Determining the Molecular Mechanisms of Vascular Sympathetic Innervation

描述（由申请人提供）：交感神经系统通过外周血管阻力的变化对血压调节不可或缺。交感神经通常支配主要供血动脉，并延伸穿过毛细血管前小动脉网络，但不进入毛细血管、小静脉或集合静脉。在发育过程中，随着神经元接近并支配靶器官，轴突延伸紧密地跟随动脉。这种与血管的紧密接近表明血管衍生分子可能在轴突生长和靶神经支配中起作用。由于前毛细血管的选择性交感神经支配，我们的假设是，分子线索，可以促进动脉和神经元之间的化学吸引或静脉和神经元之间的化学排斥存在，并可能由血管细胞表达。 我们的具体目标是使我们能够确定候选分子，可能会驱动选择性血管交感神经支配，并在体外评估其功能。我们将（1）在胚胎晚期和出生后动物的几条血管中通过免疫荧光来表征交感神经支配模式和突触形成，（2）进行神经支配和非神经支配血管的消减杂交以产生差异表达的候选分子的列表，并通过蛋白质印迹和免疫荧光验证它们的表达，以及（3）使用三维（3-D）共培养模型，使用具有神经支配和非神经支配的上级颈神经节外植体，受神经支配的血管节段，以评估响应于将鉴定的候选分子应用于培养基的定向神经突生长。 相关性：血管交感神经的改变与高血压（神经支配过度）和心脏移植后的病理后遗症（去神经支配）等疾病有关。例如，移植后交感神经元的损失损害了调节外周血流量和充分耐受运动、温度波动和其他身体压力的能力。通过了解支配血管交感神经支配的分子机制，可以开发允许修复外周血管交感神经模式并因此允许患者恢复正常功能的疗法。

DESCRIPTION (provided by applicant): The sympathetic nervous system is integral to blood pressure regulation through changes in peripheral vascular resistance. Sympathetic nerves typically innervate major feed arteries and extend through the precapillary arteriolar network, but not into capillaries, venules or collecting veins. During development, axon extension closely follows arteries as neurons approach and innervate target organs. This close proximity to blood vessels suggests that vessel derived molecules may play a role in axon outgrowth and target innervation. Due to the selective sympathetic innervation of pre-capillary vessels, our hypothesis is that molecular cues that could promote chemoattraction between arteries and neurons or chemorepulsion between veins and neurons exist and may be expressed by vascular cells. Our specific aims are designed to allow us to identify candidate molecules that may drive selective vascular sympathetic innervation and evaluate their functions in vitro. We will (1) characterize sympathetic innervation patterns and synapse formation by immunofluorescence in several blood vessels in late embryonic and postnatal animals, (2) conduct a subtractive hybridization of innervated and non-innervated vessels to generate a list of differentially expressed candidate molecules and validate their expression through western blot and immunofluorescence and (3) evaluate the functional impacts of any identified candidate molecules using a three-dimensional (3-D) co-culture model using superior cervical ganglion explants with innervated and non-innervated vessel segments to assess directed neurite outgrowth in response to the application of identified candidate molecules to the culture medium. Relevance: Alterations in vascular sympathetic has been implicated in diseases such as hypertension (hyperinnervation) and the pathological sequalae following cardiac transplant (denervation). For example, loss of sympathetic neurons after transplant compromises the ability to regulate peripheral blood flow and to adequately tolerate exercise, temperature fluctuations and other physical stresses. By understanding the molecular mechanisms governing vascular sympathetic innervation, therapies may be developed that would allow for the repair of peripheral vascular sympathetic nerve patterning and therefore allow patients to regain normal function.