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Effect of photodynamic therapy based on indocyanine green on expression of apoptosis-related genes in human gingival fibroblast cells
Accepted Manuscript Title: Effect of photodynamic therapy based on indocyanine green on expression of apoptosis-related genes in human gingival fibroblast cells Authors: Samira Gharesi, Maryam Pourhajibagher, Nasim Chiniforush, Reza Raoofian, Mehrdad Hashemi, Sima Shahabi, Abbas Bahador PII: DOI: Reference:
S1572-1000(17)30217-X http://dx.doi.org/doi:10.1016/j.pdpdt.2017.04.007 PDPDT 942
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
21-2-2017 4-4-2017 9-4-2017
Please cite this article as: Gharesi Samira, Pourhajibagher Maryam, Chiniforush Nasim, Raoofian Reza, Hashemi Mehrdad, Shahabi Sima, Bahador Abbas.Effect of photodynamic therapy based on indocyanine green on expression of apoptosis-related genes in human gingival fibroblast cells.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2017.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of photodynamic therapy based on indocyanine green on expression of apoptosis-related genes in human gingival fibroblast cells Samira Gharesi1, Maryam Pourhajibagher2,3,4, Nasim Chiniforush3, Reza Raoofian5,6, Mehrdad Hashemi1, Sima Shahabi3,4, Abbas Bahador,3,4,2* 1
Department of Genetics, Islamic Azad University, Tehran medical Branch, Theran, Iran. Department of Microbiology, School of Medicine, Tehran University of Medical Science, Tehran, Iran. 3 Laser Research Center of Dentistry (LRCD), Tehran University of Medical Sciences, Tehran, Iran. 4 Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 5 Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran. 2
6
Innovative Research Center, Islamic Azad University, Mashhad branch, Mashhad, Iran.
Running title: ICG-PDT effects on expression of apoptosis-related genes *Correspondence Address: Abbas Bahador, Ph.D., Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. Tehran University of Medical Sciences, Keshavarz Blvd, 100 Poursina Ave., Tehran, Iran. 14167-53955. Tel.: +9821 6405 3210; Fax: +98218895 5810. E-mail: [email protected]; alternate address: [email protected] Highlights Photodynamic therapy (PDT) induced the significant expression of BAX in human gingival fibroblast cells. Laser irradiation and indocyanine green (ICG) alone revealed no significant effects on the expression of BAX gene. Treatment with laser irradiation, ICG alone, and ICG-PDT caused no observable BCL-2 gene expression.
Abstract: Background: Periodontal diseases refer to inflammation of the gingiva, induction of apoptosis in human gingival fibroblast cells, destruction of the surrounding tissues, and early bone loss resulting in infections due to the pathogenic activity of the microorganisms and the host immune inflammatory responses. Recent investigations have suggested that antimicrobial photodynamic therapy (aPDT) can be an adjunct treatment therapy for periodontal infections. Aim: To prove the lack of side effects of PDT on periodontal tissues, we investigated the expression of BAX and BCL-2 genes that are involved in apoptosis after the PDT on human gingival fibroblast (HGF) cells. Materials and Methods: In this study the effect of PDT based on indocyanine green (ICG) as a photosensitizer with the diode laser were tested on the expression of BAX and BCL-2 genes in monolayers of HGF cells. The effects of PDT on the expression of BAX and BCL-2 genes were evaluated by real-time quantitative reverse transcription PCR.
Results: The results of the genes expression analysis revealed that ICG-PDT at concentrations 1000 μg/mL, induced the significant expression of BAX in HGF cells; however, the laser irradiation as well as ICG showed no significant effects on the expression of these genes. Treatment with ICG alone, laser irradiation and ICG-PDT caused no observable BCL-2 gene expression changes between the tested and control group. Conclusion: Our findings indicate that ICG-PDT at 1000 µg/mL of ICG with the exposure time of 60 s for the diode laser would appear to be an inducer of apoptosis in HGF at transcriptome level. Keywords: Antimicrobial photodynamic therapy; ; ; ; ; ; , Apoptosis, Gene expression, Human gingival fibroblast cells, Indocyanine green, Periodontal disease, Real-time quantitative reverse transcription PCR 1 Introduction
Periodontal diseases refer to the inflammatory diseases of the soft and hard tissues that support the teeth and the bacterial biofilm present on the subgingival tooth surface [1]. Recent concepts are based on mechanical scaling and root planing, to remove plaque and tartar deposits on the tooth and root surface for the treatment of periodontal disease [2]. Mechanical scaling and root planing might not be sufficient for removal of complete plaque in difficult-to-access areas. An alternative therapeutic strategy, such as use of systemic and local antibiotics, which relies solely on decontamination, brings adverse complications and development of pathogenic bacterial strains resistant to multiple drugs [3, 4]. Antimicrobial photodynamic therapy (aPDT) is used as an alternative treatment for eliminating the microorganisms [5]. aPDT is based on the principle that a photosensitizing compound binds to the targeted bacteria, and then can be activated by light of a suitable wavelength in the presence of oxygen. During this process, cytotoxic products such as singlet oxygen are generated [6]. aPDT as a localized treatment procedure can be applied into deep periodontal pockets without endangering the distant cells [7, 8]. The near-infrared (NIR) spectral region is, in particular, ideally associated with aPDT due to its excellent penetrating ability through tissues without causing significant heating. To date, the most important NIR-photosensitizing compound is indocyanine green (ICG). Essentially, ICG has very low toxicity and a strong peak in the vicinity of 775–810 nm range, which is thought to be an effective photosensitizer for aPDT [9, 10]. The increased singlet oxygen levels following aPDT may depolarize the mitochondrial membrane potential to further increase the mitochondrial permeability transition pore. Subsequently, it may cause the release of intermembrane proteins like cytochrome c into the cytosol, which allows the activation of the caspase 9 and production of the apoptosome. Thus, the production of cytotoxic products from the aPDT may lead to cell apoptosis [11]. BAX and BCL-2 are two proteins of BCL-2 family that have been best studied in apoptosis by aPDT. BAX is a necessary protein for the release of cytochrome c from mitochondria, and it plays a key role in inducing apoptosis; BCL-2 is an antiapoptotic protein [12]. The cellular uptake of the photosensitizer in mammalian cells could be due to the incubation time. When the incubation time is short, the result is reduced cell uptake and the photosensitizer is concentrated in the plasma membrane. Thus, no injury to the periodontal cells indicate that the cytotoxic products generated at the surface will not spread over to another intracellular location opposite to the one inhabiting the bacteria [13].
