The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

이 논문에 기술 된 동물과 관련된 모든 절차는 스위스 법률에 따라 취리히, 스위스의 캔톤의 동물 관리의 승인을 실시 하였다. 1. 버퍼 준비 <ol><li> (4)과 항 이뇨 호르몬 (ADH) 용액 (표 1) - 솔루션 (1)을 준비합니다. </li><li> 투석 완충액 (표 1)을 준비한다. 주 : 이것은 재관류 동안 투석 버퍼로서 사용되는 버퍼이다. 나중에, 적혈구 최종 관류 액을 형성하는 버퍼에 희석한다. </li><li> 적혈구 준비. <ol><li> 인간의 적혈구 농축액 투석 버퍼 500 ml의 (지역 혈액 은행에서 얻은 시험 물질) 250 ml에 희석. 8 분 동안 2,000 XG에 원심 분리기. 어떤 적혈구를 제거하지 않도록주의, 버퍼를 제거합니다. 배를 반복합니다. </li></ol></li><li> 완충액, 알부민 (BSA 소 혈청 알부민)를 준비한다. <ol><li> dialysi 200 ㎖에서의 버퍼는 교반 막대를 사용하여 BSA의 44g을 녹인다. 필터 종이와 솔루션을 필터링합니다. </li></ol></li><li> 관류를 준비합니다. <ol><li> BSA에 버퍼에 여과지를 통해 단계 1.3.1에서 적혈구를 필터링합니다. 투석 버퍼 800 ML의 총 부피까지 입력합니다. 주 :이 최종 관류이다. 헤마토크릿 이제 8과 12 % 사이 여야합니다. 관류 12 시간까지 4 ℃에서 저장할 수 있습니다. </li></ol></li></ol> 2. 시작 투석 및 산소화 <ol><li> 더 큰 버퍼 저수지, 작은 버퍼 저장하고 촉촉한 챔버를 37 ° C로 (작은, 이중벽 챔버 나중에 신장을 유지하기 위해 37 ° C와 100 % 습도하게) 주변의 물을 욕조를 켜십시오 (그림 2) . </li><li> 투석 버퍼로 큰 버퍼 저수지와 관류와 작은 저수지를 입력합니다. </li><li> 투석 버퍼에 5 % CO에 2 / 95 % O 2 가스 유입을 돌립니다. </li><li> 투석 버퍼에 대한 관류의 연속 투석 전환합니다. 저 유량 투석 튜브를 사용하도록주의하십시오. 3 단계로 진행합니다. </li></ol><img alt="그림 2" src="/files/ftp_upload/54712/54712fig2.jpg" /> 그림 2 : 관류 회로의 개략도. 반응식은 관류 회로의 주요 구성 요소와 버퍼 흐름의 방향을 나타낸다. 진한 파란색에 둘러싸여 모든 구성 요소는 물 목욕 / 온도 37 ° C로 유지된다. 1 : 재관류 버퍼의 적어도 3 배의 용적의 투석 버퍼 연속 95 % O 2 / 5 % CO 2로 버블 링된다. 2 : 투석 완충액 관류 완충액 연속적 롤러 펌프에 의해 투석 튜브에서 서로에 대해 투석한다. 3 :이 투석, 관류 완충액 9 % O 풍부 인해 2 / 5 % CO 2 및 전해질 농도가 일정하게 유지된다 througho유타 관류. 4 : 롤러 펌프는 신장 방향으로 관류 완충액 추진. 5 : windkessel가 연동 파도를 제거하고 버블 트랩 역할을합니다. 6 : 압력 변환기 (자유롭게 교류의 흐름을 허용하면서 4 (롤러 펌프에 연결)은 지속적인 압력을 유지하기 위해). 7 : 관류 전반에 걸쳐, 신장은 100 % 공기 습도 및 37 ° C의 신장 온도 촉촉한 챔버에 남아 있습니다. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 3. 수술 1 부 (모든 합자의도를 들어, 그림 3 참조) 참고 : 5-0 수술 스레드를 사용하여 모든 합자를 수행합니다. <ol><li> 복강 내 주사하여 마우스 (0.9 % 염화나트륨에 용해 체중, 20 ㎎ / ㎖ 케타민 및 1 ㎎ / ㎖의 자일 라진 10 μL / g)를 마취. anesth의 충분한 깊이를 확인후방 발 반사의 유무를 테스트하여 ESIA. </li><li> 습한 챔버에서 부정사 위치에 마우스를 고정합니다. 수의사 연고와 눈을 보호합니다. 요추 혈관을 상승 척추 아래 1 ML의 주사기를 놓습니다. </li><li> 가위로 먼저 흉골 개방 피부, 후 복부 근육에 음모 문장에서 중간 개복술을 수행합니다. </li><li> 장을 제거하고 복부에서 마우스 측면의 왼쪽에 놓습니다. </li><li> 결합 조직에서 방광을 확보하고 요관 및 요도 모두를 탐구한다. </li><li> 왼쪽 요관 (합자 I)의 주위에 끈을 놓으십시오. 닫습니다. </li><li> 요도 (합자 II) 주위에 끈을 놓으십시오. 