이 작품은 NIH RO1 AI079253 및 보훈학과로 운영되었습니다.

1. 동물 모델 <ol><li> 마우스 : p47 phox-/를 사용하여 - 마우스와 연령 및 성별 - 일치 C57BL6/DBA 마우스입니다. 기관 동물 케어 및 사용위원회의 실험에 대한 승인을 얻습니다. </li><li> 마취 : 마취를 유도하기 위해 지속적인 isoflurane 관리 시스템을 사용합니다. vaporizer 시스템 (VetEquip)는 (2-3 %) isoflurane으로 가득합니다. 쥐가 외부 자극에 대한 응답으로 호흡, 운동, 그리고 각막 반사를 관찰하여 완전히 anesthetized되어 있는지 확인. </li><li> 수술 절차 : 70 %의 에탄올과 수술 영역 (benchtop)을 씻어. 멸균 장갑과 깨끗한 수술 가운과 마스크를 착용하십시오. 머리를 클리핑하고 절개가 이루어집니다 영역에 70 %의 에탄올과 교류 betadine을 적용하여 마우스를 준비합니다. 멸균 표면에 수술을 수행하고 멸균 악기를 사용합니다. 모니터 마우스는 사후 operatively까지 깨어 자유롭게 이동. </li><li> Intratracheal zymosan 관리 <ol><li> 마취를 관리로 1.2에 설명되어 있습니다. </li><li> betadine과 70 %의 에탄올과 수술 영역 (목) 소독 및 수술 절개로 기관을 쉽게받을 수 있습니다. </li><li> 피어스 27 게이지 바늘로기도하고 멸균 PBS에 용해 0.5 μg / μl 솔루션을 (25g 마우스에 대한 총 판매량 50 μl)를 사용하여 1 μg / g의 용량에 zymosan (세인트 루이스, MO)를 주입. </li><li> 닫기 쥐 목 무균 조건 하에서 5-0 멸균 실크 봉합과 상처. </li><li> 4 시간, 24 시간 후 이미지. </li></ol></li><li> Cecal 결합과 주사 <ol><li> 1.2에 설명 된대로 마취를 관리 할 수​​ 있습니다. </li><li> 수술 영역 (복부)에 머리를 클립하고 betadine과 70 % 에탄올로 소독. </li><li> 중간 선 개복술을 수행하고이게 맹장을 식별합니다. </li><li> 4-0 실크 봉합사와 21 게이지 바늘 중 하나를 패스 결합으로 말초 찔린이게 맹장과 노출이게 맹장의 말초 50 %를 Ligate. </li><li> 4-0 멸균 실크 봉합과 절개를 닫습니다. </li><li> 4 시간 후 이미지와24 시간. </li></ol></li><li> 구강 탄소 tetrachloride (CCL 4) 관리 <ol><li> 1.2에 설명 된대로 마취를 관리 할 수​​ 있습니다. </li><li> 구두 gavage으로 옥수수 기름에 용해 CCL 네 (2 μg / g, 세인트 루이스, MO)를 관리 할 수 있습니다. CCl4 행정 절차는 IBC / EHS 정책에 따라 지정된 지역에서 수행해야합니다. </li><li> 4 시간, 24 시간 후 이미지. </li></ol></li></ol> 2. 발광 생물 이미지를 취득 <ol><li> 발광 모드로 설정 IVIS 200 (Xenogen 공사, 알라 메다, CA) 이미징 시스템을 초기화하고, 전하 결합 소자 (CCD) 잠글 온도 기다립니다. </li><li> 노출 시간, binning (중간) 및 F / 정지 (8) 설정을 선택합니다. </li><li> 정상적인 체온을 유지하기 위해 온수 무대에서 이미징 챔버에서 위로 향한 위치에 배치 마우스. 쥐가 IVIS 이미징 챔버에있는 동안 코 콘을 통해 1.2에 설명 된대로 마취를 관리 할 수​​ 있습니다. </li><li> L-012를 관리<html> (20 μg / g) 이미지에 대해 선택한 각 시점에서 역 궤도 분사를 통해 intravenously 멸균 PBS (10 μg / μl)에 용해. </li><li> L-012 주입 후 2 분에 시작 이미지를 캡처합니다. 우리는 일반적으로 40 초 노출을 사용합니다. </li></ol> 3. 관심 지역을 사용하여 데이터 분석 <ol><li> 생활 이미지 소프트웨어에게 v.4.2을 (Xenogen 공사, 알라 메다, CA)를 사용하여 데이터를 분석합니다. </li><li> 이미지를 열고 관심 툴의 지역 측정을 선택합니다. </li><li> 가슴 또는 / 및 마우스의 사진 이미지 위에 의사 색 디지털 이미지 (광자 검출을 대표하는) 오버레이 한 ​​후 복부 이상 관심 모양과 크기의 지역을 선택합니다. </li><li> 관심 지역에서 신호 강도 (광자 플럭스)를 정량화. </li></ol> 4. 통계 <ol><li> 개인 시간 지점에서 Bonferroni 사후 테스트를 양방향 ANOVA를 수행하는 통계 분석 소프트웨어 패키지를 사용합니다. </li></ol>tle &quot;&gt; 5. 대표 결과 Zymosan는 프로 염증성 효모 세포벽 제품 및 NADPH 산화 효소 3의 강력한 활성입니다. 