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Histomorphological and Angiogenic Analyzes of Skin Epithelium After Low Laser Irradiation in Hairless Mice.

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THE ANATOMICAL RECORD 294:1592–1600 (2011)
Histomorphological and Angiogenic
Analyzes of Skin Epithelium After Low
Laser Irradiation in Hairless Mice
JULIANE CAROLINE LEÃO, JOÃO PAULO MARDEGAN ISSA,
DIMITRIUS LEONARDO PITOL, ELLEN CAMARGO RIZZI,
FERNANDO JOSÉ DIAS, SELMA SIÉSSERE,
SIMONE CECÍLIO HALLAK REGALO, AND MAMIE MIZUSAKI IYOMASA*
Department of Morphology, Stomatology and Physiology, School of Dentistry at
Ribeirão Preto, University of São Paulo, Brazil
ABSTRACT
It is not well-understood how low-laser therapy affects the skin of the
applied area. This study analyzes skin of the masseteric region of mice
from the HRS/J strain after three different application regimens (three,
six or ten applications per regimen) of low intensity laser at 20 J/cm2 and
40 mW for 20 sec on alternate days. Three experimental groups according
to the number of laser applications (three, six or ten) and three control
groups (N ¼ 5 animals for each group) were used. On the third day after
the last irradiation, all animals were sacrificed and the skin was removed
and processed to analyze the relative occupation of the test area by each
epithelial layer and the aspects of neovascularization. Data were submitted to statistical analyzes. The irradiated groups compared to their respective controls at each period of time, showed no significant difference
in relative occupation of the test area by the layers and epithelium areas
for three and six applications, but for ten applications, a significant
decrease (P < 0.05) in the basal and granulosum layers, and epithelium
areas were found. From the comparisons of the three irradiated groups
together, the group with six laser applications showed statistical difference (P < 0.05) in total epithelium and on the layers. Vascular endothelial growth factor (VEGF) and VEGFR-2 immunoreactivities were similar
for the control and irradiated groups. Results suggested a biostimulatory
effect with low risks associated with superficial tissues, when the treatment aims the deeper layers after six applications. Anat Rec, 294:1592–
C 2011 Wiley-Liss, Inc.
1600, 2011. V
Key words: laser; skin; epithelium; light microscopy; endothelial growth factor
INTRODUCTION
In dentistry, lasers are widely used for treatment of
patients with temporomandibular disorders, which
manifest problems in joint structures of the masticatory muscles and results in radiating pain of the face,
neck, and shoulders (Fricton, 2004). Laser treatment
has been successful due to its antiinflammatory effect
(Reis et al., 2008) its analgesic and muscle relaxant
actions (Núñez et al., 2006), its fatigue reduction during tetanic contractions (Lopes-Martins et al., 2006),
and its promotion of a larger bite force and decrease in
orofacial pain (De Medeiros et al., 2005; Emshoff
C 2011 WILEY-LISS, INC.
V
Grant sponsor: FAPESP (Fundação de Amparo à Pesquisa do
Estado de São Paulo); Grant number: 2007/54385-0.
*Correspondence to: Prof. Dr. Mamie Mizusaki Iyomasa, Faculdade de Odontologia de Ribeirão Preto, USP, Departamento
de Morfologia, Estomatologia e Fisiologia, Avenida do Café, s/n,
Bairro, Monte Alegre, 14040-904, Ribeirão Preto, SP. Fax: 55 16
36024794. E-mail: mamieiyo@forp.usp.br
Received 21 September 2010; Accepted 14 June 2011
DOI 10.1002/ar.21451
Published online 1 August 2011 in Wiley Online Library
(wileyonlinelibrary.com).
ANALYZES OF THE EPITHELIUM USING LASER IN HRS/J
et al., 2008). Despite thestudies demonstrating laser
therapy efficacy, the biological effects on target tissues
are not well-understood.
According to Pontinen (1992), electromagnetic radiation, known as infrared radiation, has a longer wavelength than red light, which makes it invisible.
