QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Piñero, D. J. Articles by Beard, J. L. Search for Related Content PubMed PubMed Citation Articles by Piñero, D. J. Articles by Beard, J. L.

---

Article

Variations in Dietary Iron Alter Brain Iron Metabolism in Developing Rats

Department of Neuroscience and Anatomy, The Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, PA 17033 and ^* Graduate Program in Nutrition, The Pennsylvania State University, University Park, PA 16814

¹To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Measurements RESULTS DISCUSSION REFERENCES

 
The rat has been widely used as a model for the study of iron deficiency (ID), but the differences in the timing of development of humans and rats must be taken into account to derive appropriate conclusions from the animal model. This study was designed to evaluate the effects of dietary ID and iron excess on rat brain iron and the iron metabolism proteins, transferrin (Tf), transferrin receptor (TfR) and ferritin. The experimental design is developmentally sensitive and permits control of the timing as well as the duration of the nutritional insult. Iron-deficient and iron-supplemented (SU) rats between postnatal day (PND) 10 and 21, PND 21 and 35 and PND 10 and 35 were used to study the effects of early, late, and long-term iron deficiency and supplementation. Some ID rats were iron repleted between PND 21 and 35. These experiments demonstrated several new findings: 1) Early ID/SU (PND 10-21) altered brain iron, TfR, Tf and ferritin concentration in many regions different from those observed in the later period (PND 21-35). 2) Two weeks of iron repletion were adequate for correcting the overall Fe concentration of the brain and of individual brain regions, although larger amounts of iron were necessary to fully normalize iron and its regulatory proteins. 3) Long-term ID/SU resulted accordingly in the continued decrease or increase in brain iron concentration in some brain regions and not others. In conclusion, brain regions regulate their iron concentration in response to local needs when faced with alterations in systemic iron delivery.

KEY WORDS: • iron deficiency • iron excess • rats • brain • development

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Measurements RESULTS DISCUSSION REFERENCES

 
Iron deficiency (ID)² and anemia are the most prevalent nutritional disorders in the world (Baynes and Bothwell 1990 ). The association of iron deficiency with biochemical, behavioral and cognitive alterations is well documented, although the specific mechanisms are not yet understood. Iron is unevenly distributed in the brain, with some regions (i.e., substantia nigra, globus pallidus and ventral pallidus) having high concentration of iron (Beard et al. 1993a ). These iron-rich regions are also associated with dopamine (DA) metabolism (Beard et al. 1993a and 1994 , Youdim et al. 1989 ). Researchers have shown a decrease in the total amount of iron in the brain (Chen et al. 1995b , Erikson et al. 1997 ), as well as alterations of DA metabolism (Chen et al. 1995a , Nelson et al. 1997 ) in studies of the effects of ID in rats. The involvement of excess iron in neurologic disease has received more attention in recent years, especially in reference to the increased iron concentration in the brains of patients with Alzheimer’s disease and Parkinson’s disease (Beard et al. 1993b , Connor et al. 1992a , 1992b and 1995 ).

The effects of iron deficiency or iron supplementation on neurologic function are likely to be affected by the neurologic maturity of the animals. That is, the age of onset of the dietary iron deficiency may have a vast effect on what has been reported with regard to how much and where brain iron is lost, and on reversibility with subsequent iron repletion. The effects of iron excess have not been studied with the use of a developmentally sensitive design, although it is common to find reports in the literature in which the so-called control diet is really a diet with supplemental iron. It must be noted that the development of the nervous system in rats does not occur over the same time course relative to the development of humans. Nonetheless, the process is assumed to be similar in the sequence of events. Understanding the timing of the brain growth spurt in different species is crucial for hypotheses that relate to vulnerability (Dobbing and Sands 1979 ). The human brain growth spurt peaks between -2 and + 3 mo around birth, but in rats, it peaks around postnatal days (PND) 7 and 10 (Dobbing and Sands 1979 ). These periods of growth spurt cannot be taken as a general rule because there is regional heterogeneity of the timing of developmental events in the brain, with each small region, tract or system having its own growth spurt and period of vulnerability. The myelination process, an important one for brain function, coincides roughly with the brain growth spurt for humans and rats (Davison and Dobbing 1966 ). Another important difference between humans and rats is that the bulk of iron acquisition during early stages of development occurs mainly during the third trimester of pregnancy in humans, and postnatally in rats, coinciding with the peak of the myelination process.

There are no data on the response that iron deficiency and iron excess elicit on iron acquisition and distribution by the different brain regions of rats during early development. To investigate further the relationship between neurologic maturity and the effects of iron deficiency, excess and repletion, a set of experiments was designed with the rat as the animal model, and taking into account the neurodevelopmental differences between humans and rats mentioned above, as well as the differences in terms of early iron acquisition, so that more valid conclusions could be drawn from the results.

The overall hypothesis of these experiments is that iron deficiency and iron supplementation induce changes in brain iron, that these changes are not regionally uniform, and that they are age dependent, as well as duration and severity dependent. That is, early onset iron deficiency (early ID) or supplementation (early SU) affect brain iron metabolism in a different fashion than late onset and long-term ID and SU. Moreover, it is hypothesized that recovery from early ID is not complete after 2 wk of iron repletion.

The specific aims of the project were to determine the following:

The effects of early vs. late iron deficiency on brain iron metabolism.

The reversibility of effects of early iron deficiency.

The differential effects of late (or short) vs. long-term iron deficiency.

Whether the brain adaptive strategies [increase in transferrin (Tf) and transferrin receptor (TfR) and down-regulation of ferritin], previously observed in postweaning models of iron deficiency, are also functional in these early ID studies and whether there are regional differences in these strategies.

The effects of early vs. late iron supplementation on brain iron metabolism.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Measurements RESULTS DISCUSSION REFERENCES

 
Research design.

