kainic acid Our previous study Ara et al demonstrated the pr

Our previous study (Ara et al., 2011) demonstrated the protective efficacy of hypoxic-preconditioning against hypoxic–ischemic injury in newborn piglet model. We showed that hypoxic exposure for 3h, 24h before hypoxia–ischemia, was effective in reducing kainic acid damage in newborn piglets while itself causing no morphologic evidence of neuronal injury. Preconditioning performed 24h prior to severe hypoxia–ischemia reduced the neuronal loss in all the brain regions studied at 3 and 7days after cerebral hypoxia–ischemia (Ara et al., 2011). Here, we studied the induction of neurogenesis in response to hypoxic-preconditioning treatment at 1day, 3days and one week after neonatal HI brain injury. To address the question of the potential role of ischemic tolerance in progenitor cell proliferation, we used BrdU, an analog of thymidine. After 1, 3days or one week of recovery from PC or PC performed 24h prior to HI insult; there was an increased proliferation of cells in the SVZ, striatum and white matter as revealed by increased BrdU uptake, as well as by increased numbers of nestin-positive cells in the entire SVZ. These new cells were found to express neuronal and astrocytic markers, double cortin or GFAP. This increase in cells in S-phase within the region of the SVZ that harbors the stem/progenitor cells correlates with a doubling of the number of tripotential, self-renewing NSPs as measured using the neurosphere assay. BrdU incorporation assay in vitro demonstrated that hypoxic treatment enhanced BrdU incorporating into NSPs, and the cells positive for the neuronal marker Tuj1 or astrocytic marker GFAP incorporated BrdU during differentiation, indicating that both neuronal and glial precursors are de novo-generated from proliferating progenitor cells. Though BrdU may potentially indicate DNA repair (Rakic, 2002), the mildness of the hypoxic episode, the long delay between exposure to hypoxia and BrdU injection, the dose of BrdU administered as well as the marked augmentation of its incorporation rate into the germinative SVZ strongly suggest that BrdU labeling actually reflects proliferating cells. The present study shows that PC also induces neurogenesis in newborn piglet brain. Our data show that dividing Dcx positive cells are restricted to the SVZ, and that Dcx positive cells with morphologies of migrating neuroblasts were found in striatum and also migrated to neocortex, suggesting that the SVZ is the main source of new neurons in the neocortex. Nonetheless, we cannot exclude the possibility that some of the newly generated neurons are derived from local precursors or from glial progenitors that dedifferentiate and become respecified as neurons. Furthermore, prior PC augmented the increase in neurogenesis that occurred in the post-hypoxic–ischemic brain. These findings are largely consistent with previous studies unequivocally showing that various stresses, including hypoxia, can induce neurogenesis. Global and focal ischemic brain injuries in adult animals have been demonstrated to trigger compensatory neurogenesis from neural stem cells or progenitor cells located in germinative areas such as the hippocampal dentate gyrus or the SVZ (Kokaia and Lindvall, 2003). According to Nakatomi et al. (2002), these cells originating from the SVZ are capable to migrate and to incorporate the hippocampal circuitry. Further studies have shown that neonatal hypoxia for 5min strongly stimulated the generation of new cells in the SVZ within the ensuing three weeks, and demonstrated that newly formed cells gave rise to additional functional neurons (Pourie et al., 2006). A study by Lee et al. (2007) also observed increased neurogenesis in adult Sprague–Dawley rat brain at 4–7days of reperfusion following PC induced by a 10min transient middle cerebral artery occlusion. Considering our findings that greater number of neurons were newly generated after PC treatment, PC-induced neurogenesis might offer a potential means of replacing damaged neurons. In addition, the presence of more robust neurogenesis in the PC+HI group despite a lower level of tissue injury than in the HI group indicates that neurogenesis may not be correlated with tissue injury. This is consistent with the conclusion drawn by Arvidsson et al. (2001) who outlined that enhanced neurogenesis in adult rats following stroke was not correlated to the extent of brain damage.