닫습니다. </li><li> 전체 방광 (합자 III)의 주위에 &quot;올가미&quot;합자를 놓습니다. </li><li> 블래 1 mm의 절개. </li><li> 2cm PE (50) 튜브와 개방을 Cannulate. </li><li> 튜브 주위 닫기 합자 III. </li><li> 합자에서 왼쪽 요관 및 요도의 말단을 잘라. BLAD데르는 이제 자유롭게 이동 오른쪽 요관에 연결되어 있습니다. </li><li> 결합 조직과 지방의 복부 대동맥을 취소합니다. </li><li> 복부 중간 대동맥 결찰 (합자 IV)를 놓습니다. </li><li> 장간막 동맥과 복강 트렁크 (합자 V) 사이의 다이아 프램 아래의 대동맥 주위 합자를 놓습니다. </li><li> 장간막 동맥 (합자 VI)의 주위에 끈을 놓으십시오. </li><li> 직접 오른쪽 아래 왼쪽 신장 동맥 (합자 VII) 위의 대동맥 결찰를 놓습니다. </li><li> 꼬리 정맥 패키지 (맥) (합자 VIII)의 주위에 끈을 놓으십시오. 4 단계로 진행합니다. </li></ol><img alt="그림 3" src="/files/ftp_upload/54712/54712fig3.jpg" /> 그림 3 : 수술 중에 배치 된 합자의 개략도. 개복술 후 오픈 복부보기. 소장은 왼쪽 밖으로 이동합니다. L 및 R이 좌측 및 우측 신장을 나타낸다. 그만큼검은 색 선은 각각의 봉합사의 면적을 나타낸다. 합자이 먼저 배치하고 텍스트에 주어진 순서에 폐쇄된다. X는 동맥 삽관에 대한 절개의 위치를 표시한다. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 관류 회로 4. 프라이밍 <ol><li> 로터리 펌프를 시작하고 관류와 튜브를 입력합니다. 그것에서 모든 공기 방울을 비우주의하십시오. </li><li> 관류 대략 중간 수준에 windkessel 장치를 채 웁니다. </li><li> 모든 튜브가 작성 흐름이 시간 동안 신장 수준에서 관류 바늘을 유지하다 0 0 mmHg로에 압력 변환기 보정. </li><li> 일정한 최소 수준 (0.6 ml / 분)에서 흐름을 유지하고 5 단계로 진행합니다. </li></ol> 5. 수술 2 부 <ol><li> 합자 IV와 왼쪽 레나의 분기 사이에 클램프를 놓습니다리터 동맥. </li><li> 지느러미 벽을 절단하지 않도록주의하면서 합자 IV의 대동맥 꼬리에 작은 절개를합니다. </li><li> 용기와 확장기 대동맥의 팽창 개구. </li><li> 바늘로 대동맥을 Cannulate 단지 클램프로 끝을 밀어 (2cm 길이, PE 50 당겨). </li><li> 클램프를 엽니 다. </li><li> 이 오른쪽 신장 동맥 및 대동맥의 교차점에 도달 할 때까지 바늘 두개쪽으로의 끝을 밀어 넣습니다. </li><li> 닫기 합자 VII. </li><li> 닫기 합자 IV. </li><li> 다이어프램을 해부하여 가위로 가슴을 엽니 다. 하나의 컷으로, 대동맥, 대정맥, 마음과 식물 신경을 분리합니다. 이 단계로, 동물 연속 깊은 마취 빠른 방혈로 희생된다. </li><li> 관류 펌프의 압력 제어를 시작합니다. 80 ~ 100 mmHg의 사이의 평균 압력을 유지한다. </li><li> 닫기 합자 V. </li><li> 닫기 합자 VI. </li><li> 닫기 합자 VIII. </li><li> 결합 tissu 무료 오른쪽 신장전자와 가위로 지방 캡슐로의 삽입. </li><li> V.를 합자하는 근위 대동맥을 잘라 </li><li> VI를 합자하기 위해 원심 장간막 동맥을 잘라. </li><li> 혈관 자체로 절단하지 않도록주의하면서 신장지지 용기 번들을 잘라. </li><li> 신장에 대한 연결에 간을 잘라. 신장을 확보하는데주의를 기울여야하지만, 대정맥이 그것에 의해 개방 유지되도록, 그것에 간 접착의 작은 부분을 둡니다. </li><li> 마우스에서 신장 번들을 가져 가라. 습한 챔버에서 마우스를 제거합니다. </li><li> 간 및 신장 (합자 IX)의 연결 주위에 &quot;올가미&quot;합자를 놓습니다. </li><li> 정맥 라인 (2cm의 PE 50)와 대정 맥을 Cannulate. </li><li> 닫기 합자 IX. 정맥 라인을 통해 정맥 유출을 즉시 시작해야합니다. </li><li> 촉촉한 챔버를 닫습니다. </li></ol> 6. 다운 스트림 분석 <ol><li> 다음 시간 동안 연속적으로 혈류 intravascul 모니터링아칸소 압력 15. 예를 들면 분석을 위해 사용할 수있는 정맥 유출, 신장 레닌 방출 7 수집. 전해질 농도와 사구체 여과율 (14)의 분석을 위해 소변을 수집합니다. 관류 1 시간 후, 신장 서부 블로 팅에 대한 스냅인을 동결 할 수 있습니다 또는 이미지에 대한 고정 (16)에 접근한다. </li></ol>

