Background
It is important the knowledge on the molecules involved in the trophic mechanisms
of motoneurons in order to develop therapeutic targets to peripheral nerve disorders
which are the case of facial nerve in the Bell’s palsy. The disease usually does not
last long and undergoes spontaneous recovery in many cases but sometimes therapeutic
interventions are necessary to reduce the symptoms or when amelioration is not achieved.
In the disorder, the compromised facial nerve swells up and presses against its trajectory
inside the temporal bone, being squashed and functionally/anatomically impaired [[1 ]]. Around one in five people will suffer long lasting symptoms. In patients presenting
incomplete facial palsy and probably bearing only functional impairments, the prognosis
for recovery is very good and treatment may be unnecessary. On the other hand, patients
presenting complete paralysis, marked by an inability to close the eyes and mouth
on the involved side, that received early treatment might show a favorable response
by 3-12 months [[2 ]]. This indicated that injured facial neurons can be rescued and might undergo regeneration,
a process that takes time considering the distance to facial muscle targets. However,
some cases are resistant to current proposed treatments which are mainly based on
antiinflammatory drugs and local neuromuscular manipulations [[3 ]].
Different from peripheral sensory neurons that seem to be less resistant to axotomy
probably because of a high dependence of trophic support from their innervation targets,
the majority of adult peripheral motoneurons survive after an injury of their fibers.
Motoneuron trophism is probably a result of autocrine/paracrine mechanisms which take
place at cell perykaria that are able to the rescue axotomized cells. Moreover, the
protection of neuronal cell bodies from degeneration is essential for axonal regeneration
and similar cell signaling might be involved in both events [[4 ]].
Basic fibroblast growth factor (FGF-2, bFGF) is a mitogenic protein capable of acting
on multiple cell types such as neurons and glial cells [[5 ]]. FGF-2 protein and messenger RNA (mRNA) have been found in the cytoplasm of neurons
and in the nuclei of astrocytes of many brain regions [[5 ],[6 ],[7 ],[8 ]]. FGF-2 plays a role in the neuronal development in prenatal life and also influence
survival and plasticity of mature central nervous system (CNS) neurons [[9 ],[10 ]]. Furthermore, paracrine actions of the astroglial FGF-2 have been described following
postnatal CNS lesions [[11 ],[12 ]].
Lesions to the CNS have been described to induce a strong expression of FGF-2 mRNA
and protein in activated astroglial cells in the area of the injury [[11 ],[12 ],[13 ],[14 ]]. Although an increasing number of studies have pointed out the role of FGF-2 following
cellular lesion, few works have attempted to investigate cellular regulation of FGF-2
in response to axotomy of the peripheral motoneurons. It is likely that the ability
of adult peripheral motoneurons to survive after axotomy is probably due to multiple
cellular sources of trophic support [[15 ],[16 ],[17 ],[18 ]]. This feature must be better interpreted in order to achieve effective therapeutic
targets leading to benefits for those patients with impaired functional recovery after
Bell’s palsy.
The present work analyzed the neuronal and glial responses as well as cellular FGF-2
regulation in the facial nucleus following a cervical crush or transection, with amputation
of nerve branches, of facial nerve of the adult Wistar rat. We have also examined
the effects of systemic corticosterone and functional electrical stimulation applied
in a facial muscle on FGF-2 expression in non axotomized facial nuclei.
Methods
Animals and experimental procedures
Specific pathogen-free adult male Wistar rats (University of São Paulo, Medical Scholl)
of 250 g body weight (b.w.) were used in the experiments. The animals were kept under
standardized lighting conditions (light on at 7:00 h and off at 19:00 h), at a constant
temperature of 23°C and with free access to food pellets and tap water. The study
was conducted according protocols approved by the Animal Care and Use of Ethic Committee
at the University of São Paulo and in accordance with the Guide for Care and Use of
Laboratory Animals adopted by the National Institutes of Health.
Facial nerve injury
In the first set of experiments, rats (n = 18) were submitted to a sham-operation,
a crush or a transection of the facial nerve as described. Briefly, under sodium pentobarbital
(45 mgkg-1 , Cristalia, São Paulo, Brazil) anesthesia, the rat facial skin of the right side
was opened near the ear and the facial nerve of that side was isolated. Following,
the facial nerves were crushed (n = 6) twice with a pair of Dumont #5 forceps for
30 sec, 3 mm from the stylomastoid foramen or completely transected (n = 6) with delicate
tweezers being the distal and proximal nerve stumps inverted and tied. In the sham-operated
animals (n = 6) the facial nerves were exposed and isolated in an identical manner
but they were not axotomyzed. Animals were sacrificed 7 days after the surgery and
their brain processed for immunohistochemistry.
Systemic drug injection and functional electrical stimulation
In a second set of experiments employing unlesioned rats, effects of systemic corticosterone
injection or local functional electrical stimulation were evaluated on non axotomized
facial nuclei. In a group of rats, animals received systemic daily injections of corticosterone
(10 mg × kg-1 b.w., ip., n = 6) or solvent (n = 6) for seven days. Corticosterone (Sigma, USA)
was suspended in deionized water solution containing carboxymethylcellulose natrium
salt (0.25% w/v; Sigma) and polyoxyethylene sorbital mono-oleate (tween 80, 0.2% v/v;
Sigma). All injections were made in the afternoon to mimic the endogenous peak of
corticosterone secretions and the solvent was given in the same volume and in the
same time as the corticosterone injections. This high dose of corticosterone was chosen,
since it is a standard dose used to mimic the stress level of corticosterone [[19 ]].
