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Published in final edited form as:
Trends Neurosci. 2012 March ; 35(3): 156–163. doi:10.1016/j.tins.2011.10.006.

Sensory axon regeneration: rebuilding functional connections inthe spinal cord
George M. Smith1, Anthony E. Falone2, and Eric Frank2,*
1Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY
40536-0509; Present address: Shriners Hospital Pediatric Research Center, Temple University,
Philadelphia, PA, 19140-5104
2Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine,

136 Harrison Ave., Boston, MA 02111

Abstract
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Functional regeneration within the adult spinal cord remains a formidable task. A major barrier toregeneration of sensory axons into the spinal cord is the dorsal root entry zone. This region
displays many of the inhibitory features characteristic of other central nervous system injuries.
Several experimental treatments, including inactivation of inhibitory molecules (such as Nogo andchondroitin sulfate proteoglycans) or administration of neurotrophic factors (such as nerve growth
factor, neurotrophin3, glial derived neurotrophic factor and artemin), have been found to promoteanatomical and functional regeneration across this barrier. There have been relatively few
experiments, however, to determine if regenerating axons project back to their appropriate target
areas within the spinal cord. This review focuses on recent advances in sensory axon regeneration,
including studies assessing the ability of sensory axons to reconnect with their original synaptic
targets.

Introduction

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Recovery after injuries to the spinal cord is severely limited. Even 3000 years ago,
physicians noted that the prognosis for improvement was poor, as evidenced by this quotefrom Edwin Smith Surgical Papyrus, circa 2500-1600 BC: “Diagnosis: You should say ofhim: ‘One having a crushed vertebra in his neck; he is unconscious of his two arms, his twolegs, he is speechless. An ailment not to be treated.”. During the last several decades,
various therapies have been found to promote some degree of anatomical and functional
recovery in animal models of spinal cord injury (SCI) [1-4], yet the prognosis for recovery
in humans remains poor [5]. Although patients with severe spinal cord injuries now often
have nearly normal life spans [6], there is little or no improvement in function after the firstfew months following injury.
For many years it was not known if neurons in the central nervous system (CNS) were
intrinsically incapable of regeneration or if an unsupportive environment was responsible for
their lack of growth within the CNS. Seminal experiments utilizing peripheral nerves as© 2011 Elsevier Ltd. All rights reserved.
*
Corresponding Author: eric.frank@tufts.edu.
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bridges within the injured spinal cord provided convincing evidence that at least some CNS
neurons were capable of regenerating [7, 8]. Spinal cord neurons extended axons for several
centimeters into a peripheral nerve bridge, suggesting that peripheral nerves contain factorsthat up-regulate growth processes in CNS neurons and/or that the CNS environment
contains factors that inhibit axonal growth. Since that time, research has focused on theidentification and blockade of inhibitory factors in the CNS as well as the administration ofexogenous neurotrophic factors to stimulate axon growth.

The dorsal root crush model of spinal injuries
One experimental model that is particularly well suited to study axon regeneration within the
CNS is the regrowth of sensory axons into the spinal cord after crush lesions of the dorsal
root. Of the various axon tracts within the cord, sensory axons are among the ones that
respond best to experimental therapies to promote regeneration [4]. Dorsal root disruption
occurs commonly in brachial plexus injuries and leads to disuse of the affected arm, evenwhen motor roots remain intact. In these injuries, regenerating sensory axons are normallyunable to grow back into the spinal cord when they reach the transition between the
peripheral nervous system (PNS), and the CNS, termed the dorsal root entry zone (DREZ)[9]. Over the past ten years, various treatments have been found to promote growth past the
DREZ and into the spinal cord; this model has therefore provided a useful system to study
the relative advantages of different therapies (Table 1).

