For each well, phase-contrast images of four random fields of DRG axons were obtained using a 20× objective lens and an inverted microscope (Eclipse TE 300; Nikon). The images were captured using a 20 ms exposure on a CCD camera (Cool SNAP ES; Nikon) with MetaMorph image analysis software (Molecular Devices). For computer-based image analysis, it is important that the axon tracts be well separated; we therefore randomly selected fields of axons that were 1.5–3 mm away from the edge of the neuronal cell body cluster. To obtain images with an uniform background intensity, the gray level of the images was adjusted using the auto level function of Adobe Photoshop.
To quantify the axonal degeneration, we measured the area occupied by axons (total axon area) and by degenerated axons at various times after injury. The phase-contrast images were binarized such that pixel intensity of regions corresponding to axons were converted to black and all other regions were converted to white. The total number of black pixels was then defined as the total axon area. Healthy intact axons show a continuous tract, whereas degenerated axons have a particulate structure due to axonal fragmentation or beading. To detect degenerated axons, we used the particle analyzer module of ImageJ and counted the area of the small fragments or particles (size = 20–10,000 pixels) derived from the degenerated axons. A degeneration index (DI) was calculated as the ratio of fragmented axon area over total axon area. The production of the binary images and the function of the particle analysis as well as the accuracy of the DI in detecting axonal degeneration were optimized using multiple images of intact versus degenerating axons obtained from a large number of experiments using our DRG in vitro axotomy system.
To quantify the axonal degeneration, we measured the area occupied by axons (total axon area) and by degenerated axons at various times after injury. The phase-contrast images were binarized such that pixel intensity of regions corresponding to axons were converted to black and all other regions were converted to white. The total number of black pixels was then defined as the total axon area. Healthy intact axons show a continuous tract, whereas degenerated axons have a particulate structure due to axonal fragmentation or beading. To detect degenerated axons, we used the particle analyzer module of ImageJ and counted the area of the small fragments or particles (size = 20–10,000 pixels) derived from the degenerated axons. A degeneration index (DI) was calculated as the ratio of fragmented axon area over total axon area. The production of the binary images and the function of the particle analysis as well as the accuracy of the DI in detecting axonal degeneration were optimized using multiple images of intact versus degenerating axons obtained from a large number of experiments using our DRG in vitro axotomy system.
