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Iron deficiency anemia (IDA) has been associated with altered cognitive, motor, and social-emotional outcomes in human infants. We recently reported that rats with chronic perinatal IDA, had altered regional brain iron, monoamines, and sensorimotor skill emergence during early development.
To examine the long-term consequences of chronic perinatal IDA on behavior, brain iron and monoamine systems after dietary iron treatment in rats.
Sixty dams were randomly assigned to iron-sufficient (CN) or low-iron (EID) diets during gestation and lactation. Thereafter, all offspring were fed the iron-sufficient diet, assessed for hematology and behavior after weaning and into adulthood and for brain measures as adults (regional brain iron, monoamines, dopamine and serotonin transporters, and dopamine receptor). Behavioral assessments included sensorimotor function, general activity, response to novelty, spatial alternation, and spatial water maze performance.
Hematology and growth were similar for EID and CN rats by postnatal day 35. In adulthood, EID thalamic iron content was lower. Monoamines, dopamine transporter, and dopamine receptor concentrations did not differ from CN. EID serotonin transporter concentration was reduced in striatum and related regions. EID rats had persisting sensorimotor deficits (delayed vibrissae-evoked forelimb placing, longer sticker removal time, and more imperfect grooming chains), were more hesitant in novel settings, and had poorer spatial water maze performance than CN. General activity and spatial alternation were similar for EID and CN.
Rats that had chronic perinatal IDA showed behavioral impairments that suggest persistent striatal dopamine and hippocampal dysfunction despite normalization of hematology, growth and most brain measures.
Iron deficiency anemia (IDA), during infancy in humans, has been associated with poorer performance on mental and motor measures and behavioral alterations such as wary, hesitant behavior [1,2]. Longitudinal studies report cognitive and behavioral alterations persisted into childhood and adolescence despite iron treatment in infancy [1,3,4]. The basic neurobiological alterations that occur with iron deficiency early in life have not been fully delineated and cannot be directly studied in the human .
Investigators have explored the central nervous system effects of iron deficiency anemia (IDA) in rodent models  induced by pre- and post-weaning periods of dietary iron restriction [6–17]. To relate the studies in rodents to the human condition, it is important to recognize that the timing of birth relative to brain development is different between species. While there are important differences by brain region and system, a general framework is that the 10-day-old rat brain approximates that of the full-term human neonate and by weaning (postnatal day 21), rat brain development approximates that of the late toddler age child .
Iron deficiency anemia has been demonstrated to reduce brain iron. However, the pattern and degree of such changes appear to depend on the timing and severity of anemia suggesting that the processes of regional acquisition of iron are developmentally bound [5,13,17]. Brain iron deficiency has direct and indirect, and immediate and long-term effects that likely include processes of morphogenesis , cell growth and differentiation , cellular bioenergetics , biochemistry [5,15], myelin biology [9,16,21,22], and neurotransmitter systems. The dopamine (DA) system has been studied most extensively with regard to IDA [6,9,14,16,23–25].
Investigations of IDA and dopamine biology, brain iron, iron management proteins, and behavior have documented effects that depend on time of nutritional deficit and the time of iron treatment. When severe IDA was induced after weaning, extracellular striatal dopamine and its metabolites increased, dopamine receptors decreased in striatum and nucleus accumbens, and dopamine transporter densities decreased. In this model, dopamine receptor and brain iron concentrations were directly correlated, and alterations of iron regulatory proteins were observed [13,14,23–25]. In addition, rats made IDA after weaning had reduced activity (at baseline [16,23,26–28] and in response to cocaine administration ), evidenced fewer exploratory and stereotypic behaviors , and had poorer performance in a Y-swim maze . Treatment with iron appeared to reverse some of the biochemical and behavioral findings with post-weaning IDA . Studies of IDA induced in lactation have found reduced regional brain iron, altered iron regulatory proteins, and altered dopamine measures. Within several weeks of iron treatment at weaning, brain iron content was nearly normalized, and there were improvements in extracellular dopamine concentration and dopamine receptor densities that correlated with improvements in brain iron concentration. However, despite iron treatment, there were persistent alterations in dopamine system measures (receptors and transporters) in the terminal fields and related behaviors (exploration and stereotypes) [11–13,23]. Recent studies have explored even earlier periods of iron deficiency and iron treatment. With perinatal IDA and iron treatment by mid-lactation, altered performance was observed on a brief spatial learning task . Persistent changes of iron regulatory proteins, hippocampal structure, and hippocampal metabolism have also been observed [19,20,30]. Thus, the effects of iron deficiency on brain and behavior and the reversibility of these effects appear to depend on the timing of IDA and iron treatment in rats.
