ORIGINAL ARTICLE
See related Commentary on page 4 and 6
Helium^Neon Laser Irradiation Stimulates Migration and
Proliferation in Melanocytes and Induces Repigmentation in
Segmental-Type Vitiligo
Hsin-Su Yu, Chieh-Shan Wu, Chia-Li Yu,n Ying-Hsien Kao, and Min-Hsi Chiou
Department of Dermatology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; and nDepartment of Medicine and Institute of
Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
Low-energy helium^neon lasers (632.8 nm) have been
employed in a variety of clinical treatments including
vitiligo management. Light-mediated reaction to low-
energy laser irradiation is referred to as biostimulation
rather than a thermal e¡ect. This study sought to deter-
mine the theoretical basis and clinical evidence for the
e¡ectiveness of helium^neon lasers in treating vitiligo.
Cultured keratinocytes and ¢broblasts were irradiated
with 0.5^1.5 J per cm2 helium
neon laser radiation.
The e¡ects of the helium^neon laser on melanocyte
growth and proliferation were investigated. The results
of this in vitro study revealed a signi¢cant increase in ba-
sic ¢broblast growth factor release from both keratino-
cytes and ¢broblasts and a signi¢cant increase in nerve
growth factor release from keratinocytes. Medium
from helium^neon laser irradiated keratinocytes
stimulated [3H]thymidine uptake and proliferation
of cultured melanocytes. Furthermore, melanocyte
migration was enhanced either directly by helium^neon
laser irradiation or indirectly by the medium derived
from helium^neon laser treated keratinocytes. Thirty
L
ow-energy laser is capable of producing an energy
density so low that any biologic alterations are the re-
sults of a direct irradiation e¡ect, not thermal events. In
this system, the temperature elevations in irradiated
tissues are limited to less than 0.11C^0.51C (Basford,
1989; Yu et al, 1994; Babapour et al, 1995). Unlike traditional
high-powered surgical lasers (their energy output can be up to
100 W), such as carbon dioxide, ruby, and neodymium^yt-
trium^aluminum^garnet (Nd-YAG) lasers, low-energy lasers are
compact, low cost devices with an output power measured in
milliwatts (Walsh, 1997). Recent studies demonstrated that low-
energy lasers are potential therapeutic instruments for rheuma-
toid arthritis management (Goldman et al, 1980), modulation
of wound healing (Lyon et al, 1987), postherpetic neuralgia
(Kemmotsu et al, 1991; Yaksich et al, 1993), and recovery of nerve
Manuscript received March 23, 2001; revised August 30, 2002; accepted
for publication September 21, 2002
Reprint requests to: Yu, Hsin-Su, M.D., Department of Dermatology,
College of Medicine, Kaohsiung Medical University, 100 Shih Chuan 1st
Road, Kaohsiung 807, Taiwan; Email: dermyu@kmu.edu.tw
Abbreviations: ET-1, endothelin-1; HGF, hepatocyte growth factor; SCF,
stem cell factor.
0022-202X/03/$15.00 Copyright r 2003 by The Society for Investigative Dermatology, Inc.

56
patients with segmental-type vitiligo on the head and/
or neck were enrolled in this study. Helium
neon laser
light was administered locally at 3.0 J per cm2 with
point stimulation once or twice weekly. The percentage
of repigmented area was used for clinical evaluation of
e¡ectiveness. After an average of 16 treatment sessions,
initial repigmentation was noticed. Marked repigmen-
tation (450%) was observed in 60% of patients with
successive treatments. Basic ¢broblast growth factor is
a putative melanocyte growth factor, whereas nerve
growth factor is a paracrine factor for melanocyte
survival in the skin. Both nerve growth factor and basic
¢broblast growth factor stimulate melanocyte migra-
tion. It is reasonable to propose that helium
neon laser
irradiation clearly stimulates melanocyte migration
and proliferation and mitogen release for melanocyte
growth and may also rescue damaged melanocytes,
therefore providing a microenvironment for inducing
repigmentation in vitiligo. Key words: basic ¢broblast
growth factor/biostimulation/melanocyte/nerve growth factor/
phototherapy. J Invest Dermatol 120:56 ^64, 2003
injury (Khullar et al, 1996). The continuous wave helium^neon
(He^Ne) laser (632.8 nm) has been employed most commonly
for these clinical treatments (P˛ntinen, 1992a). Recently in vitro
studies have shown that low-energy lasers induce biostimulatory
e¡ects on cultured cells. Low-energy lasers induced macrophages
to release factors that stimulate ¢broblast proliferation (Young
et al, 1989). An increase in production of pro-collagen, collagen,
basic ¢broblast growth factors (bFGF) and proliferation of ¢bro-
blasts after exposure to low-energy laser irradiation were noticed
(Abergel et al, 1987; Yu et al, 1994). He^Ne laser treatment stimu-
lated interleukin-8 and interleukin-1a release from cultured ker-
atinocytes and induced an increase in the rate of keratinocyte
migration and proliferation (Haas et al, 1990; Yu et al, 1996).
Functional melanocytes in patients with vitiligo vulgaris disap-
pear from the involved skin by a mechanism(s) that has yet to be
identi¢ed. Segmental-type vitiligo is associated with a dysfunc-
tion of the sympathetic nerves in the a¡ected skin (Koga, 1977;
Wu et al, 2000). This type of vitiligo is relatively resistant to con-
ventional therapies such as psoralens and ultraviolet-A (UVA)
light (PUVA), UVB, and systemic and topical steroids (El Mofoy
and El Mofoy, 1980; Koga and Tango, 1988; Hann and Lee, 1996).
