Motor Control, 2009, 13, 412-441
© 2009 Human Kinetics, Inc.
Brief Electrical Stimulation Accelerates
Axon Regeneration in the Peripheral
Nervous System and Promotes
Sensory Axon Regeneration
in the Central Nervous System
Tessa Gordon, Esther Udina, Valerie M.K. Verge,
and Elena I. Posse de Chaves
Injured peripheral but not central nerves regenerate their axons but functional recovery is often poor. We demonstrate that prolonged periods of axon separation from
targets and Schwann cell denervation eliminate regenerative capacity in the peripheral nervous system (PNS). A substantial delay of 4 weeks for all regenerating axons
to cross a site of repair of sectioned nerve contributes to the long period of separation.
Findings that 1h 20Hz bipolar electrical stimulation accelerates axon outgrowth
across the repair site and the downstream reinnervation of denervated muscles in rats
and human patients, provides a new and exciting method to improve functional recovery after nerve injuries. Drugs that elevate neuronal cAMP and activate PKA promote
axon outgrowth in vivo and in vitro, mimicking the electrical stimulation effect. Rapid
expression of neurotrophic factors and their receptors and then of growth associated
proteins thereafter via cAMP, is the likely mechanism by which electrical stimulation
accelerates axon outgrowth from the site of injury in both peripheral and central nervous systems.
Keywords: neurophysiology, neuroscience, muscle function, neuromuscular
disease
The capacity of the nervous system for repair is a poignant issue for those
suffering central and/or peripheral nerve injuries, stroke, and diseases that include
demyelinating diseases and neuropathies. For those paralyzed by spinal cord injuries for example, experimental demonstration of some functional recovery have
not as yet translated into substantial mobility despite worldwide research efforts.
Challenges toward achieving long-distance regrowth of axons are particularly formidable in the injured central nervous system (CNS) despite promising findings
Gordon and Udina are with the Division of Physical Medicine and Rehabilitation, University of
Alberta, Edmonton, Alberta. Verge is with the Dept. of Anatomy and Cell Biology, Cameco MS/
Neuroscience Research Center, University of Saskatchewan, Saskatoon, Saskatchewan. Posse de
Chaves is with the Dept. of Pharmacology, University of Alberta, Edmonton, Alberta.
412
Motor and Sensory Axon PNS and CNS Axon Regeneration 413
with combinational approaches in animal models of spinal cord injury (Moon &
Bunge, 2005; Fouad & Pearson, 2004; Bunge & Pearse, 2003; Verma, GarciaAlias, & Fawcett, 2008). These approaches include strategies to digest extracellular matrix components of the scar tissue such as chondroitin sulfate proteoglycans (CSPGs) with the enzyme chondroitinase ABC, and to overcome the
inhibitory environment of the central myelin produced by the oligodendrocytes.
The latter approaches have used combinations of neurotrophic factors, inhibitors
of Rho, which is the small GTPase activated downstream of the p75 and NgR
receptor complex of central myelin, chondroitinase ABC, and rolipram to pharmacologically elevate intracellular cAMP to bypass the receptor complex and
Rho-associated inhibition of axon growth in the CNS (Grimpe et al., 2005; Pearse
et al., 2004; Lehmann et al., 1999; Fenrich & Gordon, 2004; Fitch & Silver, 2008;
Gonzenbach & Schwab, 2008; Lu & Tuszynski, 2008; Fawcett, 2006).
Central myelin contains several myelin associated proteins including Nogo-66
and Nogo-A, myelin associated glycoprotein (MAB), and oligodendrocyte myelin
glycoprotein (OMgp). These proteins are currently believed to act as growth-inhibitory molecules that, via the common receptor subunit, NgR on the neuronal
membranes, activate Rho to mediate collapse of growth cones (Fitch & Silver,
2008; Cao et al., 2007; Zheng et al., 2005; Ferraro, Alabed, & Fournier, 2004;
Schwab, 2004; Bandtlow, 2003; Grados-Munro & Fournier, 2003; Yiu & He,
2006; Filbin, 2006; Hannila, Siddiq, & Filbin, 2007; Hunt, Coffin, & Anderson,
2002; Liu, Fournier, GrandPre, & Strittmatter, 2002). In contrast Schwann cells of
the peripheral nervous system (PNS) do not express the NgR receptors although
the macrophages that infiltrate during Wallerian degeneration do (Fry, Ho, &
David, 2007). Schwann cells proliferate and co-operate with infiltrating macrophages to phagocytose and remove myelin debris. They line the endoneurial
tubes of the distal nerve stumps to form the bands of Bungner that guide and support the regenerating axons (Fu & Gordon, 1997; Sulaiman, Midha, & Gordon,
2009). The presence of the growth supportive Schwann cells in the growth pathways of the distal nerve stumps and the regenerative capacity of the motor and
sensory neurons in the PNS have been highlighted and contrasted with the inability of axons to regenerate in the CNS (Fu & Gordon, 1997). These have, however,
masked the clinically recognized frequency of poor functional outcomes of surgical repair of injured peripheral nerves in human subjects (Sulaiman & Gordon,
2008; Fenrich & Gordon, 2004; Sulaiman, Midha, & Gordon, 2009). The poor
outcomes have too often been attributed, incorrectly, to irreversible atrophy and
replacement of denervated skeletal muscles and sense organs by fat (Kline &
Hudson, 1995; Sulaiman & Gordon, 2009; Gordon et al., 2009; Sulaiman, Midha,
& Gordon, 2009; Gordon, Sulaiman, & Boyd, 2003; Gordon, Boyd, & Sulaiman,
2005; Sunderland, 1968).
In this review, we first briefly consider the factors that account for poor functional recovery after nerve injury and repair in the PNS of human patents. We
describe how we modeled nerve injury in animals to replicate delays in axons
regenerating and reaching their denervated targets—chronic axotomy—and the
chronic denervation of Schwann cells in the growth pathway during the long periods of axon regeneration at rates of 1–3mm/day. These experiments demonstrate
the relatively short time window of opportunity when axon regeneration is optimal after which regenerative capacity declines progressively with time and
414 Gordon et al.
distance. Second, we review experiments that demonstrate the effectiveness of
brief electrical stimulation in accelerating axon outgrowth from the proximal
nerve stumps of injured neurons and thereby, axon regeneration and target reinnervation. The electrical stimulation also promotes axon outgrowth in the CNS.
The mechanisms of the effectiveness of the electrical stimulation are explored and
the data to date are reviewed.
