탁월한 기술 지원에 대해 Daniel Bukala, Natalie Altmeyer 및 Funda Cay에게 감사드립니다. 원고를 편집 한 Jonathan Cotton, Greg Bowden 및 Paul Soubiran에게 감사드립니다. 이 작업은 Werner Siemens-Foundation과 Eberhard Karls University Tübingen의 의학 교수 ( &quot;Promotionskolleg&quot;) 및 CRC 156 (프로젝트 C3)을 통한 DFG의 지원을 받았다.

이 문서에 설명 된 모든 절차는 지침과 관리 및 실험 동물의 사용의 국제 표준을 따라 국가위원회 튀빙겐, 독일의 지역 동물 복지와 윤리위원회에 의해 승인되었다. 12 시간 광 : - 8 ~ 12 주령 BALB / C 및 C57BL / 6 마우스는 12 시간에 보관 하였다 암주기를 2 그룹으로 22 ± 1 ℃에서 IVCs 표준화 된 환경 조건에 수납 된 - 물 (5) 음식 액세스 수의. 1. 재료 준비 <ol><li> 사출 직전 각 데이터 시트에 따른 근 적외 형광 영상을위한 OI 염료 희석. 생체 내에서 MMP의 측정을 기동 OI 염료 (680 nm에서 여기 상업용 프로브)를 1.5 ml의 PBS 1X 20 nmol의 농도로 사용할 수 있습니다. 부드럽게 흔들거나 사용하기 전에 솔루션을 소용돌이. 빛으로부터 보호 할 때 최대 6 개월 동안 11 °의 C - OI 염료 2에 저장 될 수있다. </li&gt; <li> 정맥 내 (IV) 주사는 정맥 카테터를 준비한다. 20 U (50 ㎖ 헤파린 10 개 분사 유니트를 포함)는 0.9 % 식염수 용액으로 채워진 (0.5 mL) 중 인슐린 주사기 및 폴리에틸렌 카테터에 부착 된 30 게이지 바늘을 사용한다. OI가 염료의 마우스 당 2 nmol의 권장 복용량을 주입한다. </li></ol> 류마티스 관절염 및 만성 연락 과민 반응 2. 유도 <ol><li> 류마티스 관절염: <ol><li> RA를 유도하는 1X PBS (200 μL)를 1 (K / BxN 마우스 (10)로부터 얻은) 혈중 글루코스 -6- 포스페이트 이소 머라 제 (GPI)에 대해 1 함유 항체 (AB)의 100 μL 희석. -80 ° C에서 희석 된 혈청을 저장합니다. RA의 유도에 대한 상세 절차는 Monach 동부 등에 의해 설명된다. 8. </li><li> RA를 유도하기 위해 꼬리에 의해 부드럽게 각 마우스를 올려 t의 일 0에 희석 된 혈청 복강 내 (IP)의 200 μL를 주입그는 실험했다. 주입 후 마우스를 직접 케이지에 넣으십시오. </li><li> 선택적으로, AB 주입 전에 각 발목의 발목 직경을 측정하고 &quot;관절염 점수&quot;( 그림 1A )를 정의하십시오. 기계적 측정 장치 (마이크로 미터)를 사용하여 GPI- 혈청 후 ​​6 일째까지 매일 발목 부종을 측정하십시오. </li><li> RA 생쥐의 꼬리 정맥 에 생체 내 MMP 활성을 측정하기 위해 활성화 가능한 OI 염료 (단계 1.1 - 1.2)를 주사 GPI- 혈청 주사 후 5 일째 및 대조군 마우스의 꼬리 정맥 내로 투여 하였다. 꼬리 정맥 주사 후 24 시간 동안 광학 이미징 실험을 수행하십시오. </li></ol></li><li> 과민 반응에 연락하십시오 : <ol><li> C57BL / 6 생쥐의 sensitization 들어, 아세톤 / 오일의 4 : 1 혼합물에 용해 5 % TNCB 솔루션을 준비합니다. </li><li> 100 % 산소 (1.5 L / 분)로 증발 된 1.5 % 이소 플루 란을 사용하여 C57BL / 6 마우스를 마취시킨다. 