Name: Application of Optical Coherence Tomography to a Mouse Model of Retinopathy (Video) | JoVE Journal
Uploaded: 2022-01-12T14:30:00.000+00:00
Duration: 8 min 22 s
Description: Learn optical coherence tomography for retinopathy diagnosis in mouse models using non-invasive, high-resolution imaging. This protocol enables quantitative measurement of retinal thickness and lesion detection in vivo.

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5 minutes

Project Source: Natural Science Foundation of Guangdong Province (2018A0303130306). The authors would like to thank the Ophthalmic Research Laboratory, Joint Shantou International Eye Center of Shantou University and the Chinese University of Hong Kong for funding and materials.

The operations were performed following the Statement on the Use of Animals in Ophthalmic and Vision research from the Association for Research in Vision and Ophthalmology. The experimental design was approved by the institutional animal Ethics Committee (Medical Ethics Committee of JSIEC, EC 20171213(4)-P01). Two-month-old C57BL/6J mice and Vldlr knockout mice were used in this study. There were 7 mice in each group, all of which were female and weighed 20 g to 24 g.
1. Experimental conditions
<ol>
	<li>Assign the mice to two groups: an experimental group consisting of Vldlr knockout mice and a control group consisting of C57BL/6J mice.</li>
	<li>Feed the mice with food and water conventionally.</li>
	<li>Raise the mice in the animal laboratory under stable conditions of room temperature (22 &#176;C), humidity (50-60%), light-dark cycle (12 h-12 h), and room light intensity (350-400 lux).</li>
	<li>Prepare the experimental equipment: optical coherence tomography with confocal scanning laser ophthalmoscope (cSLO) for small animals (Figure 1A).</li>
	<li>Prepare all materials required for the experiment (Figure 1B) and weigh the mice (Figure 1C).</li>
</ol>
2. Information records
<ol>
	<li>Record the information: group, code, date of birth, age, sex, weight, and anesthetic dosage.</li>
</ol>
3. Instrument startup and testing
<ol>
	<li>Switch on the computer and start up the software.</li>
	<li>Click the Test program button to complete the test program.</li>
	<li>Turn on the thermostat and preheat it to the temperature of 37 &#176;C.</li>
	<li>Start the OCT module procedure after the program testing.</li>
	<li>Create a new subject and fill in the mouse information.</li>
	<li>Preheat the electric blanket and cover it with surgical towels.</li>
</ol>
4. Anesthesia
<ol>
	<li>Use lyophilized anesthetic powder containing Tiletamine and Zolazepam to prepare the anesthetic mixture. 
	NOTE: Follow local animal ethics committee recommendations for the choice, dosage, and route of anesthesia administration. Anesthetize the animal with an anesthetic that will provide immobility and loss of pain perception for at least 1 hour, after which the animal recovers quickly. Dosage should be based on the length of experiment time, animal weight, and other factors.</li>
	<li>Anesthetize the animal using the prepared anesthetic mixture. Ensure to keep the animal warm during the entire procedure until recovery.</li>
</ol>
5. Application of mydriatic drops
<ol>
	<li>Achieve manual restraint of the mouse by the scruff, make the eyeball protrude slightly, and rotate the mouse head with one eye facing upward.</li>
	<li>Apply the mydriatic drops to dilate the pupils (Figure 2A).</li>
	<li>Check for pupil dilation after 10 min.</li>
</ol>
6. Placement of the mouse
<ol>
	<li>Place a mouse on an electric blanket platform.</li>
	<li>Coat both eyes with medical sodium hyaluronate gel (Figure 2B).</li>
	<li>Screw a 60 D double spherical lens (preset lens) on the cSLO device (Figure 1A-5, 6).</li>
	<li>Place a 100 D contact lens on the mouse cornea with the concave side touching the sodium hyaluronate gel on the corneal surface (Figure 2C, D and Figure 3A-II).</li>
	<li>Place the mouse on the small, constant-temperature animal platform and keep the eye 1-2 mm away from the lens of the cSLO device (Figure 3A).</li>
	<li>Adjust the angle of the contact lens with forceps to keep the pupil in the center of the lens.</li>
	<li>Fine-tune the adjustments to the head to make the eye face straight ahead.</li>
</ol>
7. Confocal Scanning Laser Ophthalmoscope (cSLO)
<ol>
	<li>Click the OCT button, choose the mouse module, and start the cSLO program (Figure 4B).</li>
	<li>Select the IR mode (light source: red light), and adjust the parameter (range: 2047, Figure 4D).</li>
	<li>Select the eye to be examined (right eye: Figure 4C-1; left eye: Figure 4C-2).</li>
	<li>Control the lever and move the preset lens towards the contact lens slowly.</li>
	<li>Adjust the diopter value until the posterior pole imaging is clear (Figure 4E).</li>
	<li>Make further adjustments to align the image of the retinal posterior pole, centering it at the optic nerve head.</li>
</ol>
8. Optical coherence tomography (OCT)
<ol>
	<li>Start the OCT program (Figure 4G).</li>
	<li>Click the progress bar up and down until the OCT image appears (Figure 4H).</li>
	<li>Adjust parameters: Range Min (Figure 4I) = 0-20, Range Max (Figure 4J) = 40-60.</li>
