Histopathological examination
We examined the histopathologic changes on day 6. The hearts were fixed in 10% formalin, and embedded in paraffin. The left ventricles (LV) were sliced horizontally to the long axis, and stained with hematoxylin - eosin, and Masson's trichrome for light microscopy examinations. The extent of myocardial necrosis was evaluated by measuring the ratio (%): myocardial necrosis area/total LV area on a microscopic slide, using Microanalysis (Ather Coporation, Tokyo, Japan), which can measure areas of different colors. We calculated the area of myocardial necrosis, as indicated by the loss of red Masson's trichrome stain. Two investigators determined the histologic score, which were averaged. The analyses were blinded. To determine the number of mast cells, the hearts were stained with toluidine blue. The total number of mast cells in a given section (whole heart) was calculated as cells/mm2.
Quantitative reverse transcriptase polymerase chain reaction analysis
A total of 18 of the 20 mice were studied for gene expression. One mouse of the cetirizine 10 mg/kg group and one control mouse died before day 5 and thus were not appropriate for study because of post-mortem changes.
Total RNA was isolated from the LV using the acid guanidinium thiocyanate-phenol-chloroform method and the RNA concentration was measured spectrophotochemically. First-strand cDNA was synthesized using SUPERSCRIPT Preamplification System for First-Strand cDNA Synthesis (GIBCO BRL). Real-time quantitative PCR (TaqMan PCR) using an ABI PRISM 7700 Sequence Detection System and TaqMan PCR Core Reagent Kit (Perkin-Elmer Corp, Foster City, CA) was performed according to the manufacturer's protocol. We used 2 μl of the First-strand cDNA, and the following forward (F) and reverse (R) oligonucleotides, and probes (P) were used for the quantification of tumor necrosis factor (TNF) α, interleukin (IL)-6, and matrix metalloproteinases (MMPs) 2and 9.
TNF-α F, 5'-CATCTTCTCAAAATTCGAGTGACAA;
TNF-α R, 5'-TGGGAGTAGACAAGGTACAACCC;
TNF-α P, 5'-CACGTCGTAGCAAACCACCAAGTGGA;
IL-6F, Based on TaqMan produt No.4331348
IL-6R, Based on TaqMan produt No.4331348
IL-6P, Based on TaqMan produt No.4331348
Inducible Nitric Oxide Synthase (iNOS)
iNOSF, 5'- CAGCTGGGCTGTACAAACCTT-3'
iNOSR, 5'-CATTGGAAGTGAAGCGTTTCG-3'
iNOSP, 5'-CGGGCAGCCTGTGAGACCTTTGA-3'
MMP-2 F, 5'-ACTGACCTGCATGGAATCAGC-3'
MMP-2 R, 5'-GGTTACTTGAGTGTTCTAGCCCA-3'
MMP-2 P, 5'-TCTTTCTGGTGGCCGTGCATGA-3'
MMP-9 F, 5'-TTGTGGTCTTCCCCAAAGACC-3'
MMP-9 R, 5'-TATCCACCGAGCCATCTGTCTA-3'
MMP-9 P, 5'-AAAACCTCCAACCTCACGGACACCCA-3'
GAPDH F, 5'-TTCACCACCATGGAGAAGGC-3';
GAPDH R, 5'-GGCATGGACTGTGGTCATGA-3';
GAPDH P, 5'-TGCATCCTGCACCACCAACTGCTTAG-3'.
The conditions for the TaqMan PCR were: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min.