The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

참고 :이 프로토콜에 설명 된 모든 동물 관련 절차 이전에 듀크 대학 기관 동물 관리 및 사용위원회 (DUIACUC)에 의해 승인되었습니다. 1. 암 세포 배양 및 사출 <ol><li> 형광 표지 된 전이성 암 세포를 배양 (예를 들어 인간의 MDAMB231-GFP 유방암 세포, 박사 패트리샤 STEEG, NCI 및 YFP 표지 마우스 육종 세포, 박사 데이빗 커쉬, 듀크 대학 의료 센터, 방사선 종양학 부서의 선물에서 선물) 적절한 배양 배지 (예 : 둘 베코 변형 이글 중간 10 % 소 태아 혈청과 1 % 페니실린 / 스트렙 트 아비딘 (DMEM)) 37 ° C에서 약 90 % 컨 플루 언트 될 때까지. </li><li> , 세포를 Trypsinize PBS로 2 회 반복한다, 계산 한 다음, 27 G 바늘과 주사기를 사용, 동물 당 5 백만 세포에서 이소 플루 란 - 마취 10 주 오래 된 여성 누드 쥐의 꼬리 정맥로를 주입. 수술 수준 anesthesIA는 발가락 핀치에 반응의 부족에 의해 확인됩니다. </li></ol> 전이 사용 MicroCT 2. 모니터링 <ol><li> 전이 직경 직경으로 약 2mm의 존재를 검출하기 위해, 마이크로 - CT / 마이크로 조사기를 사용하여 래트를 일주일에 한 번 검사. 이전 12 설명한 바와 같이 마이크로 CT를 의뢰한다. <ol><li> 이미징 이전에 3 % 이소 플루 란 마취에 따라 쥐. 발가락 핀치에 의해 깊은 마취를 확인합니다. </li><li> 마취 개시 후 신속 촬상 챔버 촬상 크래들 래트를 전송 및 2.5-3 %의 이소 플루 란 - 공기 혼합물에 노즈 콘 통해 연결. 영상 크래들에 외부 위치 제어 및 레이저 구분 기호를 사용하여, 그 가슴이 MicroCT 스캐너의 광자 빔에 위치하는 방식으로 크래들에 쥐의 위치를​​ 조정합니다. 감마선에서 조사를 보호하기 위해, 영상 실의 문이 닫혀 있는지 확인합니다. </li><li> 다시 사용하여 동물의 위치를​​ 제어컬러 비디오 카메라. 저해상도 CT 이미징 테스트 실행을 수행하고 흉강의 XYZ 치수의 시야를 조정하기 위해 생성 된 화상을 사용한다. </li><li> 이미지 쥐의 40 KVP에서 2mm 알루미늄 필터를 사용하여 흉부, 2.5 mA, 7 FPS에서 0.008 복셀과는 케이지에 동물을 반환합니다. 한 동물의 이미지를 잘 작성하지 최근 15 또는 20 분 소요됩니다. 완전히 회복 될 때까지 다른 동물의 회사에 수술을 한 동물을 반환하지 않습니다. 이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 방치하지 마십시오. </li><li> 흉부 내 혈관에 의해 설명 될 수없는 무선 비교적 불투명 오브젝트의 외관을 확인하여 전이 (도 1a) </li></ol></li></ol> 3. 윈도우 상공 회의소 수술 <ol><li> 마취, 활력 징후 및 꼬리 정맥 카테터 <ol><li> 전이성 질환의 존재와 동물을 선택합니다. 복강 D와 동물을 주입50 ㎎ / ㎏ 펜 토바 비탈의 OSE. 진행하기 전에 발가락 핀치에 의한 수술 수준의 마취를 확인합니다. 주 : 마취 프로토콜은 각각의 실험 장치와 일치해야합니다. 용이 한 재 투여 선택권을 제공하는 동안은 펜 토바 비탈, 긴 절차 깊은 마취를 유도하기 위하여, 긴 작용 마취제 여기 선택되었다. 그러나, 과다 복용에 동물의 손실은 펜 토바 비탈 마취 일반적인 문제입니다. 펜 토바 비탈보다 더 큰 정도의 자율 반사 신경을 보존 또 다른 옵션은 그러나 단 하나의 재 투여주기 허용 자일 라진 (xylazine) 또는 medetomidine, 같은 진정제와 함께 케타민입니다. </li><li> 클리퍼를 사용하여, 전이성 질환이 있고, 목 영역에서 본체의 측면에 동물을 면도. 피부의 모든 남아있는 느슨한 머리를 닦아냅니다. 탈모 제거한 후 마르지 않도록, 눈 연고 수의학 적용. 