Effect Of Heterotherapy For Homopathy On The Excitability In Brain Regions And The Glutamate Metabolism System In The Pentetrazole-kindled Chronic Epileptic Rats

BackgroundEpilepsy, a functional disturbance of the CNS and induced by abnormal electrical discharge, manifestes by recurrent seizure. According to the WHO statistics, The prevalences of epilepsy in developed countries, economies in transition countries, developing countries and least developed countries were 5.0‰,6.1‰,7.2‰and 11.2%o.It was estimated that there were about 50 million epilepsy patients worldwide. In 2001,the national epidemiological survey of epilepsy demonstrated that the morbidity rate of epilepsy was 7.0‰,and the number of epileptic patients has risen to as many as 5-6 million in our country.Common clinical antiepileptic drugs such as phenytoin, sodium valproate, carbamazepine, B Hu amines have been the main drug treatment for epilepsy, but the pharmacokinetic limitations and the side effect on the bone density, the cognitive function and so on limited their application.Beside, there were about 20% of intractable epilepsy can not be controlled with drugs. Traditional Chinese medicine had been used to cure epilepsy for a long time and had some certain advantages.The pathogenesis of epilepsy was extremely complicated.With the helping of experimental animal models to simulate the human epileptic seizures and to study the pathogenesis of epilepsy had become one of the research focus in Neuroscience.PTZ kindling model had been recognized as one of the seizure models,which was the represent model of full force clonic seizures and could more objectively reflect the role of antiepileptic drugs.The excitability of the neuron increasing and the synchronously electric releaseing excessive had been recognized as the basic condition of epilepsy currently. The occurrence of epilepsy was related to the excitability of the inhibited amino acid neurons and the excited amino acids neurons in the CNS.And so if the excitatory neurotransmitter excessive accumulating, the excitability of neurons would increase and the neurons would be damaged. The energy metabolism and the functional activity throughout the brain are closely related, and during the epilepsy the glucose metabolism around the epileptogenic focus showed a obviously high metabolic comparing with the interictal.Glutamate (Glu) the most important excitatory neurotransmitter in CSN, excessive accumulating within the synaptic cleft would result in neuronal excitability increasing and neurotoxicity, which was one of the resons for seizure.And during the seizures, the Glu continued to accumulate which would further increase the toxicity of the nervous system. GLT-1 (EAAT2) was a glial cell glutamate transporter, which could uptake 90% Glu in synaptic cleft to local glial cells.At the promotion of the glutamine synthetase (GS) in the glial cells Glu was transformed to glutamine, which removed the excitability toxicity of Glu."The same disease with different treatment" was one of the characteristics of traditional Chinese medicine. Epilepsy belonged to Chinese "Epilepsy" category and the syndrome differentiation and treatment therapies was diffenrent. According to pathogenesis, the treatment therapies of Traditional Chinese Medicine for epilepsy involved "from the sputum of governance","from the Liver" and others.In order to explore if there was some different between the two treatment methods in the feature and mechanism on the same type of seizure, we selected Chaihushugantang and Dingxianwan to treat chronic epilepsic rats kindinged by PTZ, observed and compared the effects on the epileptic seizure, the level of glucose metabolism in different brain regions and excitatory neurotransmitter and Glu metabolism pathway in the hippocampus of the two compounds on PTZ-kindling rats.Method and content1.To establish the model of epileptic rats kindled by PTZAfter selecting 9 Wistar rats from the whole 150 rats as the control group, the surplus rats were injected intraperitoneal with PTZ 35mg/kg every morning and the control group rats with normal saline for 21 days.Following the Racine classification standard, the seizure level was evaluated.The rats which seizured up to the forth degree or over at least 3 times during the 18±3 injected days were injected again with the same dose of PTZ after one week, and those rats seizured again up to the forth degree or over were considered as kindling successfully.2.Therapeutical effect of the two compounds on PTZ-kindled rats and the EEG of the epileptic rats60 Fully kindled rats were randomly divided into 6 groups, that were normal sodium group(group A), Valproategroup(VPA, group B),DXW group(group C), large-dose CHSGT group(group D), medium-dose CHSGT group (group E)and low-dose CHSGT group(group F), and very group were administrated continuously for 28 days.According to the Racine standard, the seizures of epileptic rats including the seizure potential period,the seizure serious degree and the rate of tetanic were observed on the 7th,the 14th,the 21th and the 28th day after injected with PTZ 35mg/kg again. On the 14th and the 28th days two epileptic rats were randomly selected from each group for EEG tracings with 16 physiological signal acquisition system.3.Effect of the two profounds on the excitability in Cortex and Hippocampus (different brain regions) of PTZ-kindled rats54 Fully kindled rats were randomly divided into 6 groups, that were epileptic model group(group B), Valproate group(VPA, group C), DXW group(group