For a periodontist, a possible disadvantage of aPDT use for the treatment is not available. The possibility of adverse effects of aPDT on host cells should be considered [8]. In this study, we sought to investigate the apoptotic effects of a combination of ICG and diode laser on the human gingival fibroblast cells. We used cells under in vitro condition to eliminate any possible interference caused by various periodontopathic bacteria. 2 Materials and Methods 2.1 Study design Following four groups were tested: 1. ICG alone (S+ L−; S and L stand for “sensitizer” and “light,” respectively); 2. Laser group was irradiated without ICG: laser irradiation (S−L+); 3. aPDT (combined ICG and laser treatment; S+ L+); and 4. Control (no exposure to either ICG or laser irradiation; S−L−). 2.2 Preparation of ICG ICG dry powder (2 mg) (Serva, Heidelberg) was dissolved in 1.0 mL sterile distilled water and then filtersterilized by using 0.22-μm-pore-size membrane filters as a fresh stock solution (2000 μg/mL). Before each experiment, ICG stock was kept in the dark [14]. 2.3 Light source An 808 nm diode laser (DX62 Konftec, Taiwan) with output of 250 mW in continuous mode was used in our experiments. Laser power was checked with a power meter (Laser Point Srl, Milano, Italy) before and after each treatment. 2.4 Cells culturing Primary human gingival fibroblast cells (HGF, IBRC C10459) were obtained from the Iranian Biological Resource Center (Tehran, Iran). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Sigma, USA) with 1% penicillin and streptomycin (10,000 Unit/mL penicillin and 10 mg/mL streptomycin). This medium was also supplemented with 2 mM L-glutamine and 100 μg/mL of amphotericin B. The cell cultures were then incubated under standard cell culture conditions (37°C, 95% humidity, 5% CO2). Culture media was changed after every 3 days. After reaching confluency, the cells were treated with 0.25% trypsin and 0.02% ethylene diamine tetraacetic acid (EDTA). Furthermore, 3 × 105 cells (100 µL) per well were added in 96well cell culture microplates (Greiner Bio-One, Germany) [15]. 2.5 PDT HGF cells were incubated with 100 µL of ICG (1000 μg/mL) for 5 min in a humidified incubator at 37°C, 95% air, and 5% CO2 in dark condition. To prevent laser transmission to test wells in 96-well cell culture microplates, the test wells were separated from each other by wells that were filled by indian ink. At the end of the incubation period, the cells in the microplate wells containing ICG were immediately exposed to the diode laser at 250 mW for 60s (39 J/Cm2) at room temperature (25 ± 2°C) [16]. Three volumes of RNAlater® (Thermo Fisher Scientific, US) were then added to stabilize the RNA. The mixture was then
treated immediately for RNA extraction. The probe of laser was positioned perpendicular to the center of each well at a distance of 1 mm from the wells by a microphone stand to ensure an equal exposure of the entire well. We placed black paper sheets under the microplate to avoid beam reflection from the table top during the experiment [17]. The diameter of the irradiated area was the same as the well diameter at bottom of the microplate (i.e., 6.39 mm). The control group did not receive any treatment. Control wells with plain balanced salt solution (BSS, pH 7.4) were used to reveal potential dye independent apoptotic effects. The treated HGF cells were quickly harvested (cell suspension collected) after treatment to avoid binding and were washed twice with sterile phosphate-buffered saline (PBS, pH 7.4). 2.6 RNA extraction HGF cells after PDT treatment were collected for extarction of RNA. Total RNA was extracted using the Hybrid-RTM Total RNA Purification Kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s instruction. The concentration and purity of RNA sample were determined by reading the optical density at 260 and 280 nm wavelengths. For this purpose, a NanoDrop® spectrophotometer (Thermo Fisher Scientific, US) was used to assess the purity of the extracted RNA [18].
2.7 cDNA synthesis and real-time quantitative RT-PCR For real-time PCR analysis, cDNA was synthesized from 1.0 μg/µL of total extracted RNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Fermentas, Waltham, MA, USA) according to the manufacturer’s instruction. The cDNA was controlled by PCR with housekeeping GAPDH gene primers. Real-time PCR analysis of BCL-2 and BAX genes expression was carried out using the SYBR green PCR Master Mix (Takara, Shiga, Japan), in a total volume of 20 μL with Line-Gene K Real-Time PCR Detection System and Software (Bioer Technology, Hangzhou, China). The primers used were: GAPDH forward, 5′CGC TCT CTG CTC CTC CTG TT -3′ and reverse, 5′- ACG ACC AAA TCC GTT GAC TCC -3′; BAX forward, 5′- TTC TGA CGG CAA CTT CAA CTG G -3′ and reverse, 5′- AGG AAG TCC AAT GTC CAG CC -3′; and BCL-2 forward, 5′- AGG CTG GGA TGC CTT TGT GG -3′ and reverse, 5′- GGG CAG GCA TGT TGA CTT CAC -3′. GAPDH and BCL-2 primers were designed via gene runner software version 3.05 (Hastings Software Inc. Hastings, NY, USA) and BAX was used from a paper [19]. For each reaction of Real-time PCR, 10 μL of SYBR green PCR Master Mix and 7 μL of double distilled water were used. Moreover, 17 μL of the mixture was transferred to test tubes, and finally, 1 μL of the synthesized cDNA was added to each reaction tube with 2 μL of primers. The real-time PCR analysis was performed with the following thermal conditions: 5 min at 95°C for initial denaturation step, 35 cycles of 15 s at 95°C for denaturation step, 30 s at 59°C for annealing, and 15 s at 72°C for the extension, respectively, and 10 min final extension at 72°C was carried out for completion of amplicons. All samples were analyzed in triplicate, and GAPDH served as a reference gene for normalization. Melting curve analysis was performed according to the dissociation stage data and reactions to ensure that the fluorescent signal observed is from the desired PCR product. 2.8 Statistical analysis All of the experiments were carried out in triplicate. The data are represented as means plus or minus standard deviation (means ± SD). The main fold change of the target gene at each group was calculated using 2−ΔΔCT method, that is, relative to control cells. The expression levels of mRNA were shown as n-fold differences relative to the calibrator. A variation of >2-fold changes in expression level of the target gene was interpreted as significant [20].
