IA models of rats and histological analysis of induced IA
10-week-old female Sprague−Dawley rats were purchased from Japan SLC (Slc:SD, n=20, 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 [10, 18, 26], 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 inhalation of Isoflurane (induction; 5.0 %, maintenance; 1.5~2.0 %, #IYESC-0001, Pfizer Inc., New York, NY). Immediately after surgical manipulations, animals were fed the chow containing 8 % sodium chloride and 0.12 % 3-aminopropionitrile (#A0408, Tokyo Chemical Industry, Tokyo, Japan), an irreversible inhibitor of Lysyl Oxidase catalyzing the cross-linking of collagen and elastin. At 16 weeks after above surgical manipulations, animals were deeply anesthetized by the inhalation of Isoflurane (5.0 %, #IYESC-0001, Pfizer Inc.), 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.
Tissue transparency and immunohistochemistry
Tissue transparency was done using paraformaldehyde-fixed specimens and CUBIC-L and CUBIC-R solutions (#T3740 or #T3741, TCI chemicals, Tokyo, Japan) as manufacturer’s instructions. Immunohistochemistry to visualize medial smooth muscle cells was done using a CUBIC-HV 3D Immunostaining Kit and mouse monoclonal anti-a-smooth muscle actin antibody conjugated with Cy3 (#C6198, clone 1A4, Sigma, St. Louis, MI) as manufacturer’s instructions. The images were acquired by a confocal laser microscopy (FV3000, Olympus, Tokyo, Japan).
In specimens from animals, 5-µm-thick frozen sections were prepared from dissected IA lesions or anterior cerebral – olfactory artery bifurcations, aorta or liver prepared as described above. After blocking with 3 % donkey serum (#AB_2337258, Jackson ImmunoResearch), slices were incubated with primary antibodies followed by incubation with secondary antibodies conjugated with a fluorescence dye (Jackson ImmunoResearch). In some experiments, the primary antibody conjugated with a fluorescence dye was used. Finally, fluorescent images were acquired on a confocal fluorescence microscope system (FV3000, Olympus, Tokyo, Japan).
In specimens from human cases, dissected human specimen was fixed in formalin solution and embedded in paraffin. 4-µm-thick slices were then prepared for immunohistochemical analysis. After deparaffinization and blocking with 10 % donkey serum (Jackson ImmunoResearch), slices were incubated with primary antibodies followed by incubation with secondary antibodies conjugated with fluorescence dye (Jackson ImmunoResearch). Finally, fluorescent images were acquired on a confocal fluorescence microscope system (FV3000, Olympus).
The antibodies used were as follows; mouse monoclonal anti-a-smooth muscle actin antibody (#M0851, Dako, Agilent, Santa Clara, CA), mouse monoclonal anti-a-smooth muscle actin antibody conjugated with Cy3 (#C6198, Sigma), mouse monoclonal anti-CD68 antibody conjugated with Alexa Fluor 647 (#sc-20060AF647, Santa Cruz Biotechnology, Dallas, TX), mouse monoclonal anti-VEGF antibody (clone VG1, #NB100-664, Novus Biologicals, LLC, Centennial, CO), mouse monoclonal anti-Pimonidazole antibody (clone MAb1, #HP1-100, Hypoxyprobe, Inc., Burlington, MA) , Alexa Fluor 488-conjugated donkey anti-mouse IgG H&L antibody (#A21202, Thermo Fisher Scientific, Waltham, MA), Alexa Fluor 594-conjugated donkey anti-mouse IgG H&L antibody (#A21203, Thermo Fisher Scientific).
Detection of hypoxic cells and tissues in vivo
To detect hypoxic cells or tissues in vivo, Hypoxyprobe was used (Hypoxyprobe, Inc.). This probe is the derivative of Pimonidazole which binds to hypoxic cells and can be detected in immunohistochemistry using the specific antibody for Hypoxyprobe delivered from the company. 60 mg/kg of Hypoxyprobe was intraperitoneally injected in rats and after 165 min the specimens were harvested subjecting to immunohistochemistry as in the above section.
Induction of vasa vasorum from arteries at brain surface by VEGF
Burr hole was prepared in the skull of rats by high-speed drill system under general anesthesia as described above and the sheets for the slow-release of VEGF was placed on the brain surface (MedGel II from Nitta-gelatin (♯PI9, Osaka Japan) and recombinant human VEGF (1 μg/head, #564-RV-010/CF, lot #CWC0620081) from R&D systems, Inc. (Minneapolis, MN) resolved in phosphate buffered saline containing 0.1% human serum albumin (#A1653-5G, lot #SLBG2676V, Sigma). The induction of vasa vasorum was then assessed by histological examination and by immunohistochemistry on 6th day as described above.
