Photodynamic therapy using topically applied 5-aminolevulinic acid for treatment of cervical cancer / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to investigate the treatment effects of a topical application of 5-aminolevulinic acid (5-ALA) for photodynamic therapy (PDT) to treat cervical cancer. METHODS: We first investigated the effects of 5-ALA cream according to application time. And to find the effective 5-ALA concentration and the distribution times in vivo, 20% 5-ALA cream was topically applied to the tumor of the nude mouse. We then observed the distribution of 5-ALA via fluorescence measurement with using a 532 nm diode laser. 25 nude mice were divided into Control, ALA, Laser, and PDT group. To evaluate the PDT effect at cancer lesion, we applied 20% 5-ALA cream to the tumor by the same method, and the PDT was done by using a 632 nm diode laser at the time of the peak level of fluorescence. We checked the changes of the volume of cancer for 30 days, and then biopsy was done. RESULTS: The effective post-irradiation time after topical ALA application was 9 hours. In the PDT group, 40% (4/10) of the mice showed decreased tumor size. CONCLUSION: The maximum PpIX fluorescence at 9 hours after local applicationof 5-ALA cream was checked. And PDT group did not show any statistical difference than control group in the growth of tumor size than control group. However responding cases (4/10) of PDT group showed the meaningful decrease of tumor size than control group (P<0.05).

Viability of Chondrocytes in the Crushed Septal Cartilage Depending on Degree of Crushing and Their Mechanism of Cell Death / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: To analyze the viability of chondrocytes according to different degrees of crushing and to investigate the mechanism of cell death in the crushed cartilage. SUBJECTS AND METHOD: Septal cartilages were obtained from 22 patients and cartilage pieces were allocated to four groups; normal, mildly crushed, moderately crushed and severely crushed. The cartilage specimens were stained with hematoxylin-eosin and examined under light microscope. The viability of the chondrocytes and the mechanism of cell death were assessed using confocal laser scanning microscopy. RESULTS: As crushing intensity increased, chondrocyte viability significantly decreased. The mechanism of cell death was mainly due to necrosis rather than apoptosis. CONCLUSION: The viability of chondrocytes in the crushed cartilage depends on the degree of crushing. The mechanism of cell death after crushing is mainly necrosis. Therefore, for the clinical use of the crushed cartilage, slight overcorrection and standardization of the degree of crushing are recommended.

Antioxidant and Anti-Apoptotic Effect of Melatonin on the Vestibular Hair Cells of Rat Utricles

OBJECTIVES: Aminoglycosides are commonly used antibiotic agents, and they are known to generate free oxygen radicals within the inner ear and to cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Melatonin, a pineal secretory product, has the properties of being a powerful direct and indirect antioxidant. The aim of the present study was to prove the antioxidant effect of melatonin against gentamicin-induced ototoxicty. METHODS: The utricular maculae of Sprague-Dawley rats were prepared from postnatal day 2-4, and these maculae were were divided into 6 groups as follows: 1) control, 2) melatonin only, 3) gentamicin only, and 4), 5), and 6) gentamicin plus melatonin (10, 50, and 100 micrometer, respectively). To count the number of hair cells, 5 utricles from each group were stained with phalloidin-FITC on the 1st, 4th, and 7th days after drug administration. Reactive oxygen species (ROS) was assessed by using the fluorescent probe hydrofluorescent diacetate acetyl ester. The caspase-3 activity was also examined with using the fluorescent caspase-3 substrate and performing Western blotting. RESULTS: The result of this study showed that gentamicin induced the loss of utricular hair cells, and this loss of hair cells was significantly attenuated by co-administration of melatonin. Melatonin reduced ROS production and caspase-3 activation in the gentamicin treated utricular hair cells. CONCLUSION: Our findings conclusively reveal that melatonin has protective effects against gentamicin-induced hair cell loss in the utricles of rat by inhibiting both ROS production and caspase-3 activity.

