The Effects of

High-Frequency and

Low-Frequency Sonophoresis

on Epidermal Permeability Barrier

Hyun Jung Kim

Department of Medicine

The Graduate School, Yonsei University

The Effects of

High-Frequency and

Low-Frequency Sonophoresis

on Epidermal Permeability Barrier

Directed by Professor Seung Hun Lee

The Master’s Thesis

Submitted to the Department of Medicine

and the Graduate School of Yonsei University

in partial fulfillment of the requirements for the

degree of Master of Medical science

Hyun Jung Kim

June 2006

This certifies that the Master’s Thesis of

Hyun Jung Kim is approved.

Thesis Supervisor: Prof. Seung Hun Lee

Thesis Committee Member: Prof. Wook Lew

Thesis Committee Member: Prof. Soon Jung Park

The Graduate School

Yonsei University

June 2006

감사의감사의감사의감사의 글글글글

저의 부족한 능력에도 이 논문을 완성하게 해 준 모든

분들께 감사를 드립니다. 피부 장벽이라는 세계를 일깨워주신

이승헌 교수님과 이 논문을 다잡아 주신 유욱 교수님, 박순정

교수님께 감사의 글 올립니다.

이 논문의 큰 힘이 되어준 전정은 연구원, 최기주 연구원의

공에 높이 감사 드립니다.

또한 피부 장벽 모임의 모든 구성원들 특히, 원주 피부과학

교실의 최응호 선생님의 지도와 애경 중앙 연구소의 정세규

연구원, 김민정 연구원, 최선영 연구원의 도움에 고마운 마음

보냅니다.

항상 열심히 일을 해 나갈 수 있게 도와주는 우리 가족, 여보,

나의 보물 혜리, 엄마, 하늘에서 저를 지켜 보고 보호

해주시는 우리 아빠, 그리고 어머님, 아버님께 저의 모든

사랑으로 감사 드립니다.

저자 씀

i

Table of Contents

ABSTRACT··································································1 I. INTRODUCTION·······················································3 II. MATERIALS AND METHODS··································7

1. Animals ······················7 2. Methods ·····················7

A. Sonophoresis treatment··············7 B. Measurement of transepidermal water loss······7 C. Electron microscopy examination··········8

i. Calcium capture cytochemistry·········8 ii. Quantitative EM analysis···········8

D. Nile red staining ················ 9 E. Immunohistochemical stain ···········9 F. Real t ime RT-PCR·············10

3. Stat is t ica l analys is··············11 III. RESULTS·····················12

1. Transepidermal water loss·············12 2. Epidermal calcium gradient by ultrastructural calcium capture

cytochemistry···············································13 3. Assessment of Lamellar body densities·····················14 4. Nile red stain after sonophoresis treatment················ 15 5. Immunohistochemical stain with cytokines after the treatment

with high and low-frequency sonophoresis········16 6. The results of RT-PCR and real time RT-PCR······17

A. cytokines···················17 B. Lipid synthetic enzymes·············18

IV. DISCUSSION……………………………………….....19 V. CONCLUSION…………………………………...…….26 REFERENCES……………………………………………..27 ABSTRACT IN KOREAN………………………………...32

ii

List of Figures

Figure 1. The effects of sonophoresis and iontophoresis on epidermal

permeability barrier…………………………………….…….…..6

Figure 2. TEWL after treatment…..…………………….…….…..12

Figure 3. Visualization of epidermal calcium gradient by ultrastructural

calcium capture cytochemistry……………………………………... 13

Figure 4. Assessment of Lamellar body densities…………...14

Figure 5. Nile red stain after sonophoresis treatment......……15

Figure 6. Immunohistochemical stain with primary antibodies to IL-1α

and TNF-α after the treatment with high and low-frequency

sonophoresis…………………………………………………...16

Figure 7. The expression of cytokines IL-1α and TNF-α mRNA at

3hrs after sonophoresis on skin surface was analyzed by RT-PCR and

real-time quantitative RT-PCR ………………………………....17

Figure 8. The expression of lipid synthetic enzymes HMG-CoA

reductase and fatty acid synthetase(FAS) mRNA at 3hrs and serine

palmitoyl transferase (SPT) at 6hrs after sonophoresis was analyzed by

RT-PCR and real-time quantitative RT-PCR………………………....18

List of Tables

Table 1.Sequences of primers in RT-PCR………………………… ………………12

iii

Abstract

The Effects of High-Frequency and Low-Frequency Sonophoresis

on Epidermal Permeability Barrier

Hyun Jung Kim

Department of Medicine

The Graduate School, Yonsei University

(Directed by Professor Seung Hun Lee)

Sonophoresis is the application of ultrasound enhances the skin permeability

to a variety of molecules. The enhancement induced by ultrasound is

particularly significant at low-frequencies (f < 100 kHz). We have already

reported that iontophorhesis and high-frequency (1<f <3 mHz) sonophoresis

at energies that do not provoke a barrier abnormality stimulate epidermal

cytokine expression, lamellar body secretion and lipid synthesis, which are

linked to altered epidermal calcium levels. Attenuation of an acoustic wave is

inversely proportional to its frequency, and thus the more the frequency

increases, the less deeply the ultrasound penetrates into and under the skin.

