Prior to surgery, animals were anesthetized using a small mammal isoflurane inhalation system (Handlebar Anesthesia, Austin, TX). Anesthesia was induced initially with a 4% isoflurane/oxygen mixture at a 1.5 L/min flow rate, and then maintained with a 1.5–2% mixture at 1 L/min for the duration of the surgery. The surgical area (lateral aspect of the left hindlimb) was trimmed of fur and disinfected using 10% povidone-iodine. A 2–3 cm long incision was made in the skin above the thigh muscles of the left hindlimb of each rat. The biceps femoris muscle was split parallel to the muscle fibers using dissection scissors to expose the sciatic nerve. The sciatic nerve was trimmed free of any connective tissue using micro-dissection scissors. Crush- or cut-severances were made in either hypotonic Ca2+-free or isotonic Ca2+-containing saline as specified for a given protocol.
We completely crush-severed 1–2 mm lengths of sciatic nerves using Dumont #5 micro-forceps placed perpendicular to the nerve by applying enough force to cause the underlying axons to separate while leaving the epineurium intact. After crush-severance, a small slit was made in the epineurium with micro-scissors to allow solutions to diffuse more readily to axons at the injury site. Crush excision repairs were made by completely excising the crushed nerve segment with dissection scissors. This typically resulted in a 2–3 mm gap between the severed nerve ends, which were then closely re-apposed with micro-sutures similar to our cut-severance protocol (see below). For details of these and other crush-severance surgical procedures, see previous publications (Britt et al., 2010 (link); Bittner et al., 2012 (link)).
Cut-severances were made by transecting the entire sciatic nerve with a single stroke of dissection scissors to completely sever all PNAs as well as their endo-, peri-, and epineural sheaths. Cut axonal ends and sheaths completely separated by 1–3 mm. Bundles of axons would sometimes swell out of the epineural sheath at the cut ends. In some animals as specified, these axonal ends and epineural sheaths were carefully trimmed so that the cut ends formed smooth flat planes that could be closely apposed with minimal gaps or axonal protrusions. Similar axonal trimming was performed, but not always noted, in previous publications (Bittner et al., 2012 (link); Sexton et al., 2012 (link); Rodriguez-Feo et al., 2013 (link); Riley et al., 2015 (link)). Cut-severed nerves were closely re-apposed with 10–0 micro-sutures (Ethicon, Somerville, NJ), leaving enough space between sutures to allow close positioning of a micropipette for diffusion of sterile solutions of PEG, antioxidants, etc., to the lesion site. Unless stated otherwise, cut axonal ends were typically re-apposed within 5–15 min after severance. For additional details of this cut severance surgical procedure, see Bittner et al., (2012) (link).
In this paper unless explicitly stated otherwise, the term PEG-fusion always denotes the following sequence of bio-engineered solutions applied to the lesion site of crush-severed sciatic nerves or cut-severed and micro-sutured sciatic nerves: (1 ) exposure of cut- or crush-severed axonal ends to hypotonic Ca2+-free saline (PlasmaLyte-A: Baxter, Deerfield, IL), ( (2 ) application of 1% MB (Acros Organics, Morris Plains, NJ) dissolved in double-distilled water (ddH2O) for 1–2 min, (3 ) application of 50% by weight PEG (3.35kD; Sigma Aldrich, St. Louis, MO) in sterile ddH2O for 1–2 min, and (4 ) extensive rinsing with Ca2+-containing isotonic saline (Lactated Ringers: Hospira, Lake Forest, IL). [See Figure 4 , Bittner et al., 2015 .] The term “negative control” describes the same procedures and solution applications as above, but without the addition of PEG to a crush- or cut-severed nerve. Note that the ends of cut negative controls are apposed by micro-sutures, the “gold standard” for clinical repair (Isaacs et al., 2010 ; Wolfe et al., 2010 ). As another negative control for dye diffusion experiments, PEG was applied to cut-severed nerves that were not micro-sutured, i.e., cut ends not closely apposed by micro-sutures do not PEG-fuse, but rather PEG seals off the cut ends (“PEG-sealing”. See below and Spaeth et al., 2012b (link)). PEG-fused and negative control animals to be tested for behavioral recovery by SFI tests received 5 mg/kg subcutaneous injection of carprofen after surgery.
We completely crush-severed 1–2 mm lengths of sciatic nerves using Dumont #5 micro-forceps placed perpendicular to the nerve by applying enough force to cause the underlying axons to separate while leaving the epineurium intact. After crush-severance, a small slit was made in the epineurium with micro-scissors to allow solutions to diffuse more readily to axons at the injury site. Crush excision repairs were made by completely excising the crushed nerve segment with dissection scissors. This typically resulted in a 2–3 mm gap between the severed nerve ends, which were then closely re-apposed with micro-sutures similar to our cut-severance protocol (see below). For details of these and other crush-severance surgical procedures, see previous publications (Britt et al., 2010 (link); Bittner et al., 2012 (link)).
Cut-severances were made by transecting the entire sciatic nerve with a single stroke of dissection scissors to completely sever all PNAs as well as their endo-, peri-, and epineural sheaths. Cut axonal ends and sheaths completely separated by 1–3 mm. Bundles of axons would sometimes swell out of the epineural sheath at the cut ends. In some animals as specified, these axonal ends and epineural sheaths were carefully trimmed so that the cut ends formed smooth flat planes that could be closely apposed with minimal gaps or axonal protrusions. Similar axonal trimming was performed, but not always noted, in previous publications (Bittner et al., 2012 (link); Sexton et al., 2012 (link); Rodriguez-Feo et al., 2013 (link); Riley et al., 2015 (link)). Cut-severed nerves were closely re-apposed with 10–0 micro-sutures (Ethicon, Somerville, NJ), leaving enough space between sutures to allow close positioning of a micropipette for diffusion of sterile solutions of PEG, antioxidants, etc., to the lesion site. Unless stated otherwise, cut axonal ends were typically re-apposed within 5–15 min after severance. For additional details of this cut severance surgical procedure, see Bittner et al., (2012) (link).
In this paper unless explicitly stated otherwise, the term PEG-fusion always denotes the following sequence of bio-engineered solutions applied to the lesion site of crush-severed sciatic nerves or cut-severed and micro-sutured sciatic nerves: (
