A total of 85 male Mongolian gerbils (3-months-old; 50–60 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were housed under standard conditions with adequate temperature (22°C) and humidity (60%) control, a 12 h light/12 h dark cycle and had free access to food and water. The handling and care of the animals conformed to the guidelines established to comply with current international laws and policies [NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85–23, 1985, revised 1996 (18)], and were approved by the Institutional Animal Care and Use Committee of Seoul National University (Seoul, Korea). All of the experiments were conducted with an effort to minimize the number of animals used and the suffering caused by the procedures employed in the present study.

The animals were anesthetized with a mixture of 2.5% isoflurane (Hana Pharmaceutical Co., Ltd., Seoul, South Korea) in 33% oxygen and 67% nitrous oxide. Bilateral common carotid arteries were isolated and occluded using non-traumatic aneurysm clips. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope (HEINE K180^®; Heine Optotechnik, Herrsching, Germany). Following 5 min of occlusion, the aneurysm clips were removed from the common carotid arteries. Blockade of blood flow by occlusion of the common carotid arteries was not performed in the animals of the sham-operated group. The body (rectal) temperature under free-regulating or normothermic (37±0.5°C) conditions was monitored with a rectal temperature probe (TR-100; Fine Science Tools GmbH, Foster City, CA, USA) and maintained using a thermometric blanket prior to, during and following surgery until the animals completely recovered from anesthesia. Thereafter, animals were kept on the thermal incubator to maintain the body temperature of animals until the animals were euthanized.

For histology, sham- and ischemia-operated animals (n=5 per group) were anesthetized with 1 g/kg urethane (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at various time points following ischemia/reperfusion and perfused transcardially with 0.1 M PBS (pH 7.4), which was followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4). The brains were removed and post-fixed in 4% paraformaldehyde for 12 h at 25°C. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight at 4°C. Brain sections (30 µm) in coronal plane were serially cut using a cryostat (Leica Microsystems GmbH, Wetzlar, Germany). The sections were collected into 6-well plates containing PBS for further processes.

To ensure that the immunohistochemical data were comparable between groups, the sections were carefully processed under the same conditions. The tissue sections were selected between 1.4 mm and 2.0 mm posterior to the bregma in reference to a gerbil brain atlas (19) for each animal. A total of 5 sections per group, 90 µm apart from each other, were sequentially treated with 0.3% hydrogen peroxide in PBS for 30 min and 10% normal goat serum (Vector Laboratories, Inc., Burlingame, CA, USA) in 0.05 M PBS for 30 min at 25°C. They were subsequently incubated with rabbit anti-MCT4 antibodies (1:200; catalog no. sc-50329; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 25°C, followed by treatment with biotinylated goat anti-rabbit IgG for 2 h at 25°C (1:200; catalog no. BA-1000; Vector Laboratories, Inc.) and a streptavidin-peroxidase complex (catalog no. PK-6100; Vector Laboratories, Inc.). Sections were subsequently visualized by reaction with 3,3′-diaminobenzidine tetrachloride (Sigma-Aldrich; Merck KGaA) in 0.1 M Tris-HCl buffer (pH 7.2) and mounted on gelatin-coated slides. Sections were mounted in Canada Balsam (Kanto Chemical Co., Inc., Tokyo, Japan) following dehydration.

Analysis of the regions of interest in the stratum oriens, stratum pyramidale and stratum radiatum of the hippocampal CA1 region was performed using an image analysis system and ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA). Images were calibrated into an array of 512×512 pixels corresponding to a tissue area of 140×140 µm (x40 magnification). Each pixel resolution was 256 gray levels. The intensity of MCT4 immunoreactivity was evaluated by relative optical density (ROD), which was obtained following transformation of the mean gray level using the following formula: ROD=log (256/mean gray level). ROD of background was determined in unlabeled portions and this value was subtracted for correction, yielding high ROD values in the presence of preserved structures and low values following structural loss using ImageJ software version 1.50 (National Institutes of Health). A ratio of the ROD was calibrated as percentage compared with the control.

To confirm the co-localization of MCT4 and glial fibrillary acidic protein (GFAP) in the brain, sections taken 7 days following ischemia were processed under the same conditions as the immunohistochemistry analysis prior to addition of the primary antibody. Double immunofluorescence staining for rabbit anti-MCT4 (1:50) and mouse anti-GFAP (1:500; catalog no. AB5804; EMD Millipore, Billerica, MA, USA) was performed. The sections were incubated in the mixture of antisera overnight at 25°C. Following three washes for 10 min each with PBS, they were subsequently incubated in a mixture of Cy3-conjugated donkey anti-rabbit IgG (1:600; catalog no. 711-165-152; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and FITC-conjugated donkey anti-mouse IgG (1:600; catalog no. 715-095-151; Jackson ImmunoResearch Laboratories, Inc.) for 2 h at 25°C. The immunoreactions were observed under a confocal microscope (LSM 510 META NLO; Zeiss GmbH, Jena, Germany).

