Polyethylene glycol (PEG) and other bioactive solutions with neurorrhaphy for rapid and dramatic repair of peripheral nerve lesions by PEG-fusion

Abstract

Background: Nervous system injuries in mammals often involve transection or segmental loss of peripheral nerves. Such injuries result in functional (behavioral) deficits poorly restored by naturally occurring 1-2 mm/d axonal outgrowths aided by primary repair or reconstruction. "Neurorrhaphy" or nerve repair joins severed connective tissues, but not severed cytoplasmic/plasmalemmal extensions (axons) within the tissue.

New method: PEG-fusion consists of neurorrhaphy combined with a well-defined sequence of four pharmaceutical agents in solution, one containing polyethylene glycol (PEG), applied directly to closely apposed viable ends of severed axons.

Results: PEG-fusion of rat sciatic nerves: (1) restores axonal continuity across coaptation site(s) within minutes, (2) prevents Wallerian degeneration of many distal severed axons, (3) preserves neuromuscular junctions, (4) prevents target muscle atrophy, (5) produces rapid and improved recovery of voluntary behaviors compared with neurorrhaphy alone, and (6) PEG-fused allografts are not rejected, despite no tissue-matching nor immunosuppression.

Comparison with existing methods: If PEG-fusion protocols are not correctly executed, the results are similar to that of neurorrhaphy alone: (1) axonal continuity across coaptation site(s) is not re-established, (2) Wallerian degeneration of all distal severed axons rapidly occurs, (3) neuromuscular junctions are non-functional, (4) target muscle atrophy begins within weeks, (5) recovery of voluntary behavior occurs, if ever, after months to levels well-below that observed in unoperated animals, and (6) allografts are either rejected or not well-accepted.

Conclusion: PEG-fusion produces rapid and dramatic recovery of function following rat peripheral nerve injuries.

Keywords: Allograft; Neuromuscular junction re-innervation; Neurorrhaphy; Peripheral nerve injury; Polyethylene glycol fusion; Rat sciatic nerve repair; Wallerian degeneration.

Conflict of interest statement

Declaration of Interests

Dr. Jackson is CEO of Neuraptive Therapeutics. Neuraptive has exclusively licensed the PEG-fusion patent estate from the University of Texas at Austin invented by, and based on, research performed by Dr. Bittner. Dr. Bittner has assigned all of his economic interests in the licensed PEG-fusion patent estate to a third party. Neither potential conflict has affected in any way any data analyses or text in this manuscript.

Copyright © 2019 Elsevier B.V. All rights reserved.

Figures

Figure 1.

Schematic diagram showing the stepwise protocol of PEG-fusion. (A) As an initial test of nerve viability, extracellular CAPs are recorded from the intact nerve. (B) The nerve is completely severed in physiological, isotonic Ca2+ containing saline. (C) Nerve axons are carefully trimmed with sharp microscissors in hypotonic, Ca2+-free saline. (D) 1-2 drops of 1% MB dissolved in double-distilled H2O is applied to each end of the nerve. (E) The nerve ends are carefully sutured with minimal damage to the proximal and distal stumps. Additional drops of hypotonic Ca2+-free saline are applied as needed to prevent dehydration of the nerve during the surgical repair. (F) A 50% w/w solution of PEG is applied directly to the coaptation site(s) for 1-2min. (G) The nerve is irrigated with isotonic, Ca2+-containing saline. (H) CAPs are taken as a positive control to confirm initial success of the PEG-fusion procedure. EPS: epineural sheath; PN: perineurium; EN: endoneurium; CAPs: compound action potentials; ECF: extracellular fluid; MB: methylene blue; PEG: polyethylene glycol.

Figure 2.