참고 : 무 흉선 누드 쥐 requi 잔류 머리를 가질 수있다수술 절차를 진행하기 전에 입술 제거. 이 수술 절차 및 이미징을 방해 할 수 있으므로 그것은 철저하게 모든 머리를 제거하는 것이 매우 중요합니다. </li><li> 37 ° C의 물 순환 가열 패드에 배치되는 금속 접시에 앙와위에서 동물을 고정합니다. 앞과 뒤의 사지 테이프와 함께 접시에 고정되어 있습니다. 주 : 제어 및 수술 및 실험 절차 전반에 걸쳐, 맥박 산소 측정기를 사용하여 심장 요금과 동맥혈 산소와 같은 생체 신호를 기록하는 데 유용합니다. </li></ol></li><li> 기관 삽관 <ol><li> 기관에 길이 복부 근육의 중앙값 분리 하였다 횡 자궁 피부 절개를, 먼저, 동물의 환기를 위해 카테터를 배치하기 위해서는. </li><li> 기관의 등쪽을 통해 봉합하는 통로를 만들 날카로운 집게 반복 개방에 폐쇄 조치를 사용합니다. </li><li> t의 기관에 작은 절개를합니다그 복부 측면 약 두 번째 및 세 번째 기관 링 사이, 반원형보다는. 기관 카테터의 고정을 사용하려면, 등의 표면에 노출 된 기관의 충분히 긴 부분을 남겨주세요. </li><li> 기관에 2.5-3.0 mm &quot;Y&quot;기관 캐뉼라를 삽입하고 4-0 모노 필라멘트 봉합사 조입니다. 양성 폐 압력을 유지하기 위해 캐 뉼러 물의 6cm로 채워진다 만료 덕트에 접속 병 압력 순환 호흡기에 연결되어 있는지 확인. 실험적으로 달리 언급되지 않으면, 유입 가스는, 산소 100 %이어야한다. </li><li> 25 ~ 27 G 바늘 카테터를 삽입하고 쥐의 꼬리 정맥 중 하나에 헤파린 식염수로 가득하고, 테이프를 제자리에 고정합니다. 주 : 반복 꼬리 정맥 내로 헤파린 염수 소량 주입 과정 전반에 걸쳐 꼬리 정맥 카테터의 개방성을 보장. 또한, 오로 - 기관 내 삽관은 기관의지도를 즉마취 된 동물의 입을 통해 및 기관에 후두 과거 튜브는 여기에 설명되어있는 기관 절개 절차에 대한 가능한 대안이다. 그러나,이 방법은 기관의 손상을 방지하고, 또한 식도 우발적 삽관을 배제하기 위해 특별한 훈련과 경험을 요구한다. </li></ol></li><li> 폐 윈도우의 응용 프로그램 <ol><li> 무딘 절개를 이용하여 피부를 분리 한 후 절개를 생성 의해, 전이성 질환이있는 가슴 측으로부터 피부를 제거한다. 참고 : 피부 절제와 초연 이후 제거 할 수 있습니다 </li><li> 오버레이 근육 (pectoralis, 앞 톱니와 넓은 등의 dorsi)의 2 층 해부,하지만 그대로 늑간 근육을 두어 진행합니다. 일반적으로 두 개의 인접한 갈비뼈의 일부를 제거하여, 직경 약 1.5 cm의 흉강에 구멍을 만듭니다. 이상적으로, 6, 7 R의 영역에 천공을 찾습니다IBS. </li><li> 절골술 : <ol><li> 폐 표면에 출혈과 손상을 최소화하기 위해 긴밀하게 절삭 가공시에 톱니 수술 집게로 절단 할 수있는 리브를 개최합니다. 수술 용 가위를 이용하여 외부를 가리키는 나머지 갈비뼈의 뾰족한 측 떠나 약 45 °의 각도로, 제 리브의 내측부를 잘랐다. </li><li> 이어서, 다시 폐 표면 손상을 방지하기 위해, 바깥쪽으로 가리키는 갈비뼈의 뾰족한 측 남게 유사한 방식 갈비뼈의 측면을 자른다. </li><li> 다음 늑간 근육을 잘라 적출 조각을 제거, 인접 리브에 대해이 절차를 반복합니다. 이 과정 중, 폐 표면 및 흉곽 사이의 기계적 상호 작용을 최소화하는 방법으로 폐의 압력을 유지한다. 적절하게 인공 호흡기에 영감 압력을 조절하여이 작업을 수행합니다. </li></ol></li><li> 창 삽입 : <ol><li> 인 커버 글라스로 구성, 사용자 정의 만든 폐 창을 삽입플렉시 소켓 (그림 1B)에 연결합니다. 아교 접착 또는 진공 그리스의 매우 작은 양을 적용하여 소켓 첨부 창. 전이 표면이 확대 윈도우의 중심에 위치하도록하는 방식으로 윈도우를 삽입한다. 필요한 경우, 각각의면에 구멍을 약간 크게함으로써 윈도우의 중심에 미세 전이를 가지고 삽입 구멍을 조정한다. 