【作者】 池志宏；

【导师】 王占友；

【作者基本信息】 中国医科大学 ， 人体解剖与组织胚胎学， 2008， 博士

【摘要】 目的癫痫是一种常见的神经系统疾病。动物实验和临床资料表明,脑内锌稳态的变化和癫痫的发生发展是相关的。脑内可以被组织化学染色的锌离子为游离锌离子,又称可螯合锌离子（chelatable zinc ion）。这些锌离子主要位于含锌神经元（zinc-enriched neurons,ZEN）轴突末端的突触小泡内。当神经活动时,这些锌离子通过胞吐作用释放到突触间隙与某些受体结合或者穿越突触后膜进入到突触后神经元进而发挥其调节作用。近年来,锌转运体家族成员（Zinc transporter familymembers,ZNTs）被学者们一一发现,促进了锌稳态研究的发展。锌转运体3（zinctransporter 3,ZNT3）是锌转运体家族中的一个成员,是参与锌离子转运相关的蛋白质。ZNT3表达在ZEN轴突终末的突触小泡膜上,其功能是转运锌离子进入突触小泡内。目前,有关锌离子及其锌转运体ZNT3与癫痫发病机理的关系还不十分清楚。但有研究表明两者与癫痫的发生发展有着非常密切的联系。本实验旨在研究锌离子在癫痫发作过程中分布变化以及ZNT3在模型动物海马MFS的分布表达情况。实验方法一、匹罗卡品小鼠癫痫模型的建立模型组CD-1小鼠经腹腔注射匹罗卡品（300mg/kg,ip）。对照组小鼠注射等量的生理盐水。动物于注射匹罗卡品前半小时预先腹腔注射阿托品（1mg/kg）以阻滞外周胆碱能反应。小鼠在注射匹罗卡品后观察其行为学变化,实验动物痫性发作等级评价标准采用经Becker等修改后的Racine（1972年）分级标准。二、EEG记录麻醉后,脑立体定位仪固定小鼠,于前囟点（Bregma）向后X=2.30mm,矢状缝（sagittal suture）旁Y=2.10mm,硬膜下Z=2.0mm定位海马,钻孔并埋藏电极,动物饲养一周后,于腹腔注射匹罗卡品的同时,观测并记录实验动物海马EEG波形。三、甲酚紫染色与海马细胞计数动物麻醉后,常规灌流、固定、取材、切片。甲酚紫染色,常规脱水透明,光镜下观测,并进行海马DG、CA1、CA3区神经元细胞计数。四、TUNEL染色实验动物麻醉后,常规灌流、固定、取材、切片。切片浸入3%H₂O₂ PBS液5min以消除内源性过氧化物酶反应。0.01M PBS（pH 7.4）冲洗5min×3次。滴加20%小牛血清和3%小牛血清白蛋白孵育15min,封闭非特异性反应。滴加反应液20μl（TdT 2μ1,荧光素连接的核苷酸混合缓冲液18μl）,切片置于湿盒内（37℃,1h）。PBS洗5min×3次。然后滴加反应终止液（10min,RT）,抗地高辛过氧化物酶抗体（30min,37℃,每次换液前均用PBS冲洗3次,每次5min）。最后DAB显色,光镜下观察,并进行海马DG、CA3的TUNEL阳性细胞计数。五、硫化锌AMG染色实验动物麻醉后立刻灌流,每只实验动物首先灌注0.3%硫化钠溶液10min约150ml,然后灌注生理盐水10min约150ml,最后灌注2.5%戊二醛10min约150ml。常规取材、固定、切片后,AMG孵育液孵育（26℃）,常规脱水、透明、封片、光镜下观察。电镜部分:灌流、固定、取材与前相同,震动切片50μm,AMG孵育液孵育后,常规电镜切片制作,电镜下观察。部分电镜包埋块制作1μm半薄切片,进行AMG阳性反应颗粒图象分析。六、硒化锌AMG染色实验动物腹腔注射0.1%硒酸钠（20mg/kg）。动物存活1.5小时后,腹腔注射4%戊巴比妥钠麻醉。手术打开胸腔,暴露心脏,用蠕动泵经左心室灌注生理盐水150ml和2.5%戊二醛150ml。取材、固定、切片染色等过程同上。电镜部分的制作过程同上。七、TSQ荧光染色过量麻药处死动物,迅速取出大脑,放入液氮中冷冻,将样品置于冰冻切片机中,冰冻切片20μm。避光条件下,切片浸入4.5μM TSQ溶液中孵育60-90秒,然后用0.9%生理盐水冲洗60秒,荧光显微镜观察。八、Zinquin荧光染色过量麻药处死动物,迅速取出大脑,放入液氮中冷冻,然后用冰冻切片机制备20μm厚的冰冻切片。冷丙酮固定10min,Zinquin荧光溶液（1:50）孵育2h（避光）,0.1M PBS冲洗3次各10min,荧光显微镜观察。九、ZNT3免疫组织化学染色实验动物麻醉后,常规灌流、固定、取材、切片,3%H₂O₂ 10min,BSA封闭1h,ZNT3一抗孵育过夜（4℃）,二抗常温孵育2h,SABC常温孵育1h,DAB显色,常规脱水透明后光镜下观察。电镜部分:取材、灌流（灌流液为4%多聚甲醛/0.025戊二醛）、固定同上,震动切片50μm,免疫组织化学染色步骤同上,锇酸染色,后常规电镜包埋块制作,超薄切片,醋酸铀染色,电镜下观察。十、ZNT3 Western Blot分析过量麻药处死动物,迅速取大脑并剥离海马,样品剪碎后加入裂解液4℃过夜。考马斯亮蓝法进行蛋白定量,各组取等量蛋白,聚丙酰胺凝胶电泳分离,并转至PVDF膜上;脱脂奶粉溶液封闭3—7h;一抗孵育过夜（4℃）;二抗常温下孵育2h;后进行显色、曝光、胶片扫描、定量分析。结果一、匹罗卡品致SE小鼠行为观察实验动物匹罗卡品腹腔注射后,经历5-15min的潜伏期,动物表现为:凝视不动、咀嚼、吸鼻或探索行为、湿狗样震颤、反复头颈上仰、吸鼻加强并伴眨眼、面肌痉挛、点头等。此后,动物开始出现Ⅲ级以上发作,50%的实验动物经多次Ⅲ级以上发作后,进入不能自行停止的持续发作状态,即SE。实验动物进入SE的时间大约为25—35min。大约50%的实验动物被诱发出SE,20%左右的模型组动物经历SE后死亡。最终30%左右的模型组动物经历SE并存活。经过15—25天的静息期后,大约90%的成模小鼠观察到1-4次SRS。二、EEG观测结果对照组实验动物,清醒状态下,记录到的EEG以低幅α,β波为主,背景活动较低。潜伏期,模型动物EEG偶尔记录到单个中等幅度的尖波。实验动物开始痫性发作的时候,EEG显示为爆发的锋电位丛,这些锋电位丛被正常背景脑电图所间隔。实验动物SE发作的时候,EEG锋电位丛表现为有规律的,节奏几乎一致的尖波/棘波群。三、甲酚紫染色及海马神经元计数光镜下可见,对照组实验动物海马未见明显的神经元变性改变。模型组实验动物CA1区和CA3区及DG区可见许多神经元发生变性、坏死。SE后24h神经细胞变性坏死最为严重。海马各区细胞计数:模型组海马各区细胞计数减少,与对照比较均有统计学意义（P＜0.05）。四、TUNEL染色结果对照组实验动物在海马DG和CA3区TUNEL阳性反应细胞很少。SE后48h,TUNEL阳性细胞数量在模型动物DG和CA3区明显增多。SE后72h,阳性反应细胞的数量达到高峰。TUNEL阳性细胞计数结果:SE后海马DG和CA3区TUNEL阳性细胞增多,与对照组进行比较均有统计学意义P＜0.01。五、硫化锌AMG与对照组小鼠相比,SE后1h至SE后2h小鼠海马的AMG染色逐渐变浅。与此相反,SE后1h至SE后2h小鼠海马的血管AMG染色逐渐变深。SE后24h小鼠海马AMG染色强度基本恢复到对照组的水平,其AMG血管染色仍较对照组略微增强。电镜下可见各组实验动物苔藓纤维末端突触间隙内均能见到AMG阳性反应颗粒。SE 1h、2h突触间隙出现AMG阳性反应颗粒的几率较多,且间隙内AMG反应颗粒数目也较多。在对照组,电镜下可见有AMG颗粒存在的突触间隙占总数的3.6%,SE 1h组AMG颗粒阳性的突触间隙占总数的14%,SE 2h组,AMG颗粒阳性的突触间隙占总数的17.6%,SE 24h组AMG颗粒阳性的突触间隙占总数的4.8%。