The role of reactive oxygen species in capsaicin-induced mechanical hyperalgesia and in the activities of dorsal horn neurons

Abstract

Previous findings that reactive oxygen species (ROS) are involved in neuropathic pain, mainly through spinal mechanisms, suggest that ROS may be involved in central sensitization. To investigate the possible role of ROS in central sensitization, we examined in rats the effects of ROS scavengers on capsaicin-induced secondary hyperalgesia, which is known to be mediated by central sensitization. We used two different ROS scavengers: phenyl N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL). Intradermal capsaicin injection (20 microg in 20 microl olive oil) into the hind paw produced primary and secondary hyperalgesia. A systemic administration of PBN (100mg/kg, i.p.) or TEMPOL (200mg/kg, i.p.) alleviated capsaicin-induced secondary, but not primary, hyperalgesia. Intrathecal injection of PBN (1mg inof veterinary Surgery/anesthesiology, College of veterinary Medic 50 microl saline) greatly reduced hyperalgesia, whereas intracerebroventricular or intradermal injection of PBN produced only a minor analgesic effect, suggesting that PBN takes effect mainly through the spinal cord. Electrophysiological recordings from wide dynamic range (WDR) neurons in the dorsal horn showed that intradermal capsaicin enhanced the evoked responses to peripheral stimuli; systemic PBN or TEMPOL restored the responses to normal levels. Removal of ROS thus restored the responsiveness of spinal WDR neurons to normal levels, suggesting that ROS is involved in central sensitization, at least in part by sensitizing WDR neurons.

Fig. 1

A: Drawing showing the sites of capsaicin injection and behavioral testing in the rat hind paw. For capsaicin injection, a 27-gauge hypodermic needle was inserted subcutaneously at the heel of the foot (X) and advanced to the injection site (I), and capsaicin (20 μg in 20 μl olive oil) was injected intradermally. Mechanical thresholds to von Frey filament stimuli were measured at site P for primary hyperalgesia and at site S for secondary hyperalgesia. B and C: The effect of systemic PBN pretreatment on capsaicin-induced hyperalgesia. Panels B and C show the effects of PBN on primary and secondary hyperalgesia, respectively. Mechanical thresholds shown in B and C were measured at sites labeled “P” and “S” in panel A, respectively. After measuring baseline mechanical threshold (at 1 hour before capsaicin treatment), PBN (100 mg/kg, i.p.) was injected, and then capsaicin was injected 0.5 hours later (Cap i.d., 20 μg in 20 μl of olive oil). The same volume of saline was injected instead of PBN for the control group. Data are expressed as mean ± SEM, and asterisks indicate values significant different from corresponding values in the saline group according to Duncan’s post hoc test after two-way repeated ANOVA. PBN 100: rats received 100 mg/kg PBN and capsaicin (n = 9); Saline: rats received saline and capsaicin (n = 8); i.p.: intraperitoneal injection of either PBN or saline; Cap i.d.: intradermal capsaicin injection.

Fig. 2

The effect of systemic PBN post-treatment on capsaicin-induced hyperalgesia. Panels A and B show the effects of PBN on primary and secondary hyperalgesia, respectively. Mechanical thresholds shown in panels A (n = 6 for both groups) and B (n = 6) were measured at sites labeled “P” and “S” in panel A of Fig. 1, respectively. Immediately after measuring the baseline mechanical threshold, we injected capsaicin intradermally at time 0 (Cap i.d., 20 μg in 20 μl of olive oil) and then injected PBN (100 mg/kg, i.p.) 2.5 hours later. The same volume of saline was injected instead of PBN for the control group. Data in panel C (n = 6 for PBN 20 mg/kg, n = 7 for PBN 50 mg/kg, n = 9 for PBN 100 mg/kg, and n = 10 for the saline control group) are multiple groups of graded doses of PBN. Data are expressed as mean ± SEM, and asterisks indicate values significantly different from the corresponding values in the saline group according to Duncan’s post hoc test after two-way repeated ANOVA.

Fig. 3

The effect of systemic TEMPOL post-treatment on capsaicin-induced hyperalgesia. Panels A and B show effects of TEMPOL on primary and secondary hyperalgesia, respectively. Mechanical thresholds shown in panels A (n = 6 for both groups) and B (n = 6) were measured at sites labeled “P” and “S” in panel A of Fig. 1, respectively. Immediately after measuring the baseline mechanical threshold, we injected capsaicin intradermally at time 0 (Cap i.d., 20 μg in 20 μl of olive oil) and then injected TEMPOL (200 mg/kg, i.p.) 2.5 hours later. The same volume of saline was injected instead of TEMPOL for the control group. Data are expressed as mean ± SEM, and asterisks indicate values significantly different from the corresponding values in the saline group according to Duncan’s post hoc test after two-way repeated ANOVA.

Fig. 4

The effects of PBN injections given at three different locations on capsaicin-induced hyperalgesia. All data were collected by measuring the threshold at a site labeled “S” in panel A of Fig. 1 to examine secondary hyperalgesia. Panels A, B, and C show the effects of post-treatment with PBN (1 mg in 50 μl of saline) 2.5 hours after capsaicin injection. PBN was given intradermally at the peripheral injection site (i.d., n = 11), intrathecally to the lumbar spinal segments (i.t., n = 11), and intracerebroventricularly into the lateral ventricle (icv., n = 5), respectively. The same volume of saline was injected in the control group. Data are expressed as mean ± SEM, and asterisks indicate values significantly different from the corresponding values in the saline group according to Duncan’s post hoc test after two-way repeated ANOVA.

Fig. 5

The responses of the spinal dorsal horn WDR neurons to mechanical stimuli in normal, after capsaicin and then PBN treatments. Panels A and B show examples of extracellular recordings of spinal WDR neuron responses to peripheral mechanical stimuli (brush and 1, 2, and 20 g von Frey filaments). In panel A, activities were recorded before and after capsaicin (300 μg in 100 μl olive oil, i.d.; time of injection indicated by “Cap i.d.”). The evoked responses were greatly enhanced by 60 and 90 minutes after the injection. In panel B, activities were recorded before and after capsaicin treatment and then again after PBN treatment (100 mg/kg, i.p.).The enhanced evoked responses induced by capsaicin injection were reduced by systemic injection of PBN. Panel C shows group data of all recorded WDR neurons after intraperitoneal (i.p., n = 7) and intravenous (i.v., n=5) injection of PBN. Data are expressed as mean ± SEM, and asterisks indicate values significantly different from the values after capsaicin treatment of the same group according to Duncan’s post hoc test after one-way repeated ANOVA.

Fig. 6

The effect of systemic PBN on hyperalgesia induced by capsaicin injection at a distal site of the plantar surface. Panel A shows the needle insertion (X) and capsaicin injection site (I) and behavioral testing locations for primary (P) and secondary (S) hyperalgesia. Panels B and C show the effects of PBN on primary and secondary hyperalgesia, respectively. Mechanical thresholds shown in panels B (n = 6 for both groups) and C (n = 6) were measured at sites labeled “P” and “S” in panel A, respectively. Immediately after measuring the baseline mechanical threshold, we injected capsaicin intradermally at time 0 (Cap i.d., 20 μg in 20 μl of olive oil) and then injected PBN (100 mg/kg, i.p.) 2.5 hours later. We injected the same volume of saline instead of PBN for the control group. Data are expressed as mean ± SEM, and asterisks indicate values significantly different from the corresponding values in the saline group according to Duncan’s post hoc test after two-way repeated ANOVA.