Ablation of adhesion molecule L1 in mice favours Schwann cell proliferation and functional recovery...

BRAINA JOURNAL OF NEUROLOGY

Ablation of adhesion molecule L1 in mice favoursSchwann cell proliferation and functional recoveryafter peripheral nerve injuryDaria Guseva,1 Doychin N. Angelov,2 Andrey Irintchev1,3 and Melitta Schachner1,4,5

1 Zentrum fur Molekulare Neurobiologie, Universitat Hamburg, Hamburg, Germany

2 Anatomical Institute I, University of Cologne, Cologne, Germany

3 Department of Otorhinolaryngology, Friedrich-Schiller-University Jena, Jena, Germany

4 W. M. Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, NJ, USA

5 Center for Neuroscience, Shantou University Medical College, Shantou, China

Correspondence to: Melitta Schachner,

Zentrum fur Molekulare Neurobiologie,

Universitat Hamburg,

Martinistrasse 52,

D-20246 Hamburg,

Germany

E-mail: [email protected]

Correspondence may also be addressed to: Andrey Irintchev,

Department of Otorhinolaryngology,

Friedrich-Schiller-University Jena,

Lessingstrasse 2, D-07740 Jena,

Germany

E-mail: [email protected]

The adhesion molecule L1 is one of the few adhesion molecules known to be beneficial for repair processes in the adult central

nervous system of vertebrates by promoting axonal growth and neuronal survival. In the peripheral nervous system, L1 is

up-regulated by myelination-competent Schwann cells and regenerating axons after nerve damage but its functional role has

remained unknown. Here we tested the hypothesis that L1 is, as in the central nervous system, beneficial for nerve regeneration

in the peripheral nervous system by performing combined functional and histological analyses of adult L1-deficient mice

(L1y/�) and wild-type (L1y/+) littermates. Contrary to our hypothesis, quantitative video-based motion analysis revealed

better locomotor recovery in L1y/� than in L1y/+ mice at 4–12 weeks after transection and surgical repair of the femoral

nerve. Motoneuron regeneration in L1y/� mice was also enhanced as indicated by attenuated post-traumatic loss of motoneur-

ons, enhanced precision of motor reinnervation, larger cell bodies of regenerated motoneurons and diminished loss of inhibitory

synaptic input to motoneurons. In search of mechanisms underlying the observed effects, we analysed peripheral nerves at short

time-periods (3–14 days) after transection and found that Schwann cell proliferation is strongly augmented in L1y/� versus

L1y/+ mice. L1-deficient Schwann cells showed increased proliferation than wild-type Schwann cells, both in vivo and in vitro.

These findings suggest a novel role for L1 in nerve regeneration. We propose that L1 negatively regulates Schwann cell

proliferation after nerve damage, which in turn restricts functional recovery by limiting the trophic support for regenerating

motoneurons.

Keywords: adhesion molecule L1; femoral nerve; Schwann cells; peripheral nerve regeneration; motoneuron

doi:10.1093/brain/awp160 Brain 2009: 132; 2180–2195 | 2180

Received December 14, 2008. Revised April 25, 2009. Accepted May 12, 2009. Advance Access publication June 18, 2009

� The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

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IntroductionThe neural cell adhesion molecule L1 is a glycoprotein of the

immunoglobulin superfamily expressed in most, if not all, neurons

in the central nervous system (CNS). During CNS development,

L1 is targeted to the surface of developing axons and growth

cones and mediates outgrowth, adhesion, fasciculation and

guidance of axons as well as neuronal migration and survival

(Hortsch, 2003; Maness and Schachner, 2007). Perturbations

of these processes by mutations in the L1 gene lead to brain

malformations and dysfunctions in humans and mice (Maness

and Schachner, 2007). L1 is also implicated in CNS regeneration

in adult vertebrates. After spinal cord injury in zebrafish, the

expression of L1.1, a homolog of the mammalian L1, is increased

in successfully regenerating descending axons but not in ascending

projections that fail to regenerate (Becker et al., 2005). Also,

morpholino-mediated reduction of L1.1 expression leads to

deficient regeneration (Becker et al., 2004). Similar to poorly

regenerating neurons in zebrafish, mammalian neurons fail to

up-regulate L1 expression after CNS trauma (Styren et al., 1995;

Mason et al., 2003) but L1 expression is up-regulated in CNS

neurons when a peripheral nervous system (PNS) nerve bridge is

introduced (Anderson et al., 1998; Woolhead et al., 1998) or L1 is

overexpressed in Purkinje cells (Zhang et al., 2005). When L1

expression in neurons and glia is induced by viral transduction

(Chen et al., 2007), when L1 overexpressing embryonic stem

cells are transplanted (Chen et al., 2005), or when the axonal

growth-inhibiting environment is enriched in exogenous L1

(Roonprapunt et al., 2003), recovery from spinal cord injury in

rodents is enhanced. These findings clearly indicate that L1 is a

molecule promoting CNS regeneration.

In the PNS, L1 is expressed in axons and Schwann cells during

embryonic and early postnatal development and remains

expressed by non-myelinating Schwann cells in the adult

(Faissner et al., 1984; Nieke and Schachner, 1985; Martini and

Schachner, 1986, 1988). Constitutive ablation of L1 in mice

does not lead to apparent PNS disorders. Neural crest cell

migration is reduced in the developing gut of L1y/� mice but in

the adult the entire gastrointestinal tract is colonized by enteric

neurons (Anderson et al., 2006). It has, thus, been suggested that

L1 mutations contribute to Hirschsprung’s disease, a human con-

dition in which enteric neurons are absent from the distal bowel.

L1y/� mice have mild impairments in locomotion and severely

reduced pain sensitivity, but these deficits have been attributed

to abnormal function of the CNS (Thelin et al., 2003; Jakeman

et al., 2006). Sprouting of calcitonin gene-related peptide positive

sensory axons into the lesion site, which spontaneously occurs

after spinal cord injury and is associated with development of

pain, is reduced in L1y/� mice (Deumens et al., 2007). In periph-

eral nerves of adult L1y/� mice, myelination is normal but

the ensheathment of non-myelinated axons is disturbed

(Dahme et al., 1997; Haney et al., 1999). Peripheral nerve

injury in adult rodents leads to up-regulation of L1 expression in

myelination-competent Schwann cells distal to the injury site and

enhanced expression in regenerating axons (Daniloff et al., 1986;

Martini and Schachner, 1988; Martini, 1994). However, the

functional relevance of this up-regulation has not been elucidated.

We speculated, considering the regeneration-promoting properties

of L1 in the CNS, that L1 is beneficial also for PNS regeneration.

To test this hypothesis, we analysed peripheral nerve regeneration

in L1y/� mice and wild-type (L1y/+) littermates using a well-

established paradigm, femoral nerve transection and repair in

adult mice combined with quantitative video-based analysis of

motor recovery and cellular correlates of the functional outcome

(Irintchev et al., 2005a; Eberhardt et al., 2006; Simova et al.,

2006; Ahlborn et al., 2007). Contrary to our expectation, how-

ever, we found superior outcome of nerve repair in L1y/� mice

suggesting that ablation of L1 leading to enhanced proliferation of

Schwann cells is beneficial for peripheral nerve regeneration.

Materials and Methods

AnimalsWe used 12-week-old L1-deficient (L1y/�) mice from a colony

generated by insertion of a tetracycline-controlled transactivator into

the second exon of the L1 gene (Rolf et al., 2001; Saghatelyan et al.,

2004). The L1y/� mice were males carrying the mutant allele of the

X-chromosome-linked L1 gene. The wild-type (L1y/+) mice used as

controls were male littermates of the L1y/� mice carrying the

wild-type L1 allele. Unlike the L1y/� mice generated initially in

our laboratory, which express residual amounts of L1 (Dahme

et al., 1997), these L1y/� mice were L1 null mice. Importantly,

the L1y/� mice used here were maintained on a 129SvJ/NMRI

genetic background which attenuates some abnormal features

seen in L1y/� mice bred on a C57BL/6J background such as overt

hydrocephalus, low body mass (60–80% of L1y/+ mice), poor breed-

ing and high mortality of the L1y/� offspring within the first 2 months

after birth. Before and after the experiments, the mice were kept on

standard laboratory food and tap water ad libitum, with an artificial

12 h light/dark cycle. All treatments of the animals were conducted

in accordance with the German and European Community laws on

protection of experimental animals.

