Name: gold nanorod-assisted optical stimulation of neuronal cells
Uploaded: 2015-04-27T14:00:00
Description: Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Författarna vill tacka för NanoVentures Australien för resor finansieringsstöd och Prof. John Haycock för att ha delvis värd denna forskning vid University of Sheffield och Ms Jaimee Mayne för hennes hjälp under inspelningen.

1. Au NR Framställning Anm: Au NR kan syntetiseras genom ett antal recept 16, eller köpas från kommersiella leverantörer. <ol><li> Mät den initiala optiska densiteten (OD) för Au NR lösningen via UV-Vis-spektroskopi, genom att registrera absorptions- värden från 300 nm till 1000 nm med en upplösning av 0,5-2 nm. Variera volymen av lösningen som skall användas med den tillgängliga kyvetten. </li><li> Utvärdera den inledande NP molkoncentration med en lämplig teknik...

<ol>
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M., Pralle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+ion+channels+and+neurons+through+magnetic-field+heating+of+nanoparticles.">Remote control of ion channels and neurons through magnetic-field heating of nanoparticles.</a> Nat. Nanotechnol. 5 (8), 602-606 (2010).</li><li>Farah, N., 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=Holographically+patterned+activation+using+photo-absorber+induced+neural-thermal+stimulation.">Holographically patterned activation using photo-absorber induced neural-thermal stimulation.</a> J. Neural. 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De protokoll som beskrivs i denna presentation beskriver hur kulturen, differentiera och optiskt stimulera nervceller använder yttre absorbenter. NR egenskaperna (till exempel dimensioner, form, plasmonresonans våglängd och ytkemi) och laserstimuleringsparametrarna (såsom våglängd, pulslängd, repetitionshastighet etc.) kan varieras för att passa olika experimentella behov. Effekterna på cellbeteende kan övervakas med användning av standard biologiska analyser och material. Övergripande meto...

