| Objective⑴To investigate the primary culture of neural stem cells (NSCs) and induction differentiation of gamma aminobutyric acid(GABA) neurons.⑵To explore a better method to establish ananimal model of temporal lobe epilepsy in rats.⑶To explore feasibility of NSCs were transplanted into hippocampus of rat model of temporal lobe epilepsy induced by Kainic acid to treat EP and examined their survival, differentiation, and observe the changes of rats behavior and pathology and concentration of intracellular Calcium and electroencephalogram ,to provide a theoretical foundation and technical method for treatment of temporal lobe epilepsy.Methods⑴The whole brain of new born SD rat was taken and underwent primary culture .The cultured NSCs and theirs inducted and differentiated GABA neurons were identified by immunofluorescence stain.⑵Kainic acid(KA)with the dosage calculated according to the weight was injected into rat's hippocampus by stereotactic operation. To conform it,s confidence ,the changes of its behavior were observed in Racine,s standard , the changes of pathology were observed by HE and Nissl staining and Image transmission electron microscope , the changes of Imageology were observed by CT and MRI,the changes of electroencephalogram were observed by equipment for video electroencephalogram.⑶NSCs labeled by Brdu were transplanted to KA-injected rat models of temporal lobe epilepsy at right hippocampus CA3 .In 3 months , to examined survival, differentiation and physiologic function of transplanted NSCs, the changes of its behavior of space orientation ability and remembrace were observed by morris water maze , the changes of pathology were observed by HE and Nissl staining and and Immunohistochemistry twice stain and Image of transmission electron microscope, the changes of electroencephalogram were observed by equipment for video electroencephalogram ,the changes of concentration of intracellular Calcium were observed by Laser scanning confocal microscop.Results⑴After culturing for 24h ,neuropheres containing 2 to 4 cells appeared from a cell,and then they became bigger and bigger. Immunofluorescence stain Nestin positive cells in neuropheres.One day differentiation later after differentiation liquid with 10% blood serum was used ,long synapse grew from the wall of neurophere ,which could connect with the synapses from the surrounding neuropheres .A great number of bipolar cells or multipolar appeared around the neuropheres. Immunofluorescence stain showed NSE,GABA,GFAP ,Glu positive cells.The GABA positive cell differentiation rate was (30.25±2.12)% in the experimental group.While (1.14±1.39)% in control group ,p<0.01.⑵After the experimental rats awoke from anesthesia,they presented typical epileptic seizures relapse: wet-dog shakes,lips and head self-movement,Convulsion of forelimbs and hind limbs occurred successively. The seizures were paroxysmal with increased intervals especially rotation seizures were manifest.Their behavior gradually recovered to normal after 8 hours.Then the seizure occurred 1-3times everyweek. They various epileptic dischange in parietal cortex and hippocampus electroencephalogram and its pathology and Imageology.⑶Immunohistochemistry stain shows that Brdu positive reaction and both Brdu and GABA positive cells in rat models,cerebrum,which was transplanted NSCs after transplantation . A significant improvement of transplanted NSCs rat models in behavior and pathology and electroencephalogram and Cutting down concentration of intracellular Calcium .Conclusion⑴The neural stem cells isolated and cultured from new born SD rat by using the present methods,.A greatly increasing the differentiation rate from NSCs to GABA neurons is found in the experience.⑵The seizure pattern and pathological basis and Imageology of temporal epilepsy in the rat model established by stereotactic management were almost the same as those in human beings. The kainic acid induced model is an ideal animal model simulating human temporal lobe epilepsy.⑶The transplantation of NSCs may effectively improve neurological function of rats with temporal lobe epilepsy. Symptoms of rat model of temporal lobe epilepsy induced by Kainic acid can be lessened by transplantation of NSCs,as well as GABA antigen was Expressed , pathology and electroencephalogram was improvemented, concentration of intracellular Calcium was cutted down... |
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