RAW264.7 cell line, NIH3T3 cell line and U937 cell line were purchased from ATCC (#TIB-71 for RAW264.7, #CRL-1658 for NIH3T3, #CRL-1593.2 for U937, Manassas, VA). Cells were maintained in Dulbecco's Modified Eagle Medium (#044-29765, FUJIFILM Wako Chemicals, Osaka Japan) supplemented with 10 % fetal bovine serum (#FB-1365/500, lot#11953, biosera, Nuaille, France) (RAW264.7 cells and NIH3T3 cells) or RPMI-1640 (#189-02025, FUJIFILM Wako Chemicals) with 10 % fetal bovine serum (#FB-1365/500, lot#11953, biosera) (U937 cells).
Loading hypoxia on cultured cells
To load hypoxia, cells were incubated in a hypoxia chamber system (0.5 % or 5 %, #BIONIX-2, SUGIYAMA-GEN CO., LTD., Tokyo, Japan) for 8 h.
RNA purification and quantitative real time (RT)-PCR analysis
Total RNA was purified from treated cells and reverse-transcribed using a RNeasy Mini Kit (#74106, QIAGEN, Hilden, Germany) and a High-capacity cDNA Reverse Transcription Kit (#4368813, Life Technologies Corporation, Carlsbad, CA), according to the manufacturers’ instructions. For quantification of gene expression, quantitative RT-PCR was performed on a LightCycler 480 (Roche, Indianapolis, IN) with a TB Green Premix Ex Taq II (#RR820, TAKARA BIO INC., Shiga, Japan). Expression of Actb (a gene coding β-actin) was used as internal controls. For quantitation, the second derivative maximum method was used for determining the crossing point.
Primer sets used are listed as follows: 5′-ACGACCAGAGGCATACAGGGA-3′ and 5′-CCCTAAGGCCAACCGTGAAA-3′ for Actb; forward 5′-GGTCTGTTGGGAGTGGTATCC-3′ and reverse 5′-TTCCTCTCTGCAAGAGACTTCC-3′ for Il6; forward 5′-AAAGGGAGCTCCTTAACATGC-3′ and reverse 5′-CTTCCTGGGAAACAACAGTGG-3′ for Il1b; forward 5′-AATGATGTGTACGGCTTCAGG-3′ and reverse 5′-CTGTACAAGCAGTGGCAAAGG-3′ for Ptgs2 (cyclooxygenase-2 (COX-2)); forward 5′-GCACAGACCTCTCTCTTGAGC-3′ and reverse 5′-ACCTGCTGCTGCTACTCATTCACC-3′ for Ccl2 (also known as MCP-1; monocyte chemoattractant protein-1); forward 5′-GCAGTGCATACCACTTCAACC-3′ and reverse 5′-CTGATGGTCAAGATCCAGAGG-3′ for Nos2; forward 5′- AAAGCAGGTCAGTCACTTTGC-3′ and reverse 5′-TCACTCCCTCAAATCACTTCG-3′ for Vegf exon 1; forward 5′- GGAGAGATGAGCTTCCTACAGC-3′ and reverse 5′-GGATTTCTTGCGCTTTCG-3′ for Vegf exon 4-6; forward 5′- TCACATCTGCAAGTACGTTCG-3′ and reverse 5′- ACTGTGAGCCTTGTTCAGAGC-3′ for Vegf exon 7-8; 5′-ACCTCTCTTCCCACAGAAAGC-3′ and 5′-CTTAGGCTCAGGCCATTATGC-3′ for Areg (Amphiregulin); 5′-GCACGAAGATCAAGAACAACG-3′ and 5′-AGGAGCCTCTAGCTTCACACC-3′ for Ang (Angiogenin); 5′-ACAACAACCACCACAATCACC-3′ and 5′-GGTATGTGCCGACATTACAGG-3′ for Angpt1 (Angiopoietin-1); 5′-CAGTCTCTGAAGGTGGTTTGC-3′ and 5′-CAGCATGACCTAATGGAGACC-3′ for Angpt2 (Angiopoietin-2); 5′-TTGGTGGACAGTGAGTTCTCC-3′ and 5′-AACCAGACCAAAGCTCAGACC-3′ for Angpt4 (Angiopoietin-4); 