The effect of low-power laser light at different doses on gastric mucosa sensitized with methylene blue, haematoporphyrin derivative or toluidine blue

Helicobacter pylori has been associated with peptic ulcers, gastric cancer and various other gastroduodenal pathologies. Conventional antibiotic treatment is not entirely satisfactory, whereas photodynamic therapy (PDT) is a possible alternative. Although PDT has been shown to be effective in killing helicobacter on native gastric mucosa, the success of the technique will depend upon the mucosa underlying the bacteria remaining unharmed. This study examined the effect of increasing doses of low-power laser light on rat gastric mucosa, sensitized with either methylene blue (MB), haematoporphyrin derivative (HpD) or toluidine blue (TBO). No damage was detectable with any of the photosensitizers when a light dose of 250 Jcm^-2 was used. Mucosal damage was seen consistently with TBO (1 mg ml^-1) and a light dose of 500 J cm^-2. The same light dose of 500 J cm^-2 using MB caused inconsistent damage to the mucosa, whereas HpD had no effect even with the highest dose of laser light (500 J cm^-2). As the energy doses required to kill bacteria (50 and 200 Jcm^-2 for MB and TBO, respectively) are well below the levels shown to damage normal gastric mucosa, PDT forH. pylori should not be harmful to the underlying epithelium.     

Comparison of the photodynamic fungicidal efficacy of methylene blue, toluidine blue, malachite green and low-power laser irradiation alone against Candida albicans

This study was to evaluate specific effects of photodynamic therapy (energy density 15.8 J/cm², 26.3 J/cm² and 39.5 J/cm²) using methylene blue, toluidine blue and malachite green as photosensitizers and low-power laser irradiation on the viability of Candida albicans. Suspensions of C. albicans containing 10⁶ cells/ml were standardized in a spectrophotometer. For each dye, 120 assays, divided into four groups according to the following experimental conditions, were carried out: laser irradiation in the presence of the photosensitizer; laser irradiation only; treatment with the photosensitizer only; no exposure to laser light or photosensitizer. Next, serial dilutions were prepared and seeded onto Sabouraud dextrose agar for the determination of the number of colony-forming units per milliliter (CFU/ml). The results were subjected to analysis of variance and the Tukey test (P < 0.05). Photodynamic therapy using the photosensitizers tested was effective in reducing the number of C. albicans.. The number of CFU/ml was reduced by between 0.54 log₁₀ and 3.07 log₁₀ and depended on the laser energy density used. Toluidine blue, methylene blue and malachite green were effective photosensitizers in antimicrobial photodynamic therapy against C. albicans, as was low-power laser irradiation alone.     

Tumour marking properties of different haematoporphyrins and tetrasulfonated phthalocyanine—A comparison

Several photosensitizers were screened for their tumour-marking ability using laserinduced fluorescence in Wistar/Furth rats bearing subcutaneous adenocarcinomas inoculated in muscle. Of the studied photosensitizers, dihaematoporphyrin ether appeared to exhibit the best tumour-demarcation properties. Polyhaematoporphyrin ester and tetrasulfonated phthalocyanine were almost as good although the fluorescence yield was much lower. Monomeric haematoporphyrin also showed some tumour-marking qualities. By forming fluorescence intensity ratios, information from both the blue and the red spectral regions were used to provide the highest tumour-to-muscle contrast. Two excitation wavelengths were used, of which 337 nm rather than 405 nm excitation light seemed to yield a better tumour demarcation, due to a greater difference in the superimposing autofluorescence between tumour and surrounding tissue. The study included measurements on many inner organs in an attempt to gain a better understanding of the interaction between the drugs and various kinds of tissue.     

Time-resolved confocal fluorescence microscopy of porphyrins for phototherapy

The application of a novel time-resolved confocal fluorescence microspectrometer to studies of the distribution and speciation of porphyrin photosensitizers in rat C6 cerebral glioma cells is described. The instrument combines a mode-locked argon ion laser excitation source with time-correlated single photon counting fluorescence detection and has sub-micron spatial and sub-nanosecond temporal resolution. The porphyrins studied were haematoporphyrin derivative (HpD), haematoporphyrin IX (HP), porphyrinc (Pc) and the tetrakiscarborane carboxylate ester of 2,4-(,-dihydroxyethyl) deuteroporphyrin IX (BOPP). From the heterogeneous emission observed in vitro, assignments and spatial location of various porphyrin species are proposed.     

