In Vivo Brain Dialysis of Neuropeptides and Neuroimmune Signaling Proteins

描述（由申请人提供）：临床意义。神经元间的交流涉及复杂的、耦合的不同化学物质网络，包括神经肽、神经递质和被称为细胞因子的信号蛋白。趋化因子（趋化因子）和细胞因子在中枢神经系统（CNS）中起免疫调节和神经调节的作用，被认为是第三种神经通讯系统。这些重要的蛋白质影响许多不同的疾病状态。如果不解码这种化学信号网络，就无法全面了解神经系统。阻止解码这些化学网络的主要障碍之一是缺乏适当的化学分析工具，可以应用于研究啮齿动物模型的体内生物化学。特别是，这些重要的细胞因子和肽的微透析取样一直难以实施。神经肽和细胞因子ECF浓度通常是未知的，它们通过受体介导摄取（细胞基受体和细胞因子可溶性受体）的过程也知之甚少。与进行这项研究相关的风险被一些高影响的好处大大抵消了。1)将获得关于真实蛋白质浓度的重要生化信息，而不仅仅是mRNA水平的上调及其组成（例如，哪种细胞因子）。2)将进行真实的体内测量，而不是死后测量。实验方法。我们假设，对现有的脑透析方法进行修改，包括游离抗体或固定在小微球上的抗体，将显著提高蛋白质在植入大鼠大脑的透析探针中的相对回收率。此外，新的流动方式，包括循环流动和流动逆转与新的泵包括作为方法，以增加相对回收的目标蛋白质。我们的主要重点将是选择一组与癫痫相关的细胞因子和神经肽（细胞因子{CCL2 [MCP-1]， CCL3 [MIP-1a], CCL4 [MIP-12], CCL5 [RANTES], KC/GRO, IL-1b, IL-6， IL-10和TNF-a}和神经肽{血管紧张素IV， 2-淀粉样蛋白，胆囊收缩素，促肾上腺皮质激素释放激素和神经肽Y）。这些多重分析将允许同时测量从大鼠大脑收集的低至25 5L的透析液中不同的肽。

DESCRIPTION (provided by applicant): Clinical Significance. Neuronal communication involves complex and coupled networks of different chemicals including neuropeptides, neurotransmitters, and signaling proteins called cytokines. Chemokines (chemoattractant cytokines) and cytokines act as immunoregulators and neuroregulators in the central nervous system (CNS) and are considered a third neurocommunication system. These important proteins affect many different disease states. A comprehensive understanding of the nervous system cannot be achieved without decoding this chemical signaling network. One of the primary barriers preventing decoding these chemical networks is the lack of appropriate chemical analysis tools that can be applied to study in vivo biochemistry in rodent models. In particular, microdialysis sampling of these important cytokines and peptides has been difficult to implement. Neuropeptide and cytokine ECF concentrations are generally unknown and their processing via receptor meditated uptake (cell-based receptors and for cytokines - soluble receptors) is poorly understood. The risks associated with performing this research are greatly offset by several high-impact benefits. 1) Significant biochemical information about the real protein concentrations and not just upregulated mRNA levels and their makeup (e.g., which cytokines) in the CNS will be gained. 2) True in vivo measurements will be made rather than post-mortem measurements. Experimental Approach. We hypothesize that modification to existing brain dialysis methods of either inclusion of a free antibody or antibodies immobilized to small microspheres will significantly improve protein relative recovery into dialysis probes that are implanted into the rat brain. Additionally, new flow regimes including recycled flow and flow reversal with new pumps are included as methods to increase relative recovery of the targeted proteins. Our primary focus will be on a select group of cytokines and neuropeptides related to epilepsy (cytokines {CCL2 [MCP-1], CCL3 [MIP-1a], CCL4 [MIP-12], CCL5 [RANTES], KC/GRO, IL-1b, IL-6, IL-10, and TNF-a} and neuropeptides {angiotensin IV, 2-amyloid, cholecystokinin, corticotropin releasing hormone, and neuropeptide Y). These multiplexed assays will allow for the simultaneous measurement of different peptides in volumes as low as 25 5L of dialysate collected from the rat brain. PUBLIC HEALTH RELEVANCE: Proteins called cytokines as well as other neuropeptides are known to affect different human diseases related to the brain including, alcoholism, anxiety, appetite, depression, epilepsy, multiple sclerosis, pain, sleep, and various psychiatric disorders. These peptides and proteins are difficult to measure in the living brain. This work seeks to develop analytical chemistry methods to collect and detect these proteins in rodents to allow for rapid translational medical treatments for humans.