M. Wintzer et al- Strategies for identifying genes that play a role in spinal cord regeneration

8/3/2019 M. Wintzer et al- Strategies for identifying genes that play a role in spinal cord regeneration

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J. Anat. (2004) 204, pp311

Anatomical Society of Great Britain and Ireland 2004

BlackwellPublishing, Ltd.

REVIEW

Strategies for identifying genes that play a role in spinalcord regenerationM. Wintzer,* M. Mladinic, D. Lazarevic, C. Casseler, A. Cattaneo and J. Nicholls

Sissa, Trieste, Italy

Abstract

A search for genes that promote or block CNS regeneration requires numerous approaches; for example, tests can

be made on individual candidate molecules. Here, however, we describe methods for comprehensive identification

of genes up- and down-regulated in neurons that can and cannot regenerate after injury. One problem concerns

identification of low-abundance genes out of the 30 000 or so genes expressed by neurons. Another difficulty is

knowing whether a single gene or multiple genes are necessary. When microchips and subtractive differential

display are used to identify genes turned on or off, the numbers are still too great to test which molecules are actually

important for regeneration. Candidates are genes coding for trophic, inhibitory, receptor and extracellular matrixmolecules, as well as unknown genes. A preparation useful for narrowing the search is the neonatal opossum. The

spinal cord and optic nerve can regenerate after injury at 9 days but cannot at 12 days after birth. This narrow

window allows genes responsible for the turning off of regeneration to be identified. As a next step, sites at which

they are expressed (forebrain, midbrain, spinal cord, neurons or glia, intracellular or extracellular) must be deter-

mined. An essential step is to characterize proteins, their levels of expression, and their importance for regenera-

tion. Comprehensive searches for molecular mechanisms represent a lengthy series of experiments that could help

in devising strategies for repairing injured spinal cord.

Key words

CNS lesions; CNS repair; molecular analysis.

Introduction

The adult CNS of birds and mammals has little or no

capacity for functionally useful regeneration. Although

it has been known since the time of Ramon y Cajal

that some damaged CNS neurons can send out sprouts

(Ramon y Cajal, 1928), they do not grow through lesions

or make connections. By contrast, peripheral nerves

regenerate successfully after injury, as do connections

in the CNS of fish, amphibians, reptiles and invertebrates.

To try to repair the injured spinal cord has become

a challenging problem in neuroscience. Strategies

employed to enhance regeneration in the mammalian

CNS include the neutralization of potential growth

inhibitory molecules (Caroni & Schwab, 1988; Sims &

Gilmore, 1994; Dyer et al. 1998), the transplantation of

cells or tissue that support axonal elongation (Bernstein-

Goral & Bregman, 1997; McDonald et al. 1999; Ramon-

Cueto et al. 2000; Ito et al. 2001), and the delivery of

factors that are known to promote axonal growth,

such as neurotrophic factors (Xu et al. 1995; Nakahara

et al. 1996; Zhang et al. 1998). Those approaches are

showing promising results, although the growth and

functional recovery that can be observed are often

limited. Knowledge of the molecules that are involved

and the way they interact to promote or prevent

regeneration is far from complete.

Two different strategies

Sustained growth of axons involves participation by

the neuronal cell body. Axon regeneration might require

that injured neurons up-regulate a specific set of

Correspondence

Dr J. Nicholls, Sissa, Via Beirut 2, Trieste 34014, Italy. E: [email protected]

*

Present address: Department of Neuroscience, Karolinska Institute,

Stockholm 171 77, Sweden.

Accepted for publication28 October 2003


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Genes for CNS regeneration, M. Wintzer et al.


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growth-associated genes. Some of the genes that are

up-regulated or constitutively expressed in associa-

tion with axonal growth have been identified. Their

products include: (1) transcription factors, such as c-jun,

which mediates subsequent gene expression (Jenkins

et al. 1993; Herdegen et al. 1997); (2) cytoskeletal

proteins involved in axonal extension, such as T

1-tubulin

(Miller et al. 1989; Fernandes et al. 1999); (3) cell adhe-

sion molecules, such as N-CAM involved in growth cone

guidance (Jung et al. 1997); and (4) cytoplasmic growth

cone proteins involved in mediating signal transduction,

such as GAP-43 (Frey et al. 2000).

