Laser photobiomodulation (830 and 660 nm) in mast cells,VEGF, FGF,and CD34 of the musculocutaneous flap in rats submitted to nicotine

The aim of this study was to investigate the effect of laser photobiomodulation (PBM) on the viability of the transverse rectus abdominis musculocutaneous (TRAM) flap in rats subjected to the action of nicotine. We evaluated 60 albino Wistar rats, divided into six groups of ten animals. Group 1 (saline) underwent the surgical technique to obtain a TRAM flap; group 2 (laser 830 nm) underwent the surgical technique and was irradiated with a laser 830 nm; group 3 (laser 660 nm) underwent the surgical technique and was irradiated with a laser 660 nm; group 4 was treated with nicotine subcutaneously (2 mg/kg/2×/day/4 weeks) and underwent surgery; group 5 (nicotine?+?laser 830 nm) was exposed to nicotine, underwent the surgical technique, and was irradiated with a laser 830 nm; group 6 (nicotine?+?laser 660 nm) was exposed to nicotine, underwent the surgical technique, and was irradiated with a laser 660 nm. The application of PBM occurred immediately after surgery and on the two following days. The percentage of necrosis was assessed using the AxioVision® software. The number of mast cells (toluidine blue staining) was evaluated, and immunohistochemistry was performed to detect vascular endothelial growth factor expression (anti-VEGF-A), fibroblasts (anti-basic FGF), and neoformed vessels (anti-CD34). PBM with a wavelength of 830 nm increased the viability of the TRAM flap, with a smaller area of necrosis, increased number of mast cells, and higher expression of VEGF and CD34. PBM increases the viability of musculocutaneous flaps treated with to nicotine.     

Lasers in surgery and medicine reviewers. November 1, 1992 through October 31, 1993

Chloro-aluminum sulfonated phthalocyanine (CASPc) is a photo-chemically active dye employed in photodynamic therapy (PDT). CASPc is a potent generator of singlet oxygen when irradiated with 675 nm light and is also capable of fluorescence, allowing visualization of the dye in tissues. We devised an angiography system using CASPc fluorescence to determine its localization in experimental choroidal neovascularization in monkeys and then investigated the ability of CASPc to produce photochemical closure of neovascularization upon irradiation with 675nm laser light. Fluorescent imaging indicated that CASPc localized angio-graphically in areas of neovascularization for at least 24 hours. Irradiation with 675 nm laser light 5–30 minutes after CASPc injection produced complete closure of choroidal neovascularization with minimal damage to overlying retina. We conclude that CASPc localizes in neovascular choroidal vessels and that CASPc photodynamic therapy can produce closure of these choroidal vessels. © 1994 Wiley-Liss, Inc.     

Discrimination of normal and atherosclerotic aorta by laser-induced fluorescence

Precise targeting of laser energy to atherosclerotic plaque is crucial for the safe performance of laser angioplasty. The present study was designed to evaluate whether laser-induced fluorescence could distinguish atherosclerotic from normal aorta. Segments of human aorta obtained at necropsy were classified grossly and histologically as normal aorta (n = 7), thin yellow fatty plaque (n = 5), and thick white atheromatous plaque (n = 9), and analyzed by laser-induced fluorescence spectroscopy using a pulsed nitrogen laser. Fluorescence spectra were recorded over a wavelength range from 385 to 725 nm. Normal specimens had maximal fluorescence intensity at 514 nm. A prominent fluorescence peak at 448 nm was present in specimens characterized as white atheromatous plaque and at 538 nm in specimens characterized as yellow fatty plaque. The ratios of fluorescence intensity at 448 nm/514 nm and at 538 nm/514 nm correctly classified all specimens according to their gross and histologic type (p < .001). Thus, a “smart” laser angioplasty catheter system might incorporate low-power laser radiation for arterial fluorescence spectroscopy to guide delivery of high-power laser radiation for plaque ablation.     

