Ischemic stroke occurs when the blood supply to the brain is suddenly interrupted by a blood clot blocking a blood vessel in the brain, and is a major cause of disability. There has been very limited progress in developing effective therapeutic approaches for ischemic stroke, although numerous agents have been tested in animal models and in clinical trials. Among many factors, incomplete understanding of the mechanism responsible for ischemia-caused neuronal injuries is a major hurdle. Cerebral ischemia induces a series of molecular pathways involving signaling mechanisms, gene transcription, and protein modification. Free radicals and oxidative stress have been suggested to be involved in each of the steps in the injury cascade 
. Iron is the most important metal element that mediates free radical generation via a Fenton-type reaction. Furthermore, hypoxia and free iron appear to interact with each other in causing subsequent neuronal death 
Our present results demonstrate that the iron-storage proteins L-ferritin and H-ferritin are remarkably elevated in the cerebral cortex and the hippocampus. Previous studies have shown that ischemic preconditioning initiates synthesis of ferritin in the heart 
and retina 
. It has been proposed that this increase in ferritin might protect cells from iron-mediated oxidative damage during ischemia-reperfusion. Our present data show that L-ferritin and H-ferritin are increased in ischemic cerebral cortex and hippocampus (), and may provide a mechanism to reduce iron-mediated injury by reducing free iron. Our results reveal that both total iron and ferritin-bound iron are significant increased in the ischemia side of the cerebral cortex and hippocampus (). Although we cannot conclude that ferritin plays a protective role based on these results, the increased levels of ferritin indicate that free iron levels in these tissues should have been reduced by binding.
Effect of ischemia/reperfusion on the total iron and ferritin binding iron in the brain.
Functional studies have demonstrated that hepcidin is the central regulator of systemic iron homeostasis by regulating FPN1, the only protein known to release iron from cells 
. Recently, data from our laboratory and others have shown that hepcidin is widely expressed in murine brain and has a very important role in regulating iron levels in the brain by down-regulating FPN1 expression 
. However, the role of hepcidin has not been studied in the ischemic brain. The present study demonstrates that ischemia-reperfusion increases hepcidin expression and down-regulates FPN1 protein expression in the cerebral cortex and the hippocampus. Knockdown hepcidin reversed the FPN1 decrease and ferritin increase in the ischemic brains of mice. Our present results demonstrated that down-regulated expression of FPN1 by hepcidin may be one important mechanism responsible for the ischemia-induced iron increase in the brain. In other words, reduced iron release from ischemic brain cells due to down-regulation of FPN1 increases intracellular iron levels 
It is known that inflammation up-regulates hepcidin expression and that cytokine IL-6 is a major mediator of the inflammatory response. It has been reported that IL-6 leads to increase in hepcidin production and decrease in serum iron within a few hours in humans and experimental animals 
. Previous studies have revealed the mechanism responsible for IL-6 induced hepcidin expression 
. Briefly, IL-6 can activate the JAK/STAT signaling pathway that upregulates the level of p-STAT3. p-STAT3 binds to the HAMP
promoter and thus increases hepcidin expression. It is well established that inflammation is involved in stroke and that ischemia increases IL-6 concentrations in brain tissues. Indeed, in our ischemic model we observed that cerebral ischemia-reperfusion increased IL-6 production significantly. Furthermore, the ratio of p-STAT3/STAT3 in ischemic tissues was much higher than in control tissues. This implies that the increased hepcidin in ischemic tissues may be regulated through the JAK-STAT pathway.
HIF-1 is one of the factors activated in early ischemia that can induce vascular endothelial growth factor, erythropoietin, etc. 
. Those downstream genes of HIF-1 promote cell survival by generating new blood vessels and providing new oxygen supply channels. HIF-1 is composed of HIF-1α and HIF-1β protein subunits. HIF-1β is constitutively expressed and relatively stable. The HIF-1α level is dependent on tissue oxygen levels and primarily determines HIF-1 activation. Tang et al. have studied the relationship between HIF-1α and transferrin. They have observed that the activation of HIF-1α can induce TfR1 expression, which, in turn, enhances iron accumulation and eventually increases oxidative damage and lipid peroxidation 
. Moreover, previous studies have clearly shown that deferoxamine, an iron chelator, can reduce injury caused by cardiac ischemia and reperfusion 
and cerebral ischemia 
. In the present study, we observed that the HIF-1α expression level is increased in the ischemic brain, and this result is in agreement with previous reports 
. The increased HIF-1α may play a key role in up-regulating TfR1 expression in the cerebral cortex and hippocampus of the ischemic brain. Thus, the increased TfR1 may be another pathway by which the iron and ferritin levels are elevated in ischemic brain cells. The sources of the iron might be extracellular fluid, which is potentially involved in both intracellular iron storage and intravascular iron transport and delivery to the brain 
Surprisingly, we did not observe increased total iron and H-ferritin in ischemic corpus striatum. The proportion of total iron and ferritin-bound iron in ischemic and control areas was different in the cerebral cortex, the hippocampus, and the corpus striatum (). These results imply that the ischemia-induced iron change is region-dependent. The mechanisms of the area-dependent iron changes are likely complex although the hepcidin-FPN1 and Tf-TFR pathways may play a role.
In summary, the current observations revealed two pathways that contribute to iron overload in ischemic brain tissues as outlined in . First, ischemia-reperfusion increases the expression of IL-6 that up-regulates hepcidin by the JAK/STAT3 pathway. Hepcidin induces FPN1 internalized degradation, reduces the efflux of iron, and thus causes iron accumulation. Second, ischemia-reperfusion up-regulates the HIF-1α level, which upregulates TfR1 expression and accelerates iron accumulation in the ischemic tissues.
Proposed pathways for the regulation of the iron level in cerebal ischemia.