Regeneration and functional recovery following peripheral nerve injury

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Drug Discovery Today: Disease Models Vol. 1, No. 2 2004

Editors-in-Chief

Jan Tornell – AstraZeneca, Sweden

Denis Noble – University of Oxford, UK

Peripheral nervous system diseases

Regeneration and functional recoveryfollowing peripheral nerve injuryFrancisco J. Rodrıguez1,y, Antoni Valero-Cabre1,2,y, Xavier Navarro1,*1Department of Cell Biology, Physiology and Immunology, and Institute of Neurosciences, Universitat Autonoma de Barcelona,

Edif M Campus UAB, E-08193 Bellaterra, Spain2Department of Neurology, Harvard Medical School, Boston, MA 02155, USA

Peripheral nerve injuries result in loss of neural control

in denervated segments of the body, and severe dis-

abilities for the patients. Nerve regeneration usually

does not allow for adequate target reinnervation and

functional restitution. Neuronal response and axonal

regeneration imply a complex interaction of cell types

and changes in the expression of many molecules. Many

experimental models have been used to gain knowl-

edge on nerve regeneration and to develop strategies

to promote recovery.

*Corresponding author: (X. Navarro) [email protected] F.J. Rodrıguez and A. Valero-Cabre contributed equally to this paper and

both share first authorship.

1740-6757/$ � 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmod.2004.09.008

Section Editor:Kathryn Albers – Department of Medicine, University ofPittsburgh, Pittsburgh, PA 15261, USA

Significant interest lies in determining how to efficiently restore motor,

sensory and autonomic nerve function following peripheral nerveinjury. In this review, Dr Xavier Navarro and colleagues provide a

comprehensive overview of current experimental models being used tostudy the regenerative response following peripheral nerve injury. Many

different injury paradigms are available that offer a wide range of

experimental approaches in which to test novel approaches for nerverepair, drug delivery and in vivo tissue engineering. These model systems

also provide a means in which to identify genes and signaling pathwaysimportant for functional nerve recovery.

Introduction

Injuries to peripheral nerves (PNs) result in partial or total loss

of motor, sensory and autonomic functions in the involved

segments of the body. Reinnervation (see Glossary) of dener-

vated targets can be achieved by regeneration of injured

axons or by collateral branching of undamaged axons in

the vicinity. Nevertheless, these mechanisms do not provide

for satisfactory functional recovery, especially after severe

injuries. PN problems are common and encompass a large

spectrum of traumatic injuries, diseases, tumors and iatro-

genic lesions. The incidence of traumatic injuries is estimated

as >500,000 new patients annually.

After injuries to PNs, axons and myelin sheaths distal to the

lesion are degraded. The degenerative products are elimi-

nated by the cooperative action of denervated Schwann cells

(SCs) and infiltrating macrophages. Wallerian degeneration

serves to create a microenvironment favoring axonal

regrowth. SCs within the endoneurial tubes of the distal

nerve dedifferentiate towards a non-myelinating proliferative

phenotype that over-express growth factors, cell adhesion

molecules and extracellular matrix. The axotomized neurons

shift from a ‘transmitter’ state to a ‘regenerative’ state, so their

axons generate growth cones that progress from the proximal

stump into the distal nerve. Axonal regeneration requires an

adequate substrate of trophic and tropic factors, provided by

reactive SCs, macrophages and the extracellular matrix

within the degenerated nerve. The regenerative process, how-

ever, does not usually reconstitute a normal nerve structure

neither allows for normal distal reconnection after severe

lesions.

Neuronal response and axonal regeneration require a com-

plex interaction of several cell types and changes in the

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Drug Discovery Today: Disease Models | Peripheral nervous system diseases Vol. 1, No. 2 2004

Glossary

Artificial nerve graft: device designed to repair nerve gaps, as a

substitute of a natural nerve grafts.

Axonal regeneration: regrowth and elongation of the cut axon tips.

Axotomy: rupture or section of the axon, leading to loss of continuity

and of impulse conduction through the distal axon.

Iatrogenic lesion: lesion or secondary complication due to medical or

surgical procedures.

Nerve fascicle: bundle of nerve fibers encircled by perineurial sheath.

Nerve graft repair: interposition of a nerve segment between the

stumps of a transected nerve.

