SEARCH

SEARCH BY CITATION

Keywords:

  • laser;
  • skin;
  • epithelium;
  • light microscopy;
  • endothelial growth factor

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

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 = 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, 2011. © 2011 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

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 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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

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 = 5) and irradiated (N = 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 μm 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).

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
LayersGroup3 (%)t-test6 (%)t-test10 (%)t-test
BasalControl7.310.507.890.308.050.001
Experimental7.478.157.37
SpinousControl10.460.368.990.769.870.12
Experimental9.549.198.87
GranulosumControl4.390.334.800.656.040.00
Experimental3.964.653.91
KeratinousControl6.480.4610.240.978.820.55
Experimental5.6510.288.32
EpitheliumControl28.620.2131.930.8532.780.003
Experimental26.6232.2728.48
Table 2. Mean and standard error of the points from layers in the experimental groups after three, six, and ten laser applications
ApplicationsLayerPMean (%)Standard error
  1. Data from different sessions numbers of laser application in each layer were analyzed by ANOVA (P < 0.05).

3Layer basal0.0017.470.16
6  8.140.18
10  7.370.14
3  9.530.57
6Layer spinous0.6709.190.49
10  8.870.50
3  3.960.28
6Layer granulosum0.0504.650.21
10  3.910.20
3  5.640.58
6Layer keratinous0.00110.280.67
10  8.320.73
3  26.621.05
6Epithelium0.00132.271.15
10  28.471.07

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.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

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.

thumbnail image

Figure 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×.

Download figure to PowerPoint

thumbnail image

Figure 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×.

Download figure to PowerPoint

thumbnail image

Figure 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×.

Download figure to PowerPoint

thumbnail image

Figure 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 immunohistochemical 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×.

Download figure to PowerPoint

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.

Ten Laser Applications

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 connective 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).

Three, Six, and Ten Laser Applications

From the comparisons of the three irradiated groups together (three, six, and ten), and using ANOVA with 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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