3 Results ICG-PDT at 1000 µg/mL of ICG with the exposure time of 60 s for the diode laser induced significant expression of BAX in HGF cells as compared to the control group. In the illuminated ICG-1000 µg/mL cells, the expressions of BAX were increased to approximately 5.2-fold at 5 min; however, under the same treatment of cells revealed no significant changes in the expression of BCL-2 gene on the cells (Figure 1). Under the same light intensity and duration, there was no significant difference in the expression of BAX/BCL-2 genes in HGF cells as compared to the control group. Treatment with ICG at 1000 µg/mL, void of illumination, caused no observable changes in the expression of BAX/BCL-2 genes between the tested and control groups. 4 Discussion aPDT has been widely used in treating some cancers, in particular, those of the skin, age-related macular degeneration , and other diseases; it has also been used in treating infections [21-23]. aPDT as an adjunct to periodontal therapy has been evaluated by recent studies [24]. There is some evidence that aPDT not only kills the periodontal pathogens but also might inactivate the virulence factors of bacteria, improving outcome of the treatment [25]. Resolving the possible defect of aPDT in the clinical application needs emphasization that the toxic effect of aPDT on normal adjacent tissue is minimal; however, it has been indicated that the dosage of light used to kill bacteria in aPDT is unable to damage the fibroblast cells. Human periodontal cell studies gave preliminary evidence of biological effects of the aPDT on soft tissue [24, 26]. Thus, we aimed to evaluate the effects of ICG-PDT on human gingival fibroblast. ICG as a fluorescent agent with low toxicity has been widely used in biomedical fields since its first use [27-30]. Stalmans et al. stated that the toxicity of the ICG solution on the retinal pigment epithelial (RPE) cells depends on the hypo-osmolarity of the solvent [31]. Wu et al. found that ICG must be dissolved in pure water and solubilized again in BSS to avoid a hypo-osmolar solution [32]. Ho et al. reported that removal of Na+ from the ICG solution was associated with its reduced toxicity in RPE cells [33]. The absorption and emission properties of ICG are determined by the type of solvent and the concentration of ICG. When ICG is diluted in an aqueous solution by increasing the ICG concentrations the absorption peak shifts from 780 to 695 nm. This alteration of absorption peak depends on aggregation of the ICG molecules from monomer to oligomer [34]. Rezai et al. [35] have demonstrated that all the concentrations of ICG (1000, 5000, and 20,000 µg/mL) with 30 min incubation time, induced apoptosis in RPE cells. Our results showed that ICG, in the absence of light, did not induce any significant apoptosis in HGF cells when compared with the control group. The lack of induction of apoptosis could be explained by the relatively short time exposure of our study. Yam et al. [36] reported that the damage by ICG on RPE cells was determined by the concentration, change in osmolarity, pH, and incubation time. They showed that the optimum dose of ICG-PDT is as low as 250 µg/mL. In the present study, light exposure with a low dose of ICG as low as 1000 µg/mL, induced HGF cells apoptosis. Penha et al. [37] found that ICG induced apoptosis in ARPE-19 cells after 3 min of exposure at all concentrations (5, 50, 250, 500, and 1000 µg/ mL). In this study, we expected that ICG 1000 µg/mL would cause apoptosis, whereas we observed that ICG at this concentration may has a protective effect over HGF cells, since it increased the expression of BCL-2 gene. In agreement with our data, Kreisler et al. [26] showed that diode laser at 810 nm after 60 s irradiation time, did not reduce the survival rate of HGF cells. Furthermore, in vivo investigations are necessary for an overall evaluation of PDT’s applicability in periodontics and for recommendation of ideal ICG concentration in pocket decontamination. 5 Conclusion
The results of genes expression analysis reveals that ICG-PDT at concentrations of 1000 μg/mL induced expression of apoptosis-related gene, BAX, in HGF cells; however, laser irradiation as well as 1000 μg/mL of ICG revealed no significant apoptotic effects on expression of this gene in HGF cells. Acknowledgements This research has been supported by Laser Research Center of Dentistry (LRCD), Dentistry Research Institute, Tehran University of Medical Sciences & Health Services grant No. 94-04-97-30540. References 1. A. Cekici, A. Kantarci, H. Hasturk, D.E. Van Dyke, Inflammatory and immune pathwaysin the pathogenesis of periodontal disease, Periodontol. 2000. 64 (2014) 57-80. 2. J.Y He, G.G Qi, W.J. Huang, X.D. Sun, Y. Tong, C.M. Peng, X.P. Zhou , H. Chen, Short-term microbiological effects of scaling and root planing and essential-oils mouthwash in Chinese adults, J. Zhejiang. Univ. Sci. B. 14 (2013) 416-425. 3. K.R. Raj, S. Musalaiah, M. Nagasri, P.A. Kumar, P.I. Reddy, M. Greeshma, Evaluation of efficacy of photodynamic therapy as an adjunct to nonsurgical periodontal therapyin treatment of chronic periodontitis patients: A clinico-microbiological study, Indian. J. Dent. Res. 27 (2016) 483-487. 4. A. Kapoor, R. Malhotra, V. Grover, D. Grover, Systemic antibiotic therapy in periodontics, Dent. Res. J. 9 (2012) 505-515. 5. L.M. Baltazar, A. Ray, D.A. Santos, P.S. Cisalpino, A.J. Friedman, J.D. Nosanchuk, Antimicrobial photodynamic therapy: an effective alternative approach to control fungal infections, Front. Microbiol. 13 (2015) 202. 6. B.P. De Oliveira, C.M. Aguiar, A.C. Câmara, Photodynamic therapy in combating the causative microorganisms from endodontic infections, Eur. J. Dent. 8 (2014) 424-430. 7. V.K. Kamath, J.B. Pai, N. Jaiswal, Periowand: photodynamic therapy in periodontics, Univ. Res. J. Dent. 4 (2014) 133–138. 8. M. Wainwright, Photodynamic antimicrobial chemotherapy, J.Antimicrob.Chemother. 42 (1998) 13-28. 9. C. Beltes, N. Economides, H. Sakkas, C. Papadopoulou, T. Lambrianidis, Evaluation of Antimicrobial Photodynamic Therapy Using Indocyanine Green and Near-Infrared Diode Laser Against Enterococcus faecalis in Infected Human Root Canals,Photomed. Laser. Surg. 20 (2016) 1–6. 10. H.J. Lim, C.H. Oh, Indocyanine green-based photodynamic therapy with 785nm lightemitting diode for oral squamous cancer cells,Photodiagn. Photodyn. Ther. 8 (2011) 337-42. 11. P.R. Wei, Y. Kuthati, R.K. Kankala, C.H. Lee. Synthesis and Characterization ofChitosan-Coated Near-Infrared (NIR) Layered Double Hydroxide-Indocyanine Green Nano composites for Potential Applications in Photodynamic Therapy, Int. J. Mol. Sci.16 (2015)20943-68. 12. A. Chiaviello, I. Postiglione, G. Palumbo, Targets and Mechanisms of Photodynamic Therapy in Lung Cancer Cells: A Brief Overview, Cancers. 3 (2011) 1014-1041. 13. N.S. Soukos, L.A. Ximenez-Fyvie, M.R. Hamblin, S.S. Socransky, T. Hasan, Targeted Antimicrobial Photochemotherapy, Antimicrob. Agents.Chemother. 42 (1998) 2595-2601.
