cytochrome-c-t and Seizures

Epilepsy is a common neurological disorder characterized by abnormal and recurrent neuronal discharges that result in epileptic seizures. The dentate nuclei of the cerebellum receive excitatory input from different brain regions. Purkinje cell loss due to chronic seizures could lead to decreased inhibition of these excitatory neurons, resulting in the activation of apoptotic cascades in the dentate nucleus.. The present study was designed to determine whether there is a presence of apoptosis (either intrinsic or extrinsic) in the dentate nucleus, the final relay of the cerebellar circuit, following kindling-induced seizures.. In order to determine this, seizures were triggered via the amygdaloid kindling model. Following 0, 15, or 45 stimuli, rats were sacrificed, and the cerebellum was extracted. It was posteriorly prepared for the immunohistochemical analysis with cell death biomarkers: TUNEL, Bcl-2, truncated Bid (tBid), Bax, cytochrome C, and cleaved caspase 3 (active form). Our findings reproduce results obtained in other parts of the cerebellum.. We found a decrease of Bcl-2 expression, an anti-apoptotic protein, in the dentate nucleus of kindled rats. We also determined the presence of TUNEL-positive neurons, which confirms the presence of apoptosis in the dentate nucleus. We observed the expression of tBid, Bax, as well as cytochrome C and cleaved caspase-3, the main executor caspase of apoptosis.. There is a clear activation of both the intrinsic and extrinsic apoptotic pathways in the cells of the dentate nucleus of the cerebellum of rats subjected to amygdaloid kindling.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Cerebellar Nuclei; Cytochromes c; Epilepsy; Kindling, Neurologic; Proto-Oncogene Proteins c-bcl-2; Rats; Seizures

Autophagy alterations have been observed in a variety of neurological disorders, however, very few studies have focused on autophagy alterations in epilepsy. The ketogenic diet (KD) likely ameliorates neuronal loss in several seizure models. However, whether this neuroprotective function occurs via starvation-induced autophagy and its prevalence in chronic kindled seizures remains unknown. The aim of this study was to determine the role of autophagy following seizure under KD, and the potential mechanism involved. Pentylenetetrazol (PTZ)-kindled rats, which were fed a Normal diet (ND) or KD, were pretreated with intraventricular infusions of saline, autophagy inducer rapamycin (RAP), or inhibitor 3-methyladenine (3-MA). KD alleviated seizure severity, decreased the number of Fluoro-jade B (FJB)-positive cells in the hippocampus of kindled rats. These effects were abolished by 3-MA pretreatment. RAP pretreatment did not affect seizure severity, but decreased the number of FJB-positive cells in ND group. KD decreased the percentage of damaged mitochondria in kindled group. Hippocampal Beclin-1 was increased by KD in vehicle group. The autophagy proteins Atg5, Beclin-1 and the ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3-I in kindled KD-fed rats were higher, and the autophagy substrate P62 was lower than those in the kindled ND-fed rats, indicating an increase in autophagy following KD. Pretreatment with RAP increased the level of LC3-II/LC3-I, and pretreatment with 3-MA increased the level of P62 in KD-fed rats. To further clarify the mechanism of autophagy protection, the levels of key mitochondria related molecules were examed. The results showed that mitochondrial cytochrome c was up-regulated, cytosolic cytochrome c and the downstream cleaved caspase-3 was down-regulated in KD-fed rats, indicating a decrease in mitochondrial apoptosis. Taken together, our results indicated that KD activates autophagic pathways and reduces brain injury during PTZ-kindled seizures. The neuroprotective effect of KD is likely exerted via a reduction of mitochondrial cytochrome c release.

Topics: Adenine; Animals; Apoptosis; Autophagy; Caspase 3; Cytochromes c; Diet, Ketogenic; Epilepsy; Hippocampus; Male; Mitochondria; Neurons; Neuroprotective Agents; Pentylenetetrazole; Rats; Rats, Sprague-Dawley; Seizures; Sirolimus

