Effects Of Alcohol On Self-renewal And Differentiation In Neural Stem Cells From Fetal Rat Cerebral Neocortex

Prenatal alcohol exposure can cause developmental abnormalities, growth retardation and dysfunction involved in multiple organs and systems, among which brain is one of the major target organs. Previous clinical study demonstrated that the structural and functional brain developmental abnormalities induced by prenatal alcohol exposure were characterized by neuronal morphologic and functional abnormalities. Since neuroglia can support neurons, most studies focused on the adverse effects of alcohol on neurons and neuroglia.The discovery and isolation of neural stem cells provide a powerful tool for toxicological research. Neural stem cell (NSC) has self-renewal and differentiation ability. NSC renews NSC pool through self-renewal, and produces neurons and neuroglia through differentiation. Disruption of NSC self-renewal and differentiation can affect normal development and function of nervous system. The current project using NSC isolated from fetal rat cerebral neocortex investigated effects of alcohol on NSC self-renewal and differentiation in order to provide scientific proofs for elucidating mechanisms.1 Research on in vitro isolation and culture method of neural stem cells from fetal rat neocortexNeocortex of fetal SD rats was isolated on gestational day 14. Mechanical trituration was used to improve cell dissociation status and conditions of serum-free media were optimized. Three test methods including neural stem cell marker protein (nestin and SOX2), proliferation and clonogenic ability and the multi-lineage differentiation potential were employed to identify neural stem cells. Nestin of cells, cultured with this method, was expressed with strong positive. And more than 99% cells were SOX2-positive. BrdU was incorporated into the cells, and the amount of the cells was 10.55 times as much as the initial cell number after 3 days of culture. The ratio of clonal neurosphere was (33.00±4.40)% after 6 days of culture. Staining with specific protein markers in the corresponding cells confirmed that neural stem cells could form neurons (MAP2), astrocytes (GFAP) and oligodendrocytes (04) after differentiation induction. Neural stem cells obtained with this method have high purity and cell output, potent self-renewal and multi-lineage differentiation ability, which will provide a good model for toxicological research. 2 Effects of alcohol on ultrastructure of NSCs from fetal rat neocortexUsing transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to observe ultrastructure of NSC. The structure of neurospheres was slightly loose, and the majority cells were those with high nuclear/cytoplasma ratio. Neurosphere cells were heterogeneous, and can be divided into lucent (low electron density) and dark ones (high electron density). NSCs derived from neurospheres were represented in all phases of the cell cycle, several cells were apoptotic, and some cells have obvious phagocytosis effects. There were many processes and microvilli on the surface of NSCs, which might be critical structure for sphere formation, substance and information exchange.Alcohol impaired NSC mitochondria with a dose-effect relationship. Alcohol at 25mM induced slight swelling of mitochondria. Alcohol at 50mM induced moderate swelling of mitochondria and broken cristae. Alcohol at 100mM further impaired mitochondria. Through SEM, alcohol made neurospheres loose. Mitochondria damage induced by alcohol can cause apoptosis.3 Effects and mechanisms of alcohol on NSC self-renewal of from fetal rat neocortex3.1 Effects of alcohol on NSC self-renewal from fetal rat neocortexUsing trypan blue exclusion test of cell viability, neurosphere forming assay (number and diameter of neurospheres), BrdU incorporation assay to assess self-renewal ability of NSCs. Live cell number decreased with the increase of alcohol, which accounted for 78% of the control at 100 mM. Number and diameter of neurospheres also decreased with the increase of alcohol, which were dose dependent. BrdU incorporation rate can reflect DNA synthesis status, which decreased with the increase of alcohol with a dose-effect relationship. The results demonstrated that alcohol can inhibit self-renewal of NSCs3.2 Effects of alcohol on cell cycle and mRNA expression of cell cycle related genes of NSCs from fetal rat neocortexUsing flow cytometry (FCM) to analyze cell cycle of NSCs. Alcohol reduced the amount of cells in S and G2/M phase, and increased the amount of cells in G0/G1 phase, no apoptotic peak was observed. The results demonstrated that alcohol induced G0/G1 block, and delayed cell cycle progression. Using Real-time RT-PCR to detect mRNA expression of cell cycle inhibition genes p53,p16,p21 and p27. Alcohol induced mRNA expression of p16 and p21, no significant change of p53 and p27mRNA expression was observed (P>0.05). The results demonstrated that alcohol inhibited G1/S checkpoint transition through upregulation p16 and p21. The block of cell cycle may be mean transition of NSC fate to precusors.3.3 Effects of alcohol on mRNA expression of markers of NSCs from fetal rat neocortexUsing Real-time RT-PCR to detect mRNA expression of NSC markers Nestin,Sox2 and CD133, using FCM to detect SOX2 protein expression. With the increase of alcohol, nestin mRNA expression decreased, there was no change in mRNA expression of Sox2 and CD133, and the abundance of SOX2 protein increased. Alcohol inhibiting nestin and inducing SOX2 expression may be have relations to the initiation of differentiation.3.4 Effects of alcohol on mRNA expression of Egfr and Fgfrl of NSCs from fetal rat neocortexUsing Real-time RT-PCR to detect mRNA expression of NSC growth factor receptors Egfr and Fgfr1. Alcohol induced expression of the two genes, which may be have relations to differentiation of NSCs to precursors.3.5 Effects of alcohol on expression of genes related to Wnt signaling of NSCs from fetal rat neocortexUsing Real-time RT-PCR to detect mRNA expression of Wnt signaling related genes (β-catenin,c-myc and CyclinD1. Alcohol upregulated c-myc mRNA, and had no effect onβ-catenin and CyclinD1 mRNA expression. Besides inducing proliferation, c-myc can induce apoptosis. The results demonstrated that alcohol could induce apoptosis through c-myc.4 Effects and mechanism of alcohol on apoptosis of NSCs from fetal rat neocortex4.1 Effects of alcohol on apoptosis of NSCs from fetal rat neocortexUsing Annexin V/PI staining to detect apoptosis induced by 3d and 4d alcohol administration. Early apoptosis can be induced at 50mM and 100mM after 3 or 4 days.Alcohol induced decrease of mitochondria membrane potential at 100mM after 3 or 4 days. But alcohol didn't alter late apoptosis.4.2 Effects of alcohol on expression of apoptosis related genes of NSCs from fetal rat neocortexUsing Real-time RT-PCR to detect mRNA expression of apoptosis related genes Bcl-2, Survivin, Bax, Bad and Caspase3. Alcohol inhibited survivin expression at 50mM and 100mM after 2 days' administration, induced bax expression at 25-100mM after 3 days' administration, and no change was observed after 4 days' administration. Using colorimetric kit to detect aspase3 activity, there was no significant increase of caspase 3 activity after 2-4d administration.Combined with previous results that alcohol induced c-myc expression, alcohol induced early apoptosis at 25-100mM after 2-4d administration. Apoptosis inhibition gene suvivin was down regulated, proapoptosis gene c-myc was upregulated, and bax was upregulated accordingly, bcl-2/bax ratio was reduced. Mitochondria played a role in alcohol induced neural stem cell apoptosis.5 Effects of alcohol on differentiation of NSCs from fetal rat neocortexUsing Real-time RT-PCR to detect mRNA expression of mRNA expression markers of neurons, astrocytes and oligodendrocytes produced from NSC differentiation. Expression of Tuj1 (neuronal marker), Gfap (astrocyte marker) and Mbp (oligodendrocyte marker) were increased after 4d administration, and no change was observed after 8d administration. The results demonstrated that alcohol influenced mRNA expression of differentiated cell markers mainly at the early NSC differentiation stage.