Silvia Regina de Almeida Reis

The influence of low-level laser therapy on biomodulation of collagen and elastic fibers

The study of low-level laser therapy upon extracellular matrix elements is important to understand the wound healing process under this agent. However, little is known about the interference of laser light in relation to collagen and elastic fibers. Cutaneous wounds were performed on the back of 72 Wistar rats and a Ga-Al-As low-level laser was punctually applied with different energy densities. The animals were killed after 24, 48, 72 hours and 5, 7 and 14 days. Tissues were stained with hematoxilin-eosin, sirius red fast green and orcein and then analyzed. It was observed that the treated group exhibited larger reduction of edema and inflammatory infiltrate. The treated animals presented a larger expression of collagen and elastic fibers, although without statistical significance (p > 0.05). Treatment with a dosage of 4 J/cm(2) exhibited more expressive results than that with 8 J/cm(2). In this study, the authors concluded that low-level laser therapy contributed to a larger expression of collagen and elastic fibers during the early phases of the wound healing process.

INFLUENCE OF LASER PHOTOBIOMODULATION UPON CONNECTIVE TISSUE REMODELING DURING WOUND HEALING

The modulation of collagen fibers during experimental skin wound healing was studied in 112 Wistar rats submitted to laser photobiomodulation treatment. A standardized 8mm-diameter wound was made on the dorsal skin of all animals. In half of them, 0.2ml of a silica suspension was injected along the border of the wound in order to enhance collagen deposition and facilitate observation. The others received saline as vehicle. The treatment was carried out by means of laser rays from an aluminum-gallium arsenide diode semiconductor with 9mW applied every other day (total dose=4J/cm2) on the borders of the wound. Tissue sections obtained from four experimental groups representing sham-irradiated animals, laser, silica and the association of both, were studied after 3, 7, 10, 15, 20, 30 and 60 days from the laser application. The wounded skin area was surgically removed and submitted to histological, immunohistochemical, ultrastructural, and immunofluorescent studies. Besides the degree and arrangement of collagen fibers and of their isotypes, the degree of edema, the presence of several cell types especially pericytes and myofibroblasts, were described and measured. The observation of Sirius-red stained slides under polarized microscopy revealed to be of great help during the morphological analysis of the collagen tissue dynamic changes. It was demonstrated that laser application was responsible for edema regression and a diminution in the number of inflammatory cells (p<0.05). An evident increase in the number of actin-positive cells was observed in the laser-treated wounds. Collagen deposition was less than expected in silica-treated wounds, and laser treatment contributed to its better differentiation and modulation in all irradiated groups. Thus, laser photobiomodulation was able to induce several modifications during the cutaneous healing process, especially in favoring newly-formed collagen fibers to be better organized and compactedly disposed.

Effect of 670-nm Laser Therapy and Dexamethasone on Tissue Repair: A Histological and Ultrastructural Study

OBJECTIVE In this study we investigated the role of extracellular matrix elements and cells during the wound healing phases following the use of low-level laser therapy (LLLT) and anti-inflammatory drugs. BACKGROUND DATA There are few scientific studies that characterize the possible interactions of LLLT and anti-inflammatory medications. MATERIALS AND METHODS Thirty-two rats submitted to a wound inflicted with a 6-mm-diameter punch. The animals were divided into four groups: sham treated, those treated with the GaAlAs laser (4 J/cm(2), 9 mW, lambda = 670 nm, spot size 28.27 x 10(2) cm(2)), those treated with dexamethasone (2 mg/kg), and those treated with both LLLT and dexamethasone. After 3 and 5 d, the cutaneous wounds were assessed by histopathology using polarized light and ultrastructural assessment using transmission electron microscopy. Changes seen in polymorphonuclear inflammatory cells, edema, mononuclear cells, and collagen fiber deposition were semi-quantitatively evaluated. RESULTS The laser-treated group demonstrated increased collagen content and better arrangement of the extracellular matrix (p < 0.05). Fibroblasts in these tissues were increased in number and were more synthetically active. In the dexamethasone group, the collagen was shown to be non-homogenous and disorganized, with a scarcity of fibroblasts. In the group treated with both types of therapy, fibroblasts were more common and they exhibited vigorous rough endoplasmic reticulum, but they had less collagen production compared to those seen in the laser group. CONCLUSION LLLT alone accelerates post-surgical tissue repair and reduces edema and the polymorphonuclear infiltrate even in the presence of dexamethasone.

