Psychological stress induces dysregulation of iron metabolism in rat brain

Neuroscience 155 (2008) 24 –30

PSYCHOLOGICAL STRESS INDUCES DYSREGULATION OF IRON METABOLISM IN RAT BRAIN L. WANG, W. WANG, M. ZHAO, L. MA AND M. LI*

reasons that disrupt the normal distribution of brain iron. Previous studies have showed that sports and fatigue could change iron content in some organs of the body [Nikolova-Todorova and Troic, 2003; Smith et al., 2005]; evidences have indicated that psychological stress (PS) is a risk factor for neurodegeneration [Kaufer and Soreq, 1999; Miyashita et al., 2006]; reports also found that longterm exposure to stress caused necrosis of brain tissues and morphological damage to the neurons in hippocampus CA2 and CA3 regions [Friedman et al., 1996; Endo et al., 1999]. Whether these neurodegenerative diseases are associated with PS-induced disorder of brain iron metabolism still remains unclear. The aim of this study is to determine whether PS is a risk factor of disorder in cerebral iron metabolism. We determined the iron contents, the related iron-metabolism indices, and the oxidative indices in different cerebral regions of PS-exposed rats; and we conclude that PS exposure can cause increase in iron demand in some brain regions and subsequently leads to iron deposition there, suggesting that PS might be an important reason for iron deposition.

Department of Military Hygiene, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China

Abstract—Oxidative damage induced by abnormal iron accumulation in the brain is a primary cause of many neurodegenerative diseases, while the reason for iron deposition remains unclear. A previous study reported that various kinds of stress could cause a change in iron level and psychological stress (PS) was a risk factor for neuron death. In the present study we investigated the influence of PS on iron metabolism in rat brain. The results showed that both total iron and non-protein-bound-iron (NPBI) levels were higher in the cerebral cortex, hippocampus and striatum of PS rats. The levels of iron regulatory factors, including transferrin receptor 1 (TfR1), ferritin (Fn), and iron regulatory protein1 (IRP1), were all changed in the iron deposition regions of the PS-exposed rat brain, accompanied by intensified oxidative stress. It is concluded that PS can increase the intake of iron in some regions of brain and subsequently causes regional iron accumulation, indicating PS might be an important reason for iron deposition– caused neurodegenerative diseases. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: brain, iron metabolism, psychological stress, oxidative stress.

EXPERIMENTAL PROCEDURES Animals

Abnormal iron accumulation in the brain is reported to be associated with Parkinson’s disease, Alzheimer’s disease, Huntington’s chorea and neurodegeneration with brain iron accumulation (NBIA) [Zhou et al., 2001; Berg and Youdim,2006]. Previous studies suggested that increase in brain iron is an initial cause of neuronal death in neurodegenerative disorders [Rouault, 2001; Qian and Shen, 2001; Ke and Ming Qian, 2003]. The cause for disorder of iron metabolism in the brain remains elusive. Increased iron deposits may be attributable to local changes of the normal iron-regulatory systems [Qian and Shen, 2001; Ke and Ming Qian, 2003]. Excess of redox active iron produces oxidative damage and subsequently leads to tissue degeneration. Abnormal increase of iron only occurred in some specific regions of the brain of patients with neurodegenerative disorders [Lee et al., 2006], and we believe that there are some undetermined

Twenty-four male Sprague–Dawley rats, weighting 120⫾5 g (Shanghai-BK Co., Ltd. Shanghai, China), were housed individually in cages at a temperature of 24⫾1 °C, a humidity of 55⫾5% in a 12-h light/dark cycle, and were given normal chow and free access to water. After 7 days’ adaptation, the animals were evenly divided into an experiment group and a control group. All animal experimental procedures were approved by the Animal Care and Use Committee of the Second Military Medical University. The policies concerning the use and care of laboratory animals were followed constantly and conformed to local and international guidelines on the ethical use of animals. Efforts have been made to minimize the number and suffering of the animals used during our experiment procedures.