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Sensory neurons are located in dorsal root ganglia (DRG), which lie within the PNS,
allowing access to the neurons without disrupting the spinal cord. Their cell bodies arepseudo unipolar, with a peripheral and a central branch. The peripheral location of sensory
axons makes them easily available for anatomical labeling and electrophysiologicalstimulation to test for functional synapses within the spinal cord. The DRG contain many
functional classes of sensory neurons that convey specific sensory modalities, innervate avariety of peripheral targets, and project to specific locations within the spinal cord (Box 1).
These characteristics make it possible to assess whether different classes of regenerating
axons project back to their original target areas. Regeneration following dorsal root crush
occurs rapidly until the axons reach the DREZ, where a variety of inhibitory factors block
further growth (Figure 1). Most of these factors are the same ones responsible for blocking
regeneration in the cord itself, but this model avoids the mechanical trauma associated withspinal lesions, thus allowing experimental manipulation of these components to better
understand inhibitory mechanisms within the CNS. Following treatments that allow sensory
axons to grow through the DREZ, these axons need to regenerate only a relatively shortdistance to project to their original targets and form synapses that support functional
recovery of well-defined behavioral outcome measures. This makes it possible to assess
functional regeneration directly with electrical recording and to address issues of specificity
in terms of the anatomical projections of different classes of sensory neurons within the
spinal cord. Recent studies have shown that regeneration of individual axons can befollowed over time in living mice in which subsets of DRG neurons are genetically labeledwith a green fluorescent protein. [10]. That study suggests that some regenerating axons
form synaptic-like terminals on glia that stabilize axon terminals at the DREZ, similar to
those observed previously [11, 12]. The present review will discuss the advantages of using
the dorsal root crush model in detail and explore how this model is being used to provide
insights into axon regeneration within the CNS and potential development of moleculartherapies.

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Page 3Blockade of inhibitory factors in the DREZ
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The DREZ provides a unique environment to study mechanisms that induce axon
regeneration within the spinal cord in the absence of a zone of mechanical trauma, extensive
cellular disruption and the formation of a dense glial scar [13]. Following dorsal root lesionsin adult rodents, severed axons terminating within the spinal cord degenerate and induce an
injury cascade that includes recruitment of macrophages [14, 15] and proliferation of
Schwann cells [16]. Astrocytes at the transition zone become reactive, increasing
proliferation rates, expression of glial fibrillary acidic protein (GFAP), and secretion of proinflammatory cytokines, causing them to undergo hypertrophy and extend processes deeper
into the dorsal roots [17]. Reactive astrocytes also up-regulate extracellular molecules (i.e.
chondroitin sulfate proteoglycans, hyaluronic acid and tenascin) that are associated withregeneration failure in damaged axonal areas [18].

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Lesioned myelinated sensory afferents degenerate, releasing several myelin-associatedproteins that inhibit axon growth. These include Nogo, myelin-associated glycoprotein(MAG) and oligodendrocyte myelin glycoprotein (OMgp) [19]. These proteins bind andsignal through a receptor complex on neurons that includes the Nogo receptor (NgR),
leucine-rich repeat and Ig domain containing 1 (LINGO-1), and p75/Troy [20]. The myelin
inhibitory proteins also contain several growth inhibitory domains that may signal throughother receptors such as Paired-immunoglobulin-like-receptor-B (PirB) [21]. Individually or
together, these myelin inhibitory proteins dramatically reduce the outgrowth of axons from
DRG neurons in vitro [22]. Multiple reagents have been deployed to block signaling frommyelin inhibitory proteins including the blocking antibody IN-1 [23] and the soluble Nogoreceptor peptide (sNgR) [24]. Treatment of the injured spinal cord with sNgR promotes
sprouting of several axonal populations [25-27], and intrathecal application of sNgR after
dorsal root lesions leads to regeneration through the DREZ of large myelinated, but notunmyelinated, axons [28]. Regenerating sensory axons arborize both within the white matterof the dorsal columns and in the dorsal horn, but their pattern of arborization is disorganized,
unlike the normal patterning of sensory projections (compare Figures 1A and 2C).
Nevertheless, stimulation of peripheral nerves evokes synaptic potentials within the spinal
cord and there is a progressive functional return of forepaw sensory function over several
weeks. These results have recently been repeated using herpes simplex virus to express thesoluble Nogo-receptor in DRG neurons [29].

