Name: bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury
Uploaded: 2018-05-21T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

저자는 관심 없음 충돌 선언 하 고 있다.

전체 프로토콜에는 패 혈 증 유도의 첫날 및 동물, 및 마우스 BALF exosomes 절연에 대 한 두 번째 날에서 발 액체의 절연을 포함 하 여 2 일 필요 합니다. 모든 절차를 검토 하 고 기관 동물 관리 및 사용 위원회 애틀랜타 VA 의료 센터에 의해 승인 되었습니다.
1. 마우스 급성 패 혈 증 폐 상해 모델
<ol><li>사용 6-8 주-오래 된 남성 야생-타입 C57BL/6J 마우스 (20-22 g 중량) 동물에 대 한 모델, 그리고 무 균 기술 및 장비를 사용 하 여 모든 절차를 수행 합니다. 오토 클레이 브 집게와가 위 121 ° c.에 20 분을 포함 한 모든 수술 기구</li>
	<li>임의로 실험에 쥐 및 제어 그룹을 배포 합니다. 외과 보드에 쥐를 제 지. 복 경로 의해 대장균 lipopolysaccharide (LPS, 15 mg/kg)와 쥐를 취급 합니다. 개별 몸 무게에 따라 적절 한 LPS 복용량 마우스, 당 0.1 mL PBS에 희석 다음 LPS 솔루션 27 G 바늘 1 mL 주사기를 사용 하 여 주입 하 고 주입 제어 동물 PBS의 해당 볼륨.</li>
	<li>동물 BSL2 시설, 적절 한 실내 온도에서 깨끗 한 케이지로 구성 되어 마우스를 유지.</li>
	<li>24 h 후, CO2 흡입 된 마우스를 안락사.</li>
	<li>마우스 목의 중앙에 수술 영역을 선택 합니다. 작은 절 개 (약 5 m m), 고 기도 노출까지 무딘 집게를 사용 하 여 기도에 대 한 액세스에 대 한 근육을 부드럽게 분리 합니다.</li>
	<li>27 G 바늘으로 마우스 발 액체 10 mL의 멸 균 주사기를 철회. 첫째, 부드럽게, 기관지에 바늘을 삽입 및 기도, 살 균 PBS의 1 mL를 천천히 주입 다음 3 mL 일회용 주사기에 27g 바늘으로 BALF 그릴. 완료 되 면 절차, 주사기와 바늘 biohazard sharps 컨테이너에서 삭제 합니다.</li>
	<li>앞에서 설명한 Chauret 그 외 여러분 에 의해 이산화 염소와 수술 도구를 치료 27, 121 ° c.에 20 분 동안 압력가 마로 소독 하 여 모든 수술 기구를 소독</li>
</ol>2. Exosomes 절연 직렬 울트라-원심 분리 및 특성
<ol><li>15 mL 원뿔 튜브에 마우스 발 액체를 수집 합니다. 치경 세포, 4 ° c.에 1000 g에서 10 분 원심 분리기 샘플 수확</li>
	<li>새로운 15 mL 원뿔 튜브에는 상쾌한 전송 후 원심 4000 x g 세포 파편을 제거 하 4 ° C에서 30 분에 대 한 샘플.</li>
	<li>Ultracentrifuge 튜브에 더 큰 세포 입자를 제거 하 4 ° C에서 10000 x g에서 60 분 동안 원심 분리기 샘플 상쾌한을 수집 합니다.</li>
	<li>새로운 원뿔 관에는 상쾌한 전송 다음 0.2 µ m 주사기-남아 있는 더 큰 입자를 제거 하려면 필터를 통해 샘플을 필터링 합니다.</li>
	<li>Ultracentrifuge 튜브에서 샘플을 수집 하 고 튜브를 균형. 샘플은 exosomes 펠 렛을 4 ° C에서 140000 x g에서 2 h에 대 한 원심 참고: exosomes의 크기는 20-200 nm을 다릅니다. 0.2 µ m 필터는 이상의 200는 더 큰 입자를 제거 사용 nm 크기에서.</li>
	<li>삭제는 supernatants 부드럽게 합니다. 100 µ L PBS 또는 세포 솔루션 및 전송 exosomes 샘플 1.5 mL 튜브에 펠 릿을 녹이 고 사용까지-80 ° C에서 저장. 참고: 전송 전자 현미경은 고립 된 exosomes를 확인 것이 좋습니다.</li>
