Metabolic syndrome comprises a constellation of morbidities such as insulin resistance, hyperinsulinemia, atherogenic
dyslipidemia, dysglycemia and obesity (especially abdominal). Metabolic alterations are observed in major insulin target
organs, increasing the risk of cardiovascular diseases, type-2 diabetes and therefore mortality. Tissue alterations are
characterized by immune cells infiltrates (especially activated macrophages). Released inflammatory mediators such as
TNF-α induce chronic inflammation in subjects with metabolic syndrome, since inflammatory pathways are activated in
the neighboring cells. The intra-abdominal adipose tissue appears to be of particular importance in the onset of the
inflammatory state, and strategies contributing to modulate the inflammatory process within this adipose tissue can
mitigate the metabolic syndrome consequences. Considering the low level light therapy (LLLT) recognized benefits in
inflammatory conditions, we hypothesized this therapeutic approach could promote positive effects in modulating the
inflammatory state of metabolic syndrome. That being the scope of this study, male C57BL/6 mice were submitted to a
high-fat/high-fructose diet among 8 weeks to induce metabolic syndrome. Animals were then irradiated on the
abdominal region during 21 days using an 850 nm LED (6 sessions, 300 seconds per session, 60 mW output power,
~6 J/cm2 fluence, ~19 mW/cm2 fluence rate). Before and during treatment, blood was sampled either from the retroorbital
plexus or from tail puncture for glucose, total cholesterol and triglycerides analysis. So far our results indicate no
alterations on these metabolic parameters after LLLT. For further investigations, blood was collected for plasma
inflammatory cytokine quantification and fresh ex vivo samples of liver and intra-abdominal adipose tissue were
harvested for immunohistochemistry purposes.
Over the last few years, low-level light therapy (LLLT) has shown an incredible suitability for a wide range of applications for central nervous system (CNS) related diseases. In this therapeutic modality light dosimetry is extremely critical so the study of light propagation through the CNS organs is of great importance. To better understand how light intensity is delivered to the most relevant neural sites we evaluated optical transmission through slices of rat brain point by point. We experimented red (λ = 660 nm) and near infrared (λ = 808 nm) diode laser light analyzing the light penetration and distribution in the whole brain. A fresh Wistar rat (Rattus novergicus) brain was cut in sagittal slices and illuminated with a broad light beam. A high-resolution digital camera was employed to acquire data of transmitted light. Spatial profiles of the light transmitted through the sample were obtained from the images. Peaks and valleys in the profiles show sites where light was less or more attenuated. The peak intensities provide information about total attenuation and the peak widths are correlated to the scattering coefficient at that individual portion of the sample. The outcomes of this study provide remarkable information for LLLT dose-dependent studies involving CNS and highlight the importance of LLLT dosimetry in CNS organs for large range of applications in animal and human diseases.
Due to the great number of applications of Low-Level-Laser-Therapy (LLLT) in Central Nervous System
(CNS), the study of light penetration through skull and distribution in the brain becomes extremely
important. The aim is to analyze the possibility of precise illumination of deep regions of the rat brain,
measure the penetration and distribution of red (λ = 660 nm) and Near Infra-Red (NIR) (λ = 808 nm)
diode laser light and compare optical properties of brain structures. The head of the animal (Rattus
Novergicus) was epilated and divided by a sagittal cut, 2.3 mm away from mid plane. This section of rat's
head was illuminated with red and NIR lasers in points above three anatomical structures: hippocampus,
cerebellum and frontal cortex. A high resolution camera, perpendicularly positioned, was used to obtain
images of the brain structures. Profiles of scattered intensities in the laser direction were obtained from
the images. There is a peak in the scattered light profile corresponding to the skin layer. The bone layer
gives rise to a valley in the profile indicating low scattering coefficient, or frontal scattering. Another
peak in the region related to the brain is an indication of high scattering coefficient (μs) for this tissue.
