Association of Drusen Phenotype in Age-Related Macular Degeneration from Human Eye-Bank Eyes to Disease Stage and Cause of Death.

PURPOSE: To stage maculopathy, assess and quantify drusen, determine drusen subtype frequency, and compare subtypes with age-related macular degeneration (AMD) stage and cause of death using an eye-bank model of AMD. DESIGN: Cross-sectional study. PARTICIPANTS: Two thousand ninety-two human eyes from 1067 eye-bank donors, selected from a population at risk for AMD. METHODS: We analyzed donor eye tissue images (2005-2020) using both the 4- and 9-step Minnesota Grading System (MGS), an AMD grading system for eye-bank eyes corresponding to the Age-Related Eye Disease Study classification. The 9-step MGS quantifies total drusen area, hyperpigmentation, and depigmentation. We analyzed reticular pseudodrusen (RPD), basal laminar drusen (BLD), and calcified drusen (CaD) frequency within this population and explored associations with AMD stage, donor age, gender, and cause of death. Statistical analyses were performed using Wilcoxon rank-sum and chi-square tests. Testing encompassed staging eye-bank eyes using MGS analysis. MAIN OUTCOME MEASURES: Drusen subtype frequency associations with AMD stage and cause of death. RESULTS: We detected RPD in 228 (13%), BLD in 131 (7%), and CaD in 84 (5%) of the examined eyes (n = 1777). All subtypes were associated with advanced AMD (RPD: odds ratio [OR], 3.4 [95% confidence interval (CI), 2.5-4.5; P < 0.0001]; BLD: OR, 2.2 [95% CI, 1.5-3.2; P < 0.0001]; and CaD: OR, 39.1 [95% CI, 16.8-91.0; P < 0.0001]). Only the RPD subtype was associated statistically with cardiovascular death when compared with those without cardiovascular death (48% vs. 32%; OR, 2.0 [95% CI, 1.4-2.9]; P = 0.0002). CONCLUSIONS: In a large group of eye-bank eyes selected from a population at risk for AMD and graded using the 4-step and 9-step MGS, RPD, BLD, and especially CaD were associated strongly with advanced AMD. The RPD subtype was associated with a cardiovascular cause of death and may represent an ophthalmologic biomarker for cardiovascular disease.

The Nine-Step Minnesota Grading System for Eyebank Eyes With Age Related Macular Degeneration: A Systematic Approach to Study Disease Stages.

Purpose: To refine the Minnesota Grading System (MGS) using definitions from the Age-Related Eye Disease Studies (AREDS) into a nine-step grading scale (MGS-9). Methods: A nine-step grading scale descriptive analysis using three key phenotypic features (total drusen area, increased, and decreased pigmentation) of human eyebank eyes that were graded according to definitions from the AREDS criteria in order to harmonize studies of disease progression for research involving human tissue. From 2005 through February 2017, we have analyzed 1159 human eyes, procured from two eyebanks. Each macula was imaged using high-resolution, stereoscopic color fundus photography with both direct- and transillumination. Fundus images were digitally overlaid with a grading template and triangulated for foveal centration. Results: We documented and stratified risk for each globe by applying the AREDS nine-step grading scale to the key clinical features from the MGS-9. We found a good distribution within the MGS categories (1-9) with few level eight globes. Eyes were processed within 12.1 ± 6.3, hours from the time of death through imaging, dissection, and freezing or fixation. Applying the MGS-9 to 331 pairs (662 eyes were simultaneously graded), 84% were within one-grading step and 93% within two steps of the fellow eye. We also document reticular pseudodrusen, basal laminar drusen, and pattern dystrophy. Conclusions: The MGS nine-step grading scale enables researchers using human tissue to refine the risk assessment of donor tissue. This analysis will harmonize results among researchers when grading human tissue using MGS criteria. Most importantly, the MGS-9 links directly to the known risk for progression from the AREDS.

Corneal Endothelial Cell Integrity in Precut Human Donor Corneas Enhanced by Autocrine Vasoactive Intestinal Peptide.

PURPOSE: To demonstrate that vasoactive intestinal peptide (VIP), a corneal endothelial (CE) cell autocrine factor, maintains the integrity of corneal endothelium in human donor corneoscleral explants precut for endothelial keratoplasty. METHODS: Twelve paired human donor corneoscleral explants used as control versus VIP-treated explants (10 nM, 30 minutes, 37°C) were shipped (4°C) to the Lions Eye Institute for Transplantation and Research for precutting (Moria CBM-ALTK Keratome), shipped back to the laboratory, and cultured in ciliary neurotrophic factor (CNTF, 0.83 nM, 37°C, 24 hours). Trephined endothelial discs (8-8.5 mm) were analyzed for differentiation markers (N-cadherin, CNTF receptor α subunit [CNTFRα], and connexin 43) by Western blot after a quarter of the discs from 4 paired explants were cut away and stained with alizarin red S for microscopic damage analysis. Two additional paired explants (6 days in culture) were stained for panoramic view of central CE damage. RESULTS: VIP treatment increased N-cadherin and CNTFRα levels (mean ± SEM) to 1.38 ± 0.11-fold (P = 0.003) and 1.46 ± 0.22-fold (P = 0.03) of paired controls, respectively, whereas CE cell CNTF responsiveness in upregulation of connexin 43 increased to 2.02 ± 0.5 (mean ± SEM)-fold of the controls (P = 0.04). CE damage decreased from (mean ± SEM) 10.0% ± 1.2% to 1.6% ± 0.3% (P < 0.0001) and 9.1% ± 1.1% to 2.4% ± 1.0% (P = 0.0006). After 6 days in culture, the damage in whole CE discs decreased from 20.0% (control) to 5.5% (VIP treated). CONCLUSIONS: VIP treatment before precut enhanced the preservation of corneal endothelium.

The effect of postmortem time on the RNA quality of human ocular tissues.

PURPOSE: Profiling gene expression in human ocular tissues provides invaluable information for understanding ocular biology and investigating numerous ocular diseases. Accurate measurement of gene expression requires high-quality RNA, which often is a challenge with postmortem ocular tissues. METHODS: We examined the effect of various death to preservation (DP) times on the RNA quality of ten different ocular tissues. We used 16 eyes from eight different human donors. The eyes were preserved immediately in RNAlater or preserved after initial storage at 4 °C to create a range of DP times from 2 to 48 h. Ten ocular tissues were dissected from each eye. After total RNA was extracted from each dissected ocular tissue, the RNA integrity number (RIN) was determined using an Agilent Bioanalyzer. RESULTS: The RIN values from corneal and trabecular meshwork tissues were significantly (p<0.05) higher than those from the ciliary body at an earlier DP time (<6 h), but were not different among all tissues after 8 h. Interestingly, the RIN values from non-vascularized tissues were significantly (p=0.0002) higher than those from vascularized ocular tissues at early DP times (<6 h). The RIN value from the cornea was significantly (p<0.05) higher at short DP times compared to longer DP times. The RIN values from corneal tissues were significantly correlated to DP time according to regression analysis (p<0.05). CONCLUSIONS: In this study, we determined RNA quality from postmortem ocular tissues with various DP times. Our results emphasize the need for rapid preservation and processing of postmortem human donor eye tissues, especially for vascularized ocular tissues.