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C., Endre, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+facilitates+autoregulation+in+the+perfused+mouse+kidney:+An+optimized+in+vitro+model+for+assessment+of+renal+vascular+and+tubular+function.">Angiotensin II facilitates autoregulation in the perfused mouse kidney: An optimized in vitro model for assessment of renal vascular and tubular function.</a> Nephrology. 9 (5), 288-296 (2004).</li></ol>

The authors have no competing financial interests and nothing else to disclose.

관류 신장 절연 마우스 수많은 전신 요인의 영향에 의해 결함이 될 수있다 손상 동물 생체 실험 간의 격차를 1 시간 동안 제어 된 환경 생체 신장 기능을 연구하기위한 도구이고, 시험 관내 실험에서 격리 된 네프론 세그먼트 또는 반드시 기능에 손상 장기 구조의 영향을 무시 배양 된 세포. , 저자의 지식과 대안 기술이 특정 작업을이 수행하지 않는 것입니다. 신장 조직의 생?...

기관의 고립 된 관류는 수십 년 1 생리 학자들 사이에서 지속적인 노력의 대상이되고있다. 기술은 혈압, 호르몬 또는 신경 조직 등의 영향없이, 기관의 기능을 가능하게 검토한다. 칼 에두아르 Loebell는 1849 2, 고립 된 신장의 성공적인 재관류을 설명했습니다 첫 번째로 간주됩니다. 그 이후로, 관류 장치에 상당한 정련을 실시하고있다. 프레이와 그루버는 지속적인 관류 2 산소와 타악기 펌프 용 인공 폐를 소개했다. 초기 연구는 주로 큰 포유 동물 - 즉, 돼지 2 개 3 쥐 신장의 사용 - 첫번째 보고서, 와이즈 등으로의 신장을 공부하는 동안. , 작은 포유 동물 기관 관류 (4)의 연구에서 획기적인 사건이었다. Schurek 등. 충분한 신 세뇨관 경우 관류에 포유류의 적혈구를 추가의 필요성을보고산소는 5를 달성 할 수 있었다. 장기간 실험 긴급 동일한 연구 그룹 (6)에 의해 버퍼의 연속 투석의 도입이었다. 2003 년 SCHWEDA 등. 나중에 Rahgozar 등에 의해 정제 기능 마우스 고립 관류 신장 (MIPK) 7을보고 처음이었다. 18 Lindell 등의 알. 14. 절연 신장 관류 래트보다 ​​기술적으로 어려운 반면, MIPK의 사용은 유전자 변형 마우스의 다양한 배열의 사용을 가능하게하는 장점을 맺는다. 이 논문은 1 시간 동안 고립 된 마우스 신장 관류에 대한 저자의 방법의 세부 사항을 제공합니다. 상기 방법은 신장 유량 혈관 저항 호르몬 방출 혈액 가스 분석, 소변 분석, 약물의 애플리케이션의 지속적인 평가를 허용한다. 절차에 따라, 신장은, 분자 및 생화학 분석을 위해 처리 할 수있는 현미경에 대한 고정, 또는받는 사람 마우스에 이식 (그림 1). <img alt="그림 1" src="/files/ftp_upload/54712/54712fig1.jpg" /> 그림 1 : 절연 관류 신장에 가능한 입 / 출력의 개요. BGA : 혈액 가스 분석. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 많은 혁신적인 응용 프로그램 (또는 항 거부 또는 게놈 편집 약물의 응용 프로그램없이) 8, 9, 이전 이식에 장기간 정상 체온 신장 관류의 새벽 논의되는 바와 같이이 기술은 가능성, 10, 향후 몇 년에 걸쳐 관심을 증가 받게됩니다 11, decellularized 발판 (12)의 전체 신장의 생명 공학 및 광자 이미징 13 형광 염료의 고용량의 적용 </sup&gt;. 또한 급성 신부전 (14) 중 특정 유전자의 역할을 연구와 이상적인 모델이다. 단계별 프로토콜은 다른 실험실에서 성공적으로 격리 된 마우스 신장 재관류를 수행 할 수 있도록 설명한다. 첫째, 조성물 및 완충액의 제조 지정된다. 그리고, 수술 상세히 설명되며 중요한 단계가 도시되어있다. 셋째, 데이터가 성공적으로 제조 나타내는 것을 제시되어 신장 혈류, 혈관 저항, 사구체 여과율 및 분수 전해질 배설 모두 관류 신장 다른 네프론 세그먼트 형태의 생존 및 투과형 전자 현미경의 기능적 측정 관류의 1 시간 후 고정.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Perfusion Circuit:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Moist chamber 834/8</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2901</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Cannular with basket and side port</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2947</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Thermostat TC120-ST5&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-4544</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">ISM 827/230V Roller Pump Reglo Analogue</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-0114</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 1L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3438</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 0.5L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3436</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Pressure Transducer APT300&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3862</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">TAM-D Plugsys Transducer</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-1793</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">SCP