Other group of rats with unlesioned facial nerve was submitted to a functional electrical
stimulation according to protocols of Miles [[20 ]], Pilyavskii [[21 ]] and of Blum [[22 ]] adapted for facial muscles by our group. Briefly, a thread electrode for stimulation
(1.0 cm long/0.7 mm thickness) made of stainless steel fixed in a silicone-isolated
copper thread was connected to an electrical stimulator. Twenty-four hours prior first
stimulation, animals were anaesthetized (a combination of S (+)-ketamin cloridrate,
62.5 mg × kg-1 and xilazine cloridrate, 10 mg × kg-1 , respectively from Cristalia and Vetbrands, São Paulo, Brazil) and submitted to a
surgical procedure in order to expose the right side of levator labii superioris muscle
and to perform local implantation of a thread electrode which was fixed by means of
a surgical 10-0 mononylon thread. After a short trajectory through the subcutaneous
layer, the silicone-isolated copper thread was exteriorized through a small aperture
in the dorsal surface of the rat neck. The tip of that exteriorized thread was daily
connected to the electrical stimulator only during the period of stimulation sections.
Furthermore, a second electrode was fixed in the skin/subcutaneous layer to ground
the stimulation. The procedure was validated by examining the muscle response after
stimulation. Animals not showing visible contractions or vibrissal movements, or requiring
currents higher that 1 mA were discarded. Twenty-four hours later, awake and free
moving animals were submitted to the electrical stimulation protocol by means of a
4-channels-electrical stimulation (Vif FES 4, Quark, Brazil). The stimuli consisted
of a 1 mA current, 30 Hz frequency with a square wave (5 sec on/10 sec off), which
was applied daily, for 30 min in the beginning of the morning. Control rats were submitted
to electrode surgical implantations, daily connected to stimulator without receiving
the electrical stimulation.
Animals of the second set of experiments were also sacrificed 7 days after the beginning
of the procedures and their brain processed for immunohistochemistry.
Tissue processing
After the experimental procedures described above, rats were deeply anaesthetized
with sodium pentobarbital 10% (420 mg/kg/b.w., i.p.) and euthanized by a perfusion
through a cannula inserted in the ascending aorta with 50 ml of isotonic saline at
room temperature followed by 350 ml of fixation fluid (4°C) during 6 min as described
previously [[23 ],[24 ]]. The fixative consisted of 4% (w/v) paraformaldehyde and 0.2% (v/v) picric acid
in 0.1M phosphate buffer (pH 6.9). The brains were dissected out and kept in the fixative
solution for 90 min. The fixed brains were washed in 10% sucrose dissolved in 0.1M
phosphate buffered saline (PBS pH 7.4) for 2 days, frozen in ice-cold isopentane and
stored at -70°C. Coronal brain sections (14 μm thick) were made through the facial
nucleus from bregma level -11.60 mm to -10.3 mm, according to the atlas of Paxinos
& Watson [[25 ]], using a Leica cryostat (CM 3000, Germany). Sections were sampled systematically
and six series in a rostrocaudal order including every sixth section were used for
immunohistochemistry. The analyses were performed in the facial nuclei bilaterally.
The series of thaw-mounted sections were incubated overnight at 4°C in a humidified
chamber with one of the following antisera: a rabbit polyclonal FGF-2 antiserum against
the bovine FGF-2 [[26 ]] (diluted 1:800), a rabbit polyclonal antiserum against the glial fibrillary acidic
protein (GFAP, 1:1500, Dakopats, Danmark) or a mouse monoclonal antiserum against
the neurofilament (NF, only in the experiments of facial nerve injury) of molecular
weight 200 kDa (1:1000) (Sigma, USA). The antibodies were diluted in PBS containing
0.3% Triton X-100 (Sigma) and 0.5% bovine serum albumin (Sigma). The detection of
the antibodies was achieved by the indirect immunoperoxidase method using the avidin-biotin
peroxidase (ABC) technique as previously described [[27 ],[28 ],[29 ]]. After washing in PBS (3 × 10 min), the sections were incubated with a biotinylated
goat anti-rabbit or biotinylated horse anti-mouse antibodies (both diluted 1:200,
Vector, USA) for one hour. In a third step, sections were washed in PBS and incubated
with avidin-biotin peroxidase complex (both diluted 1:100, Vectastain, Vector) during
45 min. The staining was performed using 0.03% of 3,3’-diaminobenzidine tetrahydrochloride
(DAB, Sigma) as a chromogen and 0.05% (v/v) of H2 O2 (Sigma) during 6-8 min, which gave a brownish color to the immunoreaction. Duplicate
series of NF and GFAP immunoreactive sections from the facial nerve injury were stained
by cresyl violet (CV) for interalia visualization of Nissl substance. For standardization of the immunohistochemical
procedure we have used a dilution of the primary antibody and a DAB concentration
far from saturation and an incubation time adjusted so that the darkest elements in
the brain sections were below saturation. The FGF-2 antiserum is a well characterized
polyclonal antiserum raised against the n terminal (residues 1-24) of the synthetic peptide of bovine FGF-2 (1-146) [[26 ]]. This antiserum does not recognize acidic FGF (cross reactivity less than 1%) [[11 ]]. As control, sections were incubated overnight at 4°C with the FGF-2 antiserum
(diluted 1:800) pre-incubated with human recombinant FGF-2 (50 μg/ml, for 24 h at
4°C). For a further analysis of the immunostaining specificity, sections were also
incubated with the solvent of the primary or secondary antibody solutions as well
as the solvent of the avidin-biotin solution and processed simultaneously in the experimental
sections.