One widely used strategy consists of testing individ-

ual candidates for their potential to enhance fibre

outgrowth. Such experiments aim to find the

gene that

will promote or prevent spinal cord regeneration as a

first step toward a cure for injury in patients. Studying

genes one by one can provide interesting cues as

to their role in the process of regeneration. Because

regenerating growth cones make complex interactions

with their local environment, it seems unlikely that a

single gene is responsible for promoting or preventing

growth. For example, regeneration of spinal-cord axons

of dorsal root ganglia occurs after transgenic expres-

sion of GAP-43 and CAP-23 in combination, but not

when either one of these genes was expressed on its

own (Bomze et al. 2001). Nevertheless, the existence

of a Mastergene switching on a specific genetic pro-

gram cannot be excluded.

A more ambitious approach is to make a survey of all

the genes that are expressed in regenerating and non-

regenerating tissue. Differentially expressed genes

can then be fished out by comparing various samples,

such as adult and immature spinal cord, PNS and CNS,

injured and uninjured tissue. Such comparisons provide

a global view of the genes that might enhance or

inhibit neurite outgrowth.

Rarity of genes involved in regeneration

Although it may seem to be a rather simple procedure,

in practice tracking those genes that play a part in CNS

regeneration is like looking for a needle in a haystack;

indeed it is worse because neither the shape of the

needles or their numbers are known. The human brain

and spinal cord have the greatest complexity of gene

expression of any region of the body, reflecting the

diverse functions of neurons and glia. Saturation and

kinetic studies indicate that the mRNA population

expressed by a single cell is made up of 20 00030 000

distinct mRNAs, with approximately 99% being rare

(Hahn & Laird, 1971; Grouse et al. 1972; Hahn et al.

1978; Croizat et al. 1979). These 99% represent less than

30% of the total mRNA mass. Most of the mRNA

species that are isolated from a brain or a spinal-cord

sample are therefore medium- to high-abundance

transcripts. Their corresponding genes are likely to

encode proteins necessary for basic biochemical path-

ways, housekeeping proteins that are common to every

cell in the body. Thus, it is reasonable to suppose that

genes involved in the process of regeneration are of

low abundance.

A further dilution of the genes of interest is likely to

occur when experiments are performed in immature

preparations (see below). Tremendous changes in

gene expression occur in the developing CNS. They are

responsible for the myriad processes that may again be

different from regeneration.

Methods for studying differential gene

expression

A number of techniques have been developed in recent

years for the identification of differentially expressed

genes that could be involved in CNS regeneration.

In the following we describe briefly the underlying

principles for techniques such as: differential message

display, serial analysis of gene expression (SAGE),

large-scale generation of expressed sequence tags (ESTs)

from cDNA libraries, cDNA microarray screening, and

various subtractive hybridization strategies.

Differential display (DD) has been widely used to

detect and isolate genes that respond to growth

factors, developmentally regulated genes and genes

whose expression correlates with certain diseases

(Pazman et al. 2000; Fu, 2002). The general strategy is to

amplify partial cDNA sequences from subsets of mRNA

by reverse transcription and PCR, and then to display

these short cDNA fragments on a sequencing gel to

obtain an mRNA fingerprint. Differential display is

one of the most suitable methods for tracking novel

genes (Gratsch, 2002), although the generation of false

positives remains one of its major drawbacks (Broude,

2002). Su et al. (1997) used this method to demonstrate

the up-regulation of axonal transport molecules dur-

ing motor nerve regeneration in the mouse.

Large-scale sequencing of expressed sequence

tags (ESTs) is another approach for studying mRNA


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expression (Okubo et al. 1992). ESTs are randomly

selected clones sequenced from cDNA libraries. They are

short, partial DNA sequences, as opposed to full-length

clones. The first collection of ESTs was initiated in 1991

as part of the human genome project (Adams et al.

1991). Each cDNA library is constructed from total

RNA or poly(A)+ RNA derived from a specific tissue or

cell, and thus the library represents genes expressed

in the original cellular population. Overlapping EST

sequences can be assembled, allowing the complete

mRNA sequence to be determined. The analysis of EST

data revealed answers to fundamental questions such

as which genes are most abundantly expressed (e.g.

-actin,

- and

-tubulin) and the amount of regional

variation of expression within brain regions. There are

currently over 1.5 million human EST sequences depos-

ited in the publicly available database of ESTs provided

by the National Center for Biotechnology Information

(NCBI).

