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The Journal of Neuroscience, March 1, 2000, 20(5):1822-1830
Impaired Axonal Regeneration in  7 Integrin-Deficient Mice
Alexander
Werner1,
Michael
Willem2,
Leonard L.
Jones1,
Georg W.
Kreutzberg1,
Ulrike
Mayer2, and
Gennadij
Raivich1
1 Department of Neuromorphology, Max-Planck-Institute
of Neurobiology, and 2 Department of Protein Chemistry,
Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany
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ABSTRACT |
The interplay between growing axons and the extracellular substrate
is pivotal for directing axonal outgrowth during development and
regeneration. Here we show an important role for the neuronal cell
adhesion molecule  7 1 integrin during peripheral nerve
regeneration. Axotomy led to a strong increase of this integrin on
regenerating motor and sensory neurons, but not on the normally
nonregenerating CNS neurons. 7 and 1 subunits were present on the
axons and their growth cones in the regenerating facial nerve.
Transgenic deletion of the 7 subunit caused a significant reduction
of axonal elongation. The associated delay in the reinnervation of the
whiskerpad, a peripheral target of the facial motor neurons, points to
an important role for this integrin in the successful execution of axonal regeneration.
Key words:
axonal regeneration; reinnervation; facial nerve; growth
cone; motoneuron; integrin; knock-out mice
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INTRODUCTION |
Changes in adhesion properties of
transected axons and their environment are essential for regeneration.
In the proximal part, the tips of the transected axons transform into
growth cones that home onto the distal part of the nerve and enter the
endoneurial tubes on their way toward the denervated tissue (Fawcett,
1992 ; Bisby, 1995). The distal part of the nerve undergoes Wallerian degeneration, a process involving the removal of the disconnected axons
and their myelin sheaths from the associated Schwann cells. The
denervated Schwann cells proliferate, attach to each other, and form
bands of Büngner, which serve as a permissive substrate for
axonal regrowth. These Schwann cells increase synthesis of adhesion
molecules, such as L1 and laminin, which are inserted into their cell
surface and the surrounding extracellular matrix (ECM) of the
endoneurial tubes (Cornbrooks et al., 1983; Salonen et al., 1987;
Martini, 1994). The process is mirrored by regenerating axons that
upregulate receptors for endoneurial ECM molecules (Lefcort et al.,
1992; Kloss et al., 1999).
The integrins are a large family of receptors for ECM molecules (Haas
and Plow, 1994; Luckenbill-Edds, 1997) that consists of >20 different
heterodimers formed by an and a subunit. Although many
integrins, particularly 1 family members, are important for neurite
outgrowth in vitro (Toyota et al., 1990; Letourneau et al.,
1992; Tomaselli et al., 1993; Weaver et al., 1995; Condic and
Letourneau, 1997), little is known about their physiological role
during axonal regeneration in vivo. In this study we
examined the regulation and function of the 71 integrin, a
receptor for the basement membrane proteins laminins-1, -2, and -4 (Kramer et al., 1991; von der Mark et al., 1991; Yao et al., 1996).
This integrin is mainly expressed in skeletal, cardiac, and smooth muscle (Song et al., 1992; Ziober et al., 1993; Martin et al., 1996),
but it is also present in the developing brain (Van der Flier et al.,
1995; Kil and Bronner-Fraser, 1996; Velling et al., 1996). In the adult
nervous system, we now show that 7 is strongly upregulated in
axotomized neurons in various injury models during peripheral nerve
regeneration, but not after CNS injury. The deletion of the 7
subunit leads to an impairment in axonal outgrowth and a delayed target
reinnervation of regenerating facial motoneurons.
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MATERIALS AND METHODS |
Animals and surgical procedures. Adult homozygous
7 / and littermate controls (6-month-old) on a 129 Sv background
used in this study were obtained from heterozygous crossings (Mayer et
al., 1997). Normal adult C57Bl6 mice were obtained from Charles River
(Sulzfeld, Germany). The animal experiments and care protocols were
approved by the Regierung von Oberbayern (AZ 211-2531-10/93 and AZ
211-2531-37/97); all surgical procedures were performed under
anesthesia with intraperitoneal injection of 150 µl of 2.5% avertin/10 gm of body weight. The facial nerve was cut at the foramen
stylomastoideum, the hypoglossal nerve just before bifurcation, the
vagal nerve at the midcervical level, and the sciatic nerve at the
sciatic notch. For optic nerve crush, the eyeball was gently pushed
forward, and the optic nerve was crushed repeatedly with fine tweezers
for 10 sec. For a direct cerebral trauma, a 2.5-mm-deep, 2.5-mm-long,
parasagittal cortical incision was performed on the dorsal forebrain
(1.0 mm lateral of the midline, beginning 1.0 mm posterior of bregma,
right side), which transected the cerebral cortex, the corpus callosum,
and the fimbria fornix. The regeneration and reinnervation studies were
performed after a facial nerve crush at the stylomastoid foramen.
