Animals and treatments
Male Wistar rats were obtained from Charles River Laboratories (Charles River Laboratories, Le Bois d’Arbresle, France). The rats weight range from 280 to 300 g at the beginning of the experiments. The rats had a free access to food and water during the treatment period. All animal experiments were performed within the frameworks of the French legislation and received the approval of the local ethic committee (Ethic committee of Nord-Pas-de-Calais for animal experimentation, CEEA, Lille, France). A group of Wistar rats (n = 12) was submitted to a treatment by PPARα agonist. This group was fed with a 0.2% fenofibrate-enriched diet during 14 days. A second group (n = 12) was submitted to a treatment by statin. Animals (n = 12) were fed during 14 days with 0.1% atorvastatin-enriched diet. At the same time, a control group was fed with standard diet. At the end of the two weeks period, one half of the rats of each group was submitted to a middle cerebral artery occlusion. For proteomics analyses, 6 animals in each group were used. Animals were sacrificed by injection of a lethal dose of pentobarbital, followed by a transcardial perfusion of ice cold 10 mM phosphate buffered saline (PBS, pH 7.4). Brain were harvested, immersed in ice cold PBS, forebrain and cerebrum were discarded, cortex and striatum were dissected, sliced in small pieces and frozen into liquid nitrogen. Brain samples were stored at -80°C until use. For immunohistochemistry analyses, 6 animals in each group were used. The animals were transcardialy perfused with cold heparinized (500 U/L) saline solution. Thereafter, animals were perfused with 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains were removed and post-fixed in the same fixative solution during 4 hrs at 4°C, then processed for paraffin embedding and cut into 6 μm thick sections.
Middle cerebral artery occlusion method
Animals were anesthetized with chloral hydrate (300 mg/kg, I.P.) (Sigma-Aldrich, Saint Quentin Fallavier, France). Cerebral infarcts were produced by a 60 min medial cerebral artery (MCA) occlusion followed by a 24 h-reperfusion period . We introduced into the internal carotid artery a 4–0 nylon monofilament suture with its tip rounded by flame heating. Under surgical microscope control, the suture was gently advanced into the internal carotid artery to occlude the origin of MCA (25 to 30 mm distal to the carotid bifurcation). After 60 min, the suture was carefully removed to allow reperfusion. A rectal probe was inserted and the core temperature was maintained at ~ 37°C by the use of a heating pad and a heating lamp.
Two-dimensional gel electrophoresis
For 2D analysis, frozen brain samples were homogenized in 2D sample buffer using Wheaton glass homogenizer in 2D sample buffer containing 7 M urea, 2 M thiourea, Triton X-100 4% v/v, DTT 20 mM. 400 μg of protein were diluted to a final volume of 400 μL and 0.6% carrier ampholytes (pH 4–7) and 10 mM Tris (final dilution) were added immediately prior to protein load. IPG isoelectrofocusing (IEF) was carried out in a Protean IEF cell (Biorad, Hercules, CA), using a 18 cm pH 4–7 Amersham ready strip. The IPG strips were rehydrated overnight with the protein lysate. For subsequent IEF, voltage was increased gradually to 10.000 V to reach a total of 60.000 V/hr. Immediately after focusing, strips were equilibrated for 3×15 minutes in a solution containing 50 mM Tris, 2% SDS, 20% glycerol, and 1% DTT w/v, and 0.01% w/v Bromophenol blue as tracking dye. SDS-PAGE was performed in a Protean IIXi Cell (Biorad) on a 12% polyacrylamide gel, until dye track reach the end of the gels. Gels were Coomassie’s Brillant Blue-stained for a first analysis, followed by a silver-staining procedure. Thereafter, stained gels were digitized and processed using Melanie III two-dimensional gel analysis software (Genebio, Geneva, Switzerland) for spot detection and analysis. Spots were edited manually to remove technical artefacts such as gel distortion and polypeptides were identified by mass spectrometry as previously described .
For western blot analysis, frozen brain samples were sonicated in RIPA lysis Buffer (150 mM NaCl, 6 mM sodium deoxycholate, 1 mM EGTA, 1 mM Igepal CA-630, 1% ICN proteases inhibitors cocktail kit, Tris–HCl 50 mM pH 7.4), mixed with Laemmli reducing sample buffer and heated at 100°C for 5 minutes. 50 μg of protein were loaded onto a 4-12% SDS-PAGE gradient gel and submitted to an overnight electrophoresis at 20 mA per gel. At the end of the procedure, proteins were transferred on a nitrocellulose membrane. After blocking the non specific interaction sites using 5% milk in 20 mM Tris buffered saline 0.05% Tween-20 (TBST), pH 7.4, membrane was incubated overnight at 4°C with specific primary antibodies solution at 1 μg/mL TBST 5% milk (mouse anti-PDI (clone 1D3), SPA-891 from Stressgen, mouse anti-alpha-synuclein BD610786 from BD bioscience, rabbit anti- 14-3-3 Zeta SC-1019 from SantaCruz Biotechnologies, rabbit anti-TUC-4 AB5454 and pan-TUC ABN108from Chemicon and mouse anti-actin A2413 from Sigma). Thereafter, antibodies were detected with species-specific HRP coupled secondary antibodies (Goat anti-rabbit IgG-HRP AP132P, Goat anti-mouse IgG-HRP AP127P, from Chemicon) 1/50000 in TBST. Detection was performed using ECL chemiluminescent system (Amersham) and LAS3000 lumi-imager (FujiFilm, Japan).
Paraffin embedded sections were successively deparaffinized with toluene and rehydrated using a classical procedure of successive ethanol decreasing grade baths. Sections were permeabilized by immersion in 0.3% Triton X-100 in PBS (pH 7.4). Thereafter, endogenous peroxidases were blocked with 0.3% H2O2 and 10% methanol in PBS (pH 7.4). Protein nonspecific binding was blocked with 5% normal horse serum in PBS (pH 7.4). Sections were then incubated over night at 4°C with a mouse anti-Protein Disulfide Isomerase monoclonal antibody (5 μg/mL final dilution, mouse anti-PDI, SPA-891 from Stressgen) and/or a rabbit anti-Neuron Specific Enolase (5 μg/mL, NA 1247 from Biomol International) in PBS. Antibodies were visualized with an anti-goat Vectastain ABC Elite kit (Vector laboratories) and DAB/H2O2 exposure (Sigma Fast DAB tablets set, Sigma) according to the manufacturers’ instructions. For double labelling, antibodies were visualized using an Alexa Fluor-422 labelled goat anti-mouse antibody (A11001) and an Alexa Fluor 565 labelled goat anti-rabbit antibody (A11011) (1/500 in PBS, Molecular Probes).
For statistical data comparison, a standard software package (PRISM GraphPad for Windows) was used. Variables were compared between different groups with a one-way variance analysis followed by least significance difference tests. Statistical tests are considered as significant when p ≤ 0.05. All values are given as mean ± sem.
The biomolecular interactome and gene ontology analysis was performed using Cytoskape shareware , .