Peripheral nerve regeneration and intraneural revascularization

Grade I

Grade I most often corresponds to peripheral nerve compression. This type of injury is caused by direct pressure on a nerve trunk or root compression (Sunderland, 1951). Moreover, nerve compressive injuries most commonly affect nerves that cross over bony surfaces or pass between rigid structures. Nerve compression syndrome, also named compression neuropathy, can be cited in a number of disorders, for example, cubital tunnel syndrome (ulnar nerve) (Lauretti et al., 2017), carpal tunnel syndrome (median nerve) (Wahab et al., 2017), meralgia paraesthetica (lateral cutaneous nerve) (Khalil et al., 2012), and the tarsal tunnel syndrome (tibial nerve) (Doneddu et al., 2017). Similar lesions are found in sciatica syndrome, which is mostly due to compression of the sciatic nerve by a herniated disc (Markman et al., 2018). Compression neuropathies may also occur in patients who hold a single position for an extended period. These injuries include the “Saturday night palsy”, in which the upper arm of a patient is injured, usually as a result of sleeping with the affected arm over the back of a chair. A similar problem can be recognized during the postoperative setting when a limb is held in the same position for a prolonged time (Burnett and Zager, 2004). It is known that mechanical compression may lead to secondary ischemic injury. When the intra-epineural pressure exceeds intra-arterial pressure, nerve ischemia results in axon-loss lesions of variable severity. These two pathological mechanisms, mechanical compression and ischemia, are believed to be involved in grade I injury. Grade I injury is accompanied by morphological alteration of the myelin sheath or segmental demyelination. Electrophysiological examination may reveal demyelinating conduction blocks and sometimes axonal loss. Grade I is clinically associated with a, most often transient, deficiency of motor function, sometimes leading to neuropathic pain. Generally, there is not total loss of both motor and sensory functions, because complete nerve continuity is maintained (Burnett and Zager, 2004).

Different models have been used to study degenerative/regenerative mechanisms involved after grade I and to evaluate potential therapies.

The oldest compression-induced neuropathy model used was direct pressure applied to the sciatic nerve of cats, brought about by use of a bag or by a tourniquet (Denny-Brown and Brenner, 1944a, b). Later this compression-induced neuropathy model was also applied to baboons (Fowler et al., 1972; Ochoa et al., 1972). In this compression model, there is a demyelination of the nerve fibers that then leads to a block of peripheral nerve conduction with a moderate axonal loss. More recently, chemical models have been developed to mimic focal demyelination. For instance, it has been shown that an intraneural injection of phenol into rabbit sciatic nerve induces conduction blocks (Sung et al., 2001). Perineural injection of lysophosphatidylcholine (LPC) was also demonstrated to cause focal demyelination in rodent nerves (Hall and Gregson, 1971). Cold injury models have also been reported in which non-freezing cold injuries caused local failure of nerve conduction at the site of cooling (Kennett and Gilliatt, 1991). However, neuropathy models, induced by chemical products or cold injury, are difficult to compare with mechanically-induced neuropathy since the mechanisms involved are certainly different. Bennett and Xie (1988) developed an animal nerve injury model that appears to mimic many features of neuropathic disorders that occur in humans. The injury is caused, in rodents, by a loose ligation of the sciatic nerve termed chronic constriction injury (CCI). The animals display a block of nerve conduction with a moderate axonal loss, altered spontaneous behavior associated with neuropathic pain and allodynia to thermal and mechanical stimuli.

Grades II, III and IV

Grades II, III and IV are more severe than grade I. As mentioned above, the different grades described in Sunderland’s classification depend on the severity of the lesion, with loss of axon continuity and demyelination (grade II), with damage of the endoneurium (grade III) and the perineurium (grade IV) (Sunderland, 1951). Grade II, III and IV types of injuries include different causes of lesions, i.e., crush and stretch. In the following section, we will discuss the mechanisms involved in crush and stretch damage and the animal models that mimic these two types of lesions.

