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EFFECT OF LOW-INTENSITY PULSED ULTRASOUND ON THEEXPRESSION OF NEUROTROPHIN-3 AND BRAIN-DERIVEDNEUROTROPHIC FACTOR IN CULTURED SCHWANN CELLS
HUA ZHANG, M.D.,1 XIN LIN, M.D.,1 HONG WAN, M.D., Ph.D.,2 JUN-HUA LI, B.Sc.,2 and JIA-MOU LI, M.D., Ph.D.1*
It is generally known that low-intensity pulsed ultrasound (LIPUS) accelerates peripheral nerve tissue regeneration. However, the precisecellular mechanism involved is still unclear. The purpose of this study was to determine how the Schwann cells respond directly to LIPUSstimuli. Thus, we investigated the effect of LIPUS on cell proliferation, neurotrophin-3 (NT-3), and brain-derived neurotrophic factor (BDNF)mRNA expression in rat Schwann cells. Schwann cells were enzymatically isolated from postnatal 1–3 day rat sciatic nerve tissue and cul-tured in the six-well plate. The ultrasound was applied at a frequency of 1 MHz and an intensity of 100 mW/cm2 spatial average temporalaverage for 5 minutes/day. The control group was cultured in the same way but without the administration of ultrasound. Immunohisto-chemistry demonstrated that more than 98% of the experimental and control cells were positive for S-100, NT-3, and BDNF. With 5-bromo-20-deoxyuridine (BrdU) assay, the stimulated cells also exhibited an increase in the rate of cell proliferation on days 4, 7, 10, and 14. Fur-ther investigation found that mRNA expression of NT-3 was significantly upregulated in experimental groups compared with the control 14days after the LIPUS stimulation (the ratio of NT-3/b-actin was 0.56 6 0.13 vs. 0.41 6 0.09, P < 0.01), whereas the mRNA expression ofBDNF was significantly downregulated in experimental groups compared with the control (the ratio of BDNF/b-actin was 0.51 6 0.05 vs.0.60 6 0.08, P < 0.05). These results demonstrated that the application of LIPUS promotes cell proliferation and NT-3 gene expression inSchwann cells, and involved in the alteration of BDNF gene expression. VVC 2009 Wiley-Liss, Inc. Microsurgery 29:479–485, 2009.
Low-intensity pulsed ultrasound (LIPUS) is one of the
physical agents that is known to accelerate bone and tis-
sue regeneration following injury.1,2 Consequently, it has
been accepted as an effective therapy for nonunion frac-
tures and fresh fracture healing through an easy and non-
invasive application.3,4 Previous studies indicate that
LIPUS has positive effects on axonal regeneration by in
vivo peripheral nerve injury trials.5,6 Raso et al.7 have
demonstrated that the locally applied ultrasound stimuli
on the injured sciatic nerve rather than the untreated
nerves of rats can effectively enhance the number of
Schwann cell nuclei. LIPUS has been used in conjunction
with tissue-engineered nerves in repairing peripheral
nerve defect. Chang et al.6 demonstrated that applying
low-intensity ultrasound on seeded Schwann cells within
poly(DL-lactic acid-co-glycolic acid) conduits has a signif-
icantly greater number and area of regenerated axons
compared with the sham groups. Although this secondary
response by Schwann cells has been well characterized,
there is still limited information as to how Schwann cells
would directly respond to LIPUS stimulation. Therefore,
we cultured Schwann cells in culture plate as an in vitro
model and applied LIPUS in the model to demonstrate
the direct effects of physical stimulation on Schwann
cells.
Both neurotrophin-3 (NT-3) and brain-derived neuro-
trophic factor (BDNF) are key neurotrophins constituents
in peripheral nervous system, NT-3 is an important regu-
lator of neural survival, development, function, and neu-
ronal differentiation.8,9 Hess et al.10 observed that NT-3
expression may modulate the number of Schwann cells at
neuromuscular synapses. Otherwise, NT-3 is an important
autocrine factor supporting Schwann cell survival and dif-
ferentiation in the absence of axons.8 Schwann cells also
contribute to the sources of BDNF during nerve regenera-
tion, and the deprivation of endogenous BDNF results in
an impairment in regeneration and myelination of regen-
erating axons.11 BDNF also plays a role in activity-
dependent neuronal plasticity.12 The exogenous adminis-
tration of these factors has protective properties for
injured neurons and stimulates axonal regeneration.13
Based on these properties, these molecules may be used
as therapeutic agents for treating degenerative diseases
and traumatic injuries of both the central and peripheral
nervous system.
