Effects Of Hepcidin On Iron Metabolism And Neurological Outcomes In Intracerebral Hemorrhage

Intracerebral hemorrhage(ICH) is a major public health problem which occurs when a blood vessel in part of the brain becomes weak and bursts open, causing blood to leak into the brain with or without into Subarachnoid space or ventricle. ICH is a particularly devastating stroke subtype which carries a mortality rate of 30–50% and has not improved over the last two decades. Moreover, ICH imparts some form of disability in close to 90% of its survivors. Hospital admissions for ICH have increased by 18% in the past 10 years and Chinese people have higher incidences than do white Americans.At present, there is no specific treatment for ICH beyond supportive medical care.Recent studies show that the increased iron resulting from erythrocyte lysis and hemoglobin degradation is linked to delayed brain damage after ICH. There is extensive preclinical evidence, in vitro and in vivo, by different investigators and in different species to show that the iron chelator, deferoxamine can reduce hemoglobin- and iron-mediated neurotoxicity, leading to good neurologic outcomes after ICH. However, based on our current understanding of brain iron metabolism and deferoxamine, and the findings from our preliminary study on hepcidin, we believe that hepcidin might be a better agent in reducing iron and iron-mediated neurotoxicity in ICH brain, compared with deferoxamine. We hypothesize that the increase in concentration or expression of hepcidin in the brain should be able to reduce iron accumulation resulting from erythrocyte lysis and hemoglobin degradation in ICH,significantly improving neurologic outcomes.MethodsTo test the hypothesis, we investigate:1. Effects of hepcidin on iron concentration, expression of iron transport proteins(Tf R1, DMT1 and Fpn1) in the different brain regions and neurologic outcomes in a rat models of ICH(injection of autologous whole-blood to induce intracerebral bleeding)(a in vivo study); Cell apoptotic was assessd by TUNEL staining at 1, 3, 7, 14 and 28 days after ICH. Neuronal cell survival quantification was performed by NeuN Immunofluorescence staining at 28 days after ICH induction or sham surgery. Neuronal degeneration were assessd by Fluoro-Jade B staining at 1, 3, 7, 14 and 28 days after ICH. Motor dysfunction were assessed by forelimb placing, forelimb use asymmetry and corner turn tests at 1d, 3d, 7d, 14 d and 28 d after ICH. Neurocognitive function were assessed by Morris Water-Maze test at 28 d after ICH.2. Effects of hepcidin on cortical neurons treated with hemin(a in vitro study to see whether hepcidin could protect neurons from iron-depedent neurotoxicity of hemin).3. We will also investigate the effcets of ICH on brain iron metabolism by examining concentrations of iron, ferritin, and Tf in CSF or blood of ICH patents as well as concentrations of iron, hepcidin, ferritin, Tf R1, DMT1 and Fpn1 in different brain regions of rat model of ICH.Results:1. Clinic trial showed that higher stroke severity, platelet concentration in the blood, deep location of hematoma, subtentorial of hematoma and intraventricular extension of hemorrhage at day1(baseline) were associated with poor clinical outcome. We provided the further evidence that serum ferritin contents were associated with relative hematoma edema volume and could predict clinical outcome in patients with ICH. Our findings implied that not only the increased serum ferritin but also the reduced serum iron and Tf are associated with functional outcome as well as hematoma volume in patients with ICH. We found that intraventricular hemorrhage, hematoma volumes and surgery treatments have different effects on the contents of iron, ferritin, Tf and CP in the serum at different stages after ICH in patients.2. Ad-Hepcidin reduced non-heme iron in brain and CSF via inhibiting expression of iron-related proteins.As compared with the sham group, ICH induced a significant increase in non-heme iron content in the striatum, hippocampus, cortex, and CSF, peaked at 7-14 d and last at least 28 d, whereas treatment with ad-hepcidin led to a significant reduction in non-heme iron in all three regions and CSF in ICH model rats. There were a significant reduction in serum iron at 1d-14 d and increased at 28 d after ICH induction, pre-treatment with ad-hepcidin could reverse these change. Enhanced Perl’s Staining intensity was apparently increased in the perihematoma region after ICH induction and was significantly reduced by treatment with ad-hepcidin. The expression of DMT1, TfR1, Fpn1, Ft-L and Ft-H were significantly down-regulated by ad-hepcidin administration compared with the saline/ICH groups.3. Ad-Hepcidin attenuated autophagy induced by ICH/Hemin.The expression of LC3 II was significantly down-regulated and the he expression of P62 was significantly up-regulated by ad-hepcidin administration.4.Ad-Hepcidin attenuated ICH/Hemin-induced neurological damageThe LDH release induced by hemin were significantly reduced by administration of ad-hepcidin and hepcidin peptide in vivo. Ad-Hepcidin attenuated Hemin-induced apoptosis in vitro. The brain water content induced by ICH was reduced in the ad-hepcidin/ICH group compare to the saline/ICH group at 3, 7, 14 and 28 days after ICH. Ad-Hepcidin significantly reduced the loss of Neu N-immunoreactive cells, decreased the number of FJB-positive cells and Tunel-positive cells compared to saline/ICH group. Ad-Hepcidin attenuates behavioral deficits after ICH induction.ConclusionThe non-heme iron content in the brain is significant increased after ICH, and the degree and time course of nerve damage were consistent with the change of brain non-heme iron. Administration of Ad-Hepcidin could reduced non-heme iron in brain and CSF, and attenuated ICH-induced neurological damage. The effects on iron reduction and nerve protection of Ad-Hepcidin were mediated by inhibited expression of iron related protein and iron induced-autophagy.