Misdirection and guidance of regenerating axons after experimental nerve injury and repair

A review

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Misdirection of regenerating axons is one of the factors that can explain the limited results often found after nerve injury and repair. In the repair of mixed nerves innervating different distal targets (skin and muscle), misdirection may, for example, lead to motor axons projecting toward skin, and vice versa—that is, sensory axons projecting toward muscle. In the repair of motor nerves innervating different distal targets, misdirection may result in reinnervation of the wrong target muscle, which might function antagonistically. In sensory nerve repair, misdirection might give an increased perceptual territory. After median nerve repair, for example, this might lead to a dysfunctional hand.

Different factors may be involved in the misdirection of regenerating axons, and there may be various mechanisms that can later correct for misdirection. In this review the authors discuss these different factors and mechanisms that act along the pathway of the regenerating axon. The authors review recently developed evaluation methods that can be used to investigate the accuracy of regeneration after nerve injury and repair (including the use of transgenic fluorescent mice, retrograde tracing techniques, and motion analysis). In addition, the authors discuss new strategies that can improve in vivo guidance of regenerating axons (including physical guidance with multichannel nerve tubes and biological guidance accomplished using gene therapy).

Abbreviations used in this paper:BDNF = brain-derived neurotrophic factor; DY = diamidino yellow; FB = fast blue; GC = growth cone; GDNF = glial cell line–derived neurotrophic factor; LV = lentiviral vector; NGF = nerve growth factor.

Misdirection of regenerating axons is one of the factors that can explain the limited results often found after nerve injury and repair. In the repair of mixed nerves innervating different distal targets (skin and muscle), misdirection may, for example, lead to motor axons projecting toward skin, and vice versa—that is, sensory axons projecting toward muscle. In the repair of motor nerves innervating different distal targets, misdirection may result in reinnervation of the wrong target muscle, which might function antagonistically. In sensory nerve repair, misdirection might give an increased perceptual territory. After median nerve repair, for example, this might lead to a dysfunctional hand.

Different factors may be involved in the misdirection of regenerating axons, and there may be various mechanisms that can later correct for misdirection. In this review the authors discuss these different factors and mechanisms that act along the pathway of the regenerating axon. The authors review recently developed evaluation methods that can be used to investigate the accuracy of regeneration after nerve injury and repair (including the use of transgenic fluorescent mice, retrograde tracing techniques, and motion analysis). In addition, the authors discuss new strategies that can improve in vivo guidance of regenerating axons (including physical guidance with multichannel nerve tubes and biological guidance accomplished using gene therapy).

Functional recovery after nerve injury and repair is often disappointing, despite the capacity of the peripheral nervous system to regenerate. Several factors can explain this incomplete recovery. First of all, the timing of surgery is an important factor. The best chances for recovery are when nerve repair is performed directly after the injury, because 1) the capacity of regeneration has been shown to decrease with time (fewer neurons from which axons regenerate); and 2) changes occur in the distal nerve and targets due to the prolonged period of denervation (such as fragmentation of the basal lamina tubes and decrease in the number of Schwann cells22,23,25). The type of injury and possibilities for repair may also influence the functional outcome: the recovery following graft repair, for example, is reduced compared with direct coaptation repair. If the patient is older the chance of functional recovery will be decreased.60

Another factor that can explain poor recovery after nerve injury and repair is misdirection or misrouting of regenerating axons. Misdirection can explain the difference in recovery for different types of nerves (mixed, motor, and sensory nerves). In the repair of mixed nerves that innervate different distal targets (skin and muscle), misdirection may lead to motor axons projecting toward skin, and sensory axons projecting toward muscle. In the repair of a motor nerve that innervates different target muscles, motor axons may be misdirected to antagonistic muscles; for example following repair of the sciatic nerve, which distally divides into the tibial and peroneal nerve branches involved in ankle plantar and dorsiflexion, respectively.12 In sensory nerve repair misdirection may limit outcome: after repair of the median nerve at the wrist, patients may experience painful sensations in other median nerve–innervated fingers, even years after the repair.18

Different factors are involved in the misdirection of regenerating axons. Moreover, different biological mechanisms have been shown to exist that can later correct for this misdirection. In the first part of this review, these factors and mechanisms are discussed. Subsequently, several recently developed methods are reviewed that, in our opinion, have provided valuable insight into the process of regeneration. Finally, new strategies are presented that may improve in vivo guidance of regenerating axons, namely physical guidance with multichannel nerve tubes and molecular guidance with gene therapy.

The Course of the Regenerating Axon

After nerve injury and repair, the course of the regenerating axon starts at the coaptation site. At this site, multiple cellular events have taken place after the injury, including clearance of the debris of the distal axonal bodies by macrophages and Schwann cells, a process called Wallerian degeneration. The proximal axon extends its course across this injury site by sending out multiple sprouts (Fig. 1). At the tip of these sprouts are growth cones (GCs) that continuously create cell protrusions called filopodia and lamellipodia. The GCs act as antennae for neurotrophic signals that can attract regenerating axons in a certain direction by stimulating actin dynamics inside the GC, leading to axonal elongation.38 Different neurotrophic factors have been identified, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),49 and glial cell line–derived neurotrophic factor (GDNF).3 These factors are produced and secreted by Schwann cells that remain inside the distal basal lamina tubes after Wallerian degeneration. The diffusion of these factors from the distal nerve stump attracts axons to regenerate in the direction of an increasing gradient, an effect which has been called neurotropism.20 Besides attractive stimuli, repulsive factors exist that might divert regenerating axons or induce GC collapse (for example, semaphorins61). In addition, axons might be physically guided by the formation by fibroblasts of a collagen matrix between the nerve ends after the injury, along which Schwann cells migrate from the proximal and distal nerve stump and present specific cell adhesion molecules to guide regenerating axons.59

Fig. 1.
Fig. 1.

