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: 7614165
• 负责人: William Montgomery Reichert
• 金额: $ 13.65万
• 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 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7614165
• 关键词: Animals; Architecture; Area; Astrocytes; Behavior; Biocompatible; Biocompatible Materials; Biological Assay; Brain; Cell Culture System; Cell Culture Techniques; Cells; Characteristics; Chronic; Cicatrix; Clinical Trials; Coculture Techniques; Collaborations; Collection; Computer software; Computers; Computers and Advanced Instrumentation; Data; Data Set; Deposition; Development; Devices; Diagnosis; Electrodes; Embryo; Ensure; Environment; Enzyme-Linked Immunosorbent Assay; Exhibits; Extracellular Matrix; FDA approved; 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; National Institute of Environmental Health Sciences; Neurobiology; Neuroglia; Neurons; Paralysed; Patients; Phagocytes; Principal Investigator; Process; Protein Analysis; Rattus; Reaction; Research; Research Personnel; Robotics; Role; Sampling; Scientist; Screening procedure; Serum; Serum-Free Culture Media; Signal Transduction; Site; Subcutaneous Tissue; System; TNF gene; Technology; Testing; Therapeutic; Thinking; Time; Tissues; Toxin; Training; Treatment Protocols; Universities; Up-Regulation; Upper arm; Validation; Video Microscopy; Wolves; Work; Wound Healing; aged; base; biomaterial compatibility; brain cell; brain machine interface; brain tissue; cell type; clinically significant; computerized data processing; cytokine; design; glial cell development; glucose sensor; graduate student; immunocytochemistry; implant material; implantable device; implantation; implanted sensor; in vitro Assay; in vitro Model; in vitro testing; in vivo; instrumentation; leucine methyl ester; migration; mind control; molecular marker; neuroinflammation; neuronal circuitry; postnatal; prevent; public health relevance; 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）：在这里，我们提出了一个功能性的体外筛查模型，该模型是一种生物材料测试，对J.S.过去15年中开发的良好细胞培养系统的适应性适应Hong在北卡罗来纳州研究三角公园的Niehs。 我们的模型提供了一种神经电极组织反应测试方案，即（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）启用了在体内正常进行的免疫细胞化学测量，以及用于蛋白质和基因表达表达和实时的，和真实的蛋白质表达和实时的分析。 所有这些都是（3）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失败背后的复杂机制所必需的，从而使我们能够检验有关大脑对异物反应的特定假设。 该项目有两个具体的目标。 特定目的1：通过表征对位于各种CNS培养系统中的异物的反应来开发基于体外细胞培养的模型。 该目标试图在四种不同的大鼠细胞培养系统中重现围绕异物周围的神经胶质疤痕：（1）早期的胚胎中脑培养系统被证明是在体内观察到的良好体外神经炎症模型，（2）晚期的胚胎皮质培养系统，（3）早期的早期皮质胶质培养系统和（4）早期的皮质皮层培养系统和（4）ag cortical Cortut。 每个系统对培养物培养物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进行测定，以实现体内神经胶质性疤痕行为。 我们希望至少繁殖小胶质细胞迁移以及对生物材料的生物材料和GFAP+星形胶质细胞过程包裹的附着。 具体目标2：利用体外模型来测试血清因子，炎症细胞因子和小胶质细胞对胶质疤痕发育的影响。 AIM 1中开发的最佳体外模型将用于测试以下假设：血清因子通过将各种血清因子添加到无血清培养基中以挽救疤痕形成（在无血清中形成无血清）。 ）。 另外，我们将在含有培养基的血清中添加无血清中发现的因素，以测试抑制疤痕的抑制。 我们还将检验以下假设：通过免疫挑战（LPS，IL-1B和/或TNF-A给药）刺激培养系统，炎症是形成神经胶质疤痕所必需的。 将进行阻断实验，以确定IL-1B和TNF-A在神经胶质上的影响。 最后，我们将通过选择性地通过用磷酸细胞毒素亮氨酸 - 甲基酯治疗从培养物中切除小胶质细胞来检验小胶质细胞参与神经胶质疤痕的形成。 公共卫生相关性对人类使用神经假体设备的重要障碍是神经炎症反应对降解CNS中记录的微电极信号的忠诚度的影响。 该建议描述了一个细胞培养系统，该系统由中枢神经系统的所有主要细胞类型组成，该细胞类型概括了脑组织对植入材料的反应的许多标志。 该系统将用于还原性地表征对微电极材料的组织反应，以及旨在减轻此反应的策略的筛选。

项目成果

Soluble factor effects on glial cell reactivity at the surface of gel-coated microwires.

• DOI: 10.1016/j.jneumeth.2010.05.002
• 发表时间: 2010-07-15
• 期刊: JOURNAL OF NEUROSCIENCE METHODS
• 影响因子: 3
• 作者: Polikov, Vadim S.;Hong, Jau-Shyong;Reichert, William M.
• 通讯作者: Reichert, William M.

Control protocol for robust in vitro glial scar formation around microwires: essential roles of bFGF and serum in gliosis.

• DOI: 10.1016/j.jneumeth.2009.05.002
• 发表时间: 2009-07-30
• 期刊: JOURNAL OF NEUROSCIENCE METHODS
• 影响因子: 3
• 作者: Polikov, Vadim S.;Su, Eric C.;Ball, Matthew A.;Hong, Jau-Shyong;Reichert, William M.
• 通讯作者: Reichert, William M.