There is evidence that He^Ne laser irradiation leads to biologic
e¡ects such as an improvement in nerve injury (Rochkind et al,
VOL. 120, NO. 1 JANUARY 2003
1989; Khullar et al, 1996). Furthermore, a report from Russia
(Mandel, 1984) and a preliminary report from us (Yu, 2000)
reveal that low-energy laser treatment induces repigmentation
responses in patients with vitiligo. Therefore, the evidence of the
He^Ne laser as a potentially useful instrument in the treatment of
segmental-type vitiligo validated our project. The purposes of
this study were to clarify the biologic basis of He^Ne laser e¡ects
on melanocyte growth and to con¢rm the e¡ectiveness of
He^Ne laser therapy on segmental-type vitiligo repigmentation.
MATERIALS AND METHODS
Cell culture and He^Ne laser irradiation Healthy adult foreskins
were the source of keratinocytes, ¢broblasts, and melanocytes.
Keratinocytes, ¢broblasts, and melanocytes were isolated and cultured as
previously described (Jones, 1990; Yu et al, 1993). Keratinocytes were
maintained in keratinocyte-SFM complete medium, supplemented with 2
ng per ml of recombinant human epidermal growth factor and 25 mg per
ml of bovine pituitary extract (Gibco BRL, Gaithersburg, MD).
Fibroblasts were maintained in Dulbecco’s minimal essential medium
supplemented with 10% fetal bovine serum, 100 U per ml penicillin G,
and 100 mg per ml streptomycin sulfate (Gibco). Melanocytes were
maintained in melanocyte medium kit (Sigma, St. Louis, MO), which
contains growth supplements. The second or third passage of these three
cells were used in the following experiments. The method for He^Ne
laser irradiation was described in our previous study (Yu et al, 1996). The
He^Ne laser used (Lasotronic MED-1000, Lasotronic, Switzerland) had
an output of 10 mW with a diverging lens that delivered 7.0 mW (as
measured by a power meter, POW-105, Lasotronic) to a platform 17 cm
under the lens where the dishes were placed. The cultured cells were
rinsed with phosphate-bu¡ered saline (PBS) and then irradiated in PBS to
minimize the loss of laser energy through absorption by colored culture
medium. All irradiation experiments were repeated in triplicate and
all dishes within an experiment (including controls) were maintained in
PBS at room temperature and atmosphere during the period of
experimentation.
Measurement of melanocyte mitogens and growth factors from
culture supernatant Cultured keratinocytes and ¢broblasts were
seeded in dishes (3.5 cm in diameter) with a density of 5 105 cells and
incubated overnight. The plating e⁄ciency was higher than 90%. Then
cells were irradiated with 0, 0.5, 1.0, or 1.5 J per cm2 of He^Ne laser
radiation. Melanocyte mitogens and growth factors in the culture
supernatant were assayed by commercially available enzyme-linked
immunosorbent assay (ELISA) kits according to the manufacturer’s
speci¢cations. The measurements included nerve growth factor (NGF)
(Promega, Madison, WI), bFGF, stem cell factor (SCF), hepatocyte
growth factor (HGF), and endothelin-1 (ET-1) (R&D Systems,
Minneapolis, MN).
Total RNA extraction and reverse transcription polymerase chain
reaction (RT-PCR) Total RNA was extracted from keratinocytes or
¢broblasts using the TRIzol method (Gibco) and processed as
recommended by the manufacturer. Single-stranded cDNA was reverse
transcribed from total cellular RNA using the BcaBEST RNA PCR kit
(Takara Shuzo, Kyoto, Japan) (Frohman et al, 1988). For ampli¢cation of
b-actin, NGF, and bFGF, the following primer pairs were synthesized: b-
actin (sense) 50 -TCC TGT GGC ATC CAC GAA ACT-30 and (antisense)
50 -GAA GCA TTT GCG GTG GAC GAT-30; NGF (sense) 50 -GGT GGT
GCT GCC CCC TTC AA-30 and (antisense) 50 -CAA AGG TGT GAG
TCG TGG TA-30 (Nilsson et al, 1997); and bFGF (sense) 50 -AGA GAG
AGG AGT TGT GCT-30 and (antisense) 50 -GGT CCT GTT TTG GAT
CCA-30 (Tripathi et al, 1996). The annealing temperatures for the above-
mentioned primer pairs were 601C, 551C, and 501C, respectively. The
pro¢le of the ampli¢cation cycle was as follows: 30 s melting at 941C, 45 s
at annealing temperature, and 60 s extending at 721C. PCR products were
titrated to establish standard curves for documenting linearity and
permitting semiquantitative analysis of density. The preliminary test for
conditioning PCR cycle number determined that the PCR product
reached the saturation point after 33 cycles. Therefore, 30 cycles was
chosen for gene ampli¢cation. The sizes of the ampli¢ed fragments were
314 bp for b-actin, 302 bp for NGF, and 222 bp for bFGF. The PCR
products were electrophoresed through 1.8% agarose gels, stained with
ethidium bromide, and further visualized by UV illumination. The
visualized gels were recorded and analyzed on a digital imaging system
(Alpha Imager 2000, Alpha Innotech, San Leandro, CA). Levels of gene
He^Ne LASER INDUCES PIGMENTATION IN VITILIGO 57
expression were expressed as the integrated density value of PCR
products normalized to that of the b-actin in the same sample.
Melanocyte migration assay CELLocate (Eppendorf, Hamburg,
Germany) slips were coated with 10 mg per ml collagen type I (Roche,
Mannheim, Germany) and incubated at 371C for 1 h. Melanocytes were
seeded on CELLocate slips in a 24 -well culture plate at a density of
1 104 cells and incubated for 2 h until attachment. The cells were
irradiated with He^Ne laser (1.0 J per cm2) or treated with supernatant
(10% in ¢nal concentration) derived from He^Ne laser (1.0 J per cm2)
irradiated keratinocytes. For the blocking test, 1 mg per ml of neutralizing
antibody against bFGF (Ab-3, Oncogene Research Products, Boston, MA)
or NGF (Chemicon, Temecula, NJ) was added to the supernatants to
clarify the functional importance of these released growth factors in
melanocyte migration. Then microphotographs were taken at indicated
time points within 24 h and 15^20 cells for each group were selected to
measure the migrating distances (Horikawa et al, 1995).
