CRMP-2在背根节内交感神经芽生中的作用及机制研究

Neuropathic pain following periphery injury is partially due to increased excitatory connectivity between sympathetic nerves and dorsal root ganglia (DRG) neurons via sympathetic efferent fiber sprouting into DRG. So inhibition of sympathetic nerve sprouting dramatically attenuates neuropathic pain. However, since the molecular mechanism of sympathetic nerve sprouting remains unclear, the problem how to inhibit sympathetic nerve sprouting is yet unsolved. Collapsin response mediator protein 2(CRMP-2) is an axonal growth/guidance protein. CRMP-2 facilitates neuron guidance, growth and polarity, thus is very important in nerve development and central nerve regeneration after injury. And CRMP-2 also plays a role in synaptic signaling through interactions with N-type calcium channels. CRMP-2 is known increasing N-type calcium channels' cell surface trafficking then enhancing both N-type calcium channels-mediated currents and transmitter release. We proposes CRMP-2 may play an important role in sympathetic nerve sprouting into DRG and contribute to sympathetic transmitter Norepinephrine(NE) release. So this study firstly test if CRMP-2 facilitates sympathetic nerve growth and enhance NE release in cultured sympathetic neuron, we plan to apply Immunofluorescence to observe the expression of CRMP-2 on sympathetic nerve growth cone; then transfected sympathetic neuron with CRMP-2 to record axon growth; patch clamp to record calcium current; and high-performance liquid chromatigraphy coupled to electrochemical detection to analyze NE release. Secondly we test if CRMP-2 contributes to sympathetic nerve sprouting in neuropathic pain model after injury, we apply immunohistochemistry to observe the expression of CRMP-2 in sympathetic nerve sprouting. We already found novel antiepileptic Lacosamide (LCM) selectively impairs CRMP-2-mediated microtubule polymerization, which underlies neurite outgrowth and branching. So in order to confirm the role of CRMP-2 in sympathetic nerve sprouting, we apply LCM to both neuropathic pain model and CRMP-2 transfected sympathetic neuron to observe the inhibition of both sympathetic nerve sprouting and nerve growth，and the suppression of neuropathic pain. Finally, we investigate the CRMP-2 signal passway in enhancing sympathetic nerve sprouting, we apply the inhibitors of GSK-3β and RhoA kinease. Collectively, the study throws a light on the molecular mechanism of sympathetic nerve sprouting, and suggests CRMP-2 contribute to neuropathic pain via enhancing sympathetic nerve sprouting and is a new target for neuropathic pain therapy.

阻断背根节内交感芽生能有效地治疗神经病理痛，但缺乏可行方法，急需解决。芽生机制的研究有助于解答该难题。我们前期发现塌陷反应介导蛋白2（CRMP-2）与神经生长密切相关，参与脑损伤后神经芽生，增强突触信号。故提出CRMP-2可能参与交感芽生并促进去甲肾上腺素（NE）释放。我们拟培养交感神经元、细胞转染使CRMP-2过表达、建立大鼠神经病理痛模型，采用sholl分析神经突起长度、免疫荧光、免疫组化及行为学实验，观察转染细胞神经生长；背根节CRMP-2表达；观测抑制CRMP-2的药物LCM对转染细胞神经生长，大鼠交感芽生及痛行为的抑制；以膜片钳、HPLC电化学检测转染细胞钙电流及NE水平；给予转染细胞GSK-3β和RhoA抑制剂，记录对神经生长的改变，探索CRMP-2胞内信号通路。通过本研究阐明CRMP-2在交感芽生中的重要作用及分子机制；提出抑制CRMP-2可止痛，为治疗神经病理痛提供新靶点。