Establishment of PS model in rats PS model was created in rats as described previously [Noguchi et al., 2001]. Briefly, a communication box was divided into Room A and Room B with a transparent acrylic board. Room A included 10 little rooms with a plastic board– covered floor and Room B included 10 little rooms with a metal grid– exposed floor for electric conduction. Rats in Room B were randomly given electrical shock (0.6 mA for 1 s) for 30 min (60 times) through the floor and exhibited nociceptive stimulation-evoked responses, such as jumping up, defecation and crying; rats in Room A were only exposed to the responses of rats in Room B to establish PS model. PS was given to rats for 30 min every morning (10:00 – 10:30 h) for 7 days. At the end of the exposure, the rats were kept

*Corresponding author. Tel: ⫹86-21-25070352; fax: ⫹86-21-25070350. E-mail address: [email protected] (M. Li). Abbreviations: BPS, bathophenanthroline disulfonate; CORT, corticosterone; Fn, ferritin; IRE, iron response element; IRP1, iron regulatory protein 1; Lf, lactoferrin; L-NAME, NG-nitro-L-arginine methyl ester; MDA, malondialdehyde; NADPH-d, NADPH-diaphorase; NO, nitric oxide; NOS, nitric oxide synthase; NPBI, non-protein-bound-iron; PS, psychological stress; SOD, superoxide dismutase; TfR1, transferrin receptor 1.

0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.03.091

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L. Wang et al. / Neuroscience 155 (2008) 24 –30 in the cages for another 4 min before they were taken out. Animals in the control group were only kept in the cages for 4 min without receiving any stress.

Brain tissue sampling and preparation At the end of PS exposure all rats were deeply anesthetized by i.p. injection of 7% chloral hydrate, and then perfused through the left cardiac ventricle with ice-cold phosphate buffered saline (PBS; pH 7.4) to flush out the plasma. The whole brain, or the right frontal cortex, hippocampus, striatum and cerebellum were quickly removed and snap frozen in liquid nitrogen, and kept in a ⫺80 °C freezer till use. Only a part of the cortex was collected and analyzed in this study due to their size. Perfused brains were sectioned at 30 ␮m on a sliding microtome into free-floating tissue sections. Determination of total iron and non-protein-bound-iron (NPBI) concentrations. Iron concentrations were determined using a Varian SpectrAA-220G graphite furnace atomic absorption spectrometer equipped with a GTA 110 atomizer, programmable sample dispenser, and deuterium background correction. Rat brain samples were digested with concentrated nitric acid and incubated at 60 °C for 2 h. The digested samples were well mixed and further diluted. A blank sample was included for baseline check for every run. Accuracy was checked by an internally prepared solution. Standard addition method was used for calibration. Standards and control samples were prepared in an identical manner to the experimental samples. NPBI levels were analyzed by a method using bathophenanthroline disulfonate (BPS) to chelate ferrous iron, thus forming a complex that could be analyzed with spectrophotometry [Nilsson et al., 2002]. Dissected brain tissues were homogenized in a glass homogenizer in 10 vol of 50 mmol/L phosphate buffer, pH 7.4. The homogenates were centrifuged for 20 min at 3000 r.p.m. and the supernatants were collected. The sample BPS and ferrous ammonium sulfate [(NH4)2 Fe(SO4)2] of the highest purity commercially available (Sigma) were used. Perl’s iron staining. For Perl’s staining, sections were processed through a series of graded alcohols, into xylene, and rehydrated back to water. Sections were incubated in a 1:1 solution of 2% HCl and potassium ferrocyanide (Sigma) for 30 min and rinsed in water. Sections were counterstained with Neutral Red, dehydrated in increasing concentrations of ethanol, cleared in xylene, and mounted on slides. Determination of transferrin receptor 1 (TfR1) and ferritin (Fn) mRNA/protein levels. RNA extractions were carried out using TRIzol reagent following the manufacturers’ recommendations. All the RNA samples were treated with RNAse free DNAse I (IQ5 Real-Time PCR Detection System) according to manufacturer’s recommendations and stored at ⫺70 °C until use. The quality and quantity of RNA were assessed by using a spectrophotometer (Model 6300 Spectrophotometer, Jenway) before it was used. Subsequently, all the samples were diluted using nuclease free water at a concentration of 10 ng/␮l. Real time Q-RT-PCR was performed using IQ5 Real-Time PCR Detection System. Two step RT-PCR method was performed using Real Time PCR Master Mix (TOYOBO Biotech Co., Ltd.). Primers were as follows. Fn sense: 5=-TCTCCTCAAGTTGCAGAACGAA-3=, antisense: 5=-CAGGGTTTTACCCCACTCATCT-3=, probe: FAM-5=-CCAGGATGTGCAGAAGCCATCTCAA-3=-TAMRA; TfR1 sense: 5=-TTTCATAATGCTGAGAAAACAAACA-3=, antisense: 5=-GGAAAGGAGACTCTCTTGGAGATAC-3=, probe: FAM-5=TTCGTCATGAGGGAAATCAATGATCGT-3=-TAMRA. Primers used to analyze all the transcripts have been reported else where. The Q-RT-PCR data were analyzed by 2⫺⌬⌬CT method as described [Livak and Schmittgen, 2001]. The concentrations of TfR1 and Fn in the cortex, hippocampus and striatum samples were assessed using a commercially

