats (SC,
subcortical white matter; Hi, hippocampus; Cx, cortex; Fi, fimbria of hippocampus; LV, lateral ventricle).

in rats at PND 25. Fig. 3 shows representative photomicrographs more time on negative geotaxis reflex task than CN rats
(×100) depicting mid-brain sections from CN and ID rats. By and there were significant differences between the two
contrast, ID rats showed weaker definition in subcortical white groups at most of the time points (Mann–Whitney U test,
matter formation than CN rats at 25 days of age; indicative of P < 0.001).
delayed maturation of the white matter tracts. Fig. 4 shows that
the integrated optical density (IOD) of CNPase in subcortical
white matter of iron-deficient rats was significantly lower than
that of the controls (P < 0.001). But there was no significant
difference in IOD of fimbria of hippocampus between the two
groups.

3.4. Behavioral results

3.4.1. Neuroreflex development

3.4.1.1. Surface righting reflex. Fig. 5 shows the development
of surface righting reflex in iron-deficient and control rats at
PND 6 and 9. The ID rats had significantly lower scores than
that of controls which indicated prolonged latency of reflex in
ID rats.
Fig. 4. Integrated optical density (IOD) × 1000 of CNPase in subcortical white
3.4.1.2. Negative geotaxis reflex. Table 2 shows the devel- matter and fimbria of hippocampus of the two groups of rats at PND 25. Val-
opment of negative geotaxis in iron-deficient rats and ues are mean ± S.D. for each group and region. Significant difference from the
control rats during the pre-weaning period. ID rats spent control group (* P < 0.001).
 L.-l. Wu et al. / Behavioural Brain Research 188 (2008) 263–270 267

Fig. 5. Scores of control rats and iron-deficient rats on surface righting reflex
test at PND 6 and PND 9. Values are mean ± S.D. for each group and age.
Significant difference from the control group (* P < 0.01). Significant difference
from the control group (** P < 0.001).
Fig. 7. Exploratory preference for the novel object of the two groups of rats
at PND 25 and PND 35. The broken line represents chance performance
(50%). Values are mean ± S.D. for each group and age. Significant difference
from the chance level (* P < 0.05). Significant difference from the chance level
(** P < 0.001).

3.4.3. Novel object recognition task

Fig. 7 shows the mean exploration preference for the novel
object during the test. The percentage indicated the degree
of discriminated exploration of the sample and novel objects.
One-sample t-test were used to determine whether the explo-
ration percentages were significantly different from chance
levels (50%). In this test, all rats demonstrated a preference
for the novel object versus the familiar one. But only control
rats showed significantly differences as compared to the famil-
iar block. The control rats exhibited a strong preference for the
novel object at P25 [chance = 50%, t(16) = 5.097, P < 0.001] and
at P35 [chance = 50%, t(15) = 2.779, P < 0.05]. There was no
significant difference for iron-deficient rats compared to chance
levels (P > 0.05).

Fig. 6. Percent unilateral placing of control rats and iron-deficient rats in
vibrissae-evoked forelimb placing test at PND 24 and PND 29. Values are
mean ± S.D. for each group, side and age. Significant difference from the control
group (* P < 0.05). Significant difference from the control group (** P < 0.01).
3.4.2. Vibrissae-evoked forelimb placing
As shown in Fig. 6, iron-deficient rats had delayed emergence
of vibrissae-evoked forelimb placing after weaning as compared
to control rats.
4. Discussion
This study examined the consequences of gestational and lac-
tational iron deficiency on myelination in specific brain regions
and a battery of behavioral tasks depending on that system.
We observed that perinatal iron deficiency produced abnormal
myelination of subcortical white matter and altered behaviors,
some of which were irreversible consequences. The neurological
changes correlated with and accounted for the delayed functional
behaviors.

Table 2
Time spent on negative geotaxis reflex task of CN and ID rats during pre-weaning period
Age (PND) (median and 25th–75th CN ID Z p
interquartile range, s)