Light microscopic immunohistochemistry. The animals were
killed in ether, perfusion-fixed in 4% formaldehyde (FA) in
PBS (4% FA-PBS), the tissue was removed, post-fixed in 1%
FA-PBS for 2 hr, and cryoprotected with 30% sucrose overnight and
frozen on dry ice, as previously described (Raivich et al., 1998a).
Briefly, 20-µm-thick sections from brainstem, spinal cord, dorsal
root ganglia, retina, septum, and cerebral cortex and 10 µm
longitudinal sections of the facial and optic nerve were cut at
15°C, collected on gelatin-coated slides, spread in distilled
water, fixed in formalin, defatted in acetone, and pretreated with 5%
goat serum (Vector, Wiesbaden, Germany) in phosphate buffer (PB). The
sections were incubated with primary polyclonal rabbit antibodies
against 7 (1:10,000 dilution), galanin (1:400; Penninsula),
calcitonin gene-related peptide (CGRP; 1:1000; Penninsula) or glial
fibrillary acidic protein (GFAP; 1:5000; Dako, Hamburg, Germany),
monoclonal rat antibodies against 1 (1:6000; Chemicon, Palo Alto,
CA), M (1:6000; Serotec, Oxford, UK), and MHC-1 (1:100; Dianova,
Hamburg, Germany), or with a monoclonal Syrian hamster antibody against CD3 (1:3000; PharMingen, Hamburg, Germany). Then the sections were
incubated with a biotinylated goat anti-rabbit, anti-rat (1:100;
Vector) or anti-hamster (1:100; Dianova) secondary antibody, followed
by incubation with the ABC reagent (Vector), visualization with
diaminobenzidine/H2O2 (DAB;
Sigma, Deisenhofen, Germany), dehydration in alcohol and xylene, and
mounted with Depex (BDH Chemicals, Poole, UK). Double
immunofluorescence was performed with a combination of the primary
antibodies against 7 and 1, then biotinylated donkey anti-rabbit
and FITC-conjugated goat anti-rat secondary antibodies (1:100;
Dianova), followed by Texas Red-Avidin (1:100; Vector) and a
FITC-donkey anti-goat tertiary antibody (Dianova), respectively, and
scanned in a Leica (Nussloch, Germany) TCS 4D confocal laser microscope
with a 10 and 100× objective (pinhole 30/100).
Electron microscopic immunohistochemistry. Perfusion with 40 ml of PBS and 10 mM MgCl2 (Mg-PBS)
was followed by 100 ml of 0.5% glutaraldehyde and 4% FA in Mg-PBS
and by 100 ml of 4% FA and Mg-PBS, nerve dissection, and a 2 hr
immersion in 1% FA-PBS. Vibratome cross sections of 80 µm were
obtained at the level of the growth front (5-6 mm distal to the
crush), followed by pre-embedding immunohistochemistry as described
(Möller et al., 1996). Briefly, free-floating sections were
preincubated with goat serum for 4 hr, followed by incubation with the
primary antibodies against 7- or 1-subunit. The biotinylated,
secondary antibody was applied for 8 hr, followed by ABC reagent
overnight and DAB staining, intensified with cobalt and nickel
sulfates. After immunostaining, sections were fixed with
glutaraldehyde, osmicated, embedded in Araldite (Fluka, Basel,
Switzerland), cut (100 nm), counterstained with uranyl acetate and lead
citrate, and examined in a Zeiss EM 10 electron microscope.
Quantification of light microscopic immunohistochemistry.
Digital images from the stained sections were obtained using a Sony 3 CCD video camera (AVT-Horn, Aachen, Germany) and analyzed with the
OPTIMAS 6.2 imaging system (Bethell). Luminosity values for the
antibody staining intensity (SI) for each individual facial nucleus
were determined using the Mean-SD algorithm as previously described
(Kloss et al., 1999) and subsequent subtraction of the SI of the
adjacent midline (n = 4 animals per time point).
Cell counts. To quantify microglial proliferation, the
animals were injected with 200 µCi of
[3H]thymidine (Amersham, Braunschweig,
Germany) 3 d after facial nerve axotomy and 2 hr before killing by
perfusion. Fixed brainstem sections were obtained as described,
autoradiographed (Raivich et al., 1994), and labeled cells were counted
for the whole facial motor nucleus (six sections per animal).
GFAP-positive stellate astrocytes and CD3-positive lymphocytes were
counted in the facial motor nuclei of two sections per animal.
