Mechanism Of Iron On The Brain Injury After Intracerebral Hemorrhage

Experimental studies have demonstrated that iron overload occurs after intracerebral hemorrhage(ICH) and contributes to ICH-induced brain injury.Our previous study showed that non-heme iron increases about 3-fold after ICH in a rat model.The major source of iron accumulation in the brain is hemoglobin after erythrocyte lysis.However,a recent study found that iron bound to transferrin in the plasma also results in brain injury after ICH.Deferoxamine,an iron chelator,attenuates acute perihematomal brain edema and oxidative stress.Free iron can cause free radical formation and oxidative brain damage.The natural history of free iron accumulation following ICH is still not clear.We investigated the time course of flee,total iron in the brain and levels of HO-1(key enzyme of free iron metabolism) after ICH.The effects of deferoxamine on free iron in cerebrospinal fluid(CSF),total iron in the brain and HO-1, and behavioral outcomes following ICH were also examined. 1 Materials and methods(1) Experimental animal180 male Sprague-Dawley rats(300 to 350 g) were randomly assigned to three groups:control,ICH+Vehicle and ICH+DFX group,each containing 60 rats.(2) Experimental model of ICHRats were anesthetized with pentobarbital(50 mg/kg,i.p.).The right femoral artery was catheterized for blood pressure monitoring and blood sampling.Blood was obtained from the catheter for analysis of pH,PaO2,PaCO2,hematocrit and glucose and as the source for the intracerebral blood infusion.Body temperature was maintained at 37.5℃using a feedback-controlled heating pad.The animals were positioned in a stereotactic frame(Model 500,Kopf Instruments,Tujunga,CA,USA) and a cranial burr hole(1 mm) was drilled in the right coronal suture 4.0 mmlateral to the midline.Either 100 ml autologous blood(as a model of ICH),or 100 ml saline(as a model of control) were infused into the right basal ganglia through a 26-gauge needle at a rate of 10 mL/min using a microinfusion pump(Harvard Apparatus Inc.,Holliston,MA,USA). The coordinates were 0.2 mm anterior and 3.5 mm lateral to the bregma and a depth of 5.5 mm.After intracerebral infusion,the needle was removed,the burr hole was filled with bone wax,and the skin incision was closed with suture.(3) Grouping and treatmentICH rats were divided to 2 groups.In the first group,rats received deferoxamine treatment(100 mg/kg,i.p.,2 hours after ICH and at 12-hour intervals thereafter).The second group received the same amount of vehicle.The rats(12 rats/group/time point) were then killed at 1,3,7,14,or 28 days later for CSF free iron and total brain tissue iron determination.All animals underwent behavioral testing until sacrificed.(4) Free iron determination The rats were anesthetized with pentobarbital.CSF was obtained by puncture of the cisterna magna 1,3,7,14,and 28 days after ICH and stored at -80℃before determination.Free iron in CSF was determined according to the method described by Nilsson et al.(5) Total brain tissue iron determinationRats were killed at 1,3,7,14,and 28 days after ICH.Brains were perfused with saline before decapitation and then removed.A coronal slice 4 mm thick around the injection needle tract was cut,divided into ipsilateral and contralateral sides,and weighed.The brain was then homogenized with 2 ml 0.1 M phosphate-burrered saline and stored at -80℃before determination.Total brain tissue iron(μg/g tissue weight) was determined according to the method described by Fish et al.(6) Investigation of the expression of HO-1 in the brain after ICHâ‘ Western Blot Analysis:Rats were anesthetized and underwent intracardiac perfusion with 0.1 mol/1 phosphate-buffered saline(pH 7.4).The brains were removed and a 3-mm-thick coronal brain slice was cut approximately 4 mmfrom the frontal pole. The slice was separated into ipsilateral and contralateral basal ganglia.Western blot analysis was performed as previously described.Protein concentration was determined using a Bio-Rad Laboratories protein assay kit.A 50μg portion of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a hybond-C pure nitrocellulose membrane.The membranes were blocked in carnation nonfat milk and probed with primary and secondary antibodies.The primary antibody was rabbit anti-rat HO-1 antibody(1:1500). The secondary antibody was goat anti-rabbit IgG(1:2000).The antigen-antibody complexes were visualized with a chemiluminescence system(Amersham) and exposed to a Kodak X-OMAT film.Relative densities of bands were analyzed with the NIH Image program.