小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性
Summary
Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.
Abstract
Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-α) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.
Introduction
环境污染，特别是那些纳米粒子（NP）范围（1 – 20纳米的直径），已与肥胖症和由于跨越血脑屏障1-3的能力其它神经变性疾病。升高的接触污染可能诱发炎症在中枢神经系统，包括下丘脑1。在发生这种情况可能是通过小胶质细胞的纳米颗粒诱导的激活（脑免疫细胞）4-一个潜在机制。以前的研究已经在体内模型中用于研究纳米颗粒对脑健康这是费时的，昂贵的影响，并且不直接回答的纳米颗粒如何影响小胶质细胞的问题。小胶质细胞在中枢神经系统中发挥着多方面的作用，包括维护脑微环境，并通过的分泌因子和细胞因子的释放周围的神经元进行通信。根据不同的刺激，小胶质细胞可激活到M1的公关邻炎症或M2抗炎状态。例如，M1激活的小胶质细胞释放促炎细胞因子如肿瘤坏死因子α（TNF-α），而M 2活化的小胶质细胞释放抗炎细胞因子，包括白细胞介素-4（IL-4）。为了验证我们的体外生物传感器替代确定空气污染物的毒性，我们测量到20纳米的银纳米颗粒（AGNPS）小胶质细胞的反应。本文的目的是描述一种体外小胶质细胞系如何可以用作用于测试小鼠小神经胶质响应于NP和如何小胶质细胞活化的影响下丘脑的细胞的替代生物传感器标记。长期打算在验证模型的应用是在测试大脑健康和神经退行性疾病现实世界的污染物的影响。我们提供一种在体外 96孔格式测定的详细说明，用于测量小胶质细胞活化和下丘脑细胞存活麦克风的曝光以下 roglial条件培养基。
测定小胶质细胞活化使用酶联免疫吸附测定（ELISA）以下的TNF-α的酶下列AgNP曝光。以确定对下丘脑细胞中激活的小胶质细胞的效果，AGNPS被使用的过滤装置的小胶质细胞上清液（条件培养基）中除去。该过滤装置保持细胞因子，同时排除根据大小AGNPS。简要地说，从具有或不AGNPS治疗小神经胶质细胞收集上清液，加至过滤器，并在14000×g离心15分钟。那时我们能够确定对下丘脑细胞活力小胶质细胞分泌的细胞因子的影响。如前所述5,6-暴露于条件培养基（含有细胞因子）的细胞毒性通过基于刃天青的测定法来确定。代谢活性细胞减少刃天青并产生正比于活细胞7的数量的荧光信号。
核苷酸“>有使用这种技术比其他（如共培养，反式好设置，或在体内实验）的多个优点。我们的模型提供了直接激活的小胶质细胞，并确定是否分泌的因子是有毒的神经元8的能力。目前的协议采用永生BV2小胶质细胞刺激与20纳米的直径的纳米颗粒，和永生化的鼠下丘脑细胞（指定mHypo-A1 / 2）9，用于测定随后的反应。虽然该协议已经为这些特定的条件进行了优化，该方法可以是改变，以在小胶质诱导的细胞死亡的其它模型一起使用，或与其它类型的细胞包括原代小神经胶质细胞和神经元。Protocol
Representative Results
Discussion
Recent studies support that environmental exposure contributes to obesity and other neurodegenerative diseases 11,12. However, techniques used in previous studies are time consuming and expensive. Economic considerations, physiologically relevant delivery systems, ethical issues with extensive use of in vivo animal models, and difficulty translating findings into meaningful health advisories are a few of the major challenges that have impeded advancements in studying NP-induced neurotoxicity 13</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by the US Department of Veterans Affairs BLR&D IK2 BX001686 (to TAB), and grants from the University of Minnesota Healthy Foods, Healthy Lives Institute (to CMD, JPN, and TAB) and the Minnesota Veterans Medical Research & Education Foundation (to TAB). We thank Drs. Philippe Marambaud (Feinstein Institute for Medical Research, Manhasset, NY) and Weihua Zhao (Methodist Hospital, Houston, TX) for providing the BV2 cell line.
Materials
| Cells/Reagents | |||
| Mouse microglial cell line (BV2) | Interlab Cell Line Collection (Genoa, Italy) | ATL03001 | |
| Adult Mouse Hypothalamus Cell Line mHypoA-1/2 | Cellutions Biosystems Inc. | CLU172 | |
| Dulbecco’s Modified Eagle’s Medium | Invitrogen | 10313-039 | |
| Fetal bovine serum | PAA Labs | A15-751 | |
| Penicillin/Streptomycin/Neomycin | Thermo Fisher Scientific | 15640-055 | |
| Trypsin-EDTA | Thermo Fisher Scientific | 25200056 | |
| Silver nanoparticles (20nm) | Sigma-Aldrich | 730793 | |
PrestoBlue Cell Viability Reagent |
Invitrogen | A13262 | |
| Mouse TNF-α ELISA Max Delux | Biolegend | 430904 | |
| Lipopolysaccharide | Sigma-Aldrich | L4391 | |
| Sodium Citrate | Sigma-Aldrich | S4641 | |
| Equipment | |||
| 96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile | Thermo Fisher Scientific | 165305 | |
| Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane | EMD Millipore | UFC501008 | |
| SpectraMax M5 Multi-Mode Microplate | Molecular Devices | M5 | |
| Falcon 50mL Conical Centrifuge Tubes | Corning, Inc | 14-432-22 |
|
| Falcon Cell Strainers 70 μm | Corning, Inc | 08-771-2 | |
| Tabletop centrifuge 5430 | Eppendorf | 22620560 |
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