铁离子在脑出血后继发性脑损伤中的作用及机制研究
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摘要
第一部分大鼠脑出血后脑内铁离子水平变化及其与神经功能损害相关性的研究
铁过载(iron overload)是各种急慢性脑退行性疾病(诸如脑缺血、脑外伤及早老性痴呆等)常见的病理现象,研究表明由铁离子催化的过氧化反应是造成脑组织损伤的重要原因,目前认为这种反应主要由组织内低分子量的二价铁(Fe~(2+))化合物(又称游离铁)介导产生。先前的研究证实在缺血性脑卒中,游离铁的积聚伴随着氧自由基、脂过氧化物的大量释放是导致脑缺血/再灌注损伤的重要因素。最近的研究发现在脑出血(intracerebral hemorrhage,ICH)中同样存在严重的铁过载现象,而这种铁过载主要来自血肿内融解红细胞产生的各种降解产物,伴有显著的各类氧自由基的积聚,由此导致脑组织细胞蛋白质、DNA的过氧化损伤;但目前尚缺乏对脑出血后铁离子积聚、变化趋势及其作用的进一步认识。
本次实验通过建立大鼠自体血注入脑出血模型,采用去铁敏干预的措施,动态观察脑出血后脑组织内总铁水平、脑脊液内游离铁水平变化以及铁离子生成的关键酶HO-1表达水平的变化,评价去铁敏对脑内铁离子过载的干预效果及其对脑出血后神经功能的保护作用。
1材料和方法
(1)实验动物:本研究采用随机对照动物实验,采用清洁级Sprague-Dawley雄性成年大鼠180只,体重300g～350g。实验大鼠分为生理盐水注射对照组、脑出血对照组及脑出血+去铁敏干预组,每组60只。
(2)大鼠自体血注入脑出血模型的建立:大鼠采用苯巴比妥(50 mg/kg)腹腔注射麻醉成功后,右侧股动脉中置入导管用来测量动脉血压并取血用于颅内注射。检测血pH、PaO_2、PaCO_2、红细胞压积以及血糖。术中利用恒温加热板使大鼠肛温保持在37.5。C。大鼠被固定在立体定位架上,并且于右侧冠状缝旁开中线3.5mm处钻孔。用26号针头通过立体定向插入右侧基底节(在前囟前方0.2mm,腹侧5.5mm,旁开3.5mm处)。自体全血(100μl)以10μl/min的速度通过微泵注入,留针5分钟,后拔除针头,无菌骨腊封闭颅骨小孔,缝合皮肤。在空白对照组中,采用等量生理盐水取代自体血以外,其余操作相同。
(3)实验大鼠分组和治疗:模型建立成功后,脑出血大鼠随机分为两组(n=60),一组给予去铁敏治疗(100mg/kg,腹腔内注射,出血后2小时给药,随后间隔12小时给药一次),另一组给予同等量的生理盐水(给药方式同去铁敏组)。脑出血两组与空白对照组均分别于术后第1、3、7、14及28天处死同等数量模型(n=12),用于脑脊液游离铁含量、脑组织总铁含量测定以及分别用蛋白印迹和免疫组化的方法检测脑组织内HO-1表达水平。
(4)脑脊液采取及游离铁检测:大鼠经苯巴比妥麻醉后,穿刺枕大池采取脑脊液,并冻存于-80℃冰箱用于统一检测。脑脊液内游离铁的检测采用Nilsson法。
(5)脑组织总铁含量检测:大鼠经苯巴比妥麻醉后,用生理盐水灌流取脑,沿针道前后2mm切去脑组织块,取同侧及对侧基底节、称重,然后用0.1M PBS液将脑组织打成匀浆并保存于-80℃冰箱用于统一检测。组织内总铁(μg/g)测定采用Fish法。
(6)检测血肿周围组织HO-1表达水平:
①蛋白印迹法(western blot):实验大鼠经苯巴比妥麻醉后开胸暴露心脏,用生理盐水灌流取脑,沿针道前后2mm切去脑组织块,分离同侧与对侧基底节。蛋白浓度测定使用Bio-Rad蛋白测试盒。每个标本取50μg蛋白质,通过凝胶电泳法使之分离,再将其转移到硝化纤维膜(Amersham)上。应用一抗的1:1500的稀释液(兔抗大鼠多克隆HO-1)以及二抗的1:2000稀释液(过氧化物酶共轭的山羊抗兔抗体。抗原抗体复合物通过化学发光系统而显影,应用Kodak X-OMAT胶片摄片。条带的相对密度应用NIH Image(Version 1.61)分析。
②免疫组织化学(immunohitochemistry)法:实验大鼠经苯巴比妥麻醉后开胸暴露心脏,先用生理盐水后用4%多聚甲醛/0.1MPBS灌流取脑,取出脑组织并保存在4%多聚甲醛0.1MPBS(PH 7.4)中24小时,然后移入30%蔗糖-0.1MPBS(PH 7.4)缓冲液中置4℃冰箱中过夜至沉底,取出脑组织采用OCT胶包埋并冻存于深低温冰箱。切片时取出冰冻组织块入恒冷切片机,行18μm连续冠状面切片,收集血肿周围连续切片,裱于多聚赖氨酸处理载玻片上,风干,-20℃保存备用。免疫组化检测采用ABC法,采用一抗为兔抗大鼠多克隆HO-1(1:400)、二抗为生物素化的山羊抗兔IgG(1:800)。
(7)行为学检查:所有实验大鼠在术前及术后(直至处死前)均进行行为学检查,用于评估神经功能损害及恢复情况。检查者对实验大鼠情况并不了解,从而保证检查的公正性。我们采用3种行为学检查手段:转身实验、前肢上抬实验及前肢应用协调性实验