Drawing showing local cellular response to nerve transection. Sprouting occurs at the cut axonal ends in the proximal nerve segment (left). Sprouts (SPR) arising from 1 myelinated axon form a regenerating unit surrounded by common basal lamina. At the tip of each sprout there is a GC. Sprouts advance over the zone of injury in immediate association with Schwann cells (SCHW). In the injury zone there are macrophages, fibroblasts (FB), mast cells (MC), and blood corpuscle elements. In the distal segment (right), sprouts attach to the band of Bügner and become enclosed in the Schwann cell cytoplasm. Axonal misdirection is frequent. Reprinted from Journal of Hand Surgery (American Volume), volume 25, Lundborg G, “A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance,” pp 391–414, copyright 2000, with permission from Elsevier.

In an ideal situation this combination of factors results in a straight course of the regenerating axon back toward its original basal lamina tube (as illustrated in Fig. 1). In reality, however, it has been shown that axons frequently travel laterally before choosing a distal pathway (Fig. 2).69 This dispersion of axonal regeneration may lead to inappropriate target reinnervation, despite surgically correct alignment of the fascicles during nerve repair, because once the axon has entered a distal basal lamina tube, the rest of the course is determined by the original pathway of that endoneural tube. Whether the axon will eventually also reach the end of the basal lamina tube may still depend on several factors. An important factor is the amount of neurotrophic support, which is again determined by the Schwann cells in the distal nerve stump. As recently demonstrated, Schwann cells only produce these neurotrophic factors for a certain period of time (Fig. 3). In addition, the profile of growth factor expression might differ for Schwann cells inside motor versus sensory nerves.31 Another factor that might determine successful regeneration of the axon across the basal lamina tube is the interaction of the GC with specific cell adhesion molecules. Examples of such factors are L2 and HNK-1,43,44 and PSA-NCAM.21

Fig. 2.
Fig. 2.

Left: Silver staining performed by Ramon y Cajal demonstrating “chaotic” regeneration across the injury site. Right: Similar image obtained using transgenic fluorescent mice. Left panel reprinted from Ramon y Cajal S, translated and edited by Raoul M. May 1928: Degeneration and Regeneration of the Nervous System. London: Oxford University Press, 1928. By permission of Oxford University Press. (original magnification unavailable). Right panel reprinted with permission from Pan YA, Misgeld T, Lichtman JW, and Sanes JR: Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo, Journal of Neuroscience, vol. 23, no. 36, pp 11479–11488, copyright 2003 by the Society for Neuroscience.

Fig. 3.
Fig. 3.

Graphic depiction of upregulation and decline in the expression of growth factors (GDNF and BDNF) and receptors (TrkB, ret, GFR-α) by motoneurons and in the distal nerve stump 0–60 days after nerve injury. Reprinted with permission from Furey MJ, Midha R, Xu Q-G, Belkas J, Gordon T: Prolonged target deprivation reduces the capacity of injured motoneurons to regenerate, Neurosurgery, vol. 60, no. 4, pp 723–733, copyright 2007 by Wolters Kluwer Health. MN = motoneuron.

Finally, the regenerating axon has to make a functional reconnection with the target at the end of the basal lamina tube. This last step has mainly been investigated for motor axon reinnervation of the motor endplate. Also in this process, selection may occur. After nerve injury and repair, it has been shown that motor endplates are initially polyinnervated, but that later the motor axon/endplate ratio again returns to a normal 1:1 innervation.26,34,41 This initial poly- or hyperinnervation may be a mechanism for improving the accuracy of reinnervation. Feedback mechanisms such as treadmill exercise62 and manual stimulation may influence this selection process.58

Methods to Investigate Accuracy of Regeneration

A number of different methods have been developed to investigate the accuracy of regeneration and reinnervation. The experiments by Ramon y Cajal at the beginning of the 20th century are legendary55 (Fig. 2 left). His silver staining of regenerated nerve fibers already provided us with most of the insight we have today on the accuracy of regeneration across the coaptation site. Also of historical interest are the experiments on the attractive effect of the distal stump (neurotropism), in which Y-shaped tubes were used with different types of tissue in the distal forks. Based on these experiments, the theory of neurotropism was first rejected by Weiss and Taylor,68 but later the experiment was repeated by others,1,5,8,39,50 and since then this theory has been generally accepted. In this review we will not discuss the results of these experiments in detail. Rather, we will present more recently developed methods that in our opinion have provided valuable insight into specificity and accuracy of regeneration, including the use of fluorescent transgenic animal models, retrograde tracing techniques, and functional methods based on motion analysis.

Fluorescent Transgenic Animal Models

An exciting, relatively new evaluation method in research on peripheral nerve regeneration is the development of a fluorescent transgenic animal model (in the beginning only fluorescent mice were used, but more recently a transgenic rat model has been developed45). In these transgenic animals, a thy-1 promotor construct is used to direct the expression of green fluorescent protein (GFP) or a yellow variant (yellow fluorescent protein [YFP]) selectively in neuronal cells (and not in other cell types such as, for example, Schwann cells, fibroblasts, and muscle fibers).35 Subsequently, selection of a transgenic animal line in which the expression of this fluorescent protein is limited to only a subset of axons, has made it possible to visualize the pathway of an individual regenerating axon in vivo. This live imaging can be performed at multiple time intervals, before and after the nerve injury. In this way the accuracy of axon regeneration can be determined. In vivo analysis of regeneration toward the platysma muscle (that can easily be accessed and viewed) has, for example, shown that the accuracy of regeneration is high after crush injury, with individual regenerating axons entering their original path and reestablishing branches at nearly every original branching point. This specificity is completely lost after transection injury and repair.47 In addition, this technique can be used to investigate the accuracy of regeneration at the coaptation site and to investigate reinnervation of the motor endplate at the neuromuscular junction by simultaneous labeling of the acetylcholine receptors with α-bungarotoxin35 (Fig. 4). Up to now, experiments in which these transgenic animal models were used have mainly confirmed previous observations, including the relatively chaotic process of axon regeneration across a coaptation site (as demonstrated by Cajal; see Fig. 2 left) and hyperreinnervation at the motor endplate.41 In the future these animals may also be used to investigate new strategies to improve axonal regeneration and target reinnervation in vivo, providing the additional advantage that the same animals could be used for analysis with other evaluation methods (such as those mentioned below).

