The Study Of Zinc Levels, Two Kinds Of Zinc Transporters Expressions And Mossy Fibers Sprouting In The Hippocampus Of Tremor Rats

IntroductionEpilepsy is a common ailment.The alternation of zinc homeostasis in the brain may be associated with the genesis of epilepsy.Zinc transporters play very important roles in maintaining cellular zinc homeostasis.ZnT-1 was shown to be localized to the plasma membrane.Evidences in vivo and vitro experiments have proved there is relationship between cellular zinc levels and ZnT-1 expression.The expression of ZnT-3 protein in brain is highest in mossy fibers of hippocampus.The zinc level in mossy fiber synaptic vesicles is determined by ZnT-3 expression.In this study,we have analysis zinc levels and two kinds of zinc transporters(ZnT-1 and ZnT-3)expressions in the hippocampus of tremor rats,a genetic absence-like epileptic model,to provide more evidences to evaluate the relationships between zinc homeostasis in the brain and absence-like epilepsy.Mossy fiber sprouting(MFS)in hippocampus and neurons deficiency in hippocampus are general pathological phenomena exist in epilepsy.MFS in hippocampus has close relationship with recurrent seizure and it is an important point for researching of epilepsy.In the present study,mossy fibers sprouting were detected in the hippocampus of tremor rats.Materials and Methods1.Animals:tremor rats group,wild type control rats(WTC rats)group and normal Wistar rats group.Each group includes 24 rats and all the rats were 10 weeks old.2.Detect of tremor rats and wild type control rats by PCR. Total DNA of rats was prepared.Tm gene was amplified by PCR with specific tm gene primers.Tremor rats have the positive reactive products.3.Study the zinc levels in the hippocampus of tremor rats,WTC rats and normal Wistar rats with atomic absorption spectrophotometer method.4.Membrane proteins of rats' hippocampus were prepared.Analyze the ZnT-1 and ZnT-3 protein expressions in the hippocampus of tremor rats,WTC rats and normal Wistar rats with semi-qualitative Western blotting method.5.Total RNA of rats' hippocampus was prepared.Analyze the ZnT-1 mRNA expression in the hippocampus of tremor rats,WTC rats and normal Wistar rats with semi-qualitative RT-PCR method.6.Comparing observe mossy fibers sprouting in the hippocampus of tremor rats, WTC rats and normal Wistar rats with zinc selenide autometallography(ZnSe^AMG) technique.Results1.Results of gene detect:Tremor rats have the positive reactive products(the length of product is 197bp).2.The zinc levels in the hippocampus of tremor rats with recurrent absence-like seizures were apparently higher than those of WTC rats(p＜0.05)and Wistar rats (p＜0.05).There was no significant difference of zinc levels between WTC rats and Wistar rats(p＜0.05).3.The ZnT-1 but not ZnT-3 protein expression in the hippocampus of tremor rats with recurrent absence-like seizures was apparently higher than those of WTC rats (p＜0.05)and Wistar rats(p＜0.05).There was no significant difference of ZnT-1 protein expression between WTC rats and Wistar rats(p＜0.05).There was no significant difference of ZnT-3 protein expressions among the three groups of rats.4.The ZnT-1 mRNA expression in the hippocampus of tremor rats with recurrent absence-like seizures was apparently higher than those of WTC rats(p＜0.05)and Wistar rats(p＜0.05).There was no significant difference of ZnT-1 mRNA expression between WTC rats and Wistar rats(p＜0.05).5.The ZnSe^AMGstaining scores of mossy fiber sprouting in dentate gyrus and area CA3 of tremor rats' hippocampus with recurrent absence-like seizures were apparently higher than those of WTC rats(p＜0.05)and Wistar rats(p＜0.05).There was no significant difference of the ZnSe^AMGstaining scores of mossy fiber sprouting between WTC rats and Wistar rats(p＜0.05).Conclusion1.The zinc levels in the hippocampus of tremor rats with recurrent absence-like seizures were enhanced.2.The ZnT-1 expression increased at both protein and mRNA levels and ZnT-3 protein expression didn't change in the hippocampus of tremor rats with recurrent absence-like seizures.3.Mossy fibers sprouting were observed in dentate gyrus and area CA3 of tremor rats with recurrent absence-like seizures.