Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

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

Environmental pollutions, specifically those of the nanoparticle (NP) range (1 - 20 nm diameter), have been linked to obesity and other neurodegenerative diseases due to the ability to cross the blood brain barrier ^1-3. Elevated exposure to pollution may induce inflammation in the central nervous system including the hypothalamus ¹. One potential mechanism in which this occurs could be through nanoparticle induced activation of microglia (brain immune cells) ⁴. Prior studies have used in vivo models to study the effects of NPs on brain health which are time-consuming, expensive, and do not directly answer the question of how N....

Protocol

1. Microglial Cell Culture Maintenance

1. Warm cell culture medium (Dulbecco's Modified Eagle Medium; DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/neomycin (PSN) to 37 °C.
2. Obtain frozen stock of BV2 microglial cells at passage 18 - 25 from storage at -80 °C. Rapidly thaw cells in 37 °C water bath.
3. Gently transfer cells to a 75 cm² vented flask containing 10 ml cell culture media.
4. Incubate flask in 5% CO₂ at 37 °C. Aspirate media after 24 hr and replace with fresh media. Grow cells until approximately 70 - 80% confluency.

Results

We show that microglia function as a surrogate biosensor for brain response to nanoparticles using the protocol above. Our results include measurement of the toxic effects of microglial activation on downstream neuronal cell death. Figure 1 demonstrates a workflow of the protocol to activate microglia and determine if secreted cytokines reduce viability of hypothalamic neurons. TNF-α secretion was significantly increased following AgNP exposure (Figure 2).......

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 (20 nm)	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
Name	Company	Catalog Number	Comments
Equipment
96 W 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 50 ml Conical Centrifuge Tubes	Corning, Inc	14-432-22
Falcon Cell Strainers 70 μm	Corning, Inc	08-771-2
Tabletop centrifuge 5430	Eppendorf	22620560