Prog Neurobiol 2007 NavarroNeural plasticity after peripheral nerve_.pdf

8/19/2019 Prog Neurobiol 2007 NavarroNeural plasticity after peripheral nerve_.pdf

1/39

Neural plasticity after peripheral nerve injury and regenerationX. Navarro a ,b ,*, Meritxell Vivó a, Antoni Valero-Cabre ´ c ,d

a Group of Neuroplasticity and Regeneration, Institute of Neurosciences and Department of Cell Biology, Physiology and Immunology,Universitat Auto ` noma de Barcelona, E-08193 Bellaterra, Spain

b Institut Guttmann, Badalona, Spainc Department of Anatomy and Neurobiology, School of Medicine, Boston University, Boston, MA 02118, USA

d CNRS Unit 5105-ERT-Treat Vision, Department of Neurology, Fondation Ophtalmologique Rothschild, Paris, France

Received 3 October 2006; received in revised form 18 February 2007; accepted 14 June 2007

Abstract

Injuries to the peripheral nerves result in partial or total loss of motor, sensory and autonomic functions conveyed by the lesioned nerves to thedenervated segments of the body, due to the interruption of axons continuity, degeneration of nerve bers distal to the lesion and eventual death of axotomized neurons. Injuries to the peripheral nervous system may thus result in considerable disability. After axotomy, neuronal phenotypeswitches from a transmitter to a regenerative state, inducing the down- and up-regulation of numerous cellular components as well as the synthesisde novo of some molecules normally not expressed in adult neurons. These changes in gene expression activate and regulate the pathwaysresponsible for neuronal survival and axonal regeneration.

Functional decits caused by nerve injuries can be compensated by three neural mechanisms: the reinnervation of denervated targets byregeneration of injured axons, the reinnervation by collateral branching of undamaged axons, and the remodeling of nervous system circuitryrelated to the lost functions. Plasticity of central connections may compensate functionally for the lack of specicity in target reinnervation;plasticity in human has, however, limited effects on disturbed sensory localization or ne motor control after injuries, and may even result inmaladaptive changes, such as neuropathic pain, hyperreexia and dystonia.

Recent research has uncovered that peripheral nerve injuries induce a concurrent cascade of events, at the systemic, cellular and molecularlevels, initiated by thenerve injuryand progressing throughout plasticchanges at the spinal cord, brainstemrelay nuclei, thalamus andbrain cortex.Mechanisms for these changes are ubiquitous in central substrates and include neurochemical changes, functional alterations of excitatory andinhibitory connections, atrophy and degeneration of normal substrates, sprouting of new connections, and reorganization of somatosensory andmotor maps. An important direction for ongoing research is the development of therapeutic strategies that enhance axonal regeneration, promoteselective target reinnervation, but are also able to modulate central nervous system reorganization, amplifying those positive adaptive changes thathelp to improve functional recovery but also diminishing undesirable consequences.# 2007 Elsevier Ltd. All rights reserved.

Keywords: Axonal regeneration; Peripheral nerve; Nerve injury; Neuroplasticity; Neurorehabilitation; Cortical reorganization; Spinal cord

www.elsevier.com/locate/pneurobioProgress in Neurobiology 82 (2007) 163–201

Abbreviations: 5-HT, serotonin; ATF, activating transcription factor; BDNF, brain-derived neurotrophic factor; CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; ChAT, choline acetyltransferase; CMAK, calcium/calmodulin-dependent kinase; CNS, central nervous system; CNTF, ciliary neurotrophic factor;CREB, cAMPresponsiveelement binding protein;DRG,dorsal rootganglia; EPSP, excitatory postsynapticpotential;FGF,broblast growth factor; fMRI,functionalmagnetic resonance imaging; GAP, growth-associated protein; GDNF, glial cell line-derived neurotrophic factor; IB4, isolectin B4; IEG, immediate early gene; IGF,insulin-like growth factor; IL, interleukin; JNK, c-jun N-terminal kinase; LIF, leukemia inhibitory factor; LTP, long-term potentiation; LTD, long-term depression;MAPK, mitogen-activated protein kinase; MN, motoneuron; NFkB, nuclear factor kappa B; NGF, nerve growth factor; NO, nitric oxide; NPY, neuropeptide Y; NT,neurotrophin; PAS, paired associative stimulation; PET, positron emission tomography; PK, protein kinase; STAT, signal transducer and activator of transcription;TMS, transcranial magnetic stimulation; TTX, tetrodotoxin; VIP, vasoactive intestinal polypeptide; VPM, ventral-posterior medial nucleus of the thalamus; VPL,ventral-posterior lateral nucleus of the thalamus

* Corresponding author at: Faculty of Medicine, Universitat Auto `noma de Barcelona, E-08193 Bellaterra, Spain. Tel.: +34 935811966; fax: +34 935812986.E-mail address: [email protected] (X. Navarro).

0301-0082/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pneurobio.2007.06.005

mailto:[email protected]://dx.doi.org/10.1016/j.pneurobio.2007.06.005http://dx.doi.org/10.1016/j.pneurobio.2007.06.005mailto:[email protected]


2/39

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162. Cellular and molecular bases of peripheral nerve regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653. Neuronal survival and reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

3.1. Neuronal reaction and chromatolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1674. Genotypic and phenotypic changes in axotomized neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

4.1. Axonal injury signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1684.2. Activation of transcription factors and gene regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1684.3. Changes in neuropeptide expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1694.4. Changes in ion channels in injured neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1714.5. Changes in neuronal excitability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

5. Structural and synaptic plasticity of axotomized neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1735.1. Functional changes in central synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

6. Facilitation of spinal reexes after nerve injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1747. Remodeling of spinal cord circuitry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

7.1. Central sprouting of afferent projections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1767.2. Changes in spinal cord neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1787.3. Changes in intraspinal inhibitory pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

8. Plastic changes and reorganization at cortical and subcortical levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

8.1. Reorganization of somatosensory cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1798.2. Reorganization of motor cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1828.3. Reorganization at subcortical levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1838.4. Mechanisms of cortical and subcortical plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

9. Reshaping CNS plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1. Introduction

Injuries to the peripheral nerves result in partial or total loss

of motor, sensory and autonomic functions conveyed by thelesioned nerves to the denervated segments of the body, due tothe interruption of axons continuity, degeneration of nervebers distal to the lesion and eventual death of axotomizedneurons. Injuries to the peripheral nervous system can result insubstantial functional loss and decreased quality of life becauseof permanently impaired sensory and motor functions andsecondary problems, such as neuropathic pain, and have majorsocial consequences in terms of health care and long periods of sick-leave ( Jaquet et al., 2001; Rosberg et al., 2005 ).

Functional decits caused by nerve injuries can becompensated by three neural mechanisms: the reinnervationof denervated targets by regeneration of injured axons, thereinnervation by collateral branching of undamaged axons inthe vicinity, and the remodeling of nervous system circuitryrelated to the lost functions. However, clinical and experimentalevidences usually show that these mechanisms by themselvesdo not allow for a satisfactory functional recovery, especiallyafter severe injuries ( Sunderland, 1991; Kline and Hudson,1995; Lundborg, 2000a ). After peripheral nerve injuries, thecapabilityof severed axons to regenerate and recover functionalconnections is dependent on the age of the subject, the nervetrunk affected, the site and type of lesion, the type and delay of surgical repair, and the distance over which axons must regrowto span the injury. Thus, if a nerve transection resulting in a gap

between nerve stumps is left unrepaired or repaired with long

grafts, the probability of effective reinnervation of muscle andsensory receptors is poor. It is generally considered that inhumans, for nerve gaps of less than 2 cm neurological recovery

is moderate, but for gaps longer than 4 cm recovery is minimalto non-existent ( Reyes et al., 2005 ). On another hand, collateralreinnervation by undamaged axons is limited to temporal andspatial constraints, especially for large sensory and motor axons(Jackson and Diamond, 1984; Brown et al., 1980 ), and it isusually only helpful to recover protective pain sensibility andmotor strength in partially denervated muscles.

The peripheral and central nervous systems are functionallyintegrated regarding the consequences of a nerve injury: aperipheral nerve lesion always results in profound and long-lasting central modications and reorganization ( Kaas, 1991;Wall et al., 2002; Kaas and Collins, 2003 ). Neuronalconnections along the nervous system play an important rolein regulating the expression of adequate neuronal character-istics, including morphology, dendritic and axonal arborization,membrane electrical properties and production of transmittersand metabolic molecules. The mechanisms of plasticity andreorganization of spinal and brain circuits linked with theaxotomized peripheral neurons are complex; they may result inbenecial adaptative functional changes or contrarily causemaladaptive changes resulting in positive symptoms, such aspain, disesthesia, hyperreexia and dystonia, that worsen thepatient’s clinical outcome. Nowadays, there are no repairtechniques that can ensure the recovery of normal sensorimotorfunctions of an adult patient following severe nerve trauma, and

it is generally considered that a plateau has been reached for the

X. Navarro et al. / Progress in Neurobiology 82 (2007) 163–201164

on

ee

Even pain free patients can have an unhealed nerve (Taylor, 2010)


3/39

renement of surgical repair techniques ( Lundborg, 2000a,2003). Therefore, new strategies that simultaneously potentiateaxonal regeneration, promote selective target reinnervation andmodulate central reorganization are needed. In this review, wefocus on the developed plastic changes that follow peripheralnerve injuries and axonal regeneration, with structural,molecular and functional consequences at the level of thecentral nervous system (CNS), from the injured neuronal cell tothe brain.

