之后，正常小鼠70％部分肝切除血管和实质再生的可视化
Summary
Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using µCT and histological serial sections.
Abstract
一种改进硅氧烷注射步骤，用于肝血管树的可视化。此过程包括在体内注射硅氧烷化合物组成，经由＆G导管，进入门或肝静脉。硅胶注射后，器官植及离体微CT（μCT）扫描准备。硅胶注射过程在技术上具有挑战性。取得成功的，需要从医生丰富的经验显微。之一的这个过程的挑战涉及确定为聚硅氧烷化合物的适当的灌注速率。需要基于感兴趣血管系统的血流动力学被定义为聚硅氧烷化合物的灌注速率。不适当的灌注率可导致一个不完整的灌注，人工扩张和破裂的血管树。
血管系统的三维重建是基于CT扫描和使用达到临床前的软件如HepaVision。重构的血管树的质量直接与硅氧烷灌注的质量。表示血管生长的随后计算血管的参数，如总血管体积，基于所述血管重建计算。对比用硅酮血管树允许试样μCT扫描后后续的组织学后处理。试样可以进行连续切片，组织学分析和整个幻灯片扫描，并在其后的三维重建基于组织学图像中的血管树。这是用于检测分子事件和它们相对于分配给所述血管树的前提。此改性硅氧烷注入步骤也可以用于可视化和重建其它器官的血管系统。该技术具有的潜力可广泛应用于研究关于在各种动物血管解剖学和生长第二疾病模型。
Introduction
肝再生通常通过测量肝脏重量和体积的增加和通过评估肝细胞增殖率16确定。然而，肝再生不仅诱导再生实质，而且血管再生6。因此，血管生长应进一步相对于调查其在肝再生的进展的作用。肝血管系统的可视化是推进我们的血管再生的理解是至关重要的。许多间接方法已被开发，研究肝血管再生的分子机制。传统上，检测的细胞因子（血管内皮生长因子，血管内皮生长因子）14，趋化因子和它们的受体（CXCR4 / CXCR7 / CXCL12）4已经用于研究血管再生的支柱。然而，共同的3D模型与血管的定量分析将增加关键解剖信息更好地了解肝实质和血管再生的重要关系。
为了显现肝血管系统，这需要对比血管树，小鼠用不透射线的聚硅氧烷橡胶的造影剂直接注射到门户或肝静脉血管树。硅氧烷和器官的外植的聚合后，将肝脏样品用CT扫描仪进行μCT扫描。该扫描导致体素图像表示 硅氧烷喷射的试样9。
在质量控制方面，对血管系统最早是在使用3D软件的临床前可视化。分割通过设定所述软组织的强度和容器强度之间的阈值执行的。由此产生的容器面膜使用面绘制可视化。该软件还允许手工测定vascul两个参数AR增长：最大船舶长度和半径。
那么临床前软件进行三维重建血管树木和供给或引流血管分布区13后续计量。此外，该软件自动确定血管生长的某些参数，所有可见的血管结构也被称为总边缘长度或总容器体积的总长度，如。
在幼稚小鼠和在该行的70％部分肝切除（PH）的小鼠进行硅酮灌注过程。肝脏在不同的观察时间点切除使用上述可视化和定量技术分析血管和实质肝再生后收集。
该薄膜的主要目标是：（1）表明，以达到最佳反衬和（2）示出的潜在利益所得FR所需的微妙的注射技术嗡使用μCT和组织学连续切片所得样品的详细分析。看完这部片子后，读者应该有一个更好的了解如何注入聚硅氧烷化合物为特定的血管系统和技术的实用性和适用性。
Protocol
Representative Results
Discussion
通过对比硅胶注射和μCT扫描血管树已在肿瘤模型和神经系统疾病模型中引入经常研究进展血管5,7,8,10。在硅氧烷喷射的方法的改进，在本研究中提出的可视化和小鼠肝部分切除后定量血管生长。
有许多的关键步骤注意以达到良好的灌注质量。首先，全身肝素高度用肝素或盐水冲洗肝脏，以避免肝脏内凝血前推荐的。从管路气泡消除也是重要的实现硅氧烷灌注的优良品…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge funding by the German Ministry of Education and Research (BMBF) via the systems biology network “Virtual Liver”, grant numbers 0315743 (ExMI), 0315765 (UK Jena), 0315769 (MEVIS).The authors also thank Frank Schubert for technical support.