Neuritogenesis: growth of neurites, thin dendrite-like prolongations

from neurons cultured in vitro.

Neuroma: enlargement of the proximal stump of a cut nerve composed

by twisting regenerating sprouts and connective tissue.

Neurotmesis: complete transection of a peripheral nerve.

Reinnervation accuracy: adequate reinnervation of a target end organ

by axons that originally served that organ.

Root avulsion: rupture by stretch of a spinal root at its entrance in the

spinal cord.

Tubulization or tube repair: implantation of a nerve guide to bridge a

gap created in the peripheral nerve.

Vibrissa: mystacial hair in the rodents, moved by piloerector muscles for

exploratory behavior.

Wallerian: degeneration: process of degradation of axonal and myelin

debris after axotomy.

expression of many molecules with variable spatial and tem-

poral patterns. Therefore, a wide variety of methods are used

in experimental studies, depending on the specific goals of

each study. This review provides an overview of different

experimental models used to gain knowledge on the regen-

erative response after PN injuries and to assess repair strate-

gies to promote functional recovery (Table 1).

In vitro models of neuronal survival and

neuritogenesis

Neuronal cell cultures

The most simple and traditional in vitro model is the culture

of dissociated neuronal cells, mostly from dorsal root ganglia

(DRG) because of their easier access compared to other popu-

lations [1] (Fig. 1). This preparation, however, disrupts the

normal relationships between axons, glia and connective

tissue cells. Primary cell cultures need the sacrifice of a large

number of animals and allow for a limited number of cells. An

alternative model is cell lines, which are available from cell

banks (American Type Culture Collection, http://www.atc-

c.org), can be easily expanded, and provide a more homo-

genous cell population than primary neuronal cultures. One

cell line extensively used is PC12, a rat pheochromocytoma

line that when exposed to nerve growth factor (NGF) differ-

entiates into cells resembling adult sympathetic neurons.

However, because they are not real sympathetic neurons,

the genetic background – as with any cell line – can yield

results not representative of physiological roles in vivo. These

models are adequate to evaluate factors with direct impact on

178 www.drugdiscoverytoday.com

neurons for either survival or neuritogenesis, such as drugs,

growth factors or substrates, and, if co-cultured with other

cell types, such as SCs, to assess mechanisms of interaction.

Neuronal culture methods represent a suitable platform to

test strategies based on genetic manipulation of either neu-

rons or supporting cells. Ex vivo engineering of glial cells or in

vivo transduction of neurons by means of viral vectors are

becoming important tools to modulate the response after PN

injury [2,3].

Tissue-engineered 3-dimensional cultures

Tissue-engineered models of PN regeneration to study neurite

growth are generally based on the culture of dissociated

neurons on top of a 3-dimensional artificial tissue based on

extracellular matrix elements. Particular interest has received

the study of composition and geometry of the matrix and

addition of attachment peptides to investigate contact gui-

dance cues for neurite outgrowth [4,5].

In a recent study addressing how skin vasculature modu-

lates innervation by sensory neurons [6], the authors used an

artificial skin composed of a collagen–chitosan sponge filled

with human fibroblasts and endothelial cells. Endothelial

cells spontaneously formed a network of capillary-like tubes

that were followed by neurites. In addition, the 3-dimen-

sional structure of the construct allows for gradients of nutri-

ents and growth factors that might favor neurite outgrowth

from top to bottom of the sponge. The use of confocal

microscopy coupled to immunofluorescence permits visua-

lization of the neurite network and its interaction with other

components of the construct.

Organotypic cultures

Organotypic cultures have several advantages compared

with dissociated neuronal cultures. First, there is no need

for cell dissociation, a major cause of cell stress and death.

Second, the tissue cytoarchitecture and the interactions

between different cell types are both preserved, allowing

for a more physiological evaluation. Third, because the

culture preserves the whole anatomical structure it allows

for the generation of spatial interactions with other ele-

ments, such as a PN slice, or chemotropic interactions, such

as gradients for a growth factor from slow-releasing gels, or

an adjacent compartment filled with other unmodified or

engineered cell types.

Although the most commonly used structure is DRG, slices

of rodent spinal cord have also been successfully cultured.

