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 other OCT devices and animal models. Further, in our lab, these protocols are commonly used in mice and rats; however, the overall approach can be adopted to different animal models and SD-OCT devices provided an individual has the correct lens and capabilities on their device.
1. Set up the optical coherence tomography equipment
<ol>
	<li>Open the SD-OCT software (Table of Materials).</li>
	<li>Define who is taking the OCT, the study, and the treatment arm (if relevant). Name these categories in a way that will help researchers search for the desired scans later during data analysis.
	<ol>
		<li>In the Patient/Exam tab, click Test Examiner. Select the name of the examiner. Use the Setup Examiners &#38; Physicians button to add new examiners.</li>
		<li>Click Study Name to define the study. Click the Study tab to add a new study or modify treatments in an existing study. Click to the right of Select Treatment Arm to select a treatment arm.</li>
	</ol>
	</li>
	<li>Click the Add Patient button, which is used to add a new timepoint for an entire group. When the window appears, enter ID number, First name, and Last name. Select Male or Female. Enter the Date of Birth.</li>
	<li>Click the Add Exam button to add the individual rats. To identify the rats, click on an exam. Click Edit Exam. Enter the ID number into the Enter Notes box. Click the Save Changes button.</li>
	<li>Attach the proper lens to the device (Figure 1B), select the corresponding Configuration in the software, and dial in the associated reference arm position. 
	NOTE: The SD-OCT system described has customized lenses, preset scan patterns, and reference arm settings specific to the animal species and region of eye being imaged (retina or cornea, mouse or rat). Some of these details are specific to the SD-OCT system described (see table of materials). For example, not all devices offer manual adjustment of reference arm pathlength.</li>
	<li>In the Patient/Exam tab, double click the highlighted exam to proceed to the Imaging tab and begin imaging or simply click the Imaging tab. If there is a default scan, right click to delete it.</li>
	<li>Load a pre-set Scan Protocol by clicking the Select a protocol from the list button. Alternatively, add individual scans.</li>
	<li>For rat models of glaucoma and diabetic retinopathy and mouse models of retinal degeneration, choose a pre-set that consists of four images: 2 OD and 2 OS scans. For mouse myopia, choose a pre-set that consists of 8 images: 4 OD and 4 OS scans. 
	NOTE: Pre-set imaging will be explained in more detail in Section 3. This is something each laboratory makes for themselves or with the manufacturer during on-site installation.</li>
</ol>
2. Anesthetize the animal
<ol>
	<li>Administer anesthetic.
	<ol>
		<li>Anesthetize rats with ketamine (60 mg/kg) and xylazine (7.5 mg/kg) via intraperitoneal injection.</li>
		<li>Anesthetize mice with ketamine (80 mg/kg) and xylazine (16 mg/kg) via intraperitoneal injection.</li>
		<li>Wait until animals are fully anesthetized and do not respond to toe pinch.</li>
	</ol>
	</li>
	<li>Administer pupil dilation drops (1% tropicamide). Wait for pupils to dilate before imaging. 
	NOTE: Dilation of pupils increases the field of view but is not a requirement. Local (corneal) anesthetic drops (0.5% tetracaine) to numb the eye should also be used if anything will be touching the eye (for example, if applying contact lenses or using a guide). A guide is a device that is placed over the scan head and helps beginners to line up the eye and the scan head.</li>
	<li>After anesthetizing the rodent, place the rodent in a rodent alignment system that can rotate the animal in 3-dimensional space (Figure 1A, 1C, &#38; 1D). Provide thermal support. 
	NOTE: Currently, we use rodent alignment systems for mice and rats designed and sold with the SD-OCT device.</li>
	<li>Apply liquid (e.g., saline or artificial tears) to keep the eyes lubricated. Ensure that the eye does not dry out during imaging so that the optical properties of the eye are maintained between scans (when the cornea is wet, the retina can be seen clearly).
	<ol>
		<li>Make sure to maintain moisture in the opposite eye when scanning the first eye so it does not dry out.</li>
	</ol>
	</li>
	<li>Use a delicate task wipe to wick away excess saline just before imaging, as too much or too little lubricant on the eye will affect the image quality. 
	NOTE: The use of sterile lubricant gel is not recommended during OCT since it can interfere with imaging. If required, sterile lubricant gel can be used after the procedure. A contact lens can also be applied to ensure adequate moisture on the eye throughout the test. In our experience, a contact lens did not provide a marked improvement in image quality, but contact lenses do help reduce the risk of cornea drying during the imaging session.</li>
</ol>
3. Rodent OCT imaging
<ol>
	<li>Begin with one eye (OS or OD) and image the contralateral eye after.
	<ol>
		<li>Position the animal using the two rotational motions of the rodent alignment system, such that the gaze is horizontal and looking down the axis of the OCT Lens (Figure 1D).</li>
		<li>Use the OCT in Free Run mode to orient the retina for data collection. Use the Aim mode (by clicking the Aim button) initially for a continuous display of both horizontal and vertical B-scans in real time.</li>
		<li>Move the scan head closer to the eye until the retina is visible (as mouse and rat retina lenses are fixed-focus, moving the lens toward the eye focuses deeper into the retina). Then use the rodent alignment system to adjust the animal position up/down and swivel/twist to position the optic nerve head in the center, make the horizontal scan horizontal, and the vertical scan vertical (Figure 1A).</li>
		<li>Adjust the working distance such that the retinal image is flat and not curved.</li>
		<li>Adjust the reference arm position to keep the image near the top of the display window. Be careful not to push in too far or the eye image will flip back on itself.</li>
	</ol>
	</li>
	<li>Retinal imaging
	<ol>
		<li>For glaucoma, retinal degeneration, and diabetic retinopathy models: Define a volume scan that consists of 1000 x 100 x 1 (A scans x B scans x repeated B scans) for averaging. In rats, take a volume scan that is 3 x 3 mm. In mice, take a 1.5 x 1.5 mm volume scan.</li>