The binding and photosensitization effects of tetrabenzoporphyrins and texaphyrin in bacterial cells

A new class of photosensitizers which inactivate efficiently Gram-positive bacterial cells is described. The photosensitizers used in this study were Mg and Zn-tetrabenzoporphyrin (MgTBP and ZnTBP), which possess strong absorption bands at 630 nm and Cd-texaphyrin (CdTX), whose longest absorption band is at 770 nm. These sensitizers thus have optical properties that are superior to those of haematoporphyrin. It is demonstrated that MgTBP and CdTX have similar photosensitizing efficiencies as haematoporphyrin on Gram-positiveStaphylococcus aureus cells but are better on the Gram-negativeEscherichia coli in the presence of polymyxin nonapeptide, a membrane permeabilizing material. It seems, from the elemental content of the photosensitized Gram-positive and Gram-negative bacterial cells, that there is a difference in the killing mechanism of haematoporphyrin as compared to that of MgTBP or CdTX.     

Helium-neon laser: A contraindication for infected wounds? In vitro study on pseudomonas aeruginosa

The purpose of this investigation was to examine the susceptibility ofPseudomonas aeruginosa to helium-neon (He-Ne) laser in order to gain an insight into the clinical implications for this type of low-level laser therapy (LLLT) in the treatment of infected wounds. Suspensions, in the presence and the absence of methylene blue (MB, 0.001% w/v), were exposed to the light from a 9 mW He-Ne laser for 5 and 50 min. Exposure of samples without MB did not affect the viability of this organism based on the lack of significant differences in the number of colony forming units (CFU) between irradiated and control samples. In the presence of MB, statistically significant mean reductions in CFU of 39.5% (22.6 J cm^-2) and 58.2% (226 J cm^-2) were found, and growth-free zones in irradiated confluent platings could be observed. Since no logarithmic reductions were achieved, the clinical implementation of He-Ne laser as a bactericidal agent seems to be of no relevance yet. Otherwise, no stimulation of bacterial growth was observed. Therefore, infection as a contraindication for LLLT should be excluded.     

Bactericidal effects of different laser wavelengths on periodontopathic germs in photodynamic therapy

 This study was an attempt to clarify whether the bactericidal effects of photodynamic therapy (PDT) are wavelength or dose-dependent. We also attempted to create an optimised protocol for a light-based bactericidal modality to eliminate periodontal pathogens. Cultures of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphromonas gingivalis, Prevotella intermedia, and Streptococcus sanguis, were exposed to a He-Ne laser (632.8 nm) with a 30 mW power output, a 100 mW diode laser at 665 nm, or a 100 mW diode laser at 830 nm, in the presence or absence of methylene blue (MB) as a photosensitiser. A control group was also used with exposure to MB alone without laser exposure. The cultures were analysed by viable counts. The results indicated that exposure to the 100 mW laser light could eliminate up to 40% of bacteria on average. In particular, the most effective killing occurred with exposure to laser light in combination with the MB photosensitiser. The results of kinetic studies indicated that the best PDT response rate was achieved with a 60 s (energy density 21.2 J/cm²) exposure to the 665 nm wavelength diode laser in the presence photosensitiser. In this condition, approximately 95% of A. actinomycetemcomitans and F. nucleatum, and 99–100% of the black-pigmented bacteria (P. gingivalis and P. intermedia) and S. sanguis were eliminated. These results showed that both wavelength and energy density are important factors, and that a low power laser of optimal wavelength and dosage, in combination with an appropriate photosensitiser, is a practical bactericidal modality. We concluded that using a diode laser of proper power and wavelength to deliver 60 s of irradiation could be a useful adjunct with mechanical debridement in the prevention of the re-colonisation of subgingival lesions by pathogenic microorganisms. Received: 29 November 2001 / Accepted: 18 June 2002     

Effect of environmental factors on the lethal photosensitization ofCandida albicans in vitro

Suspensions ofCandida albicans in saliva, serum, broth or saline were treated with toluidine blue O or methylene blue and exposed to light from a helium/neon orgallium aluminium arsenide laser, respectively, for 120 s and survivors enumerated. Experiments were also carried out in broth at a pH ranging from 4.0 to 7.0. Appreciable kills (>10⁶ cfu ml⁻¹) of the yeast were obtained regardless of the nature of the suspending fluid. Lethal photosensitization of the yeast was also possible at a pH as low as 4.0 Only short-term (3 min) incubatin of the photosensitizer with the yeast was necessary to render it susceptible to killing by laser light.     

