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Neurobiology of Disease 21 (2006) 138 – 153
Differentially promoted peripheral nerve regeneration by grafted
Schwann cells over-expressing different FGF-2 isoforms
Kirsten Haastert,a,b,*,1 Esther Lipokatic,a,1 Martin Fischer,c
Marco Timmer,a and Claudia Grothea,b,*
a
Department of Neuroanatomy, Hannover Medical School, OE 4140, Carl-Neuberg-Str.1, 30625 Hannover, Germany
Center for Systems Neuroscience (ZSN), Hannover, Germany
c
Department of Neurophysiology, Hannover Medical School, OE 4140, Carl-Neuberg-Str.1, 30625 Hannover, Germany
b
Received 8 April 2005; revised 27 June 2005; accepted 27 June 2005
Available online 24 August 2005
Artificial nerve grafts are needed to reconstruct massive defects in the
peripheral nervous system when autologous nerve grafts are not
available in sufficient amounts. Nerve grafts containing Schwann cells
display a suitable substrate for long-distance regeneration. We present
here a comprehensive analysis of the in vivo effects of different
isoforms of fibroblast growth factor-2 (FGF-2) on peripheral nerve
regeneration across long gaps. FGF-2 isoforms were provided by
grafted, genetically modified Schwann cells over-expressing 18-kDaFGF-2 and 21-/23-kDa-FGF-2, respectively. Functional tests evaluated
motor and sensory recovery. Additionally, morphometrical analyses of
regenerated nerves were performed 3 and 6 months after grafting.
Distinct regeneration promoting effects of the different FGF-2 isoforms
were found. 18-kDa-FGF-2 mediated inhibitory effects on the grade of
myelination of regenerating axons, whereas 21-/23-kDa-FGF-2 mediated early recovery of sensory functions and stimulation of longdistance myelination of regenerating axons. The results contribute to
the development of new therapeutic strategies in peripheral nerve
repair.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Fibroblast growth factor-2; Isoforms; Peripheral nerve regeneration; Cell therapy; Neurotrophic therapy; Schwann cells; Genetic
modification
Introduction
A complete nerve transection is the most severe peripheral
nerve injury and is primarily seen in obstetrical and traumatic
brachial plexus lesions, but can also be seen in advanced extremity
injuries. Depending on the distance between the nerve stumps,
* Corresponding authors. Fax: +49 511 532 2880.
E-mail addresses: [email protected] (K. Haastert),
[email protected] (C. Grothe).
1
Contributed equally to this work.
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2005.06.020
treatment typically consists of either direct end-to-end anastomosis
of the cut nerve ends or the use of an autologous nerve graft
(Schmidt and Leach, 2003). The availability of autologous nerve
transplants is especially limited when a large amount of grafting
material is needed as in massive peripheral nerve lesions, because
it requires sacrifices of healthy nerves (Lundborg, 2004). There are
experimental and clinical approaches to use synthetic guidance
channels in peripheral nerve regeneration (Nakamura et al., 2004).
However, the golden standard is still transplantation of autologous
nerve grafts as they provide a scaffold which contains Schwann
cell basal laminae and growth factor constituting optimal growth
substrate and environment for regrowing axons (Ansselin et al.,
1997; Bunge, 1993; Lundborg, 2004). For development of an
optimal artificial nerve graft as alternative to autologous ones, it is
of high interest to combine synthetic nerve guides with preferentially autologous Schwann cell transplants and the respective
biologically active molecules.
Artificial nerve grafts filled with physiological Schwann cells
from neonatal (Hadlock et al., 2000; Mosahebi et al., 2002) and
adult rats (Ansselin et al., 1997; Guenard et al., 1992) stimulated
nerve fiber regeneration. Enhanced morphological peripheral nerve
regeneration has also been seen after treatment with nerve growth
factor (NGF) either slowly released from synthetic nerve guides
(Fine et al., 2002) or from NGF-containing polymeric microspheres within synthetic nerve guides (Fine et al., 2002; Xu et al.,
2003) or to a even better extent after treatment with glial-derived
neurotrophic factor also slowly released from synthetic nerve
guides (Fine et al., 2002). Furthermore, it has been shown
previously that entrapment of the low molecular weight (18-kDa)
isoform of fibroblast growth factor-2 (FGF-2) in synthetic nerve
guidance channels is able to enhance growth of myelinated and
unmyelinated axons across long gaps significantly (Aebischer et
al., 1989). With regard to the low and high molecular weight
FGF-2 isoforms, it has been shown that the isoforms are differentially regulated following peripheral nerve injury, indicating
differential physiological functions during peripheral nerve regeneration (for review, see: Grothe and Nikkhah, 2001).
K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
Non-resorbable silicone tubes were introduced as an experimental model for tubulization in peripheral nerve repair. Sciatic
nerve gaps exceeding 10 mm in rats resulted in no regeneration at
all across the tube (Francel et al., 1997; Lundborg et al., 1982) and
provide in this way optimal conditions to test new cell transplantation strategies in peripheral nerve repair across long gaps.
Recently, we have shown that genetically modified Schwann cells
are a useful tool to bridge long gaps (15 mm) after peripheral nerve
injury (Timmer et al., 2003). Furthermore, over-expression of the
21- and 23-kDa-FGF-2 isoforms by the transplanted Schwann cells
improved both lengths and number of regenerating myelinated
axons in a short 4-week observation time (Timmer et al., 2003).
Knowledge of specific functions of FGF-2 isoforms and other
growth factors within the regeneration scenario could contribute to
the establishment of new therapeutic strategies after peripheral
nerve lesion. In the present study, nerve guides filled with
genetically modified Schwann cells over-expressing different
FGF-2 isoforms were used to combine the necessary presence of
Schwann cells in artificial nerve transplants with the effects of
added growth factors. The objective of the present study was to
investigate specific functions of the low and high molecular weight
FGF-2 isoforms over-expressed by grafted Schwann cells on
peripheral nerve repair across long gaps in a long time period
139
(3 and 6 months). Grade and quality of peripheral nerve
regeneration were determined by functional and morphometrical
parameters as well as retrograde labeling of regenerating sensory
and motor neurons.
Materials and methods
Animals and overview of experimental design
Adult female Sprague – Dawley rats weighing approximately
180 g (Central Animal Laboratory Medical School Hannover,
Germany and Charles River Wiga, Germany) were kept under
standard conditions (room temperature 22 T 2-C, humidity 55 T
5%, light/dark cycle LD 12:12) with food and water ad libitum.
Animal care, housing, and surgery followed the guidelines of the
German law on the protection of animals and were approved by the
local animal care committee.
The animals were distributed into two groups concerning
different observation periods, a 3-month and a 6-month group.
Silicone tubes were implanted to the transected left sciatic nerve of
each rat. The tubes were filled with different ingredients to build
further experimental subgroups (Fig. 1): (1) Matrigel (Matrigel,
Fig. 1. The experimental groups and experimental design of the groups observed for 3 and 6 months. Division in experimental groups referred to the differently
filled silicone tubes. Walking track analysis and electrophysiological tests were carried out to check for motor recovery and the withdrawal test to evaluate
sensory recovery. Semi-thin and ultra-thin cross sections of the explanted, regenerated tissue cables were evaluated at several section points. Neuron tracing by
DiI was performed to reveal the quality of regenerating neurons projecting into the regenerated nerves.
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K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
only in the 3 months group: n = 10) or cells resuspended in Matrigel
as follows: (2) physiological Schwann cells (SCphysiol, 3 months
group: n = 13), (3) Schwann cells transfected with control vector
alone (SCvector alone, 3 months group: n = 13) or Schwann cells
genetically modified (4) to over-express the 18-kDa-FGF-2 isoform
(SC18-kDa-FGF-2 ov ex, 3 months group: n = 13) or (5) to over-express
the 21/23-kDa-FGF-2 isoforms (SC21/23-kDa-FGF-2 ov ex, 3 months
group: n = 11). Subgroups in the 6 months observation time
consisted of n = 9 animals each.
