Name: visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle
Uploaded: 2016-10-19T13:00:00.000+00:00
Duration: 6 min 10 s
Description: Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.

이 연구는 싱가포르 기관 동물 관리 및 사용위원회 (승인 된 프로토콜 없음. R15-0225)의 국립 대학에 따른다. 동물 모델 1. 수술 준비 <ol><li> 선박 삽관 <ol><li> 남성 흰쥐 마취 - 무게 (6 7 주)를 (203 ± 20) 복강을 통해 케타민 (37.5 ㎎ / ㎖) 및 자일 라진 (5 ㎎ / ㎖) 칵테일 g (IP) 주입 (2 ㎖ / kg) . 바늘을 정리해 또는 주입 후 주사기에서 제거하지 마십시오. </li><li> 동물이 마취되면 (핀칭 발가락으로 확인) 37 ℃에서의 체온을 유지하기 위해 가열 패드에 배치. 조심스럽게 견갑골, 전방 경부, 복부, 중간 뒷다리 및 음낭 주머니에 머리를 면도. 조심스럽게 접착 종이 테이프를 사용하여 다리를 억제. </li><li> 미세 절제 가위와 직각 집게를 보는 동안을 사용하여 모든 수술 절차를 수행실체 현미경. 수술하는 동안 부상을 방지하기 위해 구멍 방지 트레이에있는 모든 날카로운 수술 도구를 놓습니다. </li><li> 절개를 수행하기 전에 수술 부위를 교대 요오드, 70 % 에탄올로 3 회 문질러. 30 IU / ml의 헤파린 생리 식염수 용액으로 모든 카테터를 플래시합니다. </li><li> 오른쪽 경정맥을 통해 수술 가위를 사용하여 견갑골에 1.5 cm 중간 선에 피부 절개 - 1을 만듭니다. 경정맥을 노출 무딘 절개하여 밴드를 분리하고,이를 폴리에틸렌 관 (PE-50) 5-0 실크 봉합사를 이용하여 헤파린 염수로 가득 cannulate. 추가 마취를 주입 할 때 필요한 (1/3 초기 용량의 1/2, 정맥 주사 (IV)) 수술과 실험의 과정을 통해. </li><li> 기도 명백을 유지하기 위해 기관 절개술을 수행합니다. 전방 경추 지역에 1.5 cm의 절개 - 1을 만듭니다. 대신에 카테터를 고정 2-0 실크 봉합사로 폴리에틸렌 관 (PE-205)를 이용하여 기관을 Cannulate. </li><li> 혈압 모니터대퇴 동맥에 삽관을 통해. 뒷다리의 왼쪽 내측 표면에서 1.5 cm의 절개 - 1을 만듭니다. 무딘 절개에 의한 대퇴 동맥을 분리합니다. 헤파린 염수 5-0 실크 봉합사를 이용하여 충전 된 폴리에틸렌 관 (PE-10)를 대퇴 동맥 Cannulate. </li></ol></li><li> Cremaster 근육 준비 및 흐름 시각화 <ol><li> 를 확장하기 위해 음낭 주머니의 정점을 5-0 실크 봉합사를 삽입합니다. 음낭 주머니의 복부 표면을 따라 절개를합니다. 노출 된 근육, 정기적으로 따뜻한 등장 성 용액 (pH 7.4의 37 ° C)를 적용합니다. </li><li> 신중하고 철저하게 결합 조직을 둘러싸고있는 면봉을 사용하여 제거합니다. </li><li> cremaster 근육의 꼭대기를 통해 5-0 실크 봉합사를 삽입합니다. 같은 길이의 두 조각으로 봉합사를 잘라 양쪽에 매듭을 묶어. 두 매듭 사이의 근육을 잘라 조심스럽게 봉합을 잡아 당겨 사용자 정의 투명한 플렉시 글라스 플랫폼에 그것을 스트레칭. 고치다블루 압정와 플랫폼 상에 봉합사의 끝. 참고 : 결합 조직을 주변의 철저한 제거는 최적의 이미지 대비를 얻는데 매우 중요하다. </li><li> 5 ~ 6 고착이 이루어 될 때까지 단계를 반복 1.2.3. 