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.

REFERENCES

1. Heckman JD, Ryaby JP, McCabe J, Frey JJ, Kilcoyne RF. Accelera-tion of tibial fracture-healing by non-invasive, low-intensity pulsedultrasound. J Bone Joint Surg Am 1994;76:26–34.

2. Lu H, Qin L, Fok P, Cheung W, Lee K, Guo X, Wong W, Leung K.Low-intensity pulsed ultrasound accelerates bone-tendon junctionhealing: A partial patellectomy model in rabbits. Am J Sports Med2006;34:1287–1296.

3. Schortinghuis J, Bronckers AL, Stegenga B, Raghoebar GM, deBontLG. Ultrasound to stimulate early bone formation in a distractiongap: A double blind randomised clinical pilot trial in the edentulousmandible. Arch Oral Biol 2005;50:411–420.

4. Azuma Y, Ito M, Harada Y, Takagi H, Ohta T, Jingushi S. Low-intensity pulsed ultrasound accelerates rat femoral fracture healingby acting on the various cellular reactions in the fracture callus.J Bone Miner Res 2001;16:671–680.

5. Crisci AR, Ferreira AL. Low-intensity pulsed ultrasound acceleratesthe regeneration of the sciatic nerve after neurotomy in rats. Ultra-sound Med Biol 2002;28:1335–1341.

6. Chang CJ, Hsu SH, Lin FT, Chang H, Chang CS. Low-intensity-ultrasound-accelerated nerve regeneration using cell-seeded poly(D,L-lactic acid-co-glycolic acid) conduits: An in vivo and in vitro study.J Biomed Mater Res B Appl Biomater 2005;75:99–107.

7. Raso VV, Barbieri CH, Mazzer N, Fasan VS. Can therapeutic ultra-sound influence the regeneration of peripheral nerves? J NeurosciMethods 2005;142:185–192.

8. McAllister AK, Katz LC, Lo DC. Neurotrophins and synaptic plas-ticity. Annu Rev Neurosci 1999;22:295–318.

9. McAllister AK, Lo DC, Katz LC. Neurotrophins regulate dendriticgrowth in developing visual cortex. Neuron 1995;15:791–803.

10. Hess DM, Scott MO, Potluri S, Pitts EV, Cisterni C, Balice-GordonRJ. Localization of TrkC to Schwann cells and effects of neurotro-phin-3 signaling at neuromuscular synapses. J Comp Neurol 2007;501:465–482.

11. Zhang JY, Luo XG, Xian CJ, Liu ZH, Zhou XF. Endogenous BDNFis required for myelination and regeneration of injured sciatic nervein rodents. Eur J Neurosci 2000;12:4171–4180.

12. Schmidhammer R, Hausner T, Hopf R, Zandieh S, Redl H. In pe-ripheral nerve regeneration environment enriched with activity stim-ulating factors improves functional recovery. Acta Neurochir Suppl2007;100:161–167.

13. Lykissas MG, Batistatou AK, Charalabopoulos KA, Beris AE. Therole of neurotrophins in axonal growth, guidance, and regeneration.Curr Neurovasc Res 2007;4:143–151.

14. Cai F, Campana WM, Tomlinson DR, Fernyhough P. Transforminggrowth factor-b1 and glial growth factor 2 reduce neurotrophin-3mRNA expression in cultured Schwann cells via a cAMP-dependentpathway. Brain Res Mol Brain Res 1999;71:256–264.

15. Takayama T, Suzuki N, Ikeda K, Shimada T, Suzuki A, Maeno M,Otsuka K, Ito K. Low-intensity pulsed ultrasound stimulates osteo-genic differentiation in ROS 17/2.8 cells. Life Sci 2007;80:965–971.

484 Zhang et al.

Microsurgery DOI 10.1002/micr

16. Funk D, Fricke C, Schlosshauer B. Aging Schwann cells in vitro.Eur J Cell Biol 2007;84:207–219.

17. Hatami H, Oryan S, Semnanian S, Kazemi B, Bandepour M, Ahma-diani A. Alterations of BDNF and NT-3 genes expression in thenucleus paragigantocellularis during morphine dependency and with-drawal. Neuropeptides 2007;41:321–328.

18. Nahirnyak V, Mast TD, Holland CK. Ultrasound-induced thermalelevation in clotted blood and cranial bone. Ultrasound Med Biol2007;33:1285–1295.

19. Choi JJ, Pernot M, Brown TR, Small SA, Konofagou EE. Spatio-temporal analysis of molecular delivery through the blood-brain bar-rier using focused ultrasound. Phys Med Biol 2007;52:5509–5530.

20. Ikeda K, Takayama T, Suzuki N, Shimada K, Otsuka K, Ito K.Effects of low-intensity pulsed ultrasound on the differentiation ofC2C12 cells. Life Sci 2006;79:1936–1943.

21. Danialou G, Comtois AS, Dudley RW, Nalbantoglu J, Gilbert R,Karpati G, Jones DH, Petrof BJ. Ultrasound increases plasmid-medi-ated gene transfer to dystrophic muscles without collateral damage.Mol Ther 2002;6:687–693.

22. Mukai S, Ito H, Nakagawa Y, Akiyama H, Miyamoto M, NakamuraT. Transforming growth factor-b1 mediates the effects of low-inten-sity pulsed ultrasound in chondrocytes. Ultrasound Med Biol 2005;31:1713–1721.

23. Iwashina T, Mochida J, Miyazaki T, Watanabe T, Iwabuchi S, AndoK, Hotta T, Sakai D. Low-intensity pulsed ultrasound stimulates cellproliferation and proteoglycan production in rabbit intervertebraldisc cells cultured in alginate. Biomaterials 2006;27:354–361.

