Name: in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy
Uploaded: 2017-11-11T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

Dit werk werd gesteund door een subsidie van de Zwitserse National Science Foundation (SNSF; #320030_156019). De auteurs ontvangen nietfinanciële steun in Heidelberg Engineering GmBH, Duitsland.

in alle procedures, waren de BALB/c volwassen mannelijke en vrouwelijke muizen die express gfp onder de promotor van Cx3cr1 gebruikte 24. Muizen werden behandeld volgens de ARVO verklaring voor het gebruik van dieren in Ophthalmic en visie onderzoek en alle procedures goedgekeurd van de Zwitserse regering volgens de federale Zwitserse verordeningen inzake het welzijn van dieren. Muizen zijn verdoofd door een subcutane injectie van medetomidine hydrochloride (0,75 mg/kg) en ketamine (45 ...

<ol>
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Dit artikel toont een protocol voor de verwerving van retinale B-scans en beeldvorming van gfp positieve microglia distributie in het netvlies van de muis in dezelfde imaging-sessie. SD-OCT en SLO worden steeds meer gebruikt in diermodellen van retinale ziekte om informatie te verstrekken van retinale wijzigingen over tijd10,14,17,18,21. Met dit protocol, ...

In experimentele oogheelkunde, wordt behandeling van retinale pathologie meestal geëvalueerd met histologische technieken. Echter, histologie dierlijke euthanization vereist en kan leiden tot wijziging van de werkelijke eigenschappen van het weefsel. SD-OCT en SLO worden routinematig gebruikt in klinische oogheelkunde voor diagnostische doeleinden en voor het toezicht van verschillende retinale ziekten zoals diabetische macula oedeem9, anterieure ischemische optische neuropathie10of retinitis pigmentosa11 . SD-OCT en SLO zijn niet-invasieve technieken die het genereren van hoge resolutie beelden van het netvlies, die worden gevisualiseerd door de verwijde pupil zonder verdere tussenkomst. SD-OCT biedt informatie van het netvlies structuur en de dikte van het netvlies door backscattering gegevens verzamelt om te maken van transversale beelden van het netvlies, terwijl SLO verzamelt fluorescentie gegevens om te produceren van stereoscopische hoge-contrast beelden van het netvlies. Tegenwoordig, beide technieken worden steeds vaker gebruikt in experimentele oogheelkunde met kleine knaagdieren12,13,14,15 (of zelfs zebrafish16,17) en kan bieden beide kwalitatieve en kwantitatieve informatie12,17,18,19,20,21.
Accumulatie van endogene fluorophores zoals lipofuscins of de vorming van drusen in het netvlies kan worden gevisualiseerd door SLO als auto fluorescent signaal. Deze functie maakt SLO een waardevolle techniek voor diagnose en bewaking van retinale ziekten zoals leeftijdsgebonden maculaire degeneratie of retinitis pigmentosa22,23. In experimentele oogheelkunde, kan auto fluorescentie imaging (AF) worden gebruikt voor het opsporen van specifieke celtypes in verslaggever muis lijnen. Bijvoorbeeld zijn muizen heterozygoot voor de uitdrukking van gfp onder de promotor van Cx3cr124 voordelig voor in vivo visualisatie van microglial cellen in de normale retina en voor het onderzoek van microglia/macrofaag dynamiek in netvlies ziekte21. Microglia zijn de ingezetene macrofagen van het netvlies, die een cruciale rol spelen op de weefsel homeostase en weefselherstel na verwonding1,25,26. Microglia activering in het netvlies is gemeld in netvlies letsel, ischemie en degeneratie, suggereren een rol van deze cellen in het netvlies ziekte2,3,4,5, 6.
Het doel van dit protocol is voor het beschrijven van een relatief eenvoudige methode voor retinale beeldvorming en meting van retinale dikte met SD-OCT en visualisatie van gfp positieve microglia cellen in het Cx3cr1gfp/gfp muis netvlies met SLO (Heidelberg Spectralis HRA + OCT systeem). Dit protocol kan worden gebruikt voor beeldvorming en dikte metingen van gezonde of zieke netvlies in verschillende lijnen van de muis. Daarnaast kunnen Morfometrische analyses worden uitgevoerd voor de identificatie en kwantificering van microglia nummers en microglia activering in de retina met behulp van SLO21. Microglia cellen worden geassocieerd met degeneratieve ziekten in het centrale zenuwstelsel (CNS), met inbegrip van het netvlies27,28,29. Dus, door het combineren van de twee methoden die worden gebruikt in het huidige protocol, correlatie van microglia distributie en Retina degeneratie kan worden gemaakt, die kan vergemakkelijken toezicht ernst van de ziekte of de doeltreffendheid van therapeutische benaderingen in vivo.

