Microglia como um Biosensor Surrogate determinar nanopartículas neurotoxicidade
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
Poluições ambientais, especialmente aqueles da gama de nanopartículas (NP) (1 – 20 nm de diâmetro), têm sido associadas com a obesidade e outras doenças neurodegenerativas, devido à capacidade de atravessar a barreira hemato-encefálica 1-3. Exposição elevada a poluição pode induzir a inflamação do sistema nervoso central, incluindo o hipotálamo 1. Um potencial mecanismo no qual isto ocorre pode ser através da activação induzida por nanopartículas de microglia (células imunes do cérebro) 4. Estudos anteriores têm utilizado em modelos in vivo para estudar os efeitos de NPs sobre a saúde do cérebro, que são consumidoras de tempo, é caro, e não responde directamente à questão de como o NPS influenciar microglia. Microglia desempenhar um papel multifacetado no sistema nervoso central, incluindo a manutenção do microambiente do cérebro e comunicando com circundante neurónios através da libertação de factores secretados e citocinas. Dependendo dos estímulos, microglia pode ser activado a uma pr M1o-inflamatória ou um estado anti-inflamatório M2. Por exemplo, a microglia activada M1 libertar citocinas pró-inflamatórias, tais como o factor de necrose tumoral alfa (TNF-α), enquanto M2 activado libertação microglia citocinas anti-inflamatórias, incluindo a interleucina-4 (IL-4). Para validar o nosso substituto no biossensor vitro para determinar a neurotoxicidade dos poluentes do ar, medimos microglial 20 nanopartículas de prata nm (AGNPS). O objetivo deste artigo é descrever como um in vitro linha de células microgliais pode ser usado como um marcador biossensor substituto para testar a resposta microglial murino para o NPS e como microglial ativação afeta as células do hipotálamo. A longo prazo aplicação pretendida para este modelo validado é testar efeitos dos poluentes do mundo real para a saúde do cérebro e doenças neurodegenerativas. Fornecemos uma descrição detalhada de um ensaio in vitro em formato de 96 poços para a medição da activação da microglia e sobrevivência das células do hipotálamo após a exposição de MIC roglial meios condicionados.
activação da microglia foi determinada após a exposição AgNP usando uma enzima de TNF-α linked immunosorbent assay (ELISA). Para determinar o efeito da microglia activada em células do hipotálamo, os AGNPS foram removidas a partir do sobrenadante da microglia (meio condicionado), utilizando um dispositivo de filtração. O dispositivo de filtração mantém citocinas, excluindo os AGNPS com base no tamanho. Resumidamente, o sobrenadante da microglia tratados com ou sem AGNPS foi recolhido, adicionado aos filtros, e centrifugado a 14000 xg durante 15 min. Foram então capaz de determinar a influência de citocinas secretadas microgliais sobre a viabilidade das células do hipotálamo. Toxicidade celular após a exposição ao meio condicionado (contendo citocinas) foi determinada através de um ensaio baseado em resazurina 5,6 como anteriormente descrito. Metabolicamente activas células reduzir resazurina e produzir um sinal fluorescente proporcional ao número de células viáveis 7.
nt "> Existem várias vantagens de usar esta técnica em detrimento de outros (como co-cultura, configurações trans-bem, ou in vivo). O nosso modelo fornece a capacidade de ativar diretamente microglia e determinar se fatores secretados são tóxicos para os neurônios 8 . O protocolo atual usa imortalizado microglia BV2 estimulados com nanopartículas de 20 nm de diâmetro, e imortalizado células do hipotálamo murino (designado mHypo-A1 / 2) 9 para a determinação da resposta subseqüente. Embora este protocolo foi optimizado para essas condições específicas, os métodos podem ser alterada para ser usado em outros modelos de morte celular induzida por microglia, ou com outros tipos de células, incluindo neurónios e microglia primária.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</…
Divulgations
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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