To confirm the changes in the expression of MCT4 in the hippocampus, animals in sham- and ischemia-operated animals (n=5) were sacrificed and western blot analysis was performed. Following sacrifice and removal of tissues, the tissues were cut into 500 µm thick sections on a vibratome (Leica Microsystems GmbH) and the hippocampus was cut out using a surgical blade. The hippocampal tissues were homogenized in 50 mM PBS (pH 7.4) containing 0.1 mM EGTA (pH 8.0), 0.2% Nonidet P-40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM sodium fluoride, 150 mM sodium chloride, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol (DTT). Following centrifugation for 5 min at 16,000 × g at 4°C, the protein concentration was determined in the supernatants using a Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Aliquots containing 20 µg of total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 3 mM DTT, 6% SDS, 0.3% bromophenol blue and 30% glycerol. Each aliquot was subsequently loaded onto a 12% polyacrylamide gel. Following electrophoresis, the gels were transferred to nitrocellulose membranes (Pall Life Sciences, Port Washington, NY, USA). To reduce background staining, the membranes were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween-20 for 45 min at 25°C, which was followed by incubation with peroxidase-conjugated rabbit anti-MCT4 (1:500; catalog no. sc-50329, Santa Cruz Biotechnology, Inc.) and rabbit anti- β actin (1:2,000; catalog no. ab8227; Abcam, Cambridge, UK) overnight at 4°C and an ECL chemiluminescent kit (Pierce; Thermo Fisher Scientific, Inc.). The blot was densitometrically scanned for the quantification of ROD of each band using Scion Image software version 4.0.3 (Scion Corporation., Frederick, MD). Expression was normalized against β-actin.

Data are presented as the mean + standard error of the mean. The differences among means were analyzed by one-way analysis of variance followed by Bonferroni's post-hoc correction to compare the time-dependent changes in MCT4 immunoreactivity. Analysis was performed using GraphPad Prism 5.01 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Microscopy images are presented in Fig. 1 and quantification of immunoreactivity are presented in Fig. 2. MCT4 immunoreactivity was observed in the stratum pyramidale of the CA1 region of the sham-operated group (Fig. 1A). At 30 min and 3 h after ischemia, reduced MCT4 immunoreactivity was observed in the stratum pyramidale compared with the immunoreactivity observed in the sham-operated group (Figs. 1B and C and 2). Between 6 and 24 h after ischemia, MCT4 immunoreactivity levels increased time-dependently in the CA1 region, however, the difference in the MCT4 immunoreactivity was not statistically different between sham and these groups (Figs. 1D-F and 2). MCT4 immunoreactivity was significantly increased 2 days following ischemia in the stratum pyramidale compared with the sham-operated group (Figs. 1G and 2). At 3 days following the treatment, MCT4 immunoreactivity decreased significantly in the stratum pyramidale compared with at 2 days following ischemia and was barely detectable in this region (Figs. 1H and 2). At days 4 and 5 following ischemia, MCT4 immunoreactivity increased in the strata oriens and radiatum compared with sham-operated animals (Fig. 1I and J). However, few MCT4 immunoreactive structures were detected in the stratum pyramidale (Fig. 1I and J). At 7 and 10 days following ischemia, MCT4 immunoreactivity was observed in the strata oriens, radiatum and pyramidale (Fig. 1K and L). In these groups, MCT4 immunoreactivity levels significantly increased compared with the group at 5 days following ischemia (P<0.05; Fig. 2). Numerous MCT4 immunoreactive structures were colocalized with activated (exhibiting a hypertrophied cytoplasm) GFAP immunoreactive astrocytes in the CA1 region of the hippocampus (Fig. 3).

MCT4 protein levels decreased significantly in hippocampal CA1 homogenates 3 h following ischemia compared with CA1 homogenates sampled from the sham-operated group (P<0.05). The protein levels of MCT4 exhibited a significant 2.9-fold increase 2 days following ischemia compared with the sham-operated group, and a 4.5-fold increase at 2 days compared with the group at 3 h following ischemia. Only weak MCT4 bands were detected 3 days after ischemia and MCT4 levels were 25% of the sham-operated group levels. At 10 days after ischemia, MCT4 levels were marginally higher in the experimental group compared the sham-operated group (Fig. 4).