Laser-induced fluorescence detection of atherosclerotic plaque with hematoporphyrin derivative used as an exogenous probe

Clinical use of laser angioplasty is limited by the lack of an adequate guidance system. As a first step toward developing a reliable guidance system, laser-induced surface fluorescence and a fluorescent probe were used to differentiate plaque from normal arterial wall. The aortas from four normal New Zealand white rabbits and six atherosclerotic rabbits were studied in vitro. Rabbits from each group received 2.5 mg/kg of hematoporphyrin derivative intravenously 24 hours before death. Segments of the aortas were irradiated with a helium-cadmium laser, and the tissue surface fluorescence spectra were recorded with an optical multichannel analyzer. A plaque index, based on the resulting spectra, was calculated for each specimen of aorta. The spectra from normal aorta without hematoporphyrin derivative, normal aorta with hematoporphyrin derivative, and from plaque of atherosclerotic rabbits without hematoporphyrin derivative showed the same wavelength dependence. The plaque index values were not significantly different from one another. However, in plaque from atherosclerotic rabbits given hematoporphyrin derivative, the spectrum was markedly different, showing a broad spectral peak at 632 nm. This spectral peak corresponds to the spectral peak of hematoporphyrin derivative and was only seen in the plaque of atherosclerotic rabbits given hematoporphyrin derivative. The plaque index for these specimens was significantly different from all other specimens (p less than 0.001). This difference in fluorescence spectra and plaque index could be incorporated into a guidance system for laser angioplasty.     

Quantitative assessment of chondrocyte viability after laser mediated reshaping: a novel application of flow cytometry

BACKGROUND AND OBJECTIVES: Lasers can be used to reshape cartilage by accelerating mechanical stress relaxation. In this study, fluorescent differential cell viability staining and flow cytometry were used to determine chondrocyte viability following laser heating. STUDY DESIGN/MATERIALS AND METHODS: Porcine septal cartilages were irradiated with an Nd:YAG laser (lambda = 1.32 microm, 25 W/cm(2)) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 seconds). Irradiated samples were then divided into two groups analyzed immediately and at 5 days following laser exposure. Chondrocytes were isolated following serial enzymatic digestion, and stained using SYTO/DEAD Red (Molecular Probes, Eugene, OR). A flow cytometer was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post-irradiation debris in each treatment population. RESULTS: Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after one irradiation, were viable while non-irradiated controls were 100% viable. Specimens irradiated two or three times demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 and 16% after two and three exposures, respectively. In those samples maintained in culture medium and assayed 5 days after irradiation, viability was reduced by 28-88%, with the least amount of deterioration in untreated and singly irradiated samples. CONCLUSIONS: Functional fluorescent dyes combined with flow cytometric analysis successfully determines the effect of laser irradiation on the viability of reshaped cartilage.     

Thermal laser probe angioplasty: influence of constant tip temperature, plaque composition, and probe/vessel diameter ratio

Thermal laser angioplasty uses constant laser power, producing widely variable tip temperatures in vivo. Results have been suboptimal. We studied the effect of 50-400 degrees C tip temperatures on depth of ablation at 192 sites on plaqued and normal human aorta in vitro, and the angiographic and histologic response in vivo of 300-400 degrees C at probe/vessel ratios of 0.5-1.0, in 40 normal canine femoral artery segments. In vitro, there was a direct relationship between tip temperature and depth of ablation, r = 0.71 (all segments), r = 0.74 for fibrous plaque, but a poor correlation in fatty plaque r = 0.35. In fibrous plaque, there was proportionately more ablation at tip temperatures greater than 300 degrees C, mean depth 0.62 mm, than at 150-300 degrees C, mean 0.37 mm, (P less than .001). Ablation was similar in plaqued and normal aorta. In vivo, 300 degrees C, 350 degrees C, and 400 degrees C produced similar effects. At probe/vessel ratios less than 0.8, only disruption of internal elastic lamina was observed. At ratios greater than or equal to 0.8, spasm occurred in 39% (7/18), transmural damage in 28% (5/18), and perforation in one of 18. Ablation is not selective for plaque and is highly variable in fatty plaque. Tip temperatures above 300 degrees C produce greater ablation than at lower temperatures. In clinical applications, probe/vessel rations less than or equal to 0.7 may be most appropriate, and it appears that thermal remodeling may contribute more to outcome than plaque ablation.     

