In Vivo Brain Dialysis of Neuropeptides and Neuroimmune Signaling Proteins

描述(申请人提供)：临床意义。神经元通讯涉及不同化学物质的复杂和耦合网络，包括神经肽、神经递质和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作用，被认为是第三神经通讯系统。这些重要的蛋白质影响许多不同的疾病状态。如果不破译这种化学信号网络，就不可能全面了解神经系统。阻碍破译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，可以用于研究啮齿动物模型的体内生物化学。特别是，对这些重要的细胞因子和多肽进行微透析采样一直很难实现。神经肽和细胞因子ECF的浓度通常是未知的，它们通过受体介导的摄取(基于细胞的受体和细胞因子可溶性受体)的处理也知之甚少。与进行这项研究相关的风险被几个高影响的好处大大抵消了。1)将获得关于中枢神经系统中真实蛋白质浓度的重要生化信息，而不仅仅是上调的mRNA水平及其组成(例如，哪些细胞因子)。2)将进行真正的活体测量，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，包括加入游离抗体或固定在小微球上的抗体，将显著提高植入大鼠大脑的透析探针中蛋白质的相对回收率。此外，还包括新的流动体系，包括循环流动和带有新泵的流动反转，作为提高目标蛋白质相对回收率的方法。我们将主要关注与癫痫相关的一组细胞因子和神经肽(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不同的多肽，其体积低至从大鼠脑中收集的透析液的25.5L。

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.