마우스가 anesthetized되면, 자기 미친에 넣어<html> e 코 콘 (1.5 vol % 이소 플루 란 관에 연결된 5 ml 폴리에틸렌 주사기를 자르십시오)을 마취 유지하십시오. Anesthetized 동안 마른 동물의 눈 건조를 방지하기 위해 수의사 용 연고를 바르십시오. </li><li> 작은 동물 머리 트리머를 사용하여 배를 조심스럽게 (2 x 2 cm) 면도하십시오. 동물의 비특이적 인 형광 신호를 유발할 수 있고 연구 결과에 영향을 줄 수 있으므로 피부 손상을 피하십시오. </li><li> 동물을 sensitizing 100 μL 피펫을 사용하여 면도 복부 영역에서 천천히 5 % TNCB 솔루션의 80 μL를 적용합니다. 각막 손상을 방지하고 회복기 동안 낮은 체온을 피하기 위해 마우스의 눈이 침구와 닿지 않도록주의하면서 새장에 부드럽게 다시 놓습니다. </li><li> 민감화 6 일 후에 1 % TNCB 용액 (아세톤 / 오일의 9 : 1 혼합물에 용해)을 준비하고 급성 및 만성 CHSR을 유도하기 위해 피펫을 사용하여 오른쪽 귀에 20 μL를 적용합니다.&quot;&gt; 1 <ol><li> 100 μL 피펫을 사용하여 1 % TNCB 솔루션의 20 μL와 오른쪽 귀 양쪽에 1 차 도전 과제로 시작하고 CHR ( 그림 1B )를 이끌어 내기 위해 매 5 일 (감작 후 15 일) . 마이크로 미터 측정 장치를 사용하여 매일 귀 두께를 측정하십시오. </li></ol></li><li> 챌린지 12 시간 후 CHR 마우스의 생체 내 에서 MMP 활성을 측정하기 위해 활성화 가능한 OI 염료 (680 nm에서 여기가있는 상용 프로브)를 주입합니다 (1.1-1.2 단계). 꼬리 정맥 주사 24 시간 후 광학 이미징 실험을 수행하십시오 ( iv ). </li></ol></li></ol> 3. 광학 이미징을위한 동물 준비 <ol><li> 사용 된 OI 요원의 형광 신호와 자동 형광 (약 700 nm)의 간섭을 피하기 위해 낮은 또는 비 형광 chow ( 예 : 망간 무료)에 마우스 chow (적어도 이미징하기 전에 3) 스위치. </li><li> 동물을에 넣으십시오.(1.5 L / 분) (산소는 공기로 대체 될 수 있지만 하나의 실험 내에서 표준화 될 필요가 있음)로 증발 된 1.5 부피 %의 이소 플루 란을 사용하여 마취시킨다. </li><li> 마우스가 눈으로 마취되면, 자기 - 만든 코 콘 (1.5 vol % isoflurane 튜브에 연결된 절단 5 ML 폴리에틸렌 주사기에 의해 빌드)에 배치하고 마취를 유지합니다. </li><li> 작은 동물 머리 트리머를 사용하여 대상 부위 (2 cm x 2 cm)에 동물을 면도하십시오. 동물의 비특이적 인 형광 신호를 유발할 수 있고 연구 결과에 영향을 줄 수 있으므로 피부 손상을 피하십시오. 참고 : Hairs는 관심 부위 (ROI)에 따라 OI (마우스 스트레인에 따라 다르지만 흡수가 많거나 적음) 동안 형광 신호를 흡수 할 수 있습니다. </li><li> iv 주사의 경우, 따뜻한 물에 마우스의 꼬리를 두어 혈관 확장을 유도하고 주사 부위의 피부를 알코올로 부드럽게 씻고 카테터를꼬리의 말초 사이트. (가) 꼬리 정맥에 20 °의 각도에있는 바늘의 끝을 &quot;잘라&quot;주사기를 다시 현탁 카테터의 정확한 배치를 테스트 놓는다. </li><li> 카테터가 올바르게 위치되면, 주사기를 교체하고 OI 프로브 (2 nmol의)을 분사. 