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Another important class of inhibitory proteins is the family of chondroitin sulfate
proteoglycans (CSPGs), which are up-regulated after dorsal root crush [30-34]. Cytokines
produced by activated microglia, particularly Transforming Growth Factor β1 (TGF-β1),
contribute to the up-regulation of CSPGs and tenascin by astrocytes [35-37]. CSPGs aremajor inhibitors of axonal regeneration, as their presence halts sensory axon growth past the
DREZ [38]. Although the mechanism by which CSPGs inhibit regeneration is unclear [39],they may act by signaling through the protein tyrosine phosphatase-sigma receptor [40] orinactivating integrin's [41], which are involved in axon guidance and regeneration. To date,the most successful method for blocking CSPG-mediated inhibition is the use of a bacterial
enzyme, chondroitinase ABC (ChABC) that cleaves the reactive glycosaminoglycan (GAG)
side chains from the core protein. Treatment with ChABC has been particularly successful
in promoting sensory axon regeneration, although in most cases it must be combined withanother treatment to promote growth [42, 43]. For example, ChABC administration
following pretreatment with zymosan promotes anatomical and functional regeneration of
sensory axons into the spinal cord [44]. In this case, zymosan acts like a conditioning lesion
to increase the intrinsic growth capacity of the sensory axons. Likewise, following dorsal
root crush in brevican/Neurocan knockout mice, few axons grow through the DREZ unless aconditioning lesion is also performed [45]. In these examples, conditioning lesions alone

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promote only minimal regeneration through the DREZ [46, 44, 45], further reinforcing theconcept that axonal regeneration requires interventions that affect both the growth state of
the environment and the intrinsic growth state of the neuron.

Up-regulation of growth pathways in sensory neurons

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Earlier experiments using peripheral nerve grafts to bridge lesions of the transected spinal
cords suggested not only the possibility that the CNS might contain factors that actively
inhibit axonal growth, but also that PNS grafts might contain factors not present in the CNS
that stimulate axonal growth [7, 8]. Many neurotrophic factors (NTFs) are known to be upregulated in peripheral nerves in response to axonal injury, and treatment with these factors
has been particularly successful in promoting regeneration of specific sensory axon
populations. For example, a four week intrathecal infusion of nerve growth factor (NGF),
neurotrophin-3 (NT-3) or glial-derived neurotrophic factor (GDNF), but not brain-derived
neurotrophic factor (BDNF), leads to increased regeneration of sensory afferents through the
DREZ and into superficial layers of the spinal cord (Figure 2) [47]. Electrical stimulation of
peripheral nerves evokes activity in dorsal horn neurons, whose firing patterns suggest that
individual NTFs preferentially induced regeneration of axons expressing their requisite NTF
receptors. Further anatomical and behavioral tests also suggested the action of individual
NTFs on specific classes of sensory neurons. In a separate study, delayed treatment with
NT-3 (one week after root crush) resulted in significantly less regenerative capacity [48],perhaps related to the up-regulation of inhibitory factors in the DREZ. Interestingly, theseaxons initially grow through the DREZ but stop growing when they encounter a region withnumerous macrophages. Such macrophages have been observed in tissue culture to inhibit
axon growth and induce dystrophic growth cones [49]. These observations suggest that twozones of inhibition occur at the DREZ: an initial transition zone where axons leave thegrowth-supportive environment of Schwann cells to enter the growth-poor CNS
environment, and a second, slightly deeper zone, composed of macrophages anddegenerating myelin [48].

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Robust regeneration of several classes of sensory axons is also promoted by treatments with
the GDNF-family member artemin (ARTN). Systemic injections of ARTN over a two-weekperiod following crush of all brachial dorsal roots results in extensive regeneration of
myelinated and unmyelinated sensory axons (Figure 2E) [50]. Stimulation of peripheral
forelimb nerves evokes short-latency synaptic potentials in the spinal cord, indicating thereformation of functional synaptic connections by regenerated axons, and a variety of
behaviors involving proprioceptive and nociceptive function are restored. Moreover, therecovery is persistent, lasting at least six months following root lesions. The extent andpersistence of functional recovery and the ease of systemic application suggest that ARTN
has a potential for long-term therapeutic application, although at the present time, thetreatment must be initiated shortly after injury to be effective.
Sensory axon regeneration can also be induced with NTFs expressed locally within the cord
via viral expression. An early report demonstrated that viral expression of NT-3 within theventral spinal cord induced regeneration of lesioned sensory axons into the area expressing
NT-3, although behavioral recovery was not assessed [51]. The expression of fibroblast
growth factor-2 or NGF within the dorsal horn also induces massive growth of nociceptive
axons expressing calcitonin gene-related peptide (CGRP+) into the spinal cord following
dorsal root crush (Figure 2A) [52]. Robust growth was observed despite a two-week delay ininjection of the virus, suggesting that inhibitory factors in either zone of the DREZ are notsufficient to block regeneration of these fibers into the cord. In this study, the expression of
NGF lead to the regeneration of peptidergic-nociceptive axons but no other sensory
populations, most likely due to restriction of neurotrophic tyrosine kinase receptor type 1

