The Roles of Superoxide on At-Level Spinal Cord Injury Pain in Rats

Abstract

Background: In the present study, we examined superoxide-mediated excitatory nociceptive transmission on at-level neuropathic pain following spinal thoracic 10 contusion injury (SCI) in male Sprague Dawley rats.

Methods: Mechanical sensitivity at body trunk, neuronal firing activity, and expression of superoxide marker/ionotropic glutamate receptors (iGluRs)/CamKII were measured in the T7/8 dorsal horn, respectively.

Results: Topical treatment of superoxide donor t-BOOH (0.4 mg/kg) increased neuronal firing rates and pCamKII expression in the naïve group, whereas superoxide scavenger Tempol (1 mg/kg) and non-specific ROS scavenger PBN (3 mg/kg) decreased firing rates in the SCI group (* p < 0.05). SCI showed increases of iGluRs-mediated neuronal firing rates and pCamKII expression (* p < 0.05); however, t-BOOH treatment did not show significant changes in the naïve group. The mechanical sensitivity at the body trunk in the SCI group (6.2 ± 0.5) was attenuated by CamKII inhibitor KN-93 (50 μg, 3.9 ± 0.4) or Tempol (1 mg, 4 ± 0.4) treatment (* p < 0.05). In addition, the level of superoxide marker Dhet showed significant increase in SCI rats compared to the sham group (11.7 ± 1.7 vs. 6.6 ± 1.5, * p < 0.05).

Conclusions: Superoxide and the pCamKII pathway contribute to chronic at-level neuropathic pain without involvement of iGluRs following SCI.

Keywords: CamKII; at-level; ionotropic glutamate receptors; neuropathic pain; reactive oxygen species; spinal cord injury.

Figure 1

The contribution of superoxide on thoracic WDR neuronal firing rates. (A) The single WDR neuronal firing rates show increasing patterns to 5 different graded-intensity stimuli of VFFs prior to t-BOOH treatment (Before) and after (60 min) in naïve rats. Each VFF stimulation was applied for 10 s at the dorsal body trunk. (B) Cumulated firing rates show significant increases up to 60 min in the naïve group. (C) Prior to drug application, SCI-induced neuronal firing rates (Before) show significant increases in both the vehicle and SCI 1mg groups compared to the sham control (^# p < 0.05). Application of Tempol 1 mg significantly attenuates WDR neuronal firing rates from 10 min to 120 min, whereas the vehicle shows no changes in SCI rats (* p < 0.05). (D) Application of PBN 3 mg significantly attenuates WDR neuronal firing rates from 30 min to 120 min (* p < 0.05).

Figure 2

The contribution of ionotropic glutamate receptors on WDR neuronal firing rates after SCI. (A) The treatment of t-BOOH does not show significant changes in the expression of AMPA (GluR1 and GluR2/3) and NMDA (NR2A and NR2B) receptors, whereas the SCI group shows significant increases of GluR1, NR2A, and NR2B expression. Scale bar: 300 μm. (B) The treatment of glutamate (20 ug) does not show significant changes of superoxide production. Scale bar: 50 μm. (C) The treatment of NBQX (1 μg) and MK801 (50 μg) significantly attenuate WDR neuronal firing rates compared to SCI and vehicle groups (* p < 0.05).

Figure 3

Superoxide-mediated activation of CamKII in the thoracic dorsal horn neurons. (A) The double immunofluorescence staining shows expression of pCamKII among the sham, SCI, and t-BOOH groups, respectively. The treatments of t-BOOH and SCI show an increase of pCamKII expression in the T7/8 thoracic dorsal horn neurons compared to the sham control group (* p < 0.05). (B) In Western Blot, t-BOOH and SCI groups show significant increases of pCamKII expression compared to the sham group. Scale bar: 300 μm.

Figure 4

The contribution of pCamKII on mechanical sensitivity in the dorsal body trunk after SCI. (A) Dorsal trunks were divided by 10 square grids and 5.88 mN VFF was applied for 2 seconds at each square. Vertical bar: the incision of the back for the SCI surgery. Black circles: VFF stimulation dermatomes. (B) SCI (vehicle group) shows a significant increase of mechanical sensitivity compared to the sham group (* p < 0.05). However, KN-93 (50 μg/10 μL/kg) and Tempol (1 mg) treatments show significant decreases (^# p < 0.05), whereas KN-92 (50 μg/10 μL/kg) shows no significant changes compared to the vehicle group. However, all SCI groups show significant increases of mechanical sensitivity compared to the sham group (^$ p < 0.05). (C) Histograms (upper) and waveforms (bottom) represent the changes of WDR neuronal firing rates. (D) KN-93 treatment shows significant decrease of neuronal firing rates at 30 min compared to the before treatment (* p < 0.05).

Figure 5

Increase of superoxide production in the thoracic T7/8 spinal dorsal horn after T10 SCI. (A) The immunofluoresence staining images represent the neuronal marker neuron (green), superoxide marker Dhet (red), and merged signal, respectively. (B) Rats with T10 thoracic spinal cord contusion injury show a significant increase of Dhet intensity in the T7/8 spinal dorsal horn compared to the sham control (* p < 0.05). Scale bar: 50 μm.

Figure 6

The schematic flow of ROS–CamKII interaction and facilitation of nociceptive transmission in SCI pain. The cytosolic calcium ions in ER are triggered by activation of sarco/endoplasmic reticulum Ca²⁺-ATP (SERCA). Activation of RyRs increase calcium ion efflux into cytoplasm and initiates calcium-dependent processes. (1) Calcium ions trigger the activation of calcium-dependent phospholipase A₂ (PLA₂) to cause the production of ROS via breakdown of membrane phospholipid (PLs, ER-lipid metabolism). (2) Following the activation of mitochondrial calcium uniporter/voltage-dependent anion channel (MCU/VDAC), mitochondrial calcium ions generate superoxide via mitochondrial respiratory chain complexes. Outflux of superoxide via permeability transition pore (PTP) in mitochondria activates ryanodine receptors (RyRs) in ER that result in increases of calcium ions outflux. (3) Mitochondrial calcium ions outflux via sodium/calcium exchanger (NCX) increase cytosolic calcium ions. (4) Calcium/CamKII-mediated activation of mitogen activates protein kinase family (p38MAPK, ERK, and Jun) that initiates the gene/protein expression, which is result in the facilitation of excitatory nociceptive transmission.