Mechanism Of Brain Injury After Secondary Intraventricular Hemorrhage In Rats And Intervention Study Of Simvastatin

BackgroundIntracerebral hemorrhage(IVH)refers to the excessive bleeding inside or around the ventricles,where the cerebrospinal fluid is produced and circulates through towards the subarachnoid space.IVH can result from physical trauma,hemorrhaging in stroke,or an expansion of an existing intraparenchymal or subarachnoid hemorrhage.IVH is a subtype of intracerebral hemorrhage(ICH),an important public health issue that associated with high rates of death and disability in adults.IVH occurs in approximately 40% of spontaneous non-traumatic ICH patients,of whom 51–89% develop hydrocephalus.Both IVH and secondary hydrocephalus are independent risk factors for poor outcomes of ICH,thus IVH and hydrocephalus have become new targets for ICH treatment.However,the above animal models of IVH only display the characteristics of primary IVH.Clinical IVH,in contrast,is always secondary to ICH.Retrospective studie s have suggested that secondary IVH could lead to a higher risk of long-term shunt-dependent hydrocephalus compared with primary IVH.To date,the mechanisms responsible for this relationship remain poorly characterized.Based on this,we for the first time to established a secondary IVH(ICH combined with IVH)rat model,and evaluated the reliability,stability and repeatability of this model.In addition,we also compared the secondary IVH rat model with the primary IVH rat model in terms of brain injury and hydrocephalus for further examine the above clinical situation.Recent studies have indicated that iron may play an important role in post-hemorrhagic hydrocephalus and brain injury.Thus,we hypothesized that disruption of the brain–cerebrospinal fluid(CSF)barrier because of IVH,intracerebral hematoma may provide a source of iron for release into the ventricular system via this breached barrier,then increase accumulation of iron in brain parenchyma and CSF,eventually leading to brain injury and hydrocephalus.Statin,3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors,is associated with improved outcome among patients with ischemic stroke.In addition to lowering cholesterol,statins also have pleiotropic effects such as protecting blood-brain barrier,immunoregulation,anti-oxidation,and promoting angiogenesis.Over the past few years,several large retrospective cohort studies have reported that inpatient statin use is associated with improved outcomes following acute ICH.However,to date,no randomized controlled trials have proved its neuroprotective effect on ICH,especial in ICH patients with ventricular extension.As a fermentation-derived natural statin,simvastatin owns the properties of high lipophilicity and blood-brain barrier permeability.Recently,researchers have suggested that simvastatin may have the ability of accelerating hematoma absorption.However,its underlying mechanism remains elusive and the potential efficacy in the treatment of IVH was also unknown.Therefore,we hypothesized that simvastatin could ameliorate brain injury post-IVH via enhancing hematoma absorption.In a word,the present study consists of three parts as following: First,evaluat ing the stability,reliability and repeatability of this new established rat model of secondary IVH.Second,to test the clinical situation,we compared the secondary IVH rat model with the primary IVH rat model in terms of brain injury and hydrocephalus.In the meantime,we further explore the role of intracerebral hematoma in the development of hydrocephalus after secondary IVH.Finally,we examined whether simvastatin could accelerate hematoma removal,relieve hydrocephalus and promote neurological recovery after secondary IVH.Part Ⅰ The establishment and assessment of novel rats model of secondary intraventricular hemorrhage.ObjectivePrimary spontaneous intracerebral hemorrhage(ICH)with secondary intraventricular hemorrhage(IVH)is an important clinical problem of which little is known.However,so far,there is no ideal animal model effectively reflected the clinical situation of ICH with IVH.The aims of this study were,therefore,to establish a rat model of ICH with ventricular extension and investigate the occurrence of post-hemorrhagic chronic hydrocephalus and perihematomal tissue injury.Based on our previous rat model of IVH,we adjusted the injection coordinates and 200 μl autologous blood was stereotaxically infused into the right striatum(coordinates: 0.2 mm posterior,2.2 mm lateral,and 5.0 mm depth to the bregma).At 24 h post infusion,hematoma consequences and perihematomal tissue injury were evaluated on the acute phase.At 4 weeks,ventricular