Experimental designIn Experiment 1 (see ‘Nerve regeneration’ below), we analysed func-

tional recovery in L1y/� mice and L1y/+ littermates during a 12-week

recovery period following femoral nerve injury (Fig. 1A). Subsequently,

these animals were subjected to retrograde labelling of regenerated

motoneurons (Fig. 1B) and, following a survival period of 1 week,

spinal cords and both injured and intact contralateral femoral nerves

were sampled for histological analyses (Fig. 1A). In addition to these

‘injured’ mice, i.e. subjected to femoral nerve transection and surgical

repair, groups of control ‘non-injured’ L1y/� and L1y/+ mice

were included in this experiment. These mice were subjected only to

retrograde tracing (Fig. 1C) and used for morphological analysis of

quadriceps motoneurons. In Experiment 2 (‘Cell proliferation during

Wallerian degeneration’, Fig. 1D), the femoral nerve of L1y/� and

L1y/+ mice was transected and 3, 5, 7 and 14 days later the distal

nerve segments were harvested for analyses of cell proliferation.

Finally, in Experiment 3 an in vitro analysis was performed to

investigate the proliferation of Schwann cells derived from 7-day-old

L1y/� and L1y/+ mice.

L1 and nerve regeneration Brain 2009: 132; 2180–2195 | 2181



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Nerve regeneration

Transection and surgical repair of the femoral nerve

Surgery was performed as described previously (Simova et al., 2006).

The mice were anesthetized by intraperitoneal injections of 0.4 mg/kg

fentanyl (Fentanyl-Janssen, Janssen, Neuss, Germany), 10 mg/kg

droperidol (Dehydrobenzperidol, Janssen) and 5 mg/kg diazepam

(Valium 10 Roche, Hoffman – La Roche, Grenzach-Wyhlen,

Germany). The right femoral nerve was exposed and transected

approximately 3 mm proximal to the bifurcation of the saphenous

and quadriceps branches (Fig. 1B). The cut ends of the nerve were

inserted into a polyethylene tube (3 mm length, 0.58 mm inner dia-

meter, Becton Dickinson, Heidelberg, Germany) and fixed with a single

epineural 11-0 nylon stitch (Ethicon, Norderstedt, Germany) so that a

2 mm gap was present between the proximal and distal nerve stumps.

Figure 1 Schematic representation of the experimental design. In Experiment 1, L1y/� and L1y/+ mice were subjected to unilateral

transection and repair of the femoral nerve (at 0 weeks in A) proximal to the bifurcation of the sensory saphenous branch and the

mixed quadriceps branch (‘Nerve transection’ in B). Recovery of function was followed by repeated video recordings over a 12-week

period (A, arrowheads). At 12 weeks after injury, retrograde labelling of regenerated motoneurons was performed by transecting the

two nerve branches distal to the bifurcation and application of two different fluorescence dyes, fluoro-ruby and Fast Blue to the

saphenous and the quadriceps branch, respectively (B, ‘Retrograde labelling’). One week later, femoral nerves and spinal cords were

sampled for morphological analyses (A, elbow arrow). Additional control mice were subjected to retrograde labelling only (C) and

served as controls in the morphological analyses. The circles on the left hand side in C represent motoneurons with axons targeting the

quadriceps branch only and thus retrogradely labelled by Fast Blue but not with fluoro-ruby. After nerve regeneration, motor axons

regenerate correctly to the quadriceps branch or aberrantly to the saphenous branch, or both nerve branches. Application of the two

tracers allows the detection of both aberrantly and correctly projecting motoneurons (circles on the left hand side in B). The arrow at

‘Analysis of axons’ points to the approximate level at which axons were analysed in intact and regenerated nerves. In Experiment 2,

mice of both genotypes were subjected to femoral nerve transection (0 day, D) and femoral nerves were sampled for analysis of

Schwann cell proliferation at 3, 5, 7 and 14 days after injury (elbow arrows). Quantitative assessment of Schwann cell proliferation was

performed at the level indicated in C. For further details see text.

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The tube was filled with phosphate buffered saline (PBS, pH 7.4) and

the skin was closed with 6-0 sutures (Ethicon). To prevent hypo-

thermia, the mice were then kept in a warm room (35�C) until full

recovery from anaesthesia.

Analysis of motor function

Functional analysis was performed over a time-period of 12 weeks

(Fig. 1A) using a quantitative video-based approach developed in

our laboratory (single-frame motion analysis, Irintchev et al., 2005a).

To evaluate locomotor function, mice were accustomed, in 3–4 trials,

to beam-walking prior to operation. In this test, the animal walks

voluntarily from one end of a horizontal beam (length 1000 mm,

width 40 mm) towards its home cage located at the other end of

the beam. For all mice, a rear view of one walking trial was captured

once prior to the operation and at different time-points after surgery

(Fig. 1A) with a high-speed camera (A602fc, Basler, Ahrensburg,

Germany) at 100 frames per sec and stored on a personal computer

in Audio Video Interleaved (AVI) format. The video sequences were

examined using SIMI-Motion 7.0 software (SIMI Reality Motion

Systems, Unterschleissheim, Germany). Selected frames in which the

animals were seen in defined phases of the step cycle were used for

measurements of two parameters: the heels-tail angle (HTA, Fig. 2A

and C) and the foot-base angle (FBA, Fig. 2B and D) as described

previously (Irintchev et al., 2005a). Both parameters are directly

related to the ability of the quadriceps muscle to keep the knee joint

extended during contralateral swing phases. As a relative measure

of functional recovery at different time-points after nerve injury, we

calculated the stance recovery index (RI), which is a mean of the RI for

the heels-tail angle and the foot-base angle. The index for each angle

is calculated in percent as:

RI ¼ ½ðXreinn � XdenÞ=ðXpre � XdenÞ� � 100,

where Xpre, Xden and Xreinn are values prior to operation, during the

state of denervation (7 days after injury), and at any given time-point

during reinnervation, respectively.

A third parameter, the limb protraction length ratio (PLR), was

evaluated from video-recordings of voluntary pursuit movements of

the mice (Irintchev et al., 2005a). The mouse, when held by its tail

and allowed to grasp a pencil with its forepaws, tries to catch the

object with its hindpaws and extends both hindlimbs simultaneously.

In non-injured animals, the relative length of the two extremities, as

estimated by lines connecting the most distal mid-point of the extrem-

ity with the anus, is approximately equal and the PLR (ratio of

the right to left limb length) is close to 1.0. After denervation,

the limb cannot be extended maximally and the PLR becomes

significantly 41.0.

Retrograde labelling of motoneurons

At 12 weeks after nerve transection the animals were anaesthetized

with fentanyl, droperidol and diazepam for retrograde labelling

of regenerated motoneurons (Fig. 1A, Simova et al., 2006). After

exposure of the right femoral nerve, a piece of Parafilm (Pechiney

Plastic Packaging, Chicago, IL, USA) was inserted underneath

the nerve and the two nerve branches were transected approxi-

mately 5 mm distal to the bifurcation (Fig. 1B). Fluorescence retro-

Figure 2 Analysis of motor function. (A–D) Single video

frames from recordings of beam walking of an L1y/� and an

L1y/+ mouse at prior to femoral nerve repair (0 day). In A and

C the animals are seen at mid-stance of the right hind limb and

maximum altitude of the contralateral swing. Such video

frames are used to measure the heels-tail angle as shown by

the lines drawn in both panels. Note the lower position of right

heel in A compared with C. This difference results in a larger

heels-tail angle (measure from the dorsal aspect) in the L1y/�

mouse compared with the L1y/+ mouse. B and D show video

frames in which the right paw of the same mice at take-off

position. Such frames are used for measuring the foot-base

angle shown by lines drawn in the panels. A more pronounced

external rotation of the paw in B compared with D results in a

smaller foot-base angle (measured from the medial aspect) in

the L1y/� mouse compared with the L1y/+ mouse. (E–H)

Time-course of motor recovery after femoral nerve lesion.

