Correlation Between Epilepsy And Glutamate Expression And Its Metabolism In Cortex And Hippocampus Of Rats

Epilepsy is one of the most common neurologic problemsworldwide, which is badly endangering the health of people. Itis characterized by recurrent seizures and normal inparoxysmal diapauses. Enhanced activity of excitatorysynapses, produced by an imbalance of excitation andinhibition, has been considered as a possible mechanism forsustained epileptic hyperexcitability. Glutamate (Glu) is themajor excitatory neurotransmitter in the mammalianCNS.Astrocytes play a predominant role in energy metabolismand in the catabolism of γ-aminobutyric acid (GABA) andglutamate, neurotransmitters critically involved in epilepticprocesses. Glutamate uptake by astrocytes is quantitativelymost important both for maintaining normal extracellularglutamate concentrations and shaping the kinetics ofglutamatergic synaptic activity. Neuroglial cells massivelyproliferated and demonstrated positive glutamateimmunoreactive in the excised human epileptic focuses hadbeen observed. The result hinted that glial cells may beactivated in epileptic status and release glutamate afterstimulation, and the enhance excitatory of neurons, finally leadto recurrent seizures. So the glutamate immunoreactivity(Glu-IR) positive glial cells should be focused on duringepilepsy research. Based on these points of views, presentstudy is mainly about Glu-IR positive neurons and glial cellsand glutamate metabolism in astrocytes during epileptogenesis.Objective:1 To observe the change of Glu-IR positive cells inepileptic rats brain.2 To study the variety of glutamate in astrocytes inepileptic rats.3 To explore the activity of glutamate dehydrogenases(GLDH) in cortex and hippocampus of epileptic rats.Methods: The study was made up of three parts.1 Part one Preparation of acute convulsive and chronicepileptic rats models. Selected 85 healthy male Sprague-Dawley rats,age 3~4m,weight 240~260g. Acute convulsivemodel was induced by intraperitoneal injection ofpentylenetetrazol(PTZ) 60mg/kg once.By PTZ kindling inchemical method,the chronic epileptic model was built.Ratswere intraperitoneally injected 35mg/kg PTZ every otherday.Then we observed behavior and electroencephalogram(EEG) recording. Expression of FOS protein in cortex andhippocampus was simultaneously observed by immuno-histochemistry at different time after acute convulsive seizure.2 Part two Investigation of glutamate expression inneurons and astrocytes of cortex and hippocampus of theepileptic rats by LSCM.26 SD rats were divided into fivegroups randomly,6 for normal saline group(NS group),5 foracute convulsive group and 15 for chronic epilepticgroup.Among the chronic epileptic group ,there were threegroups,respectively 0h(n=5),7d(n=5)and 60d(n=5).By meansof immunohistochemistry Glu-IR positive cells were observedin different groups.Using LSCM immunofluorescence labelingmethod,we investigated Glu-IR positive and GFAP-IR positivecells.3 Part three The activity of GLDH in cortex andhippocampus of epileptic rats.41 SD rats were divided into NSgroup(n=6,) convulsive group(n=20) and chronic epilepticgroup(n=15) radomly.According to different stage afterseizure,acute convulsive group was divided into 0h,4h,24hand 7d four groups;chronic epileptic group was divided intokindling 0h,7d and 60d three groups.By means of the DGKCmethod,the activity of GLDH was measured in cortex andhippocampus.Results:1 Part one ⑴Behavior observation: After administrationof PTZ 3~5 minutes,convulsive group rats and kindledepileptic group rats were induced IV~V class seizureregularly,even lasting for one hour.While there was no seizurein NS group.⑵EEG recording: NS group showed αrhythmwaves; while epileptic rats recorded high amplitude sharpwaves and spike waves.During paroxysmal diapause periodEEG of chronic epileptic groups had great difference with thatof acute convulsive group,which hinted kindling models wereprepared successfully. ⑶c-fos immunohistochemistry:FOSprotein expression had its own distribution and time rule.Afteracute convulsive seizure ,FOS protein expressed from piriformcortex,entorhinal cortex to dentate gyrus and hippocampus.2 Part two ⑴Glu immunohistochemistry:There weretwo kinds of Glu-IR positive structures;one was Glu-IRpositive neurons,the other was Glu-IR positive glial-like cells.Glu-IR positive neurons in epileptic group were bigger thanthose of NS group obviously(P<0.05).After kindledsuccessfully, Glu-IR positive glial-like cells were found inwhole brain section,especially in cortex and hippocampus,denetate gyrus.But in the kindled 60d group, Glu-IR positiveglial-like cells decreased and had no difference with NSgroup(P>0.05). ⑵LSCM immunofluorescence labelingobservation:①Results of FITC labeling Glu: Glu-IR positiveneurons were green,whose results were consistent with Gluimmunohistochemistry. ②Results of Cy3 labeling GFAP:GFAP-IR positive astrocytes distributed in the wholebrain.Compared with control group, GFAP-IR positiveastrocytes of kindled group showed actively functionalmorphology manifestation,such as the increasement ofbranches and the enlargement of cell body.③Results ofGlu-FITC and GFAP-Cy3 double labeling.When GFAP-IRpositive astrocytes were also Glu-IR positive,the cells wereyellow;so we called those cells Glu-IR positive astrocytes.Thenumber of Glu-IR positive astrocytes in kindled group wassignificantly more than that of NS group.Our findingssuggested that the amount of glutamate in astrocytes increased.3 Part three Compared with NS group,the GLDH activityin cortex and hippocampus of convulsive group and epilepticgroup decreased obviously(P<0.05). ⑴The activity of GLDHin rats of convulsive groups(cortex:0h:1.2904±0.0624;4h:0.9525±0.0247;24h:0.7574±0.1239;7d:0.8550±0.0660;hippocampus:0h:1.0523±0.0664;4h:1.0005±0.0967;24h:0.7253±0.0426;7d:0.8294±0.0478) , compared withthat of NS controls(cortex:1.5145±0.1326;hippocampus:1.2979 ±0.1292), decreased obviously(P<0.05). ⑵Theactivity of GLDH in rats of chronic epileptic groups(cortex:0h:0.9398±0.1143;7d:0.8088±0.0437;60d:0.9518±0.0847; hippocampus:0h:0.9935±0.0419;7d:0.6003±0.0556;60d:0.8531±0.0767), compared with that of NScontrols(cortex:1.5065 ±0.1317;hippocampus:1.2875 ±0.1202), decreased obviously(P<0.05).Conclusion:1 PTZ can induce seizure definitely;we prepareconvulsive and epileptic rat models kindled by PTZsuccessfully.2 There are not only Glu-IR positive neurons but alsoGlu-IR positive glial-like cells in rat brain.3 Glu-IR positive glial-like cells in epileptic group...