Increase in reactive oxygen species and activation of Akt signaling pathway in neuropathic pain

Abstract

Neuropathic pain occurs as a result of peripheral or central nervous system injury. Its pathophysiology involves mainly a central sensitization mechanism that may be correlated to many molecules acting in regions involved in pain processing, such as the spinal cord. It has been demonstrated that reactive oxygen species (ROS) and signaling molecules, such as the serine/threonine protein kinase Akt, are involved in neuropathic pain mechanisms. Thus, the aim of this study was to provide evidence of this relationship. Sciatic nerve transection (SNT) was used to induce neuropathic pain in rats. Western blot analysis of Akt and 4-hydroxy-2-nonenal (HNE)-Michael adducts, and measurement of hydrogen peroxide (H(2)O(2)) in the lumbosacral spinal cord were performed. The main findings were found seven days after SNT, when there was an increase in HNE-Michael adducts formation, total and p-Akt expression, and H(2)O(2) concentration. However, one and 15 days after SNT, H(2)O(2) concentration was raised in both sham (animals that were submitted to surgery without nerve injury) and SNT groups, showing the high sensibility of this ROS to nociceptive afferent stimuli, not only to neuropathic pain. p-Akt also increased in sham and SNT groups one day post injury, but at 3 and 7 days the increase occurred exclusively in SNT animals. Thus, there is crosstalk between intracellular signaling pathways and ROS, and these molecules can act as protective agents in acute pain situations or play a role in the development of chronic pain states.

Fig. 1

Hydrogen peroxide (H₂O₂) concentration in lumbosacral spinal cord of naïve, sham, and SNT groups 1, 3, 7, and 15 D. * P < 0.05 (one way ANOVA followed by Student-Newman-Keuls post hoc test) compared to the naïve group. H₂O₂ values are expressed as nmol/mg of tissue. Data represent the means ± SEM (n = 5 for each group). SNT, sciatic nerve transection; 1, 3, 7, and 15 D, day(s) after surgery

Fig. 2

Western blot analysis for HNE-modified protein. (a) Representative Western blots showing HNE-protein adduct formation in lumbosacral spinal cord homogenates of naïve, sham, and SNT groups 1, 3, 7, and 15 D detected with anti-HNE antibody. (b) Mean ± SEM value percentages of pixel optical density of 75 KDa protein positive for anti-HNE antibody (expressed in arbitrary units) in spinal cord homogenates of naïve, sham, and SNT groups 1, 3, 7, and 15 days after surgery. * P < 0.05 (one way ANOVA followed by Student-Newman-Keuls post hoc test) in relation to naïve and sham (n = 5 for each group). OD, optical density; SNT, sciatic nerve transection; 1, 3, 7, and 15 D, day(s) after surgery

Fig. 3

Western blot analysis for phospho-Akt (p-Akt). (a) Representative Western blots showing expression of p-Akt (ser657) in lumbosacral spinal cord homogenates of naïve, sham, and SNT groups 1, 3, 7, and 15 D. (b) Mean ± SEM value percentages of pixel optical density of p-Akt (expressed in arbitrary units) in spinal cord homogenates of naïve, sham, and SNT groups 1, 3, 7, and 15 days after surgery. * indicates P < 0.05 between 1 D SNT and naïve. ** indicates P < 0.05 among 3 D SNT compared to 3 D sham and naïve, and 7 D SNT compared to 7 D sham and naïve. The statistical test employed was one way ANOVA followed by Student-Newman-Keuls post hoc test (n = 5 for each group). OD, optical density; SNT, sciatic nerve transection; 1, 3, 7, and 15 D, day(s) after surgery

Fig. 4

Western blot analysis for total-Akt. (a) Representative western blots showing expression of total-Akt in lumbosacral spinal cord homogenates of naïve, sham, and SNT groups 1, 3, 7, and 15 days after surgery. (b) Mean ± SEM value percentages of pixel optical density of total-Akt (expressed in arbitrary units) in spinal cord homogenates of naïve, sham, and SNT groups 1, 3, 7, and 15 D after surgery. * indicates P < 0.05 in relation to naïve and sham (one way ANOVA followed by Student-Newman-Keuls post hoc test) (n = 5 for each group). OD, optical density; SNT, sciatic nerve transection; 1, 3, 7, and 15 D, day(s) after surgery