Noxa (Latin for damage) is another BH3 family pro-apoptotic gene that is induced by p53 transcriptional activity [18,19]. Puma and Noxa have been implicated to directly and indirectly activate Bax (Bcl-2-associated X protein) and Bak (Bcl-2-antagonist/killer), resulting in permeabilization of the outer mitochondrial mem- brane, release of cytochrome c and induction of apoptosis [20,21]. Although both Puma and Noxa have been shown to mediate neuronal cell death, only Puma deficiency was significantly protective against apoptosis, stressing the importance of this factor in the apoptotic cascade [22–24]. With respect to cerebral ischemia, work by Chan and coworkers [25] demonstrated that cerebral ischemia induced Puma upregulation in the hippocampal CA1 region was inhibited by the p53 inhibitor, PFTa, which was correlated with significant neuroprotection. Due to its critical role in inducing apoptotic cell death, there has been intense study on the mechanisms of regulating p53 stability and activation, including post-translational modifications. Along these lines, recent work in cancer cells has shown that p53 activity and stability can be enhanced by acetylation at the Lysine373 residue (and possibly Lysine382 residue) on the C-terminal of the p53 protein, which leads to enhanced susceptibility of cells to stress [26–30]. This activation and acetylation of p53 is achieved by the CBP/p300 family of acetyl transferases [31–33]. A role for p53 acetylation in enhancing apoptosis of cortical neurons in vitro after exposure to various cell stressors has also been reported [34,35]. However, it is currently unknown whether p53 acetylation state is altered in the brain in vivo after cerebral ischemia. Since a change in the acetylation state of p53 following cerebral ischemia could affect its stability and activity and influence neuronal susceptibility to ischemic stress, a major goal of the current study was to address this key deficit in our knowledge. Previous work by our group and others has shown that the steroid hormone, 17b2Estradiol (17b2E2) can exert potent anti- apoptotic and neuroprotective effects in the hippocampal CA1 region following GCI, in part through an anti-oxidant effect to attenuate activation of NADPH oxidase and ROS generation [36,37]. Thus, an additional goal of our studies was to determine whether 17b2E2 and NADPH oxidase play a critical role in regulating p53 acetylation and corresponding induction of p53- regulated factors in the hippocampus following cerebral ischemia. Methods Induction of Global Cerebral Ischemia Adult Sprague-Dawley female rats (3 months of age) were bilaterally ovariectomized (OVX) and immediately implanted with placebo (Pla) or 17b-estradiol (17b2E2) Alzet minipumps (0.025 mg; 14–21 day release), producing a low physiological diestrus I level of 10–15 pg/ml of 17b2E2 in the blood [10]. In some animals, long-term 17b2E2 deprivation was performed in which the animals were ovariectomized and 10 weeks later (10 W), placebo or 17b2E2 minipumps were implanted as previously described [36]. A 4-vessel occlusion (4-VO) model of global cerebral ischemia (GCI) was performed one week after initiation of 17b2E2 replacement, as described previously [36,38]. GCI is a well known model of delayed neuronal cell death in which hippocampal CA1 pyramidal neurons are highly susceptible to damage and cell death [11]. Briefly, for induction of GCI, animals were first anesthetized with chloral hydrate (350 mg/kg, i.p.). The vertebral arteries were then electrocauterized and the common carotid arteries (CCA) were exposed. Wound clips were then used to close the incision and the rats were allowed a 24 h recovery period. Following 24 h, the animals were anesthetized using 3% isoflurane anesthesia and the CCA were re-exposed and clipped Acetylation of p53 via aneurysm clips. GCI occlusion was performed for 10 min. duration. Rats which lost their righting reflex within 30 sec. and whose pupils were dilated and unresponsive to light during ischemia were selected for the experiments. Carotid artery blood flow was restored by releasing the clips and allowing reperfusion. Rectal temperature was maintained at 37uC using a thermal blanket throughout the experiment and 2 h thereafter. Sham controls underwent the same surgical exposure procedures, except that the arteries were not occluded. All procedures were approved by the Georgia Health Science University institutional committee for care and use of animals (AUP# 09-03-174) and were in accordance with National Institutes of Health guidelines. Drug Administration To determine whether crosstalk occurs between NADPH oxidase-induced membrane superoxide (O22) production and downstream pro-apoptotic factors, Gp91ds-Tat, a competitive NOX2 inhibitor and its scrambled peptide control were used. Gp91ds-Tat is a competitive inhibitor consisting of a 9 amino-acid peptide sequence of the p47phox subunit docking site on NOX2, thereby preventing p47phox from forming a complex with NOX2 and activating the enzyme [39]. Gp91ds-Tat and the scrambled peptide control were administered into the lateral ventricles of the hippocampus CA1, at a dose of 100 ng in 5 ml. The dose of gp91ds-Tat was chosen based on previous studies by our laboratory showing its effectiveness in inhibiting NADPH oxidase and lack of side effects [36]. For intracerebroventricular (icv) injections, anesthetized rats were placed on ear bars of a stereotaxic instrument. Drug infusion was performed using a stepper motorized microsyringe (Stoelting, Wood Dale, IL, USA) at a rate of 1 mL/min into the cerebral ventricle (from the bregma: anteroposterior,60.8 mm; lateral, 1.5 mm; depth, 3.5 mm). When necessary, curcumin (200 mg/kg, sc-294110, Santa Cruz Biotechnology, Inc.) or DMSO vehicle was injected intraperito- neally, 30 min prior to the induction of GCI. DAB Staining Sections for DAB staining were incubated with 10% normal donkey serum in PBS containing 0.1% Triton X-100 and 0.3% H2O2 to block nonspecific surfaces for 1 h at room temperature. Sections were then incubated with the primary antibodies overnight at 4u Celsius (Cu) in PBS containing 0.1% Triton X- 100. The antibodies used were as follows: goat anti-Noxa (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti- Acetyl p53-lysine373 (1:200, Millipore, Billerica, MA). Afterwards, sections were washed with the same buffer, followed by incubation with a secondary biotinylated anti-goat or rabbit antibodies (Molecular Probes, Eugene, OR, USA) at a dilution of 1:500 in PBS containing 0.1% Triton X-100 for 1 h at room temperature. Sections were then washed, followed by incubation with ABC reagents for 1 h at room temperature in the same buffer. Sections were rinsed with the same buffer and incubated with DAB reagent for 2–10 min., according to the instructions of the manufacturer (Vector Laboratories). Sections were briefly washed with distilled water and dehydrated in graded alcohols, cleared in xylene, and mounted using xylene-based mounting medium following DAB staining. Images were captured on an Axiophot-2 visible microscope using an AxioVision4Ac software system (Carl Zeiss, Germany) at a magnification of 10X or 40X, respectively. The number of NOXA or Acetyl p53-lysine373-positive cells was counted, as described previously [36]. Briefly, for quantitative analyses, neurons which stained positively for Noxa or acetylated p53 were counted bilaterally per 250 mm length of medial CA1 pyramidal cell layer, in four to five sections per animal to provide a PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e27039