주입 후 주사기를 교체하고 0.9 % 생리 식염수 폴리에틸렌 관의 완전히 명확 죽은 볼륨의 25 μL를 주입. 주 : 사용 OI 염료의 조직 반감기 시간 생체 MMP 활성을 측정 (680 nm에서 여기와 상용 프로브)는 72 시간이다. 완전한 통관을 보장하기 위해, 염료의 재 주입은 이전에 주입 한 후 이전 칠일 이상 사용하지 않는 것이 좋습니다. </li></ol> 4. 광학 이미징 <ol><li> 시야 (FOV)의 중심에 OI 스캐너의 블랙 박스 검은 플라스틱 또는 종이 시트를 배치했다. </li><li> 측정 프로토콜을 설정하고 적절한 파장을 선택합니다 (여기 : 680 ± 10 nm의 전자임무 : 700 ± 10 nm) 및 영상 매개 변수. 참고 : 일부 OI 시스템에서는 몇 가지 이미징 염료에 대한 설정이 미리 정의되어 있습니다. </li><li> 형광 이미징을위한 올바른 프로토콜을 선택하려면 이미징 소프트웨어 (제조업체가 제공함)를 열고 시스템을 초기화하십시오. 대부분의 CCD 카메라는 작동 온도까지 냉각해야하며 10 분 정도 걸릴 수 있습니다. 안정적인 결과를 얻으려면 시스템이 준비 될 때까지 기다리십시오. </li><li> 생체 내 이미징 시스템 수집 컨트롤 패널 팝업을 관찰하면 선택한 각 필터 쌍이 시퀀스의 한 이미지를 나타냅니다. 이 경우 각각 680 ± 10 nm 및 700 ± 10 nm의 여기 및 방출 파장을 가진 상업용 염료에 대한 필터 쌍으로 하나의 이미지를 수집하고 측정을 시작하십시오 ( &quot;Acquire sequence&quot;). 자세한 내용은 제조업체 설명서를 참조하십시오. </li><li> 이미지에 적절하게 라벨을 지정하고 &quot;이미지 편집&quot;획득 순서 &quot;레이블&quot;는 다음 나타납니다 창 &quot;. </li><li> OI가 염료를 주입하기 전에, 각 동물의 기준 검사를 취하거나, 배경 신호를 구별하기 나이브 대조군을 사용한다. </li><li> OI가 염료의 꼬리 정맥 주사 후 시간은 OI 시스템에서 가장 높은 신호를 측정 할 수있는 위치에서, FOV의 중심에 동물을 배치하고, 측정을 시작한다. 참고 : 중요 : 저체온증 크게 이미징 에이전트의 분포에 영향을 미칠 수있다. 무대는 동물의 저체온증을 방지하기 위해 37 ° C까지 가열되어 있는지 확인합니다. 영상은 1 내지 5 개의 마우스의 동물 군에서 동시에 수행 될 수있다. 동시에 측정하는 동물의 수에 따라 FOV의 크기를 선택합니다. 각 마우스의 표시 여부를 확인하기 위해 밝은 필드 이미지를 취할 수 있습니다. </li></ol> 5. 데이터 분석 참고 : 다음 이미지 소프트웨어를 사용하여 데이터 분석을 수행제조업체의 프로토콜. <ol><li> RA의 측정을 위해서는 그림 2 와 같이 보정 된 광자 방출 단위를 사용하는 반면, 만성 CHSR은 신호 세기 (%)로 묘사됩니다. </li><li> ROI를 수동으로 그리려면 툴 플레이트를 사용하십시오. RA 이미지의 분석을 위해 각 동물의 모든 발목과 발에서 가장 높은 신호 주위에 배치 된 표준화 된 원을 사용하십시오. CHSR을 분석하려면 밝은 영역 이미지에 따라 ROI를 전체 오른쪽 귀와 왼쪽 귀 주위에 배치하십시오. </li><li> 특정 그린 ROI에서 광자 방출 또는 신호 강도 값을 측정하려면 &quot;측정&quot;을 누릅니다. 이 시스템은 설명적인 통계 분석을 위해 그려진 ROI에 값을 제공합니다. </li></ol>

