Although both a loss of spinal inhibitory neurotransmission and the involvement of oxidative stress have been regarded as important mechanisms in the pathogenesis of pain, the relationship between these two mechanisms has not been studied. To determine whether reactive oxygen species (ROS) involvement in pain mechanisms is related to the diminished inhibitory transmission in the substantia gelatinosa (SG) of the spinal dorsal horn, behavioral studies and whole cell recordings were performed in FVB/NJ mice. Neuropathic pain was induced by a tight ligation of the L5 spinal nerve (SNL). Pain behaviors in the affected foot were assessed by behavioral testing for mechanical hyperalgesia. Pain behaviors developed by three days and lasted over eight weeks. Both systemic and intrathecal administration of an ROS scavenger, phenyl-N-tert-butylnitrone (PBN), temporarily reversed mechanical hyperalgesia up to two hours one week after SNL. In non-ligated mice, an intrathecal injection of an ROS donor, tert-butyl hydroperoxide (t-BOOH), dose-dependently induced mechanical hyperalgesia for 1.5 hours. In whole cell voltage-clamp recordings of SG neurons, perfusion with t-BOOH significantly decreased the frequency of mIPSCs, and this effect was reversed by PBN. Furthermore, t-BOOH decreased the frequency of GABAA receptor-mediated mIPSCs without altering their amplitudes but did not affect glycine receptor-mediated mIPSCs. In SNL mice, mIPSC frequency in SG neurons was significantly reduced as compared to that of normal mice, which was restored by PBN. The anti-hyperalgesic effect of PBN on mechanical hyperalgesia was attenuated by intrathecal bicuculline, a GABAA receptor blocker. Our results indicate that the increased ROS in spinal cord may induce pain by reducing GABA inhibitory influence on SG neurons that are involved in pain transmission.
Highlights d Different neuroimmune signaling pathways cause neuropathic pain in males and females d Downregulation of KCC2 is equally important for pain pathogenesis in each sex d KCC2 is regulated in many ways but is uniquely responsible for regulating chloride d KCC2 is a conceptually better therapeutic target than sexspecific mechanisms
Neuropathic pain remains notoriously difficult to treat despite numerous drug targets. Here, we offer a novel explanation for this intractability. Computer simulations predicted that qualitative changes in primary afferent excitability linked to neuropathic pain arise through a switch in spike initiation dynamics when molecular pathologies reach a tipping point (criticality), and that this tipping point can be reached via several different molecular pathologies (degeneracy). We experimentally tested these predictions by pharmacologically blocking native conductances and/or electrophysiologically inserting virtual conductances. Multiple different manipulations successfully reproduced or reversed neuropathic changes in primary afferents from naïve or nerve-injured rats, respectively, thus confirming the predicted criticality and its degenerate basis. Degeneracy means that several different molecular pathologies are individually sufficient to cause hyperexcitability, and because several such pathologies co-occur after nerve injury, that no single pathology is uniquely necessary. Consequently, single-target-drugs can be circumvented by maladaptive plasticity in any one of several ion channels.DOI: http://dx.doi.org/10.7554/eLife.02370.001
Recent studies suggest that reactive oxygen species (ROS) are functional messenger molecules in central sensitization, an underlying mechanism of persistent pain. Because spinal cord long-term potentiation (LTP) is the electrophysiological basis of central sensitization, this study investigates the effects of the increased or decreased spinal ROS levels on spinal cord LTP. Spinal cord LTP is induced by either brief, high-frequency stimulation (HFS) of a dorsal root at C-fiber intensity or superfusion of a ROS donor, tert-butyl hydroperoxide (t-BOOH), onto rat spinal cord slice preparations. Field excitatory postsynaptic potentials (fEPSPs) evoked by dorsal root stimulations with either Abeta- or C-fiber intensity are recorded from the superficial dorsal horn. HFS significantly increases the slope of both Abeta- and C-fiber evoked fEPSPs, thus suggesting LTP development. The induction, not the maintenance, of HFS-induced LTP is blocked by a N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonopentanoic acid (D-AP5). Both the induction and maintenance of LTP of Abeta-fiber-evoked fEPSPs are inhibited by a ROS scavenger, either N-tert-butyl-alpha-phenylnitrone or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. A ROS donor, t-BOOH-induced LTP is inhibited by N-tert-butyl-alpha-phenylnitrone but not by D-AP5. Furthermore, HFS-induced LTP and t-BOOH-induced LTP occlude each other. The data suggest that elevated ROS is a downstream event of NMDA receptor activation and an essential step for potentiation of synaptic excitability in the spinal dorsal horn.
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