참고 : 흉막 표면에 전이성 질환은 균열을 따라 주로 나타나는 폐 표면의 다른 핑크 - 투 - 연어 색 건강한 부분 내에서 명확하게 인식 할 수있는 흰색 점 또는 지역으로 식별 할 수 있습니다. 미세 전이가 흉막 외부의 다른 영역에서 발생할 수 있지만, 조사 세포주 거의 항상 전이성 질환, 방사선 학적 검출 할 수있게되면, 천공 영역에 미세 전이를 표면화 표시. </li><li> 천공 소켓 내로 삽입하고, (L)의 내장 흉막과 직접 접촉을 생성 한 후웅은 4.0 모노 필라멘트 봉합사 (도 1C)를 사용하여, 주변 늑간 근육 창틀의 가장자리를 봉합. 탈출과 도장을 만들 잔여 공기를 돕기 위해 인공 호흡기에 영감 압력의 약간의 증가를 사용합니다. 주 : 쥐의 호흡률 그것은 70 ~ 90 BPM 사이의 호흡 속도를 조정하기를 권장 등 마취, 흥분하거나 불안 상태, 영감 공기의 산소 농도 (FIO 2)의 상태에 따라 크게 달라질 수있다. 영감 압력은주의 조정해야하지가 약보다 이상으로 설정 될 수있다. 8cm는 H 2 O (0.6 mmHg로), 폐 표면의 손상을 방지한다. </li></ol></li><li> 형광 현미경, 자동 온도 조절 (전기) 난방 담요에 위치하는 강판에 Z 방향의 운동 (그림 1D)를 제거하기 위해 설계된 맞춤 설계된 제한 수단에 동물을 배치합니다. 동물의 몸을 제어직장 서미스터를 사용하여 온도. 동물의 제한 수단의 나사를 조정하고 인공 호흡기에 영감 압력은 측면 운동의 최적 제어를 달성하기 위해. 참고 : 양자, dorso - 복부 및 cranio-꼬리 : 포유 동물에서 자연 호흡은 폐의 움직임과 가슴 확장의 세 가지 방향을 포함한다. 최대한 자연스러운 호흡 운동을 유지하기 위해, 필요한 정도까지 Z 방향 압축을 최소화하는 것이 중요하다. Z 차원 구속 혈류 및 다른 매개 변수에 영향을 미칠 수있는 아티팩트를 도입 할 가능성을 가지고 있기 때문에, 동일한 동물에 반복 된 일련의 측정 동안 일정 상태를 유지하는 것이 바람직하다. </li></ol></li></ol> 4. 이미징 및 측정 미세 혈류의 <ol><li> 심장 구멍을 통해 적혈구를 수집하고 전술 한 바와 같이, DII (1,1 = -dioctadecyl-3,3,3- = 3 = 테트라 메틸 indocarbocyanine 과염소산 염)로 레이블 &lt;SUP&gt; 10. </li><li> 윈도우 챔버 수술 완료되기 전에 유리 창에 제 1 패스 접착 효과를 피하기 위해, 쥐의 꼬리 정맥에 표시된 적혈구의 300 μl를 주입한다. 정맥 내로 공기의 도입이 더 주입을 억제하므로, 주사기 또는 카테터 내의 공기 방울을 제거한다. </li><li> 이미지 혈액의 온도를 냉각 -40 ° C 칩에 현미경 CCD 카메라와 흐름, 약 100 배 전체 해상도 (10 배 현미경 목적으로 즉, 10 배 전 카메라의 눈). 표준 로다 민 / TRITC 필터 세트 (여기 450-490 nm의 방출&gt; 515 nm의)를 사용합니다. 결과 이미지 시퀀스의 실제 프레임 속도 및 픽셀 해상도를 기록한다. 스택 당 기록 최소 200 (이상적으로 약 300) 이미지는 유속의 성공적인 분석을 보장합니다. </li><li> 식염수 IP의 약 1 ml의 모든 시간을 주입하여 동물의 유체 손실을 보충. 참고 : 개입을 포함 실험 설정, 예를 들어,암전 폐 혈류 속도를 수정 약물은 폐 전이성 암 혈류 속도의 반복 측정이 필요하다. 이러한 확장 실험을 위해, 충분한 유체와 동물을 보충하는 것이 중요하다. </li><li> 꼬리 정맥에 3N의 KCl 3 ml를 주입하여 동물을 안락사 </li><li> 모든 혈액 흐름에 대한 유속 그레이 스케일과 컬러 인코딩지도 (10, 11)를 추적 만듭니다 게시, 공개적으로 사용할 수 matlab에 기반 컴퓨터 알고리즘을 사용하여 이미지 스택을 평가합니다. 그 후, 혈액 세포의 어떤 움직임을 나타 내기 활성 혈관을 즉 없습니다 값을 임계 화 한 후, 같은 이미지 J 같은 상용 또는 공개 된 이미지 분석 소프트웨어를 사용하여 생성 된 그레이 스케일 이미지를 평가합니다. </li></ol>