Mean values� SEM of heels-tail angles (E), foot-base angles

(F) and limb PLRs (G) at different time-points after femoral

nerve transection in L1y/� and L1y/+ mice. Preoperative

values are plotted at 0 day. Number of animals studied per

group are indicated in E. (H) Shows individual animal values of

the stance RI calculated for the heels-tail and the foot-base

angles (see Materials and methods section for details) at 12

weeks after injury. A RI of 100% indicates complete recovery.

Asterisk indicates significant difference (P50.05, one-way

ANOVA with Tukey’s post hoc test) between the genotypes at

given time-point.




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grade tracers were applied to the cut nerve ends in powder form:

fluoro-ruby (tetramethylrhodamine dextran, Molecular Probes,

Leiden, The Netherlands) to the saphenous (cutaneous) branch (exclu-

sively sensory branch), Fast Blue (EMS-Chemie, Großumstadt,

Germany) to the quadriceps (muscle) branch (mixed nerve containing

sensory and motor axons). Thirty minutes after dye application, the

nerve stumps were rinsed with PBS and the wound was closed. This

labelling procedure allows visualization of all femoralis motoneurons

that have successfully regenerated their axons beyond the lesion

site. In addition, it is possible to differentiate between motoneurons

with axons correctly projecting into the quadriceps branch (blue circle

in Fig. 1B) and aberrantly projecting motoneurons sending axons to

the saphenous branch only or to both the saphenous and the quad-

riceps branch (red and purple circle in Fig. 1B, respectively). The same

labelling procedure applied to ‘non-injured’ L1y/� and L1y/+ mice, i.e.

mice which had not been subjected to nerve transection and repair

(Fig. 1C), allows to estimate the normal number of quadriceps

motoneurons all of which project to the quadriceps branch only

(blue circles in Fig. 1C).

Preparation of tissue for morphological analyses

One week after retrograde labelling (Fig. 1A) the mice were

anesthetized by intraperitoneal injection of 16% sodium pentobarbital

solution (Narcoren�, Merial, Hallbergmoos, Germany, 5 ml/g body

weight) and were transcardially perfused with 4% formaldehyde in

0.1 M sodium cacodylate buffer, pH 7.3. The lumbar spinal cord was

removed, post-fixed overnight at 4�C and then immersed in 15%

sucrose solution in 0.1 M cacodylate buffer, pH 7.3, for 1 day at

4�C. Afterwards the tissue was frozen for 2 min in 2-methyl-butane

(isopentane, Carl Roth, Karlsruhe, Germany) pre-cooled to �30�C.

For sectioning, the spinal cord segment was attached to a cryostat

specimen holder using TissueTek� (Sakura Finetek Europe,

Zoeterwoude, The Netherlands). Serial transverse sections of 25 mm

thickness were cut on a cryostat (Leica CM3050, Leica Instruments,

Nußloch, Germany) and picked up on SuperFrost�Plus glass slides

(Roth, Karlsruhe, Germany). The sections were air-dried for at least

1 h at room temperature (RT) and mounted in anti-quenching medium

(Fluoromount G, Southern Biotechnology Associates, Biozol, Eching,

Germany). These sections were used for counting retrogradely labelled

motoneurons.

Both injured and intact contralateral femoral nerves were dissected

from perfusion fixed animals. The tissue samples were post-fixed in

1% osmium tetroxide (Polysciences Europe, Eppelheim, Germany) in

0.1 M sodium cacodylate buffer, pH 7.3, for 1 h at RT, dehydrated and

embedded in resin according to standard protocols. One-mm thick

cross-sections from the motor and sensory nerve branches were cut

at a distance of �3 mm distal to the bifurcation (Fig. 1B) and stained

with 1% toluidine blue/1% borax in distilled water for analysis of axon

numbers, diameters, degree of myelination (g-ratios) in regenerated

and intact nerve branches.

Counting of retrogradely labelled motoneurons

Serial transverse sections of spinal cord were examined under a

fluorescence microscope (Axiophot 2, Zeiss, Oberkochen, Germany)

with appropriate filter sets. Each section was examined using a

40� objective by focusing through the section thickness starting

from the top surface. All profiles, except those visible at the top

surfaces of sections, were counted (Simova et al., 2006). The applica-

tion of this simple stereological principle prevented double counting of

labelled cells and allowed precise evaluation of cell numbers.

Immunofluorescence staining of perisomatic axonterminals

Immunofluorescence staining was performed as described (Irintchev

et al., 2005b) using commercial antibodies at optimal dilutions

(Table 1). Sections of spinal cord containing retrogradely labelled

motoneurons were freed from coverslips and mounting medium by

soaking in PBS. Antigen retrieval was performed by immersion into

0.01 M sodium citrate solution, pH 9.0, heated to 70�C in a water

bath for 30 min. Blocking of non-specific binding sites was then

Table 1 Primary antibodies used in this study

Antibody Cellular phenotypes/structures recognized

Host Code/clone Dilution Source

S-100 Mature Schwann cells Rabbit Z0311 1:15 000 DakoCytomation, Hamburg, Germany

S-100 Mature Schwann cells Mouse MAB079-1 1:1000 Chemicon, Hofheim, Germany

p75 NGF receptor Mature Schwann cells Rat ab27007 1:1000 Abcam, Cambridge, UK

Glial fibrillary acidicprotein (GFAP)

Mature Schwann cells Mouse MAB3402 1:500 Chemicon

Laminin B2/�1 Basal laminae Rat RT-795-P1ABX YYY 1:100 NeoMarkers, Fremont, CA, USA

Class III b-tubulin Axons Rabbit PRB-435P-100 1:2000 Covance, Berkeley, CA, USA

Macrophage antibodyF4/80

Macrophages Rat MCA497R 1:300 AbD Serotec, Raleigh, NC, USA

L1 Regenerating axons,non-myelinatingSchwann cells

Rat 555 1:500 Antibody produced in our laboratoryand purified by protein G affinitychromatography (Appel et al., 1995)

Caspase-3 activated Apoptotic cells Rabbit AF835 1:2000 R&D Systems, Minneapolis, MN, USA

Ki67 Proliferating cells Rabbit AB15580 1:500 Abcam

Bromodeoxyuridine(BrdU)

Proliferating cells Mouse G3G5 1:100 Developmental Hybridoma Bank, Iowa City,IA, USA

Vesicular GABAtransporter (VGAT)

Inhibitory (GABAergicand glycinergic)synaptic terminals

Mouse 131 011 1:1000 Synaptic Systems, Gottingen, Germany

Choline acetyltransferase(ChAT)

Cholinergic synapticterminals

Goat AB144P 1:100 Chemicon




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performed using PBS containing 0.2% Triton X-100 (Sigma), 0.02%

sodium azide (Merck, Darmstadt, Germany) and 5% normal goat or

normal donkey serum for 1h at RT. Incubation with primary antibodies

against VGAT or ChAT (Table 1), diluted in PBS containing 0.5%

lambda-carrageenan (Sigma) and 0.02% sodium azide, was carried

out at 4�C for 3 days. For a given antigen, all sections were stained

in the same solution kept in screw-capped staining plastic jars (capacity

35 ml, 10 slides, Roth). After washing in PBS, appropriate

Cy3-conjugated secondary antibodies (Jackson ImmunoResearch

Europe, Suffolk, UK) diluted 1:200 in PBS-carrageenan solution were

applied for 2 h at RT. After a subsequent washing in PBS, cell nuclei

were stained for 10 min at RT with bis-benzimide solution (Hoechst

33258 dye, 5 mg/ml in PBS, Sigma) and the sections were mounted in

anti-quenching medium. Specificity of staining was controlled by omit-

ting the primary antibody or replacing it by an equivalent amount

of non-immune IgG or serum derived from the same species as the

specific antibody. These controls were negative.