[3H]thymidine uptake in treated melanocytes Melanocytes were
seeded in 24 -well plates with a density of 4  104 cells per well and
cultured for 24 h as previously described. Melanocytes were treated either
by He^Ne laser (1.0 J per cm2) irradiation or with keratinocyte medium
obtained from He^Ne laser (1.0 J per cm2) irradiated or sham-irradiated
keratinocytes (10% in ¢nal concentration). Forty-eight hours later, the
cells were labeled with 1.0 mCi [methyl-3H]thymidine (Amersham
Pharmacia Biotech, Buckinghamshire, U.K.) per ml for 6 h. After
washing three times with PBS, the cells were lyzed with 2 N NaOH and
then neutralized with 2 N HCl. Acid-insoluble material was precipitated
with four volumes of 10% trichloroacetic acid, collected on glass ¢lters,
washed three times with 10% trichloroacetic acid and once with ethanol,
and then dried. The radioactivity on the ¢lters was determined in a liquid
scintillation counter (Imokawa et al, 1992).
Assessment of melanin content in melanocytes Melanocytes (1 106
cell) were plated in a 6 cm dish and incubated overnight. Then the cells
were treated with He^Ne laser irradiation or cultured with irradiated
conditioned medium (10% in ¢nal concentration) as described previously
for 72 h. The treated cells were detached by trypsin^ethylenediamine
tetraacetic acid solution. Cell suspension was centrifuged, and the pellet
was dissolved in 1 N NaOH. Melanin concentration was assessed by
spectrophotometry (Hu et al, 1995). Optical density (OD) at 475 nm was
measured and compared with a standard curve obtained from synthetic
melanin (Sigma). Melanin content was expressed as picograms per cell.
Melanocyte proliferation measured by mitochondrial enzymatic
activity assay A colorimetric method for determining the activity of
mitochondrial enzymes was used to detect melanocyte proliferation. The
CellTiter cell proliferation assay kit was purchased from Promega.
Melanocytes were seeded on 96 -well plates with a density of 3 103 cells
per well and incubated overnight. Then melanocyte growth supplements
were deprived of culture medium for 24 h. The cells were treated with
He^Ne laser (1.0 J per cm2) irradiation or keratinocyte conditioned
medium collected from laser-irradiated or nonirradiated keratinocytes
(10% in ¢nal concentration) and incubated for 48 h. Ten microliters
of CellTiter reagent containing tetrazolium compound and elec-
tron coupling reagents were added into each well to be catalyzed
by mitochondrial dehydrogenase enzymes in metabolically active
melanocytes. The plates were incubated for 4 h and the colorimetric
absorbance was recorded at 490 nm by microtiter plate reader (MRX-II,
Dynex Technology, Chantilly, VA) (Scudiero et al, 1988).
Patients Thirty patients with segmental-type vitiligo over the head and
neck were recruited in this study. Their background, treatment course, and
response are shown in Table I. There were 11 female and 19 male patients,
aged 8^43 y with mean age 22711 y. The lesions occurred in eight cases
when the patients were children (o13 y old), in seven cases when
adolescent (13^18 y old), and in 15 cases when adult (419 y old). The
duration of vitiligo ranged from 0.5 to 30 y (average 4.676.1 y). Twenty-
six patients were in a stable stage of the disease (lesion without active
progression in the previous 3 mo) (Moellmann et al, 1982), two patients
were in an active stage (lesion displaying progression in the previous 3
mo), and another two patients were in an unstable stage (lesion changed
from stable to active stage during the course of treatment). As for lesional
locations, there were 15 patients with the vitiliginous lesions on the cheek
(eight left side and seven right side), ¢ve patients on the neck (two left side
and three right side), seven on the chin (three left side and four right side),
and three on the left frontal area. Leukotrichia was noticed on lesions in the
case of 11 patients (36.7%). All the patients had been cleared of the
possibility of autoimmune diseases, such as thyroid diseases (Hashimoto’s
58 YU ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table I. Background data of patients

No. Sex/Age History of Stage Location Dermatomal Largest diameter of Leucotrichiaa First % of Total treatment
vitiligo (y) distribution vitiliginous lesion (cm) repigmentation repigmentation sessions

1 F/28 1 Stable L Chin V3b 2 N 8 100 20

2 F/30 0.6 Stable L Cheek V2 2 N 7 100 24
3 M/42 0.5 Stable R Cheek V2 1 N 6 100 16
4 M/39 1.5 Active R Frontal V1, V2 2.5 N 40 90 142
5 M/13 5 Stable L Cheek V1, V2 4.5 Y 16 78 132
6 M/30 8 Stable R Chin V3, C3b 3 N 18 78 137
7 F/30 1 Stable R Chin V3, C3 4 N 28 72 148
8 M/16 12 Stable L Cheek V2 4 Y 8 71 84
9 M/12 2 Stable R Chin V3, C3 3.5 N 10 65 139
10 M/15 4 Stable L Frontal V1 4 Y 15 62 120
11 F/19 5 Stable L Cheek V2 2.5 N 14 61 130
12 M/16 2 Stable L Neck C2, C3 4 Y 8 58 58
13 M/25 7 Stable R Neck V3, C2 2 Y 20 55 130c
14 F/8 1 Stable L Cheek V2 1 N 8 54 23c
15 M/20 8 Stable L Neck C3 4 Y 16 53 116
16 F/12 0.5 Stable L Cheek V2 1 N 18 52 64
17 F/16 3 Stable R Cheek V2 2 Y 34 52 134
18 M/13 1 Stable R Cheek V3 1.5 N 10 51 43
19 M/42 0.8 Unstable L Cheek V1, V2 2.5 Y 20 35 122d
20 M/12 4 Unstable R Cheek V1, V2 4 N 48 35 180
21 F/12 2 Stable L Chin V3 2.5 N 12 33 74
22 M/12 0.5 Stable R Chin V3 2 N 6 33 32
23 M/12 8 Stable L Frontal V1 4 Y 7 30 39c
24 M/10 3 Stable L Frontal V1 2.5 N 20 30 54
25 F/25 7 Stable R Neck V3, C3 3 N 14 30 105d
26 F/21 2 Stable R Cheek V2, V3 2 N 16 24 37
c,e
27 F/17 0.5 Stable R Neck V3, C2 2 N 15 23 100
d, f
28 M/43 30 Stable R Cheek V2 5 Y 0 68
f,g
29 M/32 0.5 Active L Cheek V2 4 N 0 42
e, f
30 M/40 16 Stable L Chin V3 3.5 Y 0 50
a
N, negative; Y, positive.
