Propagation speed of sound assessment in the layers of the guinea-pig esophagus in vitro by means of acoustic microscopy
Introduction
The advancement of science in recent years follows a trend of needing to know greater details in physiology, medicine and tissue engineering. Obtaining morphometric and biomechanical data from several adjacent layers of tissue is a step towards the next level in the hierarchy of structure of living tissue. Such data are needed in a mechanical analysis of a composite biological structure. Thus far, Yu et al. [1] presented a two-layer model based on bending experiments for determination of stress–strain properties. Gregersen et al. [2] studied the zero-stress state of the layers of guinea-pig esophagus and found significant differences in residual strain between the muscle and mucosa–submucosa layers. Realizing that obtaining mechanical data on individual layers of biological tissues are useful, we need to develop new methods to characterize layered organs to a greater extent. In this study we employed a scanning laser acoustic microscope (SLAM) to obtain a better understanding of the composite properties of the esophagus.
The practical application of ultrasound for imaging was demonstrated by the French physicist Paul Langevin during World War I [3]. In the early 1930s Sokolov explored the usefulness of ultrasound for imaging internal structures in optically opaque objects [3], [4] and he was first to suggest ultrasound at 3 GHz for imaging small objects. This technique is now known as acoustic microscopy, which is defined as a general term for high resolution, high frequency ultrasonic inspection techniques that produce images of features beneath the surface of the sample. Further developments led to the SLAM which was introduced by Korpel and coworkers [5]. The SLAM is a transmission mode instrument that creates real-time acoustic images of a sample throughout its entire thickness. A collimated continuous-wave ultrasound beam at frequencies from 10 to 500 MHz is produced by a piezoelectric transducer located beneath the sample (for this study, 100 MHz was used). When the ultrasound wave propagates through the sample, the wave is affected by mechanical inhomogeneities in the material. A scanned laser beam is used as the ultrasound detector (Fig. 1). The ability of the SLAM to produce simultaneously optical and acoustic images from which the acoustic properties of the specimen can be calculated make easy its use in this field of biology. The ultrasonic attenuation and propagation speed can be estimated from the obtained information. Conventional tissue fixation and staining are not required for the SLAM imaging; this allows for studies of living cells and tissues [6]. The SLAM has been found useful for the in vitro assessment of acoustic properties in biological materials such as skin [7], kidney [8] and liver [9].
The SLAM technique was thus used as the method for estimating propagation speed in the simple layered structure of the guinea-pig esophagus. The esophagus consists of an innermost mucosa–submucosal layer (hereafter referred to as the mucosal layer) that mainly consists of connective tissue with blood vessels and nerves, and outermost longitudinal and circumferential muscle layers. The esophagus is an important organ to study due to its mechanical function and composite structure. Furthermore, diseases can cause structural and biomechanical remodeling in the esophagus.
The propagation speed in the individual layers of the esophagus has, to the best of our knowledge, not been presented in the literature. The aim of this study was to determine the propagation speed in the individual layers of esophagus in vitro in the no-load state with all external forces removed, in the distended state and in the zero-stress state. The distended state corresponds to the physiological state where a bolus of fluid or food passes through the esophagus. The no-load state is the conditions where no external forces are applied, i.e., zero pressure from outside and inside. The no-load state was for many years considered to be the reference state for mechanical analysis, i.e., the reference length for strain. However, we know now that residual stresses may reside in the no-load state. The cut-open state, also called the zero-stress state, is the condition where also the residual (internal) forces have been released by making a radial cut through the wall. The zero-stress state is important in mechanics because it is the state to where all the stresses and strains refer.
Section snippets
Specimen preparation
Twenty-six 700–900-g female guinea pigs were euthanized using pentobarbital, and a long midline cut was made in the neck and chest. Calcium-free Krebs solution was poured into the chest cavity. The esophagus was separated from adjacent structures from the tongue to the stomach. A 4-cm-long segment beginning 2 mm from the root of the tongue was excised. The tissue was cleaned and snap-frozen in one of four different states (see below) in liquid nitrogen and stored at −80°C.
At the time of sample
Results
The propagation speed from the different layers and preparations are given in Table 1. It was not possible to distinguish the circular and longitudinal muscle layers in any of the preparations, though the sound beam direction was parallel to the longitudinal muscle layer and perpendicular to the circumferential muscle layer. It was a general finding that the propagation speed was higher in the muscle layer than in mucosa (p<0.001 for the distended, no-load and zero-stress states). The most
Discussion
In this paper we show that it is possible with SLAM to image sections of the guinea-pig esophagus and quantitatively distinguish its layered topography. As far as we know there are no other acoustic microscope studies that have investigated the layered structures in esophagus. The wall can be quantitatively characterized and its layers distinguished by use of the SLAM propagation speed profile. We aimed to study the properties at different states of mechanical loading. The distended state
Acknowledgements
This work is partially supported by a grant from The Danish Research Councils (9501709), Karen Elise Jensens Foundation, and the US National Institutes of Health (CA09067). The authors acknowledge the help from James F. Zachary, DVM, Ph.D., Veterinary Pathobiology, and Ann C. Benefiel, Biological Resources, Beckman Institute for Advanced Science and Technology, both at the University of Illinois.
References (24)
Reference liquid for ultrasonic attenuation
Ultrasonics
(1989)- et al.
Neutral axis location in bending and Young's modulus of different layers of arterial wall
Am. J. Physiol.
(1993) - et al.
Strain distribution in the layered wall of the esophagus
J. Biomech. Engng.
(1999) - F.V. Hunt, Origins of Acoustics New Haven, Yale University Press, New Haven, CT...
- G. Wade, Acoustic Imaging, Plenum Press, New York,...
- et al.
Acoustic microscope operating 100 MHz
Nature
(1971) - et al.
Amphibian limb regeneration curves generated by the scanning laser acoustic microscope
J. Histochem. Cytochem.
(1986) - et al.
Measurement uncertainty assessment of the scanning laser acoustic microscope and application to canine skin and wound
IEEE Trans. Ultra Ferroelec. Freq. Contrl.
(1988) - et al.
Acoustic microscope of mammalian kidney
J. Clin. Ultrasound
(1974) - et al.
Ultrasound attenuation and velocities in rat liver as a function of fat concentration: a study at 100 MHz using a scanning laser acoustic microscope
J. Acoust. Soc. Am.
(1985)
Simultaneous acoustic and optical microscopy of biological specimens
Nature
Attenuation coefficient measurement technique at 100 MHz with the scanning laser acoustic microscope
IEEE Trans. Sonics Ultrason.
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