2. Cellular and molecular bases of peripheral nerveregeneration

After nerve injuries, axons distal to the lesion site aredisconnected from the neuronal body and degenerate. The somaof axotomized neurons undergoes a series of phenotypicchanges, known as neuronal reaction and chromatolysis.Whereas Wallerian degeneration serves to create a micro-environment distal to the injury site that is favorable for the

axonal regrowth of surviving neurons, neuronal reactionrepresents the metabolic changes necessary for regenerationand axonal elongation. The functional signicance of regeneration is to replace the distal nerve segment lost duringdegeneration, allowing reinnervation of target organs andrestitution of their corresponding functions. The main events

that occur at the peripheral nerve level are summarized next(Fig. 1). For more information, recent reviews can be consulted(see Fawcett and Keynes, 1990; Sunderland, 1991; Fu andGordon, 1997; Verdu´ and Navarro, 1998; Hall, 2001; Makwanaand Raivich, 2005 ).

After injuries that cause rupture of peripheral nerve bers,axons and myelin sheaths distal to the lesion site are degradedby a process of Wallerian degeneration. Degeneration alsoaffects retrogradely a short segment of the proximal nervestump. The degenerative end products are eliminated by thecooperative action of Schwann cells and inltrating macro-phages (Perry and Brown, 1992; Stoll and Mu¨ ller, 1999 ). Therst signs of degeneration are observed within 24 h after nerveinjury, and they are prolonged for about 1–2 weeks following aproximo-distal progression. Elimination of myelin sheathsallows for clearance of regeneration-inhibitory factors asso-ciated to myelin, mainly myelin-associated glycoprotein in theperipheral nerve. Schwann cells rapidly initiate detachment of myelin sheaths after axotomy, probably by activation of

receptor tyrosine kinase erbB2 ( Guertin et al., 2005 ).Denervated Schwann cells are able to phagocyte myelin debristo some extent; however, recruitment of hematogenousmacrophages is the main pathway for phagociting myelinand axonal debris ( Tanaka et al., 1992 ). From 2 to 3 days afterinjury there is an important inltration of macrophages into the

Fig. 1. Schematic of the main events of degeneration and regeneration after peripheral nerve injury. (A) Normal nerve ber, maintaining synaptic contact with targetcells. (B) Transection of the ber results in distal fragmentation of axon and myelin sheaths. Macrophages and Schwann cells phagocyte degraded materials.Chromatolysis at the neuron soma and dendritic arbor retraction occur. (C) Fine sprouts emerge from the proximal axonal end, and elongate in association with theproliferated Schwann cells in the distal segment, that line up in bands of Büngner. (D) Axonal reconnection with target cells and maturation of the nerve ber. Theregenerated axon remains of smaller caliber and with shorter internodes than normal. The neuron returns to a normal transmitting phenotype. Taget cells may suffer

atrophy and phenotypic changes during denervation.

X. Navarro et al. / Progress in Neurobiology 82 (2007) 163–201 165


4/39

degenerating nerve, attracted by cytokines, such as monocytechemoattractant protein 1, leukemia inhibitory factor (LIF) andinterleukin (IL)-1 secreted by reactive Schwann cells ( Tofariset al., 2002). Schwann cells in the distal nerve are stimulated bythe loss of axonal contact, probably through proteins releasedby disintegrating axons ( Karanth et al., 2006 ), and later bycytokines secreted by macrophages, to proliferate after injury.De-differentiated Schwann cells line up within the endoneurialtubes to form the bands of Bü ngner, that later provide supportfor regenerating axons. The highest rate of Schwann cellmultiplication is reached by 3 days after lesion, and thencontinues with decreasing frequency for 2–3 weeks to reach athreefold increase in number ( Salonen et al., 1988 ).

Proximal to the lesion, growth cones emerge from thesevered axons, induced by local factors ( Kato and Ide, 1994 ),and elongate if they nd a favorable terrain. In the absence of aguiding structure, such as the distal nerve stump, regeneratingaxons make a tortuous course and form a neuroma, composedof immature axonal sprouts and connective tissue ( Fried et al.,

1991). If regenerating axons gain the distal nerve, they elongatewithin the endoneurial tubes, in association with the Schwanncell and the basal lamina, constituting regenerative units. Thematerials for axonal growth are mainly provided by the cellbody via axonal transport ( Hoffman and Lasek, 1980;McQuarrie and Lasek, 1989 ), but more recently the contribu-tion of local axonal synthesis and degradation of proteins hasbeen identied ( Verma et al., 2005; Willis et al., 2005 ). The rateof axonal regeneration is initially very slow, and reaches aconstant value by 3–4 days after injury, that is about 2–3 mm/ day. The factors that stimulate and control axonal regenerationoriginate from multiple sources, but the most important

inuences derive from the local environment of the lesion.Axonal elongation requires an adequate substrate of trophic andtropic factors, provided by reactive Schwann cells and theextracellular matrix within the degenerated nerve stump ( Verdúand Navarro, 1998 ).

Several sprouts emerge initially from each parent axon(Witzel et al., 2005 ) and may advance in the distal nerve;thereby the total number of axons in the distal segment mayexceed the number of parent axons in the proximal nerve forlong time ( Jenq and Coggeshall, 1985 ). When axons reachsynaptic loci at peripheral tissue, supernumerary axonal sproutsare withdrawn gradually. Nerve bers that regenerate througherroneous distal pathways to targets that cannot reinnervate,such as motor axons to the skin, are preferentially eliminated(Brushart, 1993; Brushart et al., 1998 ). The functionalsignicance of regeneration is to replace the distal nervesegment lost during degeneration and to reinnervate targettissues. However, the regenerative process usually does notreconstitute a normal nerve structure neither allows for anormal function, especially when the lesion is severe. Afternerve injury and repair, the diameter of regenerated axons, theirconduction velocity and excitability remain below normallevels for a long time ( Fields and Ellisman, 1986a,b ), andconsequently recovery of reinnervated organs is incomplete andoften inadequate. The limitation of nerve regeneration is more

marked when the lesion creates loss of continuity in the nerve,

and the amount of regeneration is lower with increasinginterstump gap length in a given nerve either when leftunrepaired or after surgical repair ( Butı́ et al., 1996; Krarupet al., 2002).

In spite of the fact that peripheral axons can regeneratethrough the injury site towards distal territories, reinnervation of targets does not always lead to adequate recovery of motor andsensory functions. The selectivity of axon-target reconnectionplays an important role in the impairment of function after nerveinjury and regeneration. Target organ specicity, or adequatereinnervation of each type of end organ (muscle, sensoryreceptor, etc.) by their original axons is far from perfect afternerve transection even if rened repair is applied ( Molander andAldskogius, 1992; Bodine-Fowler et al., 1997; Valero-Cabre ´et al., 2004), although preferential motor reinnervation has beenobserved ( Brushart, 1993 ). Cell adhesion molecules differen-tially expressed by motor or sensory Schwann cells may play alimited role for preferential guidance of regenerating axons inadult animals ( Martini et al., 1992; Eberhardt et al., 2006 ), but

later improvement in target specicity is the result of progressive withdrawal or pruning of misdirected axons, whichsuffer retrograde atrophy and degeneration when no appropriatedistal reconnection is established.

Factors contributing to poor long-term functional recoveryafter peripheral nerve injuries include: (1) Damage to theneuronal cell body due to axotomy and retrograde degenera-tion, excluding the possibility of regeneration. (2) Inability foraxonal growth due to the nerve lesion or to underlying diseases.Laceration of the nerve with an associated tissue gap or distaldegeneration due to generalized neuropathy may both impederegeneration. (3) Poor specicity of reinnervation by regen-

erating axons, when target organs become reinnervated bynerve bers with different function. Aberrant reinnervation ispronounced during regeneration over long distances. Plasticityof central connections may compensate functionally for thislack of specicity; in man plasticity has, however, limitedeffecton disturbed sensory localization or ne motor control afterinjuries.

3. Neuronal survival and reaction

The success of nerve regeneration and functional reinnerva-tion of targets depends at a rst instance on the capacity of axotomized neurons to survive and shift towards a regenerativephenotype. One of the sequelae that follow transection of aperipheral nerve is the death of a number of axotomizedneurons. The proportion of neuronal death among sensoryneurons of the dorsal root ganglia (DRG) after sciatic nerveinjury has been reported between 10 and 30%, affecting moresmall than large neurons ( Arvidsson et al., 1986; Ygge, 1989;Vestergaard et al., 1997; Groves et al., 1999; Tandrup et al.,2000). Regarding death of motoneurons, a non-signicant lossof 0–10% has been found after sciatic nerve injury ( Vanden-Noven et al., 1993; Lowrie et al., 1994; Valero-Cabre ´ et al.,2001). In contrast, avulsion or transection of ventral roots in theadult leads to retrograde cell death of 50–80% of motoneurons

in a few weeks (Koliatsos et al., 1994; Martin et al., 1999;



5/39

Natsume et al., 2002; Hoang et al., 2003 ). Sympathetic neuronsin the superior cervical ganglion undergo dramatic changesafter cutting the postganglionic nerves, with a neuronal loss of about 50% in 3 days ( Hou et al., 1998 ). A 40–60% decrease inthe population of preganglionic sympathetic neurons in theintermediolateral nucleus of spinal cord was found aftersurgical transection of the cervical sympathetic trunk ( Tang andBrimijoin, 2002 ). Neuronal death postaxotomy depends onseveral factors, mainly including age, severity of injury andproximity of injury to the cell body. Neurons in the adult areless susceptible to die than immature neurons ( Snider et al.,1992; Lowrie et al., 1994 ), whereas lesions near the cell bodiescause a higher proportion of neuronal death than distal lesions(Ygge, 1989 ). The process of neuronal death is prolonged andmore severe if axonal regeneration is prevented ( Törnqvist andAldskogius, 1994 ). The loss of motoneurons reaches signicantlevels, about 50%, in chronic human amputees ( Kawamura andDyck, 1981 ). Neuronal vacuolation and apoptotic deathfollowing nerve injury has a progressive increase during the

rst month and then a gradual decline over a period of 6 monthsin the rat (Groves et al., 1997, 1999 ).