①转身实验(corner test):观察大鼠在30°夹角转身并记录方向,向左或向右,重复12次。当右侧基底节受损时,大鼠将倾向于向右转身,比较向右转身的百分比。
②前肢上抬实验(Forelimb Placing test):观察大鼠在轻触患侧触须时,对侧前肢上抬并成功触及桌面的情况,重复10次。当右侧基底节受损时,大鼠左侧前肢出现无力症状,比较左侧上肢成功上抬的比例。
③前肢应用协调实验(Forelimb use asymmetry test):将大鼠至于透明塑料圆筒内,观察它在3～10分钟内(取决于大鼠的活跃程度)两侧前肢分别或同时扒在圆筒壁上情况,并分别记录健侧前肢(I)、患侧前肢(C)以及两侧同时上扒(B)的次数,总共计数20次,统计前肢应用协调评分=[I/(I+C+B)]-[C/(I+C+B)]。
(8)统计学检查:计量资料以平均数±标准差表示,运用Statview 5.0.1统计软件进行处理,数据采用Anova检验、t检验以及pearson相关性分析,显著性差异设置为p＜0.05。
2结果
(1)实验大鼠生理参数:所有实验大鼠的生理参数在术前及术后1小时立即进行检测。平均动脉压、血PH、PaO_2、PaCO_2、红细胞压积(Hematocrit,Hct)以及血糖均控制在正常范围(平均动脉压:70～100mmHg、血PH:7.4～7.5、PaO_2:80～120mmHg、PaCO_2:35～45mmHg、Hct:38%～43%、血糖:80～130mg/dl)。
(2)脑出血后大鼠脑脊液内游离铁水平变化
正常情况下大鼠正常脑脊液内游离铁水平非常低(1.1±0.4μmol/L),脑出血后,游离铁水平在一天内(8.5±1.3μmol/L)迅速上升,在3天后达到高峰(14.2±5.0μmol/L),此后直至术后28天维持在一个较高的水平(6.2±1.1μmol/L)。去铁敏治疗能显著降低出血后每个时间点脑脊液内游离铁水平的上升(如在术后第3天:去铁敏治疗组为6.7±2.0μmol/L:对照组14.2±5.0μmol/L,p＜0.05)。
(3)脑出血后大鼠脑组织内总铁水平变化
脑出血后患侧脑组织总铁水平同样明显升高(如术后第1天:患侧为264±55μg/g:健侧87±13μg/g,p＜0.01),且在出血后28天内始终维持一个较高的水平。去铁敏治疗并不能降低各个时间点患侧总铁水平(如术后第3天:治疗组为227±41μg/g:对照组243±46μg/g,p＞0.05)。
(4)脑出血后血肿周围组织HO-1表达水平
在正常脑组织以及生理盐水注射对照组中HO-1的免疫活性非常低,而脑出血后HO-1蛋白表达水平显著升高。免疫组化检测显示:脑出血后1d即可在血肿周围组织内发现HO-1阳性细胞显著增多,蛋白印迹检测显示:较之生理盐水注射对照组,脑出血后1d血肿周围HO-1蛋白表达水平显著升高(1652±384 versus 208±72),在出血后3天达到高峰,此后HO-1表达水平虽逐渐下降,但直至出血后28天仍可检测出HO-1蛋白活性。去铁敏治疗能轻微上调脑出血后脑组织内HO-1蛋白的表达水平。
(5)脑出血后神经功能损害及去铁敏的干预效果
脑出血后神经功能明显损害,而去铁敏治疗则显著改善了神经功能的损害程度。三种行为学检查均显示在各个时间点去铁敏治疗的有效性。如转身实验第7天:治疗组为69.6±20.0%:对照组83.9±16.8%,p＜0.05;前肢上抬实验第3天:治疗组为73.3±30.5%:对照组46.7±33.6%,p＜0.05;前肢应用协调实验第1天:治疗组为26.8±17.2%:对照组47.2±17.4%,p＜0.01。
3结论
脑出血后脑组织内存在长时间的铁离子过载现象,尤其是游离铁水平持续维持在高水平状态,可能是导致长时间铁离子介导的过氧化损伤、出现持久性神经功能损害的重要原因。铁离子螯合剂去铁敏能显著降低大鼠脑出血后脑脊液内游离铁水平,但并不能促进脑组织内总铁水平的下降,由此可以推测,去铁敏主要是通过结合及抑制铁离子活化(游离铁形成)而非促进铁离子外排出脑组织,达到减轻游离铁介导的过氧化损伤作用。脑出血后的铁离子过载与血红素加氧酶(HO-1)表达水平上调显著相关,去铁敏治疗在抑制游离铁水平上升的同时也轻微上调了HO-1的表达,表明去铁敏除了作为游离铁螯合剂外,可能还具有其它神经保护作用。
第二部分丝裂原活化蛋白激酶在大鼠脑出血后继发性脑损伤中的作用及其与铁离子过载关系的实验研究
丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)属于丝/苏氨酸蛋白激酶,广泛存在于哺乳动物细胞的胞质中,参与调节细胞生长、发育、分裂、死亡以及细胞间的功能同步等一系列生命活动;目前研究得最广泛的是ERK1/2、JNK和p38 MAPK三种MAPK。大量研究发现在各种脑退行性疾病(诸如脑淀粉样变性、Alzemer's病等)以及缺血性脑卒中、蛛网膜下腔出血的脑损伤反应过程中,均有上述三种MAPKs激活的身影,而采用ERK1/2、JNK或p38 MAPK抑制剂能拮抗缺血再灌注损伤所诱导的细胞凋亡和神经元损伤。但是,有关MAPKs激活与否及其在另一种常见的脑血管意外——脑出血中的作用则鲜有报道。此外我们先前的研究已证实铁离子过载尤其是游离铁的积聚是大鼠脑出血后继发性脑损伤以及神经功能损害的重要因素,然而由铁离子介导的脑出血后继发性脑损伤的细胞内信号途径目前并不清楚,铁离子是否能激活脑出血后MAPKs信号通路表达及其作用效应尚需要得到实验的证实。本研究将对脑出血后MAPKs的激活情况及其与铁离子过载之间的关系进行探讨。
1材料和方法
(1)实验动物及分组:本研究采用随机对照动物实验,采用清洁级Sprague-Dawley雄性成年大鼠36只,体重300g～350g。研究分为两个部分,第一部分探讨成年大鼠脑出血后脑组织MAPKs激活状态及变化情况;第二部分探讨铁离子对MAPKs激活的作用以及铁离子螯合剂去铁敏干预治疗对MAPKs激活的影响。
(2)大鼠脑出血后脑组织MAPKs激活状态及变化的研究:该部分对照组为生理盐水脑内注射组(n=3),实验组为脑出血组(n=9)。大鼠自体血注入脑出血模型的建立办法参见摘要第一部分。在对照组中,除采用等量生理盐水取代自体血以外,其余操作相同。模型建立成功后,分别于术后1、3、7天随机处死实验组大鼠,每时相位点各3只大鼠。应用蛋白印迹分析检测脑组织内磷酸化MAPKs表达水平变化。
(3)铁离子对MAPKs激活的作用以及去铁敏干预作用的研究:该部分对照组为生理盐水脑内注射组(n=6),实验组为脑出血组(n=12)和氯化亚铁(Fecl_2)脑内注射组(1mmol/L,n=6)。大鼠自体血注入脑出血模型的建立办法参见摘要第一部分。在对照组中,除采用等量生理盐水取代自体血以外,其余操作相同;氯化亚铁(Fecl_2)脑内注射组先行将Fecl_2溶解于用生理盐水,制备成1mmol/L溶液,然后将30μl 1mmol/L Fecl_2注入大鼠脑内,注射方法同脑出血模型建立方式。对脑出血组再随机分成两组(n=6),一组给予去铁敏治疗(100mg/kg,腹腔内注射,出血后2小时给药,随后间隔12小时给药一次),另一组给予同等量的生理盐水(给药方式同去铁敏组)。各组模型均于术后1天处死,分别采用蛋白印迹分析及免疫组织化学方法检测磷酸化ERK1/2、JNK和p38 MAPK蛋白表达,每组各3只。
(4)蛋白印迹分析(Western Blot Analysis)
蛋白印迹分析步骤参考第一章摘要,采用的一抗分别有的Mouse anti-phospho-ERK1/2(1:1000),Rabbit anti-phospho-JNK(1:1000)以及Rabbit anti-phospho-p38MAPK(1:1000)的稀释液,二抗分别选用的1:2000稀释液的过氧化物酶共轭的山羊抗兔抗体或兔抗鼠抗体。抗原抗体复合物通过化学发光系统而显影,应用Kodak X-OMAT胶片摄片。条带的相对密度应用NIH Image(Version 1.61)分析。
(5)免疫组织化学分析(Immunohistochemistry Analysis)
免疫组化分析步骤参考第一章摘要,应用亲和素-生物素复合物法进行检测,采用的一抗分别有的Mouse anti-phospho-ERK1/2(1:200),Rabbit anti-phospho-JNK(1:200)以及Rabbit anti-phospho-p38 MAPK(1:200)的稀释液,二抗分别选用的1:400稀释液的生物素化山羊抗兔抗体或兔抗鼠抗体。阴性对照采用正常兔IgG或缺失一抗的办法。
2结果
(1)实验大鼠生理参数:所有实验大鼠的生理参数在术前及术后1小时立即进行检测。平均动脉压、血PH、PaO_2、PaCO_2、红细胞压积(Hematocrit,Hct)以及血糖均控制在正常范围(平均动脉压:70～100mmHg、血PH:7.4～7.5、PaO_2:80～120mmHg、PaCO_2:35～45mmHg、Hct:38%～43%、血糖:80～130mg/dl)。
(2)大鼠脑出血后脑组织内ERK1/2、JNK及p38 MAPK均显著被激活:免疫组化显示脑出血后1天即可在血肿周围脑组织内发现磷酸化ERK1/2、JNK或p38MAPK阳性细胞显著增多;蛋白印迹检测发现磷酸化JNK及p38 MAPK的表达水平在出血早期较高(1天和3天),在出血后期(7天)下降;而磷酸化ERK1/2的表达水平则相反,在出血后期(7天)达到高峰。
(3)氯化亚铁脑内注射1天后能显著上调大鼠脑组织内磷酸化JNK、p38MAPK的表达,但并不能刺激ERK1/2的激活。
(4)去铁敏干预能降低脑出血后脑组织内磷酸化JNK、p38 MAPK的表达,却能上调磷酸化ERK1/2的表达水平。
3结论
