Table 1 PEG-fusion protocol and rationale to immediately repair (join) cut axonal ends
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Protocol Steps #1–5 | Completely sever and trim nerve ends | Prepare nerve ends for neurorrhaphy and PEG repair/fusion |
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1. Priming Solution #1 | Irrigation of surgical field with hypotonic Ca2+-free saline for 1–2 min | Increase axoplasmic volume. Open cut axonal ends. Expel intracellular membrane-bound vesicles/organelles |
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2. Protection Solution #2 | Administer 0.5–1% methylene blue (MB; an antioxidant) in distilled water for 1–2 min to opened cut ends | Prevent formation of intracellular vesicles/organelles that interfere with PEG-fusion of cut ends and can seal-off each apposed cut end rather joining/fusing them |
| 3. Co-apt cut nerve ends | Perform neurorrhaphy | Provide mechanical strength to epineurium to prevent PEG-fused axons from pulling apart. Closely appose cut axonal ends |
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4. PEG-fuse many axons Solution #3 | Apply 50% w/w 3.35 kDa PEG in ddH2O for 1–2 min to the coaptation site | Remove bound cell water to induce closely apposed, open, axonal membranes to non-specifically fuse |
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5. Membrane repair Solution #4 | Irrigation of coaptation site with isotonic Ca2+—containing saline | Induce vesicle formation to plug/seal any axolemmal holes after PEG-induced annealing of open cut axonal ends |