Currently, nonsurgical lasers use a large portion of the
visible and invisible spectrum, utilizing a wavelength
range of 700–980 nm with pulsed or continuous wave
emission (Pontinen, 1992; Genovese, 2000; Nicola and
Nicola, 2002) at powers that range up to hundreds of
milliwatts. Consequently, variations in the parameters
used have been reported in the current literature. For
example, helium–neon irradiation, with an energy density up to 7.2 J/cm2, does not have a deleterious effect
on keratinocyte differentiation, which is necessary for
functional epidermis development (Rood et al., 1992).
However, the low level laser therapy (LLLT) at 15 mW
of power and a dose of 3.8 J/cm2 for 15 sec induced apoptosis during the tissue healing process after three applications (Rocha Júnior et al., 2009). Additionally, the
laser may be more harmful at 585 nm than at 595 nm
in the vascular response, as shown using normal human
skin ‘‘in vivo’’ (Pikkula et al., 2005). Variations in the
type of apparel, semiconductor medium, wavelength, intensity, exposure time, and treatment duration used
make the comparison of different published studies very
difficult.
According to Karu et al. (1995, 2008), each wavelength has different types of interactions, depending on
the specific target tissue. Muscular tissues and joints
are located deeper than epithelial layers of the skin,
and each presents with different characteristics. The
current literature presents studies about laser effects
on the healing process of muscle and connective tissues
in experimental animals (Iyomasa et al., 2009), but
there are few studies about the effect of laser treatment
on the surface tissues when laser treatment aims to
reach deeper layers. Based on increased laser intensities used for muscular therapies and reports relating
that lower doses of phototherapy increase proliferation
and cell viability (Hawkins and Abrahamse, 2006), we
hypothesize that laser therapy for muscle pain treatment may also affect skin epithelium of the treated
area.
At present, there are increased practice of phototherapy in medicine and dentistry, but there are a relative
small number of basic researches of LPLI. Currently,
the wavelengths, dosage schedules, and appropriate
conditions of laser irradiation are not well established
(Gao and Xing, 2009), and the literature have demonstrated the need for further studies in animal models
with similar characteristics to human skin for better
laser standardization application (Posten et al., 2005;
Winstanley and Uebelhoer, 2008). However, phototherapy is a possible alternative treatment that may be
used in many cases to minimize the frequency of invasive procedures currently used (Wu and Wong, 2008).
Thus, to facilitate the physician and dentist to match
optimally the laser in clinical practices, this study analyzes the effects of laser treatment on masseteric skin
under various application conditions in the HRS/J
mouse strain with the laser density usually indicated
for human muscular pain and temporomandibular disorder therapy.
1593
MATERIALS AND METHODS
Laser
The Twin Laser—Mm Optics (São Carlos, São Paulo,
Brazil), device was used at 780 nm with a gallium–
aluminum–arsenide semiconductor in the Department of
Morphology, Stomatology and Physiology at the Faculty
of Dentistry in Ribeirão Preto.
Animals
Three-month old male mice of the HRS/J strain (hairless),
weighing 35 g, were obtained from the vivarium of the Faculty of Dentistry of Ribeirão Preto—University of São Paulo
and randomly divided into groups according to the number
of laser applications (three, six, and ten). Each group consisted of control (N ¼ 5) and irradiated (N ¼ 5) mice. Animals were maintained in polyethylene boxes under
controlled room temperature conditions between 24 C and
25 C and 12 hr of daylight. They received food and water
‘‘ad libitum’’. All procedures of this study were approved by
the Local Ethics Committee (Number: 7.1.879.53.1) in accordance with international laws of animal use.
Treatment of Animals
Mice from the experimental groups were anesthetized
with halothane and irradiated by laser at an energy density equivalent to 20 J/cm2 (Continous wave, 40 mW, 20
sec, spot area 0.04 cm2) on skin of the middle region of
the left masseter muscle every other day (Meireles,
2005). The control group also received the same type of
treatment, but with no irradiation. Seventy-two hours
after the last application (Maciel, 2006), animals were
anesthetized with intramuscular (i.m.) injections of xylazine (10 mg/Kg) and ketamine (75 mg/Kg) and then perfused with buffered formalin for posterior skin resection
of the masseteric region. Skin fragments were immersed
in 10% formaldehyde and histologically processed for
paraffin sections 6 lm of thick.