In past years much attention focused on the role of neurotrophic factors in the maintenance and survival of neuronsand in promoting axonal regeneration after nerve injury (forreviews, see Persson and Ibá ñez, 1993; Terenghi, 1999; Markuset al., 2002; Boyd and Gordon, 2003 ). After peripheral nerveinjury, the adult peripheral nervous system responds byincreasing the availability of neurotrophic factors, either byautocrine or paracrine sources. Additional exogenous supply of neurotrophic factors may enhance the regenerative response of peripheral neurons. The spectrum of neurotrophic factors

reported to stimulate axonal regeneration includes nerve growthfactor (NGF), brain-derived neurotrophic factor (BDNF),neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5), insu-lin-like growth factors (IGF-I and IGF-II), ciliary neurotrophicfactor (CNTF), basic broblast growth factor (FGF-2) and glialcell line-derived neurotrophic factor (GDNF). Thus, it may bepostulated that the potentiation of neuronal reaction andregeneration can be better explained by a complex mixture of different neurotrophic factors and neurotropic molecules ratherthan an individual component, especially considering thevariable dependency of different neuronal populations onneurotrophic factors.

3.1. Neuronal reaction and chromatolysis

If Wallerian degeneration serves to create a microenviron-ment distal to the injury that favors axonal regrowth of surviving neurons, the neuronal retrograde reaction representsthe metabolic changes necessary for regeneration.

The most consistent morphological changes in the neuronalbody after axotomy are dissolution of the Nissl bodies(chromatolysis), nuclear eccentricity, nuclear and nucleolarenlargement, cell swelling, and retraction of dendrites ( Lieber-man, 1971; Kreutzberg, 1995 ). The disappearance of theprominent basophilic-stained Nissl granules is particularly

evident. These granules are ribosome clusters and ordered

arrays of rough endoplasmic reticulum, which are no longerobserved after axotomy, when they become disorganized,freeing polyribosomes and ribonucleotides into the cytoplasm.The intensity and the time course of the neuronal response aremainly inuenced by severity of the injury, distance of lesion tocell body, type of neuron, and age. Quantitative image analysisrevealed that chromatolysis started signicantly as early as 8 hfollowing cranial nerves section and was not fully reversed by 3months after axotomy. The reaction was more intense andlonger lasting following axotomy without reinnervation thanwith reinnervation ( Guntinas-Lichius et al., 1996 ). Markedchromatolysis was found in motoneurons after nerve avulsionand eventually some of them underwent apoptotic death,suggesting that a neuronal response continuum may exist afteraxotomy beginning as chromatolysis and evolving into eithersurvival and regeneration or apoptosis of neurons ( Martin et al.,1999). The transition between chromatolysis and apoptosiscoincided with accumulation of metabolically active mito-chondria within the perikaryon and oxidative damage tonucleic

acids and proteins in axotomized neurons. On another hand,when their axons are able to effectively regenerate andreinnervate targets, neurons slowly return to a normal functionand morphology. Thus, spinal motoneurons that regeneratedtheir axons showed a normal distribution and morphologicalproperties at 5 months after sciatic nerve crush in the rat.Nevertheless, by 10–12 months these motoneurons exhibitedseveral morphological alterations, such as cell body enlarge-ment, dendritic thickening and clustering in the ventral horncompared with the contralateral side ( Bowe et al., 1992 ). Thosealterations likely represent delayed and progressive changes inthe neurons that successfully reinnervated peripheral targets.

Disorganization of ribosome clusters is related to increasedprotein synthesis, suggesting that chromatolysis represents themorphological changes in the injured neuron soma associatedwith an anabolic response ( Lieberman, 1971; Fu and Gordon,1997). This interpretation is supported by biochemical changesin axotomized neurons, showing reduced DNA repression,increased RNA synthesis with nuclear RNA transferred to thecytoplasm, and increased cellular protein content ( Watson,1974). Biochemical changesdevelopwithin hours afteraxotomy,coincident with the early chromatolytic morphological changes.Theconcentration ofenzymesof theoxidativepentosephosphateshunt, required for RNA synthesis, is also raised ( Härkönen andKauffman, 1974 ), whereas production of neurotransmitters andcytoskeleton proteins is decreased ( Frizell and Sjö strand, 1974;Heiwell et al., 1979; Hoffman andLasek, 1980 ). The axotomizedneurons shift from a ‘‘transmitting’’ state to a ‘‘regenerative’’state, underlied by prominent changes in gene expression,whichlead to a decrease in the synthesis of neurotransmission-relatedproducts and an increased synthesis of growth-associatedproteins and structural components of the membrane.

4. Genotypic and phenotypic changes in axotomizedneurons

Injury to neurons results in complex sequences of molecular

responses that play an important role in the successful



6/39

regenerative response and the eventual recovery of function. Inthe axotomized neurons, the rapid arrival of injury-inducedsignals is followed by the induction of transcription factors,adhesion molecules, growth-associated proteins and structuralcomponents needed for axonal regrowth.

4.1. Axonal injury signaling

Signals responsible for the initiation and maintenance of the regenerative neuronal response include a variety of mechanisms acting at sequential time phases ( Cragg, 1970;Ambron and Walters, 1996; Perlson et al., 2004; Hanz andFainzilber, 2006 ). During seconds after a nerve lesion, theaxoplasm of lesioned axons is in continuity with theextracellular medium before plasmatic membrane is sealed.The rapid inux of calcium and sodium ions causes electricalresponses that propagate retrogradely. The rst signal to reachthe neuronal body after axonal injury is thus a high frequencyburst of action potentials generated at the lesion site; this

discharge promotes an inux of calcium through voltage-dependent ion channels, that can contribute to activate severalprotein kinase pathways, including calcium/calmodulin-dependent kinase 2 (CMAK2), protein kinase (PK) A, PKC,and mitogen-activated protein kinase (MAPK) ( Ghosh andGreenberg, 1995 ). In addition, the intense excitation causesthe release of transmitters, such as glutamate, neuropeptidesand BDNF at the spinal cord. These in turn may activate alsoPKA, PKC and MAPK.

Later on, a second set of signals are conveyed by retrogradeaxonal transport, these include the early deprivation of target-derived trophic factors ( Raivich et al., 1991; Lee et al., 1998 )

and the arrival of activating signals from the own injured axonsand from non-neuronal cells. Pioneering work on Aplysia(reviewed in Ambron and Walters, 1996; Perlson et al., 2004 )and more recent ndings in mammals ( Hanz and Fainzilber,2006) has led to renewed interest on activated proteinsemanating from the injury site during the second phase of signaling. These activated proteins, termed ‘‘positive injurysignals’’, are endogenous axoplasmic proteins that undergopost-translational modications at the lesion site uponaxotomy, and then incorporate to the retrograde transportsystem for trafcking to the cell body. In addition, axonalprotein synthesis is needed for growth cone formation andmaintenance of the regrown axon ( Willis and Twiss, 2005 ).Several transcription factors have also been identied inperipheral axons and may participate in positive retrogradesignaling ( Lee et al., 2004; Lindwall and Kanje, 2005; Agthonget al., 2006 ). Recent works suggest that this retrograde transportrequires the local synthesis of carrier proteins, includingimportins and vimentin, that interact with the retrogradetransport motor dynein ( Hanz et al., 2003). During thefollowing days inltrating macrophages and reactive Schwanncells in the degenerating nerve release cytokines andneurotrophic factors that reach the injured neuronal body bya general increase of retrograde transport ( Curtis et al., 1993,1994, 1998 ), and would contribute to sustain the regenerative

program of the neuron.

4.2. Activation of transcription factors and gene regulation

The axonal signals induced in response to nerve injuryactivate several signaling pathways genes in neuronal cellbodies that may lead to two opposing consequences: cell deathor regenerative response. Amongst these are some kinases suchas the MAPKs Erk1 and Erk2, c-jun N-terminal kinase (JNK)and p38 kinase. Following axotomy, Erk activation has beenobserved in transected sciatic nerve and DRG ( Sheu et al.,2000; Obata et al., 2004a ). The increased activity of Erk isinuenced by endogenous neurotrophic factors such as NGFand GDNF( Wiklund et al., 2002 ). JNKis rapidly activated afternerve injury and persists elevated for weeks until eitherneuronal death or axonal regeneration occurs ( Kenney andKocsis, 1998; Herdegen and Waetzig, 2001 ). Inhibition of JNK rescued in vitro motoneurons from death by withdrawal of trophic factors ( Maroney et al., 1998 ). Recently, an effectivecross-talk between the Erk1/2 and JNK pathways coordinatedby MEKK1 has been shown to participate in neurite regrowth in

PC12 cells ( Waetzig and Herdegen, 2005 ). Similarly, p38 istransiently activated in axotomized neurons ( Murashov et al.,2001) and in small-to-medium DRG neurons after chronicconstriction injury ( Obata et al., 2004b ). The expression of p38may interfere with mitotic arrest but its role on neuritegrowth issmaller ( Herdegen and Waetzig, 2001 ). Moreover, inhibition of p38 leads to enhanced axonal regeneration ( Myers et al., 2003 ).