Serial analysis of gene expression (SAGE) is a sequence-

based strategy that allows the simultaneous analysis

of a large number of transcripts (Velculescu et al. 1995;

Scott & Chrast, 2001; Yamamoto et al. 2001). Typically,

cDNA fragments are generated with short tags (usually

from 8 to 11 base pairs) at the 3-end of each transcript,

concatenated and sequenced. Analysis of tags allows

the cataloging of thousands of genes expressed from a

tissue source, including a quantitative estimate of gene

expression. However, this straightforward technique

requires high-throughput sequencing facilities. SAGE

has been successfully applied to analyse changes in

gene expression in hippocampus during the early epil-

eptic phase in the rat (Hendriksen et al. 2001). Reports

of its use to isolate genes differentially expressed

after spinal cord injury are few to date.

DNA microarrays represent a highly effective tool for

studying gene expression profiles and genome compo-

sition. The principle is to immobilize hundreds or thou-

sands of cDNAs or nucleotides corresponding to ESTs

or known genes on a solid support, such as nylon mem-

brane or glass microscope slide (Lockhart et al. 1996;

Freed & Vawter, 2001; Lobenhofer et al. 2001). To

determine the difference in gene expression, labelled

cDNAs or oligonucleotides are hybridized to the array.

After hybridization and imaging, the pattern of gene

expression is quantified. The principal advantages of

this approach are the immediate interpretation of the

results and the possibility of clustering genes into tem-

poral and spatial expression patterns. One drawback is

the difficulty in calibrating and interpreting hybridiza-

tion signals of weakly expressed genes. Microarray

analysis after spinal cord injury (Carmel et al. 2001;

Fan et al. 2001) or brain injury (Matzilevich et al. 2002)

has proven useful to describe global patterns of gene

expression and to cluster genes into temporal and

spatial expression groups.

Subtractive hybridization (SH) is a valuable tech-

nique for the isolation of differentially expressed genes.

Although there are numerous protocols for SH, the

principles remain the same (Diatchenko et al. 1996).

Two cDNA populations are generated from two dif-

ferent RNA sources. For example, in suppressive PCR-

based subtractive hybridization, adaptors are ligated

to a tester pool of cDNA. A second pool (driver

cDNA) is added in excess to hybridize, and only genes

that are up-regulated in the tester pool can then be

selectively amplified by PCR. This technique requires

only small amounts of RNA, and is well suited for iden-

tifying rare transcripts and novel genes. Subtractive

hybridization is one of the most commonly used meth-

ods for identifying differentially expressed mRNAs

and has been successfully applied in studies of develop-

ment, cancer, growth-factor-stimulated cells (Feng

& Liau, 1993; Cho et al. 1998; Davidson & Swalla, 2001;

Shridhar et al. 2002). Few laboratories have used it

to analyse genes expressed after injury to the CNS.

An exception is the series of subtractions made from

regenerating identified nerve cells in the leech. Black-

shaw and colleagues (Korneev et al. 1997) have made

a subtractive library from regenerating Retzius cells of

the leech, which revealed novel sequences as well as

genes such as alpha-tubulin, synapsin and calmodulin,

up-regulated in other species, during nerve regenera-

tion. A remarkable feature of this study was that the

differences in expression were analysed in individual

identified neurons from lesioned and unlesioned nerve

cords.

A common feature of all methods for analysis of

differences in gene expression is the generation of

large amounts of data (often hundreds or thousands

of genes). The over- or under-expressed sequences are

then compared with sequences from public databases.

Genome sequencing programmes carried out for sev-

eral organisms including human and mouse have pro-

vided an immense amount of sequence information in

databanks. Techniques are now available for studying

the expression profiles of previously insufficiently char-

acterized genes.


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Methods for selecting genes that are relevant

for regeneration

A major difficulty arises once the genes that are different-

ially expressed have been identified. Which of the genes

that stand out are actually important and how can one

distinguish genes that play a part in regeneration from

unrelated genes that have slipped through the subtrac-

tion procedure? With several hundred candidates, the

number to be tested experimentally must be narrowed

down. Procedures for focusing on relevant genes include

multiple screenings and hybridizations, or repeated

experiments under various conditions and with different

tissue samples. For example, a specific sequence may be-

come significant if it appears in screens made of an inver-

tebrate and a vertebrate, or in regenerating spinal cord

and optic nerve. An interesting approach for focusing

on putative candidates is to combine some of the tech-

niques described above. For example, the hundreds

of clones obtained after subtractive hybridization can

be spotted onto microchips and can then be analysed

using microarray procedures. This strategy has proved

to be useful in identifying genes in the nervous system

(Colantuoni et al. 2001; Kornblum & Geschwind, 2001;

Dougherty & Geschwind, 2002). We describe in greater

detail methods for narrowing the search in a later

section devoted to work on the neonatal opossum.