조심스럽게 고온 소작을 사용하여 부고환에서 cremaster 근육을 제거합니다. 조직의 탈수를 방지하기 위해 노출 된 근육에 따뜻한 등장 솔루션을 Superfuse. <ol><li> 거즈의 접힌 부분과 cremaster 근육을 둘러싸고 있습니다. 비닐 막을 노광 근육 커버. 영화와 함께 거즈 조각 물 침수 현미경 목표 (그림 1A) 따뜻한 등장 솔루션을 보유 얕은 분지를 형성한다. </li></ol></li><li> 생체 내에 현미경 (그림 1C)의 동물 무대에 동물을 전송합니다. 지속적인 압력 모니터링 (그림 1E)에 대한 생리 학적 데이터 수집 시스템에 동맥 삽관을 연결합니다. </li><li> 근육 temperat 유지동물 플랫폼 (그림 1B) 아래에 부착 된 발열체와 35 ° C에서 URE. 발열체 (도 1D)의 전력 제어기에 네거티브 피드백을 제공하도록 근육 옆에 온도 센서를 배치했다. </li><li> 15 분은 환경과 평형을 무대에 동물을 둡니다. </li><li> 40 배의 물 액침 대물 긴 작동 응축기와 생체 내에 현미경 혈류 시각화. </li><li> 분지 세동맥 적혈구 코어 사이에 명확한 이미지 초점 및 콘트라스트에 기초하여 (&lt;60 μm의)을 선택 CFL 및 용기 벽이 순서 혈관의 직경 평면 현미경을 집중한다. 수직으로 혈관 벽을 정렬하기 위해 현미경에 장착 카메라를 돌립니다. </li><li> 1 초 동안 3000 / 초의 프레임 레이트로 고속 비디오 카메라를 사용하여 혈액의 흐름을 기록한다. 이미지 품질을 보존하기 위해 비 압축 8 비트 그레이 스케일 AVI 형식으로 녹화 된 영상을 저장한다. 노트:<html> / 초 3000 프레임의 최소 기록 프레임 레이트는 CFL 측정 생리 동맥 흐름 조건 하에서 적어도 한번 RBC 당 수행 될 수 있도록 권장한다. </li><li> 적혈구와 플라즈마 사이의 대비를 향상시키기 위해 510 나노 미터 - 310에서 394 nm의 스펙트럼 대역의 파장에서 최대 전송과 푸른 필터를 사용합니다. 참고 : 현미경 광원 (100 W 할로겐 램프)에서 파란색 필터를 통과하는 빛의 스펙트럼이 잠재적 인 조직 손상을 방지하기 위해 낮은 조명 강도의 확인하십시오. </li><li> 실험의 끝에, 펜토 바르 비탈 나트륨의 과량으로 동물을 안락사. </li></ol></li></ol> 2. 이미지 분석 <ol><li> CFL 폭 측정 전처리 <ol><li> MATLAB을 열고 &#39;CFL_pre.m&#39;파일을 실행합니다. (이 다른 MATLAB 파일은 찾을 수 있습니다IP &quot;&gt; 기업 MATLAB 보관.) </li><li> 분석 할 비디오 파일을 선택하려면 &#39;파일 열기&#39;를 클릭하십시오. </li><li> 수직으로 혈관 벽을 정렬하기 위해 &#39;회전&#39;슬라이더를 조정합니다. 주 : 사용자는 &#39;격자에&#39;버튼을 선택하여 용기 정렬 어시스트 그리드 라인을 표시하고, &#39;축소&#39;슬라이더를 밀어 화상의 줌 레벨을 조정할 수있다. </li><li> 용기 정렬을 확인하는 &#39;확인 편집&#39;버튼을 클릭합니다. </li><li> 관심 (ROI)의 영역을 정의하려면 &#39;설정 ROI 작물에 대한&#39;버튼을 클릭합니다. 정렬 된 이미지가 팝업 창에 표시됩니다. 이미지에 직사각형 목표를 조정하고 두 번 투자 수익 (ROI)을 확인합니다. 이미지 자르기가 필요하지 않은 경우이 단계를 건너 뜁니다. 참고 : 전용 용기로부터의 CFL 폭을 분석 할 수있는 ROI에서 하나의 용기를 포함한다. 