D), large-dose CHSGT group(group E),medium-dose CHSGT group(group F) and low-dose CHSGT group(group G), and another 9 rats as control group(group A). Every group were administrated continuously for 28 days.6 rats randomly selected from each group after administrated 1.5 hours on the 28th day were injected with PTZ35mg/kg or normal sodium again. With 10% chloral hydrate anesthesia, the brains of the rats were removed on ice, and the cortex and hippocampus were separated and reserved at-80℃frozen.3.1 Effect of the two profounds on the level of glucose metabolism in different brain regions of PTZ-kindled ratsApplicated fluorescence imaging to detect brain areas 2-NBDG content. On the 28th day, after intragastric administration for 1 hour the remaining three rats of each group were intravenous injected with the 0.1mg/0.1ml 2-NBDG 0.2ml.After that the rats were intraperitoneal injected with PTZ35mg/kg or normal sodium again.After anesthesia the breasts of the rats were opened and the rats were perfusion fixed by the ice saline and 4% paraformaldehyde. Reference to "brain stereotactic atlas" (George Paxinos the People Health Publishing House), the coronal slices which were thick 500μm were cut on the freezing microtome.at the light condition of the excitation 475nm, emission 550nm, the slices were imaging and analyzed in the vivo fluorescence imaging device conditions.2 slices of each rat were selected to analyze, and according to the formulaV/B=(the ROI of left cortex or hippocampus and the ROI of right cortex or hippocampus) divide with the ROI of the whole brain slice to calculate the V/B value.3.2 Effect of the two profounds on the content of the Glu and the Asp in different brain regions of PTZ-kindled ratsThe contents of the Glu and the Asp were detected by way of high performance liquid chromatograph (HPLC).The cerebral cortex and hippocampus accurately weighed were placed in a pre-cooling glass homogenizer to homogenate, in which was added with 80% ethanol at the ratio of 1:4 (w/v).After centrifuged the supernatant of the homogenate was shift into the beaker and evaporated. Adding 0.1 mol/1 HCL solution to dissolve the residue in the beaker under the super sound condition. Shifted the residue solution into a 5ml dissolved bottle and after centrifuged, the supernatant of which was shift into the sample bottle through 0.45μm membrane filter. And then the contents of the Glu and the Asp which were detected 6 times of each group were detected through the HPLC.4. Effect of the two profounds on Glu metabolism pathway in the hippocampus of PTZ-kindled rats54 Fully kindled rats were randomly divided into 6 groups, that were epileptic model group(group B), Valproate group(VPA, group C), DXW group(group D), large-dose CHSGT group(group E), medium-dose CHSGT group(group F) and low-dose CHSGT group(group G), and another 9 rats as control group(group A). Every group were administrated continuously for 28 days.6 rats randomly selected from each group after administrated 1.5 hours on the 28th day were injected with PTZ35mg/kg or normal sodium again. With 10% chloral hydrate anesthesia, the brains of the rats were removed on ice, and the cortex and hippocampus were separated and reserved at-80℃frozen.4.1 Effect of the two profounds on the expression of GLT-1 in the hippocampus of PTZ-kindled ratsThe expression of GLT-1 in the hippocampus of PTZ-kindled rats was detected by way of western blotting. The Hippocampus accurately weighed was grinded in liquid nitrogen, and then the protein was extracted by the tissue protein extraction kit. The concentration of the extracted protein was determined by BCA protein kit. Ploting the standard curve and calculate the sample volume of the interest protein, after which the protein was transferre onto the PVDF membrane by way of western blotting. Finally, with the help of the HRP-ECL luminescence kit the interest protein was exposured through the KODAK imaging systems,and the images were evaluated by the gray value analysis program.The interest protein of each group was dectected 6 times.The expression of the protein was measured by the ratio of the value of gray scale of GLT-1 with the value of gray scale ofβ-actin.4.2 Effect of the two profounds on the the activity of GS in the hippocampus of PTZ-kindled rats The activity of GS in the hippocampus of epileptic rats was detected by the colorimetric method.The protein concentration of the homogenate of the hippocampus was determined by BCA protein kit. Following the instructions of Glutamine synthetase kit for color reaction, the reaction product was transfered into 96-well plate and detected the OD value on the 201-Microplate Reader 6 times for each group.Finally, the activity of GS was calculated in accordance with the formula in the instructions of the kit.5.Statistical methodThe data analysis and treatment was used the SPSS 13.0 statistics software. The measurement data indicated by the mean value gentleman standard deviation, and the result comparison of each group about the seizure potential period at diffenrent time was by the Repeater Measure variance analysis and the multiple comparisons with the LSD law, the the seizure serious degree by Nonparametric Test,the rate of tetanic convulsion by R X C Chi-Square Test, and the detected results of HPLC, the Fluorescence imaging, the western blotting, and the colourimetry were by the single factor variance analysis (One-way ANOVA).P<0.05 expressed that the difference has statistics significance.Result1.To establish the model of epileptic rats kindled by PTZThe