b
V, the ¢fth cranial nerve; C, the cervical branch of the vertebral column.
c
Discontinued the treatment due to schoolwork.
d
Discontinued the treatment due to busy job.
e
Discontinued the treatment due to distant residence.
f
Discontinued the treatment due to poor treatment response.
g
Discontinued the treatment for other compelling reasons.

thyroiditis and Graves’ disease), Addison’s disease, pernicious anemia,
insulin-dependent diabetes mellitus, and alopecia areata. All of them were
healthy except for one patient with acute nonlymphocytic leukemia for 1.5
y. None of the participants had received any medical treatment in the
preceding 3 mo. The study was approved by the Ethics Committee of the
Kaohsiung Medical University Hospital.
Treatment of vitiliginous lesions with He^Ne laser A continuous
wave He^Ne laser (OMNIPROBEt Laser Biostimulation System, Physio
Technology, Topeka, KS) with an average 1.0 mW power output was used
for treatment. It was designed for point stimulation, i.e., irradiation point
by point on the skin (P˛ntinen, 1992b; Allendorf et al, 1997). When the
He^Ne laser was used for the treatment of vitiligo, the probe was placed
directly on the vitiliginous lesion. According to the manufacturer’s
instructions, the calculation formula for He^Ne laser point stimulation is
used for a beam surface area of 0.01 cm2 (direct e¡ect) and assumes only
one application point per square centimeter of tissue. The balance of tissue
is untreated directly but receives a peripheral e¡ect, thus creating islands of
biologically active tissue in the area (indirect e¡ect) (Earle, 1985). The
number of treatment points depends on the size of the lesions. The power
output was measured with a power meter (POW-105, Lasotronic). The
irradiating £ux for each treatment point in our patients was 3.0 J per cm2
with a stimulation time of 30 s (Kovacs, 1981; Yu et al, 1996). The calculation
formula for treatment is
Ta ¼ ðEa=PavÞAt
where Ta is the treatment time for a given area, Ea is the millijoules of
energy required per square centimeter, Pav is the average laser power in
milliwatts, and At is the area to be treated in square centimeters. It is
0.01 cm2 in point stimulation per centimeter of treatment area.
All our vitiligo patients received He^Ne laser treatment once or twice a
week.
Evaluation of repigmentation Vitiliginous lesions in all patients were
traced and the square centimeters of involvement were counted on an
overlaid grid; the lesions were recorded regularly by photography before,
during, and after the He^Ne laser therapy. The percentage of
repigmentation after He^Ne laser treatment was de¢ned as follows: (area
of repigmented skin7area of vitiliginous lesion prior to He^Ne laser
therapy)  100% (Kao et al, 1995).
Statistical analysis The software of the SPSS system for Windows 10.0
version (SPSS, Chicago, IL) was used for statistical analysis. The results
were expressed as mean7standard deviation (mean7SD). The Student’s t
test was also used for statistical evaluation between control and
experimental groups in the in vitro study. The two-way ANOVA one
factor repeated method was used to analyze the results of the cell
migration assay. The Student’s t test for unpaired observations was used to
detect di¡erences of sex and leukotrichia in the He^Ne laser treatment of
segmental-type vitiligo patients. A p-value of o0.05 is considered to be
statistically signi¢cant. The one-way ANOVA method for various
independent groups was used to contrast the e¡ects of age, age of onset,
duration, stage, location, and the size of lesion in the He^Ne laser therapy
of the segmental-type vitiligo patients (signi¢cant level o0.05).
VOL. 120, NO. 1 JANUARY 2003
RESULTS
A signi¢cant increase in bFGF release by He^Ne laser treated
keratinocytes and ¢broblasts The assay for bFGF release from
cells was performed 30 min after He^Ne laser treatment. Under
the same experimental conditions, He^Ne laser irradiation
stimulated ¢broblasts to release a signi¢cantly higher quantity of
bFGF than it did keratinocytes. Under 0, 0.5, 1.0, and 1.5 J per cm2
He^Ne laser irradiation, the stimulatory e¡ect of bFGF in
keratinocytes and ¢broblasts was dose-responsive, i.e.,
85.91715.50 pg per ml, 362.04719.13 pg per ml, 398.71724.50
pg per ml, and 445.53727.72 pg per ml in keratinocytes and
160.92721.11 pg per ml, 512.92725.27 pg per ml, 558.69731.30
pg per ml, and 609.13735.66 pg per ml in ¢broblasts,
respectively (Fig 1). Results of RT-PCR revealed that He^Ne
laser irradiation was unable to induce bFGF mRNA expression
in either keratinocytes or ¢broblasts (data not shown).
A signi¢cant increase in NGF release from He^Ne laser
treated keratinocytes The assay for NGF release from cells
was performed 24 h after He^Ne laser treatment. A signi¢cant
increase in NGF release from keratinocytes was noticed in He^
Ne laser treated material compared with nonirradiated controls.
The stimulatory e¡ect of He^Ne laser treatment was dose-
dependent. Irradiations of 0, 0.5, 1.0, and 1.5 J per cm2 brought
about progressively greater stimulation of NGF release, i.e.
