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 NPs influence microglia. Microglia play a multifaceted role in the central nervous system, including maintenance of the brain microenvironment and communicating with surrounding neurons via the release of secreted factors and cytokines. Depending on the stimuli, microglia can be activated to an M1 pro-inflammatory or an M2 anti-inflammatory state. For example, M1 activated microglia release pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), while M2 activated microglia release anti-inflammatory cytokines including interleukin-4 (IL-4). To validate our surrogate in vitro biosensor for determining neurotoxicity of air pollutants, we measured microglial response to 20 nm silver nanoparticles (AgNPs). The goal of this article is to describe how an in vitro microglial cell line can be used as a surrogate biosensor marker for testing murine microglial response to NPs and how microglial activation affects hypothalamic cells. The long-term intended application of this validated model is to test effects of real-world pollutants on brain health and neurodegenerative disease. We provide a detailed description of an in vitro 96-well format assay for measuring microglial activation and hypothalamic cell survival following the exposure of microglial conditioned media.

Microglial activation was determined following AgNP exposure using a TNF-α enzyme linked immunosorbent assay (ELISA). To determine the effect of activated microglia on hypothalamic cells, the AgNPs were removed from microglial supernatant (conditioned media) using a filtration device. The filtration device retains cytokines while excluding the AgNPs based on size. Briefly, supernatant from microglia treated with or without AgNPs was collected, added to the filters, and centrifuged at 14,000 x g for 15 min. We were then able to determine the influence of microglial secreted cytokines on hypothalamic cell viability. Cell toxicity following exposure to conditioned media (containing cytokines) was determined via a resazurin-based assay as previously described ^5,6. Metabolically active cells reduce resazurin and produce a fluorescent signal proportional to the number of viable cells ⁷.

There are several advantages of using this technique over others (such as co-culture, trans-well setups, or in vivo experiments). Our model provides the ability to directly activate microglia and determine if secreted factors are toxic to neurons ⁸. The current protocol uses immortalized BV2 microglia stimulated with 20 nm diameter nanoparticles, and immortalized murine hypothalamic cells (designated mHypo-A1/2) ⁹ for determination of subsequent response. While this protocol has been optimized for these specific conditions, the methods can be altered to be used in other models of microglial-induced cell death, or with other cell types including primary microglia and neurons.

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.

2. Counting and Plating Cells

1. In a 37 °C water bath, warm DMEM and 1x-trypsin-EDTA solution.
2. Aspirate media from cells and add 500 µl of trypsin. Incubate at 37°C for 2 - 5 min.
3. Using a scraper, remove cells from the flask and suspend in 5 ml of DMEM. Pass cells through a 70 µm strainer into a 50 ml tube three times.
4. Count cells using a hemocytometer and seed 8,000 cells/well in a black walled clear bottom plate in a final volume of 200 µl DMEM supplemented with 10% FBS and 1% PSN. Incubate plate in 5% CO₂ at 37 °C for 24 hr.
5. Remove old DMEM and replace with 200 µl of warmed DMEM supplemented with 1% PSN to serum starve microglia. Incubate plate in 5% CO₂ at 37 °C for 24 hr.

3. Activating Microglia

1. In separate tubes, dilute AgNPs (0.01, 0.05 or 0.1 µg/ml) and vehicle/neutral control (sodium citrate, 0.04 mM) in serum-free DMEM supplemented with 1% PSN to final working concentrations.
2. Remove 100 µl of media from each well and replace with 100 µl of appropriate treatment compound.
3. Incubate plate in 5 % CO₂ at 37 °C for 24 hr.

4. Filtering Conditioned Media

1. Collect 200 µl of media supernatant from each well and transfer into a filter (molecular weight cutoff of 10 kDa) with collection tube (1.5 to 2 ml capacity).
2. Centrifuge at 14,000 x g at room temperature for 15 min.
3. Discard the flow-through (supernatant with particles) and place the filter upside-down into a new collection tube.
4. Centrifuge at 1,000 x g at room temperature for 2 min.
   NOTE: The resulting filtered media (containing secreted cytokines) is concentrated by six-fold.
5. Bring the volume of the concentrate to 400 µl with fresh DMEM supplemented with 10% FBS and 1% PSN and store on ice.
   NOTE: To ensure residual AgNPs are removed from filtered media, generate an AgNP concentration based on the characteristic peak at about 390 - 420 nm ¹⁰. Briefly, prepare aliquots of unfiltered AgNPs (0, 0.01, 0.025, 0.05, 0.1, and 0.2 µg/ml as described in step 3.1) and filtered vehicle control (sodium citrate, 0.04 mM) in serum-free DMEM. Aliquot 50 µl of filtered samples and standards into a black walled clear bottom plate. Using a spectrophotometer, measure absorbance at 390 - 410 nm and compare readings to the AgNP concentration curve. If filtered samples have the same absorbance value as the vehicle control sample, it can be assumed residual AgNPs are removed.
6. Use half of the filtered media to determine TNF-α secretion following instructions from a commercially available kit.

5. Hypothalamic Cell Culture Maintenance

1. Warm DMEM cell culture medium supplemented with 10% FBS and 1% PSN to 37 °C.
2. Obtain frozen stock of hypothalamic (mHypo-A1/2) cells at passage 18 - 25 stored 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 at 70 - 80% confluency.

6. Determining Hypothalamic Cell Toxicity

1. Warm DMEM and 1x-trypsin-EDTA solution in a 37 °C water bath.
2. Aspirate media from hypothalamic cells and add 500 µl of trypsin. Incubate at 37 °C for 2 - 5 min. Remove cells from flask using a scraper as described above in step 2.
3. Count cells using a hemocytometer and plate hypothalamic cells at 5,000 cells/well in a black walled clear bottom plate in a final volume of 200 µl DMEM supplemented with 10% FBS and 1% PSN and incubate overnight in 5% CO₂ at 37 °C.
4. Remove 100 µl of old media using a multichannel pipetter and add 100 µl of filtered concentrated conditioned media, to a final concentration of 1x.
5. Incubate plate in 5% CO₂ at 37 °C for 24 hr.
6. Add 22 µl of resazurin reagent and incubate plate for 20 min in 5% CO₂ at 37 °C.
7. Using a multimode spectrophotometer, record fluorescence (560 nm EX/590 nm EM) to measure cell viability. Report results as relative fluorescence units (RFU).

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