	<li>프로토콜28처리 표준 편 샘플을 따릅니다. 전송 전자 현미경 검사 법, 그리고 촬영할, 눈금 200 막대를 사용 하 여 exosomes 샘플을 분석 nm.</li>
</ol>3. 미르와 공연 미르-q-RT-PCR의 준비
참고: 미르-q-RT-PCR miRNAs의 식 수준 분석 하는 것이 좋습니다.
<ol><li>표준 miRNAs 격리 프로토콜에 따라 고 정화 RNAs의 동일한 금액을 사용 하 여 단일 가닥 반전 녹음 방송24에 확인.</li>
	<li>발 액체에서 exosomal 예측에 관한 레벨을 감지 하는 표준 실시간 PCR 프로토콜을 따릅니다. 미르-155, 미르-146a, 그리고 U6 SnRNA 특정 실시간 PCR 뇌관을 사용 합니다. PCR 반응 정량 PCR 장치를 실행 하 고 내부 통제 U6 snRNA에 식 값을 정상화. 참고: 각 그룹 triplicate 샘플24이루어져 있다.</li>
</ol>4. Exosomes 라벨 및 정화
<ol><li>1.5 mL 폴 리 프로필 렌 튜브에 100 µ L 정지 솔루션 exosomes 펠 릿을 일시 중지 다음 정지 솔루션의 100 µ L로 다른 튜브에 exosomes 염료의 0.4 µ L를 추가 합니다.</li>
	<li>신속 하 게 염료 솔루션 exosomes 솔루션에 추가 하 고 pipetting으로 철저 하 게 혼합. 실 온에서 5 분에 대 한 샘플을 계속, 빛 으로부터 보호 합니다.</li>
	<li>한 exosome 스핀 열 차입 1.5 mL 튜브에 넣어. Exosome 스핀 열 센터 솔루션 혼합물을 전송 합니다.</li>
	<li>실 온에서 800 x g 2 분에 대 한 열을 회전 합니다.</li>
	<li>열을 삭제 하 고 사용까지-20 ° C에서 eluted 샘플을 저장 합니다.</li>
</ol>5. 받는 사람 세포에 의해 Exosomes의 유효성 검사
<ol><li>1 x 104 8 잘 챔버 슬라이드에 37 ° C, 5% CO2에 문화 세포 MLE 12 /cell 씨앗</li>
	<li>셀에 10 µ L 형광 표시 된 exosomes를 추가 합니다. 1 h 37 ° c.에 품 어</li>
	<li>문화 미디어와 PBS 가진 세포를 씻어 다음 실 온에서 10 분 동안 1 %paraformaldehyde 셀을 수정.</li>
	<li>DAPI와 마운트 중간에 슬라이드를 탑재 합니다.</li>
	<li>형광 현미경을 통해 샘플을 분석 하 고 이미지, 40 배 확대.</li>
</ol>6. Exosomes와 받는 사람 세포 사이 상호 작용의 확인
<ol><li>시드 2 x 105 MLE12/잘 적절 한 문화 매체, 및 37 ° C, 5% CO2에 문화 세포를 포함 하는 12-잘 문화 접시.</li>
	<li>MLE12 셀에 마우스 발 액체에서 20 µ g exosomes를 추가 하 고 실험적인 그룹 당 복제 설정.</li>
	<li>24 h 후 PBS로 세포를 세척 하 고 잘으로 600 µ L 세포의 용 해 버퍼를 추가. 부드럽게 접시를 흔들어 하 고 피펫으로 1.5 mL 튜브에 세포 샘플을 수집 합니다.</li>
	<li>표준 총 RNA 격리 프로토콜 및 cDNA 반전 녹음 방송 프로토콜 따라, 단일 가닥 cDNA 반전 녹음 방송29를 완료.</li>
	<li>받는 사람 셀29에 mRNA 표현 레벨을 감지 하는 표준 실시간 PCR 프로토콜을 따릅니다. TNF-α, 일리노이 대 한 특정 실시간 PCR 뇌관을 사용 하 여-6, 그리고 조 1. PCR 반응 정량 PCR 장치를 실행 하 고 내부 제어 GAPDH 식 값 정상화. 참고: 각 그룹 triplicate 샘플 구성 됩니다.</li>
</ol>