<ol>
	<li>Veale, D. J., Ritchlin, C., FitzGerald, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopathology+of+psoriasis+and+psoriatic+arthritis.">Immunopathology of psoriasis and psoriatic arthritis.</a> Ann Rheum Dis. 64, 26 (2005).</li><li>Harris, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheumatoid+arthritis.+Pathophysiology+and+implications+for+therapy.">Rheumatoid arthritis. Pathophysiology and implications for therapy.</a> N Engl J Med. 322 (18), 1277-1289 (1990).</li><li>Lee, D. M., Weinblatt, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheumatoid+arthritis.">Rheumatoid arthritis.</a> Lancet. 358 (9285), 903-911 (2001).</li><li>Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+mechanisms+in+the+pathogenesis+of+rheumatoid+arthritis.">Immunologic mechanisms in the pathogenesis of rheumatoid arthritis.</a> J Clin Rheumatol. 11, S39-S44 (2005).</li><li>Pap, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+pattern+of+membrane-type+matrix+metalloproteinases+in+rheumatoid+arthritis.">Differential expression pattern of membrane-type matrix metalloproteinases in rheumatoid arthritis.</a> Arthritis Rheum. 43 (6), 1226-1232 (2000).</li><li>Firestein, G. S., Paine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromelysin+and+tissue+inhibitor+of+metalloproteinases+gene+expression+in+rheumatoid+arthritis+synovium.">Stromelysin and tissue inhibitor of metalloproteinases gene expression in rheumatoid arthritis synovium.</a> Am J Pathol. 140 (6), 1309-1314 (1992).</li><li>Schwenck, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+optical+imaging+of+matrix+metalloproteinase+activity+detects+acute+and+chronic+contact+hypersensitivity+reactions+and+enables+monitoring+of+the+antiinflammatory+effects+of+N-acetylcysteine.">In vivo optical imaging of matrix metalloproteinase activity detects acute and chronic contact hypersensitivity reactions and enables monitoring of the antiinflammatory effects of N-acetylcysteine.</a> Mol Imaging. 13, (2014).</li><li>Monach, P. A., Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+K/BxN+arthritis+model.">The K/BxN arthritis model.</a> Curr Protoc Immunol. 15, 22 (2008).</li><li>Zelmer, A., Ward, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+fluorescence+imaging+of+small+animals.">Noninvasive fluorescence imaging of small animals.</a> J Microsc. 252 (1), 8-15 (2013).</li><li>Kouskoff, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+disease+provoked+by+systemic+autoimmunity.">Organ-specific disease provoked by systemic autoimmunity.</a> Cell. 87 (5), 811-822 (1996).</li><li>Fuchs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+cell+proliferation+enables+the+detection+of+the+extent+of+experimental+rheumatoid+arthritis+by+3'-deoxy-3'-18f-fluorothymidine+and+small-animal+PET.">In vivo imaging of cell proliferation enables the detection of the extent of experimental rheumatoid arthritis by 3'-deoxy-3'-18f-fluorothymidine and small-animal PET.</a> J Nucl Med. 54 (1), 151-158 (2013).</li><li>Schwenck, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+and+Cerenkov+luminescence+imaging.+Applications+in+small+animal+research.">Fluorescence and Cerenkov luminescence imaging. Applications in small animal research.</a> Nuklearmedizin. 55 (2), 63-70 (2016).</li><li>Mahling, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparative+pO2+Probe+and+[18F]-Fluoro-Azomycinarabino-Furanoside+([18F]FAZA)+PET+Study+Reveals+Anesthesia-Induced+Impairment+of+Oxygenation+and+Perfusion+in+Tumor+and+Muscle.">A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle.</a> PLoS One. 10 (4), 0124665 (2015).</li><li>Fuchs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+breathing+affects+3'-deoxy-3'-18F-fluorothymidine+uptake+in+mouse+models+of+arthritis+and+cancer.">Oxygen breathing affects 3'-deoxy-3'-18F-fluorothymidine uptake in mouse models of arthritis and cancer.</a> J Nucl Med. 53 (5), 823-830 (2012).</li><li>Fuchs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+anesthetics+on+3'-[18F]fluoro-3'-deoxythymidine+([18F]FLT)+uptake+in+animal+models+of+cancer+and+inflammation.">Impact of anesthetics on 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) uptake in animal models of cancer and inflammation.</a> Mol Imaging. 12 (5), 277-287 (2013).</li><li>Liu, N., Shang, J., Tian, F., Nishi, H., Abe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+optical+imaging+for+evaluating+the+efficacy+of+edaravone+after+transient+cerebral+ischemia+in+mice.">In vivo optical imaging for evaluating the efficacy of edaravone after transient cerebral ischemia in mice.