Animals
All procedures were carried out according to the Kumamoto University Committee for Laboratory Animal Care and use. All of the experimental procedures were performed in accordance with guidelines on animal science. Male db/db mice and nondiabetic C57BL/6 J mice were purchased from Japan Charles River Laboratories Japan Inc. (Yokohama, Japan). All animals were housed in an animal facility with a 12-hour light–dark cycle and were given the standard chow and water ad libitum.
Drugs
Linagliptin was kindly provided by Boehringer Ingelheim.
Experiment I protocol
The detail of Experimental I protocol is shown in Fig. 1. Eight-week-old male db/db mice were subjected to 17 min of transient global cerebral ischemia, as described below. Following transient global cerebral ischemia, db/db mice were randomly assigned 2 groups, and were given (1) the standard diet (MF diet, ORIENTAL YEAST Co., Ltd, Tokyo, Japan) or (2) the standard diet containing linagliptin (0.083 g/kg diet), for 8 weeks. This dose of linagliptin (0.083 g/kg diet) was established to sufficiently inhibit the circulating DPP-4 activity [19, 20]. In our preliminary experiments, we measured diet intake of db/db mice and found that oral intake of linagliptin in our present study was roughly 10 mg/kg/day. This dose of linagliptin is regarded as an appropriate dose because 3–10 mg/kg/day of linagliptin by oral administration is most often used to examine the effect of linagliptin in vivo [10, 21, 22]. Sham-operated db/db mice were served as the control. Non-fasting blood sugar was periodically measured throughout the experiment. At specified time points, cerebral blood flow, passive avoidance test, water maze test, oral glucose tolerance test, and blood pressure measurement were performed according to the method described below (see Fig. 1). At the end of the treatment (8 weeks after BCCAO), the animals were anesthetized with 3 % isoflurane and then sacrificed for histological examination. The brain was removed quickly and cut from bregma. An 8 μm-slice was made from the level of bregma −1.43 mm to bregma −2.43 mm for the following histological evaluations including volume of hippocampus and cortex, neuronal cell number.
Experiment II protocol
The protocol of the Experiment II was the same as that of Experiment I, except for the difference in duration of linagliptin administration. db/db mice were subjected to BCCAO for 17 min and then were given (1) the standard diet (MF diet, ORIENTAL YEAST Co., Ltd, Tokyo, Japan) or (2) the standard diet containing linagliptin (0.083 g/kg diet) for 1 week. The effect of short-term (7 days) linagliptin administration on cerebral IgG extravasation, cerebral activated microglia, and oxidative stress was examined.
Transient global cerebral ischemia model
db/db mice were anesthetized with 3 % isoflurane and maintained with 2 % isoflurane via a facemask. The rectal temperature was controlled at 37.0 ± 0.5 °C during surgery with a feedback-regulated heating pad. Transient global ischemia was induced by bilateral common carotid artery occlusion (BCCAO). Briefly, BCCA were isolated and occluded with a microvascular clip. After 17 min, the clips were removed to allow reperfusion. The mice were kept in a single cage and allowed free access to food and water.
Measurement of cerebral blood flow
Cerebral blood flow was measured using a laser speckle blood flow imager (Omega Zone; Omegawave, Tokyo, Japan) as previously described [23]. The blood flow at the times of pre-operation, post-operation, reperfusion and sacrifice were measured. The value of blood flow was expressed as a percentage of the pre-operation.
Passive avoidance test
The passive avoidance test was performed as two sections including (1) training section and (2) memory test section. On the training section, we performed the test as two steps. In Step 1, we put the db/db mice into the white side of the test box and let the mice be free movement for 60 s. In Step 2, 30 min after the step one, put the mice into the white side of the test box and recorded the second until the mice went into the dark place (4 paws into the dark side). After the door was closed, the mice were given the electric shock (1.6 mA, 3 s) and kept in the dark side of the test box for 60 s. Repeat the step two until the mouse did not go into the dark side for 300 s. On the memory test section, we put the mice into the white side of the test box one day and three days after the training section, then recorded the seconds when the mouse went into the dark place (upper limit for 300 s).
Morris water maze tests
The Morris water maze was performed as previously described [24]. The test was included four sessions: 1) training session, 2) hidden platform session, 3) visible platform test. After the training session, hidden platform test was performed five sessions per day on the following four days (day 1 to 4). On the visible platform test the platform was cued and the mice were released from different points.
Measurement of blood pressure
The blood pressure was measured in the conscious mice at 6 weeks after BCCAO, using the tail cuff method (BP-98A; Softron Co, Tokyo, Japan).
