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

Dette arbejde blev støttet af en bevilling af den schweiziske National Science Foundation (SNSF, #320030_156019). Forfatterne fik støtte fra Heidelberg Engineering GmBH, Tyskland.

i alle procedurer, BALB/c voksne mandlige og kvindelige mus som udtryk for normal god landbrugspraksis promotor af Cx3cr1 blev brugt 24. Mus blev behandlet ifølge ARVO erklæring om brug af dyr i Ophthalmic og Vision forskning og alle procedurer blev godkendt fra den schweiziske regering Federal schweiziske forordninger om dyrevelfærd. Mus var bedøvede af en subkutan injektion af medetomidine hydrochlorid (0,75 mg/kg) og ketamin (45 mg/kg). Ordentlig anæstesi blev bekræftet ved at ...

<ol>
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Denne artikel viser en protokol for erhvervelse af retinale B-scanninger og billedbehandling af normal god landbrugspraksis positive mikroglia distribution i mus nethinden i samme imaging-session. SD-OCT og SLO bruges i stigende grad i dyremodeller af retinal sygdom for at give oplysninger af retinale forandringer over tid10,14,17,18,21. Med denne protokol, <e...

Undersøgelse af retinale patologi vurderes i eksperimentel oftalmologi, normalt ved hjælp af histologiske teknikker. Men histologi kræver dyre euthanization og kan medføre ændring af de faktiske egenskaber af væv. SD-OCT og SLO anvendes rutinemæssigt i klinisk oftalmologi til diagnostiske formål og til overvågning af flere retinal sygdomme såsom diabetisk makulaødem9, forreste iskæmisk optisk neuropati10eller retinitis pigmentosa11 . SD-OCT og SLO er non-invasiv teknik, der genererer høj opløsning billeder af nethinden, der er visualiseret gennem det forstørrede elev uden yderligere indgriben. SD-okt indeholder oplysninger af retinale struktur og retinal tykkelse ved at indsamle backscattering data for at oprette tværsnit billeder af nethinden, mens SLO indsamler fluorescens data til at producere stereoskopiske billeder med høj kontrast af nethinden. I dag er begge teknikker bruges i stigende grad i eksperimentel oftalmologi ved hjælp af små gnavere12,13,14,15 (eller endda zebrafisk16,17) og kan give både kvalitative og kvantitative oplysninger12,17,18,19,20,21.
Ophobning af endogene fluorophores som lipofuscins eller dannelsen af drusen i nethinden kan visualiseres ved SLO som auto fluorescerende signal. Denne funktion gør SLO en værdifuld teknik til diagnose og overvågning af retinale sygdomme, såsom aldersrelateret makuladegeneration eller retinitis pigmentosa22,23. I eksperimentelle oftalmologi, kan auto fluorescens imaging (AF) anvendes til påvisning af specifikke celletyper i reporter mus linjer. F.eks er mus heterozygous for udtryk for normal god landbrugspraksis promotor af Cx3cr124 fordelagtige for i vivo visualisering af microglial celler i den normale nethinden og for undersøgelse af mikroglia/makrofag Dynamics i retinal sygdom21. Mikroglia er de bosiddende makrofager af nethinden, der spiller en afgørende rolle på væv homøostase og væv reparation efter skade1,25,26. Mikroglia aktivering i nethinden er blevet rapporteret i retinal skade, iskæmi og degeneration, tyder på en rolle for disse celler i retinal sygdom2,3,4,5, 6.
Formålet med denne protokol er at beskrive en relativt enkel metode for nethinde billedbehandling og måling af retinale tykkelse ved hjælp af SD-okt, og visualisering af normal god landbrugspraksis positive mikroglia celler i Cx3cr1normal god landbrugspraksis/NGL mus nethinden bruger SLO (Heidelberg Spectralis HRA + OCT system). Denne protokol kan udnyttes til billedbehandling og tykkelse målinger af sunde eller syge nethinder i forskellige mus linjer. Derudover kan morfometrisk analyser udføres for identifikation og kvantificering af mikroglia numre og mikroglia aktivering i nethinden ved hjælp af SLO21. Mikroglia celler er associeret med degenerative sygdomme i centralnervesystemet (CNS), herunder nethinden27,28,29. Således, ved at kombinere de to metoder, der anvendes i denne protokol, korrelation af mikroglia distribution og retinal degeneration kan gøres, som kan lette overvågning sygdommens sværhedsgrad eller effektiviteten af terapeutiske tilgange i 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