14. J.D. Ho, R.J. Tsai, S.N. Chen, H.C. Chen, Removal of sodium from the solvent reduces retinal pigment epithelium toxicity caused by indocyanine green: implications for macular hole surgery, Br. J.Ophthalmol. 88 (2004) 556-9. 15. M. Pourhajibagher, N. Chiniforush, S. Parker, S. Shahabi, R. Ghorbanzadeh, M.J. Kharazifard, A. Bahador, Evaluation of antimicrobial photodynamic therapy with indocyanine green and curcumin on human gingival fibroblast cells: An in vitrophotocytotoxicity investigation, Photodiagn. Photodyn. Ther. 15 (2016) 13-8. 16. S.S. Bhojwani, M.K. Razdan, Plant Tissue Culture: Theory and Practice, revised edn, Elsevier. Science. B.V. (1996) 467. 17.
R. Klein, Laser Welding of Plastics: Material properties of plastics, Wiley-VCH. 43 (2011) 3-69.
18. P. Desjardins, D. Conklin,NanoDropMicrovolume Quantitation of Nucleic Acids,J. Vis. Exp. 45 (2010) e2565. 19. G.M. Saed, M.P. Diamond, Apoptosis and proliferation of human peritoneal fibroblasts in response to hypoxia, Fertil.Steril. 78 (2002) 137-43. 20. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) Method. Methods. 25 (2001) 402-8. 21. M.T. Wan, J.Y. Lin, Current evidence and applications of photodynamic therapy in dermatology, Clin.Cosmet.Investig.Dermatol. 7 (2014) 145-163. 22. V. Rapozzi, E. Della Pietra, Bonavida B. Dual roles of nitric oxide in theregulation of tumor cell response and resistance to photodynamic therapy,Redox. Biol. 6 (2015) 311-7. 23. A. Azizi, S. Shademan, M. Rezai, A. Rahimi, S. Lawaf. Effect of photodynamictherapy with two photosensitizers on Streptococcus mutants: In vitro study,Photodiagn.Photodyn.Ther. 16 (2016) 66-71. 24. T. Kikuchi, M. Mogi, I. Okabe, K.Okada, H. Goto, Y. Sasaki, T. Fujimura, M. Fukuda, A. Mitani, Adjunctive Application of Antimicrobial Photodynamic Therapy in Nonsurgical Periodontal Treatment: A Review of Literature, Int. J. Mol. Sci. 16 (2015) 24111-24126. 25. S. Rajesh, E. Koshi, K. Philip, A. Mohan. Antimicrobial photodynamic therapy: An overview. J. Indian. Soc.Periodontol. 15 (2011):323-327. 26. M. Kreisler, M. Daubländer, B. Willershausen-Zönnchen, B. d’Hoedt, Effect of diode laser irradiation on the survival rate of gingival fibroblast cell cultures, Lasers. Surg. Med. 28 (2001) 445-50. 27. N.Y. Hong, H.R. Kim, H.M. Lee, D.K. Sohn, K.G. Kim, Fluorescent property of indocyanine green (ICG) rubber ring using LED and laser light sources, Biomed Opt Express. 7 (2016) 1637-44. 28. J. Meyer, A. Cunea, D. Sonntag-Bensch, P. Welker, K. Licha, F.G. Holz, S. Schmitz-Valckenberg, In vivo imaging of a new indocyanine green micelle formulation in an animal model of laser-induced choroidal neovascularization, Invest Ophthalmol Vis Sci. 55 (2014) 6204-12. 29. J.C. Kraft, R.J. Ho, Interactions of indocyanine green and lipid in enhancing near-infrared fluorescence properties: the basis for near-infrared imaging in vivo, Biochemistry. 53 (2014) 1275-83.
30. B. Jung, V. I. Vullev, and B. Anvari, “Revisiting indocyanine green: effects of serum and physiological temperature on absorption and fluorescence characteristics”, IEEE J. Sel. Top. Quantum Electron. 20 (2014) 149–157. 31. E.F. Costa, N.M. Barros, L.P. Coppini, R.L. Neves, A.K. Carmona, F.M. Penha, E.B. Rodrigues, E. Dib, O.Jr. Magalhães, M.N. Moraes-Filho, A.A. Lima Filho, M. Maia, M.E Farah, Effects of light exposure, pH, osmolarity, and solvent on the retinal pigment epithelial toxicity of vital dyes, Am J Ophthalmol. 155 (2013)705-12. 32. W.C. Wu, D.N. Hu, J.E. Roberts, Phototoxicity of indocyanine green on human retinal pigment epithelium in vitro and its reduction by lutein, Photochem.Photobiol. 81(2005) 537–540. 33. F.M. Penha, E.B. Rodrigues, M. Maia, C.H. Meyer, P. Costa Ede, E. Dib, E. Bechara, A. Lourenço, A.A. Lima Filho, E.H. Freymüller, M.E. Farah, International Chromovitrectomy Collaboration.. Biochemical analysis and decomposition products of indocyanine green in relation to solvents, dye concentrations and laser exposure, Ophthalmologica. 230 (2013) 59-67. 34. B. Yuan, N. Chen, Q. Zhu, Emission and absorption properties of indocyanine green inIntralipid solution. J. Biomed. Opt. 9 (2004) 497-503. 35. K.A. Rezai, E. asyna, B.L. Seagle, J.R. Norris, K.A. Rezaei, AcrySof Natural filter decreases blue light-induced apoptosis in human retinal pigment epithelium. Graefes. Arch. Clin. Exp. Ophthalmol. 246 (2008) 671-6. 36. H.F. Yam, A.K. Kwok, K.P. Chan, T.Y. Lai, K.Y. Chu, D.S. Lam, C.P. Pang, Effect of indocyanine green and illumination on gene expression in human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 44 (2003) 370-7. 37. F.M. Penha, M. Pons, E.F. Costa, E.B. Rodrigues , M. Maia , M.E. Marin-Castaño, M.E. Farah, Effect of vital dyes on retinal pigmented epithelial cell viability and apoptosis: implications for chromovitrectomy, Ophthalmologica. 230 (2013) 41-50. Figure legend: Figure 1. Changes in mRNA expression levels of BAX and BCL-2 genes following treatments. Values are presented as means ± SD. S+ L−; S and L stand for “sensitizer” and “light,” respectively.