Plugsys servo controller</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2806</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Windkessel&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3717</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">HSE-USB data acquisition</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Low-Flux Dialysator Diacap Polysulfone</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">7203525</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:447px;">PE-Tubing for aorta cannulation 1.19mm I.D. x 1.70mm O.D.</td>
			<td style="width:173px;">Scientific Commodities Inc.</td>
			<td style="width:119px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:447px;">Name</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Buffer reagents:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Aminoplasmal 10%</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">134518064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium pyruvate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2256-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-Glutamic acid monosodium salt hydrate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1626-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-(-)-Malic acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1125-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium-L-Lactate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">L7022-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">alpha-Ketoglutaric acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">K1875-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaHCO3</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">60130-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">U5378-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatinine</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4255-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td style="width:173px;">Roche</td>
			<td align="right" style="width:119px;">10835242001</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2 * 6H2O</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2393-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">D-Glucose</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2 * 6H2O&#160;</td>
			<td style="width:173px;">Riedel-de-Ha&#235;n</td>
			<td align="right" style="width:119px;">12074</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaH2PO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9638-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Na2HPO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0876-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Antidiuretic Hormone dDAVP</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">V2013-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">FITC-Inulin</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Filter used for erythrocyte filtration</td>
			<td>Macherey-Nagel</td>
			<td style="width:119px;">MN 615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BGA Analysis:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">ABL 80 flex</td>
			<td style="width:173px;">Radiometer Medical ApS</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Electron Microscope:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Philips CM100 TEM</td>
			<td style="width:173px;">FEI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the mouse isolated perfused kidney technique