Detection of 71 integrin mRNA. To study the mRNA, the
facial motor nuclei, the gastrocnemius, and the heart muscle were excised immediately after killing and frozen on dry ice. Individual tissue samples were homogenized and processed using Tristar (Angewandte Gentechnologie Systeme AGS, Heidelberg, Germany) according to the
manufacturer's protocol. RNA extracts (1 µg of total RNA) were
reverse-transcribed with random hexamer primers using superscript II
Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Eggenstein, Germany). Thirty-five cycle
thermoenzymatic amplification of integrin mRNA was performed in a
MJ Research DNA Engine (Peltier Thermocycler, Biometra,
Göttingen, Germany), using the following primers: 7-sense
5'-tgctcagagatgcatcc-3', 7-antisense 5'-caccggatgctcatcaggac-3' and 1-sense 5'-ggcaacaatgaagctatcgt-3', 1-antisense
5'-ccctcaacttcggattgac-3'. Amplification products were analyzed with
gel electrophoresis.
Regeneration rate in the facial nerve. Four days after
facial nerve crush, the animals were killed, followed by a brief, 5 min
perfusion-fixation with 4% FA-PBS and then by a slow, 60 min perfusion with 1% FA-PBS, followed by immediate dissection and freezing on dry ice. Nerves were cut longitudinally, and the
regenerating axons were visualized by immunostaining for galanin or
CGRP. Every fifth section was used per antibody, with an interval of 50 µm, and the distance between the most distal-labeled growth cone and the crush site measured using light microscopic grid scaling. The
average distance for each animal was calculated from four or five
tissue sections.
Reinnervation of the whiskerpad. Under avertin anesthesia, a
flat cut was performed under the skin of the right and left whiskerpad, a gelatin sponge (whiskerpad size, 1-mm-thick) filled with 15 µl of
4% FluoroGold (FG; Fluorochrome, Denver, CO) in
H2O was inserted under the pad, removed after 20 min, and the wound tissue was rinsed with PBS before suture. The
animals were perfused 48 hr after instillation of the retrograde
tracer, and the brainstem sections were spread and dried for 10 min.
The retrograde-labeled motoneurons within the facial motor nucleus (six
sections per animal) were immediately counted under a fluorescence
microscope with UV-light illumination. For illustrations (see Fig.
5B) the sections were covered with Vectorshield (Vector) and
scanned with a Leica TCS 4D confocal laser microscope (488 nm
excitation, 590 nm longpass filter).
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RESULTS |
Regulation of 7 integrin subunit in the regenerating facial
motor nucleus
Low levels of 7 were already observed in the normal brainstem
(Fig. 1a,b). Transection of
the facial nerve led to a massive increase of 7 immunoreactivity on
the axotomized facial motoneurons (Fig. 1a-c). The increase
of 7 was already apparent 1 d after axotomy, reached a maximum
at day 7, and was followed by a decrease at the start of reinnervation
at day 14. A very similar time course was previously shown for neuronal
1 immunoreactivity in the axotomized facial motor nucleus (Kloss et
al., 1999). The specificity of the polyclonal rabbit anti-7 antibody
is shown by the disappearance of the 7 immunoreactivity on
axotomized motoneurons in the 7-deficient mice (Fig. 1c).
The mRNA for the 7 integrin subunits is known to undergo alternative
splicing resulting in 7A and 7B isoforms; both isoforms associate
with 1A and 1D integrin variants (Collo et al., 1993; Ziober et
al., 1993; Van der Flier et al., 1995; Velling et al., 1996). However,
only the mRNA for the 7B and 1A variants was detected both in the
normal and axotomized facial motor nuclei by RT-PCR (Fig.
1d).
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Figure 1.
7 integrin subunit in the facial motor
nucleus after peripheral nerve transection. a, 7
immunoreactivity is weak in the normal brainstem (0 d), but rapidly
increases after transection of the facial nerve (1-42 d).
b, Quantification of 7 staining intensity in the
normal (Co) and axotomized facial motor nucleus
(Ax) shows a strong increase at day 1 and is maximum at
day 7 (n = 4 animals; mean values; error bars
indicate SEM). c, Specificity of the polyclonal rabbit
anti-7 antibody is demonstrated by the disappearance of motoneuronal
staining (at day 3) in the 7/ mouse, compared to the wild-type
animal (7+/+). Scale bars, 125 µm. d, Analysis of
the mRNA isoforms of 7 and 1 integrin subunits by RT-PCR. The
normal and axotomized facial nucleus (Fn-co,
Fn-7d) only contains 7B and 1A mRNA splice isoforms.
Gastrocnemius muscle (GCM) and heart were used as
positive controls; the GCM contains both 7A and 7B, and the heart
contains both 1A and 1D isoforms. L, 100 bp
ladder.
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Expression of 71 integrin in different central and peripheral
injury models
The increase of 7 immunoreactivity on axotomized neurons was
consistently present in different models of peripheral regeneration, but not after CNS injury. Four days after peripheral nerve injury, strong 7 staining was found on the axotomized motoneurons of the
vagal and hypoglossal nuclei (Fig.