</a> Brain Res. 1397, 66-75 (2011).</li><li>Sheth, R. A., Maricevich, M., Mahmood, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+optical+molecular+imaging+of+matrix+metalloproteinase+activity+in+abdominal+aortic+aneurysms+correlates+with+treatment+effects+on+growth+rate.">In vivo optical molecular imaging of matrix metalloproteinase activity in abdominal aortic aneurysms correlates with treatment effects on growth rate.</a> Atherosclerosis. 212 (1), 181-187 (2010).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescent+imaging+of+matrix+metalloproteinase+activity+after+myocardial+infarction.">Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction.</a> Circulation. 111 (14), 1800-1805 (2005).</li><li>Wallis de Vries, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Images+in+cardiovascular+medicine.+Multispectral+near-infrared+fluorescence+molecular+imaging+of+matrix+metalloproteinases+in+a+human+carotid+plaque+using+a+matrix-degrading+metalloproteinase-sensitive+activatable+fluorescent+probe.">Images in cardiovascular medicine. Multispectral near-infrared fluorescence molecular imaging of matrix metalloproteinases in a human carotid plaque using a matrix-degrading metalloproteinase-sensitive activatable fluorescent probe.</a> Circulation. 119 (20), e534-e536 (2009).</li><li>Weissleder, R., Tung, C. H., Mahmood, U., Bogdanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+tumors+with+protease-activated+near-infrared+fluorescent+probes.">In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.</a> Nat Biotechnol. 17 (4), 375-378 (1999).</li><li>Cortez-Retamozo, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+assessment+of+inflammation+and+treatment+response+in+a+mouse+model+of+allergic+airway+inflammation.">Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation.</a> J Clin Invest. 118 (12), 4058-4066 (2008).</li><li>McIntyre, J. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+fluorogenic+proteolytic+beacon+for+in+vivo+detection+and+imaging+of+tumour-associated+matrix+metalloproteinase-7+activity.">Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity.</a> Biochem J. 377, 617-628 (2004).</li><li>Scherer, R. L., VanSaun, M. N., McIntyre, J. O., Matrisian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+matrix+metalloproteinase-7+activity+in+vivo+using+a+proteolytic+nanobeacon).">Optical imaging of matrix metalloproteinase-7 activity in vivo using a proteolytic nanobeacon).</a> Mol Imaging. 7 (3), 118-131 (2008).</li><li>Olson, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+characterization+of+activatable+cell+penetrating+peptides+for+targeting+protease+activity+in+cancer.">In vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer.</a> Integr Biol (Camb. 1 (5-6), 382-393 (2009).</li><li>Duijnhoven, S. M., Robillard, M. S., Nicolay, K., Grull, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+targeting+of+MMP-2/9+activatable+cell-penetrating+imaging+probes+is+caused+by+tumor-independent+activation).">Tumor targeting of MMP-2/9 activatable cell-penetrating imaging probes is caused by tumor-independent activation).</a> J Nucl Med. 52 (2), 279-286 (2011).</li><li>Schafers, M., Schober, O., Hermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-metalloproteinases+as+imaging+targets+for+inflammatory+activity+in+atherosclerotic+plaques.">Matrix-metalloproteinases as imaging targets for inflammatory activity in atherosclerotic plaques.</a> J Nucl Med. 51 (5), 663-666 (2010).</li><li>Wagner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+18F-labelled+derivative+of+the+MMP+inhibitor+CGS+27023A+for+PET:+radiosynthesis+and+initial+small-animal+PET+studies.">A new 18F-labelled derivative of the MMP inhibitor CGS 27023A for PET: radiosynthesis and initial small-animal PET studies.</a> Appl Radiat Isot. 67 (4), 606-610 (2009).</li><li>Waschkau, B., Faust, A., Schafers, M., Bremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+new+fluorescence-labeled+MMP+inhibitor+to+image+tumor+MMP+activity+in+vivo+in+comparison+to+an+MMP-activatable+probe.">Performance of a new fluorescence-labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP-activatable probe.</a> Contrast Media Mol Imaging. 8 (1), 1-11 (2013).</li><li>Vogl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alarmin+S100A8/S100A9+as+a+biomarker+for+molecular+imaging+of+local+inflammatory+activity.">Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity.</a> Nat Commun. 5, 4593 (2014).</li></ol>

