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Postmortem changes in geometry, density, and sound speed within organs and tissues (melon, bone, blubber, and mandibular fat) of the dolphin head were evaluated using computed tomography (CT) scans of live and postmortem bottlenose dolphins (Tursiops truncatus). Specimens were classified into three different treatment groups: live, recently dead, and frozen followed by thawing. Organs and tissues in similar anatomical regions of the head were compared in CT scans of the specimens to identify postmortem changes in morphology. In addition, comparisons of Hounsfield units in the CT scans were used to evaluate postmortem changes in the density of melon, bone, blubber, and mandibular fat. Sound speed measurements from melon, blubber, connective tissue, and muscle were collected from fresh and frozen samples in the same specimen to evaluate effects due to freezing and thawing process on sound speed measurements. Similar results in tissue and organ geometry, density, and sound speed measurements suggested that postmortem material is a reliable approximation for live melon, bone, blubber, muscle, connective tissue, and mandibular fat. These results have implications for examining viscoelastic properties and the accuracy of simulating sound transmission in postmortem material. Anat Rec, 290:1023–1032, 2007. © 2007 Wiley-Liss, Inc.
The internal structure of complex forms can be studied using X-ray computed tomography (CT). Like most remote imaging techniques, one of the main benefits is that geometric relationships between structures are maintained in the images (Cranford, 1996; Marcucci et al., 2001; Marino et al., 2004; Summers et al., 2004). Furthermore, models of biological systems can be built using accurate morphological descriptions extracted from CT scans and tissue property data gathered from CT images and other techniques (Aroyan et al., 1992; Richardson et al., 1995; Terheyden et al., 2000; Aroyan, 2001; Soldevilla et al., 2005a).
Capturing CT data from live animals is common in humans and a variety of small terrestrial mammals for clinical studies; however, obvious challenges arise when attempting to scan live aquatic mammals; only one study to date has successfully overcome these challenges (Houser et al., 2004). Remote imaging techniques are usually performed on recently dead, frozen, and/or thawed specimens (Cranford, 1996; Maisano et al., 2002; Carpenter et al., 2004). Stranded postmortem marine mammals are often transported to laboratories and sometimes frozen until CT scanning, experiments, or observations can be made. This report is the first study to examine the accuracy of using postmortem toothed whales as representation of live tissue structure and physical properties (density and sound speed).
We are particularly interested in looking at postmortem changes in the acoustic anatomy of odontocetes (toothed whales). The anatomy of sound production/propagation (Fig. 1) is a well investigated topic in odontocetes (Mead, 1975; Heyning, 1989; Cranford, 1992, 1996; Au, 1993; Cranford et al., 1997, 2000). It is generally accepted that click generation begins by action of the palatopharyngeal muscle complex, as it forces the larynx up into the inferior bony nares and pressurizes the air in the bony nasal passages. The pressurized air passes through lips formed by a narrow slit in the spiracular cavity, causing vibrations in adjacent ellipsoid fat bodies. These vibrations are reflected forward by the skull and air sacs functioning as acoustic mirrors. The sound vibrations propagate through the melon anteriorly and emerge into the environment as a click. The melon is a fat and connective tissue organ that functions to focus sound and decrease acoustic attenuation at the tissue–water boundary (Cranford and Amundin, 2004).
Figure 1. Three-dimensional computed tomography image of the sound production anatomy in the bottlenose dolphin head. sk, skull; np, nasal passages; lx, larynx; rbc, right bursae complex; lbc, left bursae complex; me, melon; bc, brain case; ey, eye; bl, blowhole; raon, right anterior orbital notch.
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The geometry and elastic properties within the structures of the forehead will determine the sound propagation and reception pathways. CT provides a means to characterize the geometry and density topography within these organs and tissues (Cranford, 1996; McKenna, 2005; Au et al., 2006). When these data are combined with sound speed measurements and finite element tools, acoustic simulations of the organs are possible (Soldevilla et al., 2005a).
