Localization And Expression Of Zn²⁺ And ZNT3 In Pilocarpine Epileptic Mouse Hippocampus

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.