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available ELISA kits (R&D Systems Inc., USA) with the absorbance read on a microplate reader (BioRad) at a wavelength of 450 nm. All experimental procedures for these assays were performed according to the manufacturer’s instructions. TfR and Fn concentrations of each region were normalized to wet tissue weight (mg) and expressed as ␮mol/mg and ng/mg, respectively. Western blotting analysis of iron regulatory protein 1 (IRP1) and lactoferrin (Lf) expression. Dissected tissues from the cortex, hippocampus and striatum were homogenized separately by a dounce homogenizer in lysis buffer, and the protein extracts were quantitated and normalized using the Bradford assay (Dojindo Laboratories). Proteins were incubated overnight at 4 °C with a primary antibody against IRP1 (monoclonal, 1:1000, Santa), Lf (rabbit polyclonal, 1:500, Santa), or ␤-actin (rabbit polyclonal, 1:10,000, Sigma). The blots were developed by incubation in ECL chemiluminescence reagent (Amersham Life Science, Arlington Heights, IL, USA) and subsequently exposed to BioMax Light Film (Eastman Kodak Co., USA). Measurement of superoxide dismutases (SOD) activity and malondialdehyde (MDA) concentrations. To evaluate the oxidative status in PS-exposed rat brain, we also measured the SOD activity and MDA concentration. Tissue samples were preserved in 50 ␮l of 5 mM butylated hydroxytoluene (BHT; Oxis Research) to prevent further lipid peroxidation. SOD activity was measured using WST-1 kit from Dojindo Laboratories with the absorbance read on a microplate reader at a wavelength of 450 nm. MDA concentration was assessed using a commercially available colorimetric MDA assay kit (Nanjing, Jiancheng) with the absorbance read on a microplate reader at a wavelength of 586 nm. All experimental procedures were performed according to the manufacturer’s instructions. SOD activity and MDA concentration of each region were normalized to wet tissue weight (mg) and expressed as ␮mol/mg and ␮g/mg, respectively. Measurement of the number and activity of nitric oxide synthase (NOS) positive neurons. Nitric oxide (NO) plays an important role in the metabolism of iron, so we also determined number and activity of NOS positive neurons. The brain tissues were cryoprotected with sucrose, frozen, cut into 15-␮m sections, and mounted on gelatin-coated glass slides. The sections were processed using the NADPH-diaphorase (NADPH-d) histochemical method [Vincent and Kimura, 1992]. Six rats were selected from each of the two groups and five sections were randomly selected from each rat. NOS positive neurons were counted in four fields of the cerebral cortex, hippocampal CA3 and caudate putamen. Data were analyzed by one-way ANOVA.