Organotypic cultures of spinal cord have been mainly used to

investigate the effects of neuroprotective agents against

trauma or neurodegeneration [7,8]. Co-cultures of spinal

cord slices over a monolayer of muscle cells have been

applied to study neuromuscular innervation [9]. From the

several models described in the literature, the two described

below represent interesting examples for PN regeneration.

http://www.atcc.org/


Vol. 1, No. 2 2004 Drug Discovery Today: Disease Models | Peripheral nervous system diseases

Figure 1. In vitro methods to study peripheral nerve regeneration. Primary cell cultures, neurons and glial cells can be used to evaluate factors expressed by

themselves or necessary to promote survival, neuritogenesis and/or proliferation. Also, if co-cultured they enable the study of cell–cell interactions. The

dissociated cells box shows a culture of embryonic spinal cord with neurons immunostained against b-Tubulin III (a) and Schwann cells immunopurified by

magnetic beads covered with p75-NGFr antibody (b). Chemotaxis chambers are used to evaluate the activity of soluble factors to promote cell migration

through a porous membrane. Tissue-engineered 3-dimensional cultures based on a sponge composed of extracellular matrix and cells that after culturing

neurons on top allows for the study of neurite growth. Organotypic cultures can be done from dorsal root ganglia, sympathetic ganglia, spinal cord slices and

peripheral nerve slices. Possibilities to manipulate the environment include placing gels containing cells or growth factors, a monolayer of unmodified or

engineered cells, and a growth-promoting substrate.

The co-culture of DRG with a whole nerve segment or a nerve

cryosection [10] enables the study, in vitro, of the interaction

between the neurites and their normal growth substrate in

vivo, with the advantage conferred by the option to modify

the culture medium. The development of artificial nerve

grafts based on 3-dimensional structures resembling the

distal denervated nerve to support axonal growth benefits

from the aligned distribution that collagen molecules adopt

when exposed to magnetic fields during polymerization.

Tranquillo and colleagues developed an in vitro model con-

sisting on a tube filled with collagen that allows for the

placement of a DRG on a small chamber located on one

side of the rod and the analysis of neurite growth extent

and direction [11]. The field is exploring the use of other

elements of the extracellular matrix, such as fibrin and

fibronectin [12], and the incorporation of neuritotropic

oligopeptides [13].

Glial cell cultures

The de-differentiation and migration of SCs is a crucial step

for regeneration after PN injury. Two to six months after

injury, denervated SCs become atrophic and lose their cap-

ability to promote axonal regeneration. When surgery is

delayed and/or the distance axons have to regrow is long,

the distal nerve stump might remain denervated for months.

SC cultures helped to characterize and to compare the mole-

cular phenotypes of acutely and chronically denervated cells

in vitro [14,15].

SC migration can be promoted by either gradients of

soluble chemotropic factors or contact-guidance substrates.

A common technique is the use of chemotaxis chambers, a

cell culture system designed to count the number of cells able

to migrate through a porous insert in response to soluble

factors. A recent study has shown that SCs increase their

kinetic activity when exposed to NGF, but not to brain-



Table 1. Comparison summary table

In vitro models In vivo models In silico models

Pros Simplicity Similar models to human lesions Combining experimental information

from molecular to organ levels

Short time studies Different types of models and injuries Multiple hypothesis testing

Testing of multiple factors Large experience in physiological and

pathological experiments

Available cell lines Available transgenic and knockout animals

Cons Physiological differences

with in vivo models

Cost and time consuming Results should be assessed on in vitro

and in vivo models

Missing influences from

host factors

Complexity Limited models available

Accurate methodologies for evaluation

Ethical considerations

Best use of model Initial testing of factors

and drugs

Developing and testing new

therapeutic strategies

Simulation modeling of multiple

variables influencing nerve regeneration

Preservation of cytoarchitecture

in organotypic cultures

How to get access

to the models

Literature Literature Literature

Cell banks

References [1–17] [18–44] [45–54]

derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3)

[16]. A second method is the determination of maximal

distance of migration of SCs dissociated in culture or from

a PN explant in response to factors added to the medium,

attached to the culture plate surface or secreted by engineered

cells in a confluent monolayer [17]. Finally, in vitro tests are

also used to assess the ability of extracellular matrices or nerve

guides to promote SC migration [12].