Motor and Sensory Axon Regeneration in the
Peripheral and Central Nervous Systems: Problems
Axons in the Peripheral but not the Central Nervous Systems
Regenerate
Peripheral nerves regenerate their axons after crush or transection injuries outside
of, but not within the spinal cord and brainstem. Crush injuries leave the endothelium surrounding each axon intact. Axons regenerate within the endothelial tubes
where the denervated Schwann cells support and guide the regenerating axons to
their original targets (Fu and Gordon, 1997; Zochodne, 2008). Injuries that disrupt
the endothelium, such as those that transect the nerve, necessitate that Schwann
cells enter the injury site and that extracellular matrix proteins which include the
growth-promoting molecules of laminin and fibronectin, reorganize before the
regenerating axons navigate through to enter the endoneurial tubes of the distal
nerve stump (Witzel, Rohde, & Brushart, 2005). However, in contrast to crush
injuries, transected motor and sensory axons that regenerate are frequently misdirected into inappropriate distal nerve pathways to randomly reinnervate denervated targets (Mesulam & Brushart, 1979; Thomas, Stein, Gordon, Lee, & Elleker,
1987).
Peripheral nerves include the axons of motoneurons of the spinal cord and
lower brainstem that supply the musculature of the body and head, and sensory
axons of dorsal root ganglion (DRG) neurons that transmit sensory information
from the periphery to the CNS. Injury to the central process of the DRG neurons
also leads to axon regeneration but the rate of regeneration is slower and the
regenerating axons proceed only as far as the white matter of the spinal cord
(Richardson & Verge, 1987; Zhang et al., 2007; Fenrich & Gordon, 2004). Myelination of axons within the white matter of the CNS is produced by the envelopment of several axons by the oligodendrocytes as opposed to the envelopment of
single axons in peripheral nerves by Schwann cells (Elder, Friedrich, & Lazzarini,
2001; Weinberg & Spencer, 1978). Oligodendrocytes express many inhibitory
myelin-associated proteins (Yiu & He, 2003; Mukhopadhyay, Doherty, Walsh,
Crocker, & Filbin, 1994; Filbin, 2003; DeBellard, Tang, Mukhopadhyay, Shen, &
Filbin, 1996; Shen, DeBellard, Salzer, Roder, & Filbin, 1998) which, together
with the components of the glial scar, constitute the CNS growth environment that
is nonpermissive to the regeneration of the central axons from the DRG neurons
and injured central axons (Grados-Munro & Fournier, 2003; Tang, 1997; Busch &
Silver, 2007; Silver & Miller, 2004).
Motor and Sensory Axon PNS and CNS Axon Regeneration 415
Poor Functional Recovery After Peripheral Nerve Injuries
Clinical experience of poor functional recovery in human patients after surgical
repair of peripheral nerve injuries, particularly those of brachial and lumbosacral
plexi, has too often been interpreted as failure of regenerating axons to reinnervate
target tissues which have undergone irreversible atrophy (Kline, D. G. & Hudson,
A. R, 1995; Noble, Munro, Prasad, & Midha, 1998). Slow rates of regeneration of
~1 and ~3mm/day in human and animal species (Sunderland, 1947a; Sunderland,
1947c; Sunderland, 1947b; Gutmann, Guttmann, Medawar, & Young, 1942),
respectively, necessitate months and even years for axons to regenerate over the
long distances. The regeneration rate of ~3mm per day was originally determined
for rabbit sensory and motor axons. The distance at which the “pinch” of the
regenerated nerve elicited a reflex and the rate of return of response to skin nociceptive stimuli were used to calculate sensory nerve regeneration rates. Motor
nerve regeneration rates were calculated from the time for recovery of a given
motor function such as the reflex placing of the paw after crush injuries at different distances along the nerve (Gutmann et al., 1942). This slow rate of regeneration corresponds with the rate of slow axonal transport of cytoskeletal proteins
such as actin and tubulin (McLean, 1985; Watson, Hoffman, Fittro, & Griffin,
1989; Kline & Hudson, 1995; Sulaiman & Gordon, 2003).
Gutmann and Young used a cross-suture surgical paradigm to examine the
capacity of regenerating nerve to reinnervate chronically denervated peroneus
muscles in the rabbit (Gutmann & Young, 1944). The muscles were chronically
denervated for 17 months by section of the common peroneal nerve. Tibial nerve
was cut and cross-sutured to distal stump of the cut peroneal nerve and the
morphology of the muscle examined 9m later. The authors described the presence
of axons that were and were not successful in making neuromuscular connections,
concluding that highly atrophic muscles become progressively resistant to
reinnervation by regenerating axons. We used the same experimental paradigm in
a rat model of nerve injury to systematically reinvestigate the effects of chronic
denervation as well as the effects of chronic disconnection of neurons from targets
by nerve section–chronic axotomy (Fu & Gordon, 1995a; Fu & Gordon, 1995b;
Sulaiman et al., 2002; Sulaiman, Voda, Gold, & Gordon, 2002; Sulaiman &
Gordon, 2002; Gordon, Sulaiman, & Boyd, 2003; Gordon, Sulaiman, & Boyd,
2003). To examine the effects of chronic axotomy, we cut the tibial nerve to
chronically axotomize tibial neurons for up to 12m after which a cross-suture of
the tibial and freshly cut common peroneal nerves encouraged regeneration of the
axons and reinnervation of the denervated muscles (Fu & Gordon, 1995a). We
also chronically denervated the common peroneal nerve stump for periods of time
of up to 12m before cross-suturing the freshly axotomized tibial nerves to the
common peroneal nerve stumps. The cross-sutured nerves were allowed at least 4
months to regenerate and reinnervate the tibialis anterior muscle when the number
of reinnervated motor units, their isometric contractile force and the number of
muscle fibers reinnervated by each motor unit was determined (Fu & Gordon,
1995b). Consistent with the results of Gutmann and Young, the number of
reinnervated motor units declined dramatically. However the decline to less than
416 Gordon et al.
10% within 6 months of the cross-suture of the chronic denervated nerve could
not be accounted for by the failure of regenerating axons to reinnervate the
atrophic muscles that Gutmann and Young had described. This is because those
few motoneurons that regenerated their axons reinnervated up to 5 times their
normal number of muscle fibers to generate increased motor unit contractile
forces (Fu & Gordon, 1995b). Later counts of motoneurons that regenerated their
axons into chronically denervated common peroneal distal nerve stumps revealed
that indeed, the chronic denervation of the Schwann cells and not the muscle
fibers themselves, had progressively reduced the regenerative capacity of the
axons within the nerve sheaths (Sulaiman & Gordon, 2000). The reduction in
regenerative capacity through chronically denervated nerve sheaths was associated
with a parallel decline in the growth supportive state of the chronically denervated
Schwann cells within the sheaths (Gordon, Sulaiman, & Boyd, 2003; You, Petrov,
Chung, & Gordon, 1997; Hall, 1999; Li, Terenghi, & Hall, 1997). Importantly, the
few axons that did regenerate successfully became fully remyelinated,
demonstrating sustained capacity of the surviving Schwann cells to remyelinate
axons (Sulaiman & Gordon, 2000).
