Adult Sprague-Dawley rats (n=12) were anesthetized using chloral hydrate (400 mg/kg, i.p). All procedures were approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center, and were compliant with the guidelines of the National Science Council on Animal Care.

Temporary ischemia was induced by clipping the bilateral common carotid artery (CCA) and ligating the right-side middle cerebral artery (MCA), as suggested by Chen et al.11 The bilateral CCA was isolated via a midline cervical incision, and then temporarily clipped with arterial clips. A right-side middle squamous craniotomy with a diameter of 2.0-3.0 mm was performed, and the MCA was ligated using a 10-O suture. The suture was then released after 60 min of ligation, and the craniotomy site was covered with Gelfoam and bone wax. Individual animals were perfused intracardially at 0, 6, or 24 h (n=3 animals per time point) after ligation for reperfusion protein assay.12

Animals were anesthetized and decapitated, and their brains then microdissected on ice under a surgical microscope to initially retrieve the cerebrocortices. The harvested brain tissue was cut into several 500-µm-thick sections. Punch separation of the cerebrocortex sections was then performed. The tissues were homogenized on ice with a tissue tearer in 300 µL of sample buffer {58 mM dithiothreitol, 65 mmol/L 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic, 7 M urea, 1.9 mol/L thiourea, 1.75% pH 3-10 carrier ampholytes}. The mixture was then mixed for 1 h and centrifuged at 18000×g for 10 min. Protein concentration was determined in the soluble supernatant using the RC-DC-Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) to avoid interference from ampholytes and reducing agents present in the buffer.

A 2-DE system was used to separate proteins obtained from the cerebrocortex samples (250 µg/sample). An isoelectric focus over a pH range from 3 to 10 and a second dimension slab gel were used to separate the proteins by molecular weight while a first dimension gel was loaded on a large format (22×22 cm). The gels were then stained using fluorescent SYPRO Ruby (Molecular Probes, Eugene, OR, USA) to quantify the proteins, and images were obtained using HT 2-D image analysis software (Genomic Solutions Inc., Ann Arbor, MI, USA).13

Samples were prepared using a modification of a previously described technique.14 Water (18 mol/L) was then used to wash the stained gel slabs extensively, excising the spot with a volume around 1-3 mm³. The gel pieces were mixed with ammonium bicarbonate (0.1 mol/L) to double the volume and then incubated at room temperature (20℃) for 15 min. The gel pieces were dried using a high-speed vacuum centrifuge after the solvent had been removed, rehydrated with 20 µL of 20 mmol/L dithiothreitol in 0.1 mol/L NH₄HCO₃, and then incubated at 56℃ for 45 min to reduce the protein. When the tube was cooled to room temperature, the dithiothreitol solution was removed before 20 µL of 55 mmol/L iodoacetamide in 0.1 mol/L NH₄HCO₃ was added, and the gel incubated at room temperature in the dark for 30 min, followed by further incubation for 15 min in 0.2 mL of 50 mmol/L NH₄HCO₃, before adding 0.2 mL of acetonitrile. At the end of another 15-min incubation period at room temperature, the solvent was removed and the gel pieces were dried in a vacuum centrifuge. The gel pieces were rehydrated with 5 µL of 20 ng/µL modified trypsin (Promega, Madison, WI, USA) in 50 mmol/L NH₄HCO₃ and then covered with 50 mmol/L NH₄HCO₃ solution and incubated overnight at 37℃.

A nitrocellulose solution was made by dissolving a nitrocellulose membrane in 1:1 acetone/isopropanol solvent. Alpha-cyano-4-hydroxycinnamic acid (-CN) was washed with 50 µL of acetone and the acetone phase was discarded. The -CN was dissolved in acetone to a concentration of 10 mg/mL, and then the nitrocellulose and -CN solutions were mixed at a ratio of 1:4; 1 µL of this mixture was deposited onto a 96-well MALDI target plate.

A sample was prepared for addition to the plate by adding 2 µL of sample to 2 µL of a solution of acetone-washed -CN dissolved in 0.1% trifluoroacetic acid and added to a 1:1 H₂O/acetonitrile mix to a final -CN concentration of 10 mg/mL. An aliquot (1 µL) of the sample mixture was loaded onto each thin film. After the sample mixture was dried, 1.5 µL of 2% formic acid in 18-MΩ water was added to each spot. The formic solution was removed by gentle blotting. This washing step was performed twice. The samples were then dried at room temperature. Fragment sizes were determined by MALDI-TOF MS.

