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

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 v....

Protocol

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 syringe after the injection.
   2. Once the animal has been anesthetized (confirmed by toe pinching), place it on a heating pad to maintain its body....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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 (5,000 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 x 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