Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12): 본 론 포유류 후각상피의 구조 - Toxicity model

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1 online ML Comm Review Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12): / pissn / eissn Mouse Model for the Research of Sinusitis Induced Olfactory Dysfunction Yong Gi Jung Department of Otorhinolaryngology-Head and Neck Surgery, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon, Korea 부비동염에 의해 유발된 후각소실 연구를 위한 생쥐 모델 정 용 기 성균관대학교 의과대학 삼성창원병원 이비인후과학교실 Received June 26, 2015 Accepted July 20, 2015 Address for correspondence Yong Gi Jung, MD, PhD Department of Otorhinolaryngology- Head and Neck Surgery, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, 158 Paryong-ro, Masanhoewon-gu, Changwon 51353, Korea Tel Fax ent.jyg@gmail.com 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-α) in sustentacular cell of olfactory epithelium. The production of TNF-α 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. Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12): Key WordsZZ3-methylindole ㆍBromomethane ㆍycycline ㆍMice ㆍOlfaction ㆍSinusitis ㆍ Transgenic animal ㆍTumor necrosis factor. 서 론 - Copyright 2015 Korean Society of Otorhinolaryngology-Head and Neck Surgery 815

2 Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12): 본 론 포유류 후각상피의 구조 - Toxicity model

3 Mouse Model for Olfactory Dysfunction Jung YG Transgenic model - - TNF-α in vitro 817

4 Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12): Sustentacular cell Cyp2g1 promoter rtta rtta Tetracycline response element TNF- gene TNF- TNF- TNF- Fig. 1. Signaling pathway of tumor necrosis factor receptor 1 (TNFR1). There are three main sub-pathway following the binding of TNF-α to TNFR1. Dashed grey line following caspase 3 represents multiple step. TNF: tumor necrosis factor, JNK: Jun N-terminal kinase, RIP: receptor interacting protein, TRADD: TNF-associated death domain, FADD: fas-associated death domain, NF: Nuclear factor, TRAF: TNF receptor associated factor. Cyp2g1-rtTA Escherichia coli TRE-TNF-α and advantage of Tet-on system Fig. 2. Diagram of inducible olfactory inflammation mouse. Tet-on gene is knocked into the cyp2g1 locus as homologous insertion, resulting in cell-specific expression of reverse tetracycline transactivator (rtta) in sustentacular cell of olfactory epithelium. Tetracycline response element introduced by random insertion is followed by tumor necrosis factor-alpha (TNF-α) gene. TNF-α is expressed only in the presence of both rtta and doxycycline (). Research with IOI mouse

5 Mouse Model for Olfactory Dysfunction Jung YG 하며 이후 필요한 실험을 진행할 수 있다. 염증을 유발하고 2 적 변화만 발생한다(Fig. 5). 염증을 4주간 계속 지속시키면 주가 경과하면 IOI 생쥐의 후각 기능이 저하된다. 생쥐의 후각 후각상피의 변화가 발생하기 시작하며 기저막하에 염증세포 기능 분석을 위해 생쥐 후각상피 부분을 분리하여 electro 의 침윤이 관찰되고 염증세포들에 의해 축삭이 눌려 작아지 olfactogram을 시행하여 측정할 수 있다(Fig. 4). 그러나 아직 게 되며 상피내 ORN의 크기도 따라서 감소한다. 6주가 지나 이 시기는 후각상피의 형태적 변화는 발생하지 않으며 기능 면 ORN을 포함한 상피내 세포들의 손상이 더욱 진행하여 상 피층이 매우 얇아지는 것을 관찰할 수 있다(Fig. 6). 이러한 과 Fig. 3. ycycline pellet for inducible olfactory inflammation mouse (BiosServ, Flemington, NJ, USA). The dose of doxycycline is 200 mg/kg. Fig. 5. Immunofluorescence on olfactory epithelium cryosection with anti-omp and anti-dapi (4,6-diamidino-2-phenylindole) antibody after 2-weeks of induced inflammation in IOI mouse. The thickness of olfactory epithelium was not decreased ( 250). OMP: olfactory marker protein, IOI: inducible olfactory loss. A B Fig. 4. The equipment for electro-olfactogram of mouse. After bisecting head of mouse, two electrodes were placed on mucus blanket of 2nd and 3rd olfactory turbinate, and a ground electrode earths to Ringer lactate solution (A). Normal summation potential of olfactory turbinate in wild type mouse is about 10 mv (B). Fig. 6. Immunofluorescence on olfactory epithelium cryosection with anti-cytokeratin 18 and anti-dapi (4,6-diamidino-2-phenylindole) antibody after 6-weeks of induced inflammation in IOI mouse. The thickness of olfactory epithelium was significantly decreased ( 250). IOI: inducible olfactory loss