The purpose of this study is to compare the effects of high-frequency (3MHz)

and low-frequency (25kHz) sonophoresis on epidermal permeability barrier

at the energy levels that do not provoke a barrier abnormality.

Our results show the high- frequency sonophoresis and low-frequency

iv

sonophoresis effects on epidermal permeability barrier. Lamellar body

secretion was accelerated in stratum granulosum shortly after the treatment of

high and low frequency sonophoresis and there were no siginificant

differences. Lipid deposition was more prominent in high-frequency treatment

then low-frequency treatment and stronger intensities were also shown in Nile

red stain. The stimulation of expression of epidermal cytokine(ex. IL-1α and

TNF-α) and epidermal lipid synthesis enzymes(ex. HMG-CoA reductase, fatty

acid synthetase and serine palmitoyl transferase) in real time RT-PCR and

immunohistochemical staining showed the more increased patterns in the high

-frequency sonophoresis treated skin compared to the low frequency treated

skin as well as non treated control. In conclusion, the different frequency of

sonophoresis shows the different effect on epidermal permeability barrier

homeostasis. These differences were predicted from not differences in

energies but differences in penetration powers which are inversely

proportional to its frequency.

These results can suggest the experimental basis to choose the high

frequency sonophoresis as a barrier reinforcement therapy.

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Key Words : High-Frequency sosnophoresis , Low-Frequency Sonophoresis ,

Epidermal Permeability Barrier

１

The Effects of High-Frequency and Low-

Frequency Sonophoresis

on Epidermal Permeability Barrier

Hyun Jung Kim

Department of Medicine The Graduate School, Yonsei University

(Directed by Professor Seung Hun Lee)

I. INTRODUCTION Ultrasound, which is routinely used for diagnostic imaging applications, is

now being adopted in various drug delivery and other therapeutic

applications1. Sonophoresis is the application of ultrasound for enhancing the

skin permeability to a variety of molecules. There are two distinct sets of

sonophoresis based on frequency range and applications; High frequency

sonophoresis(1-3MHz) and Low frequency sonophoresis (20-100KHz)2.

The epidermis, which forms the uppermost, multilayered compartment of the

skin, has evolved to provide a physical and permeability barrier, which is

essential for survival as an adaptation to terrestrial life in mammals. This

barrier against the environment—which excludes foreign substances and

organisms and prevents the loss of vital fluids— is provided, and continuously

regenerated, by terminally differentiating keratinocytes3. Application of

ultrasound has been shown to enhance transdermal transport of various drugs

２

including macromoleculs4. In this method, we must consider the effects on

epidermal permeability barrier. The effects on epidermal permeability barrier

of high frequency sonophoresis were shown as follows. In morphological

view, it induced extensive cleft formation and domain separation in the

intercellular bilayers both within stratum corneum and at the SG-SC interface.

Nonlamellar domains filled with electron-dense amorphous material appeared

within intercellular spaces of the mid- to upper SC was also shown5. In

functional view, sonophoresis using proper ultrasound can induce changes in

the epidermal calcium that increase LB secretion without increasing

transepidermal water loss (TEWL) and stimulate generation of primary

cytokines(interleukin-1alpha (IL-1alpha), interleukin-6(IL-6), tumor necrosis

factor-alpha (TNF-alpha), transforming growth factor-beta(TGF-beta)) in the

epidermis. It also increase the synthesis of sphingolipids and neutral lipids in

the SC and lipid synthetic enzymes (Figure 1) 6-8.

The enhancement for transdermal drug delivery induced by ultrasound is

particularly significant at low-frequencies (f < 100 kHz). Significant attention

has been devoted to understand the mechanisms of low-frequency

sonophoresis. A consensus has been reached that acoustic cavitation, the

formation and collapse of gaseous cavities, is responsible for low-frequency

sonophoresis. Disruption of SC lipid bilayers due to bubble induced shock

waves or microjet impact may enhance skin permeability by at least two

mechanisms. First, a moderate level of disruption decreases the structural

order of lipid bilayers and increases solute diffusion coefficient. Second, lipid

extraction also plays a role in low-frequency sonophoresis. A porous pathway

model described transdermal transport of hydrophilic solutes during low-

frequency sonophoresis. Other mechanisms are thermal effects, convectivity,

and mechanical effects4.

The effects on skin of low-frequency sonophoresis have been suggested by

various studies. Boucaud et al. performed a microstructural analysis of skin

samples exposed to ultrasound. They reported no detectable changes in the

３

structures of human skin at an intensity of 2.5W/cm2. Hairless rat skin

exposed to the same intensity showed slight and transient erythema and

dermal necrosis at 24h9. Morimoto et al. examined a relationship between

hydrophilic solute and water (vehicle) transports in the excised hairless rat

skin in the presence of ultrasound (41 kHz, 60–300 mW/cm2) application and

also conducted skin surface observation using confocal microscopy. They

concluded that 41 kHz ultrasound can increase the transepidermal transport of

hydrophilic solutes by inducing convective solvent flow probably via both

corneocytes and SC lipids10.

Low-frequency ultrasound has been shown to synergistically enhance skin

permeability with chemical enhancers and iontophoresis11.

The purpose of this study was to determine the effects on epidermal

permeability barrier of low-frequency sonophoresis comparing to the effects

of high -frequency sonophoresis. Even though various studies showed that

application of low-frequency ultrasound enhances skin permeability more

effectively than high-frequency ultrasound, the study of the effects on

epidermal permeability barrier of low-frequency sonophoresis was rare.

Attenuation of an acoustic wave is inversely proportional to its frequency,

and thus the more the frequency increases, the less deeply the ultrasound

penetrates into and under the skin. Based on this theory, this study suggests

the differential effects of high-frequency and low-frequency sonophoresis on

epidermal permeability barrier. For this purpose we used both high(3MHz)

and low(25kHz) frequency sonophoresis treatment as methods at energy

levels that induce changes in the epidermal calcium gradient without altering

TEWL and then applied electron microscopy to show the morphological

changes and epidermal calcium ion gradients, nile red staining to show the

neutral lipid deposition, and real-time, quantitative RT-PCR and

immunohistochemical staining to demonstrate changes in expression of

epidermal cytokines and lipid synthetic enzymes.

４

Fig 1. The effects of sonophoresis and iontophoresis on epidermal

permeability barrier

５

II. Materials AND METHODS

1. Animals

Adult hairless mice were purchased from the animal laboratory of

Yonsei University and were 812-wk-old females at the time of study.

2. Methods

A. Sonophoresis treatment

After anesthesia with chloral hydrate, the treatment sites of the back

received 2min of 800mW per ㎠, 3MHz continuous wave ultrasound

(Junghun engineering, Korea) treatment as high-frequency sonophoresis

and 2min of 800mW per ㎠, 25kHz continuous wave ultrasound(Junghun

engineering, Korea) treatment as low-frequency sonophoresis as in our

previous study in which TEWL was not increased after treatment, and the

negative control sites were only dabbed with the transmission gel (Biosonic,

Amite,USA) which is generally applied before ultrasound treatment.

B. Measuremnet of Transepidermal water loss(TEWL)8

TEWL checked by Tewameter TM210( Courage+, Khazaka, Germany)

was measured at before treatment. Following anesthesia with

intraperitoneal injection of chloral hydrate, sonophoresis treated to mice

and we checked TEWL at 15min, 3hr, 6hr and 24hr in temperature of 22℃

and in humidity of 40-60%.

６

C. Electron microscopy examination8

i. Calcium capture cytochemistry

The biopsy specimens were immediately cut into small pieces (0.5

mm3) in a drop of ice cold fixative containing 2% glutaraldehyde, 2%

formaldehyde, 90 mM potassium oxalate, 1.4% sucrose and fixed

overnight on ice. The fixative was removed and the samples post fixed

in osmium/pyroantimonate for 2 h on ice and washed for 10 min in ice

cold distilled water at pH 10. All post fixed tissues were rinsed in 0.1 M

cacodylate buffer for 10 min, dehydrated in a graded ethanol series,

respectively, and embedded in epon epoxy resin. Ultrathin sections

(Leica UCT) were cut,double stained with uranyl acetate and lead citrate,

and examined with a transmission electron microscope(Hitachi H-7600).

Each section was incubated with ethylenediamine tetraacetic acid as a

control.

ii. Quantitative EM analysis

In order to exclude subjective bias in these morphologic studies, we

quantitated LB number (=density) and secretion in EM pictures by an

objective method. We used three of four EM pictures taken at low

magnification(x5000) from each sample to cover large sample areas

to further diminish bias and to improve statistical sampling.

The numbers of protrusions (the SC-SG interface and the SG inside)

were quantitated, and assessed planimetrically as the number per unit

length of SC-SG interface and SG inside. To assess LB densities, LB

images in the cytosol of the uppermost two layers of the SG were

counted and expressed as average number per unit area of cytosol.

７

D. Nile red staining6

Nile red, a fluorescence probe for lipids, was used to demonstrate

the distribution and content of lipids in the stratum corneum (SC).

Tissue samples, quick-frozen and fixated in liquid nitrogen, were

placed in a cryomold filled with OCT compound and quick-frozen

with isopentane. The frozen tissue samples were then sliced into

sections 5um thick with a cryostat. Two drops of nile red stock

solution(nile red 500㎍/㎖ acetone) were dropped onto each sample

slide and allowed 10min to stain in the dark. The slides were then

mounted and observed under the confocal microscope with 40X

objective lens and with intensity profile of the image analyzer.

E. Immunohistochemical stain8

For immunohistochemical stains, the skin biopsy was taken 15min,

1, 3, 6, 9, 12 and 24h after treatment for 2min. Tissue samples,

quick-frozen and fixated in liquid nitrogen, were placed in a

cryomold filled with OCT compound and quick-frozen with

isopentane. The frozen tissue samples were then sliced into sections

6 to 8㎛ thick with a cryostat, fixed in gelatin-coated slides, and

fixed for 5min with acetone chilled at 20℃. After being rinsed with

TBS buffer solution, the tissue sections were cultured in 3% H2O2

for 30 min and rinsed once more with TBS buffer solution. Serum

solution(DAKO protein block serum-free cat. No x0909) were

administered to the sample slides and 30min was allowed for

reactions to proceed. A total of 100 to 150㎕ of primary antibodies

for IL-1α(Santacruz, CA, USA) and TNF-α(Endogen, Woburn,

MA) was dropped on the slides placed inside a wet box. The slides

were left to sit through the reaction for 1h at room temperature and

rinsed with TBS buffer. The samples then received administration of

８

secondary antibodies (EnVision, DAKO, Carpinteria, CA) sat for

30min, and were rinsed with TBS buffer solution. Two drops of

diaminobenzidine solutions (DAKO) were dropped onto each sample

slide and allowed 5min to stain. The slides were then mounted and

observed under the optical microscope with 40X objective lens and

with intensity profile of the image analyzer.