脑出血后脑组织内存在显著的ERK1/2、JNK以及p38 MAPK激活现象表明MAPKs信号通路可能在脑出血后继发性脑损伤及保护机制中发挥重要作用;氯化亚铁脑内直接注射后亦显著激活大鼠脑组织内磷酸化JNK及p38 MAPK表达,但却不能上调大鼠脑组织内磷酸化ERK1/2的表达;去铁敏干预能降低脑出血后脑组织内磷酸化JNK及p38 MAPK的表达,同时能上调磷酸化ERK1/2的表达。根据这些现象,我们推测铁离子过载所导致的脑出血后脑损伤机制应主要是通过激活JNK及p38 MAPK信号通路所介导的致损伤反应,而去铁敏通过螯合脑出血后游离铁、抑制铁离子介导的各种脂质过氧化反应,可能也直接或间接地激活了ERK1/2信号通路所介导的脑出血后脑组织内在的自身保护作用。有关MAPKs信号通路在脑出血后继发性脑损伤机制中的作用尚有待进一步研究加以明确。
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 animal
180 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 ICH
Rats 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 treatment
ICH 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 determination
Rats 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 ICH
①Western 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.
②Immunohistochemistry 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 tests
All 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.
①Corner-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.
②Forelimb-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.
③Forelimb-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 analysis
Student 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,PaO_2:80 to 120 mmHg, PaCO_2:35 to 45 mmHg,hematocrit:38 to 43%,blood glucose level:80 to 130 mg/dl).
(2) Free iron accumulation in CSF following ICH
Free 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 ICH
The 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 ICH
HO-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 ICH
Deferoxamine 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 Conclusions
The 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 animal
A 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 set
Rats(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 Analysis
The 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 Analysis
The 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,PaO_2:80 to 120 mmHg, PaCO_2:35 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 blood
Phospho- 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 Conclusions
The 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.