Cytologic alterations in the oral mucosa after chronic exposure to ethanol

The effects of ethanol alone on the oral mucosa are still poorly understood, especially because there are few non-smoking chronic consumers of alcoholic beverages. The aim of this study was to evaluate the frequency of micronucleus, abnormal nucleus/cytoplasm ratio, pyknosis, karyorrhexis and karyolysis in exfoliated cells from the buccal mucosa and from the lateral border of the tongue in 36 non-smoker alcoholics (ethanol group) and 18 non-smokers and non-drinkers (control group). The Papanicolaou method was used. Since alcoholics generally have hepatobiliary involvement, the association between serum gamma-glutamyl transpeptidase (GGT) and some of the analyzed oral mucosa alterations was also investigated. The ethanol group showed a significant increase in the frequency of all alterations analyzed in the tongue cells when compared with the control group (p < 0.01; Mann-Whitney). However, the presence of these changes in buccal mucosa cells was not statistically significant (p > 0.05; Mann-Whitney). In the ethanol group, the correlation between serum GGT and the frequency of micronucleus and abnormal nucleus/cytoplasm ratio in oral mucosa cells was not significant (p > 0.05; Spearman). In conclusion, chronic exposure to ethanol may be associated with carcinogenic cytologic changes in the oral mucosa, even in the absence of tobacco smoking. These alterations were not correlated with hepatobiliary injury.

Effect of Low-Level Laser Therapy (660 nm) on Angiogenesis in Wound Healing: A Immunohistochemical Study in a Rodent Model

The aim of the present investigation was to evaluate the angiogenesis on dorsal cutaneous wounds in a rodent model treated with λ660 nm laser light. New vessel formation is a multistep process involving vessel sprouting, endothelial cell migration, proliferation and tube formation. Although several in vivo studies have shown that laser phototherapy influences tissue repair, a fully understanding of angiogenesis mechanisms are not yet known. Twenty-four young adult male Wistar rats weighing between 200 and 250 g were used. Under general anesthesia, one excisional wound was created on the dorsum of each animal and they were randomly distributed into two groups: one control and one treated with laser (λ660 nm, 16 mW, 10 J/cm2). Each group was subdivided into three subgroups according to the animal death timing (2, 4 and 6 days). Laser irradiation started immediately after surgery and was repeated every other day during the experiment and marked with Sirius Red, specific for collagen, and immunomarked with anti-TGF-β and anti-von Willebrand factor. Marked sections underwent histological analysis by light microscopy and the mean area of the wound of each animal was calculated and analyzed by ANOVA and Tukeys test (α=0.05). Although at some death periods, collagen expression and number of blood vessels on irradiated animals were higher than in the control ones, no significant differences were found at any time in relation to TGF-β expression (p>0.05). It was concluded that laser treatment (λ660 nm) contributed to increase angiogenesis.

p53 and MDM2 protein expression in actinic cheilitis

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

Expression of basement membrane laminin in oral squamous cell carcinomas

Summary The basement membrane is a dynamic structure that undergoes quantitative and qualitative changes during the progression of squamous cell carcinoma, which is essencially important in tumoral invasion and metastasis. Aim This study is aimed at investigating the behavior of the basement membrane in oral squamous cell carcinomas with different malignancy scores, which were obtained through the immunohistochemical expression of the laminin, a glycoprotein present in the basement membrane. Study design History cross-sectional cohort. Material and method Thirty-one cases of oral squamous cell carcinoma were subjected to histological grading of malignant tumors. The immunohistochemical expression of the laminin in lesions bearing different scores of malignancy was evaluated according to intensity and integrity, using the Streptavidin-Biotin complex method. Results We noticed significant differences in the media between intensity and continuity laminin expression in relation to different grades of malignancy. Conclusion Different expressions of laminin, a glycoprotein present in basement membranes were evident in oral cell carcinomas within different grades of histological malignancy.