In most animal studies of IDA, peripheral iron status has been severely reduced, by 60% or more, and significant growth retardation was often coincident (body weight reduced ~30%) [7,23–25]. Although severe IDA does occur in human infants, the more common condition world-wide is chronic iron deficiency that is not typically associated with severe reductions of iron status or growth . We were interested in whether biologically meaningful alterations would be documented in rats that had chronic iron deficiency, with a less severe level of anemia than previous rodent studies.
The rodent model we developed  differed from previous investigations in three respects. First, the design was based on the concept of the chronic iron deficiency that human infants around the world often experience—from in utero development into toddler age (18–24 months), followed by improved iron status due to dietary factors . Second, the model was designed to maintain a stable and less severe level of anemia during lactation, the developmental period generally equivalent to late gestation through toddler age in the human infant. Third, growth retardation was less than in previous models. The first report of this model documented the effects during the period of anemia. Hematologic measures were reduced by approximately 40% at both postnatal day (P)10 and P25. Brain weight was reduced by 5% at P10 and 10% at P25 and body weight was reduced by 15% at P10 and 25% at P25 for the IDA pups. Regional brain iron was lower for IDA pups at P25. Alterations in dopamine and serotonin metabolism were observed even earlier (P10). In addition, iron-deficient pups had reduced activity and delayed emergence of sensorimotor behaviors that specifically assessed for the integrity of the striatal dopamine system . Thus, chronic perinatal IDA was associated with significant alterations of brain iron, dopamine and serotonin biology and specific behavioral deficits in rats during anemia.
This study examined the long-term effects of chronic perinatal IDA in rats using a comprehensive battery of behavioral tasks and assessments of brain iron and monoamine systems. The objective was to determine whether dietary iron rehabilitation would correct the regional brain iron, monoamine and behavioral alterations observed during lactation with this model. Based on the studies of more severe IDA in rodents and the human infancy studies, we predicted that iron treatment at weaning would not be sufficient to normalize all brain and behavioral measures. In particular, we hypothesized that behaviors involving striatal dopamine and hippocampal systems would show persistent deficits. This prediction was based on the fundamental behavioral deficits we observed in these iron-deficient pups during anemia and the persistent effects of early IDA on hippocampal anatomy and metabolism observed by Georgieff and colleagues [19,20].
The dietary protocol has been previously described . Briefly, 60, 7-week-old Sprague Dawley female rats were obtained from Harlan, Sprague Dawley and fed a 40 mg/kg iron diet (TD89300, Harlan Teklad Nutritionals, Madison, WI) for 2 weeks prior to mating. Pregnant dams were randomly assigned to early iron deficiency (EID) and control (CN) groups at gestation day 7. Control dams continued the 40 mg/kg iron diet throughout gestation and lactation. EID group dams received a 4 mg/kg Fe diet (TD80936, Harlan Teklad Nutritionals, Madison, WI), from gestation day 7 to P7. EID group dams then received a 10 mg/kg iron diet (TD01094, Harlan Teklad Nutritionals, Madison, WI) from P7 to P20 to maintain a stable level of IDA and prevent significant growth faltering in the pups. At P20, all dams and pups received the 40 ppm iron diet. All pups continued on this diet after weaning at P23 until sacrifice. At P1–2, litters were culled to 10 pups per litter, retaining a balance of males and females as able. Litter mates remained together until P30. Thereafter, offspring were pair-housed by sex. Animals were housed in a temperature-controlled animal facility with a reversed, 12:12-h light/dark cycle. The experimental protocol was approved by the University Committee for the Care of Animals at the University of Michigan.
Body weight was measured at P35, P60, and at sacrifice (P120). Brain and liver were weighed at P120. Blood was assessed at P35 and P60 for hemoglobin and hematocrit and at P120 for hemoglobin, hematocrit, and serum iron by standard methods .
The behavioral assessments were chosen to systematically assess hypotheses about effects on specific neurobiological systems likely to be affected by early IDA based on previous studies (e.g., striatal dopamine system, hippocampus). Behavioral testing was performed between 0900 and 1400 h (during the dark cycle), under red light. Individual rats from each litter received 2–3 behavioral tests. Testers were blind to diet group status at the time of testing.