Fig. 4.
Fig. 4.

Dispersion of regenerating motor axons after autograft, single lumen, and multichannel nerve tube repair and technique of simultaneous retrograde tracing. Simultaneous tracing: FB and DY are applied to the tibial and peroneal nerve branches, respectively. The FB is transported retrogradely to the cell body of the motoneuron and the DY goes to the nucleus. A: After autograft repair, regenerating axons originating from the same motoneuron are contained by the basal lamina tubes, and both end up in the same (tibial) nerve branch. B: After single-lumen nerve tube repair, axons originating from the same motoneuron disperse and end up separately in the tibial and peroneal nerve branches. C: After multichannel nerve tube repair, axons originating from the same motoneuron are contained in the inside of a channel and end up in the same (tibial) nerve branch. D and E: Other examples of dispersion across the single-lumen nerve tube, which does not cause double labeling, such as dispersion of a single projection from a motoneuron (D), and double projections to the same branch but toward different fascicles inside this branch (E). Modified with permission from de Ruiter GC, Spinner RJ, Malessy MJ, Moore MJ, Sorenson EJ, Currier BL, et al: Accuracy of motor axon regeneration across autograft, single-lumen, and multichannel poly(lactic-co-glycolic acid) nerve tubes, Neurosurgery, vol. 63, no. 1, pp 144–155, copyright 2008 by Wolters Kluwer Health.

Retrograde Tracing Techniques

Retrograde tracing techniques are also useful for analyzing the specificity of regeneration, especially because these methods can be applied to quantify the accuracy of regeneration. Retrograde tracing is based on the uptake of a fluorescent dye that is retrogradely transported to the nucleus and/or cell body of the neuron (located in the anterior horn in the case of motoneurons, or dorsal root ganglion in the case of sensory neurons). This label can be applied anywhere along the course of the nerve or directly into the target muscle, by tracer injection or by cup application to the proximal nerve end (after nerve transection, the proximal end of the nerve is placed in a cup containing the tracer). Different technical issues must be considered in the use of retrograde tracers, including labeling efficiency, possible fading of the tracer, dye interactions (when using multiple tracers), potential toxicity,54 and persistence of the tracer, when multiple tracers are used as in sequential tracing.53

Sequential tracing is an especially useful technique for investigating the accuracy of regeneration toward a specific nerve branch by application of the first tracer before injury to label the original neuronal pool, and the second tracer at a certain interval after the injury (and possibly repair) to label the neurons from which axons have regenerated toward this branch. Different combinations of tracers have been used in different models. An example is the combination of diamidino yellow (DY) and fast blue (FB) tracers, which has been shown by Puigdellívol-Sánchez et al.52 to result in little persistence of the first tracer (DY) and interaction with the second one (FB). By sequential application of these tracers, the percentage of motoneurons from which axons are correctly directed back to the original target branch can be calculated by dividing the number of double-labeled motoneurons by the number of neurons labeled with the first tracer (single- and double-labeled) (Table 1).

TABLE 1:

Techniques of sequential and simultaneous retrograde tracing—use and indications

Retrograde Tracing TechniqueUseIndications
sequentialapplication of 2 different tracers to the same nerve branch: 1 before & 1 after nerve injury & repairto determine the percentage of original neurons from which axons have regenerated back into the correct nerve branch
simultaneousapplication of different tracers to multiple nerve branches at the same time after nerve injury & repair1) to determine the numbers of neurons from which axons have regenerated toward the different nerve branches (for example to investigate preference for a certain nerve branch); 2) to determine degree of axonal dispersion from the percentage of double-labeled neurons (for example to investigate dispersion across a nerve tube)

Puigdellívol-Sánchez et al.51 have used this technique to investigate the accuracy of motor axon regeneration after transection and repair in the rat sciatic nerve model. The first tracer (DY) was injected into an intact tibial nerve 5 days before injury, and FB tracer was applied 2 months after repair. These investigators found that 88% of the tibial motoneurons were correctly directed to the tibial nerve 2 months after transection and repair of the sciatic nerve (calculated by dividing the number of double-labeled [FB-DY] motoneurons by the total number of DY-labeled profiles), and concluded that epineurial suture repair leads to a high degree of topographically correct directional axon regeneration. In their study, however, onethird of the motoneurons were only labeled by the second tracer, which indicates that a large number of peroneal motoneurons had regenerated to the tibial nerve branch. We have used the same model and retrograde tracing technique to investigate the accuracy of regeneration to the peroneal nerve branch and found that only 42% of the peroneal motoneurons were correctly directed to this branch 2 months after sciatic nerve transection and repair.12 Considering the difference in size of the tibial and peroneal motoneuron pool (ratio 2:1), this difference in percentage of correct direction for tibial and peroneal motoneurons is not surprising and indicates that regeneration up to this point has occurred largely at random.

Another possibility for retrograde tracing is to apply multiple tracers at the same time to different nerve branches (Fig. 4, Table 1). This technique has also been used in different animal models, including the sciatic nerve, femoral nerve, and facial nerve model. In the femoral nerve model, simultaneous retrograde tracing has been used to investigate the accuracy of motor versus sensory regeneration by applying different tracers to the distal cutaneous and motor branches. Experiments in which this model was used have shown that motor axons initially grow equally into both branches, with similar numbers of retrogradely labeled motoneurons at 2 weeks, and also a large number of axons innervating both branches. With time (at 3 and 8 weeks), increasingly more motoneurons projected to the motor branch and fewer motoneurons to the cutaneous branch or both branches,6 a phenomenon which was termed “preferential motor reinnervation.” Several mechanisms may explain this phenomenon of preferential motor reinnervation,40 including the pruning of misdirected axon collaterals in favor of correctly directed ones.

