QuPath Automated Analysis of Optic Nerve Degeneration in Brown Norway Rats.

PURPOSE

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.

METHODS

QuPath software was adapted to assess axon and gliotic morphology in toluidine blue-stained, Brown Norway rat ON light micrographs. QuPath axon numbers, density, size distributions, and gliotic areas were obtained from test images and ON cross-sections separated by damage grade. QuPath results were compared with manual counting, AxonJ, and electron microscopy axon estimates.

RESULTS

QuPath-derived axon number, density, and diameter decreased with increasing ON damage. Axon density negatively correlated with gliotic areas in test images (= 0.759; 2 µm, which increased in moderately but not severely damaged images. QuPath axon counts correlated strongly with manual counts of test images (= 0.956;< 0.0001). QuPath outperformed AxonJ on test images and total ON axon counts. Compared to electron microscopy analysis, QuPath undercounted ON axons; however, correlation between the methods was robust (= 0.797;< 0.001;= 10).

CONCLUSIONS

QuPath analysis reliably identified axon loss, axon morphology changes, and gliotic expansion that occurred in degenerating ONs.

TRANSLATIONAL RELEVANCE

QuPath is a valuable tool for rapid, automated, analysis of healthy and degenerating ONs. Reproducible preclinical studies for new glaucoma treatments depend on unbiased in-depth analysis of ON pathology. This was provided by the QuPath approach.