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Loss of viable neurons was determined by Nissl (cresyl violet) staining, and thus it reveals the structural features of neurons. The spinal cord sections described above were used to stain with Nissl as previously described (Sakakima et al., 2012). Cells that contained Nissl substance were considered to be viable neurons. Condensed fragmented staining indicates neuronal degeneration. The viable neurons with visible nucleoli were manually counted using a BX-60 microscope equipped with a DP70 camera unit (Olympus).
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