Figr-1
S1572-1000(17)30217-X http://dx.doi.org/doi:10.1016/j.pdpdt.2017.04.007 PDPDT 942
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
21-2-2017 4-4-2017 9-4-2017
Please cite this article as: Gharesi Samira, Pourhajibagher Maryam, Chiniforush Nasim, Raoofian Reza, Hashemi Mehrdad, Shahabi Sima, Bahador Abbas.Effect of photodynamic therapy based on indocyanine green on expression of apoptosis-related genes in human gingival fibroblast cells.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2017.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of photodynamic therapy based on indocyanine green on expression of apoptosis-related genes in human gingival fibroblast cells Samira Gharesi1, Maryam Pourhajibagher2,3,4, Nasim Chiniforush3, Reza Raoofian5,6, Mehrdad Hashemi1, Sima Shahabi3,4, Abbas Bahador,3,4,2* 1
Department of Genetics, Islamic Azad University, Tehran medical Branch, Theran, Iran. Department of Microbiology, School of Medicine, Tehran University of Medical Science, Tehran, Iran. 3 Laser Research Center of Dentistry (LRCD), Tehran University of Medical Sciences, Tehran, Iran. 4 Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 5 Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran. 2
6
Innovative Research Center, Islamic Azad University, Mashhad branch, Mashhad, Iran.
Running title: ICG-PDT effects on expression of apoptosis-related genes *Correspondence Address: Abbas Bahador, Ph.D., Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. Tehran University of Medical Sciences, Keshavarz Blvd, 100 Poursina Ave., Tehran, Iran. 14167-53955. Tel.: +9821 6405 3210; Fax: +98218895 5810. E-mail: [email protected]; alternate address: [email protected] Highlights Photodynamic therapy (PDT) induced the significant expression of BAX in human gingival fibroblast cells. Laser irradiation and indocyanine green (ICG) alone revealed no significant effects on the expression of BAX gene. Treatment with laser irradiation, ICG alone, and ICG-PDT caused no observable BCL-2 gene expression.
Abstract: Background: Periodontal diseases refer to inflammation of the gingiva, induction of apoptosis in human gingival fibroblast cells, destruction of the surrounding tissues, and early bone loss resulting in infections due to the pathogenic activity of the microorganisms and the host immune inflammatory responses. Recent investigations have suggested that antimicrobial photodynamic therapy (aPDT) can be an adjunct treatment therapy for periodontal infections. Aim: To prove the lack of side effects of PDT on periodontal tissues, we investigated the expression of BAX and BCL-2 genes that are involved in apoptosis after the PDT on human gingival fibroblast (HGF) cells. Materials and Methods: In this study the effect of PDT based on indocyanine green (ICG) as a photosensitizer with the diode laser were tested on the expression of BAX and BCL-2 genes in monolayers of HGF cells. The effects of PDT on the expression of BAX and BCL-2 genes were evaluated by real-time quantitative reverse transcription PCR.
Results: The results of the genes expression analysis revealed that ICG-PDT at concentrations 1000 μg/mL, induced the significant expression of BAX in HGF cells; however, the laser irradiation as well as ICG showed no significant effects on the expression of these genes. Treatment with ICG alone, laser irradiation and ICG-PDT caused no observable BCL-2 gene expression changes between the tested and control group. Conclusion: Our findings indicate that ICG-PDT at 1000 µg/mL of ICG with the exposure time of 60 s for the diode laser would appear to be an inducer of apoptosis in HGF at transcriptome level. Keywords: Antimicrobial photodynamic therapy; ; ; ; ; ; , Apoptosis, Gene expression, Human gingival fibroblast cells, Indocyanine green, Periodontal disease, Real-time quantitative reverse transcription PCR 1 Introduction
Periodontal diseases refer to the inflammatory diseases of the soft and hard tissues that support the teeth and the bacterial biofilm present on the subgingival tooth surface [1]. Recent concepts are based on mechanical scaling and root planing, to remove plaque and tartar deposits on the tooth and root surface for the treatment of periodontal disease [2]. Mechanical scaling and root planing might not be sufficient for removal of complete plaque in difficult-to-access areas. An alternative therapeutic strategy, such as use of systemic and local antibiotics, which relies solely on decontamination, brings adverse complications and development of pathogenic bacterial strains resistant to multiple drugs [3, 4]. Antimicrobial photodynamic therapy (aPDT) is used as an alternative treatment for eliminating the microorganisms [5]. aPDT is based on the principle that a photosensitizing compound binds to the targeted bacteria, and then can be activated by light of a suitable wavelength in the presence of oxygen. During this process, cytotoxic products such as singlet oxygen are generated [6]. aPDT as a localized treatment procedure can be applied into deep periodontal pockets without endangering the distant cells [7, 8]. The near-infrared (NIR) spectral region is, in particular, ideally associated with aPDT due to its excellent penetrating ability through tissues without causing significant heating. To date, the most important NIR-photosensitizing compound is indocyanine green (ICG). Essentially, ICG has very low toxicity and a strong peak in the vicinity of 775–810 nm range, which is thought to be an effective photosensitizer for aPDT [9, 10]. The increased singlet oxygen levels following aPDT may depolarize the mitochondrial membrane potential to further increase the mitochondrial permeability transition pore. Subsequently, it may cause the release of intermembrane proteins like cytochrome c into the cytosol, which allows the activation of the caspase 9 and production of the apoptosome. Thus, the production of cytotoxic products from the aPDT may lead to cell apoptosis [11]. BAX and BCL-2 are two proteins of BCL-2 family that have been best studied in apoptosis by aPDT. BAX is a necessary protein for the release of cytochrome c from mitochondria, and it plays a key role in inducing apoptosis; BCL-2 is an antiapoptotic protein [12]. The cellular uptake of the photosensitizer in mammalian cells could be due to the incubation time. When the incubation time is short, the result is reduced cell uptake and the photosensitizer is concentrated in the plasma membrane. Thus, no injury to the periodontal cells indicate that the cytotoxic products generated at the surface will not spread over to another intracellular location opposite to the one inhabiting the bacteria [13].
