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

Detta arbete stöds av ett bidrag från schweiziska National Science Foundation (SNSF; #320030_156019). Författarna fick icke-finansiella stöd från Heidelberg Engineering GmBH, Tyskland.

BALB/c vuxna manliga och kvinnliga möss som uttrycker gfp enligt arrangören av Cx3cr1 var i alla förfaranden, används 24. Möss behandlades enligt ARVO uttalande om användning av djur i oftalmologiska och Vision forskning och alla förfaranden godkändes från den schweiziska regeringen enligt den federala schweiziska förordningar för djurs välbefinnande. Möss var bedövas genom en subkutan injektion med medetomidin hydroklorid (0,75 mg/kg) och ketamin (45 mg/kg). Ordentlig ane...

<ol>
	<li>Madeira, M. H., Boia, R., Santos, P. F., Ambrosio, A. F., Santiago, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+microglia-mediated+neuroinflammation+to+retinal+degenerative+diseases.">Contribution of microglia-mediated neuroinflammation to retinal degenerative diseases.</a> Mediators Inflamm. , 673090 (2015).</li><li>Ng, T. F., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+migration+of+retinal+microglia+into+the+subretinal+space.">Light-induced migration of retinal microglia into the subretinal space.</a> Invest Ophthalmol Vis Sci. 42 (13), 3301-3310 (2001).</li><li>Langmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+activation+in+retinal+degeneration.">Microglia activation in retinal degeneration.</a> J Leukoc Biol. 81 (6), 1345-1351 (2007).</li><li>Joly, S., 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=Cooperative+phagocytes:+resident+microglia+and+bone+marrow+immigrants+remove+dead+photoreceptors+in+retinal+lesions.">Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.</a> Am J Pathol. 174 (6), 2310-2323 (2009).</li><li>Arroba, A. I., Alvarez-Lindo, N., van Rooijen, N., de la Rosa, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia-mediated+IGF-I+neuroprotection+in+the+rd10+mouse+model+of+retinitis+pigmentosa.">Microglia-mediated IGF-I neuroprotection in the rd10 mouse model of retinitis pigmentosa.</a> Invest Ophthalmol Vis Sci. 52 (12), 9124-9130 (2011).</li><li>Zhang, C., Lam, T. T., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+populations+of+microglia/macrophages+in+the+retina+and+their+activation+after+retinal+ischemia+and+reperfusion+injury.">Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury.</a> Exp Eye Res. 81 (6), 700-709 (2005).</li><li>Geng, Y., 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=Optical+properties+of+the+mouse+eye.">Optical properties of the mouse eye.</a> Biomed Opt Express. 2 (4), 717-738 (2011).</li><li>Lozano, D. C., Twa, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rat+schematic+eye+from+in+vivo+biometry+and+the+correction+of+lateral+magnification+in+SD-OCT+imaging.">Development of a rat schematic eye from in vivo biometry and the correction of lateral magnification in SD-OCT imaging.</a> Invest Ophthalmol Vis Sci. 54 (9), 6446-6455 (2013).</li><li>Vaz-Pereira, S., 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=Optical+Coherence+Tomography+Features+Of+Active+And+Inactive+Retinal+Neovascularization+In+Proliferative+Diabetic+Retinopathy.">Optical Coherence Tomography Features Of Active And Inactive Retinal Neovascularization In Proliferative Diabetic Retinopathy.</a> Retina. 36 (6), 1132-1142 (2016).</li><li>Kokona, D., Haner, N. U., Ebneter, A., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+macrophage+dynamics+with+optical+coherence+tomography+in+anterior+ischemic+optic+neuropathy.">Imaging of macrophage dynamics with optical coherence tomography in anterior ischemic optic neuropathy.</a> Exp Eye Res. , (2016).</li><li>Makiyama, Y., 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=Macular+cone+abnormalities+in+retinitis+pigmentosa+with+preserved+central+vision+using+adaptive+optics+scanning+laser+ophthalmoscopy.">Macular cone abnormalities in retinitis pigmentosa with preserved central vision using adaptive optics scanning laser ophthalmoscopy.</a> PLoS One. 8 (11), e79447 (2013).</li><li>Paques, 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=High+resolution+fundus+imaging+by+confocal+scanning+laser+ophthalmoscopy+in+the+mouse.">High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse.</a> Vision Res. 46 (8-9), 1336-1345 (2006).</li><li>Joshi, R., 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=Spontaneously+occurring+fundus+findings+observed+using+confocal+scanning+laser+ophthalmoscopy+in+wild+type+Sprague+Dawley+rats.">Spontaneously occurring fundus findings observed using confocal scanning laser ophthalmoscopy in wild type Sprague Dawley rats.</a> Regul Toxicol Pharmacol. 77, 160-166 (2016).</li><li>Muraoka, Y., 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=Real-time+imaging+of+rabbit+retina+with+retinal+degeneration+by+using+spectral-domain+optical+coherence+tomography.">Real-time imaging of rabbit retina with retinal degeneration by using spectral-domain optical coherence tomography.</a> PLoS One. 7 (4), e36135 (2012).</li><li>Fischer, M. D., 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=Noninvasive,+in+vivo+assessment+of+mouse+retinal+structure+using+optical+coherence+tomography.">Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography.</a> PLoS One. 4 (10), e7507 (2009).</li><li>Bell, B. A., 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=Retinal+vasculature+of+adult+zebrafish:+in+vivo+imaging+using+confocal+scanning+laser+ophthalmoscopy.">Retinal vasculature of adult zebrafish: in vivo imaging using confocal scanning laser ophthalmoscopy.</a> Exp Eye Res. 129, 107-118 (2014).</li><li>Bailey, T. J., Davis, D. H., Vance, J. E., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral-domain+optical+coherence+tomography+as+a+noninvasive+method+to+assess+damaged+and+regenerating+adult+zebrafish+retinas.">Spectral-domain optical coherence tomography as a noninvasive method to assess damaged and regenerating adult zebrafish retinas.</a> Invest Ophthalmol Vis Sci. 53 (6), 3126-3138 (2012).</li><li>Huber, G., 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=Spectral+domain+optical+coherence+tomography+in+mouse+models+of+retinal+degeneration.">Spectral domain optical coherence tomography in mouse models of retinal degeneration.</a> Invest Ophthalmol Vis Sci. 50 (12), 5888-5895 (2009).</li><li>Dysli, C., Enzmann, V., Sznitman, R., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+Mouse+Retinal+Layers+Using+Automated+Segmentation+of+Spectral+Domain+Optical+Coherence+Tomography+Images.">Quantitative Analysis of Mouse Retinal Layers Using Automated Segmentation of Spectral Domain Optical Coherence Tomography Images.</a> Transl Vis Sci Technol. 4 (4), 9 (2015).</li><li>Sim, D. A., 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=A+simple+method+for+in+vivo+labelling+of+infiltrating+leukocytes+in+the+mouse+retina+using+indocyanine+green+dye.">A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye.</a> Dis Model Mech. 8 (11), 1479-1487 (2015).</li><li>Bosco, A., Romero, C. O., Ambati, B. K., Vetter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+of+retinal+microglial+activation+during+neurodegeneration:+confocal+ophthalmoscopic+imaging+and+cell+morphometry+in+mouse+glaucoma.">In vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma.</a> J Vis Exp. (99), e52731 (2015).</li><li>Acton, J. H., Cubbidge, R. P., King, H., Galsworthy, P., Gibson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+detection+in+retro-mode+imaging+by+a+scanning+laser+ophthalmoscope.">Drusen detection in retro-mode imaging by a scanning laser ophthalmoscope.</a> Acta Ophthalmol. 89 (5), e404-e411 (2011).</li><li>Greenstein, V. C., 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=Structural+and+functional+changes+associated+with+normal+and+abnormal+fundus+autofluorescence+in+patients+with+retinitis+pigmentosa.">Structural and functional changes associated with normal and abnormal fundus autofluorescence in patients with retinitis pigmentosa.</a> Retina. 32 (2), 349-357 (2012).</li><li>Jung, S., 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Wang, X., 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=Requirement+for+Microglia+for+the+Maintenance+of+Synaptic+Function+and+Integrity+in+the+Mature+Retina.">Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina.</a> J Neurosci. 36 (9), 2827-2842 (2016).