Analyses of motoneuron size and perisomatic synapticterminals

Sections containing retrogradely labelled motoneurons and additionally

stained for ChAT and VGAT were used to estimate soma areas and

motoneuron perisomatic coverage (Apostolova et al., 2006; Simova

et al., 2006). Linear density (number per unit length) of perisomatic

terminals was estimated for motoneurons that correctly projected to

the motor nerve branch of the femoral nerve (identified by Fast Blue

back-labelling). Stacks of images of 1 mm thickness were obtained on

a confocal microscope (Leica DM IRB, Leica, Wetzlar, Germany) using

a 63� oil immersion objective and digital resolution of 1024�1024

pixels. One image per cell at the level of the largest cell body cross-

sectional area was used to measure soma area, perimeter and number

of perisomatic puncta. These measurements were performed using the

Image Tool 2.0 software program (University of Texas, San Antonia,

TX, USA, http://ddsdx.uthscsa.edu/dig/).

Analyses of axons in the regenerated nerve branches

Total numbers of myelinated axons per nerve cross-section were esti-

mated on an Axioskop microscope (Zeiss) equipped with a motorized

stage and Neurolucida software-controlled computer system

(MicroBrightField Europe, Magdeburg, Germany) using a 100� oil

objective (Simova et al., 2006). Axonal and nerve fibre diameters

were measured in a random sample from each section. For sampling,

a grid with line spacing of 60mm was projected into the microscope

visual field using the Neurolucida software. Selection of the reference

point (0 coordinates) of the grid was random. For all myelinated axons

crossed by or attaching the vertical grid lines through the sections,

mean orthogonal diameters of the axon (inside the myelin sheath)

and of the nerve fibre (including the myelin sheath) were measured

(Fig. 6A). The mean orthogonal diameter is calculated as a mean of

the line connecting the two most distal points of the profile (longest

axis) and the line passing through the middle of the longest axis at

right angle (Irintchev et al., 1990). The degree of axonal myelination

was estimated by the ratio axon to fibre diameter (g-ratio).

Analysis of cell proliferation duringWallerian degenerationFemoral nerve surgery was performed as described above (Fig. 1B)

avoiding the use of polyethylene tubes. The mice (n = 2–3 per

genotype and time-point) were perfused 3, 5, 7 or 14 days after

injury (Fig. 1D) and the femoral nerve segments of �15 mm length,

including the proximal and distal stumps, were post-fixed and

cryoprotected by sucrose infiltration as described above. For indirect

immunofluorescence, the tissue samples were immersed in TissueTek�

medium and frozen in liquid nitrogen. Longitudinal (5mm thick)

and cross (10 mm thick) cryostat sections were used in single and

double-labelling immunofluorescence experiments using antibodies

listed in Table 1. Indirect immunofluorescence was performed as

described above for perisomatic terminals using the primary antibodies

alone for single labelling or mixed at optimal dilutions for double

labelling. Appropriate Cy3- and Cy2-conjugated antibodies pre-

absorbed with normal sera from diverse species to prevent cross-

reactions (Multiple Labeling antibodies, Jackson ImmunoResearch)

were used in double labelling experiments. Specificity of staining was

controlled by omitting the primary antibody or replacing it by an

equivalent amount of non-immune IgG or serum. These controls

were negative.

Proliferating cells were quantified using nerves sampled 5 days

after femoral nerve transection (Fig. 1D), a time-point of maximal

cell proliferation as indicated by Ki67 staining (Fig. 8D–F0). Cell

counts were performed on an Axioskop microscope equipped with

a motorized stage and Neurolucida software-controlled computer

system using a 100� oil objective. Ten spaced-serial cross-sections

(125 mm apart) cut at approximately 3 mm distal to the site of nerve

transection were used for the analysis per animal. These sections

were stained with anti-p75 NGF receptor and anti-Ki67 antibodies to

visualize Schwann cells and proliferating cells, respectively, and with

bis-benzimide solution to reveal cell nuclei. Four types of nuclei were

distinguished (Fig. 10A): Ki67+ nuclei in p75+ cells (type Ia, proliferat-

ing Schwann cells), Ki67� nuclei in p75+ cells (Ib, non-proliferating

Schwann cells), Ki67+ nuclei in p75� cells (IIa, proliferating non-

Schwann cells) and Ki67� nuclei in p75� cells (IIb, non-proliferating

non-Schwann cells). The total number of nuclei per section and the

numbers of Ia, Ib, IIa, IIb nuclei were quantified. The extent of

Schwann cell proliferation was estimated by the ratio of type Ia to

type IIa nuclei (proliferating Schwann cells to proliferating non-

Schwann cells) and numbers of Ia nuclei normalized to all nuclei in

a tissue section and nerve cross-sectional area.

Analysis of Schwann cell proliferation in vitro

Sciatic and femoral nerves were dissected from 7-day-old L1y/+ and

L1y/� mice, freed of connective tissue and transferred into Petri dishes

with ice-cold Ham’s F-12 medium (PAA, Colbe, Germany). The nerves

were treated with 0.25% trypsin (Sigma, Deisenhofen, Germany) and

0.03% collagenase (Sigma) in Dulbecco’s modified Eagle medium

(DMEM, PAA) for 30 min at 37�C. Enzymatic digestion was stopped

by rinsing 2�with F-12 medium. The tissue samples were then

transferred into complete (DMEM/Ham’s F-12) medium containing

0.01% DNase I (Sigma). The complete medium consisted of equal

parts DMEM and F-12 media and was supplemented with 60 ng/ml

progesterone, 16 mg/ml putrescine, 5 mg/ml insulin, 400 ng/ml

L-thyroxine, 160 ng/ml sodium selenite, 10.1 ng/ml triiodo-thyronine,

38 ng/ml dexamethasone (all from Sigma), 7.9 mg/ml glucose (VWR,

Darmstadt, Germany), 0.01% bovine serum albumin, 100 IU/ml

penicillin/streptomycin, 2 mM L-glutamine, 100 mM sodium pyruvate

(all from PAA) and 20% horse serum (Invitrogen, Karlsruhe,

Germany). Mechanical dissociation was performed by gentle trituration

with a fire-polished Pasteur pipette. After adding of DMEM, the cell

suspension was centrifuged at 500g for 10 min at 4�C in a centrifuge

tube on top of a 4% BSA cushion. The supernatant was discarded

and the cells were resuspended in 1 ml fresh pre-warmed (37�C)

complete medium, counted and plated at a density of 2� 106

cells/ml on 0.01% poly-L-lysine-coated coverslips. After 4 h, 20 mM




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http://ddsdx.uthscsa.edu/dig/


5-bromo-20-deoxyuridine (BrdU, Sigma) was applied to the cultures

and the cells were grown for additional 48 h at 37�C in a

CO2-enriched (5%) humidified atmosphere. Finally, the culture

medium was removed and, after rinsing with PBS, the cells were

fixed with 4% formaldehyde in PBS for 30 min at RT, rinsed 3�

with PBS and incubated with 2N HCl for 30 min at 37�C to denature

DNA. After rinsing 2�with PBS, non-specific binding was blocked by

incubation for 30 min with 5% normal goat serum (see below) in PBS

at RT. The fixed cells were incubated with a mouse monoclonal anti-

BrdU antibody (see below) overnight at 4�C, washed 3�5 min with

PBS, incubated with a Cy3-conjugated goat anti-mouse secondary

antibody (see below) for 1 h at RT, washed once with PBS and

mounted in anti-quenching medium.

Analysis of Schwann cell proliferation was performed using confocal

images of randomly selected fields obtained on a confocal microscope

(Olympus FV10-ASW, Hamburg, Germany). The percentage of

proliferating Schwann cells (ratio of BrdU+ to all Schwann cells) was

estimated in 20 fields, each containing 100–200 cells, per cell culture

preparation. At least three cultures were analysed per genotype.