In summary, the effects of the long-term chronic axotomy and Schwann cell
denervation account for progressive and complete failure of axon regeneration
and reinnervation of the more distal musculature and sense organs after repair of
nerve injuries such as those of the brachial plexus.
Staggered Axon Regeneration
An initial latent period of a few days before axons regenerate within the endoneurial
tubes of the distal nerve stumps has been determined by the “pinch test” that
evaluates rate of sensory axon regeneration and by determining the time for
recovery of a given motor function such as toe spreading after lesions at different
distances from the muscles (Holmquist, Kanje, Kerns, & Danielsen, 1993; Bisby
& Keen, 1984; Sunderland, 1947a; Gutmann, Guttmann, Medawar, & Young,
1942). The latent period was first defined in 1942 as the period of “retrograde
degeneration, branching, and the relatively slow process of outgrowth across the
suture scar” although this “scar delay” or period of time before arrive of new axon
tips in the distal nerve stump was only slightly shorter after crush injury than after
transection and suture repair (Gutmann, Guttmann, Medawar, & Young, 1942).
This period is actually much more protracted than has been appreciated. Counting
dye-labeled motoneurons that had regenerated their axons into the distal nerve
stump at progressively longer periods after nerve section and surgical repair
revealed a slow progressive regeneration of axons where only half of the
motoneurons had regenerated their axons over a distance of 25 mm in 2 weeks
(Figure 1A). This is different from the prediction of all the motoneurons
regenerating their axons at 3mm/day after a short latent period of days. We coined
the term staggered axonal regeneration for the progressive axon regeneration over
an 8–10 week period for all the motoneurons to regenerate their axons over a
25mm distance (Figure 1A; Al-Majed, Neumann, Brushart, & Gordon, 2000).
Interestingly, a report of a protracted time course of slow axonal transport of
several proteins including cytoskeletal proteins, had actually anticipated but not
recognized staggered axonal regeneration (Forman, & Berenberg, 1978) and the
early studies of Gutmann and colleagues had recognized that the regeneration
Figure 1 — Low frequency (20Hz) bipolar electrical stimulation, irrespective of duration,
accelerates outgrowth of regenerating motor axons into appropriate motor pathways after
nerve injuries. A) The femoral nerve in Sprague Dawley rats was cut and the stumps resutured with microsurgical technique. At 2 weeks and up to 10 weeks after nerve repair, the
regenerated axons in the motor nerve to the quadriceps muscle and the sensory nerve to the
skin 25 mm from the suture repair site were exposed to fluorescent dyes fluororuby and
fluorogold that were taken up by the motoneurons. The mean (± SE) of the number of dyelabeled motoneurons that regenerated their axons in the motor (M), sensory (S) and both
branches (B) of the femoral nerve are plotted as histograms as a function of the time after
nerve repair. At 2 and 3 weeks, there were as many motooneurons regenerating their axons
into motor and sensory branches, the quadriceps and the saphenous nerves, respectively.
Thereafter there was no change in the number of backlabeled motoneurons and therefore
the number that regenerated inappropriately into the saphenous nerve but the progressive
increase in numbers of backlabeled motoneurons from the quadriceps nerve branch demonstrated the preferential motor reinnervation originally described by Brushart in 1993
(Brushart, 1993) and the staggered axon regeneration described by Al-Majed et al. (2000).
B) Counts of motoneurons that regenerated their axons 25 mm into motor and sensory
pathways after 2 weeks 20Hz electrical stimulation, proximal to the site of nerve section
and surgical repair. The stimulation mediated a very rapid onset of preferential motor reinnervation at 2 weeks after surgical repair of the transected femoral nerve. In addition, all
the motoneurons had regenerated their axons by 3 rather than 8 weeks after surgical repair.
C) Counts of motoneurons that regenerated their axons into motor and sensory pathways as
in A and B at 3 weeks after surgical repair of the transected femoral nerve with and without
1h electrical stimulation immediately after nerve section and repair. Application of a blocking dose (60mg/ml) of tetrodotoxin (TTX) proximal to the site of electrical stimulation
effectively prevented the stimulation-induced increase in the number of motoneurons that
regenerated their axons into the motor branch. D) Effect of 1hr 20Hz electrical stimulation
on the number of sensory neurons in the L4–6 dorsal root ganglia that regenerated their
axons into the motor (M), sensory (S) and both (B) branches of the femoral nerve after 3
weeks. The electrical stimulation accelerated appropriate regeneration of sensory axons in
the appropriate saphenous nerve branch of the femoral nerve.
417
418 Gordon et al.
rates that they had reported for rabbit hindlimb nerves represented the fastest
regenerating axons (Gutmann, Guttmann, Medawar, & Young, 1942).
The technique of retrograde labeling for counting neurons that regenerate
their axons into the distal nerve stump after nerve injury has been used extensively
and has been validated in several laboratories including our own, has been validated (Harvey, Grochmal, Tetzlaff, Gordon, & Bennett, 2005; Obouhova, Clowry,
& Vrbova, 1994; Richmond et al., 1994; Swett, Wikholm, Blanks, Swett, &
Conley, 1986; Eberhardt et al., 2006; Brushart et al., 2002; Brushart, Gerber, Kessens, Chen, & Royall, 1998). These neuronal counts are always much less than the
counts of regenerated axons in the distal nerve stumps from histological examination of nerve cross-sections. The latter include multiple regenerated axons from
each axotomized neuron as each axon proximal to the nerve injury emits many
regenerated axons, the reported ratio of regenerating axons to proximal axons
being as high as 20:1 (Aitken, Sharman, & Young, 1947) and as low as 5:1 (Mackinnon, Dellon, & O’Brien, 1991). Therefore, simple counts of regenerated axons
in the distal nerve stumps may be misleading. For example, the effects of exogenous neurotrophic factors on axon regeneration can be misleading if these are
based solely on axon counts: brain derived neurotrophic factor (BDNF) and glial
cell derived neurotrophic factor (GDNF) both stimulate sprouting of new axons
but they do not increase the number of motoneurons that regenerate their axons
(Boyd, & Gordon, 2002; Boyd & Gordon, 2003a; Boyd & Gordon, 2003b).
We predicted that the ‘staggered axon regeneration’ occurred at the suture
site with a more constant rate of regeneration of 3mm/day within the distal nerve
stumps. To test this prediction, we backlabeled femoral motoneurons that had
regenerated their axons across the surgical site of resutured proximal and distal
stumps. We injected a fluorescent dye at a crush site close to the surgical site
(Figure 2). The dye is transported back to the cell bodies from the injection site.
