Human specimen and immunohistochemistry
Human IA samples and control arterial walls (superficial temporal artery or middle meningeal artery) were dissected during microsurgical clipping of unruptured IAs with the written informed consent. Dissected specimens were fixed in formalin solution and embedded in paraffin. Four-micrometer-thick slices were then prepared for histopathological and immunohistochemical analyses. After deparaffinization and blocking with 3% donkey serum (Jackson ImmunoResearch, West Grove, PA), slices were incubated with primary antibodies overnight at 4 °C in a humidified chamber, followed by incubation with secondary antibodies conjugated with fluorescence dye (Jackson ImmunoResearch) for additional 1 h at room temperature in a humidified chamber. Finally, fluorescent images were acquired on a confocal fluorescence microscope system (FV1000, Olympus, Tokyo, Japan). Scanning was performed using a UPLSAPO 40×/NA 0.9 objective (Olympus) at a resolution of 1024 × 1024 pixels with a field of view of 317 μm square. Maximal intensity projections along the Z-axis of at least five images at optimal thickness from each slide were obtained using a FLUOVIEW (version 4.02, Olympus). Images were processed in brightness and contrast using an Adobe Photoshop Elements14 (Adobe Systems, San Jose, CA).
In some experiments, secondary antibodies conjugated with horseradish-peroxidase (Vector Laboratories, Burlingame, CA) were used and immune complexes were detected with either a Vectastain Elite ABC kit (Vector Laboratories) or an EnVision kit (DAKO, Glostrup, Denmark) using 3,3′-diaminobenzidine (DAB) as a substrate. Images were acquired using a light microscope (IX71, Olympus) equipped with a UPlanFLN 4×/NA 0.13 objective (Olympus), UPlanFLN 10×/NA 0.3 objective (Olympus), or LUCPlanFLN 40×/NA 0.6 objective (Olympus) at a resolution of 1360 × 1024 pixels with a field of view of 4.4 × 3.3 mm, 1.8 × 1.3 mm, or 438.6 × 330.2 μm, respectively. Images were captured using a digital camera (DP72, Olympus) and a cellSens Standard (version 1.6, Olympus). The brightness and contrast of the images were adjusted in an Adobe Photoshop Elements14 (Adobe Systems).
Primary antibodies used are as follows: rabbit polyclonal anti-GPR120 antibody (dilution 1:900, #LS-A2003, LifeSpan BioSciences, Inc., Seattle, WA), mouse monoclonal anti-CD31 antibody (1:100, #M0823, DAKO), mouse monoclonal anti-smooth muscle α-actin (SMA) antibody (1:200, #M0851, DAKO), and mouse monoclonal anti-CD68 antibody (1:100, #M0814, DAKO).
The secondary antibodies used are as follows: Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H + L) antibody (1:100, #A21206, Invitrogen, Carlsbad, CA) and Alexa Fluor 594-conjugated donkey anti-mouse IgG (H + L) antibody (1:100, #A21203, Invitrogen).
Rodent IA models and histological analysis of induced IA
Male Sprague–Dawley rats were purchased from Japan SLC (n = 84, Shizuoka, Japan). Animals were maintained on a light/dark cycle of 12 h/12 h, and had a free access to chow and water.
A total of 24 rats were used to examine the pharmacokinetics of eicosapentaenoic acid (EPA). The whole blood was transcardially collected from four rats before EPA administration under intraperitoneal injection of a lethal dose of pentobarbital sodium (200 mg/kg). The remaining 20 rats were randomly divided into the two groups; vehicle-treated group and the EPA-treated group. Ten rats in each group were administrated 1000 mg/kg EPA or olive oil as a vehicle. The whole blood was then collected transcardially at 1 h (n = 3), 6 h (n = 3), or 24 h (n = 4) after the administration.
