Moderate Low Temperature Preserves the Stemness of Neural Stem Cells (Methods)

Abstract

Moderate hypothermia has been known to protect the brain from inflammation and free radicals after brain injury. However, the cellular mechanisms underlying the amelioration of brain injury by moderate therapeutic hypothermia have not been established. It is considered that neural stem cells reside in the subventricular zone (SVZ) and subgranular zone (SGZ) of the adult mammalian brain and act to regenerate damaged brain tissues. We investigated the effects of moderate low temperature and the contribution of a cold-inducible molecule towards the stemness of neural stem cells. The MEB5 mouse neural stem cell line was cultured in the presence or absence of EGF, and apoptosis, mRNA expression, and immunocytochemistry of the differentiation markers, nestin and GFAP, were evaluated at 37 or 32°C. We investigated the contribution of the cold-inducible RNA binding protein (CIRP) on apoptosis and differentiation of MEB5 cells at 32°C. EGF deprivation increased the number of apoptotic cells, decreased expression of nestin, and increased expression of GFAP. Moderate low temperature prevented apoptosis and decreases in expression of GFAP in MEB5 associated with EGF deprivation. The moderate low temperature significantly increased expression of CIRP. siRNA against CIRP significantly increased the apoptotic cell population of MEB5 cells via EGF deprivation at 32°C. These findings suggest that moderate low temperature preserved the stemness of neural stem cells and prevented cell apoptosis via the stimulation of CIRP. One of the mechanisms of rescue from brain injury via moderate hypothermia is associated with the preservation of neural stem cells.