Mechanisms and Pathways of Plasmalemma Repair

The four papers listed below were the first- to show that plasmalemmal lesions in all eukaryotic cells are sealed by accumulation of vesicles, often by formed by endocytosis, that require calcium calpain, and other substances also used in exocytosis, Golgi trafficking and synaptic release. Our laboratory has been the leader in determining the cellular and biochemical pathways and mechanism for such sealing – and to show that PEG bypasses these natural pathways to directly induce plasmalemmal collapse and sealing, aka “PEG-sealing”. We have published about 10 papers in the last 5 years documenting such phenomena and biochemical pathways. We have used these data to develop our well-specified sequence of bio-engineered solutions to successfully PEG fuse transected PNS axons or ablated PNS axonal segments repaired by PEG-fused interposition autografts or allografts (see C5 below).

C.S. Spaeth, E.A. Boydston, L.A. Figard, A. Zuzek and G.D. Bittner. 2010. A model for sealing plasmalemmal damage in neurons and other eukaryotic cells. J. Neurosci. 30:15790-15800.

C.S. Eddleman, M.L. Ballinger, M.E. Smyers, H.M.Fishman, G.D. Bittner (1998). Endocytotic formation of vesicles and other membranous structures induced by Ca2+ and axoplasmic injury. J Neurosci,18, 4029-4041.  

C.S. Eddleman, M.L. Ballinger, M.E., Smyers ,C.M. Godell, H.M., Fishman, G.D.Bittner (1997).  Repair of plasmalemmal lesions by vesicles.  PNAS, 94, 4745-4750.

T.L. Krause, H.M. Fishman, M.L. Ballinger, G.D. Bittner (1994).  Extent and mechanism of sealing by vesicles, often formed by exocytosis transected giant axons of squid and earthworms.  J Neurosci, 14, 6638-6651.


PEG-Induced Axonal Repair

The four papers listed below were all the first to show that PEG can be used to repair severed invertebrate giant axons ex vivo, then in vivo, then mammalian peripheral nerve axons (PNAs) and spinal axons ex vivo, then crushed or cut mammalian PNA axons in vivo in Ca2+-free or Ca2+-containing solutions, then ablated mammalian PNA axons in Ca2+-containing solutions. That is, from our papers listed below and our recent papers, we have demonstrated successful PEG-fusion of cut, crushed or ablated mammalian axons mostly from a rat sciatic small animal model of acute repair of mixed motor and sensory PNAs. We have also demonstrated that our PEG-fusion technology is translatable to rabbit or guinea pig PNAs and to rat or guinea pig spinal axons, as well as giant axons from three invertebrate phyla.  We have also demonstrated that severed distal rat sensory and motor PNAs can be viably maintained for 5-10 days by coolingref or cyclosporine A injectionsref and that PNAs chronically maintained for at least 3 days can be successfully PEG-fused as measured by CAP conduction through the lesionref, including for chronically maintained interposition autografts or allografts. Considered together, these data strongly suggest that this PEG-fusion technology using micro-sutures and well-specified sequences of solutions is translatable repair acutely or chronically (1-3d) severed nerves in animals from many different phyla and different mammals, at least for nerves up to 3 mm in diameter with gaps up to 10mm. This current PEG-fusion technology has in fact taken decades to develop with conceptual and practical contributions.

D.C. Riley, G.D. Bittner, M.A. Mikesh, N.L. Cardwell, A.C. Pollins, C.L. Ghergherehchi, S.R. Bhupanapadu Sunkesula, T.N. Ha, B.T.D. Hall, A.D. Poon, M. Pyarali, R.B. Boyer, A.T. Mazal, N. Munoz, R.C. Trevino, T.Schallert,  W.P. Thayer. 2015.  Polyethylene glycol--fused allografts produce rapid behavioral recovery after ablating sciatic nerve segments. J Neurosci Res. 2015 Apr;93(4):572-83. doi: 10.1002/jnr.23514. Epub 2014 Nov 25.

G.D. Bittner, C.P. Keating, J. R. Kane, J.M. Britt, C.S. Spaeth, J. D. Fan, A. Zuzek R.W. Wilcott, W. P. Thayer, J.M. Winograd, F. Gonzalez-Lima and T. Schallert. 2012.  Rapid, effective and long-lasting behavioral recovery produced by microsutures, methylene blue and polyethylene glycol after complete cut of rat sciatic nerves. J Neurosci Res. 90:967-980.

A.B.Lore, J.A.Hubbell,  D.S. Bobb Jr., M.L.Ballinger, K.L. Loftin, , J.W.Smith, M.E. Smyers,  H.D.Garcia and G.D. Bittner. 1999.  Rapid induction of functional and morphological continuity between severed ends of mammalian or earthworm myelinated axons. J Neurosci.  19: 2442 – 2454.

T.L. Krause and G.D. Bittner.  1990.  Rapid morphological fusion of severed myelinated axons by polyethylene glycol.  PNAS. 87:1471-1475.  


The following papers published in the last four years give details on these two research interests.

