Experimental Study On The Influences Of Ketamine On The Proliferation, Apoptosis, And Differentiation Of Cultured Neural Stem Cell Of Rats

Ketamine is an antagonist of glutamate N-methyl-d-aspartate receptors (NMDA-R) which takes effect on the PCP site of NMDA-R. As a non-competitive antagonist, its effects can not be reversed even by high dose of NMDA or glutamic (Glu). NMDA-R non-competitive antagonist combines with the PCP site in the deep of NMDA-R ion channel, blocking the Ca2+ channel linked NMDA-R to ruduce the inflow of Ca2+, which influences or reverses the adjustment of excitability amino acid (such as Glu,Gly) in developing central nervous system.Recent study showed that neuronal apoptosis can be triggered by the transient blockade of NMDA-R in spurt period in the immature mammalian brain. In humans, the spurt period of developing brain extends from the 6th month of gestation to several years after birth. There is basis for concern that ketamine be extensive used in pediatric and obstetrical medicine for purposes of sedation and anesthesia but ketamine is particularly effective in triggering widespread apoptotic neurodegeneration during this vulnerable period leading to reducing of brain mass and neusrobehavioral disturbances. The influence of ketamine on neural stem cell (NSC) has not been demonstrated. In our study, we isolated a cell line which has abilities to form clones and differentiate into neurons, astrocytes, and oligodendrocytes from hippocampus of embryonic rats by using serum-free cell culture and single cell clone. After identified, the cells was incubated with ketamine in different concentrations.The influence of ketamine on NSC proliferation was assayed by MTT method, its effect on NSC apoptosis was determinated with TUNEL method and its effect on NSC differentiation was analysed by flow cytometry (FCM).Result and discussion:1. The cells separated from hippocampus of embryonic rats posses the ability to form clones and differentiate into neurons, astrocytes, and oligodendrocytes and it can express Nestin antigen. They belong to the forepart embryo cell which have all the attributes of NSC .2. When the concentration is over 50mmol/L, ketamine greatly inhibit the proliferation of NSC. The effect was enhanced with the increasing of ketamine concentration. The IC50 is 467.15mmol/L. the inhibition effect of ketamine on NSC may have relation with thedecreased intracellular Ca2+ concentracion caused by ketamine.3. Ketamine trigger the apoptosis of NSC in vitro and vivo and the apoptosis ratio rose with the increasing of ketamine concentration which showed there exist obvious relativity between them. The mechanism still need more study.4. Ketamine will influence the NSC differentiation in its high concentration (500mmol/L) which decrease the neuron differentiation ratio and increase the astrocytes differentiation ratio. Ketamine reduce the Ca2+ spike potential by decreasing intracellular Ca2+ concentracion and trigger the apoptosis of neuron in great deal which lead to the decrease of neuron ratio differentiated from NSC.