Name: optical coherence tomography: imaging mouse retinal ganglion cells in vivo
Uploaded: 2017-09-22T13:00:00.000+00:00
Duration: 8 min 17 s
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 was performed on mouse retinas using the spectral domain (SD) ophthalmic imaging system (Figure 1A). The SD-OCT apparatus consists of a base and an animal imaging mount (AIM) with a rodent alignment stage (RAS) (Figure 1B). The base includes the computer, the OCT engine, the SD-OCT probe, and the mouse-specific lens. The probe is mounted onto the AIM, which includes the Z-translator. The RAS is used for mouse positioning thanks to the table with the X- and Y-translator, the cassette that can be rotated and swiveled, and the removable bite bar with the nose band. The software provided by the manufacturer allows for the acquisition and analysis of OCT files, although the latter may also be done with an open-source image processing program.
<ol>
	<li>Firmly place the probe in the AIM.</li>
	<li>Connect the mouse-specific lens to the probe.</li>
	<li>Adjust the reference arm settings for the specific lens (here, power at 086 and position at 964).</li>
	<li>Making sure that the mouse-specific lens is sufficiently distant from the cassette, attach the bite bar with the nose band onto the cassette. 
	NOTE: The distance between the lens and the cassette may be adjusted with the Z-translator screw, located at the back of the AIM. The nose band is a commercially available elastic band, and its tension should be adjusted depending upon the size of the mouse.</li>
	<li>Turn on the power supply (lower right corner of the cart) and then the computer.</li>
	<li>To launch the imaging program, double-click on the appropriate shortcut on the screen. 
	NOTE: This depends on the type of lens; here, Mouse Retina.</li>
	<li>Create a new Clinical Study and add the desired Protocols. Otherwise, use the existing Clinical Study and/or Protocols.
	<ol>
		<li>Add a new study by choosing the &#34;Study Name&#34;; &#34;ID&#34;; &#34;Treatment Arm Specifications&#34; (here, &#34;Uncategorized&#34;); and predefined &#34;Scan Protocols&#34;, if they exist.</li>
	</ol>
	</li>
	<li>Choose an &#34;Examiner&#34; in the &#34;Clinical Study&#34; section. 
	NOTE: For a new &#34;Examiner&#34;, go to &#34;Setup Examiners &#38; Physicians&#34; to define it.</li>
	<li>Add a new Patient in the Patient/Exam section by click on Add Patient; entering the &#34;ID&#34;, &#34;name&#34;, &#34;surname&#34;, &#34;sex&#34;, and &#34;date of birth&#34; (optional). 
	NOTE: Do this just before testing each mouse, if more convenient. Make sure that the ID is no longer than 10 characters. For the mouse, the refractive error for both eyes is 0 and the axial length is 23.0.</li>
	<li>Click on &#34;Add Exam&#34; and select the desired &#34;Protocol&#34; by adding &#34;Preset Scans&#34; from the list, starting with the eye that will be measured first (here, the right eye), or by customizing the scans.</li>
	<li>To customize a scan, either use an existing scan as a template and edit it, or create it from scratch through the &#34;Add Custom Scan&#34; option. After adding all the scans to the list, define a new protocol using the &#34;Enter &#34;New Protocol Name&#34; option (OS: oculus sinister, left eye; OD: oculus dexter, right eye). 
	NOTE: Here, the protocol involves three scans: (i) a radial scan, with a 1.4 mm diameter, 0 mm horizontal and vertical offsets, 1,000 lines of A-scans/B-scan, 100 B-scans/volume, 1 frame/B-scan, 80 lines of inactive A-scans/B-scan, and 1 volume; (ii) a rectangular scan, with a 1.4-mm length and width, 0&#176; angle, 0-mm horizontal and vertical offsets, 1,000 lines of A-scans/B-scan, 100 B-scans, 1 frame/B-scan, 80 lines of inactive A-scans/B-scan, and 1 volume; and (iii) an annular scan, with a 0.4-mm minimum and 0.6-mm maximum diameter, 0-mm horizontal and vertical offsets, 1,000 lines of A-scans/B-scan, 3 B-scans/volume, 48 frames/B-scan, 80 lines of inactive A-scans/B-scan, and 1 volume.</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Eye dilation and immobilization