마우스 절연 신장 관류 (MIPK)를 관류하고 1 시간 동안 기능성 생체 조건 마우스 신장을 유지하기위한 기술이다. 이 분리 된 기관 및 관류 신장 생명 공학에 대한 탈세 포화 또는 항 거부 또는 소수 신장에 고용량의 게놈 편집 약물의 투여를 포함한 미래에 가능할 수있다 많은 혁신적인 애플리케이션에 대한 생리 연구를위한 전제 조건입니다 이식. 재관류 시간 동안 신장은 신장 기능이 평가 될 수 있고, 조작, 및 다양한 제약으로 투여 될 수있다. 절차 후 신장 이식 될 수있다 또는 분자 생물학, 생화학 분석, 또는 현미경에 대한 처리. 본 논문은 관류 및 마우스 신장의 생체 외 관류에 필요한 수술 기법에 대해 설명합니다. 관류 장치의 상세에 주어진 데이터는 V를 도시 제시신장의 제조 iability : 형태소 판독 및 분자 판독 상이한 네프론 세그먼트 수송 단백질의 웨스턴 블랏 상이한 네프론 세그먼트 기능, 투과 전자 현미경과 같은 신장 혈류, 혈관 저항 및 소변 데이터.

방법 설명과 함께, 고립 된 마우스 신장은 적어도 1 시간 동안 가능한 남아있을 수 있습니다. 우리는 기능 (신장 혈류 및 혈관 저항 정맥 유출 사구체 여과율 비뇨기 분수 나 + 및 K + 배설 혈액 가스 분석, 소변 삼투압) 및 형태학 (투과형 전자 연속 재관류 1 시간 후 조직 생존력을 시험 야생형 C57BL / 6 마우스의 네 신장 현미경, TEM) 방법. 또한 다른 세관 세그?...

Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The Mouse Isolated Perfused Kidney Technique

마우스 절연 신장 관류 (MIPK)를 관류하고 1 시간 동안 기능성 생체 조건 마우스 신장을 유지하기위한 기술이다. 버퍼 수술 기법은 상세하게 설명된다.

마우스입니다 관류 신장 기법

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

the-mouse-isolated-perfused-kidney-technique

Research

JoVE Journal

Biology

21.5K 조회수.  University of Zürich.  마우스 절연 신장 관류 (MIPK)를 관류하고 1 시간 동안 기능성 생체 조건 마우스 신장을 유지하기위한 기술이다. 버퍼 수술 기법은 상세하게 설명된다. 

비디오: The Mouse Isolated Perfused Kidney Technique

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Perfused 마이크로 bioreactors의 칩 기반의 입체 세포 배양

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

연구

생물학

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Langendorff - perfused 랫 하트의 광학 매핑

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

배아 마우스 신장의 Nephrogenic 지대의 세포의 분리 및 문화

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

절연, 환기 및기구 마우스 폐의 특성화는 타악기 흐름 Perfused

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

직접 성인 Zebrafish의 신장에 세포의 이식

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

성인 Zebrafish의 신장의 해부

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

미토콘드리아 절연 마우스 중심부에 투과율 전환 기공 영업의 다중 매개 변수 측정

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

고립 된 혈액 관류 쥐 폐 준비에 단일 미세 혈관 투과성의 양을

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...