</li><li>Ebneter, A., Casson, R. J., Wood, J. P., Chidlow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+the+visual+pathway+in+experimental+glaucoma:+spatiotemporal+characterization+and+correlation+with+axonal+injury.">Microglial activation in the visual pathway in experimental glaucoma: spatiotemporal characterization and correlation with axonal injury.</a> Invest Ophthalmol Vis Sci. 51 (12), 6448-6460 (2010).</li><li>Ebneter, A., Kokona, D., Schneider, N., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Activation+and+Recruitment+of+Circulating+Macrophages+During+Ischemic+Experimental+Branch+Retinal+Vein+Occlusion.">Microglia Activation and Recruitment of Circulating Macrophages During Ischemic Experimental Branch Retinal Vein Occlusion.</a> Invest Ophthalmol Vis Sci. 58 (2), 944-953 (2017).</li><li>Lin, Y. L., Potter-Baker, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+theoretical+models+from+adult+stroke+recovery+to+improve+use+of+non-invasive+brain+stimulation+for+children+with+congenital+hemiparesis.">Using theoretical models from adult stroke recovery to improve use of non-invasive brain stimulation for children with congenital hemiparesis.</a> J Neurophysiol. , (2017).</li><li>Combadiere, C., 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=CX3CR1-dependent+subretinal+microglia+cell+accumulation+is+associated+with+cardinal+features+of+age-related+macular+degeneration.">CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration.</a> J Clin Invest. 117 (10), 2920-2928 (2007).</li><li>Bermudez, M. A., 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=Time+course+of+cold+cataract+development+in+anesthetized+mice.">Time course of cold cataract development in anesthetized mice.</a> Curr Eye Res. 36 (3), 278-284 (2011).</li><li>Toth, C. A., 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=A+comparison+of+retinal+morphology+viewed+by+optical+coherence+tomography+and+by+light+microscopy.">A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy.</a> Arch Ophthalmol. 115 (11), 1425-1428 (1997).</li><li>Ebneter, A., Kokona, D., Jovanovic, J., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+Effect+of+Oral+CSF-1R+Kinase+Inhibitor+on+Retinal+Microglia+Revealed+by+In+Vivo+Scanning+Laser+Ophthalmoscopy.">Dramatic Effect of Oral CSF-1R Kinase Inhibitor on Retinal Microglia Revealed by In Vivo Scanning Laser Ophthalmoscopy.</a> Transl Vis Sci Technol. 6 (2), 10 (2017).</li><li>Gabriele, M. L., 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=Reproducibility+of+spectral-domain+optical+coherence+tomography+total+retinal+thickness+measurements+in+mice.">Reproducibility of spectral-domain optical coherence tomography total retinal thickness measurements in mice.</a> Invest Ophthalmol Vis Sci. 51 (12), 6519-6523 (2010).</li><li>Nakao, S., 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=Wide-field+laser+ophthalmoscopy+for+mice:+a+novel+evaluation+system+for+retinal/choroidal+angiogenesis+in+mice.">Wide-field laser ophthalmoscopy for mice: a novel evaluation system for retinal/choroidal angiogenesis in mice.</a> Invest Ophthalmol Vis Sci. 54 (8), 5288-5293 (2013).</li><li>Wang, N. K., 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=Origin+of+fundus+hyperautofluorescent+spots+and+their+role+in+retinal+degeneration+in+a+mouse+model+of+Goldmann-Favre+syndrome.">Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome.</a> Dis Model Mech. 6 (5), 1113-1122 (2013).</li><li>Wang, N. K., 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=Cellular+origin+of+fundus+autofluorescence+in+patients+and+mice+with+a+defective+NR2E3+gene.">Cellular origin of fundus autofluorescence in patients and mice with a defective NR2E3 gene.</a> Br J Ophthalmol. 93 (9), 1234-1240 (2009).</li><li>Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sick+photoreceptors+attract+activated+microglia+from+the+ganglion+cell+layer:+a+model+to+study+the+inflammatory+cascades+in+rats+with+inherited+retinal+dystrophy.">Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy.</a> Brain Res. 588 (1), 21-28 (1992).</li><li>Hughes, E. H., 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=Generation+of+activated+sialoadhesin-positive+microglia+during+retinal+degeneration.">Generation of activated sialoadhesin-positive microglia during retinal degeneration.</a> Invest Ophthalmol Vis Sci. 44 (5), 2229-2234 (2003).</li></ol>

Denna artikel visar ett protokoll för förvärvet av retinal B-skanningar och avbildning av gfp positiva mikroglia distribution i mus näthinnan i den samma bildsession. SD-okt och SLO används alltmer i djurmodeller av retinal sjukdom ge information av retinala förändringar över tid10,14,17,18,21. Med detta protokoll, Cx3cr1gfp/gfp el...

I experimentell oftalmologi utvärderas undersökning av retinal patologi vanligtvis med histologiska tekniker. Dock histologi kräver djur dödshjälp och kan orsaka förändring till faktiska egenskaper i vävnaden. SD-okt och SLO används rutinmässigt i kliniska oftalmologi för diagnostiska ändamål och för övervakning av flera retinala sjukdomar såsom Diabetiskt makulaödem9, främre ischemisk optikusneuropati10eller retinitis pigmentosa11 . SD-okt och SLO är icke-invasiv teknik som genererar högupplösta bilder av näthinnan, som visualiseras genom dilaterade pupillen utan inblandning. SD-okt ger information av näthinnans struktur och retinal tjocklek genom att samla backscattering data för att skapa tvärsnittsdata bilder av näthinnan, medan SLO samlar fluorescens data för att producera stereoskopisk bilder med hög kontrast av näthinnan. Numera både tekniker används alltmer i experimentell oftalmologi med smågnagare12,13,14,15 (eller ens zebrafiskar16,17) och kan ge både kvalitativ och kvantitativ information12,17,18,19,20,21.
Ackumulation av endogena fluorophores som lipofuscins eller bildandet av drusen i näthinnan kan visualiseras genom SLO som auto fluorescerande signal. Denna funktion gör SLO en värdefull teknik för diagnos och övervakning av retinala sjukdomar såsom åldersrelaterad makuladegeneration eller retinitis pigmentosa22,23. I experimentell oftalmologi användas auto fluorescens imaging (AF) för påvisande av specifika celltyper i reporter mus linjer. Till exempel är möss som är heterozygota för uttrycket av gfp enligt arrangören Cx3cr124 fördelaktiga för i vivo visualisering av mikrogliaceller i normala näthinnan och för utredning av mikroglia/makrofag Dynamics i retinal sjukdom21. Mikroglia är bosatta makrofager av näthinnan, som spelar en avgörande roll på vävnad homeostas och vävnad reparera vid skador1,25,26. Mikroglia aktivering i näthinnan har rapporterats i skada, ischemi och degeneration, vilket tyder på en roll av dessa celler i näthinnesjukdom2,3,4,5, 6.
Syftet med detta protokoll är att beskriva en relativt enkel metod för retinal imaging och mätning av retinal tjocklek med hjälp av SD-okt och visualisering av gfp positiva mikroglia celler i Cx3cr1gfp/gfp mus näthinnan med SLO (Heidelberg Spectralis HRA + OCT system). Detta protokoll kan utnyttjas för imaging och tjocklek mätningar av friska eller sjuka näthinnor i olika mus linjer. Dessutom kan morfometriska analyser utföras för identifiering och kvantifiering av mikroglia nummer och mikroglia aktivering i näthinnan med SLO21. Mikroglia celler är associerade med degenerativa sjukdomar i det centrala nervsystemet (CNS), inklusive näthinnan27,28,29. Således, genom att kombinera de två metoder som används i detta protokoll, korrelation av mikroglia distribution och retinal degeneration kan göras, vilket kan underlätta övervakning sjukdomens svårighetsgrad eller effektiviteten av terapeutiska metoder 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

Spektrala domänen optisk koherenstomografi (SD-okt) och scanning laser Oftalmoskopi (SLO) flitigt i experimentell oftalmologi. I detta protokoll, möss att uttrycka grönt fluorescerande protein (gfp) enligt arrangören av Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) användes till bild mikroglia celler i vivo i näthinnan. Mikroglia är bosatta makrofager av näthinnan och har varit inblandade i flera retinala sjukdomar1,2,3,4,5,6. Detta protokoll ger en detaljerad strategi för generering av retinal B-skanningar, med SD-okt och imaging mikroglia cell spridning i Cx3cr1gfp/gfp möss med SLO i vivo, använder ett system med oftalmologiska imaging i plattformen. Protokollet kan användas i flera reporter mus linjer. Det finns dock vissa begränsningar i protokollet som presenteras här. Först, både SLO och SD-okt, när den används i högupplöst läge, samla in data med hög axiell upplösning men lateral upplösning är lägre (3,5 µm och 6 µm, respektive). Fokus och mättnad nivån i SLO är dessutom starkt beroende av parametern urval och korrigera uppriktningen av ögat. Dessutom använder enheter utformade för mänskliga patienter hos möss är utmanande på grund av den högsta totala optiska effekten på möss jämfört med det mänskliga ögat; Detta kan leda till laterala förstoring felaktigheter7, som också är beroende av förstoringen med muslinsen det bland annat. Men trots att den axiella scan ställning är beroende av laterala förstoring, axial SD-okt mätningarna är korrekt8.

Med hjälp av protokollet som presenteras här, SD-okt File och SLO bilder erhölls från Cx3cr1gfp/gfp möss i den samma bildsession. Figur 3 innehåller representant SD-okt enda skanningar erhålls med en 30 ° eller en 55 ° lins (figur 3A) och representativa SLO bilder erhålls med en 55 ° eller en 102 ° lins, där god jordbrukarsed positiva mikroglia celler visualiseras. Högre reflektivitet i åderhin...

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

Det här protokollet beskriver hur högupplösta avbildningstekniker såsom spektrala domänen optisk koherenstomografi och scanning laser Oftalmoskopi kan utnyttjas i små gnagare, använder ett oftalmologiska imaging platform system, för att få information om retinal tjocklek och mikrogliala cell distribution, respektive.

In Vivo Avbildning av Cx3cr1gfp/gfp Reporter möss med Spectral-domän optisk koherenstomografi och 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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In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

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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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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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