	<ol>
		<li>At least 15 min before data acquisition, instill 10% phenylephrine eye drops into the mouse eyes, remove the excess, and instill 0.5% tropicamide eye drops. 
		NOTE: The first dilatation can be done at once for all the mice that will be tested in the session. Make sure that the liquids are at room temperature.</li>
		<li>Immediately after inducing general anesthesia (step 2.2), administer 0.4% oxybuprocaine hydrochloride eye drops, keeping them in place for 3 s to anesthetize and immobilize the eyes. Afterwards, wipe off the drops and repeat the instillation of 10% phenylephrine and 0.5% tropicamide to ascertain that the eyes are well dilated. 
		NOTE: Make sure that the liquids are at room temperature and that the mouse does not swallow the oxybuprocaine.</li>
	</ol>
	</li>
	<li>General anesthesia
	<ol>
		<li>Prepare an anesthetic solution of ketamine (20 mg/mL) and xylazine (1.17 mg/mL) in saline. Store at 4 &#176;C for a maximum of 2 weeks.</li>
		<li>Approximately 5 min before testing, intraperitoneally inject 5 - 10 &#181;L of anesthetic solution per 1 g of body mass, depending upon the age and size of the mouse. 
		NOTE: Young and/or thin mice need less anesthetic, take less time to anesthetize, but also awaken faster. Here, 8 &#181;L/g (160 mg/kg of ketamine, 9.33 mg/kg of xylazine).</li>
		<li>Optionally, lubricate the eyes with viscous eye drops or an ophthalmic gel to avoid corneal dryness.</li>
	</ol>
	</li>
</ol>
3. Mouse Positioning
<ol>
	<li>Lubricate the eyes with viscous glycol-based eye drops to provide corneal hydration. 
	NOTE: If the eyes seem dry during the exam, re-apply the eye drops.</li>
	<li>Wrap the mouse in a sheet of surgical gauze to keep it warm.</li>
	<li>Using a sponge or cotton wick, apply a thin layer of an ophthalmic gel with 0.3% hypromellose onto each eye, to minimize light refraction, avoid opacities, and secure corneal hydration. While doing so, move the eyelashes and whiskers aside.</li>
	<li>Position the mouse in the cassette, with the head straight and pointing forward.</li>
	<li>Gently open the bite bar clamp and place the bar in the mouth; use the nose band to secure the position. 
	NOTE: Ensure that the bite bar is in the middle of the cassette.</li>
	<li>Place a cotton roll under the right (left) side if the right (left) eye is being tested.</li>
	<li>While keeping the clip-on aiming tip on the mouse-specific lens, bring it towards the eye by rotating the Z-translator screw counterclockwise.</li>
	<li>By rotating and swiveling the cassette and by turning the bite bar and the X-translator screws, pre-set the position of the mouse; the goal is to have the right eye look directly into the lens, and thus to align the optical axes of the eyes and the lens. 
	NOTE: If the mouse is too high or too low, the Y-translator screw should be used first.</li>
	<li>Choose the first scan in the &#34;Exam&#34;, click on &#34;Start Aiming&#34;, and make any further manual adjustments for retinal imaging.
	<ol>
		<li>Using the Z-translator screw, move the retina vertically on the left panel (Figure 2, Horizontal B-scan Alignment) and horizontally on the right panel (Figure 2, Vertical B-scan Alignment).</li>
		<li>Rotate the cassette to bring the ONH in the middle of the right panel by moving the ONH up or down. Use the bite bar screw to straighten the retina on the right panel. Swivel the cassette to position the ONH in the middle of the left panel.</li>
		<li>Use the X-translator screw to level the retina on the left panel. Keeping in mind the major function of each modulator, further adjust the position of the retina to centralize the ONH. 
		NOTE: Use the Y-translator screw to move the ONH up and down on the right panel, if necessary. The position may be refined at any time between scans.</li>
	</ol>
	</li>
</ol>
4. SD-OCT Imaging of the ONH and Retina
<ol>
	<li>Once content with the adjustments, click on &#34;Start Snapshot&#34; to begin SD-OCT scanning.
