| OBJECTIVE To study(1)the change of iron concentration and iron distribution in the cortex and hippocampus of cerebral ischemic rats;(2)the expression of proteins related to iron transport including ferroportin 1(FP 1),divalent metal transporter- 1(DMT 1)and ceruloplasmin (CP)in the cortex and hippocampus of cerebral ischemic rats;(3)the expression of interleukin-6(IL-6)and hepcidin(Hep)in the cortex and hippocampus of cerebral ischemic rats; (4)the change of circulation iron level of cerebral ischemic rats.Furthermore,to investigate the relationship between brain iron deposition and the change of the proteins related to brain iron metabolism and circulation iron level in cerebral ischemia.MATERIALS AND METHODS Male Wistar rats weighed 250-300g were randomly divided into 5 groups:the cerebral ischemia for 1 day,3 days,7 days,28 days group and the sham operated group;There were 12 rats in every group.The cerebral ischemic model of rats was made by ligating bilateral common carotid arteries with silk.The sham operated group were received the same operation without occlusion of the arteries.A half of all animals were perfused with 4%paraformaldehyde after the bloods were collected from heart.The brains of rats were collected and fixed with 4%paraformaldehyde.Brains were embedded in paraffin and sliced. Serum was obtained by centrifugating bloods.Serum iron concentration of rats was measured by colorimetric method,and serum ferritin concentration of rats was measured by radioimmunity. Iron deposition of brain was shown by DAB enhanced Perl's reaction.The expression of FP1, DMT1,CP and IL-6 in the cortex and hippocampus of rats was shown by immunohistochemistry. The fresh tissue of brains of another half of rats was collected after animals were perfused with cold normal sodium,the cortex and hippocampus was separated and freezed in deep freeze refrigeration.The iron concentration in the cortex and hippocampus of rats was measured by graphite stove atomic absorption spectrometry.The expression of FP1 mRNA,DMT1 mRNA, CP mRNA and Hep mRNA in the cortex and hippocampus of rats was measured by reverse transcription polymerase chain reaction(RT-PCR).RESULTS 1.The iron concentration of the cortex and hippocampus of rats suffering from cerebral ischemia for 1 day and 3 days was not significantly different with sham group (P＞0.05).The iron concentration of the cortex and hippocampus of rats suffering from cerebral ischemia increased at the 7th day and increased more significantly at the 28th day (P＜0.01).2.Iron staining granula deposition was not seen in neuron of the cortex and hippocampus of sham rats and the rats who were suffering from cerebral ischemia for 1 day,3 days and 7 days.Abundant iron granula deposition was shown in neurons ofⅡ-Ⅴlayer of the cortex and whole hippocampus of the rats suffering from cerebral ischemia for 28 days.3.The expression of FP1 mRNA was shown in the cortex and hippocampus of sham rats. There are a obviously decrease of expression of FP1 mRNA in the cortex of rats suffered from cerebral ischemia for 1 day,3 days,7 days and 28 days compared with sham group(P＜0.01), and the longer ischemic times was,the more decrease.The expression of FP1 mRNA in the hippocampus of the rats suffering from cerebral ischemia for 1 day obviously decreased. Whereafter,the difference of expression of FP1 mRNA in the hippocampus of cerebral ischemic rats gradually decreased with ischemic time.The expression of FP1 mRNA in the hippocampus of rats suffering from cerebral ischemia for 28 days was not significantly different as compared with sham group(P＞0.05).Immunohistochemistry showed the expression of FP1 was shown in neural cells,including epithelial cell of choroid plexus,ependymal cell, pyramidal cell and granulosa cell of the cortex and hippocampus.The expression of FP1 in the cortex and hippocampus of rats suffering from cerebral ischemia for 1 day and 3 days was not significantly different with sham group(P＞0.05).However,the expression of FP1 of cerebral ischemic rats was weakened at the 7th day(P＜0.05),and a remarkable weakening was seen at the 28th day compared with sham rats(P＜0.01).4.The expression of DMTI+ iron response element(IRE)mRNA and DMT1-IRE mRNA were shown in the cortex and hippocampus of sham rats.The expression of DMT1+IRE mRNA and DMT1-IRE mRNA in the cortex of rats suffering from cerebral ischemia for 7 days and 28 days increased as compared with sham