Preparation of transfected cells and nerve guides
Primary/secondary physiological Schwann cells were prepared as described before (Haastert et al., 2005; Timmer et al.,
2003). In brief, sciatic nerves were dissected from neonatal
Sprague – Dawley rats. After enzymatic dissociation and primary
seeding, most of contaminating fibroblasts were removed by
addition of arabinoside C (1 mM) for 2 days. Remaining
fibroblasts were removed from the Schwann cell cultures using
a-Thy1 antibody-coupled magnetic beads (Dynabeads, Dynal,
Denmark). The protocol resulted in about 99% pure Schwann
cell cultures.
Stable transfection of cells was performed using Metafectenei
(Biontex, Germany) (Mauritz et al., 2004) and plasmids constructed as follows. The 18-kDa-FGF-2 isoform was cloned
previously in pCI-neo resulting in clone pCI-18 kDa (MullerOstermeyer et al., 2001). For the construction of a vector for the
expression of FGF-2 high molecular weight isoforms (21-kDaFGF-2 and 23-kDa-FGF-2), the complete coding sequence of
FGF-2 was cut out from RSVp.metFGF (Pasumarthi et al., 1994)
and cloned into pCI-neo. The resulting clone pCI-neo13-HMW
is able to express 21- and 23-kDa-FGF-2 isoforms, but not the
18-kDa-FGF-2 isoform.
All cell populations transplanted in this study were newly
prepared and transfected. As described before (Timmer et al.,
2003), transfected cells were positively selected for their coexpressed resistance to Geneticin (G418) and successful transfection was monitored in Western blot analysis with regard to
FGF-2 isoform expression in the different cell groups: SCphysiol,
SCvector alone, SC18-kDa-FGF-2 ov ex, and SC21/23-kDa-FGF-2 ov ex (Fig.
2). Transfected cells were grown to confluence, withdrawn from
10% serum to 5% serum for 2 days, and cultured for 12 h in
serum-free, N1-Supplement substituted medium. Cells were
detached from culture flasks and appropriate cell numbers (end
concentration: 114 106 cells/ml) were prepared for each
transplantation and stored on ice. Directly prior to implantation
of the nerve guides, cells were resuspended in growth factorreduced Matrigel (v/v, 70:30, Becton Dickinson, Germany) and
injected in sterile silicone tubes.
Cell tracing
To ensure stable presence of transplanted cells at the
implantation side, in three rats the sciatic nerve gap (15 mm)
was bridged by silicone tubes filled with pre-labeled physiological
Schwann cells by incubation with PKH26-GL cell linker (Sigma,
Germany). 4 weeks post-operation, the regenerated tissue was
explanted, fixed in 4% paraformaldehyde solution. Longitudinal
cryostat sections were cut at 50 Am and viewed under an
epifluorescence microscope (BX60, Leica, Germany) using appropriate filters to visualize the red-fluorescent PKH26-GL cell
surface staining (Verdu et al., 1999).
Surgical procedures
Rats were anesthetized by intraperitoneal application of Chloral
hydrate (370 mg/kg body weight). Aseptic techniques were used to
ensure sterility. To avoid a decrease in body temperature during
anesthesia, the animals were kept on an electric pad and body
temperature was repeatedly controlled. The animals’ left hind legs
were shaved and on each animal the sciatic nerve was exposed by a
skin incision along the femur followed by the separation of the
biceps femoris and superficial gluteal muscles. The nerve was
transected at mid-thigh and the length of the gap to be covered was
15 mm. One suture (9/0 EthilonRII, Ethicon, Germany) was
attached to each end of the conduit (18 mm in length) and then to
the lumen of the conduit in order to pull the proximal nerve end 2
mm and the distal nerve end 1 mm into the lumen of the conduit. The
muscle layers were sutured (4/0 EthilonRII, Ethicon, Germany) and
finally, the skin was sutured with 3/0. DexonR (B. Braun-Dexon,
Germany). Transplantation experiments were performed on consecutive days to pool sufficient animal numbers in every group. The rats
were monitored for functional recovery for a period of 3 and 6
months, respectively. The animals were observed every second day
to check for indications of automutiliation and Altosol (EuroVet,
WDT, Germany) or Leovet Anti Bite (Reitsport Biro, Germany) was
applied to their left paws as necessary to prevent automutiliation.
FGF-2 ELISA
FGF-2 ELISA was performed 1 week after transplantation of
different cell types to reveal levels of free FGF-2 in the grafted
silicone tubes. In each group (SCphysiol group, SCvector alone group,
SC18-kDa-FGF-2 ov ex group, SC21/23-kDa-FGF-2 ov ex group), 3 animals
were sacrificed and the silicone tubes were explanted. The tubes’
contents were extracted, pooled, and diluted in 100 Al PBS. To
dissolve all bound FGF-2 without cell lyses, 10 Al Heparinase III
(1 U/ml [Sigma, Germany]) was added and the mixture incubated
at 37-C. After centrifugation, the supernatant was coated overnight
Fig. 2. Representative photographs of Western blot analysis of cell populations selected for transplantation.
K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
at 4-C on a 96-well plate. FGF-2 protein (b-FGF-2 [Tebu/
Peprotech, Germany]) was coated in 6 defined concentrations in
PBS to build a standard. The next day, unspecific antibody binding
was blocked using 1% horse serum in PBS. Samples were
incubated overnight at 4-C with a-FGF-2-antibodies (1:300 in
PBS, [Transduction Lab., Germany]). After washing with PBS,
detection of FGF-2 was performed by using mouse IgG-VectastainKit (Alexis, Germany) and ABTS-Substrate-Kit (Alexis). Absorption measurement was done at 405 nm with the help of ELX-800
multiplate reader (BioTek Instruments, Software: Mikrowin,
Version 3.27, 1999 [Mikrotek Laborsysteme GmbH, Germany]).
Functional assessment of nerve recovery
Within the 3 months group, functional assessment of motor
recovery (walking track analysis) and – after sacrificing the
animals – morphometrical analysis were performed. In the 6 months
group, additional evaluation of sensory nerve recovery (withdrawal
test), electrophysiological recordings, and retrograde labeling using
the neuron marker DiI were carried out. A summary of the
experimental design is given in Fig. 1.
The number of rats tested in this approach differed at each time
point because automutiliation could not be completely prevented in
some animals.
Motor recovery—walking track analysis
Analyses of a rat’s walking pattern by recording its footprints
and calculating the Sciatic Function Index (SFI) is a wellestablished and commonly used method for the assessment of
motor nerve recovery after sciatic nerve injury (Bain et al., 1989;
de Medinaceli et al., 1982; Hare et al., 1993; Meek et al., 1997).
To evaluate sciatic nerve recovery, walking track analysis was
carried out before implantation and 6 weeks and 12 weeks (3
months group) or 12, 16, and 24 weeks (6 months group) after
implantation.
To obtain the walking pattern, the hind paws of the rats were
pressed onto an inkpad (Pelikan, Hannover, Germany) and to reach
a dark compartment, the rats were allowed to walk up a small
inclining gangway (slope 20-, length 1 m, width 14 cm), which
was lined with white paper (modified from Klapdor et al., 1997;
Ozmen et al., 2002). All rats had a few pre-training runs.
The footprints were digitized and the following parameters
were measured by using a special computer program called
‘‘Footprint’’: (1) The print length (PL, distance from the heel to
the third toe), (2) the toe spread (TS, distance from the first to the
fifth toe), and (3) the intermediate toe spread (ITS, distance of the
second to the forth toe). All these measurements were taken from
the left experimental paw (EPL, ETS, EITS) as well as from the
right non-operated paw (NPL, NTS, NITS) of each rat tested. By
means of these data, the SFI, which results from the differences
between the injured and the intact contralateral paw, was calculated
by the modified formula from Bain et al. (1989):
SFI ¼ ð 38:3 ðEPLNPLÞ=NPLÞ
þ ð109:5 ðETSNTSÞ=NTSÞ
þ ð13:3 ðEITSNITSÞ=NITSÞ 8:8
An SFI of nearly 0 is normal and an SFI of 100 indicates total
impairment of the sciatic nerve.
141
Electrophysiological recordings
To analyze target organ reinnervation and to evaluate the
conductivity of regenerated tissue cables in vivo, compound
muscle action potentials (CMP) were recorded. To avoid
decrease of body temperature during anesthesia and for stabilizing it at 37-C for unbiased recordings, animals were kept on an
electric pad and body temperature was repeatedly controlled. The
electric pad was switched off only during recording. 6 months
after implantation, the rats were anesthetized (Chloral hydrate,
370 mg/kg body weight) and the left sciatic nerve with the
implanted silicone guide was exposed. Empty nerve guides were
explanted and the animals sacrificed. Electrophysiological recordings of regenerated nerves (bridging tissue cables across the
gaps) were performed comparably to Klinge et al. (2001). The
nerves and nerve guides were dissected microsurgically and
electrically shielded against the surrounding tissue using latex
patches. Bipolar hook electrodes (steel) were contacted with the
nerves proximally or distally to the nerve guides, respectively,
and pulsed with single rectangular stimuli of 0.1 ms duration by
a software-controlled stimulus generator (KeypointR Portable,
Medtronic Functional Diagnostics A/S, Denmark). Filters were
set at a bandwidth of 20 – 3000 Hz and stimulus intensities of
0 – 10 mA were applied. Stimulus intensity was gradually raised
from above the threshold of a minimum response to a level of
30% above the maximum response. The evoked responses were
recorded by bipolar EMG needle electrodes inserted into tendon
and belly of the gastrocnemius muscle. Recording of the CMP
was accomplished using an electromyograph for clinical applications (KeypointR Portable), which was connected to a notebook. Following a first recording after stimulation of the nerve
proximally to the nerve guide, a second recording was done after
stimulation distally to the silicone tube. The nerve conduction
velocity was estimated by the different latencies and the distance
between the proximal and distal stimulation point (20 mm).
Afterwards, the right sciatic nerve was exposed, the stimulation
electrode was placed above the bifurcation, the recording
electrodes were inserted into tendon and belly of the right
gastrocnemius muscle, and an evoked CMP was recorded as
control.
Sensory recovery—withdrawal test
To evaluate the recovery of the sensory nerve function in the
6 months group, we used the withdrawal reflex elicited by a hot
water stimulus (Derby et al., 1993; Young et al., 2001) before and
1, 4, 8, and 12 weeks after implantation, respectively. For testing,
the respective rat was held above a hot water bath (50-C). One hind
leg was fixed close to the animal’s body while the other hind leg
was hanged down. The paw from the rat’s hanging leg was
submerged into the water and the time until retraction of the paw
was monitored. If there was no retraction within 5 s, the hind paw
was removed from the water to avoid tissue damages. The first test
was performed at the left experimental paw followed by the right
contralateral paw as a control.
Retrograde labeling of regenerated neurons—tissue preparation
and fluorescence microscopy
To determine the quality of regeneration (motor or sensory) in
the 6 months group, sensory and motor neurons projecting into the
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K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
regenerated nerve were retrogradely labeled using the fluorescent
tracer DiI (1,1V-dioctadecyl-3,3,3V,3V-tetramethyl-indocarbocyanine
perchlorat, Molecular Probes, Netherlands).
After electrophysiological investigations of the left and the right
sciatic nerve, the right-sided wound was sutured as above. The left
sciatic nerve was transected distally to the silicone tube and DiI
crystals were instilled with a forceps over the cross section. After 1
h, the crystals were removed carefully by 0.9% NaCl and the
wound was closed by muscle and skin sutures. Two weeks after
tracer application, the animals were sacrificed by CO2 and the
tubes were explanted very quickly. The animals were then
transcardially perfused (0.9% NaCl in distilled water) and fixated
with 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.4). The
spinal cord and the ipsilateral left dorsal root ganglia (DRGs) L4 –
L6 were excised.
The dissected spinal cord and DRGs were fixed by immersion
24 h in 4% paraformaldehyde, freeze protected in sucrose (30%),
and cut into 14-Am (DRGs) and 25-Am (spinal cord) serial cryostat
sections (Leica, Germany), respectively. The sections were
mounted on uncoated slides and evaluated immediately.
Sections were examined by an epifluorescence microscope
(BX60, Leica, Germany) using U-MWB fluorescence filter (BP
450 – 480. DM 500. BA 515, Olympus, Germany); retrogradely
labeled cells showed the red-orange DiI-fluorescence. To visualize
the punctuated yellow appearing DiI crystals located in the
cytoplasm of each DRG neuron, we used a second fluorescence
filter (U-MWU fluorescence filter, BP 330 – 385, DM 400. BA
420, Olympus).
For each animal, the total number of DiI-labeled motoneurons
was counted on every fifth section at a 20 or 40 magnification.
The sum of the total number of labeled neurons in every fifth
section was calculated and interpolated for all sections (from first
to last section, which contained labeled neurons).
The number of DiI-positive sensory neurons in the DRGs of
each animal was expressed as the percentage of the total neuron
number. Five random sections were selected for each DRG and the
total cell number as well as the number of labeled neurons was
counted. To avoid double counting, only neurons with a wellrecognizable nucleus were recorded. To avoid bias of the
evaluation, the observer was blinded.
Analysis of regenerated sciatic nerve tissue
Tissue preparation
Analyses of silicone nerve guides and quantification of nerve
fibers were in the regenerated tissue cables done as described
before (Timmer et al., 2003). In brief, at the end of the
observation period (3 or 6 months after transplantation), the
animals were sacrificed and the silicone tubes together with
incorporated tissue werer explanted. The tubes were fixed in a
fixative according to Karnovsky (2% paraformaldehyde, 2.5%
glutaraldehyde in 0.2 M sodium cacodylat buffer, pH 7.3) for
24 h. Afterwards, the tubes were longitudinally opened, tissue
cables bridging the distance between the stumps were removed
and rinsed three times with 0.1 M sodium cacodylat buffer
containing 7.5% saccharose. Postfixation was performed in 1%
OsO4 for 1.5 h. Staining of myelin sheaths was done by a
modified protocol according to Schultze (Schultze, 1910) in 1%
potassium dichromate followed by a 24-h ethanol (25%) step and
an incubation in hematoxylin (0.5% in 70% ethanol) for 24 h.
After dehydration, tissue was epon embedded. Semi-thin (1 Am)
and ultra-thin (50 nm) transverse sections of the regenerated
tissues were cut with glass knives or a diamond knife, respectively.
Semi-thin sections were mounted on uncoated glass slides and
additionally stained for myelin with toluidine blue. These sections
were observed by light microscopy (BX60, Leica) at 400
magnification. Ultra-thin sections were placed on 0.5% formvarcoated copper grids followed by staining with uranyl acetate (5
min) and Reynold’s lead citrate (3 min). Ultra-structures of the
regenerated tissue were analyzed using an EM 9 2S electron
microscope (Zeiss, Germany).
Semiautomatic morphometry of regenerated
tissue—quantification of myelinated nerve fibers
In both experimental groups (3 months and 6 months), only gap
bridging tissue cables were evaluated on defined levels distally to
the former proximal nerve stump, 3 months group: +1.0 mm,
+5.0 mm, +7.0 mm and the endpoint of regenerated myelinated
axons (which varied among the animals), 6 months group: +7.0
mm, +15.0 mm). For electron microscopy, point +1.0 mm and the
endpoint of regenerated myelinated axons were considered in the
3 months group (>7.0 mm) and in the 6 months study, point +15.0
mm was ultra-structurally analyzed.
At the respective levels, number, diameter, and g-ratio of the
regenerated myelinated axons were calculated. The number of
regenerated myelinated axons was set in relation to the whole
cross-section caliber of the tissue cables to determine the nerve
density, fibers/mm2. Because of the large size of the cross sections
at point +1.0 mm, the myelinated axons in an area of 10,000 Am2
were counted followed by measuring the cross-section area and
interpolating the total axon number. Counting of total fiber
numbers was carried out on entire cross-section area at point
+5.0 mm, +7.0 mm, and >7.0 mm. The g-ratio, an index for the
grade of axon myelination, which is determined by the axon
diameter divided by the total fiber diameter, was evaluated for 100
axons of each section. The smaller the g-ratio, the better the
myelination of the axon.
For analyzing, digitized images of the sections were used (CCD
camera, Olympus Photomicrographics System PM20 and AnalySIS ProR, Version 3.1, Soft imaging System GmbH, Germany).
Quantification of the sections was performed using a computer
macro that was developed on the basis of AnalySIS ProR as
described before (Timmer et al., 2003). To avoid bias of the
evaluation, the observer was blinded.
Statistical analysis
All results are expressed as mean T SEM. The functional,
morphometrical, and electrophysiological assessments were analyzed using a computer program for statistical evaluation (StatView for Windows, Version 5.0; SAS Institute Inc., USA). In
case of a normal distribution, the Student’s t test (uncorrelated
data, two groups), the t test (correlated data, two parameters
within a group), or a one-factor analysis of variance (ANOVA,
uncorrelated data, more than two groups) followed by a FisherPLSD post hoc test were performed, respectively. Unless there
was no normal distribution of the data, the Mann – Whitney U test
(uncorrelated data, two groups) or the Kruskall – Wallis test
(uncorrelated data, more than two groups) were carried out.
Categorical data were analyzed by the Chi-square test. Only data
from subgroups with n = 3 samples were included into statistical
analyses; for groups with n = 2 samples, the original single
results were presented.
K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
Results
Functional assessment of nerve recovery
Sensory recovery—withdrawal test (6 months group)
To analyze the sensory nerve recovery, we used the
withdrawal reflex elicited by a hot water stimulus. 1, 4, 8, and
12 weeks after sciatic nerve lesion and tube implantation, the
latency of retraction of the experimental paw from hot water was
monitored. The right intact hind paw of the same animal served
as control. Figs. 3A – C summarize the results; 1 week after tube
implantation, the retraction time of the left experimental paw
was about 2 s longer and significantly (P < 0.05) increased
143
compared to all controls in all experimental groups. First signs
of sensory recovery could be monitored after 4 weeks in the
SC21/23-kDa-FGF-2 ov ex group (Fig. 3B). There was still a
significant difference compared to the control sides in all groups
but the latency of the withdrawal of the left experimental paw in the
SC21/23-kDa-FGF-2ov ex group was significantly (P < 0.05) shortened
compared to the SCphysiol group and the SC18-kDa-FGF-2 ov ex
group. There were no changes between the mean latency values
observable 4 and 8 weeks after implantation (data not shown).
However, after 12 weeks, the thermoreception has returned also in
the SC18-kDa-FGF-2 ov ex whereas the SCphysiol group showed still
no recovery of sensory function, compared to the control latency
(Fig. 3C).
Fig. 3. Withdrawal times elicited by a hot water stimulus. (A) The individual contralateral side with uninjured sciatic nerves served as control for each
experiment. Mean withdrawal latency T SEM depicted for the experimental paw in the respective group compared to the mean value of the intact paws
(Control) of all animals in all groups after 1, 4, and 12 weeks. (B and C) Mean withdrawal latencies T SEM, 4 (B) and 12 weeks (C) after tube grafting,
respectively. Asterisks indicate significant differences between the group mean values compared to the groups marked by an arrow (*P < 0.05, ANOVA and
Fisher-PLSD test). (D) Representative photograph of retrogradely labeled DiI-positive dorsal root ganglion (DRG) cells projecting into the regenerated
nerves, scale bar: 100 Am. (E) The mean percentage T SEM of labeled sensory neurons in the DRGs L4 to L6 is shown. Asterisk indicates significant
differences between the marked group mean values compared to the groups marked by an arrow (*P < 0.05 Student’s t test). Number of evaluated DRGs per
group: SCphysiol: n = 9, SCvector alone: n = 6, SC18-kDa-FGF-2 ov ex: n = 12, SC21/23-kDa-FGF-2: n = 6.
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K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
Motor recovery—walking track analysis and electrophysiological
recordings
To assess the functional nerve recovery, walking track analysis
was carried out before and 6, 12, 16, and 24 weeks after sciatic
nerve lesion and silicone tube implantation.
There was no impairment of the rat’s posture of the paws and
the gait before tube implantation. The mean SFI of all different
animal groups was nearly 0. According to the sciatic nerve lesion,
the walking pattern of the animals changed, because of the changed
posture of the experimental hind paw. Resulting from an increased
print length and a decreased toe spread, the calculated SFI was
significantly declined in all groups 6 weeks after implantation with
a mean value of approximately 100. Indicating no successfully
functional muscle reinnervation, there was no significant improvement of the SFI recognizable neither in the following 6 weeks
(3 months group) nor in the following 24 weeks after implantation
(6 months group) (data not shown).
Electrophysiological investigations were just carried out for
animals with a regenerated tissue cable bridging the gap between
the proximal and distal nerve stump 6 months after implantation.
The intact sciatic nerve of the right hind paw was used as
control (Table 1). Compound muscle action potentials (CMPs)
and the nerve conduction velocity of regenerated nerve fibers
were calculated by recording the latency of a CMP after
stimulation of the nerve proximally and distally to the tube
(Table 1). The amplitudes of the CMPs following stimulation
with supermaximal stimulation intensities of the regenerated
nerves (range: 0.28 – 14.66 mV) were significantly smaller than
the maximal CMP amplitudes of the control sides (range:
24.41 – 74.09 mV) (Table 1). Latencies from stimulation at the
beginning of the CMPs were larger at the regenerated sides
(range: 1.80 – 4.30 ms) as compared to the control sides (range:
0.90 – 1.80 ms), even though the distances of AP propagation
along the nerve fibers were approximately similar for both sides.
As evidence for successful muscle reinnervation after 6 months
observation time, CMPs were recorded in the SCphysiol group
and the SC18-kDa-FGF-2 ov ex group. However, there was no
significant difference in nerve conduction velocities between both
groups (SCphysiol 12.53 m/s T 5.33 m/s, n = 3; SC18-kDa-FGF-2 ov ex
10.97 m/s T 3.83 m/s, n = 4). In the regenerated nerves, conduction
velocities were dramatically reduced as compared to the normal
conduction velocities of the uninjured rat sciatic nerves, which were
reported to range at 25 – 30 m/s (Tham et al., 1997).
In summary, functional assessment indicated a more
pronounced regeneration of sensory function (thermoreception)
by grafted Schwann cells over-expressing different FGF-2
isoforms as compared to physiological Schwann cells. Furthermore, over-expression of the high molecular weight 21/23kDa-FGF-2 isoforms by grafted Schwann cells resulted in
earlier signs of recovery of thermoreception as over-expression
of 18-kDa-FGF-2. In contrast, motor recovery after the 6
months observation period was only present after overexpression of 18-kDa-FGF-2 or transplantation of physiological
Schwann cells as revealed by recording of compound muscle
action potentials elicited by stimulation of the regenerated
nerve tissue.
Additional experiment
FGF-2 ELISA
One week after implantation of the different types of Schwann
cells, different amounts of free FGF-2 were measured in the
silicone tube content supernatants of 3 tubes out of each
experimental group as listed below: SCphysiol group: 0.56 ng/Al,
SCvector alone group: 0.56 ng/Al, SC18-kDa-FGF-2 ov ex group: 0.90 ng/Al,
SC21/23-kDa-FGF-2 ov ex group: 0.66 ng/Al. These results suggest
that more free FGF-2 is located at the side of transplantation in
tubes containing FGF-2 over-expressing cells (SC18-kDa-FGF-2 ov ex
group as well as SC21/23-kDa-FGF-2 ov ex group) compared to the
control groups (SCphysiol group, SCvector alone group). To clarify
whether FGF-2 is actively secreted by the transplanted cells and
whether the FGF-2 isoforms are biologically active over
prolonged periods, more detailed studies are needed in the
future.
Table 1
Results from electrophysiological recordings
Experimental group
Latency (ms)
Control side
SCphysiol, n = 3
Mean T SEM
SCvector alone, n = 2
SC18-kDa-FGF-2
ov ex,
Mean T SEM
SC21/23-kDa-FGF-2
n=4
ov ex,
n=2
1.3
1.8
1.9
1.7 T 0.2
0.8
1.0
1.7
1.4
0.9
1.2
1.3 T 0.2
1.3
1.1
Amplitude (mV)
Exp side
Control side
Prox stim
Dist stim
3.0
13.3
3.5
6.6 T 3.4
2.0
4.3
2.2
2.8 T 0.7
4.2
7.0
4.9
3.0
4.8 T 0.8
1.8
3.0
2.5
2.1
2.4 T 0.3
24.4
64.1
64.8
51.1 T 13.3
63.3
50.7
41.6
69.8
37.4
74.1
55.7 T 9.5
59.5
66.0
Exp side
Prox stim
Dist stim
15.4
0.2
4.8
6.8 T 4.5
11.6
0.3
5.1
5.6 T 3.3
8.5
4.3
2.4
15.0
7.6 T 2.8
10.2
4.4
4.7
14.7
8.5 T 2.5
Evoked compound muscle potentials (CMPs) were recorded from the gastrocnemius muscle of animals with regenerated tissue cables 6 months after
transplantation. CMPs from the experimental side (Exp side) were compared to that recorded at the contralateral uninjured control side of the same animals.
Stimulation of the of regenerated nerve tissue was performed proximal (Prox stim) and distal (Distal stim) to the implanted tube (distance 20 mm) to enable
estimation of nerve conduction velocities.
K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
Morphological results
Cell tracing
Longitudinal cryostat sections of regenerated nerve tissue 4
weeks after transplantation of pre-labeled Schwann cells showed
the presence of the transplanted cells and their integration into the
regenerated host tissue (Fig. 4).
Quality of neurons projecting into the regenerated nerve tissue
To determine the quality of regenerating neurons, the number of
regenerated sensory and motor neurons was evaluated using
retrograde tracing by tracer DiI. Tracing was carried out 6 months
after tube implantation. Fig. 3D shows representative photographs of
retrogradely labeled sensory dorsal root ganglion (DRG) neurons.
Tracing was only performed in animals with a regenerated tissue
cable bridging the gap between the proximal and distal nerve stumps
(SCphysiol: n = 3, SCvector alone: n = 2, SC18-kDa-FGF-2 ov ex: n = 4,
SC21/23-kDa-FGF-2 ov ex: n = 2). The number of labeled sensory
neurons was recorded from the DRG L4, L5, and L6 in each animal.
Five random sections were selected for each DRG and the total, as
well as the number of labeled neurons was counted, and the
145
percentage of labeled sensory neurons was calculated. Within the
experimental groups, the mean was calculated from all evaluated
DRGs. Labeled sensory neurons were found in the DRGs of all
experimental groups. As summarized in Fig. 3E, significantly more
sensory neurons were labeled in the SC21/23-kDa-FGF-2 ov ex (49.3 T
7.8 neurons) and the SC18-kDa-FGF-2 ov ex (46.9 T 5.7 neurons) groups
as compared to the group receiving physiological Schwann cells
(SCphysiol: 36.3 T 3.6 neurons). The number of labeled sensory
neurons in the SCvector alone group was in between (41.72 T 8.09
neurons).
Labeled motoneurons were counted on every fifth spinal cord
longitudinal section. The total number of labeled neurons in every
fifth section was added up and interpolated. Due to a high SEM,
there was no significant difference in the average number of labeled
motoneurons in the SCphysiol group (number of labeled motoneurons: 0 in n = 2 and 605 in n = 1) and a mean of 240.0 T 97.36 labeled
motoneurons in the SC18-kDa-FGF-2 ov ex group (n = 4 with labeled
motoneurons in all animals). No labeled motoneurons were detected
in the SCvector alone group (n = 2) and the SC21/23-kDa-FGF-2 ov ex group
(n = 2).
Again, these data indicate promoting effects on sensory
recovery for grafted Schwann cells over-expressing FGF-2 isoforms as compared to physiological Schwann cells. This effect
seems to be more pronounced in the SC21/23-kDa-FGF-2 ov ex group.
In addition, no motoneurons contributed to the regenerated nerves
in the SC21/23-kDa-FGF-2 ov ex group, whereas motoneurons
projecting into the regenerated tissue cables were found in the
SC18-kDa-FGF-2 ov ex group.
Morphometrical results
After 3 months observation time, the percentage of tissue cables
containing myelinated axons crossing the midline of the tube was
calculated. The total number of regenerated myelinated axons at
defined levels of the tissue cables as well as the nerve density and
the g-ratio and diameter of the regenerated fibers was determined.
The number of tissue cables containing regenerated myelinated
axons that reached the distal nerve was calculated after 6 months
observation time. Furthermore, tissue cables of the 3 months
group’s blood vessels, with a minimal area of 10 Am2, were
calculated at section point +5.0 mm. Fig. 1 summarizes the
intersection levels and the evaluated data.
Fig. 4. Longitudinal sections through regenerated nerve tissue 4 weeks after
sciatic nerve transection and repair by transplantation of cell surface labeled
Schwann cells. (A) Phase-contrast of the regenerated tissue showing an
outer layer of connective tissue and residing cells in between. (B)
Immunofluorescence produced by grafted cells pre-labeled with PKH26GL cell linker. Scale bar: 200 Am.
Lengths of regenerated myelinated axons
3 months after implantation of the silicone tubes, morphometrical analysis of regenerated tissue cables was carried out. 4
bridging tissue cables could be found in the Matrigel group (n =
10), herein 2 cables contained regenerated myelinated axons,
which, however, never crossed the midline (0% tissue cables
containing of midline crossing myelinated axons, Fig. 5). In the
Matrigel group, 2 tissue cables did not contain regenerated
myelinated axons at all. In the SCphysiol group (n = 13), 7 out of
8 bridging tissue cables contained myelinated axons, which crossed
the midline in 4 cases (57%, Fig. 5) and reached the nerve distal to
the tube (+15 mm) in one case. In the SCvector alone group (n = 13),
6 bridging tissue cables all with myelinated axons could be found.
In the SCvector alone group, myelinated regenerated axons distally to
the midline reached the distal nerve stump in 5 cases (83%, Fig. 5).
In the SC18-kDa-FGF-2 ov ex group (n = 13), the containing
myelinated axons crossed the midline of the gap in 3 out of
5 bridging tissue cables (60%, Fig. 5), and in 1 out of the 3 cables
the axons reached the distal nerve. As a significant difference
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K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
Fig. 5. Percentage of tissue cables containing myelinated axons, which grew over the midline of the silicone tube 3 months after implantation. Asterisks
indicate significant differences between the group mean values of the groups marked by the asterisks compared to the groups marked with arrows (*P < 0.05,
Chi-square test).
(P < 0.05) to all other groups, in the SC21/23-kDa-FGF-2 ov ex group,
all regenerated tissue cables contained myelinated axons (7 out of
n = 11 implantations) crossing the midline of the tube (100%,
Fig. 5). Additionally, in 4 out of the 7 cases the myelinated axons
reached the distal nerve. These data indicate promoting effects on
long-distance outgrowth of regenerating myelinated axons of
grafted Schwann cells over-expressing 21/23-kDa-FGF-2 as
compared to all other experimental setups.