The present study compares CT scans of postmortem and live bottlenose dolphin heads in an effort to understand postmortem effects on tissue and organ structure and physical properties, specifically density. Changes in sound speed are also measured in one specimen before and after the freezing and thawing process. The resulting data will help identify the limitations of using postmortem material to represent living biological systems.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
This study demonstrates that X-ray computed tomography on postmortem material can be used to capture intricate in situ morphology without sacrificing structural integrity, corroborating previous studies investigating postmortem material (Stern and Webb, 1993; Estes et al., 1998; Schroeder, 2001). We believe that the significance of the study is that the two independent analyses (Hounsfield and morphological comparisons) showed little difference between live and postmortem biological material. In fact, the internal morphological structures were highly correlated. Based on these findings, accurate descriptions or models of internal structures and their geometry are possible from postmortem animals, with a general recommendation of CT scanning as soon after death or thawing as possible.
The comparisons of forehead and melon structure between the CT scans of THAWED and FRESH versus the LIVE specimen showed similar anatomic organization, although some external deformities were introduced by the disarticulation of the head from the body. THAWED B showed the greatest degree of external deformation (Fig. 3). The degree of deformation may be correlated with the length of time from death before placement in the freezer; THAWED B was put in the freezer 6–8 hr from death. Freezing the head immediately after disarticulation is important for maintaining the least disturbed external structure. We assume that similar external deformation as we found in THAWED B would be apparent if the CT scan of a FRESH specimen was not performed less than 6 hr after disarticulation from the body; however, additional specimen comparisons are needed to make specific time frame recommendations.
The structure and geometry of deeper anatomic structures remained intact in both the THAWED and FRESH specimens, when the CT images were compared with a scan of a LIVE animal. This study demonstrated the strong correlation between line profiles through both the forehead and melon (Table 2). This study did not determine a time from death when significant deformation can be observed in internal structures, but to observe structural integrity in the internal tissues, CT scanning or freezing should happen within 10 hr from death based on the maximum amount of time our samples were left before scanning. However, additional specimens are needed to make any specific recommendation.
Comparisons of H values within different tissue groups (bone, melon, blubber, mandibular fat) did vary slightly, suggesting postmortem effects on tissue properties, namely density. However, we did not control for potential differences in scanner parameters, and it is difficult to determine whether the changes were related to actually postmortem changes or difference in scanner settings. The importance of these findings is that the difference in the H values is small, suggesting that postmortem changes and/or scanner effects are minimal.
The largest variations in the H units were found in the bone, both within each specimen and across treatment groups. A possible explanation for this difference is related to the composition and structure of odontocete bones. The density of the odontocete skull varies with specific bones and regions, and in general, the bones contain a significant amount of fat molecules (Rommel, 1990). Because of this matrix of material, the variation measured in this study might reflect the sensitivity of H unit measurements.
Melon, blubber, muscle, and mandibular fat are more homogenous materials and the mean H values showed a lesser degree of discrepancy among the three treatment groups (LIVE, FRESH, and THAWED). The differences between the mean melon, blubber, muscle, and mandibular fat H values were within the standard deviations of a previous study examining physical properties of soft tissues in whales (Soldevilla et al., 2005b), suggesting that there is little electron density difference between melon, blubber, muscle, and mandibular fat for live and dead specimens.
Some of the differences in each anatomic region might be a result of temperature variation during the scanning process. Because H values are calibrated based on small changes in the temperature of water, one potential source of error could result from temperature differences between specimens. The LIVE animal was removed from the water for the scanning process, so the temperature profiles might have been effected by elective circulation, as mentioned in the original study (Houser et al., 2004). Assuming the LIVE was scanned at body temperature and the FRESH and THAWED at room temperature, a 6 H unit difference is expected (Kreel and Bydder, 1979). The additional differences between the LIVE and FRESH with the THAWED values suggest alterations caused by the freezing and thawing process and/or differences between scanners.
It is noteworthy that the smallest changes in H values occurred in the tissues we know to be associated with sound propagation (melon), which is contrary to previous studies that suggested the melon in odontocetes may be particularly sensitive to postmortem effects because it contains specialized acoustic fats (Malins and Varanasi, 1975; Varanasi et al., 1975). In addition, the morphological comparisons showed little change in melon structure. The difference between the highest density in the THAWED specimens and the lowest H values in the LIVE specimen can be explained by the specific composition of the biological material. Water comprises 75% of most soft tissues, and the physical properties of the tissue are consequently dominated by its presence (Duck, 1990). Thawed postmortem specimens must be kept from dehydrating if reliable information is to be extracted using CT. Flash freezing, although not investigated in this study, might produce the most reliable results when working with frozen postmortem biological material.
In the freezing and thawing experiments, the sound speed measurements in the tissue groups (melon, connective tissue, blubber, and muscle) remained fairly constant until approximately 120 hr after death (Fig. 6). The insignificant effect on sound speed from the freezing and thawing process found in this study is consistent with the results of previous studies. Ultrasonic sound speeds through frozen and thawed tissues were compared with fresh tissues, and no significant difference was reported for the human breast (Foster et al., 1984), myocardial tissues (Dent et al., 2000), and mammalian tissues (Van der Steen et al., 1991).
The small changes in sound speed observed in all tissue types at 25 hr after death should be investigated further using a larger sample size. The measured changes might be a result of the handling process and/or loss of fluid from the tissues. The large variance in the blubber samples near the surface of the heads in this study and in that of Fitzgerald (1975) may be a result of the loss of water from the collagen fiber matrix from dehydration in the freezing process. There is also a potential for changes to happen in the first 6 hr after death before the first data points were collected for this study (Kremkau et al., 1981).
The results of this study suggest that postmortem biological material can represent live tissues both in morphology and tissue properties, but within certain limits. The limits established in this study combined with previous studies provide a framework for quantifying the viscoelastic properties of mammalian tissues that are not easily measured in vivo. Physical properties of noncontractile soft tissues have been investigated as a function of time after death. Kremkau et al. (1981) found a decrease in ultrasonic sound speed in the human brain within the first 24 hr of death, but then found sound speed to be constant thereafter. For the human lens and vitreous, no change in ultrasonic sound speed 70 hr after death was found (Jansson and Kock, 1962). Investigation into the viscoelastic properties of canine intervertebral discs, whale blubber, beef fat, and human bone, however, show a dramatic change within the first 12 hr of death (Fitzgerald, 1975; Fitzgerald and Fitzgerald, 1995). Although the response of specific tissue types and organs from postmortem effects is important, when modeling a functioning biological system, treatment of the specimens should be conservative and measurements should always be taken as soon as possible after death based on the results of this study.
Although live tissue offers the most reliable results to answer physiological questions, the results of this study support the use of postmortem CT data to extract anatomic geometry and some tissue properties. Furthermore, our results have important implications for modeling the acoustic function of cephalic tissues in odontocetes. The potential for extracting data from CT images from postmortem animals to estimate tissue properties for finite element models is promising. These modeling techniques can be used to simulate propagation of biologically relevant sounds and noise produced in the surrounding environment and the interactions with the tissues.
Can the tissue properties measured in this study be expanded to understand additional properties? Longitudinal wave velocity is determined by the bulk modulus, rigidity modulus, and density of the tissue (Duck, 1990). Although this study did not measure the elasticity of tissues, the combined consistency of density (Fig. 5) and sound speed (Fig. 6) indicate that the bulk modulus for these tissues were conserved through each treatment. Furthermore, the mechanical properties of various connective tissues were not found to be significantly different from fresh tissue after freezing and thawing (van Brocklin and Ellis, 1965; Woo et al., 1986; Quirinia and Viidik, 1991). In contrast, Krag and Andreassen (2003) found a 20% decrease in the tensile elastic modulus of the porcine eye lens. In a study that compared the elastic shear modulus and dynamic viscosity of fresh dog vocal fold tissues to thawed tissues that were frozen at different rates, no difference for tissues that were quick frozen but significant changes for slowly frozen tissues were found (Chan and Titze, 2003). Although further analyses are needed to determine whether additional properties can be drawn from those measured in this study, the results provide a framework for future analyses.
This study supports the legitimacy of using CT data from postmortem specimens for addressing research questions investigating morphology, tissue properties, and biologically functioning tissues. At the same time, workers should be cognizant of the limits of these techniques and design experiments conservitively based on procedures presented in this study.