Neuroprotective Effect of Inhaled Nitric Oxide on Excitotoxic-Induced Brain Damage in Neonatal Rat Julien Pansiot 1,2 , Gauthier Loron 1,2 , Paul Olivier 1,2 , Romain Fontaine 3 , Christiane Charriaut-Marlangue 1,2 , Jean-Christophe Mercier 4 , Pierre Gressens 2,5 , Olivier Baud 1,2,5,6 * 1 INSERM, Ho ˆ pital Robert Debre ´, Paris, France, 2 INSERM, UMR 676, Ho ˆ pital Robert Debre ´, Paris, France, 3 INSERM UMR 711, Universite ´ Pierre et Marie Curie, Faculte ´ de Me ´decine, Ho ˆ pital de la Salpe ˆtrie `re, Paris, France, 4 APHP, Emergency Department, Ho ˆ pital Robert Debre ´, Paris, France, 5 APHP, Neonatal Intensive Care Unit Ho ˆ pital Robert Debre ´, Paris, France, 6 PremUP Foundation, Paris, France Abstract Background: Inhaled nitric oxide (iNO) is one of the most promising therapies used in neonates. However, little information is known about its impact on the developing brain submitted to excitotoxic challenge. Methodology/Principal Findings: We investigated here the effect of iNO in a neonatal model of excitotoxic brain lesions. Rat pups and their dams were placed in a chamber containing 20 ppm NO during the first week of life. At postnatal day (P)5, rat pups were submitted to intracranial injection of glutamate agonists. At P10, rat pups exposed to iNO exhibited a significant decrease of lesion size in both the white matter and cortical plate compared to controls. Microglia activation and astrogliosis were found significantly decreased in NO-exposed animals. This neuroprotective effect was associated with a significant decrease of several glutamate receptor subunits expression at P5. iNO was associated with an early (P1) downregulation of pCREB/pAkt expression and induced an increase in pAkt protein concentration in response to excitotoxic challenge (P7). Conclusion: This study is the first describe and investigate the neuroprotective effect of iNO in neonatal excitotoxic-induced brain damage. This effect may be mediated through CREB pathway and subsequent modulation of glutamate receptor subunits expression. Citation: Pansiot J, Loron G, Olivier P, Fontaine R, Charriaut-Marlangue C, et al. (2010) Neuroprotective Effect of Inhaled Nitric Oxide on Excitotoxic-Induced Brain Damage in Neonatal Rat. PLoS ONE 5(6): e10916. doi:10.1371/journal.pone.0010916 Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan Received March 11, 2010; Accepted May 7, 2010; Published June 1, 2010 Copyright: ß 2010 Pansiot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The experimental research program of Dr. Olivier Baud and his coworkers concerning iNO is partially supported by Ikaria, Inc. This study was financially supported by the INSERM, the INSERM AVENIR Program and the Mairie de Paris. Paul Olivier and Gauthier Loron were financially supported by Ikaria (iNOTherapeutics). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The experimental research program of Dr. Olivier Baud and his coworkers concerning iNO is partially supported by Ikaria, Inc. However, this does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: olivier.baud@rdb.aphp.fr Introduction Brain injury in the premature infant is a problem of major importance. Approximately 10 percent of the survivors from very preterm birth later exhibit cerebral palsy (CP) and an additional 25 to 50 percent exhibit cognitive, attentional, and/or behavioral deficits. These neurologic disabilities observed relate in consider- able part to cerebral white matter injury [1]. Factors that seem involved in the pathophysiology of CP in these models include hypoxia and ischemia, infection and inflammation, excitotoxicity, accumulation of reactive oxygen species, and deficiencies in growth factors [2,3]. These factors seem to act in combination to cause damage to the developing white matter. Glutamate accumulation may be a mechanism common to many risk factors for CP. Glutamate, the major excitatory neurotransmitter, acts via several groups of receptors, namely, N-methyl-D-aspartate (NMDA) receptors, alpha-3-amino-hy- droxy-5-methyl-4-isoxazole (AMPA) receptors, kainate receptors, and metabotropic receptors (mGluRs). Excessive activation of glutamate receptors may cause cell vulnerability, in part as a result of intracellular calcium influx [4,5]. Intracerebral injection of glutamate agonists into the neocortex and white matter of newborn rodents produces histological lesions that mimic the brain damage observed in preterm neonates [6–8]. In addition to excitotoxicity, nitric oxide is recognized as being a key modulator of risk factors involved in CP, by regulating vascular tone, reperfusion, inflammation and oxidative stress [9,10]. Despite considerable advances in our understanding of the pathophysiology of brain damage during development, therapeutic options are still extremely limited. Inhaled nitric oxide (iNO) is one of the most commonly used therapies, promising but also controversial, in neonatal intensive care units. This molecule is thought to have only a local effect, limited to the vascular tone of the lungs, and has been proposed to treat pulmonary hyperten- sion-related hypoxemia and chronic lung disease. However, increasing experimental and clinical evidences suggest that iNO could also have an impact on the developing central nervous system [11,12]. Here, we describe the neuroprotective effect of iNO in neonatal excitotoxic-induced brain damage. This effect appears to be mediated through pAkt-pCREB pathway and subsequent modu- lation of glutamate receptor subunits expression. PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e10916 ,