Schwann cells were identified by phase contrast microscopy as elon-

gated bi- or tripolar cells with an oval soma (Fig. 11A–C). Fibroblasts

appeared as flattened, polymorphic cells with large, round nuclei

(Fig. 11A–C).

Results

Gait abnormalities in non-injuredL1y/� miceFrame-by-frame analyses of video sequences recorded prior

to nerve transection (Fig. 1A) revealed differences between

L1y/� and L1y/+ littermates. First, during the mid-stance phase,

at maximum altitude of the contralateral swing, almost the entire

planar paw surface of L1y/� mice contacted the ground (Fig. 2A).

In contrast, only the digits of L1y/+ mice were on the ground

during this gait phase (Fig. 2C). Second, the external rotation of

the paw at take-off position was more pronounced in L1y/� than

in L1y/+ mice (Fig. 2B and D). Consequently, L1y/� mice had

different heels-tail angles and the foot-base angles (Fig. 2A–F),

two measures used to estimate quadriceps muscle function after

femoral nerve lesion (Irintchev et al., 2005a). However, the

observed deviations of the angle values from normal in uninjured

L1y/� mice were opposite to the changes induced by femoral

nerve injury (Fig. 2E and F) and, thus, could not be attributed

to quadriceps muscle weakness.

Enhanced functional recovery afternerve repair in L1y/� miceDespite genotype-related differences in gait prior to operation,

damage of the femoral nerve-induced changes in the heels-tail

and the foot-base angles similar in magnitude in L1y/� and

L1y/+ mice (compare values at 0 and 1 week after injury in

Fig. 2E and F). These alterations were caused by impaired extensor

function of the quadriceps muscle leading to abnormally high

heel position and external rotation of the ankle at gait cycle posi-

tions similar to those seen in Fig. 2A and C and Fig. 2B and D,

respectively. Importantly, at 1 week after injury, no significant

differences were found between L1y/� and L1y/+ mice (Fig. 2E

and F, ANOVA with Tukey’s post hoc test). After the first week,

the angles in L1y/+ mice gradually returned to normal but

recovery was deficient, as compared to normal values, even

after 12 weeks (Fig. 2E and F, P50.05, ANOVA with Tukey’s

post hoc test). Locomotor improvement in L1y/� mice was

superior compared with L1y/+ mice at 4–12 weeks after injury

(Fig. 2E and F). Also, as estimated by the stance RI, a measure

of the individual degree of recovery calculated for both angles, the

final outcome of femoral nerve repair was significantly better in

L1y/� than in L1y/+ mice (87� 14% versus 60� 8.5% recovery,

P50.05, t-test, Fig. 2H).

In addition to ground locomotion, we also evaluated the ability

of the animals to extend the knee joint during movements without

body weight support using the ‘pencil’ test (Irintchev et al.,

2005a). This ability, estimated by the limb PLR (length of the

non-injured hindlimb/length of injured limb during maximum

extension), was similar in L1y/� and L1y/+ mice before operation

(Fig. 2G). Also, the degree of disability at 1 week after operation

and the recovery thereafter were similar in the two genotypes

(Fig. 2G). In conclusion, the combined results show that L1

ablation leads to a superior functional outcome.

Reduced numbers of quadricepsmotoneurons in non-injuredL1y/� miceThe number of retrogradely labelled quadriceps motoneurons was

significantly lower, by 29%, in non-injured L1y/� mice compared

non-injured L1y/+ littermates (Fig. 3). In both phenotypes, only

Fast Blue labelled motoneurons were observed indicating that all

motoneurons project to the quadriceps nerve branch only

(Fig. 1C). No small (microglial) cells loaded with Fast Blue, as a

result of phagocytosis of retrogradely labelled motoneurons

(Simova et al., 2006), were detected in spinal cord sections from

L1y/� and L1y/+ mice indicating that motoneuron death in

L1y/� mice during the 1 week labelling period was not the

reason for the reduced number of labelled motoneurons. The find-

ing of reduced number of motoneurons in L1y/� mice, in con-

junction with a lower than normal number of myelinated axons in

non-injured nerves (see following section) and normal body mass

in L1y/� mice (35� 1.7 g versus 36� 2.8 g in L1y/� and L1y/+

mice, respectively, P40.05, t-test), indicates that L1 ablation leads

to a deficient development and/or maintenance in the adult of the

quadriceps motoneuron pool.

Better motoneuron survival andregeneration in injured L1y/� miceAt 12 weeks after nerve repair, the total number of motoneurons

retrogradely labelled through the motor (quadriceps) or the

sensory (saphenous) branch only, or through both branches of

the femoral nerve, was significantly reduced (�32%) in injured

compared with non-injured L1y/+ mice (Fig. 3). In contrast to

L1y/+ mice, the total number of regenerated motoneurons in




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injured L1y/� mice was similar to the number in non-injured L1y/

� animals (Fig. 3). In both genotypes, small fractions of all labelled

motoneurons projected aberrantly to the sensory branch only, or

to both the motor and the sensory branches (Fig. 3). Also in both

genotypes, the majority of regenerated motoneurons had axons

which had re-grown only into the appropriate motor nerve

branch—a feature of preferential motor reinnervation (PMR,

Fig. 3) (Brushart, 1988). However, the degree of PMR, estimated

as index of PMR (motoneurons projecting to the motor branch/

motoneurons projecting to the sensory branch), was significantly

higher in L1y/� mice compared with L1y/+ mice (4.4� 1.1 versus

2.0� 0.52, respectively, P50.05, t-test). In conclusion, the results

show better motoneuron regeneration in L1y/� compared with

L1y/+ mice: motoneuron loss and/or failure of regeneration are

reduced and precision of motor reinnervation is improved.

L1 deficiency does not influenceaxonal regenerationNext we analysed non-injured and regenerated nerves morpho-

logically to assess axonal numbers, axonal diameters and degree

of myelination. In both the motor and sensory branches of the

femoral nerve, we found a significant reduction in the total

number of myelinated axons in non-injured L1y/� mice (by 17

and 26%, respectively) compared with L1y/+ littermates (Fig. 4A

and B). At 12 weeks after injury, the numbers of regenerated

axons in the motor and sensory branches of the femoral

nerve were reduced compared with non-injured nerves in both

L1y/� (by 27 and 34%, respectively) and L1y/+ mice (by

26 and 35%, respectively, Fig. 4A and B). The axonal numbers

in the regenerated nerve branches were not significantly different

between L1y/� and L1y/+ mice. Also, no differences between the

genotypes were found for axonal diameters and relative degree of

myelination (g-ratio) in non-injured or regenerated nerves (Figs 5

and 6) with one exception. The frequency distribution of axonal

diameters in the non-injured motor branch of L1y/� mice was

partially shifted towards larger values (P50.05, Kolmogorov–

Smirnov test) indicating that a fraction of the axons in L1y/�

mice had larger than normal diameters (Fig. 5). With regard to

nerve repair, we can conclude that L1 ablation in both axons

and Schwann cells does not influence axonal regrowth and

remyelination as estimated at 12 weeks after injury.

Larger soma size and attenuated loss ofinhibitory synaptic input to regeneratedL1y/� motoneuronsThe soma size of motoneurons in L1y/� and L1y/+ mice with

or without preceding injury of the femoral nerve (‘Injured’ and

‘Non-injured’ groups in Fig. 7D, respectively) was evaluated only

for motoneurons projecting to the quadriceps muscle as detected

by retrograde labelling (blue cells in Figs 1B and 7A). Both

non-injured and injured L1y/� mice had larger motoneurons

compared with the respective groups of L1y/+ mice (by 20 and

16%, respectively, Fig. 7D).

