Of the 10 to 15 toluidine blue-stained ON cross-sections on each slide, one was chosen for light microscope imaging based on the presence of even staining and absence of artifacts. Average IOPs for bead and contralateral eyes were calculated as area under the IOP versus time curve divided by total time of experiment. IOP was monitored two or three times per week with a handheld iCare tonometer (iCare Finland Oy, Vantaa, Finland) in awake, non-anesthetized rats. Naïve eyes were from rats receiving no injection in either eye. Acute IOP elevation (50–60 mm Hg) sometimes accompanied bead injections but dropped to 20 to 40 mmHg within 24 hours. The beads and viscoelastic were rapidly discharged over a period of approximately 5 seconds into the anterior chamber. The syringe was loaded from behind with 10 µl of 6-µm beads, 10 µl of 10-µm beads, and 10 to 20 µl of HEALON5 viscoelastic (Johnson & Johnson Vision Care, Inc., Jacksonville, FL). A 100-µl SGE Gas Tight Syringe, Luer Lock 100F-LL-GT (World Precision Instruments, Sarasota, FL), was coupled to a pre-pulled 30-µm inner diameter glass micropipette tip (#TIP30TW1, World Precision Instruments). The eye was numbed with one drop of proparacaine HCl, 0.5% (Akorn Pharmaceuticals). Rats were anesthetized with a cocktail of 60 mg/kg ketamine (Zoetis, Kalamazoo, MI) and 7.5 mg/kg xylazine (Akorn Pharmaceuticals, Lake Forest, IL). 36 Briefly, microbeads (10 µm and 6 µm 2.7% solid suspension) were sterilized in 100% ethanol, washed three times in sterile phosphate-buffered saline (PBS), and resuspended in sterile PBS at one to four times the manufacturer's supplied concentration, resulting in a final concentration 2.7% to 10.8% by weight. 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. If you add to rock, it will mark as a triangle.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. Angle (orange) Gives the angle between two points. Right click to other options (straight line/ segmented line/ freehand line/ arrow tool, and double click to adjust the parameters. Then click on the Rock classifier.ġ9 Freehand (green) Make a form of your choice and it will fill automatically after you Add to Rock. Polygon (red) It makes straight lines connected to each other to form a polygon. If you double click, appears a tool to adjust the parameters. If you right click, two other options are available: elliptical selections and selection brush tool. Circle (yellow) - Click and drag at the image. If you right click, two other options are available: rounded rectangle and rotated rectangle. Rectangle (blue) - Click and drag at the image. Tools Once the classifiers are settled, use the best segmentation tool for your objective.
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