Histomorphological and Angiogenic Analyzes of Skin Epithelium After Low Laser Irradiation in Hairless Mice.код для вставкиСкачать
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: [email protected] 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). 1594 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. 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