A Clonogenic Survival Assay of Neural Stem Cells in Rat Spinal Cord after Exposure to Ionizing Radiation

Abstract

Neural stem cells play an important role in neurogenesis of the adult central nervous system (CNS). Inhibition of neurogenesis has been suggested to be an underlying mechanism of radiation-induced CNS damage. Here we developed an in vivo/ in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation. Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal growth factor and basic fibroblast growth factor. The survival of the proliferating cells was determined by their ability to form neurosphere colonies. The number and size of neurospheres were analyzed quantitatively at day 10, 12, 14 and 16 after plating. Plating cells from 5, 10 and 15 mm of the cervical spinal cord resulted in a linear increase in the number of neurospheres from day 10-16. Compared to the nonirradiated spinal cord, there was a significant decrease in the number and size of neurosphere colonies cultured from a 10-mm length of the rat spinal cord after a single dose of 5 Gy. When dissociated neurospheres derived from a spinal cord that had been irradiated with 5 Gy were allowed to differentiate, the percentages of neurons, oligodendrocytes and astrocytes as determined by immunocytohistochemistry were not altered compared to those from the nonirradiated spinal cord. Secondary neurospheres could be obtained from cells dissociated from primary neurospheres that had been cultured from the irradiated spinal cord. In conclusion, exposure to ionizing radiation reduces the clonogenic survival of neural stem cells cultured from the rat spinal cord. However, neural stem cells retain their pluripotent and self-renewing properties after irradiation. A neurosphere-based assay may provide a quantitative measure of the clonogenic survival of neural stem cells in the adult CNS after irradiation.