The effect of low-intensity laser irradiation in the blue, green, and red spectral ranges on healing of experimental skin wounds in rats

The effects of low-intensity laser irradiation in the red (632.8 nm), green (532 nm), and blue (441.2 nm) spectral ranges on wound healing has been studied in rats. The effect of the traditionally used red laser irradiation has been compared with the effect caused by laser irradiation in other spectral ranges, aiming to support the provisional hypothesis that a similar healing effect could be achieved at lower doses of wound irradiation by lasers emitting in the blue and green spectral ranges. The following parameters have been used to characterize healing of the experimental wounds: the functional activity of phagocytes in the wound exudate, which was determined from luminol-dependent chemiluminescence, the phagocyte number; the wound exudates’ antioxidant activity; and the rate of healing, which was determined as the change of the wound surface area. It was found that in all cases the laser irradiation accelerated the healing of wounds. Exposure to red laser irradiation at the dose of 1.5 J/cm²), and to blue or green laser irradiation at a dose of 0.75 J/cm² shortened the time of the wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes in irradiated groups increased by day 5 after surgery, whereas in the control group it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after surgery. Laser irradiation in the red spectral range at a dose of 1.5 J/cm² resulted in a larger increase in superoxide dismutase activity, as compared to that found after exposure to laser irradiation in the blue and green spectral ranges at a dose of 0.75 J/cm².     

Evaluation of bone healing following Er:YAG laser ablation in rat calvaria compared with bur drilling

The Er:YAG laser is currently used for bone ablation. However, the effect of Er:YAG laser irradiation on bone healing remains unclear. The aim of this study was to investigate bone healing following ablation by laser irradiation as compared with bur drilling. Rat calvarial bone was ablated using Er:YAG laser or bur with water coolant. Er:YAG laser effectively ablated bone without major thermal changes. In vivo micro‐computed tomography analysis revealed that laser irradiation showed significantly higher bone repair ratios than bur drilling. Scanning electron microscope analysis showed more fibrin deposition on laser‐ablated bone surfaces. Microarray analysis followed by gene set enrichment analysis revealed that IL6/JAK/STAT3 signaling and inflammatory response gene sets were enriched in bur‐drilled bone at 6 hours, whereas the E2F targets gene set was enriched in laser‐irradiated bone. Additionally, Hspa1a and Dmp1 expressions were increased and Sost expression was decreased in laser‐irradiated bone compared with bur‐drilled bone. In granulation tissue formed after laser ablation, Alpl and Gblap expressions increased compared to bur‐drilled site. Immunohistochemistry showed that osteocalcin‐positive area was increased in the laser‐ablated site. These results suggest that Er:YAG laser might accelerate early new bone formation with advantageous surface changes and cellular responses for wound healing, compared with bur‐drilling.      

A study of the effect of low-intensity laser radiation of the blue, green, and red spectral regions on the healing of experimental skin wounds in rats

The effect of low-intensity laser radiation of the blue (441.2 nm), green (532 nm), and red (632.8 nm) spectral regions on the healing of experimental skin wounds in rats has been studied. The effect of the traditionally applied laser radiation in the red region has been compared with the effect of laser radiation in the other spectral regions, assuming that, upon irradiation of wounds by lasers emitting in the blue and green regions, a similar effect can be achieved at lower doses. The following parameters characterizing the healing of experimental wounds were used: the functional activity of phagocytes of wound exudates, which was determined by luminol-dependent chemiluminescence, and their number; the antioxidant activity of wound exudates; and the rate of healing, which was determined as a change in the wound area. It was shown that irradiation with laser accelerated the healing of wounds in all cases. The exposure to laser radiations in the red (1.5 J/cm), blue, and green (0.75 J/cm2) spectral regions shortened the time of wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes after the exposure increased on day 5 after the infliction of the wound, whereas in the control it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after the operation. A maximum increase in the superoxide dismutase activity occurred after the exposure to laser radiation in the red region at a dose of 1.5 J/cm and in the blue and green spectral regions at a dose of 0.75 J/cm2.     

