This work was supported by Inserm, Universit&#233; Montpellier, Retina France, Union National des Aveugles et D&#233;ficients Visuels (UNADEV), Association Syndrome de Wolfram, Fondation pour la Recherche M&#233;dicale, Fondation de France, and the Laboratory of Excellence EpiGenMed program.

The experimental protocol was approved by the Institut national de la sant&#233; et de la recherche m&#233;dicale (Inserm; Montpellier, France), is consistent with the European directives, and complies with the ARVO Statement for the Use of Animals in Ophthalmic Research. It was carried out under the agreement of the Languedoc Roussillon Comity of Ethics in Animal Experimentation (CEEALR; nuCEEA-LR-12123).
1. Equipment Setup and Pre-imaging Preparation
NOTE: Here, OCT...

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	<li>Drexler, W., Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-of-the-art+retinal+optical+coherence+tomography.">State-of-the-art retinal optical coherence tomography.</a> Prog Retin Eye Res. 27 (1), 45-88 (2008).</li><li>Grenier, J., 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=WFS1+in+Optic+Neuropathies:+Mutation+Findings+in+Nonsyndromic+Optic+Atrophy+and+Assessment+of+Clinical+Severity.">WFS1 in Optic Neuropathies: Mutation Findings in Nonsyndromic Optic Atrophy and Assessment of Clinical Severity.</a> Ophthalmology. 123 (9), 1989-1998 (2016).</li><li>Zmyslowska, 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+thinning+as+a+marker+of+disease+progression+in+patients+with+Wolfram+syndrome.">Retinal thinning as a marker of disease progression in patients with Wolfram syndrome.</a> Diabetes Care. 38 (3), e36-e37 (2015).</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>Grieve, K., Thouvenin, O., Sengupta, A., Borderie, V. M., Paques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+the+Retina+With+Full-Field+Optical+Coherence+Tomography.">Appearance of the Retina With Full-Field Optical Coherence Tomography.</a> Invest Ophthalmol Vis Sci. 57 (9), OCT96-OCT104 (2016).</li><li>Chang, B., 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+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42 (4), 517-525 (2002).</li><li>Mustafa, S., Pandit, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approach+to+diagnosis+and+management+of+optic+neuropathy.">Approach to diagnosis and management of optic neuropathy.</a> Neurol India. 62 (6), 599-605 (2014).</li><li>Sarzi, E., 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=The+human+OPA1delTTAG+mutation+induces+premature+age-related+systemic+neurodegeneration+in+mouse.">The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.</a> Brain. 135 (Pt 12), 3599-3613 (2012).</li><li>Sarzi, E., 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=Increased+steroidogenesis+promotes+early-onset+and+severe+vision+loss+in+females+with+OPA1+dominant+optic+atrophy.">Increased steroidogenesis promotes early-onset and severe vision loss in females with OPA1 dominant optic atrophy.</a> Hum Mol Genet. 25 (12), 2539-2551 (2016).</li><li>Delettre-Cribaillet, C., Hamel, C. P., Lenaers, G., Pagon, R. 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=Optic+Atrophy+Type+1.">Optic Atrophy Type 1.</a> Gene Reviews. 7 (2007), (2007).</li><li>Lenaers, 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=Dominant+optic+atrophy.">Dominant optic atrophy.</a> Orphanet J Rare Dis. 7 (46), (2012).</li><li>Delettre, 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=Nuclear+gene+OPA1,+encoding+a+mitochondrial+dynamin-related+protein,+is+mutated+in+dominant+optic+atrophy.">Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy.</a> Nat Genet. 26 (2), 207-210 (2000).</li><li>Liu, 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=Monitoring+retinal+morphologic+and+functional+changes+in+mice+following+optic+nerve+crush.">Monitoring retinal morphologic and functional changes in mice following optic nerve crush.</a> Invest Ophthalmol Vis Sci. 55 (6), 3766-3774 (2014).</li></ol>

The OCT system, a non-invasive in vivo imaging method, provides high-resolution retinal cross-section-like scans. Thus, its main advantage is its potential for detailed analysis, with the wonderful opportunity to transpose protocols routinely applied to humans to mouse models.
In the example of Opa1delTTAG mutant mice, SD-OCT results showed an increase of RNFL and GC complex layer thickness, which allowed for further exploration of DOA pathophysiology<sup class="xr...

