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>

<ol>
	<li>Frombach, 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=Serine+protease-mediated+cutaneous+inflammation:+characterization+of+an+ex+vivo+skin+model+for+the+assessment+of+dexamethasone-loaded+core+multishell-nanocarriers.">Serine protease-mediated cutaneous inflammation: characterization of an ex vivo skin model for the assessment of dexamethasone-loaded core multishell-nanocarriers.</a> Pharmaceutics. 12 (9), 862 (2020).</li><li>Osiac, E., S&#259;ftoiu, A., Gheonea, D. I., Mandrila, I., Angelescu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+and+Doppler+optical+coherence+tomography+in+the+gastrointestinal+tract.">Optical coherence tomography and Doppler optical coherence tomography in the gastrointestinal tract.</a> Journal of Gastroenterology. 17 (1), 15-20 (2011).</li><li>Xiong, Y. Q., 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=Diagnostic+accuracy+of+optical+coherence+tomography+for+bladder+cancer:+A+systematic+review+and+meta-analysis.">Diagnostic accuracy of optical coherence tomography for bladder cancer: A systematic review and meta-analysis.</a> Photodiagnosis and Photodynamic Therapy. 27, 298-304 (2019).</li><li>Andrews, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+of+the+aging+kidney.">Optical coherence tomography of the aging kidney.</a> &amp; Clinical Transplantation. 14 (6), 617-622 (2016).</li><li>Terashima, M., Kaneda, H., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+optical+coherence+tomography+in+coronary+intervention.">The role of optical coherence tomography in coronary intervention.</a> The Korean Journal of Internal Medicine. 27 (1), 1-12 (2012).</li><li>Avital, Y., Madar, A., Arnon, S., Koifman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+coronary+calcifications+in+optical+coherence+tomography+imaging+using+deep+learning.">Identification of coronary calcifications in optical coherence tomography imaging using deep learning.</a> Scientific Reports. 11 (1), 11269 (2021).</li><li>Huang, 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=Optical+coherence+tomography.">Optical coherence tomography.</a> Science. 254 (5035), 1178-1181 (1991).</li><li>Tsai, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+in+gastroenterology:+a+review+and+future+outlook.">Optical coherence tomography in gastroenterology: a review and future outlook.</a> Journal of Biomedical Optics. 22 (12), 1-17 (2017).</li><li>Chen, 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=Relationship+between+optical+intensity+on+optical+coherence+tomography+and+retinal+ischemia+in+branch+retinal+vein+occlusion.">Relationship between optical intensity on optical coherence tomography and retinal ischemia in branch retinal vein occlusion.</a> Scientific Reports. 8 (1), 9626 (2018).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+retinal+layer+optical+intensities+on+three-dimensional+optical+coherence+tomography.">Quantitative analysis of retinal layer optical intensities on three-dimensional optical coherence tomography.</a> Investigative Opthalmology &amp; Visual Science. 54 (10), 6846-6851 (2013).</li><li>Cruz-Herranz, 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=Monitoring+retinal+changes+with+optical+coherence+tomography+predicts+neuronal+loss+in+experimental+autoimmune+encephalomyelitis.">Monitoring retinal changes with optical coherence tomography predicts neuronal loss in experimental autoimmune encephalomyelitis.</a> Journal of Neuroinflammation. 16 (1), 203 (2019).</li><li>Podoleanu, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography.">Optical coherence tomography.</a> Journal of Microscopy. 247 (3), 209-219 (2012).</li><li>Augustin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+findings+in+the+retinas+of+SOD1+knockout+mice.">Optical coherence tomography findings in the retinas of SOD1 knockout mice.</a> Translational Vision Science &amp; Technology. 9 (4), 15 (2020).</li><li>Berger, 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=Spectral-domain+optical+coherence+tomography+of+the+rodent+eye:+highlighting+layers+of+the+outer+retina+using+signal+averaging+and+comparison+with+histology.">Spectral-domain optical coherence tomography of the rodent eye: highlighting layers of the outer retina using signal averaging and comparison with histology.</a> PLoS One. 9 (5), 96494 (2014).</li><li>Burns, M. 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=New+developments+in+murine+imaging+for+assessing+photoreceptor+degeneration+in+vivo.">New developments in murine imaging for assessing photoreceptor degeneration in vivo.