â‘¡Immunohistochemistry Analysis:The rats were anesthetized and subjected to intracardiac perfusion with 4%paraform-aldehyde in 0.1M phosphate-buffered saline (pH 7.4).The brains were removed and kept in 4%paraformaldehyde for 12 h,then immersed in 25%sucrose for 3-4 days at 4℃.Brains were then placed in embedding compound and sectioned on a cryostat(18 mm thick).Immunohisto- chemistry staining was then performed using the avidin-biotin complex technique.The primary antibody was rabbit anti-rat HO-1 antibody(1:400).The secondary antibody was goat anti-rabbit IgG(1:800).Either normal rabbit IgG or the absence of primary antibody was used as negative controls.(7) Behavioral testsAll animals were tested before and after surgery and scored by investigators who were blinded to both neurological and treatment conditions.Three behavioral assessments were used:forelimb placing,forelimb-use asymmetry,and corner-turn tests.â‘ Corner-turn test:Each rat was allowed to proceed into a 30 degree corner.To exit the corner,the rat could turn either left or right.The direction was recorded.The test was repeated 10 to 15 times,with at least 30 seconds between each trial,and the percentage of right turns calculated.Only turns involving full rearing along either wall were included.The rats were not picked up immediately after each turn so they did not develop an aversion for turning around.â‘¡Forelimb-placing test:Forelimb placing was scored using a vibrissae-elicited forelimb placing test.Independent testing of each forelimb was induced by brushing the vibrissae ipsilateral to that forelimb on the edge of a tabletop once per trial for 10 trials. Intact animals placed the forelimb quickly onto the countertop.Percent of successful placing responses were determined.A previous study showed a reduction in successful responses in the forelimb contralateral to the site of injection after ICH.â‘¢Forelimb-use asymmetry test:Forelimb use during explorative activity was analyzed by videotaping rats in a transparent cylinder for 3 to 10 minutes depending on the degree of activity during the trial.Behavior was quantified first by determining the occasions when the non-impaired ipsilateral(1) forelimb was used as a percentage of total number of limb-use observations on the cylinder wall.Second,the occasions when the impaired forelimb contralateral(C) to the blood-injection site were used as a percentage of total number of limb-use observations on the wall.Third,the occasions when both(B) forelimbs were used simultaneously as a percentage of total number of limb-use observations on the wall.A single overall limb-use asymmetry score was calculated as:Limb use asymmetry score=[I/(I+C+B)]-[C/(I+C+B)].(8) Statistical analysisStudent t test and Mann-Whitney U test were used to compare brain iron and behavioral data.Values are mean±SD.Statistical significance was set at p＜0.05.2 Results(1) All physiological variables were measured immediately before and 1 hour after intracerebral infusion.Mean arterial blood pressure,blood pH,PaO2,PaCO2, hematocrit,and blood glucose level were controlled within normal ranges(mean arterial blood pressure:70 to 100 mmHg,blood pH:7.40 to 7.50,PaO₂:80 to 120 mmHg, PaCO₂:35 to 45 mmHg,hematocrit:38 to 43%,blood glucose level:80 to 130 mg/dl).(2) Free iron accumulation in CSF following ICHFree iron levels in the normal CSF were very low in the rat(1.1±0.4μmol/L).After ICH,free iron levels in CSF were increased at the first day(8.5±1.3μmol/L) and peaked at the third day(14.2±5.0μmol//L).CSF free iron remained at high levels for at least 28 days(6.2±1.1μmol//L).Deferoxamine treatment initiated 2 hours after ICH reduced free iron in CSF at all time points(e.g.,day 3:6.7±2.0μmol//L versus 14.2±5.0μmol//L in the vehicle-treated group,p＜0.05).(3) Total brain tissue iron accumulation following ICHThe levels of total brain tissue iron also increased in the ipsilateral hemisphere after ICH(e.g.,day 1:264±55μg/g versus 87±13μg/g in the contralateral side,p＜0.01), and remained elevated for at least 4 weeks(255±61μg/g versus 85±17μg/g in the contralateral side,p＜0.01).Deferoxamine treatment initiated 2 hours after ICH did not reduce total brain tissue iron in the ipslateral hemisphere following ICH at all time points(e.g.,day 1:257±41μg/g versus 264±55μg/g in the vehicle-treated group, p＞0.05;day 3:227±41μg/g versus 243±46μg/g in the vehicle-treated group,p＞0.05).