Possible discrepancies between RNA andprotein levels

Differences in abundance of a specific mRNA between

two samples do not necessarily reflect equivalent quantita-

tive differences in protein levels. Although it is generally

true that the level of a protein follows a change of

its encoding mRNA, the extent and kinetics are often

unpredictable. The activity of the proteins encoded by

mRNAs is regulated at several levels beyond their mRNA

or protein expression by their subcellular localization and

by the extent to which they are post-translationally mod-

ified. Neither of these parameters is revealed by measure-

ments of mRNA abundance. Twiss et al. (2000) showed

that under some circumstances neurons can be primed to

rapidly regenerate injured processes independently of new

gene expression, translating already existing messeng-

ers. Such regulation at the level of translation would

not be detected in assays of differential gene expression.

Large-scale analysis similar to that made at the mRNA

level can be undertaken directly with proteins, by

comparing patterns obtained from two-dimensional

(2D) protein gels. One strength of 2D gel electrophoresis

is its ability to measure quantitatively several thousand

proteins in a single sample, with subsequent identifica-

tion by mass spectrometry (reviewed by Fey & Larsen,

2001; Lilley et al. 2002). In this way one can compare quan-

tities of proteins in related samples, such as injured and

uninjured tissue. A major difficulty that remains is the

detection of low-abundance proteins by 2D gels, until

the resolution and sensitivity of the method are

improved.

In the near future one can expect to have available

protein microarrays to measure the properties of

thousands of proteins simultaneously. Proteinprotein

interactions analysed with fluorescent probes will be

analogous to DNA microarrays. An important applica-

tion of this powerful technique will be to profile the

proteins in cells under different conditions, as for example

in regenerating and non-regenerating neurons.

Localization in CNS of candidate genes

relevant for regeneration

Analysis of differential gene (or protein) expression

is a first step in the search for genes that play a part in

promoting or blocking regeneration. Identification

of putative candidates does not, however, indicate at

what sites they are expressed in the original tissue. In

situ

hybridization is important for answering funda-

mental questions of distribution: Does the gene show

a widespread expression? If not, in which areas of

the CNS can the product be found? Is it expressed by

neurons or by glial cells and in which structures (mem-

branes, extracellular space, internal organelles) does

the pattern of expression change after an injury?

The procedure for in situ

hybridization involves

screening sections or the entire CNS by labelled probes

complementary to the mRNA of interest. A risk is that

one might discard genes that at first seem uninterest-

ing. This is exemplified by Nogo, a member of the retic-

ular family of transmembrane proteins. Nogo mRNA is

widely expressed in fetal, developing and adult nervous

system of rat and human (Josephson et al. 2001), and

its levels of expression are not significantly changed

after lesions to the cortex or spinal cord (Huber et al.

2002). From those characteristics one might exclude

Nogo from a list of interesting candidates, and yet it is

known to be an inhibitor of regeneration in the CNS

(Huber & Schwab, 2000; Woolf, 2003).


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Assessment of the functional role of unknown

genes and proteins

Many genes that are detected by screening of regener-

ating and non-regenerating preparations have no known

function. The analysis of the full-length sequence of

such candidates with bioinformatic techniques is a first

step towards understanding what role they might play

in regeneration. Valuable information can be obtained

from the gene sequence that is responsible for direct-

ing the encoded protein to its cellular or extracellular

location (e.g. nucleus, organelles, plasma membrane).

Translation of the DNA sequence of a gene into the

amino acid sequence of the encoded protein can

provide clues to its structure and function.

To gain further insight into the functional role of

a candidate, numerous tests can be performed on cells

in culture. One approach is to measure effects of a

candidate gene on growth, either by over-expressing

genes that promote regeneration (gain of function)

or by blocking expression of genes that inhibit regenera-

tion (loss of function).

