Published: October 19th, 2016
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.
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.
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 v....
This study is in accordance with the National University of Singapore Institutional Animal Care and Use Committee (approved protocol no. R15-0225).
1. Surgical Preparation of the Animal Model
The visualization of the CFL in vivo is largely dependent on the surgical preparations of the animal. Excessive blood loss or extended surgery duration may subject the animal to shock and blood flow aberrations. Maintenance of tissue temperature using a heating pad as well as a customized platform during the surgery and experiment is also crucial for maintaining the physiological conditions of the rat. By using a 100 W halogen lamp in the microscope system, no discernible tissue damage was observed even at the e.......
The measurement of CFL width is essential for a better understanding of the hemodynamics in the microcirculation. In particular, the measurement of CFL widths has been performed in mesenteric6, spinotrapezius24 and cerebral25 microcirculations. Conventional measurement of in vivo CFL widths was restricted to estimations by manual inspection of the recorded video frames. The manual measurements required the averaging of several successive video frames before visually identifying t.......
|High speed camera
|Pressure sensor &biopac system
|Plasma Lyte A
|Warm in 37 °C water bath before use
|Heparin (5000 IU/ml)
|PE-10 polyethylene tube
|.024" OD X .011" ID
|PE-50 polyethene tube
|.038" OD X .023" ID
|PE-205 polyethene tube
|.082" OD X .062" ID
|2-0 non-absorbable silk suture
|5-0 non-absorbable silk suture
|Water circulating heating pad
|Sterile Cotton Gauze
|Micro Dissecting forceps
|Iris forceps 1x2 teeth
|Vessel cannulation forceps
|Gemini cautery system
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