This work was supported by the Department of Veterans Affairs Rehab R&#38;D Service Career Development Awards (CDA-1, RX002111; CDA-2; RX002928) to RSA, Merit Award (RX002615) and Research Career Scientist Award (RX003134) to MTP, Career Development Award (CDA-2, RX002342) to AJF, EY028859 to MTP, NEI Core Grant P30EY006360, Research to Prevent Blindness, and Foundation Fighting Blindness.

All procedures described were approved by the Atlanta Veterans Affairs Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications, 8th edition, updated 2011).
NOTE: The SD-OCT system used to develop the protocol below is described in the Table of Materials. While some of the procedures are specific to this particular system, the overall approach can be adapted for ...

<ol>
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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=Altered+ocular+parameters+from+circadian+clock+gene+disruptions.">Altered ocular parameters from circadian clock gene disruptions.</a> PLoS One. 14 (6), 0217111 (2019).</li><li>Chakraborty, R., 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=Circadian+rhythms,+refractive+development,+and+myopia.">Circadian rhythms, refractive development, and myopia.</a> Ophthalmic &amp; Physiological Optics. 38 (3), 217-245 (2018).</li><li>Davies, E. 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=Retinal+ganglion+cell+layer+volumetric+assessment+by+spectral-domain+optical+coherence+tomography+in+multiple+sclerosis:+application+of+a+high-precision+manual+estimation+technique.">Retinal ganglion cell layer volumetric assessment by spectral-domain optical coherence tomography in multiple sclerosis: application of a high-precision manual estimation technique.</a> Journal of Neuro-ophthalmology. 31 (3), 260-264 (2011).</li><li>Carnevali, 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=Optical+coherence+tomography+angiography+analysis+of+retinal+vascular+plexuses+and+choriocapillaris+in+patients+with+type+1+diabetes+without+diabetic+retinopathy.">Optical coherence tomography angiography analysis of retinal vascular plexuses and choriocapillaris in patients with type 1 diabetes without diabetic retinopathy.</a> Acta Diabetologica. 54 (7), 695-702 (2017).</li><li>Springelkamp, 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=Population-based+evaluation+of+retinal+nerve+fiber+layer,+retinal+ganglion+cell+layer,+and+inner+plexiform+layer+as+a+diagnostic+tool+for+glaucoma.">Population-based evaluation of retinal nerve fiber layer, retinal ganglion cell layer, and inner plexiform layer as a diagnostic tool for glaucoma.</a> Investigative Ophthalmology &amp; Visual Science. 55 (12), 8428-8438 (2014).</li><li>Allen, R. 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High resolution imaging of ocular structures in vivo allows for the assessment of retinal and ocular changes over time. In this protocol, SD-OCT was demonstrated to capture differences in ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.
The most critical aspect when performing SD-OCT is obtaining a clear image of the retina or other ocular structure of interest. It is important to take time to make sure the retina is perfectly centered an...

Spectral-domain optical coherence tomography (SD-OCT) is a precise, high-resolution imaging modality that allows clinicians and researchers to examine ocular structures noninvasively. This imaging technique is based on interferometry to capture three-dimensional retinal images in vivo on a micrometer scale1,2. It has become one of the most frequently used imaging modalities in vision research and in the clinic due to the easy detection and accuracy of pathological features such as structural defects and/or thinning of retinal layers and subretinal fluid3. In research using animal models of vision-related disorders, SD-OCT has provided essential noninvasive analyses of relationships between structure and function and their histopathological origins4. Due to its resolution (up to 2-3 microns, depending on the depth into the eye5), SD-OCT has the capability to detect even small changes in retinal layer thickness. This type of analysis can provide essential information for disease progression and assess the efficacy of neuroprotective methods and treatments for vision-related disorders.
SD-OCT is a noninvasive alternative to examining structure histologically, and the two have been shown to be correlated6. While SD-OCT does not reach cellular resolution, it does allow for longitudinal studies in animals. This is advantageous because disease progression can be tracked in individual animals over time as opposed to having to euthanize animals at specific time points. As imaging techniques continue to improve, SD-OCT technology will also progress, providing enhanced image quality as well as the capability to assess biological processes such as retinal blood vessel function in fine detail. Even since its advent in 1991, SD-OCT technology has seen huge advances in resolution, speed, and sensitivity7.
The present study utilizes an SD-OCT system to quantify changes in retinal layers in rodent models of retinal degeneration, glaucoma, and diabetic retinopathy. The SD-OCT system used here is a Fourier-domain OCT-system that utilizes low-power, near-infrared light to acquire, process, and store depth-resolved images in real time. The SD-OCT system has extended depth-imaging capability in the 800 nm wavelength band, providing 8 mm depth and 4 &#181;m resolution. In Fourier domain detection, the interference signal between scattered light from the tissue and a reference path is Fourier transformed to construct axial scans and/or axial depth profiles of scattered intensity8. For the studies here, the OCT beam is scanned over the desired retinal structure while serially acquiring axial scans. Typically, a scan pattern acquires the two-dimensional grid (B-Scans) as a collection of linear one-dimensional scan lines (A-Scans), which correspond to 2D cross-sectional images using a raster scan pattern. For studies focused on myopia in mice, this system is also used to measure dimensions of ocular structures (e.g., cornea thickness, lens thickness, vitreous chamber depth, and axial length).
The current system allows users to design their own protocols, creating scans that can be tailored and selected based on the ocular structures of interest. The principal scans featured in these user defined protocols makes this imaging technique user-friendly. For image analyses, we have developed customized programming in a mathematic modeling program. SD-OCT is a powerful tool to non-invasively identify and quantify pathomorphological changes in ocular structures and monitor vision-related disease progression.