For a periodontist, a possible disadvantage of aPDT use for the treatment is not available. The possibility of adverse effects of aPDT on host cells should be considered [8]. In this study, we sought to investigate the apoptotic effects of a combination of ICG and diode laser on the human gingival fibroblast cells. We used cells under in vitro condition to eliminate any possible interference caused by various periodontopathic bacteria. 2 Materials and Methods 2.1 Study design Following four groups were tested: 1. ICG alone (S+ L−; S and L stand for “sensitizer” and “light,” respectively); 2. Laser group was irradiated without ICG: laser irradiation (S−L+); 3. aPDT (combined ICG and laser treatment; S+ L+); and 4. Control (no exposure to either ICG or laser irradiation; S−L−). 2.2 Preparation of ICG ICG dry powder (2 mg) (Serva, Heidelberg) was dissolved in 1.0 mL sterile distilled water and then filtersterilized by using 0.22-μm-pore-size membrane filters as a fresh stock solution (2000 μg/mL). Before each experiment, ICG stock was kept in the dark [14]. 2.3 Light source An 808 nm diode laser (DX62 Konftec, Taiwan) with output of 250 mW in continuous mode was used in our experiments. Laser power was checked with a power meter (Laser Point Srl, Milano, Italy) before and after each treatment. 2.4 Cells culturing Primary human gingival fibroblast cells (HGF, IBRC C10459) were obtained from the Iranian Biological Resource Center (Tehran, Iran). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Sigma, USA) with 1% penicillin and streptomycin (10,000 Unit/mL penicillin and 10 mg/mL streptomycin). This medium was also supplemented with 2 mM L-glutamine and 100 μg/mL of amphotericin B. The cell cultures were then incubated under standard cell culture conditions (37°C, 95% humidity, 5% CO2). Culture media was changed after every 3 days. After reaching confluency, the cells were treated with 0.25% trypsin and 0.02% ethylene diamine tetraacetic acid (EDTA). Furthermore, 3 × 105 cells (100 µL) per well were added in 96well cell culture microplates (Greiner Bio-One, Germany) [15]. 2.5 PDT HGF cells were incubated with 100 µL of ICG (1000 μg/mL) for 5 min in a humidified incubator at 37°C, 95% air, and 5% CO2 in dark condition. To prevent laser transmission to test wells in 96-well cell culture microplates, the test wells were separated from each other by wells that were filled by indian ink. At the end of the incubation period, the cells in the microplate wells containing ICG were immediately exposed to the diode laser at 250 mW for 60s (39 J/Cm2) at room temperature (25 ± 2°C) [16]. Three volumes of RNAlater® (Thermo Fisher Scientific, US) were then added to stabilize the RNA. The mixture was then
treated immediately for RNA extraction. The probe of laser was positioned perpendicular to the center of each well at a distance of 1 mm from the wells by a microphone stand to ensure an equal exposure of the entire well. We placed black paper sheets under the microplate to avoid beam reflection from the table top during the experiment [17]. The diameter of the irradiated area was the same as the well diameter at bottom of the microplate (i.e., 6.39 mm). The control group did not receive any treatment. Control wells with plain balanced salt solution (BSS, pH 7.4) were used to reveal potential dye independent apoptotic effects. The treated HGF cells were quickly harvested (cell suspension collected) after treatment to avoid binding and were washed twice with sterile phosphate-buffered saline (PBS, pH 7.4). 2.6 RNA extraction HGF cells after PDT treatment were collected for extarction of RNA. Total RNA was extracted using the Hybrid-RTM Total RNA Purification Kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s instruction. The concentration and purity of RNA sample were determined by reading the optical density at 260 and 280 nm wavelengths. For this purpose, a NanoDrop® spectrophotometer (Thermo Fisher Scientific, US) was used to assess the purity of the extracted RNA [18].
2.7 cDNA synthesis and real-time quantitative RT-PCR For real-time PCR analysis, cDNA was synthesized from 1.0 μg/µL of total extracted RNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Fermentas, Waltham, MA, USA) according to the manufacturer’s instruction. The cDNA was controlled by PCR with housekeeping GAPDH gene primers. Real-time PCR analysis of BCL-2 and BAX genes expression was carried out using the SYBR green PCR Master Mix (Takara, Shiga, Japan), in a total volume of 20 μL with Line-Gene K Real-Time PCR Detection System and Software (Bioer Technology, Hangzhou, China). The primers used were: GAPDH forward, 5′CGC TCT CTG CTC CTC CTG TT -3′ and reverse, 5′- ACG ACC AAA TCC GTT GAC TCC -3′; BAX forward, 5′- TTC TGA CGG CAA CTT CAA CTG G -3′ and reverse, 5′- AGG AAG TCC AAT GTC CAG CC -3′; and BCL-2 forward, 5′- AGG CTG GGA TGC CTT TGT GG -3′ and reverse, 5′- GGG CAG GCA TGT TGA CTT CAC -3′. GAPDH and BCL-2 primers were designed via gene runner software version 3.05 (Hastings Software Inc. Hastings, NY, USA) and BAX was used from a paper [19]. For each reaction of Real-time PCR, 10 μL of SYBR green PCR Master Mix and 7 μL of double distilled water were used. Moreover, 17 μL of the mixture was transferred to test tubes, and finally, 1 μL of the synthesized cDNA was added to each reaction tube with 2 μL of primers. The real-time PCR analysis was performed with the following thermal conditions: 5 min at 95°C for initial denaturation step, 35 cycles of 15 s at 95°C for denaturation step, 30 s at 59°C for annealing, and 15 s at 72°C for the extension, respectively, and 10 min final extension at 72°C was carried out for completion of amplicons. All samples were analyzed in triplicate, and GAPDH served as a reference gene for normalization. Melting curve analysis was performed according to the dissociation stage data and reactions to ensure that the fluorescent signal observed is from the desired PCR product. 2.8 Statistical analysis All of the experiments were carried out in triplicate. The data are represented as means plus or minus standard deviation (means ± SD). The main fold change of the target gene at each group was calculated using 2−ΔΔCT method, that is, relative to control cells. The expression levels of mRNA were shown as n-fold differences relative to the calibrator. A variation of >2-fold changes in expression level of the target gene was interpreted as significant [20].
3 Results ICG-PDT at 1000 µg/mL of ICG with the exposure time of 60 s for the diode laser induced significant expression of BAX in HGF cells as compared to the control group. In the illuminated ICG-1000 µg/mL cells, the expressions of BAX were increased to approximately 5.2-fold at 5 min; however, under the same treatment of cells revealed no significant changes in the expression of BCL-2 gene on the cells (Figure 1). Under the same light intensity and duration, there was no significant difference in the expression of BAX/BCL-2 genes in HGF cells as compared to the control group. Treatment with ICG at 1000 µg/mL, void of illumination, caused no observable changes in the expression of BAX/BCL-2 genes between the tested and control groups. 4 Discussion aPDT has been widely used in treating some cancers, in particular, those of the skin, age-related macular degeneration , and other diseases; it has also been used in treating infections [21-23]. aPDT as an adjunct to periodontal therapy has been evaluated by recent studies [24]. There is some evidence that aPDT not only kills the periodontal pathogens but also might inactivate the virulence factors of bacteria, improving outcome of the treatment [25]. Resolving the possible defect of aPDT in the clinical application needs emphasization that the toxic effect of aPDT on normal adjacent tissue is minimal; however, it has been indicated that the dosage of light used to kill bacteria in aPDT is unable to damage the fibroblast cells. Human periodontal cell studies gave preliminary evidence of biological effects of the aPDT on soft tissue [24, 26]. Thus, we aimed to evaluate the effects of ICG-PDT on human gingival fibroblast. ICG as a fluorescent agent with low toxicity has been widely used in biomedical fields since its first use [27-30]. Stalmans et al. stated that the toxicity of the ICG solution on the retinal pigment epithelial (RPE) cells depends on the hypo-osmolarity of the solvent [31]. Wu et al. found that ICG must be dissolved in pure water and solubilized again in BSS to avoid a hypo-osmolar solution [32]. Ho et al. reported that removal of Na+ from the ICG solution was associated with its reduced toxicity in RPE cells [33]. The absorption and emission properties of ICG are determined by the type of solvent and the concentration of ICG. When ICG is diluted in an aqueous solution by increasing the ICG concentrations the absorption peak shifts from 780 to 695 nm. This alteration of absorption peak depends on aggregation of the ICG molecules from monomer to oligomer [34]. Rezai et al. [35] have demonstrated that all the concentrations of ICG (1000, 5000, and 20,000 µg/mL) with 30 min incubation time, induced apoptosis in RPE cells. Our results showed that ICG, in the absence of light, did not induce any significant apoptosis in HGF cells when compared with the control group. The lack of induction of apoptosis could be explained by the relatively short time exposure of our study. Yam et al. [36] reported that the damage by ICG on RPE cells was determined by the concentration, change in osmolarity, pH, and incubation time. They showed that the optimum dose of ICG-PDT is as low as 250 µg/mL. In the present study, light exposure with a low dose of ICG as low as 1000 µg/mL, induced HGF cells apoptosis. Penha et al. [37] found that ICG induced apoptosis in ARPE-19 cells after 3 min of exposure at all concentrations (5, 50, 250, 500, and 1000 µg/ mL). In this study, we expected that ICG 1000 µg/mL would cause apoptosis, whereas we observed that ICG at this concentration may has a protective effect over HGF cells, since it increased the expression of BCL-2 gene. In agreement with our data, Kreisler et al. [26] showed that diode laser at 810 nm after 60 s irradiation time, did not reduce the survival rate of HGF cells. Furthermore, in vivo investigations are necessary for an overall evaluation of PDT’s applicability in periodontics and for recommendation of ideal ICG concentration in pocket decontamination. 5 Conclusion
The results of genes expression analysis reveals that ICG-PDT at concentrations of 1000 μg/mL induced expression of apoptosis-related gene, BAX, in HGF cells; however, laser irradiation as well as 1000 μg/mL of ICG revealed no significant apoptotic effects on expression of this gene in HGF cells. Acknowledgements This research has been supported by Laser Research Center of Dentistry (LRCD), Dentistry Research Institute, Tehran University of Medical Sciences & Health Services grant No. 94-04-97-30540. References 1. A. Cekici, A. Kantarci, H. Hasturk, D.E. Van Dyke, Inflammatory and immune pathwaysin the pathogenesis of periodontal disease, Periodontol. 2000. 64 (2014) 57-80. 2. J.Y He, G.G Qi, W.J. Huang, X.D. Sun, Y. Tong, C.M. Peng, X.P. Zhou , H. Chen, Short-term microbiological effects of scaling and root planing and essential-oils mouthwash in Chinese adults, J. Zhejiang. Univ. Sci. B. 14 (2013) 416-425. 3. K.R. Raj, S. Musalaiah, M. Nagasri, P.A. Kumar, P.I. Reddy, M. Greeshma, Evaluation of efficacy of photodynamic therapy as an adjunct to nonsurgical periodontal therapyin treatment of chronic periodontitis patients: A clinico-microbiological study, Indian. J. Dent. Res. 27 (2016) 483-487. 4. A. Kapoor, R. Malhotra, V. Grover, D. Grover, Systemic antibiotic therapy in periodontics, Dent. Res. J. 9 (2012) 505-515. 5. L.M. Baltazar, A. Ray, D.A. Santos, P.S. Cisalpino, A.J. Friedman, J.D. Nosanchuk, Antimicrobial photodynamic therapy: an effective alternative approach to control fungal infections, Front. Microbiol. 13 (2015) 202. 6. B.P. De Oliveira, C.M. Aguiar, A.C. Câmara, Photodynamic therapy in combating the causative microorganisms from endodontic infections, Eur. J. Dent. 8 (2014) 424-430. 7. V.K. Kamath, J.B. Pai, N. Jaiswal, Periowand: photodynamic therapy in periodontics, Univ. Res. J. Dent. 4 (2014) 133–138. 8. M. Wainwright, Photodynamic antimicrobial chemotherapy, J.Antimicrob.Chemother. 42 (1998) 13-28. 9. C. Beltes, N. Economides, H. Sakkas, C. Papadopoulou, T. Lambrianidis, Evaluation of Antimicrobial Photodynamic Therapy Using Indocyanine Green and Near-Infrared Diode Laser Against Enterococcus faecalis in Infected Human Root Canals,Photomed. Laser. Surg. 20 (2016) 1–6. 10. H.J. Lim, C.H. Oh, Indocyanine green-based photodynamic therapy with 785nm lightemitting diode for oral squamous cancer cells,Photodiagn. Photodyn. Ther. 8 (2011) 337-42. 11. P.R. Wei, Y. Kuthati, R.K. Kankala, C.H. Lee. Synthesis and Characterization ofChitosan-Coated Near-Infrared (NIR) Layered Double Hydroxide-Indocyanine Green Nano composites for Potential Applications in Photodynamic Therapy, Int. J. Mol. Sci.16 (2015)20943-68. 12. A. Chiaviello, I. Postiglione, G. Palumbo, Targets and Mechanisms of Photodynamic Therapy in Lung Cancer Cells: A Brief Overview, Cancers. 3 (2011) 1014-1041. 13. N.S. Soukos, L.A. Ximenez-Fyvie, M.R. Hamblin, S.S. Socransky, T. Hasan, Targeted Antimicrobial Photochemotherapy, Antimicrob. Agents.Chemother. 42 (1998) 2595-2601.
