| Neuropathic pain refers to chronic pain that originates from pathology of the nervous system, usually accompanying with sensory deficit on affected skin region and disorders of autonomic nervous system. Nerve trauma, cancer, infection, chemotherapy, diabetes, stroke, autoimmune disease are examples that may cause neuropathic pain. Because of its long-standing painful experiences, profound underlying mechanisms and lack of effective treatment strategy, neuropathic pain has been a major cause of decreased quality of life even suicide of patients. Recent studies show that astrocyte is implicated in pain regulation, information processing in the nervous system and actively participates in brain physiology. A persistent activation of astrocytes in spinal cord dorsal horn was found in neuropathic pain model. Glutamate is a neurotransmitter critical for spinal excitatory synaptic transmission and pain processing in central nervous system. An increase in neuronal excitability and activation of astrocytes within the central nervous system may be initiated and maintained after excessive activation of central glutamate receptors by glutamate and aspartate after peripheral nerve injury. The excitatory amino acid transporter (EAAT) system is responsible for the reuptake of synaptically released glutamate and the maintenance of glutamate homeostasis. Nerve injury and other etiological factors of neuropathic pain could change the expression of EAATs.Tricyclic antidepressants, whose analgesic and antidepressant effects act via different mechanisms, are considered to be first-line drugs for the treatment of neuropathic pain. Recent studies on pharmacological mechanism indicate that amitriptyline could block the increase of aspartate and glutamate levels and upregulate EAATs expression in spinal cord of morphine-tolerant rats. We assume the alterations in the expression and function of gliacytes and EAATs caused by amitriptyline might be another mechanism of analgesic action. Research into its mechanisms of action will help to illustrate the underlying mechanisms of neuropathic pain, find new action target of antidepressants, and facilitate the development of new drugs and pain treatment strategy.Main Methods and Techniques:1. SD rats were randomly divided into 4 groups with 30 rats in each : control (A),SNI (B),amitriptyline (C),SNI+amitriptyline (D) groups, which respectively were treated with intraperitoneal injections of 0.2ml saline (A and B),10mg/kg amitriptyline (C and D), bid. The L3～L6 segment of the spinal cord was isolated respectively in one, three and five days after surgery. Expression of GFAP, marker of astrocyte, was determined by immunofluorescence,western blot and semiquantitative RT-PCR. Also, changes of mechanical pain threshold were measured.2. Male Sprague-Dawley rats were divided into four groups: control (A),SNI (B),amitriptyline (C),SNI+amitriptyline (D) groups, which respectively received intraperitoneal injections of 0.2ml saline (A and B),10mg/kg amitriptyline (C and D), bid. The L3～6 segments of the spinal cord were isolated in 1, 3 and 5 days after surgery. Expressions of GLAST and GLT-1 were determined by western blot and RT-PCR. Main Results and Conclusions:Compared to control, a markedly decreased rat mechanical pain threshold (P<0.05), the number of activated astrocytes per square millimeter in ipsilateral spinal dorsal horn increase in a time-dependent manner (86±20.1,187±22.0,382±55.6 per square millimeter) and an obviously high expression of GFAP at both protein (0.38±0.241,0.84±0.306,1.36±0.433) and mRNA (0.55±0.135,0.86±0.163,1.32±0.317) levels was observed in group B. However, there were no changes in group C. Mechanical pain threshold of rats in group D did not change any more at the 3, 5 days after surgery (4.5±1.50 g,4.9±1.41 g) and the number of astrocytes in ipsilateral spinal dorsal horn increase(216±44.6 per square millimeter), but was less than that of group B when compared in postoperative day 5. In addition, the expression of GFAP at both protein (0.79±0.254,0.77±0.227) and mRNA (0.63±0.065,0.64±0.174) levels in group D was higher than control but lower than group B(P<0.05) in 3, 5 days after surgery. It suggests that amitriptyline could inhibit the activation of astrocytes in spared nerve injury animals, which maybe one of its mechanisms in the treatment of neuropathic pain.Analysis of the expression of EAATs in dorsal horn of SNI rats indicates that the expression of GLAST in group B at both protein and mRNA level increased firstly and decreased later, compared to control, the expression of GLAST in group C increased gradually, and the expression of GLAST in group D was higher than control group and kept stable. The changes of expression of GLT-1 in each group are similar to that of GLAST. The expression of GLT-1 at both protein and mRNA level in group B was higher than control on day 1 after operation followed with a decrease in expression on day 3 and 5. In group C, the expression of GLT-1 increased gradually. The expression of GLT-1 in group D was higher than control and did not change on days 1, 3 and 5. All these manifestations suggest that amitriptyline could reverse the downregulation of EAATs and alleviate the pain in SNI rats which may be one of its mechanisms in the treatment of neuropathic pain.
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