SE 1h和SE 2h组有AMG颗粒存在的突触间隙计数与对照相比有显著性差异（P＜0.01）。此外,对照组毛细血管内皮基膜AMG反应颗粒较少,而SE后1h组和2h组,毛细血管内皮基膜内AMG阳性反应颗粒分布逐渐增多,SE后24h组的毛细血管基膜内AMG阳性反应颗粒数量则与对照组类似。半薄切片油镜下CA3区苔藓纤维以及CA1放射层锌离子在各组实验动物海马内的分布情况:与对照组相比,SE后1h、2h后AMG染色强度逐渐递减,而SE后24h,AMG染色强度则与对照组类似。图象分析结果:SE后1h组CA3区AMG阳性反应总面积与对照组相比有显著性差异（P＜0.05）。SE后2h组CA3区AMG阳性反应总面积与对照组相比有显著性差异（P＜0.01）。SE后1h、2h组CA1区放射层AMG阳性反应总面积与对照组相比有显著性差异（P＜0.01）。六、硒化锌AMG染色与ZNT3免疫组织化学染色对照组小鼠海马齿状回颗粒细胞层以及IML未见异常AMG与ZNT3染色。SE 30d组,异常穿越颗粒细胞层的苔藓纤维数量增加,同时,IML内的AMG与ZNT3染色比较密集,形成了一条环绕颗粒细胞层的条带。SE后60d,穿越颗粒细胞层的芽生苔藓纤维继续增多,其在IML形成的致密染色条带且颜色继续加深。电镜下可见,SE后30d模型动物海马内分子层可见被AMG及ZNT3染色阳性的突触结构。AMG颗粒呈现黑点状,大小不等,位于突触小泡的分布区域。而ZNT3免疫染色则主要定位于突触小泡膜上。SE后60d的实验动物,电镜下表现与SE 30d组类似,但突触前部AMG阳性颗粒与ZNT3免疫染色数量和强度明显增强。七、TSQ和Zinquin荧光染色对照组,两种荧光染色均能较好的标记苔藓纤维,而颗粒细胞层及内分子层未见荧光染色。SE 15d组,两种荧光染色阳性反应开始出现在颗粒细胞层。SE 30d组除了颗粒细胞层有荧光反应外,IML开始出现荧光反应。SE 60d组IML出现荧光染色条带。八、Western blot结果应用ZNT3多克隆抗体检测,约在42KD处可见特异性染色条带。对条带进行灰度测定和分析,发现模型组动物海马ZNT3含量明显增加,与对照组相比SE15d组（P＜0.05）,SE30/60d组（P＜0.01）。ZNT3的表达量与SE后动物存活时间呈正相关。结论1、在癫痫发作过程中,模型动物海马苔藓纤维末端突触前部的锌离子可以进入突触间隙内发挥作用。2、在癫痫发作过程中,模型动物海马锌稳态发生明显变化,苔藓纤维内外锌离子出现同步性减少,同时海马内毛细血管向组织中锌转运加强作为一种代偿反应。3、TSQ荧光和Zinquin荧光可以大致体现出MFS的染色特征,但不能显示出神经纤维染色的细节结构。4、SE后30d、60d实验动物海马MFS有ZNT3的表达,且ZNT3在MFS上的分布与锌离子在MFS上的分布存在着空间以及时间上的一致性。说明ZNT3可以作为一种新的检测MFS的标记物。5、ZNT3在海马内表达含量有随着实验动物SE后存活时间延长而增加的趋势。说明ZNT3可能在癫痫的发生发展过程中起着某种作用。更多还原

【Abstract】 ObjectiveEpilepsy is a common type of nervous system disease.The animal experiments and clinical data confirm that zinc homeostasis is involved in the development of epilepsy.Free zinc ion(Zn²⁺)in brain can be detected histochemically and it is also named chelatable zinc ion.Zn²⁺in brain is located in the synaptic vesicles of zinc-enriched neurons（ZEN）.There is a hypothesis that Zn²⁺releases into synaptic clefts and binds with some receptors or cross the postsynaptic membrane,then play its control action under the nervous activitiy.Recently,zinc transporter family members （ZNTs）have been cloned and characterized.The findings of these ZNTs promote the study of zinc homeostasis.Zinc transporter 3（ZNT3）is one of the members of ZNTs.It is a synaptic-vesicle-specific zinc transporter,is essential for the accumulation of zinc within synaptic vesicles,and appears to be limited to the ZEN terminals.The functional significance of vesicular zinc ions and ZNT3 protein in epilepsy is not very well understood.But some researchers point out that the changes of vesicular zinc and ZNT3 can influence the development of epilepsy.The aim of this study is to investigate the distribution of vesicular zinc in seizures and the distribution of ZNT3 in MFS.Methods1.Mouse pilocarpine modelMice were administered an intraperitoneal injection（i.p.）of Atropine at a dose of 1 mg/kg body weight 15-30 min prior to injection of pilocarpine to reduce the peripheral cholinergic effects.Experimental animals were then injected（i.p.）with a single dose of 300 mg/kg of pilocarpine in sterile saline vehicle（0.9%NaCl）.Control mice were administered a comparable volume of vehicle or were not injected after the initial methylscopolamine treatment.Onset of SE was determined by the presence of continuous class 4-5 level seizures as assessed using the Racine scale（Racine,1972）.2.EEG-detectionFor electrode implantation,mice were deeply anesthetized with sodium pentobarbital（50 mg/kg,i.p.）