HCN Channels Antagonist ZD7288 Ameliorates Neuropathic Pain And Associated Anxiety- Depression Via Up-regulation GAD

Chronic neuropathic pain and associated anxiety-depression was one of the most annoying diseases that threat to human health. However, the pathogenesis was complicated. New research shows that after peripheral nerve injury, the expression of Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) was significantly changed and was involved in anxiolytic, antidepressant behavioral characteristics. But the underlying mechanisms remain unclear.Through the method of lateral ventricles, we observed HCN channelsantagonist ZD7288 for neuropathic pain, anxiety-depression and other behavioral changes in WKY rats by using the spared nerve injury model (SNI).Mainly to solve the following three questions:1. Whether ZD7288 can amelioratepain and anxiety-depressionacts? 2. The expression changes of HCN and is HCN channel auxiliary subunit Trip8b involved in the regulation?3. Change levels of GABA in brain and is the rate-limiting enzyme GAD involved in?Methods:128 WKY rats were divided into three parts of experiments.40 WKY rats were randomly divided into Veh, ZD7288 (2.5μg/kg), ZD7288 (5μg/kg), ZD7288 (10μg/kg) 4 groups, spontaneous activity was observed in rats after intracerebroventricular administration of ZD7288.28 WKY rats were randomly divided into Sham, ZD7288 (5μg/kg), ZD7288 (10μg/kg), and Saline 4 groups, to test anxiety-depression behaviors after single administration of ZD7288.60 WKY rats were randomly divided into Sham, SNI, SNI+ZD7288 (5μg/kg), SNI+ZD7288 (10μg/kg), SNI+Saline 5 groups, to observe the changes of pain thresholdand anxiety-depression behaviors. After behavior test, detectedthe changes of HCN channels and auxiliary subunits TRIP8b by western blot.Then tested the expression of GAD and GABA. Investigate the effects of GABA secretion of ZD7288 (10μM) by whole cell patch clamp and record mIPSCs.Results:In the first part,the behavior of SNI rats showed that after 14 days continuous administration of ZD7288, the touch threshold improvedin varying degrees, but the 10μg/kg effect wassignifficant. In the tail suspension test, the 10μg/kg group was significantly increased in activity, which means depression was eased. After administration of single dose in WKY rats,theresidence time was increased in the elevated plus maze. The spontaneous activity time of ZD7288 groupsboth significantly improved in the forced swimming test. The second part showed that after SNI, HCN2 expression was significantly increased in the thalamus and decreased after the administration, but the auxiliary subunits TRIP8bwas not change significantly. In the hippocampus, SNI surgery upregulated HCN1, after administration of10μg/kgZD7288, HCN1 expression was significantly decreased butthe change of TRIP8bwas inversedto HCN1. The third part showed that compared to SNI group, theGABA expression in thalamus tended to increase in both ZD7288 groups, but the 10μg/kg group was significantly elevated. In the hippocampus, both ZD7288 groupscould significantly increase the expression of GABA. Western blot results suggested that changes in GABA might cause by the increased of GAD.In thalamus and hippocampus, ZD7288 could upregulate the expression of GAD. In order to exclude the effect of ZD7288, we found ZD7288 did not change the amplitude and frequency of mIPSC of GABA by using whole cell patch clamp.Conclusion:Through the above experiments, we found ZD7288 can play an analgesic, anxiolytic, antidepressant effect mediate by HCN channelsand up-regulaiton of the level of GAD.