Preventive and Therapeutic Effects of Low Level Laser Irradiation on Gentamicin-Induced Vestibulotoxicity in Rat Utricles / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The purpose of this study was to investigate the effects of low level laser for the prevention and treatment of aminoglycoside-induced vestibular ototoxicity. MATERIALS AND METHOD: An organotypic culture of 2 to 4 days old rat utricular maculae was established. Rats were divided into 6 groups according to the treadtment of the utricles: G (gentamicintreated), L (laser-irradiated), LG (laser-irradiated and gentamicin-treated), GL (gentamicin-treated and laser-irradiated), LGL (gentamicin-treated during laser-irradiated) and C (control). After organotypic culture, the utricles of 6 groups were examined by confocal laser scanning electron microscope and scanning electron microscope. The results of each group were compared with each other by statistical methods. RESULTS: The number of vestibular hair cells of the group G was smaller compared to that of the group C. The group L had no difference compared with the group C. The groups LG and GL showed more vestibular hair cells compared with the group G. The group LG showed more vestibular hair cells than the group GL. The group LGL showed most vestibular hair cells compared to that of the groups G, LG, and GL. CONCLUSION: The most effective treatment of aminoglycosideinduced vestibular otoxicity is the irradiation of low level laser before and after the insult of the aminoglycoside. Further clinical studies using low level laser were needed to prevent aminoglycoside-induced ototoxicity and to promote the regeneration of vestibular hair cells.

Change of Nitric Oxide Synthase Expression and Dynamic Ciliary Activity Responding to Nitric Oxide in Sinusitis Mucosa / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: There are conflicting results about ciliary activity in chronic rhinosinusitis (CRS). Dynamic movements reacting to various stimuli in CRS mucosa have been rarely studied. This study was designed to investigate the dynamic ciliary activity in response to nitric oxide stimulation in sinusitis. We aimed to identify the difference in the expression of nitric oxide synthase (NOS) in normal and CRS mucosa. SUBJECTS AND METHOD: Nasal mucosal samples were obtained from 25 sinusitis and 15 normal subjects. We measured ciliary beat frequency (CBF) in the basal and activated status. Immunohistochemial staining was used to evaluate the expression of NOS in the sinusitis. RESULTS: The CRS mucosa showed marked differences in the CBF changes stimulated by NO compared to the normal mucosa; both the maximal increase and duration of increase of CBF by NO were significantly reduced in CRS mucosa. The results of immunohistochemical stain showed that eNOS expression was evident in the normal nasal mucosa and iNOS expression was markedly increased in CRS mucosa. CONCLUSION: Dynamic ciliary activity responding to NO was markedly attenuated in the CRS mucosa. The L-NAME markedly attenuated the duration of increase and maximal increase of CBF by ATP both in the normal and CRS mucosa. eNOS expression was relatively evident in the normal mucosa, whereas iNOS expression was relatively increased in the CRS mucosa, implicating different actions of NO on CBF.

A Promotive Effect of Low-Level Laser on Hair Cell Regeneration Following Gentamicin Induced Ototoxicity in Postnatal Organotypic Culture of Rat Utricles / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: In normal postnatal mammalian inner ear sensory epithelium, regeneration of hair cells is a very rare event, but there is hair cell regeneration with partial restoration of the vestibular sensory epithelium following ototoxic damage. In this study, the effects of low-level laser on hair cell regeneration following gentamicin exposure in postnatal organotypic culture of rat utricles were investigated. MATERIALS AND METHOD: A long term organotypic culture of 2 to 7 day old rat utricular maculae was established to study aminoglycoside-induced vestibular hair cell renewal. The utricles were exposed to 1 mM of gentamicin for 48 hr and allowed to recover in a culture medium only or in a medium with daily irradiation of low-level laser, whereas the control group was not exposed to gentamicin. Whole-mount utricles were stained with FM1-43, which are known to be an efficient marker, to identify live hair cells in cultured tissues. RESULTS: Loss of hair cells was nearly stopped from 2 days after exposure to gentamicin ; a peak of regeneration was reached after 18 days and sustained for two weeks in the medium with the irradiation of low-level laser. CONCLUSION: These results suggest that low-level laser promotes spontaneous hair cell regeneration following gentamicin damage in utricular explants.

The Effect of IP3 Receptor Inhibition Using 2-APB on Gentamicin Ototoxicity in Cochlear Sensory Cell / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Elevated intracellular calcium level is known to play important roles in the apoptotic pathway. IP3 receptor (ligand-gated channels that release Ca2+ from intracellular stores) is emerging as a key site for regulation of apoptosis. 2-Aminoethoxydiphenyl borate (2-APB) is one of the reliable IP3 receptor antagonists. We examined the effect of 2-APB on gentamicin ototoxicity in vitro, using the HEI-OC1 cell line. MATERIALS AND METHOD: HEI-OC1 cells were trWWeated with 100micrometer gentamicin. Using a CaspACE assay, we measured the caspases-3 activity in the gentamicin treated hair cells with and without 2-APB pre-incubation. We also observed intra-cellular calcium concentrations in HEI-OC1 cells using a confocal microscopy (calcium green-1 stain). Live cell imaging was performed by using fluorescence video-time lapse system. RESULTS: Cytosolic calcium elevation by gentamicin was remarkably inhibited by 2-APB. Caspases-3 activities of gentamicin treated cells were higher than those of the control. After incubation with 2-APB, caspases-3 activities and cell death of gentamicin treated cells were shown to decrease. CONCLUSION: 2-APB reduces Caspases-3 activity in the gentamicin treated HEI-OC1 cells by inhibition of cytosolic calcium increase.