High-power helium–neon laser irradiation inhibits the growth of traumatic scars in vitro and in vivo

This study explored the inhibitory effect of the high-power helium–neon (He–Ne) laser on the growth of scars post trauma. For the in vitro study, human wound fibroblasts were exposed to the high-power He–Ne laser for 30 min, once per day with different power densities (10, 50, 100, and 150 mW/cm²). After 3 days of repeated irradiation with the He–Ne laser, fibroblast proliferation and collagen synthesis were evaluated. For in vivo evaluation, a wounded animal model of hypertrophic scar formation was established. At postoperative day 21, the high-power He–Ne laser irradiation (output power 120 mW, 6 mm in diameter, 30 min each session, every other day) was performed on 20 scars. At postoperative day 35, the hydroxyproline content, apoptosis rate, PCNA protein expression and FADD mRNA level were assessed. The in vitro study showed that the irradiation group that received the power densities of 100 and 150 mW/cm² showed decreases in the cell proliferation index, increases in the percentage of cells in the G0/G1 phase, and decreases in collagen synthesis and type I procollagen gene expression. In the in vivo animal studies, regions exposed to He–Ne irradiation showed a significant decrease in scar thickness as well as decreases in hydroxyproline levels and PCNA protein expression. Results from the in vitro and in vivo studies suggest that repeated irradiation with a He–Ne laser at certain power densities inhibits fibroblast proliferation and collagen synthesis, thereby inhibits the growth of hypertrophic scars.     

Photodynamic antimicrobial chemotherapy (PACT) with methylene blue increases membrane permeability in Candida albicans

Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy that combines light and a photosensitizing drug, promoting a phototoxic effect on the treated cells, in general via oxidative damage. In this work we studied the effect of PACT, using methylene blue (MB), on the permeability of Candida albicans membrane. Our results demonstrated that the combination of MB and laser (684 nm) promoted a decrease in Candida growth. The inhibition was more pronounced in the presence of 0.05 mg/ml MB and with an energy density of 28 J/cm². The decrease in Candida growth was associated with an increase in membrane permeabilization. Thus, we suggest that a PACT mechanism using MB can be related to damage in the plasma membranes of the cells.     

Safety assessment of oral photodynamic therapy in rats

Photodynamic therapy (PDT) is based on the synergism of a photosensitive drug (a photosensitizer) and visible light to destroy target cells (e.g., malignant, premalignant, or bacterial cells). The aim of this study was to investigate the response of normal rat tongue mucosa to PDT following the topical application of hematoporphyrin derivative (Photogem®), Photodithazine®, methylene blue (MB), and poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with MB. One hundred and thirty three rats were randomly divided in various groups: the PDT groups were treated with the photosensitizers for 10 min followed by exposure to red light. Those in control groups received neither photosensitizer nor light, and they were subjected to light exposure alone or to photosensitizer alone. Fluorescent signals were obtained from tongue tissue immediately after the topical application of photosensitizers and 24 h following PDT. Histological changes were evaluated at baseline and at 1, 3, 7, and 15 days post-PDT treatment. Fluorescence was detected immediately after the application of the photosensitizers, but not 24 h following PDT. Histology revealed intact mucosa in all experimental groups at all evaluation time points. The results suggest that there is a therapeutic window where PDT with Photogem®, Photodithazine®, MB, and MB-loaded PLGA nanoparticles could safely target oral pathogenic bacteria without damaging normal oral tissue.     