  • Bittner GD, Spaeth CS, Poon AD, Burgess ZS, McGill CH (2016) Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells. Neural Regen Res In Press. pMC in progress.
  • Bittner GD, Sengelaub DR, Trevino RC, Ghergherehchi CL, Mikesh M (2016) Robinson and Madison have published no data on whether polyethylene glycol fusion repair prevents reinnervation accuracy in rat peripheral nerve. J Neurosci Res in Press. pMC in progress.
  • McGill CH, Bhupanapadu Sunkesula SR, Poon AD, Mikesh M, Bittner GD (2016) Sealing Frequency of B104 Cells Declines Exponentially with Decreasing Transection Distance from the Axon Hillock. Exp Neurol 279:149-158. doi:10.1016/j.expneurol.2016.02.001. pMC in progress.
  • Bittner GD, Mikesh M, Ghergherehchi CL (2016) PEG-fusion retards Wallerian degeneration and rapidly restores behaviors lost after nerve severance. Neural Regen Res 11:217-219. Doi 10.4103/1673-5374.177716.  PMC4810973
  • Ghergherehchi CL, Bittner GD, Hastings RL, Mikesh M, Riley DC, Trevino RC, Schallert T, Thayer WP , Raju Bhupanapadu Sunkesula S,Ha TN, Munoz N, Pyarali M, Bansal A, Poon AD, Mazal AT, Smith TA, Wong NS, Dunne PJ (2016) Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by PEG-fusion following complete cut- or crush-severance of rat sciatic nerves. J Neurosci Res 94:231-235. Doi. 10.1002/jnr23704 . Epub Jan 5, 2016 PMC4715960
  • Bittner GD, Sengelaub DR, Trevino RC, Peduzzi JD, Mikesh M, Ghergherehchi CL, Schallert T, Thayer WP (2015) The curious ability of PEG-fusion technologies to restore lost behaviors after nerve severance. J Neurosci Res 94: 207-230. online 3 Nov.2015. doi. 1002/jnr 23685 PMID26525605. pMC in progress.
  • Bittner GD, Rokkappanavar KK, Peduzzi JD (2015) Application and implications of PEG-fusion as a novel technology to repair injured spinal cords. Neural Regeneration Research 10:1406-1408. PMC4625502
  • Riley DC, Bittner GD, Mikesh MA, Cardwell NL, Pollins AC, Ghergherehchi CL, Bhupanapadu Sunkesula SR, Ha TN, Hall BTD, Poon AD, Pyarali M, Boyer RB, Mazal AT, Munoz N, Trevino RC, Schallert T, Thayer WP (2015) Polyethylene glycol--fused allografts produce rapid behavioral recovery after ablating sciatic nerve segments. J Neurosci Res 93(4):572-83. doi: 10.1002/jnr.23514. PMC4329031.
  • Rodriguez-Feo CL, Sexton KW, Boyer RB, Pollins AC, Cardwell NL, Nanney LB, Shack RB, Mikesh MA, McGill CH, Driscoll CW, Bittner GD, Thayer WP (2013) Blocking the P2X7 Receptor Improves Outcomes After Axonal Fusion. J Surgical Res 184(1):705-13. doi: 10.1016/j.jss.2013.04.082.  PMC3759537
  • Zuzek A, Fan JD, Spaeth CS, Bittner GD (2013) Sealing of transected neurites of rat B104 cells requires a diacylglycerol PKC-dependent pathway and a PKA-dependent pathway. Cell Molec Neurosci 33:31-46. doi: 10.1007/s10571-012-9868-5. Epub 2012 Aug 3. PMC22865002
  • Sexton KW, Pollins AC, Cardwell NL, Del Corral GA, Bittner GD, Shack RB, Nanney LB, Thayer WP (2012) Hydrophilic polymers enhance early functional outcomes after nerve autografting. J Surgical Res 177:392-400. doi: 10.1016/j.jss.2012.03.049.  PMC4096106.
  • Bittner GD, Keating CP, Kane JR, Britt JM, Spaeth CS, Fan JD, Zuzek A, Wilcott RW, Thayer WP, Winograd JM, Gonzalez-Lima F, Schallert T (2012) Rapid, effective and long-lasting behavioral recovery produced by microsutures, methylene blue and polyethylene glycol after complete cut of rat sciatic nerves. J Neurosci Res 90:967-980. doi: 10.1002/jnr.23023.  PMID:22302646 pMC in progress.
  • Spaeth CS, Robison T, Fan JD Bittner GD (2012) Cellular mechanisms of plamalemmal sealing and axonal repair by polyethylene glycol and methylene blue. J Neursosci Res 90:955-966. doi: 10.1002/jnr.23022.  PMID 22302626. pMC in progress.
  • Spaeth CS, Spaeth EB, Wilcott RW, Fan JD, Robison T, Bittner GD (2012) Pathways for plasmalemmal repair mediated by PKA, Epac and cytosolic oxidation in rat B104 cells in vitro and rat sciatic axons ex vivo. Devel Neurol 72:1399-1414. doi: 10.1002/dneu.20998.  PMID 22076955. pMC in progress.
  • Spaeth CS, Fan JD, Spaeth FB, Robison T, Wilcott RW, Bittner GD (2012) Neurite transection produces cytosolic oxidation which enhances plasmalemmal repair. J Neurosci Res 90:945-954. doi: 10.1002/neu.20005. PMID 15266646. pMC in progress.