Downstream events inuenced by axotomy-activatedkinases include up-regulation or activation of several transcrip-tion factors. Activated JNK induces up-regulation andphosphorylation of the transcription factors c-Jun, JunD andFos into the nucleus, leading to formation of AP-1 complexes

that activate many downstream genes ( Kenney and Kocsis,1998; Raivich and Behrens, 2006 ). The expression of immediate early genes (IEGs) after axotomy has been shownto be induced according to the nerve and the type of injury(Herdegen et al., 1992; Haas et al., 1993; Kajander et al., 1996;Kenney and Kocsis, 1997 ). After sciatic nerve lesions in rats,there is a substantial increase in c-jun protein in DRG cells atearly (3 h) (Kenney and Kocsis, 1997 ) and later (24 h) stages(Jenkins and Hunt, 1991 ), compatible with biphasic activationby axonal signals and by loss of target tissue-derived NGF.Neuronal deletion of c-jun hinders the expression of regenera-tion-associated genes, reduces the speed of target reinnervationand also completely blocks central axonal sprouting ( Raivichet al., 2004). The activation of transcription factor cAMPresponsive element binding protein (CREB) in the early stagesafter injury is mediated by multiple protein kinase pathwaysand some of them (PKA, PKC and CMAK2) regulate CREBphosphorylation partially through the Erk pathway. Phosphor-ylation of CREB in the rat spinal dorsal horn is induced byneurotrophins, such as BDNF, and reduced by blocking tyrosinekinase receptors (trk) ( Miletic et al., 2004; Miyabe and Miletic,2005). Another transcription factor affected by nerve injury isactivating transcription factor (ATF)-3. ATF-3 is induced in allDRG neurons after peripheral, but not central axotomy, whatmakes it a reliable nerve injury marker ( Tsujino et al., 2000 ),

and its pattern of induction correlates with the increased



7/39

sensory axon regeneration promoted by a conditioning lesion(Seijffers et al., 2006 ). ATF-3 expression in subpopulations of DRG cells can be modulated by exogenous trophic factors,suggesting that its expression is induced by the loss of target-derived NGF and GDNF ( Averill et al., 2004 ). The members of the signal transducer and activator of transcription (STAT) genefamily are the main mediators in the signal transductionpathway of cytokines. After phosphorylation, STAT proteinsare transported into the nucleus and produce transcriptionalactivity. STAT3 probably acts as a retrograde signalingtranscription factor promoting the survival and regenerationof both sensory and motor neurons after nerve injury ( Lee et al.,2004). Following axotomy in rat facial and hypoglossal nerves,a signicant increase of STAT3 protein was found. In addition,activation of STAT3 by phosphorylation was observed within3 h in neurons and persisted for more than 3 months ( Schwaigeret al., 2000). The increased axonal regeneration rate of DRGneurons and the improved axonal growth in the spinal cord aftera conditioning injury requires also the activation of STAT3 ( Qiu

et al., 2005 ). The PI3K-Akt pathway, which is a growth factorreceptor signaling pathway, is probably vital for neuronalsurvival after injury. Aktactivation was enhanced in response tohypoglossal nerve injury in the adult (surviving neurons),whereas its activity was down-regulated in injured neonatemotoneurons (dying neurons). In addition, it enhanced axonalregeneration in vivo ( Namikawa et al., 2000 ). The transcriptionfactor nuclear factor kappa B (NF kB) has been dened as aneuronal survival signal due to its up-regulation in mousedorsal horn neurons in response to peripheral nerve transection(Pollock et al., 2005 ). NFkB has been suggested to be amediator in the regulation of the glutamate transporter EAAC1

expression by glucocorticoid receptors ( Wang et al., 2006 ). Incontrast, NF kB expression was decreased after nerve crush in Aplysia (Povelones et al., 1997 ).

The modications in the activity of transcription factorsresult in characteristic changes of gene expression in the injuredand regenerating neurons. The introduction of gene chip arraytechnologies has led to a dramatic increase in the identiednumber of regulated genes in neurons and in associated glialcells following several types of peripheral nerve injuries(Costigan et al., 2002; Hu et al., 2002; Kubo et al., 2002; Xiaoet al., 2002; Cameron et al., 2003; Schmitt et al., 2003;Boeshore et al., 2004; Vogelaar et al., 2004; Bosse et al., 2006;Del Signore et al., 2006; Yang et al., 2006 ). Hundreds of geneshave been found either up- or down-regulated by axotomy, butthe function of the majority of them remains unknown. Geneexpression changes affect the encoding of transcription factors,cytoskeletal proteins, cell adhesion and guidance molecules,trophic factors and receptors, cytokines, neuropeptides andneurotransmitter synthesizing enzymes, ion channels, andmembrane transporters. Table 1 summarizes relevant modica-tions in gene and protein expressions at the neuronal leveldescribed following sciaticnerve lesion in adult murine models.More detailed analysis of this issue is out of the scope of thisreview.

One of the prominent changes after peripheral axotomy

consists in the increase of regenerative-associated proteins,

amongst them, the most characterized is growth-associatedprotein (GAP)-43 ( Skene, 1989 ) that is up-regulated from therst day after axonal lesion, and is usually labeled to identifygrowing axonal proles. GAP-43 is rapidly transported alongthe axon to the growth cones, where the protein accumulates.Axonal regeneration is characterized by a distinct regulation of cytoskeletal proteins. Tubulins are signicantly increased inaxotomized motor and sensory neurons whereas neurolamentproteins NF-L, NF-M and NF-H are decreased, probablyreecting an increase in axoplasm uidity in order to facilitateaxonal transport ( Hoffman et al., 1987; Wong and Oblinger,1990; Tetzlaff et al., 1996 ). The reduced axonal caliber andimpulse conduction velocity have been correlated with thetransport of neurolaments in the regenerated nerve bers.Molecules associated with both anterograde and retrogradeaxonal transport, such as kinesin light chains and dynein, areup-regulated in their expression during nerve regeneration ( Suet al., 1997).

The main studies on neuronal injury and regeneration

programs have followed analyses of proteins associated withaxonal growth and cytoskeleton components, growth factors,the expression of specic regulated proteins such asneuropeptides and neurotransmitter systems, or the changesin expression of ion channels and receptors potentially involvedalso in the development of neuropathic pain (see reviews byBosse et al., 2001; Goldberg, 2003; Makwana and Raivich,2005; Hö kfelt et al., 2006; Zhou and Snider, 2006 ).

4.3. Changes in neuropeptide expression

One of the most relevant changes in the neuronal phenotype

after axotomy is the down-regulation of neurotransmitters andgenes encoding transmitter-related proteins. However, there arealso marked changes in the expression of neuropeptides in theaxotomized neurons and in their axonal projections, as well asin the spinal cord ( Table 2 ). This is in agreement with thegeneral view of the reaction of neurons in response to axotomy,when the synthetic machinery of the neuron is reprogrammedfrom transmitter synthesis to production of molecules of importance for survival and regeneration. A characteristic of neuropeptides is the plasticity in their expression, reecting thefact that release has to be compensated by de novo synthesis atthe neuronal body. Neuropeptides may exert different actionsand contribute to neural signaling when the nervous system isstressed or injured ( Palkovits, 1995; Hö kfelt et al., 2000 ).

In axotomized motoneurons, the most characteristic changesinvolve the opposite changes in choline acetyltransferase(ChAT) and calcitonin gene-related peptide (CGRP) immunor-eactivity ( Calderó et al., 1992; Borke et al., 1993; Piehl et al.,1993). Maximal reduction in ChAT immunostaining occurredat 7 days after nerve damage and the amount of the decreasewas related to the severity of the injury. The recovery of ChATto normal levels was related to the timing of reinnervation afternerve crush, but not after transection or resection injuries. Incontrast, a rapid increase in CGRP immunoreactivity, maximalat 3–10 days after lesion, preceded the decrease in ChAT levels.

Later changes were dependent on the type of injury. Increased


In Cthe

musstres

(injur

th

coalso

incrin

supp


8/39

CGRP staining persisted longer after nerve transection thanafter crush and was still evident when nearly completereinnervation was achieved. These observations suggest thatdifferent mechanisms regulate ChAT and CGRP in response tonerve injury. Transient up-regulation of galanin, vasoactive

intestinal polypeptide (VIP) and substance P mRNAs has been

observed within subpopulations of motoneurons ipsilateral tonerve lesion for periods lasting 2–3 weeks after injury ( Zhanget al., 1993a ).

Sympathetic neurons also exhibit a notable plasticity in theirneuropeptide phenotype after injuries in adult animals

(Zigmond, 1997 ). Sympathetic neurons that normally express

Table 1Summary of the changes in the molecular expression in axotomized neurons after peripheral nerve injury

Lesion Location References

Neurotrophic factors/receptorsBDNF " Transection DRG Kashiba and Senba (1999)

" CCI DRG Obata et al. (2003)" Crush DRG Ernfors et al. (1993)" Ligation DRG Fukuoka et al. (2001)" Transection MN Gu et al. (1997)" Crush DRG Tonra et al. (1998)" Spinal l igation DRG Shen et al. (1999)

Trk = Crush DRG Ernfors et al. (1993)

Ret (GDNF receptor) " Transection MN Hammarberg et al. (2000)= Transection DRG Bennett et al. (2000)

GFRalpha2 # Transection DRG Bennett et al. (2000) , Hoke et al. (2000)GFRalpha1, GFRalpha3 " Transection DRG Bennett et al. (2000)Ret and GDNFalpha1 " Crush DRG/MN Naveilhan et al. (1997)

NT-3 = Spinal ligation DRG Shen et al. (1999)= Transection SC Funakoshi et al. (1993)

trkB " Transection MN Hammarberg et al. (2000)" Crush SC/DRG Ernfors et al. (1993)

NGF = Transection MN Gu et al. (1997)" Spinal l igation DRG Shen et al. (1999)