(4) The expression of HO-1 after ICHHO-1 immunoreactivities were very low in the cerebral hemispheres of the control rat.However,HO-1 protein levels were increased markedly in the ipsilateral basal ganglia the first day after ICH(1652±384 versus 208±72 pixels in the control,P＜0.01). HO-1 positive cells were found in the perihematomal zone.By Western blot analysis, the time course study of HO-1 showed that HO-1 was increased at day 1,peaked at day 3,and was still detectable at day 28 after ICH.DFX treatment could slightly upregulate the expression of HO-1 after ICH.(5) Deferoxamine treatment ameliorates neurological deficits after ICHDeferoxamine treatment reduced ICH-induced neurological deficits in rats. Corner-turn scores were improved at all time points in the deferoxaminetreated group compared with the vehicle group(e.g.,day 7:69.6±20.0%versus 83.9±16.8%,p＜0.05). Forelimb-placing scores were also improved in the deferoxamine-treated group compared with the vehicle group(e.g.,day 3:73.3±30.5%versus 46.7±33.6%,p＜0.05). There was also an improvement in ICH induced forelimb-use asymmetry associated with deferoxamine therapy(e.g.,day 1:26.8±17.2%versus 47.2±17.4%,p＜0.01).3 ConclusionsThe present study shows that free iron levels in CSF increase at the first day,peak on the third day,and remain high for at least 28 days after ICH.The changes of free iron levels were correlated with the expression of HO-1.Systemic administration of deferoxamine,an iron chelator,reduces free iron contents in CSF and improves functional outcomes after ICH in rats.However,deferoxamine has little effect on brain total iron after ICH,which suggesting that deferoxamine cannot enhance iron export after ICH,at least in this model.In addition,deferoxamine could upregulate the expression of HO-1 levels,which indicates that there may be other mechanism of the protective effect of deferoxamine on ICH. Mitogen-activated protein kinase(MAPK)family consists of three major groups, including extracellular signal regulated kinase 1/2(ERK1/2),c-Jun N terminal kinase (JNK) and p38 MAPK,has already been widely investigated for its active actions in response to various stimuli and co-ordinate a broad range of intracellular activity from metabolism,motility,mitosis,inflammation anddifferentiation to cell death or survival. A growing number of studies have demonstrated that MAPK family was involved in various forms of brain insults,such as cerebral ischemia and subarachnoid hemorrhage, and there inhibition blocks apoptosis in many neuronal death paradigms and attenuates brain injury in cerebral ischemia.However,the involvement of MAPK family in ICH is still far less understood.One potential stress that might induce MAPK family activation after ICH is iron.Experimental studies have demonstrated that iron overload occurs after ICH and contributes to ICH-induced brain injury.The present study investigated whether MAPKs is activated in the brain after ICH and whether intracerebral injection of iron can activate MAPKs.The effects of DFX on MAPKs activation following ICH were also examined.1 Materials and Methods(1) Experimental animalA total of 36 male Sprague-Dawley rats(300 to 350 g) were used in this study. Rats were divided into two sets,in the first set,we investigated the activation of MAPKs in a rat model following intracerebral hemorrhage,while in the second set,we investigate the role of iron in the activation of MAPKs and the effect of deferoxamine, an iron chelator,on the activation of MAPKs after intracerebral hemorrhage.(2) The first set Rats(n = 3 each group) had either an intracerebral infusion of 100μl saline (control) or an infusion of 100μl autologous blood(ICH rats) and were killed at 1,3 and 7 days later for investigation of phospho-ERK1/2,phospho-JNK and phospho-p38 MAPK by western blot analysis.The method of ICH model establishment was according to which described as Chapter One.