In the 6 months group, semi-thin sections were made at the
distal end of the regenerated tissue cables (section point +15.0
mm), to evaluate the percentage of regenerated myelinated axons,
which reached the distal nerve stump 6 months after implantation.
In the SC18-kDa-FGF-2 ov ex group, 40% tissue cables contained
myelinated axons reaching the distal nerve, and in the SCphysiol
group, significantly (P < 0.05) more tissue cables contained
myelinated axons (66%). Whereas, no myelinated axons could be
found in the distal nerve stump of the SC21/23-kDa-FGF-2 ov ex group
and the SCvector alone group.
Number of regenerated myelinated axons and g-ratio
Morphometrical analysis of the semi-thin sections was carried
out at different section points of the tissue cables by using a
semiautomatic morphometry program on the basis of AnalySIS
(Soft Imaging System). Fig. 6 summarizes the data on total fiber
number and g-ratio as well as data on tissue cable caliber and nerve
density (fibers/mm2) of the 3 months group.
As shown in Fig. 6A, there were no significant differences
between the respective groups with regard to number of myelinated
axons at section point +1.0 mm in the 3 months group due to high
SEM or small sample number (Matrigel group). At section point
+5.0 mm, there were significantly (P < 0.05) less regenerated
myelinated axons in all groups as compared to the proximal level
of the tissue cables (Fig. 6A). Furthermore, compared to the section
point +5.0 mm, fewer myelinated axons were found at section
point +7.0 mm (Fig. 6A). At the most distal point containing
myelinated axons, less myelinated axons were found as compared
to section point +7.0 mm in all groups, except the SCvector alone
group (Fig. 6A). Within the SC21/23-kDa-FGF-2 ov ex group compared
to the other groups, in tendency, most regenerated myelinated
axons were detected at distal levels (+5.0 mm, +7.0 mm, and
>7.0 mm, Fig. 6A). In contrast, the number of regenerated
myelinated axons at distal levels of the tissue cables was
dramatically decreased in the SC18-kDa-FGF-2 ov ex group and after
transplantation of physiological Schwann cells, whereas transplantation of transfected control Schwann cells (SCvector alone
group) resulted in vector-mediated distal sprouting of myelinated
axons (Fig. 6A). Distal sprouting of myelinated axons is not
wanted in the context of reinnervation of single targets by single
neurons. As described above, these vector-mediated effect is no
longer seen after over-expression of the FGF-2 isoforms by the
transplanted cells and it did not influence the effects of the FGF-2
isoforms.
With regard to the g-ratio, a trend to poorer myelination of
regenerated axons in the SC21/23-kDa-FGF-2 ov ex group at section
level +1.0 mm, section point +5.0 mm, and section point +7.0 mm
compared to all other groups was found (Fig. 6B). However, at the
endpoint of the regenerated myelinated axons, the g-ratio of the
SC21/23-kDa-FGF-2 ov ex group was smaller, indicating a better
myelination, than in the other groups (Fig. 6B). Significantly
different (P < 0.05) to the SC21/23-kDa-FGF-2 ov ex group, poorest
myelination of regenerated axons at section point >7.0 mm was
observed in the SCvector alone group (Fig. 6B). Evaluating the gratio within each animal group at the different section points,
myelination of the regenerated axons showed continuous improvement in distal direction in the SC21/23-kDa-FGF-2 ov ex group in
contrast to the SC18-kDa-FGF-2 ov ex group where a more irregular
and impaired myelination was found (decreasing myelination at
mid-tube levels and re-increase >7.0 mm, Fig. 6B).
Analyzing the tissue cable calibers, they became significantly
(P < 0.05) smaller in mid-tube levels (+5.0 mm and +7.0 mm) as
compared to the proximal levels (+1.0 mm) in the SCphysiol,
SCvector alone, and the SC18-kDa-FGF-2 ov ex group (Fig. 6C).
Reaching the distal nerves, tissue cable calibers re-increased except
in the SCphysiol group (Fig. 6C). Only in the SC21/23-kDa-FGF-2 ov ex
group there were no significant changes in tissue cable calibers at the
different levels (Fig. 6C).
Nerve density is related to the tissue cable caliber (Fig. 6D).
Indicating a vector-mediated sprouting of regenerated myelinated
K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
147
Fig. 6. (A, B) Results of myelinated axon number counts and calculation of the g-ratios at defined levels of gap bridging tissue cables (3 months group, data
represent mean T SEM). (A) Number of regenerated myelinated axons in tissue cables after 3 months observation time at the different cross-section levels. Note
the different scaling of y axes. Section point +1 mm represents the interpolated axon number. Section points +5 mm, +7 mm, and >7 mm show the total axon
number of the whole cross-section area. Asterisks indicate significant differences between the section point mean values for all groups at section point +1 mm
compared to all other section points as well as the significant differences in the SC18-kDa-FGF-2 ov ex and SC21/23-kDa-FGF-2 ov ex group at section point +5 mm
compared to section point +7 mm (*P < 0.05, t test). (B) g-ratio of the regenerated myelinated axons in tissue cable cross sections 3 months after implantation.
Asterisks indicate significant differences between the groups marked by the asterisks compared to the groups marked by an arrow (*P < 0.05, Student’s t test).
(C, D) Tissue cable caliber and results for calculating nerve densities of the regenerated myelinated axons at defined levels of gap bridging tissue cables (3
months group, data represent mean T SEM). (C) Changes in tissue cable caliber are depicted. The tissue cables became significantly smaller at mid-tube levels
(+5 mm and +7 mm) as compared to the proximal beginning of the tissue cables in the SCphysiol, SCvector alone, and the SC18-kDa-FGF-2 ov ex group. Towards the
distal nerve, tissue cable calibers re-increased except in the SCphysiol group. (D) Nerve density (fibers/mm2) is related to tissue cable caliber. Asterisks indicate
significant differences between the group mean values compared to the groups marked by an arrow (*P < 0.05, ANOVA and Fisher-PLSD test [difference
between groups] or t test [differences between section points]).
axons in the SCvector alone group, the nerve density at the most distal
levels (>7.0 mm) is not decreasing as in the other groups (Fig. 6D).
In addition, distal tissue cable calibers remained relatively thin and
the total number of myelinated axons (Fig. 6A) increased at distal
levels (>7.0 mm) compared to mid-tube levels (+7.0 mm) in the
SCvector alone group (Fig. 6C).
In the 6 months group, axon numbers were only measured at
section point +7.0 mm. The SCphysiol and the SC18-kDa-FGF-2 ov ex
group contained considerably more myelinated axons than
the SCvector alone and SC21/23-kDa-FGF-2 ov ex group (SCphysiol:
2501 T 435 regenerated myelinated axons [n = 3 tissue cables],
SC18-kDa-FGF-2 ov ex: 1822 T 312 [n = 4], SCvector alone: 299 and 173
[n = 2], SC21/23-kDa-FGF-2 ov ex: 111 and 54 [n = 2]), but because of
small tissue cable numbers, the differences were not significant.
These data indicate a potential degeneration of previously wellgrown axons. Morphological signs of myelin degeneration as onion
bulb formations could be seen in the ultra-structure of regenerated
nerves at section point +15.0 mm of the SC18-kDa-FGF-2 ov ex and the
SC21/23-kDa-FGF-2 ov ex group. At this section point, a low amount of
onion bulb formations and missing axons accompanied by massive
connective tissue was characteristic in the SC21/23-kDa-FGF-2 ov ex
group, whereas onion bulb formations accompanied by unmyelinated as well as myelinated axons in the SC18-kDa-FGF-2 ov ex group
were found (Fig. 7). Reflecting the high axon numbers at section
points >7.0 mm in the SC21/23-kDa-FGF-2 ov ex group 3 months after
implantation (Fig. 6A), these findings indicate a different time
course of the postulated degeneration.