PSD-007介导的光动力疗法治疗小鼠骨肉瘤的实验研究

目的 观察光敏剂PSD-007对小鼠骨肉瘤细胞LM-8的体外及体内光动力效应.方法 PSD-007与LM-8细胞共同孵育后以激光照射,应用MTT法测定光密度(OD540)值,计算抑制率.40只C3H小鼠接种LM-8细胞,皮下瘤块直径7～8 mm时随机分为:(1)对照组,空白对照、生理盐水加光照、注射PSD-007不光照;(2)光动力治疗组,分别注射5mg/kg、10mg/kg PSD-007,6 h后以激光照射.1周后测量瘤体大小、重量,计算抑瘤率并行病理学检查.C3H小鼠30只建立肿瘤模型,肿瘤直径达10～12mm时,分别行肿瘤边缘切除无光动力治疗(对照组)、边缘切除后240 J/cm2光动力治疗及边缘切除后360 J/cm2光动力治疗.4周后比较肿瘤复发率.结果 体外只光照或只注射PSD-007对LM-8细胞均无杀伤作应.PSD-007浓度越高、激光照射强度越大,LM-8细胞OD540值越小.PSD-007浓度>4μg/ml,光照强度>6 J/cm2时,抑制率>50%.光镜下细胞形态呈坏死或凋亡样改变.体内实验显示光动力治疗组的肿瘤体积及瘤重均减小,肿瘤边缘切除高强度激光照射组的复发率较对照组低.结论 PSD-007对LM-8细胞有明确的光动力抑制效应,其作用大小取决于其浓度和激光照射强度.光动力疗法可以降低肿瘤边缘切除后的复发率.

Abstract：

Objective To evaluate the PSD-007-mediated photodynamic effect on mouse osteosarcoma cell line LM-8, both in vitro and in vivo. Methods LM-8 cells were incubated with different concentrations of PSD-007 for 4 hours and then followed different laser irradiations. After photodynamic therapy (PDT), cell viability was measured using MTT assay and the optical density in each experiment was measured at 450 nm with a micro plate reader. The inhibition rate of cell growth was calculated. Four-week-old female C3H mice were used for implantation of LM-8 cells. When the diameter of tumor reached up to 7-8 mm, the mice were randomly divided into following groups: 1) control group, including untreated control, saline with laser irradiation, PSD-007 without laser irradiation; 2) PDT group, PSD-007 (5 and 10 mg/kg) was injected intravenously into the mice, and the tumor site was irradiated with laser light 6 hours after injection. Seven days after PDT, the size and weight of the tumors were measured. The inhibition rate of tumor was calculated, and all tumor specimens were taken for pathologic examination. After the diameter of tumor was 10-12 mm, the tumors were performed a marginal resection and subsequently followed 3 different treatments: without PDT (control), PDT with 240 J/cm2 or 360 J/cm2 laser irradiation. After 4 weeks treatment, the tumor recurrence rates were analyzed. Results MTT assay revealed that the cytotoxic effect of PDT on the LM-8 cells was positively correlated with the concentration of PSD-007 and the level of laser irradiation. When the concentration exceeded 4μg/ml, and the energy exceeded 6 J/cm2, the inhibition ratio was over 50%. No anti-tumor effect was observed in the cells treated with only laser irradiation or PSD-007 injection. Compared with the control group, the size and weight of the tumors were obviously decreased after PDT. PDT performed after marginal resection of the tumor reduced the rate of local recurrence. Conclusion PDT with PSD-007 showed cytotoxic effect on the LM-8 cells, and which performed after marginal resection of the tumor reduced the rate of local recurrence.     