A longitudinal design that used dietary and cross-fostering strategies to create iron deficiency, iron excess and control groups of rats was assembled. Such a design produces animals that are iron deficient (ID), control (CN) or iron supplemented (SU) between PND 10 and 21, 21 and 35, or 10 and 35, with appropriate groups to test for reversibility of iron depletion from the first period. To produce dietary manipulations during the late lactational period (between PND 10 and 21) the pups from control dams were fostered to iron-deficient or supplemented dams. The dietary manipulations after weaning (after PND 21) were created by weaning the rats to the corresponding control, iron-deficient or supplemented diet. The design is illustrated graphically in Figure 1 .

View larger version (20K): [in this window] [in a new window]   Figure 1. Experimental design. This diagram illustrates the time frame for the different dietary treatments during the experimental period. PND, postnatal day.

Male and female Sprague-Dawley rats (250-270 g and 150-170 g, respectively) were purchased from Harlan Sprague Dawley (Indianapolis, IN). The female rats were fed a control diet (CN, see below) upon arrival; the males were fed a commercial pelleted diet (Laboratory Rodent Diet, PMI Nutrition International, Brentwood, MO). At 200-220 g of body weight, the females were mated with a male for a period of 5 d (two females and one male), after which the rats were separated and the females placed in individual cages. Pregnancy was checked by the appearance of vaginal plugs under the cages during the mating period, and by recording the animal weight every other day.

At ~d 10 of pregnancy, counted from the appearance of vaginal plugs, some pregnant dams were randomly switched to ID or SU diets. This date was selected to allow for the dams receiving the ID diet to become iron deficient by d 10 postpartum and to be used as foster dams for pups born to CN dams. The first morning that pups appeared in the cage was considered PND 1. At PND 4, the litter size was reduced to eight pups per dam and, if possible, four males and four females. At PND 10, the pups from CN dams were randomly assigned to ID, SU or another CN dam. Hence, at PND 21, there were three groups of rats, i.e., CN, ID and SU. At PND 21, the rats were randomly assigned to be weaned to a CN, ID or SU diet, or to be killed, except for the SU group, which was assigned either to a SU diet or killed (see Fig. 1 ). As a result, at PND 35, when the rest of the rats were killed, the following groups of rats were generated: 1) IDID, rats that were ID from PND 10; 2) IDCN, rats that were ID from PND 10 to 21, and were iron repleted with a CN diet between PND 21 and 35; 3) IDSU, rats that were ID from PND 10 to 21, and were iron repleted with a SU diet between PND 21 and 35; 4) CNID, rats that were CN from PND 10 to 21, and were iron depleted with an ID diet between PND 21 and 35; 5) CNCN, rats that were CN from PND 10 to 35; 6) CNSU, rats that were CN from PND 10 to PND21, and iron supplemented between PND 21 and 35; and 7) SUSU, rats that were SU from PND 10 to 35.

The diets were mixed in our laboratory, following the defined composition of the AIN-93G diet (Reeves et al. 1993 ), but using cornstarch as the only source of carbohydrates in the mixture.

The amount of iron in the mineral mix was manipulated by the addition of iron citrate to produce a diet containing 3.5 mg Fe/kg diet (ID diet), 35-40 mg Fe/kg diet (CN diet) or 400 mg Fe/kg diet (SU diet).

Upon analysis of the diets by flame atomic absorption spectrophotometry after wet digestion with nitric acid, the iron content of the diet was measured to be (mean ± SEM) 39.7 ± 0.8 mg/kg (CN diet), 3.04 ± 0.4 mg/kg (ID diet) and 420 ± 8.1 mg/kg (SU diet).

All of the rats were housed individually under controlled environmental conditions (0600-1800 h light cycle and 25°C) and were provided free access to food and water. Food intake and body weight were recorded every other day. All animal procedures were approved by The Pennsylvania State University Animal Care and Use Committee.

The dams’ iron status was closely followed by measuring hemoglobin (Hb) and hematocrit (Hct) before mating, and at midpregnancy, delivery, midlactation and weaning. The pups Hb and Hct were determined at PND 10, 14, 17, 20, 27 and 35. The blood samples were collected via tail prick.

Specimen collection.

The rats were anesthetized with Halothane; blood samples were collected directly from the heart with a heparinized syringe after a laparatomy was performed, and aliquots were analyzed for Hb and Hct. The remaining blood was refrigerated and plasma separated by centrifugation at 4°C and 1000 x g for 15 min. Plasma was frozen at -20°C before analysis for Fe and total iron-binding capacity (TIBC).

The rats were perfused with ice-cold 0.1 mol/L PBS solution, pH 7.3, through the left ventricle, with drainage through the right atrium, until the effluent was clear. The brain, liver and spleen were removed, frozen on dry ice and kept at -70°C for further analyses. Some brains from each group and sex were randomly selected and fixed by immersion in 40 g/L neutral buffered paraformaldehyde overnight, then cryoprotected by floating in sucrose (0.29, 0.58 and 0.87 mol/L) sequentially, and refrigerated at 4°C in 0.87 mol/L sucrose for histochemical determinations.

The brains perfused with PBS and frozen were dissected into the following regions: cerebral cortex (CX), deep cerebellum nuclei (DCB), superficial cerebellum (SC), hippocampus (HC), pons (PS), striatum (ST), substantia nigra (SN) and thalamus (TH) according to published methods (Focht et al. 1997 ). The regions were dissected on a cold aluminum block and frozen at -70°C. These samples were later diluted with 0.32 mol/L sucrose (S 7903, Sigma, St. Louis, MO) 1:4 (wt/v) and homogenized with the use of a sonicator with a microtip (Branson Sonifier 250, Branson, Danbury, CT) on pulse mode. Aliquots for the different analyses were separated and frozen at -70°C.