<ol>
	<li>Billingsley, K. G., 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=Pulmonary+metastases+from+soft+tissue+sarcoma:+analysis+of+patterns+of+diseases+and+postmetastasis+survival.">Pulmonary metastases from soft tissue sarcoma: analysis of patterns of diseases and postmetastasis survival.</a> Ann Surg. 229, 602-610 (1999).</li><li>Rashid, O. M., Takabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+the+role+of+surgery+in+the+management+of+breast+cancer+lung+metastasis.">The evolution of the role of surgery in the management of breast cancer lung metastasis.</a> J Thorac Dis. 4, 420-424 (2012).</li><li>Mayer, A., Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia,+lactate+accumulation,+and+acidosis:+siblings+or+accomplices+driving+tumor+progression+and+resistance+to+therapy.">Hypoxia, lactate accumulation, and acidosis: siblings or accomplices driving tumor progression and resistance to therapy.</a> Advances in experimental medicine and biology. 789, 203-209 (2013).</li><li>Okunieff, P., O'Dell, W., Zhang, M., Zhang, L., Maguire, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+oxygen+measurements+and+personalized+medicine.">Tumor oxygen measurements and personalized medicine.</a> Advances in experimental medicine and biology. 765, 195-201 (2013).</li><li>Palmer, G. 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=In+vivo+optical+molecular+imaging+and+analysis+in+mice+using+dorsal+window+chamber+models+applied+to+hypoxia,+vasculature+and+fluorescent+reporters.">In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters.</a> Nature protocols. 6, 1355-1366 (2011).</li><li>Palmer, G. 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=Optical+imaging+of+tumor+hypoxia+dynamics.">Optical imaging of tumor hypoxia dynamics.</a> Journal of biomedical optics. 15, (2010).</li><li>Funakoshi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+lung+metastasis+for+intravital+studies.">A new model of lung metastasis for intravital studies.</a> Microvasc Res. 59, 361-367 (2000).</li><li>Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+assessment+of+pulmonary+vascular+responses.">Real-time imaging assessment of pulmonary vascular responses.</a> Proc Am Thorac Soc. 8, 458-465 (2011).</li><li>Tabuchi, A., 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=Precapillary+oxygenation+contributes+relevantly+to+gas+exchange+in+the+intact+lung.">Precapillary oxygenation contributes relevantly to gas exchange in the intact lung.</a> Am J Respir Crit Care Med. 188, 474-481 (2013).</li><li>Hanna, G., 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=Automated+measurement+of+blood+flow+velocity+and+direction+and+hemoglobin+oxygen+saturation+in+the+rat+lung+using+intravital+microscopy.+American+journal+of+physiology.">Automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using intravital microscopy. American journal of physiology.</a> Lung cellular and molecular physiology. 304, 86-91 (2013).</li><li>Fontanella, A. N., 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=Quantitative+mapping+of+hemodynamics+in+the+lung,+brain,+and+dorsal+window+chamber-grown+tumors+using+a+novel,+automated+algorithm.">Quantitative mapping of hemodynamics in the lung, brain, and dorsal window chamber-grown tumors using a novel, automated algorithm.</a> Microcirculation. , (2013).</li><li>Newton, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissioning+a+small-field+biological+irradiator+using+point,+2D,+and+3D+dosimetry+techniques.">Commissioning a small-field biological irradiator using point, 2D, and 3D dosimetry techniques.</a> Medical physics. 38, 6754-6762 (2011).</li><li>Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironmental+physiology+and+its+implications+for+radiation+oncology.">Tumor microenvironmental physiology and its implications for radiation oncology.</a> Seminars in radiation oncology. 14, 198-206 (2004).</li><li>Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> J Appl Physiol. 104, 338-346 (2008).</li><li>Fingar, V. H., Taber, S. W., Wieman, T. J. <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+model+for+the+study+of+pulmonary+microcirculation:+determination+of+pulmonary+edema+in+rats.">A new model for the study of pulmonary microcirculation: determination of pulmonary edema in rats.</a> J Surg Res. 57, 385-393 (1994).</li><li>Manzoor, A. A., Schroeder, T., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-stop-shop+tumor+imaging:+buy+hypoxia,+get+lactate+free.">One-stop-shop tumor imaging: buy hypoxia, get lactate free.</a> The Journal of clinical investigation. 118, 1616-1619 (2008).</li><li>Evans, S. 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=Imaging+and+analytical+methods+as+applied+to+the+evaluation+of+vasculature+and+hypoxia+in+human+brain+tumors.">Imaging and analytical methods as applied to the evaluation of vasculature and hypoxia in human brain tumors.</a> Radiation research. 170, 677-690 (2008).</li><li>Radiloff, D. R., 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=The+combination+of+theophylline+and+endothelin+receptor+antagonism+improves+exercise+performance+of+rats+under+simulated+high+altitude.">The combination of theophylline and endothelin receptor antagonism improves exercise performance of rats under simulated high altitude.</a> Journal of applied physiology. 113, 1243-1252 (2012).</li></ol>