.The animals were placed into a stereotaxic frame.The skull surface was exposed and two small stainless steel screw electrodes were screwed into the skull.The stereotaxic coordinates relative to bregma were:lateral 2.30 mm; posterior 2.10 mm.Ventral meninx fibrosa:2.00mm.After pilocarpine treatment, EEG-detection was performed.3.Cresol purple staining and hippocampal cell countMice were deeply anesthetized with sodium pentobarbital（50 mg/kg,i.p.）,and were perfused transcardially with isotonic saline,followed by 4%paraformaldehyde in 0.1 M phosphate buffer（PB,pH 7.4）.The brains were carefully removed and further post-fixed by immersion into the corresponding fixatives for 3 h at 4℃.The samples were cut into 10μm coronal sections in a cryostat and mounted on glass slides.After Cresol purple staining,sections were observed under light microscope and performed the CA1,DG,CA3 subareas of hippocampus cell count.4.TUNEL assayBriefly,following post-fixation in 4%paraformaldehyde,permeabilization with proteinase K treatment,4%paraformaldehyde again and PBS washes,samples were incubated in biotinylated nucleotide and TdT enzyme at 37℃for 30 min.Following washes and blocking endogenous peroxidase activity using H₂O₂,the biotin labels were detected with streptavidin-HRP（horseradish peroxidase）and a DAB（diaminobenzidine tetrahydrochloride dihydrate）color reaction.Under light microscope,the CA3,DG TUNEL positive cell count was performed. 5.ivZnSAMGAfter mutilation of cervical vertebrae,animals were perfused transcardially with 3%sodium sulfide solution 150 ml,followed by isotonic saline 150 ml,finally by 2.5% glutaraldehyde in 0.1M PB 150 ml.Samples were cut into 10μm coronal sections in a cryostat and mounted on glass slides.Sections were then incubated in the AMG developer in a water bath for 1 h at 26℃.The sections were then dehydrated and coverslipped with neutral balsam for light microscopy.For electron microscopy, samples were cut into 50μm thick slices in a vibratome.Other empirical procedures were same with light microscopy.Some Semithin sections were prepared with an ultramicrotome,and performed image analysis for AMG grains.6.ZnSeAMGMice were injected intraperitoneally with sodium selenite（20 mg/kg）.After 1.5 h survival time,mice were deeply anesthetized with sodium pentobarbital（50 mg/kg, i.p.）,and were perfused transcardially with 2.5%glutaraldehyde in 0.1M PB.For light microscopy,samples were cut into 10μm coronal sections in a cryostat and mounted on glass slides.For electron microscopy,samples were cut into 50μm thick slices in a vibratome.Other empirical procedures were described as above.7.TSQ fluorescenceMice were killed by an i.p.injection of an overdose of sodium pentobarbital and decapitated.The brains were immediately removed.The samples were then immersed in liquid nitrogen.After 10 minute,samples were cut into 20μm coronal sections in a cryostat and mounted on glass slides.The sections were immersed 4.5μM TSQ solution 60-90 s,followed by being washed with isotonic saline 60s.Sections were then examined in a fluorescence microscope.8.Zinquin fluorescenceAnimals were killed by an i.p.injection of an overdose