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.

这项研究是按照新加坡的机构动物护理和使用委员会（批准的协议没有。R15-0225）的国立大学。 1.手术动物模型的制备<ol><li> 血管插管 <ol><li>麻醉雄性Sprague-Dawley大鼠（6 - 7周龄）称重（203±20）G用氯胺酮（37.5毫克/毫升）和甲苯噻嗪（5毫克/毫升）的鸡尾酒通过腹膜内（IP）注射（2毫升/千克） 。不要回顾一下针头或注射后注射器将其删除。 </li><li>一旦动物被麻醉（趾捏证实），把它放在一个加热垫，以保持其体温在37℃。轻轻地刮在肩胛骨，前宫颈，小腹，内侧后腿和阴囊的头发。轻轻地克制使用胶粘纸带的腿。 </li><li>采用显微剪刀，一边欣赏角度的，通过执行镊子所有外科手术立体显微镜。放置在一个抗穿刺托盘中的所有锋利的外科手术工具，以防止手术过程中受伤。 </li><li>在进行切开前擦洗手术部位交替碘和70％乙醇3次。同花顺30 IU / ml肝素盐水解决方案的所有导管。 </li><li>做一个1 - 使用一双手术剪过右颈静脉肩胛骨1.5厘米中线皮肤切口。钝性分离分离筋膜，露出颈静脉，用聚乙烯管（PE-50）充满利用5-0丝线缝合肝素盐水导管插入它。注入补充麻醉必要时（1/3到初始剂量的1/2，静脉内（IV））在整个手术和实验过程。 </li><li>执行气管切开，以保持呼吸道通畅。做一个1 - 在颈前区有1.5厘米的切口。使用的聚乙烯管（PE-205）用2-0丝线缝合以固定导管代替导管插入气管。 </li><li>监测血压通过在股动脉插管。使1 - 在后腿的左内侧表面1.5厘米切口。分离钝性分离股动脉。导管插入填充用5-0丝线缝合肝素盐水的聚乙烯管（PE-10）的股动脉。 </li></ol></li><li> 提睾肌准备和流量可视化 <ol><li>将5-0丝线穿过阴囊的顶点来扩展它。使沿阴囊腹面切口。定期应用暖等渗溶液（37℃; pH 7.4）中到暴露的肌肉。 </li><li>删除认真彻底周围结缔组织使用棉签。 </li><li>通过提睾肌的顶点将5-0丝线缝合。切缝合线成相等长度的两件，并在每一侧打结。切两个节点之间的肌肉，并轻轻拉动缝线拉伸它到一个定制的透明有机玻璃平台。固定缝合年底到具有粘性的蓝色平台。 注:彻底清除周围结缔组织的是获得最佳图像对比度的关键。 </li><li>重复步骤1.2.3至5〜6注视制成。小心取出使用高温烧灼附睾提睾肌。 Superfuse温暖等渗溶液到暴露肌肉，以防止组织的脱水。 <ol><li>围绕提睾肌，用纱布折叠块。盖上聚乙烯薄膜的露出肌肉。纱布片与薄膜形成浅盆保持为浸水显微镜物镜（ 图1A）温暖等渗溶液。 </li></ol></li><li>转移动物到一个活体显微镜（ 图1C）的动物阶段。动脉插管连接至连续压力监测（ 图1E）的一种生理数据采集系统。 </li><li>保持肌肉TEMPERATURE在35℃下与附着在动物的平台（ 图1B）下方的加热元件。放置一个温度探头肌肉旁，以提供给加热元件（ 图1D）的功率控制器负反馈。 </li><li>离开舞台上的动物15分钟以与环境平衡。 </li><li>可视化的活体显微镜下血流40倍水浸泡的目的和长时间工作冷凝器。 </li><li>选择无支链的小动脉（&lt;60微米）的基础上的RBC芯之间的清晰图像的焦点和对比度，CFL和血管壁，以便聚焦显微镜上的血管的直径平面。旋转安装到显微镜垂直对齐血管壁的相机。 </li><li>用的高速视频摄像机以3000 /秒的帧速率1秒记录血流量。保存所记录的视频作为未压缩的8位灰度AVI格式保存的图像质量。 注意:<html>推荐3000帧/秒的最小记录帧速率，以确保所述CFL测量可生理动脉流动条件下每红细胞至少执行一次。 </li><li>使用具有峰值发送一个蓝色滤色器在310 394纳米和光谱带通波长 - 510毫微米，以提高红细胞和血浆之间的对比度。 注:确保光谱通过从微观光源（100瓦卤素灯）蓝色的过滤器是低光照强度，以防止任何潜在的组织损伤。 </li><li>在实验结束时，安乐死动物用戊巴比妥钠的过量。 </li></ol></li></ol> 2.图像分析 <ol><li> 预处理的CFL宽度测量 <ol><li>打开MATLAB并运行"CFL_pre.m&#39;文件。 （这和其他MATLAB文件可以在找到IP"&gt;补充MATLAB归档）。 </li><li>点击"打开文件"，选择视频文件进行分析。 </li><li>调整"旋转"滑块垂直对齐血管壁。 注:用户可以通过选择"网格"单选按钮，显示该容器对准辅助网格线，并通过滑动"缩放"滑块来调整图像的缩放级别。 </li><li>点击"确认编辑"按钮，确认船舶对齐。 </li><li>点击"设置ROI作物"按钮来定义感兴趣区域（ROI）的区域。对准的图像将显示在一个弹出窗口。调整图像上的矩形目标，并双击以确认投资回报率。跳过此步骤如果不需要图像的裁切。 注意:仅包括在以分析从容器中的CFL宽度将ROI的单一容器中。点击"重置图片"按钮将图像恢复到其原来的形式，如果有必要。 </li><li>点击9，提取图像"按钮，提取所有编辑的视频帧到连续的位图图像（8位灰度"BMP"格式）。所提取的图像可以具有相同的名称作为所选择的视频文件的文件夹中找到。 </li></ol></li><li> CFL宽度的测量 <ol><li>打开MATLAB并运行"CFL_measure.m&#39;文件。 </li><li>点击"选择文件夹"，选择包含提取图像的文件夹。 </li><li>单击包含图片的文件夹，然后单击"选择文件夹"。该文件夹中的第一个图像帧将会被加载，并在"灰度图像"面板中显示，随着"图像直方图"面板的灰色强度直方图一起。 </li><li>从列表框中选择所需的图像帧进行分析，否则第一图像帧将被选中。 </li><li>点击"查找容器壁"，以识别图像，其在该位置确定在内部容器壁，其中从暗光强度分布的峰过渡到点亮了两个像素。 </li><li>检查&#39;中值滤波器"以中值滤波应用到图像，以减少"椒盐"噪音。 </li><li>检查&#39;自动对比度&#39;以数字方式调整图像强度，以提高图像对比度。 </li><li>选择在列表框中自动确定阈值（τ）一个阈值化的算法，将所述灰度级分为两类 - 白像素与上述τ灰度级（CFL），并用下面τ灰度级（RBC芯）黑色像素。 注:另一种方法是，使用手动阈值，如果没有自动阈值处理算法提供了一个合适的图像阈值。点击"手动"单选按钮，调节滑块定义手动阈值。 </li><li>为了测量宽度CFL的空间变化，在"像素分辨率"框中输入像素分辨率（分辨率这个实验装置为0.42微米/像素）。 </li><li>点击"计算"按钮来获得CFL宽度的空间变化。点击"导出的.csv&#39;的CFL宽度导出数据以表格的格式。 </li><li>为了测量宽度CFL在沿着容器具体分析线的时空变化，点击"时空变化"单选按钮，然后输入帧速率信息（在本实验装置使用的帧速率为3000帧/秒）。 </li><li>在&#39;开始帧&#39;和&#39;末帧&#39;盒，输入分别在第一帧和用于分析图像的最后一帧。 </li><li>选择沿滑动"分析线"滑杆容器内的分析线的位置。确认分析线，这是双方"灰度图像"和"二值图像"上所示的位置。 </li><li>点击"计算"，以获得CFL宽度的时间变化。 CLICK"导出的.csv&#39;的CFL宽度导出数据以表格的格式。 </li></ol></li></ol>