The two-color immunoperoxidase method was employed in a series of sections for a simultaneous
detection of the FGF-2 and GFAP immunoreactivities. The FGF-2 immunoreactivity was
firstly demonstrated as described above. Following the DAB reaction, the sections
were rinsed several times in PBS and were incubated during 48 h in a humidified chamber
with the rabbit polyclonal antiserum against GFAP described above (1:500). After several
rinses in PBS, the sections were incubated with biotinylated goat anti-rabbit immunoglobulins
(1:200, Vector) for 1 h at room temperature and with an avidin and biotin peroxidase
solution (both diluted, 1:100; Vectastain, Vector) for 45 min at room temperature.
The staining was performed using 4-chloro naphthol 0.05% (Sigma) as a chromogen and
0.05% (v/v) H2 O2 (Sigma) during 10 min. This procedure gave a brownish color to the FGF-2 immunoreactivity
and a bluish a color to the GFAP immunoreaction. The immunoreactivities were also
analyzed qualitatively and photographed in an Olympus AX70 photomicroscope (USA).
Quantitative analysis
Cell Counting
The NF+CV neuronal profiles, GFAP+CV astroglial profiles and the glial FGF-2 immunoreactive
profiles from the facial nerve injury experiment were counted under camera lucida microscopy at 16× magnification mounted in a Zeiss microscope (Germany). An area
of 116.39 μm2 was sampled in the central region of the right side (lesioned side)
and the left side (control side) of the facial nucleus and the profiles were counted.
The cytoplasmatic and nuclear localization of the FGF-2 immunoreactivity [[9 ]] were taken into account in the discrimination of the neuronal and glial FGF-2 cell
profiles. In order to minimize individual variability, the data were presented and
evaluated statistically as the quotient of ipsi vs contralateral sides.
Semiquantitative microdensitometric image analysis
FGF-2 immunoreactivity in sections from experiments of systemic corticosterone injection
and facial functional electrical stimulation of unlesioned facial nerve rats was submitted
to semi quantitative image analysis measurements. We have not performed a cell counting
in the unlesioned animals because the qualitative evaluations showed a major change
in the intensity of FGF-2 immunoreactivity per cell profiles and not in the number
of profiles. To maximize the intensities of the FGF-2 immunoreactivity on neuronal
and glial profiles, this analysis was performed on sections of rat brains from the
rostro-caudal levels described above [[29 ],[30 ]]. Fields of measurements were sampled in the central regions of the facial nuclei
bilaterally. The procedures using a Kontron-Zeiss KS400 image analyzer (Germany) have
been described previously [[9 ],[30 ],[31 ],[32 ]]. Briefly, a television camera acquired images from the microscope (40× objectives).
After shading correction, a discrimination procedure was performed as follows: the
mean gray value (MGV) and S.E.M. of white matter was measured in an area of the medulla
oblongata devoid of specific labeling (background, bg). Gray values darker than bgMGV-3
S.E.M. were considered specific labeling. The specific (sp) MGV was then defined as
the difference between the bgMGV value and the MGV of the discriminated profiles.
The size of the sampled field was 2.56 × 10-2 mm2 . This parameter reflects the immunoreactive intensities in the discriminated profiles
(spMGV) and indicates, semiquantitatively, the amount per profile of the measured
immunoreactivity. The area of discriminated profiles within the sample fields was
also registered and reflects the amount of profiles processing the immunoreactive
product. The glass value was kept constant at 200 MGV. The procedure was repeated
for each section to correct every specific labeling measurement for background. Moreover,
DAB and H2 O2 were used in optimal concentrations and FGF-2 antibody dilution was far from saturation.
Under these conditions, the steric hindrance of peroxidase complex does not appear
to disturb the linear relationship between antigen content and staining intensity.
However, in the absence of a standard curve, the relationship between antigen content
and staining intensity is unknown, and the results must be considered as semiquantitative
evaluations of the amount of antigen present in the sampled field. Thus, spMGV only
gives semiquantitative evaluations of the intensity of FGF-2 immunoreactivity [[33 ]]. In the corticosterone experiments, the data represent mean of the bilateral measurements
and in the functional electrical stimulation experiments, the data represent the quotient
of ipsilateral vs contralateral sides.