In vivo models of peripheral nerve regeneration and

recovery

A wide variety of animal and nerve models have been used in

PN regeneration research. Rodents, particularly mice and rats,

have become the most frequently utilized model. Their inex-

pensive housing costs, their distribution of nerve trunks

similar to humans, and the large availability of genetic,

cellular and system physiology data, makes them particularly

suitable. Furthermore, numerous transgenic and knockout

rodent models (not covered here) have been developed prov-

ing to be an essential approach to study regeneration-related

mechanisms and pathways. Non-human primates have been

occasionally used. In spite of their proximity to human

models, their high cost and sophisticated housing explains

its limited use. Unless long-term behavioral and electrophy-

siological assessment of fine motor and sensory functions are

carried out or drug and/or device testing is at final stages

preceding clinical application, the benefits of monkey over

rodent models are at least questionable.

In rodent models, lesions to a large variety of nerves have

been assayed. Sciatic, femoral, facial and median nerves, and

brachial and lumbar spinal roots are among the most fre-

quently found in the literature. Some of these models emu-


late clinically relevant lesions occurring in the same or similar

nerves in humans. Others enable researchers to tackle specific

molecular, cellular or behavioral problems that might help to

expand knowledge in the field.

Sciatic nerve model

The sciatic nerve model is most commonly used in PN

regeneration studies. It provides a nerve trunk with adequate

length and space at the mid-thigh for surgical manipulation

and introduction of grafts or guides (see below). The sciatic

nerve divides above the popliteal fossa into three branches:

tibial, peroneal and sural nerves. Each of these branches

conveys different proportions of motor, sensory and auto-

nomic axons towards muscles, skin receptors and vessels or

sweat glands, located at defined territories of the hindlimb.

Injury in one but not other sciatic branches yields to paralysis

and anesthesia of particular regions. Motor and sensory

neurons are orderly distributed at different levels and depths

in the spinal cord and DRG [18,19]. Although sciatic nerve

injuries themselves are rare in humans, this model provides a

very realistic testing bench for lesions involving plurifasci-

cular mixed nerves with axons of different size and types

competing to reach distal endoneurial tubules and reinner-

vate targets. Comparisons with the normal distribution

enable the accuracy of reinnervation to be addressed through

electrophysiological and retrograde tracing methods (Fig. 2).

Behavioral recovery of walking has been extensively assessed

by detailed analysis of the paw prints during gait [20].

Femoral nerve model

The femoral nerve of the rat is a relevant model to study the

accuracy of reinnervation of motor and sensory axons onto


Figure 2. Schema of retrograde tracing of motoneurons in the sciatic nerve and its branches that can be applied after injury and regeneration to assess

topographic accuracy of muscle reinnervation. Different tracers are injected in different nerves near or at the entrance in the muscle. After a few days,

corresponding motor nuclei can be visualized on either transversal or longitudinal spinal cord sections, the numbers of motoneuron bodies counted, and the

outline and location of the motor nuclei calculated. Abbrevistions: GCm, gastrocnemius medialis muscle; GCl, gastrocnemius lateralis muscle; TA, tibialis

anterior muscle; PL, plantar muscles.

appropriate targets (i.e. muscle or skin receptors). It has

terminal division onto two different branches of similar size:

a muscular branch to the quadriceps muscle (containing

�300–500 motor axons), and a cutaneous branch conveying

sensory innervation to the skin of the anterolateral region of

the thigh (�1500 myelinated sensory axons). Preferential

motor or sensory reinnervation [21,22] has been demon-

strated using this particular model. Preferential reinnervation

refers to the higher probability of regenerating motor axons

to reinnervate muscle than skin, and vice versa for sensory

axons. The relevance of pathway-related guidance molecules

can be investigated in this model.

Median nerve model

Median nerve lesions are relevant in humans, particularly in

the context of carpal tunnel syndrome. The median nerve

provides a model in which motor and sensory axons segregate

in different nerve branches distal to the wrist. Rodent models

of median nerve injury are seldom used, due to limited

possibilities of testing fine finger motor or sensory functions

in small animals. The narrow space available in the forearm of

rodents makes surgery challenging. In spite of those difficul-

ties, electrophysiological assessment of median and ulnar

nerves and behavioral testing of grasping have been reported

after injuries and nerve graft repair [23,24]. In monkeys,

regeneration after different length resections of the median

nerve repaired by either direct suture, sural grafts or collagen

guides has been studied in detail [25,26]. Nerve gaps, com-

parable with those considered limiting in humans, can be

studied in the monkey model. Electrophysiological techni-

ques similar to those used in clinical evaluation to explore

reinnervation can be applied.

Facial nerve model

In rodents, as in humans, the facial nerve is a motor nerve

innervating face muscles. Its main trunk divides into the

marginal–mandibular and the buccal branches, the latter

innervating the rows of whisker muscles. Owing to its super-

ficial location, surgical access to the facial nerve is simple.

Highly variable numbers of motoneurons (3200–6500) have

been localized in the facial nucleus by retrograde tracers.

They are organized in three different regions, depending

on the vibrissa they innervate and the projecting branch.

Rodent facial nerve lesions have been primarily used to study

molecular and cellular aspects of degeneration and regenera-

tion of motoneurons [27]. Human facial nerve lesions are



Figure 3. Micrographs of the mouse sciatic nerve exposed at the mid-

thigh and subjected to (a) a crush lesion, (b) a resection of 6 mm repaired

by autografting the same segment resected, and (c) a resection repaired

by tube implantation leaving a 6 mm gap.

common and have devastating effects, not only when no

regeneration occurs, but frequently as a result of exuberant

sprouting, hyperinnervation and misdirected reinnervation

leading to dyskinesiae [27]. To tackle this particular problem,

transections of the buccal branch in rats have been used for

testing therapies aimed to limit misdirected reinnervation

[28]. Multitracing studies, electrophysiology and behavioral

assessment of vibrissae movements during exploratory beha-

vior have been successfully implemented in this model.

Spinal root models

Depending on the level the roots are severed from the spinal

cord, plexus lesions can be classified as preganglionic or post-

ganglionic. The dorsal sensory root, the ventral motor root or

both can be involved. Ventral root avulsion results in signifi-

cant death of motoneurons in adult animals. A crush-traction

of the spinal nerves has similar effects but maintains the

continuity of the roots with the spinal cord. Surgical, pharma-

cological and gene transfer approaches have been considered

to rescue motoneurons and to promote axonal regeneration in

lesions at or close to the spinal cord interface [29,30]. Regard-

ing dorsal roots, preganglionic lesions induced by avulsion or

sectionhavea bad prognosis,because the centralaxons can not

penetrate the dorsal root entryzone into the spinal cord,which

exhibits a highly inhibitory ambiance for regeneration. Sever-

ing dorsal roots of lumbar or brachial plexuses has generated

rat models of dorsal root lesions. Local application of neuro-

trophic factors and cell transplantation combined with surgi-

cal repair has been found to overcome the central inhibition

and achieve regeneration and limited behavioral recovery in

some studies [31,32].

In vivo models of nerve injury and repair

After PN injuries the capability of severed axons to regenerate

and recover functional connections is dependent on the site,

type of lesion and the distance over which axons must regrow

to span the injury. The nerve trunk injured, the type and the

severity of the lesion determine the need for surgical repair

and the prognosis for functional return. This is why the

traditional classifications of nerve lesions in the clinical field

are based upon the morphology of the lesion and the extent

of nerve sheath damage. The most common types of injury

performed in the experimental setting are: focal crush or

freeze injury that induces axonal interruption preserving

connective sheaths (axonotmesis), complete transection dis-

rupting the whole nerve trunk (neurotmesis) and resection of

a nerve segment inducing a gap of certain length (Fig. 3).

After nerve crush regeneration is usually successful thanks

to the favorable terrain provided by reactive SCs and the

preservation in continuity of the endoneurial tubules. Both

facts enhance axonal elongation and facilitate adequate tar-

get reinnervation [19]. After a short (one to two days) latency

to cross the injury site, axons regenerate at linear rate along


the distal nerve. Crush injuries are adequate to investigate the

intrinsic cellular and molecular events that intervene in PN

regeneration, and to assess factors, such as drugs [33] that

might enhance the speed of regeneration and the effective-

ness of reinnervation. Crush injuries exerted previously or

proximally to the nerve lesion site increase the rate of regen-

eration. The effect of conditioning injuries is explained by

accelerating the shift to the neuronal ‘regenerative’ state or

through early activation of SCs.

