Role Of Iron In Brain Injury After Intraventricular Hemorrhage

BackgroudIntracerebral haemorrhage (ICH) is a common stroke type that is associated with highmortality (up to50%at1month) and patients who survive typically have major neurologicalimpairments. As yet, the mechanisms involved in the brain injury following ICH isn’tcompletely clear, and no proven medical as well as operative treatment for ICH exists.Intraventricular hemorrhage (IVH) exists in up to50%of patients with primary ICH, whichmay result in both injuries to the brain as well as secondary hydrocephalus. Recent studieshave confirmed that IVH is an independent factorr of poor outcome after ICH. A betterlearning of the pathogenesis and mechanisms of the brain injury following IVH may lead toimproved treatment efficacy for ICH.The mechanisms of IVH-related brain injury and hydrocephalus are not completely clear.The proposed mechanisms of brain injury after IVH may include acutely developinghydrocephalus with increased intracranial pressure, intraventricular hematoma compression,toxity from erythrocyte lysis and chronic hydrocephalus. The relationship betweenerythrocyte lysis and IVH-induced brain injury and hydrocephalus remains unclear. Recently,increasing evidence suggests that released component from the haematoma is a majorcontributor to brain injury induced by ICH. Erythrocyte lysis associated with clot resolutionhappens several days after ICH in human and animal models. Iron overload occurs in thebrain after ICH, and increased brain iron levels contribute to brain edema, oxidative injury,and neuronal loss following ICH. Further, deferoxamine, an effective iron chelator, has beenshown to be protective in several kinds of animal species with ICH. Iron chelation might be apromising choice for patients with ICH.The role of iron in brain injury and chronic hydrocephalus following IVH is still notquite clear. Considering that erythrocyte lysis after IVH may are more widely distributedthroughout the cerebral ventricules and subarachnoid space, we suppose that iron may play a important role in brain injury following IVH. We hypothesize that iron overload developedafter IVH cause oxidative brain injury, and contribute to brain injury and chronichydrocephalus.The main research aspect of the present project is to study: the characteristic changes ofbrain injury and iron overload following IVH; the efficacy of iron chelator and antioxidantagainst the brain injury following IVH. Three specific aims are proposed to address ourhypothesis:①investigate the role of iron overload in ventricular system enlargement andneuronal loss in a rat model of IVH, and determine the effect of deferoxamine onIVH-induced brain injury;②investigate the iron-induced brain injury by directintraventricular iron infusion, and determine if minocycline reduce iron-related brain injury ina rat model;③investigate the whether systemic edaravone treatment could attenuate theIVH-induced oxidative stress and acute brain injury, and thus improve behavioral deficitsafter IVH in rat IVH model.Part Ⅰ The role of iron in brain injury after experimental intraventricularhemorrhage and the protection of deferoxamine against IVH-induced brain injuryObjectiveTo investigated the role of iron in ventricular dilatation and neuronal death in a rat modelof intraventricular hemorrhage.MethodsThere were3parts in this study. First, male Sprague-Dawley rats had a200μl injectionof saline, autologous blood or heparinized autologous blood into the right lateral ventricle.Rats were euthanized at different time. Rats had MRI and then the brain samples were usedfor Western blot analysis, histology, and immunohistochemistry. Second, rats received aninjection of packed or lysed RBCs into the right lateral ventricle, and underwent magneticresonance imaging (MRI) at24hours and were then euthanized for immunohistochemistry.Third, rats received blood injection and then deferoxamine or vehicle treatment. The rats wereeuthanized4weeks later for measurement of hippocampal and ventricular volumes.Results1. Intraventricular infusion of autologous blood resulted in persistent ventricular dilationfrom day1to day28. Heparinized autologous blood infusion caused less ventricular exlargement at5time points, compared with none-heparinized blood. Increased brain tissueiron, dilation of bilateral ventricles, and hippocampal tissue loss were observed followingIVH. Iron