		<li>Center the optic nerve in the horizontal and the vertical access so that the volume scan is in the center. Take time to make sure the optic nerve head is at the center of the scan and straight along the nasal-temporal and superior-inferior axes (Figure 2). Scan and re-center to make sure it is exactly in the center, if needed. Repeat this scan as necessary until the optic nerve head is centered and aligned along both axes. Click the Snapshot button to take a photo. 
		NOTE: Some SD-OCT devices have the option of optically manipulating the curvature of the eye (e.g., the image is flattened) by adjusting the distance of the eye from the light source with the reference arm. We recommend flattening and centering the images when taking direct thickness measurements through the retinal layers to improve accuracy along the anterior-posterior direction.</li>
		<li>Click the Save button to save the image.</li>
		<li>Take a radial scan centered at the optic nerve head that is 1000 x 4 x 20 (A-scan x B-scan x repeated B-scans). Use repeated B scans to enhance image clarity of the eye or retina, which will help to interpret regions of the eye or layers of the retina during data analysis. 
		NOTE: Again, in rats this radial scan is 3 mm, while in mice the radial scan is 1.5 mm.</li>
		<li>Save the image.</li>
		<li>Repeat steps 3.1 through 3.2.5 in the contralateral eye.</li>
	</ol>
	</li>
	<li>Axial length measurements
	<ol>
		<li>For projects that involve imaging the whole eye, such as mouse myopia, take three scans of the entire eye and one retina scan for each eye. Choose a pre-set that consists of a radial scan that is 500 x 20 x 1 and encompasses the full diameter of the eye. 
		NOTE: This setting provides an image of the entire length of the mouse eye from the cornea to the choroid.</li>
		<li>Center the middle of the eye and retina in the field of view. Take three radial scans (whole eye scans): a linear B scan that is 1000 x 5 x 2 and two additional linear B scans of 1000 x 5 x 2 at the same location. Save the images.</li>
		<li>Afterward, if desired, zoom in and take a volume or rectangular scan (retina scan) similar to the description in 3.2 that consists of 1000 x 20 A scans x B scans. Save the volume scan.</li>
		<li>Repeat steps 3.3 through 3.3.3 in the contralateral eye. 
		NOTE: Axial length measurements are only possible on small eyes (mouse or smaller) since the imaging window of current systems is not large enough to capture a larger eye.</li>
	</ol>
	</li>
</ol>
4. Post-imaging steps
<ol>
	<li>Store saved data on a cloud, which is good practice for data management and allows for easy access for later analysis. Perform data analysis with custom software developed in a mathematical modeling program (Table of Materials).</li>
	<li>Remove the rodent from the rodent alignment system and give an intraperitoneal injection of atipamezole (1 mg/kg for rats and mice) to reverse the effects of the xylazine, so that the rodent will wake up more quickly.</li>
	<li>Allow rodents to recover on a heating pad on low heat. Give additional saline drops as needed. Return rodents to their home cage when they have regained full ambulation.</li>
	<li>Close program and turn off the OCT.</li>
</ol>
5. Post-processing of OCT images
<ol>
	<li>Process the images using custom software developed in a mathematical modeling program to suit specific OCT needs (e.g., measure the thickness of areas of interest by manually marking the images).</li>
	<li>Depending on the purpose of the image (mouse retina, rat retina, or myopia/axial length), use one of three different programs:
	<ol>
		<li>For processing the retina, select the OCT scans to load. First, define the center of the optic nerve head with a simple click.</li>
		<li>Watch as the program generates vertical lines defining distances on either side of the optic nerve head. Note that in the rat retina, these lines are at 0.5 mm and 1.2 mm away from the center of the optic nerve head, for a total of 4 vertical lines representing the nasal-temporal and inferior-superior axes of the eye depending on the radial B scan currently analyzed. 
		NOTE: In the mouse retina, these vertical lines are at 0.25 mm and 0.5 mm from the optic nerve head center.</li>
		<li>Delineate the following layers along each line: 
		The retinal nerve fiber layer (RNFL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), external limiting membrane (ELM), inner segments/outer segments (IS/OS), retinal pigment epithelium (RPE), and total retinal thickness. 
		NOTE: The radial scan does not typically have nasal/temporal and superior/inferior labels when it is opened. Scans may be created such that they have an n/t and s/I orientation, and those scans in particular are analyzed later.</li>
		<li>After an image has been delineated and the program closed, export these measurements into a spreadsheet software for data analysis.</li>
	</ol>
	</li>
	<li>Use these length and thickness values from step 5 to make comparisons between groups, for example, determining if there are regional differences (n/t/s/i), or longitudinal changes.</li>
	<li>For retinal measurements, first determine if there are any differences in the nasal-temporal and inferior-superior axis at the 0.5 mm and 1.2 mm distances. 
	NOTE: If differences in quadrants are not observed, the 0.5 mm and 1.2 mm measurements may be averaged together. This is a similar approach for the mouse retinal scans only at 0.25 mm and 0.5 mm.</li>
	<li>For myopia studies, use this program to assess the ocular parameters along the optical axis of the eye. Open the mathematical modeling program. First, select an image to load.
	<ol>
		<li>After loading the image, manually mark each scan (radial and B scans). Mark the anterior and posterior edges of the cornea, lens, vitreous chamber, and retina, so that the program will calculate corneal thickness, lens thickness, anterior and vitreous chamber depth, total retinal thickness, total axial length.</li>
		<li>After marking, exit the program which prompts a save menu. Save the delineated values in a spreadsheet software and average the three separate scans together.</li>
	</ol>
	</li>
</ol>

