Ketamine, an NMDA receptor antagonist has been widely used as an anesthetic, analgesic, and sedative in pediatric settings, and also is an illicit drug consumed increasingly by young drug abusers. Recently, tremendous studies confirm that a high dose of ketamine induces significant cell death of neurons in the developing brain. Neural stem progenitor cells (NSPCs) have been realized as an efficient experimental model to explore the toxic effect of chemicals or drugs on the brain development in the field of developmental neurotoxicity. This study is designed to further evaluate potential negative effects of ketamine on the early development of the brain, using NSPCs isolated from the cortex of embryonic brains at embryonic day 17 and primarily cultured in vitro. Dose- and time-dependent effects of ketamine on the apoptosis and the necrosis were determined using active Caspase-3 immunostaining and LDH assay, respectively. We found NSPCs had a resistance to ketamine-induced cell death expect an extreme high dose of ketamine. To detect the change of the proliferation capacity of NSPCs, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and Ki-67 staining were employed in this study. Results showed that ketamine dose- and time- dependently inhibited the proliferation of cultured NSPCs in vitro. Moreover, an in vivo study using BrdU incorporation method also demonstrated that ketamine dose-dependently reduced BrdU positive cells in the neurogenic region (ventricular zone and subventricular zone) of the developing cortex, which is consistent with the result obtained from in vitro experiments. Tuj-1, a newborn neuron marker was used to test the neuronal differentiation of NSPCs. Data indicated that ketamine enhanced the neuronal differentiation of NSPCs in dose- and time- dependent manners. Finally, to explore the potential mechanism of ketamine-induced changes of the neurogenesis of NSPCs, Akt, the key factor of Phosphoinositide-3 kinase (PI3K) signaling pathway and p27, a downstream factor of Akt, were analyzed using western-blot assays. Results indicated that PI3K signaling was involved in the ketamine-induced the change of the neurogenesis of NSPCs. Taken together, all data suggest that ketamine disturbs the normal neurogenesis of NSPCs via PI3K/Akt-27 signaling, whereas NSPCs are resistant to ketamine-induced cell death.
|Advisor:||Anand, Kanwaljeet S.|
|Commitee:||Al-Chaer, Elie D., Bhutta, Adnan T., Davies, David L., Kane, Cynthia J.M., Phelan, Kevin D., Wang, Cheng|
|School:||University of Arkansas for Medical Sciences|
|Department:||Neurobiology and Developmental Science|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 72/08, Dissertation Abstracts International|
|Subjects:||Neurosciences, Cellular biology, Pharmacology|
|Keywords:||Apoptosis, Ketamine, Neural stem progenitor cells, Neurogenesis, Neuronal differentiation, Proliferation|
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