Nerve crush injury is caused by a sudden significant force applied to the nerve, for instance with a blunt object. These lesions can also be induced by a surgical clamp during surgery, by blows resulting from an assault, or by a vehicle impact or accident (Martyn and Hughes, 1997). There are different severities of crush injury, but axon loss is consistently observed. Functional recovery in humans is very heterogeneous and may require microsurgery. However, functional recovery is generally complete, though often requiring considerable time. Pathophysiologically, nerve crush produces an interruption of the continuity of axons leading to Wallerian degeneration. However, there is no interruption of the nerve connective tissue scaffold and, for that reason, regrowth of the nerve fibers is easier and thus allows for better functional recovery (Geuna et al., 2009). However, formation of a traumatic neuroma is frequently observed at injury sites, which can limit or even prevent nerve regrowth.

The nerve crush model is commonly used by researchers studying peripheral nerve regeneration. Generally, experiments are performed on the sciatic nerves of rodents using serrated or non-serrated surgical forceps to inflict the injury. However, various other methods have been described for producing the crush injury. There is therefore a strong heterogeneity in studies, mainly due to the use of different surgical instruments (Chen et al., 1993; Kingery et al., 1994; Savastano et al., 2014). Beer et al. (2001) described the use of a non-serrated clamp aimed at standardizing the pressure exerted to the nerve. This device has provided good reproducibility in different rodent species (Beer et al., 2001; Varejão et al., 2004a, b). In animal models, it is usually accepted that inter-individual variability in tissue regeneration and functional recovery is limited. This feature makes the sciatic nerve crush injury model particularly suitable for the study of the biology of peripheral nerve regeneration as well as for the development of treatment strategies to improve it. However, spontaneous axon regeneration, observed in laboratory animals after crushing the nerve, does not often occur in humans due to frequent extensive fibrosis that is observed at the lesion site. Therefore, in many severe cases, crush lesions in humans require surgery to remove the damaged tissue and replace it with a conduit (Tos et al., 2012). A chemical model has also been developed to mimic grades II and III. For example, chemical neuropathy using an intraneural injection of glycerol into the sciatic nerve of rats showed a complete destruction of nerve fibers and the myelin sheath but maintenance of the connective scaffold, including perineurium and epineurium (Vallat et al., 1988). This model, which was used in the present study to evaluate vascularization during nerve regeneration, will be described in greater detail below.

Peripheral nerves are also vulnerable to excessive stretch. As the stretching force increases, the elastic properties of the nerve are surpassed, and myelin sheaths, axons, and some of the connective tissue may rupture. Experimental nerve stretch studies have shown that an elongation by 8%, of the rat sciatic nerve, increases intraneural pressure and reduces the blood flow by half without reducing nerve conduction velocity (Driscoll et al., 2002). An example of stretch injury in humans is neonatal Erb-Duchenne palsy, a paralysis of the arm that most commonly arises from shoulder avulsion of the brachial plexus during a complicated or difficult birth (Teixeira et al., 2015). In humans, another stretch injury may be produced by a heavy object falling onto a person’s shoulder; this results in avulsion of the brachial plexus, and pulling out of the spinal cord rootlets. These avulsion injuries are most often due to vehicle accidents. Avulsion is associated with particularly severe and long-lasting neuropathic pain. In accordance with this feature, experimental models of brachial plexus avulsion show bilateral mechanical and cold allodynia, that last significantly longer than that observed in chronic constriction injury (grade I model) and crush models (Rodrigues-Filho et al., 2003).