The statistical analysis was performed using the non-parametric Mann-Whitney U -test [[34 ]]. The number of each animal represents the Mean ± S.E.M. obtained in each side of
the facial nuclei of the sampled sections.
Results
Axotomy of facial nerve
Increases in the number of the FGF-2 immunoreactive nuclear glial profiles were found
in the ipsilateral facial nuclei seven days after both methods of axotomy, however
significance was reached after nerve transection with amputation of nerve stumps (57.97%,
[Figure 1A ], illustrated in [Figure 2A-D ]). Moreover, no statistical differences were obtained between crush and transection
regarding the number of FGF-2 immunoreactive profiles ([Figure 1A ]). Despites facial nerve crush and transection have promoted no changes in the number
of the FGF-2 immunoreactivity of neuronal profiles in the lesioned side ([Figure 1B ]), the intensity of the FGF-2 immunoreactivity increased slightly in the cytoplasm
of neuronal profiles seven days after the axotomy as evaluated qualitatively by means
of a direct microscopic analysis ([Figure 2A-D ]).
Figure 1 Effects of the unilateral crush or transection of facial nerve on FGF-2 immunoreactive
data . Ratio number of fibroblast growth factor-2 (FGF-2) immunoreactive glial (A ) and neuronal (B ) profiles, of glial fibrillary acidic protein (GFAP) immunoreactive astrocytic profiles
(C ), neurofilament plus cresyl violet immunoreactive neuronal profiles (D ) of the facial nucleus of the rats. The vertical axis represents the ratio of the
number of immunoreactive profiles in the ipsilateral versus contralateral nucleus.
Animals were studied 7 days after injury (means ± S.E.M., n = 6). *p < 0.05 according
to the non-parametric Mann-Whitney U test.
Figure 2 Microphotographs showing fibroblast growth factor (FGF-2) immunoreactivity
in coronal sections of rat facial nucleus . Animals were submitted to the following procedures and sacrificed 7 days later:
a transection of the facial nerve (with amputation of the nerve stumps) (B , D) or a sham operation (A , C) ; a 7-days systemic treatment of corticosterone (daily ip. injection of 10 mg × kg-1 , corticosterone) (E ) or solvent (F ); a 7-days unilateral functional electrical stimulation of the levator labii superioris
muscle after a local implantation of a mononylon thread electrode (1 mA current, 30
Hz frequency square wave) (G ) or without current as control (H ). The facial nerve was not lesioned in the corticosterone and electrical stimulation
experiments. The figures C and D represent higher magnification of areas inside the nuclei showed in figure A and B , respectively. The FGF-2 immunoreactivity is seen in the cytoplasm of neurons (arrows)
and in the nuclei of glial cells (arrowheads), respectively. It is observed that the
transection of the facial nerve and also systemic corticosterone increased the FGF-2
immunoreactivity in the nuclei of glial cells in facial nuclei ipsilateral to the
injury and bilaterally after drug injection. The functional electrical stimulation
of the levator labii superioris led to increase of FGF-2 mainly in the cytoplasm of
neurons of facial nucleus ipsilateraly. Bars = 50 μm (A , B ), 25 μm (C-H ).
The number of the GFAP immunoreactive glial profiles increased in the ipsilateral
facial nuclei of the crushed (193.41%) and transected (277.53%) animals 7 days after
axotomy ([Figure 1C ]). The intensity of the GFAP immunoreactivity per cell was also elevated in the lesioned
facial nuclei ([Figure 3A-D ]). The astrocytic reaction in the facial nuclei induced by the nerve crush or transection
was also observed by the increased size of the cytoplasm and processes of the GFAP
immunoreactive profiles ([Figure 3A-D ]).
Figure 3 Microphotographs showing rat facial nuclei submitted to immunohistochemistry
of different markers . Animals were submitted to the transection of the facial nerve (with amputation of
the nerve stumps) (B , D , F , H) or submitted to a sham operation (A , C , E , G) , 7 days before the sacrifice. The figures A-D show glial fibrillary acidic protein (GFAP) immunoreactivity, figures E-H show neurofilament (NF) ones in coronal sections of the facial nucleus of rats. The
figures C , D and G , H represent higher magnification of areas inside the nuclei showed in figure A , B and E , F , respectively. Arrowheads show GFAP immunoreactive astrocytes and arrows point to
NF immunoreactive neurons. The GFAP immunoreactivity is increased in the cytoplasm
and processes of astrocytes of the facial nucleus of the lesioned rats (B , D ). Furthermore, NF immunoreactivity is increased in the cell body of neurons and neuropil
of the facial nucleus of the lesioned rats (F , H ). Bars = 100 μm (E , F) , 50 μm (A , B , G , H) , 25 μm (C , D ).
Nerve injuries did not promote changes in the number of NF+CV neurons of the lesioned
side of the seven day-axotomized facial nuclei compared to sham rats (1 ± 0.04, 0.91
± 0.052, 1.08 ± 0.06 of the control, crushed and transected rats, respectively, [Figure 1D ]). Despites of that, the NF immunoreactivity increased in the perykaria, as well
as axonal and dendritic fibers of the ipsilateral facial nuclei of both crushed and
transected animals ([Figure 3E-H ]).