NGF-R " Crush MN Ernfors et al. (1989)16 kDa pancreatitis-associated protein (Reg-2) " Transection DRG Averill et al. (2002)

Transcriptional factorsc-fos " Transection MN Gu et al. (1997)

" Transection DH Kajander et al. (1996)" CCI DH Kajander et al. (1996) and Ro et al. (2004)

c-jun " Transection DRG Jenkins and Hunt (1991) , Broude et al. (1997)and Kenney and Kocsis (1997)

" Crush MN Jenkins and Hunt (1991)

Signal transd. and activ. of transc. 3 (STAT3) "(A) Transection DRG Qiu et al. (2005)Activating transcription factor 3 (ATF-3) " Transection MN/DRG/DH Tsujino et al. (2000)NFkB " Transection DH Pollock et al. (2005)

" (A) PNSL/CCI DRG Ma and Bisby (1998)

Isl 1 " Crush DRG Vogelaar et al. (2004)DRG11, LmX1b, Pax3 = Crush DRG Vogelaar et al. (2004)

OthersTNF-alpha receptor 1 (p55) " Crush DRG Ohtori et al. (2004)Nectin-3 " Transection MN Zelano et al. (2006)P38 mitogen-activated protein kinase (MAPK) "(A) CCI DRG Obata et al. (2004a)Monocyte chemoattractant protein-1 (MCP-1) " CCI DRG/MN/DH Zhang and De Koninck (2006)Fibroblast growth factor 2 (FGF-2) " Ligation DRG Madiai et al. (2003)Glutamate transporter EAAC1 # CCI DH Wang et al. (2006)cAMP response element binding protein (CREB) "(P) CCI DH Miletic et al. (2004)

All the injuries were performed on the sciatic nerve in murine adult animals. The changes indicated are mainly based on studies on mRNA expression orimmunoreactiviy; some are based on phosphorylation (P) or activity (A). Measurements were performed in sensory neurons of dorsal root ganglia (DRG) ormotoneurones (MN) of the ventral horn; in some cases, determinations were described as expression in dorsal horn (DH) or spinal cord (SC). Note that for eachmolecule, references are grouped according to type of injury, as in some cases the expression varies depending on the injury model or the cell type. The arrows ( " :increase; #: decrease) represent up- or down-regulation.



9/39

neuropeptide Y (NPY) show a reduced expression of NPYandtyrosine hydroxylase after axotomy, while they overexpressVIP, galanin and substance P. The NPY, VIP and galaninexpressions are regulated by both the removal of target-derived

NGF and the exposure to LIF, whereas the expression of substance P after axotomy occurs primarily because of theeffects of LIF ( Zigmond and Sun, 1997; Shadiack et al., 2001 ).

Primary sensory neurons change their phenotype both withregard to messengers, receptors and function after peripheralnerve injury, so that DRG neurons adapt to the new situation bysuppressing excitatory transmitters, enhancing inhibition andpromoting survival and regenerative mechanisms. Particularly,substance P and CGRP are present in many small DRG neuronsunder normal circumstances. They contribute to synaptictransmission in the dorsal horn, in addition to efferent effects inthe periphery (vasodilation, permeability). Substance P and

CGRP are markedly down-regulated after axotomy ( McGregoret al., 1984; Nielsch et al., 1987; Noguchi et al., 1990; Villaret al., 1991), a phenomenon that is dependent upon interruptionof target-derived NGF to the neuronal body ( Eriksson et al.,1997). However, substance P expression increases in A largeneurons ( Noguchi et al., 1995 ) and there are controversialresults regarding the total content and redistribution of substance P in DRG and dorsal horn after partial injuries(Cameron et al., 1997; Ma and Bisby, 1998; Swamydas et al.,2004).

Other peptides such as VIP, peptide histidine-isoleucine,galanin and NPY are normally expressed at low levels insensory neurons, but are dramatically increased after axotomy(McGregor et al., 1984; Shehab and Atkinson, 1986; Villaret al., 1989, 1991; Wakisaka et al., 1991 ). These changes seemto be involved in dorsal horn regenerative and/or compensatoryprocesses following peripheral nerve damage. Under normalconditions, galanin occurs in a small population of small sizesensory neurons in the DRG as well as in spinal dorsal horninterneurons. However, following peripheral nerve injury,expression of galanin in primary afferents and terminals in thesupercial dorsal horn has been found to be up-regulated(Wiesenfeld-Hallin and Xu, 1998 ). Galanin synthesis ispromoted by increased production of LIF after injury, aneffect that may be mediated by interleukin-6 ( Murphy et al.,

1999). In parallel, galanin-R1 receptor mRNA was down-

regulated in DRG neurons after axotomy, and a small decreasein galanin-R2 receptor mRNA levels was also seen ( Zhanget al., 1998). It has been suggested that under normal conditionsgalanin exerts tonic inhibition of nociceptive input to the centralnervous system. After peripheral nerve injury the inhibitorycontrol exerted by galanin, released from DRG neurons, isincreased, acting on galanin-R1 receptors at the dorsal horninterneurons ( Hökfelt et al., 2006 ). In normal DRG neuronsNPY is only barely detected, whereas NPY Y1-receptor isstrongly expressed at the cell soma membrane and Y2-receptormRNA levels are low. After axotomy there is a marked increaseof NPY in large and medium DRG neurons, in their centralprojections to dorsal column nuclei, as well as in some dorsalhorn neurons ( Wakisaka et al., 1991; Ohara et al., 1994 ). NPYreceptors show a complex regulation; there is a decrease in Y1-receptor and an increase in Y2-receptors that are transportedcentrifugally. Interestingly, after sciatic nerve axotomy, thedeletion of Y1-receptor protected knockout mice from theaxotomy-induced loss of DRG neurons and increased their

release and synthesis of CGRP and substance P ( Shi et al.,2006). Immunoreactivity to NPY is also present in theterminations of A b afferent bers in the nuclei gracile andcuneate, where it may mediate changes in mechanosensorysecond-order neurons ( Ossipov et al., 2002 ). These ndingssuggest that NPYergic mechanisms participate in the adaptivechanges of sensory neurons in response to injury ( Hökfelt et al.,1998). Cholecystokinin (CCK) reduces the antinociceptiveeffect of opioids. The levels of CCK and CCK receptors, as wellas CKK release, exhibit considerable plasticity early after nervelesion and inammation ( Zhang et al., 1993b; Afrah et al.,2001; Wiesenfeld-Hallin et al., 2002 ). In particular, neuropathic

pain after injury to the peripheral and central nervous systemdoes not respond well to opioids, which has been attributed toincreased activity in the endogenous CCK system.

4.4. Changes in ion channels in injured neurons

Axotomy leads to the focal accumulation of anterogradelytransported vesicles containing proteins, including ion chan-nels. Some of these may insert in the membrane of the injuredaxons, increasing the density of channels and altering the localexcitability properties. Voltage sensitive channels, particularlyfor sodium and potassium, have been shown to accumulate inneuroma formed after nerve ligation in animal models and inhumans ( Devor et al., 1993; England et al., 1994, 1998;Kretschmer et al., 2002 ). Axotomy also triggers plastic changesin the expression of several ion channels, transducers andreceptors in the injured neurons. The altered gene expression isrelated with the modications of neuronal phenotype, includingelectrical hyperexcitability. Neurotrophic signaling andimpulse trafc appear to be key factors in ion channelregulation. In fact, delivery of exogenous neurotrophins is ableto prevent or modify the lesion-induced neuronal phenotypicchanges.

Following injury to their axons within peripheral nerve,DRG neurons down-regulate some sodium channel genes and

up-regulate others. As a result, a different repertoire of sodium

Table 2Characteristic changes in the expression of neuropeptides in neurons afteraxotomy

Neuropeptide Motoneurons Sympatheticneurons

Sensoryneurons

ChAT # –a –CGRP " – #CCK " / # " " / #Galanin " " "NPY " # "Somatostatin " # "Substance P " # "VIP " " a "

a Decrease of ChATand VIP in the subpopulation of sudomotor sympatheticneurons.



10/39

channels is inserted into the neuron membrane following injury,a molecular change that contributes to hyperexcitability inthese cells. Following nerve injury, it has been found that theexpression of tetrodotoxin (TTX) sensitive sodium channelsNav1.3, normally expressed only during development, is up-regulated in primary sensory neurons, whereas channelsNav1.1, Nav1.2, Nav1.6, Nav 1.7, Nav 1.8 and Na v 1.9 aredown-regulated ( Waxman et al., 1994; Dib-Hajj et al., 1996;Sleeper et al., 2000; Xiao et al., 2002; Lai et al., 2003 ). Thesendings have deserved considerable interest as potentialcontributors to the genesis and maintenance of neuropathicpain post-injury (for reviews, see Waxman et al., 2000; Woodet al., 2004). Similar, but less extensive changes in sodiumchannels have been observed after partial injury models of neuropathic pain ( Dib-Hajj et al., 1999 ). Interestingly, thosechanges in ion channels expression do not occur aftertransection of the central axon of sensory neurons. Sodiumcurrents in injured neurons are concomitant with changes ingene expression after axonal transection. The slowly inactivat-

ing and the persistent TTX-resistant sodium currents producedby the Nav 1.8 and Na v1.9 channels are attenuated, whereas arapidly repriming sodium current produced by the newlysynthesized Na v1.3 channels, which recovers rapidly frominactivation, emerges within axotomized small DRG neurons(Cummins and Waxman, 1997; Sleeper et al., 2000 ). Thechanges in large sensory neurons with A b bers are slightlydifferent; they express tetrodotoxin-sensitive sodium currents,but with a faster repriming kinetics than in small neurons bothbefore and after axotomy, and also down-regulate thetetrodotoxin-resistant current following nerve section ( Everillet al., 2001). The accelerated recovery from inactivation of the

inappropriately expressed Na v1.3 channels produces a decreasein refractory period that contributes to hyperexcitability of DRG cells following axonal transection. These changes insodium currents persist for at least 60 days after axotomy,consistent with the long-lasting changes in sodium channelgene expression ( Dib-Hajj et al., 1996 ). However, the abnormalexpression of sodiumchannels and their corresponding currentscan be reversed by the intrathecal administration of NGF andGDNF (Lefer et al., 2002 ), and by neuronal activity promotedby electrical stimulation via a mechanism independent of trophic factors ( Klein et al., 2003 ).