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NIH-PA Author Manuscript(TrkA receptor) expression to these neurons. CGRP+ axons regenerated throughout theentire dorsal horn, showing no target specificity, which typically is confined to laminae I
and II. Whether this absence of topographic specificity results from high levels ofgeneralized expression of NTFs disrupting growth cone guidance or from neurotrophintrapping remains to be determined (see Box 2).
Many NTFs can guide growing axons as well as stimulating the growth process directly; thatis, they can act as chemoattractive or neurotropic agents. For example, sensory axons growup gradients towards sources of NGF and NT-3. In contrast, other guidance molecules, suchas semaphorin 3A, can act as chemorepulsive factors, causing axons to turn and grow awayfrom the source of expression. The poor targeting of regenerating CGRP+ axons to dorsal
spinal laminae (see above) can be improved, for example, by establishing slightly
overlapping gradients of NGF (dorsally) and semaphorin 3A (ventrally) [53]. Therefore,
local expression of these agents by viral delivery can be used to guide regenerating axons tomore appropriate target areas (Figure 2B). Interestingly, although the addition of semaphorinsignificantly reduced the overall number of axons that regenerated, the targeting of synapses
to more appropriate laminae resulted in a similar extent of functional recovery. These results
emphasize the utility of targeting regenerating axons to more appropriate synaptic targets,particularly for primary sensory afferents that can induce detrimental behavioral effects if
misdirected [54, 55].

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Neurotrophic factors also display neurotropic effects on other regenerating axon
populations. Viral delivery of NT-3 in either the nucleus gracilis or the reticular formation
combined with injection of bone marrow stromal cells expressing NT-3 into the lesion site
and a conditioning lesion of the sciatic nerve stimulate and direct the regeneration of
proprioceptive sensory axons across a lesion of the dorsal columns and back to these targets
in the brainstem [56]. For either site of viral injection, axons grew into the source of NT-3
regardless of whether it was an appropriate target (nucleus gracilis) or an inappropriate
target (reticular formation). Furthermore, the density of regenerating axons within the
nucleus gracilis was robust, about 27% of the original axon density. Although synapses wereidentified morphologically, sciatic nerve stimulation did not evoke synaptic potentials, most
likely due to conduction blockade from lack of myelination of these regenerated axons.
Interestingly, this triple combination of treatments induced sensory axon regeneration whenadministration started either 6 weeks or 15 months after mid cervical lesions [57],suggesting the potential therapeutic usefulness of this technique.

Specificity of regeneration
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An issue that is seldom addressed in studies of CNS regeneration is whether the formation of
new synaptic connections, either by regenerating or sprouting axons, results in functionallyappropriate neural circuits. Studies of regeneration of sensory fibers lend themselves
particularly well to this issue. There are many well-characterized functional classes of
sensory neurons, which innervate a variety of peripheral targets and make central projections
to specific populations of target neurons in the spinal cord. Systemic ARTN treatments
result in regeneration that is topologically specific: three different classes of sensory axons
each regenerate to their appropriate area in the spinal cord (compare Figures 1A and 2E).
Unmyelinated CGRP+ afferents are restricted to the most dorsal laminae of the dorsal horn,
myelinated cutaneous afferents project to the topographically correct location in deeper
dorsal horn laminae and myelinated muscle afferents project through the dorsal horn
towards the ventral horn [58]. In contrast, although sensory regeneration promoted by the
Nogo receptor peptide sNgR is also functional, different classes of axons do not project totheir former locations in the cord [28, 58]. NGF expression within the spinal cord also leads
to widespread distribution of regenerating CGRP+ axons rather than restricted targeting to

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Page 6dorsal laminae [52]. The patterns of regeneration promoted by these different treatments areillustrated schematically in Figure 2.

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These findings suggest that molecular guidance cues persist in the adult mammalian spinal
cord, but these cues can guide regenerating axons correctly only with certain therapeutic
treatments or by targeting the treatment to the appropriate location (for example to the cellbodies vs. the site of regeneration; see Box 1). A more general implication of these results isthat other guidance cues within the gray matter also may be available to guide theregeneration of other classes of spinal axons, such as those damaged in contusion injuries.
Therapeutic strategies that act directly on the cell body of regenerating axons, such as the
up-regulation of the mammalian target of rapamycin (mTOR) protein pathway incorticospinal neurons [59], rather than modifying the environment of the growing axons,
may be useful in promoting regeneration without disrupting the action of intrinsic guidancecues.