25.40711.20 pg per ml, 45.81720.10 pg per ml, 72.50717.22 pg
per ml, and 133.50723.50 pg per ml, respectively (Fig 2). The
stimulatory e¡ect of He^Ne laser treatment was also con¢rmed
on mRNA expression by RT-PCR (Fig 3). He^Ne laser
treatment showed no stimulatory e¡ect on NGF release by
¢broblasts (data not shown).
He^Ne laser treatment showed no signi¢cant stimulatory
e¡ects on SCF, HGF, and ET-1 release by keratinocytes or
¢broblasts.
Medium from He^Ne laser irradiated keratinocytes
signi¢cantly increases DNA synthesis and mitochondrial
enzymatic activity, but not melanin content, in
melanocytes To clarify the roles of direct laser irradiation and
laser-irradiated keratinocytes in stimulating DNA synthesis,
800
700
bFGF Conc. (pg/ml)
600
500
400
300
200
100
0 performed by time-lapse microphotography. A direct stimulatory
0 0.5 1.0 1.5
2 shown in Fig 4(A). The migration distances were 26.3721.6
Laser Dosage (J/cm )
Figure 1. E¡ects of He^Ne laser irradiation on bFGF release from
cultured human keratinocytes and ¢broblasts. Cultured human kera-
tinocytes and ¢broblasts were irradiated with 0, 0.5. 1.0, and 1.5 J per cm2 of
He^Ne laser radiation, respectively. The total amounts of bFGF released in
the supernatants (including during irradiation and 30 min after irradiation)
were measured with a commercially available bFGF ELISA kit. The stimu-
latory e¡ect on bFGF release from both keratinocytes (open bar) and ¢bro-
blasts (solid bar) by He^Ne laser irradiation showed a dose-responsive e¡ect.
He^Ne LASER INDUCES PIGMENTATION IN VITILIGO 59
180
160
NGF Conc. (pg/ml)
140
120
100
80
60
40
20
0
0 0.5 1.0 1.5
Laser Dosage (J/cm2)
Figure 2. Dose-dependent e¡ect of He^Ne laser irradiation on NGF
release from cultured human keratinocytes. Dishes of cultured human
keratinocytes were irradiated with 0, 0.5, 1.0, and 1.5 J per cm2 of He^Ne
laser radiation, respectively. At 24 h after irradiation, NGF concentrations
of the collected supernatants were measured with an NGF ELISA kit. The
pattern of NGF release by keratinocytes showed a dose-dependent e¡ect
produced by He^Ne laser irradiation.
mitochodrial enzyme activity, and melanin content in
melanocytes, cultured melanocytes were treated with 1.0 J per
cm2 laser irradiation or mock treatment. Additionally, they were
treated with supernatants either from keratinocytes without
treatment (blank media) or conditioned media from laser-
irradiated keratinocytes. The results are shown in Table II.
[3H]thymidine uptake of melanocytes treated with medium
from laser-irradiated keratinocytes increased signi¢cantly
(2181.07506.4 cpm) compared to normal control (1325.5747.4
cpm) (po0.05), whereas there was no di¡erence in radioactivity
counts between the laser irradiation group (987.97164.2 cpm)
and mock treatment (1085.37379.4 cpm). CellTiter assay
revealed that mitochondrial enzyme activity was signi¢cantly
higher in melanocytes treated with laser-irradiated keratinocyte
supernatant (0.4970.05 OD) than in the normal control
(0.2670.09 OD), but no di¡erence was detected between the
laser irradiation group (0.3070.02 OD) and the mock treatment
group (0.2970.02 OD). There were no di¡erences in melanin
contents between conditioned media and control media groups,
but laser irradiation slightly suppressed (but not statistically
signi¢cant) the melanin content. In contrast, He^Ne laser
irradiation showed no signi¢cant e¡ects on DNA synthesis,
mitochondrial enzymatic activity, and melanin content in
melanocytes (Table II).
Melanocyte migration was signi¢cantly stimulated by
both He^Ne laser irradiation and He^Ne laser treated
keratinocyte derived medium The melanocytes were seeded
on collagen type I coated CELLocate slips and treated either
with He^Ne laser irradiation or He^Ne laser treated
keratinocyte derived media as described in Materials and Methods.
Measurements of migration distance in treated melanocytes were
e¡ect of He^Ne laser radiation on melanocyte migration is
mm, 53.9729.6 mm, 76.5736.6 mm, 119.1743.1 mm, and
146.1745.8 mm at 3, 6, 9, 18, and 24 h after He^Ne laser (1.0 J per
cm2) irradiation, respectively. In contrast, in mock treatment the
migration distances were 19.2715.6 mm, 33.8719.6 mm, 50.2728.1
mm, 77.9726.9 mm, and 99.2730.3 mm at 3, 6, 9, 18, and 24 h after
treatment, respectively. A signi¢cant stimulatory e¡ect of He^Ne
laser irradiation on melanocyte migration was initiated at 6 h
(po0.01) and throughout 24 h monitoring compared to the
mock treatment groups. An indirect stimulatory e¡ect of laser
60 YU ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table II. E¡ects of media derived from laser-irradiated keratinocytes and He^Ne laser irradiation on DNA synthesis, mitochon-
drial enzymatic activity, and melanin content in cultured melanocytes
Control media Mock treatment Supernatants of irradiated keratinocytes He^Ne laser irradiation

[3H]thymidine uptake (cpm) 1325.5747.4 1085.37379.4 2181.07506.4n 987.97164.2

CellTiter assay (OD) 0.2670.09 0.2970.02 0.4970.05n 0.3070.02
Melanin content (pg per cell) 121.772.3 120.370.9 118.471.5 102.979.1
Cultured human melanocytes were seeded on a 24 -well plate at 4  104 cells per well for assessment of DNA synthesis. Melanocytes (1 106 cells) were plated in a 6 -
CM dish for assessment of melanin content. Melanocytes were seeded on a 96 -well plate at 3 103 cells per well for mitochondrial enzymatic activity assay. Melanocytes
were treated with media derived from He^Ne laser (1.0 J per cm2) irradiated keratinocytes (10% in ¢nal volume) and He^Ne laser irradiation (1.0 J per cm2).
n
po0.05 compared to normal controls and mock treatments (n ¼ 3).