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P., 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=Chlorine+dioxide+inactivation+of+Cryptosporidium+parvum+oocysts+and+bacterial+spore+indicators.">Chlorine dioxide inactivation of Cryptosporidium parvum oocysts and bacterial spore indicators.</a> Applied and environmental microbiology. 67, 2993-3001 (2001).</li><li>Cizmar, P., Yuana, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+Characterization+of+Extracellular+Vesicles+by+Transmission+and+Cryo-Transmission+Electron+Microscopy.">Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy.</a> Methods in molecular biology. , 221-232 (2017).</li><li>Yuan, Z., 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=HIV-related+proteins+prolong+macrophage+survival+through+induction+of+Triggering+receptor+expressed+on+myeloid+cells-1.">HIV-related proteins prolong macrophage survival through induction of Triggering receptor expressed on myeloid cells-1.</a> Scientific reports. 7, 42028 (2017).</li><li>Jackson, M. 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=Analysis+of+Mitochondrial+Transfer+in+Direct+Co-cultures+of+Human+Monocyte-derived+Macrophages+(MDM)+and+Mesenchymal+Stem+Cells+(MSC).">Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC).</a> Bio-protocol. 7, (2017).</li><li>Fujita, Y., 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=Extracellular+Vesicles:+New+Players+in+Lung+Immunity.">Extracellular Vesicles: New Players in Lung Immunity.</a> American journal of respiratory cell and molecular biology. 17, 474-484 (2017).</li><li>Gunasekaran, 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=Donor-Derived+Exosomes+With+Lung+Self-Antigens+in+Human+Lung+Allograft+Rejection.">Donor-Derived Exosomes With Lung Self-Antigens in Human Lung Allograft Rejection.</a> American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 17, 474-484 (2017).</li><li>Kalra, H., 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=Comparative+proteomics+evaluation+of+plasma+exosome+isolation+techniques+and+assessment+of+the+stability+of+exosomes+in+normal+human+blood+plasma.">Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.</a> Proteomics. 13, 3354-3364 (2013).</li><li>Baranyai, 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=Isolation+of+Exosomes+from+Blood+Plasma:+Qualitative+and+Quantitative+Comparison+of+Ultracentrifugation+and+Size+Exclusion+Chromatography+Methods.">Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.</a> PloS one. 10, e0145686 (2015).</li><li>Nordin, J. 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이 작품은 버지니아 장점 검토 2I01BX001786 06A1 rs 자금에 의해 지원 되었다.

질병 마우스 모델은 특정 유전자의 생리 기능을 평가 하 고 실험2의 비용을 줄이기 위해 일반적으로 사용 됩니다. 설명 된 급성 패 혈 증 폐 상해 여기 아즈와 인간에서 선 동적인 응답을 싫어 하지. 이 모델은 관련 분자 병 인, 바이오 마커의 개발 조사 하 고 잠재적인 새로운 치료5를 테스트 합니다.
공동 문화 시스템은 RNA, DNA, 지질, 및 단백...