	Measurements

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Measurements RESULTS DISCUSSION REFERENCES

 
Hematology and liver nonheme iron.

Hemoglobin was measured colorimetrically by the cyanmethemoglobin method (procedure # 525, Sigma, St. Louis, MO). Hematocrit was determined by centrifugation of blood collected into heparinized microcapillary tubes.

Plasma iron and TIBC were determined by the colorimetric method described by Cook (1980a) modified to use 50 µL of sample. Transferrin saturation was calculated by expressing the ratio plasma Fe/TIBC as a percentage. Liver nonheme iron was determined by the standard colorimetric technique described by Cook (1980b) with ferrozine as the color reagent.

Brain total iron.

This was measured according to the method described by Erikson et al. (1997) .

Total protein.

The brain tissue homogenates were diluted 1:50 with 10 mmol/L PBS, pH 7.4, and their protein concentration was determined by the micro-Lowry method (P5656, Sigma, St. Louis, MO) modified for the use of 96-well plates and 30 µL of sample.

Ferritin.

Ferritin concentration in the different brain regions was measured by immunoblot according to the immunosorbent technique previously described (Roskams and Connor 1994 ). The total protein concentration loaded into the slots was 5 µg/100 µL. The primary antibody was used at a 1:2000 dilution, and the secondary antibody was diluted 1:5000. The primary antibody was an mouse anti-rat liver ferritin monoclonal antibody (a generous gift of James Cook, University of Kansas Medical Center, Kansas City, KS); the secondary antibody was sheep anti-mouse immunoglobulin G (IgG) alkaline phosphatase conjugated (A5324, Sigma). The ferritin standard used was rat liver ferritin (F-7005, Sigma). An internal standard composed of a pool of brain homogenate was analyzed on each membrane to ensure quality control between assays. All measurements were performed using a Eagle-Eye Densitometer (Stratagene, San Diego, CA). Regression analyses were performed on the acquired densities of the standard curves on each membrane, and the concentration of samples was determined.

Transferrin.

Transferrin concentrations in the different brain regions were measured by ELISA, using a method recently developed in our laboratory. The procedure was as follows: on d 1 of the assay, 96-well plates (flat bottom, Nunc MaxiSorp) were coated with the standards [1 ng to 31.25 pg of rat transferrin (A-3937, Sigma)], and the samples (5 µg of total protein for CX, DCB, SC, ST and TH, and 3 µg for HC, PS and SN) in triplicate, sealed and incubated at 4°C overnight. On d 2, the plates were washed three times with Tris-buffered saline solution (TBS)-Tween buffer (200 µL/well); 100 µL of 10 g/L bovine serum albumin (BSA) in 0.15 mol/L TBS was added to each well, and then the plates were incubated for 1 h at room temperature. After the incubation, the wells were aspirated and 100 µL rabbit anti-rat transferrin polyclonal antibody (a generous gift of Richard Fine, Boston University, Boston, MA) diluted 1:2000 in 10 g/L BSA-TBS was added. The plates were sealed and incubated overnight at 4°C. On d 3, the plates were washed three times with TBS-Tween buffer (200 µL/well), and 100 µL of secondary antibody (goat anti-rabbit IgG alkaline phosphatase conjugated antibody diluted 1:4000 in 10 g/L BSA) was added to each cell. The plates were then sealed and incubated at room temperature for 1 h. After the incubation, the plates were washed again three times with TBS-Tween buffer, and 100 µL substrate buffer [p-nitrophenyl phosphate disodium (N-2765, Sigma) dissolved in 1 mol/L diethanolamine buffer, 0.5 mmol/L MgCl₂, pH 9.8] was added to each well. The plates were sealed and incubated at room temperature until color developed (~40 min). The reaction was then stopped by adding 100 µL of 2.5 mol/L NaOH per well, and the plate read in a plate reader (Model EL340, Bio-Tek Instruments, Winooski, VT) with dual wavelength at 405/570 nm. A blank of reagents and an internal standard composed of a mixture of brain homogenates were analyzed on each plate. Regression analyses were performed for the standard curve on each plate and Tf concentration calculated for the samples accordingly. The coefficient of determination (r²) for the standard curves was always >0.97. An interassay CV of 4.7% was estimated by using the internal standard values from each plate.

Transferrin receptor.

The concentration of TfR in the different brain regions was measured by means of the same ELISA procedure described for transferrin, with the following differences: the primary antibody was mouse anti-rat transferrin receptor (MCA-155, Serotec, Oxford, England) diluted 1:2000. The secondary antibody was sheep anti-mouse IgG alkaline phosphatase conjugate (A-5324, Sigma) diluted 1:4000. Sample (5 µg) was added per well in triplicate for each one of the regions. The substrate reaction was extended to 90 min for optimal color development. It was not possible to construct standard curves for this ELISA because there is no commercial rat TfR standard available. Instead, CN, ID and SU brain homogenate mixtures were used on each plate as internal standard to correct for plate-to-plate variability. The interassay CV for the internal standard was 7%. For the purpose of comparisons among groups, the results were expressed as absorbance per microgram of protein.

Statistical analyses.

Data were analyzed using the SAS System for Windows v6.12 statistical analysis package (SAS Institute, Cary, NC). Factorial ANOVA with two between-group factors was used to evaluate the interactions between sex and groups. One-way ANOVA with one between-group factor followed by Dunnett’s post-hoc test was used to evaluate whether the experimental treatments were different from their adequate controls for the different variables. The -level for the analyses was set at P < 0.05, and the data were tested for normality. Data are reported as means ± SEM All means presented are for males and females combined because sex and group x sex interactions were not significantly different in any analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Measurements RESULTS DISCUSSION REFERENCES

 
Effects of iron deficiency on brain iron metabolism

    Early iron deficiency. Rat pups were made iron deficient by cross-fostering them to an iron-deficient dam at PND 10. Some of these pups were killed at PND 21 (ID group). Brain iron concentration was ~20-25% lower in ID rats compared with age-matched CN rats (P < 0.001) in most, but not all brain regions (Table 1 ). The clear exception was the thalamus, which was virtually unaffected by low iron intake for 11 d. Transferrin receptor (Table 1) was up-regulated 20-25% (a level comparable to the drop in iron concentration) in most but not all brain regions in response to iron deficiency; HC was not responsive. Striatum and superficial cerebellum had a TfR concentration 50% greater than that of controls, but this did not prevent the 25% lower iron concentration. Immunohistochemical evaluations of brains from these rats revealed that much of this change was in endothelial cells, and some in microgial cells (photomicrographs not shown).

