Euthanasia, tissue processing, and histology

At the conclusion of all studies, animals were deeply anesthetized and subsequently euthanized by Euthasol (rodents) or transcardial perfusion with formalin and phosphate-buffered saline (PBS; pigs). For the rodent experiments, subjects were anesthetized, and the graft region and the distal sciatic nerve were harvested at terminal time points. Nerves were postfixed in formalin at 4°C overnight. Formalin-fixed nerves were cryoprotected for 48 hours in 30% sucrose in PBS, frozen in optimal cutting temperature media, and cryosectioned either longitudinally (20 μm) along the graft region or axially (10 μm) starting 0.5 cm distal to the graft zone. Rodent histological analyses were performed up to 16 weeks after transection to determine distal nerve Schwann cell presence, cytoarchitecture, graft survival, and graft axon integration with distal nerve.

At the terminal time points of porcine studies, ipsilateral and contralateral hindlimbs were postfixed in formalin at 4°C overnight, and then nerves were harvested and postfixed in formalin at 4°C overnight. For the short-gap study (1-cm graft; 2 weeks after repair) and the early regenerative phase cohort in the long-gap study (5-cm graft; 1 and 3 months after repair), nerves were cryoprotected and frozen. Sections were taken longitudinally (20 μm) across the graft or axially (10 μm) at 1-cm intervals along the length of the nerve beginning at 0.5 cm distal to the graft zone. Frozen sections were washed three times in PBS, blocked, and permeabilized in 4% normal horse serum with 0.3% Triton X-100 for 1 hour. All subsequent steps were performed using blocking solution for antibody dilutions. Axons were labeled with anti-NF200 (1:200; Sigma-Aldrich, N0142) and/or anti-SMI31/32 (1:1500; Millipore, NE1022/NE1023). Schwann cells and myelin were labeled with anti-S100 (1:250; Dako, Z0311) and anti–myelin basic protein (MBP; 1:1500; Encor, CPCA-MBP), respectively. Primary antibodies were applied overnight at 4°C followed by the appropriate fluorophore-conjugated secondary antibody (1:1000; Alexa Fluor, Invitrogen) for 2 hours at room temperature. For animals enrolled in the long-gap cohort studying the chronic regenerative phase (5-cm defects, 6 to 12 months after repair), formalin-fixed nerves were paraffin-embedded, and axial sections (8 μm) were taken at 1-cm intervals along the length of the nerve beginning at 0.5 cm distal to the graft zone. Deparaffinized sections were rehydrated in descending ethanol gradient, and high-heat and pressure antigen retrieval was performed in tris/EDTA buffer. Sections were blocked with 4% normal horse serum in OptiMax (BioGenex, HK583-5K). All subsequent steps were performed using blocking solution for antibody dilutions. Sections were incubated with primary antibodies for axons, Schwann cells, and myelination as described above.