The two-color immunoperoxidase procedure for the simultaneous detection of the FGF-2
and GFAP immunoreactivities revealed that the vast majority of the nuclear FGF-2 immunoreactive
cell profiles were GFAP positive astrocytes in the rat facial nuclei ([Figure 4 ]). Furthermore, a higher amount of FGF-2 was found in the nucleus of the reactive
astrocytes of axotomized facial nuclei ([Figure 4 ]).
Figure 4 Color microphotographs showing FGF-2 and GFAP immunoreactivities in coronal
sections of rat facial nucleus . Animals were submitted to the transection of the facial nerve (with amputation of
the nerve stumps, A , or sham operation, B ), 7 days before sacrifice. The two-color immunoperoxidase method employing different
chromogens was used. The diaminobenzidine (brownish color) and the 4-chloro-naphthol
(bluish color) were used for detection of the fibroblast growth factor-2 (FGF-2) and
glial fibrillary acidic protein (GFAP) immunoreactivities, respectively. Arrowheads
show FGF-2 immunoreactivity in the nuclei of the GFAP immunoreactive astrocytes. It
is also seen the FGF-2 immunoreactivity in the cytoplasm of neurons (arrows). Bars
= 10 μm.
The control sections incubated with FGF-2 antibody preadsorbed with human recombinant
FGF-2 showed no specific labeling. The control sections incubated with the solvent
of the primary and secondary antisera or with the solvent of the avidin-biotin solution
showed no immunoreactivity (data not shown).
FGF-2 in the facial nucleus after systemic corticosterone treatment
As shown in the [Figure 5A-B ], a seven days-systemic injections of corticosterone resulted in a significant increase
of FGF-2 immunoreactivity in the rat facial nuclei as seen from the measurements of
FGF-2 immunoreactive area (75.8%) and spMGV (16.4%). The qualitative analysis of the
FGF-2 immunoreactivity revealed an increased number of putative glial profiles processing
higher amount of the immunoreaction product and only few neurons showing an elevation
of the FGF-2 immunoreactivity in the facial nuclei of corticosterone treated rats
compared to control animals (illustrated in [Figure 2E, F ]). Procedures for GFAP and FGF-2 double labeling showed the presence of FGF-2 immunoreactivity
in the nuclei of astrocytes as demonstrated in the facial nerve injury experiments,
however astrocytes have not become reactive after corticosterone treatment (data not
shown).
Figure 5 Effecs of corticosterone or functional electrical stimulation on FGF-2 immunoreactive
data of rat facial nuclei . Figure shows area (A, C ) and specific mean gray value (spMGV; B, D ) of FGF-2 immunoreactive profiles in the sampled fields of the rat facial nuclei
after systemic corticosterone or solvent injection (A, B ) and functional electrical stimulation (C, D ). Measurements were performed in the facial nuclei bilaterally in the corticosterone
experiment and ipsilaterally to the levator labii superiors muscle electrode implantation
in the functional electrical stimulation experiments. The control animals for functional
electrical stimulation received electrode without electrical current. Morphometric/microdensitometric
image analysis was used. The measurements represent the FGF-2 immunostaining area
(within a 2.56 × 10-2 μm2 sampled field) and intensities (spMGV, arbitrary values) and reflect the number and
amount per profile of the measured immunoreactivity, respectively (see text for details).
Values are means ± S.E.M.; n = 4-5;*p < 0.05 according to the non-parametric Mann-Whitney
U test.
FGF-2 in the facial nucleus after functional electrical stimulation of the levator
labii superioris muscle
A seven days-functional electrical stimulation promoted increases of FGF-2 immunoreactivity
in the rat facial nuclei as seen from the measurements of FGF-2 immunoreactive area
(127%, quotient of ipsi vs contralateral sides) and spMGV (18%, quotient of ipsi vs contralateral sides, but without statistical significance) ([Figure 5C, D ]). The qualitative analysis of the FGF-2 immunoreactivity revealed a higher amount
of the immunoreaction product mainly in neurons and only few astrocytes showing elevation
of the FGF-2 immunoreactivity in the facial nuclei of electrical stimulated rats compared
to non stimulated control animals ([Figure 2G, H ]). In this experiment, FGF-2 immunoreactivity was located in the nucleus of astrocytes
in the same manner that was found in the other two experiments, however astrocyte
have not become reactive after functional electrical stimulation (data not shown).
Discussion
Retrograde reactions to axotomy leading to morphological and biochemical changes in
the neuronal perykaria [[35 ],[36 ]] compose a set of responses to maintain the neuronal trophism/plasticity and to
trigger axonal regeneration [[37 ],[38 ],[39 ]].
Axotomy of facial nerve applied in this work did not promote changes in the number
of NF+Nissl substance stained facial motoneurons either after a crush lesion, which
allows immediate fiber growth, or a transection lesion with amputation of nerve stumps.