2a,b). In the spinal cord, transection of the sciatic nerve caused an increase of 7
immunoreactivity on the spinal motoneurons and in the substantia
gelatinosa (Fig. 2c). Dorsal rhizotomy abolished the
staining in the dorsal but not the ventral horn, suggesting the
localization of 7 on the primary sensory afferents in the substantia
gelatinosa reacting to sciatic injury (data not shown). Axotomized
dorsal root ganglia (DRG) showed a redistribution of 7
immunoreactivity (Fig. 2d). As in previous studies (Velling
et al., 1996), normal, uninjured DRG showed strong 7 immunostaining
on perineuronal satellite cells. This 7 immunostaining disappeared
from the satellite cells after axotomy, but increased on the injured,
small sensory neurons. Interestingly, small caliber sensory neurons
show a particularly robust and rapid neurite outgrowth after axotomy
(Brown et al., 1992). In the normal sciatic nerve, weak 7 was only
detected in the perineurium. It increased strongly on the regenerating axons after crush (Fig. 2e).
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Figure 2.
Enhanced 7 and 1 integrin immunoreactivity
(IR) in different models of peripheral but not of central axotomy.
a-e, Peripheral nerve transection. a, b,
Axotomy of the vagal (a) or hypoglossal nerve
(b) leads to a strong increase of 7-IR in the
corresponding nucleus (4d Ax). c-e,
Transection of the sciatic nerve also leads to strong 7-IR on the
axotomized motoneurons and in the substantia gelatinosa in the lumbar
spinal cord (4d Ax). d, In the normal DRG
(Co), 7 is localized to satellite cells surrounding
the sensory neurons. Sciatic axotomy leads to a disappearance of this
staining and a strong increase of 7-IR on the small sensory neurons
(4d Ax). e, 7 is also upregulated on
the regenerating axons in the sciatic nerve (4d Ax).
f-j, Transection of CNS axons. f, g,
Immunofluorescence double labeling in the axotomized retina revealed a
colocalization of 7 (red) and 1
(green) on blood vessels (yellow
profiles), but no staining of both subunits on normal
(Co) and axotomized retinal ganglion cells (4d
Ax, asterisks). h, i, Cortical incision with
resulting transection of the underlying corpus callosum
(h; cc, arrows) and the septohippocampal
tract (i) induced 7-IR on peritraumatic blood
vessels but not on the axotomized pyramidal cells and septal neurons.
j, 7 is also absent from the axons of normal
(Co/7) and axotomized (4d
Ax/7) RGCs. The arrow points to the
transversal accumulation of peroxidase-positive leukocytes at the
lesion site (4d Ax/7). The optic nerve crush
is also demarcated by the accumulation of M2-positive macrophages
in an adjacent section (4d Ax/M, arrow). Scale bars:
c (also applicable to a, b), h,
j, 250 µm; d, e, i, 100 µm;
f, 50 µm; g, 10 µm.
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In addition to the upregulation of the 7 subunit during
regeneration, there was a parallel increase in immunostaining of the
1 subunit in the axotomized facial nucleus (see Fig. 7; Kloss et
al., 1999). A similar increase was also present in regenerating hypoglossal, vagal, and spinal motoneurons and in the DRGs (data not shown).
Central axotomy did not induce the expression of the 71 integrin
(Fig. 2f-j). In the retina, 7 (red) and 1 (green)
colocalized on the retinal blood vessels (Fig. 2f,g,
yellow profiles), similar to the presence of 71 on the
cerebral blood vessels (Velling et al., 1996). However, both integrin
subunits were absent from the retinal ganglion cells in the normal
retina and 4 d after optic nerve crush (Fig. 2g,
asterisks). Similar lack of 7 immunoreactivity was also
observed in the crushed optic nerve (Fig. 2j). The local accumulation of M2-positive macrophages was used to demarcate the
lesion site (Fig. 2j, arrows) and served as a positive control.
Cortical incision with ensuing transection of the cerebral cortex and
the underlying corpus callosum and fimbria fornix caused moderate 7
labeling on blood vessels around the wound (Fig. 2h, arrows)
but not on the adjacent cortical neurons, including the widely
projecting pyramidal cells in the third cortical layer. No 7
increase was observed on the septal neurons affected by the transection
of the fimbria fornix (Fig. 2i).
In the distal part of the crushed facial nerve, the specific
immunoreactivity to the cytoplasmic part of the 7 subunit was restricted to regenerating axons, with moderate submembranous staining
of growth cones and strong immunoreactivity in fine axonal sprouts
(Fig. 3a-c). This
immunoreactivity was not detected on the associated Schwann cells (s),
irrespective of the stage of axon and myelin (m) detachment.
Immunoreactivity against the extracellular part of the 1 subunit
(Fig. 3d) showed a cell surface staining of axons (n) and
the Schwann cells (s). Specific 1 staining was most pronounced at
the axon-axon (arrow) and axon-Schwann cell contacts (arrowhead).