The purpose of this study was to evaluate how sus-
tained LIPUS directly affects Schwann cell function. By
evaluating for the expression of the pan-specific Schwann
cell marker S-100 with immunohistochemistry, we deter-
mined whether Schwann cells dedifferentiated after
LIPUS stimulation. Schwann cell proliferation was
explored with 5-bromo-20-deoxyuridine (BrdU) uptake
assays to ascertain if direct LIPUS stimulation is mito-
genic for Schwann cells in cultured plate. Finally, we
measured how LIPUS affects Schwann cells neurotrophic
function by evaluating the mRNA expression of NT-3
1Department of Orthopaedics, Beijing Tiantan Hospital, Capital MedicalUniversity, Beijing, China2Beijing Neurosurgical Institute, Beijing, China
Grant sponsor: Natural Science Foundation of Beijing, China; Grant number:5072020.
*Correspondence to: Jia-Mou Li, M.D., Ph.D., Department of Orthopaedics,Beijing Tiantan Hospital, Capital Medical University, No.6 Tian tan xi li,Chongwen District, Beijing 100050, China. E-mail: lis@bjmu.edu.cn
Received 3 August 2008; Accepted 26 January 2009
Published online 23 March 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/micr.20644
VVC 2009 Wiley-Liss, Inc.
and BDNF, two members of the neurotrophic factor fam-
ily of the Schwann cells.
MATERIALS AND METHODS
Schwann Cells Culture
Schwann cells were prepared using a method previ-
ously described,14 with some modifications. Briefly, sci-
atic nerves were dissected from Wistar rats (n 5 30) at
postnatal day 1–3. The epineural sheath was removed.
Thereafter, the sciatic nerves were chopped into 200-lmpieces and enzymatically digested (collagenase/trypsin,
1 mg/ml, 1 hour, 378C). The resulting cell suspensions
were plated onto a six-well plate and cultured in
Schwann cell medium (DMEM/10% heat-inactivated
FCS/2 mM glutamine/pen/strep). Two different cell den-
sities were prepared for subsequent experiments, a cell
density of 5,000 cells/1.77 cm2 for proliferation assay
and immunohistochemistry assays, and a cell density of
100,000 cells/1.77 cm2 for semiquantitative RT-PCR. The
fibroblasts were eliminated by 10 lM cytosine arabino-
side and complement-mediated cytolysis with the fibro-
blast-specific antibody Thy1.1 in conjunction with baby
rabbit complement (Cedarlane, Burlington, NC). The me-
dium was changed every other day by adding 2 lM for-
skolin and glial growth factor (100 lg/ml) for expansion
of Schwann cells for up to 14 days of cells culture. The
purity of cultures was monitored by immunostaining
using the Schwann cell marker S-100 and the fibroblast
marker Thy-1.1.
Low-Intensity Pulsed Ultrasound Treatment
Schwann cells cultured within a medium and were
subjected to LIPUS with modifications as previously
described.15 This device (Nexson-The-P41, Nexus Bio-
medical Devices, Hangzhou, China) generated LIPUS
with a pulse width of 200 microseconds, repetition rate
of 1.5 KHz, operation frequency of 1 MHz, spatial aver-
age temporal average of 100 mW/cm2, 5 minutes/day.
The LIPUS treatment was started 24 hours after initiation
of cells culture and repeated for 14 consecutive days. In
the experimental group, LIPUS was transmitted from 35-
mm diameter LIPUS transducers to the bottom of the cell
culture plate via a coupling gel (Smith & Nephew, Okla-
homa, CA) and was administered in an incubator (see
Fig. 1). In the control group, plates were placed on the
same transducers for the same duration, but the LIPUS
was not administered.