These findings are in agreement and extend previous reports that have demonstrated
the resistance of mature motoneurons to axotomy of their fibers [[40 ]]. The present findings showing an increased amount of 200 kDa NF immunoreactivity,
the major protein of the neuronal cytoskeletal intermediate filament, in the cell
bodies and neuropil of axotomized facial neurons are in accordance with previous publications
that have demonstrated a remarkable regenerative capacity of motoneurons following
axotomy in adult rodents and human beings [[41 ]]. Tetzlaff and co-workers [[42 ],[43 ]] have demonstrated increased syntheses of the cytoskeletal proteins actin and tubulin
after axotomy of the rat facial nerve simultaneously to the enhanced NF contents and
a low regulation of NF synthesis. Differential regulation of expression and accumulation
of the cytoskeletal proteins in axotomized cell bodies and fibers could be due to
their different timing regarding turnover, phosphorilation and participation in specific
cell restoration, plasticity and regeneration processes [[44 ]].
The retrograde phenomenon following axotomy was also observed by the astrocytic reaction
in the injured facial nuclei. Activation of astrocytes has been demonstrated after
neuronal lesion [[45 ]], electrical stimulation [[46 ]], cytokine administration [[47 ]] by means of the increases of GFAP immunoreactivity or mRNA. The astrocytic activation
has been described to be related to local ionic homeostasis as well as to production
of neurotrophic factors [[48 ]]. In fact, the paracrine actions leading to neuronal trophic support promoted by
the CNS astrocytes have been considered to be important for maintenance and plasticity
of the injured neurons [[49 ]].
Our findings of increased GFAP immunoreactivity in the facial nucleus following crush
or transection lesions of facial nerve are in agreement and extend previous observations
which have described retrograde astroglial reactivity after axotomy of cranial motoneurons
[[15 ]] and also lesions of spinal nerves containing sensory and motor fibers [[18 ],[45 ]].
It is well known that the peripheral sensory neurons require a target-derived trophic
support [[50 ]] and the axotomy of their fibers leads to a partial disappearance of the cell bodies
located in the peripheral ganglia [[51 ]]. Moreover, axotomy of peripheral motor fibers does not trigger apoptosis of damaged
neurons acutely, however a certain degree of a long term cell body atrophy and cell
death might take place in the axotomized motoneurons in the cases of regeneration
failure [[52 ]]. These considerations already underline the importance of autocrine/paracrine mechanisms
in the trophic regulation of motoneuron perikarya.
The search for sources of trophic support for peripheral neurons after axotomy has
led to descriptions of increased synthesis of neurotrophic factors in the proximal
and distal stumps of the injured nerve [[53 ]]. Moreover, Heumann and co-workers [[54 ]] have observed an increased level of nerve growth factor protein and mRNA in non
neuronal cells surrounding the axons of sensory and motor neurons and Levy and co-workers
[[18 ]] showed increased levels of FGF-2 in the dorsal root ganglia satellite cells surrounding
the cell bodies of axotomyzed peripheral sensory neurons. In fact, local production
and release of neurotrophic molecules in different parts of compromised neurons may
be related to specific functions such as wound repair, trophic support for neuronal
maintenance and nerve fiber sprouting/outgrowth [[55 ]], this late resembling the reinervation of the distal nerve stump and target [[56 ]] when regenerative conditions are offered.
An important finding of the present work was the substantial increases of FGF-2 in
the reactive astrocytes of axotomized facial nuclei. It is known that the FGF-2 is
a potent survival factor for neurons from different parts of the nervous system and
that the molecule can also protect neurons from several types of injury [[9 ],[14 ],[57 ],[58 ],[59 ]]. It is the first time that an upregulation of a neurotrophic factor has been described
in the reactive astrocytes close to axotomized facial neuronal cell bodies thus highlighting
the importance of paracrine trophic mechanisms to those injured neurons as it has
been extensively described for others CNS lesioned regions [[60 ]]. Astroglial FGF-2 upregulation in reactive astrocytes of transected facial nucleus
was similar to that recently published by our group in the hypoglossal nucleus after
injury of their fibers [[15 ]]. It is possible that the upregulated astroglial FGF-2 in the axotomized facial
nucleus may act as a paracrine factor for cell body maintenance and probably influence
axonal regeneration as it has been described for certain types of central neurons[[61 ]].
The present study, using a polyclonal antiserum, has also shown the presence of FGF-2
immunoreactivity in the cytoplasm of facial motoneurons, which is in agreement with
previous observations that have demonstrated FGF-2 immunoreactivity in neurons of
several brainstem nuclei using different polyclonal antibodies [[6 ],[8 ],[16 ],[62 ]].
In the present paper we have described moderate elevations of the FGF-2 immunoreactivity
in the cytoplasm of neurons without changes in the number of those immunopositive
cells following facial nerve axotomy indicating a possible additional autocrine role.
It was reported that the FGF-2 synthesized in the tongue may be retrogradaly transported
to the hypoglossal nucleus thus acting as a target derived neurotrophic factor. Actually,
a transient down regulation of neuronal FGF-2 immunoreactivity in the ipsilateral
axotomized hypoglossal nucleus was described [[63 ]], however major events showed by our group have been the upregulation of astroglial
FGF-2 in the axotomized hypoglossal nucleus [[15 ]] and facial nucleus (this paper).
