Ionic channel expression in primary afferent Aβ fibers:analysis of regulatory mechanism and therapeutic application to chronic pain

离子通道表达的改变和交感神经的萌发被认为是慢性疼痛的基础，如复杂的区域疼痛综合征。损伤后几周，节后交感神经末梢萌发，包围背根神经节(DRG)神经元胞体，并与突触发生异常接触。感觉和交感神经元之间的异常相互作用也位于周围部位，包括横断神经和皮内区域的神经瘤。然而，尽管进行了广泛的研究，人们对促进交感神经生长的分子知之甚少。为了鉴定慢性疼痛相关蛋白，我们筛选了一个消减文库，该文库富含大鼠坐骨神经近端残端增加的mRNAs。在鉴定的蛋白质中，VI型胶原在残肢近端被显著诱导。VI型胶原包被的塑料板可使分离的成年大鼠背根和颈上神经节神经元、NSC34脊髓运动神经元样细胞和PC12嗜铬细胞瘤细胞长出健壮的轴突。与I型胶原、层粘连蛋白、聚D-鸟氨酸和聚L赖氨酸涂层钢板相比，这种促进神经突起的作用更持久。此外，VI型胶原涂层可以防止PC12和NSC34细胞死亡，其程度与层粘连蛋白相当。对损伤坐骨神经的免疫组织化学分析显示，VI型胶原在非髓鞘雪旺细胞、巨噬细胞和成纤维细胞以及沿血管和酪氨酸羟化酶(TM)阳性神经纤维中有较强的免疫反应。这些结果表明，VI型胶原不仅能促进运动神经和体感神经的再生，而且还能促进交感神经的萌发。我们计划研究与VI型胶原接触的体感和/或交感神经元是否会改变离子通道的表达水平。

Altered ionic channel expression and sympathetic nerve sprouting have been thought to underlie a condition of chronic pain, such as complex regional pain syndrome. Following a few weeks after injury, postganglionic sympathetic nerve endings sprout and surround cell bodies of dorsal root ganglion (DRG) neurons, and make abnormal synaptic contacts. Aberrant interactions between sensory and sympathetic neurons are also located in the peripheral sites including neuroma at a transected nerve and intradermal region. Despite extensive studies, however, little is known about the molecules that promote the outgrowth of sympathetic nerve. To identify chronic pain-associated proteins, we screened a subtractive cDNA library enriched for mRNAs increased in the proximal stump of transected rat sciatic nerve. Among the identified proteins, collagen type VI was induced prominently in the proximal stump. Collagen type VI-coated plastic plate allowed robust neurite outgrowth of dorsal root and superior cervical ganglion neurons dissociated from adult rats, NSC34 spinal motor neuron-like cells, and PC12 pheochromocytoma cells. The neurite-promoting effect was more long-lasting than those in collagen type I-, laminin-, poly-D-ornithine-, and poly-L-lysine-coated plates. In addition, coating with collagen type VI prevented death of PC12 and NSC34 cells to the extent comparable to laminin. Immunohistochemical analysis of damaged sciatic nerve revealed that intense immunoreactivity for collagen type VI was localized in nonmyelinating Schwann cells, macrophages and fibroblasts, and along vessels and tyrosine hydroxylase (TM-positive nerve fibers. These results suggest that collagen type VI promotes not only regeneration of motor and somatosensory nerves, but also sprouting of sympathetic nerve. We are planning to examine whether somatosensory and /or sympathetic neurons in contact with collagen type VI alter expression levels of ionic channels.