For example, immortalized cell lines such as PC12 cells

can be engineered to produce a protein by introducing

its coding gene into cells in culture. One method for trans-

fecting cells is to use a gene gun, which bombards

them with small gold particles coated with the DNA of

interest (McAllister, 2000; Sato et al. 2000). Alterna-

tively, genes can be delivered by trapping the DNA into

liposomes, which are taken up by the cell (Kofler et al.

1998), or by precipitating DNA with calcium phosphate.

Proteins can be studied in greater detail by introducing

the recombinant gene into cells together with green

fluorescent protein (GFP) (reviewed by Van Roessel &

Brand, 2002). The label makes it possible to follow the

gene fusion product in the living cell. Specific localiza-

tion and co-localization with other known proteins can

provide information about the role of the protein in

specific pathways of neurons and glial cells. For exam-

ple, a recombinant protein expressed in a defined cell

line may promote cell cycle arrest or loss of motility.

Methods by which to block the expression of a gene

include the introduction of antisense oligonucleotides

that hybridize with the RNA in the cell and hinder their

translation into proteins (reviewed by Lebedeva et al.

2000; Sazani et al. 2002). Whenever they are available,

antibodies that specifically block the action of an iden-

tified protein can provide clear evidence for function.

A recent effective method for silencing the expression

of a target gene is to introduce vectors that express

small interfering RNAs (known as siRNAs). siRNAs are

short (about 20 nucleotides), double-stranded mole-

cules. Once inside the cell the siRNA strands unwind

and become associated with complementary RNA

molecules. This provokes the cleavage and destruction of

target mRNA and inhibition of protein synthesis by the

cell (Scherr et al. 2003; Sorensen et al. 2003).

A further step is modification of specific sequences to

identify functional domains of the protein. For exam-

ple, deletions or mutations can define regions of genes

important for nuclear importexport or degradation.

A two-hybrid system in yeast can also be used to pro-

vide information about function. By this method it

is possible to investigate proteinprotein interactions

and to identify molecular partners (reviewed by

Causier & Davies, 2002). The two-hybrid system exploits

properties of transcription factors that have two sepa-

rate functional domains. One is a DNA binding domain

that binds to the DNA of the promoter and the other

an activation domain that binds to the transcription

apparatus. Each separate domain can be fused as a

hybrid to a second protein without changing the basic

properties of the transcription factor. When an interac-

tion occurs, the DNA binding domain and the activa-

tion domain of the transcription factor come close

together and can activate transcription of a reporter

gene encoding for a selection marker. In this way the

protein of interest can be tested for interactions with

several possible partners.

Experiments performed on cells in vitro

are indispen-

sable to assay the role of candidate molecules and their

possible involvement in regeneration. Observation of

fibre outgrowth in vitro

does not, however, imply that

similar events will occur in the animal, let alone that

growth will lead to functional recovery. Although most

tests for regeneration are made in vivo

in rats and mice,

experiments performed in Monodelphis

have demon-

strated the usefulness of this mammal for studying

regeneration.

The opossum as a preparation for molecular

studies of regeneration

Properties of neonatal opossum CNS

In the following sections we show how the approaches

described above have been applied to the CNS of the

newborn opossum, Monodelphis domestica.

The emphasis


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is on work in progress with certain clear advances but

no defined end-point in sight.

In newborn opossum pups the immature CNS is so

small that it can be removed in its entirety and main-

tained in tissue culture for up to 2 weeks (Stewart et al.

1991; Treherne et al. 1992; Mllgard et al. 1994).

During that time in culture, cell division goes on, the

isolated CNS maintains reflex responses to electrical

stimulation, and the fine structure of the nervous

system is preserved (Nicholls et al. 1990; Eugenin &

Nicholls, 1997). Axons regenerate rapidly and reliably

through spinal cord lesions (Nicholls et al. 1994;

Saunders et al. 1995; reviewed by Nicholls & Saunders,

1996). Amino acids, transmitters and larger molecules

such as antibodies rapidly penetrate the isolated tissue

from the bathing fluid (Zou et al. 1991; Varga et al.

1995). These properties facilitate detailed analysis of

mechanisms that promote or block regeneration.

After a lesion to the spinal cord in the immature animal,

regeneration is so complete that functions such as coord-

inated walking and swimming are virtually normal

(Saunders et al. 1998). In adult opossums, as in other

mammals, the CNS does not regenerate. The transition

point has been shown to occur in the early days of post-

natal life. At 9 days of age regeneration occurs reliably.