Spektrale domæne optisk kohærens tomografi (SD-OCT) og scanning laser oftalmoskopi (SLO) er flittigt brugt i eksperimentel oftalmologi. I denne protokol, mus udtrykker grøn fluorescerende proteiner (NGL) promotor af Cx3cr1 (BALB/c-Cx3cr1normal god landbrugspraksis/normal god landbrugspraksis) blev brugt til at afbilde mikroglia celler i vivo i nethinden. Mikroglia er bosiddende makrofager af nethinden og har været impliceret i flere retinal sygdomme1,2,3,4,5,6. Denne protokol giver en detaljeret metode til generering af retinale B-scanninger, med SD-OCT og billeddannelse af mikroglia celle distribution i Cx3cr1normal god landbrugspraksis/NGL mus med SLO i vivo, ved hjælp af en oftalmologiske billedbehandlingssystem for platform. Protokollen kan bruges i flere reporter mus linjer. Der er dog nogle begrænsninger i protokollen præsenteres her. Først, både SLO og SD-OLT, når det bruges i tilstanden høj opløsning indsamle data med høj aksial opløsning, men den laterale opløsning er lavere (3,5 µm og 6 µm, henholdsvis). Desuden, fokus og mætning niveau i SLO er stærkt afhængigt af parameteren udvælgelse og korrekt justering af øjet. Derudover bruger udstyr designet til menneskelige patienter i mus er udfordrende på grund af den højere samlede optiske effekt af musen øjet i forhold til det menneskelige øje; Dette kan føre til lateral forstørrelse unøjagtigheder7, som er også afhængige af forstørrelse af musen linsen bl.a. Men på trods af at den aksial scanning holdning er afhængig af laterale forstørrelse, aksial SD-okt målingerne er præcise8.

Ved hjælp af protokollen præsenteres her, SD-okt scanner og SLO billeder blev fremstillet af Cx3cr1normal god landbrugspraksis/NGL mus i samme imaging-session. Figur 3 omfatter repræsentant SD-OCT enkelt scanninger opnås med en 30 ° eller en 55 ° linse (figur 3A) og repræsentative SLO billeder opnås med en 55 ° eller en 102 ° linse, hvor normal god landbrugspraksis positive mikroglia celler er visua...

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

Denne protokol beskriver hvordan høj opløsning Billeddannende teknikker såsom spektrale domæne optisk kohærens tomografi og scanning laser oftalmoskopi kan udnyttes i små gnavere, ved hjælp af en oftalmologiske billedbehandlingssystem for platform, for at indhente oplysninger om retinal tykkelse og microglial celle distribution, henholdsvis.

In Vivo Billeddannelse af Cx3cr1normal god landbrugspraksis/NGL Reporter mus med røde-domæne optisk kohærens tomografi og Scanning Laser oftalmoskopi

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

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Medicine

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

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intrakraniel Implantation med efterfølgende 3D In Vivo Bioluminescent Scanning af murine gliomer

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Doppler Optical Coherence Tomography of Retinal Circulation

Doppler Optical Coherence Tomography af Retinal cirkulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Segmentering og måling af tykke bind i murine Fedme-modeller vha. X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

Generation og 3-Dimensional Kvantificering af Arteriel Læsioner i mus Brug Optisk Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

In Vivo Optical Imaging af hjernetumorer og Gigt Brug Fluorescent SAPC-DOPS Nanovesicles

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

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

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

Ikke-invasiv billeddannelse og analyse af cerebral iskæmi i levende rotter Brug Positron Emission Tomography med<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined In vivo Optical and Ãƒâ€šÃ‚ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Kombineret<em&gt; In vivo</em&gt; Optisk og μCT Imaging Monitor infektion, inflammation og Bone Anatomi i en Ortopædisk Implant Infektion i mus

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

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Vurdering af virksomhedens rentabilitet for Human Fat Injektion i nøgne mus med 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 ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Validering af Nanobody og antistofbaserede<em&gt; In Vivo</em&gt; Tumor xenograft NIRF-imaging Forsøg på mus ved brug<em&gt; Ex vivo</em&gt; Flowcytometri og mikroskopi

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...