Name: method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro
Uploaded: 2015-03-21T15:00:00
Description: Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1. Optisk kalibrering: Måling Light Output <ol><li> For at forberede lyset apparat levering, skal du tilslutte en bredbåndsforbindelse lyskilde (f.eks Xenon eller wolfram lampe) til en passende strømforsyning. Placer en kollimatorlinse foran lyskilden til at producere en kollimeret lysstråle. Før lys gennem en flydende varme filter til at fjerne det meste af varmen fra lysstrålen. Afhængigt af programmet, fokusere kollimeret stråle på indgangen blænde af et flydende lysleder, som giver mere fleksibe...

<ol>
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T., 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+low-level+laser+therapy+in+neurorehabilitation.">Role of low-level laser therapy in neurorehabilitation.</a> PM&amp;R. 2, S292-S305 (2010).</li><li>Fitzgerald, 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=Metallothionein-IIA+promotes+neurite+growth+via+the+megalin+receptor.">Metallothionein-IIA promotes neurite growth via the megalin receptor.</a> Experimental Brain Research. 183, 171-180 (2007).</li><li>Fitzgerald, 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=Near+infrared+light+reduces+oxidative+stress+and+preserves+function+in+CNS+tissue+vulnerable+to+secondary+degeneration+following+partial+transection+of+the+optic+nerve.">Near infrared light reduces oxidative stress and preserves function in CNS tissue vulnerable to secondary degeneration following partial transection of the optic nerve.</a> Journal of neurotrauma. 27, 2107-2119 (2010).</li><li>Ando, T., 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=Low-level+laser+therapy+for+spinal+cord+injury+in+rats:+effects+of+polarization.">Low-level laser therapy for spinal cord injury in rats: effects of polarization.</a> Journal of biomedical. 18, 098002 (2013).</li><li>Lanzafame, R. 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Vi har med succes tilpasset en præcis og kalibreret lys leveringssystem at tilvejebringe en mekanisme til undersøgelse af optimering af R / NIR-LT in vitro. Bølgelængde og intensitet parametre R / NIR-LT er i stand til at blive manipuleret præcist og effektivt ved hjælp af dette system. Vi konstaterede, at lysbehandling af cellerne førte ikke til celledød, selvom ROS ikke blev reduceret ved de bølgelængder og doser først leveres i celletyper testet. De maksimale intensiteter opnås ved den nuværende...

Kræves reaktive oxygenarter (ROS) for en række signaltransduktionsveje og normale reaktioner af cellulær metabolisme, herunder neurobeskyttelse 1. Men når endogene antioxidant mekanisme ikke er i stand til at kontrollere produktionen af ROS kan celler bukke under for oxidativt stress 2,3. Efter skade på CNS, er de tilhørende stigninger i forekomsten af ROS og oxidativt stress menes at spille en væsentlig rolle i udviklingen af skader 4,5. På trods af den omfattende række strategier for formildende oxidativ stress, der er blevet vurderet, er der i øjeblikket ingen fuldstændig effektiv, klinisk relevante antioxidant strategier for formildende ROS produktion og tilhørende oxidativt stress i klinisk brug efter neurotrauma 6. Derfor dæmpningen af oxidativt stress er fortsat et vigtigt mål for terapeutisk intervention 7. Forbedringer følgering R / NIR-LT er blevet rapporteret i en bred vifte af skader og sygdomme, herunder reduktioner i Cardial infarktstørrelse, nyre- og lever komplikationer under diabetes, retinal degeneration, CNS skade og slagtilfælde 8, måske ved at reducere oxidativ stress. Især med hensyn til CNS skade, har prækliniske undersøgelser af effekten af 670 nm lys vist gode effekter i modeller for retinal degeneration 9-11, rygmarvsskade 12, neuronal død 13. Kliniske forsøg er blevet udført for tør aldersrelateret makuladegeneration og er i øjeblikket i gang for slagtilfælde 14 dog resultaterne af disse forsøg ikke synes lovende, måske på grund af en manglende ansætte effektiv behandling parametre 15. Som sådan R / NIR-LT er ikke blevet udbredt som led i almindelig klinisk praksis neurotrauma, trods en nem at administrere, ikke-invasiv og forholdsvis billig behandling. Hindringer for klinisk oversættelse omfatter manglen på en cltidligt forstod virkningsmekanisme og fraværet af en standardiseret effektiv behandling protokol 16,17. Aktuel litteratur om lysterapi afslører et væld af variation i behandling parametre med hensyn til bestråling kilder (LED eller laser), bølgelængde (fx 630, 670, 780, 810, 830, 880, 904nm), total dosis (joule af bestråling / arealenhed), varighed (eksponeringstid), timing (præ- eller post-fornærmelse), behandlingshyppighed og leveringsmåde (puls eller kontinuerlig) 8. Variabiliteten i behandling parametre mellem studier gør sammenligning vanskelig og har bidraget til skepsis over for effekten 16. Derfor er optimering af R / NIR-LT klart påkrævet, med cellekultur systemer, der kan levere den high-throughput screening mekanisme nødvendigt at sammenligne de mange variable. Men der er få kommercielt tilgængelige belysningssystemer, der kan give tilstrækkelig fleksibilitet og kontrol over wavelength og intensitet til at udføre sådanne optimering eksperimenter. Kommercielt tilgængelige LED-enheder er generelt ikke i stand til at levere flere bølgelængder eller intensiteter, hvilket resulterer i efterforskere beskæftiger flere LED enheder fra forskellige producenter, som kan variere ikke kun i intensitet, men også det spektrum af bølgelængden af ​​lys, der udsendes. Vi har behandlet dette spørgsmål ved at anvende en bredbåndsforbindelse Xenon lyskilde filtreres gennem smalbånd interferensfiltre, hvorved der genereres en række bølgelængder og fluenser af lys, så tæt på, nøjagtig styring af parametrene for R / NIR-LT. Det er vigtigt at bemærke, at den terapeutiske dosis af behandlingen er defineret ved antallet af fotoner, der interagerer med photoacceptor (kromofor), som i tilfælde af R / NIR-LT postuleres at være cytochrom c-oxidase (COX) 18. Foton energi leveret varierer med bølgelængden; betyder lige store doser af energi ved forskellige bølgelængder vil være comværdsat af forskellige antal fotoner. Derfor blev det udsendte lys fra indretningen kalibreres til at udsende et tilsvarende antal fotoner for hver af de valgte bølgelængder der skal testes. Vi har udviklet et system, der kan anvendes til at levere R / NIR-LT ved en række bølgelængder og intensiteter til celler in vitro og demonstreret evne til at måle virkningerne af den leverede R / NIR-LT på ROS produktion i celler udsat for glutamat stress.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td>
			<td>Cell Biolabs</td>
			<td>STA-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed Reagent</td>
			<td>Molecular Probes</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>300 Watt Xenon Arc Lamp</td>
			<td>Newport Corporation</td>
			<td>6258</td>
			<td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td>
		</tr>
		<tr>
			<td>USB4000-FL Fluorescence Spectrometer</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CC-3-UV Cosine Corrector for Emission Collection</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;m diameter quartz fibre optic</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Spectroscopy Platform</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Acute toxicity, wear gloves when handling.</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>142-47-2 (anhydrous)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pheochromocytoma rat adrenal medulla (PC12) cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI1640) Media</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Horse Serum, New Zealand origin</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100X MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Retinal Muller (rMC1) cells</td>
			<td>University of California, San Diego</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 Flasks</td>
			<td>BD Biosciences</td>
			<td>B4-BE-353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>165U Papain</td>
			<td>Worthington</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well plates, clear bottom, black</td>
			<td>Corning</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td>
			<td>Costar</td>
			<td>07-200-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Seesaw Rocker</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Density Filter Paper (0.3)</td>
			<td>THORLABS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (1.0)</td>
			<td>THORLABS</td>
			<td>NE10B</td>
			<td></td>
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method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

