The video demonstrates a detailed procedure for preparing and documenting eye globes for research. It includes steps for removing anterior segments, marking orientation, using a phosphate buffer, inserting a ruby bead for scale, and capturing high-quality images of the eye's interior using specialized photography techniques.

﻿tissue preparation for ex vivo color fundus photography

הדמיה מולטימודאלית מבוססת OCT של עיני תורם אנושי עבור מחקר AMD

Fifteen years of managing neovascular age-related macular degeneration (AMD) with anti-VEGF therapy under the guidance of optical coherence tomography (OCT) has offered new insights into the progression sequence and microarchitecture of this prevalent cause of vision loss. A key recognition is that AMD is a three-dimensional disease involving the neurosensory retina, retinal pigment epithelium (RPE), and choroid. As a result of the OCT imaging of trial patients and the fellow eyes of treated clinic patients, the features of pathology beyond those seen by color fundus photography, a clinical standard for decades, are now recognized. These include intraretinal neovascularization (type 3 macular neovascularization1, formerly angiomatous proliferation), subretinal drusenoid deposits (SDDs, also called reticular pseudodrusen)2, multiple pathways of RPE fate3,4, and intensely gliotic M&#252;ller cells in atrophy5,6.
Model systems lacking maculas (cells and animals) recreate some slices of this complex disease7,8,9. Further success in ameliorating the burden of AMD could come from the discovery and exploration of primary pathology in human eyes, understanding the unique cellular composition of the macula, followed by translation to model systems. This report portrays a three decade collaboration between an academic research laboratory and an eye bank. The goals of the tissue characterization methods described herein are two-fold: 1) to inform evolving diagnostic technology by demonstrating the basis of fundus appearance and imaging signal sources with microscopy, and 2) to classify AMD specimens for targeted (immunohistochemistry) and untargeted molecular discovery techniques (imaging mass spectrometry, IMS, and spatial transcriptomics) that preserve the cone-only fovea and rod-rich para- and perifovea. Such studies could accelerate the translation to clinical OCT, for which a progression sequence and longitudinal follow-up are possible through eye-tracking. This technology, which is designed to monitor treatment effects, registers scans from one clinic visit to the next using retinal vessels. Linking eye-tracked OCT to laboratory results obtained with destructive techniques could provide a new level of prognostic value to molecular findings.
In 1993, the research laboratory captured color photographs of postmortem fundus on film10. This effort was inspired by the superb photomicroscopy and histology of the human peripheral retina by Foos and colleagues11,12,13&#160;and the extensive AMD clinicopathologic correlations by Sarks et al.14,15. Starting in 2009, ex vivo multimodal imaging (MMI) anchored on spectral domain OCT was adopted. This transition was inspired by the similar efforts of others16,17&#160;and especially by the realization that so much of the ultrastructure described by the Sarks was available in three dimensions, over time, in the clinic18,19. The goal was to acquire eyes with attached maculas in a reasonable time frame for well-powered studies of cellular-level phenotypes in the retina, RPE, and choroid. The intent was to move beyond &#34;per eye&#34; statistics to &#34;per lesion type,&#34; a standard influenced by the &#34;vulnerable plaque&#34; concepts from cardiovascular disease20,21.
The protocol in this report reflects experience with nearly 400 pairs of donor eyes accessioned in several streams. In 2011-2014, the Project MACULA website of AMD histopathology was created, which includes layer thicknesses and annotations from 142 archived specimens. These eyes were preserved from 1996-2012 in a glutaraldehyde-paraformaldehyde fixative for high-resolution epoxy-resin histology and electron microscopy. All the fundi had been photographed in color when received and were reimaged by OCT just prior to histology. An eye holder originally designed for optic nerve studies22&#160;was used to accommodate an 8 mm diameter full-thickness tissue punch centered on the fovea. OCT B-scans through the foveal center and a site 2 mm superior, corresponding to histology at the same levels, were uploaded to the website, plus a color fundus photograph. The choice of the OCT planes was dictated by the prominence of AMD pathology under the fovea23&#160;and the prominence of SDDs in rod-rich areas superior to the fovea24,25.
Starting in 2013, eyes imaged with OCT-anchored MMI during life were available for direct clinicopathologic correlations. Most (7 of 10 donors) involved patients at a retina referral practice (author: K.B.F.), which offered an advanced directive registry for patients interested in donating their eyes after death for research purposes. The eyes were recovered and preserved by the local eye bank, transferred to the laboratory, and prepared in the same way as the Project MACULA eyes. Pre-mortem clinical OCT volumes were seamlessly read in the laboratory, thus aligning the pathology features seen during life with the features seen under the microscope26.
Starting in 2014, prospective eye collection began by screening for AMD in donor eyes without a clinical history but preserved during a defined time limit (6 h). For this purpose, the eye holder was modified to accommodate a whole globe. This reduced the chance of detachment around the cut edges of the previously used 8 mm punch. The eyes were preserved in 4% buffered paraformaldehyde for immunohistochemistry and transferred to 1% the next day for long-term storage. In 2016-2017 (pre-pandemic), 184 eyes from 90 donors were recovered. The statistics and images in this report are generated from this series. During the pandemic era (2020 lockdowns and aftermath), prospective collections for transcriptomics and IMS collaborations continued at a reduced pace, essentially using the 2014 methods.
Other methods for donor eye assessment are available. The Minnesota Grading System (MGS)27,28&#160;is based on the AREDS clinical system for color fundus photography29. The limitations of this method include the combining of atrophic and neovascular AMD into one stage of &#34;late AMD&#34;. Further, the MGS entails the removal of the neurosensory retina before the photo-documentation of the RPE choroid. This step dislodges SDDs to varying degrees30,31&#160;and removes the spatial correspondence of the outer retina and its support system. Thus, efforts to link metabolic demand and signaling from the retina to pathology in the RPE-choroid may be impeded. The Utah System implemented MMI using ex vivo color photography and OCT to categorize eyes destined for dissection into regions for RNA and protein extractions32. Although preferable to whole eyecup extractions, the 3 mm diameter area at the highest risk for AMD progression33,34&#160;represents only 25% of a 6 mm diameter fovea-centered punch. Thus, techniques that can localize findings in reference to the fovea, such as serial sectioning for immunohistochemistry, are advantageous.