Analysis of inhibitory (GABAergic and glycinergic, VGAT+)

terminals and modulatory cholinergic (ChAT+) terminals around

motoneuron perikarya revealed differences between the geno-

types in mice without injury (‘Non-injured’ groups in Fig. 7E and

F). Density (number per mm) of VGAT+ terminals was lower and

that of ChAT+ puncta higher in L1y/� compared with L1y/+ mice

(20 and 22%, respectively). After nerve injury (‘Injured’ groups in

Fig. 7E and F), densities of ChAT+ terminals in both genotypes

were not changed compared with the respective non-injured

Figure 3 Analysis of motoneuron regeneration. Mean

numbers of motoneurons (�SEM) labelled through the motor

or sensory branch only (grey and dark grey bars, ‘Motor’ and

‘Sensory’, respectively), through both branches (light grey bars,

‘Both’), and the sum of neurons in the first three categories

(black bars, ‘Total’) 12 weeks after femoral nerve repair in

L1y/� and L1y/+ mice (right-hand half of the graph, ‘Injured’,

6/6 animals of L1y/� and L1y/+ mice, respectively) and

non-injured mice (‘Non-injured’ on the left-hand side, 10/13

animals of L1y/� and L1y/+ mice, respectively). In non-injured

mice retrograde tracing but no previous nerve injury was

performed. Asterisks and hash indicate differences between

non-injured and injured groups of the same genotype and

between L1y/+ and L1y/� animals under the same

experimental conditions, respectively (P50.05, one-way

ANOVA with Tukey’s post hoc test).

Figure 4 Analysis of myelinated axons in non-injured and

regenerated motor (A) and sensory (B) nerve branches at

12 weeks after injury in L1y/� and L1y/+ mice (‘Injured’,

n = 11 and 10, respectively) and in non-injured contralateral

nerves (‘Non-injured’, n = 13 and 6, respectively). Asterisks

and hash indicate differences between non-injured and injured

groups of the same genotype and between L1y/+ and L1y/�

animals under the same experimental conditions, respectively

(P50.05, one-way ANOVA with Tukey’s post hoc test).




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group as also were VGAT+ boutons in L1y/� mice. In contrast to

L1y/� mice, VGAT+ terminals were reduced in injured compared

with non-injured L1y/+ animals (�22%). We can conclude that

synaptic coverage related to nerve injury differs in L1y/+ and L1y/

� mice. Particularly interesting from a functional point of view is

the finding that nerve repair-related loss of inhibitory terminals in

L1/+ mice was not found in L1y/� mice.

Enhanced Schwann cell proliferationin L1y/� mice after nerve damageWe analysed sections from femoral nerves 3, 5, 7 and 14 days

after injury in L1y/+ and L1y/� mice (Fig. 1D) using the L1 anti-

body 555 and antibodies recognizing Schwann cell and axonal

antigens, S-100 and ß-tubulin, respectively (data not shown).

While L1 could not be detected in L1y/� samples, L1 was

up-regulated after nerve transection. The number of S-100-

positive cells was higher, at any time-point studied (2–3 animals

studied per time-point and genotype), in L1y/� versus L1y/+ mice

(Fig. 8A–C). A similar difference between the genotypes was

found for another Schwann cell marker, GFAP (not shown).

These observations suggested that L1 deficiency influences

Schwann cell proliferation after nerve injury. We used an antibody

against the Ki67 antigen to assess cell proliferation. At any time-

point after injury studied, the number of Ki67-positive cells in

longitudinal sections of L1y/� mice exceeded by at least two-fold

the numbers in L1y/+ mice (Fig. 8D–F0). To verify the possibility

that most of these proliferating cells are Schwann cells, we

performed co-localization experiments in sections of the femoral

nerve 5 days after injury using antibodies against Ki67

and Schwann cell markers, S-100 and GFAP (Fig. 1D). No

co-localization of Ki67+ nuclei and S-100+ (Fig. 9A–A0) or GFAP+

(not shown) cell profiles was found. However, most Ki67+ nuclei

in both genotypes appeared to co-localize with laminin, a basal

Figure 5 Analysis of axon diameters in regenerated and

non-injured nerves. Shown are normalized frequency

distributions of axon diameters in non-injured (A and B) and

regenerated (C and D) motor (A and C) and sensory (B and D)

nerve branches of the femoral nerve in L1y/� and L1y/+ mice.

Regenerated nerves were studied 12 weeks after injury.

Number of axons and nerves studied per group are shown in

the panels. A significant difference between the genotypes was

detected only for the non-injured motor nerve branches (A,

P50.05, Kolmogorov–Smirnov test). This difference indicates

increased proportion of large-diameter axons in the motor

branch of L1y/� mice. Abnormally thick axons (413 mm) were

not present in L1y/� animals as illustrated by images of

semithin sections from the motor nerve branches of an L1y/�

and an L1y/+ mouse (E and F).

Figure 6 Analysis of myelination in regenerated and

non-injured nerves. Mean orthogonal diameters of the axon

(A, black arrows) and of the nerve fibre (A, white arrows) were

measured and the degree of myelination was estimated by the

ratio axon to fibre diameter (A, g-ratio). Shown are normalized

frequency distributions of g-ratios in non-injured (B and C) and

regenerated (D and E) motor (B and D) and sensory (C and E)

nerve branches of the femoral nerve in L1y/� and L1y/+ mice.

Regenerated nerves were studied 12 weeks after injury.

Number of axons and nerves studied per group are identical

with those shown in Fig. 4. No differences between the

genotypes were detected for any pair of samples (P40.05,

Kolmogorov–Smirnov test).




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lamina component produced by Schwann cells (Fig. 9B–B0).

No co-localization of Ki67+ nuclei and the macrophage marker

F4/80 was observed (Fig. 9C–C0). Finally, analysis of apoptotic

cells using an antibody against activated caspase-3 did not reveal

differences between the genotypes at any time-point studied

(not shown).

Since the differentiation-associated proteins S-100 and GFAP

did not co-localize with Ki67, Schwann cell proliferation was quan-

tified using an antibody against the p75 NGF receptor which is

expressed in proliferating Schwann cells. At 5 days after femoral

nerve transection (Fig. 1D), when cell proliferation was estimated

to be maximal (Fig. 8D–F0), the number of proliferating Schwann

cells (both p75+ and Ki67+, designated as type Ia in Fig. 10A) was

higher in L1y/� than in L1y/+ mice irrespective of whether this

number was normalized to proliferating non-Schwann cells (Ki67+,

p75�, type IIa, Fig. 10B)—by 26%, to all nuclei in a nerve cross-

section (by 36%, Fig. 10C) or nerve cross-sectional area (by 20%,

Fig. 10D). Thus, L1 deficiency leads to enhancement of Schwann

cell proliferation after nerve injury.

Enhanced proliferation of Schwann cellsfrom L1y/� mice in vitroImmunofluorescence detection of BrdU and phase contrast

analysis revealed BrdU labelling in nuclei of two types of

cell in the cultures: elongated bi- or tripolar cells with an

oval soma and small nuclei, considered to be Schwann cells

(Brockes et al., 1979; Scarpini et al., 1986; Honkanen et al.,

2007), and fibroblast-like cells with flattened, polymorphic appear-

ance and large round nuclei (Fig. 11A–C). Quantitative analysis

revealed that a higher proportion of the cells with Schwann

cell morphology had incorporated BrdU in cultures derived from

L1y/� mice compared with L1y/+ cultures (57 and 39%, respec-

tively, Fig. 11D), a finding supporting the observations made

in vivo.

DiscussionThe results of this study show that L1 deficiency in mice has a

favourable impact on functional recovery after peripheral nerve

damage. Improved functional outcome was paralleled by positive

effects on regeneration including attenuated post-traumatic loss of

motoneurons, enhanced precision of reinnervation of the femoral

motor, and thus appropriate, nerve branch by motoneuron axons,

larger cell bodies of regenerated motoneurons and diminished loss

of inhibitory synaptic input to motoneurons. These phenomena are

associated with enhanced proliferation, and thus higher numbers,

of the growth-supportive denervated Schwann cells in injured

nerves of L1y/� mice.

