Neonatal hypoxic-ischemic injury (HI) results in widespread cerebral encephalopathy and affects structures that are essential for neurocognitive function, such as the hippocampus. The dentate gyrus contains a reservoir of neural stem and progenitor cells (NSPCs) that are critical for postnatal development and normal adult function of the hippocampus, and may also facilitate the recovery of function after injury. Using a neonatal mouse model of mild-to-moderate HI and immunohistochemical analysis of NSPC development markers, we asked whether these cells are vulnerable to HI and how they respond to both injury and hypothermic therapy. We found that cleaved caspase-3 labeling in the subgranular zone, where NSPCs are located, is increased by more than 30-fold after HI. The population of cells positive for both proliferating cell nuclear antigen and nestin (PCNA+Nes+), which represent primarily actively proliferating NSPCs, are acutely decreased by 68% after HI. The NSPC population expressing NeuroD1, a marker for NSPCs transitioning to become fate-committed neural progenitors, was decreased by 47%. One week after HI, there was a decrease in neuroblasts and immature neurons in the dentate gyrus, as measured by doublecortin (DCX) immunolabeling, and at the same time PCNA+Nes+ cell density was increased by 71%. NSPCs expressing Tbr2, which identifies a highly proliferative intermediate neural progenitor population, increased by 107%. Hypothermia treatment after HI partially rescues both the acute decrease in PCNA+Nes+ cell density at 1 day after injury and the chronic loss of DCX immunoreactivity and reduction in NeuroD1 cell density measured at 1 week after injury. Thus, we conclude that HI causes an acute loss of dentate gyrus NSPCs, and that hypothermia partially protects NSPCs from HI.
Bibliographical noteAcknowledgements: The authors are very grateful to Dr. Francis Northington and the members of her laboratory for providing instruction on the murine adaptation of the Vannucci model of neonatal HI, as well as for their generosity in providing advice and assistance with this project. Additionally, we wish to thank the organizers and participants of the Ninth Hershey Conference on Developmental Brain Injury for providing a forum in which to discuss our findings and place them in the broader context. We are also grateful to R. Paige Mathena for assistance with manuscript preparation, to Che-Jui Chang for technical assistance, and to Dr. Daniel Berg for advice related to neurogenesis markers. Helpful commentary on the manuscript was provided by Dr. Allan Gottschalk and Dr. Roger Johns.
Funding for this study was provided by a chair grant from the Department of Anesthesiology and Critical Care Medicine at Johns Hopkins University to C.D.M. Also, E.K. was partially supported by a Young Investigator Award from NARSAD. H.S. was supported by a grant from NIH (NS047344).
- Hypoxic-ischemic injury
- Neural stem cell