    Late iron deficiency. Some rats fed the control diet between PND 10 and 21, were then fed an iron-deficient diet for 14 d after weaning to create a state of postweaning or late iron deficiency (CNID). Brain iron was significantly lower for all brain regions except HC. Compared with the CNCN group, the CNID group lost 36 and 33% of iron for DCB and SC, respectively, and ~20% for all other regions except HC (8%) (Table 2 ). Transferrin receptor concentrations were significantly higher for all regions except DCB compared with the control group (Table 2) . The two regions with the largest drop in iron concentration, DCB and SC, are also the two with the smallest increase in TfR concentration. Transferrin concentrations increased significantly for all regions (Table 2) , up to 92, 90 and 72% for CX, SC, and DCB, respectively, and to 35% for HC, SN and TH. The L-ferritin was not as responsive to the dietary iron deficiency in this period (Table 2) . Although the overall brain ferritin concentration was lower in the CNID rats, the only significant regional change was in ST, the region with the lowest concentration of ferritin in the CNCN group.

Recovery from iron deficiency

Some rats that were iron deficient from PND 10 to 21 were repleted with iron after PND 21 by weaning them onto either the control diet (IDCN) or the supplemented diet (IDSU). Brain iron concentration increased significantly in all brain regions of ID rats within 14 d of repletion with either the CN or SU diet (Table 4 ). In comparing the iron concentration in each region with that of rats who had not been iron deficient (CNCN), we observed normalized iron concentrations in CX, HC and SC with control diet repletion, and further normalization in PS, ST and TH with supplemented diet repletion. Only DCB and SN continued to have lower iron concentrations than those of CNCN at the end of this brief repletion period.

Brain L-ferritin concentrations were affected only modestly in most brain regions during the early ID period; thus, it is not surprising that iron therapy resulted in the normalization of concentration with the control repletion (Table 4) . Interestingly, ST was quite unresponsive to CN or SU repletion.

Effects of iron supplementation on brain iron metabolism

    Early iron supplementation. Iron supplementation during the late period of lactation produced a larger iron increment in DCB and HC (45 and 38% higher than CN, respectively) than any other brain region analyzed (Table 1) . Striatum, in contrast to other brain regions, did not show any change associated with iron loading during this period. Iron loading led to a highly variable response in the TfR concentration during this period of early iron deficiency, with no down-regulation in ST, and yet a large response in SN (>50% lower than CN) (Table 1) .

The concentration of transferrin decreased only mildly in response to iron supplementation. In PS, HC, SC and SN, Tf concentrations were < 5% lower compared with CN. Striatum was also the most responsive region to iron loading (25% lower Tf than CN) (Table 1) .

Iron and ferritin did not increase in a consistent fashion with iron supplementation. The region with the largest increase in iron concentration (DCB), for example, did not have the largest increase in ferritin (SC).

    Late iron supplementation. Iron supplementation during the postweaning period (CNSU) produced a smaller increment in iron concentration than during the previous period (SU) (Table 2) for every region analyzed. Striatum, SN and SC showed only a small change associated with iron loading during this period. The down-regulation of the transferrin receptors was not very substantial in most regions (Table 2) , with the exception of SC, which showed a marked increase in the response compared with the change that occurred during the early supplementation period.

Transferrin concentrations were between 10 and 20% lower than controls in most regions except ST (40%) during this late iron-loading period (Table 2) . Iron supplementation induced higher ferritin concentrations in practically all regions except HC and TH (Table 2) .

    Long-term iron supplementation. A number of rats from the supplemented group (SU) also were fed an iron-supplemented diet after PND 21 up to PND 35. These rats, therefore, were provided high dietary iron from PND 12 to 35 (SUSU). The iron concentration of the different regions of the brain responded differently to this cumulative iron loading. Cortex, HC and SC accumulated between 20 and 30% more iron than their counterpart CNCN, but DCB and SN had less than a 10% increase (Table 3) .

The transferrin receptor was down-regulated to a similar degree in most brain regions as it was during the early iron supplementation (Table 3) . The exceptions, HC and SN, demonstrated only half of the ability to decrease that they had earlier. The amount of TfR in striatum was not responsive to supplementation during any period. However, ST had the largest decrease in Tf of all regions, almost a 50% change, whereas SN did not show any change

Superficial cerebellum and TH had a large increase in ferritin concentration associated with long-term supplementation with iron (>40%). The other regions accumulated ~20% more ferritin than the control, except CX, which had only a 10% increase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Measurements RESULTS DISCUSSION REFERENCES

 
These experiments yielded several new findings regarding iron metabolism in the brains of rats from PND 10 to 35. First, iron deficiency and supplementation during the late lactational period, PND 10-21, alter brain iron, TfR, Tf and ferritin concentration in many regions, and these effects are not identical to those observed when the nutritional injury occurs in the postweaning period (PND 21-35). Second, 2 wk of iron repletion is adequate for correcting the overall concentration of the brain after early iron deficiency and for normalizing the iron concentration of individual brain regions. Adaptive processes (TfR and Tf levels) are still ongoing in some regions at this time, however, and further repletion is necessary to normalize in full all of the iron regulatory proteins. Larger amounts of iron provided by the iron-supplemented diet (420 mg/kg) did fully replete iron and its regulatory proteins in all regions. Third, long-term iron deficiency resulted in the continued decrease in brain iron concentration in some brain regions but not others, suggesting that the manifestations of nutritional iron deficiency are tightly associated with the brain developmental patterns. Conversely, long-term iron supplementation resulted in a differential increase in regional iron in the brain. Last, iron and the iron regulatory proteins, Tf, TfR and ferritin, are distributed heterogeneously in the young rat brain (PND 21) and in the older rat brain (PND 35) and respond to iron repletion in a heterogeneous fashion. In other words, brain regions have the apparent capacity to regulate their iron concentration in response to local needs when faced with an alteration in systemic iron delivery. The substantial TfR response suggests that this is a primary mechanism for local regulation.