מחבר זרקור: Ex Vivo OCT מבוסס הדמיה מולטימודאלית של עיני תורם אנושי למחקר על ניוון מקולרי תלוי גיל  

אקס ויוו הדמיה מולטימודאלית מבוססת OCT של עיני תורם אנושיות למחקר על ניוון מקולרי תלוי גיל

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. Eye-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 maculas 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 before 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.

Watch this Scientific Journal Video about ﻿Tissue Preparation for Ex Vivo Color Fundus Photography at JoVE.com

Tissue Preparation for Ex Vivo Color Fundus Photography  

הכנת רקמות לצילום Ex Vivo Color Fundus

כדי להתחיל, מניחים את כל הגלובוסים שנמצאו מתורם העין בצלחת פטרי מלאה בשעווה. לאחר מזעור ההפרעה של טבעת הזגוגית העבה המחוברת לגוף הריסי, יש להסיר את שאריות המקטע הקדמי. לאחר סימון המוט העליון להתמצאות, מניחים את הגלובוס כאשר הצד הקדמי פונה כלפי מטה בצלחת.לאחר מציאת החיבורים המגמתיים לרקטוס העליון ולשרירים האלכסוניים העליונים, יש למרוח דיו סימון בקו 10 מ"מ בכיוון קדמי-אחורי באמצעות מוליך העץ. מלאו את הפונדוס בחיץ פוספט קר של סורנסן לפני הצילום. הכנס חרוז אודם של מילימטר אחד לתוך הפונדוס כסרגל קנה מידה פנימי.הכנס את הקוטב האחורי של גלובוס העין לתוך כור היתוך קוורץ 30 מיליליטר, באמצעות מלקחיים. עם מצלמת רפלקס עדשה יחידה המורכבת על מיקרוסקופ סטריאו, קבל תמונות צבעוניות באמצעות תאורת Trans, EPI ו- FLASH בשלוש הגדלות סטנדרטיות כדי לצלם תמונות המדגישות אזורים ספציפיים.

tissue-preparation-for-ex-vivo-color-fundus-photography

Research

JoVE Journal

Neuroscience

﻿Tissue Preparation for Ex Vivo Color Fundus Photography

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

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...

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 fundus 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 FLASH illumination at three standard magnifications to capture images highlighting specific areas.

Preparation for Ex Vivo Color Fundus Photography 

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 stereomicroscope 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 o'clock position, the optic nerve head, and the fovea five degrees below the optic nerve head.

הכנה לצילום Ex Vivo Color Fundus 

Image Acquisition for Ex Vivo Optical Coherence Tomography (OCT) and Scanning Laser Ophthalmoscopy (SLO) 

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 an internal code number will appear in the left column. Select the new patient icon and complete the patient data information before clicking Okay. 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 plus OCT on the control module. Allow the laser to acquire a live SLO image of the fundus and OCT-B 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 OCT-B scan appears in the top third 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 an unremarkable macula and hyporeflective, large choroidal vessels with a reflective RPE BL 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 hyper reflective focus, subretinal drusenoid deposits and vitelliform change.Similarly, the macula with atrophic AMD and the macula 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.