Clovis Grecco

Possibility for a full optical determination of photodynamic therapy outcome

The efficacy of photodynamic therapy (PDT) depends on a variety of parameters: concentration of the photosensitizer at the time of treatment, light wavelength, fluence, fluence rate, availability of oxygen within the illuminated volume, and light distribution in the tissue. Dosimetry in PDT requires the congregation of adequate amounts of light, drug, and tissue oxygen. The adequate dosimetry should be able to predict the extension of the tissue damage. Photosensitizer photobleaching rate depends on the availability of molecular oxygen in the tissue. Based on photosensitizers photobleaching models, high photobleaching has to be associated with high production of singlet oxygen and therefore with higher photodynamic action, resulting in a greater depth of necrosis. The purpose of this work is to show a possible correlation between depth of necrosis and the in vivo photosensitizer (in this case, Photogem®) photodegradation during PDT. Such correlation allows possibilities for the development of a real time ...

Single LED-based device to perform widefield fluorescence imaging and photodynamic therapy

Photodynamic therapy (PDT) is a treatment modality that can be indicated for several cancer types and pre-cancer lesions. One of the main applications of PDT is the treatment of superficial skin lesions such as basal cell carcinoma, Bowen’s disease and actinic keratosis. Three elements are necessary in PDT, a photosensitizer (PS); light at specific wavelength to be absorbed by the PS, and molecular oxygen. A typical PS used for skin lesion is protoporphyrin IX (PpIX), which is an intrinsic PS; its production is stimulated by a pro-drug, such as 5-aminolevulinic acid (ALA). Before starting a treatment, it is very important to follow up the PpIX production (to ensure that enough PS was produced prior to a PDT application) and, during a PDT session, to monitor its photodegradation (as it is evidence of the photodynamic effect taking place). The aim of this paper is to present a unique device, LINCE (MMOptics - São Carlos, Brazil), that brings together two probes that can, respectively, allow for fluorescence imaging and work as a light source for PDT treatment. The fluorescence probe of the system is optically based on 400 nm LED (light emitting diodes) arrays that allow observing the fluorescence emission over 450 nm. The PDT illumination probe options are constituted of 630 nm LED arrays for small areas and, for large areas, of both 630 nm and 450 nm LED arrays. Joining both functions at the same device makes PDT treatment simpler, properly monitorable and, hence, more clinically feasible. LINCE has been used in almost 1000 PDT treatments of superficial skin lesions in Brazil, with 88.4% of clearance of superficial BCC.

Non-homogeneous liver distribution of photosensitizer and its consequence for photodynamic therapy outcome

BACKGROUND Photodynamic therapy is mainly used for treatment of malignant lesions, and is based on selective location of a photosensitizer in the tumor tissue, followed by light at wavelengths matching the photosensitizer absorption spectrum. In molecular oxygen presence, reactive oxygen species are generated, inducing cells to die. One of the limitations of photodynamic therapy is the variability of photosensitizer concentration observed in systemically photosensitized tissues, mainly due to differences of the tissue architecture, cell lines, and pharmacokinetics. This study aim was to demonstrate the spatial distribution of a hematoporphyrin derivative, Photogem, in the healthy liver tissue of Wistar rats via fluorescence spectroscopy, and to understand its implications on photodynamic response. METHODS Fifteen male Wistar rats were intravenously photosensitized with 1.5mg/kg body weight of Photogem. Laser-induced fluorescence spectroscopy at 532 nm-excitation was performed on ex vivo liver slices. The influence of photosensitizer surface distribution detected by fluorescence and the induced depth of necrosis were investigated in five animals. RESULTS Photosensitizer distribution on rat liver showed to be greatly non-homogeneous. This may affect photodynamic therapy response as shown in the results of depth of necrosis. CONCLUSIONS As a consequence of these results, this study suggests that photosensitizer surface spatial distribution should be taken into account in photodynamic therapy dosimetry, as this will help to better predict clinical results.