Histological and Immunohistochemical
Procedures
Histological sections were stained by hematoxylin and
eosin (HE) and photographed at 100 using a light
microscope with the aid of the Leica IM50 program connected to a Leica DC 300F camera, which was adapted
for use with a Leica DMLB2 microscope.
For quantitative analyzes of the relative areas of the
different tissue layers (basal, spinous, granulosum, and
keratinous) in histological sections, a point-counting system was used. Using public domain Image J software (US
National Institutes of Health), a system composed by dots
on semicircles was layered over the histological image
and points over the regions of interest were counted. For
each animal, 10 microscopic fields with 957 points were
quantified and the tissue areas were calculated as a percent of the total test area (Mandarim-de-Lacerda, 1995).
Data were analyzed by two statistical tests: t-test of irradiated groups versus controls at each period of time
(Table 1) and analysis of variance (ANOVA) with post hoc
test to compare the irradiated groups within the three
different application times (Table 2). The SPSS software
version 17.0 for Windows was used (SPSS Inc., Chicago,
IL) at (P < 0.05).
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LEÃO ET AL.
TABLE 1. Relative occupation of the test area for the layers: basal, spinous, granulosum, and keratinous
in Control and Experimental Groups (t-test P < 0.05)
Applications
Layers
Group
3 (%)
t-test
6 (%)
t-test
10 (%)
t-test
Basal
Control
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
Control
Experimental
7.31
7.47
10.46
9.54
4.39
3.96
6.48
5.65
28.62
26.62
0.50
7.89
8.15
8.99
9.19
4.80
4.65
10.24
10.28
31.93
32.27
0.30
8.05
7.37
9.87
8.87
6.04
3.91
8.82
8.32
32.78
28.48
0.001
Spinous
Granulosum
Keratinous
Epithelium
0.36
0.33
0.46
0.21
TABLE 2. Mean and standard error of the
points from layers in the experimental groups after
three, six, and ten laser applications
Applications
3
6
10
3
6
10
3
6
10
3
6
10
3
6
10
Layer
P
Layer basal
0.001
Layer spinous
0.670
Layer granulosum
0.050
Layer keratinous
0.001
Epithelium
0.001
Mean
(%)
Standard
error
7.47
8.14
7.37
9.53
9.19
8.87
3.96
4.65
3.91
5.64
10.28
8.32
26.62
32.27
28.47
0.16
0.18
0.14
0.57
0.49
0.50
0.28
0.21
0.20
0.58
0.67
0.73
1.05
1.15
1.07
Data from different sessions numbers of laser application in
each layer were analyzed by ANOVA (P < 0.05).
Vascular Endothelial Growth Factor (VEGF)
and Vascular Endothelial Growth Factor
Receptor 2 (VEGFR-2)
Slides containing skin sections were deparaffinized
and hydrated in descending alcohol concentrations.
For antigen retrieval, slides were washed by immersion in phosphate buffer solution (PBS) in a water
bath at 90 C for 30 min. To inhibit nonspecific binding, slides were incubated with PBS-bovine serum albumin (BSA) (2%) in an acrylic tub. The slides were
then incubated with primary antibody diluted in PBS–
BSA (2%) for 2 hr. After washing, endogenous peroxidase activity was blocked by immersion in a glass
containing 30% H2O2. Slides were then covered with
drops of yellow link DAKO, transferred to an acrylic
tub and then covered with link red for 30 min. After
washing and drying each slide, sections were covered
by revealed solution (DAKO kit), counter-stained with
hematoxylin and mounted in Entellan. Images were
captured using a Leica IM50 program in a computer
connected to a Leica DC 300F camera and adapted to
the Leica microscope DMLB2.
