The long-term objective of this project is to understand the relationship between seizures and brain development. During the last period of this project, we established that some types of seizures, in the absence of systemic complications, can damage the immature brain. We also have preliminary evidence that this damage is, in turn, epileptogenic. This application has two goals: first, to determine whether seizure-induced neuronal injury in the immature brain leads to neuronal apoptosis, and to understand its mechanism. Second, to find out whether seizure-induced damage in the immature brain is epileptogenic. We will study the mechanism of neuronal death in the lithum-pilocarpine model of status epilepticus in the immature rat. We will carefully monitor physiological variables such as arterial blood pressure or blood gases, to ascertain that neuronal injury in the that model is the result of the seizures themselves. We will determine the time course of neuronal injury, describe its morphology by electron-microscopy and confocal fluorescesence microscopy in the CA1 pyramids, the inner dentate granule cells, and the large neurons of the dentate hilus; we will determine whether neuronal death is dependent upon protein synthesis; we will look for evidence of early DNA breakdown by restricted endonucleases by the TUNEL method for identify double- stranded DNA breaks in individual cells; and by laddering on agarose gel electrophoresis; we will look for evidence of caspase activation by immunocytochemistry and in Western blots using antibodies against proteolytic fragments cleaved from actin and from poly ADP-ribose polymerase, by using inhibitors of caspases, by using in situ hybridization and Northern blots to detect increased expression of individual caspases. The time course of this phenomena will reveal the biochemical cascade leading to apoptosis in specific cells. We will also use specific inhibitors to study the dependency of most necrotic and apoptotic death of neurons upon the activation of muscarinic cholinergic receptors, of NMDA receptors, and upon the activity of nitric oxide synthase. Second, we will determine whether seizure induced damage in the immature brain is epileptogenic by monitoring seizures in chronic animals subjected to status epilepticus at P15, P21, P28, or as adults. We will chronically administer anticonvulsants after the end of status epilepticus, and study their effects on the development of spontaneous seizures.
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