Indeed, FGF-2 undergoes receptor-mediated internalization and retrograde transport
in the central [[64 ]] and peripheral nervous system [[63 ]]. Because the levels of astroglial reaction (seen by the increased number of GFAP
immunepositive cells) and the levels of the changes in the astroglial FGF-2 immunoreactivity
were higher after facial nerve transection (without fiber regeneration) than after
crush (leading to a favorable regeneration), it is possible that such a regenerative
failure-impairing the internalization of FGF-2 synthesized in the periphery might
have favored FGF-2 synthesis in reactive astrocytes of transected facial nucleus.
Thus, paracrine actions of the astroglial FGF-2 in the facial nucleus might help to
maintain the trophism of the facial neurons when fibers are disconnected from the
target.
In addition to neuronal lesions [[11 ],[13 ],[57 ],[65 ]], different experimental designs have been used to study the role of neurotrophic
factors in the CNS. Exogenous administration of growth factors to the brain [[9 ],[14 ],[58 ],[66 ]], neuronal stimulation [[16 ]], physical activity [[67 ]], hormonal manipulation [[32 ],[68 ],[69 ],[70 ]] and electrical stimulation [[71 ]] applied in neuronal pathways are also commonly employed.
The ability of exogenous neurotrophic factors to trigger neuroprotection and to prevent
diminution of neurotransmitter synthesis following cranial nerve axotomy in the neonatal
and adult life has been described. Cuevas and co-workers have shown that acidic fibroblast
growth factor topically applied prevents the axotomy-induced neuronal death in the
newborn rat facial nerve [[72 ]]. Brain derived neurotrophic factor also promoted the survival of the axotomized
immature facial motoneurons in vivo [[73 ]] and attenuated the lesion induced-decrease of choline acetyltransferase (ChAT)
immunoreactivity and activity in adult facial motoneurons [[74 ]]. Sendtner and co-workers have demonstrated that the vulnerability of motoneurons
to axotomy in the early postnatal life is prevented by a local application of cilliary
neurotrophic factor (CNTF) [[75 ]]. The glial-derived neurotrophic factor was demonstrated to rescue axotomy-induced
death of facial neurons and to attenuate the diminution of immunoreactivity in the
axotomized facial nucleus of neonates [[76 ]]. Finally, implantation of cell lines genetically engineered to release CNTF in
the brain of mouse with a progressive neuropathy seems to rescue motoneuron loss [[77 ]].
Besides the trophic promoting effects of molecules exogenously administered, the expression
of endogenous neurotrophic factors following other types of nerve manipulation gives
further evidences of the role of specific molecules for motoneuron survival and regeneration.
Treatment of Bell’s palsy is still a matter of controversy and there is a consensus
for the need of larger and properly designed clinical trials to evaluate the effects
of antiviral drugs, glucocorticoids and other proposed therapies for disease. It has
been said that steroids e.g. prednisolone may reduce the nerve swelling-induced damage,
leading to a potential recovery in early treatments [[3 ],[78 ]].
In fact, the role of hormones in peripheral neuropathology is unknown. It has gained
evidence the actions of steroid hormones on nervous system trophism [[79 ]] and plasticity [[80 ],[81 ],[82 ]], effects that are probably related to their ability to regulate the expression
of neurotrophic factors [[70 ],[83 ],[84 ],[85 ],[86 ],[87 ]].
We have shown in this study that systemic corticosterone for 7 days led to upregulation
of FGF-2 immunoreactivity mainly in astrocytes of rat facial nucleus. The present
findings are in line with our previous observations that adrenocortical steroid administration
can increase FGF-2 mainly in the glial cells of the rat substantia nigra [[9 ]]. Moreover, dexamethasone, a potent synthetic glucocorticoid agonist, was shown
to induce the FGF-2 gene expression in primary culture of rat astrocytes from different
CNS regions [[86 ]], and also to increase FGF-2 immunoreactivity in the substantia nigra astrocytes
[[88 ]], further emphasizing the influence of steroid hormones on astroglial FGF-2 mechanisms.
It may be possible that glucocorticoid hormones also modulate astroglial FGF-2 syntheses
in the axotomized facial nucleus, as we have described in the model of experimental
parkinsonism [[32 ],[70 ]], however that was not the matter of the present investigation on non axotomized
facial nucleus. Moreover, glucocorticoids might also be able to modulate FGF-2 expression
in the neuronal fiber surrounding Schwann cells, which may be potentially involved
in sprouting and outgrowth of lesioned axons [[18 ]]. Nevertheless, based on the gliogenic, angiogenic and fibroblastogenic actions
of FGF-2 and consequently its potential actions on wound repair, glucocorticoid hormones
may use FGF-2 signaling on its neurorepair role which is also positive for axonal
regeneration [[60 ],[89 ]]. We are presently performing experiments on axotomized facial nerve to evaluate
further this issue.
Physiotherapy might also contribute to rehabilitation of Bell’s palsy. Rather largely
employed, the efficacy of acupuncture remains unknown because the available studies
do not allow adequate conclusions. Furthermore, neuronal stimulation in general and
electrical stimulation in particular seem to improve motor recovery in patients with
Bell’s palsy [[90 ],[91 ]].