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Restoration of connectivity sufficient for functionally appropriate behavior can also occur
when regenerated projections are not topographically correct. For example, the widespread
distribution of CGRP+ axons following viral expression of NGF in the dorsal horn restores
normal nociceptive responses to thermal stimuli without causing hyperalgesia [52].
Similarly, behavioral recovery is as complete following treatment with sNgR as it is with
ARTN treatment, despite the absence of topographic specificity [28, 58]. However, it has
not been determined if these axon terminals or synaptic connections undergo refinement
over time. There is also increasing evidence for significant plasticity of synaptic connections
following CNS injuries and for the importance of neural activity in functional recovery[2-4]. Rehabilitative training or exercise increases sprouting and return of function byrefining synaptic connections [60]. Such responses are further enhanced following theapplication of ChABC, which supports sprouting and potential reorganization of synaptic
connections associated with improved functional recovery [61, 62]. The effects of enforced
neural activity via training with or without the application of ChABC have not yet been
explored for recovery of function following damage to dorsal root sensory axons, but thisissue will be important to pursue in future experiments.

Conclusions

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The recovery of significant function following injuries to the spinal cord remains a daunting
challenge. One aspect of this recovery is the regeneration of lesioned CNS axons back totheir appropriate synaptic targets. For regeneration to occur, growth programs intrinsic to theneurons must be sufficient to overcome the growth-inhibiting environment of the mature
mammalian CNS and/or the inhibitory nature of the environment needs to be attenuated.
Among different classes of CNS axons, sensory axons within the spinal cord are particularly
responsive to treatments that block inhibitory factors in the CNS environment and upregulate growth pathways within the sensory neurons. Blockade of the Nogo signaling
pathway is sufficient to promote some functional recovery of sensory function, and digestionof the sugar moieties on chondroitin sulfate proteoglycans also facilitates growth of lesioned
sensory axons back into the spinal cord. A variety of neurotrophic factors can also stimulate
sensory axon growth into and within the spinal cord. These factors can be delivered byintrathecal injection, viral expression directly within the spinal cord, or even by systemic
injection. Some of these factors also act neurotropically, so that axons can be guided back toappropriate target regions by localized viral expression. The topographically specificregeneration of sensory axons promoted by systemic ARTN treatment is particularlyinteresting in this regard, as it suggests that molecular guidance cues persist in the adultmammalian spinal cord. If other therapies are found that act similarly on other classes ofTrends Neurosci. Author manuscript; available in PMC 2013 March 1.

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CNS axons, it may be possible to develop generalized strategies for promoting
topographically specific regeneration following other types of spinal injury.

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Acknowledgments
Supported by grants from the National Institutes of Health (NIH) (NS064494), the Murray Winston Foundation,
and Biogen Idec (to E.F.) and grants from the NIH (NS060784) and the Kentucky Spinal Cord and Head Injury
Research Trust (to G.M.S.).

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Box 1

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A Diversity of Neuronal Cell Types in the Dorsal Root Ganglia
The diversity of sensory information entering the spinal cord is dependent on the wide
variety of sensory neurons within dorsal root ganglia. Multiple subpopulations of neurons
have been identified based on their peripheral and central projections, function,
sensitivity to specific NTFs, and expression of neuropeptides. The identification of these
subpopulations provides unique advantages to target the growth and regeneration of
specific neuronal types.
Nociceptive neurons are small, unmyelinated cells mediating pain sensation. Peptidergic
nociceptors all express CGRP and many also express substance-P. They also express the
TrkA receptor and respond to the neurotrophin NGF [66]. Many CGRP+ neurons also
express receptors for ARTN and GFRα3 [50]. Their central projections terminate largely
in laminae I and II [67]. Non-peptidergic nociceptive neurons respond to several GDNF
family members, including GDNF and ARTN, via GFRα receptors signaling in
conjunction with RET (a receptor tyrosine kinase) [68, 69, 50]. Their axons terminate
only in the inner portion of lamina II.

