Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender Sylvain Renolleau,* ,  Se ´bastien Fau,* ,  Catherine Goyenvalle,* Luc-Marie Joly,à David Chauvier,§ Etienne Jacotot,§ Jean Mariani* , ¶ and Christiane Charriaut-Marlangue* *UMR-CNRS and  Service de Re ´animation Ne ´onatale et Pe ´diatrique, Ho ˆpital Armand Trousseau – APHP, Universite ´ Pierre et Marie Curie-Paris, Paris, France àDe ´partement d’Anesthe ´sie-Re ´animation, CHU C, Nicolle, Rouen, France §Theraptosis Research Laboratory, Theraptosis S.A., Pasteur BioTop, Paris, France ¶APHP, Ho ˆpital Charles Foix, UEF, Ivry sur Seine, France Abstract Hypoxia–ischaemia in the developing brain results in brain injury with prominent features of apoptosis. In the present study, a third generation dipeptidyl broad-spectrum caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD- OPh), was tested in a model of unilateral focal ischaemia with reperfusion in 7-day-old rats. Q-VD-OPh (1 mg/kg, i.p.) reduced cell death, resulting in significant neuroprotection at 48 h of recovery (infarct volume of 12.6 ± 2.8 vs. 24.3 ± 2.2%, p ¼ 0.006). The neuroprotective effects observed at 48 h post-ischaemia hold up at 21 days of survival time and attenuate neurological dysfunction. Analysis by gender revealed that females were strongly protected (6.7 ± 3.3%, p ¼ 0.006), in contrast to males in which there was no signi- ficant effect, when Q-VD-OPh was given after clip removal on the left common carotid artery. Immunoblot analysis demon- strated that Q-VD-OPh inhibits caspase 3 cleavage into its p17 active form and caspase 1 up-regulation and cleavage in vivo. Following ischaemia in P7 rats, males and females displayed different time course and pattern of cytochrome c release and active p17 caspase 3 during the first 24 h of recovery. In contrast, no significant difference was observed for caspase 1 expression between genders. These results indicate that ischaemia activates caspases shortly after reperfusion and that the sex of the animal may strongly influences apoptotic pathways in the pathogenesis of neonatal brain injury. The specificity, effectiveness, and reduced toxicity of Q-VD-OPh may determine the potential use of peptide-derived irreversible caspase inhibitors as promising therapeutics. Keywords: brain injury, caspase inhibitor, cell death, gender, neonatal stroke. J. Neurochem. (2007) 100, 1062–1071. Perinatal hypoxic–ischaemic (HI) injury remains a major cause of mortality and cerebral morbidity, susceptible to generate permanent neurological sequelae (Garnier et al. 2003; Ferriero 2004; Nelson and Lynch 2004). Care of the fetus and newborn infant at risk for cerebral HI is a high priority in current health care, and an understanding of the pathophysiology of perinatal HI brain damage is essential to the design of effective interventions. Participation of apoptosis and caspase activation has been now largely documented after hypoxia–ischaemia in the developing brain. Indeed, key elements of apoptosis, such as caspase 3 (Hu et al. 2000; Blomgren et al. 2001), Bcl-2 (Merry et al. 1994) and Bax (Vekrellis et al. 1997) are up- regulated in the immature as compared with the adult brain. The development of systemically active caspase inhibitor may therefore prove useful for treatment of acute and subacute hypoxia–ischaemia during development. As yet, no systemically available caspase inhibitors showing efficacy in the CNS have been reported. Recently, a broad-spectrum Received May 11, 2006; revised manuscript received August 14, 2006; accepted October 3, 2006. Address correspondence and reprint requests to C. Charriaut-Mar- langue, UMR-CNRS 7102, HICD, case 14, 9 quai St-Bernard, 75005 Paris, France. E-mail: Christiane.Marlangue@snv.jussieu.fr Abbreviations used: BAF, boc-aspartyl-(Ome)-fluoromethyl-ketone; CCA, common carotid artery; DMSO, dimethylsulfoxide; G, group; HI, hypoxia–ischaemia; P9, postnatal day 9; Q-VD-OPh, quinoline-Val- Asp(Ome)-CH 2 -O-phenoxy; TUNEL, terminal dUTP nick-end labelling. Journal of Neurochemistry , 2007, 100, 1062–1071 doi:10.1111/j.1471-4159.2006.04269.x 1062 Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1062–1071 Ó 2006 The Authors