Proliferation Assay with 5-Bromo-2-deoxy-uridine
The percent of proliferation was determined by the
ratio of total BrdU-positive nuclei to total number of
cells (DAPI-stained nuclei) as described previously.16
Schwann cells plated at a cell density of 5,000 cells/1.77
cm2 were used for counting of each individual Schwann
cell. A total of 12 plates per time point (six experimental
plates and six control plates, respectively) were analyzed.
At days 4, 7, 10, and 14 after LIPUS treatment, the cells
in plates were treated by Brdu (Sigma, Saint Louis, MO)
for 2 hours. The cells were then fixed in methanol for 10
minutes at 48C and treated with 1.25% proteinase K in
PBS (pH 7.5) for 5 minutes. Thereafter, the cells were
treated with mouse anti-BrdU monoclonal antibody
(Sigma, Saint Louis, MO) for 1 hour and then with goat
anti-mouse IgG FITC (Sigma, Saint Louis, MO) for 1
hour. DAPI (Sigma, Saint Louis, MO) and cover slips
were added. The average proliferation percentage of the
plate was counted. The average proliferation percentage
was counted by examining four random images within
per plate using a fluorescent microscope-computer inter-
face (Zeiss, Jena, Germany).
Immunohistochemistry
Schwann cells plated at a cell density of 5,000 cells/
1.77 cm2 were used for the immunohistochemistry assays.
At day 14 after LIPUS treatment, a total of 18 plates
Figure 1. Experimental apparatus for applying low-intensity pulsed
ultrasound (LIPUS). Two transducers for the control group (sham-
LIPUS; LIPUS not turned on) and two probes for the LIPUS group.
A six-well culture plate was placed on the transducers. LIPUS was
transmitted to culture plate via an interpositioning ultrasound gel.
480 Zhang et al.
Microsurgery DOI 10.1002/micr
(nine experimental plates and nine control plates) were
analyzed for S-100, NT-3, and BDNF immunostaining,
respectively. The cells were fixed in plates for 10 minutes
with 4% paraformaldehyde solution and then blocked in
4% goat serum with 0.25% triton in PBS. Then, the cells
were incubated with either mouse anti-S100 protein
monoclonal antibody (Sigma, Saint Louis, MO), mouse
anti-neurotrophin-3 monoclonal antibody (Sant Cruz, Sant
Cruz, CA), or mouse antibrain-derived neurotrophic fac-
tor primary antibodies (Sant Cruz, Sant Cruz, CA), then
subsequently with goat anti-mouse IgG FITC (Sigma,
Saint Louis, MO) or IgG TRITC (Sigma, Saint Louis,
MO) for 1 hour and counterstained with DAPI (Sigma,
Saint Louis, MO). The percentage of fluorescently labeled
cells/DAPI-stained nuclei was counted using a fluorescent
microscope-computer interface (Zeiss, Jena, Germany).
Semiquantitative RT-PCR
Schwann cells plated at a cell density of 5,000 cells/
1.77 cm2 were used for the RT-PCR assays. At day 14
after LIPUS treatment, a total of 12 plates (six experi-
mental plates and six control plates) were analyzed for
NT-3 and BDNF mRNA expression, respectively. The
cells were then incubated for 12 hours at 378C to allow
for gene transcription. Cells were then trypsinized, col-
lected as pooled samples, and RNA was extracted using
the RNeasy Mini Kit (Qiagen, Valencia, CA) following
the protocol. The cDNA was prepared for experimental
and control samples using 3 lg of RNA with SuperScript
II RNase reverse transcriptase (Invitrogen, Oklahoma,
CA) and specific primers for NT-3 (forward: 5 0-CTTATCTCCGTGGCATCCAAGG-30, reverse: 50-TCTGAAGTCAGTGCTCGGACGT-30), BDNF (for-
ward: 50-ATGGGACTCTGGAGAGCGTGAA-30, reverse:50-CGCCAGCCAATTCTCTTTTTGC-30), and b-actin(forward: 50-CCCAGAGCAAGAGAGGCATC-30, reverse:50-CTCAGGAGGAGCAATGATCT-30).17 The PCR reac-
tion conditions were consisted of one cycle of 948C for
5 minutes, followed by 30 cycles of thermal cycling
30 seconds at 948C, 30 seconds at T08C, and 1 minute at
728C. The T0 was 608C for BDNF, 648C for NT-3, and
588C for b-actin. The final cycle was followed by a
5-minute extension at 728C. Ten microliters of PCR
product was then differentiated on a 1.5% agarose gel
and the gel image was taken with a digital camera.