Nevertheless, it has been also determined that axotomy-induced changes in sodium currents were not correlated with anaxotomy-induced change of excitability in small DRG neurons(Flake et al., 2004 ), suggesting that concomitant changes inother ionic channels occurred and it is the sum of changes inionic currents what determines neuronal excitability followingaxotomy.

Immunocytochemical investigation on the expression of potassium channels in DRG cells cultured from control andaxotomized adult rats showed that voltage-gated potassiumchannels K v1.2 and 2.1 largely decreased following axotomy,whereas K v 1.1 and 1.3 had smaller decreases, and K v 1.4 and 1.6were not altered by axotomy ( Ishikawa et al., 1999 ). Largeafferent neurons show also a reduction in overall potassium

currents after nerve ligation ( Everill and Kocsis, 1999 ), and all

types of sensory neurons show a reduction in delayed rectierpotassium current and Ca 2+ -sensitive potassium conductance(Abdulla and Smith, 2001a ). These results indicate that, inrelation to changes in sodium channel expression, there aredecreases in specic potassium channels following axotomythat may lead to changes in electrical excitability of the DRGneurons. At the peripheral neuroma level, K v1.1 channelsaccumulated in an abnormal distribution, with patches alongthe internodal segments of myelinated axons ( England et al.,1998). It has been hypothesized that such up-regulation of K v 1.1 is an adaptive mechanism to stabilize the injured axonmembrane, which has been made hyperexcitable by thechanges in sodium channels.

Voltage-gated calcium channels modulate nociceptivetransmission at the level of the neuronal synapse in the centralnervous system. The role of the L, N, and P/Q types of calciumchannels varies with the nature of neural injury ( Vanegas andSchaible, 2000 ). One indication of the important role played bythese channels is the dense expression of the N-type channels in

the supercial laminae I and II of the dorsal horn, the site of synapse for nociceptive primary afferent neurons. Withdepolarization, there is an inux of Ca 2+ ions into neuronsand release of neurotransmitters such as GABA, glutamate andnorepinephrine. Nerve injury leads to a reorganization of thehigh-voltage activated calcium current properties in a subset of cutaneous afferent neurons, which show faster inactivationkinetics than control neurons ( Baccei and Kocsis, 2000 ).Studies on peripheral nerve injury models pointed to a distinctpattern of Ca 2+ channel subunit expression, with up-regulationof the a 2d subunit that is correlated with allodynic painbehavior ( Newton et al., 2001 ).

Although less investigated, ion channel expression is alsomodied in motoneurons after axonal lesion. In the axotomizedfacial motoneurons Na v 1.1 mRNA decreased whereas Na v1.3mRNA reappeared ( Iwahashi et al., 1994; Patko et al., 2003 ).Furthermore, Ca 2+ -sensitive potassium channel subunits,small-conductance SK1 and SK3 mRNAs were more abundantin the axotomized facial nuclei motoneurons than in controls.These alterations can partly explain the increase in excitabilityobserved in the axotomizedmotoneurons following facial nervelesion.

4.5. Changes in neuronal excitability

Passive membrane properties, in particular neuronal inputresistance, remain relatively stable in the majority of neuronsafter axotomy. The major exceptions are vertebrate motoneur-ons. Several reports have demonstrated that axotomizedmotoneurons exhibit changes in membrane electrical proper-ties, consisting of increased input resistance and time constant,decreased rheobase and axonal conduction velocity, andvariable changes in afterhyperpolarization duration that short-ens in S motoneurons but is prolonged in F motoneurons ( Kunoet al., 1974; Foehring et al., 1986; Pinter and Vanden Noven,1989; Titmus and Faber, 1990 ). These changes are consistentwith dedifferentiation of motoneuronal properties following

axotomy. In experiments where muscle reinnervation was not



11/39

allowed, these changes were maintained chronically ( Pinter andVanden Noven, 1989 ), whereas following reinnervation normalelectrical properties were recovered ( Foehring et al., 1986 ),suggesting that functional contact with muscle is required forthe full expression of the normal range of neuron electricalproperties. Changes in input resistance appear to be correlatedwith structural simplication of dendritic trees, changes inspecic membrane resistance and with reduction in cell bodysize (Titmus and Faber, 1990 ). In addition, the ring propertiesare also modied in axotomized motoneurons, which show asteeper frequency–intensity curve and a low threshold for thegeneration of spikes in the soma-dendritic membrane ( Takataet al., 1980).

Increases in excitability in all types of DRG sensory neuronswithin weeks of axotomy have been found, althoughelectrophysiological changes appear to vary among types of neurons and with the type of injury. In large and medium A-cells axotomy induced by sciatic nerve section or spinal nerveligation causes a reduction of excitation threshold, an increase

in duration and in rise time of the action potential, but nosignicant changes in conduction velocity and adaptationproperties. On the other hand, in small C-cells axotomy reducedaction potential threshold but did not signicantly changeresting membrane potential, action potential duration, ormaximal depolarization rate ( Gurtu and Smith, 1988; Koerberet al., 1995; Zhang et al., 1997; Kim et al., 1998; Abdulla andSmith, 2001b; Abdulla et al., 2003; Ma et al., 2003 ). Inaddition, nerve injury led to a signicant reduction of therheobase, an index of neuronal excitability, in all types of cells.It has been suggested that axotomy would increase theexcitability of DRG neurons not by altering input resistance or

threshold potential, but by increasing apparent input resistancenear the resting membrane potential in A-cells and bydecreasing the resting membrane potential in C-cells ( Kimet al., 1998 ). The incidence of spontaneous activity, recordedextracellularly from dorsal root bers, was signicantly higherafter nerve lesions than in sham animals, and occurred in alltypes of DRG neurons ( Zhang et al., 1997; Kim et al., 1998;Abdulla and Smith, 2001b; Ma et al., 2003 ). There is somecontroversy on whether the nerve injury-induced electricalinstability is restricted to axotomized neurons ( Sapunar et al.,2005) or extends also to adjacent intact neurons that maycontribute to hyperalgesia ( Ma et al., 2003). The electro-physiological changes promoted by axotomy were more intensein animals that exhibited signs of neuropathic pain, andinterestingly the onset of autotomy coincided with alterations inthe excitability of large, putative non-nociceptive neurons(Abdulla and Smith, 2001b; Ma et al., 2003 ). Nevertheless,changes in the electrical properties of cell bodies alone may notentirely explain the increased spontaneous activity and thesigns of neuropathic pain after injuries to sensory nerves.

Axotomy produces signicant changes in several measur-able electrophysiological parameters in sympathetic ganglioncells, although mature neurons are able to maintain relativelynormal electrical activity despite injury. Thus, restingmembrane potential or input resistance remained unchanged

after axotomy. Under activation, action potential duration and

amplitude were signicantly increased, whereas duration andamplitude of the afterhyperpolarization that followed the actionpotential showed considerable reduction in axotomizedneurons, with a maximal change by 14 days after injury(Gordon et al., 1987; Kelly et al., 1988 ). The reduction inafterhyperpolarization rapidly recovered in regeneratingneurons, but, in contrast, changes in its amplitude appearedto be independent of the occurrence of axonal regeneration.

5. Structural and synaptic plasticity of axotomizedneurons

In parallel to the neuronal body reaction, there is a retractionof the dendritic tree and a reduction of the synapses received byaxotomized neurons. Such morphological changes seem toaccount for a functional isolation of the injured, non-functionalneurons from the remaining neural circuits ( Sumner andSunderland, 1973; Purves, 1975 ). These changes have beenparticularly studied in motoneurons but also in autonomic

neurons. The dendritic diameter, membrane area and volume of axotomized motoneurons were shown to decrease by a third at1–3 months after axotomy. This reduction in dendritic size isdue to a loss of preterminal and terminal dendritic segments.Following reinnervation of the target muscle, the axotomizedmotoneurons did not recover their original number of dendriticbranches, but the dendritic membrane area and volume wasnormalized due to an increase in dendritic diameters and anincreased number of dendrites per neuron ( Brannstrom et al.,1992a,b; Borke et al., 1995 ). Nevertheless, other studies basedon reconstruction of the entire dendritic trees of neck motoneurons have reported an expansion of the dendritic tree

following axotomy ( Rose and Odlozinski, 1998 ). The distaldendritic arborizations of these motoneurons exhibited axon-like characteristics, supporting the claim that axotomy leads toa remodeling of the neuronal polarity ( Rose et al., 2001 ). A keyfactor in the production of such ‘‘dendraxons’’ appears to be theproximity of the axotomy to the neuronal soma ( Linda et al.,1992; Rose and Odlozinski, 1998 ). These processes might begenerated as replacement for the injured axon, but have also thepotential to make inappropriate connections within the centralnervous system that would be detrimental for functionalrecovery.

