The Photodynamic Therapy using 5-Aminolevulinic Acid(ALA): The Study of the Anti-Tumor Effect on in vitro and in vivo Experiments / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: ALA is a precursor of heme and converted to protoporphyrin IX used as effective photosensitizer. The aim of this study was to find the ideal concentration and incubation time of ALA for PDT on in vitro and in vivo experiments and to assess the anti-tumor effect of PDT using ALA on CT-26 colon cancer cell line. MATERIALS AND METHOD: CT-26 cell was cultured with serum free media including ALA in the dark room to show the intracellular accumulation of PpIX. The fluorescence of PpIX in the cell was detected under confocal laser scanning microscope. Also CT-26 cell was incubated with various concentration of ALA (1.0-0.001 mg/ml) and was irradiated with LED at 0 hr, 3 hr, 6 hr, 9 hr, 12 hr and 24 hr after application of ALA. The cell viability was assessed by MTT assay. in vivo PDT was done with optimal treatment condition and the anti-tumor effect of PDT using ALA was measured by tumor volume change. RESULTS: The fluorescence of PpIX was saturated at 6 hour after the ALA application to CT-26 cell and the optimal incubation time with ALA for PDT was 6 hours. For in vivo Study, 632 nm laser irradiation was done around the tumor 6 hours after ALA injection. The PDT using ALA on transplanted CT-26 tumors shows 40% cure rate and 40% partial remission and significant decrease of tumor volume. CONCLUSION: The peak accumulation of PpIX in the cell and in the tumor was reached 6 hours after the application of ALA. The PDT using ALA for CT-26 cells was very effective and this findings suggest that ALA is one of candidate for photosensitizer in head and neck solid tumors.

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.

iNOS Expression in Inner Ears of Guinea Pigs Injured by PAF / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Platelet-activating factor (PAF) in middle ear effusion is thought to induce hearing loss. The purpose of this study is to determine the effects of PAF placed on round window membrane (RWM) on hearing and cochlear hair cells in guinea pigs, and we also wanted to investigate the role of nitric oxide (NO) in the mechanism of PAF-induced hearing loss by comparing its immunoreactivity to iNOS between the control group and PAF application group. MATERIALS AND METHOD: Guinea pigs were divided into 2 groups: PBS, PAF. The PBS group received phosphate buffered saline (PBS) and the PAF groups received 10, 20, and 40 mug/ml of PAF soaked in gelfoam placed on the RWM. The following three tests were performed on each animal group: hearing was tested with an auditory brainstem response (ABR) test through 24 hours. At the end of 24 hours, cochlear hair cells were examined by scanning electron microscopy (SEM) and immunohistochemistry was carried out on the cochlea to test the expression of inducible nitric oxide (iNOS). RESULTS: The PAF group developed significant elevation of ABR threshold and cochlear hair cell damage in SEM compared with the PBS control group. Strong expression of iNOS on cochlea was observed in the PAF group and lighter expression was seen in PBS group. CONCLUSION: This study demonstrated that PAF placed on the RWM induced hearing loss, and cochlear hair cell damage, and strong iNOS expression in the cochlea. These findings suggest that the PAF-induced hearing loss caused by cochlear hair cell damage may have been mediated by NO. PAF-antagonists and NOS inhibitor may have future therapeutic implications in preventing sensorineural hearing loss associated with chronic otitis media.

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.

The Ototoxicity of Cisplatin is Mediated by NO: A Study using L-NAME and MK-801 in Guinea pigs / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Nitric oxide has been suggested to play an important role in the pathogenesis of cisplatin ototoxicity. L-NAME (NG-Nitroarginine Methyl Ester) is an inhibitor of nitric oxide synthase. MK-801 (Dizocilpine Maleate) is a NMDA receptor antagonist. To evaluate a role of nitric oxide in cisplatin ototoxicity, we investigated whether L-NAME and MK-801 can block the cisplatin ototoxicity in guinea pigs. MATERIALS AND METHOD: In the Group 1, normal saline was injected intraperitoneally as a control group. Group 2, 3, 4, and 5 were injected intraperitoneally as described in the following: Group 2, cisplatin only; Group 3, L-NAME+isplatin; Group 4, MK-801+cisplatin; Group 5, L-NAME+K-801+cisplatin. Using an auditory brainstem response, hearing threshold was tested before cisplatin administration and 5 days after cisplatin injection in each group. The morphological changes of the cochlea were observed by scanning electron microscopy. RESULTS: In the Group 2, a significant hearing loss was observed comparing to Group 1. In contrast , Group 3, 4, and 5 did not demonstrate any significant hearing loss compared to Group 1. In the scanning electron microscopy, the Group 2 showed distorsion and loss of stereocilia of the hair cells. However, the Group 1, 3, 4, and 5 demonstrated well preserved cochlear hair cell morphology. CONCLUSION: Hearing loss induced by ototoxicity of cisplatin was prevented by L-NAME and MK-801. This study suggests that NO may mediate cisplatin ototoxicity.