In the sciatic and facial nerve model, simultaneous tracing has been used to investigate dispersion of axonal collaterals originating from the same motoneuron to different branches.63 As mentioned above, a regenerating axon may send out multiple sprouts across the coaptation site. These sprouts may often travel laterally before choosing a distal pathway. Sprouts originating from the same motoneuron may therefore end up in different distal target branches. Especially after facial nerve repair, a high percentage of multiple projections to the zygomatic, buccal, and marginal mandibular branches has been found (15%),28,30 compared with 2.2% double projections after sciatic nerve repair.63 When interpreting these results, however, it is important to realize that this percentage may only represent part of the dispersion that may occur, because axons within the same branch but inside different fascicles may still project toward different target muscles (such as, for example, the gastrocnemius and soleus muscles for the tibial nerve).

An important advantage of retrograde tracing techniques is thus the possibility of quantifying the accuracy of regeneration. Care should be taken, however, when interpreting the results of retrograde tracing, not only because of the factors mentioned above, but also because it does not evaluate the final step of motor endplate reinnervation.

Motion Analysis

Different methods have been applied to investigate the accuracy of reinnervation related to function, including selective tension contraction measurements,73 selective recordings of compound muscle action potential amplitude,19 and walking track analysis.2,10,11 All these approaches have been used in the rat sciatic nerve lesion model. Although these methods have provided important insight into the recovery of function after nerve injury and repair, they also have several shortcomings, particularly in the analysis of the impact of misdirection on the recovery of function. Muscle contraction measurements and compound muscle action potential recordings, for example, do not account for cocontractions, nor do they measure the actual recovery of function. The most commonly used functional evaluation method, walking track analysis, only looks at the recovery of distal intrinsic foot muscles that often do not recover as well as the more proximally located muscles (such as the gastrocnemius and anterior tibialis muscles), especially after transection injury and repair. Footprint analysis is also limited due to contractures17 and autotomy.67

Other gait parameters have therefore been investigated, including analysis of the ankle angle.36,57,64,65,72 An advantage of ankle motion analysis is that it can also be used to investigate the accuracy of reinnervation of muscles involved in ankle plantar (tibial nerve function) and dorsiflexion (peroneal nerve function),14 especially if used simultaneously with electromyography recordings in the tibialis anterior and gastrocnemius muscles.27 We have used digital video analysis to investigate the recovery of ankle motion after different types of sciatic nerve injury and repair (crush injury, direct coaptation, and autograft repair). Results showed that especially peroneal nerve function was impaired after transection injury and repair, with an angle of dorsiflexion that was decreased even further compared with the situation directly after the nerve injury, possibly due to misdirection of a significant portion of peroneal motoneurons (only 42% of peroneal motoneurons were correctly directed to the peroneal nerve branch, as determined with sequential tracing) to the tibial branch, resulting in active plantar flexion during the swing phase (normally the moment of maximum dorsiflexion).12 This finding stresses the importance of misdirection as a limiting factor in nerve repair, especially when one considers that the quantitative results of regeneration (number of axons and retrogradely labeled motoneurons) in our experiment were not significantly different from those in normal animals or after direct coaptation repair. In addition to the sciatic nerve model, motion analysis has recently also been applied in the rat facial nerve model to investigate the recovery of whisker movement.28

In Vivo Guidance of Regenerating Axons

In recent years, different strategies have been developed that may guide and direct regenerating axons toward their correct target organ. Most of these guiding strategies have been investigated in vitro by using neurite outgrowth assays of (for instance) explanted dorsal root ganglion cells. For example, physical guidance of neurites has been investigated using grooved microsurfaces. This research has shown that neurites orient parallel to the walls of microchannels.42 Other examples include research on in vitro outgrowth of neurites on polymer filaments, with different shapes and coatings56), and polymer surfaces patterned with gradients of peptides or neurotrophic factors to guide neurites in a certain direction.71 Only a limited number of studies have investigated the influence on in vivo nerve regeneration. Below we discuss several physical and molecular guidance strategies that may be applied in future nerve repair, and we also discuss our own experience with in vivo axonal guidance in which multichannel nerve tubes and gene therapy are used.

Physical Guidance of Nerve Regeneration With Modified Conduits

Single-lumen or hollow nerve tubes or conduits have been developed mainly as alternatives for repair with an autologous nerve graft.16 In addition, nerve tube or entubulation repair has also been suggested to have a favorable outcome regarding the specificity of muscle reinnervation compared with direct suture repair,19,73 especially when the fascicular topography at the time of reapproximation is less clear.19 With increasing gap size, however, the lack of an internal guiding structure inside single-lumen nerve tubes has been shown to lead to the dispersion of regenerating axons,7 also resulting in more dispersed muscle reinnervation.66 We have shown, for example (using the simultaneous tracing technique described above) that 8 weeks after repair of a 1-cm gap of the rat sciatic nerve with a single-lumen poly(lactic-coglycolic acid) nerve tube, 21.4% of the motoneurons had projections to both the tibial and peroneal nerve branch, compared with 5.9% after autograft repair.15 This difference may be partially explained by the difference in the degree of axonal branching, but also by more dispersed regeneration of axonal branches in single-lumen nerve tube repair compared with contained regeneration of axonal branches inside the basal lamina tubes in autograft repair.

Our hypothesis was that introduction of multiple channels inside the nerve conduit would decrease the percentage of axonal dispersion (Fig. 4C). Multichannel conduits that had already been developed for other purposes (increased surface area for Schwann cell attachment29 and experimental spinal cord repair46) were used to investigate this. In a pilot study with multichannel poly(lacticcoglycolic acid) nerve tubes the percentage of double projections was indeed slightly decreased, although not significantly (16.9% 8 weeks after multichannel nerve tube repair and 21.4% 8 weeks after single-lumen nerve tube). In a follow-up study of multichannel collagen conduits, however, the percentages of double labeling were significantly decreased 16 weeks after repair with 2-channel (2.7%) and 4-channel (2.4%) conduits, compared with single-lumen nerve tube repair (7.1%). Although this reduction may seem small, it is important to realize that this percentage of double projections indicates only part of the axonal dispersion that occurs during regeneration across the conduit. In addition, there may be motoneurons with single projections that have dispersed and regenerated to the wrong distal target branch (Fig. 4D), and motoneurons with double projections to the same branch, but to different fascicles inside this branch (Fig. 4E). Dispersion in these cases does not lead to double labeling.

In the studies mentioned above, the number of channels that could be fitted into the multichannel nerve tube was limited due to the techniques of fabrication.13,70 New techniques may increase the number of channels that can be fitted into the conduit. Using techniques such as freeze drying4 and 3D printing, conduits with even more complex structures can be created. A potential application would be to reconstruct the internal architecture of the damaged nerve, which often does not consist of longitudinally aligned channels, but rather of fascicles that form an intraneural plexus. Other potential modifications of the internal structure of the nerve conduit (recently reviewed by Daly et al.9) include the introduction of guiding filaments inside the conduits, the application of microgrooved internal surfaces, patterning of the surface with different gradients of peptides or neurotrophic factors, or combinations of the different approaches mentioned above.

Molecular Guidance of Nerve Regeneration With Gene Therapy

Molecular guiding cues may also be applied in the future to improve the recovery of function after nerve repair. An example already mentioned above is to apply gradients of neurotrophic factors to the surface of conduits. Another option is to genetically modify the growth factor expression of Schwann cells in the distal nerve stump by using gene therapy.33 The latter could be used, for instance, to attract different subsets of axons toward different branches, as in median nerve repair, to selectively attract sensory axons toward the digital nerve branches and motor axons toward the recurrent nerve branch (Fig. 5). Hu et al.33 recently showed that injection of an adenoviral vector encoding for NGF in the sensory branch of the femoral nerve, 1 week after transection and repair proximal to the motor-sensory nerve bifurcation, increases the ratio of sensory axons regenerating toward the sensory branch. We have found that injection of a lentiviral vector (LV) encoding for GDNF (LV-GDNF) can be used for selective overexpression of GDNF in a specific nerve branch (unpublished data). Studies are in progress to investigate whether this could result in selective motor axon regeneration into the branch transduced with the LV-GDNF.

Fig. 5.
Fig. 5.

Drawing illustrating the concept of selective injection of an LV encoding for GDNF into the motor branch of the median nerve to promote motor axon regeneration toward this nerve branch. Reprinted from Hoyng SA, Tannemaat MR, De Winter F, Verhaagen J, Malessy MJ, Journal of Hand Surgery (European Volume), vol. 36, no. 9, pp 735–746, copyright © 2011. Reprinted with permission of SAGE.

Conclusions

Several factors can explain the poor recovery of function often observed after nerve injury and surgical repair, such as the time interval between nerve injury and repair, the type of injury and possibilities for repair, age of the patient, and, as discussed in this review, misdirection of regenerating axons. As already stated by Sir Sydney Sunderland, “the core of the problem is not promoting axon regeneration, but in getting them back to where they belong.”60 We have discussed several recently developed methods that can be used to investigate the accuracy of regeneration and reinnervation after experimental nerve injury and repair, each with its own value: transgenic fluorescent mice that can be used to visualize the pathway of the regenerating axon, retrograde tracing techniques that can be used to quantify accuracy of regeneration, and motion analysis that can be used to determine the recovery of ankle plantar and dorsiflexion for tibial and peroneal nerve function, respectively, in the rat sciatic nerve model. In addition, we have presented 2 novel strategies (multichannel nerve tubes and gene therapy) for guiding regenerating axons in vivo.

Disclosure

Joost Verhaagen and Martijn Malessy are supported by a grant from the “Nederlandse Organisatie voor Wetenschappelijk Onderzoek” ([NWO]; ZonMw TOP Grant 40-00812-98-10058). The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author contributions to the study and manuscript preparation include the following. Conception and design: de Ruiter. Acquisition of data: de Ruiter. Analysis and interpretation of data: de Ruiter. Drafting the article: de Ruiter, Malessy. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: de Ruiter. Administrative/technical/material support: de Ruiter.

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    • Export Citation
  • 19

    Evans PJBain JRMackinnon SEMakino APHunter DA: Selective reinnervation: a comparison of recovery following microsuture and conduit nerve repair. Brain Res 559:3153211991

    • Search Google Scholar
    • Export Citation
  • 20

    Forssman J: Über den ursachen, welche die wachstumsrichtung der peripheren nervenfasern bei der regeneration bestimmen. Beitr Pathol Anat Allgem Pathol 24:561001898

    • Search Google Scholar
    • Export Citation
  • 21

    Franz CKRutishauser URafuse VF: Polysialylated neural cell adhesion molecule is necessary for selective targeting of regenerating motor neurons. J Neurosci 25:208120912005

    • Search Google Scholar
    • Export Citation
  • 22

    Fu SYGordon T: Contributing factors to poor functional recovery after delayed nerve repair: prolonged axotomy. J Neurosci 15:387638851995

    • Search Google Scholar
    • Export Citation
  • 23

    Fu SYGordon T: Contributing factors to poor functional recovery after delayed nerve repair: prolonged denervation. J Neurosci 15:388638951995

    • Search Google Scholar
    • Export Citation
  • 24

    Furey MJMidha RXu Q-GBelkas JGordon T: Prolonged target deprivation reduces the capacity of injured motoneurons to regenerate. Neurosurgery 60:7237332007

    • Search Google Scholar
    • Export Citation
  • 25

    Giannini CDyck PJ: The fate of Schwann cell basement membranes in permanently transected nerves. J Neuropathol Exp Neurol 49:5505631990

    • Search Google Scholar
    • Export Citation
  • 26

    Gorio ACarmignoto GFinesso MPolato PNunzi MG: Muscle reinnervation—II. Sprouting, synapse formation and repression. Neuroscience 8:4034161983

    • Search Google Scholar
    • Export Citation
  • 27

    Gramsbergen AIJkema-Paassen JMeek MF: Sciatic nerve transection in the adult rat: abnormal EMG patterns during locomotion by aberrant innervation of hindleg muscles. Exp Neurol 161:1831932000

    • Search Google Scholar
    • Export Citation
  • 28

    Guntinas-Lichius OHundeshagen GPaling TAngelov DN: Impact of different types of facial nerve reconstruction on the recovery of motor function: an experimental study in adult rats. Neurosurgery 61:127612852007