Puerarin extracted from Radix puerariae has been shown to exert neuroprotective effects. However, it is still not known whether puerarin protects hippocampal neurons against cell death in pilocarpine-induced seizures. In this study, we found that pretreatment with puerarin significantly attenuated the neuronal death in the hippocampus of rats with pilocarpine-induced epilepsy. In addition, puerarin decreased the level of seizure-induced reactive oxygen species in mitochondria isolated from the rat hippocampi. Terminal deoxyuridine triphosphate nick-end labeling staining showed that puerarin exerted an anti-apoptotic effect on the neurons in the epileptic hippocampus. Western blot analysis showed that puerarin treatment significantly decreased the expression of Bax and increased the expression of Bcl-2. Moreover, puerarin treatment restored the altered mitochondrial membrane potential and cytochrome c release from the mitochondria in the epileptic hippocampi. Altogether, the findings of this study suggest that puerarin exerts a therapeutic effect on epilepsy-induced brain injury through antioxidant and anti-apoptotic mechanisms.

Topics: Animals; Antioxidants; Apoptosis; Caspase 3; Cytochromes c; Enzyme Activation; Hippocampus; In Situ Nick-End Labeling; Isoflavones; Male; Membrane Potential, Mitochondrial; Mitochondria; Neurons; Neuroprotective Agents; Pilocarpine; Rats, Wistar; Reactive Oxygen Species; Seizures; Staining and Labeling

We have demonstrated that kainate (KA) induces a reduction in mitochondrial Mn-superoxide dismutase (Mn-SOD) expression in the rat hippocampus and that KA-induced oxidative damage is more prominent in senile-prone (SAM-P8) than senile-resistant (SAM-R1) mice. To extend this, we examined whether KA seizure sensitivity contributed to mitochondrial degeneration in these mouse strains. KA-induced seizure susceptibility in SAM-P8 mice paralleled prominent increases in lipid peroxidation and protein oxidation and was accompanied by significant impairment in glutathione homeostasis in the hippocampus. These findings were more pronounced in the mitochondrial fraction than in the hippocampal homogenate. Consistently, KA-induced decreases in Mn-SOD protein expression, mitochondrial transmembrane potential, and uncoupling protein (UCP)-2 expression were more prominent in SAM-P8 than SAM-R1 mice. Marked release of cytochrome c from mitochondria into the cytosol and a higher level of caspase-3 cleavage were observed in KA-treated SAM-P8 mice. Additionally, electron microscopic evaluation indicated that KA-induced increases in mitochondrial damage and lipofuscin-like substances were more pronounced in SAM-P8 than SAM-R1 animals. These results suggest that KA-mediated mitochondrial oxidative stress contributed to hippocampal degeneration in the senile-prone mouse.

Topics: Aging, Premature; Animals; Caspase 3; Cytochromes c; Disease Models, Animal; Enzyme Activation; Glutathione; Glutathione Disulfide; Hippocampus; Ion Channels; Kainic Acid; Lipid Peroxidation; Lipofuscin; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred Strains; Mitochondria; Mitochondrial Proteins; Nerve Degeneration; Neurons; Oxidation-Reduction; Oxidative Stress; Proteins; Proto-Oncogene Proteins c-fos; Seizures; Superoxide Dismutase; Time Factors; Uncoupling Protein 2

To explore the influence of duration of convulsion state (SC) on neuronal apoptosis, mitochondrial membrane potential (Deltapsim) and cytochrome C (cyt C) release in hippocampus in Wistar rats after SC.. SC lasting for 30 minutes or 3 hours was induced by intraperitoneal injection of lithium chloride and pilocarpine. Rats were sacrificed at 3, 6, 12 hours and on 1 day after 30 minutes SC and at 3, 6 hours and on 1 day after 3 hours SC. The apoptosis, mitochondrial Deltapsim and intracellular cyt C were determined with flow cytometry, and the correlation with SC duration was compared.. The proportion of apoptotic cells, the decrease in mitochondrial Deltapsim and the release of intracellular cyt C significantly changed at 30 minutes after SC. The peak level of apoptosis was seen at 12 th hour after SC and that of mitochondrial peaked at 6 th hour after SC in apoptosis and the two early apoptotic events, respectively. Compared with the same time point after 30 minutes SC, the levels of apoptosis and the two early apoptotic events after 3 hours SC were much higher. The neuronal apoptosis and the two early apoptotic events in hippocampus after SC showed positive correlation with the duration of SC in partial correlation analysis (all P<0.05).. Severe seizure could induce the changes in neuronal apoptosis and the early apoptotic events in hippocampus after SC. The longer the duration of SC is, the more serious change in apoptosis and early apoptotic events are.