However, it is no longer possible in cervical spinal cord

segments of animals aged 12 days or more. Owing to a

rostro-caudal gradient in development, the less mature

lumbar segments still show regeneration in animals up

to 17 days. The narrow window of time during which

regeneration stops being possible is particularly useful

for studies of differences in gene expression. The space

of time between 9 and 12 days after birth is long

enough for expression of differences in genes of inter-

est, yet short enough to avoid the over-representation

of genes related to development. The presence of both

mature (cervical) and immature (lumbar) parts in the

spinal cord at 12 days enables one to perform addi-

tional comparisons between regenerating and non-

regenerating tissue of a single animal. In this way

differences in gene expression related to regeneration

can be analysed at different times (9 days vs. 12 days)

and in different places (cervical vs. lumbar at 12 days).

Analysis of genes expressed in regenerating and non-

regenerating spinal cords

We conducted subtractive hybridization experiments

to detect genes that are up- or down-regulated in the

opossum at different ages and in different parts of the

spinal cord. Both forward and backward subtractions

were done between cervical spinal cord at 9 days (can

regenerate) and at 12 days (cannot regenerate). Other

subtractions were done between 12-day lumbar (can

regenerate) and cervical parts at the same age (cannot

regenerate). Table 1 provides a summary of the various

subtractions.

cDNAs from the two first subtractions (C9C12 and

C12C9) were cloned in E. coli, resulting in about

3000 recombinant clones. A few clones were chosen

at random from those libraries and sequenced. About

half of these represented novel genes whose function

is as yet unknown. Sequencing also revealed numerous

interesting genes that could be involved in regenera-

tion. They coded for transcription factors, receptors,

signal transducing molecules, structural proteins and

extracellular matrix proteins, among others. Other

genes, as expected, were responsible for house-

keeping functions (for example, enzymes and struc-

tural proteins).

Narrowing down the search

To separate non-relevant genes from those involved in

regeneration we designed experiments based on cross-

hybridizations between different subtractions. The aim

of those experiments was to find common clones in the

various subtractions. All clones from a pair of subtrac-

tive libraries were arrayed onto nylon membranes and

hybridized with radioactively labelled probes derived

Table 1. Subtractions to reveal genes that are differentially

expressed in opossum spinal cord regions that do and do not

regenerate


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from the other subtractions. This process reduced the

number of potentially involved genes by a factor of

about 10. Those remaining included (among others):

reelin, laminin receptor, TATA-box binding protein,

MAP1b, and these were all more abundant at 9 days.

At 12 days, genes selectively expressed included PAX-6,

semaphorin receptor and GABA(A) receptor-associated

protein. Novel genes were represented in both catego-

ries. In contrast with other studies focusing on one gene

or protein, this represents an approach based on broad

screenings that can lead to unbiased identification

of potentially interesting genes in the spinal cord at

different ages.

Comparison with other databases can help in the

functional identification of relevant molecules. A strik-

ing feature is that opossum gene sequences show high

similarities to those of mouse, rat or human deposited

in databanks. EST and microarrays are now available

for another marsupial, the Tammar wallaby.

What remains is to determine for each gene: the

location of expression, its abundance, the level of

protein and its effect on the regeneration of intact spinal

cord axons after injury.

Concluding remarks

Studies of differential gene expression in injured spinal

cord provide a picture of molecular events associated

with processes of regeneration. Many steps, however,

are necessary before it can be known whether this

approach will lead to cures for patients with spinal cord

lesions. Overoptimistic claims of cures that are just

around the corner can have devastating consequences

for patients with spinal cord injuries (Pearson, 2003).

Animal tests, and extensive clinical trials for efficacy

and side-effects involve expenditure of immense sums

of money and large numbers of skilled manpower

hours. It seems possible that treatments might

arise sooner from the approach not discussed in detail

here, namely the testing of likely molecules without

any comprehensive screen. Although the large-scale

screening described here may well produce no miracle

gene, what it will do is increase basic knowledge. Our

underlying assumption is that, just as it is easier to

repair a watch once one knows the way it works, anal-

ysis of mechanisms responsible for failure of regenera-

tion in adult mammalian spinal cord will be facilitated

by an understanding of molecular mechanisms that

promote and inhibit neuron outgrowth.

Acknowledgements

We thank Dr Marco Stebel and Mr Salvatore Guarino

for their diligent and skilled care of the animal colony.

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