Functional Neuron-Glial in Vitro Assay for Implantable Neuroelectrodes

描述(申请人提供)：在这里，我们提出了一个功能体外预筛选模型，这是一种生物材料测试，适用于过去15年由位于北卡罗来纳州研究三角公园的NIEHS的J.S.Hong开发的成熟的细胞培养系统。我们的模型提供了一种神经电极组织反应测试方案，它(1)由一个细胞培养系统组成，该系统包含所有已知参与组织反应的脑细胞类型，以及(2)能够进行通常在体内进行的免疫细胞化学测量，以及访问蛋白质和基因表达的分析、药物干预和实时监测。所有这些都是在严格控制的实验条件下进行的，这些实验条件对于剖析慢性神经电极故障背后的复杂机制是必要的，从而使我们能够测试关于大脑对异物的反应的特定假设。该项目有两个具体目标。具体目标1：建立一种基于体外细胞培养的胶质瘢痕形成模型，通过表征细胞对放置在各种中枢神经系统培养系统中的异物的反应。本研究的目的是在四种不同的大鼠细胞培养系统中重建异物周围的胶质瘢痕：(1)早期胚胎中脑培养系统，被证明是体内观察到的良好的神经炎症体外模型；(2)胚胎晚期皮质培养系统；(3)出生后早期皮质胶质细胞培养系统；(4)老年皮质培养系统。每个系统对放置在培养中的代表性生物材料的反应将通过免疫细胞化学、视频显微镜、ELISA、Bio-Plex Luminex和实时PCR进行分析，以确定体内胶质瘢痕行为的特征。我们希望，至少，再现小胶质细胞的迁移和附着到生物材料和GFAP星形胶质细胞突起包膜的生物材料。具体目的2：利用体外模型检测血清因子、炎性细胞因子和小胶质细胞在胶质瘢痕形成过程中的作用。在Aim 1中开发的最好的体外模型将通过在无血清介质中添加各种血清因素来挽救瘢痕形成(无血清介质中不形成胶质瘢痕)，来检验血清因素对胶质瘢痕形成至关重要的假设。)。或者，我们将在含血清的培养液中加入无血清培养基中的因子，以测试其对瘢痕形成的抑制作用。我们还将通过免疫刺激(内毒素、IL-1B和/或肿瘤坏死因子-α给药)刺激培养系统来检验炎症对于胶质瘢痕形成是必要的这一假设。将进行阻断实验以确定IL-1B和肿瘤坏死因子-α在胶质细胞增生症中的作用。最后，我们将通过用一种吞噬细胞毒素亮氨酸甲酯有选择地从培养物中去除小胶质细胞来检验小胶质细胞参与胶质瘢痕形成的假设。公共卫生相关性人类使用神经假体设备的一个重要障碍是神经炎性反应对植入中枢神经系统的信号记录微电极的保真度造成的影响。这项建议描述了一种由中枢神经系统所有主要细胞类型组成的细胞培养系统，该系统概括了脑组织对植入材料的反应的许多特征。该系统将被用来减少组织对微电极材料的反应，以及筛选旨在缓解这种反应的策略。

DESCRIPTION (provided by applicant): Here we propose a Functional In Vitro Pre-Screening Model that is a biomaterials testing adaptation of well- established cell culture systems developed over the last 15 years by J.S. Hong at the NIEHS in Research Triangle Park, NC. Our model provides a neuroelectrode tissue reaction testing regimen that (1) is comprised of a cell culture system that contains all of the brain cell types known to participate in the tissue reaction, and (2) enables the immunocytochemical measurements normally conducted in vivo as well as access to analysis of protein and gene expression, pharmacological intervention, and real-time monitoring. All of this is (3) performed under the rigidly controlled experimental conditions that are necessary to dissect the complicated mechanisms behind chronic neuroelectrode failure, thus allowing us to test specific hypotheses regarding the brain's response to a foreign body. The project has two specific aims. Specific Aim 1: Develop an in vitro cell culture based model of glial scarring by characterizing the cellular responses to a foreign body placed in a variety of CNS culture systems. This aim attempts to recreate glial scarring around a foreign body within four different rat cell culture systems: (1) an early embryonic midbrain culture system shown to be a good in vitro model of neuroinflammation observed in vivo, (2) a late embryonic cortical culture system, (3) an early postnatal cortical glia culture system, and (4) an aged cortical culture system. Each system's response to a representative biomaterial placed in culture will be assayed by immunocytochemistry, videomicroscopy, ELISA, Bio-Plex Luminex, and real time PCR for characteristic in vivo glial scarring behavior. We hope to, at minimum, reproduce microglial migration and attachment to the biomaterial and GFAP+ astrocyte process envelopment of the biomaterial. Specific Aim 2: Utilize the in vitro model to test the effect of serum factors, inflammatory cytokines, and microglia on the development of the glial scar. The best in vitro model developed in Aim 1 will be used to test the hypothesis that serum factors are essential for the development of a glial scar by adding various serum factors into serum-free media to rescue scar formation (glial scarring does not form in serum free media). ). Alternatively, we will add factors found in serum free media into serum containing media to test for inhibition of scarring. We will also test the hypothesis that inflammation is necessary for glial scar formation by stimulating the culture system with an immune challenge (LPS, IL-1B, and/or TNF-a administration). Blocking experiments will be conducted to determine the effect of IL-1B and TNF-a in gliosis. Finally, we will test the hypothesis that microglia are involved in glial scar formation by selectively removing microglia from the culture through treatment with leucine-methyl ester, a phagocyte toxin. PUBLIC HEALTH RELEVANCE A significant barrier to the human use of neuroprosthetic devices is the effect that the neuroinflammatory response has on degrading the fidelity of the signal recorded microelectrodes implanted in the CNS. This proposal describes a cell culture system comprised of all of the major cell types of the CNS that recapitulates many of the hallmarks of the brain tissue response to implanted materials. This system will be used to reductively characterize the tissue response to microelectrode materials, as well as screen for strategies intended to alleviate this response.