Electroacupuncture Relieves Neuropathic Pain Via Upregulation Of Glutamate Transporters In The Spinal Cord Of Rats

Electroacupuncture is a modern method to relief pain with alteration of peripheral electrical stimulation rather than hand manipulation. The research of electroacupuncture in clinic veterinary began in 1960 s. It was widely used in various types of surgery pain instant of drugs to avoid cardiac and respiratory depression. Acupuncture is not only effective for acute pain but also have a good effect for chronic pain. Neuropathic pain is a kind of chronic pain caused by disease of the nervous system. The main symptoms of neuropathic pain consist of persistent pain, spontaneous pain, hyperalgesia and allodynia. Since the neuropathic pain induced the change of central plasticity and complicated interaction of the neural active substances, the mechanisms of electroacupuncture treating neuropathic pain are not yet clearly elucidated.Glutamate, also named as excitatory amino acids, is an important neurotransmitter in the mammalian central nervous system which can excited the neurons. Excitatory amino acid neurons and synapses are widely distributed in the central nervous system, especially in the hippocampus, the brain and the spinal gray matter and participate in the conduction of central pain sensation. It is reported that excitatory amino acids play a key role in central sensitization and spinal cord conduction. After nerve injury, sensory afferent fibers increasing release glutamate and the receptors are activated resulting in increased neuronal excitability and glial cells activation. Glutamate transporters(GTs) are the only way to eliminate extracellular glutamate which are important in the termination of the excitatory signals and the protection of the nerve cell from excitotoxic damage. In addition, GTs have been reported associated with chronic pain.There are five subtypes of high-affinity GTs have been cloned. Glutamate/aspartate transporter(GLAST) glutamate transporter-1(GLT-1) are two of the most effective subtype to clear the extracellular glutamate.To explore the function of GLAST and GLT-1 in EA relief neuropathic pain, the neuropathic pain model of spared nerve injury(SNI) was established. Fifty four male SD rats were divided into 3 groups, control group, SNI group and SNI+EA group. The rats in SNI group and SNI+EA group suffered the SNI surgery and the rats in control group suffered the sham surgery. The rats in SNI+EA group was administrated with EA since the 8th day after surgery, once every two day and 7 times in total. EA was applied on “Zusanli” and “Sanyinjiao” acupoints and the stimuli were set as square waves, 2 Hz in frequency, with the amplitude of 3 mA, for 30 min. The paw withdrawal threshold(PWT) was measured before surgery and 7day, 8day, 14 day, 20 day after surgery. Six rats in each group were sacrificed at 8,14 and 20 day after the surgery. The samples of L4-6 were taken and tested with real-time PCR and western blot to observe the change of GLAST and GLT-1. Another 24 male SD rats suffered both SNI surgery and intralthecal placement were dived into 4 groups, EA group, EA+PDC5 group, EA+PDC10 group and EA+PDC 20 group. The rats were administrated with EA since the 8th day after surgery according to the method described above but different doses(0μg, 5μg, 10μg, 20μg) of GTs inhibitor were injected 30 min prior to EA. To observe the effect of the inhibitor on EA analgesia the PWT was measured before surgery and 7day, 8day, 14 day, 20 day after surgery.The results showed that the PWT of the rats in SNI group was obviously lower than the rats in control group at 7day, 8day, 14 day and 20 day after surgery. The PWT of rats in SNI+EA group was obviously higher than the SNI group at the 14 day and 20 day after the surgery and showed no difference with control group. Allodynia was observe in the rats suffered SNI surgery, meanwhile EA could significantly relief neuropathic pain and showed cumulative effect. In the present study, the mRNA level of GLAST and GLT-1 showed no difference among the three groups and the protein level of the rats in SNI group was obviously lower than control group at 8day, 14 day and 20 day after surgery. The protein level of the rats in SNI+EA group was obviously higher than SNI group at 14 day and 20 day after surgery but show no difference at 8day which demonstrated that EA could inhibit the downregulation of GLAST and GLT-1 during the development of neuropathic pain. The PWT of rats treated with EA and injection of 10μg or 20μg of GTs inhibitor was significant lower than the rats treated with EA and injection of 0μg or 5μg of GTs inhibitor. These results indicated that the GTs inhibitor reversed the EA induced analgesia effect in a dose-dependent manner and further prove that spinal glutamate transporters were involved in EA analgesia.The present study established neuropathic pain model by SNI surgery and demonstrated that EA relieved neuropathic pain via upregulation of glutamate transporters in the spinal cord of rats which is beneficial to discover the neural mechanism of EA analgesia, and promote the development of neuroscience.