F. Real time RT-PCR

Total RNA was isolated from each skin obtained after iontophoresis and

sonophoresis treatment using Trizol reagent. One microgram of total RNA

was reverse-transcribed with AMV reverse transcriptase (Promega,

Madison, WI). Pairs of primers for amplification of IL-1α and TNF-α

were designed using the Primer Express Software (Applied Biosystems,

Foster City, CA). In all experiments, primer concentrations were first

optimized to avoid unspecific binding of primers, and after running the

PCR products, a dissociation curve analysis was performed to verify the

specificity of the amplification products. Probe and primer sequences used

were on Table 1. Real time quantitative PCR was performed using the ABI

Prism 7700 sequence detector (TaqMan, Perkin Elmer/Applied Biosystems,

Foster City, CA). The TaqMan PCR conditions were as follows: 15 s at 94

and 1 min at 601C with a total of 50 to 55 cycles. Data were analyzed with

the software provided with the TaqMan. TaqMan Ct values were followed

by GAPDH normalization. To avoid contamination, all assays were

performed using the universal thermal cycling parameters (Applied

Biosystems) with AmpErase UNG. All experiments were performed in

duplicate.

９

3. Statistical analysis

Statistical analysis using t tests, multiple linear regression analysis and

ANOVA were completed using Statistical Product and Service Solutions

software (SPSS Inc, Chicago, Ill) for windows (version 12). A P value of less

than 0.05 was considered significant.

Table 1.The sequences of RT-PCR primer

Forward primer Reverse primer

IL-1α CTCTAGAGCACCATGCTACAGAC TGGAATCCAGGGGAAACACTG

TNF-α GGCAGGTCTACTTTAGAGTCATTGC ACATTCGAGGCTCCAGTGAATTCGG

HMG-coA

reductase GATCCAGGAGCGAACCAA GCGAATAGACACACCACGTT

FAS CCTCACTGCCATCCAGATTG CTGTTTACATTCCTCCCAGGAC

SPT CTGAACTCCTCAACCACTA GGTTCAGCTCATCACTCAGAATC

GAPDH AATGGTGAAGGTCGGTGTGA CTGGAAGATGGTGATGGGC

１０

III. RESULTS

1. Transepidermal water loss

There were no statistically significant alterations in TEWL occurred after

either high-frequency or low-frequency sonophoresis as well as control.

Figure 2. TEWL after treatment. No statistically significant alteration

in TEWL.

１１

2. Epidermal calcium gradient by ultrastructural calcium capture

cytochemistry

High and low-frequency sonophoresis induced a marked decrease in

calcium content in the epidermis, especially the top part but there is an

increase of calcium in stratum basale in 15min and 3hrs. After 6hrs, the

epidermal calcium gradient was recovered normally in both treatment

group. There were no significant differences between high-frequency and

low-frequency sonophoresis.

Figure 3. Visualization of epidermal calcium gradient by

ultrastructural calcium capture cytochemistry. Disappearance of the

epidermal calcium gradient with a marked increase in calcium content in

the stratum basale and dermis after 15min(a) and 3hrs(b).The recovery of

calcium gradient was seen after 6hrs(c) and 24hrs(d). There was no

different pattern in the changes of calcium gradient between high and low-

frequency treatment. H: high, L: low-frequency sonophoresis(X5k). SC, stratum

corneum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale.

2(a)L 2(b)L 2(a)H 2(b)H

SG

SB

SS

SC

SG

SS

SB 2(d)H 2(d)L 2(c)H 2(c)L

SC

１２

3. Assessment of Lamellar body densities

No significant difference in increasing of LB densities until 3hrs after

sonophoresis treatment between two groups was shown. The prolonged

effects of low-frequency sonophoresis on increasing of LB densities

were seen 6hrs after sonophoresis treatment (p<0.05).

Figure 4. Assessment of Lamellar body densities. There was no

significant difference in increasing of LB densities until 3hrs after

sonophoresis treatment. The prolonged effects of low-frequency

sonophoresis on increasing of LB densities were seen 6hrs after

sonophoresis treatment.

１３

4. Nile red stain after sonophoresis treatment

Nile red, a fluorescence probe for lipids, was used to demonstrated the

distribution and content of lipids in the stratum corneum (SC). After

15mn, this results showed the more prominent increase of neutral lipid

on SC in high-frequency sononphoresis treatment group then low-

frequency sononphoresis treatment group. These patterns continued at

3hrs and 6hrs.

Figure 5. Nile red stain after sonophoresis tratment. It showed the

normal-appearing fluorescence (a). After 15min (b), lipid deposition was

more prominent in high -frequency treatment then low-frequency

treatment and stronger intensities were also shown after 3hrs(c) and

6hrs(d)in high-frequency treatment. H; high-frequency, L; low-

frequency

１４

5. Immunohistochemical stain with cytokines after the treatment with high-

and low-frequency sonophoresis.

