The curious ability of polyethylene glycol fusion technologies to restore lost behaviors after nerve severance

Abstract

Traumatic injuries to PNS and CNS axons are not uncommon. Restoration of lost behaviors following severance of mammalian peripheral nerve axons (PNAs) relies on regeneration by slow outgrowths and is typically poor or nonexistent when after ablation or injuries close to the soma. Behavioral recovery after severing spinal tract axons (STAs) is poor because STAs do not naturally regenerate. Current techniques to enhance PNA and/or STA regeneration have had limited success and do not prevent the onset of Wallerian degeneration of severed distal segments. This Review describes the use of a recently developed polyethylene glycol (PEG) fusion technology combining concepts from biochemical engineering, cell biology, and clinical microsurgery. Within minutes after microsuturing carefully trimmed cut ends and applying a well-specified sequence of solutions, PEG-fused axons exhibit morphological continuity (assessed by intra-axonal dye diffusion) and electrophysiological continuity (assessed by conduction of action potentials) across the lesion site. Wallerian degeneration of PEG-fused PNAs is greatly reduced as measured by counts of sensory and/or motor axons and maintenance of axonal diameters and neuromuscular synapses. After PEG-fusion repair, cut-severed, crush-severed, or ablated PNAs or crush-severed STAs rapidly (within days to weeks), more completely, and permanently restore PNA- or STA-mediated behaviors compared with nontreated or conventionally treated animals. PEG-fusion success is enhanced or decreased by applying antioxidants or oxidants, trimming cut ends or stretching axons, and exposure to Ca(2+) -free or Ca(2+) -containing solutions, respectively. PEG-fusion technology employs surgical techniques and chemicals already used by clinicians and has the potential to produce a paradigm shift in the treatment of traumatic injuries to PNAs and STAs.

Keywords: Wallerian degeneration; axonal regeneration; axotomy; nerve repair; polyethylene glycol.

Conflict of interest statement

Conflict of Interest

No author has a conflict of interest.

© 2015 Wiley Periodicals, Inc.

Figures

Figure 1. Morphological (A,B,D) and electrical (C) measures showing that a combination of PEG solutions and PEG hydrogels can rapidly and completely repair (fuse, join together) cut axonal ends in vitro (A,C,D) and in vivo (B)

From Bittner at al., 2000 with permission. A. Electron micrograph of a sagittal (longitudinal) section through a crayfish medial giant axon (C-MGA) PEG-fused in vitro that conducted action potentials (APs) intra-cellularly recorded through the fusion site (arrow) beginning within 10 minutes after PEG-fusion and continuing for 6 hours prior to fixation. Sh = cytoplasmic glial sheath of this un-myelinated axon. B. Photomicrograph of a Lucifer dye-filled earthworm MGA (E-MGA) cut and PEG-fused in vivo 20 days prior to sampling. A PEG-based hydrogel was then applied to give mechanical strength in vivo. The E-MGA was completely repaired in vivo as evidenced by: (1) Its ability to conduct APs through the fusion site for 20 days; (2) These APs elicited all the behaviors evoked by intact E-MGAs in un-operated earthworms; (3) When this axon was then filled with fluorescein dye at 20 days post PEG-fusion, the dye filled the entire E-MGA which had same morphology as that shown by non-severed, control MGAs. Arrow = site of PEG-fusion of cut ends and site where the PEG-based hydrogel was applied. C. Compound action potentials (CAPs) stimulated in one end chamber and recorded in the other end chamber of a three-chambered sucrose gap recording device. CAPs were first recorded from intact control bundles of rat spinal axons (trace labeled 1) prior to replacing the physiological saline in the central chamber with hypotonic Ca2+-free saline (trace labeled 2) at ~25° C. The spinal axons were then cut in the central chamber to completely eliminate the CAP (trace labeled 3). The cut ends were PEG-fused, and the central chamber was again perfused with calcium containing physiological saline. Within 15 minutes, CAPs were again recorded from PEG fused spinal axons (traces labeled 4). CAPs continued to be recorded for over 60 minutes before the experiment was terminated by again cutting the spinal axons in the central camber to eliminate the CAP, i.e., to demonstrate that the CAP was not an artifact (trace labeled 5). D. Electron micrograph of cross sections of rat spinal axons at the site of PEG-induced fusion. APs conducted through the PEG-fusion site for 2 hours before fixation. N = PEG-fused axons of near normal morphology. P = PEG-fused axons of pathological morphology (disrupted myelin sheath and many membranous structures in the axoplasm).