필요한 경우, 원래의 형태로 이미지를 복원 할 &#39;재설정 이미지&#39;버튼을 클릭합니다. </li><li> 클릭9 &#39;&#39;(BMP &#39;형식 연속 비트 맵 이미지를 8 비트 그레이 스케일)에 모든 편집 된 비디오 프레임을 추출 버튼&#39;이미지의 압축을 풉니 다. 추출 된 이미지는 선택된 비디오 파일과 동일한 이름을 가진 폴더에 있습니다. </li></ol></li><li> CFL 폭 측정 <ol><li> MATLAB을 열고 &#39;CFL_measure.m&#39;파일을 실행합니다. </li><li> 추출 된 이미지가 들어있는 폴더를 선택 &#39;폴더 선택&#39;을 클릭합니다. </li><li> 이미지가 들어있는 폴더를 클릭하고 &#39;폴더 선택&#39;을 클릭합니다. 폴더의 첫 번째 이미지 프레임을로드하고 &#39;이미지 히스토그램&#39;패널에서의 회색 강도 히스토그램과 함께 &#39;그레이 스케일 이미지&#39;패널에 표시됩니다. </li><li> 분석, 그렇지 않으면 제 1 이미지 프레임이 선택 될을 수행하는리스트 박스에서 원하는 영상 프레임을 선택한다. </li><li> 위치에 결정되는 이미지의 내부 용기 벽을 식별하는 &#39;선박 벽 찾기&#39;를 클릭 곳어두운 빛 강도 프로파일 피크 천이 두 픽셀 위에 점등한다. </li><li> &#39;소금과 후추&#39;소음을 줄이기 위해 이미지에 중간 필터를 적용하는 &#39;중간 필터&#39;를 선택합니다. </li><li> 이미지 대비를 강화하기 위해 디지털 이미지의 강도를 조절하는 &#39;자동 명암 조정&#39;을 선택합니다. </li><li> τ 위의 그레이 레벨 (CFL), 그리고 τ 아래의 그레이 레벨 (RBC 코어) 블랙 픽셀 화이트 픽셀 - 두 개의 클래스로 계조를 분할하는 자동 임계 값 (τ)을 결정 목록 상자에서 임계 알고리즘을 선택합니다. 참고 : 자동 - 임계 알고리즘 중 어느 것도 적절한 이미지 임계 값을 제공하지 않는 경우 다른 방법으로, 수동 임계 값을 사용합니다. &#39;수동&#39;라디오 버튼을 클릭하고 수동 임계 값을 정의하는 슬라이더를 조정합니다. </li><li> (해상도는 CFL 폭의 공간 변화를 측정하고 &#39;픽셀 해상도&#39;상자에 픽셀 해상도를 입력하려면이 실험 장치와 0.42 μm의 / 픽셀)이었다. </li><li> CFL 폭의 공간 변화를 얻기 위해 &#39;계산&#39;버튼을 클릭합니다. 표로 형식으로 CFL 폭 데이터를 내보내려면 &#39;내보내기 .CSV&#39;을 클릭합니다. </li><li> 프레임 속도 정보, 선박 따라 특정 분석 라인의 CFL 폭의 시간적 변화를 측정하고 &#39;시간 변화&#39;라디오 버튼을 클릭하고 입력하려면 (이 실험 장치에서 사용되는 프레임 속도를 3000 프레임 / 초였다). </li><li> 각각 &#39;프레임 시작&#39;과 &#39;마지막 프레임&#39;상자의 첫 번째 프레임과 분석을위한 이미지의 마지막 프레임을 입력합니다. </li><li> &#39;분석 선&#39;슬라이드 막대를 밀어 혈관을 따라 분석 라인의 위치를 ​​선택합니다. &#39;그레이 스케일 이미지&#39;와 &#39;바이너리 이미지&#39;에 모두 설명되어 분석 라인의 위치를 ​​확인합니다. </li><li> CFL 폭의 시간적 변화를 얻기 위해 &#39;계산&#39;을 클릭합니다. CLICK &#39;내보내기 .CSV&#39;는 표로 형식으로 CFL 폭 데이터를 내보낼 수 있습니다. </li></ol></li></ol>

