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Biology

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

Published: October 19th, 2016

DOI:

10.3791/54550

1NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 2Department of Biomedical Engineering, National University of Singapore, 3Department of Surgery, 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.

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....

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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

  1. Vessel cannulations
    1. Anesthetize a male Sprague-Dawley rats (6 - 7 weeks old) weighing (203 ± 20) g with ketamine (37.5 mg/ml) and xylazine (5 mg/ml ) cocktail through intraperitoneal (i.p.) injection (2 ml/kg). Do not recap the needle or remove it from the syr.......

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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.......

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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.......

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

....

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Name Company Catalog Number Comments
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 1x2 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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