number of the rats which seizured up to the forth degree or over at least 3 times during the 18±3 injected days was 125,which accounted for 89% of the total number. The rats were injected intraperitoneally again of PTZ after 1 week interval. There were remain 114 rats seizuring up to the forth degree or over and the kindling rate was 87%. While the control group rats were no spontaneous seizures always.The model was successfully established.2.Therapeutical effect of the two compounds on PTZ-kindled rats and the EEG of the epileptic ratsThe incubation periods of epileptic rats in different times was gradually extended trend, the difference of which was was significant (F=24.338,P=0.000).The different of every group about the the incubation periods was significant (F=15.080, P=0.000).But there was no interaction between time and group effects (F=1.461, P=0.131).Compared with sodium group, the incubation periods of DXW group and large-dose CHSGT group was significantly longer. Compared with large-dose CHSGT group, there was no significant difference between that and DXW group, while the incubation periods of low-dose CHSGT group was significant shorter in the 14th and the 21th day.The seizure serious degree and reduced the rate of tetanic convulsion of each treated group both decreased significantly on the 28th administration day(x 2=14.270,P=0.014;x2=16.800,P=0.005).Compared with each treated group, the frequency of spikes and the amplitude were higher and the duration was longer in the EEG of the saline group.While the frequency of spikes and the amplitude decreased significantly of every treated group, the EEGs of which were becoming normal gradually. 3.Effect of the two profounds on the content of 2-NBDG, Glu and Asp in different brain regions of PTZ-kindled ratsCompared with the control group, the contents of 2-NBDG in different brain regions of the model group were increased significantly(hippocampus P=0.000, cortex P=0.000).Compared with the model group, DXW group and large-dose CHSGT group both could decrease the contents of 2-NBDG in different brain regions significantly. The difference in the contents of 2-NBDG in different brain regions between DXW group and large-dose CHSGT group was insignificant (hippocampus P=0.365,cortex P=0.052).Compared with the control group, the contents of Glu and Asp in different brain regions of the model group were increased significantly (hippocampus P=0.000, cortex P=0.000).Compared with the model group, DXW group and large-dose CHSGT group both could decrease the contents of Glu and Asp in different brain regions significantly. DXW group compared with large-dose CHSGT group, the content of Glu and Asp in cortex and the content of Glu in hippocampus of large-dose CHSGT group were lower significant. (P=0.035,0.001,0.000). 4.Effect of the two profounds on the expression of GLT-1 and the activity of GS in the hippocampus of PTZ-kindled ratsThe expression of GLT-1 in hippocampus of the model was significant lower than of the control group (P=0.000).DXW group and large-dose CHSGT group both could more increase the expression of GLT-1 than the model group (P=0.000,P=0.000).The difference of the expression of GLT-1 in hippocampus between DXW group and large-dose CHSGT group was insignificant (P=0.083).,in different brain regions significantly, the contents of Glu and Asp in different brain regions of the model group were increased significantly(hippocampus P=0.000, cortex P=0.000).Compared with the model group, DXW group compared with large-dose CHSGT group, the content of Glu and Asp in cortex and the content of Glu in hippocampus of large-dose CHSGT group were lower significant.(P=0.035,P=0.001,P=0.000).The expression of GLT-1 in hippocampus of three doses CHSGT, which of large-dose CHSGT group was increased more significantly(P=0.000,P=0.000).The activity of GS in hippocampus of the model was significant lower than of the control group (P=0.000).Compared with the model group, DXW group and large-dose CHSGT group both could increase the activity of GS in hippocampus significantly (P=0.015,P=0.000).DXW group compared with large-dose CHSGT group, the activity of GS of large-dose CHSGT group was higher(P=0.025).In the three doses CHSGT, the activity of GS of large-dose CHSGT group was increased more significantly(P=0.009,P=0.000).Conclusions1.Chaihu Shugan Tang("from the Liver")and DingXianWan("from the sputum") could both inhibit the epileptic seizure and the release of epileptic waves of epileptic rats kindled by PTZ obviously, and both had prominent antiepileptic effect.2.Chaihu Shugan Tang("from the Liver") and DingXianWan ("from the sputum") could both inhibit the excitability in different brain regions of PTZ-kindled rats, by decreasing the level of excitatory neurotransmitter maybe one of their same mechanisms of antiepilepsy, but there was some difference in the effect on the content of Glu and Asp in different brain regions between the two compounds.3.Chaihu Shugan Tang("from the Liver") and DingXianWan ("from the sputum") could both increase the expression of GLT-1 and the activity of GS in hippocampus of PTZ-kindled rats, which reduced the accumulation of glutamate in synaptic space and detoxicate its neurotoxic effect, but the effect of the two compounds on the activity of GS was different.4.Chaihu Shugan Tang("from the Liver")and DingXianWan("from the sputum") both had prominent antiepileptic effect. But there were some difference between them in the effect on the content of the glutamete and the pathway of the glumate metabolism.