online ML Comm Review Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12):815-21 / pissn 2092-5859 / eissn 2092-6529 http://dx.doi.org/10.3342/kjorl-hns.2015.58.12.815 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 +82-55-290-6066 Fax +82-55-290-6465 E-mail 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):815-21 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
Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12):815-21 본 론 포유류 후각상피의 구조 - Toxicity model - - 816
Mouse Model for Olfactory Dysfunction Jung YG Transgenic model - - TNF-α in vitro www.jkorl.org 817
Korean J Otorhinolaryngol-Head Neck Surg 2015;58(12):815-21 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 - - - 818
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. www.jkorl.org 819
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