By contrast, axonal growth is limited across a gap imposed

after transection of a nerve. If left unrepaired, or if over-

imposing a nerve ligature or a capped tube, the abortive

regenerative growth leads to neuroma formation. The neu-

roma model has been mainly applied to investigate local and

neuronal changes that contribute to the development of

neuropathic pain after injury [34,35].

Surgical repair is needed for axonal regeneration to take

place after PN section. Epineurial suturing of stumps,

attempting to coapt individual nerve fascicles, has been

the most classical method of repair. In plurifascicular nerves

identification of cutaneous and muscular fascicles might be

possible by the use of neurophysiological stimulation or

histochemical staining. Long delayed repair results in a pro-

gressive reduction of the number of neurons that regenerate

and reinnervate denervated targets. Cross suture techniques

are useful to assess the consequences of either prolonged

axotomy or denervation of the distal stump. Thus, the tibial

nerve is cut, prevented to regenerate for months to prolong

axotomy, and thereafter the proximal stump is refreshed and

sutured to the freshly cut peroneal nerve. The converse

procedure is performed to prolong SC denervation. With

these models, it has been found that low-dose BDNF or glial

cell derived neurotrophic factor (GDNF) counteract the

effects of chronic axotomy [15].


The gold standard model for repairing a long nerve gap is

the autograft. Wallerian degeneration occurs more slowly in

the graft than in the distal stump, but autografts retain the

structure for long periods and do not show immunoreaction.

Autograft repair allows for similar number of regenerated

axons and functional recovery in contrast to direct suture

repair. The use of nerve allografts or xenografts is followed by

immune rejection, which is mainly directed against SCs and

myelin in the graft. Investigations on such graft models have

evaluated immunosuppressive therapies with limited second-

ary complications and procedures that reduce immunogeni-

city without affecting regeneration [36,37]. Different

procedures have been applied to make the graft acellular

but still maintain the connective sheaths. The resulting

allograft scaffolds can be repopulated by host SCs and support

axonal regeneration. Other tissue grafts, such as acellular

muscle tubes and blood vessels, have also been modeled as

a bridge for nerve gap repair [38].

Tubulization, the implantation of a tube or guide to bridge

a nerve gap, provides a useful model for studying and manip-

ulating cellular and biochemical events during PN regenera-

tion. Proximal and distal nerve stumps are fixed a few

millimeters within the ends of a tube, leaving a measured

gap in between. The silicone tube model has become the

standard for tubulization. In the past decades intense

research has focused on development of new polymeric

materials with intrinsic properties that enhance regeneration

[39]. Nerve guides offer a closed space, where neurotrophic

elements provided by the injured nerve accumulate and

support axonal growth. A limit to regeneration exists within

nerve guides depending upon the length of the gap, that is

species-dependent [40]. Therefore, tube repair offers a general

model to different approaches. Within a short or mid-length

gap, the guide constitutes a window to investigate the role of

different molecular factors and cell types in the regenerating

process [41,42]. With a long gap, above limiting length, tube

repair constitutes the basis for development of artificial grafts

that might substitute classical autologous nerve grafting.

Many works have assayed the introduction inside the tube

of neurotrophic factors, extracellular matrix components or

transplanted cells [39,40,43].

An interesting model described by Torigoe [44] is the film

model, in which the proximal stump of a transected nerve is

sandwiched between two sheets of thin plastic film and

remained in vivo for several days. The regenerating axons

and non-neuronal cells can be labeled and visualized on

the film and different strategies applied to investigate the

effects on early axonal growth and cell migration.

In silico models of nerve regeneration

A few studies have been published using oligonucleotide and

cDNA arrays for the determination of changes in gene expres-

sion in lesioned sympathetic ganglia neurons [45], in the

distal nerve stump at different time points after axotomy

[46,47], and in the ventral horn after root avulsion [48]. A

large number of expressed genes, however, do not have a

known protein correlate, and their physiological role has to

be determined by adequate in vitro or in vivo approaches. It is

also important not to neglect the technical limitation of

microarrays to detect low-abundance genes, which become

diluted when the sample is composed of multiple cellular

types, and, because they are tightly regulated, can be the real

effectors for larger changes observed in other genes.

In silico models are computer-based simulations for the

study of biological interactions at different levels of complex-

ity ranging from genes and proteins to the whole organism.