14. J.D. Ho, R.J. Tsai, S.N. Chen, H.C. Chen, Removal of sodium from the solvent reduces retinal pigment epithelium toxicity caused by indocyanine green: implications for macular hole surgery, Br. J.Ophthalmol. 88 (2004) 556-9. 15. M. Pourhajibagher, N. Chiniforush, S. Parker, S. Shahabi, R. Ghorbanzadeh, M.J. Kharazifard, A. Bahador, Evaluation of antimicrobial photodynamic therapy with indocyanine green and curcumin on human gingival fibroblast cells: An in vitrophotocytotoxicity investigation, Photodiagn. Photodyn. Ther. 15 (2016) 13-8. 16. S.S. Bhojwani, M.K. Razdan, Plant Tissue Culture: Theory and Practice, revised edn, Elsevier. Science. B.V. (1996) 467. 17.
R. Klein, Laser Welding of Plastics: Material properties of plastics, Wiley-VCH. 43 (2011) 3-69.
18. P. Desjardins, D. Conklin,NanoDropMicrovolume Quantitation of Nucleic Acids,J. Vis. Exp. 45 (2010) e2565. 19. G.M. Saed, M.P. Diamond, Apoptosis and proliferation of human peritoneal fibroblasts in response to hypoxia, Fertil.Steril. 78 (2002) 137-43. 20. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) Method. Methods. 25 (2001) 402-8. 21. M.T. Wan, J.Y. Lin, Current evidence and applications of photodynamic therapy in dermatology, Clin.Cosmet.Investig.Dermatol. 7 (2014) 145-163. 22. V. Rapozzi, E. Della Pietra, Bonavida B. Dual roles of nitric oxide in theregulation of tumor cell response and resistance to photodynamic therapy,Redox. Biol. 6 (2015) 311-7. 23. A. Azizi, S. Shademan, M. Rezai, A. Rahimi, S. Lawaf. Effect of photodynamictherapy with two photosensitizers on Streptococcus mutants: In vitro study,Photodiagn.Photodyn.Ther. 16 (2016) 66-71. 24. T. Kikuchi, M. Mogi, I. Okabe, K.Okada, H. Goto, Y. Sasaki, T. Fujimura, M. Fukuda, A. Mitani, Adjunctive Application of Antimicrobial Photodynamic Therapy in Nonsurgical Periodontal Treatment: A Review of Literature, Int. J. Mol. Sci. 16 (2015) 24111-24126. 25. S. Rajesh, E. Koshi, K. Philip, A. Mohan. Antimicrobial photodynamic therapy: An overview. J. Indian. Soc.Periodontol. 15 (2011):323-327. 26. M. Kreisler, M. Daubländer, B. Willershausen-Zönnchen, B. d’Hoedt, Effect of diode laser irradiation on the survival rate of gingival fibroblast cell cultures, Lasers. Surg. Med. 28 (2001) 445-50. 27. N.Y. Hong, H.R. Kim, H.M. Lee, D.K. Sohn, K.G. Kim, Fluorescent property of indocyanine green (ICG) rubber ring using LED and laser light sources, Biomed Opt Express. 7 (2016) 1637-44. 28. J. Meyer, A. Cunea, D. Sonntag-Bensch, P. Welker, K. Licha, F.G. Holz, S. Schmitz-Valckenberg, In vivo imaging of a new indocyanine green micelle formulation in an animal model of laser-induced choroidal neovascularization, Invest Ophthalmol Vis Sci. 55 (2014) 6204-12. 29. J.C. Kraft, R.J. Ho, Interactions of indocyanine green and lipid in enhancing near-infrared fluorescence properties: the basis for near-infrared imaging in vivo, Biochemistry. 53 (2014) 1275-83.
30. B. Jung, V. I. Vullev, and B. Anvari, “Revisiting indocyanine green: effects of serum and physiological temperature on absorption and fluorescence characteristics”, IEEE J. Sel. Top. Quantum Electron. 20 (2014) 149–157. 31. E.F. Costa, N.M. Barros, L.P. Coppini, R.L. Neves, A.K. Carmona, F.M. Penha, E.B. Rodrigues, E. Dib, O.Jr. Magalhães, M.N. Moraes-Filho, A.A. Lima Filho, M. Maia, M.E Farah, Effects of light exposure, pH, osmolarity, and solvent on the retinal pigment epithelial toxicity of vital dyes, Am J Ophthalmol. 155 (2013)705-12. 32. W.C. Wu, D.N. Hu, J.E. Roberts, Phototoxicity of indocyanine green on human retinal pigment epithelium in vitro and its reduction by lutein, Photochem.Photobiol. 81(2005) 537–540. 33. F.M. Penha, E.B. Rodrigues, M. Maia, C.H. Meyer, P. Costa Ede, E. Dib, E. Bechara, A. Lourenço, A.A. Lima Filho, E.H. Freymüller, M.E. Farah, International Chromovitrectomy Collaboration.. Biochemical analysis and decomposition products of indocyanine green in relation to solvents, dye concentrations and laser exposure, Ophthalmologica. 230 (2013) 59-67. 34. B. Yuan, N. Chen, Q. Zhu, Emission and absorption properties of indocyanine green inIntralipid solution. J. Biomed. Opt. 9 (2004) 497-503. 35. K.A. Rezai, E. asyna, B.L. Seagle, J.R. Norris, K.A. Rezaei, AcrySof Natural filter decreases blue light-induced apoptosis in human retinal pigment epithelium. Graefes. Arch. Clin. Exp. Ophthalmol. 246 (2008) 671-6. 36. H.F. Yam, A.K. Kwok, K.P. Chan, T.Y. Lai, K.Y. Chu, D.S. Lam, C.P. Pang, Effect of indocyanine green and illumination on gene expression in human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 44 (2003) 370-7. 37. F.M. Penha, M. Pons, E.F. Costa, E.B. Rodrigues , M. Maia , M.E. Marin-Castaño, M.E. Farah, Effect of vital dyes on retinal pigmented epithelial cell viability and apoptosis: implications for chromovitrectomy, Ophthalmologica. 230 (2013) 41-50. Figure legend: Figure 1. Changes in mRNA expression levels of BAX and BCL-2 genes following treatments. Values are presented as means ± SD. S+ L−; S and L stand for “sensitizer” and “light,” respectively.
Figr-1