Cell preparation for intracellular NO and iron analysis The cerebral cortices were isolated from 1-day-old Sprague–Dawley rats and incubated with 0.25% trypsin in HBSS. The primary cultured cortical neurons were plated into in a poly L-lysine-coated six-well plates at a density of about 1⫻106 cells/ml. Cells were maintained in Neurobasal/B27 medium under a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Experiments were carried out with neurons after 10 days in culture. The cells were treated with corticosterone (CORT, 1 ␮mol/L, Sigma) or CORT/NG-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor) (CORT, 1 ␮mol/l; L-NAME 1.5 ␮mol/L, Sigma) for 24 h. Untreated neurons served as control. Counting Kit-8 (Dojindo, Japan) was used to count the cells in three groups. Briefly, the cell culture medium was discarded and Cell Counting Kit-8 (CCK-8, 100 ␮l) was added to each well. Plates were incubated at 37 °C for another 2 h, and the absorbance at 450 nm was measured with a microplate reader. NO measurement. NO production was assayed by measuring the nitrite concentrations with the Griess assays. Supernatants of (100 ␮l) were added to 100 ␮l of 1:1 mixture of 1% sulfanilamide

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Fig. 1. Levels of iron in homogenates of different brain parts in control and PS exposure rats (mean⫾S.D., n⫽12, * P⬍0.05).

dihydrochloride and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4. Plates were incubated at 25 °C for 10 min, and the absorbance at 550 nm was measured with a microplate reader. Nitrite concentrations were calculated with sodium nitrite standard curve as a reference. Analysis of intracellular iron. The experiments were carried out with a quadrupole ICP-MS X7 (Thermo Electron, Winsford, UK) equipped with collision cell technology (CCT). The Ar plasma—in which the cell sample is nebulized—is provided by a 1400 W radio frequency potential (RF). A blank sample was included for baseline check for every run. Standards and control samples were prepared in an identical manner to the samples.

Statistical analysis All results were expressed as mean⫾S.E. Statistical analysis was carried out by using SPSS 11.0. All values below the detection limits were set to zero and absolute values without correction for recovery rate were used in analyses. A P value less than 0.05 was considered statistically significant.

RESULTS PS exposure increased the concentrations of total iron and NPBI in some brain regions We found that the iron levels in the right frontal cortex, hippocampus and striatum were significantly higher in the PS exposure group than in the control group (P⬍0.05); however, no significant difference was observed in iron levels in the whole brain and the cerebellum between the two groups. Moreover, the iron level in the brain stem of

Fig. 2. Levels of NPBI in homogenates of different brain parts in control and PS exposure rats (mean⫾S.D., n⫽12, * P⬍0.05, ** P⬍0.01).

Fig. 3. Perl’s iron staining revealed weak staining in cortex of control group (A, A1; A1 is an amplification of A) and very strong staining in cortex of control group (B, B1; B1 is an amplification of B). Large scale bar⫽500 ␮m.

the PS exposure group was significantly lower than that of the control group (P⬍0.05, Fig. 1), We also found that the concentrations of NPBI in the right frontal cortex, hippocampus and striatum were significantly higher in the PS exposure group than in the control group (P⬍0.05, P⬍0.01 for hippocampus), and no significant difference was observed in NPBI concentration in the brain stem and cerebellum between the two groups (Fig. 2). Iron staining results in the cortex Perl’s iron staining revealed weak staining for iron in the cortex of control group (Fig. 3A, A1) and very strong staining in the cortex of PS rats (Fig. 3B, B1). PS exposure caused changes in TfR1 and Fn mRNA/protein Real time-PCR analysis showed that PS exposure increased TfR1 mRNA levels in the cortex, hippocampus

Fig. 4. RT-PCR analysis. Transcript levels of TfR1 mRNA using the 2⫺⌬⌬CT method in the cortex, hippocampus and striatum of the two groups (mean⫾S.D., n⫽3, * P⬍0.05, ** P⬍0.01).