Figure 2. Model for Ca2+-dependent, vesicle-mediated sealing in which vesicles form a plug at a partly constricted cut end and sites of minor plasmalemmal damage in all eukaryotic cells

Modified and used with permission from Spaeth et al., 2010, ,,. PKA, PKC: phosphokinase A or C, Epac: exchange protein activated by cAMP, DAG: diacyl-glycerol. PEG bypasses all endogenous sealing pathways and induces sealing of small holes or complete transection within a fraction of a second by collapse and fusion of plasmalemmal leaflets to produce “PEG-Sealing”.

Figure 3. Sealing probability (%) vs. post-calcium addition (PC) time of B104 cells with neurites transected >50μm or

From Spaeth et al., 2012b with permission. A,B. Neurites transected in Ca2+-free saline containing 2mM MEL (open squares) or 100μM MB (open circles) before adding Ca2+ or 10mM 2kDaPEG (open triangles) for 1min after transection and then adding Ca2+. Solid lines: exponential fit to data. Asterisks: significant differences compared to control sealing (dotted, dashed lines); * = p<0.05, ** <0.01, *** <0.001. B104 cell neurites were transected in Ca2+-free saline containing MEL or MB and then bathed in Ca2+-containing saline (without MEL or MB). Sealing probabilities at each sampling time are compared for different treatments by a Cochran-Mantel-Haenszel χ2 test (CMH); sealing rates for different treatments are compared using Fisher’s Z test (FZT). At various times after Ca2+ addition (post-Ca2+ addition time: PC time), Texas Red dextran dye was added and sealing assessed by dye exclusion. C. Sealing probability 1min after adding 2kDa PEG vs. log PEG concentration (mM) for B104 cells transected >50μM from the soma and maintained in Ca2+-containing saline (open squares) or Ca2+-free saline (gray X’s). Solid lines: sigmoidal fit to data; Dashed lines: exponential decay fit to data.

Figure 4. A–G. Bioengineered sequence of solutions and micro-sutures to produce “PEG-sealing” of cut axonal ends

Schematic diagrams showing CAP recording (A), nerve excision (B) and allograft insertion (C) from axons that constitute the sciatic nerve. Higher magnification views in A–G shows five hypothetical axons and their relation to the endoneural (EN), perineural (PN) and epineural (EPS) sheaths. SE: stimulating electrode, RE; recording electrode; SS: severance site. Proximal (central) oriented to the left in A–G. Note that cut axonal ends in the host (h) and donor (d) partially collapse and vesicles begin to seal off the cut ends. The trauma of cutting also damages the axolemma adjacent to the severance site (B). D–F. Sequence and location of (D) methylene blue (MB) administration, (E) sutures and CAP recording, and F) application of PEG in double distilled water (ddH2O) (C) and their effects on axonal morphology. Note that MB and Ca2+-free isotonic saline unseal axons and prevents vesicle formation (D,E) and that hypotonic solutions open cut axonal ends (F). PEG produces incomplete membrane fusion of apposed axonal ends, possibly connecting more than one axon of unknown specificity on the proximal side of a cut site to one or more axons on the other side of an allograft. For example, motor axons may fuse with motor axons of different specificity or may fuse with sensory axons. Micro-sutures (E) are placed though the epineural sheath to bring axonal ends together, i.e., in close apposition. G–H. Sequence and location of removal of PEG by isotonic Ca2+-containing saline (G) and recording of CAPs (H). Note that isotonic Ca2+-containing saline causes the formation of vesicles to form that seal off remaining holes in the axolemma at cut axonal ends and adjacent damaged regions (H).