6 9.47 (7.16–14.31) 26.52 (16.74–30.00) −5.866 0.000

9 5.81 (3.96–9.57) 10.67 (7.21–20.40) −5.151 0.000
12 3.27 (2.06–4.63) 5.44 (3.22–7.34) −3.982 0.000
15 3.33 (2.43–4.92) 3.87 (2.50–5.89) −0.421 0.674
18 2.23 (1.78–2.98) 3.75 (2.91–7.42) −3.472 0.001
268 L.-l. Wu et al. / Behavioural Brain Research 188 (2008) 263–270
The experimental design used in this study effectively pro-
duced maternal iron-deficient anemia which indicated low iron
status of their offspring [2]. The hemoglobin and hematocrit
variables in ID dams were significantly lower than that in CN
dams at gestational day 20. In offspring, the body weights and
forelimb muscle strength of ID rats were consistently affected
during the pre-weaning period. These results were similar to
those demonstrated by other rodent models of iron deficiency
during early development [8,43,46].
In present study, we assessed the effect of perinatal iron defi-
ciency on myelination of specific brain regions (including the
subcortical white matter and the fimbria of hippocampus) by
immunohistochemical analysis. The ID rats in this study had
significantly delayed myelination of subcortical white matter at
PND 25. Whether the alterations in current study are permanent
is not known, because the assessment did not extend by PND
25. But one previous study using perinatal model found that
ID rat pups had deficits in white matter formation at 63 days
despite of iron rehabilitation after weaning [18]. This leads to
the speculation that the hypomyelination of subcortical white
matter induced by iron deficiency during critical period of brain
maturation may be irreversible.
The mechanisms underlying the hypomyelination induced by
iron deficiency are not known with certainty. The predominant
cell type containing iron in the young brain is the white mat-
ter oligodendrocytes, with rapid accumulation of iron in these
cells at the onset of myelination [15]. And oligodendrocytes
reportedly have the highest rate of oxidative metabolic activity
in the brain [31]. Thus, iron could be involved in myelination,
both directly through synthetic pathways and indirectly as an
essential cofacotor for metabolic enzymes in oligodendrocytes.
However, there was no significant difference in density of
CNPase in fimbria of hippocampus of two groups. Whereas
extensive evidence demonstrates that iron deficiency in early
development can significantly alter hippocampal iron, fer-
ritin, transferring, iron regulatory protein and gene expression
[12,25,51]. The comparable myelination in ID rats possibly
involved compensatory mechanisms and needs much further
exploration and examination. It is unclear from the current work
whether myelination of all brain regions would be delayed or
whether regional differences may persist.
In order to assess the effect of ID on behavioral development
of the rodent, previous studies [6,27,35,41,46,54] applied vari-
ous kinds of testing methods, such as auditory startle test, open
field, vibrissae-stimulated forelimb placing, sticker test, Mor-
ris water maze and so on. And they demonstrated alternation
in motor activity, stereotypic behavior, anxiety behavior, senso-
rimotor function and cognitive function. These findings were
consistent with altered behaviors in children [20,27–29,37].
The literature on iron deficiency and brain function has been
reviewed thoroughly [5,36]. But the alternation in neuroreflex
development or in NOR task has not been reported previously.
Therefore, in current study, we examined the development of
neurological reflexes which were closely related to the speed of
processing (myelination). It was a novel finding of our investiga-
tion that the development of neurological reflexes was delayed
in ID rats during lactation period. ID rats required significantly
more time to complete surface righting reflex and negative geo-
taxis reflex tasks than CN rats. These neurological deficits may
help explain findings of motor impairments in children who
had iron deficiency during infancy [36]. Myelination of cerebral
white matter contributes to normal brain functions as essential
components of neural networks. Thinner myelin sheaths affect
axonal conduction velocity and lesions of white matter could
produce neurobehavioral alterations [21,28].
In the sensorimotor task, ID rats showed poor vibrissae-
evoked forelimb placing at PND 24 and PND 29 after a short
time of iron treatment, suggesting a continued delay in this
skill. There was no significant difference between the results of
both sides. Similarly, several other researchers reported delayed
emergence of forelimb placing with the same dietary model
and they ascribed the altered behavior largely to alterations in
dopamine metabolism of striatum [8,27,54]. However, Schallert
et al. thought that this forelimb placing test also could be used
to assess the function of sensorimotor cortex [49]. Therefore,
we speculate that the delayed sensorimotor reflex in ID rats may
have some relationship with abnormal connections to sensori-
motor cortex resulting from the hypomyelination of subcortical
white matter.
For cognitive test, we selected the NOR task which has not
been studied in the ID model. We found that ID rats had impaired
visual recognition function at PND 25, and this abnormity per-
sisted into the fifth week despite of 2 weeks’ iron supplement
(from PND 21 to 35). This result is consistent with altered recog-
nition memory development in ID infants [11] and formerly ID
children [39]. This NOR task was thought to be similar to visual
recognition tests widely used in subhuman primates and could
be considered as a “pure” working-memory test completely free
of reference memory component [23]. Moreover, the working
memory deficit was recently found to closely relate to damage of
the subcortical white matter [50] and lesions of subcortical white
matter in human could produce cognitive dysfunction [17]. Thus,
the impaired visual recognition function was due largely to the
altered myelination of subcortical white matter which was deter-
mined in current research. But we cannot exclude the potential
mechanism of impaired cortical and hippocampal function in
ID model because the visual cortex and the hippocampus play
important roles in visual recognition and memory processing,
respectively. Other literatures [27,35] demonstrated that iron
deficiency in early development could impair special learning
and memory by using a Morris water maze.
The behavioral abnormalities in neurological reflex tasks,
sensorimotor function and the NOR taskis were consistent with
delayed myelination of subcortical white matter. We specu-
lated that iron deficiency could accomplish its adverse effects
on behavioral development via its effects on myelination. But
we cannot exclude the potential association with hippocam-
pal and prefrontal cortex alterations [12,30,32,42]; disturbed
monoamine neurotransmitter metabolism [4,7,53] and with
changes in glutamate metabolism [24] in ID rodents.
In summary, iron deficiency induced delayed myelination that
may account for a battery of myelination-dependent behavioral
changes. Additional studies are necessary to determine whether
these alterations also occur in other brain areas in perinatal mod-
 L.-l. Wu et al. / Behavioural Brain Research 188 (2008) 263–270
els. Iron supplement from P21 to P35 did not normalize the
alterations in NOR task, suggesting that the brain myelination
may undergo irreversible changes due to iron deficiency during
development. By using this model, we provide strong evidence
for the critical need to ensure adequate iron nutrition during early
brain development and it may help preventing and managing
chronic iron deficiency in humans.
Acknowledgements
The technical assistance in immunostaining part of Gu
Weizhong is gratefully acknowledged. The authors thank the
department of Child Health Care, Children’s Hospital, Zhejiang
University School of Medicine.
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