Protein identification from tryptic fragment sizes was made using the Mascot search engine (www.matrixscience.com) based on the entire National Center for Biotechnology Information protein database under the assumptions that peptides are monoisotopic, oxidized at methionine residues, and carbamidomethylated at cysteine residues. Up to one missed trypsin cleavage was allowed, although most matches did not contain any missed cleavages. An accuracy of 100 ppm or greater for the tryptic sizes was required. The original protein size based on the portion of the gel from which the spot was excised was not restricted. All matches using mass values (either peptide masses or MS/MS fragment ion masses) were handled on a probabilistic basis. The total score is the probability that the observed match is a random event. The direct reporting of probabilities can be confusing, and so the probability-based molecular weight search score was used, defined as -10×log₁₀ (p), where p is the absolute probability;15 thus, a probability of 10^-20 becomes a score of 200. In this study, scores of >70 were considered significant (p<0.05), and all protein identifications were in the expected size range based on their position in the gel.

Silver staining was performed using silver nitrate in combination with formaldehyde developer. The detection sensitivity of the silver stain was 0.5 ng. SYPRO-Ruby-stained gels were scanned using a high-resolution 12-bit camera and analyzed using HT 2-D gel software (Genomic Solutions). A composite gel was formed using gels obtained from each treatment. Bioimage software was initially used to identify matching spots in each tissue sample according to the manufacturer's instructions. The accuracy of the protein-spot matching was determined manually for each spot on each gel. The spot intensity was compared for each protein identified and matched, and the integrated intensity of each spot was determined for each of the eight gels. Except where indicated otherwise, the data are presented as mean±SEM values, and a Mann-Whitney test was carried out on each to determine the significance of any differences.

Levels of dynamin-1 increased by about 2.2-fold at 6 h after ischemia in the penumbra region. The DTPase dynamin is required for late-stage endocytic clathrin-coated vesicle formation. Dynamin is targeted to coated pits on the plasma membrane in a guanine-nucleotide-independent manner, through interactions between its praline, arginine-rich COOH-terminal domain and the SH3-domain-containing protein amphiphysin. Mammals express three dynamins (dynamin-1, -2, and -3) with different expression patterns.34 Dynamin-1 is expressed exclusively in the brain35 and in neurons, and levels of dynamin-1 increase with synapse formation in parallel with the levels of synaptic vesicle proteins.36 Furthermore, dynamin-1 plays a dedicated and essential role in the recycling of synaptic vesicles, which is critical to nervous system function.37,38 A late depolarizing postsynaptic potential induced by hippocampal CA1 pyramidal neurons after transient ischemia indicates an enhancement of synaptic transmission following ischemia.39 The present data are compatible with those of reports indicating that synapse-releasing efficacy is increased during reperfusion periods.39,40

The glucose-related stress response is part of a general cellular defense mechanism, referred to as the unfolded protein response (UPR), which is induced by glucose or oxygen starvation.41,42 One characteristic of the UPR is the induction of endoplasmic reticulum (ER) resident stress proteins, referred to as the glucose-regulated proteins (GRPs), which are Ca²⁺-binding chaperone proteins with protective properties.43 The glucose-regulated protein GRP78, a 78-kDa protein (also referred to as binding immunoglobulin protein), is one of the best-characterized GRPs and is known to form complexes with heterologous proteins that are processed through the ER.44 GRP78 can bind to malfolded proteins and unassembled complexes, and can protect cells against cell death caused by disturbances of the ER homeostasis.45,46,47,48 GRP78 has also been induced in endothelial cells damaged by reductive stress caused by hyperhomocysteinemia, which is a common risk factor for thrombotic vascular events such as premature arteriosclerosis, strokes, myocardial infarctions, and thrombosis.49,50 Therefore, the induction of GRP could be an adaptive response that has evolved in mammals to protect endothelial cells against stress-induced death. GRPs are induced in response to stress, but once the stress is removed these proteins are posttranscriptionally modified into biologically inactive forms.44 GRP78 is critical for the maintenance of cell homeostasis and the prevention of apoptosis. The present data show that this protein is present at very high levels at the beginning of the reperfusion period, or 1-6 h after tMCAO; however, as shown in Fig. 4B, it was relatively down-regulated at 24 h after reperfusion on the lesioned sides. This is probably the result of cell loss on the lesioned side relative to the contralateral side during the ischemic and reperfusion periods. In addition, the down-regulation of this molecular chaperone by tMCAO may indicate the potential for it to impact immune function on multiple levels.