Guld nanorod-assisteret Optisk Stimulation af neuronal Cells
Summary
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.
Abstract
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.
Introduction
Nylige undersøgelser har vist, at forbigående opvarmning forbundet med absorptionen af infrarødt lys af vand (bølgelængde> 1.400 nm) kan anvendes til at inducere virkningspotentialer i nervevæv 1 og intracellulære calcium transienter i cardiomyocytter 2. Brugen af infrarødt lys har rejst stor interesse til anvendelser i neurale proteser, på grund af den potentielle finere rumlig opløsning, manglende direkte kontakt med vævet, minimering af stimulering artefakter og fjernelse af behovet for at genetisk modificere cellerne før stimulation ( som krævet i optogenetics) 1. På trods af alle disse fordele, nyligt udviklede termiske modeller foreslået, at target væv / celler kan blive påvirket af kumulative opvarmning effekter, når flere stimulus sites og / eller høj repetitionshastigheder anvendes 3,4.
Som svar på disse udfordringer, har forskere erkendt potentialet til at bruge ydre absormer for nerve stimulation til at producere mere lokal opvarmning effekter i vævet. Huang et al. Demonstrerede dette princip ved hjælp af superparamagnetiske ferrit nanopartikler til at aktivere fjernadgang de temperaturfølsomme TRPV1 kanaler i HEK 293 celler med en radio-frekvens magnetfelt 5. Selv om denne teknik kan give mulighed for dybere penetration (magnetfelter interagerer relativt svagt med væv), blev svarene kun registreret over perioder på sekunder i stedet for de millisekund varigheder, der kræves i bioniske enheder 5. Tilsvarende Farah et al. Påvist elektrisk stimulation af rotte corticale neuroner med sorte mikropartikler in vitro. De viste celle-niveau præcision i stimulation ved hjælp af impulser i størrelsesordenen flere hundrede mikrosekunder og energier i intervallet μJ, potentielt giver mulighed for hurtigere repetitionshastigheder 6.
Anvendelsen af ydre absorbere er også blevet anvendt til at induceremorfologiske ændringer i vitro. Ciofani et al. Viste en stigning i neuronal celleudvækst ~ 40% ved anvendelse af piezoelektriske bornitrid nanorør exciteres ved ultralyd 7. Tilsvarende har endocytoserede jernoxid nanopartikler i PC12-celler blevet rapporteret at forbedre neurit differentiering på en dosis-afhængig måde, som følge af aktivering af celleadhæsionsmolekyler med jernoxid 8.
For nylig har interessen for ydre absorbere at hjælpe neural stimulation også fokuseret på anvendelsen af guld nanopartikler (AU NP'er). Au NP'er har evnen til effektivt at absorbere laserlyset på plasmoniske top og til at sprede den ind i det omgivende miljø i form af varme 9. Blandt alle de tilgængelige partikelformer den optiske absorption af guld nanorods (Au NR'er) bekvemt passer det terapeutiske vindue af biologiske væv (nærinfrarød – NIR, bølgelængde mellem 750-1,400 nm) 10. Desuden i context af neural stimulation, anvendelse af Au NR'er giver relativt gunstig biokompatibilitet og en bred vifte af overfladefunktionalisering muligheder 11. Nylige undersøgelser har vist, at en stimulerende virkning på differentiering kan induceres efter kontinuerlig laser engagementer Au NR'er i NG108-15 neuronale celler 12. Ligeledes blev intracellulære calcium transienter optaget i neuronale celler dyrket med Au NR'er efter laser bestråling moduleret med variable frekvenser og pulslængder 13. Cellemembranen depolarisering blev også registreret efter NIR laser belysning af Au NR'er i primære kulturer af spiral ganglieneuroner 14. Den første in vivo ansøgning med bestrålede Au NR'er er påvist for nylig. EOM og medarbejdere udsat Au NR'er på deres plasmoniske top og indspillede en seks-dobling i amplituden af forbindelse nerve aktionspotentialer (CNAPs) og en tre-fold fald i tærsklen stimulation i rotte ischiadicus nerver. Den entierne svar blev tilskrevet lokale opvarmning virkninger som følge af excitation af NR plasmoniske top 15.
I det foreliggende papir, er protokoller for at undersøge virkningerne af laser stimulation i NG108-15 neuronale celler dyrket med Au NR'er angivet. Disse metoder giver en enkel, men kraftfuld, måde at bestråle cellepopulationer in vitro ved hjælp af standard biologiske teknikker og materialer. Protokollen er baseret på et fiber-koblet laser diode (LD), der tillader sikker drift og gentagelig tilpasning. AU NR forberedelse og laser prøve bestråling metoder kan yderligere udvides til andre partikel former og neuronale cellekulturer, forudsat at de specifikke syntese og kultur protokoller er kendte, hhv.
Protocol
Representative Results
Discussion
De protokoller, der er skitseret i denne præsentation beskriver hvordan kultur, differentiere og optisk stimulere nerveceller ved hjælp ydre absorbere. NR egenskaber (f.eks dimensioner, form, plasmonresonans bølgelængde og overfladekemi) og laser stimuleringsparametre (såsom bølgelængde, pulslængde, impulsfrekvens, etc.) kan varieres for at matche forskellige eksperimentelle behov. Virkningerne på celle adfærd kan overvåges ved anvendelse af standard biologiske assays og materialer. Samlet t…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne anerkende NanoVentures Australien til rejser finansiering support og Prof. John Haycock for at have delvist vært denne forskning ved University of Sheffield og Ms. Jaimee Mayne for hendes hjælp under optagelserne.
Materials
| Au NR | Sigma Aldrich | 716812 | |
| NG108-15 | Sigma Aldrich | 8811230 | |
| DMEM | Sigma Aldrich | D6546 | |
| FCS | Life Technologies | 10100147 | |
| L-glutamine | Sigma Aldrich | G7513 | |
| Penicillin/streptomycin | Life Technologies | 15140122 | |
| Amphotericin B | Life Technologies | 15290018 | |
| Formaldehyde | Sigma Aldrich | F8775 | |
| Triton X-100 | BDH | T8532 | |
| BSA | Sigma Aldrich | A2058 | |
| Anti-βIII-tubulin | Promega | G7121 | |
| TRITC-conjugated anti-mouse IgG antibody | Sigma Aldrich | T5393 | |
| DAPI | Invitrogen | D1306 | |
| Fluo-4 AM | Invitrogen | F14201 | |
| DMSO | Sigma Aldrich | 472301 | |
| Pluronic F-127 | Invitrogen | P6867 | |
| Equipment name | Company | Catalogue Number | |
| UV-Vis spectrometer | Varian Medical Systems Inc. | Cary 50 Bio | |
| Mini centrifuge | Eppendorf | Mini Spin | |
| Sonic bath | Unisonics Australia | FPX 10D | |
| Cell culture incubator | Kendro | Hera Cell 150 | |
| Cell culture centrifuge | Hettich | Rotofix 32A | |
| Laser diode | Optotech | 780 nm single mode fibre – coupled LD | |
| Optical fiber | Thorlabs | 780 HP | |
| Power meter | Coherent | Laser Check | |
| ImageJ | http://rsb.info.nih.gov/ij/index.html | ||
| Epifluorescent microscope | Axon Instruments | ImageX-press 5000A | |
| μ-slide well | Ibidi | 80826 | |
| Inverted confocal microscope | Carl Zeiss Microscopy Ltd. | LSM 510 meta-confocal microscope | |
| Oscilloscope | Tektronix | TDS210 |
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