Korean J Pain.  2010 Mar;23(1):1-10.

Superoxide and Nitric Oxide Involvement in Enhancing of N-methyl-D-aspartate Receptor-Mediated Central Sensitization in the Chronic Post-ischemia Pain Model

• Ryu TH
• Jung KY
• Ha MJ
• Kwak KH
• Lim DG
• Hong JG

Abstract

BACKGROUND
Recent studies indicate that reactive oxygen species (ROS) are involved in persistent pain, including neuropathic and inflammatory pain. Since the data suggest that ROS are involved in central sensitization, the present study examines the levels of activated N-methyl-D-aspartate (NMDA) receptors in the dorsal horn after an exogenous supply of three antioxidants in rats with chronic post-ischemia pain (CPIP). This serves as an animal model of complex regional pain syndrome type-I induced by hindpaw ischemia/reperfusion injury.
METHODS
The application of tight-fitting O-rings for a period of three hours produced CPIP in male Sprague-Dawley rats. Allopurinol 4 mg/kg, allopurinol 40 mg/kg, superoxide dismutase (SOD) 4,000 U/kg, N-nitro-L-arginine methyl ester (L-NAME) 10 mg/kg and SOD 4,000 U/kg plus L-NAME 10 mg/kg were administered intraperitoneally just after O-ring application and on the first and second days after reperfusion. Mechanical allodynia was measured, and activation of the NMDA receptor subunit 1 (pNR1) of the lumbar spinal cord (L4-L6) was analyzed by the Western blot three days after reperfusion.
RESULTS
Allopurinol reduced mechanical allodynia and attenuated the enhancement of spinal pNR1 expression in CPIP rats. SOD and L-NAME also blocked spinal pNR1 in accordance with the reduced mechanical allodynia in rats with CPIP.
CONCLUSION
The present data suggest the contribution of superoxide, produced via xanthine oxidase, and the participation of superoxide and nitric oxide as a precursor of peroxynitrite in NMDA mediated central sensitization. Finally, the findings support a therapeutic potential for the manipulation of superoxide and nitric oxide in ischemia/reperfusion related pain conditions.

Keyword

central sensitization; nitric oxide; NMDA-receptor activation; persistent pain; reperfusion injury; superoxide

MeSH Terms

Allopurinol
Animals
Antioxidants
Blotting, Western
Central Nervous System Sensitization
Horns
Humans
Hyperalgesia
Inositol Phosphates
Male
Models, Animal
N-Methylaspartate
NG-Nitroarginine Methyl Ester
Nitric Oxide
Peroxynitrous Acid
Prostaglandins E
Rats
Rats, Sprague-Dawley
Reactive Oxygen Species
Reperfusion
Reperfusion Injury
Spinal Cord
Superoxide Dismutase
Superoxides
Xanthine Oxidase
Allopurinol
Antioxidants
Inositol Phosphates
N-Methylaspartate
NG-Nitroarginine Methyl Ester
Nitric Oxide
Peroxynitrous Acid
Prostaglandins E
Reactive Oxygen Species
Superoxide Dismutase
Superoxides
Xanthine Oxidase

Figure

• 

  Fig. 1 The mechanical thresholds in the ipsilateral (A) and contralateral (B) hindpaws are measured on 1 hour before ischemia (baseline) and 3 days after reperfusion (3 days). The data are presented as average ± SEM. Sham: non invasive sham control, Vehicle: normal saline i.p., LA: allopurinol 4 mg/kg i.p., HA: allopurinol 40 mg/kg i.p., *P < 0.05 compared with sham group, †P < 0.05 compared with vehicle group, ‡P < 0.05 compared with LA group.

• 

  Fig. 2 The mechanical thresholds in the ipsilateral (A) and contralateral (B) hindpaws are measured on 1 hour before ischemia (baseline) and 3 days after reperfusion (3 days). The data are presented as average ± SEM. Sham: non invasive sham control, Vehicle: normal saline i.p., SOD: superoxide dismutase (SOD) 4,000 U/kg i.p., L-NAME: N-nitro-L-arginine methyl ester 10 mg/kg i.p., SOD + L-NAME: SOD 4,000 U/kg plus L-NAME 10 mg/kg i.p., *P < 0.05 compared with sham group, †P < 0.05 compared with vehicle group, ‡P < 0.05 compared with SOD group, §P < 0.05 compared with L-NAME group.

• 

  Fig. 3 The average gel density ratio of pNR1 and β-actin in ipsilateral spinal cord (A) and contralateral spinal cord (B) and an example of Western blot gel (bottom). The data are presented as average ± SEM. Sham: non invasive sham control, Vehicle: normal saline i.p., LA: allopurinol 4 mg/kg i.p., HA: allopurinol 40 mg/kg i.p., *P < 0.05 compared with sham group, †P < 0.05 compared with vehicle group, ‡P < 0.05 compared with LA group.

• 

  Fig. 4 The average gel density ratio of pNR1 and β-actin in ipsilateral spinal cord (A) and contralateral spinal cord (B) and an example of Western blot gel (bottom). The data are presented as average ± SEM. Sham: non invasive sham control, Vehicle: normal saline i.p., SOD: superoxide dismutase (SOD) 4,000 U/kg i.p., L-NAME: N-nitro-L-arginine methyl ester 10 mg/kg i.p., SOD + L-NAME: SOD 4,000 U/kg plus L-NAME 10 mg/kg i.p., *P < 0.05 compared with sham group, †P < 0.05 compared with vehicle group, ‡P < 0.05 compared with SOD group, §P < 0.05 compared with L-NAME group.

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