The advantage of computer models of nerve regeneration is

the ease with which different variables can be altered to

explore the interactive temporal relationships of multiple

cellular and molecular factors during regeneration. A few

reports exist on mathematical models, based upon experi-

mental data, to model growth cone shape and motility [49],

to simulate nerve regeneration after crush and section fol-

lowed by tube repair [50] or to evaluate various nerve guide

designs [51]. As in silico models become more accurate and

incorporate the huge amount of data from gene arrays, they

might become indispensable for the initial design and further

optimization of new drugs or substrates.

Substrate patterning

This technology uses photolithography to generate surfaces

that combine one or more substrates on a predefined chemi-

cal, geometrical and spatial distribution. It serves to deter-

mine their ability to influence cell adhesion, proliferation,

apoptosis, migration and orientation [52]. An example of its

application is the prove that neuronal polarity depends on

the presence of substrates with different adhesive strength;

whereas neuronal soma and dendrites develop on the most

adhesive substrate, axons extend preferentially on weaker

adhesive substrates, grow faster if they find a different sub-

strate as the one at the soma, and its adhesiveness has a

decreasing gradient from it [53]. Similar technology has

shown that substrates microgrooved with adsorbed laminin

are effective in promoting the adhesion and alignment of SCs

in vitro, with optimal widths and spacing of 10–20 mm [54].

Because cells respond differently if disposed on two or three

dimensions, future directions should address the develop-

ment of 3-dimensional in vitro systems, based on materials

suitable for topographical modification such as hydrogel

scaffoldings or agarose gels.

Conclusions

In vitro models have been and will be important for expand-

ing knowledge at the cellular level. They also help paving the

way for initially developing new potential therapies to pro-

mote cell survival, enhance regeneration and guide neurite



Links

� http://www.atcc.org

� http://pns.ucsd.edu

� http://www.hand-surg.org

� http://www.vard.org/neural/neural.htm

� http://www.integra-ls.com

� http://biomed.brown.edu/Courses/BI108/BI108_2001_Groups/

Nerve_Regeneration

Outstanding issues

� Identification of genes related to axonal regeneration, Schwann cell

response and target denervation.

� Characterization of molecular cues that guide selective growth of

different types of nerve fibers to adequate targets.

� Development of clinically useful artificial nerve grafts.

� Treatment for secondary complications of nerve injuries

(neuropathic pain, hyperreflexia, atrophy).

� Neuroprotection and axonal regeneration in proximal nerve injuries.

� Enhancement of regeneration and recovery after chronic

denervation.
outgrowth. In silico models will develop in a near future and
will have a fundamental role by combining experimental

information from molecular to tissue levels. In vivo animal

models will continue to be essential for further development

of therapies and particularly for a safe and effective transfer to

human patients. Results on in vivo models might be condi-

tioned by delivery systems, pharmacokinetic and pharmaco-

dynamic profiles, adequacy of devices, and potential

biochemical and cellular interactions, which might modify

the effects found in vitro or simulated in silico. The transfer from

successful results on in vivo models to clinical treatment of

patients might be equally uncertain because of physiological

differences and distinctions of pathologies. Although experi-

mental models have provided an adequate benchmark for

assessing pharmacological agents aimed at improving regen-

eration, the applicabilityofbiochemical ormetabolic factors to

improve regeneration in human subjects remains still spec-

ulative in most instances. Nevertheless, there are good exam-

ples of successful translation from laboratory to clinic, such as

nerve tubulization repair that has been tested with success in

human patients [55], or treatment with immunophilin ligands

that, in addition to acting as immunosuppressant for accep-

tance of allografts, also enhance nerve regeneration [37].

Future research models will focus on refining strategies to

enhance PN regeneration, to counteract factors contributing

to poor functional recovery after severe injuries, such as

damage to neuronal cells due to axotomy, inability for axonal

growth due to the nerve lesion or to underlying diseases, poor

specificity of reinnervation by regenerating axonsand second-

ary plastic changes in central connections.

Related articles

Boyd, J.G. and Gordon, T. (2003) Neurotrophic factors and their

receptors in axonal regeneration and functional recovery after

peripheral nerve injury. Mol. Neurobiol. 27, 277–323

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Regeneration and functional recovery following peripheral nerve injury