GAD-65,GABA_AR,Glu,NMDAR₁ Protein Expression And Stereological Ultrastructure Study On The Mitochondrion And Synaptic Connections In Brains Of Patients With Medically Intractable Epilepsy

The character of the temporal epilepsy is a common condition in the brain by repetitively occurring seizures caused by abnormal discharging of neurons. It divided into simple partial seizures, complex partial seizures and produce generalized seizure by clinical manifestation and electroencephalogram(EEG). It's pathogenesy very complicated, Epileptic seizures can be considered as excitatory transmitter and inhibition transmitter in the area dissequilibrium. Recently, a strong correlation is the too little inhibition transmitter among glutamic acid decarboxylization(GAD), GABA receptors(GABAAR) and too much excitatory transmitter among glutamic acid (Glu) and N-methy1-D-aspartate (NMDAR) have been paid a lot attention by researcher. To explore the expression of GAD-65, GABAAR,Glu and NMDAR1 protein and to stereological ultrastructure study the ultrastructural changes of human epileptic foci in the intractable temporal epilepsy patients and to discuss its function in the pathogenesis. The specimens were obtained from 45 patients with intractable temporal epilepsy as experiment and 8 patients with arteriovenous malformations and tumors as control. Immunohistochemistry was adopted to test the expression of GAD-65,Glu and GABAAR,NMDAR1 protein in EcoG spike foci were removed surgically . photographs were taden randomly in transmission electron–microscope according to stereology, and then the parameters of mitochondrion of normal and vacuolar degeneration were stereological ultrastructure study under electron microscope. Photographs were taken randomly in transmission electron-microscope according to stereology, and then the parameters of mitochondrion of normal and vacuolar degeneration were calculated involving volume density(Vv),surface density(Sv),numerical density (Nv),average volume(v) referring in cytoplasma, and synaptic density(SD),synaptic active area(SAA),synaptic interface curvature(SIC), surface ratio(SR),synaptic vesicles density(SVD). Mitochondrial ATP ,ADP and AMP contents and ATP production rates were measured by high performance liquid chromatogram(HPLC).Sharp wave, spike wave, spike and slow wave of EEG were measured before operation and in operation. Their difference was compared with controls. The results are as following:1.Light microscope (1) In all spiking areas of SPS TLE specimens, the cells in the cortex of temporal lobe with some degree of vacuolization and edematous. All specimens have minimal of cell loss in CA1.(2) In all spiking areas of CPS TLE specimens, the cells in the cortex of temporal lobe with clear signs of vacuolization ,edematous, pycnosis, chromatin margination, endochylema degeneration or appear vacuole and cell death. In the area of CA1 and CA3 fields and dentate gyrus appear preserved with clear signs of cell loss.(3) In all spiking areas of PGS TLE specimens, the cells in the cortex of temporal lobe with clear signs of vacuolization .The granule cell appears axon spine extending and sprouting. And cytoarchitectonically significant abnormal in horizontal cell and gitter cell.In contrast, cortex of temporal lobe and hippocampal specimens having varying minimal of cell loss and degenerative changes2.Immunohistochemistry Glu and NMDAR1 immunoreactivity is quite prevalent throughout the granular cell layer and pyramidal cell layer. In hippocampal formation with staining evident in all CA3 fields, hilus and subiculum. Glu were heavily immunolabeled and distributed in both the pyramidal cell bodies as well as their dendritic processes. Glu positive granule cell somata show moderate staining with minimal process staining and appear have dendrite hyperplasy. Statistics showed direct correlation between the number of positive reaction cells and occurring seizures frequency and EEG; and negative correlation between that and the course of disease with negative correlation. The number of positive reaction cells in SPS is more than that in CPS and PGS. The perikarya of the granule cells in the dentate gyrus were also consistently immunoreactive. Furthermore, a dense immunolabeling of principle cells was visible in the adjacent subicular complex. The intensity of GAD-65 and GABAA staining varied only slightly in all specimens. Statistics showed negative correlation between the number of positive cells and occurring seizures frequency and between that and course of disease and EEG.3.The ultrastructural changes of the foci were obvious, including the neurons decreasing in number and swelling and degeneration, nuclear pyknosis, marginal collection of heterochromatin; mitochondria swell or disintegrate or capillaries appear twisted and obstructed, or endothelia cells swell; basement membrane increases in thickness or delamination astrocytes swell.4.Under electron microscopy of the foci area, DAB reactive product demonstrating Glu appeared as needle or mass-like precipitation and were located in perikaryon, dendrites and axons, occasionally in nuclei. Whereas, DAB reactive products demonstrating GAD-65 appeared as granular precipitation and were located at rough endoplasmic reticulum, dendritic microtubules and around synaptic vesicles in axon terminals. Two kinds of reactive products were easy to be distinguished from each other in terms of electron density and morphology.5.Stereology results The difference between experiment groups and control group at the vacuolar mitochondrion was observed to reveal significances in Vv, Sv, V(P<0.05) , There was direct correlation with occurring seizures frequency; but it revealed significances at the mitochondrion inδ, Nv(P<0.05) ,and course of disease , EEG with negative correlation. The SD,SAA, SIC, SR, SVD of the synapses in the experiment groups were less than that in the control group. There was direct correlation with occurring seizures frequency, course of disease and EEG. The symmetric axo-dendritic synapses and axo-spine cleft was about 20nm in width and less than that of the control group.Several conclusions are drawn according the study, and lists as following:1. The path-characters is concerned with the epileptic morbility. In SPS spiking areas of TLE specimens, the cells with clear signs of swelling and degeneration; In CPS spiking areas of TLE specimens, the cells with clear signs of vacuolization ,edematous, pycnosis, chromatin margination, endochylema degeneration or appear vacuole and cell death. In PGS spiking areas of TLE specimens, Granule cell somata show moderate staining with minimal process staining and appear have dendrite hyperplasy.2.The studies demonstrate that the GAD-65,GABAAR,Glu,NMDAR1 protein might play an important role in brains of the three different category TLE patients. The differece of distribute and contents may contribute to the different seizure typus.3.The synaptic number was diminishing, but powerful function and fast conduction of velocity fast in PGS. The number of excitatory synapse increase was one of the reasons that cause different type seizure.4.The mitochondrial metabolism in experiment group is weaker than that in control group, especially in CPS. The mitochondrial was intensive and hyperplasy in PGS. The results show the highest energy metabolism highest in PGS of the three seizure typus.