Regulation of notch signaling pathway on proliferation and differentiation of rat’s neonatal brain-derived neural stem cells

Spinal cord injury (SCI) is an important and disabling disease globally. It has been reported that regulation of Notch signaling can affect the proliferation and differentiation of neural stem cells (NSCs). However, when γ-secretase inhibitors and paralyzed spinal cord extracts regulate Notch signaling to better promote neuronal differentiation of NSCs, although its mechanisms remain unclear. The aim of this study were to establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs and to investigate the effects of spinal cord extracts (SCEs) from paralyzed rats at different time points on the proliferation and differentiation of neonatal rat brain NSCs. First, primary rat brain NSCs were isolated and cultured. BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells. Different NSCs specific antibodies (anti-nestin, NF200, NSE and GFAP antibodies) were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining. A rat SCI model was constructed. HE staining was applied to measure the injury changes of spinal cord tissue in SCI rats. The extracts of rat spinal cords at 8, 24, 72h, 5, 7, 14 days were prepared and then co-treated with NSCs. The number of NSCs was measured by using MTT colorimetry to reflect the effect of different paralyzed SCEs on NSCs proliferative capacity. The total RNA of NSCs was respectively extracted to check the mRNA levels of Notch1 and Hes1 genes using RT-PCR. Finally, NSCs were cultured with different spinal cord extracts and DAPT and the total RNA of NSCs was respectively extracted to check the mRNA levels of Notch1 and Hes1 using RT-PCR. Brain derived cells from newborn rats (2 to 3 days) proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging. When BrdU was incorporated into the 5th generation of passaged cells, positive BrdU cells and nestin cells were observed by immunofluorescence staining. After induction of dissociation using 5% fetal bovine serum, positive NF200, NSE and GFAP cells were observed by immunofluorescence staining. The extract of paralyzed spinal cord could upregulated Notch1 and Hes1 mRNA levels in NSCs to encourage NSCs proliferation, which can mimic the microenvironment after SCI. Among them, the SCE at 7 days had the strongest effect on promoting the proliferation of rat brain NSCs and the mRNA of Notch1 and Hes1, which increased with the raise of co-culture time. DAPT can down-regulate the expression level of NSCs Notch1 and Hes1 mRNA and inhibit the proliferation of NSCs, and blocking Notch signaling at 48-72 hours can better promote the differentiation of NSCs into neurons, with the highest ratio of differentiated neurons and more residual NSCs. This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification, and it is speculated that regulating the Notch/ Hes1 signal at 7days after SCI may be the best time to encourage the proliferation of endogenous NSCs.

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