0.76
0.65
0.97
0.85
0.12
0.00
0.55
0.003
RESULTS
Three Laser Applications
In the control group, the stratified squamous epithelium occupied an area of 28.62% of the test area. Relative occupation of the test area for the layers was: basal
layer 7.31%, spinous layer 10.46%, granulosum layer
4.39%, and keratinous layer 6.48% (Table. 1). Keratinocytes were evident with a clear cytoplasm located mainly
in the basal layer. Superficial dermis was composed of
connective tissue, nuclei with varied cells, collagen
fibers, and blood capillaries (Fig. 1A). VEGF expression
was identified diffusely throughout the epidermis and
hair follicles (Fig. 3A), whereas VEGFR-2 expression
was observed in keratinocytes and endothelial cells (Fig.
4A). Figure 2A is the negative control for VEGF, and
Fig. 2B is the negative control for VEGFR-2.
In the irradiated group that received three laser applications, no significant difference was found 26.62% (P >
0.05) compared to the control group in regard to the epithelium. Similarly, the basal 7.47%, spinous 9.54%, granulosum 3.96%, and keratinous 5.65% layers did not
show significant differences (Table. 1) and maintained
similar histological characteristics between groups (Fig.
1B). VEGF (Fig. 3B) and VEGFR-2 (Fig. 4B) expression
patterns were also similar to controls.
Six Laser Applications
In this group, the control and irradiated groups,
showed stratified squamous epithelium occupied by
31.93% and 32.27% of the test area, respectively, with no
significant difference (P > 0.05). No significant difference was found too, in relative occupation of the test
area by layers: 7.89% and 8.15% (basal), 8.99% and
9.19% (spinous), 4.80% and 4.65% (granulosum), and
10.24% and 10.28% (keratinous) for control and irradiated groups, respectively (Table. 1). In both groups, cells
with clear cytoplasm were located in the basal layer,
between the keratinocytes and were histologically distinct. In connective tissue of the superficial dermis,
nuclei of varied cells, disorganized collagen fibers and
blood capillaries were observed (Fig. 1C). The experimental group displayed compact collagen fibers on the
superficial dermis (Fig. 1D). VEGF (Fig. 3C and D) and
VEGFR-2 (Fig. 4C and D) expression patterns were not
different between control and irradiated groups.
ANALYZES OF THE EPITHELIUM USING LASER IN HRS/J
1595
Fig. 1. A: Photomicrography of the control group after three laser
applications. 100. B: Photomicrography of the experimental group
after three laser applications. 100. C: Photomicrography of the control group after six laser applications. 100. D: Photomicrography of
the experimental group after six laser applications. 100. E: Photomicrography of the control group after ten laser applications. 100.
F: Photomicrography of the experimental group after ten laser applications. 100.
Ten Laser Applications
nective tissues were well characterized (Fig. 1E), with
notable differences (P < 0.05) in the smaller occupation
of the test area by the basal and granulosum layers area
in the irradiated group (Fig. 1F). VEGF and VEGFR-2
expression patterns were similar to the groups with three
and six irradiations (Fig. 3E and F) (Fig. 4E and F).
After ten laser applications, a significant decrease (P
< 0.05) in the test area occupation by the epithelium
area of the irradiated group was observed, 32.78% and
28.48% for control and irradiated groups, respectively. A
similar decrease was observed for the following layers:
basal (8.05% and 7.37%) and granulosum (6.04% and
3.91%), but were not found significant differences in spinous (9.87% and 8.87%), and in keratinous (8.82% and
8.32%) when compared control and experimental groups,
respectively (Table.1). Epithelial layers and dermis con-
Three, Six, and Ten Laser Applications
From the comparisons of the three irradiated groups
together (three, six, and ten), and using ANOVA with
1596
LEÃO ET AL.
Fig. 2. A: VEGF immunohistochemical identification in the group after ten laser applications—negative
control. 40. B: VEGFR-2 immunohistochemical identification in the group after six laser applications –
negative control. 40.
post hoc test (P < 0.05), six laser applications was
statistically different in relative occupation of the test
area by the total epithelium (26.62%, 32.27%, and
28.47%), respectively. It was also significantly different
on the layers: basal (7.47%, 8.14%, and 7.37%), granulosum (3.96%, 4.65%, and 3.91%) and keratinous
(5.64%, 10.28%, and 8.32%) area. In this parameter,
only spinous layer (9.53%, 9.19%, and 8.87%) area was
not different statistically (Table 2).
