Qupath automated analysis of optic nerve degeneration in brown Norway rats

Barbara A. Mysona, Sharmila Segar, Cecilia Hernandez, Christian Kim, Jing Zhao, David Mysona, Kathryn E. Bollinger

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


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 (R2 = 0.759; P < 0.0001; N = 40) and in ON cross-sections (R2 = 0.803; P < 0.0004; N = 10). Although axon losses occurred across most axon diameters, large axons were more susceptible to degeneration. The exception was swollen axons > 2 μm, which increased in moderately but not severely damaged images. QuPath axon counts correlated strongly with manual counts of test images (R2 = 0.956; P < 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 (R2 = 0.797; P < 0.001; N = 10). Conclusions: QuPath analysis reliably identified axon loss, axon morphology changes, and gliotic expansion that occurred in degenerating ONs.

Original languageEnglish (US)
Article number22
JournalTranslational Vision Science and Technology
Issue number3
StatePublished - 2020


  • Axons
  • Glaucoma
  • Gliosis
  • Image analysis
  • Optic nerve degeneration

ASJC Scopus subject areas

  • Biomedical Engineering
  • Ophthalmology


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