Acute Effects of Photodynamic Treatment on Elastic Fibre Network in Atherosclerotic Plaques of Rabbit Aorta

Our objective was to demonstrate the acute effects of photodynamic treatment on the elastic framework in the atherosclerotic plaque. The photosensitiser, mono-l-aspartyl chlorin 5₆ (NP5₆), was administered intravenously, at a dose of 5 mg/kg of body weight, to cholesterol-fed, atherosclerotic rabbits. Six hours later, visible atherosclerotic lesions of the abdominal aorta were irradiated with a diode laser, wavelength 664 nm. The energy administered ranged from 50 J/cm² to 200 J/cm². The abdominal aorta was then excised and digested by a modified hot alkaline method. Scanning electron microscopy revealed the destruction of the architecture of the elastic fibre network in the atherosclerotic plaque, whereas no change was seen in the non-atheromatous aortic wall. Damage to the elastic fibre network was more obvious in plaque that was exposed to higher energy levels. No significant changes were observed in laser-treated plaques in animals that did not receive the NP5₆ pretreatment. Findings suggest that atherosclerotic plaques of the abdominal aorta can be selectively degraded by laser irradiation following pretreatment with the photosensitiser, NP5₆. Received for publication 23 July 1997; accepted following revision 20 January 1998.     

Laser light influences cellular viability and proliferation in diabetic-wounded fibroblast cells in a dose- and wavelength-dependent manner

Phototherapy stimulates metabolic processes in healing wounds. Despite worldwide interest, phototherapy is not firmly established or practiced in South Africa. This study aimed to determine which dose and wavelength would better induce healing in vitro. Diabetic-induced wounded fibroblasts were irradiated with 5 or 16 J/cm² at 632.8, 830, or 1,064 nm. Cellular morphology, viability (Trypan blue and apoptosis), and proliferation (basic fibroblast growth factor) were then determined. Cells irradiated with 5 J/cm² at 632.8 nm showed complete wound closure and an increase in viability and basic fibroblast growth factor (bFGF) expression. Cells irradiated at 830 nm showed incomplete wound closure and an increase in bFGF expression. Cells irradiated at 1,064 nm showed incomplete closure and increased apoptosis. All cells irradiated with 16 J/cm² at all three wavelengths showed incomplete wound closure, increased apoptosis, and decreased bFGF expression. This study showed that diabetic-wounded cells respond in a dose- and a wavelength-dependent manner to laser light. Cells responded the best when irradiated with a fluence of 5 J/cm² at a wavelength of 632.8 nm.     

Critical parameters in the cytotoxicity of photodynamic therapy using a pulsed laser

. Photodynamic therapy (PDT) using a pulsed laser is becoming popular, but its cytotoxic effect is still not clear. We therefore studied the cytotoxicity of PDT using a pulsed laser by changing its irradiation parameters and compared the degrees of cytotoxicity with those of PDT using continuous-wave (CW) light sources. Mice renal cell carcinoma cells were incubated with PAD-S31, a water-soluble photosensitiser of which the excitation peak is 670 nm, and were then irradiated with either a tungsten lamp, a CW diode laser, or a nanosecond pulsed Nd:YAG laser-based optical parametric oscillator system. When the PAD-S31 concentration and total light dose were constant (12 μg/ml and 40 J/cm², respectively), the CW laser caused fluence rate-dependent decrease in cellular proliferation until the fluence rate reached 90 mW/cm², at which point inhibition of cellular proliferation was more than 80%. The cytotoxicity then became almost saturated at fluence rates of>90 mW/cm². On the other hand, inhibition of cellular proliferation in samples irradiated with the pulsed laser reached 80% even at the fluence rate of 15 mW/cm², and, interestingly, the cytotoxicity paradoxically decreased with increase in the fluence rate. Moreover, the cytotoxicity in the PDT using the pulsed laser depended on the repetition rate. The inhibition of cellular proliferation by PDT using 30-Hz irradiation was greater than that by PDT using 5-Hz irradiation when the same fluence rates were used. These results suggest that the efficacy of PDT using a pulsed laser depends considerably on fluence rate and repetition rate. Paper received 4 March 2002; accepted after revision 24 May 2002. Correspondence to: Yuji Morimoto, MD, PhD, Department of Medical Engineering, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Tel: +81-42-995-1596; Fax: +81-42-996-5199; e-mail: moyan@interlink.or.jp     