ImagQuant analysis software (Stratagene Company, La
Jolla, CA) was used to determine the densities of the
NT-3 and BDNF bands when compared with the b-actincontrol for both experimental and control samples.
Statistical Analysis
Data from the proliferation assays and RT-PCR
experiments were analyzed for statistical significance
using the Student’s paired t-test. The significance between
control and experimental values was determined with a
set significance value of P < 0.05.
RESULTS
Proliferation Assay
The Schwann cells that were subjected to LIPUS con-
sistently demonstrated an increase in cell proliferation.
Figure 2 shows a fluorescent microscope picture demon-
strating the difference in the percentage of BrdU-positive
cells in both experimental and control cells at day 7. The
percentage of BrdU-positive cells was significantly higher
in experimental groups than in control groups on days 4
(17.5 6 2.4% vs. 10.8 6 1.4%, P < 0.01), 7 (46.9 64.1% vs. 23.2 6 3.1%, P < 0.01), and 10 (69.4 6 7.7%
vs. 48.6 6 6.0%, P < 0.05), respectively (see Fig. 3).
However, no statistical significance was observed
between experimental and control groups on day 14 (80.2
6 9.6% vs. 76.7 6 6.4%).
Immunohistochemistry
The immunohistochemistry study showed that more
than 98% of Schwann cells were positive for the pan-spe-
cific Schwann cell marker S-100 at day 14, with or with-
out LIPUS treatment. This indicates that LIPUS has no
effect on the phenotype of Schwann cells. Immunostain-
ing for NT-3 and BDNF shows that Schwann cells were
positive in more than 98% of the evaluated cells in both
the experimental and control cells at day 14. Addition-
ally, the distribution of the positively stained cells was
uniform for both the inner and outer areas of the circular
plated region. These results further demonstrated that
LIPUS treatment does not change the phenotype of the
Schwann cell.
RT-PCR
Schwann cells that were subjected to sustained LIPUS
exhibited an increase in NT-3 mRNA expression and a
decrease in BDNF mRNA expression (see Fig. 4). The
NT-3/b-actin ratio of RT-PCR products in the experimen-
tal group was 0.56 6 0.13 and 0.41 6 0.09 in the control
group. However, the BDNF/b-actin ratio of RT-PCR
products in the experimental group was 0.51 6 0.05 and
0.60 6 0.08 in the control group. The differences in NT-
3 and BDNF products for experimental and control
groups were found to be statistically significant (P <0.01 and P < 0.05, respectively) (see Fig. 5). Reverse
transcriptase controls with no reverse transcriptase
enzyme confirmed that there was no genomic DNA
contamination.
LIPUS on the Expression of NT-3 and BDNF 481
Microsurgery DOI 10.1002/micr
DISCUSSION
Ultrasound is commonly used for diagnostic imaging
and physiotherapy and can exert biological effects
through either thermal or mechanical mechanisms in liv-
ing tissue.18,19 In contrast to high-intensity continuous
ultrasound, LIPUS (<100 mW/cm2) has much lower
intensities, which are regarded as nonthermogenic and
nondestructive.20 Mechanical strains received by cells
may result in biochemical events and increase membrane
permeability.21 Despite the wide use of LIPUS for
improving the peripheral nerve tissue regeneration in ani-
mal models,5–7 very little is known about its effects on
the glial cell of peripheral nerves. It has been reported
that Schwann cells somehow respond to LIPUS stimula-
tion.6,7 However, the results of previous investigations
were somewhat inconclusive, particularly in what refers
to the application in the precise cellular mechanism.