Findings that the number of motoneurons that regenerated their axons across the
suture site increased progressively over a protracted period of 4 weeks, a surprisingly long period of time, confirmed our hypothesis (Brushart et al., 2002). Similarly, the regeneration of sensory axons was ‘staggered’ with only a proportion of
the DRG neurons regenerating their axons initially and the number increasing
with time (Brushart, Jari, Verge, Rohde, & Gordon, 2005). Hence the backlabeling of axons that had crossed the suture site at different times after suture elucidated the ‘staggering’ of the axon as they emerged from the proximal nerve stump
to regenerate across the site of the suture.
In the same manner that elaboration of the cross-bridge theory of muscle
contraction had its origins in the original morphological studies in the 19th century (Ford, Huxley, & Simmons, 1977), the findings of staggered axon regeneration across the suture site had actually been made at the turn of the 20th century,
by Ramon Y Cajal (Cajal, 1928). Cajal had used silver staining to visualize the
progress of nerve fibers regenerating after a peripheral nerve section with or without a sequential surgical apposition of proximal and distal nerve stumps. He
described the outgrowth of axons from the proximal nerve stump that crossed the
surgical site in a varied manner. Despite apposition of nerve stumps, growing
Motor and Sensory Axon PNS and CNS Axon Regeneration 419
Figure 2 — Low frequency (20Hz) electrical stimulation for 1h accelerates, by 4 days the
sensory and motor axon outgrowth across the surgical gap between transected and surgically repaired union of the proximal and distal nerve stumps of the rat femoral nerve and
1.5mm into the distal nerve stump. Sensory and motor neurons that regenerated their axons
across the suture site to enter the distal nerve stumps were backlabeled by microinjecting
0.5µl of fluororuby 1.5mm into the stump after a brief crush. The numbers (mean ± SE) of
backlabeled sensory and motor neurons that regenerate their axons within 4 days were
significantly higher in the stimulated group.
axons often took tortuous routes to navigate the gap and, in some cases, axons
formed unusual spirals that even grew in the opposite direction within the proximal nerve stump (Cajal, 1928). In a later study, Brushart and his colleagues used
transgenic methodology to express a yellow fluorescent protein in neurons of the
mouse to visualize the regenerating axons after a femoral nerve transection and
surgical repair (Redett et al., 2005; Brushart & Mesulam, 1980). This study not
only confirmed Cajal’s findings but placed the progressive axon outgrowth that we
had discovered in the context of the initially disorganized extracellular matrix at
the suture site and the randomly placed Schwann cells that had migrated from
both proximal and distal nerve stumps. Similarly, Rafuse and colleagues described
the staggered outgrowth in their study of rat nerves regenerating across a suture
line (Franz et al., 2008). Staggered outgrowth explains the prolonged period of
crossing of regenerating axons across the site of suture of proximal and distal
nerve stumps.
420 Gordon et al.
Solutions: Motor and Sensory Axon Regeneration Is
Accelerated by Electrical Stimulation
Low Frequency Electrical Stimulation, Irrespective
of Duration, Accelerates Outgrowth of Regenerating Motor
Axons Into Appropriate Motor Pathways After Nerve Injuries
in the PNS
In the 1980s it was reported that low frequency electrical stimulation of peripheral
nerves after crush injury accelerated return of reflex foot withdrawal and contractile force in the reinnervated leg muscles (Nix & Hopf, 1983; Pockett & Gavin,
1985). These formed the basis for our hypothesis that the electrical stimulation
accelerated axon regeneration and in turn muscle reinnervation after section and
microsurgical repair of a peripheral nerve. We chose to electrically stimulate the
proximal nerve stump of a cut and repaired rat femoral nerve at the low frequency
of 20Hz continuously for 2 weeks (Al-Majed, Neumann, Brushart, & Gordon,
2000). This stimulation regimen was based on the average firing frequency of
motoneurons and that motoneurons regenerate randomly into appropriate and
inappropriate distal nerve stumps 2 weeks after nerve section and repair (Brushart, 1988; Brushart, 1993; Brushart, 1993). At progressively longer intervals after
the surgical repair, two different dyes were applied to the motor nerve branch to
quadriceps muscle and to the saphenous nerve that conveys only sensory nerves
from the skin (Figure 1A). Thereby we counted dye-labeled motoneurons that had
regenerated appropriately and inappropriately into motor and sensory nerve
branches. We found that all the motoneurons that regenerated their axons into the
distal nerve stumps, eventually regenerated into the appropriate endoneurial tubes.
These directed their axons appropriately into the muscle nerve branch and into the
denervated muscle (Figure 1A). Stimulated and nonstimulated nerve regeneration
was compared over the same 8 week period. Remarkably, the low frequency stimulation promoted the axon regeneration of all the motoneurons over a 25mm distance within 3 weeks rather than the protracted period of 8–10 weeks (Al-Majed,
Neumann, Brushart, & Gordon, 2000; Figure 1B). Again, motoneurons had likely
regenerated their axons randomly into motor and sensory nerve pathways with
axons arriving more quickly at the distal nerve stump to select the appropriate
pathways.
To establish a more clinically feasible method to accelerate axon regeneration, we progressively shortened the period of electrical stimulation to 1h just
after surgical repair of the transected femoral nerve. This short 1h period did not
diminish the effectiveness of the stimulation in accelerating motor regeneration
(Figure 1C; Al-Majed, Neumann, Brushart, & Gordon, 2000). The 1h electrical
stimulation accelerated axon regeneration when the proximal stump was stimulated before or after the suture of the proximal and distal nerve stumps in rats and
mice (Al-Majed, Neumann, Brushart, & Gordon, 2000; Franz, Rutishauser, &
Rafuse, 2008). The accelerating effect is mediated via conduction of action potentials to the cell body since the effect was eliminated by pharmacological conduction block with application of the sodium channel blocker, tetrodotoxin (Figure
1C).
Motor and Sensory Axon PNS and CNS Axon Regeneration 421
Axonal outgrowth from the proximal nerve stump and/or regeneration rate
may be accelerated to account for the stimulation effect of accelerated axon regeneration. To decide between these possibilities, we a) used a new method of applying retrograde dyes just distal to the repair site to evaluate how many neurons
regenerated their axons across the suture site (Figure 2) and b) determined regeneration rate in radiolabeling experiments (Brushart et al., 2002; Brushart et al.,
2002). Counts of both sensory neurons and motoneurons that regenerated their
axons across the suture site demonstrated that the 1h electrical stimulation significantly increased the numbers, i.e., the stimulation accelerated axon outgrowth
across the suture site (Figure 2; Brushart et al., 2002). Evaluation of the regeneration rate showed that the stimulation-induced accelerated axon regeneration was
not due to increased rate of regeneration of the axons within the distal nerve
stumps (Brushart et al., 2002). The latter accounts for accelerated regeneration
after a nerve lesion in the PNS and/or the CNS that is preceded by a conditioning
lesion of the peripheral nerve (McQuarrie, Grafstein, & Gershon, 1977; Bisby &
Pollock, 1983; Udina et al., 2008; Lankford, Waxman, & Kocsis, 1998).