    • Search Google Scholar
    • Export Citation
  • 29

    Hadlock TSundback CHunter DCheney MVacanti JP: A polymer foam conduit seeded with Schwann cells promotes guided peripheral nerve regeneration. Tissue Eng 6:1191272000

    • Search Google Scholar
    • Export Citation
  • 30

    Hizay AOzsoy UDemirel BMOzsoy OAngelova SKAnkerne J: Use of a Y-tube conduit after facial nerve injury reduces collateral axonal branching at the lesion site but neither reduces polyinnervation of motor endplates nor improves functional recovery. Neurosurgery 70:154415562012

    • Search Google Scholar
    • Export Citation
  • 31

    Höke ARedett RHameed HJari RZhou CLi ZB: Schwann cells express motor and sensory phenotypes that regulate axon regeneration. J Neurosci 26:964696552006

    • Search Google Scholar
    • Export Citation
  • 32

    Hoyng SATannemaat MRDe Winter FVerhaagen JMalessy MJ: Nerve surgery and gene therapy: a neurobiological and clinical perspective. J Hand Surg Eur Vol 36:7357462011

    • Search Google Scholar
    • Export Citation
  • 33

    Hu XCai JYang JSmith GM: Sensory axon targeting is increased by NGF gene therapy within the lesioned adult femoral nerve. Exp Neurol 223:1531652010

    • Search Google Scholar
    • Export Citation
  • 34

    Ijkema-Paassen JMeek MFGramsbergen A: Reinnervation of muscles after transection of the sciatic nerve in adult rats. Muscle Nerve 25:8918972002

    • Search Google Scholar
    • Export Citation
  • 35

    Lichtman JWSanes JR: Watching the neuromuscular junction. J Neurocytol 32:7677752003

  • 36

    Lin FMPan YCHom CSabbahi MShenaq S: Ankle stance angle: a functional index for the evaluation of sciatic nerve recovery after complete transection. J Reconstr Microsurg 12:1731771996

    • Search Google Scholar
    • Export Citation
  • 37

    Lundborg G: A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg Am 25:3914142000

    • Search Google Scholar
    • Export Citation
  • 38

    Lykissas MGBatistatou AKCharalabopoulos KABeris AE: The role of neurotrophins in axonal growth, guidance, and regeneration. Curr Neurovasc Res 4:1431512007

    • Search Google Scholar
    • Export Citation
  • 39

    Mackinnon SEDellon ALLundborg GHudson ARHunter DA: A study of neurotrophism in a primate model. J Hand Surg Am 11:8888941986

    • Search Google Scholar
    • Export Citation
  • 40

    Madison RDRobinson GAChadaram SR: The specificity of motor neurone regeneration (preferential reinnervation). Acta Physiol (Oxf) 189:2012062007

    • Search Google Scholar
    • Export Citation
  • 41

    Magill CKTong AKawamura DHayashi AHunter DAParsadanian A: Reinnervation of the tibialis anterior following sciatic nerve crush injury: a confocal microscopic study in transgenic mice. Exp Neurol 207:64742007

    • Search Google Scholar
    • Export Citation
  • 42

    Mahoney MJChen RRTan JSaltzman WM: The influence of microchannels on neurite growth and architecture. Biomaterials 26:7717782005

    • Search Google Scholar
    • Export Citation
  • 43

    Martini RSchachner MBrushart TM: The L2/HNK-1 carbohydrate is preferentially expressed by previously motor axonassociated Schwann cells in reinnervated peripheral nerves. J Neurosci 14:718071911994

    • Search Google Scholar
    • Export Citation
  • 44

    Martini RXin YSchmitz BSchachner M: The L2/HNK-1 carbohydrate epitope is involved in the preferential outgrowth of motor neurons on ventral roots and motor nerves. Eur J Neurosci 4:6286391992

    • Search Google Scholar
    • Export Citation
  • 45

    Moore AMBorschel GHSantosa KAFlagg ERTong AYKasukurthi R: A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration. J Neurosci Methods 204:19272012

    • Search Google Scholar
    • Export Citation
  • 46

    Moore MJFriedman JALewellyn EBMantila SMKrych AJAmeenuddin S: Multiple-channel scaffolds to promote spinal cord axon regeneration. Biomaterials 27:4194292006

    • Search Google Scholar
    • Export Citation
  • 47

    Nguyen QTSanes JRLichtman JW: Pre-existing pathways promote precise projection patterns. Nat Neurosci 5:8618672002

  • 48

    Pan YAMisgeld TLichtman JWSanes JR: Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo. J Neurosci 23:11479114882003

    • Search Google Scholar
    • Export Citation
  • 49

    Paves HSaarma M: Neurotrophins as in vitro growth cone guidance molecules for embryonic sensory neurons. Cell Tissue Res 290:2852971997

    • Search Google Scholar
    • Export Citation
  • 50

    Politis MJEderle KSpencer PS: Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerves. Brain Res 253:1121982

    • Search Google Scholar
    • Export Citation
  • 51

    Puigdellívol-Sánchez APrats-Galino AMolander C: Estimations of topographically correct regeneration to nerve branches and skin after peripheral nerve injury and repair. Brain Res 1098:49602006

    • Search Google Scholar
    • Export Citation
  • 52

    Puigdellívol-Sánchez APrats-Galino ARuano-Gil DMolander C: Fast blue and diamidino yellow as retrograde tracers in peripheral nerves: efficacy of combined nerve injection and capsule application to transected nerves in the adult rat. J Neurosci Methods 95:1031102000

    • Search Google Scholar
    • Export Citation
  • 53

    Puigdellívol-Sánchez APrats-Galino ARuano-Gil DMolander C: Persistence of tracer in the application site—a potential confounding factor in nerve regeneration studies. J Neurosci Methods 127:1051102003

    • Search Google Scholar
    • Export Citation
  • 54

    Puigdellívol-Sánchez AValero-Cabré APrats-Galino ANavarro XMolander C: On the use of fast blue, fluoro-gold and diamidino yellow for retrograde tracing after peripheral nerve injury: uptake, fading, dye interactions, and toxicity. J Neurosci Methods 115:1151272002

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Article Information

Address correspondence to: Godard C. W. de Ruiter, M.D., Ph.D., Department of Neurosurgery, Leiden University Medical Center, Albinusdreef 1, Leiden, The Netherlands. email: G.C.W.de_Ruiter@lumc.nl.

