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 .
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 five years give details on these two research interests.
- Spaeth, J.D. Fan, E.B. Spaeth, T. Robison, R.W. Wilcott and G.D. Bittner. 2012. Neurite transection produces cytosolic oxidation which enhances plasmalemmal repair. J Neurosci Res. 90:945-954. doi: 10.1002/neu.20005. PubMed PMID: 15266646.
C.S. Spaeth, E.B. Spaeth, R.W.Wilcott, J.D.Fan, T.Robison and G.D. Bittner. 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. PubMed PMID: 22076955.
C.S. Spaeth, T.Robison, J.D.Fan and G.D. Bittner. 2012. Cellular mechanisms of plasmalemmal sealing and axonal repair by polyethylene glycol and methylene blue. J Neursosci Res. 90:955-966. doi: 10.1002/jnr.23022. PubMed PMID: 22302626.
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. doi: 10.1002/jnr.23023. PubMed PMID: 22302646.
K.W. Sexton, A.C. Pollins, N. L. Cardwell, G. A. Del Corral, G. D. Bittner, R. B. Shack, L. B. Nanney, W. P. Thayer. 2012. Hydrophilic polymers enhance early functional outcomes after nerve autografting. J. Surgical Res. 177:392-400. doi: 10.1016/j.jss.2012.03.049. PubMed PMID: 22521220; PubMed Central PMCID: PMCPMC4096106.
A. Zuzek, J.D. Fan, C. S. Spaeth, G.D. Bittner. 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.
C.L. Rodriguez-Feo, K.W. Sexton,R. B. Boyer, A. C. Pollins,N. L. Cardwell, L. B. Nanney,R. B. Shack, M. A. Mikesh,C. H. McGill, C. W. Driscoll, G. D. Bittner, W. P. Thayer. 2013. Blocking the P2X7 Receptor Improves Outcomes After Axonal Fusion. J. Surgical Research. 184(1):705-13. doi: 10.1016/j.jss.2013.04.082. PubMed PMID: 23731685; PubMed Central PMCID:.
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. Apr;93(4):572-83. doi: 10.1002/jnr.23514. PubMed PMID: 25425242; PubMed Central PMCID: PMCPMC4329031.
G.D. Bittner, K.K. Rokkappanavar, J.D. Peduzzi. 2015. Application and implications of PEG-fusion as a novel technology to repair injured spinal cords. Neural Regeneration Research. 10:1406-1408.
G.D. Bittner, D.R. Sengelaub, R.C. Trevino, J.D. Peduzzi, M. Mikesh, C.L. Ghergherehchi, T.Schallert, W.P. Thayer. 2016. 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
C. L. Ghergherehchi, G. D. Bittner, R. L. Hastings, M. Mikesh, D. C. Riley, R. C. Trevino, T. Schallert, W. P. Thayer, S. Raju Bhupanapadu Sunkesula,, T-A. N. Ha\, N. Munoz, M. Pyarali, A. Bansal, A. D. Poon, A. T. Mazal, T. A. Smith, N. S. Wong, P. J. Dunne. 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. 94:231-245. Doi. 10.1002/jnr23704 . Epub Jan 5, 2016
G.D. Bittner, M. Mikesh, C. L. Ghergherehchi. 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
C.H. McGill, S. R. Bhupanapadu Sunkesula, A.D. Poon,M. Mikesh, G. D. Bittner. 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
G. D. Bittner, C. S. Spaeth, A. D. Poon, Z. S. Burgess, C. H. McGill. 2016. Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells. Neu. Regen. Res. 11:1033-1042. doi 10.4103/1.
G.D. Bittner, D.R. Sengelaub, R.C. Trevino, C.L. Ghergherehchi, M. Mikesh. 2017. Robinson and Madison have published no data on whether polyethylene glycol fusion repair prevents reinnervation accuracy in rat peripheral nerve. J Neurosci Res. 95:863-866
GD Bittner, DL Sengelaub, CL Ghergherehchi. 2018. Conundrums and confusions regarding how PEG-fusion produces excellent behavioral recovery after peripheral nerve injuries. Neural Regeneration Research. 13: 53-57..
Andrew D. Poon, Sarah H. McGill, Solomon Raju Bhupanapadu Sunkesula, Zachary S. Burgess, Patrick J. Dunne, Edward E. Kang and George D Bittner. 2018. CaMKII and DMSO affect the sealing frequencies of transected hippocampal neurons. J. Neurosci. Res. 96:1208-1222.
Mikesh M, Ghergherehchi CL, Hastings RL, Ali A, Rahesh S, Jagannath K, Sengelaub DR, Trevino RC, Jackson DM, Bittner GD. 2018. Polyethylene glycol solutions rapidly restore and maintain axonal continuity, neuromuscular structures and behaviors lost after sciatic nerve transections in female rats. J. Neurosci. Res. 96: 1223-1242.
Mikesh M, Ghergherehchi CL, Rahesh S, Jagannath K, Ali A, Sengelaub DR, Trevino RC, Jackson DM, Tucker HO, Bittner GD. 2018. Polyethylene glycol treated allografts not tissue matched nor immunosuppressed rapidly repair sciatic nerve gaps, maintain neuromuscular functions, and restore voluntary behaviors in female rats. J. Neurosci. Res. 96:1243- 1264.
Ghergherehchi CL, Mikesh M, Sengelaub DR, Jackson DM, Smith T, Shores JT, Bittner GD. (2018) Polyethylene glycol (PEG) and other bioactive solutions with neurorrhaphy for rapid and dramatic repair of peripheral nerve lesions by PEG-fusion. J Neurosci Methods. Submitted.