Rød / nær-infrarødt lys terapi (R / NIR-LT), leveret af laser eller lysdiode (LED), forbedrer funktionelle og morfologiske resultater i en række skader på centralnervesystemet in vivo, eventuelt ved at reducere oxidativ stress. , Effekter af R / NIR-LT på oxidativt stress har vist sig dog at variere bølgelængde eller intensitet bestråling. Undersøgelser, der sammenligner behandling parametre mangler på grund af manglen på kommercielt tilgængelige anordninger, der giver flere bølgelængder eller intensiteter, der er egnede til høj gennemløb in vitro optimering studier. Denne protokol beskriver en teknik til levering af lys ved en række bølgelængder og intensiteter for at optimere terapeutiske doser, der kræves for en given skade model. Vi antager, at en metode til at levere lys, hvor bølgelængde og intensitet parametre let kan ændres, kan lette bestemmelse af en optimal dosis af R / NIR-LT til reduktion reaktive oxygenarter(ROS) in vitro. Ikke-kohærent Xenon lys blev filtreret gennem smalbåndede interferensfiltre at levere varierende bølgelængder (center bølgelængder 440, 550, 670 og 810nm) og fluenser (8,5 x 10 -3 til 3,8 x 10 -1 J / cm2) af lys til dyrkede celler. Lyseffekt fra apparatet blev kalibreret til at udsende terapeutisk relevante, lige kvantale doser af lys ved hver bølgelængde. Reaktive arter blev påvist i glutamat stressede celler behandlet med lys, ved hjælp af DCFH-DA og H 2 O 2 følsomme fluorescerende farvestoffer. Vi succes leveret lys på en afstand af fysiologisk og terapeutisk relevante bølgelængder og intensiteter, til dyrkede celler udsat for glutamat som en model for CNS skade. Mens fluenser af R / NIR-LT anvendes i den aktuelle undersøgelse ikke udøve en virkning på ROS genereret af de dyrkede celler, hvilken fremgangsmåde lys levering kan anvendes til andre SystEMS herunder isolerede mitokondrier eller mere fysiologisk relevante organotypiske skive kultur modeller, og kan bruges til at vurdere effekter på en række resultatmål for oxidativ metabolisme.

Udgangen af lys afgivet ved en bølgelængde på 670 nm blev kalibreret under anvendelse af neutralfiltre for at bestråle cellerne med en række fluenser omfatter en dosis af 670 nm lys tidligere vist sig at være gavnlig in vivo (0,3 J / cm2) 20. Da antallet af neutralfiltre foran lyskilden forøges, intensiteten (W / m 2) faldt, så mindre lys at passere til målområdet. Tabel 1 viser data kalibrering af 670nm lys genereret fra lyskilden er udstyret med en bølgelængde filter og omfat...

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Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Metode til vurdering af effekter af en række bølgelængder og intensiteter af Rød / nær-infrarødt lys terapi på oxidativt stress<em&gt; In vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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Biology

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

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Biochemical Titration of Glycogen In vitro

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Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

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Undersøge de skadelige effekter af lav pres Plasma sterilisation på overlevelse af Bacillus subtilis sporer ved hjælp af Live celle mikroskopi

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

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Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...