Photoeradication of Helicobacter pylori using 5-aminolevulinic acid: preliminary human studies

BACKGROUND AND OBJECTIVES: Helicobacter pylori (HP) is an endemic pathogenic bacterium causing gastritis and gastroduodenal ulceration in humans and is linked to the development of gastric malignancies. These first human in vivo studies investigated the photoeradication of HP using laser and white light. STUDY DESIGN/MATERIALS AND METHODS: In 13 HP-positive volunteers, a zone of gastric antrum was irradiated with laser (410 nm, 50 J/cm(2)) or endoscopic white light (10 J/cm(2)) 45 minutes after oral 5-aminolevulinic acid (5-ALA) 20 mg/kg. HP-eradication was assessed by biopsy urease test and HP-culture from irradiated and control zones 5 minutes, 4 and 48 hours post-irradiation. RESULTS: A maximum eradication effect was achieved at 4 hours post-irradiation when 85% of biopsies in the monochromatic and 66% in the white light exposed zones, and 58 and 33% in the respective control zones were HP-negative. CONCLUSIONS: HP numbers were greatly reduced following exposure to 5-ALA and either laser or white light in vivo. Photoeradication appears feasible, but further light dosimetry and the development of convenient application methods is required.     

Cellular effects of the pulsed tunable dye laser at 577 nanometers on human endothelial cells, fibroblasts, and erythrocytes: an in vitro study

The 577-nm flashlamp-pumped tunable dye laser pulsed at 450 microseconds is rapidly becoming the treatment of choice for removal of portwine stains and other vascular ectasias. In this study, we examined the mechanisms of vessel destruction by determining the effects of laser irradiation on three types of primary target cells--erythrocytes, endothelial cells, and fibroblasts. Human endothelial cells and fibroblasts in microwell plates were irradiated at various energy densities with the laser, after which several aspects of cellular biology were determined, including 1) viability of cells by trypan blue exclusion test; 2) cell proliferation by [3H]thymidine incorporation; and 3) rate of protein synthesis using [3H]leucine incorporation as a marker. In endothelial cell cultures, both [3H]thymidine and [3H]leucine incorporations were inhibited at energy levels of 5-12 J/cm2 (P less than 0.01). In fibroblast cultures, cell proliferation was similarly inhibited, while supratherapeutic energy density (greater than or equal to 12 J/cm2) was required for inhibition of protein synthesis. The laser energy in the range of 5-8.5 J/cm2 had no effect on cell viability. Erythrocytes as target cells for laser energy demonstrated rapid, dose-dependent lysis, as determined by release of free hemoglobin into culture medium. Addition of erythrocytes into a coculture with endothelial cells abolished the direct inhibitory effect noted in cultures when endothelial cells were present alone. The results of the latter experiment imply that erythrocytes are the primary target cell absorbing the laser energy at 577 nm. However, direct laser effects on endothelial cells may also contribute to the mechanisms of ablation of the vascular ectasias by the tunable dye laser at 577 nm.     

Phototherapy promotes cell migration in the presence of hydroxyurea

Phototherapy has been shown to cause an increase in cell proliferation and migration. This study focused on viability (trypan blue), proliferation [sodium 3′-(1-(phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro)-benzene sulphonic acid hydrate (XTT) and adenosine triphosphate (ATP)] and migration of WS1 cells following irradiation in the presence of hydroxyurea (HU), which is an inhibitor of proliferation. Wounded cells were irradiated on days 1 and 4 with a fluence of 5 J/cm² with a helium–neon (He-Ne) laser at 632.8 nm. After a repair time of 24 h, cellular responses were assessed. Wounded irradiated cells without HU showed an increase in cell viability and proliferation, which was confirmed by complete wound closure by day 4. Although wounded irradiated cells treated with 5 mM HU showed incomplete wound closure, these cells showed increased migration compared with that of control cells. This study showed that laser irradiation using an He-Ne laser with a fluence of 5 J/cm² stimulates cell viability. The HU results confirmed that laser irradiation promotes cell migration and proliferation.     

