Effects of signal pathway inhibitors on the proliferation of NSCs in 4% HCM.

<p>The number of neurospheres was counted at 24 h (A), and the diameters of neurospheres were measured at 48 h (B). DMSO had no effect on NSCs proliferation. The inhibitor LY294002 resulted in a decrease of the number and diameters of neurospheres. ^**p<0.01, SP600125 compared with DMSO; ^ΔΔp<0.01, LY294002 compared with DMSO, ^##p<0.01, LY294002 compared with SP600125 (n = 3).</p

Immunofluorescence identification of differentiated NSCs.

<p>Neurons and astrocytes derived from NSCs were immunoreactive with anti-β-TubIII and anti-GFAP respectively. All of the nuclei were stained blue with Hoechst33258 (A), β-TubIII+ neurons were stained red (B), and GFAP+ astrocytes were stained green (C). Immunostaining showed that the marker β-tubIII and GFAP never co-localization at the same field (D). Scale bar  = 100 µm.</p

Effects of 4% HCM, LY294002 and SP600125 on the activities of PI3-K, Akt and JNK.

<p>The phosphorylation levels of Akt, PI3-K, and JNK significantly increased in HCM. High levels of p-Akt and p-PI3-K induced by HCM were blocked by LY294002 (B, C). The level of p-JNK was blocked in the presence of SP600125 (E). Representative results of the expressions of Akt/p-Akt, PI3-K/p-PI3-K (A), and JNK/p-JNK (D). ^##p<0.01, HCM compared with NCM; ^**p<0.01, LY and SP compared with HCM (n = 3).</p

Effects of different conditioned media on the rate of proliferation of NSCs.

<p>The number of neurospheres was counted at 24 h (A), and the diameters were measured at 48 h (B). There was no difference in the number of neurospheres between 4% HCM and NCM (A). ^**p<0.01, 4% HCM compared with NCM; ^##p<0.01, 1% HCM compared with NCM, ^ΔΔp<0.05, 1% HCM compared with 4% HCM (n = 3).</p

Effects of different hypoxic conditions on VEGF and BDNF secretions of cerebral cortical cells.

<p>The protein concentrations of VEGF (A) and BDNF (B) in different conditioned media were detected with ELISA. Hypoxia significantly enhanced the secretion of VEGF and BDNF compared with normoxia. ^*p<0.05, ^**p<0.01, 4% HCM compared with NCM; ^##p<0.01, 1% HCM compared with NCM (n = 3).</p

Identification of <i>cerebral cortical cells and</i> neural stem cells.

<p>The cerebral cortical cells were cultured with Neurobasal medium containing 2% B27 for 5 d; the spindly neurites grew out of the cell bodies and were observed as three-dimensional structures (A). Immunofluorescence staining showing nuclei stained blue with Hoechst33258, immunopositive neurons stained red with β-TubIII, and immunopositive astrocytes stained green with GFAP (B). Scale bar  = 200 µm.</p

Immunofluorescence identification of VEGF and BDNF in cerebral cortical cells.

<p>Astrocytes (GFAP+) were stained green, nuclei were stained blue with Hoechst33258, both VEGF+ and BDNF+ cells were stained red. A1 and B1 represent the cells cultured with NCM, B1 and B2 represent the cells cultured with 4% HCM. Expression of VEGF and BDNF was observed in some of the astrocytes (yellow staining in the astrocytes). Scale bar  = 100 µm.</p

Effects of different conditioned media on the differentiation of NSCs.

<p>The highest proportion of β-TubIII+ neurons and the lowest proportion of GFAP+ astrocytes were observed with 4% HCM. The reverse results were observed with 1% HCM. ^**p<0.01, 4% HCM compared with NCM: ^##p<0.01, 1% HCM compared with NCM (n = 3).</p

NSCs primary culture and Nestin identification.

<p>The neural stem cells were cultured with Neurobasal medium supplemented with 2% B27 and bFGF (20 ng/ml) for 48 h. The halos can be seen clearly around the round-shaped neurospheres (A). The neurospheres showed green fluorescence when they were stained with Nestin (B). Scale bar  = 200 µm.</p

Effects of different hypoxic conditions on VEGF mRNA and BDNF mRNA levels in cerebral cortical cells.

<p>The levels of VEGF mRNA (A) and BDNF mRNA (B) in cortical cells cultured under normoxic, 1% O₂, or 4% O₂ conditions. Fold changes were calculated using the ΔΔ<i>Ct</i> method, and the mRNA levels of VEGF and BDNF were detected by RT-PCR. ^#p<0.05, ^##p<0.01, 4% O₂ compared with normoxia; ^*p<0.05, ^**p<0.01, 1% O₂ compared with normoxia (n = 3).</p