Table 1 PEG-fusion protocol and rationale to immediately repair (join) cut axonal ends

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

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

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

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