Topics: Animals; Apoptosis; Cytochromes c; Disease Models, Animal; Hippocampus; Membrane Potential, Mitochondrial; Neurons; Random Allocation; Rats; Rats, Wistar; Seizures; Time Factors

Experimentally evoked seizures can activate the intrinsic mitochondrial cell death pathway, components of which are modulated in the hippocampus of patients with temporal lobe epilepsy. Bcl-2 family proteins are critical regulators of mitochondrial dysfunction, but their significance in this setting remains primarily untested. Presently, we investigated the mitochondrial pathway and role of anti-apoptotic Bcl-2 proteins using a mouse model of seizure-induced neuronal death. Status epilepticus was evoked in mice by intra-amygdala kainic acid, causing cytochrome c release, processing of caspases 9 and 7, and death of ipsilateral hippocampal pyramidal neurons. Seizures caused a rapid decline in hippocampal Bcl-w levels not seen for either Bcl-2 or Bcl-xl. To test whether endogenous Bcl-w was functionally significant for neuronal survival, we investigated hippocampal injury after seizures in Bcl-w-deficient mice. Seizures induced significantly more hippocampal CA3 neuronal loss and DNA fragmentation in Bcl-w-deficient mice compared with wild-type mice. Quantitative electroencephalography analysis also revealed that Bcl-w-deficient mice display a neurophysiological phenotype whereby there was earlier polyspike seizure onset. Finally, we detected higher levels of Bcl-w in hippocampus from temporal lobe epilepsy patients compared with autopsy controls. These data identify Bcl-w as an endogenous neuroprotectant that may have seizure-suppressive functions.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Caspase 7; Caspase 9; Cytochromes c; DNA Fragmentation; Electroencephalography; Electrophysiology; gamma-Aminobutyric Acid; Hippocampus; Humans; Kainic Acid; Membrane Proteins; Mice; Mice, Mutant Strains; Mitochondria; Neurons; Proteins; Proto-Oncogene Proteins; Seizures; Status Epilepticus

There is increasing evidence that neuronal cell death induced by seizures occurs via extrinsic (death receptors) and intrinsic (mitochondria) pathways. Caspase-8 cleaves Bid, which releases cytochrome c, bridging the "extrinsic" and "intrinsic" pathways. Cleavage of Bid may amplify caspase-8-induced neuronal cell death following seizures. In the present study, we explored the effect of an inhibitor of caspase-8 (z-IETD-fmk) on the release of Smac/DIABLO and cytochrome c from mitochondria. Rats received intra-amygdaloid injection of kainic acid (KA) to induce seizures for 1 h. The seizures were then terminated by diazepam (30 mg/kg). The damaged and surviving neurons in hippocampus were observed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of caspase-8, Bid, XIAP, caspase-9, cytochrome c and Smac/DIABLO were detected with immunofluorescence and Western blot. The cleavage of caspase-8 and Bid increased at 0 h, cytosolic fraction of cytochrome c and Smac/DIABLO increased by 2 h, cleavage of caspase-9 was detected by 4 h, TUNEL-positive neurons appeared at 8 h and reached a maximum at 24 h following administration of diazepam in the ipsilateral CA3 subfield of hippocampus. Inhibition of caspase-8 significantly decreased neuronal cell death, accompanied by reduction of t-Bid, cleaved caspase-9 and cytosol cytochrome c. Smac/DIABLO from mitochondria was not affected. These results suggest that seizures can lead the translocation of cytochrome c into the cytosol, and the activation of caspase-8 occurs upstream the mitochondria release of cytochrome c and Smac/DIABLO. Inhibition of caspase-8 attenuated neuronal cell death following seizures by decreasing mitochondria release of cytochrome c but not Smac/DIABLO.

Topics: Animals; Apoptosis Regulatory Proteins; Blotting, Western; Carrier Proteins; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Death; Cytochromes c; Cytosol; Enzyme Inhibitors; Functional Laterality; In Situ Nick-End Labeling; Injections, Intraventricular; Limbic System; Male; Mitochondrial Proteins; Neurons; Oligopeptides; Rats; Rats, Sprague-Dawley; Seizures; X-Linked Inhibitor of Apoptosis Protein