	<li>Adjust the preset lens distance and position direction until an ideal OCT image is obtained.</li>
	<li>Select the scanning position by moving the standard line in the cSLO (Figure 4M).</li>
	<li>Start scanning from the optic nerve head.</li>
	<li>Collect images in the same order for each eye: horizontal line: optic nerve head &#8594; superior &#8594; inferior; vertical line: optic nerve head &#8594; nasal &#8594; temporal.</li>
	<li>Collect images from four directions.</li>
	<li>Click Average to overlay the cSLO and OCT image signals (Figure 4F and Figure 4O).</li>
	<li>Click the shot button to acquire the SLO-OCT image (Figure 4P).</li>
	<li>Save and export all the images (Figure 4Q, R).</li>
</ol>
9. The end of the experiment (after the OCT examination)
<ol>
	<li>Place the mouse on the electric blanket to keep it warm until it wakes up. 
	NOTE: The mouse should be monitored until it regains sufficient consciousness to maintain sternal recumbency. Postoperative exposure to bright light should be minimized.</li>
	<li>Remove the 100 D contact lens.</li>
	<li>Apply the levofloxacin eye gel to protect the cornea.</li>
	<li>Place the mouse back in the cage after it wakes up. 
	NOTE: Ensure that the examined mouse is not returned to the company of other mice until fully recovered.</li>
	<li>Turn off the software and switch off the computer.</li>
	<li>Clean the 100 D contact lens with water; dry the lens.</li>
	<li>Clean and disinfect the environment.</li>
</ol>
10. Image analysis
<ol>
	<li>Compare the OCT images of Vldlr knockout mice with those of C57BL/6J mice.</li>
	<li>Observe multiple positions: vertical and horizontal scans passing through the optic papilla; superior, inferior, nasal, and temporal scans; and abnormal reflection site scans.</li>
	<li>Observe the thickness, shape, layering, and abnormal reflectance lesions of the retina in each image, as well as the vitreous interface of the retina and the vitreous body.</li>
	<li>Record the locations, characteristics, and numbers of lesions.</li>
</ol>
11. Retinal stratification correction
<ol>
	<li>Click Load Examination on the OCT interface (Figure 5A).</li>
	<li>Call out the OCT images of a mouse from a pop-up window.</li>
	<li>Select images: OCT image scanning through the optic papilla, horizontally or vertically.</li>
	<li>Double-click the image in the Media Container to display it on the screen (Figure 5C).</li>
	<li>Click on Layer Detection to complete automatic layering on the retina (Figure 5D).</li>
	<li>Select the dividing lines on both sides of the layer prepared for analysis (Figure 6D-10).</li>
	<li>Select a separate dividing line (Figure 6B-6) and click Edit Layer (Figure 6A-1) to activate the line when a red circle appears (Figure 6B-7).</li>
	<li>Adjust Spacing (Figure 6A-4, e.g., 50) and Limit Range (Figure 6A-5, e.g., 50).</li>
	<li>Modify the dividing line by moving the red circle (compare the green dividing line in Figure 6B and Figure 6C; Figure 6C shows the modified result).</li>
</ol>
12. Retinal lamination thickness
<ol>
	<li>Click the Measure Marker button (Figure 6D-9).</li>
	<li>Select the dividing line of the layer to be analyzed (e.g., in the outer nuclear layer, select the 4th and 5th dividing line in the list) to display the boundary of the layer on the OCT image (Figure 6D-10).</li>
	<li>Select Connect with Layer (Figure 6D-11) and Stay Connected on Move (Figure 6D-12).</li>
	<li>Select the area to display the results (the selected column is colored, Figure 6D-13).</li>
	<li>Click the position to be analyzed on the OCT image to make the measurement line appear (perpendicular to the horizontal axis and consistent with the color of the resulting area) (Figure 6D-14).</li>
	<li>Click on the next column for the next measurement and reveal the previous data (Figure 6E-15).</li>
	<li>Read the Vert value (thickness of the measured position) in the Length in &#181;m (tissue) row (Figure 6E, red rectangle).</li>
	<li>Click Delete Marker (Figure 6E-16) and New Marker (Figure 6E-17) to retest so that the results will cover the original data (if remeasurement is necessary).</li>
	<li>Press Print Scr on the keyboard to save screenshots, or click Save Examination to save directly (Figure 5H).</li>
	<li>Input the data into a spreadsheet or statistical software for statistical analysis.</li>
</ol>
13. Measurement of full retinal thickness
<ol>
	<li>Select line 1 (ILM, inner limiting membrane, Figure 7B) and line 7 (OS-RPE, OS: outer photoreceptor segments; RPE: retinal pigment epithelial layer, Figure 7C) in the list in the upper right corner. 
	NOTE: The full retinal thickness means the thickness of the retinal neurepithelium layer, which is the retina between ILM and OS-RPE on OCT).</li>
	<li>Measure the retinal thickness on both sides of the optic papilla at a specific interval.
	<ol>
		<li>For example: from the appearance of the retinal structure at the edge of the optic papilla, measure 4 values with 200 &#181;m spacing of the horizontal ruler (Figure 7G, H).</li>
	</ol>
	</li>
	<li>Record all measured values in a spreadsheet.</li>
	<li>Use multiple t-tests (one per row) to compare the measured values of each corresponding position in both groups.</li>
</ol>