</a> Advances in Experimental Medicine &amp; Biology. 854, 269-275 (2016).</li><li>Jagodzinska, 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=Optical+coherence+tomography:+imaging+mouse+retinal+ganglion+cells+in+vivo.">Optical coherence tomography: imaging mouse retinal ganglion cells in vivo.</a> Journal of Visualized Experiments: Jove. (127), e55865 (2017).</li><li>Kocaoglu, O. P., 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=Simultaneous+fundus+imaging+and+optical+coherence+tomography+of+the+mouse+retina.">Simultaneous fundus imaging and optical coherence tomography of the mouse retina.</a> Investigative Opthalmology &amp; Visual Science. 48 (3), 1283-1289 (2007).</li><li>Tode, 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=Thermal+stimulation+of+the+retina+reduces+Bruch's+membrane+thickness+in+age+related+macular+degeneration+mouse+models.">Thermal stimulation of the retina reduces Bruch's membrane thickness in age related macular degeneration mouse models.</a> Translational Vision Science &amp; Technology. 7 (3), 2 (2018).</li><li>Wang, R., Jiang, C., Ma, J., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+morphological+changes+in+the+retina+of+rhodopsin-/-+mice+with+spectral+domain+optical+coherence+tomography.">Monitoring morphological changes in the retina of rhodopsin-/- mice with spectral domain optical coherence tomography.</a> Investigative Ophthalmology &amp; Visual Science. 53 (7), 3967-3972 (2012).</li><li>Xie, 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=A+spectral-domain+optical+coherence+tomographic+analysis+of+Rdh5-/-+mice+retina.">A spectral-domain optical coherence tomographic analysis of Rdh5-/- mice retina.</a> PLoS ONE. 15 (4), 0231220 (2020).</li><li>Li, Q., 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+imaging+by+optical+coherence+tomography+to+monitor+retinal+degeneration+in+the+mouse.">Noninvasive imaging by optical coherence tomography to monitor retinal degeneration in the mouse.</a> Investigative Ophthalmology &amp; Visual Science. 42 (12), 2981-2989 (2001).</li><li>Horio, N., 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=Progressive+change+of+optical+coherence+tomography+scans+in+retinal+degeneration+slow+mice.">Progressive change of optical coherence tomography scans in retinal degeneration slow mice.</a> Archives of Ophthalmology. 119 (9), 1329-1332 (2001).</li><li>Hu, W., 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=Expression+of+VLDLR+in+the+retina+and+evolution+of+subretinal+neovascularization+in+the+knockout+mouse+model's+retinal+angiomatous+proliferation.">Expression of VLDLR in the retina and evolution of subretinal neovascularization in the knockout mouse model's retinal angiomatous proliferation.</a> Investigative Opthalmology &amp; Visual Science. 49 (1), 407-415 (2008).</li><li>Wyne, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+VLDL+receptor+in+endothelial+cells.">Expression of the VLDL receptor in endothelial cells.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 16 (3), 407-415 (1996).</li><li>Augustin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+characterization+of+spontaneous+retinal+neovascularization+in+the+mouse+eye+by+multifunctional+optical+coherence+tomography.">In vivo characterization of spontaneous retinal neovascularization in the mouse eye by multifunctional optical coherence tomography.</a> Investigative Opthalmology &amp; Visual Science. 59 (5), 2054-2068 (2018).</li><li>Fang, 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=Fundus+autofluorescence,+spectral-domain+optical+coherence+tomography,+and+histology+correlations+in+a+Stargardt+disease+mouse+model.">Fundus autofluorescence, spectral-domain optical coherence tomography, and histology correlations in a Stargardt disease mouse model.</a> The FASEB Journal. 34 (3), 3693-3714 (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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

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 ...

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Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

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 ...

application-optical-coherence-tomography-to-mouse-model

Research

JoVE Journal

Medicine

3.6K Views.  Shantou University and the Chinese University of Hong Kong. Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Video: Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...