The reduction of brain iron in specific brain regions, with lactational iron deficiency, extends the previous observations that brain iron concentration can be reduced rapidly with postweaning iron deficiency (Chen et al. 1995b , Erikson et al. 1997 and 1998 ). The quantitative decline in brain iron concentration over the 10 d of consuming low iron milk was not equal in all brain regions, nor was this sensitivity the same as that observed in studies of later iron deficiency. Cortex and HC lost less iron in the late ID than in the early ID, whereas DCB, SC and TH were more affected during the late ID; the rest of the regions did not differ substantially between the two periods. However, the most dramatic change was in TH, almost resilient to the deficiency during the early anemia, and having 20% less iron with either late or long-term anemia. These data show that the sensitivity to iron deficiency is developmentally linked. It appears that adaptive responses to protect against iron depletion were more successful in some regions. Perhaps regional needs for iron were lower in the regions with smaller change, preventing a large decline in iron concentration.

The repletion studies demonstrated that the brain is fully capable of regaining iron in a rapid fashion, despite the early reports that brain turnover (or more exactly, brain iron loss from the body) is a very slow process (Dallman et al. 1975 , Dallman and Spirito 1977 ). This capacity to replete iron in all brain regions is not supported by the accepted notion in the scientific literature that iron deficiency in the preweaning period leads to irreversible alterations in brain size and iron concentration (Dallman and Spirito 1977 , Felt and Lozoff 1996 ). Previous studies from our research group demonstrated rapid reversibility of regional brain iron concentration after postweaning iron deficiency (Erikson et al. 1997 ). Others have shown increases in iron uptake into the brain in iron deficiency and that the increased transcytosis of iron and Tf that occurs across the blood brain barrier (BBB) (Crowe and Morgan 1992 ) can account in large part for this rapid recovery of brain iron concentration. The brain obtains iron via regulation of the Tf receptor on the surface of endothelial cells on the brain microvasculature (Kalaria et al. 1992 , Morris et al. 1992 ). The data in this study and unpublished histochemical analysis concur that endothelial cells are the most identifiable cell type that responds to iron deficiency via up-regulation of the TfR. There is a large regional variation in the quantitative ELISA data, which suggests that regional regulation of movement across the BBB does in fact occur (Hill et al. 1985 ). The increase in uptake of iron observed in iron deficiency is not reflective of overall changes in BBB permeability, and is highly selective to iron and Tf. Although actual kinetic data were not collected in this experiment, it is reasonable to assume that up-regulation of transferrin receptors is responsible in part for this alteration in uptake.

The regional specificity for brain iron acquisition is of great interest because the distribution of iron in the developing brain is not the same as that in the adult brain (Connor 1994 , Focht et al. 1997 , Hill 1988 , Roskams and Connor 1994 ). In a recent quantitative study using 10-wk-old Fisher 344 rats, Focht et al. (1997) reported nearly equivalent concentrations of iron in the striatum, hippocampus, thalamus and frontal cortex, followed by brain stem, medial cortex, pons and cerebellum. Although the amount of iron was about three times the amount measured here, there is a rough similarity in iron distribution between the two studies. Systematic evaluations of strain differences in brain iron distribution in rats have not been conducted, but rat strain as well as mouse strain differences in the response to dietary iron have been reported (Morse et al. 1999 , Rao and Jagadeesan 1995 ).The substantia nigra, an iron-rich region in the adult rat brain as assessed by histochemical methods (Hill 1988 ), had the highest iron concentration in these young rat brains at PND 21 and 35. This region had TfR values very close to the control and a large increase in Tf concentration during iron repletion, suggesting that iron was somehow ‘targeted’ to go to the substantia nigra. In studies of uptake and distribution of iron in the brains of young rats, it has been suggested that mechanisms may exist for the translocation of iron from one area of the brain to another (Dwork et al. 1990 ). It is interesting to recall that iron accumulates in the substantia nigra, globus pallidus and caudate of elderly humans, in particular those with Alzheimer’s disease and Parkinson’s disease (Good et al. 1992 , Loeffler et al. 1995 ). The transferrin receptor data, although only relative because we could not calculate the actual amount of the protein, are valuable in the sense that it is probably the first time that they have been measured under these very well-controlled experimental conditions. These data show that some regions such as striatum are very responsive to iron deficiency early in development, up-regulating the synthesis of TfR, but that this is not the case if the deficiency occurs after weaning. Other regions such as HC and CX responded better to the deficiency during the late, rather than the early period of iron deficiency.

In a previous study, we found very little association between iron and ferritin concentrations in the different brain regions when an antibody not specific to a ferritin isomer was used (Erikson et al. 1997 ). In a correlation analysis using all rats in this study, ferritin concentrations paralleled those of iron in all brain regions except TH and ST. This supports the assumption that L-ferritin, even in developing brain, is designed to store nonessential amounts of iron in cells. H-Ferritin analyses are ongoing and may provide further insight into the regulation of iron movement.