EFFECT OF METHYLENE BLUE ON THE ABSORBANCE OF SOLUTIONS OF HAEMOGLOBIN

The effect of the i.v. administration of methylene blue dyeon haemoglobin oxygen saturation, measured by a pulse oximeterwas investigated. The absorption spectra of various concentrationsof haemoglobin, oxyhaemoglobin and methylene blue were compared.The amount of 660-nm light absorbed by methylene blue was sufficientto cause a factitious haemoglobin desaturation as measured bythe pulse oximeter.     

Effects of cavity preparation using a nanosecond-pulsed Nd-YAG laser on tooth-restoration interface

This study evaluated the tooth-restoration interface between composite resin or glass ionomer cement and the tooth surface in teeth where caries removal and cavity preparation were performed either with the dental drill, the dental drill and laser irradiation, or laser irradiation alone. A nanosecond-pulsed Nd-YAG laser was used at energy densities of 2–8 J cm^-2. After tooth restoration, microleakage tests were performed using methylene blue. Using general linear model procedures, no significant differences in microleakage were determined between ‘drill’ and ‘drill and laser’-prepared groups (p< 0.01) in resinand in glass-ionomer-restored teeth. These specimens showed significantly less dye penetration than the ‘laser only’ group (p< 0.0001). No correlation was found between fluence and microleakage using either restorative material (p = 0.8). Dye penetration was significantly greater in the composite-filled than in the glass-ionomer group (p< 0.05). Mechanisms influencing laser effects on bonding require further investigation before clinical application of lasers as an alternative to the dental drill can become viable.     

Photodynamic Inactivation of the Single Crayfish Nerve Cell: Dynamics of Electrophysiological Responses and Comparison of Photosensitisers

. The study of single neuron response to photodynamic effect provides a means for the study of the dynamics of cytotoxic events leading to cell death and allows comparison of the phototoxicity of different photosensitisers. Isolated crayfish stretch receptor neurons were photosensitised for 30 min, then irradiated with a He-Ne laser (632.8 nm; 0.3 W/cm²) until irreversible firing cessation. The dynamics of neuron firing frequency were continuously recorded throughout. The following photosensitisers were studied: methylene blue, janus green B, protoporphyrin IX, chlorins e ₆ and p ₆, haematoporphyrin derivative (Photoheme) and sulphonated aluminium phthalocyanine (Photosens). Nerve cells were found to be insensitive to either He-Ne laser irradiation or photosensitisation alone, but very vulnerable to the photodynamic effect: neurons changed firing rate and died at nanomolar concentrations of photosensitisers. The dynamics of neuron responses was found to depend on photosensitiser type and concentration. The current approach provides a means of evaluation of initial threshold cell membrane alteration and cytotoxic events leading to cell death. The dependence of firing acceleration rate and neuron lifetime on photosensitiser concentration additionally allowed comparison of efficiencies of different photosensitisers. Photosens, Photoheme and chlorin p ₆ were found to be the most potent photosensitisers: neurons responded to their photodynamic effects at concentrations as low as 1–5 nM. Paper received 23 February 1998; accepted following revision 7 December 1998.     

Evaluation of inflammatory biomarkers associated with oxidative stress and histological assessment of low‐level laser therapy in experimental myopathy

The objective of the present work was to study the effect of helium–neon (He–Ne) and gallium–arsenide (Ga–As) laser upon inflammatory biomarkers associated with oxidative stress: fibrinogen, nitric oxide (NO), L ‐citrulline, and superoxide dismutase (SOD). These were evaluated through histological assessment, in rats with experimental myopathy. Materials and Methods: The groups studied were: (A) control, (B) injured, (C) injured and treated with He–Ne laser, (D) injured and treated with Ga–As laser, (E) irradiated with He–Ne; and (F) irradiated with Ga–As laser. Myopathy was induced by injecting 0.05 mg/rat/day of adrenaline in the left posterior limb muscle at the same point on 5 consecutive days, in groups B, C, and D. Low‐level laser therapy (LLLT) was applied with 9.5 J/cm² daily for 7 consecutive days with each laser. The determination of the biomarkers was made by spectrophotometry. The muscles (5/8, single blinded) were stained with Gomori Trichrome and examined by optic microscopy. The quantitative variables were statistically analyzed by the Fisher's test and categorical data by the Axionvision 4.8 program. Pearson's chi‐squared test was applied, setting significant difference at P < 0.05 for all cases. Results: In group B, the biomarkers were significantly increased compared to the other groups (P < 0.001), except for NO which in group B decreased significantly (P < 0.001). In group B, there was a higher inflammatory infiltration level (80.67%) in relation to destroyed fibers. Conclusions: LLLT caused significant changes in inflammatory biomarkers and oxidative stress: decreased levels of fibrinogen, L ‐citrulline and SOD as opposed to the increase of NO in rats with experimental myopathies and significant muscle recovery. Lasers Surg. Med. 42:577–583, 2010. © 2010 Wiley–Liss, Inc.     