6 Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12): 결 론 REFERENCES 1) Fokkens WJ, Lund VJ, Mullol J, Bachert C, Alobid I, Baroody F, et al. EPOS 2012: European position paper on rhinosinusitis and nasal polyps A summary for otorhinolaryngologists. Rhinology 2012;50(1): ) Delank KW, Stoll W. Olfactory function after functional endoscopic sinus surgery for chronic sinusitis. Rhinology 1998;36(1): ) Jiang RS, Lu FJ, Liang KL, Shiao JY, Su MC, Hsin CH, et al. Olfactory function in patients with chronic rhinosinusitis before and after functional endoscopic sinus surgery. Am J Rhinol 2008;22 (4): ) Raviv JR, Kern RC. Chronic rhinosinusitis and olfactory dysfunction. Adv Otorhinolaryngol 2006;63: ) Raviv JR, Kern RC. Chronic sinusitis and olfactory dysfunction. Otolaryngol Clin North Am 2004;37(6): , v-vi. 6) Yee KK, Pribitkin EA, Cowart BJ, Vainius AA, Klock CT, Rosen D, et al. Neuropathology of the olfactory mucosa in chronic rhinosinusitis. Am J Rhinol Allergy 2010;24(2): ) Lee SH, Lim HH, Lee HM, Park HJ, Choi JO. Olfactory mucosal findings in patients with persistent anosmia after endoscopic sinus surgery. Ann Otol Rhinol Laryngol 2000;109(8 Pt 1): ) Moran DT, Rowley JC 3rd, Aiken GR, Jafek BW. Ultrastructural neurobiology of the olfactory mucosa of the brown trout, Salmo trutta. Microsc Res Tech 1992;23(1): ) Chuah MI, Zheng DR. Olfactory marker protein is present in olfactory receptor cells of human fetuses. Neuroscience 1987;23(1): ) Kern RC, Pitovski DZ. Localization of 11 beta-hydroxysteroid dehydrogenase: specific protector of the mineralocorticoid receptor in mammalian olfactory mucosa. Acta Otolaryngol 1997;117(5): ) Leung CT, Coulombe PA, Reed RR. Contribution of olfactory neural stem cells to tissue maintenance and regeneration. Nat Neurosci 2007;10(6): ) Carter LA, MacDonald JL, Roskams AJ. Olfactory horizontal basal cells demonstrate a conserved multipotent progenitor phenotype. J Neurosci 2004;24(25): ) Chen M, Tian S, Yang X, Lane AP, Reed RR, Liu H. Wnt-responsive Lgr5+ globose basal cells function as multipotent olfactory epithelium progenitor cells. J Neurosci 2014;34(24): ) Hurtt ME, Thomas DA, Working PK, Monticello TM, Morgan KT. Degeneration and regeneration of the olfactory epithelium following inhalation exposure to methyl bromide: pathology, cell kinetics, and olfactory function. Toxicol Appl Pharmacol 1988;94(2): ) Holbrook EH, Iwema CL, Peluso CE, Schwob JE. The regeneration of P2 olfactory sensory neurons is selectively impaired following methyl bromide lesion. Chem Senses 2014;39(7): ) Bakos SR, Schwob JE, Costanzo RM. Matrix metalloproteinase-9 and -2 expression in the olfactory bulb following methyl bromide gas exposure. Chem Senses 2010;35(8): ) Jang W, Youngentob SL, Schwob JE. Globose basal cells are required for reconstitution of olfactory epithelium after methyl bromide lesion. J Comp Neurol 2003;460(1): ) Schwob JE, Youngentob SL, Mezza RC. Reconstitution of the rat olfactory epithelium after methyl bromide-induced lesion. J Comp Neurol 1995;359(1): ) Youngentob SL, Schwob JE, Sheehe PR, Youngentob LM. Odorant threshold following methyl bromide-induced lesions of the olfactory epithelium. Physiol Behav 1997;62(6): ) Dehnhard M, Bernal-Barragan H, Claus R. Rapid and accurate highperformance liquid chromatographic method for the determination of 3-methylindole (skatole) in faeces of various species. J Chromatogr 1991;566(1): ) Schiestl FP, Roubik DW. Odor compound detection in male euglossine bees. J Chem Ecol 2003;29(1): ) Turk MA, Flory W, Henk WG. Chemical modulation of 3-methylindole toxicosis in mice: effect on bronchiolar and olfactory mucosal injury. Vet Pathol 1986;23(5): ) Owens JG, James RA, Moss OR, Morgan KT, Bowman JR, Struve MF, et al. Design and evaluation of an olfactometer for the assessment of 3-methylindole-induced hyposmia. Fundam Appl Toxicol 1996; 33(1): ) Peele DB, Allison SD, Bolon B, Prah JD, Jensen KF, Morgan KT. Functional deficits produced by 3-methylindole-induced olfactory mucosal damage revealed by a simple olfactory learning task. Toxicol Appl Pharmacol 1991;107(2): ) Miller MA, Kottler SJ, Ramos-Vara JA, Johnson PJ, Ganjam VK, Evans TJ. 3-methylindole induces transient olfactory mucosal injury in ponies. Vet Pathol 2003;40(4): ) Kim HY, Kim JH, Dhong HJ, Kim KR, Chung SK, Chung SC, et al. Effects of statins on the recovery of olfactory function in a 820