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The authors declare no potential conflict of interest.

In this study, OCT imaging using a small-animal retinal imaging system was applied to evaluate retinal changes in Vldlr knockout mice, which demonstrate incomplete posterior vitreous detachment, subretinal neovascularization, and retinal thickness thinning. OCT is a noninvasive imaging method to examine the condition of the retina in vivo. Most OCT devices are designed for human eye examination. The size of the hardware equipment, the setting of the focal length, the setting of the system parameters, an...

Optical coherence tomography (OCT) is an imaging technique that can provide in vivo high resolution and crosssectional imaging for tissue1,2,3,4,5,6,7,8, especially for the noninvasive examination in the retina9,10,11,12. It can also be used to quantify some important biomarkers, such as retinal thickness and retinal nerve fiber layer thickness. The principle of OCT is optical coherence reflectometry, which obtains crosssectional tissue information from the coherence of light reflected from a sample and converts it into a graphic or digital form through a computer system7. OCT is widely used in ophthalmology clinics as an essential tool for diagnosis, follow-up, and management for patients with retinal disorders. It can also provide insight into the pathogenesis of retinal diseases.
In addition to clinical applications, OCT has also been used in animal studies. Although pathology is the gold standard of morphological characterization, OCT has the advantage of noninvasive in vivo imaging and longitudinal follow-up. Furthermore, it has been shown that OCT is well correlated with histopathology in retinopathy animal models11,13,14,15,16,17,18,19,20. The mouse is the most commonly used animal in biomedical studies. However, its small eyeballs pose a technical challenge to conducting OCT imaging in mice.
Compared to the OCT first used for retinal imaging in mice21,22, OCT in small animals has now been optimized with respect to hardware and software systems. For example, OCT, in combination with the tracker, significantly reduces the signal-to-noise ratio; OCT software system upgrades allow more retinal layers to be detected automatically; and the integrated DLP beamer helps to reduce the motion artifacts.
Very-low-density lipoprotein receptor (Vldlr) is a transmembrane protein in endothelial cells. It is expressed on retinal vascular endothelial cells, retinal pigment epithelial cells, and around the outer limiting membrane23,24. Subretinal neovascularization is the phenotype of Vldlr knockout mice23. Therefore, Vldlr knockout mice are used to investigate the pathogenesis and potential therapy of subretinal neovascularization. This article demonstrates the application of OCT imaging to detect retinal lesions in Vldlr knockout mice, hoping to provide some technical reference for retinopathy research in small animal models.

