The Utah Protocol for Postmortem Eye Phenotyping and Molecular Biochemical Analysis

Abstract

Purpose: Current understanding of local disease pathophysiology in AMD is limited. Analysis of the human disease-affected tissue is most informative, as gene expression, expressed quantitative trait loci, microenvironmental, and epigenetic changes can be tissue, cell type, and location specific. Development of a novel translational treatment and prevention strategies particularly for earlier forms of AMD are needed, although access to human ocular tissue analysis is challenging. We present a standardized protocol to study rapidly processed postmortem donor eyes for molecular biochemical and genomic studies.

Methods: We partnered with the Utah Lions Eye Bank to obtain donor human eyes, blood, and vitreous, within 6 hours postmortem. Phenotypic analysis was performed using spectral-domain optical coherence tomography (SD-OCT) and color fundus photography. Macular and extramacular tissues were immediately isolated, and the neural retina and retinal pigment epithelium/choroid from each specimen were separated and preserved. Ocular disease phenotype was analyzed using clinically relevant grading criteria by a group of four ophthalmologists incorporating data from SD-OCT retinal images, fundus photographs, and medical records.

Results: The use of multimodal imaging leads to greater resolution of retinal pathology, allowing greater phenotypic rigor for both interobserver phenotype and known clinical diagnoses. Further, our analysis resulted in excellent quality RNA, which demonstrated appropriate tissue segregation.

Conclusions: The Utah protocol is a standardized methodology for analysis of disease mechanisms in AMD. It uniquely allows for simultaneous rigorous phenotypic, molecular biochemical, and genomic analysis of both systemic and local tissues. This better enables the development of disease biomarkers and therapeutic interventions.

Figure 1

Butterfly dissection of the posterior donor eye. The posterior globe is cut into the depicted butterfly pattern, including sclera, RPE/choroid, and retina. Incisions are made from the anterior opening, between the attachment points for each extraocular muscle, and continue toward the posterior of the eye until the four lobes of the eye lay flat, with macula and optic nerve in the center. Macular retinal tissue is collected using an 8-mm disposable biopsy punch centered over the fovea as depicted. Additionally, a 6-mm punch is used to cut a button of RPE/choroid from within the 8-mm punch, which minimizes retina contamination to the RPE.

Figure 2

Color fundus imaging in a normal donor eye. Following the butterfly dissection but prior to macular tissue isolation, retinal images are obtained for each eye at 0.7× (A) and 1.25× (B) magnifications using an Olympus SZX16 microscope camera illuminated with a Schott KL 1600 LED Fiber Optic Light Source Illuminator. Images are taken in the same orientation as done in a clinical setting for highest translational quality. As shown, detailed optic nerve, macula, fovea, and posterior pole vasculature can be seen.

Figure 3

Color fundus and OCT imaging of normal and diseased donor eyes provides precise visualization of AMD features. Analogous or the same eye that was used for color fundus image that is also used for the corresponding SD-OCT image as shown demonstrating (A) normal findings, (B) intermediate nonexudative AMD, (C) neovascular AMD, and (D) central geographic retinal atrophy. Color images are used to assign a classic AREDS disease classification. SD-OCT allows for clarification of drusen as seen in B or a neovascular membrane as confirmed in C and labeled with arrows. Please note the white circle present in the en face is an imaging artifact from the light source throughout.