Minocycline Protects Vestibular Hair Cells from Neomycin-Induced Ototoxicity by Inhibiting Reactive Oxygen Species Production and Caspase-3 Activity / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Aminoglycosides, a commonly used antibiotic agent, destroys the sensory hair cells in the cochlear and vestibular system leading to irreversible hearing loss and balance problem. Minocycline, a second-generation tetracycline antibiotic, has been known to possess anti-apoptotic properties in addition to its antimicrobial action. We hypothesized that minocycline would attenuate aminoglycosides induced vestibulotoxicity in rat utricles. SUBJECTS AND METHOD: Utricular maculae prepared from postnatal day 3-4 rats were treated with neomycin alone or in combination with minocycline. For hair cell count, utricles were stained with phalloidin-FITC. Reactive oxygen species (ROS) was assessed using the fluorescent probe, hydrofluorescent diacetate acetyl ester (H2DCFDA). Caspase-3 activity was also examined using the fluorescent caspase-3 substrate. RESULTS: Neomycin induced dose-dependent loss of utricular hair cells. Minocycline reduced ROS production and caspase-3 activation in neomycin treated utricular hair cells. CONCLUSION: Minocycline has protective effect in neomycin induced ototoxicity in rat utricle by inhibiting ROS production and caspase-3 activity.

ROS Formation and the Mechanism of Photodynamic Therapy Using 9-Hydroxypheophorbide-a on AMC-HN3 Human Head and Neck Carcinoma Cell Line / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: During PDT, photosensitizer accumulates in the cell and irradiation forms ROS. ROS leads to activation of apoptoticpathway and cell death. Elevated intracellular calcium is known to play important role in apoptotic pathway. There are two type of ROS formation. The type of ROS formation differs in type of photosensitizers. We designed the experiment to define the relationship of ROS and cell death in PDT. MATERIALS AND METHOD: AMC-HN3 cells were cultured. Using a CaspACE assay kit, we measured caspases-3 activity after PDT. We also observed intra-cellular calcium concentrations using confocal microscopy (calcium green-1 stain) after PDT. To determine which type of reaction occursduring ROS formation, MTT assay was performed. RESULTS: Confocal microscopy showed that ROS had formed at the site of photosensitizer formation after PDT. After PDT, intracellular calcium increased. MTT assay showed more viability increase in blocking type II reaction. Caspase assay showed highest level after 4hrs. CONCLUSION: ROS is formed at the site photosentizer formation after PDT. Type II reaction was the main type of ROS formation. Apoptosis was main pathway of cell death in low dose of photosensitizer after PDT.

A Promotive Effect of Low Level Laser on Hair Cell Viability in Postnatal Organotypic Culture of Rat Utricles

BACKGROUND AND OBJECTIVES: To culture and maintain mammalian hair cells is still a big challenge. In this study, long-term organotypic culture of rat utricular maculae was established to study vestibular hair cell. The effects of low level laser on hair cell viability in postnatal organotypic culture of rat utricles were investigated. MATERIALS AND METHOD: Uticular explants were prepared from postnatal 2 to 7 rats and cultured. To improve hair cell survival, the utricles were irradiated daily with low level laser. Whole-mount utricles were stained with FM1-43 which is known to be an efficient marker to identify live hair cells in cultured tissues. Such cells visualized directly through tissue culture dish with cover glass bottom by Confocal laser scanning microscope at specific time points. RESULTS: The explanted utricular hair cells were cultured for up to 31 days in in vitro culture system. In low level laser irradiation group, utricular hair cells were more survived at 24 DIV and 31 DIV. CONCLUSION: These results suggest that low level laser promotes hair cell viability in utricular explants.