Diameter of the regenerated myelinated axons
Evaluation of the axon diameter in tissue cables of the 3 months
group revealed a significantly ( P < 0.05) smaller axon diameter in
the SC21/23-kDa-FGF-2 ov ex group (2.50 Am T 0.07 Am) compared to
the SCvector alone group (2.95 Am T 0.21 Am) at mid-tube levels
(section point +5.0 mm). In all experimental groups, only smalldiameter myelinated axons were regenerated (mean axon diameter
<3.5 Am) (data not shown).
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K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
Fig. 7. Representative photographs of semi-thin sections (section point + 15 mm) 6 months after grafting provide an impression of tissue appearance in the
SC18-kDa-FGF-2 ov ex group (A) and SC21/23-kDa-FGF-2 ov ex group (D). Scale bar: 10 Am. Photographs of the respective ultra-thin sections at the distal nerve
(+15.0 mm) reveal onion bulb formations as signs for myelin degradation together with well-myelinated (arrow) and unmyelinated (UMA) axons in the
SC18-kDa-FGF-2 ov ex group (B, C) and only few onion-bulb-like structures surrounded by massive connective tissue in the SC21/23-kDa-FGF-2 ov ex group (E).
Scale bar: 2 Am.
Ultra-structure of the regenerated myelinated axons
According to the findings from our short-term investigations
(Timmer et al., 2003), transmission electron microscopy of
regenerated nerve fascicles in transverse sections in the distal part
of the tissue cables revealed the presence of myelinated and
unmyelinated axons associated with Schwann cells. Although
differing g-ratios of regenerated axons were found in the different
experimental groups, the axons in all groups displayed correct
myelin sheaths 3 months after transplantation (Fig. 8). Myelin
compaction is not affected by the over-expression of the different
FGF-2 isoforms by the transplanted Schwann cells compared to
transplantation of physiological Schwann cells, 3 months after
transplantation.
Percentage of vascularized area at section point +5.0 mm
To check the effects of the FGF-2 isoforms on angiogenesis, the
percentage of vascularized tissue covered by blood vessels with a
minimum area of 10 Am2 was calculated for section point +5.0 mm
in tissue cables after 3 months observation time (Fig. 9). The
SCphysiol, SC18-kDa-FGF-2 ov ex, and SCvector alone groups contained
significantly more blood vessels than the SC21/23-kDa-FGF-2 ov ex
group (SCphysiol: 2.39 T 0.16%, SCvector alone: 2.26 T 0.4%,
SC18-kDa-FGF-2 ov ex: 2.62 T 0.23%). Differences between the
Matrigel and the SC21/23-kDa-FGF-2 ov ex group were not significant
because of the small animal numbers in the Matrigel group. Indicating an effect on peripheral nerve regeneration that is independent
of the grade of vascularization, the smallest amount of blood vessels
at mid-tube levels were found in the SC21/23-kDa-FGF-2 ov ex group
(1.23% T 0.18).
Discussion
Several studies using cell free synthetic nerve grafts examined
the effects of different growth factors on peripheral nerve repair
across long gaps including FGF-2 (Aebischer et al., 1989), BDNF
and NT-3 (Xu et al., 1995), and GDNF and NGF (Fine et al.,
2002). Short-term studies of our own group showed that a cellular
substrate like transplanted genetically modified Schwann cells
over-expressing FGF-2 isoforms is a promising tool to promote
K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
149
Fig. 8. Ultra-structure of myelin sheaths of regenerated axons revealed no deficits in myelin formation, 3 months after grafting. Representative photographs (A,
C, E, G: scale bar: 2 Am) and higher magnification of the myelin compaction (B, D, F, H: scale bar: 1 Am) from the SCphysiol group (A, B), the SCvector alone
group (C, D), the SC18-kDa-FGF-2 ov ex group (E, F), and the SC21/23-kDa-FGF-2 ov ex group (G, H).
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K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
Fig. 9. Mean percentage T SEM of the vascularized cross-section area
composed of blood vessels with a minimum area of 10 Am2 at section point
+5 mm 3 months after grafting. Asterisk indicates significant differences
between the SC21/23-kDa-FGF-2 ov ex group compared to all other groups (*P <
0.05; ANOVA and Fisher-PLSD post hoc test).
nerve regeneration across long gaps (Timmer et al., 2003). In the
present study, we analyzed long-term regeneration and functional
recovery of transected rat sciatic nerves and focused on the
different effects of low and high molecular weight (HMW) FGF-2
isoforms, applied by grafted Schwann cells via somatic gene
transfer.
21-/23-kDa-FGF-2 over-expression in vivo mediates sensory nerve
recovery
The rationale to perform a distinct analysis of the in vivo effects
of the 18-kDa-FGF-2 isoform compared to the 21-/23-kDa-FGF-2
isoforms was based on previous findings of differential regulation
of endogenous FGF-2 isoforms in injured peripheral nerves
(Grothe et al., 2000a; Meisinger and Grothe, 1997). The FGF-2
protein is expressed in different isoforms, representing different
translation products from a single mRNA (Florkiewicz et al., 1991;
Florkiewicz and Sommer, 1989). In DRGs, the elevation of the
endogenous FGF-2 following injury implied a trophic function of
this molecule for sensory neurons. Furthermore, the selective
induction of different FGF-2 isoforms after nerve lesion suggested
different physiological functions at the lesion site and in DRGs
(Meisinger and Grothe, 1997). Indicating a time-dependent effect
on the regeneration process, 18-kDa-FGF-2 was enhanced 5 h after
nerve crush in the proximal and distal nerve stumps, whereas
HMW-FGF-2 isoforms displayed a stronger upregulation 7 days
after the lesion (Grothe et al., 2000a; Meisinger and Grothe, 1997).
In correlation with the differential regulation of the FGF-2
isoforms, these molecules seem to support nerve regeneration
differentially as well. In the present study, first signs of recovery
of thermoreception were seen 4 weeks after implantation of 21-/
23-kDa-FGF2 over-expressing Schwann cells. In addition, in the
SC21/23-kDa-FGF-2 ov ex group, significantly more DRG neurons
projected in the regenerated nerve tissue as compared to the
SCphysiol group. In the SC18-kDa-FGF-2 ov ex group, a retarded
recovery of thermoreception 12 weeks after implantation and less
DRG neurons projecting in the regenerated nerves 6 months after
implantation were found.
In the SC21/23-kDa-FGF-2 ov ex group, no motoneurons projecting
into the regenerated tissue cables could be detected 6 months
after implantation. This finding, together with signs of axon
degeneration at the most distal levels of tissue cables of the
SC21/23-kDa-FGF-2 ov ex group 6 months after grafting, suggests a
possible inhibitory effect on motoneuron regeneration or the
absence of a trophic factor supporting motoneurons. The possible
degeneration of myelinated axons seems to be retarded in the
SC18-kDa-FGF-2 ov ex group as compared to the SC21/23-kDa-FGF-2 ov ex
group. This different time pattern is suggested by different
amounts of onion bulb formations in the distal nerves of both
groups 6 months after grafting. Further evidence for a selective
sensory regeneration promoting effect of 21-/23-kDa-FGF-2
isoforms compared to 18-kDa-FGF-2 in vivo could be drawn
from the fact that although no functional motor recovery could be
detected in any of the experimental groups by walking track
analysis, recording of compound muscle action potentials was
found in the SC18-kDa-FGF-2 ov ex and the SCphysiol but not in the
SC21/23-kDa-FGF-2 ov ex group.