Nya studier har visat att den transienta upphettning i samband med absorption av infrarött ljus genom vatten (våglängd&gt; 1400 nm) kan användas för att inducera aktionspotentialer i nervvävnad 1 och intracellulära kalcium transienter i kardiomyocyter 2. Användningen av infrarött ljus har höjt stort intresse för applikationer inom neurala proteser, på grund av den potentiella finare rumsliga upplösningen, brist på direkt kontakt med vävnaden, minimering av stimulerings artefakter, och avlägsnande av behovet att genetiskt modifiera cellerna före stimulering ( såsom krävs i optogenetik) 1. Trots alla dessa fördelar, nyligen utvecklade termiska modeller föreslog att målet vävnaden / cellerna kan påverkas av kumulativa uppvärmningseffekter, när flera stimulans webbplatser och / eller höga repetitionsfrekvenser används 3,4. Som svar på dessa utmaningar, har forskare insett potentialen att använda yttre absormarna för nervstimulering för att producera mer lokala uppvärmningseffekter i vävnaden. Huang et al. Visade denna princip genom att använda superpara ferrit nanopartiklar för att fjärraktivera de temperaturkänsliga TRPV1 kanaler i HEK 293 celler med en radiofrekvent magnetfält 5. Även om denna teknik kan möjliggöra djupare penetration (magnetfält samverkar relativt svagt med vävnad) var synpunkterna endast noterats under perioder av sekunder, snarare än de millisekund löptider som krävs i Bionic enheter 5. Likaså Farah et al. Visade elektrisk stimulering av råtta kortikala neuron med svarta mikropartiklar in vitro. De visade cellnivå precision i stimulering med hjälp pulsvaraktig på order av hundratals ps och energier i intervallet μJ, potentiellt möjliggör snabbare repetitionsfrekvenser 6. Användningen av yttre absorbenter har även tillämpats för att induceramorfologiska förändringar in vitro. Ciofani et al., Visade en ökning av neuronal cell utväxt ~ 40% med hjälp av piezoelektriska bornitrid nanorör upphetsade av ultraljud 7. På liknande sätt har endocyteras järnoxidnanopartiklar i PC12-celler rapporterats öka neurite differentiering på ett dos-beroende sätt, på grund av aktivering av cellvidhäftningsmolekyler med järnoxiden 8. Nyligen har intresset för yttre absorbatorer att hjälpa nervstimulering också fokuserat på användningen av guld nanopartiklar (Au NP). Au NP har förmågan att effektivt absorbera laserljus vid plasmoniska topp och att avleda det till den omgivande miljön i form av värme 9. Bland alla tillgängliga partikelformer, den optiska absorptionen av guld nanostavar (Au ​​NR) matchar lämpligen det terapeutiska fönstret av biologiska vävnader (nära infraröd - NIR, våglängd mellan 750-1,400 nm) 10. Dessutom i fortsext av neural stimulering, användandet av Au NR ger relativt gynnsam biokompatibilitet och ett brett utbud av ytfunktionalisering alternativ 11. Nyligen genomförda studier har visat att en stimulerande effekt på differentiering kan induceras efter löpande laser exponeringar av Au NR i NG108-15 neuronala celler 12. Likaså var intracellulära kalcium transienter inspelad i nervceller odlade med Au NR efter laserstrålning modulerad med variabla frekvenser och pulslängder 13. Cell membrandepolarisering registrerades också efter NIR laser belysning av Au NR i primära kulturer av spiral ganglieneuroner 14. Den första in vivo ansökan med bestrålad Au NR har visats nyligen. EOM och medarbetare exponerades Au NR på deras plasmoniska topp och spelade in en sexfaldig ökning av amplituden av förening nervaktionspotentialer (CNAPs) och en tre-faldig minskning i tröskelstimulering i råttischiasnerver. Den svEnhanced svar skrevs närvärmeeffekter till följd av excitation av NR plasmoniska topp 15. I föreliggande dokument är protokoll för att undersöka effekterna av laserstimulering i NG108-15 neuronala celler odlade med Au NR anges. Dessa metoder ger en enkel, men ändå kraftfullt, sätt att bestråla cellpopulationer in vitro med användning av standardbiologiska tekniker och material. Protokollet är baserat på en fiberkopplad laserdiod (LD) som möjliggör säker drift och repeterbar uppriktning. De Au NR beredning och laserprov bestrålningstjänster metoder kan utökas ytterligare till olika partikel former och neuronala cellkulturer, förutsatt att de specifika syntes och kultur protokollen är kända, respektive.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Au NR</td>
			<td>Sigma Aldrich</td>
			<td>716812</td>
			<td></td>
		</tr>
		<tr>
			<td>NG108-15</td>
			<td>Sigma Aldrich</td>
			<td>8811230</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6546</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Life Technologies</td>
			<td>10100147</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Life Technologies</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BDH</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;III-tubulin</td>
			<td>Promega</td>
			<td>G7121</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC-conjugated anti-mouse IgG antibody</td>
			<td>Sigma Aldrich</td>
			<td>T5393</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P6867</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Varian Medical Systems Inc.</td>
			<td>Cary 50 Bio</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Eppendorf</td>
			<td>Mini Spin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic bath</td>
			<td>Unisonics Australia</td>
			<td>FPX 10D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Kendro</td>
			<td>Hera Cell 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td>Hettich</td>
			<td>Rotofix 32A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diode</td>
			<td>Optotech</td>
			<td>780 nm single mode fibre - coupled LD</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Thorlabs</td>
			<td>780 HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Power meter</td>
			<td>Coherent</td>
			<td>Laser Check</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>http://rsb.info.nih.gov/ij/index.html</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent microscope</td>
			<td>Axon Instruments</td>
			<td>ImageX-press 5000A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;-slide well</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Carl Zeiss Microscopy Ltd.</td>
			<td>LSM 510 meta-confocal microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS210</td>
			<td></td>
		</tr>
	</tbody>
</table>

gold nanorod-assisted optical stimulation of neuronal cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

Genom att använda protokollen 1, 2 och 3 som beskrivs här, var en stimulerande effekt på differentiering observerats i NG108-15 neuronala celler odlade med Au NP (Au NR, poly (styrensulfonat) -belagda Au NR och kiselbelagd Au NR) efter laser exponeringar mellan 1,25 och 7,5 W · cm -2. Konfokala bilder av rhodamineB-märkt Au NR visade att partiklarna var intern från dag 1 av inkubation 12. Lokaliseringen ades observerades främst i cellens cytoplasma, vilket tyder på att den föredragna mekan...

Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Guld nanorod Assisterad Optisk Stimulering av nervceller

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

Research

JoVE Journal

Neuroscience

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