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 ...

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

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...