In our study, we observed that LIPUS increased
Schwann cell proliferation indicating that LIPUS is in
vitro mitogenic for Schwann cells. The LIPUS treatment
could effectively improve Schwann cell proliferation in
the early stage (days 4, 7, and 10), whereas at later stage
(day 14) self-renewal ability of these cells reached to
much higher level, and there was no obvious difference
between experimental and control group. This increase in
proliferation confirmed results with previous in vitro
data,22,23 which proposes that cultured cells may be mito-
genic in response to LIPUS stimulation. Furthermore,
these data lend credence to the possibility that the LIPUS
Figure 2. LIPUS induces increased mitogenesis of in vitro cultured Schwann cells. Fluorescent images depicting the increase in the ratio
of the number of BrdU-stained nuclei (green) to DAPI-stained nuclei (blue) in experimental or control cells. Note the increased number of
BrdU-positive cells in experimental cells versus the control cells. (experimental A, B; control C, D). [Color figure can be viewed in the
online issue, which is available at www.interscience.wiley.com.]
Figure 3. Increase in proliferation of cultured Schwann cells in
response to LIPUS. The mitotic cells were identified by metabolic
BrdU labeling. The results confirmed that experimental groups dis-
play a higher proliferation rate. Statistical significance levels of P <
0.05 are denoted by * in the graph.
482 Zhang et al.
Microsurgery DOI 10.1002/micr
stimulus may directly trigger Schwann cell proliferation
in the early phase.
Previous studies have shown that LIPUS in the cul-
tured cells induced significant cellular response in nucleus
pulposus cells, endothelial cells, osteoblasts, chondro-
cytes, and fibroblasts,24–28 while little is known about
Schwann cell response to direct LIPUS stimulation. The
previous works with peripheral nerve injury have demon-
strated that the magnitude and duration of LIPUS have a
direct effect on whether this stimulus has a positive or
negative effect on nerve regeneration.6,7 Yet, there are
currently no data about the actual types and levels of the
ultrasound for the cells within the peripheral nerve.
Although using similar values from studies on chondro-
cytes, endothelial cells, and osteoblasts would allow
direct comparison between different cell lines, these val-
ues may not have any significance to neural tissue based
upon the different physiologic demands of each tissue
type. Thus, we chose the magnitude and duration of stim-
ulation for these experiments based on a previous work,
which demonstrated that ultrasound induced a biological
response in Schwann cells.6
In addition to the increased cell proliferation, LIPUS
stimulation to cell cultures has previously been demon-
strated to induce an alteration of cellular phenotype,20,29
little is known about the effects of LIPUS stimulation on
Schwann cell phenotype. Thus, our initial experiments
were directed to determine whether LIPUS would induce
phenotype alteration to the Schwann cell or not. S-100
immunostaining results evidenced that Schwann cells do
not dedifferentiate into another cell type following LIPUS
stimulation.
LIPUS stimulation of cultured Schwann cells induced
an alteration in cell function as demonstrated by pro-
moted cell proliferation and NT-3 gene expression, which
is consistent with that LIPUS enhances peripheral nerve
regeneration that was observed from in vivo models.7,30
It has been documented that NT-3 has a strong effect on
neurite outgrowth.31,32 Additionally, some studies using
genetically modified Schwann cells to overexpress the
NT-3 gene have examined the role of NT-3 in the neuron
survival and axonal regeneration/remyelination.33,34 It has
been reported that Schwann cells transduced ex vivo with
adenoviral or lentiviral vectors encoding a functional
NT-3 molecule led to the presence of a significantly
increased number of axons in the contusion site.35 The
results of Yamauchi et al.36 showed that NT-3 activation
of receptor tyrosine kinase C (TrkC) stimulates Schwann
cell migration through two parallel signaling units, Ras/
Tiam1/Rac1 and Dbs/Cdc42. Poduslo and Curran37
observed that NT-3 has a higher permeability coefficient
across the blood–nerve barrier and would contact sensory
axons soon after reaching the circulation of adult rats.
The increase in NT-3 expression might lead to an
increase in the number of nerve regeneration in the
axons. LIPUS-induced increase in NT-3 expression, as
demonstrated in our model, may create an environment
that is permissive for axonal sprouting and Schwann cells
migration after peripheral nerve injury.
