Alteration Of Mitochondrial Ultrastructure And Function In The Hippocampus Of Temporal Lobe Epilepsy Model

PART IAN STUDY OF EEG AND PATHOLOGY OF LITHIUM-PILOCARPINE MODEL OF TEMPORAL LOBE EPILEPSYObjective To investigate the alteration of behavior> electrophysiology and pathology of lithium-pilocarpine model of temporal lobe epilepsy, and so to decipher seizure-dependent changes in neuronal injury in rat hippocampus after status epilepticus (SE) induced by pilocarpine(PILO).Methods Male Wistar rats received lithium-pilocaroine to induce SE. After 1h when the rats developed SE, they were treated by diazepam to stop SE. Afterwards all rats were monitored by video recordings to assure development of spontaneous recurrent seizures (SRS), record EEG. study the pathological changes with HE,Nissl and Timm staining at 3h,7d,45d after SE induced by pilocarpine and detect NPY and GFAP immunoreactive positive cells in hippocampus by immunohistochemistry.Results After injection of lithium and pilocarpine, 89% of the rats were induced to develop SE, and after a latency phase, average about 14.5±5.9, SRS could appear. EEG showed accumulated spike waves in acute phase, normal during the latency phase and showed epileptic waves during the chronic phase. The pathologic investigation using HE,Nissl and Timm staining showed the hippocampal neuronal damage and mossy fiber sprouting(MFS). Immunohistochemistry showed that prolonged seizures induce the long-lasting neoexpression of NPY in mossy fibers and MFS pathway, also, GFAP immunoreactivity enhanced in the hippocampus after seizures, indicating astroglisis.Conclusions Epilepsy induced by lithium-pilocarpine develops in 3 phase according to alteration of behavior and EEG: acute phase(status epilepticus),latency phase and chronic phase(spontaneous recurrent seizures). Epileptic seizures induced by lithium-pilocarpine can cause neuronal damage, MFS and astroglisis in the hippocampus of rats, moreover, inhibitory actions of NPY, released after seizures, exered t a protective effect that reduced the risk of seizure recurrence.PART IIALTERATION OF MITOCHONDRIAL ULTRASTRUCTURE AND ENZYMECOMPLEXES ACTIVITY OF RESPIRATORY CHAIN IN THE HIPPOCAMPUSOF TEMPORAL LOBE EPILEPSY MODELObjective To investigate the alteration of mitochondrial ultrastruture and enzyme complexes activity of the respiratory chain in the hippocampus of temporal lobe epilepsy model, so to study the relation between mitochondria and epilepsy.Methods Male Wistar rats were divided randomly into saline control group,acute period groups (3h),silent period group (7d),chronic period group(45d) after status epilepticus(SE) and group which received pilocarpine (PILO) but did not develop SE. Mitochondrial ultrastruture damage was evaluated by electron microscope, and changes of enzyme complexes activity in the hippocampus were detected by enzymatic analysis. The four enzyme complexes of the respiratory chain are as follows: NADH:ubiquinone oxidoreductase(complex I), succinate:ubiquinone oxidoreductase(complex II), ubiquinol:ferricytochrome c oxidoreductase(complex III) and cytochrome c oxidase (complex IV) .Results (1) Mitochondrial ultrastructure was damaged and it varied from mild to profoundly severe in the hippocampus during experimental epilepsy. Mild damage was characterized by early swelling as manifested by separation of cristae. In the more severe cases, mitochondrial swelling was accompanied by clearing of matrix density and disruption of membrane integrity. The most severe damage of mitochondria was vacuolar plus rupture of inner and outer mitochondrial membranes. It should be mentioned that mitochondrial damage from mild to severe existed in all groups tested, just it became more severe as time went on, that is the utmost degree of mitochondrial damage occurred in chronic group. (2) The activity of complex I was significantly decreased 3h,7d and 45d after SE induced by pilocarpine (P<0.05 vs control group) ; the activity of complex II in the hippocampi was not changed by pilocarpine at any time tested; the activity of complex III showed slightly increase at 3h, decreased to the control level 7d after SE, and then decreased significantly 45d after SE (P<0.05 vs control group) ; COX activity in the corresponding hippocampi showed a significant increase 3h after SE (P<0.01 