Oxidative stress in the spinal cord is an important contributor in capsaicin-induced mechanical secondary hyperalgesia in mice

Abstract

Recent studies indicate that reactive oxygen species (ROS) are critically involved in persistent pain primarily through spinal mechanisms, thus suggesting ROS involvement in central sensitization. To investigate ROS involvement in central sensitization, the effects of ROS scavengers and donors on pain behaviors were examined in mice. Capsaicin- induced hyperalgesia was used as a pain model since it has 2 distinctive pain components, primary and secondary hyperalgesia representing peripheral and central sensitization, respectively. Capsaicin (25 microg/5 microl) was injected intradermally into the left hind foot. Foot withdrawal frequencies in response to von Frey filament stimuli were measured and used as an indicator of mechanical hyperalgesia. The production of ROS was examined by using a ROS sensitive dye, MitoSox. Mice developed primary and secondary mechanical hyperalgesia after capsaicin injection. A systemic or intrathecal post-treatment with either phenyl-N-tert-butylnitrone (PBN) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL), ROS scavengers, significantly reduced secondary hyperalgesia, but not primary hyperalgesia, in a dose-dependent manner. Pretreatment with ROS scavengers also significantly reduced the magnitude and duration of capsaicin-induced secondary hyperalgesia. On the other hand, intrathecal injection of tert-butylhydroperoxide (t-BOOH, 5 microl), a ROS donor, produced a transient hyperalgesia in a dose-dependent manner. The number of MitoSox positive dorsal horn neurons was increased significantly after capsaicin treatment. This study suggests that ROS mediates the development and maintenance of capsaicin-induced hyperalgesia in mice, mainly through central sensitization and that the elevation of spinal ROS is most likely due to increased production of mitochondrial superoxides in the dorsal horn neurons.

Figure 1

Sites of capsaicin injection and behavioral testing in the mouse hind foot. For capsaicin injection, a 30-gauge needle was inserted at the heel of the foot (X) and advanced to the injection site (I), and capsaicin (25 μg in 5 μl of vehicle) was injected intradermally (i.d.). Foot withdrawal frequencies in response to von Frey stimuli were measured at site P for primary hyperalgesia and at site S for secondary hyperalgesia.

Figure 2

Time course of primary and secondary hyperalgesia after intradermal (i.d.) capsaicin (25 μg in 5 μl saline) injection in the mouse hind foot. Asterisks (*), p ≤ 0.05 (n=6), indicate values significantly different from corresponding values in the vehicle treated group according to Duncan’s post hoc test after two-way repeated ANOVA. Arrowheads indicate time of capsaicin (or vehicle) injection

Figure 3

Effects of systemic (i.p.) PBN or TEMPOL post-treatment on capsaicin-induced hyperalgesia. A,D Time course of PBN or TEMPOL effects on primary hyperalgesia. B,E Time course of PBN or TEMPOL effects on secondary hyperalgesia. C,F The dose response of PBN or TEMPOL on secondary hyperalgesia 2 hr after i.d. capsaicin injection and 30 min post ROS scavenger. Asterisks (*), p ≤ 0.05 (n=6), indicate values significantly different from corresponding values in the vehicle treated group according to Duncan’s post hoc test after two-way repeated ANOVA. Arrowheads indicate time of capsaicin injection. Downward arrows indicates time of tested compound (PBN, TEMPOL, or saline) injection.

Figure 4

Effects of intrathecal (i.t.) PBN or TEMPOL treatment on capsaicin-induced hyperalgesia. A,D Time course of PBN or TEMPOL effects on primary hyperalgesia. B,E Time course of PBN or TEMPOL effects on secondary hyperalgesia. C,F The effect of PBN or TEMPOL on secondary hyperalgesia 2 hr after i.d. capsaicin injection and 30 min after either PBN, TEMPOL, or Saline treatment. Asterisks (*), p ≤ 0.05 (n=6), indicate values significantly different from corresponding values in the vehicle treated group according to Duncan’s post hoc test after two-way repeated ANOVA. Arrowheads indicate time of capsaicin injection. Downward arrows indicate time of tested compound (PBN, TEMPOL, or saline) injection.

Figure 5

Effects of i.t. t-BOOH on mechanical hyperalgesia. A: i.t. t-BOOH transiently induced mechanical hyperalgesia in normal mice in a dose-dependent manner. Saline did not show any changes in mechanical sensitivity. B: The bar graph shows the effect of t-BOOH on mechanical hyperalgesia 45 min after injection. *, p ≤ 0.05 (n=10). The open arrowhead indicates time of t-BOOH (or saline) injection.

Figure 6

Effects of systemic PBN or TEMPOL pre-treatment on capsaicin-induced hyperalgesia. A,D Time course of PBN or TEMPOL effects on primary hyperalgesia. B,E Time course of PBN or TEMPOL effects on secondary hyperalgesia. C,F The effect of PBN or TEMPOL on secondary hyperalgesia 2 hr after i.d. capsaicin injection and 2.5 hrs after either PBN, TEMPOL, or Saline treatment. Asterisks (*), p ≤ 0.05, indicate values significantly different from corresponding values in the vehicle treated group according to Duncan’s post hoc test after two-way repeated ANOVA. Arrowheads indicate time of capsaicin (or saline) injection. Downward arrows indicate time of tested compound (PBN, TEMPOL, or saline) injection.

Figure 7

MitoSOX positive cells in the deep layer of the L4-L5 spinal dorsal horn. A&B: MitoSOX labeled cells: vehicle (A) and capsaicin (B) treated mice: C&D: MitoSOX+NeuN label: NeuN label (C); MitoSOX label (D); combined (E). F&H: MitoSOX+GFAP label: GFAP label (F); MitoSOX label (G); combined (H).

Figure 8

A: Density of MitoSOX positive cells (average # of cells per 10,000 μm² of sample area ± SEM) in superficial (La I/II) and deep (La III/V) layers of the dorsal horn after vehicle (n=7) and capsaicin (n=7) treatment. *, p ≤ 0.05 (n=7). B: The average proportion of neurons and glia among MitoSOX positive cellular profiles in the deep layer (La III-IV) of the dorsal horn after i.d. capsaicin injection (n=7).