저자가 공개하는 게 없다.

OI는 전임상 연구 에서 비 침습적 생체 내 분자 이미징을위한 매우 유용하고 빠르며 저렴한 도구입니다. OI의 특별한 강점은 염증 반응과 같은 매우 역동적 인 과정을 모니터하는 능력입니다. 또한 OI는 수 일에서 수주에 이르는 장기간의 질병 경과를 추적 할 수있게 해줍니다. OI는 양전자 방출 단층 촬영 (PET) 또는 자기 공명 영상 (MRI)과 같은 다른 생체 내 ...

류마티스 성 관절염 (RA)이나 건선의 건선과 같은자가 면역 질환은 지연 형 과민 반응 (DTHR)으로 분류됩니다. 1 RA는 부식성 윤 활막염과 관절 파괴를 특징으로하는 일반적인자가 면역 질환입니다. 염증 세포의 침투와 증식, 염증 세포의 발현 증가로 판 누스 형성, 연골 및 뼈 파괴를 일으키는 염증 관절염 관절. matrix metalloproteinases (MMPs)에 의한 콜라겐과 같은 세포 외 기질 분자의 절단은 조직 전환과 혈관 신생에 필수적이며 조직 파괴를 유발합니다. 5 , 6 접촉 과민 반응 (CHR)은 호중구가 응집되어 산화가 일어난다는 특징이 있습니다. RA와 유사하게 CHR의 MMP는 변이 형이다만성 염증을 설정하기 위해 조직 변환, 세포의 이동과 혈관 신생에 VED. RA를 조사하기 위해, 글루코스 -6- 포스페이트 이소 머라 제 (GPI) -serum 주입 마우스 모델을 사용 하였다. GPI에 대한 항체를 포함하는 트랜스 제닉 K / BxN 마우스 8 혈청 류마티스 염증 GPI 혈청 주입 후 6 일째에 부은 발목 최대 24 시간 (1.1 참조)에서 개발하기 시작 후 나이브 BALB / c 마우스에 주입 하였다. 만성 CHR을 분석하기 위해, C57BL / 6 마우스는 복부에 trinitrochlorobenzene (TNCB)에 민감했다. 오른쪽 귀는 감작 후 일주를 시작하기 5 회까지 도전되었다 (도 1.1 및 1.2 참조). 비침 작은 동물 OI 주로 임상 연구에서 사용되어, 직관 형 형광등 chemiluminescent- 및 생물 발광-신호의 생체 내 연구에 기초한 기술이다. 취득한 반 정량적 데이터는 molec에 대한 통찰력을 제공합니다울라 기관의 메커니즘과 건강의 조직뿐만 아니라 병에 걸린 실험 동물 모델 및 측정을 따라 길이 방향의 수 (예를 들어 생체 내에서 치료 반응 프로파일을 평가하기 위해). 같은 동물이 아닌 시점에 따라 다른 마우스를 사용하는 여러 시점에서 후속 연구에서 측정 할 수있는 종 연구의 큰 장점은 동물 수의 감소이다. OI의 해상도는 기관의 상세한 기능 이미징 및 실험 동물에서 더 작은 조직 구조를 할 수 있습니다. -70 ° C까지 많은 장치에서 냉각되어 좁은 투과 스펙트럼, A A 차광판에 의해 산란 빛으로부터 보호 &quot;어두운 박스&quot;및 대전 성 결합 소자 (CCD) 카메라와 특정한 여기 및 방사 필터를 사용 매우 특정한 수 있고 형광 신호 민감한 측정. excitation-과 함께 형광 제를 사용하여근적외선 형광 창 (650 - 950 nm)의 방출 스펙트럼에서 신호 대 잡음비가 크게 향상 될 수 있습니다. 근적외선 형광 창은 헤모글로빈과 물에 의한 신호 흡수가 상대적으로 낮을뿐만 아니라 배경 자동 자기장이 낮다는 특징이 있습니다. 9 이것은 작은 동물의 조직에서 2cm까지 침투 깊이를 허용합니다. OI- 프로브는 표적 조직을 직접 ( 예 : 형광 표지 된 항체에 의해) 처리하거나 표적 조직에서 활성화시킬 수 있습니다 ( 예 : 프로테아제). 활성화 가능한 OI 프로브는 Förster 공명 에너지 전달 (FRET)으로 인해 비활성화 된 형태로 광학적으로 침묵하며, 이는 분자 내의 여기 에너지를 다른 영역으로 이동시키는 퀀칭 부분 (quenching moiety)으로 향하게됩니다. 염료가 (예를 들어 프로테아제에 의해) 절단되면, 에너지는 더 이상 분자 내에서 전달되지 않으며, 형광 신호는 OI에 의해 검출 될 수있다. 이것은 높은 특이성을 갖는 OI 프로브의 설계를 허용합니다뚜렷한 생물학적 과정과 탁월한 신호 대 잡음비. 다음 프로토콜은 동물의 준비, 생체 내에서 MMP-2, -3, -9 및 -13 활성 및 염증의 두 실험 모델 (RA, CHR)을 이미지화하기 위해 Activatable OI 프로브를 사용하여 OI 측정을 자세히 설명합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cornergel</td>
			<td>Gerhard Mann GmbH</td>
			<td>1224635</td>
			<td>ophthalmic ointment&#160;</td>
