The HDAC inhibitors TSA, suberoyl bis-hydroxamic acid (SBHA), scriptaid, and nullscript were purchased from Biomol International (Plymouth Meeting, PA), whereas sodium butyrate was purchased from Sigma-Aldrich (St. Louis, MO). Homocysteate (HCA), camptothecin, and hydrogen peroxide were purchased from Sigma-Aldrich. Roscovitine and olomoucine were purchased from Biomol International. pEGFP, pEGFP-p21-full, and pEGFP-p21-ΔNLS constructs were kindly provided by Dr. Asada (International Medical Center of Japan, Tokyo, Japan). The hemagglutinin (HA)-tagged ASK-1 construct was kindly provided by Dr. Houghton (St. Jude Children's Research Hospital, Memphis, TN). B6:129S2-Cdkn1a (p21 knock-out) (Brugarolas et al., 1995
) and B6:129SF2/J (wild-type control) mouse lines were obtained from the Jackson Laboratory (Bar Harbor, ME). Embryonic day 17 (E17) pregnant Sprague Dawley rats were obtained from Harlan Sprague Dawley (Frederick, MD). Adult male Sprague Dawley rats were obtained from Charles River Breeding Laboratories (Wilmington, MA).
Primary neurons and cell culture
Cell cultures were obtained from the cerebral cortex of fetal Sprague Dawley rats (embryonic day 17) or B6: 129S2-Cdkn1a (p21 knock-out) and B6:129SF2/J (wild-type control) mice (embryonic day 15) by a papain dissociation method as described previously (Murphy et al., 1990
). Cultures were plated on poly-d
-lysine (Sigma-Aldrich)-coated cell culture dishes and maintained in minimum essential medium (Invitrogen, Grand Island, NY) containing 5.5 g/L glucose, 2 mm l
-glutamine, 100 μm
cystine, and supplemented with 10% fetal bovine serum (FBS; Invitrogen). Cultures from the cortex of fetal rats or mice at this stage of development are ~85% neuronal, the balance being predominantly glial. All experiments were initiated 24 h after plating unless stated otherwise. Under these conditions, the cells are not susceptible to glutamate-mediated excitotoxicity. Longer-term cultures for adenoviral infection were maintained by switching to minimum essential medium (Invitrogen) containing 5.5 g/L glucose, and supplemented with 2 mm
GlutaMAX (Invitrogen) and 1× B27 (Invitrogen), at 3–4 d in vitro
(DIV). The HT22 murine hippocampal cell line was a kind gift from D. Schubert (Salk Institute, La Jolla, CA). B35 neuroblastoma cell line was purchased from American Type Culture Collection (Manassas, VA). Both HT22 and B35 cell lines were maintained and cultured in DMEM (Invitrogen) with high glucose, l
-glutamine, and pyridoxine hydrochloride, and supplemented with 10% FBS.
For cytotoxicity studies, cells were rinsed with warm PBS and then placed in medium containing the glutamate analog HCA (5 mm, unless stated otherwise). HCA was diluted from 100-fold concentrated solutions that were adjusted to pH 7.5. For HDAC inhibitor treatments except pulse treatments, HDAC inhibitors were added at the time of HCA treatment and present throughout experiment. Viability was assessed by calcein AM/ethidium homodimer-1 staining (live/dead assay) (Invitrogen) under fluorescence microscopy and the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method (Promega, Madison, WI).
Transfections and adenoviral infections
HT22 hippocampal neuron cells were cotransfected with a puromycin cDNA construct (pPUR; Clontech, Mountain View, CA) and either pEGFP (Clontech) vector alone or pEGFP vector containing a p21 cDNA (pEGFP-p21-full) or pEGFP vector containing a p21-ΔNLS cDNA (pEGFP-p21-ΔNLS), using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's protocol. Stably transfected HT22 neurons were selected over several weeks by the addition of puromycin (4 μg/ml) to the culture medium. Puromycin-resistant clones were pooled to avoid confounds introduced by clonal selection, and p21 or GFP expression was verified by Western blot analysis and GFP immunofluorescence under an inverted fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). Primary mixed cortical neurons were infected with adenovirus (multiplicity of infection = 100) harboring both p21 and GFP cDNAs (Adp21+GFP), or GFP cDNA (Ad-GFP) alone. To generate adenoviruses, either p21 or GFP was subcloned into the multiple cloning site of pAd5-CMV-NpA vector (ViraQuest, North Liberty, IA) and verified by sequencing. Recombinant adenovirus generation and amplification was performed by ViraQuest using RAPAd technology. Additionally, recombination by ViraQuest included the addition of HSV promoter-GFP constructs so that, in addition to CMV promoter-p21 or CMV promoter GFP, adenoviruses also harbor HSV promoter-GFP. Primary mixed cortical neurons were infected at 5 DIV and cultured for an additional 4 d before exposure to hydrogen peroxide to allow for transgene expression.
Total RNA was prepared from primary mixed cortical neurons using TriZOL (Invitrogen) and cDNA generating using a SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen), according to the manufacturer's protocol. Real-time PCRs were performed as a duplex reaction using p21 gene expression assay, which uses a FAM-labeled probe, and β-actin gene expression assay, which uses a VIC-labeled probe (Applied Biosystems, Foster City, CA), so that p21 amplification could be normalized to β-actin. Real-time PCRs were performed using a 7500 Real Time PCR System (Applied Biosystems) using standard PCR protocol and amplification conditions.