Figure 7 Analysis of motoneuron soma size and perisomatic nerve terminals. The estimation was performed only for correctly

projecting motoneurons (retrogradely labelled with Fast Blue, but not with fluoro-ruby, applied to the motor and sensory nerve branch,

respectively, A). Confocal images (1-mm thick optical slices) show VGAT+ (B) and ChAT+ (C) terminals around motoneuron cell bodies

(asterisks) in sections from an L1y/+ mouse studied 12 weeks after nerve repair. Scale bar indicates 25 mm for A–C. (D–F) Soma area of

ChAT+ and VGAT+ motoneurons (D) and linear densities of VGAT+ (E) and ChAT+ (F) puncta in non-injured mice (subjected to

retrograde tracing only) and mice studied 12 weeks after nerve repair (‘Injured’). Shown are group mean values + SEM values calculated

from individual mean values. Between 200 and 350 cells from 5 to 12 animals were analysed per group and parameter. Asterisks and

hash indicate differences between non-injured and injured groups of the same genotype and between L1y/+ and L1y/� animals under

the same experimental conditions, respectively (P50.05, one-way ANOVA with Tukey’s post hoc test).




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Functional and structural deficits inuninjured L1y/� miceAnalyses of non-injured L1y/� mice revealed deficits, as compared

with L1y/+ littermates, in numbers of quadriceps motoneurons

and numbers of myelinated axons in the motor and sensory

branches of the femoral nerve. These differences could be

caused by target factors such as, for example, reduced muscle

mass despite normal body weight. Since L1 has been found to

support neuronal survival in vitro (Hulley et al., 1998; Chen

et al., 1999; Loers et al., 2005), we favour the view that

L1 deficiency in motoneurons leads to loss of motoneurons and

sensory neurons which in turn results in lower numbers of axons in

the peripheral nerves. Deficient motoneuron numbers may also

contribute to the mild gait deficits observed in our L1y/� mice

on 129/SvJ/NMRI background and previously in L1y/� mice on

a C57BL/6J/129S7 genetic background (strain B6; 129S7-

L1camtm1Sor/J, Jackson Laboratory, Bar Harbor, ME, USA,

Jakeman et al., 2006). If numbers of muscle fibres are not reduced

by ablation of L1, motoneuron deficit would lead to an increase

in motor unit size (number of muscle fibres innervated by one

motoneuron) in L1y/� mice. In association with abnormal sensory

inputs, this could contribute to reduced precision of motor control.

In line with the idea of increased motor unit size are the observa-

tions of larger somata of retrogradely labelled quadriceps

motoneurons and increased axonal diameters in the motor

branch of the femoral nerve of L1y/� mice. In contrast to other

deficits, non-injured L1y/� mice had the same myelination as

L1y/+ animals in both branches of the quadriceps nerve estimated

by g-ratio.

Enhanced outcome of nerve injury inL1y/� micePrevious studies have shown that homophilic L1 interactions

promote neurite outgrowth on cultured Schwann cells (Bixby

et al., 1988; Kleitman et al., 1988; Seilheimer and Schachner,

1988). Therefore, our initial expectation was to find at least

delayed recovery, if not inferior final outcome in L1y/� mice.

We surprisingly observed better locomotor recovery in L1y/�

mice than in L1y/+ littermates. L1y/� mice showed subtle

abnormalities in gait prior to operation. However, the degree of

Figure 8 Analysis of Schwann cells proliferation in vivo. Longitudinal cryostat sections of 5mm thickness from distal stumps of

transected femoral nerves of L1y/� mice (A–F) and L1y/+ littermates (A0–F0) dissected 3–7 days after injury (Fig. 1D). Staining for the

Schwann cell marker S-100 (A–C0) reveals higher numbers of S-100+ cells in L1y/� mice (A–C) than in L1y/+ littermates (A0–C0).

Staining of sections serial to those shown in panels A–C0 with an antibody against the proliferation antigen Ki67 (D–F0) reveals higher

numbers of proliferating cells in L1y/� mice than in L1y/+ mice at all time-points studied. Scale bars in A and D indicate 50 and

100 mm for A–C0 and D–F0, respectively.




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impairment after injury was similar in L1y/� and L1y/+ mice as

indicated by the parallel and equal in extent changes occurring

between 0 and 7 days in both genotypes (Fig. 2E and F).

Therefore, L1y/� and L1y/+ mice suffer from the injury to the

same extent. Better functional recovery in L1y/� mice became

apparent first at 4 weeks after injury indicating lack of advantages

during the early phase of regeneration. At 12 weeks after injury,

motoneuron survival/axonal regrowth, as assessed by retrograde

labelling, was also better in L1y/� mice. Compared with non-

injured mice of the same genotype, number of regenerated

retrogradely labelled motoneurons was reduced in L1y/+ but not

in L1y/� mice. Also, PMR after nerve injury was more

pronounced and somata of regenerated motoneurons were

larger in L1y/� than in L1y/+ mice. Similar effects on motoneuron

survival and regrowth and a similar time-course of functional

improvement were found after femoral nerve injury and applica-

tion of a peptide mimic of the HNK-1 (human natural killer cell)

glycan in mice (Simova et al., 2006) which is expressed in

myelinated axons of the motor, but not sensory branches of the

mouse femoral nerve. We interpret such findings as indications of

trophic effects on motoneurons after axotomy, an interpretation in

line with the observation of enhanced Schwann cell proliferation in

L1y/� mice observed here. It is, however, also conceivable, that

the enhanced functional improvement in L1y/� mice is addition-

ally related to attenuated susceptibility to injury of motoneurons

that have survived as the most resistant ones to developmental

elimination.

L1 and synaptic connectivity ofmotoneuronsThis study provides also, for the first time, data on perisomatic

coverage of motoneurons by VGAT+ (glycinergic and

GABAergic) and ChAT+ (cholinergic) axon terminals after nerve

injury. Considering the results for L1y/+ mice, two observations

appear important. First, the number of cholinergic boutons on

motoneurons was similar before and after injury. This is in contrast

to the dramatic reduction of this synaptic input to lumbar moto-

neurons after thoracic spinal cord injury in mice which correlates

with reduced locomotor recovery (Apostolova et al., 2006;

Jakovcevski et al., 2007). These two observations suggest that

injury-induced alterations in the modulatory cholinergic innerva-

tion of motoneurons (Miles et al., 2007) do not contribute to

motor deficits after nerve regeneration. Second, regenerated

motoneurons were deficiently innervated by inhibitory terminals

at 12 weeks after injury compared with non-injured motoneurons

in L1y/+ mice. This deficit is likely related to functional

impairments after nerve regeneration since perisomatic inhibition

is crucial for normal motoneuron function. Considering genotype

specific effects, it is interesting that non-injured L1y/� motoneur-

ons were surrounded by more cholinergic and less inhibitory

boutons than L1y/+ motoneurons. At present it cannot be

judged whether these differences in synaptic coverage represent

compensations for or contribute to functional impairments in non-

injured L1y/� mice. And finally, no loss of inhibitory terminals

in regenerated L1y/� motoneurons compared with non-injured

L1y/� motoneurons was found—in contrast to L1y/+ mice—a

feature that might favour enhanced locomotor recovery. The

combined observations indicate that L1 deficiency causes altera-

tions, as compared with L1y/+ mice, in synaptic connectivity of

motoneurons in the non-injured spinal cord and after peripheral

nerve regeneration.

Enhanced Schwann cell proliferationas a mechanism underlying enhancedfunctional recovery in L1y/� miceAfter nerve injury, the Schwann cells in the distal nerve stump

switch their mode from myelination of electrically active axons

to growth support for regenerating axons (Mirsky and Jessen,

1999; Scherer and Salzer, 2001). Denervated Schwann cells

proliferate, align in chains, bands of Bungner, inside endoneurial

tubes (basal laminae deposited by Schwann cells) and act as

cellular and molecular guides for the regenerating axons.

