This work was funded by the US Department of Veterans Affairs BLR&amp;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 &amp; 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.

1. Microglial Cell Culture Maintenance
<ol>
	<li>Warm cell culture medium (Dulbecco&#39;s Modified Eagle Medium; DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/neomycin (PSN) to 37 &#176;C.</li>
	<li>Obtain frozen stock of BV2 microglial cells at passage 18 - 25 from storage at -80 &#176;C. Rapidly thaw cells in 37 &#176;C water bath.</li>
	<li>Gently transfer cells to a 75 cm2 vented flask containing 10 ml cell culture media.</li>
	<li>Incubate flask in 5% CO2...

<ol>
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L., Calderon-Garciduenas, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Air+pollution:+mechanisms+of+neuroinflammation+and+CNS+disease.">Air pollution: mechanisms of neuroinflammation and CNS disease.</a> Trends Neurosci. 32, 506-516 (2009).</li><li>Brochu, P., Bouchard, M., Haddad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+daily+inhalation+rates+for+health+risk+assessment+in+overweight/obese+children,+adults,+and+elderly.">Physiological daily inhalation rates for health risk assessment in overweight/obese children, adults, and elderly.</a> Risk Anal. 34, 567-582 (2014).</li><li>Jerrett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic-related+air+pollution+and+obesity+formation+in+children:+a+longitudinal,+multilevel+analysis.">Traffic-related air pollution and obesity formation in children: a longitudinal, multilevel analysis.</a> Environ Health. 13, 49 (2014).</li><li>Kraft, A. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-modified+gold+nanoparticles+for+the+inhibition+of+Mycobacterium+smegmatis.">Thiol-modified gold nanoparticles for the inhibition of Mycobacterium smegmatis.</a> Chem Commun (Camb). 50, 15860-15863 (2014).</li><li>Colella, M., Lobasso, S., Babudri, F., Corcelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+acid+is+associated+with+halorhodopsin+as+a+free+fatty+acid.+Radiolabeling+of+halorhodopsin+with+3H-palmitic+acid+and+chemical+analysis+of+the+reaction+products+of+purified+halorhodopsin+with+thiols+and+NaBH4.">Palmitic acid is associated with halorhodopsin as a free fatty acid. 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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</...

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-&#945;), 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-&#945; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cells/Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse microglial cell line (BV2)</td>
			<td>Interlab Cell Line Collection (Genoa, Italy)</td>
			<td>ATL03001</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult Mouse Hypothalamus Cell Line mHypoA-1/2&#160;</td>
			<td>Cellutions Biosystems Inc.</td>
			<td>CLU172</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Invitrogen</td>
			<td>10313-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>PAA Labs</td>
			<td>A15-751</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Neomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15640-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nanoparticles (20nm)</td>
			<td>Sigma-Aldrich</td>
			<td>730793</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			PrestoBlue Cell Viability Reagent</td>
			<td>Invitrogen</td>
			<td>A13262</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse TNF-&#945; ELISA Max Delux</td>
			<td>Biolegend</td>
			<td>430904</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide</td>
			<td>Sigma-Aldrich</td>
			<td>L4391</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma-Aldrich</td>
			<td>S4641</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td>
			<td>Thermo Fisher Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane</td>
			<td>EMD Millipore</td>
			<td>UFC501008</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M5 Multi-Mode Microplate</td>
			<td>Molecular Devices</td>
			<td>M5</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL Conical Centrifuge Tubes</td>
			<td>Corning, Inc</td>
			<td> 
			14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainers 70 &#956;m</td>
			<td>Corning, Inc</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop centrifuge 5430</td>
			<td>Eppendorf</td>
			<td>22620560</td>
			<td></td>
		</tr>
	</tbody>
</table>

microglia as a surrogate biosensor to determine nanoparticle neurotoxicity

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-&#945;) 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.

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-&#945; secretion was significantly increased following AgNP exposure (Figure 2</str...

