Name: in vivo two-photon microscopy of single nerve endings in skin
Uploaded: 2014-08-24T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

Forfatterne vil gerne takke Neurotar Ltd for teknisk bistand, CIMO Foundation og FGSN for finansiel støtte.

Procedurer, der involverer dyreindivider er blevet godkendt af den nationale dyreforsøg Board, Finland. 1. Animalske Forberedelse til Imaging <ol><li> Bedøver en mus ved intraperitional (IP) injektion af ketamin (0,08 mg pr legemsvægt) og xylazin (0,01 mg pr legemsvægt). Kontroller anæstesi med den bageste tå knivspids refleks. </li><li> Fordyb dyrets øjne i øjendråber (Viscotears) for at beskytte øjnene mod dehydrering. </li><li> Sæt musen på en varmepude (Supertech) ved 37 ° C for at forhindre h...

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I denne video protokol vi demonstrere fremgangsmåden til ikke-invasiv langsgående to-foton-billeddannelse af enkelt nerveender. Dynamikken i huden innervationer påvirkes ved sådanne sygdomme som psoriasis og perifer neuropati 2, og i traumatiske skader 9. To-foton billedbehandling giver mulighed for en detaljeret analyse af nervefibre strukturer i collagen matrix. Anvendelsen af ​​transgene reporter mus hjælper med at undgå de problemer vedrørende farvning af...

Kutane nerveender gennemgår dynamiske forandringer under forskellige patofysiologiske tilstande. Nervefibre kan gå gennem processen med degeneration og regeneration eller omstrukturering i løbet af sådanne sygdomme som perifer neuropati 2 eller Mortons neuroma 3. Efter traumatisk skade, en vigtig del af nerveender dynamik i huden er reinnervation af det beskadigede område. Men den fælles tilgang til undersøgelse af nerveender er ex vivo histologiske sektionering, der mangler oplysninger i realtid om de igangværende processer 4. Brug genetisk indkodede fluorescerende markører, er det muligt at spore de nerveender i huden på levende dyr, og dermed opnå rig og betydeligt mere relevante oplysninger om de strukturelle ændringer. Undersøgelsen af ​​kutane nerveender er muligt under anvendelse af konventionel fluorescensmikroskopi imidlertid stærk lysspredning af hudvævet kraftigt underminerer kvalitetenaf de data, der er erhvervet 5. Multiphoton mikroskopi tillader erhvervelse af billeder i høj opløsning i stærkt spredende væv på grund af den ikke-lineære opsummering af energi excitationslyset fotoner resulterer i emission af fluorescens kun fra brændpunktet af målet. Denne effekt fører til en solid stigning i indtrængningsdybden og forbedring af signal-støj-forholdet for måling i hudvævet 6. Anvendelse af den samme laser som til billeddannelse, er det muligt at fremstille selektiv dissektion af nervefibre 7. I den følgende protokol viser vi metode langsgående afbildning af kutane nerveender i vivo i reporter transgene mus kombineret med selektiv laser læsion under anvendelse af kommercielt tilgængelige multifoton mikroskopsystem.

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			<td height="21" style="height:21px;width:216px;">Round cover glass&#160;</td>
			<td style="width:209px;">Electron Microscopy Science</td>
			<td style="width:119px;">72296-05</td>
			<td style="width:687px;">5 mm diameter #1.5 thickness</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eye drops Viscotears</td>
			<td style="width:209px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>2mg/g</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superglue Loctite 401</td>
			<td style="width:209px;">Henkel</td>
			<td>135429</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Ketaminol</td>
			<td style="width:209px;">Intervet</td>
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			<td>Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 &#181;g per gramm of animal weight)</td>
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			<td height="21" style="height:21px;width:216px;">Rompun</td>
			<td style="width:209px;">Bayer Healthcare</td>
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			<td>Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 &#181;g per gramm of animal weight)</td>
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			<td style="width:687px;">Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS</td>
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			<td height="21" style="height:21px;width:216px;">Ethanol 70%</td>
			<td style="width:209px;"></td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Distilled water or Milli-Q water</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Syringes 1ml&#160;</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">300013</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">30G 1/2&#34; needles</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">304000</td>
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			<td height="21" style="height:21px;width:216px;">Plastic packaging material</td>
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			<td>Could be purchaized from general hardware store</td>
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			<td height="21" style="height:21px;width:216px;">FV-1000MPE microscope</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">FV-1000MPE</td>
			<td>Microscope with motorized stage and rigid metal bar for fixation</td>
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			<td height="21" style="height:21px;width:216px;">25X XLPlan objective</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">XLPLN 25XWMP</td>
			<td>Water imersion objective optimized for multiphoton imaging</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mai-Tai DeepSee laser (2W)</td>
			<td style="width:209px;">SpectraPhysics</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:209px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td>Heating pad with a temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal ring fixator</td>
			<td style="width:209px;">Neurotar Ltd.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:209px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thy1-YFPH mice strain</td>
			<td style="width:209px;">JaxLab</td>
			<td style="width:119px;">003782</td>
			<td></td>
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in vivo two-photon microscopy of single nerve endings in skin

Nerveender i huden er involveret i fysiologiske processer, såsom sensing 1, samt i patologiske processer såsom neuropatisk smerte 2. Deres tæt på overfladen positionering letter mikroskopisk billeddannelse af hudens nerveender i levende intakt dyr. Brug multifoton mikroskopi, er det muligt at opnå fine billeder overvinde problemet med stærk lysspredning af hudvævet. Reporter transgene mus, der udtrykker EYFP under kontrol af Thy-1-promotoren i neuroner (herunder periferien sensoriske neuroner) er velegnede til de langsgående undersøgelser af individuelle nerveender over længere perioder på op til flere måneder eller endda livslang. Desuden, ved hjælp af den samme femtosecond laser som til billeddannelse, er det muligt at fremstille yderst selektive læsioner af nervefibre for undersøgelser af omstrukturering nervefibrene. Her præsenterer vi en enkel og pålidelig protokol for langsgående multiphoton in vivo billeddannelse oglaserbaseret mikrokirurgi på musen hud nerveender.

Brug af den beskrevne teknik er det muligt at spore den samme fiber, efter læsionen, og at undersøge nedbrydningen af beskadigede nerveender (figur 1). Erhvervelse af stakken med en tykkelse på 120-150 um er sædvanligvis passende for den gentagne billeddannelse i adskillige dage for at holde hele fiber i synsfeltet. Læsionen kan typisk fremstilles præcist når plastemballage materiale justeres til flade huden, så kollagen lag vises med ensartet intensitet på billedet...

Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In Vivo to-foton mikroskopi af Single nerveender i hud

Protokollen til dynamisk langsgående billeddannelse og selektiv laser læsion af nerveender i reporter transgene mus præsenteres.

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Research

JoVE Journal

Neuroscience

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

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In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

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The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

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This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

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Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

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A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

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Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...