Mikroglia som et surrogat Biosensor til Bestem Nanopartikel Neurotoksicitet
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
Miljømæssige forureninger, specielt dem af nanopartikel (NP) område (1 – 20 nm i diameter), har været forbundet med fedme og andre neurodegenerative sygdomme som følge af evnen til at krydse blod-hjerne barrieren 1-3. Forhøjet udsættes for forurening kan inducere inflammation i centralnervesystemet, herunder hypothalamus 1. En mulig mekanisme, hvor dette sker kunne være gennem nanopartikel induceret aktivering af mikroglia (hjerne immunceller) 4. Tidligere undersøgelser har anvendt in vivo modeller til at studere virkningerne af NP'er på hjernens sundhed, som er tidskrævende, dyre, og som ikke direkte besvare spørgsmålet om, hvordan NP'er påvirke mikroglia. Microglia spiller en mangesidet rolle i det centrale nervesystem, herunder vedligeholdelse af hjernen mikromiljø og kommunikere med omgivende neuroner via frigivelse af udskilte faktorer og cytokiner. Afhængigt af stimuli, kan mikroglia aktiveres til en M1 pro-inflammatoriske eller en M2 antiinflammatorisk tilstand. For eksempel M1 aktiverede mikroglia frigive pro-inflammatoriske cytokiner, såsom tumornekrosefaktor-alfa (TNF-α), mens M2 aktiverede mikroglia frigivelse anti-inflammatoriske cytokiner, herunder interleukin-4 (IL-4). For at validere vores surrogat in vitro biosensor til bestemmelse neurotoksicitet af luftforurenende stoffer, vi målte mikroglial respons på 20 nm sølv nanopartikler (AgNPs). Målet med denne artikel er at beskrive, hvordan en in vitro mikroglial cellelinie kan bruges som et surrogat biosensor markør til test murine mikroglial respons på nationale parlamenter og hvordan mikroglial aktivering påvirker hypothalamus celler. Den langsigtede tilsigtede anvendelse af dette valideret model er at afprøve effekten af den virkelige verden forurenende stoffer på hjernen sundhed og neurodegenerativ sygdom. Vi giver en detaljeret beskrivelse af en in vitro format med 96 brønde assay til måling mikroglial aktivering og hypothalamus celle overlevelse efter eksponering af mic roglial konditionerede medier.
Mikroglial aktivering blev bestemt efter AgNP eksponering ved anvendelse af en TNF-α enzymbundet immunsorbentassay (ELISA). For at bestemme virkningen af aktiveret mikroglia på hypothalamus celler blev AgNPs fjernet fra mikroglial supernatant (konditioneret medium) under anvendelse af en filtreringsanordning. Filtreringen enhed bevarer cytokiner mens eksklusive AgNPs baseret på størrelse. Kort fortalt, supernatanten fra mikroglia behandlet med eller uden AgNPs blev opsamlet, tilsat til filtrene, og centrifugeret ved 14.000 xg i 15 min. Vi kunne derefter bestemme indflydelsen af microgliale secernerede cytokiner på hypothalamus cellelevedygtighed. Celletoksicitet efter udsættelse for konditionerede medier (indeholdende cytokiner) blev bestemt via en resazurin-baseret assay som tidligere beskrevet 5,6. Metabolisk aktive celler reducerer resazurin og producere et fluorescerende signal proportionalt med antallet af levedygtige celler 7.
nt "> Der er flere fordele ved at anvende denne teknik igen andre (såsom co-kultur, trans-well opsætninger, eller in vivo forsøg). Vores model giver mulighed for direkte at aktivere mikroglia og afgøre, om udskilte faktorer er giftige for neuroner 8 . Den nuværende protokol anvendelser udødeliggjort BV2 mikroglia stimuleret med nanopartikler diameter 20 nm, og udødeliggjort murine hypothalamus celler (betegnet mHypo-A1 / 2) 9 til bestemmelse af efterfølgende reaktion. Selvom denne protokol er blevet optimeret til disse specifikke betingelser, metoder kan være ændret til at blive anvendt i andre modeller af mikroglial-induceret celledød, eller med andre celletyper, herunder primære microglia og neuroner.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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