This work corroborates the use of transcranial LLLT in studies with rats which are subjected to models of
CNS diseases. The outcomes of this study point to the possibility of transcranial LLLT in humans for a
large number of diseases.
Several studies indicate that low level laser therapy (LLLT) accelerates the healing process, however, for a determined
pathology, dosimetry remains difficult to be established. To understand the tissue optical properties under different
conditions is extremely relevant since the dose delivered to the target tissue is known to be critical. The skin
pigmentation influence on the laser attenuation is not yet well established on different mice lineages or human ethnical
groups, making the dose problematic. Along the same line, inflammatory processes may cause similar problems since the
tissues in this condition change their optical properties due to inflammatory cell accumulation. This work evaluated the
attenuation pattern of a HeNe laser (λ=632.8 nm) using ex vivo skin samples from Balb/C and C57BL/6 mice under
inflammatory stages induced in their paw by local carrageenan inoculation. The samples were placed between two
microscope slides, and a CCD camera was placed orthogonal to the beam path. The intensity distribution of the scattered
light was photographed in grayscale and analyzed by ImageJ software. Our findings suggest that even slight differences
of the epithelial pigmentation could result in a relevant dose loss delivered to the deeper tissues. The increase of the
inflammatory cell density in the connective tissue indicated a highly scattering area also resulting in a dose loss for the
deeper tissues when compared to control group.
Candida albicans is the most frequent human opportunistic pathogenic fungus and one of the most important causes of
nosocomial infections. In fact, diagnosis of invasive candidiasis presents unique problems. The aim of this work was to
evaluate, by fluorescence image analysis, cellular labeling of C. albicans with CdTe/CdS quantum dots conjugated or
not to concanavalin A (ConA). Yeast cells were incubated with CdTe/CdS quantum dots (QD) stabilized with
mercaptopropionic acid (MPA) (emission peak at 530 nm) for 1 hour. In the overall study we observed no morphological
alterations. The fluorescence microscopic analysis of the yeast cells showed that the non-functionalized QDs do not label
C. albicans cells, while for the QD conjugated to ConA the cells showed a fluorescence profile indicating that the
membrane was preferentially marked. This profile was expected since Concanavalin A is a protein that binds specifically
to terminal carbohydrate residues at the membrane cell surface. The results suggest that the QD-labeled Candida cells
represent a promising tool to open new possibilities for a precise evaluation of fungal infections in pathological
conditions.
Antimicrobial photodynamic inactivation (APDI) is based on the utilization of substances that can photosensitize
biological tissues and are capable of being activated in the presence of light. Cryptococcus neoformans is an yeast
surrounded by a capsule composed primarily of glucoronoxylomannan that plays an important role in its virulence. This
yeast causes infection on skin, lungs and brain that can be associated with neurological sequelae and neurosurgical
interventions, and its conventional treatment requires prolonged antifungal therapy, which presents important adverse
effects. The aim of this study was to evaluate the protective effect of Cryptococcus neoformans capsule against reactive
oxygen species generated by APDI. Cryptococcus neoformans KN99α, which is a strain able to produce capsule, and
CAP59 that does not present capsule production were submitted to APDI using methylene blue (MB), rose bengal (RB),
and pL-ce6 as photosensitizers (PS). Then microbial inactivation was evaluated by counting colony form units following
APDI and confocal laser scanning microscopy (CLSM) illustrated localization as well as the preferential accumulation of
PS into the fungal cells. C. neoformans KN99α was more resistant to APDI than CAP59 for all PSs tested. CLSM
showed incorporation of MB and RB into the cytoplasm and a preferential uptake in mitochondria. A nuclear
accumulation of MB was also observed. Contrarily, pL-ce6 appears accumulated in cell wall and cell membrane and
minimal florescence was observed inside the fungal cells. In conclusion, the ability of C. neoformans to form capsule
enhances survival following APDI.