Voluntary-behavior analyses (SFI scores) of PEG-fusion. (A) Our historical mean±SEM SFI scores for Sham Controls, PEG-fused Allografts, PEG-fused Single Cuts, and Negative Control Allografts and Single Cuts with neurorrhaphy and all Standard PEG solutions except PEG. The two n values in the key for each curve gives the number of animals sampled at 3-42d PO, and the number sampled 3d-84d PO, respectively. Negative Control Allografts show no significant recovery at any time up to 84d PO. Animals with Negative Control Single Cuts show some functional recovery by 63-84d PO (p<0.05). PEG-fused Single-Cuts recover within 14d PO (p<0.05), increase recovery by 21-28d PO (p<0.01), and plateau around 42d PO (p>0.001). PEG-fused Allografts show significant recovery by 3-7d PO (p<0.01), and recover to near baseline by 42d PO (p>0.001). (B) Representative footprints at 42d PO for Sham, PEG-fused, and Negative Control animals after single cut repair. (C) SFI data showing decreased success of PEG-fusion by altering or omitting steps in the standard protocol (Standard PEG). Omission of MB (Standard PEG, no MB) or hypotonic Ca2+ free saline (Standard PEG, no hypotonic Ca2+-free) produces significant behavioral recovery compared to Negative Controls, but less recovery compared to the Standard Protocol. Animals receiving the Standard PEG protocol without microsutures (PEG, no sutures) show no behavioral recovery at any PO time. (D) Surgical variability exhibited by different personnel all using the Standard PEG-fusion protocol. Students with no prior surgical background sometimes attain successful PEG-fusions (p<0.05) after 0-30 practice animals during which they are mostly not successful; trained surgeons typically attain success after 0-10 practice animals.

Figure 3.

Axolemmal and axoplasmic continuity is restored within minutes after PEG-fusion repairs and axonal transport is restored within days for single transections and within 2-3 weeks for segmental ablations repaired with PEG-fusion. (A and B) Electrophysiological confirmation of sciatic nerve continuity after single cut PEG-fusion. (A) CAP (mV) recordings of intact nerves (Unop: solid red line) and after transection without (Negative Control: dashed black line) or with PEG-fusion (solid blue line). SA = stimulus artifact. CAP arrow: peak amplitude. (B) CMAP recordings using same protocols as CAPS. (C) Morphological evidence of axonal continuity only after PEG-fusion. Intra-axonal dye diffusion of Texas Red at 1d PO in an Unoperated (C1), Negative Control (C2) or PEG-fused (C3) sciatic nerve. Arrow: points to repair site. Dye diffuses from the proximal segment of the nerve into the distal segment after PEG-fusion, but does not cross the repair site in Negative Controls. (D) Transverse spinal hemisections of labeled motoneurons of an intact control animal (Unop), and an animal with a PEG-fused sciatic nerve at 5d PO after injection of BHRP into the anterior tibialis muscle. Scale bar = 500μm.

Figure 4.

NMJs and nerve fibers in Unoperated, PEG-fused and Negative Control (NC) animals. Unoperated animals (A,D,G) have NMJs whose innervating axons are not fragmented and whose shape mirrors that of the muscle acetylcholine receptor (AChR). Unoperated nerve fibers (J) have electron-lucid axoplasm and distinct electron-dense myelin sheaths. A 7d PO PEG-fused allograft NMJ (B,E,H) is also clearly innervated by a non-fragmented axons. Nerve fibers from a 14d PO PEG-fused allograft distal host nerve (K) have more extracellular space than unoperated nerves, but also have intact myelin sheaths and electron-lucid axoplasm with little or no signs of Wallerian degeneration. In contrast, a 7d PO Negative Control allograft has no axon innervating the AChR (C,F,I). A 14d PO singly cut NC (L) nerve has no myelinated nerve fibers and shows extracellular debris characteristic of Wallerian degeneration. Scale bar= 20μm for A-I, 10μm for J-L.

Figure 6.

Recovery of Sensation in PEG-treated clinical cases. (A) PEG-therapy group demonstrated statistically significant recovery at 1, 4, and 8 weeks PO (p < 0.05) by static 2-point discrimination (s2PD), shaded area represents normal range; and (B) Medical Research Council Classification (MRCC) for Sensory Recovery Scale, shaded area represents good clinical recovery.