ROLE OF GLIA CELL LINE-DERIVED NEUROTROPHIC FACTOR IN NEUROPATHIC PAIN AND ELECTROACUPUNCTURE ANALGESIA

As 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. Neuropathic pain is often intense and refractory to conventional analgesic therapy. Electroacupuncturc (EA) has long been used to relieve neuropathic pain. 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 glial cell-line supernatant as a tropic 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. 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 remain unknown. It remains unclear whether GDNF was involved in EA analgesia on neuropathic pain. By using the neuropathic pain model induced by chronic constriction injury (CCI) to the sciatic nerve of rats, we investigated the possible role of GDNF and its receptor GFRα-1 in neuropathic pain EA analgesia. For EA treatment, ‘Huan-Tiao’ and ‘Yang-Ling-Quan’ acupoints were selected. A pair of stainless steel needles of 0.3mm diameter were inserted with a depth of 5mm into the contralateral (in a respect of performed CCI) acupoints ‘Huan-Tiao’ (GB-30, located near the hip joint, on the inferior borders of muscle gluteus maximus and muscle piriformis; the inferior gluteal cutaneous nerve, the inferior nerve; deeper, the sciatic nerve) and ‘Yang- Ling-Quan’ (GB-34, located near the knee joint, anterior and inferior to the small head of the fibula, in muscle peroneus longus and brevis, where the common peroneal nerve bifurcates into the superficial and deep peroneal nerves). The two needles were connected with the output terminals of an electroacupuncture apparatus (Model G 6805-2, Shanghai Medical Electronic Apparatus Company, China). Alternating strings of dense-sparse frequencies (60 Hz for 1.05 s and 2Hz for 2.85 s alternately) were selected. The intensity was adjusted to induce slight twitch of the hindlimb (⩽1 mA, 12 V), with the intensity tasting for 30 min. EA was administered once every other day from the 7th to 28th day after CCI surgery. Therapeutic effect of chronic EA treatment on neuropathic pain of rats Repeated EA was administered on neuropathic pain rats every other day since the 7th day after CCI surgery. Ipsilateral paw withdrawal latency 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). The results demonstrated that repeated EA has potent therapeutic analgesic effect on neuropathic pain of rats. Expression of GDNF and its receptor GFRα-1 during neuropathic pain and EA treatment Immunohistochemistry and Western Blot were used to examine the expression of GDNF and GFRα-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 GFRα-1 mRNA in DRG of neuropathic pain rats. The results showed that: (1) the expression of both GDNF and GFRα-1 protein significantly increased in DRG and spinal dorsal hom of rats after CCI-induced neuropathic pain, and could be further significantly enhanced by EA treatment; (2) the expression of both GDNF mRNA and GFRα-1 mRNA significantly increased in DRG rats after CCI-induced neuropathic pain, and could be further significantly enhanced by EA treatment. These results suggested that endogenous GDNF and GFRα-1 might play a role in neuropathic pain and EA analgesia. Effects of knockdown of GFRα-1 on CCI-induced thermal hyperalgesia in rats and EA analgesia Immunohistochemistry, Western Blot and RT-PCR were used to determine the knockdown effect of antisense ODN on the expression of GFRα-1. The results showed that the expressions of GFRα-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 GFRα-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. These results indicated that endogenous GDNF and GFRα-1 might play an anti-hyperalgesic role in neuropathic pain of rats and might be involved in EA analgesia on neuropathic pain. Effects of knockdown of GFRα-1 on expression of SOM during neuropathic pain and EA analgesia. GDNF has been reported to increase the number of neurons in the DRG that can express somatostatin (SOM), and also increase the release of SOM from 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. Immunohistochemistry was used to examine the expression of SOM in dorsal root ganglion (DRG) and spinal dorsal hom 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 hom as well as ppSOM mRNA level in DRG of neuropathic pain rats; (2) SOM expression in DRG and spinal dorsal hom as well as ppSOM mRNA level in DRG decreased significantly after GFRα-1 was knocked down by antisense ODN treatment; (3) the enhancement of SOM expression by EA treatment was significantly attenuated after GFRα-1 was knocked down by antisense ODN treatment. Maintenance and activation of endogenous SOM might be a pathway through which Endogenous GDNF and GFRα-1 exert their anti-hyperalgesic role in neuropathic pain of rats and EA analgesia.