铁过载(iron overload)是各种急慢性脑退行性疾病(诸如脑缺血、脑外伤及早老性痴呆等)常见的病理现象,研究表明由铁离子催化的过氧化反应是造成脑组织损伤的重要原因,目前认为这种反应主要由组织内低分子量的二价铁(Fe~(2+))化合物(又称游离铁)介导产生。先前的研究证实在缺血性脑卒中,游离铁的积聚伴随着氧自由基、脂过氧化物的大量释放是导致脑缺血/再灌注损伤的重要因素。最近的研究发现在脑出血(intracerebral hemorrhage,ICH)中同样存在严重的铁过载现象,而这种铁过载主要来自血肿内融解红细胞产生的各种降解产物,伴有显著的各类氧自由基的积聚,由此导致脑组织细胞蛋白质、DNA的过氧化损伤;但目前尚缺乏对脑出血后铁离子积聚、变化趋势及其作用的进一步认识。
本次实验通过建立大鼠自体血注入脑出血模型,采用去铁敏干预的措施,动态观察脑出血后脑组织内总铁水平、脑脊液内游离铁水平变化以及铁离子生成的关键酶HO-1表达水平的变化,评价去铁敏对脑内铁离子过载的干预效果及其对脑出血后神经功能的保护作用。
1材料和方法
(1)实验动物:本研究采用随机对照动物实验,采用清洁级Sprague-Dawley雄性成年大鼠180只,体重300g～350g。实验大鼠分为生理盐水注射对照组、脑出血对照组及脑出血+去铁敏干预组,每组60只。
(2)大鼠自体血注入脑出血模型的建立:大鼠采用苯巴比妥(50 mg/kg)腹腔注射麻醉成功后,右侧股动脉中置入导管用来测量动脉血压并取血用于颅内注射。检测血pH、PaO_2、PaCO_2、红细胞压积以及血糖。术中利用恒温加热板使大鼠肛温保持在37.5。C。大鼠被固定在立体定位架上,并且于右侧冠状缝旁开中线3.5mm处钻孔。用26号针头通过立体定向插入右侧基底节(在前囟前方0.2mm,腹侧5.5mm,旁开3.5mm处)。自体全血(100μl)以10μl/min的速度通过微泵注入,留针5分钟,后拔除针头,无菌骨腊封闭颅骨小孔,缝合皮肤。在空白对照组中,采用等量生理盐水取代自体血以外,其余操作相同。
(3)实验大鼠分组和治疗:模型建立成功后,脑出血大鼠随机分为两组(n=60),一组给予去铁敏治疗(100mg/kg,腹腔内注射,出血后2小时给药,随后间隔12小时给药一次),另一组给予同等量的生理盐水(给药方式同去铁敏组)。脑出血两组与空白对照组均分别于术后第1、3、7、14及28天处死同等数量模型(n=12),用于脑脊液游离铁含量、脑组织总铁含量测定以及分别用蛋白印迹和免疫组化的方法检测脑组织内HO-1表达水平。
(4)脑脊液采取及游离铁检测:大鼠经苯巴比妥麻醉后,穿刺枕大池采取脑脊液,并冻存于-80℃冰箱用于统一检测。脑脊液内游离铁的检测采用Nilsson法。
(5)脑组织总铁含量检测:大鼠经苯巴比妥麻醉后,用生理盐水灌流取脑,沿针道前后2mm切去脑组织块,取同侧及对侧基底节、称重,然后用0.1M PBS液将脑组织打成匀浆并保存于-80℃冰箱用于统一检测。组织内总铁(μg/g)测定采用Fish法。
(6)检测血肿周围组织HO-1表达水平:
①蛋白印迹法(western blot):实验大鼠经苯巴比妥麻醉后开胸暴露心脏,用生理盐水灌流取脑,沿针道前后2mm切去脑组织块,分离同侧与对侧基底节。蛋白浓度测定使用Bio-Rad蛋白测试盒。每个标本取50μg蛋白质,通过凝胶电泳法使之分离,再将其转移到硝化纤维膜(Amersham)上。应用一抗的1:1500的稀释液(兔抗大鼠多克隆HO-1)以及二抗的1:2000稀释液(过氧化物酶共轭的山羊抗兔抗体。抗原抗体复合物通过化学发光系统而显影,应用Kodak X-OMAT胶片摄片。条带的相对密度应用NIH Image(Version 1.61)分析。
②免疫组织化学(immunohitochemistry)法:实验大鼠经苯巴比妥麻醉后开胸暴露心脏,先用生理盐水后用4%多聚甲醛/0.1MPBS灌流取脑,取出脑组织并保存在4%多聚甲醛0.1MPBS(PH 7.4)中24小时,然后移入30%蔗糖-0.1MPBS(PH 7.4)缓冲液中置4℃冰箱中过夜至沉底,取出脑组织采用OCT胶包埋并冻存于深低温冰箱。切片时取出冰冻组织块入恒冷切片机,行18μm连续冠状面切片,收集血肿周围连续切片,裱于多聚赖氨酸处理载玻片上,风干,-20℃保存备用。免疫组化检测采用ABC法,采用一抗为兔抗大鼠多克隆HO-1(1:400)、二抗为生物素化的山羊抗兔IgG(1:800)。
(7)行为学检查:所有实验大鼠在术前及术后(直至处死前)均进行行为学检查,用于评估神经功能损害及恢复情况。检查者对实验大鼠情况并不了解,从而保证检查的公正性。我们采用3种行为学检查手段:转身实验、前肢上抬实验及前肢应用协调性实验
①转身实验(corner test):观察大鼠在30°夹角转身并记录方向,向左或向右,重复12次。当右侧基底节受损时,大鼠将倾向于向右转身,比较向右转身的百分比。