A Promotive Effect of Low Level Laser on Hair Cell Viability in Postnatal Organotypic Culture of Rat Utricles

BACKGROUND AND OBJECTIVES: To culture and maintain mammalian hair cells is still a big challenge. In this study, long-term organotypic culture of rat utricular maculae was established to study vestibular hair cell. The effects of low level laser on hair cell viability in postnatal organotypic culture of rat utricles were investigated. MATERIALS AND METHOD: Uticular explants were prepared from postnatal 2 to 7 rats and cultured. To improve hair cell survival, the utricles were irradiated daily with low level laser. Whole-mount utricles were stained with FM1-43 which is known to be an efficient marker to identify live hair cells in cultured tissues. Such cells visualized directly through tissue culture dish with cover glass bottom by Confocal laser scanning microscope at specific time points. RESULTS: The explanted utricular hair cells were cultured for up to 31 days in in vitro culture system. In low level laser irradiation group, utricular hair cells were more survived at 24 DIV and 31 DIV. CONCLUSION: These results suggest that low level laser promotes hair cell viability in utricular explants.

Tissue Reactions of the 532 nm Flash Lamp Pumped Solid State Laser and Diode Pumped Solid State Laser on the Muscle and Skin of the Guinea Pig / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The efficiency of second harmonic generation at 532 nm is high when a pulsed Nd : YAG laseris used as a pump source of fundamental waves. The pulse durations of 532 nm-irradiation can be varied from 10 ns to 1000 ns by changing the pumping method. The purpose of this study was to compare the macroscopic and micropscopic changes in the muscle and skin after irradiation by 532 nm Flash lamp pumped solid state (FPSS) laser and Diode pumped solid state (DPSS) laser, whose pulse durations are 600 ns and 100 ns, respectively. MATERIALS AND METHOD: Two experiments were conducted on the muscle and skin of guinea pig. First, the guinea pig muscle and skin were irradiated by the 532 nm FPSS and DPSS lasers with various intensities and time duration. Macroscopic examinations were performed on the muscle lesions and immediate histopathologic examinations on the skin were carried out. RESULTS: The volume of vaporization and necrosis of the guinea pig muscle by the FPSS laser was larger than that by the DPSS laser. The vaporization and necrosis reaction of skin by the FPSS was significantly stronger than that by the DPSS laser. CONCLUSION: It appears that the DPSS laser system would be superior over the FPSS laser system in providing more accurate and precise surgery with less intense injury on the surrounding tissue.

Effect of Photostimulation Using Low-Level Laser Therapy on Wound Healing in Induced Mouse Skin Wound / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: This study was conducted to investigate the healing effect of the low-level laser irradiation on wound healing in vivo using DPSS laser (532 nm) and Diode laser (660 nm). MATERIALS AND METHOD: Each mouse received dorsal, full-thickness round incision (=2 cm) and daily laser irradiation (4 J/cm2) was done before sacrifice. On sacrifice at 3, 7, 10 days, the wound was excised, then wound closure and histologic stages were measured, and standardized. RESULTS: The percentages of wound closure in DPSS laser, Diode laser, control were 33.2+/-2.4, 34.2+/-3.5, 24.0+/-2.7 at day 3, 64.8+/-3.5, 72.2+/-2.8, 42.8+/-5.0 at day 7 and 82.2+/-7.9, 87.2+/-3.7, 71.4+/-4.0 at day 10, respectively, with p<0.05. Histological evaluation showed that laser irradiation enhanced wound epithelialization, cellular content deposition, granulation tissue formation, collagen deposition and neovascularization in the laser-treated wounds as compared to the control group. CONCLUSION: Low-level laser irradiation at 532 nm and 660 nm significantly enhanced cutaneous wound healing effect in the wounded mouse model. Further investigation of the mechanism of low-level laser therapy in primary wound healing is warranted.

Anticancer Effect and Apoptosis Mechanism of Photochemotherapy Using Photogem and 632 nm Diode Laser on Human Squamous Cell Carcinoma Cell Line / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: This study was conducted to investigate the anticancer effect of photochemotherapy in vitro using a photosensitizing agent (Photogem) and a laser therapy (632 nm diode laser). MATERIALS AND METHOD: Human squamous cell carcinoma cell (SNU-1041) was treated to laser therapy for various irradiation times, laser powers, and interval times. The treated cell was analyzed by MTT assay, DAPI staining to see apoptosis. RESULTS: The viability of cells was decreased with the increasing of the laser irradiation time and the laser power. No significant difference in cell viability was noted by various interval time. Increasing apoptosis was observed by increasing concentration of Photogem and increasing lasering time by DAPI staining. CONCLUSION: This study demonstrated anticancer effect of photochemotherapy using Photogem and 632 nm diode laser. Apoptosis was observed in the process of cancer cell death.