우리는 이전에 해당 intratracheal zymosan는보다 강력한 neutrophilic 폐 염증과 p47 phox-/에서 프로 염증성 시토 킨 생산을 유도했다 - 마우스에게 1 wildtype에 비해. zymosan 도전 다음 마우스 - 여기, 우리의 목표는 wildtype와 p47 phox-/의 폐에 ROS 생성을 비교하는 것이 었습니다. 4 H 및 24 시간에서 마우스 - 4 시간에서 intratracheal zymosan 관리 한 후, 우리는 기준뿐만 아니라 p47 phox-/에 비해 wildtype의 광자 방출의 증가에 비해 wildtype 쥐 가슴 광자 방출에 상당한 증가를 관찰했다. 반면, p47 phox-/에서 bioluminescence 신호 - 마우스 zymosan 처리 (Fig.1 AB) 이후 4 시간에서 기본 수준에서 유지. 따라서, NADPH 산화 효소는 B에 나타납니다전자 zy​​mosan 관리에 따라 폐의 ROS 생성의 주요 소스. 다음, 우리는 CLP 유발 패혈증 모델에서 ROS 생산도 평가합니다. 패혈증은 급성 폐 손상 (ALI)과 급성 호흡 곤란 증후군 (ARDS) 4, 5 등의 최종 장기 손상과 관련된 생명을 위협하는 증후군이다. ROS로 인한 부상은 패혈증 유발 다중 장기 부전을 유도하는 중요한 요소로 간주되었습니다. 우리는 ROS가 크게 가슴 (4 시간) 이상 및 기준에 비해 wildtype 쥐 CLP에 따라 복부 (24 시간) 이상 증가 된 것으로 나타났습니다. 그러나, p47 phox-/에서 ROS 수준 - 마우스는 시간 포인트 (Fig.2 AC) 모두에서 기본 수준과 비슷했다. 이러한 결과는 CLP 유발 패혈증 마우스 모델에서 ROS 생성이 산화 효소에 의존 NADPH입니다 보여줍니다. 마지막으로, 우리는 CCL 4 유발 간 손상 모드에서 ROS 생산을 감지하는 발광 생물 이미징을 사용하여리터. CCL 4 간세포 괴사가 발생하고, 급성 간 손상과 간 섬유증 6을 모두 모델링하는 데 사용되었습니다. CCL 4 행정에 따라 4 H의 마우스 - 염증과 부상 이전에 설명 모델에 대조적으로, 우리는 복부에서 ROS 생산 (35 %) p47 phox-/에서 기준이 지남에 따라 증가하는 겸손 것으로 나타났습니다. 마우스 (Fig.3 AB) - 그러나, 4 - 유도 ROS 생성 CCL의 크기는 p47 phox-/에서보다 wildtype 쥐 훨씬 더했습니다. 이러한 데이터는 모두 p47 phox 함유 NADPH 산화 효소에 의존하고 독립적 인 ROS 생성은 급성 CCL 4 유발 간 손상에서 발생하는 것이 좋습니다. <img alt="그림 1" src="/files/ftp_upload/3925/3925fig1.jpg" /> zymosan 치료 후 폐의 그림 1. ROS 생산. A) 대표 bioluminescenROS 생산의 CE 이미지. 마우스 및 복부 발광이 zymosan 치료에 의해 ​​변경되지 - 가슴뿐만 아니라, 발광는 wildtype와 p47 phox-/ 모두의 복부 이상 감지 값입니다. wildtype 및 p47 phox-/의 가슴에서 B) 광자 카운트 - zymosan의 (IT) intratracheal 단일 분사 한 후 마우스. Bioluminescence 이미징은 기본, 4 H,, 24 H에서 수행되었다. 가슴 관심 지역의 발광은 지정된 의사 색 척도를 이용하여 확인되었다. 결과는 의미로 제공됩니다 ± SE, N = 6-9 그룹 당, * = P &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig1large.jpg" target="_blank">큰 그림을 보시려면 여기를 클릭하세요</a> . <img alt="그림 2" src="/files/ftp_upload/3925/3925fig2.jpg" /> 그림 2. ROS 감지는 다음 CLP. ROS 생산, B의 A) 대표 bioluminescence 이미징) 광자 가슴에서 카운트 wildtype와 p47 phox-/의 복부에서 C) 광자 카운트 - 기준에서 마우스, 4 시간, 그리고 CLP 후 24 시간. 결과는 의미로 제공됩니다 ± SE, N = 그룹 당 4-5, * = P &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig2large.jpg" target="_blank">큰 그림을 보시려면 여기를 클릭하세요</a> . <img alt="그림 3" src="/files/ftp_upload/3925/3925fig3.jpg" /> CCL 후 복부 4 유발 간 손상의 그림 3. ROS 생산. 구두 CCL 네 치료 후 기준선에서 마우스, 4 시간, 24 시간 - ROS 생산 및 wildtype와 p47 phox-/의 복부에서 B) 광자 카운트의 A) 대표 bioluminescence 이미징. . N = 4-5 당 그룹, * = P &lt;0.05, 결과는 의미 ± SE로 표시됩니다 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig3large.jpg" target="_blank">보시려면 여기를 클릭하세요 큰 그림</a> </&gt;.

