HYPOXIA/ISCHEMIA EXPANDS THE REGENERATIVE CAPACITY OF PROGENITORS IN THE PERINATAL SUBVENTRICULAR ZONE Z. YANG AND S. W. LEVISON* Department of Neurology and Neurosciences, UMDNJ–New Jersey Medical School, Newark, NJ 07101, USA Abstract—Neurons and oligodendrocyte progenitors are highly sensitive to perinatal hypoxic–ischemic injury. As ac- cumulating evidence suggests that many insults to the hu- man infant occur in utero, and preventing brain damage to infants in utero will prove difficult, there is strong rationale to pursue regenerative strategies to reduce the morbidity asso- ciated with developmental brain injuries. The purpose of this study was to determine whether a hypoxic–ischemic insult stimulates the neural stem/progenitor cells in the subven- tricular zone to generate new neurons and oligodendrocytes. Hypoxia–ischemia was induced using the Vannucci rat model on postnatal day– 6 pups. Injections of 5=-bromo-2=-deoxyuri- dine to label cells undergoing DNA synthesis after hypoxia– ischemia revealed that there is a robust proliferative re- sponse within the subventricular zone of the injured hemi- sphere that continues for at least 1 week after the hypoxic– ischemic episode. Using the neurosphere assay to quantify the number of neural stem/progenitor cells in the subven- tricular zone, we find that there are twice as many neural stem/progenitor cells in the affected dorsolateral subven- tricular zone at 1 week of recovery and that these cells gen- erate larger spheres in response to growth factors compared with controls. Precursors from the injured hemisphere gen- erate three times as many neurons in vitro and more than twice as many oligodendroglia compared with controls. Hy- poxia–ischemia also increases neurogenesis in vivo. Dou- blecortin positive cells with migratory profiles were observed streaming from the ipsilateral subventricular zone to the stri- atum and neocortex, whereas, few doublecortin positive cells were found in the contralateral hemisphere after hypoxia– ischemia. These observations provide evidence that the so- matic neural progenitors of the subventricular zone partici- pate in the production of new brain cells lost after hypoxia–ischemia. © 2006 Published by Elsevier Ltd on be- half of IBRO. Key words: neurogenesis, oligodendrocyte, neural stem cells, hypoxia–ischemia, rat. Recent experimental data indicate that diverse forms of ischemic injury stimulate neural precursor proliferation (Jin et al., 2001; Zhang et al., 2001; Arvidsson et al., 2002; Parent et al., 2002). Most of these investigations have focused on neuronal production in the adult brain. How- ever, there are clinical situations where there is significant oligodendrocyte degeneration, such as injuries sustained by infants as a consequence of inadequate cerebral blood flow or oxygen deprivation. Advances in neonatal critical care have dramatically improved survival rates from these injuries, but approximately 50% of hypoxic–ischemic (H/I) infants will subsequently suffer neurologic sequelae includ- ing cerebral palsy, epilepsy, and cognitive deficits (Volpe, 2001). Both neurons and oligodendrocytes are highly sen- sitive to H/I injury with the specific pathology related to the developmental stage of the CNS (Hill et al., 1995; Naka- jima et al., 2000; Ness et al., 2001; Back et al., 2002; McQuillen et al., 2003). In a term infant there is a both neuronal and glial injury while in pre-term infants white matter injury predominates (Volpe, 2001). Increased neu- rogenesis alone in pre-term infants would, therefore, be inadequate to restore normal brain development. Over the past 20 years a large number of studies have evaluated neuroprotective strategies to prevent brain dam- age subsequent to developmental brain injuries. However, there is a growing body of literature supporting the view that in many cases the infant sustains brain damage in utero (Ferriero, 2004; Wu et al., 2004). As preventing brain damage to infants in utero will prove difficult, there is strong rationale to pursue regenerative strategies to re- duce the morbidity associated with these events. It is well established that neural stem cells (NSCs) and multiple classes of transit-amplifying progenitors, reside in the sub- ventricular zone (SVZ) of the mammalian brain throughout life (Reynolds and Weiss, 1992; Palmer et al., 1995; Sanai et al., 2004). As it is still not possible to discern NSCs from multipotential progenitors (which possess limited self-re- newal), especially in the neonatal SVZ, we have adopted the term neural stem/progenitor cells (NSPs) to encom- pass both populations. These NSPs provide the greatest potential to regenerate various neural cells required to reconstitute functional recovery, since NSPs in the neona- tal SVZ not only generate new neurons but also generate new oligodendrocytes (Levison and Goldman, 1993; Luskin, 1993; Levison et al., 1999; Suzuki and Goldman, 2003; Merkle et al., 2004). Ischemic insults in the adult and immature brain stimulate proliferation of precursors in the SVZ, however, there are limited data showing that there is significant generation of new neurons and oligodendro- cytes to repair and repopulate the damaged brain. Thus, *Correspondence to: S. W. Levison, Laboratory for Regenerative Neu- robiology, Department of Neurology and Neuroscience, UMDNJ–New Jersey Medical School, 185 South Orange Avenue, H-506, Newark, NJ 07101, USA. Tel: +1-973-676-10001155; fax: +1-973-395-7233. E-mail address: steve.levison@umdnj.edu (S. W. Levison). Abbreviations: ANOVA, analysis of variance; BrdU, 5=-bromo-2=-de- oxyuridine; CCA, common carotid artery; DAPI, 4=,6=-diamidino-2-phe- nylindole dihydrochloride hydrate; Dcx, doublecortin; EDTA, ethyl- enediaminetetraacetic acid; EGF, epidermal growth factor; FBS, fetal bovine serum; FGF-2, basic fibroblast growth factor-2; GFAP, glial fibrillary acidic protein; H/I, hypoxia/ischemia or hypoxic–ischemic; NeuN, neuronal nuclei; NSC, neural stem cell; NSP, neural stem/ progenitor cells; P, postnatal day; PBS, phosphate-buffered saline; SVZ, subventricular zone; SVZ DL , dorsolateral subventricular zone; Tuj1, III-tubulin. Neuroscience 139 (2006) 555–564 0306-4522/06$30.00+0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.12.059 555