vs control group) , dropped to the control level 7d after SE, and then decreased significantly 45d after SE (P<0.01 vs control group) . Rats receiving PILO but did not present SE showed similar enzyme activities with control rats.Conclusions Our results demonstrate that mitochondrial ultrastructural damage and change of mitochondrial-encoded enzyme activity in the hippocampus are associated with experimental temporal lobe epilepsy, indicating mitochondria were more vulnerable to epilepsy. Thus, the relation between mitochondria and epilepsy in function and structure may provide novel insights into the therapeutic advances that will prevent and even cure epilepsy. PART IIICHANGES OF EXPRESSION OF MITOCHONDRIAL CYTOCHROME C OXIDASE SUBUNITS III AND IV IN RATHIPPOCAMPUS OF TEMPORAL LOBE EPILEPSYObjective To investigate the effects of lithium-pilocarpine induced epilepsy on expression of cytochrome c oxidase(COX) subnuits III(COX III) and IV (COX IV) encoded by mtDNA and nDNA respectively in rat hippocampus, so to study the influence of epileptic seizure on the expression of mitochondrial genome and nuclear genome.Methods Male Wistar rats were divided randomly into saline control group,acute period groups (3h),silent period group (7d),chronic period group(45d) after status epilepticus(SE) and group which received pilocarpine (PILO) but did not develop SE. The expression of COX III and COX IV mRNA and protein in rat hippocampus were detected respectively by real-time quantitative-PCR,immunohistochemistry and Western blot,Results (1) immunohistochemistry: An significant increase in COX III staining was observed in neuronal cell bodies distributed throughout the hippocampus(CA1, CA3, dentate gyrus , and hilus) of rats killed 3h after SE, when compared to the saline-treated group (F<0. 01) . On day 7 after SE, the immunoreactivity was reduced to the control level and the immunoreactivity of cell bodies was restricted predominantly to the dentate gyrus, CA3 and CA1 regions of the hippocampus. The chronic period (45d) showed decreased staining with the findings indicating neuronal degeneration such as condensation of cell bodies, vacuolization and rearrangement of pyramidal cell layers in the CA3 of the hippocampus (P<0. 05) . The reactivity against COXIV was also visualized in all hippocampal formation of all groups studied. The major intensity (not significantly) of cytoplasms staining was found in the hippocampus of rats from acute (3h) group. The silent (7d) and chronic (45d) periods showed similar pattern of hippocampal COXIV immunoreactivity compared to saline-treated group. COXIII and COXIV protein expression showed no alteration in the hippocampus of rats that were unresponsive to pilocarpine. (2) Real-time quantitative PCR(RQ-PCR): COX III mRNA showed significantly increased expression in the group of rats presenting 3h of SE(p<0.001) when compared with the control group. The silent group presented similar levels of expression of COX III, but the chronic group showed significantly decreased level in respect to the control group (p < 0.001). Slightly increased , but not significant expression of COXIV was found in acute(3h) group , then decreased slightly and remained constant during the following days of epilepy compared with saline-treated animals. (3) Western blot: To comfirm the changes of COX III and COXIV protein expression after seizures, we performed Western blotting. Results showed that COX III protein level at 3h after SE significantly increased(p<0.01), then decreased to the control level on day 7, but dropped significantly on day 45 (p<0.01) when compared with the control group. Protein levels of COX IV had no significant differences among groups, though acute group (3h) showed slightly increased level of COX IV. COX III and COXIV level were not changed in the hippocampus of rats that received PILO but did not develop SE.Conclusions our results demonstrate that epileptic seizure can cause the damage of COXIII in gene and protein level, whereas not affect the expression of COXIV, indicating epilepsy can result in mitochondrial dysfunction, mitochondria are more vulnerable to epilepsy. Thus, the relation between mitochondria and epilepsy in function and structure may provide novel insights into the therapeutic advances that will prevent and even cure epilepsy.