<table>
	<tbody>
		<tr>
			<td>Spectralis Imaging system (HRA+OCT)</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>ophthalmic imaging platform system</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>Version 1.9.13.0</td>
		</tr>
		<tr>
			<td>78D standard ophthalmic non-contact slit lamp lens</td>
			<td>Volk Optical Inc., Ohio, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis wide angle 55&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50117-001</td>
			<td></td>
		</tr>
		<tr>
			<td>medetomidine hydrochloride 1 mg/mL (Domitor)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;590 - ATCvet: QN05CM91</td>
			<td>anesthetic/analgesic</td>
		</tr>
		<tr>
			<td>ketamine 50mg/ml (Ketalar)</td>
			<td>Parke-Davis, Zurich, Switzerland</td>
			<td>72276388</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td>
			<td>ISPI, Bern, Switzerland</td>
			<td>N/A</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td>
			<td>B. Braun Mesungen AG, Carl-Braun-Stra&#223;e, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropylmethylcellulose (Methocel 2%)</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>+4 dpt rigid gas permeable contact lens</td>
			<td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td>
			<td>N/A</td>
			<td>Base Curve: 7.20 to 8.40 mm 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			Power: -25.00 to +25.00 Diopters</td>
		</tr>
		<tr>
			<td>balanced salt solution (BSS)</td>
			<td>Inselspital, Bern, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole 5 mg/mL (Antisedan)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>N/A</td>
			<td>&#945;2 adrenergic receptor antagonist</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software, Inc, San Diego, CA, USA</td>
			<td>N/A</td>
			<td>statistical analysis software</td>
		</tr>
	</tbody>
</table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

Spectrale domein optische coherentie tomografie (SD-OCT) en scannen laser ophthalmoscopy (SLO) worden uitgebreid gebruikt in experimentele oogheelkunde. In het huidige protocol, muizen uiten groen fluorescente proteïne (gfp) onder de promotor van Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) werden gebruikt om het imago microglia cellen in vivo in het netvlies. Microglia zijn verblijfplaats macrofagen van het netvlies en zijn betrokken bij verschillende retinale ziekten1,2,3,4,5,6. Dit protocol biedt een gedetailleerde aanpak voor generatie van retinale B-scans, met SD-OCT en beeldvorming van microglia cel distributie in Cx3cr1gfp/gfp muizen met SLO in vivo, met behulp van een oogheelkundige imaging platform systeem. Het protocol kan worden gebruikt in verscheidene verslaggever muis lijnen. Er zijn echter enkele beperkingen bij het protocol hier gepresenteerd. Ten eerste, SLO én SD-OCT, wanneer gebruikt in de modus hoge resolutie verzamelen van gegevens met hoge axiale resolutie maar de laterale resolutie is lager (3,5 µm en 6 µm, respectievelijk). De focus en intensiteit niveau in SLO is bovendien sterk afhankelijk van de parameter selectie en de juiste uitlijning van het oog. Bovendien, met behulp van de inrichtingen die zijn ontworpen voor menselijke patiënten in muizen is uitdagend als gevolg van de hogere totale optische macht van het oog van de muis ten opzichte van het menselijk oog; Dit kan leiden tot het lateraal vergroting onnauwkeurigheden7, die zijn ook afhankelijk van de vergroting door de lens van de muis onder anderen. Echter, ondanks dat de axiale scan standpunt is afhankelijk van de laterale vergroting, de axiale SD-OCT-metingen zijn nauwkeurige8.

Met behulp van het protocol hier gepresenteerd, SD-OCT scant en SLO beelden werden verkregen uit Cx3cr1gfp/gfp muizen in dezelfde imaging-sessie. Figuur 3 bevat vertegenwoordiger SD-OCT één scans verkregen met een 30 ° of een 55 ° lens (Figuur 3 bis) en representatieve SLO beelden met een 55 ° of een 102 ° lens verkregen, waar gfp positieve microglia cellen worden gevisualiseerd. Hogere reflectiviteit v...

Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Dit protocol beschrijft hoe hoge resolutie beeldvormende technieken zoals spectrale domein optische coherentie tomografie en scannen van laser ophthalmoscopy kan worden gebruikt in kleine knaagdieren, met behulp van een oogheelkundige imaging platform systeem, om informatie te verkrijgen over netvlies dikte en microglial cel distributie, respectievelijk.

In Vivo Beeldvorming van Cx3cr1gfp/gfp Reporter muizen met spectrale domein optische coherentie tomografie en scannen Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

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In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Orale Biofilm Analyse van de Palatal Expanders door middel van fluorescentie in-situ hybridisatie en confocale laser scanning microscopie

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We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

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Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

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Gecombineerd<em&gt; In vivo</em&gt; Optisch en μCT Imaging aan Infectie, Ontsteking, en Bone Anatomie Monitor in een orthopedisch implantaat Infectie in Muizen

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

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Beoordeling van de levensvatbaarheid van menselijk vet Injectie in Naakt Muizen met Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

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Validatie van Nanobody en antilichamen gebaseerde<em&gt; In Vivo</em&gt; Tumor Xenograft NIRF-imaging Experimenten op muizen, waarbij<em&gt; Ex Vivo</em&gt; Flowcytometrie en Microscopie

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