<ol>
	<li>Kim, S., Kong, R. L., 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=Temporal+and+spatial+variations+of+cell-free+layer+width+in+arterioles.">Temporal and spatial variations of cell-free layer width in arterioles.</a> Am J Physiol Heart Circ Physiol. 293 (3), H1526-H1535 (2007).</li><li>Ong, P. K., Namgung, B., Johnson, P. C., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+erythrocyte+aggregation+and+flow+rate+on+cell-free+layer+formation+in+arterioles.">Effect of erythrocyte aggregation and flow rate on cell-free layer formation in arterioles.</a> Am J Physiol Heart Circ Physiol. 298 (6), H1870-H1878 (2010).</li><li>Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+uneven+red+cell+influx+on+formation+of+cell-free+layer+in+small+venules.">Effect of uneven red cell influx on formation of cell-free layer in small venules.</a> Microvasc Res. 92, 19-24 (2014).</li><li>Goldsmith, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Microcirculatory+Society.">The Microcirculatory Society.</a> Eugene M. Landis Award Lecture. The Microrheology of Human-Blood. Microvasc Res. 31 (2), 121-142 (1986).</li><li>Buerk, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+We+Model+Nitric+Oxide+Biotransport?+A+Survey+of+Mathematical+Models+for+a+Simple+Diatomic+Molecule+with+Surprisingly+Complex+Biological+Activities.">Can We Model Nitric Oxide Biotransport? A Survey of Mathematical Models for a Simple Diatomic Molecule with Surprisingly Complex Biological Activities.</a> Annu Rev Biomed Eng. 3 (1), 109-143 (2001).</li><li>Tateishi, N., Suzuki, Y., Soutani, M., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+dynamics+of+erythrocytes+in+microvessels+of+isolated+rabbit+mesentery:+cell-free+layer+and+flow+resistance.">Flow dynamics of erythrocytes in microvessels of isolated rabbit mesentery: cell-free layer and flow resistance.</a> J Biomech. 27 (9), 1119-1125 (1994).</li><li>Ong, P. K., Cho, S., Namgung, B., Kim, S. <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+cell-free+layer+formation+on+NO/O2+bioavailability+in+small+arterioles.">Effects of cell-free layer formation on NO/O2 bioavailability in small arterioles.</a> Microvasc Res. 83 (2), 168-177 (2012).</li><li>Ong, P. K., Jain, S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+NO+bioavailability+by+temporal+variation+of+the+cell-free+layer+width+in+small+arterioles.">Modulation of NO bioavailability by temporal variation of the cell-free layer width in small arterioles.</a> Ann Biomed Eng. 39 (3), 1012-1023 (2011).</li><li>Park, S. W., Intaglietta, M., Tartakovsky, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+stochastic+fluctuations+in+the+cell+free+layer+on+nitric+oxide+bioavailability.">Impact of stochastic fluctuations in the cell free layer on nitric oxide bioavailability.</a> Front Comput Neurosci. 9, 131 (2015).</li><li>Ng, Y. C., Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+transient+model+for+prediction+of+arteriolar+NO/O2+modulation+by+spatiotemporal+variations+in+cell-free+layer+width.">Two-dimensional transient model for prediction of arteriolar NO/O2 modulation by spatiotemporal variations in cell-free layer width.</a> Microvasc Res. 97, 88-97 (2015).</li><li>Sriram, K., 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+effect+of+small+changes+in+hematocrit+on+nitric+oxide+transport+in+arterioles.">The effect of small changes in hematocrit on nitric oxide transport in arterioles.</a> Antioxid Redox Sign. 14 (2), 175-185 (2011).</li><li>Hightower, C. 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=Integration+of+cardiovascular+regulation+by+the+blood/endothelium+cell-free+layer.">Integration of cardiovascular regulation by the blood/endothelium cell-free layer.</a> Wiley Interdiscip Rev Syst Biol Med. 3 (4), 458-470 (2011).</li><li>Ng, Y. C., Namgung, B., Leo, H. L., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+aggregation+may+promote+uneven+spatial+distribution+of+NO/O+in+the+downstream+vessel+of+arteriolar+bifurcations.">Erythrocyte aggregation may promote uneven spatial distribution of NO/O in the downstream vessel of arteriolar bifurcations.</a> J Biomech. , (2015).</li><li>Ong, P. K., Jain, S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+the+cell-free+layer+width+may+enhance+NO+bioavailability+in+small+arterioles:+Effects+of+erythrocyte+aggregation.">Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles: Effects of erythrocyte aggregation.</a> Microvasc Res. 81 (3), 303-312 (2011).</li><li>Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+rheology+in+microcirculation.">Erythrocyte rheology in microcirculation.</a> Jpn J Physiol. 46 (1), 1-14 (1996).</li><li>Soutani, M., Suzuki, Y., Tateishi, N., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+Flow+Dynamics+of+Erythrocytes+in+Microvessels+-+Influence+of+Erythrocyte+Aggregation.">Quantitative Evaluation of Flow Dynamics of Erythrocytes in Microvessels - Influence of Erythrocyte Aggregation.</a> Am J Physiol Heart Circ Physiol. 268 (5), H1959-H1965 (1995).</li><li>Kim, S., Kong, R. L., 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=A+computer-based+method+for+determination+of+the+cell-free+layer+width+in+microcirculation.">A computer-based method for determination of the cell-free layer width in microcirculation.</a> Microcirculation. 13 (3), 199-207 (2006).</li><li>Soutani, M., Suzuki, Y., Tateishi, N., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+Flow+Dynamics+of+Erythrocytes+in+Microvessels.">Quantitative Evaluation of Flow Dynamics of Erythrocytes in Microvessels.</a> Influence of Erythrocyte Aggregation. 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. K., 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=An+automated+method+for+cell-free+layer+width+determination+in+small+arterioles.">An automated method for cell-free layer width determination in small arterioles.</a> Physiol Meas. 32 (3), N1-N12 (2011).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Threshold+Selection+Method+from+Gray-Level+Histograms.">A Threshold Selection Method from Gray-Level Histograms.</a> IEEE Trans. Syst., Man, Cybern. 9 (1), 62-66 (1979).</li><li>Prewitt, J. M., Mendelsohn, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+cell+images.">The analysis of cell images.</a> Ann N Y Acad Sci. 128 (3), 1035-1053 (1966).</li><li>Ridler, T. 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宽度的测量是为了更好地理解微循环血流动力学是必不可少的。特别是，CFL宽度的测量已经在肠系膜6进行spinotrapezius 24和脑25微循环。 体内 CFL宽度的常规测量通过所记录的视频帧的人工检查仅限于估计。手动测量所需几个连续视频帧的平均肉眼识别红细胞核心和血管壁15,16的边界之前。在另一项研究中，异硫氰酸荧光素（FITC） -标记的RBC和罗丹明-B异硫氰酸（RITC）标记的血浆被用来确定在猫脑微血管25的平均CFL宽度。这些以前的测量方法是非常耗时的，需要为荧光标记，这限制了紧凑型荧光灯的Wi的空间和时间分辨率的附加步骤嗞嗞的测量。与此相反，由高速摄像机的记录耦合到有效图像分割和分析，这里展示的技术中允许与一个命令的空间分辨率（0.42微米）的紧凑型荧光灯的时空变化的定量小于一个红细胞的大小1/3000秒的时间分辨率。 提睾肌的正确的手术的准备是在确定的CFL宽度测量的精度是至关重要的。特别是，彻底去除相邻结缔组织是必不可少的，以确保在提睾肌微动脉的一个良好的聚焦。此外，测量的时间和空间分辨率取决于显微镜和照相机的规格。而较高放大倍数的物镜可提高空间分辨率，其降低的视场，而这又限制了获得的容器长度为量化CFL宽度的空间变化。因此，microscopiÇ配置可以根据本技术的具体应用进行修改。 图像分割为紧凑型荧光灯宽度测量的精确度的另一个重要因素。之间开发了各种技术，基于灰度直方图图像阈值提供了用于图像分割和分析了一个简单而有效的方法。因此，前景对象是从根据其灰度级的差的背景萃取。在理想的情况下，图像的直方图将双峰和在山谷底部的阈值是微不足道的。然而， 在体内实验图像并不总是表现出这种灰度级的配置文件。我们的结果表明图像质量和对比度如何可以影响图像的分割处理。使用的光学蓝色滤光片的显著增强了RBC和在动脉的血浆之间的对比度（ 图3），以及似乎applyin时是必不可少的克的CFL宽度测量不管算法（ 图4）基于直方图的阈值处理。这导致了明显的双峰图象直方图，它允许一个有效识别的阈值。然而，应该指出的是，即使是与来自体内图像获得双峰直方图的两个峰（局部极大值）非常不等方差和直方图的宽的山谷（局部最小值）仍可影响阈值选择（ 表1 ）。因此，适当的阈值算法的选择需要研究的基础上的图像质量和用户必须考虑每个阈值算法的局限性对在量化CFL宽度最好适用性。 作为所述CFL宽度很大程度上取决于流动条件，在整个实验过程中持续动脉压测量是至关重要的。为了确定局部流动条件下，血流的伪剪速率可以通过测量血液中的容器5的平均流速进行计算。 总之，对于外科手术制备大鼠提睾肌和这里所描述的定量图像分析的协议已被用来获取关于在体内 CFL宽度的动态变化的定量信息。在确保CFL宽度测量的精确度的主要挑战包括肌肉和图像分割，这两者已在上面讨论的正常手术准备。这种技术可容易地适用于其它微循环研究，以调查血液流变学和在各种生理和病理条件血流动力学像差。因此，这些研究结果有助于微血管的治疗方法和临床干预的未来发展。