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Am J Physiol Heart Circ Physiol. 268 (5), H1959-H1965 (1995).</li><li>Namgung, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+histogram-based+thresholding+methods+for+the+determination+of+cell-free+layer+width+in+small+blood+vessels.">A comparative study of histogram-based thresholding methods for the determination of cell-free layer width in small blood vessels.</a> Physiol Meas. 31 (9), N61-N70 (2010).</li><li>Ong, P. 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W., Calvard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picture+Thresholding+Using+an+Iterative+Selection+Method.">Picture Thresholding Using an Iterative Selection Method.</a> IEEE Trans. Syst., Man, Cybern. 8 (8), 630-632 (1978).</li><li>Shanbhag, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+Information+Measure+as+a+Means+of+Image+Thresholding.">Utilization of Information Measure as a Means of Image Thresholding.</a> Cvgip-Graph Model Im. 56 (5), 414-419 (1994).</li><li>Bishop, J. J., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+erythrocyte+aggregation+and+venous+network+geometry+on+red+blood+cell+axial+migration.">Effects of erythrocyte aggregation and venous network geometry on red blood cell axial migration.</a> Am J Physiol Heart Circ Physiol. 281 (2), H939-H950 (2001).</li><li>Yamaguchi, S., Yamakawa, T., Niimi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+plasma+layer+in+cerebral+microvessels.">Cell-free plasma layer in cerebral microvessels.</a> Biorheology. 29 (2-3), 251-260 (1992).</li></ol>

