Name: optical coherence tomography: imaging mouse retinal ganglion cells in vivo
Uploaded: 2017-09-22T13:00:00
Description: Watch this Scientific Journal Video about Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo at JoVE.com

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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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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		<tr height="20">
			<td height="20" style="height:20px;width:296px;">Mice</td>
			<td style="width:324px;"></td>
			<td style="width:91px;"></td>
			<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>
		</tr>
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			<td height="40" style="height:40px;width:296px;">Name</td>
			<td style="width:324px;">Company</td>
			<td style="width:91px;">Catalog Number</td>
			<td>Comments</td>
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			<td height="24" style="height:24px;width:296px;">EnVisu R2200 SD-OCT Imaging System</td>
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			<td>Spectral-Domain Optic Coherence Tomography system</td>
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			<td>Software for OCT acquisition and analysis</td>
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			<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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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 ...

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