OD, optical density.

Figure 3. Stimulatory e¡ect of He^Ne laser irradiation on NGF

gene expression in cultured human keratinocytes. Dishes of cultured
human keratinocytes were irradiated with an He^Ne laser as previously
described. At 4 h after irradiation, the cells were collected for total RNA
extraction and RT-PCR as described in Materials and Methods. The expres-
sion of NGF mRNA by keratinocytes was semiquantitatively analyzed by
normalization with b-actin mRNA expression. A stimulatory e¡ect was
observed in laser-irradiated groups. The ratios of NGF to b-actin in inte-
grated density value were 0.075 (0 J per cm2), 0.123 (0.5 J per cm2), 0.222 (1.0
J per cm2), and 0.378 (1.5 J per cm2), respectively. PCR product sizes of b-
actin and NGF were 314 bp and 302 bp. M, 100 bp ladder DNA marker.

treatment on melanocyte migration was demonstrated by the

addition of He^Ne laser treated keratinocyte culture supernatant
on melanocytes. The migration distances at 3, 6, 9, 18, and 24 h
after supernatant addition were 23.0716.6 mm, 54.0728.8 mm,
80.0731.0 mm, 127.2739.8 mm, and 158.8738.9 mm, whereas
those of normal controls were 25.0711.3 mm, 44.7720.0 mm,
64.7729.3 mm, 91.7733.5 mm, and 110.9736.3 mm, respectively
(Fig 4B). The p-value at 9 h was less than 0.05 and the p-values
after 18 h and 24 h treatments were less than 0.01 compared to
normal controls. He^Ne laser treated keratinocyte medium
induces a stimulatory e¡ect on melanocyte migration. No
signi¢cant blocking e¡ect in migration was observed when
neutralizing antibodies against bFGF and NGF were added; the
migration distances at 3, 6, 9, 18, and 24 h were 20.5712.2 mm
and 22.4719.0 mm, 66.0734.1 mm and 59.2718.5 mm, 89.5738.4
mm and 84.3716.3 mm, 114.7729.1 mm and 133.8721.4 mm, and
148.5719.3 mm and 169.1723.7 mm, respectively. The results for
the antibody blocking test revealed that bFGF and NGF in the
supernatant derived from He^Ne laser irradiated keratinocytes
have no functional signi¢cance in melanocyte migration in this
experimental system (Fig 4B).
He^Ne laser irradiation induces repigmentation in
vitiligo The He^Ne laser irradiation was applied once or twice
per week and the results are summarized in Table III. After an
average of 16710 therapeutic sessions, mostly perilesional
Figure 4. Direct and indirect e¡ects of He^Ne laser irradiation on
migration of cultured human melanocytes. Cultured human melano-
cytes were seeded on collagen type I coated Cellocate slips at 1 104 cells
per dish. Time-lapse photographs were taken at the indicated time points
and the migration distance (mm) was measured. (A) Melanocytes were trea-
ted with either mock irradiation or 1 J per cm2 He^Ne laser irradiation. (B)
Melanocytes were treated with cultured medium or 10% (vol/vol) super-
natants (derived from keratinocytes treated with 1 J per cm2 He^Ne laser
irradiation) with or without 1 mg per ml of neutralizing antibody (Ab)
against bFGF or NGF. npo0.05, nnpo0.01 compared to mock treatment
or control medium. Bars: 1 SD; n ¼ 20.
repigmentation was noticed. Three patients (10%) displayed
100% repigmentation after 2074 treatment sessions. A
maintenance treatment to prevent depigmentation was carried
out once or twice every month. After an average of 13775
treatment sessions, three of 30 patients (10%) showed 76%^99%
repigmentation response; 12 patients (40%) displayed 51%^75%
recovery after 99743 sessions; seven (23.3%) showed 26%^50%
after 87753 sessions; and two patients (6.7%) presented less than
25% improvement following 69745 treatment sessions. Three
VOL. 120, NO. 1 JANUARY 2003
Table III. E¡ectiveness of He^Ne laser treatment in 30 patients with segmental-type vitiligo
% of repigmentation No. (%) of patients
100% 3 (10%)
76^99% 3 (10%)
51^75% 12 (40%)
26^50% 7 (23.3%)
o25% 2 (6.7%)
0% 3 (10%)
Total 30 (100%)
patients showed no repigmentation response. One of them had
vitiligo for 16 y, the other for 30 y. The third one had it for only
6 mo, but in the past 1.5 y he had su¡ered from acute
nonlymphocytic leukemia. The repigmentation response after
He^Ne laser irradiation was found not to be correlated to sex,
age, stage of advancement, distribution, or poliosis of segmental
vitiligo in this study group. Size and duration of the vitiliginous
lesion was the most important factor a¡ecting the e¡ectiveness of
He^Ne laser treatment. Nine patients (30%) were lost from our
follow-up study due to schoolwork, busy jobs, distant residence,
poor treatment response, or other compelling reasons. There was
no obvious discomfort or side-e¡ects during or after the He^Ne
laser therapy.