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Tactile mechanosensitive sensory axons are larger than nociceptive axons and are well
myelinated. Peripherally they project via cutaneous nerves to a variety of specialized
receptors in skin. Their central terminations are restricted to the dorsal horn but in deeper
laminae than nociceptive axons, and these projections are arranged topographically,reflecting the specific area of skin they innervate peripherally [70]. Many of theseneurons express TrkB and respond to the neurotrophin BDNF.
The largest sensory neurons are proprioceptive, projecting peripherally via muscle nerves
and supplying muscle spindles and Golgi tendon organs. They provide the CNS withinformation regarding body position and muscle length and tension. These neurons
respond to the neurotrophin NT3 via TrkC receptors [71, 72], and many of them also
express GFRα1, the receptor for GDNF. Their central axons project to more ventral
layers of the spinal cord than mechanoreceptors do, where they provide direct excitatory
input to motoneurons (mediating the simple stretch reflex) as well as a variety ofinterneurons involved in locomotion [73].

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Box 2

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Location, Location, Location: Getting regenerating axons back to theirappropriate locations
The re-establishment of the correct patterns of synaptic connections is an importantconsideration for reconstruction of neural circuits during regeneration. During
development, spatially precise expression of targeted-derived growth factors andchemotropic molecules helps establish specific projection patterns of growing axons[74].
Following CNS injury in adult mammals, exogenous growth factors induce regenerationand sprouting, but only rarely are the new projections topographically appropriate. Many
neurotrophic factors (NTFs) have well-established chemoattractive properties; growthcones will turn and follow a concentration gradient of NGF, NT-3, or BDNF [75, 76].
Sensory axons grow into peripheral nerve grafts or transplants expressing high levels of
NTFs, but then remain in the graft rather than projecting back out and into the host tissue[77, 76]. The application of additional NTFs is required to entice growth back into thehost [78, 76, 79]. Moreover, axons grow into the region of the exogenous NTFs whether
or not these factors are placed at the appropriate target location [56].

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Does the localized expression of NTFs affect topographic organization? Following dorsal
root rhizotomy, localized infusion of NGF, NT-3, or GDNF all induce sensory axon
regeneration through the DREZ, but there has been little assessment if the regenerating
axons project correctly within the spinal cord [47]. Indeed, virally mediated expression of
NGF within the dorsal horn following lumbar root rhizotomies promotes robust
regeneration of peptidergic-nociceptive axons, but these axons grow throughout theentire region of NGF expression (Figure 2A) [52]. The application of NTFs intrathecallyor by viral-mediated expression creates a regional rather than cell-specific distribution,and the supra-physiological concentration of NTF may overwhelm cues from otherguidance molecules. For example, NGF shows a dose-dependent neutralization of
semaphorin 3A chemorepulsion both in vitro [80] and in vivo [55]. Slightly overlapping
gradients of chemoattractive (eg. NGF) and chemorepulsive (eg. Semaphorin 3A) factors,
however, can promote more appropriately targeted projections (Figure 2B) [53].
Although an improvement over expression of NGF alone, this is still a crude method torecapitulate a precise developmental guidance pattern. Intrathecal application of NT-3
promotes regeneration of myelinated sensory axons but not to specific locations in thecord (Figure 2C) [63].

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Interestingly, the systemic application of ARTN after dorsal root injury is the only known
example in which treatment with a NTF results in appropriate and precise topographicorganization of sensory input (Figure 2E) [50, 58]. Systemic ARTN does not enter the
spinal cord and presumably acts directly on the sensory cell bodies within dorsal root
ganglia [58], suggesting that its ability to promote topographically specific regeneration
may be a consequence of its site of action. A puzzling aspect of ARTN's ability topromote regeneration of myelinated sensory axons is that its best-known receptor,
GFRα3, is expressed primarily on unmyelinated sensory neurons; relatively few large
sensory neurons are immunopositive for GFRα3 [81, 82]. It is possible that GFRα3 may
be up-regulated in large neurons following dorsal root crush and/or ARTN treatment;
alternatively other receptors may mediate the action of ARTN on these neurons. Further
studies are required to explore these possibilities.

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Figure 1.