L. Wang et al. / Neuroscience 155 (2008) 24 –30

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Fig. 5. RT-PCR analysis. Transcript levels of Fn mRNA using the 2⫺⌬⌬CT method in the cortex, hippocampus and striatum of the two groups (mean⫾S.D., n⫽3, * P⬍0.05, ** P⬍0.01).

and striatum (P⬍0.05 for hippocampus and striatum), though the increase in the cortex was not significant (Fig. 4); the Fn mRNA level of PS exposure group was significantly lower than that in the control group (P⬍0.01 for cortex, P⬍0.05 for striatum), though the decrease was not significant in the hippocampus (Fig. 5). After 7 days’ exposure to PS, TfR1 levels in the cortex, striatum and hippocampus were significantly higher than those in the control group (P⬍0.05, Fig. 6); Fn concentrations in the cortex and hippocampus were significantly lower in PS exposure group than in the control group (P⬍0.05); and the Fn concentration in the striatum was also lower than that of the control group, but the difference was not significant (Fig. 7). PS exposure increased IRP1 and Lf expression in different parts of rat brain Western blot analysis showed that PS exposure increased IRP1 immunoreactivity in the cortex, hippocampus and striatum in the rat brain (Fig. 8); and the expression of Lf was also increased in the hippocampus of the PS-exposed rats (Fig. 9).

Fig. 7. Levels of Fn in homogenates of the cortex, hippocampus and striatum of control and PS rats (mean⫾S.D., n⫽12, * P⬍0.05, ** P⬍0.01).

PS exposure intensified the oxidative reaction in rat brain SOD activities in the cortex and hippocampus in the PS group were significantly lower than those in the control group (P⬍0.01 for cortex, P⬍0.05 for hippocampus); SOD activity in the striatum was significantly higher in the PS exposure group than in the control group (P⬍0.01). No significant difference in SOD activities in the cerebellums and brain stem was observed between the two groups (Table 1). We also found that MDA levels were significantly increased in the cortex, hippocampus and striatum of the PS exposure group compared with the control group (P⬍0.01, Table 2). PS exposure increased NOS positive neurons in different brain regions NADPH-d histochemical method revealed that the number of NOS positive neurons in the cerebral cortex, hippocampal CA3 and caudate putamen were significantly higher in PS-exposed rats than in the control animals (P⬍0.05) (Table 3). Effects of CORT and NOS inhibitor on NO and iron concentrations in primarily cultured nerve cells The NO production of the CORT cultured nerve cells was increased significantly compared with the control group and L-NAME cultured cells (P⬍0.05); and the NO production of the NOS inhibitor cultured nerve cells was decreased significantly compared with the control group (P⬍0.05, Fig. 10).

Fig. 6. Levels of TfR1 in homogenates of the cortex, hippocampus and striatum of control and PS rats (mean⫾S.D., n⫽12, * P⬍0.05, ** P⬍0.01).

Fig. 8. Western blot analysis shows that PS increased IRP1 immunoreactivity in the cortex, hippocampus and striatum (C, cortex; H, hippocampus; S, striatum).

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Fig. 9. Western blot analysis shows that Lf expression is increased in the hippocampus of the PS-exposed rats (H, hippocampus).

Iron concentrations of the CORT cultured nerve cells were increased significantly compared with the control group and the NOS inhibitor group (P⬍0.05); and the iron concentrations of the NOS inhibitor cultured nerve cells were decreased significantly compared with the control group (P⬍0.05, Fig. 11).

DISCUSSION The present study was to investigate the influence of PS exposure on iron metabolism in rat brain. We found that PS exposure caused an increase in iron intake and transportation in some regions of the brain and subsequently resulted in iron deposition in those regions, accompanied by intensified oxidative reaction; therefore we suggest that PS is an important cause for iron deposition–induced neurodegenerative diseases. The concentrations of iron in different parts of the brain vary greatly. Also, changes in iron contents in the neurodegenerative disorders are regionally specific. Our previous studies found that cold, hot and rotation stimuli could change iron contents in some organs of rats, and prolonged depression could cause death of neurons in the hippocampus [Sheline et al., 1999]; therefore we speculate that stress might have caused change in normal iron metabolism, and our speculation was confirmed in this study: we found that, although PS did not alter the total content of brain iron under normal dietary iron levels, the iron contents were increased in the cerebral cortex, striatum, and hippocampus (Fig. 1), which happen to be the regions involved in degeneration diseases [Ke and Ming Qian, 2003; Lee et al., 2006]. The staining result of iron also revealed iron deposition in the cerebral cortex (Fig. 3). Therefore, it can be concluded that the iron concentrations