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Figure 3.
Regenerating facial neurites contain 7- and
1-integrin subunits at the ultrastructural level.
a-c, 7- in regenerating motoneurites
(n) 4 d after nerve crush in association
with intact myelin (m) (a),
with a partially demyelinated (b), and a
completely demyelinated (c), 7-negative
Schwann cell (s). The submembranous and
cytoplasmic staining is attributable to immunoreactivity against the
cytoplasmic part of the 7-subunit. d, The immune
staining against the extracellular part of the 1-subunit is
localized on the cell surface of regenerating neurites
(n) and Schwann cells (s)
at day 7. Pronounced staining at sites of axon-axon and
axon-Schwann cell contacts (arrowhead). Scale bar,
0.45 µm.
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Effects of 7 deficiency on axonal outgrowth and
target reinnervation
To examine the functional role of 7 integrin during nerve
regeneration, we compared the regeneration rate in 7-deficient mice
and wild-type controls obtained from heterozygous crossings (Mayer et
al., 1997). The facial nerve was crushed near the stylomastoid foramen
(Fig. 4a, arrow), allowed to
regenerate for 4 d, fixed, and sectioned longitudinally. Nerve
sections were stained for the neuropeptides CGRP or galanin (Fig.
4b), and the distance between the lesion site and the
axonal growth front was measured with a light microscopic grid
scaling. At day 4, the regeneration distance for the wild-type animals
was 6.01 ± 0.35 mm for galanin- and 6.38 ± 0.29 for
CGRP-immunoreactive axons (Fig. 4c; mean ± SE,
n = 4 animals). A similar distance was also determined
for heterozygous mice (5.96 ± 0.55 for galanin and 5.84 ± 0.55 mm for CGRP-positive axons, n = 2). The homozygous
7-deficient mice (n = 4) showed a significant,
33-35% reduction to 4.04 ± 0.24 mm for galanin- and 4.15 ± 0.48 mm and for CGRP-immunoreactive axons (p < 0.01 for each axonal marker; unpaired t test).
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Figure 4.
7 deficiency leads to reduced axonal outgrowth
of axotomized facial motorneurons. a, The facial nerve
was crushed near the foramen stylomastoideum (arrow).
b, After 4 d, the nerve was cut longitudinally and
stained for galanin or CGRP, which accumulate in the terminals of the
elongating neurites. The average distance between the crush site and
the growth front was determined for each axonal marker in five tissue
sections per animal. The micrograph shows axonal regneration in a
wild-type animal. c, Both the CGRP- and galanin-positive
axonal populations show a regeneration distance of ~6 mm at day 4 in the 7+/+ and 7+/. This regeneration distance was
reduced by 33% (galanin) and 35% (CGRP) in the 7/ mice
(7/, 7+/+, n = 4 mice; 7+/,
n = 2 mice; mean values; error bar indicates SE).
Scale bar, 200 µm.
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Reduced axonal regeneration could lead to a delay in the reinnervation
of the peripheral target. We first examined the time course of
reinnervation of the whiskerpad in normal, C57Bl6 mice (Fig.
5a-c). FG was applied into
the whiskerpads on the operated and unoperated side at different time
points after facial nerve crush. The number of retrogradely labeled
facial motoneurons was counted 2 d after application of the
retrograde tracer (Fig. 5a,b) and compared to the unoperated
side. No FG-labeled motoneurons were observed in the operated facial
motor nucleus 7 d after the initial facial nerve crush. At day 9, there was a steep increase to 53 ± 10% of the contralaterally
labeled neurons, which reached a maximum at day 14 (Fig.
5c).
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Figure 5.
Lack of 7 integrin subunit causes a delay of
target reinnervation in the axotomized facial nerve.
a-c, Experimental model. a, Fluorogold
was applied into both whiskerpads 2 d before killing, retrogradely
transported by normally connected (control side, Co) and
reinnervating motoneurons (axotomized side, Ax), and
detected in the brainstem (b) at different time
points after facial nerve crush. The onset of whiskerpad reinnervation
is observed between day 7 and day 9. c, Time course of
whiskerpad reinnervation, with total number of FG-positive motoneurons
(FG + MN) on the axotomized side, expressed as percentage of the number
on the contralateral side; six brainstem sections were counted per
animal. Scale bar, 100 µm. d, Effect of 7
deficiency on whiskerpad reinnervation 9 and 21 d after facial
nerve crush. Unlike the 7+/+ mice (n = 4), no
FG-positive motoneurons were found in the axotomized, facial motor
nuclei of the 7/ animals (n = 5) at day 9 (p < 0.05%, Wilcoxon test). Both groups of
animals (n = 4) showed a similar reinnervation
index at day 21. The horizontal bar shows the mean
percentage per group.