Materials
| PERFUSOR® VI | B.BRAUN | 87 222/0 | |
| Pipetus®-akku | Hirschmann | 9907200 | |
| Pipets | Greiner | 606180 | |
| micro scissors | Fine Science Tools (F·S·L) | No. 14058-09 | |
| micro serrefine | Fine Science Tools (F·S·L) | No.18055-05 | |
| Micro clamps applicator | Fine Science Tools (F·S·L) | No. 18057-14 | |
| Straight micro forceps | Fine Science Tools (F·S·L) | No. 00632-11 | |
| Curved micro forceps | Fine Science Tools (F·S·L) | No. 00649-11 | |
| needle-holder | Fine Science Tools (F·S·L) | No. 12061-01 | |
| 1ml syringe | B.Braun | 9161406V | |
| 5ml syringe | B.Braun | 4606051V | |
| extension and connection lines | B.Braun | 4256000 | 30cm, inner ø1.2mm |
| 6-0 silk (Perma-Hand Seide) | Ethicon | 639H | |
| 6-0 prolene | Ethicon | 8711H | |
| Microfil® MV diluent | FLOW TECH, INC | ||
| Microfil® MV – 120 | FLOW TECH, INC | MV – 120 (blue) | |
| MV curing agent | FLOW TECH, INC | ||
| Heparin 2500 I.E./5ml | Rotexmedica | ETI3L318-15 | |
| Saline | Fresenius Kabi Deutschland GmbH | E15117/D DE | |
| Imalytics Preclinical software | Experimental Molecular Imaging, RWTH Aachen University, Germany | ||
| HepaVision | Fraunhofer MEVIS, Bremen, Germany | ||
| NanoZoomer 2.0-HT Digital slide scanner | Hamamatsu Electronic Press, Japan | C9600 | |
| Tomoscope Duo CT | CT Imaging GmbH, Erlangen, Germany | TomoScope® Synergy | |
References
- Bearden, S. E., Segal, S. S. Neurovascular alignment in adult mouse skeletal muscles. Microcirculation. 12 (2), 161-167 (2005).
- Brown, R. P., Delp, M. D., Lindstedt, S. L., Rhomberg, L. R., Beliles, R. P. Physiological parameter values for physiologically based pharmacokinetic models. Toxicol.Ind.Health. 13 (4), 407-484 (1997).
- Dai, D., et al. Elastase-Induced Intracranial Dolichoectasia Model in Mice. Neurosurgery. , (2015).
- Ding, B. S., et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature. 468 (7321), 310-315 (2010).
- Downey, C. M., et al. Quantitative ex-vivo micro-computed tomographic imaging of blood vessels and necrotic regions within tumors. PLoS.One. 7 (7), 41685 (2012).
- Ehling, J., et al. CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis. Gut. , (2014).
- Ehling, J., et al. Micro-CT imaging of tumor angiogenesis: quantitative measures describing micromorphology and vascularization. Am.J.Pathol. 184 (2), 431-441 (2014).
- Ghanavati, S., Yu, L. X., Lerch, J. P., Sled, J. G. A perfusion procedure for imaging of the mouse cerebral vasculature by X-ray micro-CT. J.Neurosci.Methods. 221, 70-77 (2014).
- Gremse, F., et al. Hybrid microCT-FMT imaging and image analysis. J.Vis.Exp. (100), (2015).
- Jing, X. L., et al. Radiomorphometric quantitative analysis of vasculature utilizing micro-computed tomography and vessel perfusion in the murine mandible. Craniomaxillofac.Trauma Reconstr. 5 (4), 223-230 (2012).
- Melloul, E., et al. Small animal magnetic resonance imaging: an efficient tool to assess liver volume and intrahepatic vascular anatomy. J.Surg.Res. 187 (2), 458-465 (2014).
- Schwier, M., Bohler, T., Hahn, H. K., Dahmen, U., Dirsch, O. Registration of histological whole slide images guided by vessel structures. J.Pathol.Inform. 4 ((Suppl)), 10 (2013).
- Selle, D., Preim, B., Schenk, A., Peitgen, H. O. Analysis of vasculature for liver surgical planning. IEEE Trans.Med.Imaging. 21 (11), 1344-1357 (2002).
- Shergill, U., et al. Inhibition of of VEGF- and NO-dependent angiogenesis does not impair liver regeneration. Am.J.Physiol Regul.Integr.Comp Physiol. 298 (5), 1279-1287 (2010).
- Sueyoshi, R., Ralls, M. W., Teitelbaum, D. H. Glucagon-like peptide 2 increases efficacy of distraction enterogenesis. J.Surg.Res. 184 (1), 365-373 (2013).
- Wei, W., et al. Rodent models and imaging techniques to study liver regeneration. Eur.Surg.Res. 54 (3-4), 97-113 (2015).
- Xie, C., Wei, W., Zhang, T., Dirsch, O., Dahmen, U. Monitoring of systemic and hepatic hemodynamic parameters in mice. J.Vis.Exp. (92), e51955 (2014).