		</tr>
		<tr>
			<td>Forene</td>
			<td>Abbott GmbH</td>
			<td>4831850</td>
			<td>isoflurane</td>
		</tr>
		<tr>
			<td>U40 insulin syringe</td>
			<td>Becton Dickinson and Company</td>
			<td>324876</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sintetica</td>
			<td>6093089</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Med-PE 0.28x0.61mm</td>
			<td>Reichelt Chemietechnik GmbH+Co</td>
			<td>28460</td>
			<td>polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61&#160;mm&#160;</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 30 G</td>
			<td>Becton Dickinson &#38; Co. Ltd.</td>
			<td>305106</td>
			<td>30 G injection cannula</td>
		</tr>
		<tr>
			<td>RTA-0011 isoflurane vaporizer</td>
			<td>Vetland Medical Sales and Services LLC</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Artagain drawing paper</td>
			<td>Strathmore Artist Paper</td>
			<td>446-8</td>
			<td>coal black</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Optical imaging system</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 25 G</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Chloro-1,3,5-trinitrobenzene</td>
			<td>Sigma Aldrich GmbH</td>
			<td>7987456F</td>
			<td>TNCB</td>
		</tr>
		<tr>
			<td>MMPSense 680</td>
			<td>Perkin Elmer&#160;</td>
			<td>NEV10126</td>
			<td>fluorescent imaging dye</td>
		</tr>
		<tr>
			<td>Oditest&#160;</td>
			<td>Koreplin GmbH</td>
			<td>C1X018</td>
			<td>mechanical measurment</td>
		</tr>
		<tr>
			<td>Miglyol 812</td>
			<td>SASOL</td>
			<td>-</td>
			<td>Oil</td>
		</tr>
		<tr>
			<td>&#160;BALB/C, C57BL/6</td>
			<td>Charles River Laboratories&#160;</td>
			<td>-</td>
			<td>Mice used for experiements</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich GmbH</td>
			<td></td>
			<td>For dilution of the RA serum&#160;</td>
		</tr>
		<tr>
			<td>Pipette (100&#181;l)</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td>Used for TNCB application&#160;</td>
		</tr>
		<tr>
			<td>shaver&#160;</td>
			<td>Wahl&#160;</td>
			<td>9962</td>
			<td>Animal hair trimmer</td>
		</tr>
		<tr>
			<td>Living Image&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td></td>
			<td>Imaging software to measure OI</td>
		</tr>
	</tbody>
</table>