7 Mouse Model for Olfactory Dysfunction Jung YG 3-methylindole-induced anosmia mouse model. Am J Rhinol Allergy 2012;26(2):e ) Kim JW, Hong SL, Lee CH, Jeon EH, Choi AR. Relationship between olfactory function and olfactory neuronal population in C57BL6 mice injected intraperitoneally with 3-methylindole. Otolaryngol Head Neck Surg 2010;143(6): ) Miller MA, O Bryan MA. Ultrastructural changes and olfactory deficits during 3-methylindole-induced olfactory mucosal necrosis and repair in mice. Ultrastruct Pathol 2003;27(1): ) Kern RC, Conley DB, Haines GK 3rd, Robinson AM. Pathology of the olfactory mucosa: implications for the treatment of olfactory dysfunction. Laryngoscope 2004;114(2): ) Heilmann S, Huettenbrink KB, Hummel T. Local and systemic administration of corticosteroids in the treatment of olfactory loss. Am J Rhinol 2004;18(1): ) Lane AP, Zhao H, Reed RR. Development of transgenic mouse models for the study of human olfactory dysfunction. Am J Rhinol 2005;19 (3): ) Lane AP, Turner J, May L, Reed R. A genetic model of chronic rhinosinusitis-associated olfactory inflammation reveals reversible functional impairment and dramatic neuroepithelial reorganization. J Neurosci 2010;30(6): ) Feldmann M, Maini RN. The role of cytokines in the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford) 1999;38 Suppl 2: ) Kollias G, Douni E, Kassiotis G, Kontoyiannis D. On the role of tumor necrosis factor and receptors in models of multiorgan failure, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Immunol Rev 1999;169: ) Barker V, Middleton G, Davey F, Davies AM. TNFalpha contributes to the death of NGF-dependent neurons during development. Nat Neurosci 2001;4(12): ) Scherbel U, Raghupathi R, Nakamura M, Saatman KE, Trojanowski JQ, Neugebauer E, et al. Differential acute and chronic responses of tumor necrosis factor-deficient mice to experimental brain injury. Proc Natl Acad Sci U S A 1999;96(15): ) Zúñiga-Pflücker JC, Jiang D, Lenardo MJ. Requirement for TNFalpha and IL-1 alpha in fetal thymocyte commitment and differentiation. Science 1995;268(5219): ) Suzuki Y, Farbman AI. Tumor necrosis factor-alpha-induced apoptosis in olfactory epithelium in vitro: possible roles of caspase 1 (ICE), caspase 2 (ICH-1), and caspase 3 (CPP32). Exp Neurol 2000;165(1): ) Hua ZC, Ding X. cdna cloning and heterologous expression of mouse CYP2G1. Ann N Y Acad Sci 1998;864: ) Roet KC, Verhaagen J. Understanding the neural repair-promoting properties of olfactory ensheathing cells. Exp Neurol 2014;261: ) Chou RH, Lu CY, Wei-Lee, Fan JR, Yu YL, Shyu WC. The potential therapeutic applications of olfactory ensheathing cells in regenerative medicine. Cell Transplant 2014;23(4-5): ) Ekberg JA, St John JA. Crucial roles for olfactory ensheathing cells and olfactory mucosal cells in the repair of damaged neural tracts. Anat Rec (Hoboken) 2014;297(1): 정답 및 해설 답 2 해 설 환자는 문진 및 산소 포화도 모니터링 상 central apnea에 의한 hypoventilation이 의심되는 상태이므로 central apnea 진단을 위해 수면다원검사 시행 시 nasal and oral airflow에 대한 변화와 가슴 및 복부의 호흡 노력에 대한 변화를 유의 해서 관찰해야 한다. Reference: Cumming s Otolaringology Head & Neck Surgery Fifth edition. Philadelphia, PA: Mosby Elsevier;2010. p.255, 263. 답 4 해 설 면역 요법은 집먼지 진드기, 꽃가루, 고양이 항원, Alternaria, Cladosporium 등 일부 곰팡이에 대하여 효과가 입증된 상 태이며 4세 이상의 소아에서 제한적으로 이용된다. 주로 환경요법, 약물요법에 효과가 없거나 부작용이 심할 때 이용되며, 증상이 연중 두 계절 혹은 6개월 이상 지속되고, 증상과 항원에 대한 양성 피부 반응 혹은 혈청 특이항체가 확실할 때 시 행한다. Reference: 대한이비인후과학회. 이비인후과-두경부외과학. 서울: 일조각;2009. p

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract