| Iron is essential for vital cellular activities and as such is required to be present in a readily available form.However,more and more researches indicate that excess iron accumulation in the body will bring a huge damage,especially iron overload in the central nervous system:oxidative damage is implied in the neurodegenerative diseases.Iron, especially non-protein-bound-iron(NPBI) catalyzes the formation of toxic hydroxyl radicals.Thus,iron toxicity can lead to the CNS irreversible pathological results.Previous researches including some experiments results our office did showed that motion sickness, sports and fatigue can make iron redistribution in the rat body,and we concluded previous researches and associated with modern social work tension,life pressure or study competition made people always be a more or less compressive stress.The exposure of animals to long-term stress produces deleterious effects on the brain,morphological neuronal damage in the CA2 and CA3 subfields of the hippocampus,an extensive literature demonstrate that glucocorticoids secreted during stress have a broad range of deleterious effects in the brain.Hence we guess:mental stress may be one of reasons for the brain iron metabolism disorders.ObjectiveTo study the characteristic effects of psychological stress on brain iron metabolism and to establish a useful experimental basis for further study involving how stress changes cell normal iron homeostasis and the consequent effects on physiological function of the human body.Method1.Effects of psychological stress on iron concentrations in the rat brain To divide experimental animals into groupsAll experimental procedures involving animals received the approval from the Animal Care and Use Committee of the Second Military Medicine University.Guidelines and Policy on using and caring of the laboratory animals were followed at all time.Male SD rats(120±5 g body weight) fed with a standard diet were purchased from the Shanghai-BK Ltd.Co,and were housed individually in a cage in a temperature-controlled room(24±1℃,55±5%humidity) with a 12-hour light and 12-hour dark cycle.After adaptation for 3 days,the rats were divided into the foot-shock group(FSG),psychological stress group(PSG) and the control group(CG).Each rat was exposed to stress for 30 minutes every day.To build psychological stress model of SD ratsUsing a communication box system,footshock stress(FS) and psychological stress (PS) were administered to the rats.The communication box was divided into two parts with a transparent acrylic board,i.e.,Part A including ten rooms with a plastic board-covered floor for electric insulation and part B including ten rooms with a metal grid-exposed floor.Rats in part B were administered an electrical shock through the floor (90 V,0.8 mA for 1 second) randomly for 30 min,90 times in total,and then exhibited a nociceptive stimulation-evoked response such as jumping up,defecation and crying. Thus they were exposed to systemic(physical) stress.Rats in part A were not directly administered the electrical shock,but were exposed to psychological stress in response to the actions of the rats in Room B.Measurement of serum CORT and ACTH,hypothalamus NE levels in rats under stressThe concentrations of serum CORT and ACTH were measured by the enzyme-linked immunoassays(ELISAs) kit;the hypothalamus NE levels were measured by the radioimmunofocus assays(RI) kit.Measurement of brain iron concentrations in rats 1.4.1 Determination of total iron in ratsIron 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.Standard addition method was used for calibration.Standards and control samples were prepared in an identical manner to the experimental samples.1.4.2 Determination of NPBI levels in ratsNPBI levels were analyzed by a method using bathophcnanthroline disulfonate (BPS) to chelate ferrous iron,thus forming a complex that could be analyzed with spectrophotometry.Dissected brain tissues were homogenized in a glass homogenizer in 10 vol of 50 mmol/L phosphate buffer,pH 7.4.1.4.3 Perl's iron stainingFor Perl's staining,sections were processed through a series of graded alcohols,into xylene,and rehydrated back to water.Sections were counterstained with Neutral Red, dehydrated in increasing concentrations of ethanol,cleared in xylene,and mounted on slides.2.Effects of Psychological Stress on Brain Iron Metabolism2.1 Determination of TfR1 and Fn mRNA levelsReal 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.Primers used to analyze all the transcripts have been reported else where.The Q-RT-PCR data were analyzed by 2-ΔΔAACT method as described2.2 Determination of TfR1 and Fn levelsThe concentrations of TfR1 and