Extracorporeal shockwave: mechanisms of action and physiological aspects for cellulite, body shaping, and localized fat—Systematic review

ABSTRACT Extracorporeal Shockwave Therapy (ESWT) has had a wide use in rehabilitation, and has presented positive effects in the treatment of unaesthetic affections. The objective of the present study was to search, in the literature, the mechanisms of action and the physiological aspects of shockwaves acting on the biological tissue to improve the condition of cellulite and localized fat. The systematic review of the literature was carried out in the period of September 2016 to February 2017 based on the bibliographic databases such as Lilacs, Medline, PubMed, and SciELO. Fifteen articles were identified in that systematic review, three of which were excluded as they did not make the complete access to the article available or the theme investigated did not encompass the objective of the study. The revision demonstrated that extracorporeal shockwaves present relevant effects on the biological tissue, which leads to the restructuring of skin properties and subcutaneous tissue, thus clinically improving the aspects of cellulite and localized fat.

Assembly and characterization of a nonlinear optical microscopy for in vivo and ex vivo tissue imaging

The purpose of this study is the assembly and characterization of a custom-made non-linear microscope. The microscope allows the adjustment for in vitro, in vivo and ex vivo imaging of biological samples. Two galvanometer mirrors conjugated by two spherical mirrors are used for the lateral scan and for the axial scan a piezoeletric stage is utilized. The excitation is done using a tunable femtosecond Ti: Sapphire laser. The light is focused in tissue by an objective lens 20X, water immersion, numerical aperture of 1.0, and working distance of 2.0 mm. The detection system is composed by a cut off filter that eliminates laser light back reflections and diverse dichroic filters can be chosen to split the emitted signal for the two photomultiplier detector. The calibration and resolution of the microscope was done using a stage micrometer with 10 μm divisions and fluorescent particle slide, respectively. Fluorescence and second harmonic generation images were performed using epithelial and hepatic tissue, the images have a sub-cellular spatial resolution. Further characterization and differentiation of tissue layers can be obtained by performing axial scanning. By means of the microscope it is possible to have a three dimensional reconstruction of tissues with sub-cellular resolution.

Pulmonary decontamination for photodynamic inactivation with extracorporeal illumination

Infectious pneumonia is a major cause of morbidity and mortality, despite advances in diagnostics and therapeutics in pulmonary infections. One of the major difficulties associated with the infection comes from the high rate of antibiotic resistant microorganisms, claiming for the use of alternative techniques with high efficiency and low cost. The photodynamic inactivation (PDI) is emerging as one of the great possibilities in this area, once its action is oxidative, not allowing microorganism develops resistance against the treatment. PDI for decontamination pulmonary has potential for treatment or creating better conditions for the action of antibiotics. In this study, we are developing a device to implement PDI for the treatment of lung diseases with extracorporeal illumination. To validate our theory, we performed measurements in liquid phantom to simulate light penetration in biological tissues at various fluency rates, the temperature was monitored in a body of hairless mice and the measurements of light transmittance in this same animal model. A diode laser emitting at 810 nm in continuous mode was used. Our results show 70% of leakage at 0.5 mm of thickness in phantom model. The mouse body temperature variation was 5.4 °C and was observed light transmittance through its chest. These results are suggesting the possible application of the extracorporeal illumination using infrared light source. Based on these findings, further studies about photodynamic inactivation will be performed in animal model using indocyanine green and bacteriochlorin as photosensitizers. The pulmonary infection will be induced with Streptococcus pneumoniae and Klebsiella pneumoniae.

In vitro and in vivo evaluation of the induced PDT response using a femtosecond laser

One of the limitations of PDT in the treatment of bulk tumors is the light penetration in biological tissues at the red spectrum. PDT illumination at short pulsed regime may present a higher light penetration compared to the CW regime, and potentially a higher volume of induced necrosis. The major goal of this study was an in vitro and in vivo evaluation of PDT response after illumination using a 630 nm femtosecond laser. Photogem in distilled water solution was illuminated either with CW or femtosecond laser under the same fluence and fluence rate parameters. Wistar rats weighting between 280 and 300 g were intravenously photosensitized with Photogem. Thirty minutes after drug injection, the normal liver was irradiated either with the CW or the femtosecond laser (fluence: 150 J/cm2). The in vitro results showed that, under the same conditions, the degradation rate evaluated via fluorescence spectroscopy was higher under femtosecond laser irradiation. Histological analysis of the induced necrosis showed that there was a significant higher depth of necrosis when the femtosecond laser was used. Based on these results, femtosecond lasers seem to be an alternative in PDT applications, improving results for the treatment of lesions for which a larger light penetration is required.

