Figure 4

Immunofluorescence analysis. (A) Fluorescence imaging of a longitudinal section located distally to the implants shows cells positive to Fluororuby, a neural tracer injected proximally to the implanted prosthesis. Positive cells for the neural tracer in the nervous regenerated area were identified in all rats receiving the Fluororuby injection. (B) DAPI cell nuclei staining of the neuronal tracer positive area. Confocal cross-section image (75% of the conduit length) of myelin sheets, stained with MBP and CNPase (C), wrapping regenerated axons positive to β-tubulin staining (D). Myelinated axons were noticed throughout the tissue regenerated inside the conduits. (E) Confocal merge image shows distribution of both myelinated and unmyelinated axons in the nervous area regenerated in the conduit lumen. (F) High magnification confocal image, arrows indicate myelinated axons. Cross-section immunostainings for collagen IV in healthy sciatic nerves (G) and in regenerated tissues (H) show a noticeable amount of basement membrane component distributed throughout the prosthesis lumens of all the treated rats but with a remarkably different microstructure organization. Collagen IV is also detected both nearby and within the conduit inner walls (I) where low fibroblast density (J) is observable: same field DAPI cell nuclei staining (K).