Methods for the endoscopic photographic and visual detection of helium cadmium laser-induced fluorescence of Photofrin II

Endoscopic detection of small tumors is key to the early diagnosis and treatment of malignancy. This paper describes a simple, endoscopic detection system which enables tumor localization and a permanent record based on the laser-induced fluorescence of dihematoporphyrin ether (LIFD). Spectral analysis of dihematoporphyrin ether (DHE, Photofrin II) was performed with a Perkin Elmer LS-5 scanning fluorimeter. DHE at concentrations of 50 micrograms/ml and 5 micrograms/ml in 95% ethanol were tested, demonstrating fluorescence quenching at 50 micrograms/ml DHE at 406 nm excitation. This phenomenon was not observed at 442 nm excitation. Based on this data and the availability of the helium cadmium laser, a series of endoscopic detection systems was developed and tested utilizing a LiConix 4240NB helium cadmium laser (TEMoo, 442 nm, 40 mW). A fiber with a microdiverging (MDL) lens was used. Irradiance achieved at the tip of the fiber was 31.58 mW/cm2 for MDL. A Corning 34832 (550 nm) sharp cutoff barrier filter was coupled to an Olympus OES BF2T10 bronchoscope. Successful detection of LIFD was obtained. Direct observation of LIFD is possible when wearing Laserguard argon safety goggles (OD 15 at 488 nm, OD 11 at 514 nm). Photographic recording of LIFD was performed with the following cameras and parameters: Olympus OM-2S camera (OM2) with EES135 film (ISO 1600) with a 4-second exposure (method 1) and the Olympus OES SCP-10 instant camera with Polaroid 779 (ISO 640) film and a 120-second exposure (method 2). The photographic methods demonstrate the red fluorescence of DHE on filter paper disks at concentrations of 0.5 micrograms/ml (500 ng/ml). (ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of calcified atherosclerotic plaque by laser-induced plasma emission.

The use of fluorescence spectroscopy to discriminate atherosclerotic from normal tissue is limited by a lower sensitivity for calcified than noncalcified atherosclerotic plaque (65% vs. 93%, respectively). To evaluate plasma emission as a means to detect calcified plaque, 325 normal and atherosclerotic cadaveric aortic sites were irradiated through a 100-micron silica fiber in blood by a pulsed holmium laser (lambda = 2.1 microns, fluence = 4 J/mm2). A photodiode positioned near the proximal end of the fiber detected plasma emission during a laser pulse. Plasma emission was detected at 0% (0/110) of normal, 0% (0/107) of noncalcified atherosclerotic tissue, and 91% (98/108) of calcified atherosclerotic sites. Spectroscopic analysis confirmed the presence of calcium lines in the plasma emission from calcified atherosclerotic plaque. Although ablative fluences (greater than 3 J/mm2) were required for plasma generation, a single laser pulse ablated only to a depth of 67 +/- 16 microns in normal tissue. In an additional 10 calcified atherosclerotic sites, laser ablation was continued as long as plasma emission was detected. In all cases, plaque ablation was terminated before arterial perforation. Furthermore, the adjunctive use of plasma detection improved the accuracy of fluorescence spectroscopic classification of normal and atherosclerotic tissue. In conclusion, plasma detection has a high sensitivity (91%) and specificity (100%) for calcified atherosclerotic plaque and may be a useful adjunct for laser angioplasty guidance. Furthermore, plasma detection can be implemented both simply and inexpensively.     