of sodium pentobarbital and decapitated.The brains were immediately removed.The samples were then immersed in liquid nitrogen.After 10 min,samples were cut into 20μm coronal sections in a cryostat and mounted on glass slides.The sections were fixed with acetone for 10 min at 4℃.Afterward,the sections were incubated with Zinquin solution（1:50） for 2 h at room temperature（RT）.After extensive washing in PBS,the sections were then examined in a fluorescence microscope.9.ZNT3 immunohistochemistryThe perfusion and fixation were same with above.Samples were cut into 10μm coronal sections in a cryostat and mounted on glass slides.The sections were rinsed twice in Tris-buffered saline（TBS,pH 7.4）,and treated with 3%hydrogen peroxide （H₂O₂）in PB for 10 min.After rinsing with TBS,all tissue sections were preincubated for 1 h with 5%bovine serum albumin（BSA）and 3%goat serum in TBS to reduce nonspecific staining.The sections were incubated with anti-ZNT3 serum diluted 1:100 in TBS containing 3%goat serum and 1%BSA and 0.3%Triton-X 100 overnight at 4℃.After extensive washing in TBS,sections were incubated with a 1:200 diluted biotinylated anti-rabbit IgG for 1 h at RT.The ABC Kit was then applied for 1 h at RT in order to visualize the reaction sites.The ABC solution was diluted 1:100 in TBS.A brown color appeared in the sections after incubation of the sections in 0.025% 3,3’-diaminobenzidine with 0.0033%H₂O₂ for 10 min at RT.The sections were dehydrated in graded alcohols and coverslipped with neutral balsam for light microscopy.For electron microscopy,samples were cut into 50μm thick slices in a vibratome.Other empirical procedures were described as above.10.Western BlotMice were killed by an i.p.injection of an overdose of sodium pentobarbital and decapitated.The brains were immediately removed.The resulting homogenate was centrifuged at 12,000 g for 30 min at 4℃.The supernatant was collected and total protein levels were measured by a BCA protein assay kit.Sample protein was separated on 10%SDS-polyacrylamide gels,and the proteins were transferred onto PVDF membranes in an electronasfer device.Then the membranes were blocked in 5%nonfat milk in TBS containing 0.05%Tween 20（TTBS）for 3 h and then incubated in primary antibody overnight at 4℃.The dilutions of primary antibodies were 1:500 for ZNT3 and 1:12000 for GAPDH.Membranes were washed with TTBS and followed by incubating with horseradish peroxidase-conjugated secondary antibody for 2 h at RT with constant agitation.The membranes were washed and reacted with an enhanced chemiluminescence.（ECL）kit（Pierce,CA）,and visualized by exposed to Kodak-XAR film.Results1.Pilocarpine-induced seizuresAfter pilocarpine treatment 5-15 min,mice typically displayed behavioral activity changes（e.g.wetdog shakes,slight body tremors,salivation,and stiffening of the tail）. About 50%of mice developed SE and survived.After a period of 15-25 days in which normal activity was observed,90%mice that survived SE were observed to experience spontaneous seizures.2.Electroencephalographic analysisBackground EEG in the awake animal was recorded.In control group,EEG displayed a slow voltage activity noticed,includingα,βwave.In incubation period,a single sharp wave can be observed infrequently.Electrical spike-bursts,separated by normal or slightly suppressed background activity,emerge at the beginning of the seizure activity.Under SE,the spike-bursts disappeared and a regular spike-wave pattern of activity predominates3.Cresol purple staining and hippocampal cell countUnder light microscope,there was no any cell damage in all subareas of control mice hippocampus.For pilocarpine model,cell degeneration,cell death and cellular swelling were observed in CA1/CA3/DG subareas of hippocampus.24 h onset of SE, the cell damage was most severe.After 48 h of SE,cell damage was catabatic to some extent.Cell count of CA1/CA3/DG:the survival cells in CA1/CA3/DG in the epileptic mice hippocampus were decreased than that of control mice.Cell count for all group of epileptic mice were statistically different from the control mice（P＜0.05）.4.TUNEL assayThere were few TUNEL positive cells in CA3/DG subareas of hippocampus.SE 48h,TUNEL positive cells increased obviously either in CA3 or in DG.SE 72h,the quantity of TUNEL positive cell achieved the maximum.TUNEL positive cell count: all groups of epileptic mice were statistically different from the control mice（P＜0.01）.5.ivZnSAMGCompared with control group,SE 1h group AMG staining intensity was bit lower, whereas its blood capillary’s AMG sataining intensity was higher.SE 2h,the AMG grains in hippocampus decreased more seriously,whereas its blood capillary’s AMG sataining was most intense.For SE 24h,either its hippocampal AMG staining or blood capillary’s AMG sataining were similar with that of control group.Under electron microscope,AMG grains were observed in synaptic clefts of mossy fiber terminals of all groups’ mice.For SE 1h and SE 2h,AMG reaction positive synaptic clefts were more frequent than other groups and the quantity of AMG grains were more confertim.In control group,about 3.6%synaptic clefts had AMG grains existence.For SE1h group,it reached 14%.There were 17.6%synaptic clefts were AMG positive in SE2h group.SE24h,the percentage of AMG positive synaptic clefts descented to 4.8%.Percentage of synaptic clefts which had AMG grains existence of SE1h and SE2h were statistically different from the control mice:P＜0.01.The distribution of AMG grains in blood capillary basal lamina have similar tendency with that in synaptic clefts.From SE1h to SE2h,there was a progressively increasing tendency for the quantity or intensity of AMG grains in blood capillary basal lamina,whereas it is decreased in SE24h and similar with control group.From semithin section,AMG grains in CA3 mossy fiber terminal and CA1 reflecting layer image analysis were performed.Compared with control group,the AMG staining intensity in CA3 and CA1 subareas of SE1h and SE2h mice were gradually descendent.24h after SE,the AMG staining intensity in both subareas was similar with control group.Total areas of AMG reaction in CA3 mossy fiber terminal, SE1h was statistically different from the control mice:P＜0.05.SE2h:P＜0.01.Total areas of AMG reaction in CA1 reflecting layer,SE1h and SE2h were statistically different from the control mice:P＜0.01.6.ZnSeAMG and ZNT3 immunohistochemistryIn control group,no mossy fiber outgrowth could be detected in the IML in both ZNT3 immunohistochemistry and AMG staining sections.In the group of SE15d, ectopic mossy fiber terminals stained by AMG and ZNT3 were found crossing the granular laye.But the AMG grains and ZNT3 immunostaining in IML were scarce.In the group of 30 days after pilocarpine treatment,animals were found to have significant MFS versus controls.Both ZNT3 and AMG stained sections displayed a dense band in the IML consistent with aberrant mossy fiber sprouting.At 2 months after pilocarpine treatment,there was a progressively increasing pattern of staining intensity of ZNT3 and AMG staining in the IML and granular layer.At electron microscopic level,ectopic terminals stained by AMG and ZNT3 were found in IML of hippocampi after 30 days and 60 days pilocarpine treatment.AMG and ZNT3 stained hippocampal sections revealed AMG grains and ZNT3 immunoreactivity in regions corresponding to the MFTs.Almost all membranes of synaptic vesicles were ZNT3 positive.In general,there was a progressively increasing tendency for the quantity or intensity of silver granules and ZNT3 immunoreactivity in ectopic terminals in IML from 30 days to 60 days after pilocarpine treatment.7.TSQ,Zinquin fluorescenceIn the control group,TSQ and Zinquin fluorescence was present in the regions corresponding to mossy fiber terminal fields.15 days after pilocarpine treatment, fluorescence was found in the granular layer.In the group of 30 days after pilocarpine treatment,TSQ and Zinquin fluorescence was present in IML.At 2 months after pilocarpine treatment,there was a progressively increasing pattern of staining intensity of TSQ and Zinquin fluorescence in the IML and granular layer. 8.Western blotThe expression levels of zinc transporter protein ZNT3 in the pilocarpine treated mice hippocampi were measured using Western blot analysis.The semiquantitative analysis of immuno-blots showed statistically significant elevations of ZNT3 in hippocampi of mice at 15 days after seizures compared age-matched control mice （P＜0.05）.ZNT3 contents in hippocampi of mice after 30 and 60 days treatment of pilocarpine significantly increased compared with age-matched controls（P＜0.01）.The expression of ZNT3 protein in hippocampus had an increased tendency from 15 to 60 days after pilocarpine treatment.Conclusion1.Vesicular zinc ions in mossy fiber terminal could release to synaptic clefts in seizures.2.In seizures,zinc homeostasis of hippocampus altered markedly.Hippocampal zinc content in mossy fiber decreased simultaneously.To compensate zinc consumption in seizures,transportation of zinc from blood to tissues by blood capillary increased.3.TSQ and Zinquin fluorescence staining in epileptic hippocampus roughly displayed the characteristics of MFS.But they could not describe the details of mossy fiber terminal.4.After SE 30 and 60 days,MFS of experimental animal hippocampus could be detected the expression of ZNT3.The distribution of ZNT3 and zinc in MFS had a space-time concordance,suggested that ZNT3 immunohistochemistry could be served as a new method to detect MFS.5.The progressively increasing ZNT3 content in TLE hippocampus might be relative to functional significance of ZNT3 in epilepsy.更多还原