Photodynamic therapy: Optimal treatment parameters in murine fibrosarcoma

We have investigated the efficacy of photodynamic therapy (PDT) by using two preparations of haematoporphyrin derivative (HPD) that demonstrated different biological activities against experimental murine fibrosarcoma RIF-1 in C3H/He mice. We have been able to demonstrate a minimum clonogenic survival rate of 0.25% by using the more active HPD at 20 mg/kg with a 40-h retention time and a total laser light dose of 100 J/cm². Further, we noted that clonogenic survival rates of 21.6% and 25.0% respectively could be achieved by using the less active HPD (at 20 mg/kg) with a laser light dose of 150 J/cm², or the more active HPD (at 20 mg/kg) with a laser light dose of 20 J/cm². In both cases necrosis of the surrounding normal tissue was absent. Necrosis of the normal tissue surrounding the tumour was shown to be associated with high laser energy (100 J/cm² and higher) in conjunction with a high dose (20 mg/kg) of the more active HPD. A comparison of survival curves as a function of laser energy for the RIF-1 cells following PDT with the two different preparations of HPD showed a difference in the kinetics of cell death. A curve with a shoulder region and aD _o (mean lethal dose) of 41 J/cm² was obtained when the less active HPD was used, whereas PDT using the more active HPD resulted in a curve with no shoulder and aD _o of 16 J/cm².     

Antimicrobial photodynamic therapy as a new approach for the treatment of vulvovaginal candidiasis: preliminary results

In this work, we present the efficacy of photodynamic therapy against yeast cells in an animal model. We tested two photosensitizers, methylene blue and protoporphyrin IX. Thirty-seven female BALB-c mice with a body mass of 20–25 g were used. To achieve persistent vaginitis, estrus was induced by subcutaneous injection of 0.1 mg/mL estradiol valerate applied weekly. Three days after pseudo-estrus, intravaginal inoculation with Candida albicans was performed. Mice were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection before inoculation, and antimicrobial photodynamic therapy (aPDT) was performed 5 days after fungal inoculation. Two photosensitizers were tested, methylene blue (MB; 100 μM) and protoporphyrin IX (PpNetNI; 10 μM). Two custom-made LEDs emitting light at 660 and 630 nm at approximately 800 mW each were used for irradiation. The aPDT treatment reduced the fungal colony-forming units (CFUs) by one order of magnitude for the MB (p?=?0.020) and PpNetNI (p?=?0.018) photosensitizers. Seven days after the treatment, there were significantly fewer CFUs compared to the control group (p?=?0.041 and p?=?0.035 for MB and PpNetNI, respectively), but this was not increased compared to the initial number immediately after aPDT. Using aPDT as a therapeutic option to decrease fungal infection in a vaginal candidiasis model resulted in a significant reduction in the C. albicans population. Both photosensitizers were effective for preventing reinfection within 7 days. The aPDT also had no effect on the vaginal mucosa at the ultrastructural level. In addition to the fungicide effect, we observed reduced swelling and lack of the formation of abscesses, microabscesses coating the cornified epithelial layer, and the accumulation of neutrophils in the submucosa.     