A comparative study of the effects of laser and light-emitting diode irradiation on the wound healing and functional activity of wound exudate leukocytes

The effects of coherent He-Ne laser and non-coherent light-emitting diode radiation on rat skin wound healing and functional activity of wound excudate leukocytes were compared. A comparative pathomorphological analysis showed that the He-Ne laser and light-emitting diode irradiation stimulated the transition of the inflammatory phase of the wound healing into the reparative (proliferative) and scarring phases sequentially. It was also detected that the functional activity of leucocytes changed in a dose-dependent manner. The leukocyte activity was found to be similar in the groups with laser and light-emitting diode irradiation. Thus, we can conclude that coherent laser and non-coherent light-emitting diode radiation have very close effects on wound healing and activity of wound exudate leukocytes, and coherence is not required for this activity.     

The role of endogenous porphyrins in laser therapy of experimental skin wounds

The role of endogenous porphyrins in the effect of laser irradiation on the superoxide dismutase (SOD) activity of wound exudate and rat leukocyte activity has been studied on models of aseptic incised skin wounds. Wounds were irradiated with a He-Ne laser (632.8 nm, 1.5 J/cm²) on the 2nd, 3rd, and 4th days after the beginning of the experiment. Irradiation effects were evaluated by the SOD activity (NBT test) and the activity of leukocytes of the wound exudate (as a chemiluminescent response to opsonized zymosan). It was found that in animals subjected to laser irradiation, the SOD activity sharply increased. This effect depended on endogenous porphyrin concentration and was retained throughout the experiment. The SOD activity in unirradiated animals decreased from the 2nd to the 5th day of experiment. The evaluation of the activity of wound exudate leukocytes did not reveal any distinct dependence of the effect on the concentration of endogenous porphyrins.     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: Achieving localized sub-cellular excitation in spatially-resolved experiments

Abstract

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

Photodynamic Therapy Using Systemic Administration of 5-Aminolevulinic Acid and a 410-nm Wavelength Light-Emitting Diode for Methicillin-Resistant Staphylococcus aureus-Infected Ulcers in Mice

Bacterial resistance to antibiotics has become a worldwide problem. One potential alternative for bacterial control is photodynamic therapy. 5-aminolevulinic acid is a natural precursor of the photosensitizer protoporphyrin IX. Relatively little is known about the antibacterial efficacy of photodynamic therapy using the systemic administration of 5-aminolevulinic acid; a few reports have shown that 5-aminolevulinic acid exerts photodynamic effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In this study, we evaluated the effectiveness of photodynamic therapy using 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode in vitro and in vivo for the treatment of MRSA. We found that 5-aminolevulinic acid photodynamic therapy with the light-emitting diode had an in-vitro bactericidal effect on MRSA. In vivo, protoporphyrin IX successfully accumulated in MRSA on ulcer surfaces after intraperitoneal administration of 5-aminolevulinic acid to mice. Furthermore, 5-aminolevulinic acid photodynamic therapy accelerated wound healing and decreased bacterial counts on ulcer surfaces; in contrast, vancomycin treatment did not accelerate wound healing. Our findings indicate that 5-aminolevulinic acid photodynamic therapy may be a new treatment option for MRSA-infected wounds.     

The role of lipid peroxidation products in the effect of He-Ne laser on human blood leukocytes

The role of lipid peroxidation products formed in membranes of human blood leukocytes after irradiation with He-Ne laser was studied. It was found that low-intensity laser irradiation (0.3-1.6 J/cm2) leads to both cell activation and an increase in the content of lipid peroxidation products. The intensity of lipid peroxidation was analyzed by estimating the amount of TBA reactive products and lipid diene conjugates. Irradiation in the presence of an exogenous photosensitizer (protoporphyrin IX) enhanced the phenomena observed. The use of antioxidants (tocopherol and ionol) completely eliminated the laser-induced effects (changes in leukocyte activity and accumulation of lipid peroxidation products). These results can be explained by the fact that laser irradiation leads to the activation of lipid peroxidation in leukocyte membranes, which in turn enhances the response of cells to the stimulus (priming).     