Synergistic effects of Combined Therapy: nonfocused ultrasound plus Aussie current for noninvasive body contouring

Background and objectives Nowadays, there are several noninvasive technologies being used for improving of body contouring. The objectives of this pilot study were to verify the effectiveness of the Heccus® device, emphasizing the synergism between nonfocused ultrasound plus Aussie current in the improvement of body contour, and to determine if the association of this therapy with whole-body vibration exercises can have additional positive effects in the results of the treatments. Subjects and methods Twenty healthy women aged 20–40 years participated in the study. Ten patients received Combined Therapy treatment (G1) and the other 10 participants received Combined Therapy with additional vibratory platform treatment (G2). Anthropometric and standardized photography analysis, ultrasonography, cutometry and self-adminestered questionnaires of tolerance and satisfaction levels with the treatment were used. Results Compared with baseline values, reduction of fat thickness was observed by ultrasonography in the posterior thigh area in the G1 group (P<0.05) and in the buttocks (P<0.05) and the posterior thigh areas (P<0.05) in the G2. All the treated areas in both groups showed reduction in cellulite degree in the buttocks, G1 (P<0.05) and G2 (P<0.05), and in posterior thigh areas, G1 (P<0.05) and G2 (P<0.05). Optimal improvement of skin firmness (G1, P<0.0001; G2, P=0.0034) in the treated areas was observed in both groups. Conclusion We conclude that the synergistic effects of the Combined Therapy (nonfocused ultrasound plus Aussie current) might be a good option with noninvasive body contouring treatment for improving the aspect of the cellulite, skin firmness and localized fat. If used in association with the whole-body vibratory platform, the results can be better, especially in the treatment of localized fat. Further studies with larger sample size should be performed to confirm these results.

Molecular analyses of two bacterial sampling methods in ligature‐induced periodontitis in rats

The prevalence profile of periodontal pathogens in dental plaque can vary as a function of the detection method; however, the sampling technique may also play a role in determining dental plaque microbial profiles. We sought to determine the bacterial composition comparing two sampling methods, one well stablished and a new one proposed here. In this study, a ligature‐induced periodontitis model was used in 30 rats. Twenty‐seven days later, ligatures were removed and microbiological samples were obtained directly from the ligatures as well as from the periodontal pockets using absorbent paper points. Microbial analysis was performed using DNA probes to a panel of 40 periodontal species in the checkerboard assay. The bacterial composition patterns were similar for both sampling methods. However, detection levels for all species were markedly higher for ligatures compared with paper points. Ligature samples provided more bacterial counts than paper points, suggesting that the technique for induction of periodontitis could also be applied for sampling in rats. Our findings may be helpful in designing studies of induced periodontal disease‐associated microbiota.

Comparison of two photosensitizers in photodynamic therapy using light pulses in femtosecond regime: an animal study

Photodynamic therapy is a therapeutic modality for cancer treatment based on the interaction of light with a sensitizer agent and molecular oxygen present into the target cells. The aim of this study is the evaluation of photodynamic therapy using pulsed light source in the femtosecond regime through necrosis induced in healthy rat liver. The induced necrosis profile with CW laser and pulsed laser were evaluated in animal model, which received Photodithazine (chlorine e6 derivative). The light sources used in these studies were a 660 nm CW diode laser and a Ti:Sapphire Regenerative Amplifier laser (1 kHz repetition rate and 100 fs pulse width) associated with an optical parametric amplifier (OPA) to convert to 660 nm. The results were compared with a previous study when was used a hematoporphyrin derivative (Photogem) as a sensitizer. The induced necrosis with Photogen was greater with pulsed laser (2.0 ± 0.2 mm) in comparison with CW laser (1.0 ± 0.2 mm), while in Photodithazine the induced necrosis with was greater with CW laser (2.9 ± 0.2 mm) comparing the pulsed laser (2.0 ± 0.2 mm). These results indicate dependence of PDT mechanisms with photosensitizer and the light regime applied.