Oral glucose tolerance test (OGTT)
The oral glucose tolerance test was performed as previously described [25]. Briefly, mice were fasted for overnight and then orally given glucose (1 mg/g body weight). Tail vein bloods were taken and the serum glucose concentrations were measured at 0, 30, 60 and 120 min after glucose administration using a portable glucose meter (Sanwa Kagaku Kenkyusho CO., LTD, Nagoya, Japan).
The evaluation of brain atrophy and surviving neurons
For the assessment of volume of hippocampus and cortex, the slices were selected every 200 μm from bregma −1.43 to bregma −2.43 and stained with Nissl staining. Areas of the bilateral hippocampus and cortex on each slice were calculated separately and added together and multiplied by slice thickness to give the volume. For the assessment of surviving pyramidal cells and the neurons in cortex, the selected area of hippocampus (1 field in each side of CA1 region at × 200 magnification) and cortex (2 fields were counted in each side at × 200 magnification) were counted using Lumina Vision version 2.2.0 analysis software and the average number of surviving pyramidal cells and neurons in cortex were used for evaluation of the neuronal injury.
The measurement of activated microglia
For the assessment of number of activated microglia, brain sections were immunostained with anti-ionized calcium binding adaptor molecule-1. (Iba-1(ab5076); 1:500; Abcam, Tokyo, Japan) as previously described [26]. The number of positive cells was counted in both the CA1 region of hippocampus (1 field was counted in each side of CA1 region at × 200 magnification) and the cortex (2 fields were counted in each side at × 200 magnification). The number of cells was expressed as cells/mm2.
Measurement of immunoglobulin G extravasation
Brain immunolocalization of immunoglobulin G was measured as previously described [27, 28]. IgG immunoreactivity was quantified in both sides the CA1 region of hippocampus (1 field was counted in each side of CA1 region at × 200 magnification) and the cortex (2 fields were counted in each side of cortex at × 200 magnification). The value was measured with Lumina Vision version 2.2.0 analysis software (Mitani Corporation, Tokyo, Japan) and expressed as the mean value compared with the sham-operated group.
Detection of superoxide
To detect superoxide levels in hippocampus and cortex, dihydroethidium (DHE; SIgam, St. Louis, MO, USA) staining was used in situ as previously described [29]. The frozen tissue sections were stained with DHE fluorescence and measured using Lumina Vision version 2.2.0 analysis software. The mean fluorescence was quantified in both sides the CA1 region of hippocampus (1 field was counted in each side of CA1 region at × 200 magnification) and the cortex (2 fields were counted in each side of cortex at × 200 magnification). Each value was expressed related to individual group sham-operated mouse. The results were expressed relative to values obtained for sham-operated mouse.
Western blotting
The detailed Western blot method has been previously described [30]. Briefly, the front part of brain protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The antibodies used were as follows: anti-claudin-5 (ab53765) (1:500; Abcam), GAPDH (sc-32233) (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-gp91phox (sc-20782) (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were incubated for 1 h with appropriate secondary antibodies and processed with an enhanced chemiluminescence reagent kit (Amersham ECL plus kit, GE Healthcare, UK). Mouse or rabbit secondary antibody (1:5000 or 1:30000; Cell Signaling) was used in accordance with first antibody. The antibodies were visualized by using an enhanced chemiluminescence method (ECL Plus; GE Healthcare, Buckinghamshire, UK). The intensity of the bands was quantified by using analysis software (Image J; National Institute of Health, Bethesda, MD, USA). In individual samples, each value was corrected for that of GAPDH.
Measurement of DPP-4 activity and GLP-1 in serum and brain tissue and serum insulin
DPP-4 activity, GLP-1 concentrations, and serum insulin were determined in samples from mice subjected to 8 weeks of linagliptin treatment. Serum and brain DPP-4 activities were measured using DPP4-Glo Protease Assay (Promega, Madison, WI, USA), as described [21]. Active GLP-1 concentrations in serum and brain tissue were measured by the Active form Assay Kit-IBL (Immuno-Biological Laboratories, Gunma, Japan). Serum insulin concentrations were measured by Morinaga ultra sensitive mouse insulin ELISA kit.
Statistical analysis
Data were presented as mean ± SEM. The data on time course experiments were analyzed by two-way ANOVA with repeated measures followed by Bonferroni post hoc test for multiple comparisons. Statistical significance was determined with one-way ANOVA, followed by the Tukey's multiple comparisons test between each group. When a normal distribution was not confirmed among comparison groups, data were analyzed by Dunn’s multiple comparison test. Data were analyzed by non-paired t-test when two groups were compared. All analyses were performed using GraphPad Prism 6.0 for Mac (GraphPad Software, Inc; La Jolla, CA, USA). In all tests, differences were considered statistically significant at a value of P < 0.05.