Figure 5. Electrophysiological evidence of PEG-fusion in allografts with trimmed cut ends

From Riley et al., 2015 with permission. A. Representative CAP (mV) recordings initially from a negative control (dashed lines) and a PEG-fused (solid lines) allograft pre-injury (thinner lines) and then within 5 min after ablation of a 1 cm segment, insertion of a 1 cm donor segment without (negative control: thicker dashed line) or with PEG-fusion (thicker solid line) of both micro sutured trimmed. SA = arrow points to peak of stimulus artifact. CAP: arrow points to peak amplitude of a CAP. B. CAPs (mV, mean ± SE) recorded pre injury and immediately post-repair for 4 groups: Negative controls recorded at Vanderbilt University (n=12) and University of Texas at Austin (UT) (n=6), PEG-fused at Vanderbilt University (n=13) and University of Texas at Austin (n=6).

Figure 6. Mean SFI scores for Cut or Crush treatment groups vs. post-operative week

From Bittner et al., 2012 with permission. See Bittner et al., 2012 for tabulated means+/−SEM and detailed statistical comparisons. A, B. **above cut protocol curves in A identify post-operative data points that significantly (p < 0.01) differ from the Cut (no PEG, no micro-suture) curve. For Crush Protocols (B), asterisks (*) identify individual post-operative data points for Crush+MB+PEG treatment group that differ significantly from data points for the Crush group at the same post-operative time. *= p<0.05, ** = p<0.01. Each group has at least 20 animals, over 300 total animals in the study. @@ indicates significant difference of p<0.01 of MEL group vs. negative control group curves.

Figure 7. CAP or SFI restoration after PEG-Fusion of axons crush severed- or cut-severed in Ca2+-free or Ca2+-containing salines, with or without careful trimming of cut ends)

A–D modified from Ghergherehchi et al., 2015 with permission. E modified from Riley et al., 2015 with permission. A–B. CAPs are restored in vivo within minutes after PEG-fusing sciatic PNAs crushed (A) in Ca2+-free, (B) but not in Ca2+-containing salines having various inhibitors of plasmalemmal sealing as listed in the key to the right of B. Key: the number of animals sampled (n = 4A, 3B) for each protocol plotted in panels A and B (see text). C–E. Mean SFI ± SE (upper graphs) or individual SFI measures (lower graphs) of behavioral recovery at 3d–6wk postoperatively for sciatic nerves cut-severed (C–D) or ablated (E) in Ca2+-containing salines with or without (negative controls) PEG-fusion.and without (C) or with (D) subsequently carefully trimming cut ends and micro suturing with some axonal stretching (C,D) — or after a carefully-trimmed allograft (E) is inserted and micro-sutured with no stretching of host or donor axons. Key: In upper graphs, “No PEG-fusion” curves show negative control data as black lines and symbols. “PEG-fusion” curves in blue show data for all PEG-fused nerves. “PEG-fusion -59 Recovery” curves in green show data for those PEG-fused sciatic nerves in rats having SFI scores by 6 postoperative weeks of -59 or better. In lower graphs C,D, SFI scores of individual animals with PEG-fused nerves that recovered to SFI scores of -59 or better shown as colored lines, PEG-fused no revovery animals shown as black lines and symbols. E shows individual SFI scores for rats with PEG-fused allografts in colored symbols and lines and negative controls in black symbols and lines. Note that one rat with a PEG-fused allograft did not shopw behavioral recovery (red line and symbols in lower graph of Panel E).

Figure 8. Axonal Diameters and G ratios

From Ghergherehchi et al., 2015 with permission. Cross-section EM images of un-operated (A) and single cut rat sciatic nerves with PEG-fusion repair (B) and without PEG-fusion repair (C) 6 weeks postoperatively. Scatterplots of G ratio versus axon diameter, for un-operated (A), PEG-fused (B), and non-PEG fused (C) sciatic nerves 6 weeks post-severance. Red data points: axons measured inside the sciatic epineural sheath; black data points: axons measured outside the sciatic epineural sheath.