②前肢上抬实验(Forelimb Placing test):观察大鼠在轻触患侧触须时,对侧前肢上抬并成功触及桌面的情况,重复10次。当右侧基底节受损时,大鼠左侧前肢出现无力症状,比较左侧上肢成功上抬的比例。
③前肢应用协调实验(Forelimb use asymmetry test):将大鼠至于透明塑料圆筒内,观察它在3～10分钟内(取决于大鼠的活跃程度)两侧前肢分别或同时扒在圆筒壁上情况,并分别记录健侧前肢(I)、患侧前肢(C)以及两侧同时上扒(B)的次数,总共计数20次,统计前肢应用协调评分=[I/(I+C+B)]-[C/(I+C+B)]。
(8)统计学检查:计量资料以平均数±标准差表示,运用Statview 5.0.1统计软件进行处理,数据采用Anova检验、t检验以及pearson相关性分析,显著性差异设置为p＜0.05。
2结果
(1)实验大鼠生理参数:所有实验大鼠的生理参数在术前及术后1小时立即进行检测。平均动脉压、血PH、PaO_2、PaCO_2、红细胞压积(Hematocrit,Hct)以及血糖均控制在正常范围(平均动脉压:70～100mmHg、血PH:7.4～7.5、PaO_2:80～120mmHg、PaCO_2:35～45mmHg、Hct:38%～43%、血糖:80～130mg/dl)。
(2)脑出血后大鼠脑脊液内游离铁水平变化
正常情况下大鼠正常脑脊液内游离铁水平非常低(1.1±0.4μmol/L),脑出血后,游离铁水平在一天内(8.5±1.3μmol/L)迅速上升,在3天后达到高峰(14.2±5.0μmol/L),此后直至术后28天维持在一个较高的水平(6.2±1.1μmol/L)。去铁敏治疗能显著降低出血后每个时间点脑脊液内游离铁水平的上升(如在术后第3天:去铁敏治疗组为6.7±2.0μmol/L:对照组14.2±5.0μmol/L,p＜0.05)。
(3)脑出血后大鼠脑组织内总铁水平变化
脑出血后患侧脑组织总铁水平同样明显升高(如术后第1天:患侧为264±55μg/g:健侧87±13μg/g,p＜0.01),且在出血后28天内始终维持一个较高的水平。去铁敏治疗并不能降低各个时间点患侧总铁水平(如术后第3天:治疗组为227±41μg/g:对照组243±46μg/g,p＞0.05)。
(4)脑出血后血肿周围组织HO-1表达水平
在正常脑组织以及生理盐水注射对照组中HO-1的免疫活性非常低,而脑出血后HO-1蛋白表达水平显著升高。免疫组化检测显示:脑出血后1d即可在血肿周围组织内发现HO-1阳性细胞显著增多,蛋白印迹检测显示:较之生理盐水注射对照组,脑出血后1d血肿周围HO-1蛋白表达水平显著升高(1652±384 versus 208±72),在出血后3天达到高峰,此后HO-1表达水平虽逐渐下降,但直至出血后28天仍可检测出HO-1蛋白活性。去铁敏治疗能轻微上调脑出血后脑组织内HO-1蛋白的表达水平。
(5)脑出血后神经功能损害及去铁敏的干预效果
脑出血后神经功能明显损害,而去铁敏治疗则显著改善了神经功能的损害程度。三种行为学检查均显示在各个时间点去铁敏治疗的有效性。如转身实验第7天:治疗组为69.6±20.0%:对照组83.9±16.8%,p＜0.05;前肢上抬实验第3天:治疗组为73.3±30.5%:对照组46.7±33.6%,p＜0.05;前肢应用协调实验第1天:治疗组为26.8±17.2%:对照组47.2±17.4%,p＜0.01。
3结论
脑出血后脑组织内存在长时间的铁离子过载现象,尤其是游离铁水平持续维持在高水平状态,可能是导致长时间铁离子介导的过氧化损伤、出现持久性神经功能损害的重要原因。铁离子螯合剂去铁敏能显著降低大鼠脑出血后脑脊液内游离铁水平,但并不能促进脑组织内总铁水平的下降,由此可以推测,去铁敏主要是通过结合及抑制铁离子活化(游离铁形成)而非促进铁离子外排出脑组织,达到减轻游离铁介导的过氧化损伤作用。脑出血后的铁离子过载与血红素加氧酶(HO-1)表达水平上调显著相关,去铁敏治疗在抑制游离铁水平上升的同时也轻微上调了HO-1的表达,表明去铁敏除了作为游离铁螯合剂外,可能还具有其它神经保护作用。
第二部分丝裂原活化蛋白激酶在大鼠脑出血后继发性脑损伤中的作用及其与铁离子过载关系的实验研究
丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)属于丝/苏氨酸蛋白激酶,广泛存在于哺乳动物细胞的胞质中,参与调节细胞生长、发育、分裂、死亡以及细胞间的功能同步等一系列生命活动;目前研究得最广泛的是ERK1/2、JNK和p38 MAPK三种MAPK。大量研究发现在各种脑退行性疾病(诸如脑淀粉样变性、Alzemer's病等)以及缺血性脑卒中、蛛网膜下腔出血的脑损伤反应过程中,均有上述三种MAPKs激活的身影,而采用ERK1/2、JNK或p38 MAPK抑制剂能拮抗缺血再灌注损伤所诱导的细胞凋亡和神经元损伤。但是,有关MAPKs激活与否及其在另一种常见的脑血管意外——脑出血中的作用则鲜有报道。此外我们先前的研究已证实铁离子过载尤其是游离铁的积聚是大鼠脑出血后继发性脑损伤以及神经功能损害的重要因素,然而由铁离子介导的脑出血后继发性脑损伤的细胞内信号途径目前并不清楚,铁离子是否能激活脑出血后MAPKs信号通路表达及其作用效应尚需要得到实验的证实。本研究将对脑出血后MAPKs的激活情况及其与铁离子过载之间的关系进行探讨。
