Cellular Mechanisms of Plasmalemmal Sealing and Axonal Repair by Polyethylene Glycol and Methylene Blue C.S. Spaeth, 1 T. Robison, 2 J.D. Fan, 2 and G.D. Bittner 1,2,3 * 1 Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 2 Section of Neurobiology, University of Texas at Austin, Austin, Texas 3 Institute for Neuroscience, University of Texas at Austin, Austin, Texas Mammalian neurons and all other eukaryotic cells endogenously repair traumatic injury within minutes by a Ca 21 -induced accumulation of vesicles that interact and fuse with each other and the plasmalemma to seal any openings. We have used uptake or exclusion of extracellular fluorescent dye to measure the ability of rat hippocampal B104 cells or rat sciatic nerves to repair (seal) transected neurites in vitro or transected axons ex vivo. We report that endogenous sealing in both preparations is enhanced by Ca 21 -containing solu- tions and is decreased by Ca 21 -free solutions containing antioxidants such as dithiothreitol (DTT), melatonin (MEL), methylene blue (MB), and various toxins that decrease vesicular interactions. In contrast, the fusogen polyethyl- ene glycol (PEG) at 10–50 mM artificially seals the cut ends of B104 cells and rat sciatic axons within seconds and is not affected by Ca 21 or any of the substances that affect endogenous sealing. At higher concentrations, PEG decreases sealing of transected axons and disrupts the plasmalemma of intact cells. These PEG-sealing data are consistent with the hypothesis that lower concentra- tions of PEG directly seal a damaged plasmalemma. We have considered these and other data to devise a proto- col using a well-specified series of solutions that vary in tonicity, Ca 21 , MB, and PEG content. These protocols rapidly and consistently repair (PEG-fuse) rat sciatic axons in completely cut sciatic nerves in vivo rapidly and dramatically to restore long-lasting morphological conti- nuity, action potential conduction, and behavioral functions. V V C 2012 Wiley Periodicals, Inc. Key words: calcium; axonal severance; nerve repair After injury, mammalian axons that do not rapidly (within minutes) repair plasmalemmal damage do not survive (Schlaepfer and Bunge, 1973; Bittner et al., 2000; Yoo et al., 2004; Nguyen et al., 2005), much less regenerate. Hence, rapid repair of plasmalemmal damage can be neuroprotective (Ramo ´n y Cajal, 1928; Kwon et al., 2009; Radogna et al., 2009; Spaeth et al., 2010). However, axolemmal sealing does not guarantee neuronal survival after injury. For example, neurons such as rat hippocamplal B104 cells that seal plasmalemmal damage at sites nearer the soma are less likely to survive than neurons that seal plasmalemmal damage at sites farther from the soma (Ramo ´n y Cajal, 1928; Nguyen et al., 2005; Spaeth et al., 2010). Rapid plasmalemmal repair (sealing) of small holes or complete axonal transections in mammalian neurons (including B104 cells) and other eukaryotic cells is endoge- nously produced by a Ca 21 -induced accumulation of mem- brane-bound structures (mostly vesicles) mediated by various protein isomers, many of which are Ca 21 -dependent and involved in membrane fusion at synapses or the Golgi appara- tus (Fig. 1A,C; Spaeth et al., 2010). Oxidizing agents (H 2 O 2 , thimerosal [TH]) and substances that enhance vesicular inter- actions have recently been reported to increase plasmalemmal sealing in neurons and muscles (Cai et al., 2009b; Spaeth et al., 2010, 2011). Antioxidants such as melatonin (MEL) or methylene blue (MB) and inhibitors of vesicular interactions have been reported to decrease endogenous plasmalemmal sealing (Spaeth et al., 2010, 2011). Although the cellular mechanism is as yet unknown, MB has also been reported to improve behavioral recovery slightly after damage to nerve or other tissues (Zhang et al. 2006; Radogna et al., 2009; Rojas et al., 2009). Here we describe in vitro data showing that MB decreases the probability of plasmalemmal repair (sealing) at severed ends of rat hippocampal B104 cells by endog- enous cellular mechanisms (Fig. 1A,C) that take 10–20 min to complete. The reducing agent dithiothreitol (DTT); the antioxidants MEL and MB; and various toxic substances, such as N-ethylmaleimide (NEM), bre- feldin A (Bref A), and botulinum toxins (BoNT) all decrease plasmalemmal sealing (Spaeth et al., 2011). In C.S. Spaeth’s current address is University of Texas Southwestern Medi- cal School, Dallas, TX 75390 *Correspondence to: George Bittner, Institute for Cell and Molecular Biology, The University of Texas at Austin, 1 University Station, C0920, Austin, TX 78712-0248. E-mail: bittner@mail.utexas.edu Contract grant sponsor: Lone Star Paralysis Foundation. Received 2 November 2011; Revised 4 December 2011; Accepted 15 December 2011 Published online 3 February 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23022 Journal of Neuroscience Research 90:955–966 (2012) ' 2012 Wiley Periodicals, Inc.