Qualitative and quantitative cytological evidences show areduction in number and area of coverage of synaptic terminalson chromatolyzed motoneurons from a few days after axotomy.The degree of synaptic detachment on motor neurons isinuenced by the severity of nerve injury ( Svensson et al.,1991). The reduction in number and percentage of boutons onthe surface membrane is due to detachment of synapses, whichinvolves disappearance of both pre- and postsynaptic mem-brane thickenings and widening of the synaptic cleft ( Sumnerand Sunderland, 1973; Chen, 1978 ). Recent results suggest thatduring synaptic remodelingafter nerve injury, nitric oxide (NO)acts as a signal for synaptic detachment and inhibits synapseformation ( Sunico et al., 2005 ). The separation of synapticterminals from the neuron membrane is associated with the

presence of reactive astrocytes and microglia. Most areas of



12/39

surface membrane bared of synaptic contact are occupied byastrocytic processes. Loss and recovery of presynaptic boutonsoccur in parallel with changes in astroglial ensheathment of themotoneurons, that appears to be inuenced by functionalchanges in the lesioned neurons ( Chen, 1978; Aldskogius andSvensson, 1993; Aldskogius et al., 1999; Lan et al., 2003 ).Microglial cells, although extending to neuronal surfacemembrane, cover only a small portion of it. Most microgliaare seen located at some distance from surface membrane andare frequently separated by sheets of astrocytic processes,although they may play a role in the activation of astrocytes andin elimination of dead neurons ( Aldskogius et al., 1999 ). If regeneration is allowed, synaptic restoration takes place inparallel to peripheral target reinnervation. By contrast, afterpermanent axotomy synaptic frequency and cover remainreduced to 30–50% of control values during months ( Purves,1975; Johnson et al., 1998 ). The amount of synaptic loss andrecovery is variable and affects various types of synapses todifferent degrees ( Purves, 1975; Chen, 1978; Brannstrom and

Kellerth, 1999; Lan et al., 2003 ). For example, aftermotoneuron axotomy, excitatory S type boutons are eliminatedto a larger degree than inhibitory F type terminals ( Borke et al.,1995; Linda et al., 2000 ). The preferential elimination of glutamatergic terminals onto lesioned motoneurons may reectan active reorganization of the synaptic input to diminish theexcitotoxic inuence on these neurons. The expression of themajor histocompatibility class I complex in lesioned neuronsand astroglia has been reported to play a role in the regulation of synaptic elimination, mainly inuencing the elimination of inhibitory terminals ( Zanon and Oliveira, 2006 ).

5.1. Functional changes in central synapses

The synapse made between Ia muscle spindle afferents andalpha motoneurons (Ia-MN synapse) in the spinal cord has beenby far the most widely studied model of central synapse inphysiological and pathological conditions. After nerve lesionsand regeneration three distinct phases in the Ia-MN synapticfunction have been described: an early increase in thepostsynaptic responses, followed by a longer lasting declineand a phase of recovery when the nerve is allowed toreinnervate target muscles. The excitatory postsynapticpotentials (EPSPs) induced in motoneurons by stimulation of Ia afferents are signicantly enlarged already during the rstday following peripheral nerve section in adult animals(Mendell, 1984; Miyata and Yasuda, 1988; Manabe et al.,1989; Seburn and Cope, 1998 ). This early increase in synaptictransmission strength, which lasts for about 3 days, isattributable to lack of activation of the synapse that resultswhen Ia afferents are transected, to reduced activity of homonymous motoneurons, and likely due to an increase inthe probability of transmitter release. Section of the afferentbers is necessary for the increase in synaptic transmissionstrength, but concomitant section of the motor bers ampliesthe effect (Cope et al., 2001 ).

The second phase of progressive decline in the EPSP

amplitude at the Ia-MN synapse develops by 1 week after

axotomy, which is time related with stripping of spinal cordsynapses ( Horch and Lisney, 1981; Mendell, 1984 ). Theposttetanic potentiationof EPSPs is decreasedafter axotomy, aregressive change that has been related to a change in synapticrelease properties more than to changes in the electricalproperties of motoneurons ( Gustafsson et al., 1986 ). However,theEPSPsare restored to normalsizewhen thenerve is allowedto reinnervate the muscle, even if a long delay precludesadequate recovery of muscle activity, sensory activity ormotoneuron properties ( Goldring et al., 1980; Mendell et al.,1995). The degree to which central synaptic efcacy declinesand recovers followingsection and regeneration of a peripheralnerve depends partly upon the type of motoneurons; thosesupplying slow muscles show faster and better recovery thanfast ones. Partial recovery of synaptic strength has also beenobserved when the cut Ia afferents are forced to reinnervateskin (Mendell et al., 1995 ), and with treatment of the musclenerves with exogenous supplyof neurotrophins ( Munsonet al.,1997; Mendell et al., 1999 ). The higher amplitude of evoked

EPSPs is due to a higher probability of transmitter release(Hellgren andKellert, 1989; SeburnandCope,1998 ), althoughhyperexcitability of sensory and motor neurons ( Eccles et al.,1958) may also play a role. Neuronal deprivation or expositionto neurotrophic factors, such as BDNF, NT-3, NT-4/5 andGDNF, might be the signals for regenerating neurons to modifytheir phenotype, thus enhancing or depressing synapticcontacts ( McAllister et al., 1999; Mendell and Munson,1999). Neurotrophins induce changes in the expression of Na +

and K + channels ( McAllister et al., 1999 ) and glutamatereceptors ( Chew and Gallo, 1999 ). Induced changes in thesubunit conguration of NMDA and non-NMDA receptors in

spinalmotoneurons after injurymay account for changes in theIa-MN transmission ( Popratiloff et al., 1996; Alvarez et al.,2000).

A similar process has been described for sympatheticneurons of the superior cervical ganglion ( Purves, 1975;Sánchez-Vives and Gallego, 1993 ). Within 72 h of axoninterruption, the amplitude of EPSPs recorded in principalneurons in response to preganglionic stimulation declined,being maximally reduced by 7 days following axotomy. Littleor no difference was found in the electrical properties of axotomized neurons in comparison with normal neurons.Recovery in EPSPs occurred to normal amplitude if functionalperipheral connections were re-established. On the contrary, if axons were prevented from regenerating to their target organ,neurons did not generally recover normal synaptic function, andmost affected cells died within a month.

6. Facilitation of spinal reexes after nerve injury

Marked plastic changes in the connections and function of spinal reexes occur after nerve injuries in parallel to peripheralaxonal regeneration and target reinnervation. Such changesmay play important effects on movement control and sensoryprocessing, if they remain permanent especially whenreinnervation is incomplete or defective ( Valero-Cabre´ and

Navarro, 2001, 2002b ).



13/39

Regarding motor spinal reex restitution after nerve injury,Scott (1985) and Scott and Panesar (1995) showed completerecovery of the stretch reex at 4 months after sciatic nervecrush in cats and similar to normal function of reinnervatedmuscle spindles. In contrast, a permanent absence of stretchreex responses was found even 3 years after tibial nervesection in cats, which was attributed either to stripping of central synapses or to poor muscle spindle reinnervation ( Copeand Clarck, 1993; Cope et al., 1994; Abelew et al., 2000 ). Infact, the density of reinnervating proles is decreased and thedistribution is altered with respect to control spindles afternerve lesions ( Verdú and Navarro, 1997 ), probably underlyingdecits in stimulus transduction.

To avoid the confounding effect of receptor dysfunction,electrical stimulation of either regenerated nerves or ipsilateralor contralateral intact nerves has been used in the assessment of spinal motor reexes. After nerve or ventral root section,stimulation of Ia afferent bers revealed a progressive declinein the monosynaptic response of axotomized motoneurons

(recorded from the ventral roots) and increase in the centraldelay time during the rst 2 weeks, whereas longer latencypolysynaptic responses were increased. Monosynapticresponses reappeared by about 5 weeks post-lesion and tendedto increase in amplitude thereafter ( Downman et al., 1953 ).Synaptic transmission is depressed following interruption of either limb of the segmental reex arch. The reduction in EPSPamplitude is reversed following reinnervation of the peripheraltissue by the lesioned bers, whether or not the regeneratingber nds the correct muscle ( Mendell and Munson, 1999 ).

Recovery of the H wave reex (recorded from targetmuscles), the electrical counterpart of the monosynaptic stretch

reex, by stimulation of afferent bers proximal to the injuryindicates that the spinal reex circuits are functional as soon asthe muscle is reinnervated. The H reex is highly facilitated atearly stages of reinnervation, resulting in a three- to fourfoldincrease of the H/M amplitude ratio ( Fig. 2), thus indicating arelative increase of the Ia-motoneuron synaptic response toelectrical stimulation of intact afferents. Advancing musclereinnervation reverted this facilitatory effect, and as the M waveamplitude increased with time the H/M ratio declined to nearnormal. However, levels of the H/M ratio at 3 months post-lesion remained signicantly higher than in control animals,particularly in experimental situations with a low degree of muscle reinnervation, such as following nerve resection andautograft or tube repair ( Valero-Cabre´ and Navarro, 2001,2002a ). When muscle reinnervation was hampered, the H waveamplitude remained increased during many months ( Hellgrenand Kellert, 1989 ). Interestingly, a prominent strengthening of monosynaptic reexes in the immediately adjacent spinal cordsegments to a ventral root injury has also been found. Thereexes were almost doubled in size at 6 and 12 weekspostoperatively ( Havton and Kellerth, 2004 ). The remainingmotoneurons in those adjacent segments seem to undergocompensatory synaptic rearrangements, receiving more exci-tatory boutons, leading to increased excitability and enhancedreexes ( Holmberg and Kellerth, 2000 ). Moreover, sciatic

nerve injury during the rst week in the rat, a critical

development period when axotomy causes marked neuronaldeath, leads to permanent enhancement of reex responsesfrom reinnervated muscles that is maintained during adulthood(Navarrete et al., 1990 ).

