Early events of peripheral nerve regeneration

Early regeneration of injured peripheral nerves involves a series of events that are important in the success of eventual reconnection. In many nerve injuries, such as transections with gaps, axons and Schwann cells (SCs) penetrate into new microenvironments de novo, not involving zones of Wallerian degeneration. We studied unexplored axon-SC interactions by sampling of newly forming connections through a silicone conduit across transected rat sciatic peripheral nerve gaps. Axon and SC participation in bridge formation was addressed by light microscopy, electron microscopy and by double-labeling immunohistochemistry,including confocal imaging, and several, less appreciated aspects of early regrowth were identified. There are limitations to early and widespread regeneration of axons and SCs into bridges initially formed from connective tissue and blood vessels.Regrowth is 'staggered' such that only a small percentage of parent axons sampled the early bridge. There is an intimate, almost invariable relationship between SCs and extension of axons, which challenges the concept that axons lead and SCs follow.'Naked' axons were infrequent and limited in scope. Axons did not seek out and adhere to vascular laminin but intimately followed laminin deposits associated with apposed SCs. Growth cones identified by labeling of beta III tubulin, PGP(9 x 5) and GAP(43)/B(50) were complex, implying a pause in their regrowth, and were most prominent at the proximal stump-regenerative bridge interface. There is surprising and substantial hostility to local regrowth of axons into newly forming peripheral nerve bridges.Early axon outgrowth, associated with apposed Schwann cell processes, is highly constrained even when not exposed to adjacent myelin and products of Wallerian degeneration.     

Limb muscle regeneration in peripheral nerve autografts

In a previous study we demonstrated regenerative growth of extraocular muscle within transplanted peripheral nerve autografts. The present study addresses the feasibility of inducing regeneration of limb muscle within autologous peripheral nerve implants in the gluteus medius of beagles. In six anesthetized animals, a 2-cm segment of the left infraorbital sensory nerve was removed from the nose and implanted between the cut ends of several muscle fascicles in the left gluteus medius. After 4 weeks, the nerve grafts were removed and examined by light and electron microscopy. Muscle fibers were seen surrounded by the epineurium of the implanted nerve along its entire length, growing in parallel with the long axis of the nerve. The regenerating fibers were closely associated with the basal lamina of degenerating myelinated and unmyelinated axons. This study suggests that limb muscle, like extraocular muscle, is capable of organized regenerative growth within peripheral nerve autografts.     

Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration

ABSTRACT: BACKGROUND: Nerve conduits provide a promising strategy for peripheral nerve injury repair. However, the efficiency of nerve conduits to enhance nerve regeneration and functional recovery is often inferior to that of autografts. Nerve conduits require additional factors such as cell adhesion molecules and neurotrophic factors to provide a more conducive microenvironment for nerve regeneration. METHODS: In the present study, poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]} (PLGL) was modified by grafting Gly-Arg-Gly-Asp-Gly (RGD peptide) and nerve growth factor (NGF) for fabricating new PLGL-RGD-NGF nerve conduits to promote nerve regeneration and functional recovery. PLGL-RGD-NGF nerve conduits were tested in the rat sciatic nerve transection model. Rat sciatic nerves were cut off to form a 10 mm defect and repaired with the nerve conduits. All of the 32 Wistar rats were randomly divided into 4 groups: group PLGL-RGD-NGF, group PLGL-RGD, group PLGL and group autograft. At 3 months after surgery, the regenerated rat sciatic nerve was evaluated by footprint analysis, electrophysiology, and histologic assessment. Experimental data were processed using the statistical software SPSS 10.0. RESULTS: The sciatic function index value of groups PLGL-RGD-NGF and autograft was significantly higher than those of groups PLGL-RGD and PLGL. The nerve conduction velocities of groups PLGL-RGD-NGF and autograft were significantly faster than those of groups PLGL-RGD and PLGL. The regenerated nerves of groups PLGL-RGD-NGF and autograft were more mature than those of groups PLGL-RGD and PLGL. There was no significant difference between groups PLGL-RGD-NGF and autograft. CONCLUSIONS: PLGL-RGD-NGF nerve conduits are more effective in regenerating nerves than both PLGL-RGD nerve conduits and PLGL nerve conduits. The effect is as good as that of an autograft. This work established the platform for further development of the use of PLGL-RGD-NGF nerve conduits for clinical nerve repair.     