Male and female pups from each litter were sacrificed at P120 to measure brain iron or monoamine content. After euthanasia with intraperitoneal pentobarbital (150 mg/kg), animals were perfused with phosphobuffered saline (pH 7.4) through the left ventricle and organs were rapidly removed. Eight brain regions were quickly dissected on ice for measurement of iron content: frontal cortex, caudate-putamen, hippocampus, thalamus, nucleus accumbens, pons, superficial cerebellum, and deep cerebellar nuclei. Brain regions were immediately placed in storage tubes and frozen at −80 °C. Half-brains were also reserved for autoradiography for monoamine studies and were frozen slowly in dry ice:isopentane slurry and then stored at −80 °C. Livers were rapidly removed, weighed, and immediately frozen at −80 °C. Liver non-heme iron was determined using published methods . Blood samples for serum iron were centrifuged at 3000 × g at 4 °C for 15 min and sera were frozen at −80 °C.
Brain regions were digested by published standard procedures and iron content was analyzed by atomic absorption spectrophotometry as previously described and reported as μg/g tissue [13,14]. Catecholamine analysis in striatum was conducted by HPLC as described previously . Ligand binding for density of dopamine and serotonin transporters was performed on sections from reserved half-brains as previously reported . The density of receptors and transporters were quantified for brain regions using NIH image (Bethesda, MD) as previously described .
The fundamental analyses were ANOVA with diet group or sex as the main effect variables and χ2 for categorical variables using SPSS. All biological data were examined for normal distributions and log transformed when necessary prior to ANOVA. Behavioral measures were analyzed using the mean scores for males and females by litter for vibrissae-evoked forelimb placing and for individuals for all other assessments. General linear modeling using SPSS and Proc Mix using SAS  were used to assess the contribution of other factors such as context of assessment. Interactions between main effects were examined with the level of significance set at p < 0.05. For behavioral and brain measures with effect sizes ranging from 1.5 to 3, 8–10 rats per group were sufficient to determine significant group differences.
After 2 weeks of the iron-sufficient diet and thereafter, EID body weight and hematology did not differ from CN. Liver weights were also similar at sacrifice. At sacrifice, EID absolute brain weights were lower than CN. However, when normalized for body weight, brain weight did not differ significantly by diet group Table 1.
Body weight declined to a similar degree for both experimental groups before and during testing (<5%). There was no significant difference by diet group or sex for days to criteria on this task. Control rats averaged 32.1 ± 3.8 days versus EID 32.3 ± 7.4 days to reach 80% correct responses on 2 of 3 consecutive days.
The EID rats did not differ from CN rats for regional brain iron at P120, with the exception of a marginally lower iron concentration in the thalamus. There was a significant effect of sex on iron concentration in the frontal cortex and deep cerebellar nuclei, but no diet by sex interaction for brain iron in any region (Table 3).
At P120, monoamine concentrations in the striatum (MPHG [methoxyhydroxyphenyl glycol], norepinephrine, epinephrine, DOPAC [dihyroxyphenyl acetic acid], dopamine, HVA [homovanillic acid], 5-HIAA [5-hydroxyindole acetic acid], and serotonin) did not differ significantly by diet group (Table 4). There was a trend for reduced dopamine D2 receptor in the substantia nigra for EID as compared to CN rats (Table 5) but no effect of diet group on DAT density (Table 6). Serotonin transporter concentrations (SERT) were significantly greater for CN than EID rats in caudate-putamen and related regions (Table 7).
This study examined the long-term consequences of chronic perinatal IDA in rats using a comprehensive battery of behavioral tasks and assessments of brain iron and monoamine systems. With the level and timing of IDA and iron treatment in this model, we observed that most measures of brain iron and monoamines were restored by adulthood. However, as reported in longitudinal studies of chronic iron deficiency in human infants, behavioral deficits persisted despite iron treatment and recovery of iron status measures [1,3,4].
There is growing evidence that IDA affects sensorimotor functions. We and others previously reported delayed emergence of forelimb placing with chronic perinatal IDA [17,47]. In the present study of long-term outcome, we observed delayed forelimb placing into the 4th week of life . EID rats eventually attained this skill, and at 1 month of age no deficits were noted on the sticker task. However, 2–3 months later, sensorimotor deficits (sticker removal and naturalistic grooming) were detectable again. EID rats required significantly more time to remove forelimb stickers, and grooming was notable for more imperfect chains, characterized as having inserted or omitted events in the chain sequence. The findings suggest that these EID rats had difficulty successfully coordinating patterns of upper extremity motor movements. Damage to the striatum, neonatal dopamine depletion, and specifically, damage to the dorsolateral striatum alter sticker and syntactic grooming performance [31,35,48]. Thus, one explanation for our findings is that EID caused a fundamental alteration in dopamine nigrostriatal pathways or the feedback systems that affect these pathways. These sensorimotor deficits may help explain longitudinal findings of persistent motor and senorimotor impairments in children who had IDA during infancy [49,50].