Immunohistochemical stains for IL-1α and TNF-α in epidermis

treated with sonophoresis showed increased expression compared to NC.

When comparing between high and low-frequency sonophoresis, it

showed more prominent increase of cytokine expressions in high-

frequency sonophoresis treatment group.

Figure 6. Immunohistochemical stain with primary antibodies to

IL-1αααα (a) and TNF-αααα (b) at 6hrs after the treatment with high

and low-frequency sonophoresis. It showed the stronger expressions of

cytokines in high-frequency treatment then low-frequency treatment .

C;control, H; high-frequency, L; low-frequency.(X100)

.

L (a) H(a)

C(b) L(b) H (b)

C(a)

１５

6. The results of RT-PCR and real time RT-PCR

A. Cytokines

Expressions of mRNA IL-1α and TNF-α increased in murine

epidermis at 3hrs after sonophoresis treatment compared to control.

For quantitative analysis, we applied the real time RT-PCR. High-

frequency sonophoresis treatment group showed the statistically

significant increase of cytokine expressions compared to low-

frequency treatment group as well as control group.

a. b.

Figure 7. The expression of cytokines IL-1αααα and TNF-αααα

mRNA at 3hrs after sonophoresis on skin surface was analyzed by

RT-PCR (a) and real-time quantitative RT-PCR using GAPDH as

an endogenous control. Both cytokines showed increased expression

in high-frequency sonophoresis treated skin (High) compared to the

low-frequency treated skin (Low) as well as non treated control (NC).

* : p<0.01

１６

B. Lipid synthetic enzymes

Expressions of mRNA HMG-CoA reductase and fatty acid

synthetase(FAS) increased in murine epidermis at 3hr and serine

palmitoyl transferase (SPT) at 6hrs after sonophoresis treatment

compared to control. For quantitative analysis, we applied the real

time RT-PCR. High-frequency sonophoresis treatment group showed

the statistically significant increase of HMG-CoA reductase

expressions compared to low-frequency group as well as control

group. The expression of FAS and SPT increased more in high-

frequency then low-frequency as well as control, but the increase was

not statistically significant.

a. b.

Figure 8. The expression of lipid synthetic enzymes HMG-CoA

reductase and fatty acid synthetase(FAS) mRNA at 3hrs and serine

palmitoyl transferase (SPT) at 6hrs after sonophoresis was analyzed

by RT-PCR(a) and real-time quantitative RT-PCR(b). All three

enzymes showed increased expression in the high-frequency

sonophoresistreated skin(high) compared to the low-frequency(low)

treated skin(low) as well as non treated control (NC). * :p<0.01

１７

IV. DISCUSSION

The epidermis, which forms the uppermost, multilayered compartment of the

skin, has evolved to provide a physical and permeability barrier, which is

essential for survival as an adaptation to terrestrial life in mammals. This

barrier against the environment —which excludes foreign substances and

organisms and prevents the loss of vital fluids — is provided, and

continuously regenerated, by terminally differentiating keratinocytes12. The

advantages of transdermal drug delivery route compared to oral route are no

gastrointestinal degradation, no first-pass metabolism by the liver, steady

delivery and better compliance13. Transdermal drug delivery, for examples,

chemical enhancers, iontophorhesis, electroporation, photomechanical waves,

microneedle array and sonophoresis offers an attractive alternative to the

conventional drug delivery methods of oral administration and injection14.

Sonophoresis is the use of ultrasound to increase percutaneous absorption of

a drug14. There are two distinct sets of sonophoresis based on frequency range

and applications.High-frequency sonophoresis(1-3MHz) and low-frequency

sonophoresis(20-150KHz)4. The use of low-frequency sonophoresis

(20–150 kHz) was recently shown to be more effective in enhancing

transdermal transport15.