group(P＜0.01),but not in rats suffering from cerebral ischemia for 1 day and 3 days.The expression of DMT1+IRE mRNA and DMT1-IRE mRNA in the hippocampus of rats of cerebral ischemic group also increased compared with sham group(P＜0.01),but not DMT1-IRE mRNA in rats suffering from cerebral ischemia for 1 day.Immunohistochemistry showed that the expression of DMT1 was seen in pyramidal cell and granulosa cell of cortex and hippocampus.The expression of DMT1 in the cortex and hippocampus of rats suffering from cerebral ischemia for 3 days,7 days and 28 days increased compared with sham group(P＞0.05),but not in rats suffering from cerebral ischemia for 1 day (P＞0.05).5.The expression of CP mRNA was shown in the cortex and hippocampus of sham rats. The expression of CP mRNA in the cortex and hippocampus of rats suffering from cerebral ischemia decreased compared with sham group(P＜0.01).The longer cerebral ischemic times was,the more decreased.The expression of CP was shown by immunohistochemistry in neural cells,including epithelial cell of choroid plexus,ependymal cell,astrocyte of the cortex and hippocampus and vascular endothelial cell,but not in pyramidal cell and granulosa cell of the cortex and hippocampus.The expression of CP in the cortex and hippocampus of rats suffering from cerebral ischemia for 3 days,7 days and 28 days decreased compared with sham group (P＜0.05),but not in rats suffering from cerebral ischemia 1 day.6.Immunohistochemistry showed that the expression of IL-6 was slightly shown in neuron and astrocyte of the cortex and hippocampus of sham rats.The expression of IL-6 in the cortex and hippocampus of rats suffering from cerebral ischemia began to increase at the 1st day (P＜0.01),increased to a peak level at the 3rd day(P＜0.01)and continue at the 7th day (P＜0.01).The expression of IL-6 in the cortex and hippocampus of rat suffering from cerebral ischemia returned to baseline level at the 28th day.7.The expression of Hep mRNA was less shown in the cortex and hippocampus of sham rats.The expression of Hep mRNA in the cortex and hippocampus of cerebral ischemic rats was not significantly different compared with sham group.8.Serum iron concentration of sham rats was 8.31±0.66μg/ml.Serum iron concentration of rats suffering from cerebral ischemia for 1 day decreased compared with sham group (P＜0.05).Though serum iron concentration of rats suffering from the cerebral ischemia for 3 days was still less than sham group,it was not significant(P＞0.05).Whereafter,serum iron concentration of cerebral ischemic rats began to increase,and was higher at the 28th day than sham group(P＜0.01).9.Serum ferritin concentration of sham rats was 4.17±0.39ng/ml.Though serum ferritin concentration of rats suffering from cerebral ischemia for 1 day was not significantly different with sham group(P＞0.05),serum ferritin concentration of rats suffering from cerebral ischemia for 3 days was less than sham group(P＜0.05).Subsequently,Serum ferritin concentration of cerebral ischemic rats began to increase,and was higher at the 7th and 28th day than sham group(P＜0.01).CONCLUSIONS1.In the cortex and hippocampus of rats,cerebral ischemia induced increase of iron concentration,enhance of iron staining,and abundant iron deposition in neuron.2.In the cortex and hippocampus of rats,cerebral ischemia induced an increase of the expression of DMT1,and a decrease of the expression of FP1 and CP,which may involve in the mechanism of increase of iron concentration and abundant iron deposition in neuron by induced cerebral ischemia.3.The expression of IL-6 in the cortex and hippocampus of rats increased during acute period of cerebral ischemia,whereas decreased to baseline levels during chronic period of cerebral ischemia.IL-6 play a dual role to brain in cerebral ischemia.4.The expression of Hep was less in the cortex and hippocampus of rats,and cerebral ischemia did not induce any change of the expression of Hep.5.IL-6 may induce the up-regulation of Hep expression in lever,which may decrease the iron level of organism.However,the relationship was not confirmed in brain of rat.6.Serum iron and ferritin concentration decreased during acute period of cerebral ischemia, whereas increased during chronic period of cerebral ischemia,which may involve in the mechanism of increase of iron concentration and abundant iron deposition in neuron by induced cerebral ischemia.
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