1材料和方法
(1)实验动物及分组:本研究采用随机对照动物实验,采用清洁级Sprague-Dawley雄性成年大鼠36只,体重300g～350g。研究分为两个部分,第一部分探讨成年大鼠脑出血后脑组织MAPKs激活状态及变化情况;第二部分探讨铁离子对MAPKs激活的作用以及铁离子螯合剂去铁敏干预治疗对MAPKs激活的影响。
(2)大鼠脑出血后脑组织MAPKs激活状态及变化的研究:该部分对照组为生理盐水脑内注射组(n=3),实验组为脑出血组(n=9)。大鼠自体血注入脑出血模型的建立办法参见摘要第一部分。在对照组中,除采用等量生理盐水取代自体血以外,其余操作相同。模型建立成功后,分别于术后1、3、7天随机处死实验组大鼠,每时相位点各3只大鼠。应用蛋白印迹分析检测脑组织内磷酸化MAPKs表达水平变化。
(3)铁离子对MAPKs激活的作用以及去铁敏干预作用的研究:该部分对照组为生理盐水脑内注射组(n=6),实验组为脑出血组(n=12)和氯化亚铁(Fecl_2)脑内注射组(1mmol/L,n=6)。大鼠自体血注入脑出血模型的建立办法参见摘要第一部分。在对照组中,除采用等量生理盐水取代自体血以外,其余操作相同;氯化亚铁(Fecl_2)脑内注射组先行将Fecl_2溶解于用生理盐水,制备成1mmol/L溶液,然后将30μl 1mmol/L Fecl_2注入大鼠脑内,注射方法同脑出血模型建立方式。对脑出血组再随机分成两组(n=6),一组给予去铁敏治疗(100mg/kg,腹腔内注射,出血后2小时给药,随后间隔12小时给药一次),另一组给予同等量的生理盐水(给药方式同去铁敏组)。各组模型均于术后1天处死,分别采用蛋白印迹分析及免疫组织化学方法检测磷酸化ERK1/2、JNK和p38 MAPK蛋白表达,每组各3只。
(4)蛋白印迹分析(Western Blot Analysis)
蛋白印迹分析步骤参考第一章摘要,采用的一抗分别有的Mouse anti-phospho-ERK1/2(1:1000),Rabbit anti-phospho-JNK(1:1000)以及Rabbit anti-phospho-p38MAPK(1:1000)的稀释液,二抗分别选用的1:2000稀释液的过氧化物酶共轭的山羊抗兔抗体或兔抗鼠抗体。抗原抗体复合物通过化学发光系统而显影,应用Kodak X-OMAT胶片摄片。条带的相对密度应用NIH Image(Version 1.61)分析。
(5)免疫组织化学分析(Immunohistochemistry Analysis)
免疫组化分析步骤参考第一章摘要,应用亲和素-生物素复合物法进行检测,采用的一抗分别有的Mouse anti-phospho-ERK1/2(1:200),Rabbit anti-phospho-JNK(1:200)以及Rabbit anti-phospho-p38 MAPK(1:200)的稀释液,二抗分别选用的1:400稀释液的生物素化山羊抗兔抗体或兔抗鼠抗体。阴性对照采用正常兔IgG或缺失一抗的办法。
2结果
(1)实验大鼠生理参数:所有实验大鼠的生理参数在术前及术后1小时立即进行检测。平均动脉压、血PH、PaO_2、PaCO_2、红细胞压积(Hematocrit,Hct)以及血糖均控制在正常范围(平均动脉压:70～100mmHg、血PH:7.4～7.5、PaO_2:80～120mmHg、PaCO_2:35～45mmHg、Hct:38%～43%、血糖:80～130mg/dl)。
(2)大鼠脑出血后脑组织内ERK1/2、JNK及p38 MAPK均显著被激活:免疫组化显示脑出血后1天即可在血肿周围脑组织内发现磷酸化ERK1/2、JNK或p38MAPK阳性细胞显著增多;蛋白印迹检测发现磷酸化JNK及p38 MAPK的表达水平在出血早期较高(1天和3天),在出血后期(7天)下降;而磷酸化ERK1/2的表达水平则相反,在出血后期(7天)达到高峰。
(3)氯化亚铁脑内注射1天后能显著上调大鼠脑组织内磷酸化JNK、p38MAPK的表达,但并不能刺激ERK1/2的激活。
(4)去铁敏干预能降低脑出血后脑组织内磷酸化JNK、p38 MAPK的表达,却能上调磷酸化ERK1/2的表达水平。
3结论