The first mechanism of low-frequency sonophoresis is the cavitational

effect. Cavitation is the formation of gaseous cavities in a medium

upon ultrasound exposure 4. The second is the disruption of SC lipid bilayers

due to bubble induced shock waves or microjet impact. It was based on the

study of showing that moderate level of disruption decreases the structural

order of lipid bilayers and increases solute diffusion coefficient after 1MHz

sonophoresis treatment16. Other study by Alvarez-Roman et al. showed the

extraction in significant fraction (30%) of the intercellular lipids of the

stratum corneum17. But our study used the energy level and times that do not

disrupt the epidermal permeability barrier function, so we could not see the

１８

disruption of lipid structures. Our treatment induced the changes of epidermal

calcium gradient, that is the initial signal for epidermal barrier recovery, not

the destruction of lipid structure but the increase in neutral lipid deposition

and lipid synthesis as previous our study6-8. The relationship between

sonophoresis and the disruption of SC lipid bilayers depends on the time and

energy level. Alvarez-Roman et al applied low frequency sonophoresis for 2

h with a sonicator (VCX 400 Sonics and Materials, Danbury, CT) at a

frequency of 20 KHz and an intensity of 15 W/cm2, using a 0.1:0.9 on/ off

duty cycle to minimize excessive temperature increase. That condition

contained the immersion of samples for a long time. Based on our routine

sonophoresis treatment, especially for dermartological application in which

treatment time is shorter than 10min without immersion, the lipid extraction

might not be the significant mechanism for our sonophoresis system. Third

mechanism is the porous pathway model. Transdermal transport of

hydrophilic solutes was described using a porous pathway model, which

assumes that transdermal transport occurs through hydrophilic pores in the

skin. According to the porous pathway model, solute transport across the skin

is determined by the pore radius, porosity, and tortuosity of the skin. Low-

frequency sonophoresis appears to increase skin permeability by increasing

the porosity rather than increasing the pore size16, 17. Paliwal et al suggested

that extended lacunar spaces may symbolize low frequency sonophoresis-

induced permeation pathways in the SC as shown in confocal microscopy and

transmission electron microscopy (TEM) 18. Our study and previous studies

showed that, cavitation, intracellular cavities were observed in stratum basale,

while some perinuclear cavities were shown in stratum granulosum and

spinosum after the treatment of both high- and low-sonophoresis, which is

believed to be responsible for the ultrasonic permeabilization of cells and

tissues such as skin in addition to lacunae dilatation as Paliwal et al’s report19,

20.

１９

Effect and safety of low-frequency sonophoresis on skin has been proved

by various study. Tachibana et al21 showed that the exposure of rabbit skin to

low-frequency ultrasound (105 kHz, 5000 Pa pressure amplitude) induced no

damage to the skin upon ultrasound application. Mitragotri et al22 suggested

the histological studies of hairless rat skin exposed to low-frequency

ultrasound (20 kHz, 12.5–225 mW/cm2) that showed no damage to the

epidermis and underlying living tissue. Boucaud et al9 also proved that the

effect of low-frequency ultrasound (20 kHz) on human skin that were exposed

to low-intensity ultrasound (<2.5 W/cm2), as no histological change and in

microscopic examination using transmission electron microscopy confirmed a

lack of structural modification. As one exception was the study of Yamashita

et al.23 Using scanning electron microscopy, the effects of ultrasound with

frequency of 48 kHz (0.5 W/cm2) on the surface of hairless mice and human

skin was observed that the outer layer in mice stratum corneum was totally

removed and pores were observed, whereas in human skin some removal of

keratinocytes around hair follicles. In our study, we could not find direct

damage to skin, especially epidermal permeability barrier (Figure 2)19, 20.

The signals for recovery of epidermal permeability barrier divided by the

direct changes of TEWL or not. The one group contains the acute disruption

model, that is, the skin barrier with either solvents or tape stripping produces

the direct changes of TEWL and a homeostatic response in the subjacent

nucleated layers of the epidermis, resulting in rapid restoration of normal

barrier function and the chronic essential free fatty acid deficient model24-27.

The other group means the manipulation of epidermal calcium in vivo directly

regulate recovery of epidermal permeability barrier. Iontophoresis6 using

proper electric current, sonophoresis7, 8, 26 using proper ultrasound and

application of glycolic acid28 can induce changes in the epidermal calcium

gradients that increase LB secretion without increasing transepidermal water

loss (TEWL). These are good tools, if their density or intensity did not disrupt

the skin barrier, could be used for in vivo studies to define the recovery of

２０

epidermal permeability barrier in response to changes in epidermal calcium in

vivo(Figure 2). Our study also showed that low-frequency sonophoresis has

similar effect on the change of epidermal calcium gradients as high-frequency

sonophoresis (Figure 3). The frequency of sonophoresis could not affect the

difference in calcium gradient changes.

Although the total time required for barrier recovery varies according to age

and species, there is always an initial, rapid recovery phase, followed by a

prolonged recovery phase that requires about 35 hrs for completion in rodents

29 . The first step in the repair response following barrier disruption is rapid

secretion (within minutes) of performed LB contents from cells of the outer

SG, which leaves the cytosol of these cells largely devoid of LB26, 30. Newly

formed LB then begin to reappear in SG cells by 30–60 min, and by 3–6 hr

the number (density) of LB in SG cells exceeds normal. Because of

accelerated secretion and organellogenesis between 30 min and 6hr, the

quantities of secreted LB contents increase at the SG–SC interface, and by 2 h

new, lipid-enriched lamellar bilayers begin to appear in the lower SC. Thus,

the exocytosis of LB provides a pathway by which the epidermis delivers

lipids and their respective lipid-processing enzymes simultaneously to the

extracellular spaces of the SC 25, 29. We examined the increase of lamellar

body secretion (data not shown) and lamellar body densities on both group

compared to control. The reason why the increase was maintained of both

groups at similar levels until 3hr and changed as the prolonged prominence in

low-frequency group after 6hr will be further studied (Figure 4).

The rapid formation of LB following acute barrier disruption requires

increased availability of the major lipid components of LB, i.e., cholesterol,

glucosylceramides, and phospholipid31, 32. Although the epidermis is a very

active site of lipid synthesis under basal conditions, barrier disruption

stimulates a further increase in the synthesis of cholesterol, ceramides, and

fatty acid (a major component of both PL and ceramides) 33-35. The increase in

cholesterol synthesis is associated with an increase in the activity, protein

２１

levels and mRNA levels of HMG CoA reductase and other key enzymes in

the cholesterol synthetic pathway; i.e., HMG CoA synthase, farnesyl

diphosphate synthase, and squalene synthase36,37. Whereas the increase in

fatty acid (FA) synthesis occurs because of an increase in the activity and

mRNA levels of both of the key enzymes of FA synthesis, acetyl CoA

carboxylase and FA synthase(FAS), the increase in ceramide synthesis is

because of an increase in the activity and mRNA levels of serine palmitoyl

transferase (SPT)38, the enzyme which catalyzes the first committed step in

ceramide synthesis. In contrast, glucosylceramide synthase, the enzyme which

synthesizes glucosylceramides, does not increase following barrier disruption.