18-kDa-FGF-2 over-expression in vivo mediates inhibitory
functions on myelination of regenerating axons
It is reported that 18-kDa-FGF-2 is acting as a negative
component in the control of myelin synthesis (Morgan et al.,
1994). Administration of forskolin stimulated the expression of P0,
a myelin-related protein, and this forskolin-mediated P0-induction
was prevented by 18-kDa-FGF-2 (Morgan et al., 1994). The
upregulation of FGF-2 at the lesion site suggests a physiological
role of this factor during the myelination process of regenerating
fibers (Meisinger and Grothe, 1997). Additional evidence for a
physiological significance of FGF-2 during peripheral nerve repair
comes from FGF-2 deleted mice. In the absence of FGF-2, the
axon and myelin diameter of regenerated fibers was increased,
which, however, was not accompanied by changes of the myelin
compaction (Jungnickel et al., 2004). The present study revealed a
complex situation with regard to g-ratio of regenerated fibers.
Usually, the mean g-ratio for uninjured myelinated fibers inside the
sciatic nerve ranged between 0.6 and 0.7 (Fansa et al., 1999). In the
SC18-kDa-FGF-2 ov ex group, the mean g-ratio at defined levels of the
regenerated tissue cables ranged inconstantly above 0.7. Especially
at the most distal levels, myelination was relatively low compared
to that in the SCphysiol and the SC21/23-kDa-FGF-2 ov ex groups. These
findings suggest an inhibitory function of 18-kDa-FGF-2 during
myelination in vivo.
Over-expression of 21-/23-FGF-2 isoforms stimulates
long-distance myelination of regenerating axons
In contrast to the 18-kDa-FGF-2 isoform, the 21-/23-kDa-FGF-2
isoforms showed a potent suppression of the grade of myelination
only at the beginning of regeneration, represented by high g-ratio
proximal and at mid-tube levels of the regenerated tissue, whereas
at the most distal levels, a high grade of myelination (smaller
g-ratio) was evident. Furthermore, 3 months after implantation,
only in the SC21/23-kDa-FGF-2 ov ex group all regenerated tissue
cables contained regenerated axons that were myelinated up to
K. Haastert et al. / Neurobiology of Disease 21 (2006) 138 – 153
levels distal to the mid-tube. These results suggest that HMWFGF-2 isoforms mediate remyelination of axons especially in
longer distances from the proximal nerve stump.
Differential effects of FGF-2 isoforms were also seen in other
systems
Differential effects of the FGF-2 isoforms have been shown
for cells of the central and peripheral nervous system. In cultures
of dissociated dopaminergic neurons obtained from rat mesencephalon at embryonic day 14, 18-kDa-FGF-2 supplemented to
the medium accounts for significantly increased branching points
of neurites, whereas, HMW-FGF-2 isoforms significantly
enhanced neural soma areas (Grothe et al., 2000b). Furthermore,
over-expressed HMW-FGF-2 seems to specifically affect karyokinesis in postmitotic peripheral and central neurons in culture
(Nindl et al., 2004). The over-expression of the 18-kDa-FGF-2
isoform and HMW-FGF-2 in cultured PC 12 cells and in
immortalized Schwann cells resulted in distinct altered cell
morphology (Grothe et al., 1998). Additionally, cell proliferation
is differentially affected by the different FGF-2 isoforms; 18-kDaFGF-2 over-expressing cells demonstrated significant reduction of
proliferation compared to the HMW-FGF-2 producing PC 12
cells and Schwann cells (Muller-Ostermeyer et al., 2001). In a rat
model of Parkinson’s disease, enhanced reinnervation, survival,
and functional impact of dopaminergic neurons were seen after
co-transplantation with HMW-FGF-2 over-expressing Schwann
cells compared to the 18-kDa-FGF-2 over-expressing cells
(Timmer et al., 2004).
In systems outside of the nervous system, for example, the low
and HMW-FGF-2 isoforms, when over-expressed in NIH 3T3 and
A31 cells, were mitogenic but differed with regard to invasion
potential, drug resistance, and gene amplification potential (Dini et
al., 2002). In addition, high but not low molecular weight FGF-2
isoforms induce hypertrophy in cultured neonatal cardiomyocytes
and there is also in vivo evidence linking HMW-FGF-2 to
cardiohypertrophy (Kardami et al., 2004).
These results suggest that differential functions of FGF-2
isoforms are a more common event in several cell systems.
Role of FGF-2 isoforms within the regeneration scenario
We demonstrated here differential in vivo effects of overexpressed 18-kDa-FGF-2 and 21-/23-kDa-FGF-2 isoforms produced by transplanted genetically modified Schwann cells in an
animal model of peripheral nerve repair across long gaps.
Other neurotrophic factors and growth factors also showed
distinct beneficial effects on peripheral nerve regeneration. Among
these factors, FGF-1 dispersed in collagen inside of a synthetic
graft demonstrated regeneration of myelinated axon numbers in the
same extent as autologous grafts, over a 10-mm gap in rat sciatic
nerves (Midha et al., 2003). In the same study, treatment with
brain-derived neurotrophic factor (BDNF) showed also good
results, but rather poor compared to FGF-1 treatment and
autologous nerve grafts (Midha et al., 2003). Exogenous leukemia
inhibitory factor (LIF) filled into silicone tubes leaving also a
10-mm gap in rat sciatic nerves improved the fiber regeneration of
damaged peripheral nerves as well as recovery of skeletal muscle
function (Tham et al., 1997). For LIF, it was also shown that this
cytokine filled in synthetic nerve tubes is able to promote fiber
outgrowth even after late secondary repair but to a much lesser
151
extent as compared to syngeneic nerve grafts (McKay Hart et al.,
2003). Vascular endothelial growth factor treatment of acellular
peroneal nerve grafts in rats resulted in similar muscle reinnervation as compared to peroneal autografts. In addition, nerve fiber
regeneration was also enhanced at the proximal coaptation site, but
it did not persist across the distal coaptation site of a 20-mm gap
(Rovak et al., 2004). Nerve growth factor (NGF) and glia-derived
neurotrophic factor (GDNF), respectively, continuously released
by synthetic guidance channels bridging a 15-mm gap in rat sciatic
nerves showed different effects on the regeneration of myelinated
sensory and motor axons as well as unmyelinated axons (Fine et
al., 2002). NGF entrapped in microspheres loaded into synthetic
nerve guides bridging a 10-mm gap in rat sciatic nerves showed
good regeneration of myelinated fibers also, according to fiber
diameter, fiber population, and grade of myelination (Xu et al.,
2003). Furthermore, impregnation of fibronectin tubes bridging a
10-mm rat sciatic nerve gap with BDNF, neurotrophin-3 (NT-3), or
NT-4 revealed that NT-4 preferentially supports and improves the
functional reinnervation of slow motor units, whereas BDNF and
NT-3 showed less beneficial effects on regenerating motoneurons
(Simon et al., 2003).
We show here support of sensory recovery after massive
substance loss in the peripheral nervous system by the transplantation of 21-/23-kDa-FGF-2 over-expressing Schwann cells.
Reflecting the results mentioned above, a combination of different
growth factors would be promising for a more complete therapy of
peripheral nerve injuries in the future. With regard to continuous
availability and stability of the proteins, we favor the use of
genetically modified Schwann cells. Regarding the clinical
application, autologous Schwann cells should be transplanted.
Successful genetic modification of adult rat and human Schwann
cells, respectively, has been already demonstrated (Mauritz et al.,
2004; Haastert et al., submitted for publication).
Acknowledgments
We are very grateful to Kerstin Kuhlemann, Maike Wesemann,
and Natascha Heidrich for excellent technical assistance. We
thank Dr. Peter Claus for plasmid construction, Dr. Doychin
Angelov for helpful hints on retrograde tracing, and Dr. Cordula
Matthies for helping us with devices for electrophysiological
recordings. Studies were supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (to C.G., Gr 857/15-3) and by the
Kogge-Stiftung für veterinärmedizinische Forschung, Gießen,
Germany (to K.H.).
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