Computational protocol: In Vivo Multimodal Imaging of Drusenoid Lesions in Rhesus Macaques

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Protocol publication

[…] Digital color fundus photographs were evaluated by two experienced image graders (GY, ET) using ImageJ (version 1.49v; National Institutes of Health, Bethesda, MD). Drusenoid lesions were categorized using the Age-Related Eye Disease Study 2 (AREDS2) system for classification of AMD, which has been previously validated for grading of digital fundus photographs in human clinical trials with high reproducibility. Grading involves a modified grid template adapted from the Early Treatment Diabetic Retinopathy Study (ETDRS), with a set of graduated circles used to estimate maximum drusen size and total area involved by pigment abnormalities and drusen (Fig. ). The grid template was calibrated in these animals based on the ocular dimensions of the optic disc in rhesus macaques, which measures 1400 μm vertically and 1000 μm horizontally, compared to 1800 μm in both dimensions in humans. The radii of the modified grid template circles remain as 1/3 DD, 1 DD, and 2 DD based on the vertical disc diameter, but are equivalent to 467 μm, 1400 μm, and 2800 μm respectively (Fig. ). Rhesus and human studies can be compared by applying a scaling constant of 0.778 for linear measurements, and 0.605 for area measurements. Lesions graded include area of hyperpigmentation or hypopigmentation; maximum size and area of drusen within the grid; presence of calcified drusen, drusenoid pigment epithelial detachment, or reticular drusen; presence, area, configuration, and center-involvement of geographic RPE atrophy; and presence of neovascular AMD (Supplemental Table ). The details of the grading criteria have been extensively described,. Any discrepancies in grading were resolved by open adjudication between the graders. Manual drusen segmentation of the fundus photographs was then performed by the two graders by outlining individual lesions within the inner and outer ETDRS circle using the freehand selection tool in ImageJ as previously described and calibrated to the vertical diameter of the optic disc. All measurements were determined from the mean of the two graders’ measurements, and include the number of drusenoid lesions, total area of lesions, average lesion size, and size of largest lesion (Fig. ).Figure 1 The SD-OCT images were exported from Heidelberg Explorer software (version, Heidelberg Engineering, Heidelberg, Germany) to Duke Optical Coherence Tomography Retinal Analysis Program (DOCTRAP, version 62.0),, a custom image segmentation software designed using MATLAB (Mathworks, Natick, MA). For thickness measurements of retinal and choroidal layers, segmentation boundaries were automatically determined by DOCTRAP, followed by manual refinement by two experienced graders (CM, BW) along a 3 mm horizontal segment centered on the fovea. Measured layers include nerve fiber layer (NFL), ganglion cell/inner plexiform layers (GCL/IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor inner and outer segments (IS/OS), retinal pigment epithelium (RPE) which includes any drusenoid complex between the RPE and Bruch’s membrane, as well as the choriocapillaris (CC), and outer choroid (OC). The GCL/IPL were measured as a single complex due to difficulty in distinguishing between these layers even in normal retinal scans. The total retinal thickness was measured from the internal limiting membrane to Bruch’s membrane, and total choroidal thickness was measured from Bruch’s membrane to the choroidal-scleral junction as previously described,. Measurements were determined from the mean of the two graders’ measurements, and then averaged across the central 3mm for thickness comparisons.Correlation in drusen size and area between left and right eyes was determined using two-tailed Pearson correlation. Differences in chorioretinal layer thickness measurements and differences in lesion number, size, and area were compared using linear regression analysis with generalized estimating equations to account for two eyes evaluated from each animal. The association of AREDS2 grading with segmented measurements was also determined using linear regression with generalized estimating equations. [...] Genomic DNA from blood or buffy coat was extracted with a commercial kit (DNeasy Blood and Tissue Kit; Qiagen, Hilden, Germany) following the manufacturer’s protocol, and quantified using a spectrophotometer (NanoDrop 2000c; Thermo Fisher Scientific, Waltham, MA, USA). The region of interest was amplified using the following primers HTRA1-Forward 5′-TATCACTTCACTGTGGGTCCGG-3′, HTRA1-Reverse 5′-GCGATTCGCGTCCTTCAAACTA-3′, ARMS2-Exon1-Forward 5′-GATGGCAGCTGGCTTGGCAAGG, ARMS2-Exon1-Reverse 5′-GGGGTAAGGCCTGATCATCTGCA-3′ with a high-fidelity DNA polymerase (Phusion or Q5; New England Biolabs, Ipswich, MA, USA) and purified using a PCR purification kit (QIAquick; Qiagen). Sanger sequencing of PCR products was performed by the University of California, Davis DNA Sequencing facility (Davis, CA, USA). Association of single nucleotide polymorphisms with drusen phenotype was determined using a Fisher’s Exact test. All statistical analyses were performed using SPSS Statistics (version 22, IBM). […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Macaca mulatta, Homo sapiens
Diseases Brain Diseases, Eye Diseases, Macular Degeneration, Retinal Diseases