Figure 2.

Ear skin nerve fibers in the secondary hyperalgesia region show peripheral hypersensitivity after CCI-ION in Pirt-GCaMP3 mice. (A) Schematic diagram of a mouse ear. Red or blue color indicates where V3 nerve fibers (green) or C2 nerve fibers are innervated into ear skin, respectively. White square dash lines indicate where the image was taken. (B) Representative GCaMP3 imaging of ear skin explants. Ear skin after CCI-ION in Pirt-GCaMP3 mice were activated by capsaicin (1 μM) or high KCl (100 mM). Upper rows show ipsilateral (on the CCI-ION side) V3 region. Middle rows show contralateral (uninjured side) V3 region. Lower rows show ipsilateral C2 region. Yellow arrow heads indicate hairs and hair follicles which are autofluoresecent. Red arrow heads indicate activated fibers and endings. Ipsi., ipsilateral; cont., contralateral. Scale bar: 50 μm. (C–E) In left panels time course of the amplitude of the Ca²⁺ transient that was evoked by capsaicin (C,1 μM; D, 10 μM) or KCl (E, 100 mM) application in ear skin. In right panels Ca²⁺ transient amounts with area under curve (a.u.c.) that was evoked by capsaicin (C,1 μM; D, 10 μM) or KCl (E, 100 mM) in ear skin. All population data for Ca²⁺ transient are expressed as the percentage of baseline Ca²⁺ transient (ΔF/F₀) and are presented as mean ± s.e.m. Black bars indicate when stimuli were applied. (F) Length of ear skin fibers and endings after activation by capsaicin (1 μM and 10 μM), which was normalized to the length activated by KCl. Significant increases were found in ipsilateral ear (V3) as compared to contralateral ear (V3) or ipsilateral ear (C2). *p<0.05; **p<0.01; ***p<0.001.