DISCUSSION
It is known that skin has three layers firmly adhered
to each other, the epidermis, dermis, and hypodermis.
The epidermis is formed by a stratified squamous epithelium, which acts as both, a mechanical and waterproof
barrier. In this epithelium, keratinocytes are organized
in layers: basal, spinous, granulosum, and keratinous.
Lower cell layers consist of metabolically active cells,
whereas the upper undergo a keratinization process
(Kierszenbaum et al., 2008). In our studies using hematoxylin–eosin staining, epidermis, and dermis were histologically distinct.
From comparisons of the three groups together, it was
found significant differences in relative occupation of the
test area by the epithelial area when the number of
laser applications was evaluated using an energy density
equivalent to 20 J/cm2 and 40 mW of power for 20 sec.
When the irradiated groups were compared to their respective controls at each period of time, the skin irradiated for three and six applications did not show a
significant difference in the layers and epithelium areas.
However, significant suppression in relative occupation
of the test area by basal and granulosum layers, and epithelium areas were evident for ten laser applications. After ten laser applications, a significant decrease was
revealed in relative area, when compared to respective
control and with six laser applications groups, suggesting a depression of the cellular activities. Considering
the particular conditions of this experiment, previous
reports have suggested that it is not possible to avoid
injury on human epidermis when the laser is used at
595 nm and 4 J/cm2 for more than 40 msec, even when
using a freezing spray (Dai et al., 2003; Rocha Júnior
et al., 2009) reported that, during the tissue healing pro-
cess, three LLLT applications at 15 mW of power and 3.8
J/cm2 for 15 sec resulted in apoptosis. Moreover,
Orringer et al. (2008) applied a single dose of a pulsed
laser at 595 nm with a tip diameter of 10 mm and a
dose of 7.5 J/cm2 for 10 msec on the epidermis and
observed an increase in epithelial thickness caused by
molecular and cellular changes that stimulated cell proliferation. Due to scarce studies and high parameter variation of LLLT, it is difficult to compare our results. In a
future, other well-controlled investigations, with controlled selections of lasers and treatment study parameters, using animal models with human characteristics
will contribute to a better understanding of the effects of
LLLT on the skin.
The increase in layers area of basal, granulosum and
keratinous, as well as, in total epithelium area were
statistically different in the six application times, when
compared with three and ten laser applications. However, the difference was not significant between irradiated and control groups. Then, six laser applications
could be used as a potential biomodulator, and have
lower risks associated with causing injuries to superficial tissues when the treatment is targeting deeper
layers. These results are strengthen by reports that
reveal laser action to promote stimulation and proliferation of keratinocytes ‘‘in vivo’’ (Baı̆bekov et al., 1988;
Rood et al., 1992; Grossman et al., 1998; Gál et al.,
2006). Laser potential is also expressed by increased mobility of human keratinocytes in cell culture (Haas et al.,
1990) and in other models (Medrado et al., 2003; Prado
et al., 2006; Bossini et al., 2009; Prado et al., 2009).
According to Gao and Xing (2009), improvements to the
healing process are due to the ability of low laser irradiation to promote mitochondrial respiratory chain activity. Under the conditions of this experimental study, six
laser applications for therapy of deep structures did not
cause damage to superficial tissue.
VEGF is known to be expressed on the subsarcolemmal region of skeletal muscle fibers, in vascular smooth
muscles, and on walls of capillaries, being the last one
active on endothelial cells proliferation (Hudlicka and
Brown, 2009). According to Byrne et al. (2005), VEGF is
a glycoprotein that belongs to the family of genes for
vascular growth factors, and VEGF-A is involved in
angiogenesis. This factor may bind either to heparin or
ANALYZES OF THE EPITHELIUM USING LASER IN HRS/J
1597
Fig. 3. A: VEGF immunohistochemical identification in the control
group after three laser applications. 40. B: VEGF immunohistochemical identification in the experimental group after three laser applications. 40. C: VEGF immunohistochemical identification in the control
group after six laser applications. 40. D: VEGF immunohistochemical
identification in the experimental group after six laser applications.