Withdrawal reexes elicited by electrical stimulationcharacteristically consist of three bursts of muscle activity(C1, C2 and C3) of gradually longer latency, lower amplitudeand higher threshold, which are likely mediated by differentpopulations of sensory afferent bers (A b , Ad and C bers,respectively) ( Meyerson et al., 1995; Navarro et al., 1999 ).After sciatic nerve injury, crossed extensor reex responsesdependent on myelinated afferent bers (C1 and C2), similar to

the behavior shown by the stretch reex (H wave), showed a

Fig. 2. Recordings of compound muscle action potentials in the rat gastro-cnemius muscle of (A) the intact contralateral limb, (B) the limb subjected tosciatic nerve section and suture repair at 1 month and (C) at 2 monthspostsurgery. Note the progressive recovery in amplitude of the M wave, andthe marked increase in amplitude of the H wave, resulting in an enhanced H/Mratio after nerve injury and regeneration. Horizontal scale: 2 ms/division;vertical scale: 10 mV/division.



14/39

marked increase in amplitude during the rst stages of regeneration, and tended to decline as reinnervation progressed(Valero-Cabre´ and Navarro, 2002b ). However, at the end of 3months follow-up, the reex amplitude remained at signi-cantly higher levels than in controls, more in animals presentinga low degree of sensory-motor reinnervation. In contrast,component C3 of the crossed extensor reex, mediated byunmyelinated C afferents, reappeared in a lower proportion of animals, and had an amplitude similar to baseline values(Valero-Cabre´ and Navarro, 2002b ). Thus, after nerve injuriesreexes mediated by myelinated sensory afferents appear toexhibit a higher degree of facilitation than those mediated byunmyelinated bers.

Spinal reex facilitation correlates with the severity of theinjury and the degree and time course of muscular reinnerva-tion. These observations are compatible with the fact thatneurotrophic factors released by Schwann cells and target cellsare able to modulate the physiological behavior of intact oraxotomized neurons ( McAllister et al., 1999; Mendell and

Munson, 1999 ). In adult animals, all these changes persist longtime after reinnervation and may impair motor unit activationand control of movement, and account in part for the poorclinical outcome achieved after severe nerve injuries ( Cope andClarck, 1993; De Medinaceli, 1988; Gramsbergen et al., 2000;Valero-Cabre´ and Navarro, 2002a ).

Different mechanisms mayaccount for the observed patternsof facilitation of reex responses following nerve injuries.Mechanisms that may contribute to the abnormalities reportedin reex responses following nerve injuries include increase of neuronal and synaptic excitability ( Eccles et al., 1958; King andThomson, 1995 ), decrease of the effectiveness of propiospinal

and descending inhibitory systems ( Castro-Lopes et al., 1993;Sanna et al., 1993 ), and remodeling of sensory afferentprojections within the spinal cord ( Woolf et al., 1992; Shortlandand Fitzgerald, 1994 ).

7. Remodeling of spinal cord circuitry

Peripheral regeneration following nerve section results in themismatch of the connections between sensory receptors andafferentberswith second-orderneurons in thedorsal horn of thespinal cord and alters the somatotopy of the body representationat thespinal cord level.Thesealterations result in loss ordecreaseof tactile acuity and discrimination, and underlie in part positivesymptoms such as dysesthesia and neuropathic pain. The lack of specicity in peripheral reinnervation after complete nervesection followed by surgical repair mainly arises fromtopographic intrafascicular changes generated after the injury,the random growth of regenerative sprouts and misrouting of regenerated axons ( Abernethy et al., 1992; Madison et al., 1996;Puigdellı́ vol-Sá nchez et al., 2006 ). Regenerating sensory axonscan reinnervate foreign target territories and inappropriatereceptors, including reinnervation of muscle receptors bycutaneous afferents and vice versa, or reinnervation of tactilereceptors by nociceptive afferents ( Koerber et al., 1989 ).

Studies using neuroanatomical and electrophysiological

techniques proved that central reorganization induced by nerve

lesion produces changes in the spinal cord receptive elds, thefunctional efcacy of central connections and the laminarprojections of afferent bers, so that the properties of thecentral projections of regenerated sensory neurons are not wellmatched with their peripheral receptor properties ( Koerberet al., 1995 ). Koerber and Mirnics (1996) and Koerber et al.(2006) found that, at 6 months after tibial nerve sectionand repair in the cat, the somatotopic organization in theaffected areas of thedorsal horn waslost; the receptive elds of dorsal horn cells were larger than normal and consisted of multiple small discontinuous skin areas, in agreementwith the random and disperse nature of axon regenerationfollowing nerve section. At longer intervals (9–12 months) thereceptive elds shrank, approaching normal sizes, but thesomatotopic organization remained partially scrambled.Moreover, shortly after regeneration, focal electrical stimula-tion of the skin within the receptive eld of a dorsal hornneuron revealed that numerous bers that were not connectedto the dorsal horn cell could be electrically activated. At longer

recovery times the incidence of afferent coupling increasedsignicantly. This plasticity process implies signicantchanges in synaptic efcacy. Over time some original synapticinputs would be unchanged or even strengthened while othersmight weaken or disappear, in order for receptive elds toreturn towards normal sizes; on the other hand, pre-existingsilent inputs must be activated or new synaptic inputsestablished in order to make the receptive elds morecontinuous ( Koerber et al., 2006 ).

7.1. Central sprouting of afferent projections

After a peripheral nerve lesion, DRG neurons show variableresponses regarding sprouting or regenerative growth of theircentral axonal branch. Myelinated afferent terminals labeledwith cholera-toxin beta-subunit show an advantage in collateralsprouting into the adjacent denervated neuropil in lamina IIIafter nerve lesion. Intact and unlesioned sensory neuronspositive for CGRP are able to sprout vigorously into segmentsdenervated by rhizotomy in a nonsomatotopic manner. Incontrast, isolectin B4 (IB4) labeled sensory neurons maintain asomatotopic distribution centrally, which is not altered by priornerve lesion ( Belyantseva and Lewin, 1999 ). Following nerveinjury there is a dramatic decrease in IB4 expression in thesupercial dorsal horn, that is partially recovered if peripheralregeneration is allowed ( Molander et al., 1996 ). An ultra-structural quantitative assay following constrictive injury to therat sciatic nerve has also demonstrated a signicant decrease inthe density and number of central boutons of IB4-labeledafferents during the rst 2 weeks due to degeneration, followedby recovery by regrowth of terminals, in contrast with nochanges in CGRP-labeled afferents ( Bailey and Ribeiro-da-Silva, 2006 ). These data indicate a considerably heterogeneousresponse to injury and regeneration amongst different types of afferent sensory neurons, particularly between peptidergic(NGF-dependent) and non-peptidergic (GDNF-dependent)sensory neurons, which is likely related to different expression

of neurotrophic factors (see Fig. 3).



15/39

There has been extensive controversy regarding therearrangement of the central terminals of injured and intactsensory afferents in the spinal cord following nerve lesions, andits potential contribution to neuropathic pain and mechanicalhypersensitivity. After peripheral nerve lesions, the centralaxons of injured myelinated A b bers have been found inseveral studies using retrotracing and intraber injectiontechniques to sprout from their normal termination territorywithin deep laminas III–Vof the dorsal horn to partially invadelaminas II and I, which are normally restricted to receive inputsfrom C and Ad sensory bers ( Woolf et al., 1992, 1995;Shortland and Woolf, 1993; Koerber et al., 1994 ). Intactmyelinated afferents also showed an enlarged projection intoareas normally provided by a neighbor-injured nerve, althoughreorganization was limited to the dorsoventral plane in adultanimals ( Doubell et al., 1997 ). In contrast, nerve lesions inneonatal animals induced a more extensivegrowth of uninjuredterminals outside their normal termination region, permanentlyaltering the somatotopic representation maps and the laminardistribution ( Shortland and Fitzgerald, 1994 ). The death oratrophy of a number of small sensory DRG cells and the

transganglionic degeneration of their terminations within the

spinal cord dorsal horn after nerve injuries ( White and Kocsis,2002) would induce sprouting of A b and Ad afferents from thesame spinal level as well as of A b and C bers from other spinallevels into the empty dorsal horn laminas I and II. Even if regeneration was allowed after sciatic nerve crush, collater-alization of A b bers lasted for months on the spinal cord graymatter ( Woolf et al., 1995 ). The myelinated endings that sproutinto laminas I and II, and also into laminas III–V( Koerber et al.,1994), would raise the number of synaptic contacts with correctand mismatched spinal interneurons, thus increasing thestrength and duration of polysynaptic reex responses(Valero-Cabre´ and Navarro, 2002a ) and underlying thedevelopment of abnormal sensory input and hyperalgesia(Shortland et al., 1997 ).

Nevertheless, more recent studies have raised concernsabout the specicity of the used tracer (cholera toxin B subunit)and the changes of axotomized thin bers for its transport, anddo not support the hypothesis that A b afferents sprout into thesupercial lamina after nerve section. Only a very limited

number of large A bers was found to sprout into inner laminaII, a region normally innervated by C bers, but not into outerlamina II or lamina I, and no obvious changes were found in therostrocaudal distribution or in the laminar locations of axonalcollaterals ( Bao et al., 2002; Hughes et al., 2003; Shehab et al.,2003; Koerber et al., 2006 ). When detected, A ber sproutinginto lamina II after nerve injury seems to derive mainly fromsmall Ad bers that terminate in lamina I ( Ma and Tian, 2001 ).Moreover, the suggestion that central terminals of intactmyelinated afferents sprout into regions of lamina II occupiedby adjacent nerves that have been a

Prog Neurobiol 2007 NavarroNeural plasticity after peripheral nerve_.pdf

Documents