Effects Of Edaravone, A Free Radical Scavenger, On Neuropathic Pain And Its Mechanisms After Spinal Nerve Ligation In Rats

ObjectiveNeuropathic pain is a disorder which result from primarily nervous system injure or other diseaes which induce unfunction of nervous system. It has become an area of intense research activity-largely because it represents a disorder with high unmet medical need. It is not a single entity but rather includes a range of heterogenous conditions that differ in aetiology, location and initiating cause. Until now, besides non-steroidal anti-inflammatory drug and opium preparations, no effective pharmacological treatments for neuralgia was published. At present, few drugs are effective in treating neuropathic pain,and their efficacy is only demonstrated in≤30% of patients . NSAID has many adverse effect and the opium preparations is easy to addiction.so the drugs which have merit between NSAID and opium are needed.Recent studies indicate that reactive oxygen species (ROS) are involved in persistent pain, including neuropathic and inflammatory pain. Edaravone, a free radical scavenger, that was developed in 1984 and is widely used clinically in Japan for acute cerebral infarction to prevent ischemia reperfusion injury, has been shown to inhibit inflammatory-induced pain in rats. However, it is unknown that whether Edaravone is effective on neuropathic pain up to now. In present study, we used the spinal nerve liagation(SNL)-induced neuropathic pain model of rats to investigate whether Edaravone is effective on neuropathic pain.MethodsBehavioral tests With the mechanical stimulation, to investigate the effect of edaravone treatment and preemptive edaravone treatment on neuropathic pain induced by spinal nerve ligation. To examine therapeutic analgesic effects, graded doses of edaravone(2, 4 or 8 mg/kg) or saline was repeated given intraperitoneally injections twice a day.To examine preemptive analgesic effects , edaravone was given intraperitoneally injections twice a day prior to the neuropathic lesion for one day and then continue to treat the animals as the same way for 2 days after L5 spinal nerves were ligated.Electrophysiological test In this part of the experiment, The test comprise two steps.First,we analogue the oxidative stress induced by H₂O₂ on the DRG neurons isolated in primary culture and investigated the actions of H₂O₂ on the excitability of neurons. Then we investigated effect of edaravone treatment on H₂O₂-induced injure.Second, the normal DRG cells were given preemptive edaravone treatment following H₂O₂ application.Molecularbiological study The animals were devided into sham,SNL and edaravone treatment groups.with the method of immunohistochemistry and western blot,to investigate the expression of pJNK on DRG and spinal cord and effect of edaravone treatment on the expression of pJNK.ResultsBehavioral tests In the edaravone treatment test, note the normal or almost normal thresholds after the 2 or 4 mg/kg injections. The 8 mg/kg injection merely produce tenuous analgesic effects on the rats of neuropathic pain. The analgesic effects lasted 2 day and had no difference compared to the SNL group at the 3th day after the drugs were cessation.. In the preemptive edaravone treatment test, slow decline in mechanical thresholds after the lesion and the thresholds remain elevated for 3 days on edaravone group.Electrophysiological test In this study, we first examined the effects of edaravone treatment on the the excitability of nociceptive DRG neurons. The result showed that H₂O₂ was sufficient to cause persistent alterations in voltage threshold .However ,the membrane potentials did not change after edaravone treatment.In the next step,we examined the effect of preemptive edaravone treatment on the excitability of nociceptive DRG neurons following H₂O₂ application.. After recording the control threshold potential(TP), edaravone was added to the extracellular solution.TP began to decrease after edaravone reached the cell. When it peaked, 400 U M H₂O₂ was added to the extracellular , the edaravone can inhibited the H₂O₂-induced deporlarization.Molecularbiological studyâ‘ Preemptive edaravone treatment reduced the number of pJNK-positive neurons in the injured DRG.The result showed that SNL induced very dramatic upregulation of pJNK in the nucleus of the injured (L5) DRGs and preemptive edaravone can apparently reduce the percentage of pJNK -IR neurons 24 h after SNL. Western blot analysis show that both JNK1 (46 kDa) and JNK2 (54 kDa) was phosphorylated in the DRG and increase after SNL surgery. It also confirmed that the expression of pJNK(both pJNKl and pJNK2 ) decreaseed in the edaravone group compared to SNL.â‘¡Reduction of pJNK in the spinal astrocytes after preemptive edaravone treatment.At the end of the third day,the L5 spinal cord of the rats were removed for immunohistochemistry after preemptive edaravone treatment. More pJNK (active form of JNK)-immunoreactive (IR) cells were found in the ipsilateral than the contralateral spinal cord at the third day on the SNL groups , predominantly in the superficial dorsal horn . The result also show that preemptive edaravone treatment can reduce pJNK expression in the spinal cord. Double immunofluorescence indicated that pJNK completely colocalizing with GFAP(a marker for astrocytes )in the spinal cord elevated at the third day and it is obvious that the number of pJNK-positive cells were reduced after preemptive edaravone treatment.ConclusionPreemptive treatment of edaravone by intraperitoneally administered had analgesic effects on spinal nerve ligation-induced chronic pain without inducing any behavioural side-effects or motor disturbances at the does we had given. The effects of edaravone as a treatment drug is limited and it merely produce tenuous analgesic effects on the rats of neuropathic pain.One of the analgesic mechanism of edaravone was its contribution to threshold potential and its inhibition on pJNK pathway.