The protective effect of nitirc oxide synthase inhibitor on hearing loss induced by TNF-alpha and IL-1beta injection into CSF in guinea pigs / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: One of the most serious complications of bacterial meningitis, particularly in childhood, is sensorineural hearing loss; yet the mechanisms of this hearing loss are not clearly understood. It is also known that the levels of TNF-alpha, IL-1beta in CSF are significantly elevated in bacterial meningitis patients. This study is designed to evaluate the role of nitric oxide (NO) in the ototoxic effect of TNF-alpha and IL-1beta injected into CSF of guinea pigs. MATERIALS AND METHODS: Twenty-six guinea pigs (52 ears) were randomly assigned into six groups consisting of a control group, a NG-nitro-L-arginine methyl ester (L-NAME) group, a TNF-alpha group, a TNF-alpha plus L-NAME group, a IL-1beta group and a IL-1beta plus L-NAME group. The thresh shifts of the auditory brain stem response (ABR) were measured before, 6 and 24 hours after the administration of cytokines. TNF-alpha and IL-1beta were directly injected into the cisterna magna in a dosage of 0.1 microgram in 10 microgram/ microliter concentration. L-NAME, a NO synthase (NOS) inhibitor, was injected intraperitoneally 30 minutes prior to the administration of cytokines. Scanning electron microscopy was used to find the morphological damage of the hair cells. RESULTS: In TNF-alpha group, the ABR thresh shift after injection of 6 and 24 hours was 19.2 +/- 10.2 dB, 18.3 +/- 10.3 dB, in the TNF-alpha plus L-NAME group, 3.3 +/- 2.6 dB, 8.3 +/- 4.1 dB, in the IL-1beta group, 6.9 +/- 4.6 dB, 11.25 +/- 8.3 dB, and in the IL-1beta plus L-NAME group, 1.7 +/- 2.7 dB, 1.3 +/- 2.5 dB, respectively. There was damage in the 2nd, 3rd row of the outer hair cells in the TNF-alpha and IL-1beta group. CONCLUSION: This study suggests that the sensorineural hearing loss associated with bacterial meningitis is caused in part by the actions of proinflammatory cytokines such as TNF-alpha, IL-1beta and that these are mediated by NO.

The Effects of IL-1beta in Cerebrospinal Fluid on Hearing in the Guinea Pigs / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Cytokines such as interleukin-1beta (IL-1beta) released into cerebrospinal fluid (CSF) during bacterial meningitis play an important role in causing inflammation and tissue damage. Bacterial meningitis is often complicated by a sensorineural hearing loss. The present study is to investigate the effect of IL-1beta injected into CSF on hearing in guinea pigs. MATERIALS AND METHODS: Thirty guinea pigs (60 ears) were randomly assigned to the following three groups: 1) Control group receiving intracisternal PBS injection. 2) 10 ng group receiving intracisternal injection of 10 ng/ml of IL-1beta. 3) 100 ng group receiving intracisternal injection of 100 ng/ml of IL-1beta. Auditory brainstem response (ABR) was performed before the injection, 10, and 24 hours after the injection of PBS and IL-1beta. The concentration of IL-1beta in the perilymph was measured in each group. RESULT: The ABR threshold shift at 10 and 24 hours were respectively 3.3+/-2.6 dB, 2.8+/-2.0 dB in the control group, 21.94+/-14.46 dB, 5.83+/-9.74 dB in the 10 ng group, and 21.58+/-15.99 dB, 4.74+/-9.05 dB in the 100 ng group. The ABR thresholds were significantly increased in the 10 and 100 ng groups compared to the control group at 10 hours, but they were not significantly different at 24 hours after the injection. The concentrations of IL-1beta in the perilymph at 10 hours were 2.17+/-0.6 ng/ml in the 10 ng group and 3.58+/-1.1 ng/m in the 100 ng group. Those were 0.53+/-0.1 ng/ml in the 10 ng and 0.86+/-0.2 ng/ml in the 100 ng groups at 24 hours after the injection. CONCLUSION: The results of this study showed that IL-1beta released into CSF during meningitis may play an important role in causing hearing loss.

Genetic Abnormality and Localization of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Patients with Nasal Polyps

BACKGROUND AND OBJECTIVES: Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel protein, its kinetics and localization are altered in cystic fibrosis. The purpose of this study was to evaluate whether the nasal polyp patients without phenotypic manifestation of cystic fibrosis have any form of CFTR mutations and to characterize the localization of CFTR in the nasal polyp. MATERIALS AND METHODS: The study group consisted of 71 subjects with nasal polyp who underwent an intranasal operation, and 20 normal subjects. Peripheral blood of the study groups were screened for mutation on the exon 3, 4, and 7 of the CFTR gene using single-stranded DNA conformational polymorphism (SSCP). Immunohistochemical staining for CFTR was conducted on the nasal polyps of studied subjects and normal turbinates as the control. RESULTS: While in the nasal polyp group, SSCP screening revealed two cases of mutant band on the exon 3, the normal control group did not show mutant band in all exons screened. CFTR showed the typical apical distribution in the normal turbinate mucosa, whereas in the nasal polyp, regardless of an abnormal band in exon 3, CFTR demonstrated a heterogenous pattern of localization consisting of cytoplasmic labeling, perinuclear staining, and intermingled apical location. CONCLUSION: These results suggest that an altered localization of the CFTR in the nasal polyps, based not only on the CFTR mutation but also on the acquired inflammatory process, may have an important role in the formation of nasal polyps.