Mouse Model for the Research of Sinusitis Induced Olfactory Dysfunction / 대한이비인후과학회지

Olfactory dysfunction is one of the most debilitating problem in chronic rhinosinusitis (CRS) patients, and exact mechanism underlying sinusitis induced olfactory dysfunction was not fully understood. In vivo manipulation for olfactory epithelium and fresh specimen for histopathological analysis are essential for research, but it is nearly impossible to do in human due to inaccessibility of olfactory epithelium and risk for complication. For this reason, several animal models using toxic materials, such as 3-methylindole or bromomethane, have been suggested for mimicking olfactory epithelial damage in CRS, but none of them could truly imitate the event which happens in real patient. Inducible olfactory inflammation (IOI) mouse is a transgenic mouse model selectively producing tumor necrosis factor-alpha (TNF-alpha) in sustentacular cell of olfactory epithelium. The production of TNF-alpha can be actively initiated by giving food containing doxycycline to IOI mouse, and inflammation is stopped in the absence of doxycycline. Both toxicity model and transgenic model have their own advantages and disadvantages, therefore appropriate model should be selected for optimal results.

Development of a HPLC/MS/MS methodology for determining 3-O-methyldopa in human plasma and its application in a bioequivalence study

A simple, sensitive and specific HPLC/MS/MS methodology was developed and it was validated for determining 3-O-methyldopa, the major metabolite of dopamine, in human plasma. The separation was achieved on Atlantis T3 C18 analytical column (5 μm; 150 x 4.6 mm i.d.) using a mobile phase consisted of a solution of water and methanol (85:15, v/v) and containing formic acid 0.05 %. The extraction from the analyte and the internal standard sample was performed using a simple protein plasma precipitation with perchloric acid. The detection was conducted on a triple quadrupole tandem mass spectrometer with a positive multiple reaction monitoring mode (MRM). The monitored fragmentation transitions were m/z212.0  m/z 166.0 for 3-O-methyldopa and m/z 227.10  m/z 181.0 for carbidopa (internal standard).The calibration curves were linear in the range of 50–4000 ng/mL for 3-O-methyldopa. The methodology presented a good precision and accuracy in accordance to the criteria for biomedical analysis. And it was successfully applied to the bioequivalence study of two formulations levodopa + benserazide (200 + 50mg) in plasma samples from healthy human volunteers, following the ANVISA guidelines...

Transplantation of Neural Stem Cells in Anosmic Mice

OBJECTIVES: Treating olfactory dysfunction is a challenge for physicians. One of the therapeutic options could be transplantation of stem cells. In this study, neural stem cells were transplanted into anosmic mice. METHODS: Neural stem cells were generated from the olfactory bulb of green fluorescent protein (GFP)-transgenic C57BL6 mice. Anosmia were induced by injection of intraperitoneal 3-methylindole. The neural stem cells were transplanted transnasally on the next day. The olfactory function was evaluated by a food-finding test once a week. The olfactory neuroepithelium was harvested for histologic examination and protein analysis at 4 weeks. RESULTS: Twenty-five percent (6/24) of the control mice that were not transplanted with neural stem cells survived at 4 weeks while 67% (8/12) of the transplanted mice survived (P=0.029). The food finding test showed that the transplanted mice resumed finding food at 3 weeks while the control mice resumed finding food at 4 weeks. GFP-positive cells were observed in the olfactory neuroepithelium of the transplanted mice. Western blotting revealed that the olfactory marker protein expression was significantly lower in the control mice than that in the transplanted mice. CONCLUSION: This study demonstrated that improvement of mouse survival was achieved and recovery of olfactory function was promoted by transnasal transplantation of neural stem cells in the anosmic mouse model. These results indicate that stem cells might be one of the future modalities for treating olfactory impairment.

Effect of Systemic Steroid on the Olfactory Epithelium Injured by 3-Methylindole in Mice

BACKGROUND AND OBJECTIVES: Various chemicals can affect the function of olfaction and steroids have been used for the treatment of olfactory dysfunction. In this study, we investigated the effect of chronic dexamethasone treatment on olfactory epithelium injured by 3-methylindole (3-MI). SUBJECTS AND METHODS: 0.75 mg/kg of dexamethasone and 0.15 mL of normal saline were administered to each of the 12 mice belonging to the experimental and control group respectively every other day from 1 week, before a single intraperitoneal administration of 175 mg/kg 3-MI, to 4 weeks after 3-MI injection. Three mice from each group were sacrificed every week, and olfactory epithelium was examined after H & E and immunohistochemical staining. RESULTS: On H & E staining, the height of the olfactory epithelium and the polarity of the cells showed no difference between the two groups. On olfactory marker protein (OMP) staining, the number of OMP-immunoreactive (IR) olfactory receptor cells was significantly increased in the experimental groups from 2 and 4 weeks after 3-MI injection compared with the control group. On proliferating cell nuclear antigen (PCNA) staining, PCNA-IR basal cells were significantly reduced in groups that received dexamethasone from 2 weeks to 3 weeks after injection compared with the control group. CONCLUSION: Dexamethasone shows no protective effect in early necrosis of olfactory epithelium by 3-MI, but showed positive effect on the regeneration of the olfactory receptor cells of olfactory epithelium.