	<ol>
		<li>If a 3D imaging is not required, uncheck the OCU option.</li>
	</ol>
	</li>
	<li>Save the scan and the report.</li>
	<li>Proceed with the next scans.</li>
	<li>To image the second eye, after retracting the lens, turn the cassette accordingly and repeat steps 3.6 - 4.3.</li>
</ol>
5. Acquisition Completion
<ol>
	<li>When the acquisition is complete, remove the mouse from the cassette, apply ophthalmic gel with 0.3% hypromellose to each eye, and place the mouse on a heating plate to wake up.</li>
	<li>After the last acquisition, close the software and switch off the computer and the OCT machine (power supply button).</li>
	<li>Clean the cassette with disinfectant.</li>
</ol>
6. Analysis
<ol>
	<li>For the retinal layer thickness measurement, use the automated segmentation software provided by the manufacturer.
	<ol>
		<li>Click on the &#34;Patient&#34;, choose the desired &#34;Exam&#34; from then list, and click on the &#34;Review Exam&#34; option.</li>
		<li>Load the desired OCT data onto the OCT software by right-clicking on the folder icon in the desired scan.</li>
		<li>Right click on the B-scan; configure the calipers by enabling up to 10 measuring calipers; and enter their names, angles, and colors.</li>
		<li>Choosing the desired caliper by a right-clicking on the B-scan; place it accordingly onto the retina for measurement. 
		NOTE: For the scans centered on the ONH, set 5 calipers on each side of it, equidistant from each other. For the peripapillary analysis, make sure that the caliper is not placed too far from the ONH. For radial scanning, analyze 10 pictures per scan that were chosen by the OCT software. Their numbers may be found in the &#34;Reports&#34; folder.</li>
		<li>Save the results for analysis in a spreadsheet software program by right-clicking on the B-scan and clicking &#34;Save results&#34;. 
		NOTE: The results can be found in the same folder as the scanning data.</li>
	</ol>
	</li>
	<li>Alternatively, use the homemade caliper macro, &#34;MRI Retina Tool&#34;, developed for an open-source image processing program (see the Table of Materials).
	<ol>
		<li>Make sure that the &#34;MRI Retina Tool&#34; and the &#34;Modify Polygon Section Tool&#34; macros are active. Load the image. Click on the m button to start the measurement. 
		NOTE: The macro automatically creates one 0.2 mm-long measuring cassette on both sides of the ONH. Each cassette contains 5 measuring points that function as calipers. Their lateral position is unchangeable and is customized for the peripapilla in the radial scans. The horizontal position of the cassettes is predefined to measure the RNFL/GCL thickness, but it is easily modifiable to measure the GC complex layer instead. If the scan quality is poor, the horizontal position needs to be adjusted, or the picture needs to be excluded from the analysis.</li>
		<li>For horizontal adjustment, click on the e button. In the newly opened &#34;ROI Manager&#34; window, choose the first cassette.</li>
		<li>Click on the blue polygon button and adjust the position of the first cassette by clicking on the borders of the measured layer in the picture.</li>
		<li>Repeat for the second cassette by first choosing it in the &#34;ROI Manager&#34; window and then clicking on the appropriate image. Click on the r button to re-measure. View the results in the &#34;Measurements&#34; window. 
		NOTE: The results may be copied to a spreadsheet software program at any time. They contain the following values for the right cassette (r), left cassette (l), and total: Intden - integrated density within the cassette; Area: area of the measurement, in mm2; Len: length of the caliper within the cassette, in mm; Mean: mean intensity of the signal within the cassette; and Std: standard error of the mean intensity of the signal.</li>
		<li>Proceed with the next image.</li>
		<li>Further analyze the data in a spreadsheet software program. 
		NOTE: To find the mean thickness of the layer, take the ten Len values.</li>
	</ol>
	</li>
</ol>

