Functional Neuron-Glial in Vitro Assay for Implantable Neuroelectrodes

可植入神经电极的功能性神经元胶质体外测定

• 批准号: 7464531
• 负责人: William Montgomery Reichert
• 金额: $ 20.21万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-15 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7464531
• 关键词: Age; Animals; Appendix; Architecture; Area; Astrocytes; Behavior; Biocompatible; Biocompatible Materials; Biological Assay; Biological Markers; Brain; Cell Culture System; Cells; Characteristics; Chronic; Cicatrix; Clinical Trials; Coculture Techniques; Collaborations; Collection; Computer software; Computers; Computers and Advanced Instrumentation; Condition; Cultured Cells; Data; Data Set; Deposition; Development; Devices; Diagnosis; Electrodes; Embryo; End Point; Ensure; Environment; Enzyme-Linked Immunosorbent Assay; Exhibits; Extracellular Matrix; Factor Analysis; Failure; Foreign Bodies; Future; Gene Expression; Genes; Glial Fibrillary Acidic Protein; Gliosis; Glucose; Heart; Histocompatibility; Histology; Human; Immune; Implant; Implanted Electrodes; In Situ; In Vitro; Individual; Inflammation; Inflammatory; Interleukin-1; Intervention; Knowledge; Laboratories; Left; Letters; Longevity; Measurement; Mediator of activation protein; Michigan; Microdialysis; Microelectrodes; Microglia; Midbrain structure; Modeling; Modification; Molecular; Monitor; Monkeys; Neurobiology; Neuroglia; Neurons; Numbers; Paralysed; Patients; Personal Satisfaction; Phagocytes; Placement; Polymerase Chain Reaction; Principal Investigator; Process; Protein Analysis; Public Health; Rattus; Reaction; Research; Research Personnel; Robotics; Role; Sampling; Scientist; Screening procedure; Serum; Serum-Free Culture Media; Signal Transduction; Site; Students; Subcutaneous Tissue; System; TNF gene; Technology; Testing; Therapeutic; Thinking; Time; Tissues; Toxin; Training; Treatment Protocols; United States Food and Drug Administration; Universities; Up-Regulation; Upper arm; Validation; Video Microscopy; Week; Wolves; Work; Wound Healing; base; biomaterial compatibility; brain cell; brain machine interface; brain tissue; cell type; clinically significant; computerized data processing; cytokine; day; design; glucose sensor; immunocytochemistry; implant material; implantable device; implantation; implanted sensor; in vitro Assay; in vitro Model; in vivo; instrumentation; leucine methyl ester; migration; mind control; neuroinflammation; neuronal circuitry; postnatal; prevent; research study; response; sensor; telemetering; thought control; tool

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.

描述（由申请人提供）：在此，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 J.S. 在过去 15 年中开发的成熟细胞培养系统的适应性。 Hong 在北卡罗来纳州三角研究园的 NIEHS。 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）由细胞培养系统组成，其中包含已知参与组织反应的所有脑细胞类型，并且（2）也能够进行通常在体内进行的免疫细胞化学测量如蛋白质和基因表达分析、药物干预和实时监测。 所有这些（3）都是在严格控制的实验条件下进行的，这些条件是剖析慢性神经电极故障背后的复杂机制所必需的，从而使我们能够测试有关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：通过表征细胞对放置在各种中枢神经系统培养系统中的异物的反应，开发基于体外细胞培养的神经胶质疤痕模型。 该目标试图在四种不同的大鼠细胞培养系统中重建异物周围的神经胶质疤痕：（1）早期胚胎中脑培养系统被证明是体内观察到的神经炎症的良好体外模型，（2）晚期胚胎皮质培养系统，（3）早期出生后皮质神经胶质细胞培养系统，以及（4）老年皮质培养系统。 每个系统对培养物中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，以了解特征性的体内神经胶质疤痕行为。 我们希望至少能够重现小胶质细胞的迁移和对生物材料的附着以及生物材料的 GFAP+ 星形胶质细胞过程的包封。 具体目标2：利用体外模型检测血清因子、炎症细胞因子和小胶质细胞对胶质疤痕形成的影响。 目标 1 中开发的最佳体外模型将用于测试血清因子对于神经胶质疤痕的形成至关重要的假设，方法是将各种血清因子添加到无血清培养基中以挽救疤痕形成（神经胶质疤痕不会在血清中形成）免费媒体）。 ）。 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疤痕形成的抑制。 我们还将通过免疫攻击（LPS、IL-1B 和/或 TNF-a 给药）刺激培养系统来测试炎症对于神经胶质疤痕形成所必需的假设。 将进行阻断实验以确定IL-1B和TNF-a在神经胶质增生中的作用。 最后，我们将通过用亮氨酸甲酯（一种吞噬细胞毒素）处理选择性地从培养物中去除小胶质细胞来检验小胶质细胞参与神经胶质疤痕形成的假设。 公众健康相关性人类使用神经假体装置的一个重大障碍是神经炎症反应会降低植入中枢神经系统的微电极信号记录的保真度。 该提案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反应的许多特征。 该系统将用于还原表征组织对微电极材料的反应，并筛选旨在减轻这种反应的策略。