Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

This protocol enables new treatments and preventions for age-related macular degeneration, or AMD. This is the largest disease with a central neurodegeneration, and yet, we think it's the most approachable because of the precise structure of the eye and the availability of cellular-level clinical imaging of the retina. The neovascular complications of the underlying disease of AMD have been treated successfully for 15 years in the 15%of patients who have it.

Recently, the FDA approved the first drug for the end stage of the underlying disease. Single cell RNA sequencing has revealed the molecular repertoire of the 100 plus cell types in the retina and supporting choroidal vasculature. I tracked Optical Coherence Tomography, or OCT, has enabled us to glimpse a detailed cellular level progression sequence in the clinic.

Existing cell and animal model systems for AMD research are non predictive of human pathology and progression risk. This impedes the development of new treatments and preventions. Our lab contributed many top-level findings about AMD, including the early loss of broad photoreceptors required for night vision.

It was also observed that better preserved macular with attached neurosensory retinas are directly relevant to clinical OCT imaging. Many parts of AMD pathology became visible with clinical OCT, which shows cross-sectional views of the retina and choroid. The OCT results of human donor eyes can be directly compared to findings in clinical imaging.

If laboratories could emulate clinical imaging standards by using OCT to characterize eyes or assays, and laboratory findings like gene expression or proteomics, and leverage the longitudinal timeline of clinical imaging to focus on the most informative risk indicators. To begin, place the whole globes recovered from the eye donor in the Petri dish filled with wax. After minimizing the perturbation of the ring of thick vitreous attached to the ciliary body, remove the anterior segment remnants.

After marking the superior pole for orientation, place the globe with the anterior side facing down in the dish. After finding the tendinous insertions for the superior rectus and superior oblique muscles. Apply a marking ink in a 10 millimeter line in an anterior to posterior direction using the wooden applicator.

Fill the fungus with cold Sorensen's phosphate buffer before photography. Insert a one millimeter ruby bead into the fundus as an internal scale bar. Insert the posterior pole of the eye globe into a 30 milliliter quartz crucible using forceps.

With a single lens reflex camera mounted to a stereo microscope, acquire color images using Trans-Epi and flashed illumination at three standard magnifications to capture images highlighting specific areas. To begin the ex vivo color fundus photography, set the high-definition multimedia interface camera to the manual ISO function. Arrange two light sources perpendicular to each other, and turn the epi-illumination light sources to full power.

At the dissecting microscope, insert the one millimeter ruby bead into the posterior pole. Next, insert the posterior pole of the eye globe into a 30 milliliter quartz crucible filled with buffer. When the tissue sinks, insert a bracing between the eye and the wall of the crucible.

Place the globe in the crucible onto the stereo microscope stage to observe the interior ocular fundus through the microscope. Using the lowest magnification, orient the eye by identifying the tissue mark at the 12:00 position, the optic nerve head and the fovea five degrees below the optic nerve head. To begin, switch on the laser ophthalmoscopy instrument.

Position the OCT head by moving the entire unit and raising its height. Focus the image by rotating the knob. Then, lock down the position of the head by turning the thumbscrew.

Insert the eye sample into the holder. Manually adjust the eye position in the holder, and stabilize it with spacers without indenting the sclera. Orient the holder to face the superior rectus muscle up.

After opening the proprietary visualization and analysis software for the OCT device, a patient list indexed by internal code number will appear in the left column. Select the new patient icon and complete the patient data information before clicking OK.Once done, select the operator and the study from the dropdown menu. After viewing, a blank screen, press the yellow button on the control panel to start the image acquisition.

Select IR OCT on the control module. Allow the laser to acquire a live SLO image of the fundus and OCTB scan. Manipulate the OCT laser head to acquire a relative position and focus with a black knob on the unit.

Rotate the round black disc on the control panel to adjust the intensity. Use the black disc to average 100 frames. Once the fundus is in focus, the OCTB scan appears in the top 1/3 of the display.

Use the cursor to move the blue line present on the fundus image to center the fovea before pressing Acquire. Select IR and the volume setting on the control module to acquire the OCT volumes. Adjust all the OCT control and scan settings.

Once the near infrared reflectance fundus view is covered in blue B-scan lines, recheck the OCT position in the top right window and click Acquire on the control module to complete the volume scan. Once the image acquisition is complete, select Exit to save the image. After processing, the image will appear on the screen.

To begin the scanning laser ophthalmoscopy, bring the eye globe placed in the holder to the scanning laser ophthalmoscope. On the ophthalmoscope, select the R position. Then, select the IR mode to align and orient the globe.

Orient the camera head. Focus the image by rotating the knob. Turn and lock down the position of the head with the thumbscrew.

Then, adjust the black lever in position R.Once the image is focused, move the selector knob from R to A.Select ICGA, 100%intensity, a 30-degrees field of view, and single phase imaging. After pressing the black disc for averaging, select Acquire. The ex vivo imaging of unremarkable macula obtained from a donor revealed in unremarkable macula and hyporeflective large choroidal vessels.

With a reflective RPE BBL Brooks Band between the two. The multimodal ex vivo imaging of a macula with early intermediate age-related macular degeneration, or AMD, revealed a closeup view of the fovea showing hyperpigmentation corresponding to the vitelliform lesion. In the early intermediate AMD, visible features included soft drusen with a hyporeflective line at the base, a hyporeflective focus, subretinal drusenoid deposits, and vitelliform change.

Similarly, the macula with atrophic AMD and the macular with macular atrophy secondary to neovascular AMD were studied. Also, the ex vivo OCT imaging of the donor's eyes facilitated the study of common artifacts in postmortem tissues.