| IntroductionEpilepsy is a kind of repeating, abrupt, temporary brain tissue malfunction that results from brain cells'excessive discharge. It is one of common neurologic diseases. Presently the effects of antiepileptic drugs are limited, so more and more people have begun to investigate antiepileptic traditional Chinese medicine. Animal experiments and clinical observation have proved Scolopendra, Bombyx Batryticatus, Uncaria and Arisacma Consanguineum have antiepileptic effects of suppressing the shock, expelling the wind , removing the phlegm and ventilating the gas. But whether the compound composed of them has suppressive effect on absence - like seizures of genetic tremor rats hasnt been reported. Furthermore, the animal models of investigating antiepileptic drugs were mostly induced by chemoconvulant or electrical stimulation. Since genetic factor is more primary in clinical epileptic patients, we used tremor rats fetched in from Japan as experimental animal models. We implanted electrodes in cortical and hippocampus of tremor rats and then recorded the electroencephalograms ( EEG) to observe effects of the traditional Chinese medicinal compound composed of Scolopendra, Bombyx Batryticatus, Uncaria and Arisacma Consanguineum on absence - like seizures in tremor rats. Meanwhile,we determined the content of GABA in the hippocampus of the tremor rats by paper electrophoresis to observe the effects of the traditional Chinese medicinal compound on the GABAergic inhibitory system.Methods1. Selection of the tremor ratsCut rat tail into a microfuge tube and add Tail Buffer and Protein-ase K. Vortex briefly, then incubate at 55 overnight. Centrifuge the sample added phenol and chloroform . Collect DNA. Add DNA to PCR work liquid composed of Buffer, Mgcl2, dNTP, primer, Taq DNA Polymerase and distilled water. Amplify PCR under the designed conditions. Weigh agarose and dissolve it in 1 x TAE Buffer and add EB. Fill the gel onto the horizontal board and push it into electro-phoretic groove. Mix the sample and loading buffer and add them into gel and electrophoresis. After electrophoresis analysis the gel image and select the tremor rats.2. Implanting electrode in cortical and hippocampus of tremor ratsSelect eighteen tremor rats of 14-18 weeks old. Randomly divide them into three groups; normal saline group, valproate group (0. 036 g/kg) and traditional Chinese medicinal compound group (3.42 g/kg). Each group includes 6 rats. The animals were anaesthetized with 3% sodium pentobarbital (40 mg/kg,ip). Fix the rat onto the stereotaxic apparatus, cut the skin of the midline between eyes and ears. Scratch out muscles and periosteum. Stanch and sanitize. Drill three bores at the following sites of ( -3 mm, 3 mm) (3 mm, 3 mm) ( -2 mm, -4 mm) from the bregma and implant cortical electrode, reference electrode and hippocampal electrode in turn. Finally embedthe electrodes with dental cement.3. Mensuration of the EEGAfter a 7 - d recovery period, measure the EEG of tremor rats before administration by pen - (tracing polygraph. The three group animals were orally administrated normal saline, VPA and traditional Chinese medicinal compound respectively (10 ml/kg) two times every day and added up to 6 days. And record the EEG of the rats at Ih after administration of 5 days and 6 days and 1 day, 2 days, 3 days and 4 days after stopping administration. Each was continuously recorded for 15 minutes.4. Determination of the content of GABA in the hippocampus of the tremor ratsThe rats were divided into four groups: group D was control group of normal rats, group E was model control group of no administrated tremor rats, group F was VPA group of tremor rats, group G was TC-MC group of tremor rats. Each group was six rats. Rats were decapitated and then we took out hippocampus, added chilled normal saline. Homogenize, centrifuge three times. The supernatant was used for paper electrophoresis. Drop samples on the filter paper. After electro-phoresis for 30 minutes, the filter paper w... |
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