Iron is important for normal dopamine functioning in regions connected to the substantia nigra (Chen et al. 1995a , Youdim et al. 1989 ). The substantia nigra is also rich in both iron and dopaminergic neurons (Hill 1988 ). The nigro-striatal dopaminergic pathway in the striatum has been found to be sensitive to iron deficiency with both decreases in dopamine D₂ receptors and increases in extracellular dopamine observed (Ashkenazi et al. 1982 , Chen et al. 1995a , Youdim et al. 1989 ). Recent studies from our laboratory demonstrated that extracellular dopamine concentrations in the striatum return to normal within the same time frame for restoration of ventral midbrain iron concentration (Nelson et al. 1997 ). In addition, radioligand binding studies have demonstrated alterations in dopamine transporters in striatal pathways in iron deficiency (Morse et al. 1999 ).

Although the traditional view has been that the brain is resistant to excess dietary iron, this experiment is the first to evaluate the effects of iron excess on brain iron. Our data show that dietary iron excess can increase brain iron concentration, and that this increase is developmentally linked and not uniform for the different brain regions. This is very important because many studies of the effects of iron deficiency on brain iron have used control diets comparable to our supplemented diet. This could lead to artificially large differences between the two groups because dietary iron excess does increase brain iron.

One of the strengths of our experimental design is that it addresses the developmental differences between humans and rats, and matches the initiation of the nutritional injury to a developmental stage that is similar in the two species. The greatest prevalence of iron deficiency occurs in infants after 6 mo of life, with an increased risk after the first 4 mo, especially if the infant was premature or bottle-fed and did not receive iron supplements during this period (Allen 1997 , de Andraca et al. 1997 ). The experimental design used in this study closely matches the rat to the same neurodevelopmental stage an infant would be by the time the risk for iron-deficient anemia is greater, i.e., PND 10 in rats and 4-6 mo of life in the infant, thereby defining a better animal model for the study of the pernicious effects of iron deficiency early in life.

In conclusion, the results of this study suggest that the effects of nutritional iron deficiency and excess on brain iron metabolism are tightly associated with the brain development patterns. Although the different brain regions seem to be capable of regulating their iron concentration in response to local needs, when faced with an alteration in systemic iron delivery, the substantial TfR response suggests that this is a primary mechanism for local regulation, and this response was found to be developmentally dependent.

	FOOTNOTES

 
² Abbreviations used: BBB, blood brain barrier; BSA, bovine serum albumin; CN, control; CX, cortex; DA, dopamine; DCB, deep cerebellum nuclei; Hb, hemoglobin; HC, hippocampus; Hct, hematocrit; ID, iron deficiency; IgG, immunoglobulin G; PND, postnatal day; PS, pons; SC, superficial cerebellum; SN, substantia nigra; ST, striatum; SU, iron supplemented; TBS, Tris-buffered saline solution; Tf, transferrin; TfR, transferrin receptor; TH, thalamus; TIBC, total iron-binding capacity.

Manuscript received April 12, 1999. Initial review completed July 19, 1999. Revision accepted October 11, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Measurements RESULTS DISCUSSION REFERENCES

 

1. Allen N. H. Pregnancy and iron deficiency: unresolved issues. Nutr. Rev. 1997;55:91-101[Medline]

2. Ashkenazi R., Ben-Shachar D., Youdim M.B.H. Nutritional iron and dopamine binding sites in the rat brain. Pharmacol. Biochem. Behav. 1982;17(suppl.1):43-47

3. Baynes R. D., Bothwell T. H. Iron deficiency. Annu. Rev. Nutr. 1990;10:133-148[Medline]

4. Beard J. L., Chen Q., Connor J., Jones B. C. Altered monoamine metabolism in caudate-putamen of iron-deficient rats. Pharmacol. Biochem. Behav. 1994;48:621-624[Medline]

5. Beard J. L., Connor J. R., Jones B. C. Brain iron: location and function. Prog. Food Nutr. Sci. 1993a;17:183-221[Medline]

6. Beard J. L., Connor J. R., Jones B. C. Iron in the brain. Nutr. Rev. 1993b;51:157-170[Medline]

7. Chen Q., Beard J. L., Jones B. C. Abnormal brain monoamine metabolism in iron deficiency anemia. J. Nutr. Biochem. 1995a;6:486-493

8. Chen Q., Connor J. R., Beard J. L. Brain iron, transferrin and ferritin concentrations are altered in developing iron-deficient rats. J. Nutr. 1995b;125:1529-1535

9. Connor J. R. Iron acquisition and expression of iron regulatory proteins in the developing brain: manipulation by ethanol exposure, iron deprivation and cellular dysfunction. Dev. Neurosci. 1994;16:233-247[Medline]

10. Connor J. R., Menzies S. L., St. Martin S. M., Mufson E. J. A histochemical study of iron, transferrin and ferritin in Alzheimer’s diseased brains. J. Neurosci. Res. 1992a;31:75-83[Medline]

11. Connor J. R., Snyder B. S., Arosio P., Loeffler D. A., LeWitt P. A quantitative analysis of isoferritins in select regions of aged, Parkinsonian, and Alzeimer’s diseased brains. J. Neurochem. 1995;65:717-724[Medline]

12. Connor J. R., Snyder B. S., Beard J. L., Fine R. E., Mufson E. J. Regional distribution of iron and iron-regulatory proteins in the brain in aging and Alzheimer’s disease. J. Neurosci. Res. 1992b;31:327-335[Medline]

13. Cook J.D. Iron 1980a:105-109 Churchill Livingstone New York, NY.

14. Cook J.D. Iron 1980b:32-36 Churchill Livingstone New York, NY.

15. Crowe A., Morgan E. H. Iron and transferrin uptake by brain and cerebrospinal fluid in the rat. Brain Res 1992;592:8-16[Medline]