<ol>
	<li>Segal, B., Han, W., Bushey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+limits+innate+immune+response+in+the+lungs+in+mice.">NADPH oxidase limits innate immune response in the lungs in mice.</a> PLoS ONE. 5, e9631 (2010).</li><li>Jackson, S., Gallin, J., Holland, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p47phox+mouse+knock-out+model+of+chronic+granulomatous+disease.">The p47phox mouse knock-out model of chronic granulomatous disease.</a> J. Exp. Med. 182, 751-758 (1995).</li><li>Gantner, B. N., Simmons, R. M., Underhill, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+induction+of+inflammatory+responses+by+dectin-1+and+Toll-like+receptor+2.">Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2.</a> J. Exp. Med. 197, 1107-1117 (2003).</li><li>Dejager, L., Pinheiro, I., Libert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecal+ligation+and+puncture:+the+gold+standard+model+for+polymicrobial+sepsis.">Cecal ligation and puncture: the gold standard model for polymicrobial sepsis.</a> Trends Microbiol. 19, 198-208 (2011).</li><li>Tsiotou, A. G., Sakorafas, G. H., Bramis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septic+shock;+current+pathogenetic+concepts+from+a+clinical+perspective.">Septic shock; current pathogenetic concepts from a clinical perspective.</a> Med. Sci. Monit. 11, 76-85 (2005).</li><li>Fujii, T., Fuchs, B. C., Tanabe, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+carbon+tetrachloride+induced+liver+fibrosis:+Histopathological+changes+and+expression+of+CD133+and+epidermal+growth+factor.">Mouse model of carbon tetrachloride induced liver fibrosis: Histopathological changes and expression of CD133 and epidermal growth factor.</a> BMC Gastroenterology. 10, 79-90 (2010).</li><li>Kubo, H., Morgensterm, D., Doerschuk, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+complement-induced+lung+injury+in+mice+with+deficiency+of+NADPH+oxidase.">Preservation of complement-induced lung injury in mice with deficiency of NADPH oxidase.</a> J. Clin. Invest. 97, 2680-2684 (1996).</li><li>Segal, B., Sakamoto, N., Bulkley, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xanthine+oxidase+contributes+to+host+defense+against+Burkholderia+cepacia+in+the+p47(phox-/-)+mouse+model+of+chronic+granulomatous+disease.">Xanthine oxidase contributes to host defense against Burkholderia cepacia in the p47(phox-/-) mouse model of chronic granulomatous disease.</a> Infect Immun. 68, 2374-2378 (2000).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiological reviews. 82, 47-95 (2002).</li><li>Jones, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radical-free+biology+of+oxidative+stress.+American+journal+of+physiology.">Radical-free biology of oxidative stress. American journal of physiology.</a> Cell physiology. 295, C849-C868 (2008).</li><li>Hubbard, W. J., Choudhry, M., Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecal+ligation+and+puncture.">Cecal ligation and puncture.</a> Shock. 24, 52-57 .</li><li>Wardman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+and+luminescent+probes+for+measurement+of+oxidative+and+nitrosative+species+in+cells+and+tissues:+progress,+pitfalls,+and+prospects.">Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.</a> Free Radic. Biol. Med. 43, 995-1022 (2007).</li><li>Pollock, J. D., Williams, D. A., Gifford, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+X-linked+chronic+granulomatous+disease,+an+inherited+defect+in+phagocyte+superoxide+production.">Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.</a> Nat. Genet. 9, 202-209 (1995).</li><li>Aramaki, Y., Y, ., Yoshida, H. <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+sensitive+chemiluminescence+probe,+L-012,+for+measuring+the+production+of+superoxide+anion+by+cells.">A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells.</a> Biochem. Biophys. Res. Commun. 193, 554-559 (1993).</li><li>Daiber, A., Oelze, M., August, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+superoxide+and+peroxynitrite+in+model+systems+and+mitochondria+by+the+luminol+analogue+L-012.">Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012.</a> Free Radic. Res. 38, 259-269 (2004).</li><li>Kielland, A., Blom, T., Nandakumar, K. S. <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+reactive+oxygen+and+nitrogen+species+in+inflammation+using+the+luminescent+probe+L-012.">In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012.</a> Free Radic. Biol. Med. 47, 760-766 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