Please include this information when citing this paper: published online October 11, 2013; DOI: 10.3171/2013.8.JNS122300.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Drawing showing local cellular response to nerve transection. Sprouting occurs at the cut axonal ends in the proximal nerve segment (left). Sprouts (SPR) arising from 1 myelinated axon form a regenerating unit surrounded by common basal lamina. At the tip of each sprout there is a GC. Sprouts advance over the zone of injury in immediate association with Schwann cells (SCHW). In the injury zone there are macrophages, fibroblasts (FB), mast cells (MC), and blood corpuscle elements. In the distal segment (right), sprouts attach to the band of Bügner and become enclosed in the Schwann cell cytoplasm. Axonal misdirection is frequent. Reprinted from Journal of Hand Surgery (American Volume), volume 25, Lundborg G, “A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance,” pp 391–414, copyright 2000, with permission from Elsevier.

  • View in gallery

    Left: Silver staining performed by Ramon y Cajal demonstrating “chaotic” regeneration across the injury site. Right: Similar image obtained using transgenic fluorescent mice. Left panel reprinted from Ramon y Cajal S, translated and edited by Raoul M. May 1928: Degeneration and Regeneration of the Nervous System. London: Oxford University Press, 1928. By permission of Oxford University Press. (original magnification unavailable). Right panel reprinted with permission from Pan YA, Misgeld T, Lichtman JW, and Sanes JR: Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo, Journal of Neuroscience, vol. 23, no. 36, pp 11479–11488, copyright 2003 by the Society for Neuroscience.

  • View in gallery

    Graphic depiction of upregulation and decline in the expression of growth factors (GDNF and BDNF) and receptors (TrkB, ret, GFR-α) by motoneurons and in the distal nerve stump 0–60 days after nerve injury. Reprinted with permission from Furey MJ, Midha R, Xu Q-G, Belkas J, Gordon T: Prolonged target deprivation reduces the capacity of injured motoneurons to regenerate, Neurosurgery, vol. 60, no. 4, pp 723–733, copyright 2007 by Wolters Kluwer Health. MN = motoneuron.

  • View in gallery

    Dispersion of regenerating motor axons after autograft, single lumen, and multichannel nerve tube repair and technique of simultaneous retrograde tracing. Simultaneous tracing: FB and DY are applied to the tibial and peroneal nerve branches, respectively. The FB is transported retrogradely to the cell body of the motoneuron and the DY goes to the nucleus. A: After autograft repair, regenerating axons originating from the same motoneuron are contained by the basal lamina tubes, and both end up in the same (tibial) nerve branch. B: After single-lumen nerve tube repair, axons originating from the same motoneuron disperse and end up separately in the tibial and peroneal nerve branches. C: After multichannel nerve tube repair, axons originating from the same motoneuron are contained in the inside of a channel and end up in the same (tibial) nerve branch. D and E: Other examples of dispersion across the single-lumen nerve tube, which does not cause double labeling, such as dispersion of a single projection from a motoneuron (D), and double projections to the same branch but toward different fascicles inside this branch (E). Modified with permission from de Ruiter GC, Spinner RJ, Malessy MJ, Moore MJ, Sorenson EJ, Currier BL, et al: Accuracy of motor axon regeneration across autograft, single-lumen, and multichannel poly(lactic-co-glycolic acid) nerve tubes, Neurosurgery, vol. 63, no. 1, pp 144–155, copyright 2008 by Wolters Kluwer Health.

  • View in gallery

    Drawing illustrating the concept of selective injection of an LV encoding for GDNF into the motor branch of the median nerve to promote motor axon regeneration toward this nerve branch. Reprinted from Hoyng SA, Tannemaat MR, De Winter F, Verhaagen J, Malessy MJ, Journal of Hand Surgery (European Volume), vol. 36, no. 9, pp 735–746, copyright © 2011. Reprinted with permission of SAGE.

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    • Export Citation
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    Evans PJBain JRMackinnon SEMakino APHunter DA: Selective reinnervation: a comparison of recovery following microsuture and conduit nerve repair. Brain Res 559:3153211991

    • Search Google Scholar
    • Export Citation
  • 20

    Forssman J: Über den ursachen, welche die wachstumsrichtung der peripheren nervenfasern bei der regeneration bestimmen. Beitr Pathol Anat Allgem Pathol 24:561001898

    • Search Google Scholar
    • Export Citation
  • 21

    Franz CKRutishauser URafuse VF: Polysialylated neural cell adhesion molecule is necessary for selective targeting of regenerating motor neurons. J Neurosci 25:208120912005

    • Search Google Scholar
    • Export Citation
  • 22

    Fu SYGordon T: Contributing factors to poor functional recovery after delayed nerve repair: prolonged axotomy. J Neurosci 15:387638851995

    • Search Google Scholar
    • Export Citation
  • 23

    Fu SYGordon T: Contributing factors to poor functional recovery after delayed nerve repair: prolonged denervation. J Neurosci 15:388638951995

    • Search Google Scholar
    • Export Citation
  • 24

    Furey MJMidha RXu Q-GBelkas JGordon T: Prolonged target deprivation reduces the capacity of injured motoneurons to regenerate. Neurosurgery 60:7237332007

    • Search Google Scholar
    • Export Citation
  • 25

    Giannini CDyck PJ: The fate of Schwann cell basement membranes in permanently transected nerves. J Neuropathol Exp Neurol 49:5505631990

    • Search Google Scholar
    • Export Citation
  • 26

    Gorio ACarmignoto GFinesso MPolato PNunzi MG: Muscle reinnervation—II. Sprouting, synapse formation and repression. Neuroscience 8:4034161983

    • Search Google Scholar
    • Export Citation
  • 27

    Gramsbergen AIJkema-Paassen JMeek MF: Sciatic nerve transection in the adult rat: abnormal EMG patterns during locomotion by aberrant innervation of hindleg muscles. Exp Neurol 161:1831932000

