Role Of Glial Cell Line-derived Neurotrophic Factor In Neuropathic Pain And Electoracupuncture Analgesia In Rats

Neuropathic pain is one of the most common pathologic pains in clinic. Neuropathic pain arises as a debilitating consequence of injury to the nervous system, which is characterized by combination of spontaneous burning pain, hyperalgesia and allodynia. Neuroplastic changes of a number of bioactive substances following nerve injury have been implicated in the manifestation of neuropathic pain, such as neurotransmitters as well as their receptors, ion channels, neurotrophic factors and cytokines. Unfortunately, the etiological mechanism of neuropathic pain has been poorly understood. Neuropathic pain is often intense and refractory to conventional analgesic therapy. Management of neuropathic pain is one of the most difficult challenges in clinic. Electroacupuncture (EA) has long been used to relieve pain. In clinic, EA is an established adjuvant analgesic modality for the treatment of chronic pain. Previous studies indicated that EA had potent analgesic effect on neuropathic pain in patients and rat models. However, the mechanism of EA analgesia on neuropathic pain is not clear because of the complexity of neuropathic pain and EA modulating effect.Glial cell line-derived neurotrophic factor (GDNF) was originally purified from a rat glioma cell-line supernatant as a trophic factor for embryonic midbrain dopamine neurons, and was later found to have potent survival-promoting effects on various types of neurons including primary sensory neurons. The biological action of GDNF is mediated by a two-component receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor GFRα-1 (originally named GDNFR-α), which acts as a ligand binding domain and the receptor protein tyrosine kinase Ret, which acts as the signal transducing domain. GDNF is thought to bind preferentially to GFRα-1 and GDNF fails to exert its biological action in the absence of GFRα -1. Besides its potent survival-promoting effects on diverse groups of neurons, GDNF system has been proved by previous studies to play an important role in the modulation of nociceptive signals especially during neuropathic pain. However, the expression of GDNF and its receptor during neuropathic pain and the role of GDNF in neuropathic pain have been unknown. Previous study suggested that GDNF might be involved in the therapeutic effect of EA on Parkinson's disease. It remains unclear whether GDNF is involved in EA analgesia on neuropathic pain. GDNF has been reported to increase the number of neurons in the DRG that can express somatostatin (SOM), and also increase the release of SOMfrom the central terminals of sensory neurons into the dorsal horn. Endogenous SOM might be involved in the modulating effect of endogenous GDNF on neuropathic pain. Therefore, to investigate to role of GDNF and its receptor in neuropathic pain and EA analgesia is the main target of the present study.Previous studies proved that intrathecal administration of GDNF had potent analgesic effect on neuropathic pain. However, there are difficulties for systemic delivery of GDNF on neuropathic pain patients. Chronic delivery of GDNF by means of cell grafts near the pain processing centers of the spinal cord could be a reliable alternative. Acupuncture has good modulating effects on the stability and balance of immunology and endogenous environment of the body, which are the major influence factors for the survival and proliferation of transplanted cell grafts. The present study was also aimed to explore whether synergistic therapeutic effect could be obtained by using combination of EA and transplantation of fibroblasts gene-engineered to secrete GDNF.Using neuropathic pain model of rats induced by chronic constriction injury (CCI) to the sciatic nerve, the present study was aimed to: (1) observe therapeutic effect of cumulative EA on neuropathic pain of rats; (2) examine expression of GDNF and its receptor GFR a -1 during neuropathic pain and EA treatment using immunohistochemistry, in situ hybridization, western blot and RT-PCR; (3) knock down the expression of GFRa-1 by antisense strategy to address the role of GDNF system in neuropathic pain and EA analgesia; (4) examine expression of SOM after GFR a -1 was knocked down by antisense oligodeoxynucleotide (ODN) treatment using immunohistochemistry and RT-PCR; (5) observe effect of intrathecal transplantation of fibroblasts gene-engineered to secrete GDNF on neuropathic pain of rats and EA analgesia.The results are as follows:1. Therapeutic effect of cumulative EA on neuropathic pain of ratsCumulative EA was administered on neuropathic pain rats every other day since the 7th day after CCI surgery. Ipsilateral paw withdraw latency (PWL) of the EA treatment group increased gradually compared with those of CCI group and sham-EA treatment group, and the differences were statistically significant after three times of EA treatment (from the 12th day after surgery).2. Expression of GDNF and its receptor GFRa-1 during neuropathic pain and EA treatmentImmunohistochemistry and western blot were used to examine the expression of GDNF and GFRa -1 protein in dorsal root ganglion (DRG) and spinal dorsal horn of neuropathic pain rats. In situ hybridization and RT-PCR were used to examine the expression of GDNF mRNA and GFRa -1 mRNA in DRG of neuropathic pain rats. The results showed that: (1) the expression of both GDNF and GFRa-1 protein significantly increased in DRG and spinal dorsal horn of rats after CCI-induced neuropathic pain, and could be further significantly enhanced by EA treatment; (2) the expression of both GDNF mRNA and GFRa -1 mRNA significantly increased in DRG of rats after CCI-induced neuropathic pain, and could be further significantly enhanced by EA treatment.3. Effects of knockdown of GFRa-1 on CCI-induced thermal hyperalgesia of rats and EA analgesiaImmunohistochemistry, western blot and RT-PCR were used to determine the knockdown effect of antisense ODN on the expression of GFRa -1. The results showed that the expressions of GFRa-1 in both DRG and spinal dorsal horn were significantly knocked down by antisense ODN treatment.To investigate the role of GDNF in neuropathic pain of rats, antisense ODN specifically against GFRa-1 was intrathecally administered before CCI surgery and after neuropathic pain was firmly established respectively. The results showed that the thermal hyperalgesia of neuropathic pain rats could be significantly aggravated by antisense ODN treatment, but not by NS or mismatch ODN treatment.In order to address whether GDNF signaling system was involved in EA analgesia on neuropathic pain, antisense ODN was intrathecally administered during EA treatment since the 7th day after CCI surgery. The results showed that EA analgesia was significantly attenuated by antisense ODN treatment compared with NS treatment and mismatch ODN treatment.4. Effects of knockdown of GFR a -1 on expression of SOM during neuropathic pain and EA analgesia.Immunohistochemistry was used to examine the expression of SOM in DRG and spinal dorsal horn of neuropathic pain rats. RT-PCR was used to examine the expression of preprosomatostatin (ppSOM) mRNA in DRG of neuropathic pain rats. The results showed that: (1) there was no significant change in the expression of SOM and ppSOM mRNA following CCI, while EA could significantly enhance SOM expression in DRG and spinal dorsal horn as well as ppSOM mRNA level in DRG of neuropathic pain rats; (2) SOM expression in DRG and spinal dorsal horn as well as ppSOM mRNA level in DRG decreased significantly after GFRa -1 was knocked down by antisense ODN treatment; (3) the enhancement of SOM expression by EA treatment was significantly...