알리와 아즈 생명이 형태의 약 1 백만 명 전세계에 영향을 미치는 비 cardiogenic 폐 부 종으로 인 한 심한 hypoxemia 호흡 실패는 매년1. 아즈의 병 인 폐 감염 또는 포부와 간접 모욕의 다양 한에서 직접 상해를 포함합니다. 그러나 지난 10 년간 아즈의 분자 병 인의 증가 이해 되었습니다, 그리고, 아즈에 대 한 특정 타겟된 치료는 아직 수 개발2,3.
심각한 폐 상해의 여러 동물 모델 실험 치료 인간 연구4,5번역에 대 한 다리를 제공 하는 개발 되었습니다. 일반적으로 사용 되는 모델의 올레 산, 박테리아, LPS, 및 bleomycin 로컬 설치 포함. 다른 접근 국 소 빈 혈 reperfusion, cecal 결 찰 빵 꾸, 기계 환기-유발 스트레치 부상, hyperoxia 또는 박테리아의 LPS5조직 관리 포함. 임상 가설을 테스트 하는 유용한 생물 학적 시스템을 제공 하는 이러한 모델 및 잠재적인 치료의 개발에 대 한. 인간의 아즈 시뮬레이션, 동물 모델 염증 및 폐에 장벽 기능에 결함을 가진 상피 그리고 내 피 세포에 급성 부상 재현 한다.
Exosomes 분자 내용이 포함, DNA, RNA, 단백질과 지질, 직경에서 20-200 nm와 막 소포 그리고 분자 구성 송금을 통해 조직 microenvironment에서 간 세포 커뮤니케이션을 촉진. Exosomes 여러 종류의 세포, 내 피 세포, 상피 세포, 평활 근 세포와 종양 세포 등에서 분 비 되 고 인간의 체액에 존재. 연구 나타냅니다 exosomes는 염증 성 질환, 전염 성 및 살 균 동안 잡담 stromal 세포와 면역 세포를 통제 하 고 그들의 비정상적인 릴리스 생리 기간 동안 다양 한 자연 및 실험적인 자극에 의해 통제 될 것으로 보인다 그리고 병 적인 프로세스6. 이러한 통신 네트워크 폐 질환의 병 인에 중요 한 역할을 재생할 수 있습니다 그리고 pathophysiological 진행7,8에 영향을 미칠 수 있습니다. 비 코딩 RNAs, miRNAs 조직과 체액, 플라즈마, 세라에 존재 하며 닢 레벨9,10에 mRNA 식 조절에 뉴클레오티드를로 18-22
Exosomes 패키지 miRNAs 차별화 및 여러 종류의 세포의 기능에 영향을 미칠 및 과도 한 수준의 다양 한 질병, 암, 폐 질환, 비만, 당뇨병 및 심혈 관 질환11를 포함 하 여와 관련 된 12,,1314,,1516. 받는 사람 세포와 exosomal miRNAs의 왕복으로 항목 수정 microenvironment17,18hemostasis 세포 커뮤니케이션을 촉진 한다. 심각한 폐 상해 여러 셀 형식 exosomes8통해 광범위 한 세포 통신 관련 된 복잡 한 프로세스입니다. 미르-155와 미르-146a 일반적인 transcriptional 규제 메커니즘을 공유 하 고 염증 반응과 면역 관용19,20을. 최근 연구는 모두 exosomal miRNAs 면역 세포21사이 였죠 통해 염증 반응을 조절 나타냅니다. 그러나, 밑에 치경 응답에 exosomal miRNAs의 modulatory 효과 분자 메커니즘 불분명 남아, 의심할 여 지 없이 잠재적인 임상 관련성 및 변환 의미 가치 추가 조사.
공동 문화 모델 염증과 암22,23같은 복잡 한 환경에서 특정 세포 유형의 상호 작용을 정의 하기 위해 채용 되 고 있다. 이러한 플랫폼 특히 면역 및 구조 셀에 대 한 세포 유형 사이 교차 하는 대화가 대체 전략을 제공 합니다.
내부 tracheal LPS의 aerosolized, 복 또는 조직의 관리 유도 실험 폐 상해24,,2526, 널리 사용 되 고 상피 그리고 내 피 침투성을 유도 하는 것으로 나타났습니다. 결함입니다. 여기 복 LPS 사용 하 여 마우스에 심각한 폐 상해의 정화 조 모델을 유도 하. LPS의 복 관리의 24 시간 이내 침투성 결함은 염증 세포의 모집과 폐에서 유도 된. 또한, 우리는 발에서 exosomes 포함 155 미르와 미르-146a, 그리고 발 액체에서 exosomes 프로 염증 성 cytokines 일리노이-6와 TNF-α를 포함 하 여 받는 사람 상피 세포에서 식 유도 보여줍니다. 이 데이터는 먼저 보여 그 exosomal miRNAs는 급성 패 혈 증 폐 상해의이 모델에 발에서 분 비.

