Evaluation of the long-term regenerative potential in an experimental nerve amputee model

In this study, we evaluated the long-term maintenance of regenerated axons in an experimental nerve amputee model. The sciatic nerve of adult rats was transected and repaired with a silicone tube leaving a short gap; the distal nerve segment was again transected 10 mm distally and the distal stump either introduced in a capped silicone chamber (amputee group) or connected to denervated targets (tibial branch into the gastrocnemius muscle and peroneal nerve apposed to skin) (reinnervation group). Morphological studies were performed at 2.5, 6, and 9 months after surgery. In all cases, axons regenerated across the silicone tube and grew in the distal nerve segment. In the amputee group, the morphological results show the expected features of a neuroma that is formed when regenerating axons are prevented from reaching the end organs, with a large number of axonal profiles indicative of regenerative sprouting. The number of myelinated axons counted at the distal nerve was sustained over 9 months follow-up, indicating that regenerated axons are maintained chronically. Immunohistochemical labeling showed maintained expression of choline acetyltransferase, calcitonin gene-related peptide, and growth-related peptides 43 in the distal neuroma at 6 and 9 months. Reconnection of the distal nerve to foreign targets mildly improved the pattern of nerve regeneration, decreasing the number of excessive sprouts. These results indicate that axons regenerated may be eventually interfaced with external input-output systems over long time, even if ending in the absence of distal targets as will occur in amputee limbs.