Microglia som et surrogat Biosensor fastslår partikler Nevro
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
Miljømessige forurensninger, spesielt de av nanopartikkel (NP) område (1 – 20 nm i diameter), har vært knyttet til fedme og andre nevrodegenerative sykdommer som skyldes evnen til å krysse blod-hjerne barrieren 1-3. Forhøyet eksponering for forurensning kan indusere inflammasjon i sentralnervesystemet, inkludert hypothalamus 1. En mulig mekanisme hvor dette skjer kan være gjennom nanopartikkel indusert aktivering av microglia (hjerneimmunceller) 4. Tidligere studier har brukt in vivo modeller for å studere effekter av NPs på hjernen helse som er tidkrevende, dyrt og ikke direkte svar på spørsmålet om hvordan NPs påvirke microglia. Mikroglia spille en mangesidig rolle i sentralnervesystemet, blant annet vedlikehold av hjerne mikromiljøet og kommuniserer med omgivende neuroner via frigjøring av utskilte faktorer og cytokiner. Avhengig av stimuli, kan mikroglia aktiveres til en M1 pro-inflammatorisk eller en M2 anti-inflammatorisk tilstand. For eksempel, M1 aktiverte mikroglia frigi pro-inflammatoriske cytokiner slik som tumornekrosefaktor alfa (TNF-α), mens M2 aktiverte mikroglia frigivelse anti-inflammatoriske cytokiner, inkludert interleukin-4 (IL-4). For å validere vår surrogat in vitro biosensor for å bestemme nevro av luftforurensning, vi målte microglial respons på 20 nm sølv nanopartikler (AgNPs). Målet med denne artikkelen er å beskrive hvordan en in vitro microglial cellelinje kan brukes som et surrogat biosensor markør for testing muse microglial respons på NPs og hvordan microglial aktivisering påvirker hypothalamus celler. Den langsiktige tiltenkt anvendelse av denne validert modellen er å teste effekten av virkelige forurensninger på hjernen helse og nevrodegenerativ sykdom. Vi gir en detaljert beskrivelse av en in vitro 96-brønners format undersøkelse for å måle microglial aktivering og hypotalamus celleoverlevelse etter eksponering av mikrofonen roglial klimaanlegg medier.
Microglial aktivering ble bestemt etter AgNP eksponering ved hjelp av en TNF-α enzymbundet immunosorbent analyse (ELISA). For å bestemme virkningen av aktiverte mikroglia på hypotalamus-celler, ble AgNPs fjernet fra microglial supernatant (kondisjonert medium) ved hjelp av en filtreringsanordning. Den filtrering enhet beholder cytokiner mens utelukke AgNPs basert på størrelse. I korthet supernatant fra mikroglia behandlet med eller uten AgNPs ble oppsamlet, tilsatt til filtrene, og sentrifugert ved 14 000 xg i 15 min. Vi var da i stand til å bestemme påvirkning av mikrogliaceller utskilt cytokiner på hypothalamus celle levedyktighet. Celle toksisitet etter eksponering til kondisjonert medium (inneholdende cytokiner) ble bestemt ved hjelp av en resazurin-basert assay som tidligere beskrevet 5,6. Metabolsk aktive celler redusere resazurin og frembringe et fluorescerende signal proporsjonal med antallet levedyktige celler 7.
nt "> Det er flere fordeler med å bruke denne teknikken fremfor andre (slik som ko-kultur, trans-brønners oppsett, eller in vivo-forsøk). Vår modell gjør det mulig å direkte aktivere mikroglia og bestemme om utskilt faktorer er toksisk for nevroner 8 . Den nåværende protokollen bruker udødelig BV2 microglia stimulert med 20 nm diameter nanopartikler, og udødelig murine hypothalamus celler (betegnet mHypo-A1 / 2) 9 for bestemmelse av påfølgende reaksjon. Selv om denne protokollen er optimalisert for disse konkrete forholdene, metodene kan være endret for å bli brukt i andre modeller av microglial-indusert celledød, eller med andre celletyper, inkludert primære mikroglia og nerveceller.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</…
Divulgazioni
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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