Fn in the cortex,hippocampus and striatum samples were assessed using a commercially available ELISA kitswith the absorbance read on a microplate reader at a wavelength of 450 nm.2.3 Western blotting analysis of IRP-1 and Lf expressionDissected tissues from the cortex,hippocampus and striatum were homogenized separately by a dounce homogenizer in lysis buffer.Proteins were incubated overnight at 4℃with a primary antibody against IRP-1(monoclonal,1:1000,Santa),Lf(rabbit polyclonal,1:500,Santa),orβ-actin(rabbit polyclonal,1:10000,Sigma).The blots were developed by incubation in ECL chemiluminescence reagent and subsequently exposed to BioMax Light Film. 3.Effects of Psychological Stress on Brain oxidative status3.1 Measurement of SOD activity in rat brainTissue samples were preserved in 50μl of 5 mM butylated hydroxytoluene to prevent further lipid peroxidation.SOD activity was measured using WST-1 kit with the absorbance read on a microplate reader at a wavelength of 450 nm.SOD activity of each region were normalized to wet tissue weight(mg) and expressed asμmol/mg.3.2 Measurement of GSH levels in rat brainGSH level was measured using kit with the absorbance read on a microplate reader at a wavelength of 490 nm.GSH concentration of each region were normalized to wet tissue weight(mg) and expressed asμmol/mg.3.3 Measurement of MDA concentrations in rat cerebral cortex,hippocampus and striatumMDA concentration was assessed using a MDA assay kit with the absorbance read on a microplate reader at a wavelength of 586 nm.MDA concentration of each region were normalized to wet tissue weight(mg) and expressed asμg/mg.3.4 Western blotting analysis of HO-1 expressionDissected tissues from the cortex,hippocampus and striatum were homogenized separately by a dounce homogenizer in lysis buffer.Proteins were incubated overnight at 4℃with a primary antibody against HO-1(monoclonal,1:1000,Santa) orβ-actin(rabbit polyclonal,1:10000,Sigma).4.Effects of molecular mechanisms of psychological stress on brain iron metabolism4.1 Effects stress hormones on brain iron contentsMale SD rats(120±5g body weight) were divided into the ACF-psychological stress group(P+ACF),α-CRF-psychological stress group(P+CRF),ACF-control group (P+ACF) andα-CRF-control group(P+CRF).Each rat was exposed to stress for 30 minutes every day.Iron concentrations were also determined using a Varian SpectrAA-220G graphite furnace atomic absorption spectrometer.4.2 Measurement of the number and activity of nitric oxide synthase(NOS) positive neurons The sections were processed using the NADPH-diaphorase(NADPH-d) histochemical method.NOS positive neurons were counted in 4 fields of the cerebral cortex,hippocampal CA3 and caudate putamen.Data were analyzed by One-way ANOVA.4.3 Cell preparation for intraceUular NO and iron analysisThe primarily cultured cortical neurons were plated into in a poly 1-lysine-coated 6-well plates at a density of about 1×106 cells/ml.The cells were treated with corticosterone(CORT,1μmol/L) or corticosterone/ L-NAME or corticosterone/ AG (CORT,1μmol/l;L-NAME 1.5μmol/L)(CORT,1μmol/l;AG 1.2μmol/L) for 24 h. Untreated neurons served as control.Counting Kit-8 was used to count the cells in three groups.4.4 NO measurementNO production was assayed by measuring the nitrite concentrations with the Griess assays.Plates wcrc incubated at 25℃for 10 min,and the absorbance at 550 nm was measured with a microplatc reader.Nitrite concentrations wcrc calculated with sodium nitrite standard curve as a reference.4.5 Analysis of intracellular ironThe experiments were carried out with a quadrupole ICP-MS X7 equipped with collision cell technology.A blank sample was included for baseline check for every run. Standards and control samples were prepared in an identical manner to the samples.a) Measurement of IRE binding activityWe used gel retardation to detect the IRE binding activity in the neuron,and the autoradiographic images were analyzed by immunosorbent assay method. Statistical analysisAll results were expressed as mean±SE.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. Results1.PS exposure increased the iron concentrations in some brain regions1.1 Determination of the PS model estabolishmentWe found that the levels of serum ACTH,corticosterone and hypothalamus noradrenalin in the model animals were significantly higher than those in the control animals(P＜0.05),indicating the successful of model establishment.1.2 Effects of psychological stress on iron concentrationsWe 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 the PS exposure group was significantly lower than that of the control group(P＜0.05)1.3 Effects of psychological stress on NPBI levelsWe 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.1.4 Iron staining results in the cortexPerl's iron staining revealed weak staining for iron in the cortex of control group, and very strong staining in the cortex of PS rats.2.Effects of Psychological Stress on Brain Iron Metabolism2.1 PS exposure caused changes in TfR1 and Fn mRNAReal time-PCR analysis showed that PS exposure increased TfR1 mRNA levels in the cortex,hippocampus and striatum(P＜0.05 for hippocampus and striatum),though the increase in the cortex was not significant;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. 