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

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.