<ol>
	<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 authors have nothing to disclose.

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.

<table><tbody><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Opa1&lt;em&gt;&lt;sup&gt;delTTAG&lt;/sup&gt;&lt;/em&gt; mouse</td><td>Institute for Neurosciences in Montpellier, INSERM UMR 1051, France</td><td>-</td><td>Opa1 knock-in mice carrying&amp;nbsp; OPA1 c.2708_2711delTTAG mutation on C57Bl6/J background</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EnVisu R2200 SD-OCT Imaging System</td><td>Bioptigen, Leica Microsystems, Germany</td><td>-</td><td>Spectral-Domain Optic Coherence Tomography system</td></tr><tr><td>EnVisu R2200 SD-OCT Imaging System Software</td><td>Bioptigen, Leica Microsystems, Germany</td><td>-</td><td>Software for OCT acquisition and analysis</td></tr><tr><td>ImageJ 1.48v</td><td>Wayne Rasband, National Institutes of Health, USA</td><td>-</td><td>Software for analysis, requires downloading and installing two hommade macros: http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Retina_Tool</td></tr><tr><td>Self-regulating heating plate</td><td>Bioseb, France</td><td>BIO-062</td><td>Protection against hypothermia</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nose Band</td><td>-</td><td>-</td><td>Elastic band</td></tr><tr><td>Gauze pads 3&quot; x 3&quot;</td><td>Curad, USA</td><td>CUR20434ERB</td><td>Protection against hypothermia</td></tr><tr><td>Dual Ended Cotton tip applicator</td><td>Essence of Beauty, CVS Health Corporation, USA</td><td>-</td><td>Gel application</td></tr><tr><td>Cotton Twists</td><td>CentraVet, France</td><td>T.7979C.CS</td><td>Mouse positioning</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents and Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>N&amp;eacute;osyn&amp;eacute;phrine Faure 10%</td><td>Laboratoires Europhtha, Monaco</td><td>-</td><td>Eye dilatation</td></tr><tr><td>Mydriaticum 0.5%</td><td>Laboratoires Th&amp;eacute;a, France</td><td>3397908</td><td>Eye dilatation</td></tr><tr><td>Cebesine 0.4%</td><td>Laboratoire Chauvin, Bausch&amp;amp;Lomb, France</td><td>3192342</td><td>Local anesthesia</td></tr><tr><td>Imalgene 1000</td><td>Merial, France/CentraVet, France</td><td>IMA004</td><td>General anesthesia</td></tr><tr><td>Rompun</td><td>Bayer Healthcare, Germany/CentraVet, France</td><td>ROM001</td><td>General anesthesia, analgesia, muscle relaxation</td></tr><tr><td>NaCl 0.9%</td><td>Laboratoire Osalia, France&amp;nbsp;</td><td>103697114</td><td>Physiological serum</td></tr><tr><td>Systene Ultra</td><td>Alcon, Novartis, USA</td><td>-</td><td>Hydration of eyes</td></tr><tr><td>GenTeal&#039;</td><td>Alcon, Novartis, USA</td><td>-</td><td>Ophtalmic gel to minimize light refraction and opacities</td></tr><tr><td>Aniospray Surf 29</td><td>Laboratoires Anios, France</td><td>59844</td><td>Desinfectant</td></tr></tbody></table>

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 ...

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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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JoVE Journal

Neuroscience

19.1K Views.  INSERM U1051, Institut of Neurosciences of Montpellier. 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.

Video: Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

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

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

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

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

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

Medicine

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://app.jove.com/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

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In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

In Vivo Imaging of Cx3cr1^gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

Two-Photon In Vivo Imaging of the Mouse Retina