Ability to visualize dividing stem cells in the whole brain structures could facilitate the analysis of neurogenesis dynamics, increase its accuracy, and reveal its hidden functional patterns. We used reporter mouse lines with marked neural stem cells (Nestin-GFP and Nestin-CFPnuc) to develop and validate methods for 3D visualization and quantitation of neural stem and progenitor cells and of proliferating cells in the whole brain of adult mice.
Stem and progenitor cells as well as glia and neurons were identified in isolated hemispheres and hippocampi by whole-mount immunohistochemistry (WM-IHC). The population of dividing cells was labeled by 5-ethynyl-2'-deoxyuridine (EdU) and detected by click-reaction with fluorescent azide. Images were acquired using laser-scanning confocal microscopy and 3D reconstructions and image analysis was performed using ImarisBitplane software.
Our methods of WM-IHC and click-reaction allowed staining entire mouse hemispheres and were compatible for simultaneous detection of stem cells and dividing cells. Using whole hippocampi we also developed techniques for automatic counting of stem and proliferating cells in acquired 3D images. These new approaches were validated by conventional methods for detection and quantitation of dividing stem cells.
We next applied our techniques for visualization and quantification of cells in whole hippocampi of mice at different ages and of mice irradiated with 0.34 Gy of fast neutrons. We found significant decreases in the numbers of stem cells and dividing cells during normal aging and after irradiation, demonstrating the utility of our method for quantitative analysis of neurogenesis in whole brain structures.
This study was supported by grant 11.G34.31.0071 from Russian Ministry of Education and Science and by RFBR grant no.14-04-01768.