A comparative study of the effects of laser and LED radiation on lipid peroxidation in rat wound fluid

The effect of laser (632 nm) and LED (630 nm) on lipid peroxidation in rat wound fluid (exudate) was studied with the aim of comparing the efficiency of coherent and incoherent light on the processes that take place during wound healing. The study was performed using the model of cut aseptic wounds proposed by L.I. Slutskii. It was shown that irradiation of wounds with light of both laser and LED caused a decrease in the concentration of lipid peroxidation products in wound fluid as compared with the control group. An increase in the antioxidative activity of wound fluid was observed. It was concluded that irradiation with light of both laser and LED decreases the level of oxidative stress in wound fluid and that radiation coherence does not play a significant role.     

Low-dose photodynamic therapy effect on closure of scratch wounds of normal and diabetic fibroblast cells: An in vitro study

Chronic wounds such as diabetic ulcers are a serious public health problem. Extensive research is needed to find new alternatives for wound treatment. Photodynamic therapy (PDT) is a non-invasive method, which has been studied for several decades to treat cancer, infections, and other diseases. PDT involves the administration of a photosensitizer compound followed by irradiation with using light at specific wavelength to produce reactive oxygen species (ROS) using molecular oxygen. It is possible that low dose photodynamic therapy (LDPDT) could improve wound healing and stimulates the cell repair process. This study we explored the effect of LDPDT on wound healing in vitro using normal and diabetic cellular wound models. The effects of different concentrations of 5-ALA and different energy densities (dark or light) on the cell viability of human fibroblast cells were studied using the MTT assay. After ascertaining the optimum parameters, a scratch wound assay was performed on both normal and diabetic cells and then cells treated with 1 and 5 μg/mL of 5-ALA at 1 J/cm² energy density. ROS production and morphological alteration of the cells were studied. The mortality of normal fibroblast cells increased with increasing 5-ALA concentration and also increasing energy density (up to 3 J/cm²). However, in diabetic cells, the mortality rate did not decrease. Diabetic cells showed increased migration and closure of the scratch compared to normal cells under similar conditions. A low concentration of 5-ALA (5 μg/mL) and low energy density of 1 J/cm² in both normal and diabetic cells gave a small increase in ROS levels compared to controls. This may explain the positive effects of LDPDT on wound healing. The findings of this study suggest that LDPDT may have a potential effect on the wound healing of diabetic wounds.     

Photobiomodulation alters matrix protein activity in stressed fibroblast cells in vitro

A balance is maintained between matrix synthesis and degradation, and a prolonged increase in matrix metalloproteinases (MMPs) affects healing. Photobiomodulation (PBM) speeds up healing and alters wound environment. The study aimed to determine changes in protein and gene expression of collagen type 1 (Col‐I), MMP‐3 and ‐9 and TIMP‐1 in fibroblasts irradiated at 660 or 830 nm. Commercially purchased human skin fibroblast cells were modeled into five groups namely, normal, normal wounded, diabetic wounded, hypoxic wounded and diabetic hypoxic wounded. Control cells were sham irradiated. Laser irradiation was conducted at 660 or 830 nm (108/or 94 mW, 9.1 cm², 420/or 483 s) with 5 J/cm². Forty‐eight hours post‐irradiation, protein expression of TIMP‐1, MMP‐3, ?9 and Col‐I was determined by flow cytometry and immunofluorescence, and gene expression by real‐time RT‐PCR. There was an increase in TIMP‐1 and Col‐I, and a decrease in MMP‐3 and ‐9, as well as an alteration in mRNA expression of MMP3, MMP9, TIMP1 and COL1A1 in irradiated cells. Due to the responsiveness of the diabetic hypoxic wounded model, the findings propose this model as appropriate for wound healing studies and suggest that PBM promotes the remodeling phase of wound healing by decreasing matrix degradation and upregulating synthesis.      

