Mice (6-week-old male C57/BL6JJcl) used in this study were obtained from CLEA (Tokyo,Japan). The experimental protocols used in this study were approved by the Nihon University School of Dentistry Animal Ethical Committee and conducted according to the legal requirement (AP19DEN033-1).
Unilateral common carotid artery occlusion (UCCAO)
The mice were intraperitoneally (i.p.) injected with a mixture of 0.25 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol. The right CCA was exposed and ligated with a silk suture, and the surgical wound was closed.
For minocycline experiments, minocycline (MilliporeSigma, St. Louis, MO, USA) was suspended in PBS and injected (i.p.; 50 mg/kg body weight) at 2 h before and right after UCCAO.
Cell culture and treatment
The mice microglia cell line MG6 was obtained from the RIKEN BRC cell bank [13, 14]. The cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal calf serum, 10 µg/ml insulin (MilliporeSigma, St. Louis, MO, USA) 0.1 mM 2-mercaptoethanol in a 5% CO2 incubator. The cells (2 × 105/12 well plate) were incubated in hypoxic condition (5% O2, 5% CO2, Sugiyamagiken, Tokyo, Japan) for 6, 12, and 24 h. Culture supernatants and total RNA were harvested and subjected to ELISA and real-time polymerase chain reaction (PCR), respectively. For recombinant AIF-1 (enQuire BioReagents, Littleton, CO, USA) stimulation, the cells were cultured with or without 100 ng/ml of recombinant AIF-1 for 6 h . Total RNA was subjected to real-time PCR.
Total RNA was purified from excised brain or cultured MG6 cells using RNeasy mini kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was synthesized with Superscript III™ Reverse Transcriptase (Invitrogen, San Diego, CA, USA) and subjected to real-time PCR. Real-time PCR was performed using Thermal Cycler Dice ® Real Time System TP800 (TaKaRa, Tokyo, Japan) with TB Green ® Premix Ex Taq™II (Tli RNaseH Plus) (TaKaRa, Tokyo, Japan). The primers used are as follows.For AIF-1 5’-GTCCTTGAAGCGAATGCTGG-3’ (forward) and 5’-CATTCTCAAGATGGCAGATC-3’ (reverse). For β-actin 5’-GGTCAGAAGGACTCCTATGTGG-3’ (forward) and 5’-TGTCGTCCCAGTTGGTAACA-3’ (reverse).
On Day 0, 1, 3, and 5 after UCCAO, peripheral blood was collected and incubated for 30 min at 37℃ and then at 4℃ for 18 h. The samples were centrifuged at 1,200 x g for 20 min and the supernatants were preserved at -80℃. The brain tissue was minced with scissors in a cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% TritonX-100) and then homogenized using a Dounce homogenizer (Wheaton Industries, Millville, NJ, USA). The samples were centrifuged (12,000 x g, 5 min) and the supernatants were harvested. MG6 cell culture supernatants were harvested after culture under hypoxic condition. The samples were centrifuged (12,000 x g for 3 min) and the supernatants were harvested. AIF-1 concentration was measured using ELISA kit (MyBioSource, San Diego, CA, USA).
The mice were transcardially perfused with 4% paraformaldehyde and the organs (spleen, lung, liver, kidney, and brain) were excised and further fixed with the same fixative for 18 h. The organs were sliced and embedded in paraffin. Four-micrometer-thick sections were prepared, deparaffinized in xylene, rehydrated with 100% ethanol, and subjected to HE staining. For immunohistochemical staining, the sections were incubated with Dako Proteinase K (Agilent Technologies, Santa Clara, CA, USA) for 20 min at room temperature (RT) and washed with PBS. Endogenous peroxidase activity was inactivated with 0.3% hydrogen peroxide in methanol for 20 min at RT. Non-specific binding was blocked by incubating the sections with 1% BSA-PBS for 1 h at RT. The blocking solution was removed, and the rabbit anti-mouse Iba-1 antibody (Ab) (1:100 diluted with 1% BSA-PBS, FUJIFILM Wako, Osaka, Japan) was applied and incubated for 18 h at RT. Negative controls were incubated with 1% BSA-PBS instead of the primary Ab. The sections were then incubated with Dako EnVision™+ Dual Link System-HRP (ready to use, Agilent Technologies, Santa Clara, CA, USA) for 1 h at RT. The sections were washed and were developed with freshly prepared 3,3’-Diaminobenzidine chromogen (DAB, MilliporeSigma, St. Louis, MO, USA) solution for 7 min, counterstained with hematoxylin for 30 sec, dehydrated in a series of ethanol dilutions, cleared in xylene, and mounted on glass coverslips. The images (both HE and immunohistochemical stainings) were viewed and photographed using the all-in-one microscope BZ X-810 (Keyence, Osaka, Japan). Iba-1+ cell numbers in the spleen were counted under microscope. The area were divided into red pulp, white pulp and perivascular area of the central vein. Five different area (200 µm x 200 µm) were randomly picked up and Iba-1+ cells were counted. The count was repeated three times by different individuals per specimen. Total of three specimens were counted.
Male mice were used in all experiments. All data were analyzed statistically using IBM SPSS® Statistics 20 software (International Business Machines, Armonk, NY, USA). The data are expressed as the mean ± SD. The error bars represent the SD. For ELISA measurement, One-way ANOVA with Dunnett’s test and One-way ANOVA with Tukey’s test were used. For real-time PCR, student’s t-test and One-way ANOVA with Dunnett’s test were used to analyze statistical significance. Differences were considered significant at P < 0.05 (*p < 0.05, **p < 0.01).Sample sizes are shown in the figure legends.