OCT is a diagnostic tool that facilitates the examination of retinal structures1, including the optic nerve head (ONH). Over the years it has become a dependable indicator of disease progression in humans2,3, as well as in rodents4,5. It uses interferometry to create cross-sectional images of retinal layers at a 2-&#181;m axial resolution. The innermost layer is the retinal nerve fiber layer (RNFL), containing RGC axons, which is followed by the ganglion cell layer (GCL), containing mostly RGC bodies. Next is the inner plexiform layer (IPL), where RGC dendrites meet bipolar, horizontal, and amacrine cell axons. These, together with horizontal cells, form the inner nuclear layer (INL), and their protrusions connect with photoreceptor axons in the outer plexiform layer (OPL). This is followed by the outer nuclear layer (ONL), with photoreceptor cell bodies, and is separated from the photoreceptor layer by the outer limiting membrane (OLM), also called the inner segment/outer segment (IS/OS) layer. Finally, the last observable layers in the mouse retina are the retinal pigment epithelium (RPE) and the choroid (C). The RNFL alone is normally too thin to be measured in mice; thus, analyzing the RNFL/GCL instead is preferable4,5. Another possibility is the GC complex layer, which contains the latter in addition to the IPL, making it thicker and thus even easier to measure on OCT scans4. Consequently, OCT can provide insight into the pathological status of the retina, such as in OPNs. 
Alternatively, the thickness of the mouse retina is often analyzed with post mortem histology. However, this technique faces limitations relating to tissue collection, fixation, cutting, staining, mounting, etc. Hence, some defects, such as subtle thickness changes, cannot be detected. Finally, because the same mouse cannot be tested at several time points, the number of animals per study greatly increases, unlike for OCT. All in all, the non-invasiveness, high-resolution, possibility for repetition, time monitoring in time, and ease of use of the OCT technology make it the method of choice in retinal disease studies.
Mouse models are used to identify gene defects and to elucidate molecular mechanisms underlying retinopathies6. OPN is a form of retinopathy with substantial damage to the optic nerve (ON), which is made up of approximately 1.2 million RGC axons. OPN can be focused on the ON or can be secondary to other disorders, inborn or not7, leading to visual field loss and later, blindness. Characteristic traits of OPN are RGC loss and ON damage, which can be observed in human OCT as RNFL and GCL thinning2,3. Meanwhile, the pathophysiology of OPN is still poorly understood, and hence the need to test mouse retinas remains. 
This manuscript describes the imaging and quantification of retinal layer thickness, using the example of the Opa1delTTAG mouse line8,9, a model of dominant optic atrophy (DOA)10. To assess RGC pathophysiology, radial, rectangular, and annular scans were quantified. This was done either with standard calipers provided by the OCT software or with a homemade macro developed for an open-source image processing program. The standard calipers are difficult to manipulate and often thicker than the RNFL/GCL, while the homemade calipers are easy to use, reproducible, and more precise. The macro performs a measurement for an automatically detected layer, in 5 points and at fixed positions, on both sides of the ONH in the peripapillary region. The goal of the presented protocol is to describe OCT scan acquisition to specify retinal positioning, with a focus on RGCs.

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			<td style="width:909px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Opa1delTTAG mouse</td>
			<td style="width:324px;">Institute for Neurosciences in Montpellier, INSERM UMR 1051, France</td>
			<td style="width:91px;">-</td>
			<td>Opa1 knock-in mice carrying&#160; OPA1 c.2708_2711delTTAG mutation on C57Bl6/J background</td>
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			<td style="width:324px;">Bioptigen, Leica Microsystems, Germany</td>
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			<td>Spectral-Domain Optic Coherence Tomography system</td>
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			<td style="width:324px;">Bioptigen, Leica Microsystems, Germany</td>
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			<td>Software for OCT acquisition and analysis</td>
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			<td height="24" style="height:24px;width:296px;">ImageJ 1.48v</td>
			<td style="width:324px;">Wayne Rasband, National Institutes of Health, USA</td>
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			<td>Software for analysis, requires downloading and installing two hommade macros: http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Retina_Tool</td>
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			<td style="width:324px;">CentraVet, France</td>
			<td style="width:91px;">T.7979C.CS</td>
			<td>Mouse positioning</td>
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			<td height="40" style="height:40px;width:296px;">Name</td>
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			<td height="24" style="height:24px;width:296px;">N&#233;osyn&#233;phrine Faure 10%</td>
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			<td height="20" style="height:20px;width:296px;">Mydriaticum 0.5%</td>
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			<td>Eye dilatation</td>
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			<td style="width:324px;">Laboratoire Chauvin, Bausch&#38;Lomb, France</td>
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			<td style="width:324px;">Laboratoires Anios, France</td>
			<td>59844</td>
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optical coherence tomography: imaging mouse retinal ganglion cells in vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

The SD-OCT technology enables retinal imagining and thickness analysis that is comparable to histology, but is faster and more detailed (Figure 3). As presented with wildtype C57Bl/6 mice, even though the quality of an SD-OCT scan (Figure 3A, right) is not as good as that of an image of a retinal cross-section (Figure 3A, left), it visualizes more layers (e.g., OLM). Moreover, it takes only ...