Effect of gallium arsenide diode laser on human periodontal disease: a microbiological and clinical study

BACKGROUND AND OBJECTIVE: The present study is aimed to describe short-term results on selected microbiological and clinical parameters obtained by treatment with soft laser in conjunction with methylene blue and/or mechanical subgingival debridement in human periodontal disease. STUDY DESIGN/MATERIALS AND METHODS: Ten patients, in whom each dental quadrant was randomly designated to receive one of four types of treatment procedures, were included in the study. Groups of quadrants received: scaling/root planing (SRP); laser application (L); SRP combined with L (SRP/L); oral hygiene instructions (OHI). Four single rooted teeth (one in each quadrant), having an interproximal site with a probing depth of 4 mm mesio-buccally, were selected in each patient. The selected teeth were first assessed for microbiological (one site/tooth) and then for clinical variables (six sites/tooth). Supragingival irrigation with methylene blue was performed prior to laser application. The microbiological (proportions of obligate anaerobes) and clinical measurements (plaque and gingival indices, bleeding on probing, probing pocket depth) were evaluated over a period of 32 days. RESULTS: Only the SRP/L and SRP groups provided significant reductions in the proportions of obligate anaerobes before and after treatments with no significant differences in between. Parallel to the microbiological changes, both SRP/L and SRP resulted in similar clinical improvements, whereas L alone revealed a limited effect similar to OHI. CONCLUSION: Within the limits of this study, methylene blue/soft laser therapy provided no additional microbiological and clinical benefits over conventional mechanical debridement.     

Effect of 308-nm excimer laser light on peri-implantitis-associated bacteria—an in vitro investigation

Dental implants are becoming increasingly important in prosthodontic rehabilitation. Bacterial infections, however, can induce bone loss and jeopardize clinical success. Recent literature has demonstrated that infrared CO₂ laser light is suitable for the decontamination of exposed implant surfaces. The aim of the present study was to investigate the influence of 308-nm excimer laser irradiation on peri-implantitis-associated bacteria in vitro. In this study, a XeCl excimer laser (308 nm) was used (Summit Technology, Boston, USA). Both aerobe (Streptococcus mutans, S. sanguis, Actinomyces naeslundii) and anaerobe microorganisms (A. odontolyticus, Prevotella melaninogenica) were tested. According to previous studies, a constant energy of 0.8 J/cm² and a constant frequency of 20 Hz were used for all irradiations. Colony-forming units after laser irradiation were counted. Excimer laser irradiation showed significant influence on the growth of all microorganisms. As compared to S. mutans and S. sanguis, A. naeslundii demonstrated higher sensitivity to laser irradiation. Anaerobe microorganisms, in contrast, demonstrated that a total of 200 pulses were sufficient to reduce the replication of these germs for more than 99.9%. Excimer laser irradiation (λ = 308 nm) can significantly reduce both aerobe and anaerobe microorganisms. Depending on the parameters chosen, 200 pulses are sufficient for sterilization. New studies are necessary to evaluate if this wavelength is more of value in the treatment of peri-implantitis than other wavelengths or conventional therapies.     

Laser ablation of dyed acrylic bone cement

Revision surgery of cemented implants is indicated when mechanical failure causes severe pain and/or loss of function for the patient. Successful revision arthroplasty of cemented implants requires complete removal of the existing cement. Removal of old cement is an arduous task often causing damage to the surrounding bone tissue. In this study, the authors investigate the use of an Argon laser and the addition of dyes to enhance the laser ablation of bone cement. Methylene blue and red dye #13 were each added separately to polymethylmethacrylate (PMMA) bone cement powder. A continuous wave Argon ion laser (λ = 514 nm) was used for cement ablation. Cement samples were ablated at different power levels (1.5, 2.3, and 3.0 W) and exposure times (30, 60, 90, 120 sec). The results show that the Argon laser was unable to ablate undyed PMMA. However, the addition of either methylene blue or red dye #13 greatly improved cement ablation by altering the cements' absorption characteristics. Results of Student's t-tests show a statistical difference between red and blue dyed PMMA mean ablation areas at all energy levels tested (P < .0002). As expected, all red ablation areas were greater than blue ablation areas at each energy level tested since red dye absorbs more energy at 514 nm than methylene blue dye. The results of this study suggest that by selectively altering the absorption characteristics of PMMA, laser removal of bone cement can be achieved. In addition, this study also shows that bone tissue does not absorb visible light energy at 514 nm, suggesting that bone cement may be removed with minimal damage to the surrounding bone tissue. Lasers Surg. Medicine 20:280–289, 1997. © 1997 Wiley-Liss, Inc.