Figure 9

Sciatic axons stained to identify sensory, motor or degenerating axons. A–C, E–G. From Riley et al., 2015 with permission. Sensory (A–C) or motor (E–G) myelinated axons in paraffin-embedded sections of sciatic nerves viewed at 20× stained for viability by CA II (A–C) or Co-Ach (E–G), respectively, for an unoperated control (A,D) and distal to the allograft at 6wk postoperatively for a negative control (B,F) and PEG-fused sciatic nerve (C,G) showing excellent behavioral recovery. Note that the negative control sciatic nerve (B,F) has very few deeply stained sensory or motor axons whereas the PEG-fused sciatic nerve (C,G) more closely resembles the un-operated control (A,E), i.e., further evidence that PEG-fusion retards or prevents Wallerian degeneration after myelinated axons are severed from their cell body. D,H. Longitudinal sections distal to a complete transection site stained with a marker (NPY1) for Wallerian degenerating PNAs (brown color) for a negative control (D) and PEG-fused rat sciatic axon (H).

Figure 10. Intra-axonal dye diffusion and axon morphology in allografts

From Riley et al., 2015) with permission. A, B. No intra axonal dye diffusion of Texas Red at 1d postoperatively in a negative control allograft (A) but observed for a PEG-fused allograft (B). Arrow: site of proximal cut end of host sciatic nerve micro-sutured to proximal end of donor sciatic allograft. C–G. Increased myelinated axon viability for PEG-fused allografts compared to negative controls or unsuccessful PEG-fusion at 6w postoperatively. Toluidine-blue, plastic embedded sections of sciatic nerves viewed at 20× for an un-operated control (C) and the mid-allograft region at 6wk postoperatively for a negative control (D) and PEG-fused sciatic nerves showing excellent (E), good (F) or no (G) behavioral recovery as assessed by SFI scores (see Fig. 11 and Riley et al., 2015). Note that successful PEG-fusion is correlated with survival of increased numbers of larger diameter myelinated axons within the allograft segment.

Figure 11

Counts of sensory, motor, or total number of axons in sciatic nerves repaired by PEG-fused autografts or allografts. A. From Sexton et al., 2013 with permission. B–F. From Riley et al., 2015 with permission. A. Total number of viable motor and sensory axons in cross sections of sciatic nerves for negative control (no PEG-fusion) vs. PEG-fused sciatic PNAs in autografts and allografts. Sensory PNAs were identified by immunohistochemical staining for carbonic anhydrase II (CA II) antibodies and motor PNAs by staining for choline-O-acetyl- transferase antibodies (Co-Ach). These data show no statistically significant difference for PEG-treated vs. controls in the proximal sciatic nerve but always a significantly greater number of PNAs in PEG-treated rats within the grafts or distal to the grafts. That is, PEG-fusion retards or reduces Wallerian degeneration. From Sexton et al., 2014. B. Total viable myelinated axon counts (mean, n=4) from a representative cross-section of sciatic nerves from the allograft region of unoperated control, negative control, and three PEG-fused allografts showing excellent, good or no SFI behavioral recovery 6wk postoperatively. C, D. Sensory (C) or motor (D) viable myelinated axon counts (mean ± SE, n=6) at 6wk post-operatively from a representative cross section from each of proximal, allograft, and distal regions of PEG-fused and negative control animals. E. SFI scores at 1w for each animal as listed vs. the number of surviving myelinated axons in allografts at 6wk postoperatively. R2 = 0.9946, p < 0.05. G19 = negative control animal not PEG-fused. F. SFI scores at 1–6wk individually plotted for each animal as listed vs. the number of surviving myelinated axons in allografts at 6wk postoperatively.

Figure 12. PEG-fusion significantly improves BBB scores following SCI

From Bittner et al., 2015 with permission. A MASCIS rod-drop device (set at 10 gm, 12.25 cm) was used to produce a mild contusive SCI at T9–10 in twelve male Lewis rats randomly assigned to receive PEG-fusion solutions that included MB and PEG or the same series of solutions without PEG. * BBB scores at same postoperative time that differ by p < 0.05 (two-tailed t test). *** The two curves differ by p< 0.001 according to a two-way ANOVA with Bonferroni post-test.