Watch this Scientific Journal Video about Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity at JoVE.com

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

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.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

The overall goal of this protocol is to describe an in vitro surrogate biosensor for testing microglial response to nanoparticles. We will then determine neurotoxicity following exposure to conditioned media from microglia stimulated with nanoparticles. This method can help answer key questions in the neuroscience field such as how actively screened nanoparticles for neurotoxicity.The main advantage of this technique is that our model provides the ability to directly activate microglia and determine if secretive factors are neurotoxins without the use of expensive long term rodent studies. Though this method can provide insight into microglial induced cell death following microparticle exposure. It can also be applied to other models of neurodegenerative diseases within the context of testing potential neurotoxic environmental pollutants.Generally individuals new to this method will struggle with the condition media filtration step. Because the filtration efficiency may differ depending on the size, shape, and the concentration of the nanoparticles. In preparation warm the DMEM supplemented with 10%FBS and 1%PSN to 37 degrees Celsius.Then take frozen BV2 microglial cells obtained from passages 18 25, and rapidly thaw them at 37 degrees Celsius. Next, gently transfer the thawed cells to a 75 square-centimeter vented flask containing 10 milliliters of medium and incubate the flask. After 24 hours replace the medium and continue growing the cells until they reach a confluency of 70-80%At this point aspirate the medium and add 500 microliters of warm 1 ex trypsin EDTA solution.After allowing the flask to incubate for a few more minutes use a scraper to collect the cells into 5 milliliters of warm DMEM. Then filter the cells from the flask using a 70 micron strainer. Repeat this filtration three times using the same strainer and collect the cells in a 50 milliliter tube.Once filtered, count the cells and then seed a black-walled clear bottom plate with 8000 cells per well in 200 microliters of supplemented DMEM. After 24 hours of incubation, replace the medium in the wells with serum-free DMEM to starve the microglia. After 24 more hours of incubation, proceed to activate the microglia.To do so, first dilute silver nanoparticles in serum-free DMEM to the desired working concentration. Then replace 100 microliters of medium with the treatment compound. Be sure to include a negative control of diluted vehicle solution without the nanoparticles.Now continue the incubation for 24 hours and proceed with the analysis. To begin collect 200 microliters of supernatant medium from each plate well and load it onto a filter that will remove the nanoparticles placed over a micro centrifuge tube. It's important to remember that when filtering the nanoparticles their size, shape, and the concentration are a factor for fusion efficiency.So it is advisable to test the filtration beforehand. Now spin the tubes down at 14000g at room temperature for 15 minutes. Then discard the flow-through which contains the nanoparticles and place the filters upside down in new collection tubes.Spin the filters upside down at 1000g for two minutes to collect concentrated conditioned medium containing cytokines. Next increase the volume of the collection to 400 microliters with freshly made supplemental DMEM and store the tubes on ice. To ensure residual sulfur nanoparticles are removed from filtered media, prepare unfiltered nanoparticle standards at a range of concentration from a blank to 0.2 micrograms per milliliter in serum-free DMEM.Then aliquot 50 milliliters of the filtered samples and of each standard into a black-walled clear bottom plate and measure absorbance from 390 to 410 nanometers. To measure the concentration of TNF-alpha in the samples, use half their remaining volume in a commercially available kit. For this assay establish hypothalamic cells from frozen stocks.Using the same methods described for establishing microglial cultures. Ultimately use them to seed black-walled clear bottom plates with 5000 cells per well in 200 microliters of supplemented DMEM. After 24 hours of culturing, remove 100 microliters of medium and replace it with 100 microliters of filtered and concentrated medium collected from the activated microglial cell cultures then incubate the plate for 24 hours under standard conditions.The next day add 22 microliters of resazurin reagent to each well and continue the incubation for 20 minutes. Then with a mulit-mode spectrophotometer take a florescence measure to estimate the cells'viability in terms of relative fluorescence units. With the outlined protocol microglia function can be used as a surrogate biosensor for the brain's response to nanoparticles.TNF-alfa secretion by microglia was significantly increased following sliver nanoparticle exposure. When hypothalamic neurons were exposed to conditioned medium from the silver nanoparticle activated microglia, the viability of the neurons was significantly reduced. In principle exposure to the silver nanoparticles activated the microglia to an M1 pro-inflammatory state.Then the released pro-inflammatory cytokines contribute to surrounding cell death. After watching this video you should have a good understanding of how to use microglia as a surrogate biosensor for testing response to nanoparticles and if this response is neurotoxic. Once mastered, this technique can be done in three or four days if it's performed properly.Following this procedure, other methods such as profiling other secreted cytokines following microglial activation can be performed in order to phenotype microglial activation states.

microglia-as-surrogate-biosensor-to-determine-nanoparticle

Research

JoVE Journal

Neuroscience

6.9K Views.  Minneapolis Veterans Affairs Health Care System. The overall goal of this protocol is to describe an in vitro surrogate biosensor for testing microglial response to nanoparticles. We will then determine neurotoxicity following exposure to conditioned media from microglia stimulated with nanoparticles. This method can help answer key questions in the neuroscience field such as how actively screened nanoparticles for neurotoxicity.The main advantage of this technique is that our model provides the ability to directly activate microglia...