생활 동물 반응성 산소 종의 &quot;실시간&quot;측정 (ROS)는 형광등과 chemiluminescent 프로브를 사용하여 달성 될 수있다. 형광 프로브가 약 신호 대 잡음 비율 12 문제에서 고통 있지만, 설명 된 이미징 기술은 루미놀 기반의 기판 L-012와 ROS의 화학 반응에 따라 발광을 검출에 대한 더 많은 문자를 구분합니다. 모든 발광 생물 이미징 기술과 마찬가지로,이 방법론은 기관과 조직에 의해 파장에 의?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> L-012 </td><td> 와코 화학 물질 USA 주식회사 </td><td> 120-04891 </td><td></td></tr><tr><td> Zymosan </td><td> 시그마, 세인트 루이스, MO </td><td> Z4250 </td><td></td></tr><tr><td> 탄소 tetrachloride </td><td> 시그마, 세인트 루이스, MO </td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPH 산화 효소는 항균 및 항 곰팡이 호스트 방어를 mediates 중요한 효소이다. 항균 호스트 방어에서의 역할뿐만 아니라, NADPH 산화 효소는 염증 반응 1 변조 중요한 신호 기능이 있습니다. 따라서, NADPH 산화 효소 파생 ROS 생성의 &quot;실시간&quot;동역학에 측정 할 수있는 방법의 개발은 방위, 염증, 그리고 부상을 개최 할 관련 메커니즘을 이해하는 중요한 연구 도구가 될 것으로 예상된다. 만성 granulomatous 질환 (CGD)는 심각한 감염과 과도한 염증을 특징으로 NADPH 산화 효소의 상속 장애입니다. 식세포 NADPH 산화 효소의 활성화는 cytosolic subunits (p47 phox, p67 phox, 그리고 p40 phox)과 막에 바인딩 flavocytochrome (gp91 phox와 P22 phox heterodimer로 구성)에 RAC의 translocation이 필요합니다. 손실CGD에서 이러한 NADPH 산화 효소 구성 요소 결과의의 기능 변이. CGD 환자와 마찬가지로, gp91 phox - 결함 마우스와 p47 phox - 결함 마우스에 결함이 식세포 NADPH 산화 효소 활동 및 장애인 호스트 방어 13, 14가 있습니다. 위에 설명 된 NADPH 산화 효소의 구성 요소가 포함 phagocytes,뿐만 아니라 다른 세포 유형의 다양한 NADPH 산화 효소의 다른 isoforms을 표현한다. 여기, 우리는 생활 마우스에서 ROS 생산을 수량화하고 염증과 부상의 모델에서 ROS 생성에 NADPH 산화 효소의 노력이 윤곽을 그리다 할 수있는 방법을 설명합니다. 이 방법은 전하 결합 소자 (CCD)에 의해 기록되어 발광을 방출하기 위해 L-012 (루미놀의 아날로그)와 반응 ROS에 기반을두고 있습니다. L-012 프로브의 원래 설명에, L-012에 따라 달라 chemiluminescence은 완전히이 반응에서 검색된 메인 ROS는 슈퍼이라고 표시, 초과 산화물의 dismutase에 의해 폐지되었다산화물 음이온 15. 후속 연구는 L-012은 반응성 질소 종 16, 17 등 다른 자유 래디칼을 감지 할 수 것으로 나타났습니다. Kielland 외. 17 phorbol myristate 아세테이트, NADPH 산화 효소의 강력한 활성,의 주제 응용 프로그램이 발광 프로브 L-012를 사용하여 마우스에서 발견 할 수 산화 효소에 의존 ROS 생성을 NADPH는 생각이 들어서 보여 주었다. 이 모델에서, 그들은 L-012-의존 발광는 p47 phox - 결핍 생쥐에서 폐지 된 것으로 나타났다. 우리는 wildtype 마우스와 NADPH 산화 효소 - 결함 p47 phox-/에서 ROS 생성 비교 - 다음과 같은 세 가지 모델의 생쥐 2 : zymosan, NADPH 산화 효소를 활성화 할 수 있습니다 벽 파생 제품 프로 염증 곰팡이 세포의 1) intratracheal 관리, 2) cecal 결합과 주사 (CLP), 보조 급성 폐 염증과 부상으로 간 복부 패혈증의 모델, 그리고 3) 구두 탄소 tetrachloride에게(CCL 4) ROS에 의존 간 부상의 모델입니다. 이 모델은 특히 각각, 비 전염성 염증, polymicrobial 패혈증 및 독소 유발 장기 부상의 맥락에서 NADPH 산화 효소에 의존 ROS 생성을 평가하는 선정되었다. p47에 wildtype 마우스에 bioluminescence을 비교 phox-/ - 마우스 우리는이 모델의 발광 생물 신호에 p47 phox 함유 NADPH 산화 효소에 의해 생성 된 ROS의 특정 공헌을 윤곽을 그리다 수 있습니다. p47에 비해 wildtype 쥐 증가 ROS 수준을 보여 Bioluminescence 이미징 결과는 phox-/ - 마우스는 NADPH 산화 효소는 염증 자극에 대한 응답으로 ROS 생성의 주요 원천입니다 지적했다. 이 방법은 생체 내 염증 동안 ROS 생성의 &quot;실시간&quot;모니터링을위한 최소한 침해 접근 방식을 제공합니다.