Grade V

Grade V, according to Sunderland’s classification, is the most severe nerve injury. In this type of injury, there is a total loss of nerve trunk continuity (Sunderland, 1951). The most frequent etiology is transection or laceration of peripheral nerves. Transection injuries are caused by a cutting object (for example, knife wounds, broken glass, metal shards, chainsaw blades, wood splinters, and animal bites) (Martyn and Hughes, 1997). Another example is the inadvertent transection of spinal nerve branches during a surgical intervention. While partial recovery may occur, full spontaneous recovery is impossible. In general, as in the skin and other tissues, the sharper the object that cuts, the neater the injury and the better is recovery. Furthermore, the likelihood of satisfactory functional recovery is greater when the distance between proximal and distal nerve stumps is shorter. In humans, a nerve defect requires the implantation of an autograft using, for example, a segment of a sensory nerve, usually the sural nerve (Berger and Millesi, 1978). Another solution, in the case of tissue loss, is the implantation of tubular reconstruction using either biological materials (biological nerve guides, various autologous tissues, bio-functionalized bio-materials) and/or synthetic materials (silicon, poly ε-caprolactone, chitosan…) (Tos et al., 2012; Haastert-Talini et al., 2013; Reid et al., 2013; Johansson and Dahlin, 2014; Stößel et al., 2018).

In laboratory animals, axonal regeneration occurs spontaneously after complete nerve transection (after suture). Hence, in order to mimic nerve repair in humans, researchers turned to the proximal nerve stump, suturing it to a neighboring stump. In the case of the sciatic nerve, complete transection that is not followed by surgical repair induces the loss of motor function in the ipsilateral paw. Furthermore, sciatic nerve transection also induces loss of sensory function of the paw, explaining the self-mutilation frequently observed in the early post-operative stages. The experimental transection model is a very useful approach to study denervation of distal nerve trunks and the consequences on target skeletal muscles, as well as for studying strategies to prevent muscle atrophy (Karsidag et al., 2012; Moimas et al., 2013; Blom et al., 2014; Gordon and Borschel, 2017). Since alterations in both the distal nerve segment and target muscle occur very early (1 week) after injury, the transection model can be used to study both early and late events (3 to 6 months). In addition, sciatic nerve transection can be used to study direct suture repair (end-to-end neurorrhaphy) (Geuna et al., 2009). If no tissue loss occurs, sciatic nerve transection can be used in rodent models to study tissue glues (Félix et al., 2013) and strategies for reducing post-surgical scar tissue formation (Que et al., 2013). However, even if spontaneous regeneration occurs, a nerve graft may still be necessary to avoid tension or stretch in the nerve, a condition which might limit regeneration and functional recovery (as mentioned above) (Battiston et al., 2009; Siemionow and Brzezicki, 2009).

Wallerian degeneration

Traumatic peripheral neuropathies are usually characterized by section of the axon which is then disconnected from its cell body. Axonal injury produces a complete loss of nerve conduction velocity, which in turn induces muscle weakness and loss of sensation, but also leads to neuropathic pain and adaptive responses (Stoll and Müller, 1999). In addition, nervous tissue damage can be deleterious for patients because recovery is not always complete, with the formation of scar-like tissue impairing optimal limb functionality. If the injury is large or close to the cell body, it can induce neuronal death. However, if the nerve section or the crush injury is distant from the neuron cell body, nerve fibers can regenerate. The degeneration of the distal nerve ending is known as Wallerian degeneration (Figure 2). Wallerian degeneration is a unique and structured form of axon degeneration (Stoll et al., 1989). In the first stages of Wallerian degeneration, axonal and myelin debris are produced. Resident macrophages in the nerve tissue then differentiate into activated-macrophages which can phagocytose cellular debris. Moreover, the surrounding Schwann cells (SCs) are a source of monocyte chemoattractant protein-1 which leads to the recruitment of circulating monocytes (Toews et al., 1998). In axons, activation of mRNA translation is observed in the proximal stumps. This increased translation stimulates the formation of the protein complex, importin-phosphorylated extracellular regulated protein kinase 1/2 vimentin. This complex is transported by the motor protein dynein in a retrograde direction to the cell body and this signal informs the neuron of the axonal damage (Yudin et al., 2008). The neuron then reacts by increasing its volume and by breaking up aggregates of rough endoplasmic reticulum (Nissl bodies), which promotes protein synthesis. In addition, within the first hours after injury, analysis of spinal cord tissue has shown that messenger RNAs for antioxidant proteins (Nrf2, glutathione reductase and heme oxygenase 1) are increased after, in this case, sciatic nerve crush (Renno et al., 2017). The ending of the cut axon rapidly closes and swells because of the accumulation of substances that arrive from the neuron cell body. Only a few hours after the nerve injury, the growing axonal extremity extends filipodia. At first, these are randomly oriented but thereafter, they gain unidirectionality, once the expression of myosin and actin are upregulated within the neuron cell body (Marx, 1995). Concomitantly, regeneration and reorganization of blood vessels accompany the growth of axons. This specific and important aspect of the response to damage will be further discussed below.

