Quantitative Axon Injury Evaluation

Eyes and intracranial portions of optic nerves were processed as previously reported (Smith et al., 2002 ). Nerves were stained with paraphenylenediamine (PPD), which differentially stains single damaged axons allowing sensitive detection of axon injury. Nerves were determined to have one of three damage levels that are readily distinguishable by axon counting (Fig. 3) (Anderson et al., 2005 (link); Libby et al., 2005a (link),b (link)). For the Wld^s and Bax experiment, two masked investigators assigned the same damage level to 91% of the nerves (360 out of 399). The 39 nerves that were assigned different damage levels only differed by one level. A third masked investigator assessed these 39 nerves and always agreed with one of the original damage levels. The most commonly assigned level was used. Representative images were taken on an Olympus BX50 microscope.