The effect of low level laser irradiation on adult human adipose derived stem cells

This study investigated the effect of low level laser irradiation on primary cultures of adult human adipose derived stem cells (ADSC) using a 635-nm diode laser, at 5 J/cm(2) with a power output of 50.2 mW and a power density of 5.5 mW/cm(2). Cellular morphology did not appear to change after irradiation. Using the trypan blue exclusion test, the cellular viability of irradiated cells increased by 1% at 24 h and 1.6% at 48 h but was not statistically significant. However, the increase of cellular viability as measured by ATP luminescence was statistically significant at 48 h (p < 0.05). Proliferation of irradiated cells, measured by optical density, resulted in statistically significant increases in values compared to nonirradiated cells (p < 0.05) at both time points. Western blot analysis and immunocytochemical labeling indicated an increase in the expression of stem cell marker beta1-integrin after irradiation. These results indicate that 5 J/cm(2) of laser irradiation can positively affect human adipose stem cells by increasing cellular viability, proliferation, and expression of beta1-integrin.     

Laser-induced effects in different biological samples

Experiments were carried out on cancerous HeLa cells and blood serum using a double integrating sphere and a He-Ne laser to investigate the optical properties and cellular effects due to photodynamic therapy (PDT). In the first experiment, HeLa cells were exposed to Photofrin at concentrations of 0, 10, 20, 30, 50 and 112.4 μg/ml at an irradiance of 0.2 W/cm² using diode laser light. Using a confocal microscope, cell debris and morphological changes in HeLa cells were recorded at different Photofrin concentrations. The results showed cell debris in HeLa cells at the highest concentration of Photofrin. In a second experiment, photobleaching was observed in HeLa cells in the presence of various concentrations of 5-aminolaevulinic acid ranging from 0–50 μg/ml. There was progressive degradation of the 635 nm peak during continuous laser irradiation at an irradiance of 0.2 W/cm². We conclude that cells demonstrating high initial fluorescence undergo bleaching at a faster rate than those with lower fluorescence. Finally in a third experiment, cancerous and noncancerous blood serum was irradiated at an irradiance of 0.1 W/cm² using a He-Ne laser in conjunction with a double integrating sphere system. Forward and back scattering of normal and malignant serum showed an exponential decrease in fluorescence amplitude. The results indicate that there is notable amplitude difference between malignant and normal blood serum with malignant blood serum showing decreased scattering. These results have important implications for photodiagnosis and photodynamic therapy.     

Direct evidence that induced nitric oxide production in hepatocytes prevents liver damage during lipopolysaccharide tolerance in rats

BACKGROUND: The role of nitric oxide (NO) in lipopolysaccharide (LPS) tolerance in the liver has been investigated in a number of previous studies, but it is still not clear whether NO is cytotoxic or cytoprotective. The aims of this study were to investigate whether low-dose LPS (LLPS)-induced hepatic production of NO is beneficial and to clarify the origins of cytoprotective NO-producing cells in the liver during LPS tolerance. MATERIALS AND METHODS: Male Wistar rats received saline or LLPS intraperitoneally (i.p.; 0.01-1000 microg/kg) followed by a high dose of LPS (HLPS, 5 mg/kg) at various time intervals (4-16 h). NG-nitro-L-arginine methyl ester (L-NAME) was used to investigate the effects of inhibition of NOS. 4,5-Diaminofluorescein (DAF-2) was used to identify NO-producing cells in isolated liver cells in vitro. At various time points (4-16 h) after saline or LLPS (1 microg/kg, i.p.) injection, hepatocytes and Kupffer cells were isolated, incubated in 7 microm DAF-2 diacetate, and perfused with Krebs solution. Illumination at 495 nm revealed DAF-fluorescence (515 nm) in isolated cells under confocal laser fluorescence microscopy. The NO production in hepatocytes and Kupffer cells was assessed by the number of labeled cells per 1000 cells or per 100 cells, respectively. RESULTS: Pretreatment with LLPS (0.1-100 microg/kg) resulted in a significant reduction (maximal at 8 h) of the HLPS-induced liver damage. L-NAME abolished the LLPS-induced protection. The NO production in hepatocytes was significantly increased and reached a maximum of 84% of all cells 8 h after LLPS administration. By contrast, the NO production in Kupffer cells remained constant at 95%, even following preinjection of LLPS. CONCLUSION: LLPS-induced NO in hepatocytes, but not in Kupffer cells, exhibits cytoprotective effects on HLPS-induced liver damage, suggesting that NO has a beneficial role in the induction of the early phase of LPS tolerance.     

