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 as N-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 regenerations 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 cellular and molecular basis of peripheral nerve regeneration.

Nerve injury triggers the conversion of myelin and non-myelin (Remak) Schwann cells to a cell phenotype specialized to promote repair. Distal to damage, these repair Schwann cells provide the necessary signals and spatial cues for the survival of injured neurons, axonal regeneration and target reinnervation. The conversion to repair Schwann cells involves de-differentiation together with alternative differentiation, or activation, a combination that is typical of cell type conversions often referred to as (direct or lineage) reprogramming. Thus, injury-induced Schwann cell reprogramming involves down-regulation of myelin genes combined with activation of a set of repair-supportive features, including up-regulation of trophic factors, elevation of cytokines as part of the innate immune response, myelin clearance by activation of myelin autophagy in Schwann cells and macrophage recruitment, and the formation of regeneration tracks, Bungner's bands, for directing axons to their targets. This repair programme is controlled transcriptionally by mechanisms involving the transcription factor c-Jun, which is rapidly up-regulated in Schwann cells after injury. In the absence of c-Jun, damage results in the formation of a dysfunctional repair cell, neuronal death and failure of functional recovery. c-Jun, although not required for Schwann cell development, is therefore central to the reprogramming of myelin and non-myelin (Remak) Schwann cells to repair cells after injury. In future, the signalling that specifies this cell requires further analysis so that pharmacological tools that boost and maintain the repair Schwann cell phenotype can be developed.

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The repair Schwann cell and its function in regenerating nerves

Functional recovery is often poor despite the capacity for axonal regeneration in the peripheral nervous system and advances in microsurgical technique. Regeneration of axons in mixed nerve into inappropriate pathways is a major contributing factor to this failure. In this study, we use the rat femoral nerve model of transection and surgical repair to evaluate (1) the effect of nerve transection on the speed of regeneration and the generation of motor-sensory specificity, (2) the efficacy of electrical stimulation in accelerating axonal regeneration and promoting the reinnervation of appropriate muscle pathways by femoral motor nerves, and (3) the mechanism of action of electrical stimulation. Using the retrograde neurotracers fluorogold and fluororuby to backlabel motoneurons that regenerate axons into muscle and cutaneous pathways, we found the following. (1) There is a very protracted period (10 weeks) of axonal outgrowth that adds substantially to the delay in axonal regeneration (staggered regeneration). This process of staggered regeneration is associated with preferential motor reinnervation (PMR). (2) One hour to 2 weeks of 20 Hz continuous electrical stimulation of the parent axons proximal to the repair site dramatically reduces this period (to 3 weeks) and accelerates PMR. (3) The positive effect of short-term electrical stimulation is mediated via the cell body, implicating an enhanced growth program. The effectiveness of such a short-period low-frequency electrical stimulation suggests a new therapeutic approach to accelerate nerve regeneration after injury and, in turn, improve functional recovery.

https://www.jneurosci.org/content/jneuro/20/7/2602.full.pdf

Brief Electrical Stimulation Promotes the Speed and Accuracy of Motor Axonal Regeneration

Ongoing activity in peripheral nerves: the physiology and pharmacology of impulses originating from a neuroma

Nerve regeneration in silicone chambers : influence of gap length and of distal stump components

Examination of the processes of traumatic degeneration of myelin in the proximal stump of divided rat sciatic nerves with the electron microscope reveals a complete and coherent series of Schwann cells which are progressively more advanced in the degradation of the myelin. The earlier stages appear to be a wrinkling and distortion of the myelin lamellae, followed by erosion of the circumferential lamellae with subsequent loss of cohesion and fragmentation of the myelin systems. Retraction of the myelin from the region of the node of Ranvier is observed. Myelin debris in the cytoplasm of Schwann cells occurs as vacuoles containing membranous material and pale staining globular inclusions which may develop from the membrane containing vacuoles. Cells containing similar inclusions are present ‘free’ in the endoneurium. The presence of similar cells associated with axons and only partly covered by basement membrane suggests that the cells ‘free’ in the endoneurium are ‘transformed’ Schwann cells digesting their myelin. These cells are most frequent between 3 and 6 days after nerve section and gradually disappear thereafter. We did not see any indications that cells other than Schwann cells were extensively involved in the traumatic degeneration of myelin.