The authors declare no competing financial interests.

CFL 폭의 측정은 미세 순환의 혈류 역학의 이해에 필수적이다. 특히, CFL 폭의 측정은 장간막 6에서 수행되어, 24, 25 microcirculations 대뇌를 spinotrapezius. 생체 CFL 폭의 측정은 종래의 녹화 된 비디오 프레임의 수동 검사 추정치로 제한 하였다. 수동 측정은 시각 RBC 코어 및 용기의 벽 (15, 16)의 경계를 식별하기 전에 몇 개의 연속적인 비디오 프레임의 평균화가 필요했다. 또 다른 연구에서, 형광 염료 (FITC)가 적혈구 -labelled 및 로다 B의 isothiocynate은 (RITC) 표지 된 플라즈마는 고양이 대뇌 미세 혈관 (25)의 평균 CFL 폭을 결정하는 데 사용 하였다. 이러한 이전의 측정 방법은 매우 시간 소모적이며, CFL 위스콘신의 공간 및 시간 해상도를 제한하는 형광 표식에 대한 추가 단계가 필요DTH 측정. 대조적으로, 효과적인 이미지 분할 및 분석 고속 카메라 레코딩을 결합하여, 여기에서 입증 된 기술은 작은 RBC 및 크기보다 주문의 공간 해상도 (0.42 μm의)와 CFL의 시공간 변동의 정량을 허용 1 / 3,000 초의 시간 해상도. cremaster 근육의 적절한 수술 준비가 CFL 폭 측정의 정확성을 결정하는데 중요하다. 특히, 인접한 결합 조직의 완전한 제거는 cremaster 근육의 소동맥의 좋은 초점을 보장하기 위해 필수적이다. 또한, 측정의 공간적 해상도는 현미경과 카메라 사양에 의존한다. 높은 배율 목적은 공간 해상도를 향상시킬 수 있지만, 그것은 차례로 CFL 폭의 공간적 변화를 정량화하기 위해 수득 용기의 길이를 제한하는 시야를 감소시킨다. 따라서, microscopiC 구성은 기술의 특정 어플리케이션에 따라 변경 될 수있다. 이미지 분할은 CFL 폭 측정의 정확도에 대한 또 다른 중요한 인자이다. 개발 된 다양한 기술 중에서도, 그레이 레벨 히스토그램에 기초하여 화상 임계 값은 영상 분할 및 분석을위한 간단하고 효과적인 방법을 제공한다. 따라서, 전경 오브젝트는 자신의 계조 차이에 기초하여 상기 배경으로부터 추출된다. 이상적인 경우, 화상 히스토그램이 바이 모달되며 계곡의 바닥에서 임계 값은 간단하다. 그러나 생체 내에서 실험 이미지는 항상 같은 그레이 스케일 수준의 프로파일을 나타내지 않는다. 우리의 결과는 이미지 품질 및 콘트라스트의 이미지 분할 처리에 영향을 미칠 수있는 방법을 보여준다. 광 청색 필터의 사용은 현저하게 (도 3) 적혈구 및 세동맥의 플라즈마 사이의 콘트라스트를 개선하고, 그 때 applyin 필수적인 것으로 보인다g없이 알고리즘 (도 4)의 폭 CFL 측정 히스토그램 기반 임계. 이것은 하나의 유효 문턱 값을 식별 할 수있는 고유 한 쌍봉 화상 히스토그램을 초래한다. 그러나, 심지어 생체 이미지에서 얻어진 바이 모달 히스토그램 두 피크 (로컬 최대 값)의 매우 불균등 분산 히스토그램 다양한 밸리 (극소)가 여전히 임계 선택 (표 1에 영향을 미칠 수 있음을 유의해야한다 ). 따라서, 적절한 임계 값 알고리즘의 선택 검사 화상 품질에 기초해야하고 사용자 CFL 폭 정량화에 가장 적합 각 임계 값 알고리즘의 제한 사항을 고려한다. CFL 폭이 유동 상태에 크게 의존하는 바와 같이, 실험 과정에 걸쳐 연속 동맥압 측정이 필수적이다. 로컬 유동 조건을 결정하기 위해,혈류의 pseudoshear 레이트 혈관 (5) 내의 평균 유속을 측정함으로써 계산 될 수있다. 요약하면, 래트 cremaster 근육 및 여기서 설명 정량적 화상 분석 수술 준비를위한 프로토콜은 생체 내에서 CFL 폭의 동적 변동에 대한 정량적 정보를 얻기 위해 사용되어왔다. CFL 폭 측정의 정확성을 보장하는 주요 과제는 상기 언급 한 둘의 근육 및 이미지 세분화, 적절한 수술 준비를 포함한다. 이러한 기술은 용이 hemorheological 다양한 생리 학적 및 병리학 적 상황에서 혈역학 수차를 조사하기 위해 다른 미세 연구에 적용 할 수있다. 따라서,이 연구 결과는 미세 혈관 치료 방법 및 임상 개입의 미래 발전에 기여한다.