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.

所述CFL 在体内的可视化很大程度上取决于动物的手术准备。因失血过多或延长手术时间可能使动物震惊和血流像差。使用加热垫以及手术和实验期间定制平台组织温度的维护也用于保持大鼠的生理条件的关键。通过在显微镜系统中使用一个100瓦的卤素灯，即使在实验结束时没有观察到明显的组织损伤。 图2A示出了通过在大鼠提睾肌无支链动脉，在那里可以红细胞芯和内部容器壁（ 图2C）之间可以观察到所述CFL一个典型的RBC流。在实验过程中这些部件之间的良好的对比度为确保CFL宽度测量的精度是至关重要的。图像分析的初始阶段涉及检测的内血管壁。通过获取在垂直于容器沿着分析线的光强度分布，该位置是在高峰，从暗过渡到超过两个像素（ 图2B）点亮近似。 作为RBC和紧凑型荧光灯具有不同的光透射率，在灰度级的差异可被细分为两类（二值图像）。然而，在图像的直方图的两个峰之间的精确阈值的识别可以通过图像质量差和对比度（ 图3A）的限制。为了提高红细胞和CFL，蓝色过滤器可用于（ 图3B）之间的对比。这也很明显在图4中，其中，所述红细胞芯的边界可以与使用蓝色滤光片的被更精确地确定。此外，阈值算法20-23的选择也可以影响所述CFL宽度的测量（ 图4）。它在图4A中是显而易见的，不同的阈值的算法的结果中识别不同的RBC芯边界，这可能又导致错误的CFL测量。为了更好地说明在图4B所述CFL宽度测量的阈值算法的影响，使用不同的阈值的算法获得所述CFL宽度的空间配置示于图5和表1中总结。 <img alt="图1" src="/files/ftp_upload/54550/54550fig1.jpg" /> 图 1: 活体显微系统和提睾肌准备 答:通过手术外置大鼠提睾肌乙:定制平台加热元件放置提睾肌，并在35℃ 下保持它的温度:用显微CUS系统tomized动物阶段和高速摄像机微循环血流量的提睾肌D中的可视化:负反馈温度控制器和电源E:生理数据采集系统持续压力监测。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54550/54550fig2.jpg" /> 图2:图像处理的血管壁的位置和CFL宽度 A 的测定:。在动脉RBC流的典型灰度图像（血管直径= 52微米）B:沿着分析线光强分布（面板实线） C:沿容器CFL测量结果代表。该固体和虚线箭头表示RBC芯的内部容器壁和外边缘，分别（LWB＆RWB:左，右血管壁边界，LCB和RCB:左，右RBC核心边界） <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54550/54550fig3.jpg" /> 图3:。图像对比度增强与光学滤镜蓝色无（A）和蓝色滤镜（B）得到的灰度图像的图像直方图<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig3large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54550/54550fig4.jpg" /> FIGURE 4:。（:无蓝滤光片，底排（B）:用蓝色滤镜上排（A））使用RBC核心位宽使用五个不同的阈值分割算法RBC核心和血管壁图3叠加在灰度图像的边界决心（从左至右）大津的方法，最小值法，模间法，迭代选择方法（ISODATA）和模糊熵的阈值（Shanbhag）。在实线和虚线表示内血管壁和RBC核心的外边缘，分别为。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54550/54550fig5.jpg" /> 图5:CFL宽度的空间变化对应于图4b沿左CFL宽度（A）。和右（B）血管壁，分别。 （D:在血管直径的距离） <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54550/54550table1.jpg" /> 表1:阈值和CFL宽度数据在图5 * P &lt;0.001:从大津的方法显著差异。 †P &lt;0.001:左差异显著。统计分析采用双尾配对t检验进行。

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.

在大鼠提睾肌的小动脉的可视化和量化的无细胞层

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

Research

JoVE Journal

Biology

6.3K 次观看.  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.

视频: 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.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

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

科研

医学

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.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

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

免疫与感染

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.

使用穆林精密切肺片对肺动脉内下氧肺血管收缩进行视频形态分析

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.

鼠标提睾肌肌肉制备的微循环活体成像

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

生物学

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.

使用数字免疫荧光显微镜成像在大鼠软脑膜小动脉雌激素受体α

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.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

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.

通过体内显微镜评估的小鼠中白细胞肌渗透

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.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

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.

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

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.