Neurotrophin–neurotrophin interactions are regulated
by neurotrophin levels, NT-3 and BDNF in particular can
be coexpressed and each can regulate the levels of the
other. The relative expression levels of the neurotrophins
is thought to be mediated through receptor Trk activity.38
NT-3 infusion caused a significant decrease in the level
of BDNF proteins in both kindled and nonkindled hippo-
campus, likely via downregulation of TrkA.39 Further-
more, a study of Karchewski et al.40 showed that NT-3
can act in an antagonistic fashion to NGF in the regula-
tion of BDNF expression in intact neurons and mitigate
Figure 5. Results from RT-PCR analysis are expressed relative to
b-actin mRNA expression 14 days after the LIPUS stimulation.
There was significantly upregulated in experimental groups com-
pared with the control in NT-3 mRNA expression (P < 0.01), and
significantly downregulated in BDNF mRNA expression (P < 0.05).
Figure 4. Expression of NT-3 and BDNF in cultured Schwann cells
was evaluated by semiquantitative RT-PCR. The left lane repre-
sents the experimental mRNA expression and the right lane corre-
sponds to the control mRNA expression. The b-actin in each lane
served as an internal control.
LIPUS on the Expression of NT-3 and BDNF 483
Microsurgery DOI 10.1002/micr
BDNF’s expression in injured neurons. It is also consist-
ent with a study in which, in contrast, deletion of the
NT-3 gene in transgenic mice increased BDNF and TrkB
mRNA synthesis, suggesting that decreased NT-3 may
disinhibit BDNF expression.41 Similarly, our model has
demonstrated an upregulation of NT-3 mRNA and down-
regulation of BDNF mRNA expression after the LIPUS
stimulation. Hence, it is possible that NT-3 acts in an op-
posite fashion result in a downregulation in BDNF
expression in intact Schwann cells. Further investigation
is necessary to determine the molecular mechanisms of
NT-3 and BDNF signaling pathway by the data presented
in our study.
The results suggest that LIPUS may have several dif-
ferent clinical applications in the improvement of periph-
eral nerve regeneration. First, given its nontoxicity and a
wide margin of biologic safety, it may be used as an
effective physical stimulant when engineering peripheral
nerve tissue. Schwann cell-based therapies that use trans-
plantation techniques for the treatment of nerve tissue
repairing are being widely investigated for their potential
as clinical applications.42–44 LIPUS applied in conjunc-
tion with other forms of biologic stimulation is worth
considering when optimizing an innovative ‘‘multilevel’’
form of treatment. Second, application of LIPUS in vivo
is likely to be considered to stimulate repair of damaged
peripheral nerve tissues. Some experimental studies sup-
ported the result that both end-to-side and tubulization
repair of peripheral nerves led to successful axonal regen-
eration along the severed nerve trunk as well as to a par-
tial recovery of the lost function.45,46 With the availabil-
ity of the LIPUS as an activator of Schwann cells, it can
be an effective alternative in nerve reconstruction and be
of great value in various kinds of peripheral nerve micro-
surgery. Further investigation is required to identify an
accurate and continuous application of LIPUS treatment
to achieve constant and reproducible results prior to clini-
cal use.
As demonstrated in this study, NT-3 and BDNF
mRNA expression in Schwann cell response to LIPUS
may be independent of the reciprocal regulation between
the glial cells and neurons. Normally, during development
and axonal injury, this reciprocal relationship between the
glial cells and neurons causes a response in the glial
cells, which occurs secondary to the neuron. However,
data from our in vitro model indicate otherwise. The
Schwann cells responded in the absence of neurons,
suggesting that Schwann cell responses may be directly
elicited through LIPUS stimuli in our model.
CONCLUSIONS
The findings of this study provide preliminary evi-
dence that the application of LIPUS to Schwann cells is
effective in stimulating cell proliferation and neurotro-
phins gene expression. Although further studies are cer-
tainly required in further optimizing the delivery of
LIPUS and identifying the signal-regulated mechanisms
responsible for the molecular responses, the in vitro
results presented in our study already suggest that this
approach should prove helpful for research on peripheral
nerve regeneration.
ACKNOWLEDGMENTS
The authors thank Dr. Zhang Zhiwei and Dr. Zhao
Jun for providing their expert technical assistance.
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LIPUS on the Expression of NT-3 and BDNF 485
Microsurgery DOI 10.1002/micr
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