Functional Neuron-Glial in Vitro Assay for Implantable Neuroelectrodes

描述（由申请人提供）：在这里，我们提出了一种功能性体外预筛选模型，该模型是一种生物材料，用于测试过去15年来由J.S. Hong在北卡罗来纳州三角研究公园的NIEHS开发的完善的细胞培养系统的适应性。我们的模型提供了一种神经电极组织反应测试方案，该方案(1)由一个细胞培养系统组成，该系统包含已知参与组织反应的所有脑细胞类型，并且(2)能够在体内正常进行免疫细胞化学测量，以及蛋白质和基因表达分析，药理干预和实时监测。所有这些都是在严格控制的实验条件下进行的，这是解剖慢性神经电极失效背后复杂机制所必需的，从而使我们能够测试关于大脑对异物反应的特定假设。该项目有两个具体目标。具体目标1：通过表征细胞对各种CNS培养系统中异物的反应，建立基于体外细胞培养的胶质瘢痕模型。本研究试图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)胚胎早期中脑培养系统是体内观察到的良好的体外神经炎症模型，(2)胚胎晚期皮质培养系统，(3)出生后早期皮质胶质培养系统，(4)老年皮质培养系统。将通过免疫细胞化学、视频显微镜、ELISA、Bio-Plex Luminex和实时PCR检测每个系统对培养中具有代表性的生物材料的反应，以检测体内胶质瘢痕行为的特征。我们希望，至少，再现小胶质细胞迁移和附着到生物材料和生物材料的GFAP+星形胶质细胞包膜过程。特异性目的2：利用体外模型检测血清因子、炎症因子、小胶质细胞对胶质瘢痕形成的影响。Aim 1中开发的最佳体外模型将用于验证血清因子对胶质瘢痕的发展至关重要的假设，方法是将各种血清因子添加到无血清培养基中，以挽救瘢痕的形成（胶质瘢痕在无血清培养基中不会形成）。。或者，我们将在无血清培养基中发现的因子添加到含血清培养基中，以测试对瘢痕形成的抑制作用。我们还将通过免疫刺激（LPS、IL-1B和/或TNF-a）刺激培养系统来验证炎症是胶质瘢痕形成所必需的假设。阻断实验将确定IL-1B和TNF-a在胶质瘤中的作用。最后，我们将通过亮氨酸甲酯（一种吞噬细胞毒素）选择性地从培养物中去除小胶质细胞，来验证小胶质细胞参与胶质瘢痕形成的假设。人类使用神经假体装置的一个重要障碍是神经炎症反应会降低植入中枢神经系统的微电极记录的信号的保真度。这个建议描述了一个由中枢神经系统所有主要细胞类型组成的细胞培养系统，它概括了许多脑组织对植入材料反应的特征。该系统将用于还原表征组织对微电极材料的反应，以及筛选旨在减轻这种反应的策略。

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.