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Thomas D. Olsen, Ling Luo, Bonnie Archer, Kyle Jackman, Ying Liu, Krysten Zygmunt, Ross Whitaker, Balamurali K. Ambati; A Novel Method for Determining Choroidal Neovascularization Volumes in vivo. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1687.
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© ARVO (1962-2015); The Authors (2016-present)
Choroidal neovascularization (CNV) volumes calculated by laser confocal microscopy combined with immunostaining is the most common method for CNV analysis. However, it only can be used in vitro and the CNV baseline cannot be evaluated. We developed new software, Seg3D, (University of Utah Center for Biomedical Computing) and determined if it can be used for in vivo CNV volume calculations.
Laser induced CNV and aav.shRNA.sflt subretinal injection induced CNV were developed in C57BL6/j mice as CNV models. After 2 weeks-6 months, CNV was imaged by OCT & FA using Heidelberg Eye Explorer Spectralis HRA+OCT II and subsequently exported into the Seg3D program. The scaling factors for each dimension, x, y & z (µm/pixel), were recorded and the corneal curvature standard was changed from 7.7 to 1.75. Each lesion area, on 2 dimensional images, was outlined using the provided polyline tool in Seg3D. The total number of voxels inside the identified regions were counted and reported by Seg3D. The volume of each OCT image stack was calculated and then normalized by multiplying the number of voxels by the scaling factors for each dimension. Mice were euthanized and prepared for IHC staining immediately after Spectralis images were taken. The same CNV lesions were calculated using scanning laser confocal microscope after immunohistochemistry staining (Isolectin alexa fluor@546, invitrogen), as usual. The same z stack step size was used on both methods. Microsoft Excel was used to analyze the volume calculations of each method as well as the correlation statistic and average difference.
The CNV volume calculated using Seg3D (3.03x106µm3) was, on average, 2.5 times larger than the volumes (1.21 x106µm3) calculated using the laser confocal microscope (n=19, P=0.0006). The correlation statistical analysis showed 0.76 correlation between these two methods.
Seg3D software is the singular method for CNV volume calculations in vivo to date. It can be used to analyze on the animal experiments with a normalized baseline requirement, as well as can be used to evaluate AMD in patients.
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