IA models of rats and histological analysis of induced IA
All of the following experiments, including animal care and use, complied with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and complied with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of National Cerebral and Cardiovascular Center. The present manuscript adheres to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for reporting animal experiments.
10-week-old female Sprague − Dawley (SD) rats were purchased from Japan SLC (Slc:SD, n = 69, Shizuoka, Japan). Animals were maintained on a light/dark cycle of 12 h/12 h, and had a free access to chow and water. To induce IAs, rats were subjected to the bilateral ovariectomy, the ligation of the left carotid artery, the right external carotid artery and the right pterygopalatine artery, and systemic hypertension achieved by the combination of a high salt diet and the ligation of the left renal artery  under general anesthesia by the combination of intraperitoneal injection of pentobarbital sodium (50 mg/kg) with inhalation of Isoflurane (1.5 ~ 2%). Immediately after surgical manipulations, animals were fed the chow containing 8% sodium chloride and 0.12% 3-aminopropionitrile (Tokyo Chemical Industry, Tokyo, Japan), an irreversible inhibitor of Lysyl Oxidase catalyzing the cross-linking of collagen and elastin. Dead animals within one week after surgical manipulations were excluded from the analyses. At 8 weeks in RNA sequencing analysis or 9 weeks in histopathological examination 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 circle of Willis was then stripped from brain surface and an IA lesion induced at an anterior communicating artery or a posterior communicating artery was dissected as a rupture-prone lesion if SAH was macroscopically not detected. All dead animals during the observation period were autopsied to examine the onset of SAH. If SAH was observed, the ruptured IA lesions were stripped and histopathologically examined. Histopathological examination was done after Elastica van Gieson staining or Azan staining.
G-CSF (300 ∝g/kg, MOCHIDA PHARMACEUTICAL CO., LTD., Tokyo, Japan) was subcutaneously administered to rats twice a week from the 28th day to the 56th day after surgical manipulations. The number of myeloperoxidase (MPO)-positive cells or CD68-positive cells was calculated as a cell count present within 1 mm square around the dome of induced IAs. The dose of G-CSF administered in rats were determined by the preliminary dose-response analyses.
RNA purification and RNA sequencing analysis
Dissected rupture-prone aneurysms and the remaining circle of Willis from a same animal were grinded and homogenized in liquid nitrogen. Total RNA was isolated from homogenized samples with a RNeasy fibrous tissue mini kit (QIAGEN, Hilden, Germany) with an on-column DNase treatment, according to manufacturer’s instructions. Quantity of each total RNA sample was measured with a NanoDrop (Thermofisher, Waltham, MA), and its quality was assessed by using the RNA integrity number (RIN) on an Agilent 4200 Tapestation (Agilent, Santa Clara, CA). The libraries from purified RNA samples (500 ng) for RNA sequencing analyses were then prepared using a TruSeq stranded mRNA sample preparation kit (Illumina, San Diego, CA). Paired-end sequencing (2 × 75 base pair) was performed on a NextSeq500 (Illumina). Each read was then mapped to the Rattus norvegicus reference genome (Rnor6) by CLC genomics workbench (version 11, QIAGEN). Differential expression analyses including principal component analysis and gene ontology (GO) analysis were performed using the RNA-Seq tool, one similar to the DESeq and the edgeR package, in CLC genomics workbench. Genes whose expression reaches a fold change over 1.5 in aneurysm lesions compared with that in the remaining circle of Willis were considered as over- or under-expressed ones.
All the raw data from RNA sequencing analysis was deposited to Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) (ID # Data will be deposited after the acceptance.).
At the indicated period after the aneurysm induction, 5-um-thick frozen sections were prepared. After blocking with 3% donkey serum (Jackson ImmunoResearch, Baltimore, MD), slices were incubated with primary antibodies followed by incubation with secondary antibodies conjugated with a fluorescence dye (Jackson ImmunoResearch). Finally, fluorescent images were acquired on a confocal fluorescence microscope system (FV1000 or FV3000, Olympus, Tokyo, Japan).
The following primary antibodies were used: Cy3-conjugated mouse monoclonal anti-α-smooth muscle actin (SMA) antibody (#C6198, Sigma-Aldrich, St. Louis, MO), mouse monoclonal anti-rat CD68 antibody (#ab31630, Abcam, Cambridge, UK), rabbit polyclonal anti-MMP9 antibody (#ab38898, Abcam), rabbit polyclonal anti-myeloperoxidase (MPO) antibody (#ab9535, Abcam), rabbit polyclonal anti-GRO alpha (CXCL-1) antibody (#ab86436, Abcam) and rabbit polyclonal anti-Histone H3 (citrulline R2 + R8 + R17) antibody (#ab5103, Abcam).
HL-60 cell line used as a model of neutrophils was purchased from ATCC (#CCL-240, Manassas, VA) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (Sigma-Aldrich).
Quantitative real time (RT)-PCR analysis in cultured cells
HL-60 cells were stimulated with recombinant TNF-α (100 ng/ml, R&D SYSTEMS, Minneapolis, MN) for 90 min. Total RNA was then purified from stimulated cells and reverse-transcribed by using a RNeasy Mini Kit (QIAGEN) and a High-capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Carlsbad, CA) according to the manufacturers’ instructions. For quantification of gene expression, RT-PCR was performed on a Real Time System CFX96 (Bio-rad, Hercules, CA) using a SYBR Premix Ex Taq II (TAKARA BIO INC., Shiga, Japan). Expression of ACTB was used as the internal control. For quantitation, the second derivative maximum method was used for determining the crossing point.
Primer sets used are listed in Additional file 1.
HL-60 cells were stimulated with recombinant TNF-α (100 ng/ml) for 90 min. The supernatant was then prepared and collagenolytic activity in the supernatant was examined by a gelatin zymography as the manufacture’s instructions (Gelatin Zymography Kit, Cosmo Bio Co., LTD., Tokyo, Japan). In a rat model, ruptured IA lesions were harvested and grinded in a liquid nitrogen. Specimens were then lysed and subjected to a gelatin zymography as the manufacture’s instructions (Gelatin Zymography Kit, Cosmo Bio Co., LTD.).
The concentration of TNF-α or PGE 2 in the culture supernatant
HL-60 cells were stimulated with recombinant TNF-α (100 ng/ml) or LPS (10 ∝g/ ml, SIGMA, Lot # 123M4052V) for 5 h. In some experiments, cells were pre-treated with indomethacin for 15 min (100 nM, Wako). The supernatant was then prepared and the concentration of TNF-α or PGE2 in the supernatant was examined as the manufacture’s instructions (Quantikine ELISA for human TNF-α, R&D SYSTEMS, or Prostaglandin E2 EIA Kit, Cayman Chemical, Ann Arbor, MI).
Data are shown by box-and-whisker plots. Statistical comparisons between 2 groups were conducted using a Mann − Whitney U test and comparisons among more than 2 groups were done by a Kruskal − Wallis test followed by a Steel test. The incidence of IAs or SAH was analyzed by a Fisher’s exact test. A p value smaller than 0.05 was defined as statistically significant.