05 BR MAO.indd

Hypoxia-ischemia (HI) occurring in immature brains stimulates the expression of tissue-type plasminogen activator (tPA). Neuroserpin is a selected inhibitor of tPA in the central nerves system. However, the role that neuroserpin plays and the possible mechanisms involved during neonatal HI are poorly defi ned. In this study, an oxygen-glucose deprivation and reoxygenation (OGD/R) model was generated with cultured rat cortical neurons mimicking neonatal HI injury ex vivo, and an acute neuronal excitatory injury was induced by exposure to a high concentration of N-methyl-D-aspartic acid (NMDA). Cells received either neuroserpin or MK-801, an antagonist of the NMDA receptor, during OGD/R, and were incubated with or without neuroserpin after NMDA exposure. Cell viability and morphology were detected by a Cell Counting Kit-8 and immunohistochemical staining, respectively. TPA expression and activity were also assessed. We found that MK-801 alleviated injuries induced by OGD/R, suggesting an excitatory damage involvement. Neuroserpin provided a dosedependent neuroprotective eff ect in both OGD/R and acute excitatory injuries by inhibiting the activity of tPA, without aff ecting neuronal tPA expression. Neuroserpin protected neurons against OGD/R even after a delayed administration of 3h. Collectively, our data indicate that neuroserpin protects neurons against OGD/R. mainly by inhibiting tPA-mediated acute neuronal excitotoxicity. Key terms: excitotoxicity, hypoxia-ischemia, neuroserpin, N-methyl-D-aspartic, tissue-type plasminogen activator. Abbreviations: BBB: blood brain barrier; BSA: bovine serum albumin; CCK-8: Cell Counting Kit-8; CNS: central nervous system; DMEM: Dulbecco’s modified Eagle medium; EAAs: excitatory amino acids; ELISA: enzyme-linked immunosorbent assay; FCS: fetal calf serum; HI: hypoxia-ischemia; HIE: hypoxic-ischemic encephalopathy; LTP: long-term potentiation; MAP2: microtubule-associated protein 2; NB: neurobasal medium; NMDA: N-methyl-D-aspartic; NSP: neuroserpin; OGD/R: oxygen-glucose deprivation and reoxygenation; PAI1: plasminogen activator inhibitor-1; QRT-PCR: Quantitative real-time reverse-transcriptase Polymerase Chain Reaction; tPA: tissue-type plasminogen activator. Corresponding authors: Meng Mao, West China Second University Hospital, Sichuan University, Chengdu 610041, PR China, Tel: 13608088613, Fax: +86 28 87461751, E-mail address: [email protected], Dan Yu, West China Second University Hospital, Sichuan University, Chengdu 610041, PR China, Tel: 15928623580, Fax: +86 28 85501319, E-mail address: [email protected] Received: January 19, 2012. In Revised form: August 27, 2012. Accepted: August 30, 2012 INTRODUCTION Tissue-type plasminogen activator (tPA), a serine proteinase cleaving plasminogen into plasmin, is well known as a thrombolytic enzyme in the intravascular space (Collen, 2001). However, tPA is also expressed in the central nervous system (CNS) and displays both physiological and pathological activity (Benarroch, 2007). Neuroserpin (NSP), an axonally secreted serine protease inhibitor (Osterwalder et al., 1996), is a selective inhibitor of tPA in the CNS. Both of these proteins are associated with neuronal activity and stimulated to release by neuronal depolarization (Qian et al., 1993; Berger et al., 1999). Studies propose that neuroserpin functions as a neuroprotective factor by eliminating the deleterious eff ects of tPA in some pathological conditions. The genetic knockout of tPA or administration of neuroserpin has shown better results in ischemic stroke models (Wang et al., 1998; Zhang et al., 2002; Yepes et al., 2000). During the course of cerebral ischemia, endogenous neuroserpin increases and then blocks microglial activation, decreases the number of apoptotic cells, preserves the integrity of blood brain barrier (BBB) and attenuates tPA-mediated inflammation (Yepes and Lawrence, 2004; Rodríguez-Gonzáez et al., 2011). Furthermore, tPA has been indicated to enhance N-methyl-D-aspartic acid (NMDA) receptor-mediated excitatory neuronal death, and tPAdefi cient mice show a dramatic resistance to excitotoxicity (Nicole et al., 2001; Tsirka et al., 1995). Additionally, neuroserpin protects neurons against low concentration NDMA-induced chronic excitotoxicity both in vitro and in vivo (Lebeurrier et al., 2005). A negative correlation between a decrease of serum neuroserpin level and an increase of glutamate has been found in adult ischemic stroke patients recently (Rodríguez-González et al., 2011). Although the role of neuroserpin in the adult ischemic stroke model has already been investigated, its effect in neonatal hypoxia-ischemia (HI) injury as well as in acute excitotoxicity has not been revealed. Unlike adult brains, HI only transiently impairs blood perfusion in newborn (Mujsce et al., 1990; Adhami et al., 2008). Reperfusion is accompanied by increased glucose levels, which has been shown to be detrimental (Sheldon et al., 1992), and excessive oxygen and free radicals, producing further brain injury (Harukuni and Bhardwaj, 2006). Based on this evidence, we used an oxygenglucose deprivation and reoxygenation (OGD/R) model to mimic neonatal HI injury ex vivo. An acute neuronal excitatory injury model was generated by exposure to high NMDA concentration. We tested the hypothesis that neuroserpin would display neuroprotective effects by restraining tPAmediated acute neuroexcitotoxicity in neonatal HI. 05 BR MAO.indd 357 03-01-13 16:17 MAO ET AL. Biol Res 45, 2012, 357-362 358