Irreversible changes occurring in long-term denervated Schwann cells affect delayed nerve repair

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Journal of Neurosurgery

Journal of Neurosurgery, Jenuary 2017

Irreversible changes occurring in long-term denervated Schwann cells affect delayed nerve repair

Giulia Ronchi 1,2 , Michele Cillino 3 , Giovanna Gambarotta 1 , Benedetta Elena Fornasari 1 , Stefania Raimondo 1, 2 , Pierfrancesco Pugliese 4 , Pierluigi Tos 4 , Adriana Cordova 3 , Francesco Moschella 3 and Stefano Geuna 1, 2

Multiple factors may affect functional recovery after peripheral nerve injury, among them the lesion site and the interval between the injury and the surgical repair. When the nerve segment distal to the lesion site undergoes chronic degeneration, the ensuing regeneration (when allowed) is often poor. The aims of the current study were as follows: 1) to examine the expression changes of the neuregulin 1/ErbB system during long-term nerve degeneration; and 2) to investigate whether a chronically denervated distal nerve stump can sustain nerve regeneration of freshly axotomized axons.

This study used a rat surgical model of delayed nerve repair consisting of a cross suture between the chronically degenerated median nerve distal stump and the freshly axotomized ulnar proximal stump. Before the suture, a segment of long-term degenerated median nerve stump was harvested for analysis. Functional, morphological, morphometric, and biomolecular analyses were performed.

The results showed that neuregulin 1 is highly downregulated after chronic degeneration, as well as some Schwann cell markers, demonstrating that these cells undergo atrophy, which was also confirmed by ultrastructural analysis. After delayed nerve repair, it was observed that chronic degeneration of the distal nerve stump compromises nerve regeneration in terms of functional recovery, as well as the number and size of regenerated myelinated fibers. Moreover, neuregulin 1 is still downregulated after delayed regeneration.

The poor outcome after delayed nerve regeneration might be explained by Schwann cell impairment and the consequent ineffective support for nerve regeneration. Understanding the molecular and biological changes occurring both in the chronically degenerating nerve and in the delayed nerve repair may be useful to the development of new strategies to promote nerve regeneration. The results suggest that neuregulin 1 has an important role in Schwann cell activity after denervation, indicating that its manipulation might be a good strategy for improving outcome after delayed nerve repair.

Key Words delayed nerve repair; NRG1/ErbB system; neuregulin 1; nerve degeneration; nerve regeneration; stereology; peripheral nerve

1 Department of Clinical and Biological Sciences, University of Torino
2 Neuroscience Institute Cavalieri Ottolenghi
3 Plastic and Reconstructive Surgery, Department of Surgical, Oncological and Oral Sciences, University of Palermo, Italy
4 Reconstructive Microsurgery, Centro Traumatologico Ortopedico Hospital, University of Torino.
(A-C) Transmission electron micrographs of degenerating median nerve 3 months (A) , 6 months (B) , and 9 months (C) after axotomy. Bar = 2 μm. Neither myelinated nor unmyelinated fibers were detectable. White asterisk: atrophic Schwann cells; black asterisk: fibroblastic-like cells. (D) Soluble Type I/II NRG1 isoform mRNA expression in degenerated median nerves. Values in the graphs are expressed as the mean ± SEM (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). Soluble NRG1 was strongly downregulated. (E-G) Light micrographs of regenerated nerves after immediate repair (E) , 3-month delay (F) , and 6-month delay (G) . Bar = 10 μm. After 6 months of regeneration, delayed repaired groups showed many regrowing myelinated fibers, but smaller and less numerous compared to immediate repair group. (H) Soluble Type I/II NRG1 isoform mRNA expression in the 6-month delayed repair group, after 6 months of regeneration. This result was compared with the expression obtained in the 6-month degenerating nerve and in the control nerves. An additional positive control for regeneration (end-to-end–repaired median nerve, 3 months after regeneration) is shown. Values in the graphs are expressed as the mean + SEM (**p ≤ 0.01; ***p ≤ 0.001). Soluble NRG1 (Type I/II) was still strongly downregulated after 6 months of delayed nerve regeneration.