Cell lysates were obtained by rinsing cortical neurons with cold PBS followed by lysis in NP-40 lysis buffer (Boston Bioproducts, Worcester, MA). Protein concentrations in lysates were quantified by Bradford assay (Bio-Rad, Hercules, CA). Nuclear and cytoplasmic protein extractions were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL) according to the manufacturer's protocol. Samples were boiled in Laemmli buffer and electrophoresed under reducing conditions on 12% [or 7.5% for retinoblastoma protein (pRb) immunoblots] polyacrylamide gels. Proteins were transferred to a nitrocellulose membrane (Bio-Rad) by electroblotting. Nonspecific binding was inhibited by incubation in Tris-buffered saline with Tween 20 (TBST: 50 mm Tris-HCl, pH 8.0, 0.9% NaCl, and 0.1% Tween 20) containing 5% nonfat milk for at least 1.5 h. Primary antibodies against p21 (BD Biosciences, San Jose, CA), p15 (Santa Cruz Biotechnology, Santa Cruz, CA), p16 (BD Biosciences), p27 (BD Biosciences), p57 (Millipore, Billerica, MA), pRb (BD Biosciences), GFP (Invitrogen), histone H4 (Millipore), acetyl histone H4 (Millipore), histone H3 (Millipore), phospho-JNK (Cell Signaling, Danvers, MA), total JNK (Cell Signaling), GAPDH (Millipore), NeuN (Millipore), α-tubulin (Sigma-Aldrich), and HA (Sigma-Aldrich) were diluted in TBST containing 5% milk overnight at 4°C followed by incubation with respective horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) for 2 h at room temperature. Immunoreactive proteins were detected according to the enhanced chemiluminescent protocol (GE Healthcare, Piscataway, NJ).
Animal preparation and monitoring for middle cerebral artery occlusion experiments
Adult male Sprague Dawley rats (n = 6 per treatment) weighing 250–280 g were operated on to examine the effect of pre- and post-HDAC inhibitor treatment on infarct volumes after middle cerebral artery occlusion (MCAo). Twelve- to 14-week-old male mice, B6: 129SF2/J (wild type; n = 9) and B6:129S2-Cdkn1a (p21 knock-out; n = 10) weighing 20–30 g were used to examine the endogenous role of p21 in the prevention of stroke damage. Animals were allowed ad libitum access to food and water before and after surgery. Rats were anesthetized by an intraperitoneal injection of 400 mg/kg chloral hydrate followed 45 min later by a maintenance intraperitoneal infusion at a rate of 120 mg/kg/h using a butterfly needle set. The animals were free breathing. Body temperatures were kept stable at 36.5 ± 0.5°C using a feedback-regulating heating pad and a rectal probe (Harvard Apparatus, Holliston, MA). In rats, the right femoral artery was cannulated for measurement of arterial blood gases, glucose, and mean arterial blood pressure. These physiological parameters were monitored before and after MCAo. In addition, laser Doppler flowmetry (Moor Instruments, Devon, UK) was used to monitor the regional cerebral blood flow through a burr hole 2 mm in diameter created in the right parietal bone (2 mm posterior and 6 mm lateral to bregma). Preparation and monitoring of mice were the same as for rats with the following changes. Mice were anesthetized by inhalation of a mixture of isoflurane (1.5–2%), oxygen (30%), and nitrogen (70%) via nosecone, and a laser Doppler flowmetry probe was attached directly to the parietal bone.
All rats and mice were subjected to right MCAo. Under the operating microscope, the right common carotid artery was exposed through a midline incision in the neck. A 4-0 (rats) or 6-0 (mice) nylon suture with its tip rounded by heating over a flame and subsequently coated with poly-l-lysine (Sigma-Aldrich) was introduced into the external carotid artery and then advanced into the internal carotid artery for a length of 18–19 mm (rats) or 9–10 mm (mice) from the bifurcation. This method placed the tip of the suture at the origin of the anterior cerebral artery, thereby occluding the MCA. The placement of the suture tip was monitored by laser Doppler flowmetry measurements of regional cerebral blood flow. MCAo caused a sharp drop in regional cerebral blood flow to <30% (20% for mice) of preischemic base line. The suture was left in place (rats) or removed after 20 min (mice), and the animals were allowed to awaken from the anesthesia after closure of the operation sites.
Sodium butyrate was dissolved in PBS (vehicle) for a total volume of 100 μl and was administered intraperitoneally to animals (n = 6) at a dose of 1200 mg/kg of body weight. Sodium butyrate was administered 24 and 4 h before MCAo (pretreatment experiment) or 30 min after MCAo (posttreatment experiment). The control animals received an equivalent volume of the vehicle on an identical administration schedule.
Twenty-four hours after MCAo, animals were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (50 mg/kg, i.p.) and decapitated. The brain was rapidly removed, sliced into seven 2 mm coronal sections using a rat matrix (RBM 4000C; ASI Instrument, Warren, MI), and stained according to the standard 2,3,5-triphenyltetrazolium chloride (TTC) method. Each slice was drawn using a computerized image analyzer (Scion, Frederick, MD). The calculated infarction areas were then compiled to obtain the infarct volumes per brain (in mm 3). Infarct volumes were expressed as a percentage of the contralateral hemisphere volume to compensate for edema formation in the ipsilateral hemisphere.
In vivo experiments
All experimental procedures were approved by the Harvard Medical Area Standing Committee on Animals and/or Weill Medical College of Cornell University Institutional Animal Care and Use Committee and meet the standards of the Federal and State reviewing organizations.
Statistical analyses were performed using the statistical analysis package Prism (GraphPad Software, San Diego, CA).