Optical coherence tomography is widely used in the clinical diagnosis of different retinal diseases, but it is still challenging to apply in mouse models. This protocol describe how to manage the OCT technique in animals. This technology can be applied to animals with different eyeball sizes.
It enables non-invasive, high-resolutions, and real-time retinal cross-sectional imaging, along with quantitative measurements of retinal thickness. OCT assists in retinopathy diagnosis by identifying the lesion features such as location, range, shape, and size of the lesions, as well as all suspected retinal abnormalities such as retinal neovascularization. This protocol is applicable to animal studies or fundus diseases, including and retinal diseases.
Before starting the procedure, be prepared and follow the protocol step by step. Perform the experiment when the animal is anesthetized. One should be able to obtain good quality OCT images after some practice.
To begin, switch on the computer and start up the software. Click the test program button to complete the test program. Then, turn on the thermostat and preheat it to 37 degrees Celsius.
After the program testing, start the OCT module procedure. Create a new subject and fill in the mouse information. Then, preheat the electric blanket and cover it with surgical towels.
Place the anesthetized mouse on the electric blanket platform and immediately coat both eyes with medical sodium hyaluronate gel. Next, place a 100D contact lens on the mouse cornea with the concave side touching the sodium hyaluronate gel on the corneal surface. Then, place the mouse on the small, constant-temperature animal platform of the cSLO device.
Adjust the angle of the contact lens with forceps to keep the pupil in the center of the lens. Fine-tune the adjustments to the head to make the eye face straight ahead. Click the OCT button, choose the mouse module, and start the cSLO program.
Then, use the operating lever to slowly move the preset lens towards the contact lens. Make further adjustments to align the image of the anterior-posterior pole, centering it at the optic nerve head. Next, start the OCT program and click the progress bar up and down until the OCT image appears.
Adjust the minimum range between 0 to 20 and maximum range between 40 to 60. Then, adjust the preset lens distance and position direction until an ideal OCT image is obtained. Select the scanning position by moving the standard line in the cSLO.
Then, click average to overlay the cSLO and OCT image signal and click the shot button to acquire the SLO OCT image. After the experiment, remove the 100D contact lens and apply the levofloxacin eye gel to protect the cornea. For retinal stratification correction, click load examination on the OCT interface and call out the OCT images from a pop-up window.
Then, double-click the image in the media container to display it on the screen. Next, click on layer detection to complete automatic layering on the retina. After selecting the dividing line on both sides of the layer prepared for analysis, click edit layer to adjust spacing and limit range.
Then, modify the line by moving the red circle. To measure the retinal lamination thickness, click the measure marker button and select the dividing line of the layer to be analyzed to display the boundary of the layer on the OCT image. Then, select connect with layer and stay connected on move.
Next, select the area to display the results and click the position to be analyzed on the OCT image to make the measurement line appear. Click on the next column for subsequent measurements and to reveal the previous data. To measure full retinal thickness, select line one and line seven in the list in the upper right corner.
Then, from the appearance of retinal structure at the edge of the optic papilla, measure four values at 200 micrometer-spacing intervals on the horizontal ruler. OCT images of C57BL/6 mice showed various retinal layers with clear demarcation. In contrast, Vldlr knockout mice showed abnormal hyperreflective lesions.
The OCT images showed some middle reflective bands on the retinal surfaces of the Vldlr knockout mice. These bands adhered to the retinal vessel, being consistent with the OCT characteristics of incomplete vitreous detachment. One hyperreflective lesion appeared on the subretinal space and spread to the outer nuclear layer, but did not break through the outer plexiform layer.
The OCT appearance of this lesion was consistent with pathological findings. The pathological section showed that neovascularization broke through the retinal pigment epithelium layer, photoreceptor inner-outer segments, and the external limiting membrane, invading the outer nuclear layer, but not the outer plexiform layer. Another band of hyperreflective lesion, located at the subretinal space, did not involve the outer nuclear layer.
Consistent with pathological findings, this subretinal neovascularization did not break through the external limiting membrane. Comparing the retinal thickness of the right eye in the temporal, nasal, superior, and inferior directions of the posterior polar between the two groups revealed that the retinal thickness of Vldlr knockout mice was significantly lower than that of the C57BL/6J mice. When performing the protocol, properly place a 100D contact lens on the cornea, then adjust the image position.
Select the scanning location and overlay the image before shooting. Following this procedure, anterior segment OCT is feasible, requiring changing the distance between the preset lens and cornea. It can show the cornea, anterior chamber, iris, ciliary body, and lens.
This technique provides an important tool for retinopathy research in small animal models, promoting non-invasive detection and measurement of retinopathy in basic research.