Scanning electron microscopy of isolated peripheral nerve fibres

Summary The surface morphology of normal myelinated nerve fibres prepared in different ways for scanning electron microscopy has been studied and compared with the surface features of similar fibres undergoing retrograde changes. Nodes of Ranvier, paranodal specializations, artefactual fractures of the myelin, and the endoneurial collagen sheaths are described. A regular pattern of elevations, usually with a pitted or depressed surface seen on normal myelinated fibres after certain preparative procedures are thought to be artefacts produced during preparation and to be related to the neurokeratin network.Alterations in the surface structure of fibres central to long-standing nerve transections include irregular protuberances, serial surface corrugations and large swellings, all associated with demyelination. Fibres that have undergone retrograde degeneration consist of endoneurial tubes with focal swellings occupied by macrophages or myelin debris, together with fine unmyelinated and small myelinated regenerating axons. Strict centrifugal progression of myelination of regenerating axons was not observed.We thank Mr. R. A. Willis for his collaboration and for taking the SEM photographs of normal nerve fibres, and the Cambridge Scientific Instrument Co. Ltd. for permission to reproduce the SEM photographs of experimental nerve fibres. We also thank Dr. A. Boyde for access to his SEM and for helpful comments on interpretation of the scanning electron micrographs, Prof. J. Z. Young, Dr. P. K. Thomas, and Dr. R. H. M. King for discussion, and Messrs. P. Reynolds and D. Gunn for photography.A grant from the Muscular Dystrophy Group of Great Britain is gratefully acknowledged.     

Venous graft-derived cells participate in peripheral nerve regeneration

Background

Based on growing evidence that some adult multipotent cells necessary for tissue regeneration reside in the walls of blood vessels and the clinical success of vein wrapping for functional repair of nerve damage, we hypothesized that the repair of nerves via vein wrapping is mediated by cells migrating from the implanted venous grafts into the nerve bundle.

Methodology/Principal Findings

To test the hypothesis, severed femoral nerves of rats were grafted with venous grafts from animals of the opposite sex. Nerve regeneration was impaired when decellularized or irradiated venous grafts were used in comparison to untreated grafts, supporting the involvement of venous graft-derived cells in peripheral nerve repair. Donor cells bearing Y chromosomes integrated into the area of the host injured nerve and participated in remyelination and nerve regeneration. The regenerated nerve exhibited proper axonal myelination, and expressed neuronal and glial cell markers.

Conclusions/Significance

These novel findings identify the mechanism by which vein wrapping promotes nerve regeneration.     

Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration

Abstract.  Objective : In our study, we describe the use of spider silk fibres as a new material in nerve tissue engineering, in a 20-mm sciatic nerve defect in rats. Materials and methods : We compared isogenic nerve grafts to vein grafts with spider silk fibres, either alone or supplemented with Schwann cells, or Schwann cells and matrigel. Controls, consisting of veins and matrigel, were transplanted. After 6 months, regeneration was evaluated for clinical outcome, as well as for histological and morphometrical performance. Results : Nerve regeneration was achieved with isogenic nerve grafts as well as with all constructs, but not in the control group. Effective regeneration by isogenic nerve grafts and grafts containing spider silk was corroborated by diminished degeneration of the gastrocnemius muscle and by good histological evaluation results. Nerves stained for S-100 and neurofilament indicated existence of Schwann cells and axonal re-growth. Axons were aligned regularly and had a healthy appearance on ultrastructural examination. Interestingly, in contrast to recently published studies, we found that bridging an extensive gap by cell-free constructs based on vein and spider silk was highly effective in nerve regeneration. Conclusion : We conclude that spider silk is a viable guiding material for Schwann cell migration and proliferation as well as for axonal re-growth in a long-distance model for peripheral nerve regeneration.     

Pleiotrophin cellular localization in nerve regeneration after peripheral nerve injury.

Pleiotrophin (PTN) is a member of the family of heparin-binding growth factors that displays mitogenic activities and promotes neurite outgrowth in vitro. In vivo, PTN is widely expressed along pathways of developing axons during the late embryonic and early postnatal period. Although the level of PTN gene expression is very low during adulthood, activation of the gene may occur during recovery from injury and seems to play an important role in tissue regeneration processes. In this study, we investigated whether PTN was involved in the regenerative process of injured peripheral nerves. To refer localization of the fluorescent markers to myelinated axons, we developed a specific computer tool for colocalization of fluorescence images with phase contrast images. Immunohistochemical analysis showed PTN in different types of nonneural cells in distal nerve segments, including Schwann cells, macrophages, and endothelial cells, but not in axons. Schwann cells exhibited PTN immunoreactivity as early as 2 days after injury, whereas PTN-positive macrophages were found 1 week later. Strong PTN immunoreactivity was noted in endothelial cells at all time points. These findings support the idea that PTN participates in the adaptive response to peripheral nerve injury. A better understanding of its contribution may suggest new strategies for enhancing peripheral nerve regeneration.     

The cellular and molecular basis of peripheral nerve regeneration

Functional recovery from peripheral nerve injury and repair depends on a multitude of factors, both intrinsic and extrinsic to neurons. Neuronal survival after axotomy is a prerequisite for regeneration and is facilitated by an array of trophic factors from multiple sources, including neurotrophins, neuropoietic cytokines, insulin-like growth factors (IGFs), and glial-cell-line-derived neurotrophic factors (GDNFs). Axotomized neurons must switch from a transmitting mode to a growth mode and express growth-associated proteins, such as GAP-43, tubulin, and actin, as well as an array of novel neuropeptides and cytokines, all of which have the potential to promote axonal regeneration. Axonal sprouts must reach the distal nerve stump at a time when its growth support is optimal. Schwann cells in the distal stump undergo proliferation and phenotypical changes to prepare the local environment to be favorable for axonal regeneration. Schwann cells play an indispensable role in promoting regeneration by increasing their synthesis of surface cell adhesion molecules (CAMs), such asN-CAM, Ng-CAM/L1, N-cadherin, and L2/HNK-1, by elaborating basement membrane that contains many extracellular matrix proteins, such as laminin, fibronectin, and tenascin, and by producing many neurotrophic factors and their receptors. However, the growth support provided by the distal nerve stump and the capacity of the axotomized neurons to regenerate axons may not be sustained indefinitely. Axonal regeneration may be facilitated by new strategies that enhance the growth potential of neurons and optimize the growth support of the distal nerve stump in combination with prompt nerve repair.     

The effects of cisplatinum and vincristine on peripheral nerve regeneration

Current treatment modalities for extremity sarcoma often include tumor extirpation plus neoadjuvant therapy. Limb-sparing surgery may require reconstruction of critical nerve defects. Neurotoxic side effects from adjuvant chemotherapy have been reported and raise concerns regarding the effects of chemotherapy on nerve regeneration. In an attempt to define the effects of adjuvant chemotherapy on peripheral nerve regeneration, cisplatin and vincristine were administered to rats following isografting of the posterior tibial nerve. Parameters used to assess peripheral nerve regeneration included walking track analysis and histomorphology. Sixty 250-g Sprague-Dawley rats were randomly allocated into one of three treatment groups. Each animal underwent a 15-mm reversed interposition nerve isograft from 30 donor rats into the right posterior tibial nerve. Ten animals served as control. The remaining animals were divided into two groups of 25 animals each. One group received cisplatin (75 mg/m2) and the other group received vincristine (1 mg/m2). Chemotherapy was administered at 4-week cycles for a total of six cycles (24 weeks). Walking track analysis was performed monthly. Nerve specimens were harvested from the grafted segment and the distal posterior tibial nerve for histomorphology. Walking track analysis demonstrated no statistical difference in print length between the control and chemotherapeutic groups at the conclusion of the study. The number of axons per square millimeter and nerve fiber density were not statistically different between control and chemotherapeutic groups. In the rodent posterior tibial nerve model, postoperative adjuvant therapy does not significantly alter functional outcome in peripheral nerve regeneration. The practice of immediate nerve grafting after tumor extirpation, despite planned postoperative chemotherapy, is supported.