Photodynamic antimicrobial chemotherapy (PACT) is based on the principal that microorganisms will be inactivated
using a light source combined to a photosensitizing agent in the presence of oxygen. Oxidative damage of cell
components occurs by the action of reactive oxygen species leading to cell death for microbial species. It has been
demonstrated that PACT is highly efficient in vitro against a wide range of pathogens, however, there is limited
information for its in vivo potential. In addition, it has been demonstrated that sublethal photodynamic inactivation may
alter the virulence determinants of microorganisms. In this study, we explored the effect of sublethal photodynamic
inactivation to the virulence factors of Candida albicans. Methylene Blue (MB) was used as photosensitizer for sublethal
photodynamic challenge on C. albicans associated with a diode laser irradiation (λ=660nm). The parameters of
irradiation were selected in causing no reduction of viable cells. The potential effects of PACT on virulence determinants
of C. albicans cells were investigated by analysis of germ tube formation and in vivo pathogenicity assays. Systemic
infection was induced in mice by the injection of fungal suspension in the lateral caudal vein. C. albicans exposed to
sublethal photodynamic inactivation formed significantly less germ tube than untreated cells. In addition, mice infected
with C. albicans submitted to sublethal PACT survived for a longer period of time than mice infected with untreated
cells. The oxidative damage promoted by sublethal photodynamic inactivation inhibited virulence determinants and
reduced in vivo pathogenicity of C. albicans.
The reduction of pathogenic microorganisms in supragingival plaque is one of the principal factors in caries prevention
and control. A large number of microorganisms have been reported to be inactivated in vitro by photodynamic therapy
(PDT). The purpose of this study was to develop a rat model to investigate the effects of PDT on bacterial reduction in
induced dental caries. Twenty four rats were orally inoculated with Streptococcus mutans cells (ATCC 25175) for three
consecutive days. The animals were fed with a cariogenic diet and water with 10% of sucrose ad libitum, during all
experimental period. Caries lesion formation was confirmed by Optical Coherence Tomography (OCT) 5 days after the
beginning of the experiment. Then, the animals were randomly divided into two groups: Control Group: twelve animals
were untreated by either light or photosensitizer; and PDT Group: twelve animals were treated with 100μM of methylene
blue for 5min and irradiated by a Light Emitting Diode (LED) at λ = 640±30nm, fluence of 172J/cm2, output power of
240mW, and exposure time of 3min. Microbiological samples were collected before, immediately after, 3, 7 and 10 days
after treatment and the number of total microaerophiles was counted. OCT images showed areas of enamel
demineralization on rat molars. Microbiological analysis showed a significant bacterial reduction after PDT.
Furthermore, the number of total microaerophiles in PDT group remained lower than control group until 10 days posttreatment.
These findings suggest that PDT could be an alternative approach to reduce bacteria in dental caries.
Blue light, especially from LED devices, is a tool very frequently used in dental procedures. However, the investigations
of its effects on dental enamel are focused primarily on enamel demineralization and fluoride retention. Despite the fact
that this spectral region can inhibit enamel demineralization, the consequences of the irradiation on demineralized
enamel are not known. For this reason, we evaluated the effects of blue LED on enamel remineralization. Artificial
lesions formed in bovine dental enamel samples by immersion in undersaturated acetate buffer were divided into three
groups. In the first group (DE), the lesions were not submitted to any treatment. In the second (RE), the lesions were
submitted to remineralization. The lesions from the third group (LED+RE) were irradiated with blue LED (455nm,
1.38W/cm2, 13.75J/cm2 and 10s) before the remineralization. Cross-sectional microhardness was used to assess mineral
changes induced by remineralization under pH-cycling. The mineral deposition occurred preferably in the middle portion
of the lesions. Specimens from group RE showed higher hardness value than the DE ones. On the other hand, the mean
hardness value of the LED+RE samples was not statistically different from the DE samples. Results obtained in the
present study show that the blue light is not innocuous for the dental enamel and inhibition of its remineralization can
occur.
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