By observing a subpopulation of motor and sensory neurons that express
green fluorescent protein in transgenic mice, Rafuse and colleagues reported that
each parent axon emitted more regenerating axon sprouts in response to electrical
stimulation, possibly increasing the chances of encountering the Schwann cells
within the endoneurial tube that guide and support the regenerating axons down
the distal nerve stump (Franz, Rutishauser, & Rafuse, 2008). This is consistent
with there being longer and more regenerating axons after 1h 20Hz electrical
stimulation (English, Schwartz, Meador, Sabatier, & Mulligan, 2007; English,
Meador, & Carrasco, 2005).
Insights gained from the evidence for accelerated axon outgrowth by the electrical stimulation now permits full appreciation of the early work that revealed a
more rapid reinnervation of denervated targets after electrical stimulation of
crushed nerves in rabbits and rats (Nix, & Hopf, 1983; Pockett & Gavin, 1985)
(see Gordon et al., 2009). We have now extended these early findings to experiments in carpal tunnel patients who suffered a loss of more than 50% of their
functional motor units in the median eminence (Gordon et al., 2009; Gordon,
Brushart, Amirjani, & Chan, 2007). We electrically stimulated the median nerve
proximal to and immediately following carpal tunnel release surgery. We placed
stainless steel electrodes on the nerve during the surgery for the 1h 20Hz electrical
stimulation. The electrodes were removed thereafter by pulling them through the
skin. We recorded electromyographic signals from the muscles of the thenar eminence in response to maximal electrical stimulation of the median nerve: the compound muscle action potential, and single motor unit action potentials in response
to all-or-none stimulation of single motor axons. The ratio of the compound action
potential and the mean size of the unit action potentials provided an estimate of
the number of innervated motor units in the thenar muscles before the carpal
tunnel release surgery and at 3 month intervals after the surgery. The 1h 20Hz
electrical stimulation was effective in promoting reinnervation of muscles by all
the motoneurons within 9 months as opposed to the nonsignificant rise in numbers
of functional motor units in the patients whose median nerve was not stimulated
after the surgical release (Gordon et al., 2009; Gordon, Brushart, Amirjani, &
Chan, 2007). These studies not only demonstrate that electrical stimulation that
422 Gordon et al.
we had demonstrated to accelerate axon outgrowth in rats, was also effective in
accelerating muscle reinnervation after nerve injury in human patients.
Brief but Not Prolonged Low Frequency Electrical
Stimulation Accelerates Sensory Axon Regeneration Into
Appropriate Sensory Pathways After Peripheral Nerve Injury
The same brief 1h period of 20Hz electrical stimulation to the proximal nerve
stump immediately after nerve repair also accelerates sensory axon outgrowth and
directs their axons specifically into correct sensory nerve pathways (Figure 1D)
(Brushart, Jari, Verge, Rohde, & Gordon, 2005). This effect is also mediated via
action potential conduction to the cell body since the effect was eliminated by a
tetrodotoxin blockade of the centrally conducted action potentials (Geremia,
Gordon, Brushart, Al-Majed, & Verge, 2007). The effect of the electrical stimulation on sensory neurons differed from that on the motoneurons. Motor axon outgrowth was accelerated by 20Hz electrical stimulation irrespective of the duration
of the stimulation, 1h stimulation being as effective as 1d or even 2w periods of
stimulation (Al-Majed, Neumann, Brushart, & Gordon, 2000). In contrast, a 1h
period of 20Hz stimulation but not longer, was effective in accelerating sensory
axon outgrowth (Geremia, Gordon, Brushart, Al-Majed, & Verge, 2007). The
stimulation increased the number of sensory neurons that regenerated their axons
across the suture site by a factor of 1.7, as compared with the >threefold increase
in the number of motoneurons that regenerated axons (Figure 2). It remains to be
determined whether the reduced effectiveness of the electrical stimulation in
accelerating sensory axon regeneration is linked to a more rapid outgrowth of
sensory than motor axons (Suzuki, Ochi, Shu, Uchio, & Matsuura, 1998). We
found that 27% of the sensory neurons regenerated their axons across the suture
site within 4 days as compared with 3% of the motoneurons (Figure 2). By 3
weeks, significantly more sensory neurons had regenerated their axons into the
distal nerve stumps as compared with the motoneurons (Figure 1). This may be
linked to the greater heterogeneity in sensory versus motor neuronal populations,
with a subpopulation of ~45% sensory neurons expressing NGF receptors and the
plasticity associated gene GAP-43 (Verge, Tetzlaff, Richardson, & Bisby, 1990).
The neurons appear to be primed for rapid neurite outgrowth as for example, they
emit collateral sprouts in response to exogenous NGF (Mearow, Kril, Gloster, &
Diamond, 1994; Diamond, Coughlin, Macintyre, Holmes, & Visheau, 1987)
Brief Electrical Stimulation of the Peripheral Nerve
Accelerates Axon Outgrowth in the CNS
Injury to peripheral but not central axons elicits a strong cell body response in
DRG sensory neurons, including upregulation of both growth associated proteins
(Richardson, & Issa, 1984) and the mammalian transient receptor potential calcium-permeable cation channels (canonical TRPC subfamily of 7 members). The
latter are linked to neurite outgrowth in vitro (Wu, Huang, Richardson, Priestley,
& Liu, 2008). This cell body response to a conditioning crush or transection lesion
of the peripheral nerve is sufficient to promote regeneration of cut central axons in
Motor and Sensory Axon PNS and CNS Axon Regeneration 423
the dorsal columns (Figure 3 and 4) in association with elevation of cellular cAMP
and upregulation of growth associated genes (Neumann, Skinner, & Basbaum,
2005; Neumann, Bradke, Tessier-Lavigne, & Basbaum, 2002; Neumann & Woolf,
1999; Udina et al., 2008; Richardson & Verge, 1986; Richardson, Issa, & Aguayo,
1984; Wu et al., 2007; Mason, Lieberman, Grenningloh, & Anderson, 2002;
Kenney & Kocsis, 1998; Chong et al., 1994; Wong & Oblinger, 1990).
