Male Sprague-Dawley rats (200-220 g, n=60) were purchased from Samtako Co. (Animal Breeding Center, Osan, Korea), maintained under controlled temperature (25℃) and lighting (12/12-h light/dark cycle), and were allowed free access to food. All experiments were carried out in accordance with guidelines approved by the ethics committee concerning animal research at Gyeongsang National University (GNU-141222-R0064). Rats were randomly divided into four groups: non-diabetic+sham, diabetic+sham, non-diabetic+MCAO, and diabetic+MCAO. Diabetic conditions were induced by intraperitoneal injection of streptozotocin (40 mg/kg, Sigma, St. Louis, MO, USA). Streptozotocin was dissolved in 10 mM citrate buffer (pH 4.6) and non-diabetic animals received only citrate buffer as the vehicle. Blood glucose levels were determined using a strip sensor (Accu-Chek-Roche Diagnostics, Mannheim, Germany) according to the manufacturer protocol. A fasting blood glucose of >300 mg/dL was considered diabetic. Body weight and blood glucose were measured before MCAO.

MCAO was conducted via an intraluminal procedure, as previously described, after 4 weeks of streptozotocin treatment [19]. Zoletil (50 mg/kg; Virbac, Carros, France) was used for anesthesia before MCAO. Briefly, the right common carotid artery, external carotid artery, and internal carotid artery were exposed through a midline cervical incision. The occipital and superior thyroid arteries were knotted with 6/0 black silk thread and subsequently cut. The external carotid artery was knotted with black silk thread, and a 4/0 monofilament nylon needle with a blunted round tip about 30 mm in length was inserted from the external carotid artery into the internal carotid artery until the tip blocked the origin of the middle cerebral artery. Sham-operated animals were subjected to the same procedures except for the insertion of the filament. Rats were decapitated 24 hours after the onset of permanent occlusion, and brain tissue was carefully excised. The infarction volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC; Sigma) staining. Brain tissues were cut into 2-mm coronal sections using a brain matrix (Zivic Instruments, Pittsburgh, PA, USA). Brain sections were immersed in a 1% TTC solution in phosphate-buffered saline (PBS) for 20 min at 37℃, and then fixed in a 10% formalin solution [20]. The size of the infarct area was measured by Image-ProPlus 4.0 software (Media Cybernetics, Silver Spring, MD, USA). The volume of infarct lesions was calculated as the infarction area divided by the total section area, and reported as a percentage.

Our proteomics study was performed according to a previously described method [18]. The right cerebral cortices were excised according to experimental groups and homogenized in buffer solution (8 M urea, 4% CHAPS, ampholytes, and 40 mM Tris-HCl). After homogenization, the homogenates were centrifuged at 16,000 g for 20 min at 4℃ and the supernatant was removed. The pellets were dissolved in lysis buffer and the total protein concentration was determined via the Bradford method (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Immobilized pH gradient (IPG) gel strips (pH range 4–7 and pH 6–9; 17 cm; Bio-Rad) were incubated in rehydration buffer (8 M urea, 2% CHAPS, 20 mM DTT, 0.5% IPG buffer, and bromophenol blue) for 13 hrs at room temperature. The assayed protein samples were loaded on IPG strips using the provided sample cups, and subjected to first-dimension isoelectric focusing (IEF) using the Ettan IPGphor 3 system (GE Healthcare, Uppsala, Sweden) with the following protocol: 250 V (15 min), 10,000 V (3 hours), and then 10,000-50,000 V. After first-dimension separation, the strips were incubated in equilibration buffer (6M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, and bromophenol blue) containing DTT and iodoacetamide. The strips were then loaded onto gradient gels (7.5–17.5%) and second-dimension electrophoresis was performed on Protean-II XI electrophoresis equipment (Bio-Rad) at 5 mA per gel for 2 hours followed by 10 mA per gel at 10℃ until the bromophenol blue dye migrated off the bottom of the gel.

