Research Of The Ultrastructural Changes Of Kainic Acid-Treated Astrocytes

Background:The astrocytes are the most abundant glial cells, and have a very important function for the normal activity of the central nervous system.Compared to the neurons,the understanding for AST is very poor,because the study for ASTs in vivo is more difficult than for the nerve cells.The most research methods for neurons in vivo are not fit for ASTs.So,the research in vitro become the target pursuited by many researchers.The epilepsy is a kind of familiar disease of nervous system.Although the understanding of the mechanism of the syndrome is not quietly clear, but the abnormal excitation of neurons and the disequilibrium between excibility and inhibition in CNS are considered to be the two key factors for epilepic seizure by most researchers.The recent studies indicated the ASTs not only have the supportive,nutritious and protective functions in CNS, but also play an important role in the activity of neurons through the microenvironment. They can infect the neuronic excitability through adjusting the ion concentration ,for example,ASTs can transport the excess K~+ ,which was released by the exciting neural celts, to micrangium .ASTs also are the important metabolic sites for GA and GABA,which are the capital transmitters for the equilibrium for the excibility and inhibition in CNS.So more and more researchers think highly for the role of ASTs in the epilepic seizure .Kainic Acid( KA) is a kind of acidic amino that has a pair of oxatyls , and it's structure is similar to the thing for the glutamine acid. After stereotaxic injection of KA into ventricle or directly administrated to body, the experimental animals can be found have some epilepic seizures. The clinical performances and pathologic changes of this model are very similar to represetation of mankind's temporal lobe epilepsy(TLE) ,and it has alreadybeen generally accepted by the researchers for the ideal animal model of human TLE .Discovery from the pathologic examination of epilepsy focus manifestd the main performance of the ASTs were hyperplasia,hypertrophy and even apomorphosis or necrosis. But all of these research experiment were in vivo, because of existing a factor of nerve, it is hard to say this kind of changes of the AST are the reason or the result of epilepic seizure, or both of they . The observation of the changes of ASTs after administrating with KA in vitro would be helpful to answer the tough problem.Objective:To culture the rat hippocampous ASTs in vivo, then the ultrastructural changes of the cells in respond to kainic acid exposure were observed to reveal whether the KA has a toxic effect to ASTs in vitro.Method:1. Neonate Wistar rat hippocampal ASTs were culturesd for 8 days in DMEM culture media containing fetal calf serum (FCS) and equine serum .Then the culture flasks were taked into swing bed for oscillating 14 hours. Gathered the attached cells to new culture capsules for subculture .When the cells covered the capsules on the whole(the time is about 7days) ,the immunocytochemical stain of GFAP was made to identified the ASTs purity.2. The subculture cells was randomly divided into five groups : (a) two levels of KA concentrations (A): 25umol/L(Al)and 250umol/L(A2);(b) two levels of KA treatment duration(B):10min(Bl) and 100min(B2);(c) control group was treated with the equivalent saline.3. According to the experiment design, samples were treated with different interventions, followed by fixation, washing and dessication .Then the samples can be sanned directly by atomic force microscope(AFM) ,but the samples for transmission electron microscope(TEM) must be standed for more treatments suchlike alcoholic dehydration,embedment,tinction,and so on.4. The intracytoplasm ultrastructure of the rat hippocampal ASTs were observed by TEM,and the surface ultrastructure were scanned by AFM .Result:1. The proportion of GFAP positive ASTs is over 90% in the subculture cells.2. The changes of the intracytoplasm ultrastructure.Control group: the shape of the nucleus was regular ,look like round or oval;the plasmosome was not obvious,and the chromotin was sparse;no abnormality was found in others organelle .Group AlBl:the nucleus were still normal ,but some mitochondria were swollen and the number of the ribosomes is increased.Group A1B2: some nucleus were irregular ,there were many vacuoles and ribosomes in the kytoplasm;Group A2Bl:the nucleus were regular,but the heterochromatin was increased, vacuoles and ribosomes were also very ease founded in the endochylema;Group A2B2: the shape of the nucleus was twisted , huge plasmosomes can be found,a lot of vacuoles in the endochylema ,and the autophagosomes were found in some cells.3. Outcome of AFM scanning(1) The changes of the surface ultrastructure.Control group: Somas and projections of ASTs were in good shape.When the images of soma surface were zoomed in ,regular and homogeneous light wrinkles could be seen ,but the surfaces were generally glossy.Group A1B1: no special abnormality was found in the cells,but when the images were zoomed in,the surface was round .Group A1B2: the somas were a little swollen,the projections became thicker and the surface was round.Group A2B1: the cells were swollen , the projections were increased and some of them were ruptured .When the images were zoomed in ,irregular pores could be seen on the somas surfance.Group A2B2: the shape of the cells were irregular and the somas were swollen ,their projections were ruptured. When the images were zoomed in,there were many irregular pores in the surface,even,in minority of the cells , the fissure looked like 'canyon'could be found in the somas.(2) The diameters of pores on the astrocytic surface in the Groups A2Bland A2B2 were measured.There was significant difference between the two groups(P=0.002<0.05).(3) The apparent measurement of each groups cells size.Although the analytical softwares of AFM can not measure the cell volume directly.but they can measure the diameters of the somas in the 3-D images,so we used the half of the cross product with three diameters in the three axes(X,Y,Z) to indirectly reflect the cells size.We found the apparent volume means were Group A2B2> Group A1B2> Group A2B1> Group AlBl>Control Group,but compared with Control Group ,only the Group B2 was significant increasing in statictics( P<0.05). Conclusions:1. Our results indicate the cultured rat hippocampal astrocytes have some injuried ultrastructural changes by treated with KA, and this injuried morphologic changes have a dosage-time dependence.2. The pores showed by AFM maybe the pathologic morphologic foundation of irreversible neurotoxicity.The treatment duration plays a prior role in the KA-induces astrocytic hypertrophy in vitro.