Fig. 1.
A mouse model of BCABP. (A) Assessment for hind-paw mechanical hypersensitivity in tibiae of mice by von Frey test. Picture of a mouse withdrawing left hind-leg following poking with von Frey filament (left). The dynamic plantar aesthesiometer with von Frey filament that pokes mouse hind-paw with automated increasing force (right). Mice with pain exhibit hind-paw withdrawal with lighter force than those without pain. (B) Radiograph of osteolytic lesions in tibiae associated with 4T1 BC colonization at day 15 of intratibial cell inoculation (1 × 105 cells/10 μl) in sham (left) and 4T1 mouse (right). Multiple osteolytic lesions are seen in tibiae of 4T1 mouse (right). Scale bar 1 mm. (C) Progression of hind-paw mechanical hypersensitivity in tibiae of 4T1 mice. The force of the filament by which mice withdrew their hind-paw is shown on Y-axis as paw withdrawal thresholds (g) in figure. Mice with BCABP in tibiae show decreased paw withdrawal threshold in parallel with the extent of BCABP. For example, sham mice withdrew their hind-leg by the force of 10.78 ± 1.08 g (Mean ± SD), while 4T1 mice by 5.5 ± 1.45 g (Mean ± SD) at day 9. Decreased paw withdrawal threshold in all group of mice at day 2 is due to surgical trauma. To evaluate therapeutic effects, the neutralizing antibody to HMGB1 (2 mg/kg/mouse, ip, once a day) or control IgY antibody was given at day 13, 14 and 15 (arrows) when 4T1 mice demonstrated considerable hind-paw mechanical hypersensitivity with clearly discernible osteolysis in tibiae associated with 4T1 BC colonization on radiographs. The HMGB1, but not IgY control, antibody significantly reduced hind-paw mechanical hypersensitivity in tibiae of 4T1 mice. Mice withdrew their hind-leg by the force of 4.85 ± 0.22 g (Mean ± SD) before administration of the HMGB1 antibody and by 7.07 ± 0.26 g (Mean ± SD) after administration of the HMGB1 antibody. Data are shown as mean ± SD (n = 8). * Significantly different from sham mice (p < 0.05). # Significantly different from 4T1 and 4T1 + IgY mice (p < 0.05). (D) Expression of a molecular pain marker pERK in DRGs harvested from sham and 4T1 BC mice treated with or without the HMGB1 antibody. After sacrifice at day 17, DRGs were harvested, immediately lysed and subjected to Western analysis. Expression of pERK was increased in 4T1 mice with BCABP. Administration of the HMGB1 antibody reduced pERK expression in DRGs of 4T1 mice. ERK1/2 expression was not changed.