From two days after injury (D2), circulating monocytes differentiate into activated-macrophages and contribute to the removal of axonal and myelin debris, a process persisting at least for 2 weeks (Taskinen and Röyttä, 1997; Stoll and Müller, 1999). In addition, phagocytosis is dependent on the galactoside-binding protein of macrophages galectin-3, which supports the elimination of myelin debris by SCs themselves (Reichert et al., 1994; Rotshenker, 2009). In early stages, damaged SCs are involved in protein expression changes at the site of injury. The loss of contact between SCs and damaged axons induces retraction of the myelin sheath and leads to the formation of ovoid structures. These ovoid structures appear two days after a lesion and persist for at least 2–3 weeks (Stoll et al., 1989; Reynolds et al., 1994). In parallel with the loss of axonal contact, SCs progressively acquire a non-myelinating phenotype and become proliferative. Marked down-regulation of the expression of several proteins, including peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ or P0), connexin-32 and transcription factor Krox-20 has been reported (Topilko et al., 1994; Parkinson et al., 2008).

Nerve regeneration

Successful peripheral nerve regeneration after injury relies on both injured axons and non-neuronal cells, including SCs, endoneurial fibroblasts and macrophages. Together, these produce a supportive microenvironment allowing successful regrowth of the proximal nerve fiber ending (Figure 2).

Apart from their roles in phagocytosis of debris, macrophages also secrete interleukin (IL)-1 which induces secretion of nerve growth factor (NGF) by SCs, promoting autocrine-stimulated growth and proliferation of SCs (Ronchi et al., 2009). SCs have a peak in mitotic activity at 3–4 days after injury. This event is instrumental in promoting axonal regeneration. Indeed, macrophages are also involved in the production of factors that are mitogenic for SCs and fibroblasts, such as transforming growth factor (TGF)-β and insulin-like growth factor (IGF) (Baichwal et al., 1988; Zochodne, 2000). Finally, macrophages, in damaged nerves, help to rescue valuable cholesterol and especially, produce apolipoprotein E, enabling future myelin reconstruction (Weinberg and Spencer, 1978; Baichwal et al., 1988; Rotshenker, 2003).

In neurons, the production of proteins associated with growth including brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), growth-associated protein (GAP)-43 and c-jun, and of neuropeptides such as calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), substance P and somatostatin is increased (Zochodne, 2000). Conversely, neurofilament synthesis and release of neurotransmitters (acetylcholine) are repressed (Fu and Gordon, 1997). Thus, from D3 to D7 post-injury, a growth cone is formed at the ending of the proximal axonal stump. It has been found that calcium plays a major role at the proximal stump in promoting growth cone formation (Geraldo and Gordon-Weeks, 2009). Neurotrophins play a central role in attraction and repulsion of the filipodia. For example, semaphorins, ephrins, and netrins are involved in the guidance of regrowing axons. In contrast, molecules, such as collapsin 1, which promotes growth cone collapse, inhibit the guidance of regrowing axons (Goodman, 1996; Tuttle and O’Leary, 1998). The development of the growth cone may also be inhibited by scar tissue (mainly composed of collagen) and thus, to counteract this, axonal growth cones release proteases and plasminogen activators favoring the degradation of this scar tissue (Geraldo and Gordon-Weeks, 2009). These proteases and plasminogen activators also help to clear any cell-cell or cell-matrix interactions from non-neuronal cells that are hindering progression of the growth cones (Geraldo and Gordon-Weeks, 2009). SCs are the main source of neurotrophic factors which interact with tyrosine kinase receptors to modify the neuronal gene expression profile in order to promote axonal growth. NGF is responsible for promoting axonal growth and SC proliferation (Lunn et al., 1990). At this stage, the SC nuclei become more rounded and their content in heterochromatin is reduced (Ide, 1996). SCs and endoneurial fibroblasts migrate and proliferate at the lesion site to form the neuroma, a tissue bridge that attempts to restore the junction between the proximal and the distal segments. In the distal segment, four days after cutting of the sciatic nerve in the rat, proliferating SCs are aligned in columns forming “bands of Büngner” that are involved in endoneurial regeneration (Burnett and Zager, 2004). Bands of Büngner are linear bands of interdigitating SCs that form a physical guide for axonal regrowth. Neurotrophic factors (e.g., semaphorins, ephrins, netrins…) increase cell survival, and also promote the secretion of regenerating factors (e.g., ciliary neurotrophic factor [CNTF], BDNF, GDNF, and NGF) in neurons and in SCs. Furthermore, SCs produce neurite-promoting proteins such as fibronectin and laminin, which are incorporated into the extracellular matrix (ECM). Growth cones utilize these proteins for adhesion to the basal lamina (a layer of ECM secreted by SCs, on which the SCs sit) of the endoneurium (Lunn et al., 1990).

