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

Summary

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.

Abstract

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.

Introduction

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 scale^1,2. 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 µm 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.

Protocol

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, 8^th 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

1. Open the SD-OCT software (Table of Materials).
2. 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.
   1. In the Patient/Exam tab, click Test Examiner. Select the name of the examiner. Use the Setup Examiners & Physicians button to add new examiners.
   2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. Load a pre-set Scan Protocol by clicking the Select a protocol from the list button. Alternatively, add individual scans.
8. 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.

2. Anesthetize the animal

1. Administer anesthetic.
   1. Anesthetize rats with ketamine (60 mg/kg) and xylazine (7.5 mg/kg) via intraperitoneal injection.
   2. Anesthetize mice with ketamine (80 mg/kg) and xylazine (16 mg/kg) via intraperitoneal injection.
   3. Wait until animals are fully anesthetized and do not respond to toe pinch.
2. 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.
3. After anesthetizing the rodent, place the rodent in a rodent alignment system that can rotate the animal in 3-dimensional space (Figure 1A, 1C, & 1D). Provide thermal support.
   NOTE: Currently, we use rodent alignment systems for mice and rats designed and sold with the SD-OCT device.
4. 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).
   1. Make sure to maintain moisture in the opposite eye when scanning the first eye so it does not dry out.
5. 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.

3. Rodent OCT imaging

1. Begin with one eye (OS or OD) and image the contralateral eye after.
   1. 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).
   2. 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.
   3. 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).
   4. Adjust the working distance such that the retinal image is flat and not curved.
   5. 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.
2. Retinal imaging
   1. 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.
   2. 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.
   3. Click the Save button to save the image.
   4. 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.
   5. Save the image.
   6. Repeat steps 3.1 through 3.2.5 in the contralateral eye.
3. Axial length measurements
   1. 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.
   2. 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.
   3. 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.
   4. 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.

4. Post-imaging steps

1. 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).
2. 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.
3. 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.
4. Close program and turn off the OCT.

5. Post-processing of OCT images

1. 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).
2. Depending on the purpose of the image (mouse retina, rat retina, or myopia/axial length), use one of three different programs:
   1. For processing the retina, select the OCT scans to load. First, define the center of the optic nerve head with a simple click.
   2. 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.
   3. 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.
   4. After an image has been delineated and the program closed, export these measurements into a spreadsheet software for data analysis.
3. 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.
4. 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.
5. 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.
   1. 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.
   2. 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.

Results

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...

Discussion

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...

Materials

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