But glucosylceramide synthesis could still be dependent upon the availability

of FA for both sphingoid base formation and N-acylation35.Thus, a specific,

coordinate increase in the synthesis of the key lipid constituents of LB

provides the pool of lipids, required for the formation of new LB3.

Our previous study using high frequency sonophoresis showed the increase

of cholesterol, at 3hr and continuing to 24hrs, free fatty acid and ceramide, at

6hrs and continuing to 24hrs, of epidermal lipid level compared to control

group. The expression of HMG-CoA reductase, FAS and SPT were also

increased. In conlusion, sonophorhesis changed the epidermal calcium

gradient and started the barrier recovery cascade, such as lipid synthesis8. Our

study also showed the both group induced the expression of HMG-CoA

reductase, FAS and SPT. This study showed the more prominent effects of

high-frequency sonophoresis than low-frequency sonophoresis as well as

control (Figure 8). This stronger effect on lipid synthetic enzyme during

epidermal permeability barrier recovery can be explained by the theory based

on that fact; “Attenuation of an acoustic wave is inversely proportional to its

frequency, and thus the more the frequency increases, the less deeply the

ultrasound penetrates into and under the skin.” 39 The penetration power of

high frequency sonophoresis will be weaker then low frequency sonophoresis.

But total power on epidermis is stronger in high frequency sonphoresis

２２

treatment then low frequency, because the energy can not penetrate deeply but

can concentrate on epidermis. The stronger effect on epidermal permeability

barrier recovery of high-frequency sonophoresis also was shown in the Nile

red staining results. The neutral lipid deposition was more prominent in high

frequency sonophoresis (Figure 5).

Both release of cytokines from a preformed pool and production of several

cytokines and growth factors increase with either acute or prolonged barrier

disruption, and in chronic inflammatory skin diseases, which generally

display high levels of primary cytokines, chemokines and other inflammatory

markers40-42 . The changes in epidermal calcium without barrier perturbation

such as iontophoresis and sonophoresis, regulate mRNA expression of

epidermal cytokines and then protein in vivo6, 7, 26. Our study also showed the

increase in mRNA expression and protein expression of epidermal cytokines,

IL-1α and TNF-α. In these results, we also examined the stronger effects of

high frequency sonophoresis similar to lipid synthetic enzymes. This results

also suggested as the evidence for the more concentrated effects of high-

frequency sonophoresis on epidermal permeability barrier then low-

frequency.

The concentrated effects of high-frequency sonophoresis restricted some

steps of epidermal permeability recovery such as neutral lipid deposition, lipid

synthetic enzyme and cytokine expression. The effects on calcium gradient,

that is the first signal for barrier recovery, are same in both groups and on the

increase in lamellar body densities are same until 3hrs and stronger in low-

frequency sonophoresis treatment group. We don’t know the exact mechanism

of these differences. But we suggested some hypothesis. First, both types of

sonophoresis are sufficient to make the changes of epidermal calcium gradient.

Second, the deeper penetration activity of low-frequency could make the

prolonged effects on epidermal permeability barrier even though less

concentrated power as we could see in changes of lamellar body densites. But

we must further studied the prolonged effects on other points such as lipid

２３

synthetic enzyme and cytokines. Third, as we mentioned, the more

concentrated power on epidermis of high-frequency sonophoresis could the

stronger effects on the expressions of lipid synthetic enzymes and epidermal

cytokines.

Finally we must consider the thermal effects of sonophoresis to explain the

differential effects of high-frequency and low-frequency sonophoresis on

epidermal permeability barrier. Absorption of ultrasound increases

temperature of the medium. Materials that possess higher ultrasound

absorption coefficients, such as bone, experience severe thermal effects

compared with muscle tissue, which has a lower absorption coefficient. The

increase in the temperature of the medium upon ultrasound exposure at a

given frequency varies directly with the ultrasound intensity and exposure

time. The absorption coefficient of a medium increases directly with

ultrasound frequency resulting in temperature increase. But in these days, the

importance of thermal effects as a mechanism of sonophoresis is decreasing.43

Boucaud et al reported that combined with the absence of skin changes after

heat treatment, indicate that the ultrasound- induced necrosis mechanism is a

nonthermal effect44. During our study, the difference of temprerature between

two groups were not observed (data not shown), so the thermal effects was

insufficient the differential effects of high-frequency and low-frequency

sonophoresis on epidermal permeability barrier.

The further study will be needed to prove that the different effects of

sonophoresis on different depth of skin, such as the epidermal and dermal

level, depend on the frequencies.