<table>
	<tbody>
		<tr>
			<td>1% tropicamide</td>
			<td>Sandoz</td>
			<td>Sandoz #6131403550; NDC- 24208-585-59</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% tetracaine</td>
			<td>Alcon</td>
			<td>NDC 0065-0741-12</td>
			<td></td>
		</tr>
		<tr>
			<td>AIM-RAS G3 120 V</td>
			<td>Leica Bioptigen</td>
			<td>90-AIMRAS-G3-120</td>
			<td>Specialized platform to hold the OCT Scanner Head for mice</td>
		</tr>
		<tr>
			<td>Celluvisc gel</td>
			<td>REFRESH CELLUVISC</td>
			<td>#4554; NDC-0023-4554-30</td>
			<td></td>
		</tr>
		<tr>
			<td>G3 18 mm Telecentric Lens</td>
			<td>Leica Bioptigen</td>
			<td>90-BORE-G3-18</td>
			<td></td>
		</tr>
		<tr>
			<td>G3 Mouse Lens</td>
			<td>Leica Bioptigen</td>
			<td>90-BORE-G3-M</td>
			<td></td>
		</tr>
		<tr>
			<td>G3 Rat Lens</td>
			<td>Leica Bioptigen</td>
			<td>90-BORE-G3-R</td>
			<td></td>
		</tr>
		<tr>
			<td>heating pad</td>
			<td>Fabrication</td>
			<td>11-1130</td>
			<td></td>
		</tr>
		<tr>
			<td>InVivoVue software</td>
			<td>Leica Bioptigen</td>
			<td></td>
			<td>Specialized software that pairs with the Leica Bioptigen SD-OCT system</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td>mathematical modeling program</td>
		</tr>
		<tr>
			<td>Mouse/Rat Kit</td>
			<td>Leica Bioptigen</td>
			<td>90-KIT-M/R</td>
			<td>Mouse/rat rodent alignment system</td>
		</tr>
		<tr>
			<td>saline</td>
			<td>ADDIPAK</td>
			<td>200-39</td>
			<td></td>
		</tr>
		<tr>
			<td>System Envisu R4300 VHR 120 V</td>
			<td>Leica Bioptigen</td>
			<td>90-R4300-V1-120</td>
			<td>SD-OCT system</td>
		</tr>
	</tbody>
</table>

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.

SD-OCT is considered successful if high quality images are obtained such that ocular dimensions can be reliably measured. Here, a variety of uses of SD-OCT are illustrated using models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.
In a light-induced retinal degeneration (LIRD) model, exposure to bright light (10,000 lux) induces degeneration of photoreceptor cells in the retina9. Representative SD-OCT images reveal a thinner outer nuclear layer, w...

Watch this Scientific Journal Video about In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography at JoVE.com

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

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 ...