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To determine the effects of 7 deficiency, the number of FG-labeled
motoneurons in 7/ and 7+/+ mice were compared 9 and 21 d
after facial nerve crush (Fig. 5d). At day 9, wild-type
animals showed a definite onset of reinnervation, with 25 ± 9%
of the motoneurons labeled compared to that on the contralateral side. At the same time point, there were no FG-positive motoneurons in the
operated facial motor nucleus of the 7/ mice
(p < 0.05 compared to wild-type animals,
Wilcoxon test). This effect disappeared at day 21, with both animal
groups showing a similar level of whiskerpad reinnervation.
Neuroglial response in the facial motor nucleus of normal
and 7/ mice
To determine if 7 deficiency also causes central changes in the
injured facial motor nucleus that could impair regeneration, we
examined characteristic markers of the neuroglial response using
established histology and immunohistochemistry protocols (Raivich et
al., 1994; Möller et al., 1996), shown in Figure 6. The neuronal response was assessed
with immunohistochemistry for the 1 integrin subunit, CGRP and
galanin, neuropeptides in axotomized motoneurons (Möller et al.,
1996; Klein et al., 1997), and the astrocyte response with the
number of GFAP-immunoreactive, stellate figures. The early microglial
activation was examined using autoradiography for
[3H]thymidine-labeled, proliferating
cells and immunoreactivity for the M2-integrin. For the late
response at day 14 we stained against MHC-1 on the microglia and
counted the number of CD3-positive, infiltrating lymphocytes. As shown
in Figures 6 and 7, there was no apparent
difference for the neuronal peptides, early and late glial activation
markers, and the recruitment of lymphocytes between the wild-type and
the 7-deficient mice, either with light microscopy (Fig. 6) or at
the quantitative level (Fig. 7). However, there was a clear and
statistically significant increase in the neuronal staining for the
1 integrin subunit on the regenerating motoneurons in the 7/
mice (Figs. 6, 7).
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Figure 6.
Effect of 7 deficiency on the neuroglial
response. All activation markers increase 3 d after facial nerve
transection in the regenerating facial motor nucleus
(Ax), compared to the contralateral, unoperated side
(Co). Note the stronger increase in the neuronal 1
integrin immunoreactivity after axotomy in the 7/ mice. No
apparent difference in the immunostaining for neuronal galanin
(GAL), the GFAP-positive astrocytes or the
M2-positive microglia (M) between the
7+/+ and 7/ animals. Scale bar, 200 µm.
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Figure 7.
Quantitative effects of 7 deficiency on a panel
of neuroglial activation markers. There is a significant increase in
the immunoreactivity for the neuronal (1, galanin, CGRP) and
microglial (M) markers, the number of GFAP-positive, stellate
astrocytes, and the number of
[3H]thymidine-labeled, proliferating cells in the
axotomized facial motor nucleus 3 d after nerve transection
compared to contralateral side. There is also a strong increase in
microglial MHC1 immunoreactivity and the number of CD3-positive
lymphocytes at day 14. Only the immunoreactivity for the 1 shows a
statistically significant change in the axotomized nucleus
between the two groups of animals, with an increase of 70% in the
7/ mice. Open circle, Significant increase for
the axotomized versus the contralateral side
(p < 0.03, paired t test).
Asterisk, Significant increase for the 7/ versus
the 7+/+ animals (p < 0.02, unpaired
t test).
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DISCUSSION |
Expression of 7 and 1 integrin subunits is linked to
peripheral nerve regeneration
The current study shows a highly consistent increase in 71
integrin immunoreactivity on axotomized motor and sensory neurons in
different models of successful, peripheral regeneration. In contrast,
this immunoreactivity for 7 and 1 subunits was not detected on
intact and axotomized adult retinal ganglion cells, which normally do
not regenerate. Similar lack of immunoreactivity was also observed for
axotomized pyramidal cells and the septal neurons. Both the 1
integrin and different associated -subunits are strongly expressed
during brain and retinal development (Neugebauer and Reichardt, 1991;
Tomaselli et al., 1993; Weaver et al., 1995; Kil and Bronner-Fraser,
1996; Velling et al., 1996). Previous studies also show an important
role for 1 integrin family members in the neurite outgrowth of
embryonic retinal ganglion cells (Neugebauer and Reichardt, 1991; Stone
and Sakaguchi, 1996; Ivins et al., 1998; Treubert and Brummendorf,
1998). However, blocking antibodies against the 1 integrin subunit
did not affect axonal regeneration in the adult retinal explants (Bates
and Meyer, 1997). In view of the current findings, high levels of 7
and 1 integrin expression appear not to be linked to axotomy per se
but to a successful type of axonal regeneration in vivo.