deposit was associated with upregulation of ferritin and heme oxygenase-1.2. Intraventricular infusion of lysed RBCs, rather than packed RBCs, resulted inventricular enlargement and marked increase of brain heme oxygenase-1and OX-42.3. Systemic use of deferoxamine reduced IVH-related ventricular enlargement and lesshippocampal volume, as well as iron deposition and ferritin upregulation.ConclusionRat model of IVH resulted in brain iron accumulation and persistent enlargement of thecerebral ventricles. Further, systemic deferoxamine usage reduced IVH-inducedhydrocephalus, hippocampus tissue loss and iron accumulation. These findings suggest thatiron-overload was involved in secondary brain injury after IVH. Deferoxamine may be ahopeful therapy for patients with ICH and IVH. Part II Minocycline attenuates brain injury by intraventricular injection of iron:the possible role of the iron chelating abilityObjectiveTo investigate the brain injury induced by intraventricular infusion of iron, as well as theeffect of minocycline on iron-induced injury in rat model.MethodsMale Sprague-Dawley rats had200μl of saline, FeCl2, FeCl2+Minocycline, FeCl2+macrophage/microglia inhibitory factor (MIF)、 FeCl3, FeCl3+Minocycline. Rats wereeuthanized at1day after intraventricular injection for brain water content or histologicalevaluation after MRI. Fluoro-Jade C staining was chosen to detect periventricular neuronaldegeneration, and DNA damage in the brain was detected by TUNEL and PANT staining.Iron chelating ability of minocycline was also tested by chemical methrod. Results1. FeCl2injection into right ventricle caused marked bilateral brain edema,periventricular neuronal injuries and acute ventricular dilatation. Minocycline but not MIFinjection together with iron significantly reduced brain edema and neuronal death, althoughboth of minocycline and MIF inhibited the microglia activation after iron injection.2. FeCl3injection into right ventricle caused marked ventricular dilatation and neuronaldegeneration. Minocycline reduced iron-induced neuronal degeneration but not ventriculardilatation.3. Ferrous and ferric iron chelating ability of minocycline was confirmed by chemicalmethod.ConclusionIntraventricular iron infusion could result in brain injury and ventricular dilation. Theiron chelating ability and beneficial effect of minocycline on iron-induced brain injury suggestsa new mechanism for neuroprotective effect of minocycline.Key words：Intraventricular hemorrhage Iron Minocycline HydrocephalusPart III Neuroprotective effects of edaravone after intraventricular hemorrhage inratsObjectiveTo investigated investigated whether systemic edaravone treatment could attenuate theIVH-induced oxidative stress, brain edema and delayed hydrocephalus, and improvebehavioral deficits in a rat model of IVH.MethodsThere were2parts in this study. In the first part (short-term study), rats were divided into3groups. There were intraventricular saline injection (saline) group, IVH with vehicletreatment (IVH+Veh) group, and IVH with edaravone treatment (IVH+Edv) group. Ratsreceived an injection of200μl saline or autologous whole blood into the right lateral ventricleand were treated with either edaravone or vehicle. Rats were euthanized at day1for brainwater content as well as malondialdehyde (MDA) content and superoxide dismutase (SOD)assay. In the second part of the experiment (long-term study), the animals were divided into3groups as in the first part. The rats were treated with either edaravone or vehicle every24h up to3days. Behavioral tests were carried out from day23after IVH, and MRI scan was carriedout at day28after IVH. All rats were euthanized for histological and morphologicalobservations at day28after infusion.Results1. Intraventricular infusion of autologous blood caused acute oxidative stress, brainedema, and delayed damage in learning and memory.2. Edaravone treatment can reduce IVH-caused brain edema and elevated lipidperoxidation. Furthermore, repeated edaravone treatment reduced ventricular dilatation andimproved the learning and memory damage after IVH.ConclusionOur results indicate that oxidative injury occurs after IVH, and edaravone treatmentreduced IVH-induced brain injury and neurobehavioral deficits. These effects suggest thatoxidative stress may have a role in IVH-induced brain injury and hydrocephalus. Edaravonemight be a hopeful therapeutic for patients with IVH.