The conditioned lesion not being clinically feasible, we explored whether a
1h period of 20Hz electrical stimulation that also upregulates cAMP, promotes
axon outgrowth into the same CNS lesion site as does the conditioning lesion. We
found that electrical stimulation of the intact peripheral nerve promotes axon outgrowth in the CNS concomitant with cAMP elevation but does not accelerate
axon elongation through the CNS lesion in the same manner as the conditioning
lesion (Figure 4; Udina et al., 2008). These findings are in accord with findings
Figure 3 — An in vivo method to compare the effect of 1) a conditioning lesion (CL), 2)
20Hz 1hr and 3) 200Hz 1h electrical stimulation of the sciatic nerve on the regeneration of
dorsal root ganglion (DRG) sensory axons that were transected in the dorsal columns at the
T8 level of the spinal cord at the time of the CL or electrical stimulation. At 14 weeks,
cholerotoxin B (CTB) was injected central to the site of the lesion or electrical stimulation
for anterograde labeling of the sensory axons at the lesion site in the CNS.
424
Motor and Sensory Axon PNS and CNS Axon Regeneration 425
that elevated cAMP promotes neurite and axon outgrowth of DRG sensory neurons on nonpermissive substrates of central myelin (Hannila & Filbin, 2007;
Pearse et al., 2004; Qiu, Cai, & Filbin, 2002).
Mechanisms of Accelerated Axon Outgrowth
The Cell Body Response to Axotomy
The Cell Body Response in Axotomized Neurons. While all neurons mount a
cell body response with upregulation of regeneration associated genes, the
response is more robust in neurons regenerating in the PNS (Bisby, 1980; Bulsara,
Iskandar, Villavicencio, & Skene, 2002; Verge, Tetzlaff, Richardson, & Bisby,
1990; Tetzlaff & Bisby, 1990; Miller, Tetzlaff, Bisby, Fawcett, & Milner, 1989; Fu
& Gordon, 1997; McPhail, Fernandes, Chan, Vanderluit, & Tetzlaff, 2004). Transcriptional upregulation of growth associated proteins such as GAP-43, CAP-23
and SCG10, is directly correlated with regenerative capacity of neurons in the
PNS and CNS (Mason, Lieberman, Grenningloh, & Anderson, 2002). These proteins interact with second messenger systems in growth cones to affect the cortical
cytoskeleton and in turn, promote axonal growth (Caroni, 1997; Mason, Lieberman, Grenningloh, & Anderson, 2002; Korshunova et al., 2008; Strittmatter, Igarashi, & Fishman, 1994). In situ hybridization was used effectively to demonstrate
the upregulation of growth associated and cytoskeletal proteins, including T-1tubulin and actin, in axotomized sensory neurons and motoneurons after peripheral nerve transection with or without nerve repair (Gordon, 1983; Al-Majed,
Tam, & Gordon, 2004; Fu & Gordon, 1997; McQuarrie & Lasek, 1989; Tetzlaff,
Lederis, Cassar, & Bisby, 1989; Verge, Tetzlaff, Richardson, & Bisby, 1990; Bisby
& Tetzlaff, 1992; Fenrich & Gordon, 2004; Strittmatter, Igarashi, & Fishman,
1994; Chong et al., 1992). Increased intracellular levels of tubulin and actin meet
the requirements for growth cone advancement while the concomitant reduction
of neurofilament protein accounts for the reduced caliber of the axons proximal to
the lesion from whence the axons sprout and regenerate into the distal nerve stump
(Meiri, Pfenninger, & Willard, 1986; Fenrich & Gordon, 2004; Strittmatter, Igarashi, & Fishman, 1994; Gordon, Gillespie, Orozco, & Davis, 1991; Fu & Gordon,
1997).
Figure 4 — Electrical stimulation (ES) promotes axon outgrowth into CNS lesion site but
not regeneration over long distances, in contrast to the conditioning lesion (CL) that promotes both outgrowth and elongation of axons into the site. A,C,E,G) Camara lucida drawings of 25µm spinal cord sagittal sections of cholera toxin B-labeled DRG sensory axons
that regenerated into the lesion site (shown in gray), 14 weeks after the lesion with and
without the CL or ES. These drawings indicate that the ES promotes axon outgrowth into
the lesion site but that this effect was not as dramatic as that of the CL. B,D,F,H) Comparisons of the cumulative sum of the number (± SE) of regenerating axons that advanced into
the lesion site expressed as a percentage of all the labeled axons proximal to the lesion are
plotted as a function of the distance craniad from the T8 lesion site in the spinal cord. Axon
outgrowth was significantly elevated by 20Hz but slightly but not significantly increased by
200Hz (A-C). Electrical stimulation accelerated axon outgrowth but not regeneration rate
in contrast to the CL that accelerates both to promote regeneration of axons up to 1800µm
into the lesion (D). Statistical significance vs Sham when p < .05.
426 Gordon et al.
Upregulation of Neurotrophic Factors After Axotomy. Expression of the growth
associated proteins mediating axon outgrowth in axotomized motoneurons is preceded by upregulation of neurotrophic factors and their receptors, the most prominent of which are brain derived neurotrophic factor (BDNF) and its receptors trkB
and p75 (Figure 5), and glial derived neurotrophic factor (GDNF) and its receptors. These appear to be essential for axon regeneration; trkB antibodies reducing
regenerative capacity and exogenous BDNF and/or GDNF increasing axon regeneration after prolonged axotomy (Boyd, & Gordon, 2003b; Boyd & Gordon,
2003a; Boyd & Gordon, 2002; Boyd & Gordon, 2001), The intrinsic neuronal and
Schwann cell supply of neurotrophic factors is sufficient to support axon regeneration but their progressive depletion during prolonged axotomy and/or Schwann
cell denervation requires exogenous sources of growth factors to sustain axonal
regeneration (Gordon, Sulaiman, & Boyd, 2003; Furey, Midha, Xu, Belkas, &
Gordon, 2007). Differential expression of several growth factors by Schwann
Figure 5 — Brief (1h) 20Hz electrical stimulation accelerates upregulation of neurotrophic factors
and their receptors that preceeds accelerated upregulation of growth associated genes in axotomized
motoneurons after femoral nerve transection and surgical repair. Semiquantitative in situ hybridization with synthetic oligonucleotide probes end-labeled with 135S-ATP was used to measure expression of mRNA encoding BNDF (A), its receptor, trkB (B) and the regeneration associated genes,
tubulin (C) and GAP-43 (D) for details see (Verge et al., 1992; Al-Majed, Brushart, & Gordon, 2000).
The 12µm sections of fresh-frozen lumbar spinal cord were hybridized overnight and then exposed to
emulsion for adequate resolution of the silver grains that were activated by the 135S-ATP per probe
(Kobayashi, Bedard, Hincke, & Tetzlaff, 1996). The fraction of the area occupied by the autoradiographic silver grains was multiplied by the total cell area and expressed as a percentage of the signal
(± SE).