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Spinal projection patterns of various classes of sensory axons. Unmyelinated peptidergic andnon-peptidergic nociceptive axons are shown in red and green, and myelinated cutaneous(blue) and proprioceptive (purple) axons are shown in blue and purple. A. Sensory
projections in a normal, un-injured spinal cord. Peptidergic nociceptive axons primarily
terminate in laminae I and II, whereas non-peptidergic nociceptive axons terminate more
ventrally in lamina II. Projections from large myelinated proprioceptive axons terminate
within the deeper dorsal laminae and into ventral motor neuron pools, whereas cutaneoustactile axons terminate in laminae III - V. Broken lines in the spinal cord represent different
laminae of Rexed, starting from the upper dorsal lamina I. B. Several weeks after sensory
axons are lesioned in the dorsal roots, axons regenerate up to the dorsal root entry zone
(DREZ, highlighted in yellow), but then stop growing and form dystrophic growth cones orturn around and grow distally within the nerve [9, 10]. C. Higher magnification of the DREZ
showing two regions of the dorsal root: a PNS region containing Schwann cells, and a CNS
region containing oligodendroglia (not shown), astrocytes and macrophages. The border
between Schwann cells and astrocytes is delineated by a basal limitans (orange). The DREZ
and dorsal spinal cord contain degenerative myelin, tenascin, and CSPGs which act aspresumptive inhibitory components that block further progression of growth cones. Without
treatment, growth cones remain in this dystrophic state.

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Figure 2.

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Pattern of regeneration of different sensory afferent axon populations promoted by different
therapeutic treatments after dorsal root lesions in rodents. Presumptive gradients of thehighest concentrations of neurotrophic factors or soluble Nogo receptor (sNgR) are shown inorange. A. Viral-mediated expression of NGF centered within the dorsal horn induces
regeneration of peptidergic nociceptive axons throughout the entire region of NGF
expression. Non-peptidergic and myelinated sensory axons do not regenerate [52]. B.
Slightly overlapping expression of NGF (orange) and Semaphorin 3A (SEMA, blue) restrict
the growth of these axons to the upper dorsal horn [53]. C. Intrathecal administration ofsNgR induces regeneration of myelinated but not unmyelinated sensory axons. Myelinatedaxons project to ectopic locations within the dorsal horn [28]. D. Intrathecal administrationof NT-3 induces regeneration primarily of myelinated sensory axons, with someregeneration of non-peptidergic unmyelinated axons. Myelinated axons projected to bothnormal and ectopic regions of the cord [47, 63]. E. Systemic application of artemin (ARTN)
induces robust regeneration of all sensory axon populations, and the axons project to their
topographically appropriate locations [58].

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Table 1Treatments Promoting Regeneration Through the Dorsal Root Entry Zone1

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Agent / Treatment

Axonal regeneration2

Functional recovery3

Refs

NGF / Adenovirus

Peptidergic-Nociceptive

Thermal Nociceptive[52, 55, 53]

Unmyelinated axons

Thermal Nociceptive[47]/ Intrathecal infusion

Electrophysiology
NT-3 / Adenovirus/ Intrathecal infusion

Proprioceptive axons

ND

[51]

Large myelinated,

Electrophysiology[47]

Proprioceptive axons

Beam walking, Paw placement[63]

BDNF / Intrathecal infusion

No regeneration observed

GDNF / Intrathecal infusion

Multiple sensory populations[47]
Thermal and mechanical nociception, grid
walking[47]

Electrophysiology
ARTN / Systemic

All sensory axon populations.

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Thermal/mechanical nociception andproprioception[50, 58]

Electrophysiology
sNgR / Intrathecal infusion

Cutaneous and proprioceptive myelinated axons

Paw preference and grip strength,
electrophysiology[64, 28, 29]

CTB-labeled myelinated axons

Proprioceptive function[29]/ alone – rat

Little regeneration

none (electrophysiology)[44]/ alone – mouseSome regeneration into DREZ

ND

[45]

/ rat

Dextran labeled myelinated axons

Electrophysiology[44]/ mouseSmall increase in regeneration into DREZ

ND

[45]

CTB-labeled myelinated axons

Paw placement, proprioceptive function;[65]/ Herpes Simplex Virus
Peripheral nerve lesion

with CSPG deletion

RAR / lentivirus

Electrophysiology
1

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Effects of various treatment paradigms on anatomical and functional regeneration of sensory axons into the spinal cord. All experiments were
performed on rats except those with peripheral nerve lesions, which were performed on rats and mice, as indicated. Abbreviations: CTB, cholera
toxin subunit B; RAR, retinoic acid receptor β2.
2

Axonal regeneration indicates the types of sensory axons that regenerated as determined anatomically.

3

Functional recovery was assessed behaviorally or by electrophysiological recordings from the spinal cord, as indicated.

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