    • Search Google Scholar
    • Export Citation
  • 28

    Guntinas-Lichius OHundeshagen GPaling TAngelov DN: Impact of different types of facial nerve reconstruction on the recovery of motor function: an experimental study in adult rats. Neurosurgery 61:127612852007

    • Search Google Scholar
    • Export Citation
  • 29

    Hadlock TSundback CHunter DCheney MVacanti JP: A polymer foam conduit seeded with Schwann cells promotes guided peripheral nerve regeneration. Tissue Eng 6:1191272000

    • Search Google Scholar
    • Export Citation
  • 30

    Hizay AOzsoy UDemirel BMOzsoy OAngelova SKAnkerne J: Use of a Y-tube conduit after facial nerve injury reduces collateral axonal branching at the lesion site but neither reduces polyinnervation of motor endplates nor improves functional recovery. Neurosurgery 70:154415562012

    • Search Google Scholar
    • Export Citation
  • 31

    Höke ARedett RHameed HJari RZhou CLi ZB: Schwann cells express motor and sensory phenotypes that regulate axon regeneration. J Neurosci 26:964696552006

    • Search Google Scholar
    • Export Citation
  • 32

    Hoyng SATannemaat MRDe Winter FVerhaagen JMalessy MJ: Nerve surgery and gene therapy: a neurobiological and clinical perspective. J Hand Surg Eur Vol 36:7357462011

    • Search Google Scholar
    • Export Citation
  • 33

    Hu XCai JYang JSmith GM: Sensory axon targeting is increased by NGF gene therapy within the lesioned adult femoral nerve. Exp Neurol 223:1531652010

    • Search Google Scholar
    • Export Citation
  • 34

    Ijkema-Paassen JMeek MFGramsbergen A: Reinnervation of muscles after transection of the sciatic nerve in adult rats. Muscle Nerve 25:8918972002

    • Search Google Scholar
    • Export Citation
  • 35

    Lichtman JWSanes JR: Watching the neuromuscular junction. J Neurocytol 32:7677752003

  • 36

    Lin FMPan YCHom CSabbahi MShenaq S: Ankle stance angle: a functional index for the evaluation of sciatic nerve recovery after complete transection. J Reconstr Microsurg 12:1731771996

    • Search Google Scholar
    • Export Citation
  • 37

    Lundborg G: A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg Am 25:3914142000

    • Search Google Scholar
    • Export Citation
  • 38

    Lykissas MGBatistatou AKCharalabopoulos KABeris AE: The role of neurotrophins in axonal growth, guidance, and regeneration. Curr Neurovasc Res 4:1431512007

    • Search Google Scholar
    • Export Citation
  • 39

    Mackinnon SEDellon ALLundborg GHudson ARHunter DA: A study of neurotrophism in a primate model. J Hand Surg Am 11:8888941986

    • Search Google Scholar
    • Export Citation
  • 40

    Madison RDRobinson GAChadaram SR: The specificity of motor neurone regeneration (preferential reinnervation). Acta Physiol (Oxf) 189:2012062007

    • Search Google Scholar
    • Export Citation
  • 41

    Magill CKTong AKawamura DHayashi AHunter DAParsadanian A: Reinnervation of the tibialis anterior following sciatic nerve crush injury: a confocal microscopic study in transgenic mice. Exp Neurol 207:64742007

    • Search Google Scholar
    • Export Citation
  • 42

    Mahoney MJChen RRTan JSaltzman WM: The influence of microchannels on neurite growth and architecture. Biomaterials 26:7717782005

    • Search Google Scholar
    • Export Citation
  • 43

    Martini RSchachner MBrushart TM: The L2/HNK-1 carbohydrate is preferentially expressed by previously motor axonassociated Schwann cells in reinnervated peripheral nerves. J Neurosci 14:718071911994

    • Search Google Scholar
    • Export Citation
  • 44

    Martini RXin YSchmitz BSchachner M: The L2/HNK-1 carbohydrate epitope is involved in the preferential outgrowth of motor neurons on ventral roots and motor nerves. Eur J Neurosci 4:6286391992

    • Search Google Scholar
    • Export Citation
  • 45

    Moore AMBorschel GHSantosa KAFlagg ERTong AYKasukurthi R: A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration. J Neurosci Methods 204:19272012

    • Search Google Scholar
    • Export Citation
  • 46

    Moore MJFriedman JALewellyn EBMantila SMKrych AJAmeenuddin S: Multiple-channel scaffolds to promote spinal cord axon regeneration. Biomaterials 27:4194292006

    • Search Google Scholar
    • Export Citation
  • 47

    Nguyen QTSanes JRLichtman JW: Pre-existing pathways promote precise projection patterns. Nat Neurosci 5:8618672002

  • 48

    Pan YAMisgeld TLichtman JWSanes JR: Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo. J Neurosci 23:11479114882003

    • Search Google Scholar
    • Export Citation
  • 49

    Paves HSaarma M: Neurotrophins as in vitro growth cone guidance molecules for embryonic sensory neurons. Cell Tissue Res 290:2852971997

    • Search Google Scholar
    • Export Citation
  • 50

    Politis MJEderle KSpencer PS: Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerves. Brain Res 253:1121982

    • Search Google Scholar
    • Export Citation
  • 51

    Puigdellívol-Sánchez APrats-Galino AMolander C: Estimations of topographically correct regeneration to nerve branches and skin after peripheral nerve injury and repair. Brain Res 1098:49602006

    • Search Google Scholar
    • Export Citation
  • 52

    Puigdellívol-Sánchez APrats-Galino ARuano-Gil DMolander C: Fast blue and diamidino yellow as retrograde tracers in peripheral nerves: efficacy of combined nerve injection and capsule application to transected nerves in the adult rat. J Neurosci Methods 95:1031102000

    • Search Google Scholar
    • Export Citation
  • 53

    Puigdellívol-Sánchez APrats-Galino ARuano-Gil DMolander C: Persistence of tracer in the application site—a potential confounding factor in nerve regeneration studies. J Neurosci Methods 127:1051102003

    • Search Google Scholar
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