Design,Synthesis, Biological Evaluation and Molecular Docking Studies of Quercetin-Linker-H2S Donor Conjugates for the Treatment of Diabetes and Wound Healing**

Based on the use of quercetin for treating diabetes and H₂S for promoting wound healing, a series of three quercetin-linker-H₂S donor conjugates was designed, synthesized and characterized by ¹H-NMR, ¹³C-NMR and MS. Meanwhile, in vitro evaluation of these compounds was also researched by IR-HepG2 treatment experiment, MTT assay, scratch test and tubule formation experiment. The three compounds could be used to treat insulin resistance induced by high glucose and promote the proliferation of human umbilical vein endothelial cells, wound healing, and the formation of tubules in vitro under a high-glucose environment. Our results illustrate that these compounds could be used to treat diabetes and promote wound healing at the same time. Furthermore, molecular docking study results of the compounds were consistent with the evaluated biological activity. In vivo research of compounds is underway.     

High-efficient and synergetic antibacterial nanocomposite hydrogel with quaternized chitosan/Ag nanoparticles prepared by one-pot UV photochemical synthesis

Hydrogel dressings have significant advantages such as absorption of tissue exudate, maintenance of proper moist environment, and promotion of cell proliferation. However, facile preparation method and high-efficient antibacterial hydrogel dressings are still a great challenge. In this study, a facile approach to prepare antibacterial nanocomposite hydrogel dressing to accelerate healing was explored. The hydrogels consisted of quaternized chitosan and chemically cross-linked polyacrylamide, as well as silver nanoparticles (AgNPs) stabilized by chitosan. The synthesis of the hydrogels including the formation of AgNPs and polymerization of acrylamide was accomplished simultaneously under UV irradiation in 1 hour without adding initiator. The hydrogels showed favorable tensile strength of ∼100 kPa with elongation at break over 1000% and shear modulus of ∼10⁴ Pa as well as suitable swelling ratio, which were appropriate for wound dressing. The combination of quaternized chitosan and AgNPs exhibited high-efficient and synergetic antibacterial performance with low cytotoxicity. In vivo animal experiments showed that the hydrogel can effectively prevent wound infection and promote wound healing. This study provides a facile method to produce antibacterial hydrogel wound dressing materials.     

He-Ne 激光辐照与UV-B 辐射对小麦幼苗细胞器中Na+/K+-ATP 酶活性的影响

分别采用5 mJ.s^-1.mm^-2 He-Ne激光辐照、10.08 kJ.m^-2.d^-1增强UV-B辐射及二者组合对小麦(Triticum aestivum)晋麦8号(Triticum aestivum ‘Jinmai8’)幼苗进行处理。第5 天开始测定各处理小麦幼苗叶片中线粒体、叶绿体及细胞溶质中Na⁺/K⁺-ATP酶活性的变化。结果表明, 随着处理天数的增加, 小麦幼苗叶片线粒体、叶绿体及细胞溶质中Na⁺/K⁺-ATP酶活性均在第6天下降, 第7天升高, 而后又逐渐下降。在处理的第7天, 仅He-Ne激光辐照可使小麦幼苗叶片线粒体、叶绿体及细胞溶质中Na⁺/K⁺-ATP酶活性升高; 增强UV-B辐射使各细胞器中Na⁺/K⁺-ATP酶活性下降; 复合处理后小麦各细胞器中Na⁺/K⁺-ATP酶活性均高于UV-B单独辐射处理。实验结果表明 , 一定剂量的He-Ne激光辐照能够部分修复UV-B辐射对小麦幼苗细胞器中Na^+/K⁺-ATP酶造成的损伤。     

Experimental study of photodynamic effect on bacterial wound microflora

The in vitro study of the influence of photodynamic action (with the use of photosensitizer "Photosens", laser and non-laser irradiation) on the strains of the main representatives purulent wound microflora was carried out. Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis cultures have been isolated from wound secretions and identified. The photosensitizer was shown to produce no bactericidal effect by itself. Irradiation with laser and non-laser light sources induced a sharp decrease in the number of viable cells in the cultures under study. The irradiation of the photosensitizer led to its activation, manifested in bactericidal action. The results thus obtained confirm good prospect of using photodynamic therapy for the treatment of purulent wounds.     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: achieving localized sub-cellular excitation in spatially-resolved experiments