16. Dallman P. R., Siimes M. A., Manies E. C. Brain iron: persistent deficiency following short term iron deprivation in the young rat. Br. J. Haematol. 1975;31:209-215[Medline]

17. Dallman P. R., Spirito R. A. Brain iron in the rat: extremely slow turnover in normal rats may explain the long lasting effects of early iron deficiency. J. Nutr. 1977;107:1075-1081

18. Davison A. N., Dobbing J. Myelination as a vulnerable period in brain development. Br. Med. Bull. 1966;22:40-44[Free Full Text]

19. de Andraca I., Castillo M., Walter T. Psychomotor development and behavior in iron-deficient anemic infants. Nutr. Rev. 1997;55:125-132[Medline]

20. Dobbing J., Sands J. Comparative aspects of the brain growth spurt. Early Hum. Dev. 1979;3(1):79-83[Medline]

21. Dwork A. J., Lawler G., Zybert P. A., Durkin M., Osman M., Willson N., Barkai A. I. An autoradiographic study of the uptake and distribution of iron by the brain of the young rat. Brain Res 1990;518:31-39[Medline]

22. Erikson K. M., Beard J. L., Connor J. R. Distribution of brain iron, ferritin, and transferrin in the 28-day-old piglet. Nutr. Biochem. 1998;9:276-284

23. Erikson K. M., Piñero D. J., Connor J. R., Beard J. L. Regional brain, iron, ferritin, and transferrin concentrations during iron deficiency and iron repletion in developing rats. J. Nutr. 1997;127:2030-2038[Abstract/Free Full Text]

24. Felt B., Lozoff B. Brain iron and behavior in rats are not normalized by treatment of iron deficiency anemia during early development. J. Nutr. 1996;126:693-701

25. Focht S., Snyder B. S., Beard J. L., van Gelder W., Williams L. R., Connor J. R. Regional distribution of iron, transferrin, ferritin, and oxidatively-modified proteins in young and aged Fischer 344 rat brains. Neuroscience 1997;79:255-261[Medline]

26. Good P. F., Olanow C. W., Perl D. P. Neuromelanin-containing neurons of the substantia nigra accumulate iron and aluminun in Parkinson’s disease: a LAMMA study. Brain Res 1992;593:343-346[Medline]

27. Hill J. M. The distribution of iron in the brain. Youdim M.B.H. eds. Brain Iron: Neurochemistry and Beahvioural Aspects 1988:1-4 Taylor and Francis London, UK.

28. Hill J. M., Ruff M. R., Weber R. J., Pert C. B. Transferrin receptors in rat brain: neuropeptide-like pattern and relationship to iron distribution. Proc. Natl. Acad. Sci. U.S.A. 1985;82:4453-4557[Abstract/Free Full Text]

29. Kalaria R. N., Sromek S. M., Grahovac I., Harik S. I. Transferrin receptors of rat and human brain and cerebral microvessels and their status in Alzheimer’s disease. Brain Res 1992;585:87-93[Medline]

30. Loeffler D. A., Connor J. R., Juneau P. L., Snyder B. S., Kanaley L., DeMaggio A. J., Nguyen H., Brickman C. M., LeWitt P. A. Transferrin and iron in normal, Alzheimer’s disease, and Parkinson’s disease brain regions. J. Neurochem. 1995;65:710-724[Medline]

31. Morris C. M., Keith A. B., Edwardson J. A., Pullen R.G.L. Uptake and distribution of iron and transferrin in the adult rat brain. J. Neurochem. 1992;59:300-306[Medline]

32. Morse A. C., Beard J., L & Jones B. C. A genetic developmental model of iron deficiency: biological aspects. Proc. Soc. Exp. Biol. Med. 1999;220:147-152[Medline]

33. Nelson C., Erikson K. M., Piñero D. J., Beard J. L. In vivo dopamine is altered in iron-deficient anemic rats. J. Nutr. 1997;127:2282-2288[Abstract/Free Full Text]

34. Rao J., Jagadeesan V. Development of a rat model for iron deficiency and toxicological studies: a comparison among Fischer 344, Wistar, and Sprague-Dawley strains. Lab. Anim. Sci. 1995;45:393-397[Medline]

35. Reeves P. G., Nielsen F. H., Fahey G. C., Jr AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 1993;123:1939-1951

36. Roskams A. J., Connor J. R. Iron, transferrin, and ferritin in the rat brain during development and aging. J. Neurochem. 1994;63:709-716[Medline]

37. Youdim M.B.H., Ben-Shachar D., Yehuda S. Putative biological mechanisms of the effect of iron deficiency on brain biochemistry and behavior. Am. J. Clin. Nutr. 1989;50:607-617

This article has been cited by other articles:

				K. J. Collard Iron Homeostasis in the Neonate Pediatrics, April 1, 2009; 123(4): 1208 - 1216. [Abstract] [Full Text] [PDF]

				E. L. Unger, C. J. Earley, and J. L. Beard Diurnal cycle influences peripheral and brain iron levels in mice J Appl Physiol, January 1, 2009; 106(1): 187 - 193. [Abstract] [Full Text] [PDF]

				E. L. Unger, J. A. Wiesinger, L. Hao, and J. L. Beard Dopamine D2 Receptor Expression Is Altered by Changes in Cellular Iron Levels in PC12 Cells and Rat Brain Tissue J. Nutr., December 1, 2008; 138(12): 2487 - 2494. [Abstract] [Full Text] [PDF]

				J. L. Beard, E. L. Unger, L. E. Bianco, T. Paul, S. E. Rundle, and B. C. Jones Early Postnatal Iron Repletion Overcomes Lasting Effects of Gestational Iron Deficiency in Rats J. Nutr., May 1, 2007; 137(5): 1176 - 1182. [Abstract] [Full Text] [PDF]