Hadlock and co-workers investigated the effects of a local brief electrical stimulation
(1 h, 3 V, 20 Hz square wave), a mechanical (manual) target muscle manipulation, or
both on functional recovery (whisker movement) of transected and repaired facial nerve
of rats [[92 ]]. Either therapy alone led to long last better effects than that of untreated rats
or animals submitted to an association of the two methods. It seems likely that neuronal
activation by afferent inputs triggered by manual stimulation approaches is involved
in the functional recovery of the denervated muscles since it is effective in cases
of cranial nerve lesions with preservation of the sensory fibers (facial or hypoglossal
nerve) but ineffective for the treatment of injury of peripheral nerve containing
both sensory and motor fibers [[93 ]]. Indeed, manual stimulation was shown to improve function and to reduce polyinnervation
without triggering collateral sprouting compared to acute electrical nerve stimulation
prior to reconstructive surgery after facial nerve injury in rats [[94 ]]. These findings are in line with a recent report that showed failure of whisker
functional recovery and collateral axonal branching, and also a reduced motor endplate
reinervation after facial nerve repair (end-to-end suture) treated by electrical stimulation
in rats [[95 ]]. Finally, the nature of electrical stimuli must be consider regarding a potential
damage to the nerve tissue as described recently by Sapmaz and co-workers [[96 ]] after strong and numerous electrical stimuli to the rat facial nerve ranging from
1 to 5 mA.
All in all, the above considerations seem to be in line with our results regarding
the increases of FGF-2 in the facial nuclei of rats submitted to a functional electrical
stimulation applied in a facial muscle after local implantation of an electrode.
To our knowledge, functional electrical stimulation has not been applied in rodents,
despite some reports have evaluated its efficacy for neurofunctional restoration after
facial nerve lesions in rabbits [[97 ],[98 ],[99 ]]. We can not exclude the possibility of retrograde signals may have triggered the
increases of FGF-2 in the neuronal cells of facial nucleus after the functional electrical
stimulation performed in our works, however it seems possible that proprioceptive
reflexes might have exerted an important role in that process.
We should emphasize that based on the results presented in this report, the functional
electrical stimulation led to elevation of FGF-2 mainly in the cytoplasm of neurons
of facial nucleus, which differed to the findings of systemic corticosterone that
promoted elevation of FGF-2 in the nuclei of astrocytes of facial nucleus. Taken together,
the present paper opened up new avenues for development and further analyses of therapies
for Bell’s palsy.
In fact, one possibility is the combination of both strategies: hormonal therapy promoting
mainly paracrine actions of glial neurotrophic factors to motoneurons that may be
necessary for an acute/subacute thophic support and functional electrical stimulation
leading mainly neurotrophic autocrine action that may be necessary for a subacute
maintenance.
In line with the above described possibilities, Hetzler and co-workers, by using the
rat facial nerve crush, showed positive effects of a combinatorial strategy of electrical
stimulation proximal to crush injury site and testosterone propionate (TP) administration
(in gonadectomized adult male rats) in enhancing facial nerve regenerative properties.
In their experiment, while either single treatment modality of electrical stimulation
or exposure to supraphysiologic levels of gonadal steroids has some benefit, such
improvements are transitory [[100 ]]. The application of both treatment modalities significantly accelerates the functional
recovery of multiple functional parameters, with the most important being the time
until complete recovery. This significant improvement may be attributed to the ability
of each modality to affect different aspects of cellular events associated with axonal
regeneration and also to a synergism between the two types of treatment. Whether electrical
stimulation affects axonal sprouting in the initial fiber outgrowth phases is a matter
that remains to be determined. Whereby electrical stimulation was able to reduce the
delay before sprout formation, hormone accelerated the overall regeneration rate,
and the combined treatment led to additive effects. Furthermore, the two treatments
triggered differential temporal effects on expression of genes related to neurotrophism
and neuroplasticity [[101 ]] which emphasize a possible importance of associative therapies in modulating specific
molecular pathways for neurorestoration of axotomized neurons.
Conclusion
The presence of the FGF-2 immunoreactivity in the neurons and astrocytes of the facial
nucleus indicates that the FGF-2 may be an important growth factor for peripheral
motoneurons. Expression of astroglial/neuronal FGF-2 in the facial nucleus may be
correlated to local paracrine/autocrine trophic actions to axotomized or stimulated
facial motoneurons. The FGF-2 signaling may be explored in the search of new therapeutic
target for Bell’s palsy.
Abbreviations
bg: background; CNS: central nervous system; CV: cresyl violet; FGF: Fibroblast growth
factor; GFAP: glial fibrillary acidic protein; MGV: mean gray value; NF: Neurofilament;
sp:specific
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KFM, CJCSF, AFB, CMS, RTS, GPO performed experimental procedures, surgery, drug administration,
electrical stimulation, quantitative analyses and statistics. GC wrote the paper.
The authors read and approved the final manuscript.
Cite this article as: Coracini et al.: Differential cellular FGF-2 upregulation in the rat facial nucleus following axotomy,
functional electrical stimulation and corticosterone: a possible therapeutic target
to Bell’s palsy. Journal of Brachial Plexus and Peripheral Nerve Injury 2010 5 :16.