Phenotype characterization of pericytes during tissue repair following low‐level laser therapy

Background/purpose: The action of low‐level laser therapy (LLLT) on pericytes during wound healing is not well established. The objective of this study was to identify the effect of laser treatment on pericytes during tissue repair.

Genetic risk factors for nonsyndromic cleft lip with or without cleft palate in a Brazilian population with high African ancestry

Nonsyndromic cleft lip with or without cleft palate (NSCL ± P) is the most common orofacial birth defect, exhibiting variable prevalence around the world, often attributed to ethnic and environmental differences. Linkage analyses and genome‐wide association studies have identified several genomic susceptibility regions for NSCL ± P, mostly in European‐derived or Asian populations. Genetic predisposition to NSCL ± P is ethnicity‐dependent, and the genetic basis of susceptibility to NSCL ± P likely varies among populations. The population of Brazil is highly admixed, with highly variable ancestry; thus, the genetic determinants of NSCL ± P susceptibility may be quite different. This study tested association of 8 single‐nucleotide polymorphisms (SNPs), previously identified by genome‐wide studies in other populations, with NSCL ± P in a Brazilian population with high African ancestry. SNPs rs560426, rs642961, rs1530300, rs987525, rs3758249, rs7078160, rs17085106, and rs13041247 were genotyped in 293 Brazilian patients with NSCL ± P and 352 unaffected Brazilian controls. Each sample was also genotyped for 40 biallelic short insertion/deletion polymorphic markers to characterize genetic ancestry. The average African ancestry background was 31.1% for the NSCL ± P group and 36.7% for the control group. After adjustment for ancestry and multiple testing, the minor alleles of rs3758249 (OR: 1.58, 95% CI: 1.25–2.01, P = 0.0001) and rs7078160 (OR: 1.59, 95% CI: 1.21–2.07, P = 0.0002) were significantly associated with risk of NSCL ± P. Polymorphisms located in IRF6 (rs642961) and 8q24 (rs1530300 and rs987525) showed marginal associations in this Brazilian population with high African ancestry. These results indicate that rs3758249 at 9q22 and rs7078160 at 10q25.3 represent risk loci for NSCL ± P in the Brazilian population with high African ancestry.

Effect of LED Phototherapy (λ700 ± 20 nm) on TGF-β Expression During Wound Healing: An Immunohistochemical Study in a Rodent Model

OBJECTIVE The aim of the present investigation was to evaluate transforming growth factor β (TGF-β) expression on cutaneous wounds in rodents treated or not treated with LED light. BACKGROUND TGF-β is a multifunctional cytokine that presents a central action during tissue repair. Although several studies both in vitro and in vivo have shown that LED phototherapy influences tissue repair, a full understanding of the mechanisms involved in its usage, such as in the modulation of some growth factors, remains unclear. MATERIALS AND METHODS Under general anesthesia, 24 young adult male Wistar rats weighing 200-250 g had one excisional wound created on the dorsum of each, and were randomly distributed into two groups: G0 (Control) and G1 (LED, λ700 ± 20 nm, 16 mW, SAEF = 5 J/cm(2), Illuminated Area = 2 cm(2), 8 mWcm(2), 626 s) Each group was subdivided into three subgroups according to the animal death timing (2, 4, and 6 days). LED phototherapy started immediately after surgery and was repeated every other day during the experimental time. Following animal death, specimens were removed, routinely processed to wax, cut and immunomarked with polyclonal anti-TGF-β, and underwent histological analysis by light microscopy. The mean area of expression of each group was calculated. The data were statistically analyzed using ANOVA and Tukeys test. RESULTS The area of the expression of TGF-β on LED-irradiated animals was significantly smaller than on controls at day 2 (p = 0.013). No significant difference was found at later times. It is concluded that the use of LED light, at these specific parameters, caused an inhibition of the expression of TGF-β at an early stage of the healing process.