２４

V. CONCLUSION

In conclusion, both high and low-frequency sonophorhesis at energies that

do not provoke a barrier abnormality stimulate epidermal cytokine expression

and lamellar body secretion, which are linked to altered epidermal calcium

levels. Attenuation of an acoustic wave is inversely proportional to its

frequency, and thus the more the frequency increases, the less deeply the

ultrasound penetrates into and under the skin. Our results were shown to more

in increase the neutral lipid depositions in Nile red staining and stimulate the

expression of epidermal cytokine(ex. IL-1α and TNF-α) ,epidermal lipid

synthesis enzymes(ex. HMG-CoA reductase, fatty acid synthetase and serine

palmitoyl transferase) in real time RT-PCR and immunohistochemical

staining in high-frequency sonophoresis treatment group then low-frequency

sonophoresis treatment group. Lamellar body density was increased in stratum

granulosum shortly after the treatment of high and low frequency

sonophoresis and there were no siginificant differences except the prolonged

effects of low-frequency sonophoresis after 6hr. The further study will be

needed to prove that the differential effects of sonophoresis on different depth

of skin depend on the frequencies.

２５

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３０

Abstract (In Korean)Abstract (In Korean)Abstract (In Korean)Abstract (In Korean)

저주파저주파저주파저주파 초음파초음파초음파초음파 영동법과영동법과영동법과영동법과 고주파고주파고주파고주파 초음파초음파초음파초음파 영동법이영동법이영동법이영동법이 표피표피표피표피 투과투과투과투과

장벽에장벽에장벽에장벽에 미치는미치는미치는미치는 영향영향영향영향 비교비교비교비교

<지도교수 이승헌>

연세대학교 대학원 의학과

김현정

초음파 영동법(sonophoresis)이란 다양한 분자에 대한 피부 투과를

향상시키기 위한 초음파의 사용법을 의미한다. 주파수를 기준으로

고주파 초음파 영동법(1-3MHz)과 저주파 초음파 영동법(20-

100KHz)으로 나누게 된다. 초음파 영동법은 피부 장벽을 통과하여

경피적으로 약물을 투과시키는 전달 체계이므로 피부 장벽에

미치는 영향을 고려해보아야한다. 기존의 연구에 의하면 고주파

초음파의 피부 장벽에 미치는 영향은 첫째, 형태학적인 관점에서

보면, 각질 세포 간격이 불규칙하게 넓어지는 현상과 함께 각질

세포 사이의 지질의 다층막 구조가 없어지고 electron dense 또는

lucent한 무정형의 물질로 채워지며. 확장된 lacunae, cavitation이

관찰된다. 둘째, 경피 수분 손실의 변화 없이 즉, 피부 장벽 기능의

직접적인 손상이 없이 표피 내의 칼슘 농도의 변화 및 칼슘 이온

기울기의 변화를 유발하고 이를 신호로 층판 소체의 분비 및

지질의 합성을 증가시킨다. 또한 IL-1α, IL-6, TNF-α, TGF-β 같은

사이토카인의 분비가 촉진됨으로써 피부 장벽 회복 기전의 과정을

유도한다. 저주파 초음파 영동법은 최근 고주파 초음파 영동법에

비해 약물 투과도가 더 뛰어난 것으로 알려져 있으며 그

기전으로는 공동화 현상, 열 효과, 대류 전달 유발, 기계적 효과,

３１

porous pathway 등이 제시되고 있다. 하지만 이로 인한 효과에 비해

실제 피부 장벽에 미치는 영향에 대한 연구는 충분히 이루어지지

못하였다.

본 연구에서는 저주파 초음파 영동법에 의한 피부의 구조적인

변화와 피부 장벽 회복 기전에 미치는 영향을 고주파 초음파

영동법과 비교 관찰 해보고자 한다. 초음파의 투과력은 주파수에

반비례 하므로 주파수가 증가할수록 역상관적으로 투과력은

감소하게 된다. 이러한 이론에 근거하면 고주파 초음파 영동법이

저주파 초음파 영동법에 비해 표피 투과 장벽에 미치는 영향이

더욱 클 것으로 생각된다. 실험 결과 두 방법 모두 TEWL의 변화를

유발하지 않은 채 효과적으로 표피 칼슘 이온 기울기를 변화시키게

되어 표피 손상 후 장벽 회복을 유발 하게 하였다. 이러한 TEWL의

변화나 칼슘 이온 기울기의 변화는 두 방법에서 유의한 차이를

보이지는 않았다. 표피 장벽 회복 기전의 중요한 요소인 층판

소체의 밀도에서도 3시간까지는 유의한 차이를 보이지 않다가 그

이후 저주파 초음파 영동법에서 좀 더 지속적으로 밀도가 증가되어

있음을 관찰하였다. Nile red 염색으로 관찰한 표피 각질층에서 중성

지질의 분포는 15분 후부터 고주파 초음파 영동법에서 더 현저한

증가를 보였다. 면역 조직 화학 검사와 real time RT-PCR 결과 IL-

1α와 TNF-α의 증가도 고주파 초음파 영동법이 우세함을 알 수

있었으며 이는 지질 합성 효소 결과에서도 고주파 초음파 영동법의

효과가 저주파 초음파 영동법보다 크다는 것을 알 수 있었다.

따라서 이번 연구에서 초음파 영동법이 표피 투과 장벽에 미치는

영향은 주파수에 역상관 관계로 고주파 초음파 영동법이 저주파

초음파 영동법 보다 우세함을 알 수 있었다. 이러한 가설에

기인하여 향후 진피층에서 저주파 초음파 영동법과 고주파 초음파

영동법의 효과의 차이를 규명하는 연구를 진행할 예정이다.

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

핵심되는 말 : 고주파, 저주파 초음파 영동법, 표피 투과 장벽 회복