non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent

이 논문은 서로 다른 2 가지 마우스 염증 모델 인 류마티스 관절염 (RA)과 접촉 한 생체 내 형광 광학 이미징 (OI) 을 통해 활성화 가능한 형광 프로브에 의한 매트릭스 금속 단백질 분해 효소 (MMP) - 활성을 영상화하는 비 침습적 인 방법을 설명합니다 과민 반응 (CHR) 모델. 근적외선 (NIR) 창 (650 - 950 nm)의 파장을 갖는 빛은 650 nm 이하의 파장에 비해 깊은 조직 침투와 최소한의 신호 흡수를 허용합니다. 형광 OI를 사용하는 주요 이점은 다른 동물 모델에서 저렴하고 빠르고 쉽게 구현할 수 있다는 것입니다. 활성화 가능한 형광성 프로브는 비활성 상태에서는 광학적으로 침묵하지만 프로테아제에 의해 활성화되면 높은 형광성을 갖습니다. 활성화 된 MMP는 조직 파괴를 일으키고 RA 및 CHR과 같은 지연 형 과민 반응 (DTHR)에서 질병 진행에 중요한 역할을합니다. 또한 MMP는연골 및 뼈의 파괴와의 키 프로테아제 - 염증성 사이토 카인에 반응 대 식세포, 섬유 아세포 및 연골 세포에 의해 유발된다. 여기뿐만 아니라 질병의 유도 후 6 일 MMP-2, -3 같은 키 MMP의 활성화되어 프로브, -13 및 -13를 사용하여 근처 RA에서 MMP 활성의 적외선 형광 OI 및 대조군 마우스에 대한 촬상 프로토콜을 기술 급성 (1 배 도전)과 만성 건강한 귀에 비해 오른쪽 손잡이 (5 배 도전) CHR와 마우스에있다.

경구 용 BALB / c 마우스에서 류마티스 성 관절염 (RA)을 유도하기 위해, 동물에게 0 일째에 GPI에 대한자가 항체 (1x PBS로 1 : 1로 희석)를 주사 하였다.이 GPI- 혈청에서 최대 염증 (발목 부종) RA 모델은 주사 후 6 일째 날 11에 있다. 따라서, 2 nmol의 활성화 가능한 OI 염료가 준비되었고 5 일째에 관절염 마우스 및 건강한 대조 동물의 꼬리 정맥에 주사되었다. 주사...

Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

이 논문은 염증의 두 가지 실험 모델에서 키 매트릭스 메탈의 생체 내 활성을 시각화 기동 광학 이미징 프로브를 사용하여 형광 이미지의 적용을 설명한다.

비 침습적<em&gt; In Vivo</em&gt; 활성화 가능한 형광 이미징 에이전트를 사용한 염증성 MMP 활성의 형광 광학 이미징

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

non-invasive-vivo-fluorescence-optical-imaging-inflammatory-mmp

Research

JoVE Journal

Immunology and Infection

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

8.7K 조회수.  Eberhard Karls University of Tübingen.  이 논문은 염증의 두 가지 실험 모델에서 키 매트릭스 메탈의 생체 내 활성을 시각화 기동 광학 이미징 프로브를 사용하여 형광 이미지의 적용을 설명한다. 

비디오: Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

백혈구 호밍 및 마이 그 레이션의 비침습 영상 생체내에</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

연구

면역학 및 감염병학

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

동적 라이브 셀 이미징에 대한 호스트 병원체 상호 작용 연구를위한 광학 트랩의 사용

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

다른 동물 모델에서 NADPH 산화 효소 활동의 Bioluminescence 영상

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Zebrafish 애벌레의 분해해 칸디다 증의 비침습 이미징

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

마우스의 림프 Vasculature의 비 침습적 광학 이미징

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

생체 내 이미징 방법에 급성 및 만성 염증을 구별하는

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

의 Zebrafish의 애벌레 모델의 선천성 면역 반응의 비 침습적 영상<em&gt; 연쇄상 구균 iniae</em&gt; 감염

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

에 의해 쥐 림프구 라벨링<sup&gt; (64)</sup&gt; CU-항체 수용체는 대한 타겟팅<em&gt; 생체</em&gt; PET / CT에 의한 세포 트래 피킹

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

생물 발광 시스템 비-침략 적 비보에 의해 Murine 호스트에서 Enterohemorrhagic 대장균 식민지의 검출

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...