The overall goal of this experiment is to use optical coherence tomography or OCT to quickly obtain an accurate assessment of retinal thickness. OCT can help answer key questions in the field of ophthalmology such as, optic neuropathy. The main advantage of this technique is that, it is a non-invasive method for detecting structural changes in the mouse retinal ganglion cells.

This technique is really interesting because it is non-invasive, safe and reputable method to test the effect of treatment in the optic neuropathy. To begin, first adjust the reference arm settings appropriate for the lens. Next, turn on the power supply and the computer.

When the computer is ready, launch the imaging program. Create a new clinical study and add desired protocols. Go to patient exam section and set exam defaults.

Click, add patient in the patient exam section to add a new patient and enter the relevant patient information. Then click, add exam, start exam and add preset scans from the list to select the desired protocol starting with the eye that will be measured first. At least 15 minutes before the data acquisition, instill drops of room temperature 10%phenylephrine into each eye of all the mice that will be tested.

Then remove the excess reagent and instill drops of room temperature 0.5%tropicamide. Approximately five minutes before the acquisition, induce general anesthesia. Immediately after, administer 0.4%oxybuprocaine hydrochloride to each eye, leaving the solution in place for three seconds to anesthetize and immobilize the eyes.

Then, remove the drops and repeat the 10%phenylephrine and 0.5%tropicamide installation. When the eyes are sufficiently dilated for the imaging, lubricate the eyes with viscous glycol based eye drops to provide corneal hydration and wrap the mouse in a sheet of surgical gauze to keep it warm. Using a sponge or cotton wick, apply a thin layer of ophthalmic gel supplemented with 0.3%hypromellose onto each eye.

While doing so, set the eyelashes and whiskers aside. Position the mouse in the cassette with the head straight and pointing forward and carefully place the bite bar in its mouth. Place a cotton roll under the side of the animal for the eye that is being examined.

Then, keeping the clip on aiming tip on the mouse specific lens, rotate the z translator screw counter clockwise to bring the lens towards the eye. Preset the position of the mouse by rotating and swiveling the cassette and turning the bite bar and the x-translator screws until the experimental eye is positioned to look directly into the lens. When the optical axis of the eyes and the lens are aligned, select the first scan in the exam.

Click start aiming and use the z-translator screw to move the retina vertically in the left panel and horizontally in the right panel. By rotating the cassette, move the optic nerve head up or down to bring it into the middle of the right panel. Use the bite bar screw to straighten the retina within the right panel and swivel the cassette to position the optic nerve head in the middle of the left panel.

Then use the x-translator screw to level the retina within the left panel and keeping in mind the major function of each modulator, further adjust the position of the retina for the perfect centralization on the optic nerve head. When the retina is in position, click start snapshot to begin the spectral domain optical coherence tomography scanning. Do not forget to save the scan and the report.

Adjust the centralization if necessary. When the scanning is complete, retract the lens and position the second eye as just demonstrated. After the second eye has been scanned, remove the mouse from the cassette, apply ophthalmic gel with 0.3%hypromellose to each eye and place the mouse on a heating plate with monitoring until full recovery.

To focus on retinal ganglion cells, use the MRI retina tool macro created for image J.Open the MRI retina tool and modify polygon selection tool and load the image of interest. Click M to begin the measurement, then click E to adjust the horizontal position. Select the first cassette in the newly opened region of interest manager window.

Click on the blue polygon and click on the borders of the measured layer in the picture to adjust the position of the first cassette. Select the second cassette in the region of interest manager window and adjust the second cassette. Then click R, to re-measure the retina and view the results in the measurements window.

Even though the quality of a spectral domain OCT scan is not as good as that of an image of a retinal cross section, the OCT allows more layers to be visualized. The retina layers are easily measured in an OCT scan and encompass the retinal nerve fiber in ganglion cell layers, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner and outer segment layers retina pigment epithelium and the choroid, allowing a complex study of the entire retina. OCT measurement of retinal thickness is just demonstrated, indicates that visually impaired genetically mutant OPA1 female mice exhibit a progressive thickening of the ganglion cell complex and the peripapillary retina nerve fiber layers.

A comparison of the retina nerve fiber in ganglion cell layer thickness using standard and homemade calipers, demonstrates that standard calipers measure a significantly lower thickness than do homemade calipers, as the standard calipers are much thicker and more difficult to place on a thin layer border. Once mastered, this technique can be done in 15 minutes if performed correctly. After its development, OCT paved the way for the researchers in the field of ophthalmology to analyze structural changes in the mouse retina.

After watching this video, you should have a good understanding of how to perform and analyze OCT imaging for retina ganglion cells in mice.