Quantitative methods to measure axonal regeneration
In the current study we used two separate methods to compare the
speed of nerve regeneration in normal and 7 knock-out mice: at the
early phase of axonal outgrowth and at the onset of target reinnervation. In the first method, we immunostained longitudinal nerve
sections against the axon-specific neuropeptides CGRP and galanin (Gray
et al., 1992). These peptides are synthesized in two apparently
nonoverlapping motoneuron populations (Moore, 1989) and are transported
anterogradely into the axonal growth cones. Because the facial nerve is
almost purely motoric, the staining of the growth front is specific to
motor axons. Four days after injury, the growth distance in normal mice
was 6.01 ± 0.35 mm for the galanin- and 6.38 ± 0.29 mm for
the CGRP-positive motor axons. Previous studies using
electrophysiological and autoradiographic techniques revealed a
regeneration rate of motor and sensory neurons of ~4 mm/d (Forman and
Berenberg, 1978; McQuarrie et al., 1978; Bisby and Keen, 1985; Chen and
Bisby, 1993). However, they also showed an initial delay of ~2 d
(Forman and Berenberg, 1978). If this delaying effect was included, the
resulting regeneration rate in the current study would be ~3
mm/d.
The second method addressed the effects on the late stage of axonal
regeneration, at the onset of target reinnervation. Here, we applied FG
to the whiskerpads at different time points after crush (Hirota et al.,
1996). The observed onset of FG uptake by reinnervating motor axons
after day 7 corresponds well with the beginning of whiskerpad movements
at day 9 (Chen and Bisby, 1993). Because the total distance between
crush site and whiskerpad is ~18 mm, the axons appear to grow at a
rate of 3.6 mm/d, again assuming an initial delay of 48 hr. The
slightly lower regeneration rate at the early phase of axonal outgrowth
might be attributable to the previously reported suboptimal growth
speed during the first days after injury (Forman and Berenberg, 1978).
Overall, the current data on the speed of axonal outgrowth are in
agreement with previous studies. Importantly, both methods used produce similar results and show little intragroup variation, allowing us to
observe statistically significant changes in relatively small groups of animals.
7 integrin subunit plays an important role in the regenerating
facial nerve
Both 7 and 1 integrin subunits were present on growing axons
in the distal part of the crushed facial nerve and participated in
contacts among axons and between axons and Schwann cells. The importance of the 71 integrin is also underlined by transgenic experiments. 7 null mice show a reduced rate of axonal outgrowth and
a delay in the reinnervation of the whiskerpad, a peripheral target of
facial motoneurons. This effect of 7 deficiency was present in two
nonoverlapping populations of facial motoneurons that express galanin
or CGRP (Moore, 1989), pointing to a rather general role of this
adhesion molecule in promoting regeneration.
The present experiments clearly suggest a peripheral site of 71
action. The 71 integrin is a specific receptor for laminin-1 (11 1), laminin-2 (211), and laminin-4 (221)
(Kramer et al., 1991; von der Mark et al., 1991; Yao et al., 1996). The
laminin-2 and laminin-4 isoforms are present in the normal peripheral
nerve (Kuecherer-Ehret et al., 1990; Hsiao et al., 1993), and their synthesis is increased after injury (Kuecherer-Ehret et al., 1990; Doyu
et al., 1993; LeBeau et al., 1994). At the ultrastructural level,
laminin immunoreactivity is present on the Schwann cell surface and the
basal membranes both facing the regenerating axons (Kuecherer-Ehret et
al., 1990; Wang et al., 1992; Ide, 1996). Immune neutralization of
laminin inhibits axonal growth inside the basal lamina scaffolds in
peripheral nerves (Ide, 1996); a similar effect was observed using
antibodies specific for the 2-laminin chain (Agius and Cochard,
1998). Thus, the 211 and 221 laminin isoforms are
most likely, functionally active targets for the axonal 71
integrin in the regenerating peripheral nerve. In contrast to the
periphery, antibodies against laminin failed to inhibit neurite
outgrowth on the immature spinal cord substrate (Agius et al.,
1996).
Absence of the 7 integrin subunit causes only a partial reduction in
the speed of nerve fiber regeneration. This suggests the presence of
additional axonal molecules promoting axon outgrowth, leading to a
partial functional compensation for the 7 deficiency. The
particularly strong increase of the 1 subunit after axotomy in the
7/ mice clearly supports such a compensatory mechanism via other
associated -subunits (Toyota et al., 1990; Condic and Letourneau,
1997). Other potential groups include cadherins (Seilheimer and
Schachner, 1988; Doherty et al., 1990) and the Ig superfamily of cell
adhesion molecules (Seilheimer and Schachner, 1988; Doherty et al.,
1990; Martini, 1994). Here, a conditional gene-targeting approach to
these molecules in axotomized neurons will shed more light into the
mechanisms regulating cell adhesion and the overall process of
successful axonal regeneration.