In vivo animal studies are instrumental to basic science for understanding human physiology and pathology. In particular, in vivo microhemodynamic studies can elucidate the potential impairment of microcirculatory functions altered by abnormal rheological conditions of blood. A number of previous microhemodynamic studies1 have used the rat cremaster muscle model for visualizing microvascular blood flow. The cremaster muscle is a thin layer of striated muscle surrounding the testes. Thus, the blood flow in the muscle can be visualized with a trans-illumination microscope by means of surgical exposure. This enables us to acquire the in vivo blood flow images without the use of any fluorescence or contrast agents. In addition, the entire blood perfusion of the muscle network can be controlled by reducing the upstream blood flow with abdominal aorta occlusion2. Owing to these advantages, the cremaster muscle model has been widely used to investigate the formation of cell-free layer (CFL) in microvessels1,3. 
The CFL width is a prominent hemodynamic parameter in microcirculation, which has been of great interest for its important roles in regulating microcirculatory functions. The CFL is formed by the shear-induced transverse inward migration of red blood cells (RBCs) towards the flow center4. Consequently, this migration leads to the depletion of RBCs near the vessel walls, eventually resulting in a cell-free plasma layer. Accordingly, the parietal CFL naturally becomes a diffusion barrier to oxygen (O2) delivery from the RBC core to the tissues, and to the scavenging of nitric oxide (NO) by the RBCs5,6. In addition, the production of NO can also be modulated by the dynamic variations of the CFL width7,8. Therefore, the roles of the CFL in both gas transport and the regulation of homeostasis in microcirculation need to be fully ascertained to better understand blood flow in microcirculation. Recent studies have focused on bridging the hemodynamics and gas transport functions of the CFL in the microcirculation9-12. Furthermore, a separate set of studies has also investigated how the pathological elevation in RBC aggregation modulates CFL formation and its effect on O2 and NO bioavailability in tissues13,14. 
The roles of the CFL become more significant in microcirculation where the relative size of the CFL width to the vessel diameter is prominent. This necessitates an effective approach of quantifying the CFL in in vivo blood flow. Particularly, image acquisition and image analysis are the two key components determining the accuracy of CFL width measurement. Successful visualization of tissue blood flow should be preceded by an appropriate surgical preparation of the animal model. Additionally, a proper image analysis technique is needed to overcome the limitations of conventional manual measurements that are mostly induced by human errors15,16. With advancements in optical instrumentation and computing power for digital image processing, it is now possible to achieve a more accurate and consistent measurement of the CFL width17-19. Nonetheless, the accuracy of these measurements, being image-based, still ultimately depends on the quality of the images.
Therefore, this study explores the factors influencing the measurement of the in vivo CFL width. We focused particularly on demonstrating the surgical preparation and digital image analysis for measurements of the CFL width in arterioles of the rat cremaster muscle.

<table><tbody><tr><td>Intravital microscope</td><td>Olympus</td><td>BX51WI</td><td>Equipment</td></tr><tr><td>High speed camera</td><td>Photron</td><td>1024PCI</td><td>Equipment</td></tr><tr><td>Blue filter</td><td>HOYA</td><td>B390</td><td>Equipment</td></tr><tr><td>Pressure sensor &amp;amp; biopac system</td><td>Biopac system</td><td>TSD104A, MP100</td><td>Equipment</td></tr><tr><td>Temperature controller</td><td>Shimaden</td><td>SR 1</td><td>Equipment</td></tr><tr><td>Plasma Lyte A</td><td>Baxter</td><td>NDC:0338-0221</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Saline 0.9%</td><td>Braun</td><td></td><td></td></tr><tr><td>Heparin (5,000 IU/ml)</td><td>LEO</td><td></td><td></td></tr><tr><td>PE-10 polyethylene tube</td><td>Becton Dickinson</td><td>427400</td><td>.024&quot; OD x .011&quot; ID&amp;nbsp;</td></tr><tr><td>PE-50 polyethene tube</td><td>Becton Dickinson</td><td>427411</td><td>.038&quot; OD x .023&quot; ID</td></tr><tr><td>PE-205 polyethene tube</td><td>Becton Dickinson</td><td>427446</td><td>.082&quot; OD x .062&quot; ID</td></tr><tr><td>2-0 non-absorbable silk suture</td><td>Deknatel</td><td>113-S</td><td></td></tr><tr><td>5-0 non-absorbable silk suture</td><td>Deknatel</td><td>106-S</td><td></td></tr><tr><td>Water circulating heating pad</td><td>Gaymar</td><td></td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>Isotemp 205</td><td>Equipment</td></tr><tr><td>Sterile Cotton Gauze&amp;nbsp;</td><td>Fisher Scientific</td><td>22-415-468</td><td></td></tr><tr><td>Cotton-tipped applicators</td><td>Fisher Scientific</td><td>23-400-124</td><td></td></tr><tr><td>Dumont Forceps</td><td>Kent Scientific</td><td>INS14188</td><td>Surgical instrument</td></tr><tr><td>Micro Dissecting forceps</td><td>Kent Scientific</td><td>INS15915</td><td>Surgical instrument</td></tr><tr><td>Iris forceps 1 x 2 teeth</td><td>Kent Scientific</td><td>INS15917</td><td>Surgical instrument</td></tr><tr><td>Vessel cannulation forceps</td><td>Kent Scientific</td><td>INS500377</td><td>Surgical instrument</td></tr><tr><td>Micro scissor</td><td>Kent Scientific</td><td>INS14177</td><td>Surgical instrument</td></tr><tr><td>Iris scissor</td><td>Kent Scientific</td><td>INS14225</td><td>Surgical instrument</td></tr><tr><td>Vessel clip</td><td>Kent Scientific</td><td>INS14120</td><td>Surgical instrument</td></tr><tr><td>Gemini cautery system</td><td>Braintree Scientific</td><td>GEM 5917</td><td>Surgical instrument</td></tr></tbody></table>

visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

생체 내에서 CFL의 시각화 동물의 수술 준비에 크게 의존한다. 과도한 혈액 손실 또는 확장 수술 시간은 충격과 혈류 수차 동물 대상이된다. 가열 패드뿐만 아니라 수술 실험 기간 동안 맞춤형 플랫폼을 사용하는 조직의 유지 온도는 래트의 생리적 조건을 유지하기위한 중요하다. 현미경 시스템에서, 100 W 할로겐 램프를 사용하여 식별 할 수있는 조직 손상도 실험의 끝에서 관찰되지 않았다. 도 2a는 CFL의 RBC 코어와 내부 용기 벽 (도 2c) 사이에 관찰 될 수 래트 cremaster 근육에서 분지 세동맥을 통한 전형적인 RBC 흐름을 나타낸다. 실험 기간 동안 이들 구성 요소 사이의 적절한 콘트라스트 CFL 폭 측정의 정확성을 보장하기위한 중요하다. 이미지 분석의 초기 단계가 포함내부 용기 벽의 검출. 용기에 수직 분석 라인을 따라 광 강도 프로파일을 취득하여, 위치는 다크 행 천이 개의 화소 (도 2b) 위에 불 해당 피크에 근사한다. 적혈구 및 CFL 다른 광선 투과율을 가지고 같이 그레이 레벨의 차이는 두 개의 클래스 (이진 화상)으로 세분 될 수있다. 그러나, 화상 히스토그램의 2 개의 피크 사이의 정확한 문턱 값의 식별은 불량한 화질 및 콘트라스트 (도 3a)에 의해 제한 될 수있다. 블루 필터를 사용할 수있는 적혈구 및 CFL (그림 3B) 사이의 대비를 향상시킬 수 있습니다. 이렇게하는 RBC 코어의 경계가보다 정확하게 블루 필터를 사용하여 식별 될 수 있으며,도 4에서 또한 알 수있다. 또한, 임계 값 알고리즘 20-23의 선택도 CFL 폭의 측정에 영향을 미칠 수있다 (도4). 그것은 서로 다른 임계 값 알고리즘, 확인 된 다른 RBC 코어 경계에 이어진도 4a에 알 수있는 수 잘못된 CFL 측정에 차례 리드한다. 더도 4b에서 CFL 폭 측정의 임계 값 알고리즘의 영향을 설명하기 위해 서로 다른 임계 알고리즘을 이용하여 얻어진 CFL 폭에 대한 공간 정보는도 5에 도시되고 표 1에 요약되어있다. <img alt="그림 1" src="/files/ftp_upload/54550/54550fig1.jpg" /> . 그림 1 :. 생체 내에 현미경 시스템 및 Cremaster 근육 준비 A :. 수술로 표면화 쥐 cremaster 근육 B하십시오 cremaster 근육을 배치하고 35 ° C의 온도에서 온도를 유지하기위한 가열 요소와 사용자 정의 플랫폼 : 기분이야와 현미경 시스템연속적인 압력 모니터링 생리 데이터 수집 시스템 :. 네거티브 피드백 온도 조절기 및 전원 공급 E :. 상기 cremaster 근육 D에서 미세 순환 흐름의 가시화 동물 단계 고속 카메라 tomized. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54550/54550fig2.jpg" /> 그림 2 :.. 세동맥의 RBC 흐름의 전형적인 그레이 스케일 이미지 (혈관 직경 = 52 μm의) B : 분석 선을 따라 빛의 강도 프로파일 (패널 A의 실선) 선박 벽 위치 및 CFL 폭 (A)의 결정에 대한 이미지 처리 . C : 혈관을 따라 CFL 측정의 대표적인 결과입니다. 그만큼고체 점선 화살표는 각각 RBC 코어의 내부 용기 벽과 외주를 나타낸다. (LWB &amp; RWB는 : 왼쪽과 오른쪽 혈관 벽 경계, LCB 및 RCB는 : 왼쪽과 오른쪽 RBC 코어 경계) <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54550/54550fig3.jpg" /> 그림 3 :.. 광학 블루 필터와 이미지 콘트라스트 향상 (A)없이 청색 필터 (B)로 얻은 그레이 스케일 이미지의 이미지 히스토그램 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/54550/54550fig4.jpg" /> Figur전자 4 :.. (: 블루 필터를 사용하지 않고, 아래 줄 (B) : 블루 필터 상위 행 (A))를 사용을 RBC 코어 폭은 다섯 가지 형 임계 값 알고리즘 RBC 코어와 그림 3에서 그레이 스케일 이미지에 중첩 혈관 벽의 경계를 사용하여 결정 오쓰의 방법, 최소 방법, 인터 모드 방법, 반복 선택 방법 (ISODATA) 및 퍼지 엔트로피 임계 값 (Shanbhag) (왼쪽에서 오른쪽으로). 고체 및 점선은 내부 용기 벽과 RBC 코어의 바깥 쪽 가장자리를 표시, 각각. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 5" src="/files/ftp_upload/54550/54550fig5.jpg" /> 그림 5 : CFL 폭의 공간 변화 왼쪽을 따라 4B 그림에 해당하는 CFL 폭 (A).우측 (B) 혈관벽 각각. (D : 혈관 직경의 거리) <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="1 번 테이블" src="/files/ftp_upload/54550/54550table1.jpg" /> 표 1 : 그림 5. *의 P &lt;0.001에서 임계 값 및 CFL 폭 데이터 : 오오 츠의 방법과 유의 한 차이. † P &lt;0.001 : 왼쪽에서 유의 한 차이. 통계 분석은 양측 짝 t-테스트를 ​​사용하여 수행 하였다.

Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

쥐 Cremaster 근육의 세동맥의 셀이없는 계층의 시각화 및 정량화

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

visualization-quantification-cell-free-layer-arterioles-rat-cremaster

Universidad San Sebastian

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JoVE Journal

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6.3K 조회수.  National University of Singapore. This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

비디오: Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

쥐 Cremaster 근육의 국소 빈혈 reperfusion의 - 상해에서 백혈구 - 내피 상호 작용의 실시간 디지털 이미징 (IRI)

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

연구

의학

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

라이브 마우스의 혈관 염증과 혈전 형성하는 동안 활성화 내피에 Heterotypic 혈소판 - 호중구 상호 작용의 실시간 영상

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

면역학 및 감염병학

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Murine 정밀 절단 폐 슬라이스를 사용하여 폐 내 동맥의 저산소 폐 혈관 수축의 비디오 형태 측정 분석

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

엑기스의 Intravital 이미징을위한 마우스 Cremaster 근육 준비

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

생물학

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

디지털 Immunofluorescent 현미경을 사용하여 쥐 Pial Arterioles에서 에스 트로겐 수용체 - α의 영상

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

쥐의 고립 된 저항 동맥의 비디오 현미경을 이용 하 여 혈관 제어 메커니즘의 평가

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Intravital 현미경 검사법에 의해 평가된 마우스에 있는 Cremaster 근육의 백혈구 침투

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

주변 줄기 세포 이주의 분석을위한 마우스 Cremaster 근육에있는 미세 혈관의의 intravital 현미경

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

마우스 심장의 모세혈관, 동맥 및 페리사이클의 동적 측정 및 이미징

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.