are increased in some specific regions of the brain after PS exposure. We believe that PS induces iron deposition in certain cerebral regions by changing the iron regulation factors. Brain iron is tightly regulated by the iron-binding proteins transferrin-TfR and Fn. TfR and Fn are coordinately and reciprocally controlled in response to iron levels by a posttranscriptional mechanism involving the binding between IRP and the iron response element (IRE) on TfR and Fn mRNA [Hentze et al., 2004; Pantopoulos, 2004]. Although we found that there was iron accumulation in some areas of the PS rat brain, there was no compensatory increase in Fn protein. More interestingly, there was an increase in TfR1 levels as would not be expected under these conditions (Figs. 4, 5). When TfR levels are increased, the cell can increase uptake of iron from extracellular transferrin. In this situation, if iron storage protein–intracellular Fn levels are low (Figs. 6, 7), free iron–NPBI levels into the cell will be increased (Fig. 2). Iron is known to be critical in the normal development and metabolism of brain; however, NPBI can cause great oxidative damage through generating hydroxyl radicals. The hydroxyl radical is generated from hydrogen peroxide, a reaction that requires a transitional metal, most importantly NPBI in its active ferrous form (Fenton reaction) [Gutteridge, 1992]. Our data showed that PS did induce oxidative damages in some regions of rat brain: SOD activity, which catalyzes the breakdown of superoxide radicals and provides the first line defense against oxygen toxicity, had undergone changes (Table 1), and the MDA level, which is a byproduct of the lipid peroxidation process [Millan-Plano et al., 2003; Topal et al., 2004], was increased greatly (Table 2). Under conditions of heightened oxidative stress, Fn is up-regulated and TfR1 is down-regulated to limit the availability of iron to take part in redox reactions. However, our findings in the present study suggested an opposite mechanism for the process: we found that the increase of iron might have preceded the occurrence of oxidative stress, or we can say that it is the increase of NPBI, which is caused by PS, that has led to the production of toxic free radical and subsequently led to oxidative stress. We also noticed that PS exposure also caused higher expression of Lf, another important metal transporter, in the hippocampus (Fig. 9); and the iron deposition in the hippocampus after PS exposure was significantly higher than that of other regions. Studies have found that long-term depression could lead to decreased volume and neuron death in the hippocampus. Hence we speculate that the hippocampus is the most vulnerable to the oxidative stress induced by

Table 1. SOD activity in different parts of rat brain in control and PS exposure groups (␮mol/mg, mean⫾S.D., n⫽12)

PS Control

Cortex

Hippocampus

Striatum

Brain stem

Cerebellum

0.298⫾0.034* 0.352⫾0.026

0.335⫾0.027** 0.480⫾0.027

0.353⫾0.037* 0.290⫾0.013

0.249⫾0.011 0.326⫾0.018

0.339⫾0.031 0.327⫾0.018

* P⬍0.05 vs. control group. ** P⬍0.01 vs. control group.

L. Wang et al. / Neuroscience 155 (2008) 24 –30

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Table 2. MDA concentrations in different parts of rat brain in control and PS exposure groups (␮g/mg, mean⫾S.D., n⫽12)

PS Control

Cortex

Hippocampus

Striatum

0.256⫾0.0213* 0.181⫾0.0114

0.217⫾0.0223* 0.159⫾0.0127

0.196⫾0.0200* 0.142⫾0.0119

* P⬍0.05 vs. control group.