Motor and Sensory Axon PNS and CNS Axon Regeneration 427
cells in denervated endoneurial sheaths of motor and sensory axons has been suggested to play a key role in the preferential selection of regenerating motor and
sensory axons for their appropriate pathways (Hoke et al., 2006; Brushart, Jari,
Verge, Rohde, & Gordon, 2005; Brushart, 1993).
Brief Electrical Stimulation Upregulates Expression of Neurotrophic Factors
and Growth-Associated Proteins. Findings that tetrodotoxin blockade of action
potential propagation to the cell body prevented stimulation-induced acceleration
of axon regeneration (Al-Majed, Neumann, Brushart, & Gordon, 2000; Geremia,
Gordon, Brushart, Al-Majed, & Verge, 2007), indicated that the cell body response
to axotomy was likely to be the locale of the stimulation-induced effect. The stimulation promotes a rapid and marked upregulation of BDNF and trkB receptors
that precedes the pronounced upregulation of growth associated proteins including tubulin and GAP-43 (Figure 5)(Al-Majed, Brushart, & Gordon, 2000; Geremia, Gordon, Brushart, Al-Majed, & Verge, 2007). However, the correlation of
accelerated axon outgrowth and upregulation of mRNA for growth associated
proteins suggests but does not prove a causal relationship. Findings that the stimulation failed to enhance axon regeneration in NT4/5 knockout mice (English,
Schwartz, Meador, Sabatier, & Mulligan, 2007) and that a selective functional
blocking antibody against BDNF blocked the enhanced axonal outgrowth by 1h
20Hz electrical stimulation (Tyreman et al., 2008) provided more direct evidence.
Hence, the expression, synthesis, release and binding of neurotrophins to trkB
receptors on stimulated neurons, mediate the accelerated axon outgrowth response
to electrical stimulation. A likely scenario is that the NT4/5 and BDNF synthesized by the neurons, act via auto- and paracrine routes to promote expression of
growth associated genes in response to trkB activation.
Role of cAMP in Accelerated Axon Outgrowth From
Motoneurons in Response to Brief Electrical Stimulation
It was the conditioning lesion experiments that first drew attention to cAMP as a
mediator of accelerated axon regeneration, the conditioning lesion promoting
sensory axon growth in the PNS and CNS in association with increased intracellular
cAMP and exogenous cAMP mimicking the effect (Udina et al., 2008; McQuarrie,
Grafstein, & Gershon, 1977; Carlsen, 1982; Kilmer & Carlsen, 1984; Neumann,
Bradke, Tessier-Lavigne, & Basbaum, 2002; Neumann & Woolf, 1999). The
conditioning effect of the peripheral nerve lesion can be blocked by pharmacological
block of the endogenous cAMP (Qiu et al., 2002). Given that there are basal levels
of cAMP in motoneurons (Aglah, Gordon, & Posse De Chaves, 2008) we
examined whether infusing rolipram, a specific inhibitor of the neural
phosphodiesterase type IV, would accelerate axon outgrowth. This was indeed the
case, the rolipram accelerating outgrowth of axons with increased numbers of
motoneurons that regenerated their axons across the suture site and through the
distal nerve stump, akin to the brief electrical stimulation effect. Furthermore, the
effect of the rolipram treatment was sufficient to also accelerate the reinnervation
of denervated skeletal muscles (Gordon et al., 2009).
In vitro, pharmacological elevation of cAMP promoted neurite outgrowth
from embryonic motoneurons on a growth permissive substrate (Figure 6). Forskolin
which stimulates adenylyl cyclase to increase endogenous cAMP, dibutyryl cAMP
Figure 6 — Pharmacological elevation of cAMP stimulates neurite outgrowth and in motoneurons plated on a growth permissive polyornithine/laminin-coated culture dish. Motoneurons plated at 200 cells per well of a 96–well dish or 30,000 per 24-well dish, were
treated at the time of plating with 3M forskolin or 1mM of dbcAMP (A-C) or with 10M
Rolipram (D-E). The mean (± SE) percent of motoneurons with neurites and the mean (±
SE) length of the longest neurites were determined after 24h in tissue culture. The experiments were done in triplicate and repeated three times. The mean (± SE) number of motoneurons that extended neurites (A,D) and the mean (± SE) length of the longest neuritis
(B,E) were significantly increased in medium containing 3µM forskolin, 1mM dbcAMP, or
10µM rolipram. The increase with rolipram was accompanied by a significant increase in
cAMP levels (F). Representative phase-contrast micrographs of the motoneurons with and
without Forskolin or dbcAMP are shown (C). The differences between treated and untreated controls were determined by the Student t test. Data were analyzed by One Way
ANOVA with Newman-Keuls Multiple range test. Significance is indicated: *** p < .001
(with respect to the untreated control).
428
Motor and Sensory Axon PNS and CNS Axon Regeneration 429
(dbcAMP), a soluble cAMP analog, and rolipram each increased the number of
the motoneurons that extend neurites and neurite length (Figure 6; Aglah, Gordon,
& Posse De Chaves, 2008). SQ22356, a soluble and potent adenylyl cyclase
inhibitor that reduces endogenous cAMP, evokes a dose-dependent decline in
neurons that extend neurite and their length (Figure 7).
Figure 7 — Pharmacological reduction in intracellular cAMP causes decreased neurite
outgrowth and extension in motoneurons in vitro. Motoneurons cultured at a density of 200
cells per 96-well dish or 30,000 per 24-well dish, were treated with SQ22536 at different
concentrations at the time of plating and the mean ± SE of (A) intracellular cAMP levels
and (B) percent of motoneurons which extended neurites determined 18h after treatment.
Data were analyzed by One Way ANOVA with Newman-Keuls Multiple range test. Significance is indicated with respect to the untreated control *** p < .001.
430 Gordon et al.
In line with the common downstream intracellular target of cAMP being protein kinase A (PKA), we found that forskolin and dbc-AMP phosphorylated the
PKA RIIB subunit in cultured motoneurons (Figure 8A) in parallel with the
increase in numbers of neurons with neurites and the length of the neurites (see
Figure 6AB). PKA phosphorylation and increased neurite length in response to
the elevated cAMP were both reduced by the PKA inhibitor, H89 (Figure 8).
These experimental findings indicate that cAMP acts via PKA to accelerate axon
outgrowth in response to electrical stimulation in vivo. However, unlike DRG
sensory neurons where cAMP activates ERK downstream of PKA in vitro (Cai et
al., 2001), elevated motoneuronal cAMP in response to forskolin or dbcAMP did
not phosphorylate ERK2 above basal levels or increase the pERK/Total ERK
ratio. Agents that reduced cAMP including SQ22356 and H89, did not change
ERK2 phosphorylation, consistent with basal ERK2 activation in motoneurons
being independent of cAMP (Figure 9). As a control, BDNF induced a sustained
and significant Erk activation that, taken together with the data from the Western
blot analysis in Figures 8 and 9, demonstrates that ERK activation is not an essential component of the effectiveness of cAMP in promoting motor axon growth.
