Male Mongolian gerbils (Meriones unguiculatus) were obtained from the Experimental Animal Center, Hallym University, Chuncheon, Korea. Gerbils were used at 6 months (B.W., 65 to 75 g) of age. The animals were housed in a conventional state under adequate temperature (23℃) and humidity (60%) control with a 12-hour light/12-hour dark cycle and provided with free access to food and water. All experimental procedures were in accordance with the National Institutes of Health guidelines and ARRIVE guidelines for the care and use of laboratory animals. The animal protocol used in present study was reviewed and approved based on ethical procedures and scientific care by the Kangwon National University-Institutional Animal Care and Use Committee. All of the experiments were conducted to minimize the number of animals used and the suffering caused by the procedures used. The animals were fed commercially available rodent diet, consisted of different fat concentrations as follows: normal diet (ND; D12450B, 10% kcal% fat, 20% kcal% protein, 70% kcal% carbohydrate, Research Diets, New Brunswick, NJ, USA), HFD (D12492, 60% kcal% fat, 20% kcal% protein, 20% kcal% carbohydrate, Research Diets), for 28 days. Finally, the animals were divided into four experimental groups: (1) ND-fed sham-operated group (ND-sham-group); (2) ND-fed ischemia-operated group (ND-ischemia-group); (3) HFD-fed sham-operated group (HFD-sham-group); and (4) HFD-fed ischemia-operated group (HFD-ischemia-group). On day 28, body weight was determined, and blood glucose and serum lipid profiles were measured.

On day 28 after feeding ND or HFD, blood samples were collected from each animal by orbital puncture, and blood glucose levels were analyzed using a blood glucose monitor (Ascensia Elite XL Blood Glucose Meter, Bayer, Toronto, ON, Canada). In addition, serum was separated from the blood by centrifugation and kept at -80℃ until analyzed. Triglyceride and total cholesterol in the serum was measured using an enzymatic method with commercial kits (Asan Pharmaceutical Co., Seoul, Korea).

One day after blood sampling, transient cerebral ischemia was induced. The animals were anesthetized with a mixture of 2.5% isoflurane (Baxtor, Deerfield, IL, USA) in 33% oxygen and 67% nitrous oxide. Bilateral common carotid arteries were isolated and occluded using nontraumatic aneurysm clips. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope. After 5 minutes of occlusion, the aneurysm clips were removed from the common carotid arteries. The body (rectal) temperature under free-regulating or normothermic (37℃±0.5℃) conditions was monitored with a rectal temperature probe (TR-100, Fine Science Tools, Foster City, CA, USA) and maintained using a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. Thereafter, animals were kept on the thermal incubator (Mirae Medical Industry, Seoul, Korea) to maintain body temperature until they were euthanized. Sham-operated animals were subjected to the same surgical procedures except that the common carotid arteries were not occluded.

For the histological analysis, animals were anesthetized with 40 mg/kg sodium pentobarbital and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (pH 7.4). The brains were removed and postfixed in the same fixative for 6 hours. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter, frozen tissues were serially sectioned on a cryostat (Leica, Wetzlar, Germany) into 30-µm coronal sections, and they were then collected into six-well plates containing PBS.

To examine the delayed neuronal death in the septal region after ischemia-reperfusion, the animals (n=7 at each time) were used at sham, 2, 4, and 7 days after ischemia-reperfusion. NeuN immunohistochemistry and Fluoro-Jade B (F-J B) histofluorescence staining were performed according to our previous method [22]. In brief, the sections were incubated with diluted mouse anti-NeuN (1:1,000, Chemicon, Temecula, CA, USA) and subsequently exposed to biotinylated goat antimouse immunoglobulin G and streptavidin peroxidase complex (1:200, Vector, Burlingame, CA, USA). They were visualized by staining with 3,3'-diaminobenzidine (Sigma, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer (pH 7.2).

For F-J B staining, the sections were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol and then in 70% alcohol. They were transferred to a solution of 0.06% potassium permanganate and then to a 0.0004% F-J B (Histochem, Jefferson, AR, USA) staining solution. The sections were examined using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue (450 to 490 nm) excitation light and a barrier filter.

To evaluate the effects of ND and HFD against ischemic damage, NeuN-immunoreactive (⁺) neurons and F-J B positive (⁺) cells were counted in a 250×250 µm square applied approximately at the septal region using an image analyzing system (Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA). The studied tissue sections were selected at 120-µm intervals, and cell counts were obtained by averaging the counts from each animal.

To examine the gliosis, the animals (n=7 at each time) were used at sham, 2, 4, and 7 days after ischemia-reperfusion, and immunohistochemistry for gliosis was performed according to our previous method [23]. In brief, immunohistochemical staining for rabbit antiglial fibrillary acidic protein (GFAP; 1:800, Chemicon) for astrocytes and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1:800, Chemicon) was performed according to the above-mentioned protocol. A negative control test was also carried out using preimmune serum instead of primary antibody in order to establish the specificity of the immunostaining. The negative control test resulted in the absence of immunoreactivity in all structures.

Digital images of the septal region were captured with an AxioM1 light microscope (Carl Zeiss) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. Semiquantification of the immunostaining intensities was evaluated with digital image analysis software (MetaMorph 4.01, Universal Imaging Corp., Downingtown, PA, USA). The level of immunoreactivity was scaled as -, ±, +, or ++, representing no staining (gray scale value, ≥200), weakly positive (gray scale value, 150 to 199), moderate (gray scale value, 100 to 149), or strong (gray scale value, ≤99), respectively.

The data shown here represent the mean±SEM. Differences in the means among the groups were statistically analyzed by one-way analysis of variance with a post hoc Bonferroni's multiple comparison test in order to elucidate ischemia-related differences among experimental groups. Statistical significance was considered at P<0.05.

The changes in body weight, blood glucose, serum triglyceride, and total cholesterol of the ND- and HFD-groups were measured on day 28 after feeding. No difference in body weight gain was noted between the ND- and HFD-groups. However, the levels of blood glucose, serum triglyceride, and total cholesterol were markedly increased in the HFD-group compared with those in the ND-group (Table 1).