Microglia come un biosensore surrogato per determinare nanoparticelle neurotossicità
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
Inquinamenti ambientali, in particolare quelle della gamma di nanoparticelle (NP) (1 – 20 nm di diametro), sono stati collegati a obesità e altre malattie neurodegenerative dovute alla capacità di attraversare la barriera emato-encefalica 1-3. L'esposizione elevata a inquinamento può indurre l'infiammazione del sistema nervoso centrale, tra cui l'ipotalamo 1. Un potenziale meccanismo in cui questo si verifica potrebbe essere attraverso nanoparticelle indotta attivazione di microglia (cellule immunitarie del cervello) 4. Studi precedenti hanno usato modelli in vivo per studiare gli effetti delle NP sulla salute del cervello che sono in termini di tempo, costosa, e non direttamente rispondere alla domanda di come NP influenzano microglia. Microglia svolgono un ruolo poliedrico nel sistema nervoso centrale, compreso il mantenimento del microambiente cerebrale e comunicante con circostanti neuroni tramite il rilascio di fattori secreti e citochine. A seconda degli stimoli, microglia può essere attivato per un pr M1o-infiammatorie o uno stato antinfiammatorio M2. Ad esempio, M1 attivato microglia rilasciano citochine pro-infiammatorie come il fattore di necrosi tumorale alfa (TNF-α), mentre M2 attivato rilascio microglia citochine anti-infiammatorie comprese interleuchina-4 (IL-4). Per convalidare la nostra surrogata in vitro biosensore per la determinazione neurotossicità degli inquinanti atmosferici, abbiamo misurato la risposta della microglia a 20 nm nanoparticelle d'argento (AGNPS). L'obiettivo di questo articolo è quello di descrivere come una linea di cellule microgliali in vitro può essere utilizzato come marcatore surrogato biosensore per testare la risposta della microglia murina di NP e come microglia attivazione colpisce le cellule ipotalamiche. Il lungo termine destinato applicazione di questo modello convalidato è quello di testare gli effetti delle sostanze inquinanti del mondo reale per la salute del cervello e le malattie neurodegenerative. Forniamo una descrizione dettagliata di un test in vitro formato da 96 pozzetti per la misura di attivazione della microglia e la sopravvivenza delle cellule ipotalamico dopo l'esposizione del microfono roglial mezzi condizionati.
attivazione della microglia è stato determinato a seguito di esposizione AgNP utilizzando un enzima TNF-α linked immunosorbent assay (ELISA). Per determinare l'effetto della microglia attivata su cellule ipotalamiche, i AGNPS sono stati rimossi dal surnatante microglia (mezzi condizionati) utilizzando un dispositivo di filtraggio. Il dispositivo di filtraggio mantiene citochine, escludendo i AGNPS in base alle dimensioni. Brevemente, surnatante dal microglia trattate con o senza AGNPS stato raccolto, aggiunto ai filtri, e centrifugato a 14.000 xg per 15 min. Siamo stati quindi in grado di determinare l'influenza delle citochine secrete microgliali sulla vitalità delle cellule ipotalamo. Tossicità cellulare a seguito di esposizione ai media condizionata (contenenti citochine) è stata determinata mediante un saggio resazurin basato come precedentemente descritto 5,6. Metabolicamente attive cellule riducono resazurin e producono un segnale fluorescente proporzionale al numero di cellule vitali 7.
nt "> Ci sono molti vantaggi di utilizzare questa tecnica rispetto ad altri (come co-coltura, messe a punto trans-bene, o esperimenti in vivo). Il nostro modello offre la possibilità di attivare direttamente microglia e determinare se i fattori secreti sono tossici per i neuroni 8 . L'attuale protocollo utilizza immortalato microglia BV2 stimolato con le nanoparticelle di diametro 20 nm, e immortalato cellule ipotalamiche murine (designato mHypo-A1 / 2) 9 per la determinazione della risposta successiva. Anche se questo protocollo è stato ottimizzato per queste condizioni specifiche, i metodi possono essere alterato per essere utilizzato in altri modelli di morte cellulare indotta microgliali, o con altri tipi di cellule inclusi microglia primaria e neuroni.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</…
Declarações
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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