Spectral domain optical coherence tomography is a valuable tool for imaging ocular structures in vivo, and for tracking changes in ocular dimensions over time in a variety of eye disease models. We use SDOCT to measure retinal thickness in models of retinal degeneration in diabetic retinopathy, retinal thickness and cupping in a glaucoma model, and axial length in a myopia model. The SDOCT technique is straightforward, but practice is critical in obtaining good images.And experimenting with different protocols is important for determining which protocol best fits each research need. Demonstrating the procedure will be Andrew Feola, a principal investigator from the Center for Visual and Neurocognitive Rehabilitation. Begin by opening the spectral domain optical coherence tomography software, and entering the acquisition study and treatment arm information.Under the Patient Exam tab, click test examiner and select the name of the examiner. Click Study Name to define the study and open the Study tab to add a new study or to modify the treatments in an existing study. Click to the right of Select Treatment Arm to select a treatment arm.To add a new time point for an entire group, click Add Patient. In the window, enter the ID number, first and last name. Select the sex of the patient and the date of birth.To add the individual rats, click Add Exam. To identify the rats, click on an exam and click Edit Exam. Enter the ID number into the Enter Notes box and click Save Changes.Next, attach the proper lens to the device. Select the corresponding configuration in the software and dial in the associated reference arm position. Under the Patient Exam tab, double click the highlighted exam to proceed to the Imaging tab and begin imaging.To load a preset scan protocol, click Select a protocol from the list. For rat models of glaucoma and diabetic retinopathy, select a preset that consists of two OD and two OS, for the right and left eye scans respectively. After placing the anesthetized rat into a rodent alignment system, apply lubricant to the animal's eyes.Just before imaging, use a delicate task wipe to wick away any excess saline, and use two rotational motions of the rodent alignment system to position the rat such that the gaze is horizontal, and looking down the axis of the optical coherence tomography lens. Use the Free Run mode to orient the retina for data collection. Then use the Aim mode for a continuous display of both the horizontal and vertical B scans in real time.Move the scan head closer to the eye until the retina is visible, and use the rodent alignment system to adjust the animal position until the optic nerve head is visible in the center. Make the horizontal scan horizontal, and the vertical scan vertical. Adjust the working distance such that the retinal image is flat and not curved, and adjust the reference arm position to keep the image near the top of the display window.For retinal imaging of glaucoma, retinal degeneration, and diabetic retinopathy models, define a three by three millimeter volume scan that consists of 1, 000 A scans by 100 B scans by 1 repeated B scan for averaging. Center the optic nerve in the horizontal and the vertical axis so that the volume scan is in the center straight along the nasal, temporal, and superior, inferior axes. Click Snapshot to take a photo and click Save to save the image.Obtain a radial scan centered at the optic nerve head that is 1, 000 A scans by 4 B scans by 20 repeated B scans. And use the repeated B scans to enhance the image clarity of the eye or retina to facilitate interpretation of the regions of the eye or layers of the retina during data analysis. Then save the image and repeat the scan for the contralateral eye.For post-processing of the images, open the appropriate mathematical modeling software program. For processing the retina, select the optical coherence tomography scans to load, and click to define the center of the optic nerve head. The program will generate vertical lines that define the distances on either side of the optic nerve head.In the rat retina, these lines are at 0.5 and 1.2 millimeters from the center of the optic nerve head, with a total of four vertical lines representing the nasal, temporal and inferior, superior axes of the eye, depending on the radial B scan currently analyzed. Along each line, delineate the retinal nerve fiber layer, the inner plexiform layer, the inner nuclear layer, the outer plexiform layer, the outer nuclear layer the external limiting membrane, the inner and outer segments, the retinal pigment epithelium and the total retinal thickness. When all of the layers have been delineated, export the measurements to a spreadsheet for data analysis.Representative spectral domain optical coherence tomography images reveal a thinner outer nuclear layer, which contains the photoreceptor cell bodies in retinas from light-induced retinal degeneration BALB/c mice compared to undamaged control mice. After quantifying the retinal layer thickness, a significant difference between undamaged and light-induced retinal degeneration mice is observed for the total retinal thickness, outer nuclear layer thickness and inner and outer segment thickness. In the ocular hypertension model, a distinct remodeling at the optic nerve head is observed, including optic nerve cupping after eight weeks.Quantification of the retinal nerve fiber layer thickness after eight weeks of ocular hypertension reveals a significant thinning compared to baseline measurements. Imaging of retinas from a diabetic rat model and Wistar control animals reveals that at six weeks of age, the retinal nerve fiber layer and total retinal thickness are reduced in diabetic rats compared to Wistar rats in the peripheral retina, with the greatest differences observed in the inferior and temporal quadrants of the retina. The axial length of the eye in a mouse model of myopia is visibly longer than that observed in wild type eyes at 84 days of age.This myopic eye data is from a mouse lacking a clock gene, suggesting that clock genes contribute to myopia development. The measurement of ocular structures with SDOCT can be used in conjunction with other assays, like electroretinogram and optomotor response, to detect subtle changes over time in many eye disease models.

in-vivo-structural-assessments-ocular-disease-rodent-models-using

Research

JoVE Journal

Bioengineering

2.7K visualizações.  Atlanta Veterans Affairs Medical Center. Spectral domain optical coherence tomography is a valuable tool for imaging ocular structures in vivo, and for tracking changes in ocular dimensions over time in a variety of eye disease models. We use SDOCT to measure retinal thickness in models of retinal degeneration in diabetic retinopathy, retinal thickness and cupping in a glaucoma model, and axial length in a myopia model. The SDOCT technique is straightforward, but practice is critical in obtaining good images.And experimenting...