저자가 공개하는 게 없다.

생체 내에 모델 현미경 전산 혈액 흐름 분석을 사용하여 미세 순환 혈류 및 쥐의 폐 전이에서 다른 동적 공정에서 화상 변경 가능하다 제시된다. 다른 방법은 설치류의 개방 ribcages에 노출 폐에 현미경을 수행하기 위해 존재하지만,이 모델은 폐쇄 가슴 속에서 가슴 벽의 구멍을 통해 이미지 폐 전이에 처음이다. 이 방법을 사용하여, 가능성은 폐 전이의 미세 순환 혈류에 의한 약리학 적 변화를 측?...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

고형 종양의 혈관은 굴곡의 큰도, 높은 intervascular 거리 (13)을 보여주는 일반 혈액 공급이 크게 다르지 알려져있다. 정상적인 폐 미세 (도 2A, 상부 패널)에 비해 따라서, 실험적인 폐 및 유방암 전이 육종 혈류 트랙 부정형 intervascular 큰 갭 (도 2A, 저급 두 개의 패널)을 갖는다. 이전의 연구에서는 폐 윈도우 방식의 능력은 정상 폐에서 열 혈류 속도의 변화를 ...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

쥐의 폐 전이의 미세 혈류 속도의 자동 측정

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Research

JoVE Journal

Medicine

8.4K 조회수.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

비디오: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

마우스 폐 내피 표면 층의 생체 측정

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

연구

의학

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

담배 연기에 노출 된 마우스의 폐 폐기종 및 작은기도 리모델링 자동 측정

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

증가 폐 동맥 박동 흐름 새끼 돼지의 저산소 폐 고혈압을 개선

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

조산 신생아 경피 미세 영상

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

쥐의 복부 대동맥의 수축을 통해 심장 리모델링의 모델

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

동시 심전도 기록 및 침략 적 혈액 압력 측정 쥐에

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

쥐의 심근 허혈 재관류 후 관상 동맥 흐름 예비의 평가

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

인간의 혈류를 모방하는 흐름 루프 모델에서 혈액 접촉 임플란트의 혈적합성 테스트

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.