2.2 PS exposure caused changes in TfR1 and Fn levelsTfR1 levels in the cortex,stfiatum and hippocampus were significantly higher than those in the control group(P＜0.05);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.2.3 PS exposure increased IRP1 and Lf expression in different parts of rat brainWestern blot analysis showed that PS exposure increased IRP-1 immunoreactivity in the cortex,hippocampus and striatum in the rat brain;and the expression of Lf was also increased in the hippocampus of the PS-exposed rats.3.PS exposure intensified the oxidative reaction in rat brain3.1 SOD activities in rat brain of two groupsSOD 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.3.2 MDA levels in rat brain of two groupsWe also found that MDA levels were significantly increased in the cortex, hippocampus and striatum of the PS exposure group compared to the control group (P＜0.01).4.Effects of molecular mechanisms of psychological stress on brain iron metabolism4.1 PS exposure increased NOS positive neurons in different brain regionsThe 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).4.2 Effects of CORT and NOS inhibitors on NO concentrations in primarily cultured nerve cells The NO production of the CORT cultured nerve cells was increased significantly compared to 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 to the control group(P＜0.05).4.3 Effects of CORT and NOS inhibitors on iron concentrations in primarily cultured nerve cellsIron concentrations of the CORT cultured nerve cells were increased significantly compared to 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 to the control group(P＜0.05).Conclusions1.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,which happen to be the regions involved in degeneration diseases.The staining result of iron also revealed iron deposition in the cerebral cortex.Therefore,it can be concluded that the iron concentrations are increased in some specific regions of the brain after PS exposure.2.We believe that PS induces iron deposition in certain cerebral regions by changing the iron regulation factors.Although we found that there was iron accumulation in some areas of the PS rats 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.When TfR1 levels were increased,the cell can increase uptake of iron from extra cellular transferrin.Increased IRP1 expression in the cortex,hippocampus and striatum of PS-exposed rats might have caused the changes in TfR1 mRNA and Fn mRNA.In addition,we noticed that PS exposure also caused higher expression of Lf, another important metal transporter,in the hippocampus;and the iron deposition in the hippocampus after PS exposure was significantly higher than those 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 most vulnerable brain parts to the oxidative stress induced by PS-associated disorder of iron metabolism, which deserves further study in the future.3.Our data showed that PS did induced 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,and the MDA level, which is a by-product of the lipid peroxidation process.4.We believe that NO is an important factor for the increased iron demand in local cerebral regions of PS-exposed rats.The increased number of NOS positive neurons will unavoidably lead to the increase of NO secretion.Glucocorticoids can greatly influence NO diffusion to different brain areas and NO is very important to protect the neurons under stress.Our results showed that corticosterone increased the NO production in cortex nerve cells,and NOS inhibitor decreased the NO production in the corticosterone cultured cells.Meanwhile,we also found that iron concentrations were significantly increased in the corticosterone treated cells and significantly decreased in NOS inhibitor treated ceils.Therefore,we believe that the activation NO induced by glucocorticoids under background of stress is an important reason for the upregulation of iron in some brain areas.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,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.
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