<table><tbody><tr><td>100-Dpt contact lens</td><td>Volk Optical,Inc, Mentor, OH</td><td></td><td>Accessory belonging to the RETImap</td></tr><tr><td>Double aspheric 60-Dpt glass lens</td><td>Volk Optical,Inc, Mentor, OH</td><td></td><td>Accessory belonging to the RETImap</td></tr><tr><td>Electric heating blanket</td><td>POPOCOLA</td><td>CW-DRT-01</td><td>50 x 35 cm</td></tr><tr><td>Injection syringe (1 mL)</td><td>Kaile</td><td></td><td>0.45 x 16RWLB</td></tr><tr><td>Levofloxacin Hydrochloride Eye Gel</td><td>EBE PHARMACEUTICAL Co.LTD</td><td></td><td>5 g: 0.015 g</td></tr><tr><td>Medical sodium hyaluronate gel</td><td>Alcon</td><td>16H01E</td><td></td></tr><tr><td>Microliter syringes</td><td>Shanghai high pigeon industry and trade co., LTD</td><td>Q31/0113000236C001-2017</td><td>50 &amp;micro;L</td></tr><tr><td>Povidone iodine solution</td><td>Guangdong medihealth pharmaceutical Co.,LTD</td><td></td><td>100 mL</td></tr><tr><td>RETImap</td><td>ROLAND CONSULT</td><td>19-99_50-2.1_1.2E</td><td>cSLO/ERG/VEP/FA/OCT/GFP</td></tr><tr><td>Small animal ear studs</td><td>OSMO POCKET OT110</td><td>INS1005-1S</td><td></td></tr><tr><td>Tropicamide Phenylephrine Eye Drops</td><td>Santen Pharmaceutical Co.,LTD</td><td></td><td>5 mg/mL</td></tr><tr><td>Xylazin</td><td>Sigma</td><td>X1251-5G</td><td>5 g</td></tr><tr><td>Zoletil 50</td><td>Virbac.S.A</td><td>7FRPA</td><td>Tiletamine 125 mg + Zolazepam 125 mg</td></tr></tbody></table>

application of optical coherence tomography to a mouse model of retinopathy

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.

Thanks to the high-resolution scans of OCT, the layers of the mouse retina can be observed, and abnormal reflections and their exact locations can be identified. The retinal OCT images of Vldlr knockout mice and C57BL/6J mice were compared in this study. The OCT images of all C57BL/6J mice showed various retinal layers with different reflectivity, and the demarcation was clear (Figure 8D). In contrast, all Vldlr knockout mice showed abnormal, hyperreflective lesions on the ...

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 ...

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Research

JoVE Journal

Medicine

This video demonstrates an in vivo imaging technique for detecting and quantifying eye inflammation in a mouse model of post-infectious uveitis. The technique, known as optical coherence tomography (OCT), generates high-resolution cross-sectional images of the eye for assessing disease progression.

Optical Coherence Tomography Imaging for Assessing Uveitis in a Mouse Model

Source: Jagodzinska, J., et. al.,  Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo. J. Vis. Exp. (2017)  In this video, we demonstrate the use of spectral-domain optical coherence tomography (SD-OCT) to image retinal layers and assess structural changes in a genetically modified mouse model with optic neuropathy.

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A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

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Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

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.

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

Learn optical coherence tomography for retinopathy diagnosis in mouse models using non-invasive, high-resolution imaging. This protocol enables quantitative measurement of retinal thickness and lesion detection in vivo.

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

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