To induce ON degeneration in a subset of ONs, intraocular pressure (IOP) was raised by injecting polystyrene microbeads (Polysciences, Inc., Warrington, PA) into the anterior chamber. This was provided by the QuPath approach. Reproducible preclinical studies for new glaucoma treatments depend on unbiased in-depth analysis of ON pathology. QuPath is a valuable tool for rapid, automated, analysis of healthy and degenerating ONs. QuPath analysis reliably identified axon loss, axon morphology changes, and gliotic expansion that occurred in degenerating ONs. Compared to electron microscopy analysis, QuPath undercounted ON axons however, correlation between the methods was robust ( R 2 = 0.797 P < 0.001 N = 10). QuPath outperformed AxonJ on test images and total ON axon counts. QuPath axon counts correlated strongly with manual counts of test images ( R 2 = 0.956 P < 0.0001). Axon density negatively correlated with gliotic areas in test images ( R 2 = 0.759 P 2 µm, which increased in moderately but not severely damaged images. QuPath-derived axon number, density, and diameter decreased with increasing ON damage. QuPath results were compared with manual counting, AxonJ, and electron microscopy axon estimates. QuPath axon numbers, density, size distributions, and gliotic areas were obtained from test images and ON cross-sections separated by damage grade. QuPath software was adapted to assess axon and gliotic morphology in toluidine blue-stained, Brown Norway rat ON light micrographs. A novel application of QuPath open-source digital analysis software is used to provide in-depth morphological analysis of progressive optic nerve (ON) degeneration in rats.
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