PS-associated disorder of iron metabolism, which deserves further study in the future. Increased IRP expression in the cortex, hippocampus and striatum of PS-exposed rats might have caused the changes in TfR1 mRNA and Fn mRNA (Fig. 8). The role of IRP1 in synthesis of TfR1 and Fn is regulated by changes in iron status in the cell. IRP1 is a bifunctional “sensor” of iron, switching between RNA binding and enzymatic activities as aconitase on cellular iron status. Intracellular iron concentrations act as post-transcriptional levels through iron regulatory proteins (IRPs) that interact with the mRNAs of TfR and Fn. Previous study showed that iron deposition resulting from disorder of iron metabolism was an important factor in Parkinson’s and other ND disease neuropathology. It is suggested that PS exposure causes an increase in iron demand in some parts of rat brain, which subsequently leads to increase of iron intake and transportation to maintain the normal function of the brain. We believe that NO is an important factor for the increased iron demand in local cerebral regions of PS-exposed rats. We found that PS exposure significantly increased the NOS positive neurons in the cerebral cortex, hippocampus and striatum of the rat brain, which were also where the iron deposition was found (Table 3). The increased number of NOS positive neurons will unavoidably lead to the increase of NO secretion. NO is an important signal transduction molecule and many of its functions involve its binding to iron either in the heme of guanylate cyclase or in the [Fe-S] centers of important nonheme iron proteins [Hentze and Kühn, 1996; Richardson and Ponka, 1997]. Endogenous NO production abolishes completely aconitase activity by disassembling the [Fe-S] cluster and strongly activates IRE binding by IRP1 resulting in inhibited Fn mRNA synthesis and stabilizes the TfR mRNA over a long time period [Caltagirone et al., 2001; Gonzalez et al., 2004; Mehlhase et al., 2005]. Glucocorticoids can greatly influence NO diffusion to different brain areas and NO is very important to protect the neurons under stress [Lopez et al., 1999]. Our results showed that CORT increased the NO production in cortex nerve cells, and NOS inhibitor decreased the NO production in the CORT cultured cells (Fig. 11). Meanwhile, we also found that iron concentra-

Fig. 10. Effects of CORT and L-NAME on NO concentrations in primarily cultured nerve cells (mean⫾S.D., n⫽6, * P⬍0.05 vs. control group; # P⬍0.05 vs. CORT cultured cells).

tions were significantly increased in the CORT-treated cells and significantly decreased in NOS inhibitor-treated cells (Fig. 12). Therefore, we believe that the activation NO induced by glucocorticoids under background stress is an important reason for the upregulation of iron in some brain areas. Further study is needed to clarify whether the limited distribution of NOS-positive neurons is due to the uneven distribution of glucocorticoid receptors in the brain or to the same location of iron deposition and central control area of stress. We believe that PS-induced iron deposition in certain parts of the brain is a result of the increased iron demand in the cells of those regions; the brain needs to increase iron intake and transportation to maintain its normal function under stress. Also whether this increase in iron deposition in specific brain areas translates into neurodegeneration needs further study.

CONCLUSION In conclusion, we found in the present study that the contents of iron and NPBI were both increased in the cerebral cortex, hippocampus, and striatum of rats exposed to PS,

Table 3. Number of NOS positive neurons in the cortex, hippocampus and striatum of control and PS rats (mean⫾S.D., n⫽6)

PS Control * P⬍0.05.

Cortex

Hippocampus

Striatum

5.87⫾0.81* 1.56⫾0.42

11.83⫾1.21* 4.30⫾1.05

8.47⫾0.89* 4.85⫾0.69

Fig. 11. Effects of CORT and L-NAME on iron concentrations in primarily cultured nerve cells. (mean⫾S.D., n⫽6, * P⬍0.05 vs. control group; # P⬍0.05 vs. CORT cultured cells).

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accompanied by intense oxidative stress response, which is caused by PS-induced increase of local iron demand and the subsequent activation of iron regulation system. We believe that PS-induced location iron deposition and subsequent intensification of oxidative stress response is one of the important reasons for neurodegenerative disease. Acknowledgments—This work was supported by the National Natural Foundation of China (30471463). The authors wish to thank Yu Danghui of Second Military Medical University Press for careful reading of the English language of the manuscript.

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(Accepted 31 March 2008) (Available online 25 April 2008)