				J. C McCann and B. N Ames An overview of evidence for a causal relation between iron deficiency during development and deficits in cognitive or behavioral function Am. J. Clinical Nutrition, April 1, 2007; 85(4): 931 - 945. [Abstract] [Full Text] [PDF]

				J. G. Anderson, P. T. Cooney, and K. M. Erikson Brain Manganese Accumulation is Inversely Related to {gamma}-Amino Butyric Acid Uptake in Male and Female Rats Toxicol. Sci., January 1, 2007; 95(1): 188 - 195. [Abstract] [Full Text] [PDF]

				S. J. Garcia, K. Gellein, T. Syversen, and M. Aschner Iron Deficient and Manganese Supplemented Diets Alter Metals and Transporters in the Developing Rat Brain Toxicol. Sci., January 1, 2007; 95(1): 205 - 214. [Abstract] [Full Text] [PDF]

				M. S Golub, C. E Hogrefe, A. F Tarantal, S. L Germann, J. L Beard, M. K Georgieff, A. Calatroni, and B. Lozoff Diet-induced iron deficiency anemia and pregnancy outcome in rhesus monkeys Am. J. Clinical Nutrition, March 1, 2006; 83(3): 647 - 656. [Abstract] [Full Text] [PDF]

				S. M. Patton, D. J. Pinero, N. Surguladze, J. Beard, and J. R. Connor Subcellular localization of iron regulatory proteins to Golgi and ER membranes J. Cell Sci., October 1, 2005; 118(19): 4365 - 4373. [Abstract] [Full Text] [PDF]

				K. Kordas and R. J. Stoltzfus New Evidence of Iron and Zinc Interplay at the Enterocyte and Neural Tissues J. Nutr., June 1, 2004; 134(6): 1295 - 1298. [Abstract] [Full Text] [PDF]

				R. J. Stoltzfus, H. M. Chway, A. Montresor, J. M. Tielsch, J. K. Jape, M. Albonico, and L. Savioli Low Dose Daily Iron Supplementation Improves Iron Status and Appetite but Not Anemia, whereas Quarterly Anthelminthic Treatment Improves Growth, Appetite and Anemia in Zanzibari Preschool Children J. Nutr., February 1, 2004; 134(2): 348 - 356. [Abstract] [Full Text] [PDF]

				J.R. Connor, P.J. Boyer, S.L. Menzies, B. Dellinger, R.P. Allen, W.G. Ondo, and C.J. Earley Neuropathological examination suggests impaired brain iron acquisition in restless legs syndrome Neurology, August 12, 2003; 61(3): 304 - 309. [Abstract] [Full Text] [PDF]

				S. M. Grant, J. A. Wiesinger, J. L. Beard, and M. T. Cantorna Iron-Deficient Mice Fail to Develop Autoimmune Encephalomyelitis J. Nutr., August 1, 2003; 133(8): 2635 - 2638. [Abstract] [Full Text] [PDF]

				J. Beard Iron Deficiency Alters Brain Development and Functioning J. Nutr., May 1, 2003; 133(5): 1468S - 1472. [Abstract] [Full Text] [PDF]

				J. Beard, K. M. Erikson, and B. C. Jones Neonatal Iron Deficiency Results in Irreversible Changes in Dopamine Function in Rats J. Nutr., April 1, 2003; 133(4): 1174 - 1179. [Abstract] [Full Text] [PDF]

				J. Han, J. R. Day, J. R. Connor, and J. L. Beard H and L Ferritin Subunit mRNA Expression Differs in Brains of Control and Iron-Deficient Rats J. Nutr., September 1, 2002; 132(9): 2769 - 2774. [Abstract] [Full Text] [PDF]

				L. E. Murray-Kolb, F. Takaiwa, F. Goto, T. Yoshihara, E. C. Theil, and J. L. Beard Transgenic Rice Is a Source of Iron for Iron-Depleted Rats J. Nutr., May 1, 2002; 132(5): 957 - 960. [Abstract] [Full Text] [PDF]

				D. J. Pinero, N. Li, J. Hu, J. L. Beard, and J. R. Connor The Intracellular Location of Iron Regulatory Proteins Is Altered as a Function of Iron Status in Cell Cultures and Rat Brain J. Nutr., November 1, 2001; 131(11): 2831 - 2836. [Abstract] [Full Text] [PDF]

				C. Kwik-Uribe Response to letter of Dr. Elise Malecki J. Nutr., May 1, 2001; 131(5): 1585 - 1585. [Full Text]

				D. J. Piñero, B. C. Jones, and J. L. Beard Variations in Dietary Iron Alter Behavior in Developing Rats J. Nutr., February 1, 2001; 131(2): 311 - 318. [Abstract] [Full Text]

				J. L. Beard Iron Biology in Immune Function, Muscle Metabolism and Neuronal Functioning J. Nutr., February 1, 2001; 131(2): 568S - 580. [Abstract] [Full Text]

				D. J. Pinero and J. R. Connor Iron in the Brain: An Important Contributor in Normal and Diseased States Neuroscientist, December 1, 2000; 6(6): 435 - 453. [Abstract] [PDF]

				C. L. Kwik-Uribe, D. Gietzen, J. B. German, M. S. Golub, and C. L. Keen Chronic Marginal Iron Intakes during Early Development in Mice Result in Persistent Changes in Dopamine Metabolism and Myelin Composition J. Nutr., November 1, 2000; 130(11): 2821 - 2830. [Abstract] [Full Text] [PDF]

				K. M. Erikson, B. C. Jones, and J. L. Beard Iron Deficiency Alters Dopamine Transporter Functioning in Rat Striatum J. Nutr., November 1, 2000; 130(11): 2831 - 2837. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Piñero, D. J. Articles by Beard, J. L. Search for Related Content PubMed PubMed Citation Articles by Piñero, D. J. Articles by Beard, J. L.