dilatation,brain tissue loss,hippocampus volume,and cortical thickness were measured with magnetic resonance imaging and neurocognitive function was assessed using the Morris water maze test.Results1.With blood infusion,the rats produced reproducible hematoma and ventricle expansion,which closely mimics the ICH with ventricular extension in humans.2.The secondary IVH animals demonstrated brain edema,blood–brain barrier breakdown,and marked perihematomal tissue injury on the acute phase.3.At 4 weeks,the T2 images showed remarkable hydrocephalus and tissue loss,and the Morris water maze test revealed neurocognitive deficits.ConclusionThe present ICH with the ventricular extension rat model features characteristics of both ICH and IVH rat models,which could be used for extending our pathophysiological understanding of post-hemorrhagic chronic hydrocephalus and perihematomal tissue damage.MethodsPart Ⅱ The comparasion between rats model of primary-and secondary intraventricular hemorrhage and mechanism study on brain injury after secondary intraventricular hemorrhage.ObjectiveThe secondary intraventricular hemorrhage(IVH)was reported to may have a higher incidence of hydrocephalus,which would lead to poorer outcomes for patients with ICH.However,the mechanisms related to this relationship remain unclear.Thus,this study was designed to exam the above clinical viewpoint and to clarify the underlying mechanism.MethodsRat models of ICH with ventricular extension(ICH/IVH)or primary IVH was induced by autologous blood injection.The early/ long-term brain injury,ventricular dilation,iron deposition and ferritin expression were detected using MRI,Evans blue,brain water content measure,immunohistochemistry and Western blot techniques.Then,the role of hematoma in hydrocephalus after ICH/IVH was explored by intraperitoneal injection with deferoxamine(an iron-chelator).Results1.With the same volumes of blood infusion,the ICH/IVH group caused more significant ventricular dilation,iron overload and ependymal cilia damage,more serious early brain injury and neurocognitive dysfunction when compared with the IVH group.2.Deferoxamine more significantly alleviated ventricular expansion in ICH/IVH when compared with the IVH alone.ConclusionOur data reveal that secondary IVH resulted in more severe hydrocephalus and iron deposition than primary IVH.Intracerebral hematoma contributes to hydrocephalus by increasing iron accumulation and worsening ependymal cilia harmness.Part Ⅲ Effect of simvastatin on brain injury after secondary intraventricular hemorrhage and related mechanism exploration.ObjectiveWe previously found that hematoma worsens hydrocephalus after intraventricular hemorrhage(IVH)via increasing iron deposition and aggravating ependymal cilia injury;therefore,promoting hematoma absorption may be a promising strategy for IVH.Recently,some investigations imply that simvastatin has the ability of accelerating hematoma absorption.Thus,this study was designed to examine the efficacy of simvastatin for IVH in rats.MethodsIntracerebral hemorrhage with ventricular extension was induced in a dult male Sprague–Dawley rats after autologous blood injection.Simvastatin or vehicle was administered orally at 1 day after IVH and then daily for 1 week.MRI studies were performed to measure the volumes of intracranial hematoma and lateral ventricle at days 1,3,7,14,and 28 after IVH.Motor and neurocognitive functions were assessed at days 1 to 7 and 23 to 28,respectively.Iron deposition,iron-related protein expression,ependymal damage,and histology were detected at day 28.Expression of CD36 s cavenger receptor(facilitating phagocytosis)was examined at day 3 after IVH using western blotting and immunofluorescence.In the meantime,fluorochrome labeled erythrocyte was injected into brain to investigate whether simvastatin could promote microglia-induced erythrocyte phagocytosis at day 3.Results1.Simvastatin significantly increased hematoma absorption ratio,reduced ventricular volume,and attenuated neurological dysfunction post-IVH.2.Less iron accumulation and more cilia survival was observed in the simvastatin group when compared with the control.3.Higher expression of CD36 was detected around the hematoma after simvastatin administration,and simvastatin enhanced microglia-mediated erythrocyte phagocytosis.ConclusionSimvastatin significantly enhanced brain hematoma absorption,alleviated hydrocephalus,and improved neurological recovery after experimental IVH,which may in part by upregulating CD36 expression and promoting microglia-mediated erythrocyte phagocytosis.Our data suggest that early simvastatin use may be a novel therapy for IVH patients.