Ultrastructural evidence of differential vulnerability of axons, Schwann cells, and their myelin sheaths in an ex-vivo model of peripheral nerve repair

Ex-vivo peripheral nerve preparations are widely used as experimental models of nerve repair, particularly in functional studies involving electrophysiological recordings that require prolonged maintenance of neural tissues outside their physiological environment. However, little is known about the early ultrastructural changes occurring in peripheral nerve tissue during these procedures. In particular, the effects of ex-vivo maintenance on axons and Schwann cells remain poorly characterized. In the present study, tibial nerves were explanted from the hind limbs of neonatal rats, transected, re-apposed using different conductive adhesive matrices, and subjected to electrophysiological recordings to assess the restoration of nerve function. During these procedures, nerve segments were maintained in an oxygenated saline solution for approximately 5 hours before fixation and processing for conventional transmission electron microscopy of samples collected both proximal and distal to the repair site. Ultrastructural analysis revealed comparable alterations in myelinated fibers in all samples, consisting of early degeneration of myelin sheaths characterized by widespread loss of the normal lamellar architecture, multifocal lamellar loosening, and the formation of large interlamellar spaces. Additional alterations included inward and outward ballooning of the myelin sheath, accompanied by lamellar lysis and accumulation of amorphous material. These changes were progressively associated with degeneration of Schwann cell bodies, resembling experimentally induced Wallerian-like degenerative processes in peripheral nerve fibers. In contrast, axons generally appeared well preserved, exhibiting intact mitochondria, vesicles, and cytoskeletal elements. Notably, unmyelinated fibers remained largely unaffected, with both axons and Schwann cells retaining well-preserved ultrastructural features. These findings reveal that myelinating Schwann cells are the earliest components to undergo degenerative changes during ex-vivo maintenance, indicating a particular susceptibility to ex-vivo stress rather than incompatibility with the adhesive matrices employed. Despite these alterations, axons remained largely preserved, suggesting that degenerating Schwann cells may still retain their protective functions. Residual insulating properties may also persist, potentially supporting continued impulse conduction along nerve fibers, with preservation of unmyelinated fibers likely contributing to the maintenance of detectable electrophysiological activity in explanted nerves.