脑出血后脑组织内存在显著的ERK1/2、JNK以及p38 MAPK激活现象表明MAPKs信号通路可能在脑出血后继发性脑损伤及保护机制中发挥重要作用;氯化亚铁脑内直接注射后亦显著激活大鼠脑组织内磷酸化JNK及p38 MAPK表达,但却不能上调大鼠脑组织内磷酸化ERK1/2的表达;去铁敏干预能降低脑出血后脑组织内磷酸化JNK及p38 MAPK的表达,同时能上调磷酸化ERK1/2的表达。根据这些现象,我们推测铁离子过载所导致的脑出血后脑损伤机制应主要是通过激活JNK及p38 MAPK信号通路所介导的致损伤反应,而去铁敏通过螯合脑出血后游离铁、抑制铁离子介导的各种脂质过氧化反应,可能也直接或间接地激活了ERK1/2信号通路所介导的脑出血后脑组织内在的自身保护作用。有关MAPKs信号通路在脑出血后继发性脑损伤机制中的作用尚有待进一步研究加以明确。
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 animal
180 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 ICH
Rats 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 treatment
ICH 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 determination
Rats 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 ICH
①Western 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.
②Immunohistochemistry 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 tests
All 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.
①Corner-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.
②Forelimb-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.
③Forelimb-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 analysis
Student 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,PaO_2:80 to 120 mmHg, PaCO_2:35 to 45 mmHg,hematocrit:38 to 43%,blood glucose level:80 to 130 mg/dl).
(2) Free iron accumulation in CSF following ICH
Free 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 ICH
The 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 ICH
HO-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 ICH
Deferoxamine 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 Conclusions
The 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 animal
A 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 set
Rats(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 Analysis
The 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 Analysis
The 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,PaO_2:80 to 120 mmHg, PaCO_2:35 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 blood
Phospho- 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 Conclusions
The 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.
引文
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