(3) The second setRats(n = 6 each group) had either an intracerebral infusion of 100μL saline (control),an infusion of 30μl ferrous chloride(1 mmol/L) or an infusion of 100μl autologous blood(ICH rats),and then the ICH rats received either DFX treatment(100 mg/kg,i.p.,2 h after infusion of autologous blood) or the same amount of saline (vehicle).Rat brains were sampled 1 day after intracerebral injection for investigation of phospho-ERK1/2,phospho-JNK and phospho-p38 MAPK by western blot analysis and immunohistochemistry.(4) Western Blot AnalysisThe method of western blot analysis was according to which described as Chapter One.The primary antibodies were mouse anti-phospho-ERK1/2(1:1000 dilution),rabbit anti-phospho-JNK(1:1000 dilution) and rabbit anti-phospho-p38 MAPK(1:1000 dilution).The secondary antibody was HRP-conjugated goat anti-rabbit or rabbit anti-mouse antibody in a dilution of 1:2000.The antigen-antibody complexes were visualized with a chemiluminescence system(Amersham) and exposed to a Kodak X-OMAT film(Rochester,NY,USA).Relative densities of bands were analyzed with the NIH Image program(Version 1.62,Bethesda,MD,USA).(5) Immunohistochemistry AnalysisThe method of immunohistochemistry analysis was according to which described as Chapter One.Immunohistochemistry staining was then performed using the avidin-biotin complex technique.The primary antibodies were mouse anti-phospho-ERK1/2 (1:200 dilution),rabbit anti-phospho-JNK(1:200 dilution) and rabbit anti-phospho-p38 MAPK(1:200 dilution).The secondary antibody was biotinylated goat anti-rabbit or rabbit anti-mouse antibody in a dilution of 1:400.Either normal rabbit IgG or the absence of primary antibody was used as negative controls.2 Results(1) All physiological variables were measured immediately before and 1 hour after intracerebral infusion.Mean arterial blood pressure,blood pH,PaO2,PaCO2, hematocrit,and blood glucose level were controlled within normal ranges(mean arterial blood pressure:70 to 100 mmHg,blood pH:7.40 to 7.50,PaO₂:80 to 120 mmHg, PaCO₂:35 to 45 mmHg,hematocrit:38 to 43%,blood glucose level:80 to 130 mg/dl).(2) ERK1/2,JNK and p38 MAPK were activated in the ipsilateral basal ganglia after infusion of autologous bloodPhospho- ERK1/2,JNK and p38 MAPK immunoreactivities were all very low in the cerebral hemispheres of the control rat.However,phospho- ERK1/2,JNK and p38 MAPK protein levels were increased markedly in the ipsilateral basal ganglia after ICH. Phospho- ERK1/2,JNK and p38 MAPK positive cells were found in the perihematomal zone.By Western blot analysis,the time course study of MAPKs showed that activated JNK and p38 MAPK increased markedly 1 day after infusion of autologous blood and remained at high levels at least for 7 days,while phospho-ERK1/2 was slightly increased 1 day after ICH and higher at the later period after ICH(day 7).(3) Intracerebral infusion of ferrous iron could also activate JNK and p38 MAPK but fail to activate ERK1/2 at 24 h after infusion.Phospho-JNK or p38 MAPK positive cells were detected by immunohistochemistry in the ipsilateral basal ganglia after ferrous iron infusion,while the immunoreactivity of phosphorylated-ERK1/2 in the ipsilateral basal ganglia remains very weak compared with control group.Same phenomenon was shown by western blot analysis.(4) Deferoxamine treatment suppressed the upregulation of phosphor-JNK and phosphor-p38 MAPK,but increased the expression of phosphor-ERK1/2 in the ipsilateral basal ganglia 24 h after infusion of autologous blood.3 ConclusionsThe evidence that there was significant activation of ERK1/2,JNK and p38 MAPK following intracerebral hemorrhage suggests that MAPKs signaling pathway may act as an important role in the mechanism of brain injury after intracerebral hemorrhage.The evidences that there was a marked increase in JNK and p38 MAPK activation after iron infusion,and deferoxamine,an iron chelator,could partially block JNK and p38 MAPK activation indicating that iron does play a role in JNK and p38 MAPK activation after intracerebral hemorrhage.However,the block was incomplete suggesting that other factors also result in JNK and p38 MAPK activation.The ERK1/2 could not be activated by iron infusion,but deferoxamine treatment upregulated the phospho-ERK1/2 levels after intracerebral hemorrhage indicates that the anti-oxidative effect of deferoxamine may upregulate the ERK1/2 pathway,which should be related to brain tolerance to injury after intracerebral hemorrhage.The mechanisms of MAPKs pathway in ICH-induced brain injury need to be investigated further.