Riluzole Induces Long-term Depression Of Nociceptive Signaling And Long-lasting Analgesia In Neuropathic Pain Rats

Nociceptive pain is an essential early warning mechanis m that reflects ourcapacity to detect the presence of potentially damaging stimuli. Pain has beenrecognized as the fifth vital signs in addition to temperature, respiration, pulse andblood pressure by WHO. The long-lasting neuropathic pain is emerging as a majorhealth problem, and the mechanisms of neuropathic pain development remain poorlyunderstood. In2011, the International Association for the Study of Pain（IASP）hasdefined neuropathic pain as pain arising as a direct consequence of a lesion or diseaseaffecting the somatosensory system. At present, the nonsteroidal anti-inflammatroydrugs (NSAIDs), anti-epileptic, anti-depressive and opioids are mainly used for therelief of neuropathic pain. These currently available therapeutics, however, are not veryeffective and causes serious side effects, limits their applications in the clinic.Therefore, development of novel specific analgesics alleviating neuropathic pain isurgently needed. The spinal superficia l dorsal horn (SDH, lamina I and II) is a relaystation in which sensory information is received, integrated, and rela yed to several brainregions. Thinly myelinated A-and unmyelinated C-fibers of most nociceptors innervate the SDH. The SDH is therefore regarded as the “Spinal center of pain modulation” andprovides a potential target for the development of novel specific analgesics.Riluzole has been used to treat amyotrophic lateral sclerosis (ALS) for decades.Recent studies suggested that Riluzo le may have analgesic effects, but its underlyingmechanisms are not clear. The present study is aimed to investigate the effects ofRiluzole on the synaptic thansmission of SDH nociceptive pathway in bothphysiological and pathological conditions.Experiment one: Riluzole induces LTD of C and Aδ fiber drives toneurons in the SDHObje ctive: To examine the inhibitory effects of riluzole on A and C fiber-evokedexcitatory postsynaptic currents (eEPSCs) in the spinal cord slices.Methods: Parasagittal spinal slices with a dorsal root attached were cut from thelumbosacral spina l cord of Sprague-Dawley rats (4-5weeks old). Whole-cellrecordings were made from SDH neurons. Holding voltage was set at-70mV, currentrecordings were started10min after whole-cell formation.(1). Test the effects ofriluzole on eEPSCs at different concentrations (10,20,50,100and500μM) in normalspinal slices.(2). Test the time-course of riluzole’s inhibitory effect on eEPSCs and theblocking effect of Tat-GluR2on the riluzole-induced inhibition.Results:1. Riluzole significantly suppresses A and C fiber-evoked eEPSCs (P <0.001, paired t-test) in a concentration dependent manner.2. Single application ofriluzole induces LTD of A and C fiber drives to neurons in the SDH (>60min); theinterference peptide of Tat-GluR2on endocytosis of postsynaptic glutamate AMPAreceptors significantly reverses the Riluzole-induced LTD (P <0.001, one-wayAVOVA）.Experiment two: The effect of riluzole to the endocytosis of postsynapticAMPA receptors in the SDHObjective: To study the mechanisms of riluzole-induced LTDMethods: The preparation of spinal cord slices and whole cell recordings wereperformed as mentioned in the experiments one.(1) The effects of riluzole on sEPSC, and Glu, AMPA, GABA and Gly induced currents were used to distinguise thepresynaptic and postsynaptic effect of riluzole.(2) Western blot was used to examinethe spinal expression of GluR2before and after riluzole treatement.Results:(1) Riluzole significantly suppresses both the amplitude and frequency ofsEPSC (P <0.05, K–S test) and Glu or AMPA induced currents, but has no effect onGABA or Gly induced currents, suggesting that both pre-and post-synapticmechanisms were involved in the riluzole’s action.(2) The expression of GluR2in themembrane of SDH neurons was significantly decreased after riluzole treatment (P <0.001, one-way AVOVA).Experiment three: The analgesic effect of riluzole in nerve-injuryinduced neuropathic pain modelObjective: To observe the analgesic effect of riluzole in neuropathic pain modelMethods:(1) The preparation of spinal cord slices and whole cell recordings wereperformed as mentioned in the experiments one. Strychnine (2μM) and Bicuculine (20μM) were used to making in vitro model of disinhibition. The effect of riluzole onStrychnine and Bicuculine induced hyperexcitability of SDH neurons was examined.(2)180-220g SD rats underlying SNL surgery were randomly divided into two groups(n=6, each group). Riluzole treatement group: Single intraperitoneal injection ofrilulzole (12mg/kg). Vehic le group: Single intraperitoneal injection of vehicle(DMSO). Rilulzole or vehicle was injected5days after SNL.50%mechanicalwithdrawal threshold (MWT) was measured using von Frey flaments before and afterSNL (3h,1,3,7,14,21d).Results:(1) Riluzole significantly inhibitses Strychnine and Bicuculine inducedhyperexcitability of SDH neurons.(2) A single intraperitoneal injection of Riluzole, butnot Vehicle,5days after SNL, attenuates the nerve injury-induced mechanical allodynia(P <0.01, compared to corresponding control, n=6rats, one-way ANOVA withBonferronni post-hoc test). The anti-allodynia effect lasts at least14days.Conclusions:1. Riluzole induces LTD of nociceptive signaling in SDH of rat in a concentrationdependent manner. 2. Riluzole induced LTD is associated with the endocytosis of AMPA receptors.3. Riluzole produces long-lasting anti-allodynia in nerve injury induced neuropathicpain condition.4. Riluzole is a potential therapeutic approach for the treatment of neuropathic pain.