Watch this Scientific Journal Video about Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo at JoVE.com

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

The overall goal of this experiment is to use optical coherence tomography or OCT to quickly obtain an accurate assessment of retinal thickness. OCT can help answer key questions in the field of ophthalmology such as, optic neuropathy. The main advantage of this technique is that, it is a non-invasive method for detecting structural changes in the mouse retinal ganglion cells.This technique is really interesting because it is non-invasive, safe and reputable method to test the effect of treatment in the optic neuropathy. To begin, first adjust the reference arm settings appropriate for the lens. Next, turn on the power supply and the computer.When the computer is ready, launch the imaging program. Create a new clinical study and add desired protocols. Go to patient exam section and set exam defaults.Click, add patient in the patient exam section to add a new patient and enter the relevant patient information. Then click, add exam, start exam and add preset scans from the list to select the desired protocol starting with the eye that will be measured first. At least 15 minutes before the data acquisition, instill drops of room temperature 10%phenylephrine into each eye of all the mice that will be tested.Then remove the excess reagent and instill drops of room temperature 0.5%tropicamide. Approximately five minutes before the acquisition, induce general anesthesia. Immediately after, administer 0.4%oxybuprocaine hydrochloride to each eye, leaving the solution in place for three seconds to anesthetize and immobilize the eyes.Then, remove the drops and repeat the 10%phenylephrine and 0.5%tropicamide installation. When the eyes are sufficiently dilated for the imaging, lubricate the eyes with viscous glycol based eye drops to provide corneal hydration and wrap the mouse in a sheet of surgical gauze to keep it warm. Using a sponge or cotton wick, apply a thin layer of ophthalmic gel supplemented with 0.3%hypromellose onto each eye.While doing so, set the eyelashes and whiskers aside. Position the mouse in the cassette with the head straight and pointing forward and carefully place the bite bar in its mouth. Place a cotton roll under the side of the animal for the eye that is being examined.Then, keeping the clip on aiming tip on the mouse specific lens, rotate the z translator screw counter clockwise to bring the lens towards the eye. Preset the position of the mouse by rotating and swiveling the cassette and turning the bite bar and the x-translator screws until the experimental eye is positioned to look directly into the lens. When the optical axis of the eyes and the lens are aligned, select the first scan in the exam.Click start aiming and use the z-translator screw to move the retina vertically in the left panel and horizontally in the right panel. By rotating the cassette, move the optic nerve head up or down to bring it into the middle of the right panel. Use the bite bar screw to straighten the retina within the right panel and swivel the cassette to position the optic nerve head in the middle of the left panel.Then use the x-translator screw to level the retina within the left panel and keeping in mind the major function of each modulator, further adjust the position of the retina for the perfect centralization on the optic nerve head. When the retina is in position, click start snapshot to begin the spectral domain optical coherence tomography scanning. Do not forget to save the scan and the report.Adjust the centralization if necessary. When the scanning is complete, retract the lens and position the second eye as just demonstrated. After the second eye has been scanned, remove the mouse from the cassette, apply ophthalmic gel with 0.3%hypromellose to each eye and place the mouse on a heating plate with monitoring until full recovery.To focus on retinal ganglion cells, use the MRI retina tool macro created for image J.Open the MRI retina tool and modify polygon selection tool and load the image of interest. Click M to begin the measurement, then click E to adjust the horizontal position. Select the first cassette in the newly opened region of interest manager window.Click on the blue polygon and click on the borders of the measured layer in the picture to adjust the position of the first cassette. Select the second cassette in the region of interest manager window and adjust the second cassette. Then click R, to re-measure the retina and view the results in the measurements window.Even though the quality of a spectral domain OCT scan is not as good as that of an image of a retinal cross section, the OCT allows more layers to be visualized. The retina layers are easily measured in an OCT scan and encompass the retinal nerve fiber in ganglion cell layers, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner and outer segment layers retina pigment epithelium and the choroid, allowing a complex study of the entire retina. OCT measurement of retinal thickness is just demonstrated, indicates that visually impaired genetically mutant OPA1 female mice exhibit a progressive thickening of the ganglion cell complex and the peripapillary retina nerve fiber layers.A comparison of the retina nerve fiber in ganglion cell layer thickness using standard and homemade calipers, demonstrates that standard calipers measure a significantly lower thickness than do homemade calipers, as the standard calipers are much thicker and more difficult to place on a thin layer border. Once mastered, this technique can be done in 15 minutes if performed correctly. After its development, OCT paved the way for the researchers in the field of ophthalmology to analyze structural changes in the mouse retina.After watching this video, you should have a good understanding of how to perform and analyze OCT imaging for retina ganglion cells in mice.

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Neuroscience

18.9K 조회수.  INSERM U1051, Institut of Neurosciences of Montpellier. The overall goal of this experiment is to use optical coherence tomography or OCT to quickly obtain an accurate assessment of retinal thickness. OCT can help answer key questions in the field of ophthalmology such as, optic neuropathy. The main advantage of this technique is that, it is a non-invasive method for detecting structural changes in the mouse retinal ganglion cells.This technique is really interesting because it is non-invasive, safe and reputable method to test the effect o...