在大鼠提睾肌的小动脉的可视化和量化的无细胞层
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.
Materials
| Intravital microscope | Olympus | BX51WI | Equipment |
| High speed camera | Photron | 1024PCI | Equipment |
| Blue filter | HOYA | B390 | Equipment |
| Pressure sensor &biopac system | Biopac system | TSD104A, MP100 | Equipment |
| Temperature controller | Shimaden | SR 1 | Equipment |
| Plasma Lyte A | Baxter | NDC:0338-0221 | Warm in 37 °C water bath before use |
| Saline 0.9% | Braun | ||
| Heparin (5000 IU/ml) | LEO | ||
| PE-10 polyethylene tube | Becton Dickinson | 427400 | .024" OD X .011" ID |
| PE-50 polyethene tube | Becton Dickinson | 427411 | .038" OD X .023" ID |
| PE-205 polyethene tube | Becton Dickinson | 427446 | .082" OD X .062" ID |
| 2-0 non-absorbable silk suture | Deknatel | 113-S | |
| 5-0 non-absorbable silk suture | Deknatel | 106-S | |
| Water circulating heating pad | Gaymar | ||
| Water bath | Fisher Scientific | Isotemp 205 | Equipment |
| Sterile Cotton Gauze | Fisher Scientific | 22-415-468 | |
| Cotton-tipped applicators | Fisher Scientific | 23-400-124 | |
| Dumont Forceps | Kent Scientific | INS14188 | Surgical instrument |
| Micro Dissecting forceps | Kent Scientific | INS15915 | Surgical instrument |
| Iris forceps 1×2 teeth | Kent Scientific | INS15917 | Surgical instrument |
| Vessel cannulation forceps | Kent Scientific | INS500377 | Surgical instrument |
| Micro scissor | Kent Scientific | INS14177 | Surgical instrument |
| Iris scissor | Kent Scientific | INS14225 | Surgical instrument |
| Vessel clip | Kent Scientific | INS14120 | Surgical instrument |
| Gemini cautery system | Braintree Scientific | GEM 5917 | Surgical instrument |
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