Defining Target Contribution to Orofacial Pain by Antisense Oligonucleotides

描述（由申请人提供）：慢性口面疼痛是一种常见的临床综合征，由于对慢性口面疼痛的细胞机制知之甚少，因此缺乏特异性和有效的治疗药物。基于体外生物化学研究和非口面神经损伤诱导的慢性疼痛模型的数据，我们假设三叉神经损伤导致三叉神经节和相关脑干和上颈脊髓中钙通道α-2-δ-1亚基（Cava 2d 1）和血小板反应蛋白-4（TSP 4）的表达改变，其通过促进突触发生在神经病理性疼痛的发展中发挥功能性作用。在这个探索性的建议中，我们计划测试Cava 2d 1和TSP 4诱导损伤是否在口面神经性疼痛模型中神经性疼痛的发生和/或维持中起因果作用。我们将在来自假手术和眶下神经损伤动物的三叉神经节和相关脑干/脊髓样本中进行Western印迹分析、免疫组织化学研究和实时PCR，以确定Cava 2d 1和TSP 4表达与神经病理性疼痛的发展之间是否存在可靠的相关性。此外，我们将以剂量依赖性方式，在口面疼痛状态发作之前和之后，将Cava 2d 1和TSP 4反义或错配寡核苷酸鞘内应用于神经损伤模型，以确定预防和逆转Cava 2d 1和/或TSP损伤诱导是否可以分别阻断口面神经性疼痛的发展和维持。这些研究的完成将使我们能够确定损伤诱导的Cava 2d 1和TSP表达对口面神经性疼痛的功能贡献，并为进一步研究这些蛋白质在口面疼痛发展中的作用机制奠定基础。这项研究的最终目标是确定新的目标和途径，为口面疼痛管理的新药物的发展。项目叙述：神经损伤引起的慢性口面神经痛（orofacial neuropathic pain，ONS）是一种常见的临床综合征，由于其细胞机制尚不清楚，缺乏特异有效的治疗药物。来自体外生物化学研究和非口面神经损伤疼痛模型的现有数据支持三叉神经损伤可能导致三叉神经节和相关脑干和上颈脊髓中钙通道α-2-δ-1亚基（Cava 2d 1）和血小板反应蛋白-4（TSP 4）的表达改变，其通过新机制在口面疼痛发展中发挥功能性作用。在这个探索性的建议中，我们计划测试三叉神经损伤是否会导致Cava 2d 1和TSP 4的表达改变，这有助于口面神经性疼痛模型中神经性疼痛的发生和/或维持。我们将使用生物化学和行为药理学方法来验证这一假设。本研究的完成将为了解口面神经性疼痛机制和开发下一代口面神经性疼痛治疗药物提供重要信息。

DESCRIPTION (provided by applicant): Chronic orofacial pain is a common clinical syndrome lacking specific and effective therapeutic agents due to the fact that the cellular mechanisms of chronic orofacial pain are poorly understood. Based on data from in vitro biochemical studies and non-orofacial, nerve injury-induced chronic pain models, we hypothesize that trigeminal nerve injury leads to altered expression of the calcium channel alpha-2-delta-1 subunit (Cava2d1) and thrombospondin-4 (TSP4) in trigeminal ganglia and associated brainstem and upper cervical spinal cord that plays a functional role in the development of neuropathic pain by promoting synaptogenesis. In this exploratory proposal, we plan to test if Cava2d1 and TSP4 induction by injury plays a causal role in the genesis and/or maintenance of neuropathic pain in an orofacial neuropathic pain model. We will perform Western blot analysis, immunohistochemical studies, and real-time PCR in trigeminal ganglia and associated brainstem/spinal cord samples from sham and infraorbital nerve injured animals to determine if there is a firm correlation between Cava2d1 and TSP4 expression and development of neuropathic pain. In addition, we will apply Cava2d1 and TSP4 antisense or mismatch oligonucleotides intrathecally to the nerve injury model, in a dose-dependent manner, before and after the onset of orofacial pain states to determine if preventing and reversing Cava2d1 and/or TSP induction by injury can block orofacial neuropathic pain development and maintenance, respectively. Completion of these studies will allow us to determine the functional contribution of injury-induced Cava2d1 and TSP expression to orofacial neuropathic pain and form the foundation for further investigation of the mechanisms underlying the contribution of these proteins in orofacial pain development. The final goal of the study is to identify new targets and pathways for the development of new medications for orofacial pain management. Project Narrative: Chronic orofacial pain derived from nerve injury, or orofacial neuropathic pain, is a common clinical syndrome lacking specific and effective therapeutic agents due to the fact that its cellular mechanisms are poorly understood. Existing data from in vitro biochemical studies and non-orofacial nerve injury pain models support that trigeminal nerve injury may lead to altered expression of the calcium channel alpha-2-delta-1 subunit (Cava2d1) and thrombospondin-4 (TSP4) in trigeminal ganglia and associated brainstem and upper cervical spinal cord that plays a functional role in orofacial pain development through a novel mechanism. In this exploratory proposal, we plan to test if trigeminal nerve injury causes altered expression of the Cava2d1 and TSP4 that contributes to the genesis and/or maintenance of neuropathic pain in an orofacial neuropathic pain model. We will test this hypothesis using biochemical and behavioral pharmacology approaches. Completion of this study will provide important information for the understanding of orofacial neuropathic pain mechanisms and the development of next generation of medications for orofacial neuropathic pain management.