24. Hiyama A, Mochida J, Iwashina T, Omi H, Watanabe T, SeriganoK, Iwabuchi S, Sakai D. Synergistic effect of low-intensity pulsedultrasound on growth factor stimulation of nucleus pulposus cells.J Orthop Res 2007;25:1574–1581.

25. Hill GE, Fenwick S, Matthews BJ, Chivers RA, Southgate J. Theeffect of low-intensity pulsed ultrasound on repair of epithelial cellmonolayers in vitro. Ultrasound Med Biol 2005;31:1701–1706.

26. Sena K, Leven RM, Mazhar K, Sumner DR, Virdi AS. Early generesponse to low-intensity pulsed ultrasound in rat osteoblastic cells.Ultrasound Med Biol 2005;31:703–708.

27. Parvizi J, Wu CC, Lewallen DG, Greenleaf JF, Bolander ME. Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondro-cytes by increasing aggrecan gene expression. J Orthop Res 1999;17:488–494.

28. Zhou S, Schmelz A, Seufferlein T, Li Y, Zhao J, Bachem MG. Mo-lecular mechanisms of low intensity pulsed ultrasound in humanskin fibroblasts. J Biol Chem 2004;279:54463–54469.

29. Schumann D, Kujat R, Zellner J, Angele MK, Nerlich M, Mayr E,Angele P. Treatment of human mesenchymal stem cells with pulsedlow intensity ultrasound enhances the chondrogenic phenotype invitro. Biorheology 2006;43:431–443.

30. Lowdon IM, Seaber AV, Urbaniak JR. An improved method ofrecording rat tracks for measurement of the sciatic functional indexof de Medinaceli. J Neurosci Methods 1988;24:279–281.

31. Sahenk Z, Nagaraja HN, McCracken BS, King WM, Freimer ML,Cedarbaum JM, Mendell JR. NT-3 promotes nerve regeneration and

sensory improvement in CMT1A mouse models and in patients.Neurology 2005;65:681–689.

32. Markus A, Patel TD, Snider WD. Neurotrophic factors and axonalgrowth. Curr Opin Neurobiol 2002;12:523–531.

33. Pettingill LN, Minter RL, Shepherd RK. Schwann cells geneticallymodified to express neurotrophins promote spiral ganglion neuronsurvival in vitro. Neuroscience 2008;152:821–828.

34. Zhang X, Zeng Y, Zhang W, Wang J, Wu J, Li J. Co-transplantationof neural stem cells and NT-3-overexpressing Schwann cells intransected spinal cord. J Neurotrauma 2007;24:1863–1877.

35. Golden KL, Pearse DD, Blits B, Garg MS, Oudega M, Wood PM,Bunge MB. Transduced Schwann cells promote axon growth andmyelination after spinal cord injury. Exp Neurol 2007;207:203–217.

36. Yamauchi J, Miyamoto Y, Tanoue A, Shooter EM, Chan JR. Rasactivation of a Rac1 exchange factor, Tiam1, mediates neurotrophin-3-induced Schwann cell migration. Proc Natl Acad Sci USA 2005;102:14889–14894.

37. Poduslo JF, Curran GL. Permeability at the blood-brain and blood-nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3,BDNF. Brain Res Mol Brain Res 1996;36:280–286.

38. Mallei A, Rabin SJ, Mocchetti I. Autocrine regulation of nervegrowth factor expression by Trk receptors. J Neurochem 2004;90:1085–1093.

39. Yamamoto N, Hanamura K. Formation of the thalamocortical projec-tion regulated differentially by BDNF- and NT-3-mediated signaling.Rev Neurosci 2005;16:223–231.

40. Karchewski LA, Gratto KA, Wetmore C, Verge VM. Dynamic pat-terns of BDNF expression in injured sensory neurons: Differentialmodulation by NGF and NT-3. Eur J Neurosci 2002;16:1449–1462.

41. Elmer E, Kokaia M, Ernfors P, Ferencz I, Kokaia Z, Lindvall O.Suppressed kindling epileptogenesis and perturbed BDNF and TrkBgene regulation in NT-3 mutant mice. Exp Neurol 1997;145:93–103.

42. Li Q, Ping P, Jiang H, Liu K. Nerve conduit filled with GDNFgene-modified Schwann cells enhances regeneration of the peripheralnerve. Microsurgery 2006;26:116–121.

43. Gravvanis AI, Lavdas AA, Papalois A, Tsoutsos DA, Matsas R. Thebeneficial effect of genetically engineered Schwann cells withenhanced motility in peripheral nerve regeneration: Review. ActaNeurochir Suppl 2007;100:51–55.

44. Rochkind S, Astachov L, el-Ani D, Hayon T, Graif M, Barsky L,Alon M, Odva kI, Nevo Z, Shahar A. Further development of recon-structive and cell tissue-engineering technology for treatment ofcomplete peripheral nerve injury in rats. Neurol Res 2004;26:161–166.

45. Geuna S, Nicolino S, Raimondo S, Gambarotta G, Battiston B, TosP, Perroteau I. Nerve regeneration along bioengineered scaffolds.Microsurgery 2007;27:429–438.

46. Lloyd BM, Luginbuhl RD, Brenner MJ, Rocque BG, Tung TH,Myckatyn TM, Hunter DA, Mackinnon SE, Borschel GH. Use ofmotor nerve material in peripheral nerve repair with conduits. Micro-surgery 2007;27:138–145.

LIPUS on the Expression of NT-3 and BDNF 485

Microsurgery DOI 10.1002/micr