Effect of Systemic Steroid on the Olfactory Epithelium Injured by 3-Methylindole in Rats / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Various chemicals can affect the function of olfaction and steroids have been used for the treatment of the olfactory dysfunction. In this study, we investigated the effect of chronic dexamethasone treatment on olfactory epithelium injured by 3-methylindole (3-MI). MATERIALS AND METHOD: 0.75 mg/kg of dexamethasone were administered to 12 experimental rats every other day for 1-4 weeks, following a single intraperitoneal administration of 150 mg/kg 3-MI at the first week. Three rats from each group were sacrificed every week, and olfactory epithelium were examined after H & E and immunohistochemical staining. RESULTS: On H & E staining, the height of the olfactory epithelium and the polarity of the cells showed no difference. On protein gene product 9.5 staining, the number of immunoreactive olfactory receptor cells significantly increased in the experimental groups from 2 and 4 weeks after 3-MI injection compared with the control group. On proliferating cell nuclear antigen staining, immunoreactive basal cells were significantly reduced in groups that received dexamethasone from 2 weeks to 3 weeks after injection compared with the control group. CONCLUSION: Dexamethasone shows no protective effect in early necrosis of olfactory epithelium by 3-MI and showed positive effect on the regeneration of the olfactory receptor cells of olfactory epithelium.

Olfactory Evoked Potential for Objective Olfactory Function Evaluation / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The exact diagnosis for olfactory dysfunction plays an indispensable complementary role to the proper management of olfactory dysfunction. But there is no confirmative objective method for olfactory function evaluation. Recently, olfactory evoked potential by jet steam method was introduced as an objective method for the evaluation of olfactory function. The purpose of this study is to evaluate clinical efficacy of olfactory evoked potential. SUBJECTS AND METHOD: Thirty two normal individuals were evaluated with olfactory evoked potential and their olfactory thresholds were within normal range when tested with T&T olfactometry. Odorant, skatole was introduced 1cm apart from the nostril by jet steam pulse. The results were averaged by Neuropack Four Computer. RESULTS: Twenty-nine individuals had typical positive waves with latency near 0.1ms, but three individuals had no typical positive wave. The threshold of twenty individuals in electro-olfactometry was E1 with E1 recognition or detection threshold in T&T olfactometry. The threshold of other nine individuals in electro-olfactometry was E2 with E1 recognition and detection threshold in T&T olfactometry. CONCLUSION: Olfactory evoked potential has good correlation with classic T&T olfactometry and could be used as an objective method for the evaluation of olfactory function.

A Study on Recovery from Smell Dysfunction Induced by 3-Methylindole in Rats

BACKGROUND AND OBJECTIVES: Evaluating the olfactory function is as important in animal research as morphological study. However, it is difficult to gauge the smell function in rats because of the underdevelopment of current electrophysiologic measuring devices. The aim of this study is to assess changes in smell dysfunction induced by 3-methylindole (3-MI) in rats using an 8-odor olfactometer. MATERIALS AND METHOD: Eight female Sprague-Dawley rats were used. Ethyl acetate at 10(-4.0) (v/v) concentration was used as an S+ odorant, and six different concentrations (10 (-4.0), 10(-4.5), 10(-5.0), 10(-5.5), 10(-6.0), 10(-6.5)) of butanol mixed with ethyl acetate were used as an S- odorant. S+ and S- stimuli were delivered randomly using the 8-odor olfactometer. After injection of 3-MI at a dosage of 300 mg/kg, mixed odor discrimination test was performed for five weeks. RESULTS: Normal rats were able to discriminate ethyl acetate from ethyl acetate mixed with butanol to a concentration down to 10(-6.2) (v/v). Immediately after the 3-MI injection, the rats lost all capacity for smell. From 16 days later, the smell function began improving spontaneously. At the end of the fifth week, the discrimination threshold was 10(-5.7) (v/v), which was almost equal to the original level. CONCLUSIONS: Systematically administered 3-MI caused smell loss in rats. Though not completely, the smell function was recovered spontaneously. An olfactometer is a reliable and accurate device in evaluating the olfactory function in rats.