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S., Jia, Y., Huang, D. <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:+Technical+principles+and+clinical+applications+in+ophthalmology.">Optical coherence tomography angiography: Technical principles and clinical applications in ophthalmology.</a> Taiwan Journal of Ophthalmology. 7 (3), 115-129 (2017).</li><li>Treister, A. 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=Prevalence+of+Subclinical+CNV+and+Choriocapillaris+Nonperfusion+in+Fellow+Eyes+of+Unilateral+Exudative+AMD+on+OCT+Angiography.">Prevalence of Subclinical CNV and Choriocapillaris Nonperfusion in Fellow Eyes of Unilateral Exudative AMD on OCT Angiography.</a> Translational Vision Science &amp; Technology. 7 (5), 19 (2018).</li></ol>

The authors have nothing to disclose.

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 ...

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

Research

JoVE Journal

Bioengineering

2.8K Views.  Atlanta Veterans Affairs Medical Center. 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.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

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.

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.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Neuroscience

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 ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

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 ...

Optical Coherence Tomography Imaging for Assessing Uveitis in a Mouse Model

Source: John, S., et al. <a href="https://app.jove.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a> J. Vis. Exp. (2021)
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.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Summary

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Chapters in this video

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

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

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

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

Optical Coherence Tomography Imaging for Assessing Uveitis in a Mouse Model