MitoSox Red (Molecular Probes, Eugene, OR) was dissolved in a 1:1 mixture of dimethylsulfoxide (DMSO) and saline to a final concentration of 33 μM. Under isoflurane anesthesia, 10 μl of MitoSox was injected intrathecally by using a direct transcutaneous intrathecal injection method described previously. Approximately 23 hrs after MitoSox injection, mice received an i.d. injection of either capsaicin (0.5 %, 25 μl) or the same volume of vehicle on both the plantar and dorsal surfaces of the left hind foot. Mice remained under 1.5 % isoflurane anesthesia for 30 min to suppress any side effects induced by capsaicin. Mice were perfused through the aorta with saline followed by fixative containing 4% paraformaldehyde 1 hr after capsaicin injection and the L4-L5 spinal cord segments were removed. The cord was postfixed 4-15 hr in the perfusion fixative, equilibrated in 30% sucrose, cryosectioned at 30 μm, and mounted on gelatin coated slides. The sections were examined under a fluorescent microscope with a rhodamine filter. Two different regions of the dorsal horn were photographed from 10 randomly selected sections from each animal: the lateral part of laminae I -II and laminae III-V. Photographs were taken with a Q-Imaging Retiga 2000R digital camera attached to an Olympus BX50 microscope using a 63x oil objective lens and saved as digital image files. The number of MitoSox positive cellular profiles with distinctive nuclei (dark oval shaped space surrounded by red granules) was counted from these pictures.