To induce IA, under general anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg), 7-week-old male rats were subjected to ligation of the left carotid artery and hypervolemia achieved by the combination of a high salt diet and ligation of the left renal artery. Immediately after above surgical manipulations, the chow was completely switched from a normal to the special one containing 8% sodium chloride and 0.12% 3-aminopropionitrile (Tokyo Chemical Industry, Tokyo, Japan), an inhibitor of lysyl oxidase that catalyzes the cross-linking of collagen and elastin. 3-Aminopropionitrile was added in a chow to reduce the stiffness of arterial walls and thus facilitate degenerative changes of the media. The special chow was refilled twice a week, and all rats kept free access to water and the special chow after surgical manipulations during the observation period. A total of 60 rats were subjected to surgical manipulations and 30 rats among them were used for examining the effect of EPA on IAs. Another 30 rats were used to determine the intake of EPA in the plasma. These 30 rats were randomly allocated into three groups: the olive oil-administered group (vehicle-treated group, n = 10), the 100 mg/kg/day EPA-administered group (n = 10), or the 1000 mg/kg/day EPA-administered group (n = 10). Dead animals during the observation period were excluded from the analysis. Blood pressure was measured by the tail-cuff method on the 11th day after surgical manipulations. The rats were placed in a cylindrical warmer (#THC-31, Softron, Tokyo, Japan) and kept at 37 °C during the measurement. Blood pressure were then measured using an automatic measurement device (#BP-98A, Softron) without any anesthesia and its value was defined as the average of three consecutive measurements. Body weight was measured twice; before surgical manipulations and on the day of sacrifice. At times indicated in the corresponding figure legends or results after above surgical manipulations, animals were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (200 mg/kg), and transcardially perfused with 4% paraformaldehyde solution. The bifurcation site of anterior cerebral artery (ACA)-olfactory artery (OA) including the induced IA lesion was then stripped, and serial frozen sections were made. Histopathological examination to evaluate IAs was done after Elastica van Gieson staining which visualizes internal elastic lamina (IEL) and induced IAs were histologically defined as a lesion with the disruption of IEL [8, 13]. The area of IA was measured using ImageJ software (version 1.51s, https://imagej.nih.gov/ij/index.html)  using the section including the maximum diameter of the lesions.
EPA (Epadel (ethyl icosapentate), Mochida Pharmaceutical Co., LTD., Tokyo, Japan) was commercially available and we thus purchased this drug. Immediately before administration, EPA was extracted from Epadel capsule and then diluted with olive oil to the concentration of 25 mg/ml or 250 mg/ml. Then, 4 ml/kg of EPA solution was orally administered by gavage. The dose of EPA was determined by referencing the previous reports in a rat model [15,16,17]. For the measurement of plasma concentration of EPA, docosahexaenoic acid (DHA), and also arachidonic acid (AA), arterial blood was transcardially collected with an 18-gauge needle at 24 h after the last administration of EPA or vehicle after intraperitoneal injection of a lethal dose of pentobarbital sodium (200 mg/kg). The collected blood (5–8 ml per animal) was heparinized and the plasma was then collected. The plasma samples were stored at − 80 °C until measurement. The plasma concentration of EPA, DHA, and AA was measured by gas chromatography at SRL Inc. (Tokyo, Japan).
At the indicated period after surgical manipulations, 5-μm-thick frozen sections were prepared from dissected IA lesions as described above. After blocking with 3% donkey serum (Jackson ImmunoResearch), slices were incubated with primary antibodies overnight at 4 °C in a humidified chamber, followed by incubation with secondary antibodies conjugated with a fluorescence dye (Jackson ImmunoResearch) for additional 1 h at room temperature in a humidified chamber. The slices were then mounted with by a ProLongR Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) (Invitrogen), a fluorescence dye that binds to double-stranded DNA and then visualizes nuclei. Finally, fluorescent images were acquired on a confocal fluorescence microscope system (FV1000, Olympus). Scanning was performed using a UPLSAPO 40×/NA 0.9 objective (Olympus) or 60×/NA 1.35 oil immersion objective (Olympus) at a resolution of 1024 × 1024 pixels with a field of view of 317 μm or 211 μm square, respectively. Maximal intensity projections along the Z-axis of at least five images at optimal thickness from each slide were obtained using a FLUOVIEW (version 4.02, Olympus). Images were processed in brightness and contrast using an Adobe Photoshop Elements14 (Adobe Systems).
The primary antibodies used are as follows: Cy3-conjugated mouse monoclonal anti-smooth muscle α-actin antibody (1:200, #6198-2ML, Sigma Aldrich, St. Louis, MO), mouse monoclonal anti-CD68 antibody (1:100, #31630, Abcam, Cambridge, UK), and rabbit polyclonal anti-C-C motif chemokine 2 (CCL2) antibody (1:100, #ab9779, Abcam).