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 process, 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” (Baĭbekov 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 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 carboxy-terminal 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 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 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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The authors are grateful to FAPESP for financial support.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  • Bagazgoitia L, Boixeda P, Lopez-Caballero C, Beà S, Santiago JL, Jaén P. 2008. Venous malformation of the eyelid treated with pulsed-dye-1064-nm neodymium yttrium aluminum garnet sequential laser: an effective and safe treatment. Ophthal Plast Reconstr Surg 24: 488490.
  • Baĭbekov IM, Musaev ESh, Alimov DT. 1988. Effects of gastric mucosa irradiation with helium-neon laser on epithelial cells. Biull Eksp Biol Med 105: 750752.
  • Bossini PS, Fangel R, Habenschus RM, Renno AC, Benze B, Zuanon JA, Neto CB, Parizotto NA. 2009. Low-level laser therapy (670 nm) on viability of random skin flap in rats. Lasers Med Sci 24: 209213.
  • Byrne AM, Bouchier-Hayes DJ, Harmey JH. 2005. Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J Cell Mol Med 9: 777794.
  • Dai T, Pikkula BM, Tunnell JW, Chang DW, Anvari B. 2003. Thermal response of human skin epidermis to 595-nm laser irradiation at high incident dosages and long pulse durations in conjunction with cryogen spray cooling: an ex-vivo study. Lasers Surg Med 33: 1624.
  • De Medeiros JS, Vieira GF, Nishimura PY. 2005. Laser application effects on the bite strength of the masseter muscle, as an orofacial pain treatment. Photomed Laser Surg 23: 373376.
  • Dudelzak J, Hussain M, Goldberg DJ. 2009. Vascular-specific laser wavelength for the treatment of facial telangiectasias. J Drugs Dermatol 8: 227229.
  • Emshoff R, Bösch R, Pümpel E, Schöning H, Strobl H. 2008. Low-level laser therapy for treatment of temporomandibular joint pain: a double-blind and placebo-controlled trial. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 105: 452456.
  • Fricton JR. 2004. The relationship of temporomandibular disorders and fibromyalgia: Implications for diagnosis and treatment. Curr Pain Headache Rep 8: 355363.
  • Gál P, Vidinský B, Toporcer T, Mokrý M, Mozes S, Longauer F, Sabo J. 2006. Histological assessment of the effect of laser irradiation on skin wound healing in rats. Photomed Laser Surg 24: 480488.
  • Gao X, Xing D. 2009. Molecular mechanisms of cell proliferation induced by low power laser irradiation. J Biomed Sci 16: 116.
  • Genovese WJ. 2000. Laser de baixa intensidade: aplicações terapêuticas em odontologia. Lovisa. p 130.
  • Grossman N, Schneid N, Reuveni H, Halevy S, Lubart R. 1998. 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species. Lasers Surg Med 22: 212218.
  • Haas AF, Isseroff RR, Wheeland RG, Rood PA, Graves PJ. 1990. Low-energy helium-neon laser irradiation increases the motility of cultured human keratinocytes. J Invest Dermatol 94: 822826.
  • Hawkins DH, Abrahamse H. 2006. The role of laser fluence in cell viability, proliferation, and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation. Lasers Surg Med 38: 7483.
  • Hintringer T. 2009. Treatment of haemangiomas and vascular malformations with the neodymium-YAG laser--strategy and results in over 2000 cases. Handchir Mikrochir Plast Chir 41: 8387.
  • Hudlicka O, Brown MD. 2009. Adaptation of skeletal muscle microvasculature to increased or decreased blood flow: role of shear stress, nitric oxide and vascular endothelial growth factor. J Vasc Res 46: 504512.
  • Iyomasa DM, Garavelo I, Iyomasa MM, Watanabe IS, Issa JP. 2009. Ultrastructural analysis of the low level laser therapy effects on the lesioned anterior tibial muscle in the gerbil. Micron 40: 413418.
  • Karu TI. 2008. Mitochondrial signaling in mammalian cells activated by red and near-IR radiation. Photochem Photobiol 84: 10911099.
  • Karu T, Pyatibrat L, Kalendo G. 1995. Irradiation with He-Ne laser increases ATP level in cells cultivated in vitro. J Photochem Photobiol B 27: 219223.
  • Kierszenbaum AL, Rivkin E, Tres LL. 2008. Expression of Fer testis (FerT) tyrosine kinase transcript variants and distribution sites of FerT during the development of the acrosome-acroplaxome-manchette complex in rat spermatids. Dev Dyn 237: 38823891.
  • Lopes-Martins RA, Marcos RL, Leonardo PS, Prianti AC, Muscará MN, Aimbire F, Frigo L, Iversen VV, Bjordal JM. 2006. Effect of low-level laser (Ga-Al-As 655 nm) on skeletal muscle fatigue induced by electrical stimulation in rats. J Appl Physiol 101: 283288.
  • Maciel VH. 2006. Protocolos clínicos para aplicação do LASER de baixa intensidade nos procedimentos fisioterapêuticos. São Carlos: MM Optics. P 1920.
  • Mandarim-de-Lacerda CA. 1995. Métodos quantitativos em morfologia. Uerj. p 131.
  • Medrado AR, Pugliese LS, Reis SR, Andrade ZA. 2003. Influence of low level laser therapy on wound healing and its biological action upon myofibroblasts. Lasers Surg Med 32: 239244.
  • Meireles GCS. 2005. Análise comparativa do efeito dos lasers GaAIAs de λ = 660 nm e λ = 780 nm na cicatrização de úlceras por queimadura em dorso de ratos diabéticos e não-diabéticos: estudo histológico; Comparative analysis of the effect of lasers GaAIAs of λ = 660 nm e λ = 780 nm in the cicatrization of ulcers for burning in back of diabetic and not-diabetic rats: histological study. In: Universidade Federal da Bahia. Faculdade de Odontologia e Universidade Federal da Paraíba. Faculdade de Odontologia. p 120.
  • Nicola JH, Nicola EMD. 2002. Wavelength, frequency, and color: absolute or relative concepts? J Clin Laser Med Surg 20: 307311.
  • Núñez SC, Garcez AS, Suzuki SS, Ribeiro MS. 2006. Management of mouth opening in patients with temporomandibular disorders through low-level laser therapy and transcutaneous electrical neural stimulation. Photomed Laser Surg 24: 4549.
  • Orringer JS, Hammerberg C, Hamilton T, Johnson TM, Kang S, Sachs DL, Fisher G, Voorhees JJ. 2008. Molecular effects of photodynamic therapy for photoaging. Arch Dermatol 144: 12961302.
  • Pikkula BM, Chang DW, Nelson JS, Anvari B. 2005. Comparison of 585 and 595 nm laser-induced vascular response of normal in vivo human skin. Lasers Surg Med 36: 117123.
  • Pontinen P. 1992. Low level laser therapy as a medical treatment modality. Pöntinen, Pekka J.: Art Urpo, Ltd., Tampere. p 99101.
  • Posten W, Wrone DA, Dover JS, Arndt KA, Silapunt S, Alam M. 2005. Low-level laser therapy for wound healing: mechanism and efficacy. Dermatol Surg 31: 334340.
  • Prado RP, Liebano RE, Hochman B, Pinfildi CE, Ferreira LM. 2006. Experimental model for low level laser therapy on ischemic random skin flap in rats. Acta Cir Bras 21: 258262.
  • Prado RP, Pinfildi CE, Liebano RE, Hochman BS, Ferreira LM. 2009. Effect of application site of low-level laser therapy in random cutaneous flap viability in rats. Photomed Laser Surg 27: 411416.
  • Reis SR, Medrado AP, Marchionni AM, Figueira C, Fracassi LD, Knop LA. 2008. Effect of 670-nm laser therapy and dexamethasone on tissue repair: a histological and ultrastructural study. Photomed Laser Surg 26: 307313.
  • Rocha Júnior AM, Vieira BJ, de Andrade LC, Aarestrup FM. 2009. Low-level laser therapy increases transforming growth factor-beta2 expression and induces apoptosis of epithelial cells during the tissue repair process. Photomed Laser Surg 27: 303307.
  • Rood PA, Haas AF, Graves PJ, Wheeland RG, Isseroff RR. 1992. Low-energy helium neon laser irradiation does not alter human keratinocyte differentiation. J Invest Dermatol 99: 445448.
  • Shibuya M. 2006. Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1): a dual regulator for angiogenesis. Angiogenesis 9: 225230; discussion 231.
  • Winstanley DA, Uebelhoer NS. 2008. Future considerations in cutaneous photomedicine. Semin Cutan Med Surg 27: 301308.
  • Wu EC, Wong BJ. 2008. Lasers and optical technologies in facial plastic surgery. Arch Facial Plast Surg 10: 381390.