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

Laser Impact on Bacterial ATP: Insights into the Mechanism of Laser-Bacteria Interactions

The mechanisms of laser action on bacteria are not adequately understood. Here, an attempt has been made to study the fluctuation in ATP (adenosine triphosphate) concentration following laser irradiation from a pulsed Nd:YAG laser on a marine biofilm-forming bacterium Pseudoalteromonas carrageenovora. A stationary phase bacterial suspension (density 10^7-8 ml^{m 1}) was exposed to pulsed laser irradiations at a fluence of 0.1 J cm^{m 2} (pulse width 5 ns, repetition rate 10 Hz) for different durations, ranging from 2 s to 15 min. The total viable count (TVC) and ATP concentration of the irradiated samples were determined immediately after the laser irradiation. While the maximum reduction in the TVC observed with respect to the control was 59% immediately after 15 min irradiation, the ATP concentration showed a reduction of about 86% for the same duration. The ATP concentration showed an abrupt reduction from 3 min of laser irradiation and continued to reduce significantly with increasing duration of irradiation. Thus, 3 min irradiation at a fluence of 0.1 J cm^{m 2} is considered as an approximate threshold for ATP production in this bacterium. As the decreased level of ATP production continued, bacterial mortality resulted. The reduction in ATP production could be due to damage caused by the laser irradiations on bacterial metabolic processes such as cellular respiration.     

Differential Effects of Phosphatase Inhibitors on the Calcium Homeostasis and Migration of HaCaT Keratinocytes

Changes in intracellular calcium concentration ([Ca²⁺]_i) as well as in the phosphorylation state of proteins have been implicated in keratinocyte wound healing revealed in scratch assays. Scratching confluent HaCaT monolayers decreased the number of cells displaying repetitive Ca²⁺ oscillations as well as the frequency of their Ca²⁺-transients in cells close to the wounded area and initiated migration of the cells into the wound bed. In contrast, calyculin-A (CLA) and okadaic acid (OA), known cell permeable inhibitors of protein phosphatase-1 and 2A, increased the level of resting [Ca²⁺]_i and suppressed cell migration and wound healing of HaCaT cells. Furthermore, neither CLA nor OA influenced how scratching affected Ca²⁺ oscillations. It is assumed that changes in and alterations of the phosphorylation level of Ca²⁺-transport and contractile proteins upon phosphatase inhibition mediates cell migration and wound healing.     

Expression and simple,one-step purification of fragile histidine triad (Fhit) tumor suppressor mutant forms in <Emphasis Type="Italic">Escherichia coli</Emphasis> and their interaction with protoporphyrin IX

The product of human fragile histidine triad (FHIT) gene is a tumor suppressor protein of still largely unknown cellular background. We have shown previously that it binds protoporphyrin IX (a photosensitizer) which alters its enzymatic activity in vitro. Fhit, diadenosine triphosphate (Ap₃A) hydrolase, possesses the active site with histidine triad His-φ-His-φ-His-φφ. So-called histidine Fhit mutants (His94Asn, His96Asn and His98Asn) exhibit highly reduced activity in vitro, however, their antitumor function has not been fully described yet. In this work we have cloned the cDNAs of histidine mutants into pPROEX-1 vector allowing the production of His₆-fusion proteins. The mutated proteins: Fhit-H94N, Fhit-H96N and Fhit-H98N, were expressed in Escherichia coli BL21(DE3) and purified (up to 95%) by an improved, one-step affinity chromatography on Ni-nitrilotriacetate resin. The final yield was 2 mg homogenous proteins from 1 g bacteria (wet wt). The activity of purified proteins was assessed by previously described assay. The same purification procedure yielded 0.8 mg/ml and highly active wild-type Fhit protein (K _m value for Ap₃A of 5.7 μM). Importantly, purified mutant forms of Fhit also interact with a photosensitizer, protoporphyrin IX in vitro.