Previous studies of rats and human infants showed that those with early IDA were more wary and hesitant [2,12]. In the present study, we observed similar behavioral differences. EID rats had less initial exploration in tasks assessing activity and emergence neophobia. Hesitance could have consequences on interaction with others and the progress and processes of learning . The neurobiological basis of this behavior is not clear but dopaminergic, other neurotransmitter systems and potentially hypothalamic adrenal pituitary systems could play a role [51,52]. In addition, the timing of early iron deficiency may be important. In a recent study of pre- and postnatal iron deprivation in monkeys, behavioral differences depended on the timing. Prenatal iron deprivation was associated with “less fearful” and more impulsive behaviors, whereas postnatal deprivation was associated with “more tense” and withdrawn behaviors in testing environments . The latter pattern resembles the behavior in studies of human IDA infants .
EID rats also had poorer place learning performance in the spatial water maze. This difference may relate to behavioral alterations coincident with, or apart from, learning per se. The EID rats showed more thigmotaxis, a pre-potent strategy whereby animals search the structure or the periphery for a route of escape [42,43]. The CN rats appeared to learn quickly that thigmotaxis was not a functional strategy, but the EID rats did not abandon this maladaptive strategy as readily. Deficits in hippocampal morphology may underlie the poorer performance and altered behavior of the EID rats. Morphologic and metabolic abnormalities in the hippocampus have been demonstrated with perinatal IDA [15,19,20]. However, the persistent thigmotaxis could also reflect increased emotionality and/or less cognitive flexibility in the context of the novel and mildly stressful task of place learning in the water maze . The influence of the different behavioral responsiveness on tasks of cognitive functioning is relatively un-explored and warrants further investigation [2,11,12].
We previously reported that rats in this chronic perinatal IDA model had more brain iron deficiency as they progressed through lactation, with associated changes in dopamine transporter, serotonin transporter, and D2 receptor levels in the mesolimbic and nigrostriatal systems . In the present report of long-term effects, we observed that several months of dietary iron “rescue” was sufficient to restore brain iron and most measures of dopamine and serotonin biology. These findings are similar to those in a previous study that used a shorter period of IDA during lactation and dietary recovery at weaning . When severe IDA occurred post-weaning in other studies, iron repletion did not always reverse changes in monoamine concentrations, dopamine transporter and D2 receptor levels [16,24,55]. Thus, the timing of IDA and treatment appears to be important to brain iron and monoamine outcome. We observed that EID rats still had reduced serotonin transporter density in adulthood in a number of thalamic and striatal brain regions . It is possible that this result involved compensatory mechanisms. Early injury in the striatal dopamine system has been associated with a compensatory increase in serotonergic innervation in the striatum and nucleus accumbens [56,57]. The mechanisms of this increase and the consequences behaviorally are not completely understood. However, if such mechanisms were activated with chronic perinatal IDA, it could result in a brief “compensation” period followed by long-term declines in serotonin transporter densities as observed in the current model.
One limitation in this study was that all animals underwent developmental testing every 3 days during lactation (beginning at P6) . It is know that “handling” in the postnatal period alters stress responses later in life [58,59]. Thus, it is possible that our early postnatal evaluations affected the behavioral responses we observed in adulthood. It is also important to note that the level of iron deficiency anemia in this rodent model and the effects on growth were more significant than typically observed in human infants with chronic iron deficiency where hematologic indicators are typically reduced by 20% and there is little to no retardation of growth [2,17]. However, the growth retardation was less pronounced and the degree of brain iron deficiency was more moderate in this model than other rodent models of IDA during early development .
In conclusion, this study systematically assessed the adult behavioral phenotype of rats after dietary iron rehabilitation for chronic perinatal IDA. Despite recovery of brain iron and most measures of neurotransmitter function, behavioral deficits consistent with fundamental alterations of the striatal dopaminergic and hippocampal systems persisted. We observed differences in sensorimotor abilities, response to novel settings, and performance on a spatial learning task. Development is influenced by motor, sensory, social, and cognitive experiences. If these are impoverished or slowed by chronic perinatal IDA, this effect might underlie the deficits in functional behavioral outcomes we observed in adulthood. Using rodent models to understand the neurobiologic underpinnings (neurotransmitter, myelination, anatomy and metabolism) for these behavioral findings and how they change depending on the time and degree of IDA and iron treatment may help identify optimal management of chronic iron deficiency in humans.
The entire group of investigators participating in the Brain and Behavior in Early Iron Deficiency Program Project contributed to our thinking about brain and behavioral analyses and interpretation for this paper.
Supported by a program project grant from NIH (P01 HD39386, Brain and Behavior in Early Iron Deficiency, Betsy Lozoff, Principal Investigator) and R01 NS35088 (JB).