Low-level 809 nm GaAlAs laser irradiation increases the proliferation rate of human laryngeal carcinoma cells in vitro

The aim of the study was to investigate the effect of low-level 809 nm laser irradiation on the proliferation rate of human larynx carcinoma cells in vitro. Epithelial tumor cells were obtained from a laryngeal carcinoma and cultured under standard conditions. For laser treatment the cells were spread on 96-well tissue culture plates. Sixty-six cell cultures were irradiated with an 809 nm GaAlAs laser. Another 66 served as controls. Power output was 10 mW(cw) and the time of exposure 75–300 s per well, corresponding to an energy fluence of 1.96–7.84 J/cm². Subsequent to laser treatment, the cultures were incubated for 72 h. The proliferation rate was determined by means of fluorescence activity of a redox indicator (Alamar Blue Assay) added to the cultures immediately after the respective treatment. The indicator is reduced by metabolic activity related to cellular growth. Proliferation was determined up to 72 h after laser application. The irradiated cells revealed a considerably higher proliferation activity. The differences were highly significant up to 72 h after irradiation (Mann–Whitney U test, p < 0.001). A cellular responsiveness of human laryngeal carcinoma cells to low-level laser irradiation is obvious. The cell line is therefore suitable for basic research investigations concerning the biological mechanisms of LLLT on cells.     

A pilot study of ICG laser therapy of acne vulgaris: photodynamic and photothermolysis treatment

BACKGROUND AND OBJECTIVES: The near-infrared (NIR) laser radiation due to its high penetration depth is widely used in phototherapy. In application to skin appendages, a high selectivity of laser treatment is needed to prevent light action on surrounding tissues. Indocyanine green (ICG) dye may provide a high selectivity of treatment due to effective ICG uploading by a target and its narrow band of considerable absorption just at the wavelength of the NIR diode laser. The goal of this study is to demonstrate the efficacy of the NIR diode laser phototherapy in combination with topical application of ICG suggested for soft and thermal treatment of acne vulgaris. STUDY DESIGN/MATERIALS AND METHODS: Twenty-two volunteers with facile or back-located acne were enrolled. Skin sites of subjects were stained by ICG and irradiated by NIR laser-diode light (803 or 809 nm). One mg/ml solution of ICG was applied for 5 or 15 minutes to the cleaned skin site. Untreated, only stained and only light irradiated skin areas served as controls. For soft acne treatment, the low-intensity (803 nm, 10-50 mW/cm(2), 5-10 minutes) or the medium-intensity (809 nm, 150-190 mW/cm(2), 15 minutes) protocols were used. The single and multiple (up to 8-9) treatments were provided. The individual acne lesions were photothermally treated at 18 W/cm(2) (803 nm, 0.5 seconds) without skin surface cooling or at 200 W/cm(2) (809 nm, 0.5 seconds) with cooling. RESULTS: The observations during 1-2 months showed that soft acne treatment decreased the number of active elements, reduced erythema and inflammation, and considerably improved the skin state without any side effects. At high power densities (up to 200 W/cm(2)), ICG stained acne inflammatory elements were destroyed for light exposures of 0.5 seconds. CONCLUSIONS: Based on the concept that hair follicle, especially sebaceous gland, can be intensively and selectively stained by ICG due to dye diffusion through pilosebaceous canal and its fast uptake by living microorganisms, by vital keratinocytes of epithelium of the canal and sebaceous duct, and by rapidly proliferating sebocytes, new technologies of soft and thermal acne lesions treatment that could be used in clinical treatment of acne were proposed.