40. E: VEGF immunohistochemical identification in the control group
after ten laser applications. 40. F: VEGF immunohistochemical identification in the experimental group after ten laser applications. 40.
the extracellular matrix. In the present study, the immunostaining patterns for VEGF in all control and irradiated animals after 72 hr of the last application were
similar.
According to Shibuya (2006), VEGFR-2 is expressed
mainly in vascular endothelial cells and has strong tyrosine kinase activity. It has four regions: the domain
ligand to extracellular ligand, the transmembrane
domain, the tyrosine kinase domain, and the carboxyterminal region on the intracellular side. VEGFR-2 is
the main signal transducer for the positive activation of
endothelial precursor cells to differentiate into vascular
endothelial cells and continue proliferation. VEGFR-2
was expressed in the epithelial tissues of all control
and irradiated groups, showing similar patterns for immunohistochemical identification. According to Byrne
et al. (2005), in healthy adults, VEGF action is restricted
to wound repair when activated platelets release it with
other cytokines; VEGF is also released by monocytes, keratinocytes, and endothelial cells at the wound site.
VEGFR-2 is expressed in vascular endothelial cells from
the postnatal period until adulthood (Shibuya, 2006;
Hudlicka and Brown, 2009), also these authors reported
that capillary deformation, stimulated by stress, induces
nitric oxide (NO) release, VEGF increase, and VEGFR-2
expression in early endothelial cell proliferation and
angiogenesis. Based on these data, we conclude that the
number of laser applications in our experimental
1598
LEÃO ET AL.
Fig. 4. A: VEGFR-2 immunohistochemical identification in the control group after three laser applications. 40. B: VEGFR-2 immunohistochemical identification in the experimental group after three laser
applications. 40. C: VEGFR-2 immunohistochemical identification in
the control group after six laser applications. 40. D: VEGFR-2 immu-
nohistochemical identification in the experimental group after six laser
applications. 40. E: VEGFR-2 immunohistochemical identification in
the control group after ten laser applications. 40. F: VEGFR-2 immunohistochemical identification in the experimental group after ten laser
applications. 40.
conditions did not modify capillary proliferation because
the immunohistochemical patterns for VEGF and
VEGFR-2 identification were similar between all control
and irradiated animals.
According to Gao and Xing (2009), improvement in
the healing process is due to the ability of low laser
irradiation to promote the activation of mitochondrial
respiratory chain activity, cell signaling, and synthesis
or release of many molecules, growth factors, interleukins, and cytokines, aside from angiogenic stimulation.
Despite different experimental conditions, it is important to point out that the laser has been used in vascular lesion treatments by Wu and Wong (2008),
Bagazgoitia et al. (2008), and Dudelzak et al. (2009) and
to prevent the rapid growth of hemangiomas by Hintringer (2009). According to Pikkula et al. (2005), laser
treatment was more harmful at 585 nm than 595 nm on
vascularization of normal human skin ‘‘in vivo’’. Dudelzak et al. (2009) demonstrated that shorter wavelengths
(532 nm) are effective for small vessel reduction and
longer wavelengths are effective for larger vessel
treatments.
In conclusion, using different numbers of low laser
applications (three, six, or ten), at 20 J/cm2, on skin of
the masseteric region in mice of the HRS/J strain, six
laser applications showed the layers area and total epithelium area, statistically different when compared
with three and ten laser applications, but this same
ANALYZES OF THE EPITHELIUM USING LASER IN HRS/J
parameter from the comparisons between the irradiated
and control groups was not significant. None number of
laser applications used affected activity of VEGF and
VEGFR-2 release. After ten laser applications, a depression was observed on epithelium area, with significant
difference when compared to six applications, and
between irradiated and control groups. In this way, a
biostimulatory action with low risks associated at superficial tissues, may be suggested when the treatment
aims the deeper layers using six application. Besides,
the number of effective laser applications can be less
than ten, having better cost-effective associated to the
treatment.
ACKNOWLEDGEMENTS
The authors are grateful to FAPESP for financial
support.
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