Figure 8 — PKA downstream of cAMP mediates neurite outgrowth in motoneurons in
vitro. A) Motoneurons cultured at a density of 30,000 cells per 24-well dish were treated at
the time of plating with forskolin (3µM), dbcAMP (1mM), H89 (20µM), Rp-cAMP
(500µM), as well as forskolin or dbcAMP together with H89 or Rp-cAMP. (A) PKA activation was examined by detecting the phosphorylated form of the PKA regulatory subunit
RII. (B) Motoneurons cultured at a density of 2000 cells per 96-well dish were treated at
the time of plating with H89 (20µM) or Rp-cAMP (500 M) in the absence or presence of
forskolin (3µM) or dbcAMP (1mM) and neurite length was measured after 24h. (A) Treatment of motoneurons with forskolin and dbcAMP induced PKA activation by phosphorylation. Conversely H89, a PKA inhibitor, and the cAMP antagonist Rp-cAMP inhibited
PKA basal activity and reduced forskolin- and dbcAMP-induced PKA activation. (B)
There were corresponding reductions in the mean (± SE) length of neurites with H89 and
RpcAMP in the presence of forskolin that increases cAMP and with H89 in the presence of
dbcAMP. Data were analyzed by One Way ANOVA with Newman-Keuls Multiple range
test. Significance is indicated with respect to the untreated control *** p < .001.
Motor and Sensory Axon PNS and CNS Axon Regeneration 431
Figure 9 — Cyclic AMP does not regulate Erk activation downstream of PKA in motoneurons in vitro. Motoneurons were cultured at a density of 30,000 per 24-well dish and
treated with dbcAMP (1mM), forskolin 3 M, SQ22536 (500M), H89 (20 M), RpcAMP (500 M) or BDNF (50 ng/ml). (A) Equal amounts of protein from cell lysates of
each treatment were separated by SDS-PAGE and immunoblot analysis for Erk activation
was performed. The figure is a representative of at least three independent experiments
after 15min of treatment. (B) Mean ± SE values of the ratio of pERK/Total ERK are plotted
as a function of duration of exposure to the drugs. The basal level of Erk2 phosphorylation
was present even when motoneurons were untreated and agents that increase (dbcAMP,
forkoline) or reduce cAMP (SQ22536) did not affect Erk activation by phosphorylation
(p-Erk). Accordingly, the PKA inhibitors H89 and RpcAMP were also unable to regulate
Erk activation. In contrast, the motoneurons responded robustly to BDNF (50ng.ml) with
Erk phosphorylation in the same time periods of incubation. Active Erk and total Erk were
quantified using UN-SCAN-IT software. Significance is indicated with respect to the untreated control *** p < .001.
Conclusions
There is a limited window of opportunity for functional recovery after peripheral
nerve injury and surgical repair because the growth state of the axotomized neurons and the growth-support of the Schwann cells in the denervated distal nerve
stumps deteriorate over time. Hence, it is imperative to explore methods to sustain
these states and thereby, prevent their time-dependent deterioration. The staggering of regenerating axons that proceeds at surprisingly slow rates across the surgical repair site is a key component of delayed axon regeneration. The marked
acceleration of axon outgrowth that we observed in response to a 1h period of
20Hz electrical stimulation of axotomized neurons, has considerable potential to
promote earlier reinnervation of denervated targets. The accelerated axon crossing
of the repair site could, at least in part, effectively counteract the time constraints
of the growth-permissive environment of the PNS and the capacity of the neurons
to regenerate their axons.
Transection of a sensory nerve evokes rapid depolarization at the injury site,
followed by a burst of action potentials referred to as an “injury discharge” lasting
up to minutes (Wall & Gutnick, 1974; Berdan, Easaw, & Wang, 1993; Chung,
432 Gordon et al.
Leem, & Kim, 1992; Blenk, Janig, Michaelis, & Vogel, 1996). Findings that TTXblockade of the discharge just before and during the stimulation period reduces
the sensory cell body response 3 days later, indicates that the discharge in of itself,
is important for axon outgrowth after injury (Pettersson, Gordon, Tireman, &
Verge, 2006). If this is so, the brief period of 1h 20Hz electrical stimulation would
further this promotion because the electrical stimulation accelerates outgrowth of
both motor and sensory axons. As shown in Figure 10, electrical stimulation is
effective, irrespective of the duration of stimulation, in raising cAMP to upregulate BDNF and trkB via PKA activation and, in turn, upregulates regeneration
associated genes to promote axon outgrowth. In motoneurons, rolipram by elevating cAMP effectively increases axon outgrowth in vitro. That electrical stimulation is likely to act via cAMP to promote BDNF and trkB expression and in turn,
promotes synthesis of growth associated proteins, is supported by in vitro evidence that elevated cAMP acts via PKA to promote neurite outgrowth. However,
Figure 10 — Schematic representation of the mechanism by which a brief period of low
freqnecy electrical stimulation promotes axon outgrowth in motor and sensory neurons.
Electrical stimulation increases intracellular cAMP that, via PKA leads to the transcription
and translation of neurotrophic factors. These in turn act via adenylase cyclase to sustain
the levels of intracellular cAMP that act downstream of PKA to upregulate the regeneration
associated genes to synthesize sufficient cytoskeletal proteins such as tubulin and actin and
growth associated proteins such as GAP-43 to promote the outgrowth of axons from the
proximal stump of a transected nerve.
Motor and Sensory Axon PNS and CNS Axon Regeneration 433
unlike sensory neurons, cAMP affects on neurite outgrowth in motoneurons are
not mediated via the ERK pathway.
In sensory neurons, electrical stimulation is effective in accelerating axon
outgrowth only when it is brief in duration, longer durations of stimulation downregulating trkB expression in the neurons. The electrical stimulation increases
cAMP as it does in motoneurons. The cAMP in turn, is likely to exert its effects
via PKA and downstream targets to promote the synthesis of neurotrophins. These
in turn, may be released to act in an autocrine and/or paracrine manner to promote
expression of regeneration associated genes, and in turn to promote axon outgrowth (Figure 10).
Acknowledgments
This work was supported by operating grants to TG and VMKV from CIHR and the CIHR
Regenerative Medicine Team grant that includes Drs Zochodne, Sayed, and Midha from
University of Calgary, VMKV from University of Saskatchewan, and Dr. Chan and TG
from University of Alberta. EU was a recipient of a postdoctoral fellowship from the
Spanish Ministry of Health Research. TG is an AHFMR Senior Investigator.
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