Name: microglia as a surrogate biosensor to determine nanoparticle neurotoxicity
Uploaded: 2016-10-25T14:00:00
Description: Watch this Scientific Journal Video about Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity at JoVE.com

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>
	<li>Block, M. 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. D., Harry, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+microglia+and+neuroinflammation+relevant+to+environmental+exposure+and+neurotoxicity.">Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity.</a> Int J Environ Res Public Health. 8, 2980-3018 (2011).</li><li>Duffy, C. 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=Role+of+orexin+A+signaling+in+dietary+palmitic+acid-activated+microglial+cells.">Role of orexin A signaling in dietary palmitic acid-activated microglial cells.</a> Neurosci Lett. 606, 140-144 (2015).</li><li>Butterick, T. A., 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=Use+of+a+caspase+multiplexing+assay+to+determine+apoptosis+in+a+hypothalamic+cell+model.">Use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model.</a> J Vis Exp. , (2014).</li><li>Xiao, J., 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=Monitoring+of+cell+viability+and+proliferation+in+hydrogel-encapsulated+system+by+resazurin+assay.">Monitoring of cell viability and proliferation in hydrogel-encapsulated system by resazurin assay.</a> Appl Biochem Biotechnol. 162, 1996-2007 (2010).</li><li>Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., Bistoni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+murine+microglial+cells+by+a+v-raf/v-myc+carrying+retrovirus.">Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus.</a> J Neuroimmunol. 27, 229-237 (1990).</li><li>Belsham, D. D., 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=Ciliary+neurotrophic+factor+recruitment+of+glucagon-like+peptide-1+mediates+neurogenesis,+allowing+immortalization+of+adult+murine+hypothalamic+neurons.">Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons.</a> FASEB J. 23, 4256-4265 (2009).</li><li>Paramelle, D., 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=A+rapid+method+to+estimate+the+concentration+of+citrate+capped+silver+nanoparticles+from+UV-visible+light+spectra.">A rapid method to estimate the concentration of citrate capped silver nanoparticles from UV-visible light spectra.</a> Analyst. 139, 4855-4861 (2014).</li><li>Wei, Y., 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=Chronic+exposure+to+air+pollution+particles+increases+the+risk+of+obesity+and+metabolic+syndrome:+findings+from+a+natural+experiment+in+Beijing.">Chronic exposure to air pollution particles increases the risk of obesity and metabolic syndrome: findings from a natural experiment in Beijing.</a> FASEB J. , (2016).</li><li>Levesque, S., Surace, M. J., McDonald, J., Block, M. 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+&amp;+the+brain:+Subchronic+diesel+exhaust+exposure+causes+neuroinflammation+and+elevates+early+markers+of+neurodegenerative+disease.">Air pollution &amp; the brain: Subchronic diesel exhaust exposure causes neuroinflammation and elevates early markers of neurodegenerative disease.</a> J Neuroinflammation. 8, 105 (2011).</li><li>Block, M. L., 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=The+outdoor+air+pollution+and+brain+health+workshop.">The outdoor air pollution and brain health workshop.</a> Neurotoxicology. 33, 972-984 (2012).</li><li>Carson, M. J., Crane, J., Xie, A. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+CNS+microglia:+the+quest+to+identify+predictive+models.">Modeling CNS microglia: the quest to identify predictive models.</a> Drug Discov Today Dis Models. 5, 19-25 (2008).</li><li>Valdearcos, 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=Microglia+dictate+the+impact+of+saturated+fat+consumption+on+hypothalamic+inflammation+and+neuronal+function.">Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function.</a> Cell Rep. 9, 2124-2138 (2014).</li><li>Perry, V. H., Holmes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+priming+in+neurodegenerative+disease.">Microglial priming in neurodegenerative disease.</a> Nat Rev Neurol. 10, 217-224 (2014).</li><li>Block, M. L., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+microglial+activation+and+progressive+dopaminergic+neurotoxicity.">Chronic microglial activation and progressive dopaminergic neurotoxicity.</a> Biochem Soc Trans. 35, 1127-1132 (2007).</li><li>Vincenti, J. E., 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=Defining+the+Microglia+Response+during+the+Time+Course+of+Chronic+Neurodegeneration.">Defining the Microglia Response during the Time Course of Chronic Neurodegeneration.</a> J Virol. 90, 3003-3017 (2015).</li><li>Grabert, K., 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=Microglial+brain+region-dependent+diversity+and+selective+regional+sensitivities+to+aging.">Microglial brain region-dependent diversity and selective regional sensitivities to aging.</a> Nat Neurosci. 19, 504-516 (2016).</li><li>Lull, M. E., Block, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+and+chronic+neurodegeneration.">Microglial activation and chronic neurodegeneration.</a> Neurotherapeutics. 7, 354-365 (2010).</li><li>Oeckinghaus, A., Hayden, M. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+in+NF-kappaB+signaling+pathways.">Crosstalk in NF-kappaB signaling pathways.</a> Nat Immunol. 12, 695-708 (2011).</li><li>Gifford, J. 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. Radiolabeling of halorhodopsin with 3H-palmitic acid and chemical analysis of the reaction products of purified halorhodopsin with thiols and NaBH4.</a> Biochim Biophys Acta. 1370, 273-279 (1998).</li><li>Sherry, B., Jue, D. M., Zentella, A., Cerami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+high+molecular+weight+glycosylated+forms+of+murine+tumor+necrosis+factor.">Characterization of high molecular weight glycosylated forms of murine tumor necrosis factor.</a> Biochem Biophys Res Commun. 173, 1072-1078 (1990).</li><li>. PubChem Compound Database Available from: <a target="_blank" href="https://pubchem.ncbi.nlm.nih.gov/compound/104755">https://pubchem.ncbi.nlm.nih.gov/compound/104755</a> (2004)</li><li>Koenigsknecht-Talboo, J., Landreth, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+phagocytosis+induced+by+fibrillar+beta-amyloid+and+IgGs+are+differentially+regulated+by+proinflammatory+cytokines.">Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines.</a> J Neurosci. 25, 8240-8249 (2005).</li><li>McCarthy, R. 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=Characterization+of+a+novel+adult+murine+immortalized+microglial+cell+line+and+its+activation+by+amyloid-beta.">Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta.</a> J Neuroinflammation. 13, (2016).</li><li>Schauer, J. J., 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=Source+apportionment+of+airborne+particulate+matter+using+organic+compounds+as+tracers.">Source apportionment of airborne particulate matter using organic compounds as tracers.</a> Atmos Environ. 30, 3837-3855 (1996).</li><li>Kleeman, M. J., 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=Source+apportionment+of+fine+(PM1.8)+and+ultrafine+(PM0.1)+airborne+particulate+matter+during+a+severe+winter+pollution+episode.">Source apportionment of fine (PM1.8) and ultrafine (PM0.1) airborne particulate matter during a severe winter pollution episode.</a> Environ Sci Technol. 43, 272-279 (2009).</li><li>Borm, P. J., 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=The+potential+risks+of+nanomaterials:+a+review+carried+out+for+ECETOC.">The potential risks of nanomaterials: a review carried out for ECETOC.</a> Part Fibre Toxicol. 3, (2006).</li></ol>

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 ...

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