In Fig 5 we show three medium sized typical cases of
segmental-type vitiligo in which repigmentation was induced by
He^Ne laser treatment. Case 1, a 39-y-old man, had had vitiligo
on the right forehead and paranasal area for 1.5 y. PUVA therapy
revealed a perilesional hyperpigmentation without improvement
of vitiligo (Fig 5, 1A). He had received no treatment 3 mo prior
to He^Ne laser therapy. After 142 therapy sessions, 90%
repigmentation was observed (Fig 5, 1B). In case 2 vitiliginous
lesions were located on the distribution of the ¢rst and second
branches of the left trigeminal nerve in a 13 -y-old boy; he had
had them for 5 y (Fig 5, 2A). Leukotrichia had been noticed on
the left eyebrow for about 4 y. After 16 sessions, an obvious
repigmentation response was found on the left lower cheek
lesion. After 132 treatment sessions 78% repigmentation was
seen (Fig 5, 2B). Case 3, a 12-y-old boy, had su¡ered from
depigmented lesions on his right chin and upper neck for 2 y
(Fig 5, 3A). After 14 sessions, obvious perilesional and
perifollicular repigmentation was noticed. After 139 treatment
sessions (Fig 5, 3B) 65% repigmentation response was found.
DISCUSSION
Functional melanocytes in the bulb and infundibulum of the hair
follicle are destroyed in vitiliginous skin, although inactive mela-
nocytes in the middle and lower parts of the follicle and the outer
root sheath are spared (Staricco, 1961; Cui et al, 1991; Norris et al,
1994). Melanocytes could be recruited from the outer root sheath
of the hair follicle to repigment vitiliginous skin through the
chronic action of various forms of therapy (Cui et al, 1991; Staric-
co and Miller-Milinska, 1962). Recovery of vitiligo is initiated by
the proliferation of the inactive melanocytes, followed by their
upward migration to the nearby epidermis to form perifollicular
pigment islands (Fitzpatrick, 1997). Melanocyte proliferation and
migration are controlled by the same mitogens but in di¡erent
ways. Melanocytes proliferate in response to three di¡erent classes
of mitogen: (i) leukotriene C4 (LTC4) or LTD4 (Morelli et al,
1989); (ii) ET-1 (Imokawa et al, 1992; Yohn et al, 1993); or (iii) one
of the receptor tyrosine kinase growth factors bFGF, SCF, HGF,
melanotropin (melanocyte stimulating hormone), epidermal
growth factor, and platelet-derived growth factor (Graeven and
Herlyn, 1992; Halaban et al, 1992a; 1992b; Halaban, 2000). Each
of the three major classes of melanocyte mitogen is capable of
inducing migration in melanocytes. Both keratinocytes and ¢-
He^Ne LASER INDUCES PIGMENTATION IN VITILIGO 61
First repigmentation (mean7SD) Total treatment (mean7SD)
771 2074
25713 13775
1679 99743
21715 87753
1671 69745
53713
broblasts, natural cellular neighbors of melanocytes in vivo, could
release these putative melanocyte growth factors (Hirobe, 1994).
In normal melanocytes the binding of ligands, such as bFGF,
SCF, HGF, and ET-1, to cognate receptors on melanocyte cause
phosphorylation of crucial tyrosine moieties. The binding results
in an intracellular signaling cascade that brings about the rapid
activation of the mitogen-activated protein kinase (MAPK) as
well as activation of ribosomal S6 kinases (RSKs) (B˛hm et al,
1995). These events cause rapid transcriptional induction of
selected genes such as c-fos (Hill and Treisman, 1992; Pawson,
1995; Moellmann and Halaban, 1998). In melanocytes, a single
growth factor is su⁄cient to trigger the MAPK cascade but is
not able to sustain melanocyte proliferation or viability. Activated
MAPK and RSKs become translocated into the nucleus and may
modify several transcription factors, notably cAMP-responsive-
element binding protein. At least two of the growth factors in
combination are necessary to induce melanocyte proliferation
(Moellmann and Halaban, 1998). Recent in vitro studies have
shown that low-energy laser radiation induces biostimulatory ef-
fects on cultured cells. The results of this study revealed that the
release of bFGF, but not its mRNA expression, from both
cultured keratinocytes and ¢broblasts is noticed immediately
after He^Ne laser irradiation. Because bFGF lacks a secretory se-
quence, it is presumably expressed on the surface of cells and
transferred to melanocytes by cell-to-cell contact (Halaban et al,
1988). Our ¢ndings suggest that the He^Ne laser induces im-
mediate bFGF dissociation from cell surfaces, but does not trigger
de novo synthesis. In this study, under identical experimental con-
ditions, He^Ne laser irradiation stimulated a signi¢cantly higher
bFGF release by ¢broblasts than by keratinocytes. He^Ne laser
irradiation could not stimulate keratinocytes or ¢broblasts to
release SCF, HGF, and ET-1. As He^Ne laser irradiation can
only stimulate one mitogen (i.e., bFGF) from cultured cells, the
possibility of the production of other melanocyte mitogens
from keratinocytes, ¢broblasts, and/or nerve endings (Toyoda
et al, 1999) induced by the same treatment needs to be further
investigated.
In this study, we demonstrated that low-energy He^Ne laser
radiation could enhance melanocyte migration by both direct ir-
radiation and indirect stimulation (by medium derived from irra-
diated keratinocytes). Furthermore, this medium, but not direct
irradiation, also stimulated DNA synthesis and cell proliferation
in cultured melanocytes. These results provide a theoretical basis
for the biostimulatory e¡ects of repigmentation by He^Ne laser.