7 integrin deficiency does not affect the central
neuroglial response
In contrast to axonal regeneration, the absence of the 7
integrin subunit did not lead to a change of the cellular response in
the axotomized facial nucleus, the affected part of the CNS. Nerve transection is known to cause a pronounced cellular response at
two different sites: distal to the site of axotomy, but also in and
around the cell body of the affected neurons. Distal to the lesion, the
nerve undergoes Wallerian degeneration, followed by the outgrowth of
the sprouting axons into the distal nerve toward the peripheral tissues
(Fawcett, 1992; Bisby, 1995).
At the level of the neuronal cell body, injured motoneurons increase
protein synthesis, including adhesion molecules (Martini and Schachner,
1988; Möller et al., 1996; Jones et al., 1997) and neuropeptides
(Raivich et al., 1995). Reactive astrocytes upregulate cytoskeletal
proteins like GFAP and convert to a stellate type (Graeber and
Kreutzberg, 1986; Raivich et al., 1999). Activated microglial cells
proliferate, upregulate activation markers such as M2 integrin
(Raivich et al., 1994), and transform after neuronal cell death into
phagocytes that degrade neuronal debris (Torvik and Skjorten, 1971;
Kreutzberg, 1996; Klein et al., 1997). The upregulation of antigen
presenting molecule MHC1 and costimulatory factors such as B7.2 and
ICAM-1 (Raivich et al., 1998a; Werner et al., 1998; Bohatschek et al.,
1999) on these phagocytotic cells coincides with the recruitment of
lymphocytes (Raivich et al., 1998a).
Although these different aspects of glial activation have been thought
to support nerve regeneration (Streit, 1993), recent studies have put
some doubt on this notion. Thus, the cytostatic ablation of
proliferating microglia with cytosine-arabinoside does not affect the
speed of axonal regeneration in the hypoglossal nerve (Svensson and
Aldskogius, 1993). A similar absence of effect on the regenerating
facial nerve is also observed in mice that are deficient for the
macrophage-colony stimulating factor with a severe reduction of
microglial activation and proliferation (A. Werner and G. Raivich,
unpublished observations). An alternative hypothesis is that neuronal
injury leads to two separate sets of effects: it induces a neuronal
regeneration program and at the same time, a central glial activation,
with little effect of the latter on the former. This glial response was
recently suggested to be part of the anti-infectious repertoire of the injured CNS, which could help to protect the damaged neurons and the
surrounding environment from possible infection (Raivich et al., 1999).
Interestingly, the data provided by the current study support the
notion of two separate, relatively independent sets of effects. In
addition to the reduction in axonal regeneration, the lack of the 7
subunit also causes a strong increase in the level of the associated
neuronal 1 integrin. This upregulation of 1 may be part of a
compensatory mechanism, to ensure successful regeneration in the
injured peripheral nerve. Surprisingly, the contralateral, upoperated
facial nucleus shows normal levels of 1 immunoreactivity both in the
wild-type and the 7/ mice, suggesting that the effect of 7
deficiency on the expression of 1 is triggered by the neuronal
regeneration program.
In contrast, the absence of 7 did not appear to affect the response
to injury by glia or lymphocytes, suggesting that the immune
surveillance of the injured CNS is unaffected by the absence of the 7 subunit. At present, little is known about the neuronal trigger that initiates these central reactions. However, the absence of
the 7 integrin subunit did not affect the expression of neuronal peptides like CGRP or galanin. In vitro, these neuropeptides
play an important role in the activation of astrocytes and microglia (Lazar et al., 1991; Priller et al., 1998) and might be central mediators of the glial response.
In summary, the current study suggests that the 71 integrin is an
important mediator of axonal regeneration. Axotomy leads to a highly
consistent increase of 71 integrin immunoreactivity on axotomized
motor and sensory neurons in different models of successful
regeneration. Both the 7 and 1 subunit are concentrated on growth
cones in the regenerating nerve, and the transgenic deletion of the
7 subunit led to a reduced rate of axonal elongation in the
axotomized nerve and a delayed target reinnervation. Moreover, the lack
of changes in the central neuroglial response to injury clearly
indicates a peripheral site of action for this cell adhesion molecule
in the interaction with the extracellular matrix in the injured
peripheral nerve.
| |
FOOTNOTES |
Received Oct. 5, 1999; revised Dec. 6, 1999; accepted Dec. 22, 1999.
This work was supported by grants 01KO9401/3 and 01KO9703/3 of the
Bundesministerium für Bildung und Forschung. We thank Andrea
Koppius, Dietmute Büringer, and Karin Brückner for their expert technical assistance and Dr. Jim Chalcroft for his help with
digital photography.
Correspondence should be addressed to Dr. Gennadij Raivich, Department
of Neuromorphology, Max-Planck-Institute of Neurobiology, Am
Klopferspitz 18a 82152 Martinsried, Germany. E-mail:
Raivich{at}neuro.mpg.de.
| |
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TOP
ABSTRACT
INTRODUCTION