A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. I. The traumatic degeneration of myelin in the proximal stump of the divided nerve.

SummaryBetween seven days and six weeks after division the internal architecture of rat sciatic nerves is altered, their original mono- or di-fascicular configuration being replaced by a collection of small fascicles each surrounded by perineurium. This change, called by us ‘compartmentation’, has a minimum retrograde extent of 3.5 mm and is brought about by changes in Schwann cells and endoneurial fibroblasts, which undergo circumferential elongation to surround groups of axons and so come to resemble perineurial cells. Ultrastructural changes occur in these cells during compartmentation. There is a marked rise in the number of endoneurial fibroblasts in the distal segments of the proximal stump. The stimulus to the development of compartmentation is considered to be disturbance of the endoneurial environment following rupture of the perineurium. Changes in the structure and appearance of endoneurial cells suggest that metaplasia occurs between Schwann cells, endoneurial fibroblasts and perineurial cells, and it is concluded that these cell types in the endoneurium have a common origin from embryonic ectoderm. This suggests that the surgical treatment of peripheral nerve injuries should be primarily directed to the reconstitution of the endoneurial environment.

A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. IV. Changes in fascicular microtopography, perineurium and endoneurial fibroblasts.

SummaryIn the first six days after division myelinated axons in the proximal stump of rat sciatic nerves produce collateral and terminal sprouts. These are present as circumscribed “groups” which are positively distinguishable from clusters of non-myelinated axons. Two types of “groups” are identifiable, and their distribution in some of the nerve segments is analysed. Their evolution was followed in sequential nerve segments, the initial ‘tight’ structure becoming looser between 7 and 10 days, and myelinated axons appeared in them during this time. At this stage a complete basal lamina was present surrounding the entire “group”. Some of the cells in the “groups” did not have the characteristics of Schwann cells. Between 7 and 10 days after division alveolate vesicles and densely staining material in the cisternae of the rough surfaced endoplasmic reticulum were prominent in Schwann cells in the distal part of the proximal stump. It is thought that both types of “group” are developed from single myelinated axons and the name “regenerating unit” is proposed for both types. Their relationship to “clusters”, seen in the distal stump of regenerating peripheral nerves, and “onion bulbs”, present in some peripheral neuropathies, is discussed.

A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. II. The development of the "regenerating unit".

SummaryChanges in the proximal stump of axons of divided rat sciatic nerves in the first 6 weeks after nerve section were studied, particularly in terms of alterations in the organelle content, axoplasmic ultrastructure and the diameter of the axons. A variety of organelle types were observed; quasi-membranous structures, multivesicular bodies, dense bodies, vesicles and tubules, dense cored vesicles and alveolate vesicles: their identification and the functional implications of their presence are discussed. Alterations in the ultrastructure of the “stained” elements of the axoplasm are described. Axons containing excess organelles were divided into classes, comprising myelinated axons; and “supergiant”, “giant” and “conventional” non-myelinated axons. Temporal changes in these axons are described. The characteristics of the various classes of apparently non-myelinated axon are considered in terms of their identification as regenerating terminal sprouts of myelinated axons, segmentally demyelinated axons, sections through abnormal nodes of Ranvier or merely non-myelinated axons. The structure of axons in “regenerating units” is described. Changes in the neurofilament microtubule ratio of small axons without excess organelles are demonstrated, and “spiralling” of neurofilaments in some myelinated and non-myelinated axons with normal axoplasmic ultrastructure is illustrated and discussed.

G. Weddell

Papers

A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. I. The traumatic degeneration of myelin in the proximal stump of the divided nerve.

A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. IV. Changes in fascicular microtopography, perineurium and endoneurial fibroblasts.

A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. II. The development of the "regenerating unit".

A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. 3. Changes in the axons of the proximal stump.

Nerve endings in the conjunctiva.