<table><tbody><tr><td>lipopolysaccharide</td><td>Sigma-Aldrich</td><td>Escherichia coli 055:B5</td><td></td></tr><tr><td>PBS pH7.2(1X)</td><td>Life technologies</td><td>20012-027</td><td></td></tr><tr><td>mirVana miRNA isolation kit</td><td>Thermo Fisher</td><td>449774</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Thermo Fisher</td><td>4369016</td><td></td></tr><tr><td>mmu-miR-155 (002571) primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>has-miR-146a (000468) primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>U6snRNA primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>TNF-&amp;alpha;(Mm00443258_m1 ) primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>IL-6 (Mm00446190_m1&amp;nbsp;&amp;nbsp; )primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>ZO-1 (Mm00493699_m1&amp;nbsp; )primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>minimal essential medium (MEM)</td><td>Thermo Fisher</td><td>11095-072</td><td></td></tr><tr><td>Trysin-EDTA solution(0.05%)</td><td>Thermo Fisher</td><td>25300054</td><td></td></tr><tr><td>Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&amp;nbsp;</td><td>Sigma-Aldrich</td><td>MINI67-1KT</td><td></td></tr><tr><td>Exosome Spin Columns (MW3000)</td><td>Thermo Fisher</td><td>4484449</td><td></td></tr><tr><td>Beckman Coulter Optima L-100XP ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>L-100XP</td><td></td></tr><tr><td>Ultra-clear centrifuge tubes</td><td>Beckman Coulter</td><td>344058</td><td></td></tr><tr><td>Applied Biosystems 7500 Fast Real-Time PCR System</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>transmission electron microscopes (TEM)</td><td>JEOL Ltd</td><td>120 kV TEM</td><td></td></tr><tr><td>Olympus BX41 microscope</td><td>Olympus</td><td>BX41</td><td></td></tr></tbody></table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

심각한 폐 상해 (알리) 및 급성 호흡 곤란 증후군 (아즈)는 높은 질병 률 및 사망률을가지고 계속 해 서 폐 질환의 이질적인 그룹을 나타냅니다. 알리의 분자 병 인 되 고 더 나은 정의; 그러나, 질병의 복잡 한 특성으로 인해 분자 치료는 아직 개발. 여기 급성 패 혈 증 폐 상해의 lipopolysaccharide (LPS) 유도 마우스 모델 사용 하 여 exosomes 염증 반응에서의 역할의 윤곽을 그리 다. 이 모델을 사용 하 여, 쥐 복 LPS에 노출 되는 miRNA와 염증 반응 조절 cytokines 패키지 된 폐에서 Broncho 치경 게 (BAL) 액체에 exosomes 분 비는 보여줄 수 있었습니다. 추가 공동 문화 모델 시스템을 사용 하 여, 우리는 대 식 세포에서 풀어 놓인 exosomes 기관지 상피 세포에서 꽉 접합 단백질의 표현을 방해 보여. 이 결과 건의 염증 반응에 기여 하는 크로스 토크 1) exosomal 왕복을 통해 타고 난 면역 및 구조 세포 사이 및 구조상 방 벽 및 2) 타겟팅이 miRNAs의 치료 하는 새로운 플랫폼을 제공할 수 있습니다. 알리 고 아즈입니다.

정화 조 폐 상해를 유도, 쥐 복 LPS (15 mg/kg)로 대우 되었다. 그림 1A와 같이 LPS 관리의 24 시간 이내 neutrophilic 유입 폐에서 본 했다. 마우스 발 유체, 격리 및 정화 exosomes의 그려 했다. 형태학 BALF exosomes의 전자 전송 현미경 검사 법 (그림 1B)에 의해 확인 됐다.
선택적 프로-염증 성 miRNAs ?...

Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Bronchoalveolar 게 Exosomes Lipopolysaccharide 유도 정화 조 폐 상해에

복 LPS에 드러낸 쥐에는 miRNAs와 함께 패키지 된 Broncho-치경 게 (BAL) 액체에 exosomes 분 비. 공동 문화 시스템을 사용 하 여, 우리 보여줍니다 발 액체에서 발표 하는 exosomes 기관지 상피 세포에서 꽉 접합 단백질의 표현을 방해 폐 상해를 강조 하는 프로 염증 성 cytokines의 표현을 증가.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

bronchoalveolar-lavage-exosomes-lipopolysaccharide-induced-septic

Research

JoVE Journal

Genetics

10.8K 조회수.  Atlanta VAMC. 복 LPS에 드러낸 쥐에는 miRNAs와 함께 패키지 된 Broncho-치경 게 (BAL) 액체에 exosomes 분 비. 공동 문화 시스템을 사용 하 여, 우리 보여줍니다 발 액체에서 발표 하는 exosomes 기관지 상피 세포에서 꽉 접합 단백질의 표현을 방해 폐 상해를 강조 하는 프로 염증 성 cytokines의 표현을 증가.

비디오: Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

에어로졸 리포 폴리 사카 라이드를 사용하여 생성 폐 호중구하는 방법

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

연구

면역학 및 감염병학

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

쥐 폐의 기관지 폐포 세척은 염증성 세포의 침윤을 분석하는

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

급성 호흡 곤란 증후군의 모델로서 돼지의 세척에 의한 계면 활성제 고갈 (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

의학

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular 소포 Murine Bronchoalveolar 게 유체는 한 원심 분리 기법을 사용 하 여에서 격리

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

일방적인 기관내 산 성 증착에 의한 자가 제한 급성 폐 손상의 모델

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

올레산 급성 호흡 곤란 증후군에 대 한 모델로 돼지에 주입

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

결합 된 오존 과 LPS 유도 뮤린 급성 폐 부상 동안 폐 세포 적응 시각화

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.

The present protocol describes a lung injury model in mice using oleic acid to mimic acute respiratory distress syndrome (ARDS). This model increases the inflammatory mediators on edema and decreases lung compliance. Oleic acid is used in the salt form (oleate) since this physiological form avoids the risk of embolism.

올레산 유도 급성 호흡곤란 증후군의 마우스 모델

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

직접 경포당증 주입에 의해 마우스에 있는 심각한 폐 상해를 유도하는

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome (ARDS)

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% in severe cases. Further research efforts are needed to better understand the complex pathophysiology of this disease. There are different well-established animal models to induce acute lung injury but none has been able to adequately mimic the complex pathomechanisms of ARDS. The most crucial factor for the development of this condition is the damage to the alveolar capillary unit. The combination of two well-established lung injury models allow us to mimic in more detail the underlying pathomechanism. Bronchoalveolar lavage (BAL) leads to surfactant depletion as well as alveolar collapse. The repeated instillation of fluid volumes causes subsequent hypoxemia. Surfactant depletion is a key factor of ARDS in humans. BAL is often combined with other lung injury approaches, but not with a second hit followed by oleic acid injection (OAI) yet. Oleic acid injection leads to severely impaired gas exchange, a deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The OAI mimics most of the expected effects of ARDS consisting of extended inflammation of lung tissue with an increase of alveolar leakage and gas exchange impairment. A disadvantage of the combination of different models is the difficulty to determine the influence to the lung injury caused by BAL alone, OAI alone or both together. The model presented in this report represents the combination of BAL and OAI as a new double-hit lung injury model. This new model is easy to implement and an alternative to study different therapeutic approaches in ARDS in the future.

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome ARDS

Different, complex animal models exist to study the pathophysiology of acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage and oleic acid injection induced lung injury is suitable as a new double-hit animal model for studying the acute respiratory distress syndrome.

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% ...