The results of antibody blocking tests revealed that bFGF and
NGF in the supernatant derived from He^Ne laser irradiated ker-
atinocytes have no functional signi¢cance in melanocyte migra-
tion in this experimental system. Both bFGF and NGF stimulate
melanocyte migration; the stimulatory e¡ect was reported to be
induced by nanogram per milliliter levels in vitro (Yaar et al, 1991;
Pincelli and Yaar, 1997). In our experiment, however, the levels of
bFGF and NGF in the supernatant were picograms per milliliter;
this di¡erence provides an explanation for this result. Some other
factors must exist in the supernatant and warrant further investi-
gation. In contrast, melanin content in melanocytes revealed no
di¡erences between controls and media from irradiated keratino-
cytes. The possible explanations are (i) that this in vitro experi-
62 YU ET AL
Figure 5. He^Ne laser treatment induced repigmentation in three
typical patients with segmental-type vitiligo. Figure 5,1(AA) A 39-
y-old man had vitiligo on right forehead and paranasal area for 1.5 y. Fig-
ure 5,1B After 142 sessions of He^Ne laser irradiation, 90% repigmentation
was observed. Figure 5,2(AA) A 13 -y-old boy had vitiligo on left eyebrow
and cheek areas for 5 y. Leukotrichia was noticed on the left eyebrow for
about 4 y. Figure 5,2(B) 78% repigmentation was observed after 132 ses-
sions of treatment. Figure 5,3(AA) A 12-y-old boy had depigmented le-
sions on right chin and upper neck for 2 y. Figure 5,3(B) 65%
repigmentation was found after 139 sessions of treatment.
mental model is not the optimal condition for melanin synthesis
(di¡erent from the micro milieu around the melanocytes in vivo);
(ii) the experimental duration was not enough for melanin synth-
esis in vitro; (iii) He^Ne laser radiation is not able to stimulate
melanin synthesis in cultured melanocytes; and (iv) bFGF sup-
presses melanogenesis (Bradford et al, 1994).
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Low-energy He^Ne laser radiation does induce marked photo-
protective, antioxidant, and photoreparative e¡ects in animals
(Mandel, 1984). Melanocyte destruction and repopulation can be
found in the perilesional sites of vitiligo (Norris et al, 1994; Fala-
bella, 1997). In this study, both perilesional and perifollicular re-
pigmentation were noticed in vitiligo after He^Ne laser
treatment. These results provide the evidence that He^Ne laser
radiation exhibits a biostimulatory e¡ect for both perilesional
and perifollicular melanocytes in vitiligo. Very recently Schwartz
et al. (2000) demonstrated that He-Ne laser irradiation upregu-
lated NGF mRNA levels in skeletal Muscle cultures. Our in vitro
studies revealed that He^Ne laser irradiation stimulated an in-
crease in NGF release from cultured keratinocytes and its gene
expression, but not in ¢broblasts. NGF can protect melanocytes
from UV-induced apoptosis by upregulating BCL-2 levels in the
cells (Zhai et al, 1996). NGF is a major paracrine maintenance fac-
tor for melanocyte survival in skin (Yaar et al, 1991). An increase
in NGF production induced by He^Ne laser treatment provides
an explanation for the photoprotective e¡ect and initial perile-
sional repigmentation in segmental-type vitiligo.
Segmental-type vitiligo occurs in asymmetric distribution;
and because of its earlier onset, recalcitrant course, and decreased
association with autoimmune diseases, it is considered a special
type of vitiligo (Koga and Tango, 1988; Kovacs, 1998). This type
of vitiligo showed poor response to PUVA, UVB, and systemic
and topical steroid treatment (at least in oriental patients) (Koga,
1977; EL Mofoy and EL Mofoy, 1980; Hann and Lee, 1996;
Grimes, 1997), although these therapies could inhibit the spread
of old lesions and induced repigmentation of new lesions of viti-
ligo, especially on the face (Hann and Lee, 1996). Twenty to 40
PUVA treatments are generally required before any repigmenta-
tion occurs (Plott and Wagner, 1990). In this study, repigmentation
was noticed after an average of 16 He^Ne laser therapy sessions.
Mandel et al (1997) reported that after 6^8 mo of low-energy
He^Ne laser therapy marked repigmentation was observed in
63.9% and some follicular repigmentation in 34.4% of 18 vitiligo
patients. In our study group, 60% of patients (18 in 30) presented
more than 50% repigmentation and three patients (10%) showed
100% recovery. For treatment control, 20 previously treated pa-
tients with segmental-type vitiligo over head and neck were ree-
valuated. They were age and sex matched patients (data not
shown) with He^Ne laser treated patients in this study. All of
them received topical PUVA therapy three times a week. After
an average of 1975 therapeutic sessions, initial repigmentation
was mostly perilesional and/or perifollicular. Eleven of 20 (55%)
patients showed a better than 50% repigmentation response.
Recent investigations reported 63% e¡ectiveness for UVB
(Westerhof and Nieuweboer-Krobotova, 1997), 45% for topical
PUVA, 63% for oral PUVA, and 44% for topical steroid therapy
in vitiligo treatment (including all types of vitiligo) (Plott and
Wagner, 1990). Our results con¢rm that He^Ne laser irradiation is
e¡ective in treating even segmental-type vitiligo. The e¡ectiveness
of He^Ne laser treatment was at least as good as that of conven-
tional therapies for vitiligo. Sex, age, stage of advancement, distri-
bution, and poliosis of vitiligo were not found to be statistically
related to the e¡ectiveness of He^Ne laser irradiation treatment in
our patients. It had been reported that the lesional duration and
size a¡ect the e¡ectiveness of the treatment to a great extent
(Scherschun et al, 2001). The lesional size and duration was found
to be negatively correlated to repigmentation response in our pa-
tients, however (though it was not statistically signi¢cant). As one
patient with leukemia revealed no response to treatment, the role
of malignancy in repigmentation needs to be further studied. The
laser apparatus used in this study belonged to class 2 lasers, which
are regarded as safe, although one should not stare directly into the
beam. No obvious harmful e¡ects were noticed in our treated
patients or reported in the literature (P˛ntinen, 1992c).
This study provides a theoretical basis and clinical evidence for
the e¡ectiveness of He^Ne lasers in treating vitiligo. The e¡ec-
tiveness of He^Ne laser treatment is at least as good as that
of the conventional therapies for vitiligo. Low-energy laser
VOL. 120, NO. 1 JANUARY 2003
irradiation is an easier treatment to apply, less expensive, and has
no harmful side-e¡ects compared to most conventional therapies.
The He^Ne laser is a potentially useful therapeutic instrument
for vitiligo management.
This study was supported by the Chen, Jieh-Chen Scholarship Foundation, Tainan,
Taiwan.
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