5′-ACAGCTCAACTCAGTGCTTCC-3′ and 5′-GCCTATAGGGTTACGGTTTGG-3′ for Cnn1 (Calponin 1); 5′-GTAAGATTTGGCGAACAGACG-3′ and 5′-TAATGTCCGAAGTGGTCATCG-3′ for Dll4 (Delta like canonical Notch ligand 4); 5′-GGATGGAGGTTAATCCTGACC-3′ and 5′-GGCACAGTTTGTCTTCAATGG-3′ for Egf (Epidermal growth factor); 5′-CCAGAGGGAGCTCTACAAAGG-3′ and 5′-AGATCTTTCCAGGAAGCAAGG-3′ for Eng (Endoglin); 5′-AGGACGATGTAGCTGTTGTGG-3′ and 5′-ATGGCGAACTGAAACTACTGG-3′ for Col18a1 (Collagen, type XVIII, alpha 1); 5′-CAGTACAGGCATGTTCCAAGC-3′ and 5′-CGATGAGCAGTGTTTGAAAGC-3′ for Fgf7 (Fibroblast growth factor 7); 5′-CTTTCTGGCAAGAACTTGTGC-3′ and 5′-CACAAGCAATCCAGAGGTACG-3′ for Hgf (Hepatocyte growth factor); 5′-CAGCTTTGGTTACACAATCAGC-3′ and 5′-GAAGCTTTCAGTGGGTTAGGG-3′ for Igfbp1 (Insulin-like growth factor binding protein 1); 5′-TGCTGTTCATTGACCTTCTCC-3′ and 5′-AGGTTGCAGACAGTGATGACG-3′ for Igfbp2 (Insulin-like growth factor binding protein 2); 5′-AGAAGTTCTGGGTGTCTGTGC-3′ and 5′-ATTCCAAGTTCCATCCACTCC-3′ for Igfbp3 (Insulin-like growth factor binding protein 3); 5′-GTTCGGCTCAAACTTCTCTCC-3′ and 5′-CTGAGATGAGACCCTGTGACC-3′ for Ccn3 (Cellular communication network factor 3); 5′-ACTTCTCTTCCTGCGAATGG-3′ and 5′-GAAGTCAGATCCACAGCATCC-3′ for Pdgfa (Platelet derived growth factor, alpha); 5′-GGCAAGTTGGCCATATTTAGG-3′ and 5′-TATATTCCACTCCAGCCAAGC-3′ for Pdgfb (Platelet derived growth factor, B polypeptide); 5′-TGTCCACTGCTCACATACAGC-3′ and 5′-GGCATTCAGAAGCAGAGAGG-3′ for Serpine1 (Serine (or cysteine) peptidase inhibitor, clade E, member 1); 5′-TAGTCTAGCGGGAAGGTGAGG-3′ and 5′-CAGCAAGATTACTGGCAAACC-3′ for Serpinf1 (Serine (or cysteine) peptidase inhibitor, clade F, member 1); 5′-ATGTATGGGCAGTTGTCTTGG-3′ and 5′-TGACCAGAGAGATACGGATGG-3′ for Thbs2 (Thrombospondin 2); 5’- CATACTCCTGCTTGCTGATCC-3’ and 5’-GATGCAGAAGGAGATCACTGC-3’ for ACTB; 5’-ACACCCTCTATCACTGGCATCC-3’ and 5’-AACATTCCTACCACCAGCAACC-3’ for PTGS2; 5′-TCAGCAATGAGTGACAGTTGG-3′ and 5′-ATAGGCTGTTCCCATGTAGCC-3′ for TNF; 5′-CATTTGTGGTTGGGTCAGG-3′ and 5′-AGTGAGGAACAAGCCAGAGC-3′ for IL6; 5′-ATTCAGCACAGGACTCTCTGG-3′ and 5′-CAAGCTGGAATTTGAGTCTGC-3′ for IL1B; 5’-AGCTTCTTTGGGACACTTGC-3’ and 5’-ATAGCAGCCACCTTCATTCC-3’ for CCL2; 5′-GACCTGATGTTGCCATTGTTGG-3′ and 5′-TACCAACTGACGGGAGATGAGC-3′ for NOS2
Concentration of VEGF in the supernatant from hypoxia-loaded RAW264.7 cells
The supernatant from hypoxia-loaded RAW264.7 (0.5 %, 0~24 h) was collected subjecting to ELISA (#RSD-MMV00-1, R&D Systems) to examine the concentration of VEGF according to the manufacture’s indications.
Data are shown as Mean ± S.E.M. Differences between the 2 groups were examined using a non-parametric Mann-Whitney test. Differences more than 3 groups were examined using a Kruskal-Wallis test followed by a Steel test. A p value smaller than 0.05 was defined as statistically significant.