For silver staining, the gels were first immersed in fixation solution (12% acetic acid, 50% methanol) for 2 hour, washed with 50% ethyl alcohol, and treated with 0.2% sodium thiosulfate. The gels were then washed with deionized water, followed by impregnation in a silver solution (0.2% silver nitrate), and were developed in a 0.2% sodium carbonate solution. The gel images were collected using Agfa Arcus 1200™ (Agfar-Gevaert, Mortsel, Belgium). The PDQuest 2-D analysis software (Bio-Rad) was used to analyze protein spots on gels after silver staining. Differentially expressed protein spots were determined based on the results of the PDQuest software. The protein spots were then excised and distained. The gel particles were digested in a trypsin-containing buffer. The extracted peptides were analyzed using a Voyager-DETM STR biospectrometry workstation (Applied Biosystem, Forster City, CA, USA) for peptide mass fingerprinting. Database searches were carried out using the MS-Fit and ProFound programs. SWISS-PROT and NCBI were used as the protein sequence databases.

The right cerebral cortices were isolated under sterile conditions, immediately frozen in liquid nitrogen, and stored at −70℃. The TRIzol Reagent Kit (Life Technologies, Rockville, MD, USA) was used for extraction of total RNA according to the manufacturer's instructions. Total RNA (1 µg) was reverse-transcribed into complementary DNA with a SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The reverse transcription step was carried out at 42℃ for 30 min, the inactivation of reverse transcriptase at 95℃ for 5 min, followed by cooling down of the samples at 4℃ for 5 min. The primer sequences of DLP-1 were: 5'-TCACCAAAGAT GGTGCTCCCGGAT-3' (forward primer), 5'-AATTAC AGTACACAGGAA-3' (reverse primer). The primer sequences of actin were: 5'-GGGTCAGAAGGACTCC TACG-3' (forward primer), and 5'-GGTCTCAAACATG ATCTGGG-3' (reverse primer). The reverse-transcribed cDNA was used as a template for PCR amplification. The reaction conditions for PCR were as follows: an initial step of 94℃ for 5 min; 94℃ for 30 sec, 54℃ for 30 sec, and 72℃ for 1 min, repeated for 30 cycles; and a final extension at 72℃ for 10 min. The PCR products were loaded on 1% agarose gels, and the PCR product bands were visualized under ultraviolet light.

Western blot analysis was performed as previously described [18]. Briefly, the right cerebral cortices were dissolved and homogenized in lysis buffer (1 M Tris-HCI, 5M sodium chloride, 0.5% sodium deoxycholate, 10% sodium dodecyl sulfate, 1% sodium azide, and 10% NP-40) containing 200 µM phenylmethylsulfonyl fluoride as a protein inhibitor. After sonication, homogenates were separated by centrifugation at 15,000 g for 20 min at 4℃, and the supernatants were harvested for further process. A bicinchoninic acid kit (Pierce, Rockford, IL, USA) was used for the measurement of protein concentration according to the manufacturer's protocols. An equal amount of total protein (30 µg) from each sample was mixed with the loading buffer, boiled for 5 min, cooled down, and loaded onto a 10% SDS-polyacrylamide gel, where electrophoresis was carried out. The proteins were then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with 5% non-fat dry milk for 1 hour at room temperature to minimize non-specific antibody binding, washed in Tris-buffered saline containing 0.1% Tween-20 (TBST), and then incubated with either an anti-DLP-1 antibody (diluted 1:1,000; BD Biosciences, San Jose, CA, USA) or an anti-β-actin (diluted 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4oC. After washing in TBST, the membranes were reacted with horseradish perxoxidase-conjugated goat anti-rabbit IgG (1:5,000; Pierce). The enhanced chemiluminescence Western blot analysis system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) was used according to the manufacturer's manual for the detection of immunoreactive bands.

The experimental data are expressed as mean±SEM. The intensity analysis was carried out using the SigmaGel 1.0 (Jandel Scientific, San Rafael, CA, USA) and SigmaPlot 4.0 (SPSS Inc., Point Richmond, CA, USA) programs. The relative expression levels of each group were normalized to that of the non-diabetic+sham group. The differences among various experimental groups were analyzed via two-way analysis of variance followed by post-hoc Scheffe's test. A P value of <0.05 was considered statistically significant.