Moreover, the switch in Schwann cell phenotype after injury

is associated with downregulation of myelin-associated genes,

up-regulation of several growth-associated genes, and increased

production of neurotrophic factors and extracellular matrix

proteins supporting neuronal survival and axonal regrowth

(Fu and Gordon, 1997; Scherer and Salzer, 2001; Moran and

Graeber, 2004). If this support is reduced by, for instance, long-

term denervation during adulthood or persistence of Schwann cell

immaturity in early postnatal animals, regeneration is compromised

(Fenrich and Gordon, 2004; Moran and Graeber, 2004). In light of

Figure 9 Analysis of proliferating cells in vivo. Cross-sections

from the quadriceps (motor) branch (on the left hand side in

each pair of sections) and the saphenous (sensory) branch

of the femoral nerve of L1y/� mouse (A–C) and an L1y/+

littermate (A0–C0) cut from nerve segments distal from the

site of injury at 5 days after injury (Fig. 1D). Ki67+ nuclei

co-localize with laminin (B and B0) but not with S-100

(A and A0) or with the macrophage marker F4/80 (C and C0).

Scale bar indicates 25 mm.




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this knowledge, better regeneration and functional recovery in

L1y/� mice can be explained by enhanced Schwann cell prolifer-

ation during the period of Wallerian degeneration. Higher number

of Schwann cells may provide enhanced trophic support by neu-

rotrophins, cytokines and other factors thus positively influencing,

not only motoneurons, but also sensory neurons and improve their

survival and promote the restoration of afferent connections.

A relationship between L1 expression and Schwann cell prolif-

eration has not been reported or considered previously. The only

exception is the finding that reduction of Schwann cell prolifera-

tion by serum deprivation in vitro augments L1 expression

(Takeda et al., 2001). One possible explanation for the effect of

L1 ablation on cell proliferation in vivo is delayed differentiation,

and thus prolonged proliferation, of Schwann cells after contact

with regrowing axons as a result of L1 deficiency in both

interacting cell types. Disruption of homophilic L1 interactions

(Bixby et al., 1988; Kleitman et al., 1988; Seilheimer and

Schachner, 1988) may underlie such an effect. While this hypoth-

esis has to be tested, our experiments with primary Schwann cell

cultures, which are devoid of nerve cells but contain L1-negative

fibroblast-like cells, indicate that L1-mediated control of cell

proliferation is exerted via Schwann cell—extracellular matrix

interactions or cis-interactions between L1 and other molecules

on the Schwann cell membrane. One possibility is that L1 sup-

presses cell proliferation by binding to integrins on the surface of

Schwann cells. For example, interaction of L1 with a7b1-integrin,

a receptor for laminin-2 on Schwann cells (Chernousov et al.,

2007), through RGD motifs in the sixth immunoglobulin domain

of L1, can modulate integrin-mediated signalling and thus cell

proliferation (Itoh et al., 2004, 2005; Armstrong et al., 2007;

Maness and Schachner, 2007). Another possibility is that loss of

the interaction of L1 with the fibroblast growth factor receptor 1

Figure 10 Analysis of Schwann cell proliferation in vivo. Cross-section from the quadriceps (motor) branch of the femoral nerve of an

L1y/+ mouse (A) cut �3 mm distal from the site of injury at 5 days after injury (Fig. 1D) stained for the Schwann cell antigen p75 and

the proliferation marker Ki67. Four types of cell were distinguished and quantified in 10 space-serial sections per animal: p75+ cells with

Ki67+ nuclei (type Ia, proliferating Schwann cells), p75+ cells with Ki67� nuclei (Ib, non-proliferating Schwann cells), p75� cells with

Ki67+ nuclei (IIa, proliferating non-Schwann cells) and p75� cells with Ki67� nuclei (IIb, non-proliferating non-Schwann cells). The

numbers of proliferating Schwann cells (type Ia) were significantly higher in L1y/� than in L1y/+ mice (*P50.05, t-test, n = 3 animals

per genotype) as indicated by their ratio to proliferating non-Schwann cells (type IIa, B), and numbers per section normalized to the

number of all nuclei (C) or nerve cross-sectional area (D). The values shown in B–D are group mean values (+SEM) calculated

from individual mean values (10 sections analysed per animal). Scale bar in A indicates 50 mm.




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(FGFR1) in L1y/� Schwann cells promotes proliferation (Dong

et al., 1997). Upon fibronectin type III domain-mediated binding

of L1 to FGFR1, the receptor is phosphorylated and this favours

differentiation and axonal growth in neurons rather than prolifer-

ation (Loers et al., 2005; Kulahin et al., 2008). The importance of

FGF-2 signalling in the control of Schwann cell proliferation has

previously been demonstrated using mice overexpressing FGF-2

(Jungnickel et al., 2006). These transgenic mice show enhanced

Schwann cell proliferation but, in contrast to our L1y/� mice, no

superior functional outcome of peripheral nerve injury compared

with wild-type mice was detected. These observations do not

contradict our hypothesis that increased numbers of Schwann

cells during Wallerian degeneration favour the final outcome of

nerve injury since FGF-2 overexpression stimulates signalling not

only via the regeneration-promoting FGFR1, but also via FGFR3,

which has a negative impact on nerve regeneration (Jungnickel

et al., 2004).

L1 and myelinationAlthough L1 is involved in myelination in vitro (Seilheimer et al.,

1989; Wood et al., 1990; Coman et al., 2005), peripheral nerve

myelination in L1y/� mice is normal (Dahme et al., 1997; Haney

et al., 1999). Here, we found similar degrees of myelination in

L1y/� and L1y/+ mice for both non-injured and regenerated

nerves. These findings are in agreement with the conclusion of

Martini and Schachner (1986, 1988) based on analyses of

L1 expression during nerve development and regeneration, that

L1 is not essential for the maintenance of compact myelin.

However, the expression pattern observed in these studies sug-

gested that L1 is involved in initial axon-Schwann cell interactions

and at the onset of myelination. Our findings, therefore, do not

preclude a role of L1 in myelination. During normal development

until adulthood and within a 12-week recovery period after nerve

injury, compensatory mechanisms may overcome an L1-related

delay in myelination.

In conclusion, the results of this study indicate that L1, in

contrast to its role in the injured CNS, has a negative impact on

peripheral nerve regeneration, implicating L1’s suppression of

Schwann cell proliferation and thus reduced trophic support that

proliferating, denervated Schwann cells convey to neurons.

Ensuing L1-related enhancement of differentiation may then be

counteractive to regeneration after injury in the adult PNS.

These findings warrant experiments designed to test the potential

of a functional, but transient block of L1 expression by Schwann

cells in the regenerating nerve, using, for example, neutralizing

antibodies, to improve of the outcome of nerve injury. An

alternative strategy would be to transiently stimulate Schwann

cell proliferation, and thereby improve the functional outcome

after injury, by applying Schwann cell mitogens during the

period of Wallerian degeneration.

AcknowledgementsWe are grateful to Gabriele Loers for providing L1 antibody and

help with Schwann cells culture, Peggy Puthoff for supplying

Figure 11 Analysis of Schwann cell proliferation in vitro.

Primary culture from peripheral nerves of an L1y/+ mouse

treated with BrdU for 48h to label proliferating cells (the same

field on panels A–C). Schwann cells were identified by phase

contrast microscopy as elongated bi- or tripolar cells with an

oval luminescent soma (A and C). The number of Schwann

cells containing BrdU-labelled nuclei (two cells indicated by

arrows, B and C) was estimated in cultures of both genotypes

and expressed as percentage of all Schwann cells. Large cells

with BrdU-positive nuclei (B) and barely visible cell bodies (A)

were classified as fibroblast-like cells and were not analysed.

The proportion of BrdU-labelled Schwann cells was significantly

higher in cultures derived from L1y/� mice than in L1y/+

cultures (D, *P50.05, t-test). Values shown in D are

means + SEM.




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transgenic mice, Emanuela Szpotowicz for technical assistance and

Achim Dahlmann for genotyping. M.S. is New Jersey Professor of

Spinal Cord Research.

FundingHertie Stiftung (1.01.1/06/005).

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Ablation of adhesion molecule L1 in mice favours Schwann cell proliferation and functional recovery...

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