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH 산화 효소가 phagocytes에서 반응성 산소 종의 주요 소스 (ROS)입니다. 때문에 ROS의 임시 자연의, 그것은 생활 동물의 ROS 수준을 측정하고 모니터링하기가 어렵습니다. 생활 쥐 ROS의 일련 정량화하기위한 최소한 침입 방법 설명되어 있습니다.

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 ...

bioluminescence-imaging-nadph-oxidase-activity-different-animal

Research

JoVE Journal

Immunology and Infection

16.1K 조회수.  Vanderbilt University School of Medicine. NADPH 산화 효소가 phagocytes에서 반응성 산소 종의 주요 소스 (ROS)입니다. 때문에 ROS의 임시 자연의, 그것은 생활 동물의 ROS 수준을 측정하고 모니터링하기가 어렵습니다. 생활 쥐 ROS의 일련 정량화하기위한 최소한 침입 방법 설명되어 있습니다.

비디오: Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

연구실 고양이와 마우스에 외부의 뜻과 Endoparasites의 진단

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

연구

면역학 및 감염병학

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfp 기자 마우스를 사용하여 건강하고 병에 걸린 간에서 면역 세포의 장기 생체 내에 다중 광자 현미경 이미징

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

분리 및 다른 세 쥐 흉선 이식의 이식.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

생물 발광 영상은 아프리카 트리파노소마 증의 늦은 단계의 감염을 감지하는

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

다제 내성 항 감염 블루 라이트 치료의 생체 조사에서 아시네 토 박터 바우 마니는 생물 발광 영상을 사용하여 감염을 굽기

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

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

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

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

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

에 대한 응답으로 인간의 기본 호중구와 단핵구에 의해 항진균 활성의 라이브 영상<em&gt;를했다. 투스</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

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 ...