Recently, it has been shown that crush injury of the sciatic nerve produces generalized oxidative stress with a significant increase in isoprostanes found in the urine and a decrease in the total antioxidant capacity of the blood from D7 until D14 after injury (Renno et al., 2017).

Three to four weeks after injury, maturation needs to occur before the functional connection is complete. The maturation process includes remyelination, axon enlargement, and finally, functional re-innervation. SC proliferation is then arrested and the production of C-jun promotes differentiation; furthermore, the secretion of neurotrophic factors such as NGF and CNTF occurs (Elfar et al., 2008). The outgrowing axonal endings produce neuregulin-1 (NRG1) type III and I, ATP and acetylcholine, which thus promote the change in the SC phenotype from a non-myelinating phenotype to a myelinating phenotype (Birchmeier and Nave, 2008; Vrbova et al., 2009). In rodents, at five weeks after injury, the general nerve morphology observed using light microscopy appears normal. However, using electron microscopy, a depletion of nerve fiber number (both myelinated and unmyelinated) and the development of small clusters of three or four regenerating axons called “clusters of regeneration” are observed. In addition, macrophage invasion in myelin sheaths has been observed (Vallat et al., 1988). At the molecular level, a proteomic study has shown that five weeks after crush injury in rats, the expression of proteins involved in signaling and metabolism in nerves (aldehyde reductase 1, annexin V, alpha-crystallin B, dimethyl argininase 1, sialic acid synthase, periaxin, ubiquitin C-terminal hydrolase…) are decreased (Jiménez et al., 2005). In contrast, the expression of proteins involved in lipid metabolism (apolipoprotein D, apolipoprotein E, cathepsin B…) and of those involved in cellular processes of maturation and degradation enabling future myelin reconstruction (galectin 3, tropomyosin 4-alpha, tropomyosin 3-gamma…) are up-regulated (Jiménez et al., 2005). These proteomic data indicate that axonal regrowth is complete and that the maturation phase, i.e., remyelination of fibers, is beginning.

Finally, five weeks after sciatic nerve injury in rats, an increase in reactive oxygen species (ROS) and lipoperoxidation has been reported. In nerves, the increase in ROS has been attributed to an increase in myeloperoxidase, a pro-oxidant enzyme secreted particularly by macrophages (Caillaud et al., 2018).

Three months after injury, nerve fibers appear almost normal. However, the endoneurium is still found to be divided into many micro-compartments by a profusion of fibroblasts that gradually differentiate into perineurial cells. Normal perineurium also regenerates within this time. This micro-compartmentalization slowly develops from the periphery. After several months, small newly formed fascicles at the periphery of the original sciatic fascicles are observed (Vallat et al., 1988).