Isoflurane-anesthetized mice were transurethrally catheterized (PE10, 11 mm length) and drained of urine [7 (link)]. Fifteen minutes before instillation, mice received either HMGB1 antagonist [21 (link)], glycyrrhizin (50 mg/kg, ip; Calbiochem, Billerica, MA), glycyrrhizin vehicle control (10 μM NH4OH in sterile PBS, pH 7.4; ip), or MIF antagonist, (S,R)3-(4-hy-droxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester [22 (link)] (ISO-1; 20 mg/kg, ip; EMD Bioscience, San Diego, CA; catalog 475837). Bladders were instilled with either PAR4-activating peptide (AYPGKF-NH2; 100 μM in PBS; pH 7.4, 150 μl) or a scrambled control peptide (YAPGKF-NH2; 100 μM in PBS; pH 7.4, 150 μl) and retained for 1 hour. Intravesical fluid was collected from the catheter tip, treated with protease inhibitors (Halt III; Thermo Sci., Rockford, IL), and stored at -80°C until analysis.
Twenty-four hours after instillation, mice were tested for abdominal mechanical allodynia (as above) and then anesthetized (isofluorane anesthesia). Bladders were removed, fixed in 10% formalin, and embedded in paraffin for histology (see below).
Twenty-four hours after instillation, mice were tested for abdominal mechanical allodynia (as above) and then anesthetized (isofluorane anesthesia). Bladders were removed, fixed in 10% formalin, and embedded in paraffin for histology (see below).
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