The secondary antibodies used are as follows: Alexa Fluor 488-conjugated donkey anti-mouse IgG (H + L) antibody (1:100, #A21202, Invitrogen) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H + L) antibody (1:100, #A21206, Invitrogen).
Macrophage was defined as the cell positive for CD68 in immunohistochemistry. The number of infiltrated macrophages in each lesion was calculated as a cell count present around the dome of induced aneurysms. Independent two researchers (Y.A. and I.O.) who did not know the group allocation counted the number of CD68-positive cells in each slide and the results were compared. When disagreed, the researchers checked and discussed the image together, and determined the number of positive cells.
Plasma concentration of Resolvin E1
Plasma was prepared from rats administered for 12 days with 1000 mg/kg/day of EPA. The plasma was collected at 24 h after the last administration of EPA as described above. And the concentration of Resolvin E1 was measured by a Rat Resolvin E1 ELISA Kit (#MBS2601346, MyBioSource, Inc., San Diego, CA) according to the manufacturer’s instructions.
Cell culture and treatment with compounds targeting GPR120
The Raw264.7 cell line (ATCC, Manassas, VA), a mouse monocyte/macrophage cell line, was used due to endogenous expression of functional GPR120 . Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum. In some experiments using EPA, DMEM without phenol-red (FUJIFILM Wako Pure Chemical Corporation) was used. Cells were pre-treated with GPR120 agonists, EPA (Mochida Pharmaceutical Co., Ltd.) or TUG-891 (Cayman Chemical, Ann Arbor, MI), before stimulation with lipopolysaccharide (LPS) (Lot. 123M4052V, # L2654, Sigma Aldrich).
Western blot analysis
Whole cell lysate was prepared by a RIPA buffer (Sigma Aldrich) supplemented with proteinase and phosphatase inhibitors (Roche, Indianapolis, IN). Protein concentration was, then, determined by a bicinchoninic acid (BCA) method (Pierce BCA Protein Assay Kit, Thermo Scientific, Waltham, MA). After sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), separated proteins were transferred to a PDVF membrane (Hybond-P, GE healthcare, Buckinghamshire, UK) and blocked with an ECL plus blocking agent (GE healthcare). The membranes were incubated with primary antibodies followed by incubation with an anti-IgG antibody conjugated with horseradish peroxidase (GE healthcare). Finally, the signal was detected by a chemiluminescent reagent (ECL Prime Western Blotting Detection System, GE healthcare). α-Tubulin was served as an internal control.
Antibodies used in Western blot analysis are as follows: rabbit monoclonal anti-p65 antibody (Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phospho NF-κB p65 (Ser536) antibody (Cell Signaling Technology), and mouse monoclonal anti-α-tubulin antibody (Sigma Aldrich).
Quantitative real time-PCR analysis
RNA purification from cultured cells and reverse transcription were done using a RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) and a High-capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Carlsbad, CA) according to manufacturers’ instructions. For the quantification of gene expression, RT-PCR was performed on a Real Time System a LightCycler 480 (Roche) with a TB Green Premix Ex Taq II (TAKARA BIO INC., Shiga, Japan) using the expression of α-actin (Actb) as an internal control. For quantification, the second derivative maximum method was used for crossing point determination.
Primer sets used in the present experiment are as follows: forward 5′-GTCTCTGCCGCCCTTCTGTG-3′ and reverse 5′-AGGTGACTGGGGCATTGATTG-3′ for Ccl2 which encodes monocyte chemoattractant protein 1 (MCP-1), and 5′-ACGACCAGAGGCATACAGGGA-3′ and 5′-CCCTAAGGCCAACCGTGAAA-3′ for Actb.
Data are shown as mean ± SD or by box-and-whisker plots. Differences between the two groups were examined using a non-parametric Mann-Whitney test by a JMP Pro 13 (SAS Institute Inc., Cary, NC). Statistical comparisons between more than two groups were conducted using a Kruskal-Wallis test followed by the post-hoc Steel-Dwass test by a JMP Pro 13 (SAS Institute Inc.). We used above non-parametric tests in the present study because the sample size was not enough to assess whether the data sets were normally distributed or not by a Shapiro-Wilk test. The Jonckheere-Terpstra test was performed to confirm a dose-response relationship using an EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan, http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html) . A p value smaller than 0.05 was defined as statistically significant.